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CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of Provisional Application No. 61/177,203, filed May 11, 2009, the entire disclosure of which is hereby incorporated by reference herein.
BACKGROUND
[0002] Hitch assemblies provide a connection between a device, such as a trailer, ski rack, or the like, and a vehicle. A receiver-type hitch assembly typically includes a receiver that is attached to the frame of a vehicle and a hitch that is removably inserted into the receiver. The hitch may include, for example, a conventional trailer ball that is sized to be engaged by a ball receiver on a trailer. Alternatively, the hitch may comprise a portion of a carrier, for example, a bicycle carrier, ski carrier, cargo carrier, or the like. The hitch may include additional or alternative mechanisms for engaging an apparatus to be carried or towed. A conventional SAE receiver typically comprises a rectangular tube with a rearwardly facing square opening that is 1.25 inches (32 mm), 2.0 inches (51 mm) or 2.5 inches (64 mm) square.
[0003] The insertable hitch includes a shaft having an outer dimension that is somewhat smaller than the inner dimension of the receiver so that the hitch can be relatively easily inserted into the receiver. A hitch pin (or locking pin) is inserted through holes provided in the side walls of the receiver and alignable holes in the hitch. The locking pin may be secured, for example, with a retaining clip to prevent the locking pin from inadvertently coming out during use. Exemplary prior art hitch assemblies include those disclosed in U.S. Pat. No. 6,105,989, to Linger, which is hereby incorporated by reference in its entirety, and in U.S. Pat. No. 6,382,656, to Johnson, Jr., which is hereby incorporated by reference in its entirety.
[0004] Detachable hitches are preferred for many applications. For example, a user may use one hitch for towing loads and other hitches for attaching bicycle racks, ski racks, carriers, or the like, to the vehicle. Also, hitches typically extend beyond the rear of the towing vehicle to enable attachment of a trailer to the hitch with clearance for the trailer and towing vehicle to articulate relative to each other during towing. The protruding hitch with a ball attachment can be bothersome and dangerous when the vehicle is used without the trailer attached; therefore, it is beneficial to be able to remove the hitch when it is not needed.
[0005] However, as noted above the hitch shaft is smaller than the receiver opening, and so the fit between the hitch and the receiver includes some play between the receiver and the walls of the hitch shaft. The relatively loose fit permits undesirable relative movement or play between the receiver and the hitch, which can be noisy and annoying. The play between the walls of the receiver and hitch can cause clanging noises and vibrations that can be felt by operators and passengers within the towing vehicle. The play may also be magnified by the lever arm of the hitch so that it is felt more strongly by the trailer. That same play can also increase wear and stress on various parts of the mechanisms attaching the trailer to the towing vehicle, leading to undesirable wear and fatigue.
[0006] The disadvantages of the relatively loose fit between the receiver and hitch coupling have been recognized by others. For example, in U.S. Pat. No. 6,974,147, to Kolda, which is hereby incorporated by reference, a mechanism for preventing relative movement between these members is disclosed, wherein the tow bar or mounting member is provided with a cam that is adjustably urged into the mounting member and abuts the hitch pin. The adjustment mechanism causes the cam to rotate, extending through a slot in the mounting member, and is urged against the receiver. However, the mechanism has the disadvantage that it presses against the receiver at a single position and against the opposite side of the mounting member at a single position, in addition to the hitch pin, which may still permit some movement between the mounting member and receiver.
SUMMARY
[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0008] An anti-rattle hitch is disclosed having an insert that is configured to be inserted into a receiver. For clarity, the hitch will be described with directional references, such as “upper” and “lower,” although it will be appreciated that the particular orientation may be different. The insert has an upper wall with a piston aperture, a lower wall, and two side walls. A lower wedge block and an upper wedge block are disposed in the insert. The lower wedge block has a lower surface that slidably engages the lower wall of the insert, and an upper angled surface. The upper wedge block has a lower angled surface that slidably engages the lower wedge block, and a piston that extends through the piston aperture, such that the longitudinal position of the upper wedge block is constrained by the piston. An adjustment member engages the lower wedge block, extends out of the insert, and is operable to adjust the longitudinal position of the lower wedge block, thereby adjusting the transverse position of the upper wedge block. This configuration allows adjusting the position of the piston that extends out of the insert. The hitch is configured such that the piston may be adjusted to press against the receiver, thereby locking the hitch therein and avoiding play therebetween.
[0009] In an embodiment of the invention, the piston comprises a cylinder that is attached to the upper wedge member with a screw.
[0010] In an embodiment of the invention, a second piston aperture is provided through the insert, and a second piston is attached to the upper wedge block and extends through the second piston aperture.
[0011] In an embodiment of the invention, the hitch includes a ball mount member that is configured to support a tow ball.
[0012] In an embodiment of the invention, the adjustment member is a threaded rod that threadably engages the first wedge member and a head that extends out of the tubular insert.
[0013] In an embodiment of the invention, the adjustment member includes a security feature, such as a lock or a keyed head, that hinders operation of the adjustment member without a corresponding tool.
[0014] In an embodiment of the invention, a low friction panel is provided between the angled faces and may comprise an ultrahigh molecular weight polyethylene.
[0015] In an embodiment of the invention, the wedge blocks further include second angled faces that are slidably engaged.
DESCRIPTION OF THE DRAWINGS
[0016] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
[0017] FIG. 1A is a perspective view of a hitch assembly in accordance with the present invention;
[0018] FIG. 1B is another perspective view of the hitch shown in FIG. 1A ;
[0019] FIG. 2 is an exploded view of the hitch assembly shown in FIG. 1A ; and
[0020] FIG. 3 is a cross-sectional view of the hitch assembly shown in FIG. 1A .
[0021] FIG. 4 is a fragmentary end view of the hitch shown in FIG. 1A .
DETAILED DESCRIPTION
[0022] FIG. 1A is a perspective view of a hitch assembly 100 in accordance with the present invention and showing in phantom a receiver 90 and a tow ball 101 . A three-quarter rear perspective view of the hitch assembly 100 is shown in FIG. 1B . The hitch assembly 100 includes a tubular insert 102 that is sized and configured to be inserted into a receiver 90 to cooperatively comprise a receiver-type hitch assembly. The tubular insert 102 is a substantially square tube. In an exemplary embodiment the tubular insert 102 is sized to engage an SAE standard receiver having a square opening that is 1.25 inches (32 mm), 2.0 inches (51 mm) or 2.5 inches (64 mm) on each side. The tubular insert 102 is fixedly attached to a structural member, for example a ball mount 103 that is configured to support a tow ball 101 . For example, the tow ball 101 ( FIG. 1A ) may bolt through aperture 107 ( FIG. 1B ) in the ball mount 103 . An optional gusset plate 105 reinforces the connection between the tubular insert 102 and the ball mount 103
[0023] The tubular insert 102 includes a first wall 102 A (in this case the upper wall), oppositely disposed second and third walls 102 B, 102 C (e.g., side walls), and a fourth wall 102 D (e.g., lower wall) disposed opposite the first wall 102 A. As seen most clearly in the exploded view of FIG. 2 , the first wall 102 A includes a pair of longitudinally spaced piston apertures 104 . The second and third walls 102 B, 102 C each have a locking pin aperture 106 (one visible), which are aligned to receive a conventional locking pin (not shown). Corresponding locking pin apertures 96 are provided through the receiver 90 . Also visible in FIG. 1A are a pair of adjustable pistons 110 , which are discussed in more detail below.
[0024] FIG. 2 shows an exploded view of the hitch 100 . Refer also to FIG. 3 , which shows a longitudinal cross-sectional view of the hitch 100 , taken through a centerline of the tubular member 102 . A sliding wedge mechanism is disposed in the tubular insert 102 and is operable to selectively tighten the hitch 100 within the receiver 90 , thereby reducing or eliminating play between the hitch 100 and the receiver 90 . The wedge mechanism includes a first wedge member 112 , defining a first angled face 112 A, a second angled face 112 B and a recess 112 C therebetween. A threaded aperture 113 is oriented longitudinally from a proximal end of the first wedge member 112 .
[0025] A second wedge member 114 is positioned generally adjacent the first wedge member 112 and includes a first angled face 114 A, a second angled face 114 B, and a recess 114 C therebetween. When assembled, the first wedge member first angled face 112 A is disposed adjacent the second wedge member first angled face 114 A, and the first wedge member second angled face 112 B is disposed adjacent the second wedge member second angled face 114 B to slidably engage the first wedge member 112 when the hitch 100 is assembled.
[0026] The pistons 110 are attached to an upper face 114 D of the upper block 114 . In the present embodiment the attachment is accomplished with flathead fasteners 110 A, although other attachment means may be used, including for example by forming a post (threaded or unthreaded) on the bottom of the pistons, with corresponding apertures in the second wedge member 114 . Optionally, recesses 114 E are provided in which the pistons 110 are securely seated.
[0027] A threaded adjustment fastener 120 extends through an aperture 108 in the ball mount 103 and into the tubular insert 102 to threadably engage the first wedge member 112 threaded aperture 113 . Optionally, an angled spacer 122 and spring, or other biasing member 124 , are also provided. It will now be appreciated that the longitudinal position of the second wedge member 114 is constrained within the tubular insert 102 by the pistons 110 extending through the piston apertures 104 . The position of the first wedge member 112 is adjusted with the adjustable fastener 120 .
[0028] The first and second wedge members 112 , 114 angled faces 112 A, 114 A, and 112 B, 114 B are configured to slidably engage. In this embodiment, low friction pads 118 are provided between the respective angled faces. For example, low friction pads may comprise polymeric material. In a current embodiment, the low friction pads comprise ultrahigh molecular weight polyethylene, which has a very low coefficient of friction, is self-lubricating, and is highly resistant to abrasion.
[0029] The wedge member recesses 112 C, 114 C are sized and shaped to cooperatively define an opening therebetween that is aligned with the locking pin apertures 106 in the tubular insert 102 (which are also alignable with corresponding apertures 96 in the receiver 90 ), such that the wedge members 112 , 114 will not interfere with the locking pin during use.
[0030] A fragmentary end view of the hitch 100 is shown in FIG. 4 , showing the first wedge member 112 and the lower portion of the tubular insert 102 . In this embodiment, the lower surface of the first wedge member 112 is provided with longitudinal ribs 112 D to reduce friction between the first wedge member 112 and the tubular member 102 , and thereby facilitate adjustment of the wedge mechanism. It is further contemplated that a low friction panel or other friction-reducing mechanism (not shown) may be provided between the first wedge member 112 and the tubular insert 102 .
[0031] In the present embodiment, the hitch 100 is assembled by inserting the adjustment member 120 through the aperture 108 in the ball mount 103 and inserting the angled spacer 122 and spring 124 through the open end of the tubular insert 102 to slide over the adjustment member 120 . The first and second wedge members 112 , 114 are inserted together into the tubular insert 102 and the adjustment member 120 engages the threaded aperture 113 . The second wedge member is then positioned such that the recesses 114 E are aligned with the piston apertures 104 , and the pistons 110 are inserted through the respective piston apertures 104 and attached to the second wedge member 114 .
[0032] To use the hitch 100 , the adjustment member 120 is adjusted such that the pistons 100 are approximately flush with the first wall 102 A of the tubular insert 102 . The hitch 100 may then be inserted into the receiver 90 . The adjustment member 114 is then adjusted such that the first wedge member 112 is drawn to the right in FIG. 3 , as indicated by arrow 80 . The second wedge member 114 is restrained from moving longitudinally by the pistons 110 . Due to the angled faced of the first and second wedge members 112 , 114 , the second wedge member 114 moves upwardly as indicated by arrow 81 , such that the pistons 110 move upwardly to engage and press against the receiver 90 , as indicated by arrows 82 . The locking pin (not shown) is then inserted through the locking pin apertures 96 , 106 .
[0033] To disengage the hitch 100 from the receiver 90 , the adjustment member 120 is adjusted in the reverse direction. After removing the locking pin, the adjustment member 120 is adjusted in the opposite direction. The biasing spring 124 aids in moving the first wedge member to the left in FIG. 3 , and the pistons 110 disengage from the receiver 90 , such that the tubular insert 102 can be readily pulled out of the receiver 90 .
[0034] It is also contemplated that the adjustment member 120 may include one or more security features, such as a lock or the like. In an embodiment the adjustment member incorporates an unusual head shape, such that the adjustment member is not easily adjusted without a corresponding, suitably keyed tool (not shown). This security feature provides the additional advantage that once the hitch 100 is securely locked to the receiver 90 , the hitch 100 cannot be easily removed from the vehicle without the special tool. This will provide the additional advantage of protection from theft.
[0035] Although not required for the present invention, in an exemplary embodiment the first and second wedge members 112 , 114 may be formed from a relatively soft material such as aluminum or a composite material, and the tubular insert 102 and ball mount 103 may be formed from a conventional rugged material such as steel.
[0036] As discussed above, the hitch 100 may alternatively be configured as a portion of any hitchable device, for example, a bicycle carrier, ski carrier, or the like. Also, although the current hitch 100 includes two generally cylindrical pistons 110 that engage the receiver 90 , it would be straightforward to change the number of pistons and/or to use other shapes or sizes of members for engaging the receiver. For example, it is contemplated that the second wedge member 114 may be provided with four smaller pistons or protrusions disposed generally at the corners of the second wedge member 114 , with corresponding apertures in the tubular sleeve member 102 .
[0037] Although a currently preferred embodiment has been described, many modifications may be made to this embodiment without departing from the present invention. For example, it is contemplated that the first and second wedge members 112 , 114 may be formed from some alternate material, such as a polymer or composite material. Also, where threadable connections are shown, it will be appreciated that other connection means, as are known in the art, may alternatively be used. It is also contemplated that a cover or other blocking means may be provided on the end of the tubular sleeve member 102 , to deter foreign matter from entering the member.
[0038] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
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An anti-rattle hitch ( 100 ) includes a tubular insert ( 102 ) that is configured to be inserted into a receiver ( 90 ). A wedge mechanism comprising a first wedge member ( 112 ) and a second wedge member ( 114 ) are disposed in the insert, and have slidably engaged angled faces. The second wedge member includes one or more pistons that extend through piston ( 110 ) apertures in the insert such that the longitudinal position of the second wedge member is fixed. An adjustment member ( 120 ) adjusts the position of the first wedge member, thereby adjusting the piston position which can be biased against the receiver.
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BACKGROUND OF THE INVENTION
[0001] Cranes are used for heavy lifting, but weigh several tons (since they typically use heavy counterweights) and typically are stationary, with a predefined range or so-called ‘reach.’ Some cranes are mounted on wheels, thus rendering them tow-able or even drivable, but they still suffer from the same weight and reach problems. Moreover, cranes are very expensive to own and operate. Forklifts have the same problems of high weight and cost, and suffer even more limited reach relative to their counterweighted base, but they are maneuverable. Neither cranes nor forklifts are lightweight and portable enough to be employed in rooftop installations.
[0002] Auto shop engine pullers use hoists, e.g. cable and pulley systems, or hydraulics to meet medium load lifting needs. They are generally fixed in position and do not break down easily for transporting to a different work site. Moreover, such an engine puller typically has a negative range, i.e. its effective lift range is within the perimeter of its base's footprint.
[0003] Rooftop installations, e.g. of heating/ventilation/air conditioning/refrigeration (HVAC/R), often require lifting of light to medium loads of less than approximately 1000 pounds. It is most often cost-prohibitive to do a rooftop installation or replacement, e.g. of an air conditioning unit, using a crane. A typical shop forklift weighs upwards of twelve tons, exceeding the load capacity of most rooftops. In any event, a crane would typically be required to lift the forklift onto the rooftop. Hydraulic/pneumatic lifts are heavy and difficult to transport. Moreover, a hydraulic/pneumatic lift requires power and/or a hydraulic/pneumatic source.
SUMMARY OF THE INVENTION
[0004] Manual boom lift apparatus and method involve a base having three support legs, a fulcrum configured to fixedly mount the legs, a boom member detachably mounted on the fulcrum, the boom member including on either end a counterbalance arm configured for detachably mounting one or more counterweights and a lift arm configured for hoisting a load, the lift apparatus enabling lift and placement of the load by pivotal manipulation of the boom member. Assembly of the detachable boom lift apparatus components is performed on site (in situ) and involves removably pinning aligned hole pairs to join the components and filling one or more containers with ballast to act as counterweights to the hoisted load. The apparatus is lightweight and durable, is easy to transport through small openings and can be used in rooftop installations of heating, ventilation, air conditioning and refrigeration (HVAC/R) equipment.
[0005] These and additional objects and advantages of the present invention will be more readily understood after consideration of the drawings and the detailed description of the preferred embodiment which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is an isometric view in exploded assembly form of the invented apparatus in accordance with one embodiment of the invention, with the counterbalance weights shown only in phantom.
[0007] Detail A corresponds with FIG. 1 , but shows in enlarged fragmentary, assembled, isometric view the fulcrum mechanism that forms a part of the invented apparatus in accordance with one embodiment of the invention.
[0008] FIG. 2 is a front elevation corresponding with FIG. 1 but with the boom omitted for clarity.
[0009] FIG. 3 is a side elevation corresponding with FIG. 2 .
[0010] FIG. 4 is an isometric view in exploded assembly form of the invented apparatus in accordance with a second embodiment of the invention, with the counterbalance weights shown only in phantom.
[0011] FIG. 5 is an isometric view in exploded assembly form of the invented apparatus in accordance with a third embodiment of the invention, with the counterbalance weights shown only in phantom.
[0012] FIG. 6 is a front elevation corresponding with FIG. 5 , with the boom omitted for clarity.
[0013] FIG. 7 is a side elevation corresponding with FIG. 5 , but with the boom included, and with the counterbalance weights shown only in phantom.
[0014] FIG. 8 is a flowchart illustrating the invented boom lift method of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The invented method and apparatus provide a low-cost, manual, portable, lightweight boom lift that lends itself to rooftop installations, at elevations above the rooftop of up to approximately ten feet, of light-to-medium loads up to approximately 1000 pounds. The apparatus is assembled by pivotally pinning, e.g. with one or more cotter keys, various relatively lightweight members together on site (in situ) and to charge the counterweight arm end of the boom lift opposite the lift arm also on site, thus greatly facilitating maneuverability in transport, employment and deployment. The counterweights preferably are water-chargeable containers. The boom lift leverages its load by a simple 2:1 mechanically advantaged boom lift manipulation atop its tripod support legs-and-wheels arrangement. Neither hydraulic nor pneumatic nor power conduits are required for operation. The lightweight extruded square-tubular and die-cast aluminum and steel materials and simple structural geometries render the apparatus very low cost. The apparatus is structured for ready break-down and thus has a relatively small footprint in use and an even smaller footprint in transit to and from a work site. These and other advantages will be more apparent from the detailed discussion below.
[0016] FIG. 1 illustrates in isometric view the invented manual boom lift apparatus 10 in accordance with a first embodiment of the invention. Apparatus 10 includes a base 12 having three or more support legs 14 , 16 , 18 . It further includes a fulcrum mechanism 20 configured to fixedly mount support legs 14 , 16 , 18 in a tripod configuration, as shown. Apparatus 10 further includes a boom member 22 detachably and pivotally mounted on fulcrum mechanism 20 . Boom member 22 includes fore and aft respectively a counterweight arm 24 configured for detachably mounting one or more weights 26 b, 26 c, 26 d, 26 e, 26 f (shown in phantom) and a lift arm 28 configured to hoist a load 30 (also shown in phantom). Those of skill in the art will appreciate that lift apparatus 10 enables lift and placement of the load by manual pivotal manipulation of boom member 22 .
[0017] Base 12 further includes a generally triangular, hinged, pivotal brace mechanism 32 that includes three brace members 34 , 36 , 38 ; a snapper pin 40 for fixing it in place between support legs 14 , 16 , 18 and a generally square fulcrum base plate 42 . Those of skill in the art will appreciate that support legs 14 , 16 , 18 are welded or otherwise durably and fixedly mounted to a lower surface of a tripod cap 42 . At the base of each of support legs 14 , 16 , 18 is a generally square pad such as pad 44 for mounting a wheel, as will be described below by reference to FIGS. 6 and 7 .
[0018] Fulcrum, or pivot/support, mechanism 20 will be described below in more detail by reference to detail A of FIG. 1 . From FIG. 1 , however, it may be seen to include a frame 46 defining a vertical rectangular channel 48 through which boom member 22 extends horizontally for pivotal mounting at a desired height within the channel. Those of skill in the art will appreciate that height adjustment and pivotal mounting of boom member 22 in fulcrum mechanism 20 is made possible by providing a vertical array of opposing through-hole pairs formed in the upright sidewalls of frame 46 as shown. Fulcrum mechanism 20 is fixedly and durably mounted atop tripod cap 42 as by seam or spot welding (the former being most preferred) or other suitable technique.
[0019] In accordance with one embodiment of the invention, boom member 22 includes three separate sections or components: A lift member 50 and a counterbalance member 52 receive in proximal ends thereof a pivot member 54 , with each of the lift member 50 and counterbalance member 52 pinned to pivot member 54 through corresponding receiving holes and with pivot member 54 pinned at a desired height within frame 46 . As will be better seen by reference below to Detail A, three hitch pins 56 are used to pin counterbalance member 50 to pivot member 54 , lift member 52 to pivot member 54 and pivot member 54 to pivot frame 46 . A load attachment mechanism 58 for securing load 30 is provided at a distal end of lift member 50 , and a counterweight support member 25 is provided at a distal end of counterbalance member 52 for carrying one or more counterweights 26 a, 26 b, 26 c, 26 d, 26 e, 26 f. (Those of skill in the art will appreciate that, in accordance with one embodiment of the invention, counterweight support member 25 weighs approximately 30 pounds, thus effectively causing rearward counterbalance member 52 of boom 22 to pivot downwardly toward the support surface, e.g. a roof, when the boom is charged with neither load nor counterweight.)
[0020] Invented apparatus 10 in use is operated manually to lift and maneuver load 30 as desired by manually manipulating counterbalance member 52 . Those of skill in the art will appreciate that a mechanical advantage of approximately 2:1 is obtained by a 2:1 length ratio between counterbalance member 52 and lift member 50 . Moreover, load 30 is effectively counterbalanced by one or more counterweights 26 a, 26 b, 26 c, 26 d, 26 e, 26 f to facilitate maneuvering the load into proper position and orientation. More or fewer counterweights 26 a, 26 b, 26 c, 26 d, 26 e, 26 f can be used to roughly adjust the counterbalancing effect on variable loads. Also, by virtue of the novel construction of the counterweights themselves, very fine adjustment of counterbalancing effect is possible.
[0021] This is because, in accordance with one embodiment of the invention, the counterweights are ballast-filled containers to and from which ballast can be added or subtracted. Preferably, the counterweights are made of water-fillable, sealed containers that can be simply filled in situ (at the site where the apparatus is to be employed in lifting and positioning a load) and slid onto either side of counterweight support member 25 , as shown in FIG. 1 . Thus, in accordance with one embodiment of the invention, infinite adjustment of counterbalance effect can be achieved for delicate load handling tasks. Moreover, the lightweight portability of invented apparatus 10 is not compromised by the fixed weight of an integral counterweight. Instead, the containers can remain empty (and thus as light as air) while the boom lift apparatus is positioned at the installation site, e.g. on top of a roof, and then ballast, preferably liquid and most preferably water, can be introduced into the containers to a desired fill factor and corresponding weight.
[0022] Those of skill in the art will appreciate that six 6-gallon containers when filled with water (weighing approximately 8.3 pounds/gallon) would weigh approximately 300 pounds, which when added to the 30-pound weight of counterweight support arm 25 would provide adequate counterweight to an approximately 660 pound load. This is because of the 2:1 leverage obtained by use of the invented boom lift having a longer counterbalance arm and a shorter load lift arm, as described and illustrated herein. Importantly, smaller or larger loads are accommodated as well, by simply reducing or increasing the mass of the counterweights that are secured to counterweight support member 25 (the length of which may, within the spirit and scope of the invention, be decreased or increased to accommodate fewer or more containers). Those of skill in the art also will appreciate that alternative counterweight numbers and configurations are within the spirit and scope of the invention.
[0023] After use, the water or other ballast can be dumped or siphoned from the containers and the boom lift apparatus easily transported to the next work, e.g. HVAC/R installation, site.
[0024] Those of skill in the art will appreciate that the purpose of hinged brace 32 is to permit triangular brace members 34 , 36 , 38 to be moved out of the way for easy transport of invented apparatus 10 . Those of skill will also appreciate that construction of boom mechanism 22 in sections similarly facilitates break down and reduces over dimension of invented apparatus 10 during transportation. Finally, those of skill in the art will appreciate that, in accordance with one embodiment of the invention, the bases of support legs 14 , 16 , 18 are equipped with wheels (not shown in FIG. 1 ) to facilitate positioning of invented apparatus 10 while it is in use, i.e. while a load is being positioned and oriented for installation. Thus, tripod base 12 of invented apparatus 10 provides a relatively wide and deep stance or footprint to stabilize loads while also facilitating smooth and effective load movement from one place to another. The wheels, which can be employed in any of the three embodiments described and illustrated herein, will be described in more detail below by reference to FIGS. 6 and 7 .
[0025] Detail A illustrates in fragmentary detail isometric view fulcrum mechanism 20 , load member 50 and lift member 52 as they are assembled in accordance with one embodiment of the invention. Fulcrum mechanism 20 will be understood to include frame 46 defining channel 48 , as described above. Frame 46 may be seen to include a fulcrum base plate 60 , left and right sidewalls 62 , 64 , left and right gusset pairs 66 a, 66 b, 68 a, 68 b and fulcrum cap 70 . Left and right sidewalls 62 , 64 are formed of opposing U-shaped angle members having their U-shaped openings facing outwardly, away from one another, as shown. Sidewalls 62 , 64 have formed therein four sets of opposed through hole pairs 72 , 74 , 76 , 78 spaced apart by approximately 4″ and preferably evenly spaced along vertically extending sidewalls 62 , 64 to permit height adjustment of boom mechanism 22 by selectively pinning pivot member 54 at a desired elevation within channel 48 by a hitch pin 56 .
[0026] Those of skill in the art will appreciate that proximal ends of load member 50 and lift member 52 extend slidably around pivot member 54 on either end of pivot member 56 and are pinned in place with a pair of hitch pins 56 . In accordance with one embodiment of the invention, there is a 4″ gap between the proximal ends of load member 50 and lift member 52 , so that pivot member 54 alone extends through channel 48 and so that load member 50 and lift member 52 extend respectively fore and aft of the channel. Preferably, hitch pins 56 are cotter key locked in place after they are installed, thereby to secure the affected assemblies. The same is true of snapper pin 40 in tripod base 12 (refer briefly back to FIG. 1 ). (Most preferably, each cotter key corresponding to a hitch pin or snapper pin is physically affixed to its respective pin, as is known, to prevent key loss.) It will be understood by those of skill in the art that more or fewer hole pairs may be provided, within the spirit and scope of the invention, having a greater or lesser gap therebetween. It will also be understood that the component parts of frame 46 preferably are welded, e.g. seam-welded (most preferably) or spot-welded. But those of skill in the art will appreciate that, within the spirit and scope of the invention, frame 46 may assume alternative forms made by alternative means, such as any suitably durable structure formed alternatively by one or more of extruding, machining or casting.
[0027] Any suitable materials and dimensions can be used in invented apparatus 10 , and the following description of materials and dimensions used in accordance with one embodiment of the invention is intended to illustrate but not to limit the scope of the invention. For example, boom load and lift members 50 , 52 preferably are of 2.5″ square aluminum (hollow) tubing having 0.25″ (¼″) thick walls, with load member 50 being approximately 5′ long and with lift member 52 being approximately 10′ long. Pivot member 54 preferably is of 2″ square milled steel tubing having ¼″ thick walls, with pivot member 54 being approximately 18-24″ long. Support legs 14 , 16 , 18 preferably are of 2″ square aluminum tubing (radius corner) having ¼″ thick walls, with legs 14 , 16 being approximately 5′ long and with leg 18 being approximately 82″ long. (Those of skill in the art will appreciate that preferably the triangular base of the tripod that supports the fulcrum is nominally vertically aligned with the lateral center of mass of apparatus 10 , there by to obtain maximum horizontal stability of invented apparatus 10 .) Tripod cap 42 preferably is of 10″ square flat aluminum having a thickness of ¼″. Brace members 34 , 36 , 38 preferably are 1″×1″ aluminum angle brackets having a thickness of ¼″, with members 36 , 38 being approximately 38″ long and with member 34 being approximately 15.5″ long.
[0028] Fulcrum base plate 60 is of 10″ square flat steel having a thickness of ¼″. Vertical sidewalls 62 , 64 and gussets pairs 66 a, 66 b, 68 a, 68 b are also of flat steel having a thickness of ¼″. Fulcrum cap 70 is of 5″ square flat steel having a thickness of ¼″. Those of skill in the art will appreciate that, in accordance with one embodiment of the invention, the component parts of fulcrum mechanism 20 are milled or otherwise formed steel, thus providing greater durability but slightly higher weight, whereas the remaining components of invented apparatus 10 in large part are formed of aluminum, providing adequate durability and lower weight. Nevertheless, it is contemplated as being within the spirit and scope of the invention that one or more suitable alternative materials for these component parts of the invented apparatus are within the spirit and scope of the invention.
[0029] In accordance with one embodiment of the invention, support leg base plates 44 are of 5″ square flat aluminum having a thickness of ¼″. Load member 25 is a 1″ round Schedule 40 ends-threaded pipe and includes screw-on end caps. Hitch pins 56 are of an aluminum alloy 0.5″ in diameter and 4.75″ in length. Finally, snapper pin 40 is of an aluminum alloy 0.3125″ ( 5/16″) in diameter and 3.5″ in length. As described above, preferably the hitch pins and the snapper pins are integrally (inseparably) equipped with secure, cotter-type key locks.
[0030] FIGS. 2 and 3 illustrate the first embodiment of the invention corresponding with FIG. 1 , respectively in a front and side elevation. Briefly, FIGS. 2 and 3 respectively in front and side elevation show tripod support base 12 including front support legs 14 , 16 (omitted from FIG. 2 , for the sake of clarity, is rear support leg 18 ); counterweight arm 24 including elongate counterweight member 52 ; front brace member 34 ; fulcrum mechanism 20 including elongate pivot member 54 ; boom mechanism 22 ; lift mechanism 22 ; load attachment mechanism 58 ; and load arm 28 including elongate load member 50 .
[0031] In accordance with the first embodiment of the invention described and illustrated herein, a load of up to approximately 500-1000 pounds readily can be lifted, positioned, oriented and placed. Moreover, such can be accomplished with only one or two operators, since the load is counterbalanced and leverage is increased in accordance with the invention. This capacity may, within the spirit and scope of the invention, be increased or decreased by dimensional scaling. It is contemplated as being within the spirit and scope of the invention to reinforce counterbalance member 52 (and/or lift member 50 ) along its substantial length by seam or spot welding (or otherwise affixing) a length of 1″×1″ aluminum angle having a thickness of ¼″ thereto. Such reinforcement, if deemed necessary or desirable, can be added to any of the embodiments of the invention as described and illustrated herein, and is within the spirit and scope of the invention.
[0032] FIG. 4 illustrates in isometric view the invented apparatus 10 ′ in accordance with a second embodiment of the invention. Very briefly, it will be understood that this second embodiment of the invention features a boom lift assembly that is separable from the tripod base for ease of passage through small openings. Other structural and material aspects of the second embodiment are identical with those of the first embodiment and, for the sake of brevity, will not be described in detail.
[0033] FIG. 4 illustrates an alternative embodiment of the invention at 10 ′. All particulars of invented apparatus 10 ′ are identical to those of invented device 10 described above, except that a detachable tripod cap 42 ′ is provided atop modified support legs 14 ′, 16 ′, 18 ′ and except that a triangular brace mechanism 32 ′ is removably pinned to support legs 14 ′, 16 ′, 18 . Those of skill in the art will appreciate that this alternative configuration achieves even better portability of invented apparatus 10 ′, while retaining ease of assembly on site. Access through smaller openings can be obtained using invented apparatus 10 ′ since all three support legs and the brace itself are easily removed during transportation and easily assembled for use.
[0034] Tripod cap 42 ′ is equipped with square tubular extensions 80 , 82 , 84 onto which support legs 14 ′, 16 , 18 ′ readily slide and are secured by the use of three cotter key-like locking hitch pins 56 (only one of which is shown, for the sake of clarity). Those of skill in the art will appreciate that tubular extensions 80 , 82 , 84 and support legs 14 ′, 16 ′, 18 ′ are equipped with corresponding through holes (also not shown, for the sake of clarity) for pinning purposes. Brace mechanism 32 ′ includes fixed brace member 34 ′ having through holes (also not shown) formed therein and removable brace members 36 ′, 38 ′ also having through holes formed therein on either end thereof to receive three corresponding snapper pins 40 for quick and easy assembly and employment of invented apparatus 10 ′ on site. Thus it will be appreciated by those of skill in the art that triangularly configured support legs 14 ′, 16 ′ fixed by brace member 34 ′ can readily be fitted through a smaller opening with third support leg 18 ′ and corresponding brace members 36 ′, 38 ′ and tripod cap 42 ′ removed. Thus the ‘transport footprint’ of invented apparatus 10 ′ is reduced to facilitate transportation without significant negative impact on ease of assembly on site.
[0035] FIG. 5 illustrates in isometric view the invented apparatus 10 ″ in accordance with a third embodiment of the invention. Very briefly, it will be understood that this third embodiment of the invention features an optional outrigger mechanism associated with the base and optional cable truss and crank lift mechanisms associated with the boom. Other structural and material aspects of the third embodiment are identical with those of the first and second embodiment and, for the sake of brevity, will not be described in detail.
[0036] Apparatus 10 ″ includes an outrigger mechanism indicated generally at 86 , the outrigger mechanism including two laterally opposed outrigger legs 88 , 90 that, while selectively widen the footprint and thus increase the stability of the boom lift. Those of skill in the art will appreciate that support legs 14 ″ and 16 ″ are equipped in accordance with this embodiment of the invention with mounting brackets 92 , 94 that pivotally mount outrigger legs 88 , 90 so that when employed the outrigger legs extend outwardly but generally within the plane formed by support legs 14 ″, 16 ″, as shown. Those of skill will appreciate that pivotable outrigger legs 88 , 90 permit tripod base 12 ″ to be easily transported with a reduced footprint by pivoting the outrigger legs into generally axial alignment with their corresponding support legs. Those of skill also will appreciate that, within the spirit and scope of the invention, the outrigger legs can be removably, rather than fixedly, attached to the mounting brackets, as by pivotally pinning with a pair of hitch pins. Finally, those of skill in the art will appreciate that outrigger legs 88 , 90 and mounting brackets 92 , 94 preferably are made of any suitably durable material, e.g. ¼″ tubular and/or angular aluminum.
[0037] Those of skill will appreciate that, not shown in FIG. 5 , for the sake of clarity, are wheels on pads 44 provided on the bases of support legs 14 ″, 16 ″ and outrigger legs 88 , 90 . Those of skill also will appreciate that FIG. 5 shows fixedly mounted tripod cap 42 rather than detachably mounted tripod cap 42 ′, although within the spirit and scope of the invention either can be employed with invented apparatus 10 ″. Apparatus 10 ″ also includes a square tubular steel member 96 atop fulcrum mechanism 20 , member 96 extending upwardly from and mounted on fulcrum cap 70 . Member 96 includes a cable eyelet 98 at its upper reach to accommodate a cable 100 extending therethrough. Cable 100 under predetermined tension extends through eyelet 98 , with a fore end thereof pinned to load lift member 50 ′ and with an aft end thereof pinned to counterbalance member 52 ′, as shown. Those of skill in the art will appreciate that member 96 , cable 100 , load lift member 50 ′ and counterbalance member 52 ′ thus form a cable bow truss to stabilize and support the ends of beam member 22 and to provide added lift capability of invented apparatus 10 ″.
[0038] A forward end of a second cable 102 is provided with a load hook 104 and a rearward end of cable 102 is wound around a spindle (not visible in FIG. 5 ) that forms part of a crank mechanism 106 having a manual crank handle 108 . The substantial length of cable 102 will be understood to extend through counterbalance, pivot and load lift arms 52 ′, 54 ′ and 50 ′, and to exit load lift arm 50 ′ near its distal end through a guide mechanism, e.g. an eyelet, 110 . Those of skill in the art will appreciate that the hook 104 end of cable 203 can be alternately spooled out and in to reach and secure a load (not shown in FIG. 5 ). In other words, the nominal elevation of hook 104 can be adjusted relative to the distal end of load lift member 50 ′ by manually operating crank mechanism 106 by turning crank handle 108 , thereby facilitating a load's secure attachment.
[0039] FIGS. 6 and 7 illustrate the third embodiment of the invention shown in FIG. 5 in a front and side elevation, respectively, and also show the wheels that, within the spirit and scope of the invention in all of its illustrated embodiments, preferably are included for the purpose of mobility. It can be seen from FIG. 6 that preferably all support and outrigger legs, whether three or five in number (only four of which are shown in FIG. 6 for the sake of clarity, with the fifth wheel being shown only in FIG. 7 ), are equipped with wheel mechanisms (designated 112 , 114 , 116 , 118 , 120 in FIGS. 6 and 7 ). The lateral distance between each lateral wheel mechanism and its corresponding outrigger wheel mechanism is preferably approximately 30″. In accordance with the wheeled embodiment(s) of the invention, the wheel mechanisms are seam or spot welded or otherwise affixed to pads 44 so that their pneumatic tires freely turn.
[0040] Those of skill in the art will appreciate from FIG. 7 that invented apparatus 10 , 10 ′, 10 ″ in any of its various embodiments, of which the embodiment in FIG. 7 is typical, provides a boom lift that is pivotal, as indicated by curved arrows. It will also be appreciated that the invented apparatus also provides for wheeled movement that is fore and aft, as indicated by straight arrows. These two movements facilitate on site (in situ) securement, lift, positioning, orientation and placement of a substantial load.
[0041] FIG. 8 is a flowchart that illustrates the invented boom lift method in accordance with another embodiment of the invention. The boom lift method will be understood to include a) transporting to a work site separate components including a tripod having a fulcrum at its apex and wheels at its base, a boom having a counterbalance arm and a load lift arm, one or more counterweights and one or more pins (block 800 ); assembling the separate components at the work site using the one or more pins to join the components into an assembled boom lift, said assembling including pivotally mounting the boom member on the fulcrum of the tripod (block 802 ); securing a load on a load lift end of the boom at the work site (block 804 ); securing the one or more counterweights on a counterbalance arm of the boom member at the work site (block 806 ); and manually maneuvering the counterbalance arm while wheeling the boom lift to position the load at the work site (block 808 ). Those of skill in the art will appreciate that alternative methods of using the invented boom lift apparatus are contemplated and are within the spirit and scope of the invention.
[0042] In brief summary, the advantages of the invention are many. The invention provides a simple but elegant solution to roof-top or other hard-to-reach work sites where installations of modestly heavy loads is required. It does so by configuring a boom lift in discrete, lightweight component parts that are readily transported to the work site even through narrow openings such as windows, doorways, stairways, etc. due of their narrow span when so broken down. Yet the boom lift assembles quickly using easily hitched pins to join the component parts on site into a durable boom lift configuration. Importantly, the boom lift provides mechanical advantage of leverage by the disparate lengths of its load lift arm and counterbalance arm. Also importantly, the counterbalance arm is counterweighted on site by the simple provision of a preferably ubiquitous liquid ballast such as water easily introduced into one or more sealable containers. The boom lift is just as easily disassembled, therefore, after use.
[0043] Accordingly, while the present invention has been shown and described with reference to the foregoing embodiments of the invented apparatus and method, it will be apparent to those skilled in the art that other changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
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Manual boom lift apparatus and method involve a base having three support legs, a fulcrum configured to fixedly mount the legs, a boom member detachably mounted on the fulcrum, the boom member including on either end a counterbalance arm configured for detachably mounting one or more counterweights and a lift arm configured for hoisting a load, the lift apparatus enabling lift and placement of the load by pivotal manipulation of the boom member. Assembly of the detachable boom lift apparatus components is performed on the work site (in situ) and involves removably pinning aligned hole pairs to join the components and filling one or more containers with ballast to act as counterweights to the hoisted load. The apparatus is lightweight and durable, is easy to transport through small openings and can be used in rooftop installations of heating, ventilation, air conditioning and refrigeration (HVAC/R) equipment.
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The present invention relates to improvements in cases for storing glasses. Its purpose is to provide a system to shut a case in a completely secure way and to avoid the problem of accidental opening that occurs as conventional shutting means wear out.
Different types of cases for glasses are known. One conventional case has a totally flexible constitution. Three of its edges are joined. The remaining edge has mouth to its interior by which the glasses are introduced.
The cases of this type are the most commonly used, but have as a disadvantage that, due to the open inlet mouth, it is frequent that the glasses slide out and fall, occasioning breakage and various damage.
In order to solve these problems, the present invention comprises a case with a quite safe shutting system based on a set of cams which are projected from respective flexibly ceding arms. Each can wedges at wedging ends into an opposing hole, thus preventing these type of accidents.
Another conventional case is one having a zipper disposed in one of their longitudinal walls. In this case, the intensive use of the zipper due to continuous extraction and introduction of the glasses causes it to quickly wear away allowing the case to accidently open, facilitating the dropping of the element that the case contains.
On the other hand, the case which is the object of the invention has a cam system and wedging openings in the respective ends that, due to their constitution and function, are quite durable and suffer from only minimal wear.
Conventional cases are also known that have a shutting system based on two metallic flexibly ceding arms that project from their respective longitudinal shutting walls and mutually intercrossing at their engrossed free ends when the case is closed. These type of cases have the problem that their arms suffer plastic deformations that shortens their useful life due to the great displacement that must occur when their ends intercross.
To solve this problem, the present invention has a set of clams that are forcibly wedgable in corresponding wedging openings in such a way that the top face of each cam is left exposed in said opening so it may be pressed during the opening of the case. The comparative advantage of this system over the above-mentioned prior art is that the necessary displacement of the cam to open and close the case reduced, thus the reducing wearing of the material of the flexibly cedent arms and increasing the efficiency and the useful life of the case.
There are also conventional cases which are generally rigid and have a shutting means comprising a flexibly ceding flap projected from one of the shutting walls. Said flap, is kept closed by a ledge wedgable in a wedging means provided in the opposing shutting wall. The flap is opened with a commanding rod that goes through the shutting wall. The most frequent problem that this type of case present is that the rod gets stuck which, according to the position in which it gets stuck, prevents the proper opening or closing of the case.
The present invention eliminates this because as the same cam that composes a shutting means constitutes at the same time an opening means commandable through the opening into which the cam is wedged. In this way, the retentive element is permanently exposed and may be readily released by the user, in contrast with conventional cases where the shutting element is left in an inaccessible position once the case has been shut.
Another type of conventional case has a shutting means which is engaged by wedging cams into opening located in the same side wall. This mechanism is inconvenient to open.
The present invention solves this problem placing the shutting means in two opposed lateral walls that are not the opening walls. This complementarily arrangement facilitates the handling when the case is opened.
Furthermore, the present case allows different embodiments according to different necessities. In this way, a case can be composed with shorter longitudinal lateral walls that continue themselves with respective shutting means, allowing a saving of material.
Also, in another embodiment, the present invention may comprise a case with both boxes laterally shut by means of their corresponding walls. Thus, a more appropriate case is obtained for those who require an increased protection of their glasses, as both opposing boxes define a completely shut receptacle. In this aspect, the case is equally apt to be used with contact lenses.
BRIEF DESCRIPTION OF THE DRAWINGS
For an increased clarity and comprehension of the object of the invention, the same is illustrated with various figures which represent one of the preferred embodiments. The figures are an illustrative example, not limitative:
FIG. 1 is a view in perspective of the case in a shut position, in which its general conformation can be observed;
FIG. 2 is side view with a partial section that makes evident the hinging means, while in the opposite end the shutting means are observed;
FIG. 3 is a side view with the case in an open position, in the lower box, at the end opposite to the hinging, the wedging end is seen, while in the upper box, the flexibly cedent arm ending in the cam is seen;
FIG. 4 is a front view of the transversal lateral walls with a section where the cam wedged in the opening of the wedging end is clearly seen;
FIG. 5 is a top view of the case where the section shows one of the arms and its corresponding cam wedged in the respective opening;
FIG. 6 is a posterior view where the hinging means are seen as well as their respective lateral walls;
FIG. 7 is a view in perspective that shows the general conformation of the case, in an embodiment where the longitudinal lateral walls laterally shut both boxes;
FIG. 8 is a front view of the case of FIG. 7, where the partial transversal section allow the viewing of the connected shutting means;
FIG. 9 is a side view of the case in FIG. 7 with both boxes in an open position, the partial longitudinal section allowing the viewing in detail the hinging means in both boxes;
FIG. 10 is a view similar to FIG. 9 but with both boxes in a shut position, the dotted line showing the position of the flexibly cedent arm;
FIG. 11 is a top view of the case in FIG. 7, where the partial section allows the view of the disposal of the flexibly cedent arm and the wedge of the cam in the opening; and
FIG. 12 is a posterior view of the case where the hinging means are seen, as well as the opening stop of both boxes.
In different figures, the same reference numbers indicate corresponding or equal parts, and the set of various elements have been marked with letters.
List of the main references:
(a) first box
(a') first box (shut)
(b) second box
(b') second box (shut)
(c) (c') shutting means
(d) (d') hinging means
(1) main wall of (a)
(1') opening transversal lateral wall of (a)
(1'') hinging transversal lateral wall of (a)
(1''') opening edge of (a)
(2) main wall of (b)
(2') opening transversal lateral wall of (b)
(2'') hinging transversal lateral wall of (b)
(3) wedging ends of (c)
(3') opening of (3) for the wedging of (4)
(4) cam of (c)
(5) flexibly cedent arms
(6) longitudinal lateral wall of (a)
(6') end of (6)
(7) longitudinal lateral wall of (b)
(7') end of (7)
(8) small prehensile pincers of (d)
(9) axle of (d)
(10) main wall of (a')
(10') opening transversal lateral wall of (a')
(10'') hinging transversal lateral wall of (a')
(11) main wall of (b')
(11') opening transversal lateral wall of (b')
(11'') hinging transversal lateral wall of (b')
(12) openings of (19) for the wedging of (13)
(13) cam of (c')
(14) flexibly cedent arms of (a')
(15) axle of (d')
(16) small prehensile pincers of (d')
(17) opening stopper
(18) longitudinal lateral wall of (a')
(19) longitudinal lateral wall of (b')
SUMMARY OF THE INVENTION
Improvement in cases for glasses of the type comprehending two boxes (a) and (b) that are elongated and hinged among each other and complement their respective cavities opposite to each other to define the admission receptacle for the glasses. The boxes are formed by respective main walls (1) and (2), obverse and reverse of the case, respectively. In each one of them originate the lateral walls (1) (6) (1'') and are opposed in a confronting way and include retentive means (c) disposed towards the opposite area of the hinge (d). Two of the opposed lateral walls (6) of one of the boxes (a) comprehend respective flexibly cedent arms (5). Projecting from their free ends, cams (4) which are forcibly wedged and auto-retainable in respective compatible openings (3') in the other box (b). The cam (4) and the openings (3') form a shutting means (c). The commanding means (1''') is located in the lateral (1') opposed to the hinging wall (1'').
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention refers to improvements in cases for glasses.
The present improvements have been practiced in a case that comprehends first (a) and a second (b) elongated boxes with their respective opposable cavities defining the receptacle for admission of the glasses. Boxes (a) and (b) are provided with hinging means (d) constituted by small prehensile pincers (8) and axles (9), also having a shutting means of the case.
More particularly, the first box (a) is an elongated body having a main wall (1) and lateral walls (1'), (6) and (1'') that define a cavity. These lateral walls comprise a hinging transversal wall (1'') that ends in small prehensile pincers (8), an opening transversal wall (1') ended in an opening edge (1''') and two longitudinal lateral walls (6).
Each one of these longitudinal lateral walls (6) are shorter than the box (a) and ends in an end (6') from which a flexible cedent arm (5) projects. Cedent arm (5) is ended in a cam (4).
This cam (4) is a ledge of a gradually variable thickness at an inclined plane. Its thinner section is located opposite the cavity of the second box (b) that comprises a shutting means (c). The cam (4) is forcibly wedged and auto-retained in a compatible corresponding wedging opening (3') conformed by the second box (b) when the case is closed.
The second box (b) comprises a main wall (2) and four lateral walls, a transversal hinging wall (2''), an opening transversal wall (2') and two longitudinal walls (7). The latter (7) has a length equal to the length of the longitudinal walls (6) of the other box (a), each one terminating with wedging ends (7') of the flexibly cedent arms (5).
From the wedging ends (7'), the main wall (2) continues extending, its laterals edges ending at respective wedging ends (3) for the cams (4). Wedging ends (3) have respective compatible openings (3') into which cams (4) are engaged.
According to one embodiment, between the ends (6') (7') of the longitudinal lateral walls (6) (7) and the wedging ends (3) is an inlet opening to the case which is defined so that, in a shutting position, it is at least partially blinded by the flexibly ceding arms (5).
The wedging openings (3') of the cams (4) go across the walls of the wedging end (3) in such a way that pulsation openings for each of the cams (4) are formed in the external part of the box (b). In this way, when the case is in a shut position, the openings (3') constitute opening means that leave exposed the top faces of the cams (4).
In another embodiment of the present case, the longitudinal lateral walls (18) and (19) of both boxes (a') and (b') are of the same length as the respective adjacent main walls (10) and (11). In this way, a first box (a') and a second box (b') conform with each other laterally when the case is shut.
Similar to the first embodiment both boxes (a') and (b') are provided with hinging means (d') comprising small prehensile pincers (16) and axles (15).
Both boxes (a') and (b') are provided with respective shutting means (c'). More particularly, from the internal faces of the longitudinal lateral walls (18) of the first box (a') project the flexibly cedent arms (14), finding at their free ends one of the shutting means (c') constituted by the cam (13).
In the case of the second box (b'), its longitudinal lateral walls (19) present respective openings (12) for engaging the cams (13) of the first box (a').
Functioning of the set:
When the open case according to the present invention is disposed towards the closed position, the boxes (a) and (b), which are hingedly connected by way of the axles (9) and the small pincers (8), get close until the lateral longitudinal walls (6) and (7) of both boxes (a) and (b) are oppositely confronted. In those conditions, the flexibly cedent arms (5) of the first box (a) dispose themselves in an adjacent way in the inside of the wedging ends (3) of the second box (b). As the case is closed, the top faces of the cams (4) slide by the edges of the wedging ends (3) until the cams (4) wedge in a forced and auto-retentive way in the respective wedging openings (3').
In this position, the top faces of the cams (4) are left exposed in the respective wedging openings (3'), so that when the operator pushes the cams through the openings (3'), the flexibly cedent arms (5) are displaced, allowing the unwedging of the cams (4) so they may be extracted from the wedging ends (3).
In this disposition, the opening edge (1"') is useful to effect the separation between the boxes (a) and (b) and thus to open the case.
The boxes (a') and (b') are laterally shut in a similar way. Although unlike boxes (a) and (b) with shorter longitudinal lateral walls (6) and (7), when boxes (a') and (b') are shut, lateral walls (18) and (19) cover the flexibly cedent arms (14). The latter (14) are disposed internally behind the longitudinal lateral walls (19) of the second box (b') in such a way the cams (13) wedge in the respective openings (12). In this condition, boxes (a') and (b') define a shut receptacle.
Indubitably, when this invention is put into practice, modifications may be introduced concerning details in construction and shape, without departing from the fundamental principles of the present invention.
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A case for glasses with a closing mechanism comprising a pair of flexible arms extending from the walls of the top of the case. Each flexible arm terminates with a wedge shaped cam. A pair of holes in the walls of the bottom of the case are present at points opposing the cams. When the case is closed, the cams engage the bottom walls, forcing the flexible arms inward. When the case is fully closed, the cams engage the opposing holes and thus secure the case in the closed position. The case may be opened by pressing the cams inward.
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BACKGROUND OF THE INVENTION
This is a continuation in part of application Ser. No. 504,600 filed 6-20-83 now abandoned which is a continuation of application Ser. No. 360,402 filed Mar. 22, 1982 now abandoned.
There are three basic methods by which heat is transferred from a region of high temperature to a region of lower temperature.
1. Conduction. All materials transmit heat through a given thickness of material at a rate which is dependant on its thermal conductivity. This is generally expressed by the following relationship:
k=BTU/(hour)(ft.sup.2)/(°F./ft)=BTU/(hour)(ft)(°F.)
where:
k=thermal conductivity
BTU=heat transmitted in British Thermal Units
hour=standard time period
°F.=temperature difference, degrees Fahrenheit
ft=length, feet
ft 2 =area, square feet
The values of thermal conductivity are inherent with the material and differ widely. For example copper, which is an excellent conductor has a value of k=220 whereas, k=0.025 for corkboard, an insulator. Obviously, when one chooses materials for fabrication of thermal protective clothing and items such as sleeping bags and active wear he will try to select material with low thermal conductivity.
For any given material, the thermal conductivity is rated at heat rate/area/length/temperature difference. Therefore, a very powerful method of decreasing heat transfer becomes available to the inventor if he reduces the effective area of conducting material available for the heat to flow through. The inventor uses this approach in this invention by having thin cross sections of low thermal conductivity materials between the inner and outer surfaces.
Another method of decreasing heat flow through a given material is to simply increase the thickness or length of the heat conduction path. This method was also used by the inventor, by causing the conduction path to be extremely long compared to the area available. This is done by utilizing thin plastic sheets so that heat has to flow edgewise through several thicknesses thereby providing very high ratios of length to area of heat flow and resultant high thermal resistance. Also, the effective conduction length is substantially greater than the overall material thickness dimension. This is illustrated schematically in FIG. 1, wherein the conduction heat path length is shown along the arrows. Thus the effective thickness of the material for heat transfer is much greater than the thickness between the cover sheets.
2. Convection. Heat is transferred from one point to another within a fluid (air), driven by a temperature difference, by the mixing of one portion of the fluid with another. In natural convection the motion of the fluid is entirely the result of differences of density resulting from the temperature differences. If motion is produced by mechanical means such as a fan, or by wind, the resultant mechanism of gaseous heat transfer is called forced convection. Convective heat transfer is greatly reduced by confining the air spaces between the hot and cold surfaces into tiny cells which inhibit the thermally induced flow of air and reduce or prevent convective motion. When the ratio of cell height to cell width is great enough, the boundary layer and skin friction drag damp out most of the convection heat induced motion and internal convection becomes negligible. This is well known in the art and that is why insulating foams use such small cells. This invention makes use of the cell size inhibition of convective flow by limiting the absolute size and relative dimensions of the cells formed in this material. A total cell height to cell width ratio ranging from 4 to 20 and more is effective for this purpose. Also utilizing multiple layers to achieve the desired cell height to width ratio increases the viscous friction on air flow between the layers. Open cells over porous linings are used to allow perspiration and moisture to penetrate from the inner lining to the ambient outer atmosphere, thus this material is said to "breathe".
3. Radiation. A hot body gives off heat in the form of radiant energy which is emitted in all directions. When this radiant energy strikes another body, part may be reflected, part may be transmitted through the body and the remainder is absorbed and transformed into heat. Radiant energy is transferred from a warm body to a cooler one according to the relationship:
w=k.sub.1 k.sub.2 (T.sub.1 .sup.4 -T.sub.2.sup.4)
Where:
W=energy transmitted by radiation, BTU/(hr)(ft 2 )
k 1 =Stefan-Boltzman constant, BTU/(hr)/(ft 2 )/(°F.) 4
k 2 =emissiviy factor, (a function of the surfaces of both bodies), dimensionless
T 1 =radiating surface temperature, (hot body), °F.
T 2 =absorbing surface temperature, (cold body), °F.
Wherein the emissivity factor is a function of the surface characteristics of the materials, for example, a highly reflective material such as gold has an emissivity of 0.01, whereas a good absorber (and emitter) such as black paint has an emissivity of 0.95.
Radiant energy transmission from surface 1 to surface 2 may be controlled by 2 methods:
(1) Low emissivity (highly reflective) surfaces limit the thermal energy leaving the warm surface, and/or reflect thermal energy from the colder surface, and limit its absorbtion. This reflecting principle is utilized by the familiar radiation shield type of thermal insulation.
(2) High emissivity surfaces, which are also highly absorbing, can be coupled with a particular geometry so that thermal energy leaving a warm surface is intercepted and absorbed by the insulating medium and thus cannot directly reach the cold surface. This method is utilized by this invention in that an absorbing material is used to form the insulating cellular structure.
Highly reflective surfaces on plastic films are generally achieved by use of a metallic coating which is relatively expensive, and prone to degrade and flake off with time, handling, and washing, to which it would normally be subjected to in articles such as clothing. The absorbing characteristics of the materials recommended in this invention do not substantially change with normal treatment over time and thus are thermally more stable, thereby providing longer useful article life.
ENVIRONMENTAL DESIGN
In the design of an insulating material, one has to consider the end use of the material, the thermal environment on the outside of the material and the thermal requirements for the protected or inner side of the material. Some of the parameters to be considered in selecting the most suitable materials and critical design dimensions for applying the disclosed insulation include:
Outside temperature range
Exposure to sun
Wind
Humidity
Perspiration rate of body
Precipitation
Direct contact with solid surfaces
For example, continuous exposure to moisture would preclude the use of materials such as urethane foam where one does not want to absorb water.
MECHANICAL DESIGN
This invention considers the requirement that the insulating material can be worn comfortably by a human being.
In reviewing the design of insulating materials as described above, the inventor has greatly simplified the construction of his insulating material and has improved the thermal resistance for comparable weight. For example, typical polyester fiber battings have a working density of 0.40 to 0.50 lb./cu. ft and down or feathers about 0.25 to 0.30 lb./cu. ft, while the disclosed preferred material working density is about 0.28 lb./cu. ft. The inventor has improved the flexibility and "wearability" of his design with a low cost implementation as compared to those disclosed in the references described below and on the market. He has also shown that it is not necessary to have metallic radiation reflectors as a part of his construction. The inventor has also been able to increase the vapor permeability by about an order of magnitude with his design so as to allow a person wearing insulating clothing made from his invention to be more comfortable because perspiration can readily pass through the insulating material.
DESCRIPTION OF THE PRIOR ART
In previous practice, insulating materials designed to retard heat flow into and/or out of a system have been specified by the environmental conditions to which the system is exposed, to the particular mode of thermal energy transfer to be retarded and by special requirements such as flexibility, strength, weight, cost, etc. Inexpensive cellular and fibrous materials primarily reduce heat transfer by reduction of conducting area per unit cross section, by limiting convection between the hot and cold surfaces and by scattering some of the radiant energy. Honeycomb like materials with small thin cellular structures are superior insulators because they can be made with smaller heat conducting cross sectional areas, their cells can be sized to restrict convection to a greater degree than bulk insulators and foams by limiting the onset of convection to a predesigned threshold value, also radiant energy can be reduced by being trapped within the cell walls.
Since their introduction around the turn of the century, honeycomb materials have been highly developed technically but their relatively high manufacturing cost, difficulty in application and processing, and structural rigidity have limited their use primarily to aircraft and military structural applications where rigidity and structural efficiency are more important than cost. Their use as thermal insulation has been limited by high manufacturing costs and undeveloped manufacturing technology. In addition, honeycomb insulation has not been used in clothing because of poor compression recovery, low flexibility and generally poor techniques of garment construction. The technology of this invention offers a unique solution to all the above problems.
Jonnes in U.S. Pat. No. 4,136,222 shows a honeycomb sandwich comprising a honeycomb core formed from polymeric foam cemented between two sheets, at least one of which has a vapor deposited layer of specularly reflective material. He says that his honeycomb layer is between 0.25 to 1.5 centimeters thick and each cell has a maximum span of 5 centimeters. The foam honeycomb covers or connects with between 10 to 60 percent of the cover sheets area or preferably 20 to 40 percent. It is apparent that this design will inhibit transfer of perspiration from the inside to the outside of the clothing. If the deposited metallic layers are to be effective, their surface can not be very open to the passage of moisture at atmospheric pressure. Minute fractures in the reflective layers can reduce the reflectivity as each fracture acts as a perfect black body. On the other hand, the invention described in this application has a porosity of at least 95% which will allow the free passage of water vapor. Impervious metallic radiation reflectors are unnecessary to the effective operation of the invention described in this application because the radiation is absorbed by the cell walls and not transmitted directly to the cold surface. Another problem with Jonnes' invention is that although the inner and outer layers may be flexible, and granted that the polymeric foam is flexible, when the three parts are assembled together, the moment of inertia in bending increases by an enormous amount. The assembled invention will be stiff and undrapable as compared to the invention disclosed in this application. In fact, a section of such honeycomb cloth will be self supporting and it is well known that one property of honeycomb construction is its extreme resistance to the formation of compound curves. Jonnes relies on the shearing and squeezing of his foam honeycomb cells for flexibility because his cover sheets will resist tensile deflection and shear. In contrast, the cell walls of this invention, not being normal to the cover sheets, will exhibit greater compression recovery. Also, if moisture does penetrate into the interior of the foam cells, it will tend to be absorbed and retained thereby increasing the weight of the cloth with time. If the moisture condenses, it will be retained until it evaporates or is forced out of the cells by squeezing or wringing. Also, freezing of entrapped moisture can crack the separating layers thereby increasing the structural and thermal degradation rates. In contrast the disclosed material would not retain more than 0.01% water by weight due to the low affinity of these non porous plastic film materials to moisture.
Balk, In U.S. Pat. No. 3,968,287 describes a method of making a composite laminate comprising two synthetic-resin foils having mutually transverse main stretch directions which is formed by bonding the foils together after they have been incised in rows of spaced apart incisions extending in the main stretch direction of each foil. The invention described in this application can use a similar process to provide the insulating cells of the disclosed invention but does not claim the process of forming the insulating cells. The inventor merely describes the process in accordance with his duty to disclose the best technique for forming the cells. In these applications, it is unimportant as to how the cells are formed, and they may be formed from single or multiple layers of plastic sheet. Alternatively, they can be formed by molding, by rolling through rotating dies, by extrusion or by any means known to those with ordinary skill in the art of fabricating such cells.
Crane, in U.S. Pat. No. 3,245,606, describes a packaging bag of slit transparent plastic film wherein about half the film area is unslit to provide for package strength and shape. The purpose of the slit portion is to provide package expandability and ventilation for the contained goods such as a bunch of grapes. The concept of using a slit pattern for expanding thin plastic sheet is similar to that of this invention. However, the necessary use of specific slit pattern, extent of stretch, material thickness and radiation absorption properties are among the factors that render Crane's packaging material unsuitable for use as an insulation.
Wyckoff, in U.S. Pat. No. 3,405,027 describes the manufacture of composite films wherein a system of webs and ribs imparts the desired strength characteristics to the laminated structure. The invention described herein can use a similar method for producing the insulating cells however it is not the preferred method of manufacture.
Clough, in U.S. Pat. No. 3,707,433 describes an insulating material comprising two layers of plastic film sandwiched around a reinforcement of fibers with one or both of the inner surfaces of the plastic sheets coated with a reflective metal layer. A limited amount of vapor permeability is provided by a system of transverse fibers and holes. Alternatively, he uses a foam separator and a sandwich construction which has all of the disadvantages described above for the Jonnes invention. The main method of restricting heat transmission utilizes the radiant reflective properties of metallized films. Although individual layers are thin, the required adhesive bonding within each layer, will restrict drapability, and in a multi-layer use will cause the item to be stiff, especially as compared to the flexibility inherent in this invention.
Lewis, et al. in U.S. Pat. No. 3,649,430 describes metal laminates with polymeric cores used to provide sound and vibration damping characteristics to structural materials. Here the polymeric core material is bonded to the metal foil outer layers. These cores are selected to help provide rigidity to the structure. Such materials, while perhaps suited to structural insulation uses are wholly inapplicable to clothing or similar uses where flexibility is required.
Akao in U.S. Pat. No. 4,331,725 describes a laminated wraping material comprising at least two uniaxially-stretched sheets of plastic film adhesively bonded to provide desired strength properties. The adhesive layer may be perforated in the form of a net. Use of such a material as an insulation material for clothing, or other articles that need to "breathe" or inhibit heat transmission would not be suitable due to the inherent limitations of the film, such as moisture impermeability, thickness and stiffness.
Doll in U.S. Pat. No. 3,839,525 describes a method for producing a net-like material by slitting a plastic film and stretching and heating sufficiently to provide a rounded cross section to the material elements. The method of slitting and stretching a plastic film is well known and is disclosed in this invention to indicate the preferred method of forming cellular material. The netting material of Doll would not provide adequate thermal conductive resistance as the cross sectional area per unit length is large as compared to the slit and stretched material of this invention that has not been heated to nearly the extent disclosed by Doll.
Rasmussen in U.S. Pat. No. 3,454,455 discloses a laminate wherein polymeric films having mutually transverse stretch directions are slit in a row pattern to provide a reticular structure with increased tear resistance. This material is similar to that described by Balk, above and its use and limitations relative to the present invention would be similar.
SUMMARY OF THE PRESENT INVENTION
This invention is a design for a composite insulating fabric which reduces and controls heat transfer and provides for free moisture transfer from the interior protected surface to the outer ambient surface. The invention accomplishes this by the combination of substantially non-planar or three dimensional layers of fabric formed into regular and predictable patterns of contiguous honeycomb like cells which are formed with offset walls between each layer, with a continuous, porous inner layer of fabric fastened to selected edges of the adjacent cellular layer. Each adjacent and offset cellular layer may be attached to the next cellular layer at selected points of the cellular structure. This selected and sparse system of attachment is very important because it greatly reduces heat transfer by conduction across the layers and reduces the structural rigidity of the "honeycomb" like structure. The ability of this material to stretch and shift makes garments possible which are close fitting, warm and comfortable. The cell walls being inclined give the cellular layers excellent compression recovery. Many applications will not require anything other than edge attachment or stabilization. If desired, an outer layer of flexible material may be loosely attached to the outer layer of cells to form a sandwich with little resistance to shear forces caused by deflection or crushing. The outer and inner cover sheets can be selected with characteristics determined by the user such as permeability, wearability, suitability for decoration, etc. The invention is inexpensive to fabricate, very flexible, has low bulk density and has excellent insulating properties while allowing free passage of water vapor from the inner surface to the outer surface. The insulating material will be very useful in the fabrication of lightweight outdoor equipment used in mountaineering, skiing, camping and hiking, such as sleeping bags, blankets, outdoor clothing and tents.
It should be noted however, that use of reflective surfaces on the cellular material and/or the cover sheets can be used if desired to further enhance overall thermal performance of the insulation. This use can increase cost, weight, degradation rate, and reduce breathability.
Presently available insulating materials on the market are bulky and expensive, such as down filled double walled clothing, blankets and sleeping bags of heavier synthetic substitutes for down, such as polyester fibers. Most of the sandwich materials mentioned in the description of prior art section of this application have not appeared in the commercial market due to high cost, poor comfort characteristics or other reasons cited above.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic cross section of the invention which illustrates the energy transmission paths between the two sides of the insulating material.
FIG. 2 is a schematic cross section illustrating energy transmission and an alternate aligned layer construction.
FIG. 3 is a top view of a typical slit pattern in a sheet of material prior to formation of cells by stretch forming.
FIG. 4 is a perspective view of the sheet illustrated in FIG. 11 after stretch forming.
FIG. 5 is a perspective view of the cell pattern illustrating cell dimensions.
FIG. 6 is a cross section of the invention showing a three layer embodiment.
FIG. 7 is a view of elements of an alternate embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 schematically illustrates a random oriented three cell layer thick construction of the invention illustrating the energy transmission paths between the inner cover sheet 2 and the outer cover sheet 4. Energy transfer by conduction takes a long tortuous path through each cell wall layer as shown by the arrows 6. The transfer length comprises the summation of the individual lengths of the cell walls plus the thickness of the cover sheets. Also, the thermal resistance of each of the contact points between layers is very high relative to an equivalent thickness of solid material-thus the overall conduction thermal length to area ratio is very high for the total material thickness. Convection transmission would normally take place by air currents set up with the cells as shown by arrow 8, however these currents are limited in this invention with a proper cell size configuration, where boundary layer and skin friction drag damp out this motion. Radiant energy diffuse scattering is illustrated at a single point 10 from one of the outer layers. At each location where a radiation emission arrow intercepts a solid cell boundary, that energy will be absorbed and not transmitted to the other layer.
Referring now in detail to FIG. 2, there is illustrated a non-random or aligned orientation of a three cell layer construction and schematic cross section of the invention. On one side, the insulating material outer surface 5 is exposed to thermal radiation 7, convection heat transfer 9 from the wind 11, and conduction heat transfer 13 from a contacting surface 15. On the other side of the insulating cellular material 17 is a layer of porous fabric 19 which is in close proximity to a human being 21, or some object requiring thermal insulation. This porous fabric is exposed to liquid moisture 25, humidity 27, air currents 29, and contact with a human being or object. Heat in the forms of thermal radiation 31, conduction 33, and convection 35 are transferred across this porous fabric layer. The direction of the net heat transfer depends on the temperature difference across the wall with heat always going towards the direction of lower temperature.
The heat transferred externally by convection is absorbed by the outermost layer of material 5 and then is transferred across cell walls 17 by long discontinuous paths to the inner cloth layer 19 and then to the inner protected region. Heat transfer by conduction between the inner and outer surfaces has been greatly reduced by this long path and is nearly equivalent to that of gaseous conduction through the entrapped air. Internal convective heat transfer is limited by the appropriate design and selection of cell height to width ratios. The cell height to width ratio should exceed 4 and is practical up to at least 20 or 30. Testing of a sample of this material has indicated an effective thermal conductivity of 0.036 BTU/hr ft°F. measured between temperatures of 96° F. and -7°, this material had a cell height to width ratio of 9.3. The contribution to be expected from solid and gaseous conduction represents about one third of the total heat transferred, the remainder is that due to radiation plus convection. Radiation absorption by the cell walls and convection suppression by cell size and configuration is very effective because the combined radiation plus convection transfer mechanisms, which would be difficult to separately estimate, represents only about one fourth of the theoretical black body radiation interchange between the two boundary temperatures.
The cell walls are inclined to the plane of the cover sheets to enhance the flexibility and compression recovery of this construction. The angle which the cell walls make with the normal to the cover sheets 16 is shown in FIG. 6 should exceed 10°, up to about 60°, for most applications. Cell dimensions of width 12 and height 14 are also identified in FIG. 6.
Moisture passes across the porous inner and outer layers, and through the open cells to the outer atmosphere. Moisture is not absorbed by the cell walls to any degree because they are "impervious" to water and the materials shed moisture (hydrophobic).
There are many possible methods for producing the cellular material desired, three of which include:
(1) Starting from solid planar sheets of a plastic material such as polyolefin and making a regular pattern of slits, as shown in FIG. 3, and then either by stretching in a direction normal to the slits, and/or applying heat to produce the desired cell configuration, shown in FIG. 4 this stage is determined by the slit pattern and the amount of stretch or heat applied.
(2) Applying localized pressure differences in a mold or form to the planar unslit plastic sheet causing permanent deformation of the material in a cellular pattern.
(3) Causing a liquified plastic material to be cast into a mold and allowed to solidify thereby assuming the desired shape and cellular pattern.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In one specific embodiment of this invention the material is configured for use as thermal insulation in an article of clothing outer wear such as a ski parka. This application uses a construction of twelve layers, made from slit and expanded sheets. (a simplified configuration of three layers 20, 22, 24 disposed between cover sheets 26, 28 is shown in FIG. 5).
Each layer of cells is manufactured from a planar sheet of a polymeric film, such as high density polyethylene, that can be between 0.0003 and 0.020 inches in thickness, preferably about 0.0006 inch, and maintains flexibility over the desired application temperature range of -60° F. to 130° F. The material should be absorptive to infra red radiation of 9 to 12 microns wavelength, which is the band of thermal radiation wavelengths, by having its surface treated or preferably by being opaque black. The slit pattern can be arranged to yield after stretching cell dimensions of between 0.025 to 0.75 inch wide and 0.010 to 0.5 inch high, the specific dimensions depending upon the particular application. The preferred slit pattern illustrated in FIG. 3, utilized a slit length, L1,of 0.1875 inch, an unslit spacing, L2, of 0.0625 inch and a row spacing, D1, of 0.050 inch. Expanding the slit material 100% in a direction normal to the slit and heat stabilizing while stretched, creates the desired honeycomb like cellular pattern. Each of these layers will have a cell wall height of 0.0918 inch a cell width of 0.118 inch and a layer height of 0.0795 inch due to the 60 degree angle between the cell walls and the nominal film plane. The assembly of 12 layers has an overall cell height to width ratio of 9.3, which is the critical parameter.
The desired configuration may be achieved by outer edge stabilizing the necessary number of layers and using conventional techniques such as heat sealing, bonding, stitching or stapling. Additional layers of metallized film may be used to enhance radiation energy inhibition and act as a mass transfer or vapor barrier between conventional fabric elements, when the limitations introduced by metallized films discussed above, primarily permeability and coating degradation, may be acceptable for a particular application. These metallized films can be incorporated as either part of the outer layers or as any part of the sandwich construction. Vapor permeability may be controlled by perforating the otherwise solid sheet forming the radiation/vapor barrier.
Another embodiment of this invention permits the thermal insulating effects to be varied by the garment wearer. This is accomplished by taking advantage of the natural elasticity of the sheet material and using a thicker film material for added strength and durability, such as opaque black 0.010 inch nylon, slit into the same pattern described above and stretched about 10% and heatset. This slight stretching will always keep the cells open to permit moisture transfer and help prevent the surface tension forces, due to any entrapped moisture, from causing adjacent flattened layers to adhere. Thus the cell walls will be inclined at large angles, nearly 90 degrees, to the normal of the film plane.
Used in cold weather clothing, an assembly of twenty layers are "tack" bonded at the edges to provide stabilization without unnecessary seam bulk. This assembly is less than 0.25 inch thick but may be stretched to twice its original length resulting in a 20% decrease in width and increase in thickness or loft to about 2 inches, and resulting in a greatly increased insulation effectiveness.
Specifically, such a garment might have loose fitting short sleeves and cover the upper torso, therefore providing limited thermal insulation effectiveness. When desired by the wearer, the sleeves would be drawn toward the wrists and the torso material drawn down toward the waist resulting in a much greater degree of insulating effectiveness. Thus this embodiment can provide comfort over a wide range of environmental conditions by enabling the wearer to adjust the amount of insulation provided.
A third embodiment of this invention permits use of this material in applications where a blown fill is desired. Heat stabilized layer assemblies (made by any of above the described methods) may be cut, chopped or otherwise formed into small segments and used in a manner similar to that of ordinary blown fill insulations such as feathers, down or fibers. Heat stabilization is the preferred method enabling the slit and stretched layers to maintain shape upon cutting. FIG. 7 shows how 3 such segments may be in a random arrangement. Thus all of the advantages of this invention, with non-optimimum thermal performance, can be utilized without the need for changing end article production equipment and processes.
Summarizing the advantages of this invention:
(1) Thermal efficiency--an effective overall thermal conductivity of less than three times that of air. This is accomplished without the use of reflective layers.
(2) Light Weight--an overall working density range of about 0.2 to 3.0 pounds/cubic foot.
(3) Flexibility--an extremely flexible material able to fit human contours without stiffness.
(4) Low cost--material can be made in large quantities from readily available low cost polymeric materials.
(5) Moisture permeability--the open cell structure and hydrophobic nature of the film results in an insulation that will "breathe", permitting moisture (which can break down insulation materials and increase their weight) to pass through and not be absorbed or retained by the cell walls.
(6) Simplicity--multilayer assemblies can be readily assembled and generally requires only outer edge stabilization by conventional techniques to retain its shape.
(7) Durability--the types of preferred polymeric materials are capable of withstanding long periods of normal handling without degradation.
(8) Variable thermal conductivity--an embodiment permits the user to adjust the insulation effectiveness for optimal comfort.
The disclosed embodiments of this inventions are not to be construed as limitations thereof, but are merely examples of particular uses.
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A composite insulating material which is comprised of one or more layers of open ended cells formed from flexible thin plastic sheet, and may be fastened to one or more cover sheets. The material is flexible enough to be worn as clothing and derives its insulating properties by reducing thermal conducting paths and areas, by introducing numerous controlled geometry air cells between the heat source and inner layer to further lower conductivity and inhibit convection currents and, by absorbing radiant thermal energy within the cell walls thereby reducing heat transmission by radiation. Metallized reflecting layers are not required to achieve high insulating efficiency.
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BACKGROUND OF THE INVENTION
This invention relates to a process for producing an adipic acid diester which comprises reacting butadiene, carbon monoxide and an alcohol in the presence of cobalt carbonyl catalyst and a specific reaction medium.
Processes for producing an adipic acid diester by reacting butadiene, carbon monoxide and an alcohol in the presence of cobalt carbonyl catalyst have been known in the prior art.
For example, Japanese Patent Publication No. 20177/1974 discloses a process for producing an adipic acid diester from butadiene, carbon monoxide an an alcohol which comprises
the first step of reacting butadiene, carbon monoxide an an alcohol in the presence of cobalt carbonyl catalyst and pyridine at a temperature of 120°-160° C. to form a 3-pentenoic acid ester, and
the second step of reacting the resulting 3-pentenoic acid ester in the reaction mixture, carbon monoxide and an alcohol at a temperature of 160°-180° C. without separating the 3-pentenoic acid ester, cobalt carbonyl catalyst and pyridine.
Patent Publication No. 20177/1974 also discloses the process which comprises
the first step of reacting butadiene, carbon monoxide and an alcohol in the presence of cobalt carbonyl catalyst and pyridine at a temperature of 120°-160° C. to form a 3-pentenoic acid ester, and
the second step of separating the resulting 3-pentenoic acid ester from the reaction mixture, and reacting the 3-pentenoic acid ester, carbon monoxide and an alcohol in the presence of cobalt carbonyl catalyst and pyridine at a temperature of 160°-200° C.
However, in Patent Publication No. 20177/1974, the yield of object product in the former method in which the second step is carried out without separating the 3-pentenoic acid ester, which is the reaction product in the first step, from the reaction mixture is inferior to that in the latter method in which after the 3-pentenoic acid ester, which is the reaction product in the first step, is separated from the reaction mixture before carrying out the second step reaction.
Therefore, in order to obtain an adipic acid diester in a high yield according to the invention of Patent Publication No. 20177/1974, it is necessary to separate from the reaction mixture the 3-pentenoic acid ester obtained through the hydroesterification reaction of butadiene and transfer it to another reactor, and the 3-pentenoic acid ester is hydroesterified in the another reactor. However, such process is complicated and the yield of object product in the process is not completely satisfactory. In addition, the reaction rate in the hydroesterification reaction of butadiene in the presence of pyridine solvent alone is low, so a large amount of expensive cobalt carbonyl catalyst has to be used in order to increase the reaction rate and recovery of the catalyst is costly.
SUMMARY OF THE INVENTION
The present inventors carried out research on a process for producing an adipic acid diester by hydroesterification reaction of butadiene in a high yield and with a high selectivity; that is, research was directed to enhancing the catalyst activity in such process. As a result, we found that when the reaction of butadiene, carbon monoxide and an alcohol are carried out in a specific reaction medium, an adipic acid diester can be produced in high yield by a single process that uses a small amount of a cobalt carbonyl catalyst to achieve adequate hydroesterification rate and wherein 3-pentenoic acid ester is not separated from the hydroesterified solution of butadiene but is immediately subjected to hydroesterification of 3-pentenoic acid ester after changing the reaction temperature.
This invention relates to a process for producing an adipic acid diester which comprises the first step of reacting butadiene, carbon monoxide and an alcohol in the presence of cobalt carbonyl catalyst at a temperature of from 80° to 160° C., to form a 3-pentenoic acid ester, and
the second step of reacting the 3-pentenoic acid ester in the reaction mixture, carbon monoxide and an alcohol at a temperature of from 160° to 220° C., characterized in that the first and second reactions are carried out in a reaction medium comprising at least two amine solvents selected from the group consisting of pyridine, guinoline, isoquinoline and substituted pyridine, substituted quinoline and substituted isoquinoline in which substituent or substituents are selected from the group consisting of alkyl having 1-6 carbon atoms, alkenyl having 1-6 carbon atoms, aryl, alkylaryl having 7-10 carbon atoms and aralkyl having 7-10 carbon atoms and optionally at least one solvent selected from the group consisting of hydrocarbons, esters, ethers and mixtures thereof.
DETAILED DESCRIPTION OF THE INVENTION
The amine solvents employed in the present invention include pyridine, quinoline, isoquinoline, α-picoline, β-picoline, γ-picoline, 2,3-lutidine, 2,4-lutidine, 2,5-lutidine, 2,6-lutidine, 3,4-lutidine, 3,5-lutidine, 4-benzyl pyridine, 4-vinyl pyridine, quinoline and isoquinoline. Of these compounds, pyridine, β-picoline, γ-picoline, 3,4-lutidine, 3,5-lutidine and isoquinoline are preferred.
It is critical that at least two of the above mentioned amine solvents be used. In general, any one of the at least two amine solvents is preferably used in an amount of more than 2 moles %, more preferably 5 moles %, most preferably 10 moles % on the basis of total mole of the amine solvents. The case of using at least two amine solvents in which one of the amine solvents is present in an amount of less than 2 moles % gives superior results to the case of using one amine solvent alone, but the use of a mixture of the amine solvents in which any one of the amine solvents is present in an amount of more than 2 moles % gives results superior to those obtained with the use of one amine solvent.
The amount of mixture of the amine solvents employed is not critical. In general, the mixture of the amine solvents is used in an amount of from 0.05 to 10 parts by weight on the basis of 1 part by weight of butadiene, preferably from 0.2 to 3 parts by weight. Use of the mixed amine solvents in an amount less than 0.05 parts by weight is likely to cause side-reaction. Use of the mixed amine solvents in an amount of more than 10 parts by weight suppresses hydroesterification reaction of a 3-pentenoic acid ester.
At least one solvent selected from the group consisting of hydrocarbons, esters and ethers may be used together with the above amine solvents. Hydrocarbon solvents include, for example, hexane, octane, cyclohexane, benzene, toluene and decaline. Ether solvents include, for example, aliphatic ethers, such as diethyl ether, tetrahydrofuran and dioxane. Ester solvents include aliphatic ester, such as methyl acetate.
The amount of the solvent employed is not critical. In general, the solvent is used in an amount of from 0.1 to 10 parts by weight on the basis of 1 part by weight of butadiene, preferably from 0.3 to 3 parts by weight.
The cobalt carbonyl catalyst employed in the present invention include cobalt carbonyl and cobalt carbonyl complex.
The cobalt carbonyl catalyst may be the synthetic solution obtained by reacting synthetic gas (CO and H 2 ) with cobalt compound(s) comprising inorganic cobalt compounds, such as cobalt hydroxide, cobalt carbonate and basic cobalt carbonate or organic cobalt compounds, such as cobalt salt of organic acid, cobaltocene and cobalt acetylacetonate in the alcohol employed as a starting material, or the synthetic solution obtained by reacting synthetic gas (CO and H 2 ) with cobalt compounds in the presence of pyridine, quinoline, isoquinoline, alkyl-substituted pyridine, alkyl-substituted quinoline, alkyl-substituted isoquinoline or other compound having ligand.
In the prior method for producing an adipic acid diester by hydroesterificating butadine and hydroesterificating a 3-pentenoic acid ester by using a large amount of cobalt carbonyl or cobalt carbonyl complex, the cobalt carbonyl or the cobalt carbonyl complex must be prepared in high purity and high yield by a complicated and costly process. On the other hand, since an adipic acid diester can be prepared by using a small amount of catalyst according to the present invention, the synthetic solution containing cobalt carbonyl catalyst can be prepared by a simple method from an inorganic or organic cobalt compound.
The amount of cobalt carbonyl catalyst employed is not critical. When dicobalt octacarbonyl is employed, dicobalt octacarbonyl in an amount of 0.001 to 0.05 moles per 1 mole of butadiene, preferably dicobalt octacarbonyl in an amount of 0.005 to 0.03 moles may be industrially used. The use of catalyst in an amount of less than the lower limit as mentioned above lowers the reaction speed too much. The use of catalyst in an amount of more than the upper limit merely adds to production cost, since the cost of recovering the catalyst increases.
Alcohols employed in the present invention include lower aliphatic alcohols having 1-10 carbon atoms such as methanol, ethanol, propanol and butanol. Methanol is important industrially. One of these alcohols or mixture thereof may be used. The amount of the alcohol employed is not critical. The alcohol in an amount of at least 2 moles per 1 mole of butadiene, preferably the alcohol in an amount of 2 to 10 moles per 1 mole of butadiene may be used. When the alcohol in an amount of less than 2 times of mole to butadiene is used, expensive butadiene is consumed for undesirable side reaction. The use of the alcohol in an amount of more than 10 moles per 1 mole of butadiene lowers the hydroesterification reaction speed of butadiene and a 3-pentenoic acid ester.
The partial pressure of carbon monoxide is not critical in the hydroesterification reaction of butadiene and hydroesterification reaction of a 3-pentenoic acid ester. The partial pressure of carbon monoxide may be more than 50 Kg/cm 2 , and preferably, the partial pressure is in the range of 100 to 400 Kg/cm 2 in the practice of the present invention.
The reaction temperature is in the range of from 80° to 160° C. in the esterification reaction of butadiene, and preferably is in the range of from 100° to 140° C. The reaction temperature is in the range of from 160° to 220° C. in the hydroesterification reaction of a 3-pentenoic acid ester, and preferably is in the range of from 170° to 200° C.
According to the present invention, an adipic acid diester can industrially be produced from butadiene by using a small amount of the catalyst without requiring any complicated operation.
The present invention can be carried out either as batch process or as a continuous process.
The present invention is further illustrated by non-limiting Examples.
EXAMPLES 1-10
Into a 200 ml stainless steel autoclave equipped with magnet stirrer were charged 15 grs. (0.277 moles) of butadiene, 22 grs. (0.686 moles) of methanol and 2 grs. (0.0058 mol) of dicobalt octacarbonyl catalyst and mixed amine solvents as given in Table 1. The reaction was carried out at 130° C. under carbon monoxide partial pressure of 300 Kg/cm 2 for 1.5 hours and the reaction was further carried out at 185° C. under carbon monoxide partial pressure of 300 Kg/cm 2 for additional 2 hours.
The results are shown in Table 1.
TABLE 1__________________________________________________________________________ Ex. 1 Ex. 2 Ex. 3 Ex. 4__________________________________________________________________________components butadiene g (mol) 15 (0.277) same as same as same as methanol g (mol) 22 (0.686) Ex. 1 Ex. 1 Ex. 1 Co.sub.2 (CO).sub.8 g (mol) 2 (0.0058)mixed (1) kind pyridine pyridine β-picoline pyridinesolution of g (mol) 10 (0.126) 10 (0.126) 10 (0.107) 10 (0.126)amine (2) kind isoquinoline γ-picoline γ-picoline isoquinoline g (mol) 10 (0.077) 10 (0.107) 10 (0.107) 10 (0.077)hydrocarbon solvent, kind hexaneether solvent or g (mol) 20 (0.232)ester solventreaction hydroesteri- reaction pressure of 300 same as same as same asconditions fication of CO kg/cm.sup.2 Ex. 1 Ex. 1 Ex. 1 butadiene reaction temperature 130 °C. reaction time Hr 1.5 hydroesteri- reaction pressure of 300 same as same as same as fication of CO kg/cm.sup.2 Ex. 1 Ex. 1 Ex. 1 3-pentenoic reaction temperature 185 ester °C. reaction time Hr 2.0conversion of butadiene mol % 100 100 100 100selectivity to dimethyl adipate mol % 66.4 69.7 67.1 72.5selectivity to methyl 3-pentenoate mol % 10.1 8.5 10.7 7.0selectivity to methyl n-valerate mol % 4.2 8.1 6.9 4.3selectivity to dimethyl 2-ethyl succinate mol % 1.8 1.7 1.6 2.1selectivity to dimethyl 2-methyl glutarate mol % 8.2 6.8 6.0 7.9__________________________________________________________________________ Ex. 5 Ex. 6 Ex. 7__________________________________________________________________________components butadiene g (mol) 15 (0.277) same as same as methanol g (mol) 22 (0.686) Ex. 5 Ex. 5 Co.sub.2 (CO).sub.8 g (mol) 2 (0.0058) mixed (1) kind pyridine pyridine pyridine solution of g (mol) 10 (0.126) 10 (0.126) 10 (0.126) amine (2) kind γ-picoline β-picoline β-picoline g (mol) 10 (0.107) 10 (0.107) 10 (0.093) hydrocarbon solvent, kind benzene hexane diethyl ether ether solvent or g (mol) 20 (0.256) 20 (0.232) 20 (0.170) ester solventreaction hydroesteri- reaction pressure of 300 same as same asconditions fication of CO kg/cm.sup.2 Ex. 5 Ex. 5 butadiene reaction temperature 130 °C. reaction time Hr 1.5 hydroesteri- reaction pressure of 300 same as same as cation of CO kg/cm.sup.2 Ex. 5 Ex. 5 3-pentenoic reaction temperature 185 ester °C. reaction time Hr 2.0conversion of butadiene mol % 100 100 100selectivity to dimethyl adipate mol % 75.4 77.5 73.1selectivity to methyl 3-pentenoate mol % 4.8 4.0 7.5selectivity to methyl n-valerate mol % 9.1 6.6 7.3selectivity to dimethyl 2-ethyl succinate mol % 1.7 1.8 1.9selectivity to dimethyl 2-methyl glutarate mol % 6.7 7.8 6.8__________________________________________________________________________ Ex. 8 Ex. 9 Ex. 10__________________________________________________________________________components butadiene g (mol) 15 (0.277) same as same as methanol g (mol) 22 (0.686) Ex. 8 Ex. 8 Co.sub.2 (CO).sub.8 g (mol) 2 (0.0058) mixed (1) kind pyridine pyridine β-picoline solution of g (mol) 10 (0.126) 10 (0.126) 10 (0.107) amine (2) kind 3,4-lutidine 3,5-lutidine γ-picoline g (mol) 10 (0.093) 10 (0.093) 10 (0.107) hydrocarbon solvent, kind cyclohexane decalin methyl acetate ether solvent or g (mol) 20 (0.238) 20 (0.145) 20 (0.197) ester solventreaction hydroesteri- reaction presssure of 300 same as same asconditions fication of CO kg/cm.sup.2 Ex. 8 Ex. 8 butadiene reaction temperature 130 °C. reaction time Hr 1.5 hydroesteri- reaction pressure of 300 same as same as cation of CO kg/cm.sup.2 Ex. 8 Ex. 8 3-pentenoic reaction temperature 185 ester °C. reaction time Hr 2.0conversion of butadiene mol % 100 100 100selectivity to dimethyl adipate mol % 69.8 67.3 70.5selectivity to methyl 3-pentenoate mol % 7.6 8.1 10.4selectivity to methyl n-valerate mol % 8.7 9.3 7.2selectivity to dimethyl 2-ethyl succinate mol % 2.0 1.9 1.6selectivity to dimethyl 2-methyl glutarate mol % 8.0 8.2 5.7__________________________________________________________________________
Control Tests 1 and 2
The procedures of the above examples were repeated by using starting materials and reaction conditions as shown in Table 2. The results are shown in Table 2.
TABLE 2__________________________________________________________________________ control test 1 control test 2__________________________________________________________________________components butadiene g (mol) 15 (0.277) 15 (0.277) methanol g (mol) 22 (0.686) 22 (0.686) Co.sub.2 (CO).sub.8 g (mol) 2 (0.0058) 2 (0.0058) pyridine g (mol) 20 (0.253) 20 (0.253) benzene g (mol) 20 (0.256)reaction hydroesteri- reaction pressure of 300 300conditions fication of CO kg/cm.sup.2 butadiene reaction temperature 130 130 °C. reaction time Hr 1.5 1.5 hydroesteri- reaction presssure of 300 300 fication of CO kg/cm.sup.2 3-pentenoic reaction temperature 185 185 ester °C. reaction time Hr 2 2conversion of butadiene mol % 94 97.8selectivity to dimethyl adipate mol % 42 48selectivity to methyl 3-pentenoate mol % 6.2 2.2selectivity to methyl n-valerate mol % 5.1 5.3selectivity to dimethyl 2-ethyl succinate mol % 1.6 1.5selectivtiy to dimethyl 2-methyl glutarate mol % 8.1 7.6__________________________________________________________________________
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A process for producing adipic acid diester which comprises the first step of reacting butadiene, carbon monoxide and an alcohol in the present of cobalt carbonyl catalyst at a temperature of from 80° to 160° C. to form a 3-pentenoic acid ester, and
the second step of reacting the 3-pentenoic acid ester in the reaction mixture, carbon monoxide and an alcohol at a temperature of from 160° to 220° C., characterized in that the first and second reactions are carried out in an amine solvent is disclosed.
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BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a glass substrate for use as an information recording medium such as a magnetic disk or the like which can be used as a hard disk, and a method of manufacturing such a glass substrate.
[0003] 2. Description of the Related Art
[0004] Hard disk drives has magnetic heads which are slightly lifted off corresponding magnetic surfaces of hard disks as they rotate in reading data from and storing data in the hard disks.
[0005] If a hard disk has a perfectly flat surface, then when a magnetic head is to be lifted off the hard disk surfaces from a CSS (Contact Start-Stop) mode, the magnetic head tends to adhere to the hard disk surface. Therefore, it has been customary for hard disk surfaces to have minute surface roughness referred to as texture.
[0006] Conventional texturing techniques for roughening hard disk surfaces include a film texturing process for growing a film with a rough surface on the surface of a glass substrate and a laser texturing process for applying a laser beam to form surface roughness directly on a glass substrate.
[0007] Recent higher-density recording hard disk designs require hard disk drives to reduce the height of lifted magnetic heads while operating in a seek mode.
[0008] The inventor has found that the surface roughness of a glass substrate for use as a hard disk has to satisfy certain conditions in order to avoid adhesion or sticking of the magnetic head which is lifted to a reduced height.
[0009] However, the conventional texturing processes including the film texturing process and the laser texturing process fail to produce the level of fine surface roughness which satisfies those conditions.
SUMMARY OF THE INVENTION
[0010] According to the present invention, a glass substrate for use as an information recording medium has a finely roughened surface on which a magnetic film is to be formed, said finely roughened surface having an average surface roughness (Ra) in the range of 0.3 nm≦Ra<3.0 nm and including surface irregularities shaped and distributed isotropically and arranged substantially in succession, the surface irregularities including 5 to 50,000 peaks or convexities having a height of at least 3 nm and no convexities having a height of at least 15 nm within an area of 50 μm×50 μm. The glass substrate has an acid-resistant criterion in terms of an etching rate of at least 16 nm/min. upon contact with hydrofluoric acid at a temperature of 50° C. and a concentration of 0.1 weight %.
[0011] The average surface roughness (Ra) is extended three-dimensionally such that the central-line average roughness defined by JIS B0601 is applicable to a measured surface (10 μm×10 μm), and is defined as follows:
Ra = ( 1 / n ) ∑ i = 1 n abs ( Zi - Z0 )
[0012] where n represents the number of data points of a scanning probe microscope, abs an absolute value, Zi an ith data value of the scanning probe microscope, and
Z0 = 1 / n ∑ i = 1 n Zi
[0013] When the surface of the glass substrate is chemically strengthened by an ion exchange to produce a surface compressive stress, the glass substrate is made suitable for hard disks.
[0014] If the glass substrate contains SiO 2 and Al 2 O 3 , then the difference (SiO 2 —Al 2 O 3 ) between their molar fractions (molar %) is preferably at most 59.5 molar %.
[0015] [0015]FIG. 1 of the accompanying drawings shows the relationship between the difference (SiO 2 —Al 2 O 3 ) and the average surface roughness (Ra). It can be seen from FIG. 1 that if the difference (SiO 2 —Al 2 O 3 ) between the molar fractions exceeded 59.5 molar %, the surface roughness (Ra) of the roughened surface could not exceed 0.3 nm even when the concentration of the hydrofluoric acid or sulfuric acid used in an acid treatment process.
[0016] [0016]FIG. 2 of the accompanying drawings shows the relationship between the difference (SiO 2 —Al 2 O 3 ) between the molar fractions and the number of convexities having a height of at least 3 nm in the area of 50 μm×50 μm. A study of FIG. 2 reveals that if the difference (SiO 2 —Al 2 O 3 ) between the molar fractions exceeded 59.5 molar %, the number of convexities having a height of at least 3 nm would be at most 5.
[0017] [0017]FIG. 3 of the accompanying drawings shows the relationship between the difference (SiO 2 —Al 2 O 3 ) between the molar fractions and the acid resistance of the glass substrate (the etching rate (nm/min.) upon contact with hydrofluoric acid at a temperature of 50° C. and a concentration of 0.1 weight %). A review of FIG. 3 indicates that if the difference (SiO 2 —Al 2 O 3 ) between the molar fractions exceeded 59.5 molar %, the acid resistance of the glass substrate would be less than 16 nm/min.
[0018] Therefore, the difference between the molar fractions of SiO 2 and Al 2 O 3 should preferably be at most 59.5 molar %.
[0019] From the composition of the glass, the difference between the molar fractions of SiO 2 and Al 2 O 3 has a lower limit of 42.5 molar %. The acid resistance at the time the difference between the molar fractions of SiO 2 and Al 2 O 3 is 42.5 molar % (the etching rate (nm/min.) upon contact with hydrofluoric acid at a temperature of 50° C. and a concentration of 0.1 weight %) is 2000 nm/min.
[0020] Preferable constituent proportions (molar fractions) of the glass substrate which include other constituents may be in the following ranges:
[0021] SiO 2 : 55-70 molar %
[0022] Al 2 O 3 : 1-12.5 molar %
[0023] Li 2 O: 5-20 molar %
[0024] Na 2 O: 0-12 molar %
[0025] K 2 O: 0-2 molar %
[0026] MgO: 0-8 molar %
[0027] CaO: 0-10 molar %
[0028] SrO: 0-6 molar %
[0029] BaO: 0-2 molar %
[0030] TiO 2 : 0-8 molar %
[0031] ZrO 2 : 0-4 molar %
[0032] The glass substrate according to the present invention may contain, in addition to the above constituents, colorants of Fe 2 O 3 , MnO, NiO, Cr 2 O 3 , CoO, etc., and clarifiers of SO 3 , As 2 O 3 , Sb 2 O 3 , etc. insofar as they do not impair the characteristics of the glass substrate.
[0033] Of the above constituents, SiO 2 is a major constituent of the glass. If the proportion of SiO 2 were less than 55 molar %, then the durability of the glass would be lowered, and if the proportion of SiO 2 exceeded 70 molar %, then the viscosity of the glass would be increased and the glass would not easily be melted. Therefore, the proportion of SiO 2 should preferably be in the range from 55 to 70 molar %.
[0034] Al 2 O 3 serves to increase the rate of an ion exchange and also to increase the durability of the glass. If the proportion of Al 2 O 3 were less than 1 molar %, then rate of an ion exchange and the durability of the glass would not be increased. If the proportion of Al 2 O 3 were in excess of 12.5 molar %, then the viscosity of the glass would be increased, the devitrification resistance of the glass would be lowered, and the glass would not easily be melted. Therefore, the proportion of Al 2 O 3 should preferably be in the range from 1 to 12.5 molar %.
[0035] Li 2 O is a constituent that is exchanged in an ion exchange, and serves to increase the solubility at the time the glass is melted. If the proportion of Li 2 O were less than 5 molar %, then the surface compressive stress of the glass substrate after the ion exchange would be insufficient, the viscosity of the glass would be increased, and the glass would not easily be melted. If the proportion of Li 2 O were in excess of 20 molar %, then the chemical durability of the glass substrate would be poor. Therefore, the proportion of Li 2 O should preferably be in the range from 5 to 20 molar %.
[0036] Na 2 O is a constituent that is exchanged in an ion exchange, and serves to increase the solubility at the time the glass is melted. If the proportion of Na 2 O were in excess of 12 molar %, then the chemical durability of the glass substrate would be poor. Therefore, the proportion of Na 2 O should preferably be at most 12 molar %.
[0037] K 2 O serves to increase the solubility at the time the glass is melted. If the proportion of Na 2 O were in excess of 2 molar %, then the chemical durability of the glass substrate would be poor, and the surface compressive stress of the glass substrate after the ion exchange would be lowered. Therefore, the proportion of K 2 O should preferably be at most 2 molar %.
[0038] MgO serves to increase the solubility of the glass. If the proportion of MgO were in excess of 8 molar %, then the liquid-phase temperature of the glass would be increased, and the devitrification resistance of the glass would be poor. Therefore, the proportion of MgO should preferably be at most 8 molar %.
[0039] CaO serves to increase the solubility of the glass. If the proportion of CaO were in excess of 10 molar %, then the liquid-phase temperature of the glass would be increased, and the devitrification resistance of the glass would be poor. Therefore, the proportion of CaO should preferably be at most 10 molar %.
[0040] SrO serves to increase the solubility of the glass. A large amount of SrO contained in the glass would not be preferable as it would increase the specific gravity of the glass. The proportion of SrO should preferably be at most 6 molar %.
[0041] BaO serves to increase the solubility of the glass. A large amount of BaO contained in the glass would not be preferable as it would increase the specific gravity of the glass. The proportion of BaO should preferably be at most 2 molar %.
[0042] TiO 2 is a constituent for increasing the chemical durability of the glass. If the proportion of TiO 2 were in excess of 8 molar %, then the liquid-phase temperature of the glass would be increased, and the devitrification resistance of the glass would be poor. Therefore, the proportion of TiO 2 should preferably be at most 8 molar %.
[0043] ZrO 2 is a constituent for increasing the chemical durability of the glass. If the proportion of ZrO 2 were in excess of 4 molar %, then the possibility for ZrO 2 to be separated out as fine crystals when the glass is melted would be increased. Therefore, the proportion of ZrO 2 should preferably be at most 4 molar %.
[0044] A method of manufacturing a glass substrate for use as an information recording medium according to the present invention includes an acid treatment process and an alkali treatment process. In the acid treatment process, the surface of a glass substrate is selectively dissolved to form fine pores therein. In the alkali treatment process which is carried out subsequent to the acid treatment process, the fine pores formed in the surface of the glass substrate are enlarged.
[0045] Specifically, when a glass made up of many constituents is treated by a treatment solution containing an acid, the constituents of the glass are not uniformly dissolved by the acid, but those constituents which are less resistant to the acid are dissolved preferentially. After the glass is treated in the acid treatment process, those constituents which are less resistant to the acid produce a porous region in the vicinity of the surface of the glass substrate. The constituents which are less resistant to the acid include an alkaline metal oxide, an alkaline earth metal oxide, an aluminum oxide, etc., and the constituents which are more resistant to the acid include a silica oxide, a titania oxide, a zirconia oxide, etc.
[0046] Therefore, a glass substrate which is more resistant to an acid is less likely to form a porous region after being treated with the acid, and does not produce sufficient surface irregularities when treated with an alkali after the acid treatment process.
[0047] According to the present invention, it has been found that in order for the glass substrate to achieve a surface roughness Ra of at least 0.3 nm in an alkali cleaning process after the acid treatment process, the etching rate of the glass substrate in a hydrofluoric acid bath (50° C.) having a concentration of 0.1 weight %, which represents an acid-resistant criterion, is required to be at least 16 nm/min.
[0048] The porous region produced by the acid treatment process in the glass substrate whose etching rate is at least 16 nm/min. would be completely removed if excessively etched by an alkaline solution. However, the etching process using the alkaline solution can be stopped at a stage where the pores in the porous region are enlarged by controlling conditions for the acid and alkali treatment processes.
[0049] By combining the acid and alkali treatment processes with each other, it is possible to form a finely roughened surface having an average surface roughness (Ra) in the range of 0.3 nm≦Ra<3.0 nm and including surface irregularities shaped and distributed isotropically and arranged substantially in succession.
[0050] If the glass substrate initially contains a large flaw, then the flaw tends to make some surface irregularities more visually noticeable than others or produce an undulating pattern of surface irregularities. To avoid such a drawback, it is preferable to carry out a polishing process prior to the acid treatment process for removing flaws and polishing marks having an amplitude of at least 5 nm thereby to make the average surface roughness (Ra) less than 0.5 nm.
[0051] The polishing process may be performed in any desired way. If the polishing process is carried out using an abrasive composition, then the abrasive composition may contain a cerium oxide, a silicon oxide, an aluminum oxide, a magnetite oxide, a manganese oxide, or the like.
[0052] Because the glass substrate can be polished evenly and smoothly if the abrasive composition is well dispersed in an abrasive liquid, the abrasive liquid should preferably be alkaline or neutral. To make the abrasive liquid alkaline, an additive of potassium hydroxide, sodium hydroxide, ammonia, trimethanol amine, or the like is added to the abrasive liquid.
[0053] The manganese oxide may be Mn 2 O 3, Mn 3 O 4 , or MnO 2 , for example. Since Mn 2 O 3 , Mn 3 O 4 , and MnO 2 are easily ionized and dissolved in an acidic solution containing an oxidizing agent, when the glass substrate is treated by a treating agent containing an acid in the acid treatment process subsequent to the polishing process, the abrasive composition can easily be removed if an oxidizing agent such as a hydrogen peroxide solution, an ozone solution, or the like.
[0054] If the glass substrate is used as a hard disk substrate, then it is preferable to add, after the alkali treatment process, a chemically strengthening process for increasing the surface compressive stress of the glass substrate by way of an ion exchange.
[0055] The acid used in the acid treatment process may be hydrofluoric acid, sulfuric acid, nitric acid, or phosphoric acid. If hydrofluoric acid is used, then its concentration should preferably be in the range from 0.01 weight % to 0.5 weight % because it has a large etching effect on glass. If sulfuric acid, nitric acid, or phosphoric acid is used, then its concentration should preferably be in the range from 0.1 weight % to 5 weight % because it has a small etching effect on glass.
[0056] In the alkali treatment process, it is preferable to use an aqueous solution containing an alkaline constituent, a surface-active agent, and a chelating agent as major constituents.
[0057] The alkaline constituent may comprise caustic soda, sodium hydroxide, tetramethylammonium hydroxide, sodium carbonate, or potassium carbonate. The surface-active agent may comprise a nonionic surface-active agent such as polyoxyethyne alkyl ether or a polyoxyethylene derivative, a cationic surface-active agent such as quaternary ammonium salt, e.g., lauryltrimethyl ammonium chloride, higher amine halogenate, e.g., hardened tallow amine, or halide alkyl pyridium, e.g., dodecylpyridinium chloride, an anionic surface-active agent such as ester sodium alkylsulfate, sodium fatty acid, alkyl aryl sulfonate, or the like, or an amphoteric surface-active agent such as amino acid salt, e.g., sodium lauryl aminopropionic acid. The chelating agent may comprise dimethylglyoxime, dithizone, oxine, acetylacetone, glycine, ethylenediaminetetraacetic acid, or nitrilotriacetic acid.
[0058] It is preferable that the aqueous solution contain 0.001 weight % -5 weight % of the alkaline constituent, 0.001 weight % -1 weight % of the surface-active agent, and 0.001 weight % -1 weight % of the chelating agent.
[0059] The above and other objects, features, and advantages of the present invention will become apparent from the following description when taken in conjunction with the accompanying drawings which illustrate preferred embodiments of the present invention by way of example.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] [0060]FIG. 1 is a graph showing the relationship between the difference between the molar fractions of SiO 2 —Al 2 O 3 and the average surface roughness (Ra);
[0061] [0061]FIG. 2 is a graph showing the relationship between the difference between the molar fractions of SiO 2 —Al 2 O 3 and the number of convexities having a height of at least 3 nm in an area of 50 μm×50 μm; and
[0062] [0062]FIG. 3 is a graph showing the relationship between the difference between the molar fractions of SiO 2 —Al 2 O 3 and the acid resistance of a glass substrate (the etching rate (nm/min.) upon contact with hydrofluoric acid at a temperature of 50° C. and a concentration of 0.1 weight %.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
INVENTIVE EXAMPLE 1
[0063] An aluminosilicate glass substrate for use as an information recording medium, having a thickness of 1.0 mm and a diameter of 65 mm, was evenly polished to an average surface roughness Ra of about 0.25 nm, using an abrasive composition containing CeO 2 (MIREK manufactured by Mitsui Mining & Smelting Co., Ltd.) and a suede pad.
[0064] The glass substrate was made up of SiO 2 : 65.5 molar % Al 2 O 3 : 11.5 molar %, Li 2 O: 8.0 molar %, Na 2 O: 9.0 molar %, MgO: 2.4 molar %, and CaO: 3.6 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 160 nm/min.
[0065] After the glass substrate was rinsed in a pure water bath, it was scrubbed with pure water to remove most of the abrasive composition.
[0066] Then, the glass substrate was dipped in a bath of 1.00 weight % of sulfuric acid, a bath of 0.02 weight % of hydrofluoric acid, or a bath of 0.10 weight % of hydrofluoric acid which was kept at 40° C., for 2.5 minutes. After an ultrasonic energy of 1 W/cm 2 at about 48 kHz was applied to the glass substrate for 2.5 minutes, the glass substrate was lifted out of the bath, and then rinsed in a pure water bath to remove the chemical therefrom.
[0067] Then, the glass substrate was dipped in a bath of a commercially available alkaline detergent (pH 11, RB25 manufactured by Chemical Products Co., Ltd.) kept at 40° C. for 2.5 minutes. After an ultrasonic energy of 1 W/cm 2 at about 48 kHz was applied to the glass substrate for 2.5 minutes, the glass substrate was lifted out of the bath, and then rinsed in a pure water bath to remove the chemical therefrom.
[0068] The process of dipping the glass substrate in the pure water bath to rinse the glass substrate was repeated three times. Finally, the glass substrate was dipped in a bath of isopropyl alcohol, and after an ultrasonic energy at about 48 kHz was applied to the glass substrate for 2 minutes, the glass substrate was dried in a vapor of isopropyl alcohol for 1 minute, thus producing a specimen of Inventive Example 1.
INVENTIVE EXAMPLE 2
[0069] The glass substrate was made up of SiO 2 : 66.0 molar %, Al 2 O 3 : 11.0 molar %, Li 2 O: 8.0 molar %, Na 2 O: 9.0 molar %, MgO: 2.4 molar %, and CaO: 3.6 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 113 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, thus producing a specimen of Inventive Example 2.
INVENTIVE EXAMPLE 3
[0070] The glass substrate was made up of SiO 2 : 66.1 molar %, Al 2 O 3 : 9.6 molar %, Li 2 O: 7.3 molar %, Na 2 O: 9.6 molar %, MgO: 2.9 molar %, CaO: 4.3 molar %, and K 2 O: 0.2 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 47 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, thus producing a specimen of Inventive Example 3.
INVENTIVE EXAMPLE 4
[0071] The glass substrate was made up of SiO 2 : 65.3 molar %, Al 2 O 3 : 8.1 molar %, Li 2 O: 5.2 molar %, Na 2 O: 12.3 molar %, MgO: 2.8 molar %, CaO: 4.1 molar %, K 2 O: 0.2 molar %, and SrO: 2.0 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 35 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, thus producing a specimen of Inventive Example 4.
INVENTIVE EXAMPLE 5
[0072] The glass substrate was made up of SiO 2 : 66.3 molar %, Al 2 O 3 : 7.1 molar %, Li 2 O: 5.2 molar %, Na 2 O: 12.3 molar %, MgO: 2.8 molar %, CaO: 4.1 molar %, K 2 O: 0.2 molar %, and SrO: 2.0 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 16 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, thus producing a specimen of Inventive Example 5.
INVENTIVE EXAMPLE 6
[0073] The glass substrate was made up of SiO2: 66.1 molar %, Al 2 O 3 : 9.6 molar %, Li 2 O: 7.3 molar %, Na 2 O: 9.6 molar %, MgO: 2.9 molar %, CaO: 4.3 molar %, and K 2 O: 0.2 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 47 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, except that the glass substrate was evenly polished to an average surface roughness Ra of about 0.40 nm using an abrasive composition containing CeO 2 and a suede pad, thus producing a specimen of Inventive Example 6.
INVENTIVE EXAMPLE 7
[0074] The glass substrate was made up of SiO 2 : 66.1 molar %, Al 2 O 3 : 9.6 molar %, Li 2 O: 7.3 molar %, Na 2 O: 9.6 molar %, MgO: 2.9 molar %, CaO: 4.3 molar %, and K 2 O: 0.2 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 47 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, except that the glass substrate was evenly polished to an average surface roughness Ra of about 0.31 nm using an abrasive composition containing Mn 2 O 3 (NANOBIX manufactured by Mitsui Mining & Smelting Co., Ltd.) and a nonwoven cloth and that a mixture of 1 weight % of sulfuric acid and 3 weight % of hydrogen peroxide solution was used instead of sulfuric acid and hydrofluoric acid in the acid treatment process, thus producing a specimen of Inventive Example 7.
INVENTIVE EXAMPLE 8
[0075] The glass substrate was made up of SiO 2 : 59.7 molar %, Al 2 O 3 : 3.8 molar %, Li 2 O: 14.8 molar %, Na 2 O: 1.4 molar %, MgO: 4.2 molar %, CaO: 7.2 molar %, K 2 O: 0.3 molar %, SrO: 4.2 molar %, TiO 2 : 2.9 molar %, and ZeO 2 : 1.5 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 65 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, except that the glass substrate was evenly polished to an average surface roughness Ra of about 0.25 nm using an abrasive composition containing CeO 2 and a suede pad, thus producing a specimen of Inventive Example 8.
COMPARATIVE EXAMPLE 1
[0076] The glass substrate was made up of SiO 2 : 67.3 molar %, Al 2 O 3 : 7.1 molar %, Li 2 O: 6.1 molar %, Na 2 O: 11.3 molar %, MgO: 2.4 molar %, CaO: 3.6 molar %, K 2 O: 0.2 molar %, and SrO: 2.0 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 14 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, except that the glass substrate was evenly polished to an average surface roughness Ra of about 0.25 nm using an abrasive composition containing CeO 2 and a suede pad, thus producing a specimen of Comparative Example 1.
COMPARATIVE EXAMPLE 2
[0077] The glass substrate was made up of SiO 2 : 66.1 molar %, Al 2 O 3 : 9.6 molar %, Li 2 O: 7.3 molar %, Na 2 O: 9.6 molar %, MgO: 2.9 molar %, CaO: 4.3 molar %, and K 2 O: 0.2 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 47 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, except that the glass substrate was unevenly polished to an average surface roughness Ra of about 0.40 nm to provide at least 10 polishing marks having a depth ranging from 20 nm to 30 nm and a length of at least 2 μm in an area of 50 μm×50 μm, using an abrasive composition containing CeO 2 and a suede pad, thus producing a specimen of Comparative Example 2.
COMPARATIVE EXAMPLE 3
[0078] The glass substrate was made up of SiO 2 : 66.1 molar %, Al 2 O 3 : 9.6 molar %, Li 2 O: 7.3 molar %, Na 2 O: 9.6 molar %, MgO: 2.9 molar %, CaO: 4.3 molar %, and K 2 O: 0.2 molar %. The etching rate of the glass substrate using an aqueous solution of 0.1 weight % of hydrofluoric acid at a temperature of 50° C. was 47 nm/min. The glass substrate was processed under the same conditions as in Inventive Example 1, except that the glass substrate was evenly polished to an average surface roughness Ra of about 0.50 nm using an abrasive composition containing CeO 2 and a suede pad, thus producing a specimen of Comparative Example 3.
[0079] The specimens of Inventive Examples 1-8 and Comparative Examples 1-3 were observed for substrate surface roughness (Ra) in a field of view of 50 μm×50 μm by a scanning probe microscope (SPI3700 manufactured by SPM SII). The observed results are given in the table shown below. The numbers of peaks having a height greater than 3 nm and the numbers of peaks having a height greater than 15 nm, observed in the field of view of 50 μm×50 μm are also given in the table. In the table, substrate surface irregularities as observed by the scanning probe microscope were evaluated as “uniform” if they were successive and isotropic, and “ununiform” otherwise.
Table
[0080] It can be seen from the above table that if the etching rate (with 0.1 weight % of hydrofluoric acid at 50° C.), which serves as an acid-resistant criterion, was at least 16 nm/min. as with Inventive Examples 1-8, isotropic and successive surface irregularities were formed on the glass substrates by the alkali treatment process subsequent to the acid treatment process, the surface irregularities having an average surface roughness Ra in the range of 0.3 nm≦Ra<3.0 nm and including 5 to 50000 peaks of a height of at least 3 nm and no peaks of a height of at least 15 nm in the area of 50 μm×50 μm. If the etching rate (with 0.1 weight % of hydrofluoric acid at 50° C.) was less than 16 nm/min. as with Comparative Example 1, no sufficient pores were formed in the glass substrate by the acid treatment process, but surface irregularities were formed on the glass substrates by the alkali treatment process, the surface irregularities having an average surface roughness Ra less than 0.3 nm and including 5 to 50000 peaks of a height of at least 3 nm and no peaks of a height of at least 15 nm in the area of 50 μm×50 μm. The glass substrate according to Comparative Example 1 had a smooth surface.
[0081] When the glass substrate was evenly polished and had an average surface roughness Ra less than 0.5 nm after the polishing process as with Inventive Examples 1-8, no visually noticeable polishing trace was produced by the alkali treatment process subsequent to the acid treatment process, but isotropic and successive surface irregularities having an average surface roughness Ra in the range of 0.3 nm≦Ra<3.0 nm were formed on the glass substrates.
[0082] When the glass substrate was unevenly polished to an average surface roughness Ra less than 0.5 nm to provide at least 10 polishing marks having a depth ranging from 20 nm to 30 nm and a length of at least 2 μm in the area of 50 μm×50 μm, as with Comparative Example 2, ununiform and discrete surface irregularities, including 100 or more peaks having a height of at least 15 nm, were formed on the glass substrate even though the average surface roughness Ra was in the range of 0.3 nm ≦Ra<3.0 nm.
[0083] When the glass substrate was polished to an average surface roughness Ra of at least 0.5 nm as with Comparative Example 3, ununiform and discrete surface irregularities, including 100 or more peaks having a height of at least 15 nm in the area of 50 μm×50 μm, were formed on the glass substrate.
[0084] According to the present invention, as described above, a glass substrate having a predetermined level of acid resistance is treated with an acid and an alkali under appropriate conditions to produce fine surface irregularities which have an average surface roughness Ra in the range of 0.3 nm≦Ra<3.0 nm and include 5 to 50000 peaks of a height of at least 3 nm and no peaks of a height of at least 15 nm in the observed area of 50 μm×50 μm. The surface irregularities are isotropic, i.e., not localized in any directions but present in all directions, and substantially successive.
[0085] Although certain preferred embodiments of the present invention have been shown and described in detail, it should be understood that various changes and modifications may be made therein without departing from the scope of the appended claims.
TABLE Inventive Example 1 2 3 4 5 6 Substrate Composition SiO2 65.5 66.0 66.1 65.3 66.3 66.1 mol % Al2O3 11.5 11.0 9.6 8.1 7.1 9.6 Li2O 8.0 8.0 7.3 5.2 5.2 7.3 Na2O 9.0 9.0 9.3 12.3 12.3 9.6 MgO 2.4 2.4 2.9 2.8 2.8 2.9 CaO 3.6 3.6 4.3 4.1 4.1 4.3 K2O 0.0 0.0 0.2 0.2 0.2 0.2 SrO 0.0 0.0 0.0 2.0 2.0 0.0 BaO 0.0 0.0 0.0 0.0 0.0 0.0 TiO2 0.0 0.0 0.0 0.0 0.0 0.0 ZrO2 0.0 0.0 0.0 0.0 0.0 0.0 SiO2—Al2O3 54.0 55.0 56.5 57.2 59.2 56.5 Etching rate (nm/min) 160 113 47 35 16 47 (in a bath of 0.1 weight % of HF at 50° C.) Abrasive compound type Polished substrate MIRAKE 0.25 0.26 0.24 0.25 0.25 0.40 Surface roughness uniform uniform uniform uniform uniform uniform uniformity Surface irregularity NANOBIX Ra (nm) (Acid treatment) (Alkali treatment) Treated substrate 1 wt % H2SO4 RB25 Surface roughness Surface roughness Ra (nm) 0.69 0.62 0.43 0.36 0.30 0.49 Ra (nm) Surface irregularity uniformity uniform uniform uniform uniform uniform uniform Surface irregularity Number of peaks (>15 nm) 0 0 0 0 0 0 uniformity Number of peaks (>3 nm) 8113 5608 1017 97 5 1354 Number of peaks 0.005 wt % HF RB25 (>3 nm) Surface roughness Ra (nm) 1.21 1.08 0.45 0.39 0.30 0.53 Number of peaks Surface irregularity uniformity uniform uniform uniform uniform uniform uniform (>15 nm) Number of peaks (>15 nm) 0 0 0 0 0 0 Number of peaks (>3 nm) 15110 11000 2513 210 6 3111 0.04 wt % HF RB25 Surface roughness Ra (nm) 1.4 1.30 0.5 0.42 0.31 0.59 Surface irregularity uniformity uniform uniform uniform uniform uniform uniform Number of peaks (>15 nm) 0 0 0 0 0 0 Number of peaks (>3 nm) 35050 30000 5505 321 10 6339 0.1 wt % HF RB25 Surface roughness Ra (nm) 1.98 1.85 1.22 0.71 0.33 1.37 Surface irregularity uniformity uniform uniform uniform uniform uniform uniform Number of peaks (>15 nm) 0 0 0 0 0 0 Number of peaks (>3 nm) 48250 46600 9801 957 15 1117 1 wt % H2SO4 + RB25 3% H2O2 Surface roughness Ra (nm) Surface irregularity uniformity Number of peaks (>30 nm) Number of peaks (>3 nm) Comparative Example 7 8 1 2 3 Substrate Composition SiO2 66.1 59.7 67.3 66.1 66.1 mol % Al2O3 9.6 3.8 7.1 9.6 9.6 Li2O 7.3 14.8 6.1 7.3 7.3 Na2O 9.6 1.4 11.3 9.6 9.6 MgO 2.9 4.2 2.4 2.9 2.9 CaO 4.3 7.2 3.6 4.3 4.3 K2O 0.2 0.3 0.2 0.2 0.2 SrO 0.0 4.2 2.0 0.0 0.0 BaO 0.0 0.0 0.0 0.0 0.0 TiO2 0.0 2.9 0.0 0.0 0.0 ZrO2 0.0 1.5 0.0 0.0 0.0 SiO2—Al2O3 56.5 55.9 60.2 56.5 56.5 Etching rate (nm/min) 47 65 14 47 47 (in a bath of 0.1 weight % of HF at 50° C.) Abrasive compound type Polished substrate MIRAKE 0.25 0.25 0.40 0.50 Surface roughness uniform uniform (uniniform) uniform Surface irregularity NANOBIX 0.31 Ra (nm) uniform (Acid treatment) (Alkali treatment) Treated substrate 1 wt % H2SO4 RB25 Surface roughness Surface roughness Ra (nm) 0.50 0.26 0.61 0.85 Ra (nm) Surface irregularity uniformity uniform uniform (ununiform) (ununiform) Surface irregularity Number of peaks (>15 nm) 0 1 511 597 uniformity Number of peaks (>3 nm) 2777 0 1298 1956 Number of peaks 0.005 wt % HF RB25 (>3 nm) Surface roughness Ra (nm) 0.62 0.27 1.2 1.79 Number of peaks Surface irregularity uniformity uniform uniform (ununiform) (ununiform) (>15 nm) Number of peaks (>15 nm) 0 0 369 411 Number of peaks (>3 nm) 5554 0.5 2442 3895 0.04 wt % HF RB25 Surface roughness Ra (nm) 0.73 0.27 1.96 2.53 Surface irregularity uniformity uniform uniform (ununiform) (ununiform) Number of peaks (>15 nm) 0 0 512 362 Number of peaks (>3 nm) 14757 0.3 4268 5985 0.1 wt % HF RB25 Surface roughness Ra (nm) 1.49 0.28 1.96 2.53 Surface irregularity uniformity uniform uniform (ununiform) (ununiform) Number of peaks (>15 nm) 0 0 598 651 Number of peaks (>3 nm) 24305 1.3 6933 7564 1 wt % H2SO4 + RB25 3% H2O2 Surface roughness Ra (nm) 0.38 Surface irregularity uniformity uniform Number of peaks (>30 nm) 0 Number of peaks (>3 nm) 11352
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A glass substrate for use as an information recording medium has an average surface roughness (Ra) in the range of 0.3 nm≦Ra<3.0 nm and includes surface irregularities shaped and distributed isotropically and arranged substantially in succession. The surface irregularities include 5 to 50,000 convexities having a height of at least 3 nm and no convexities having a height of at least 15 nm within an area of 50 μm×50 μm. A porous region produced by an acid treatment process in the glass substrate would be completely removed if excesively etched by an alkaline solution. However, the etching process using the alkaline solution can be stopped at a stage where the pores in the porous region are enlarged by controlling conditions for the acid and alkali treatment processes.
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This application claims the benefit of Provisional Application U.S. Ser. No. 60/881,410 filed Jan. 19, 2007.
BACKGROUND OF THE INVENTION
Use of pre-processed foods, both in homes and in restaurants, has created a demand for effective high-capacity automated food processing equipment. That demand is particularly evident with respect to hamburgers, molded steaks, fish cakes, and other molded food patties.
Food processors utilize high-speed molding machines, such as FORMAX®, MAXUM700®, F-6™, F-12™, F-19™, F-26™, or F-400™ reciprocating mold plate forming machine, available from Formax, Inc. of Mokena, Ill., U.S.A., for supplying patties to the fast food industry. High-speed molding machines are also described for example in U.S. Pat. Nos. 3,887,964; 4,372,008; 4,356,595; 4,821,376; 4,996,743, and published U.S. Patent Application 2005/0092187, U.S. Ser. No. 10/942,627 filed Sep. 16, 2004.
The FORMAX® F-26™ reciprocating mold plate forming machine has enjoyed widespread commercial success for over 35 years. A typical FORMAX® F-26™ molding machine can operate at 90 strokes per minute and produce about 32,400 patties per hour based on the standard width mold plate for the F-26™ which is about 27 inches wide and can include 6 mold cavities.
The mold plate for the FORMAX® F-26™ is mounted to the reciprocating driving mechanism by being keyed to a drawbar that is driven at opposite ends by the longitudinally reciprocating drive rods of the driving mechanism. The mold plate includes three spaced apart cylindrical holes that receive pins that are fixed to the drawbar. Side locks or cams rotate to overlie side recesses formed in the mold plate.
The FORMAX® MAXUM700® reciprocating mold plate patty forming machine was introduced in 2003. It is a larger machine that can operate at 120 strokes per minute and produce about 43,200 patties per hour based on the standard width mold plate for the MAXUM700® which is about 28.5 inches.
The mold plate for the FORMAX® MAXUM700® is also mounted to the reciprocating driving mechanism by being keyed to a drawbar that is driven at opposite ends by the longitudinally reciprocating drive rods of the driving mechanism. The mold plate includes two spaced apart oblong holes that receive oblong pins that are fixed to the drawbar. Side locks or cams rotate to overlie side recesses formed in the mold plate.
Due to the difference in lateral dimensions between the F-26™ and MAXUM700® mold plates and the difference in the shape and number of keys, the F-26™ mold plates are not currently compatible with the MAXUM700® forming machine.
A processing plant can be set up to run molding machines to mold patties of variable selected thickness, shape or food material content. Accordingly, the processing plant may already have an assortment of F-26™ mold plates to be fit into the F-26™ patty-forming machine. Currently, if the processing plant wishes to replace the F-26™ with a MAXUM700® machine, the F-26™ mold plates are not compatible. Additionally, some processing plants may wish to upgrade from one or more F-26™ machines to one or more MAXUM700® machines, or run a combination of F-26™ and MAXUM700® machines.
The present inventors have recognized that it would be desirable for a user of the MAXUM700® patty-forming to be able to reuse F-26™ mold plates, which the user may already have in inventory because of prior use of the F-26™ machine, in a MAXUM700® machine.
The present inventors have recognized that it would be desirable for a user of the MAXUM700® patty-forming machine to be able to use F-26™ mold plates, which the user already has in inventory because of current use of the F-26™ machine, interchangeably in a MAXUM700® machine.
SUMMARY OF THE INVENTION
The present invention set forth different apparatus and methods of modifying a reciprocating mold plate patty-forming machine such that a mold plate for a smaller machine can be fit into a larger forming machine. According to one aspect, a tooling set for the modification includes a dedicated drawbar configured to mount the smaller mold plate and modified wider spacers to closely meet the smaller mold plate within the forming machine. According to another aspect, the smaller mold plate is reworked to include a key pattern identical to or similar to the key pattern of the mold plate for the larger forming machine, and wider spacers are also provided.
According to the preferred embodiment of the present invention, an adapter system selectively mounts either a first mold plate having a first set of keyholes or a second mold plate having a second set of keyholes, different than the first set of keyholes to a single reciprocating mold plate patty-forming machine. The adapter system can include first and second sets of adapters and a drawbar having provision for connection at opposite ends to driving rods of the drive system. The first set of adapters have keys corresponding to the first set of keyholes, and the second set of adapters having keys corresponding to the second set of keyholes, the first and second set of adapters being selectively attached to the drawbar.
The present invention provides a method of modifying a reciprocating mold plate patty-forming machine of the type having a drawbar for connecting a mold plate and a drive mechanism connected to the drawbar for reciprocating the drawbar, for mounting either a first mold plate having a first width or a second mold plate having a smaller second width. The present invention provides a method of modifying a reciprocating mold plate patty-forming machine wherein the first mold plate comprises plural keyholes in a first pattern and the second mold plate comprises plural keyholes in a different second pattern, and wherein the mold plate forming machine comprises side spacers that closely meet the side edges of the first mold plate.
The inventive method includes the steps of: providing a first drawbar for the first mold plate and a second drawbar for the second mold plate, the first drawbar having first keys that correspond to the first pattern and the second drawbar having second keys corresponding to the second pattern; and providing replacement side spacers that have a width to closely meet the side edges of the second mold plate.
Preferably, the first and second drawbar comprise a common bar member, wherein the first drawbar comprises at least one adapter having the first keys, the adapter connectable to the common bar member.
Preferably, the second drawbar comprises at least one second adapter having the second keys, the second adapter connectable to the common bar member.
Numerous other advantages and features of the present invention will be become readily apparent from the following detailed description of the invention and the embodiments thereof, and from the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematical longitudinal cross-sectional view of a first patty-forming machine;
FIG. 2 is an enlarged sectional view taken from FIG. 1 ;
FIG. 3 is a schematical longitudinal cross-sectional view of a second patty-forming machine;
FIG. 3A is a enlarged, fragmentary longitudinal cross-sectional view taken from FIG. 3 ;
FIG. 3B is a enlarged, fragmentary cross-sectional view taken generally along line 3 B- 3 B in FIG. 3A ;
FIG. 4 is a sectional view taken generally along line 4 - 4 of FIG. 3 ;
FIG. 5 is a plan view of a first mold plate and associated spacers usable with the first patty-forming machine of FIGS. 1 and 2 ;
FIG. 6 is a plan view of a second mold plate and associated spacers usable with the second patty-forming machine of FIGS. 3 and 4 ;
FIG. 7 is a plan view of a first system for converting a first mold plate to be usable with the second patty-forming machine of FIGS. 3-4 ;
FIG. 8 is a plan view of a second system for converting a first mold plate to be usable with the second patty-forming machine of FIGS. 3-4 ;
FIG. 9 is a plan view of a third system for converting a first mold plate to be usable with the second patty-forming machine of FIGS. 3-4 ;
FIG. 10 is a plan view of a fourth system for converting a first mold plate to be usable with the second patty-forming machine of FIGS. 3-4 ;
FIG. 11 is a perspective view of a drawbar according to the fourth system of FIG. 10 set up to mount an F-26™ mold plate;
FIG. 12 is a perspective view of a drawbar according to the fourth system of FIG. 10 set up to mount a MAXUM700® mold plate;
FIG. 13 is a perspective view of one adapter used in the set up of FIG. 11 ;
FIG. 14 is a perspective view of one adapter used in the set up of FIG. 12 ; and
FIG. 15 is an exploded plan view of tooling used to convert a MAXUM700® machine to mount an F-26™ mold plate according to the fourth system of FIG. 10 .
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
While this invention is susceptible of embodiment in many different forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated.
FORMAX® F-26™ Patty-Forming Machine
The high-speed food patty molding machine 20 illustrated in FIGS. 1 and 2 generally illustrates a FORMAX® F-26™ patty-forming machine, available from Formax, Inc. of Mokena, Ill., U.S. This application incorporates by reference U.S. Pat. Nos. RE30,096; 4,996,743; 4,356,595; 4,821,376 and 4,182,003 which describe in more detail what is substantially a FORMAX® F-26™ patty-forming machine including improved configurations and operating controls.
Molding machine 20 includes a machine base 21 , preferably mounted upon a plurality of rollers or wheels. Machine base 21 supports the operating mechanism for machine 20 , and contains hydraulic actuating systems, electrical actuating systems, and most of the machine controls.
Molding machine 20 includes a supply means 24 for supplying a moldable food material, such as ground beef, fish, or the like, to the processing mechanisms of the machine. Supply means 24 comprises a large food material storage hopper 25 that opens into the intake of a food pump system 26 . The food pump system 26 includes two food pumps that continuously pump food, under pressure, into a manifold 27 connected to a cyclically operable molding mechanism 28 .
In the operation of machine 20 , a supply of ground meat or other moldable food material is dumped into hopper 25 from overhead. The floor of hopper 25 comprises a conveyor belt 31 for moving the food material longitudinally of the hopper toward the other components of the food material supply means 24 .
At the forward end of hopper 25 , the right hand end of the hopper as seen in FIG. 1 , the food material is fed downwardly by supply means 24 into the intake of the reciprocating pumps constituting pumping system 26 . The pumps of system 26 operate in overlapping alteration to each other; at any given time when machine 20 is in operation at least one of the pumps is forcing food material under pressure into the intake of manifold 27 .
The manifold 27 comprises a valving system for feeding the food material, still under relatively high pressure, into the molding mechanism 28 . Molding mechanism 28 operates on a cyclic basis, first sliding a multi-cavity mold plate 32 into receiving position over manifold 27 and then away from the manifold to a discharge position aligned with a series of knockout cups 33 . When mold plate 32 is at its discharge position, knockout cups 33 are driven downwardly, discharging the hamburgers or other molded products from machine 20 , as indicated by arrow A in FIG. 1 .
The conveyor belt 31 extends completely across the bottom of hopper 25 , around an end roller 35 and a drive roller 36 . A chain drive is provided for drive roller 36 , driven by an electric motor (not shown).
The forward end of hopper 25 communicates with a vertical pump feed opening 39 that leads downwardly into a pump intake chamber 41 . A U-shaped frame 42 is mounted on machine base 21 , extending over hopper 25 adjacent the left hand side of the hopper outlet 39 . A mounting bracket 43 is affixed to the upper portion of frame 42 , extending over the pump feed opening 39 in hopper 25 .
Multiple electric feed screw motors 45 (one shown), are mounted upon bracket 43 . Motor 45 drives a feed screw 51 that extends downwardly through opening 39 in alignment with a pump plunger 88 .
When machine 20 is in operation, one or more of the feed screw motors are energized whenever one plunger is withdrawn to the left in FIG. 1 , so that feed screws supply meat from hopper 25 downwardly through opening 39 and into one side of the intake 41 of the food pumping system 26 . Similarly, one or more of the feed screw motors actuate feed screws to feed meat to the other side of intake 41 whenever the other plunger 68 is withdrawn. In each instance, the feed screw motors are timed to shut off shortly after the plunger is fully retracted, avoiding excessive agitation of the meat. As the supply of food material in the outlet 39 of hopper 25 is depleted, conveyor belt 31 continuously moves the food forwardly in the hopper and into position to be engaged by feed screws.
The food pump system 26 comprises two reciprocating food pumps 61 (one shown) mounted upon the top 63 of machine base 21 . The first food pump 61 includes a hydraulic cylinder 64 having two ports. The piston 64 a in cylinder 64 is connected to an elongated piston rod 67 ; the outer end of piston rod 67 is connected to a large plunger 68 . Plunger 68 is aligned with a first pump cavity 69 formed by a pump cavity enclosure 71 that is divided into two chambers by a partial central divider wall 72 . The forward wall 74 of pump cavity 69 has a relatively narrow slot that communicates with the pump manifold 27 as described more fully hereinafter. The second food pump is essentially similar in construction to pump 61 .
The pump feed manifold 27 , comprises a manifold valve cylinder 101 fitted into an opening in housing 71 immediately beyond the pump cavity walls. The valve cylinder is selectively rotatable to direct food product from either pump cavity to a slot 111 in housing 71 that constitutes a feed passage for molding mechanism 28 .
The upper surface of the housing 71 that encloses the pump cavities and the manifold 27 comprises a support plate 121 that projects forwardly of the housing, and that affords a flat, smooth mold plate support surface. The mold plate support 121 may be fabricated as a separate plate bolted to or otherwise fixedly mounted upon housing 71 . It includes the upper portion of the manifold outlet passage 111 .
Mold plate 32 is supported upon plate 121 . Mold plate 32 includes a plurality of individual mold cavities 126 extending across the width of the mold plate (See FIG. 5 ) and alignable with the manifold outlet passageway 111 . A cover plate 122 is disposed immediately above mold plate 32 , closing off the top of each of the mold cavities 126 . A housing 123 is mounted upon cover plate 122 . The spacing between cover plate 122 and support plate 121 is maintained equal to the thickness of mold plate 32 by support spacers 402 , 404 ( FIG. 5 ) mounted upon support plate 121 ; cover plate 122 rests upon spacers 402 , 404 , 406 when the molding mechanism is assembled for operation. Cover plate 122 is held in place by mounting bolts.
Mold plate 32 is connected to two spaced apart drive rods 128 via a drawbar 127 . The drive rods 128 extend alongside housing 71 and are each connected at one end to a swing link 129 . The other end of each link 129 is pivotally connected to a rocker arm 131 which, with a respective second arm 132 , forms a crank pivoted on a fixed shaft 133 . The free end of crank arm 132 is provided with a lost motion connection, entailing a pin 134 in an elongated slot 135 , to a connecting rod assembly 136 that includes a hydraulic shock absorber 137 . Shock absorber 137 is connected to a mold plate crank arm 138 having a crank pin 139 linked to the output shaft 141 of a gear reducer 142 . Gear reducer 142 is driven through a variable speed drive, represented in FIG. 2 by a pulley 143 , actuated by a mold plate drive motor (not shown).
Molding mechanism 28 further comprises a knockout apparatus. The knockout apparatus comprises the knockout cups 33 , which are affixed to a mechanism within the housing 123 that is driven by the machine motor via chains and sprockets. The details of the knockout mechanism can be found in U.S. Pat. RE30,096, herein incorporated by reference. The mechanism reciprocates the knockout cups in synchronism with the movement of the mold plate. Knockout cups 33 are coordinated in number and size to the mold cavities 126 in mold plate 32 ; there is one knockout cup 33 aligned with each mold cavity 126 and the mold cavity size is somewhat greater than the size of an individual knockout cup.
FORMAX® MAXUM700® Patty-Forming Machine
The high-speed food patty molding machine 200 illustrated in FIGS. 3-4 substantially illustrates a FORMAX® MAXUM700® patty-forming machine. This application incorporates by reference published U.S. patent application 2005/0092187, U.S. Ser. No. 10/942,627 filed Sep. 16, 2004, herein incorporated by reference, which describes in more detail what is substantially a FORMAX® MAXUM700® patty-forming machine.
Molding machine 200 includes a machine base 221 which supports the operating mechanisms of the machine and contains hydraulic actuating systems, electrical actuating systems, and most of the machine controls.
The food patty molding machine 200 includes a supply mechanism 224 for storing and supplying a moldable food product, such as ground beef, fish, pork, chicken, potatoes, or the like, to the processing mechanisms of the machine. Supply means 224 includes a large food product storage hopper 225 that supplies food product to a food pump system 226 . System 226 includes two food pumps operating in alternation; other machines typically include only a single food pump. The two food pumps continuously pump food, under pressure, into a valve manifold 227 connected to a cyclically operable molding station 228 .
Molding station 228 includes a mold plate 232 that moves cyclically between a fill position, shown in FIG. 3 and a discharge position in which its mold cavities are outside of station 228 , within a knockout station 240 aligned with a set of knock-out cups 233 . Details of the knockout mechanism can be found in U.S. Ser. No. 10/942,627 filed Sep. 16, 2004, herein incorporated by reference.
Food supply means 224 includes a conveyor belt 231 that extends completely across the bottom of hopper 225 . The forward end of hopper 225 communicates with a vertical hopper outlet 239 that leads downwardly into two pump chambers; only one pump chamber 241 is shown. One or more feed screws 238 are driven in rotation to deliver food product through the outlet 239 to the pump chamber 241 . The conveyor belt 231 is driven in circulation to deliver food product in the hopper 225 to the feed screw 238 .
As illustrated in FIG. 4 , the food pump system 226 comprises two reciprocating food pumps 261 , 262 . Food pumps 261 , 262 are driven by hydraulic cylinders 264 , 284 , respectively. The piston in each cylinder is connected to a piston rod that is in turn connected to a large pump plunger 266 , 268 respectively. The respective plunger 266 , 268 is aligned with and extends into a pump cavity 269 , 289 , which is substantially enclosed by a housing 271 . The forward wall 274 of each pump cavity 269 , 289 includes a respective slot 273 , 293 that communicates with the valve manifold 227 .
As shown in FIG. 3A , valve feed manifold 227 includes a valve cylinder 301 fitted into an opening in housing 271 immediately beyond wall 274 . Valve cylinder 301 includes two intake slots 307 , 308 . One slot 307 , 308 is alignable with a corresponding outlet slot 273 , 293 in pump cavity wall 274 , depending on which pump 261 , 262 is in use. Rotation of cylinder 301 is effective to move one slot 307 , 308 into alignment and one slot 307 , 308 out of alignment with corresponding slots 273 , 293 depending on which pump is in operation and which is being refilled. Valve cylinder 301 also includes outlet slots 309 aligned with a slot 311 in housing 271 that comprises a fill passage for the molding station 228 .
As best illustrated in FIGS. 3B and 4 , mold plate 232 is connected to a drawbar 329 that is connected to drive rods 328 that extend alongside housing 271 . The mold plate 232 includes oblong keyholes 430 a , 430 b ( FIG. 6 ) that receive oblong keys 431 a , 431 b that are fastened to the drawbar 329 from below the drawbar 329 by two fasteners threaded into threaded holes in the keys 431 a , 431 b . The other end of each drive rod 328 is pivotally connected to a connecting link 331 via a coupling plate 331 a and a pivot connection. The connecting link 331 is shown in two positions (one solid, one dashed).
Each drive rod 328 is carried within a guide tube 332 that is fixed between a wall 334 and a front bearing housing 333 . The connecting links 331 are each pivotally connected to a crank arm 342 ( FIG. 3 ). The crank arm 342 is fixed to, and rotates with, a circular guard plate 335 .
The crank arms 342 are each driven by a right angle gear box 336 via a “T” gear box 337 having one input that is driven by a precise position controlled motor 338 such as a servomotor, and two outputs connected to the gearboxes 336 . The “T” gear box 337 and the right angle gear boxes 336 are configured such that the crank arms 342 rotate in opposite directions at the same rotary speed.
A tie bar 339 is connected between the rods 328 to ensure a parallel reciprocation of the rods 328 . As the crank arms 342 rotate in opposite rotational directions, the outward centrifugal force caused by the rotation of the crank arms 342 and the eccentric weight of the attached links 331 cancels, and separation force is taken up by tension in the tie bar 339 .
During most of each cycle of operation of mold plate 232 , the knockout mechanism remains in the elevated position, with knockout cups 233 clear of mold plate 232 . When mold plate 232 reaches its extended discharge position the knockout cups 233 are driven downward to discharge the patties from the mold cavities.
FIG. 3A illustrates the upper surface of the housing 271 that encloses the pump cavities and the manifold 227 comprises a support plate 381 that affords a flat, smooth mold plate support surface. The mold plate support 381 may be fabricated as a separate plate bolted to or otherwise fixedly mounted upon housing 271 . It includes the upper portion of the manifold outlet passage 311 .
Preferably a separate fill plate 381 a is fixed to the manifold housing 271 . The fill plate 381 a includes a plurality of openings 381 b for passing food product into the mold cavities 126 . A reciprocal stripper or seal-off plate 383 is sliding carried by the fill plate 381 a . The function, configuration and structure of the fill plate and stripper and seal-off plate is described in U.S. Pat. No. 4,821,376, herein incorporated by reference.
Mold plate 232 is supported upon plate 381 and 381 a . Mold plate 232 includes a plurality of individual mold cavities 126 extending across the width of the mold plate (See FIG. 6 ) and alignable with the manifold outlet passageway 311 . Cover plate 122 is held in place by mounting bolts.
A breather plate 390 can be arranged above the mold plate 232 closing off the top of each of the mold cavities 126 . The spacing between the breather plate 390 and support plate 121 is maintained equal to the thickness of mold plate 232 by support spacers 402 , 404 , 406 ( FIG. 6 ) mounted upon support plate 381 . The breather plate 390 includes air apertures 391 that communicate air into passages 392 , 393 , 394 , 395 and 396 into the opening 239 and pump cavity 241 . The passages 394 are through the rear spacer 406 .
A cover plate 322 is disposed above the breather plate 390 . A housing 323 which contains the knockout mechanism is mounted upon cover plate 322 .
FORMAX® F-26™ Mold Plate and FORMAX® MAXUM700® Mold Plate
FIG. 5 illustrates the mold plate 32 used in the forming machine 20 of FIGS. 1 and 2 . Additionally, side spacers 402 , 404 and rear spacer 406 are shown. The mold plate 32 is of conventional design for use in a FORMAX® F-26™ reciprocating mold plate patty forming machine. The mold plate includes the mold cavities 126 , food product pressure balance grooves 410 opened to pressure balance slots 412 , and food particle drop slots 416 . To mount the mold plate 32 to a drawbar, three cylindrical key holes are provided through the mold plate 32 : a center keyhole 420 a , a left keyhole 420 b and a right keyhole 420 c . Additionally, a left corner recess 422 a and a right corner recess 422 b are provided. A conventional F-26™ mold plate has a width W 1 equal to about 27 inches and a length L 1 equal to about 19.5 inches.
FIG. 6 illustrates the mold plate 232 used in the forming machine 200 of FIGS. 3-4 . Similar components or configurations to the arrangement in FIG. 5 are given the same reference number even though the components may be of different size in practice. The mold plate 232 is of typical design for use in a FORMAX® MAXUM700® reciprocating mold plate patty forming machine. The area marked 431 provides slots and grooves configured as described in U.S. Provisional Application No. 60/844,789, filed Sep. 15, 2006. Alternately the mold plate, the breather plate and spacers can be as described in U.S. patent application 2005/0092187, U.S. Ser. No. 10/942,627 filed Sep. 16, 2004, both herein incorporated by reference. To mount the mold plate 32 to a drawbar, two oblong or rounded rectangular keyholes: a left keyhole 430 a and a right keyhole 430 b are provided through the mold plate 232 . Additionally, the left corner recess 422 a and the right corner recess 422 b are provided. A conventional MAXUM700® mold plate has a width W 2 equal to about 28.5 inches and a length L 2 equal to about 20.375 inches.
Using FORMAX® F-26™ Mold Plate on a FORMAX® MAXUM700® Machine
As stated in the background of the invention, the present inventors have recognized that it would be advantageous to be able to mount an FORMAX® F-26™ mold plate onto a FORMAX® MAXUM700® patty forming machine.
One system of providing this compatibility is illustrated in FIG. 7 . According to this embodiment, a new drawbar 450 set up to be mounted in a FORMAX® MAXUM700® machine includes lock cams set inward to correspond to the width W 1 of the F-26™ mold plate. Furthermore, the drawbar 450 includes cylindrical pins 456 a , 456 b , 456 c that fit within the cylindrical keyholes through the mold plate 32 , keyholes 420 a , 420 b , 420 c , respectively. Wider side spacers 460 , 462 and a wider end spacer 464 (see FIG. 15 ) would also be required due to the smaller dimensions of the F-26™ mold plate. If a perforated fill plate and seal off stripper are used, replacement fill plates and seal off stripper plates to accommodate the different mold cavity number and pattern, may also be required.
Two cam locks 454 are used to hold the mold plate 32 to the drawbar 450 . To mount or remove a mold plate the cams are turned with flat sides inward. To lock down a mold plate to the drawbar, the cam locks are tuned to overlie the recesses. A friction washer prevents the cam locks from turning once in locked position.
One drawback to this method is that a different drawbar would be needed to thereafter re-mount a FORMAX® MAXUM700® mold plate to this FORMAX® MAXUM700® forming machine. In effect, an F-26™ mold plate compatible-drawbar and a FORMAX® MAXUM700® mold plate-compatible drawbar would be required to run both types of mold plates on this FORMAX® MAXUM700® machine.
Another method of providing this compatibility is illustrated in FIG. 8 . According to this system, the mold plate 32 is modified to include a left oblong keyhole 466 a and a right oblong keyhole 466 b for receiving a left oblong key 468 a and a right oblong key 468 b that are fastened to a drawbar 470 by screws that extend from a bottom side of the drawbar 470 . The oblong keys 468 a , 468 b provide sufficient shear strength of the connection between the mold plate 32 and the drawbar 470 . The key holes 420 a , 420 b , 420 c are retained for the purpose of reusing the mold plate 32 in an FORMAX® F-26™ molding machine. The oblong keyholes 466 a , 466 b and the oblong keys 468 a , 468 b cannot be located in the correct position to also mount a mold plate 232 according to this embodiment if the keyholes 420 a , 420 b , 420 c are to be retained. Therefore, according to this system a designated drawbar must be used for the mold plate 32 which is incompatible with mounting the mold plate 232 .
Wider side spacers 460 , 462 and a wider end spacer 464 (see FIG. 15 ) would also be required due to the smaller dimensions of the F-26™ mold plate. If a perforated fill plate and seal off stripper are used, replacement fill plates and seal off stripper plates to accommodate the different mold cavity number and pattern, may also be required.
Two cam locks 454 are used to hold the mold plate 32 to the drawbar 470 . To mount or remove a mold plate the cams are turned with flat sides inward. To lock down a mold plate to the drawbar, the cam locks are tuned to overlie the recesses. A friction washer prevents the cam locks from turning once in locked position.
Another method for providing this compatibility is shown in FIG. 9 . According to this system, the mold plate 32 is modified wherein oblong key holes 466 a , 466 b are cut through the mold plate 32 at the same positions as required for the mounting of the mold plate 232 on the keys 431 a , 431 b ( FIG. 3B ). According to this system, either the mold plate 32 or the mold plate 232 can be mounted to a common drawbar 490 that has oblong keys 431 a , 431 b fastened thereto and inserted into either the oblong keyholes 466 a , 466 b of the mold plate 32 or the oblong key holes 430 a , 430 b of the mold plate 232 ( FIG. 6 ). A drawback of the system is that once the mold plate 32 is so modified, it cannot be re-installed into an F-26™ machine.
Wider side spacers 460 , 462 and a wider end spacer 464 (see FIG. 15 ) would also be required due to the smaller dimensions of the F-26™ mold plate. If a perforated fill plate and seal off stripper are used, replacement fill plates and seal off stripper plates to accommodate the different mold cavity number and pattern, may also be required.
Two cam locks 454 are used to hold the mold plate 32 to the drawbar 490 . To mount or remove a mold plate the cams are turned with flat sides inward. To lock down a mold plate to the drawbar, the cam locks are tuned to overlie the recesses. A friction washer prevents the cam locks from turning once in locked position. When the wider mold plate 232 is used, the cam locks 454 are relocated to a wider position to accommodate the wider mold plate 232 . In this regard, four holes are provided in the drawbar 490 for the installation of the cam locks in the desired position, two inner holes 454 a , 454 a and two outer holes 454 b , 454 b.
A further method of providing this compatibility is described in FIG. 10 . According to this system a common drawbar 500 can be used for both mold plate 32 , and mold plate 232 . According to this embodiment the drawbar 500 is provided with a flat top surface 502 with rectangular recesses 504 , 506 . To mount the mold plate 32 , a first set of adapters is installed. A left adapter 510 and a right adapter 512 include respective rounded rectangular adapter bases 510 a , 510 b that are fit snugly within the recesses 504 , 506 . The adapter bases 510 a , 512 a are fastened to the drawbar 500 using fasteners which extend from a bottom surface of the drawbar 500 into threaded holes into the adapter bases 510 a , 512 a open on a bottom of each adapter base. In the illustrated embodiment, the adapter includes threaded holes for four (one being aligned with the pin) fasteners.
Key pins 516 , 518 extend upwardly from each respective adapter base 510 a , 512 a . The recess and adapter are configured such that the key pins extend in registry with the mold plate keyholes 420 b , 420 c . A fixed key pin 519 on the drawbar fits into the keyhole 420 a . The key pin 519 is preferably attached with a screw from below or can be formed with, machined into, welded to, fastened to, or otherwise connected to the drawbar 500 .
Each adapter 510 , 512 , including the adapter base and key pin, comprises a machined, unitary body, preferably composed of 17-4 stainless steel.
Two cam locks 454 are used to hold the mold plate 32 to the drawbar 500 . To mount or remove a mold plate the cams are turned with flat sides inward. To lock down a mold plate to the drawbar, the cam locks are tuned to overlie the recesses. A friction washer prevents the cam locks from turning once in locked position. When the wider mold plate 232 is used, the cam locks 454 are relocated to a wider position to accommodate the wider mold plate 232 . In this regard, four holes are provided in the drawbar 500 for the installation of the cam locks in the desired position, two inner holes 454 a , 454 a and two outer holes 454 b , 454 b (see FIG. 11 ).
The system shown in FIG. 10 also includes provision for mounting the wider mold plate 232 . As shown in FIG. 12 , a second, different set of adapters, a left adapter 526 and a right adapter 528 are fit snugly within the recesses 504 , 506 , respectively. Each of the adapters 526 , 528 includes a rounded rectangular adapter base 526 a , 528 a , respectively. An oblong key 532 , 534 extends from each base 526 a , 528 a . The adapter bases 526 a , 528 a are fastened to the drawbar 500 using fasteners which extend from a bottom surface of the drawbar 500 into threaded holes into the adapter bases 526 a , 528 a open on a bottom of each adapter base. In the illustrated embodiment, the adapter includes threaded holes for three fasteners.
The oblong keys 532 , 534 are located on the respective bases 526 a , 528 a in order to be in registry with the oblong keyholes 430 a , 430 b of the mold plate 232 when the mold plate 232 is mounted to the drawbar 500 .
The center fixed key pin 519 may remain in most cases but is not used for mounting the mold plate 232 . Preferably, it is fastened in place and can be removed if necessary. Due to the shape of the mold plate 232 , the key pin 519 may not interfere with the mounting of the mold plate 232 .
Each adapter 526 , 528 , including the adapter base and oblong key comprises a machined, unitary body, preferably composed of 17-4 stainless steel.
The drawbars illustrated above are preferably composed of 17-4 stainless steel. The spacers 460 , 462 , 464 are preferably composed of mild steel or 300 series stainless steel.
According to this embodiment, by the use of selective adapters 510 , 512 , 526 , 528 , a common drawbar can be used for both mold plates 32 , 232 on a FORMAX® MAXUM700® machine. For setting up a FORMAX® MAXUM700® machine to use an FORMAX® F-26™ mold plate 32 , the tooling shown in FIG. 15 may be needed. The tooling includes the drawbar 500 , a narrower fill plate 381 a , a narrower stripper or seal off plate 383 , wider spacers 460 , 462 , 464 to compensate for the narrower mold plate 32 , and the adapters 510 , 512 . The adapters 526 , 528 are used to return the tooling to a condition to mount a FORMAX® MAXUM700® mold plate 232 on the FORMAX® MAXUM700® machine.
From the foregoing, it will be observed that numerous variations and modifications may be effected without departing from the spirit and scope of the invention. It is to be understood that no limitation with respect to the specific apparatus illustrated herein is intended or should be inferred.
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Apparatus and methods of modify a reciprocating mold plate patty-forming machine such that a mold plate for a smaller machine can be fit into a larger forming machine. A tooling set for the modification includes a dedicated drawbar configured to mount the smaller mold plate and modified wider spacers to closely meet the smaller mold plate within the forming machine. An adapter system selectively mounts either a first mold plate having a first set of keyholes or a second mold plate having a second set of keyholes, different than the first set of keyholes to a single reciprocating mold plate patty-forming machine. The adapter system can include first and second sets of adapters and a drawbar having provision for connection at opposite ends to driving rods of the drive system. The first set of adapters have keys corresponding to the first set of keyholes, and the second set of adapters having keys corresponding to the second set of keyholes, the first and second set of adapters being selectively attached to the drawbar.
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FIELD OF THE INVENTION
The present invention is directed to a method of joining bimetallic tubulars and a weld configuration for such bimetallic tubulars.
BACKGROUND OF THE INVENTION
Bimetallic tubulars with highly corrosion resistant inner liners can be utilized for reducing drill pipe corrosion in super sour well environments containing elemental sulfur. However, when the bimetallic tubulars are joined, alloying of the two distinct materials comprising the bimetallic tubular may occur and significantly reduce the bimetallic tubular's corrosion resistance and strength at the point of the weld. What is needed in the art is a method for welding and a weld configuration which preserves the continuity of the corrosion resistant inner liners of bimetallic tubulars and which avoids alloy formation of the different materials of the tubular layers at the weld joint.
The welding method and weld configuration of the instant invention advantageously avoid alloying of the discreet materials comprising the layers of the bimetallic tubulars when the weld joints are formed. Thus the possibility of decreased corrosion resistance and strength resulting from alloying of the two materials composing the bimetallic tubular in the weld configuration is eliminated.
SUMMARY OF THE INVENTION
The present invention is directed to a method of joining bimetallic tubulars and the weld configuration resulting therefrom.
The method comprises:
(a) joining a first and a second bimetallic tubular each having an inner layer comprised of a first composition, and an outer layer comprised of a second composition, wherein said inner layer extends beyond said outer layer at the area to be joined;
(b) circumferentially butt welding said inner layers of said first and said second bimetallic tubular such that said butt weld does not contact said outer layers of said first and said second bimetallic tubular and wherein said butt weld consists essentially of said first composition;
(c) circumferentially lap welding a sleeve having a first and second end, to said outer layers of said bimetallic tubulars at each of said sleeve ends, said sleeve slidingly engaged with said bimetallic tubular and encompassing said butt weld, said sleeve being comprised of said second composition, wherein said sleeve is of a length extending beyond said butt weld such that when said lap welding occurs said lap welds do not contact said inner layers of said butt weld, and wherein said lap weld consists essentially of said second composition of said bimetallic tubulars.
The weld configuration comprises:
(a) a first and a second bimetallic tubular each having a first and second end and an inner layer of a first composition and an outer layer of a second composition wherein said inner layer extends beyond said outer layer at said first end of said bimetallic tubular and wherein said bimetallic tubulars are aligned at said first end in butting relationship;
(b) a circumferential butt weld joining said inner layers at said first ends of said bimetallic tubulars, said butt weld consisting essentially of the same material as said inner layers of said bimetallic tubulars;
(c) a sleeve having a first and second end, said sleeve consisting essentially of the same material as said outer layers of said bimetallic tubulars, said sleeve surrounding said butt weld and of sufficient length such that said first and second ends of said sleeve contact only said outer layers of said first and said second bimetallic tubulars;
(d) a circumferential lap weld joining said sleeve to said outer layers of said bimetallic tubulars and located at each of said first and second ends of said sleeve, said lap weld consisting essentially of the same material as said outer layers of said bimetallic tubulars and contacting only said outer layers of said bimetallic tubulars.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows a cross sectional view of a preferred weld joint for a bimetallic tubular. FIG. 1B shows a second weld joint for a bimetallic tubular. FIG. 1A shows the preferred weld joint wherein the bimetallic tubulars being joined are fashioned in such a way that the inner layer extends beyond the outer layer end but is tapered to provide a V-groove butt weld configuration for the inner layer when two separate lengths of bimetallic tubulars are placed end to end in a straight line. In both FIGS. 1A and 1B the bimetallic tubulars having an inner layer (1) and an outer layer (2) are butt welded (4) where the butt weld penetrates only (1), and a sleeve (3) is placed over the butt weld, which sleeve is lap welded (5) such that the lap weld penetrates only (2) and (3).
DETAILED DESCRIPTION OF THE INVENTION
Bimetallic tubulars are tubes comprised of two layers, each consisting of two distinct and separate materials wherein the second or outer layer (2) circumferentially surrounds and is bonded to the first or inner layer (1). The materials comprising the layers of the bimetallic tubulars can be pure metals or alloys. During a conventional welding procedure, the materials of the inner and outer layers (2) mix forming an alloy which is more apt to corrode and/or lose strength than the layers of the bimetallic tubulars themselves. Applicants' welding procedure prevents such alloying of the distinct materials of the two layers and maintains the integrity of the inner and outer layers (2). Maintaining the integrity means that a continuous layer is formed for both the inner and outer layers (2) following applicants' welding procedure. The sleeve (3) utilized in applicants' method further adds to the strength of the welded bimetallic tubulars at the weld joint. Applicants' weld method and weld configuration are particularly advantageous for bimetallic tubulars having a corrosion resistant inner layer (1).
The welding method begins with a bimetallic tubular wherein the ends of the inner layer (1) extend beyond the ends of the outer layer (2). The extension provides an area where a circumferential butt weld (4) can be prepared without any of the welding material contacting the outer layer (2) and forming an alloy. Preferably, the bimetallic tubulars would be available with extending inner layers (1). However, if the ends of the inner and outer layers (2) are flush, a portion of the outer layer (2) can be ground away or removed in any manner to expose a suitable length of inner layer (1). A suitable length of inner layer (1) is one allowing for the butt weld (4) to be prepared without it contacting the outer layer (2). This is easily determined by one skilled in the art. To maintain the integrity of the inner layer (1), the butt weld (4) is prepared such that upon completion of the weld, the weld consists essentially of the same material as the inner layer (1). For example, if the inner layer (1) were niobium, the butt weld (4) would also be niobium. If the inner layer (1) were an alloy of titanium, the butt weld (4) would also be the titanium alloy. By utilizing the same welding material as the composition of the inner layer (1), the integrity of the inner layer (1) is maintained and one continuous tube is formed.
During the welding of bimetallic tubulars having layers comprised of alloys, it is possible that one of the metals forming the alloy may volatilize during the welding process. In such a case, the welding material will contain an additional amount of the volatile metal such that upon completion of the weld the weld consists essentially of the same material as the layer of the bimetallic tubular being welded. The additional amount of volatile metal necessary depends upon the composition of the layer of the bimetallic tubular and is readily determinable by one skilled in the art. In such a case the initial welding material will be enhanced with the volatile metal or metals in an amount which is equivalent to the amount vaporized during the welding procedure.
The method of the present invention can be applied to bimetallic tubulars whose layers are comprised of any individual metals or alloys of any metals. All metals of the periodic table and alloys thereof are contemplated as comprising the layers of the bimetallic tubulars. Preferably the inner layers (1) will be comprised of niobium. Likewise, the outer layer (2) will preferably be comprised of carbon steel.
Preferably, the bimetallic tubulars will be fashioned such that the inner layer (1) extends beyond the outer layer (2) and is formed to overlap the contour of the outer layer's (2) end but is tapered to provide a V-Groove butt weld (4) configuration for the inner layer (1) when two separate lengths of bimetallic tubular are placed end to end in a straight line (SEE FIG. 1A).
Once the butt weld (4) is complete, a sleeve (3) composed of the same material as the outer layer (2) and slidably engaging said bimetallic tubulars, is placed around the butt weld (4). The sleeve (3) length is such that it extends beyond the butt weld (4) for a length sufficient to allow for lap welding (5) at each of its ends without the lap welds (5) formed coming into contact with the inner layer (1) or the butt weld (4). The sleeve (3) is then circumferentially lap welded (5) to the outer layer (2) using essentially the same material the sleeve (3) and outer layer (2) are made of. Again, if the outer layer is an alloy, certain metals of the alloy may vaporize during the welding procedure. In such a case the welding material will contain an additional amount of the vaporizable metal which will compensate for the loss of such metal during welding enabling the weld to consist essentially of the same material of said sleeve (3) and said outer layer (2) once the welding is complete. By utilizing the same materials as the outer layer (2) for both the sleeve (3) and lap weld (5) material, the integrity of the outer layer is maintained, and no alloying between the sleeve (3) and outer layer (2) occurs. By utilizing a sleeve (3) such that its ends contact only the outer tubing, no alloy is formed with the inner tubing upon preparation of the lap weld (5).
Both the lap weld and the butt weld (4) can be prepared using standard methods known to those skilled in the art. For example ASME Section IX weld methods can be used.
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A method for joining bimetallic tubulars and a bimetallic tubular weld configuration which ensures both a continuous corrosion resistant inner lining across the joint as well as the required mechanical strength.
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BACKGROUND OF THE INVENTION
This invention generally relates to catheters, and particularly intravascular catheters for use in percutaneous transluminal coronary angioplasty (PTCA) or for the delivery of stents.
In percutaneous transluminal coronary angioplasty (PTCA) procedures a guiding catheter is advanced in the patient's vasculature until the distal tip of the guiding catheter is seated in the ostium of a desired coronary artery. A guidewire is first advanced out of the distal end of the guiding catheter into the patient's coronary artery until the distal end of the guidewire crosses a lesion to be dilated. A dilatation catheter, having an inflatable balloon on the distal portion thereof, is advanced into the patient's coronary anatomy over the previously introduced guidewire until the balloon of the dilatation catheter is properly positioned across the lesion. Once properly positioned, the dilatation balloon is inflated with inflation fluid one or more times to a predetermined size at relatively high pressures so that the stenosis is compressed against the arterial wall and the wall expanded to open up the vascular passageway. Generally, the inflated diameter of the balloon is approximately the same diameter as the native diameter of the body lumen being dilated so as to complete the dilatation but not overexpand the artery wall. After the balloon is finally deflated, blood flow resumes through the dilated artery and the dilatation catheter and the guidewire can be removed therefrom.
In such angioplasty procedures, there may be restenosis of the artery, i.e. reformation of the arterial blockage, which necessitates either another angioplasty procedure, or some other method of repairing or strengthening the dilated area. To reduce the restenosis rate of angioplasty alone and to strengthen the dilated area, physicians now normally implant an intravascular prosthesis, generally called a stent, inside the artery at the site of the lesion. Stents may also be used to repair vessels having an intimal flap or dissection or to generally strengthen a weakened section of a vessel or to maintain its patency. A tubular cover formed of synthetic or natural material may be present on an outer or inner surface of the stent. Stents are usually delivered to a desired location within a coronary artery in a contracted condition on a balloon of a catheter which is similar in many respects to a balloon angioplasty catheter, and expanded within the patient's artery to a larger diameter by expansion of the balloon. The balloon is deflated to remove the catheter and the stent left in place within the artery at the site of the dilated lesion. See for example, U.S. Pat. No. 5,507,768 (Lau et al.) and U.S. Pat. No. 5,458,615 (Klemm et al.), which are incorporated herein by reference.
In the design of catheter balloons, characteristics such as strength, compliance, and profile of the balloon vary depending on the desired use of the balloon catheter. A variety of polymeric materials are conventionally used in catheter balloons, and the balloon material and manufacturing procedure are chosen to provide the desired balloon characteristics. Use of polymeric materials such as PET that do not stretch appreciably consequently necessitates that the balloon is formed by blow molding, and the deflated balloon material is folded around the catheter shaft in the form of wings, prior to inflation in the patient's body lumen. However, it can be desirable to employ balloons, referred to as formed-in-place balloons, that are not folded prior to inflation, but which are instead expanded to the working diameter within the patient's body lumen from a generally cylindrical or tubular shape (i.e., essentially no wings) that conforms to the catheter shaft.
Catheter balloons formed of expanded polytetrafluoroethylene (ePTFE) expanded in place within the patient's body lumen without blow molding the ePTFE tubing have been disclosed. Prior disclosed methods of forming an ePTFE balloon involved wrapping a sheet of ePTFE on a mandrel and heating the wrapped sheet to fuse the layers of wrapped material together to form a tube. The resulting ePTFE tube may be subsequently heated in one or more additional heating steps and otherwise further processed, and typically combined with a nonporous liner to complete formation of the balloon. It would be a significant advance to provide an ePTFE tube for forming a balloon or other expandable medical device or component, with improved performance characteristics and manufacturability.
SUMMARY OF THE INVENTION
This invention is directed to a catheter balloon or other expandable tubular medical device or component, having at least a first layer with a first section, and a second section longitudinally compacted by more than the first section. The second section of the first layer is typically longitudinally adjacent to the first section of the first layer, and preferably extends at least in part along a central portion of the length of the first layer. The longitudinal compaction of the material of the first layer in accordance with the invention results in a balloon or other device or component having improved performance characteristics such as compliance and dimensional stability. One aspect of the invention is directed to a method of longitudinally compacting a porous polymeric tube incrementally along the length of the tube, to compact sections of the tube.
Longitudinal compaction of a section of the first layer decreases the length of the section and preferably also decreases the porosity of the material forming the section, so that in one embodiment, the first layer first section, which is more highly longitudinally compacted than the first layer second section, has a lower porosity than the first layer second section. The degree of longitudinal compaction is expressed herein as a percentage length reduction. Thus, a section having a precompaction length (i.e., the length of the section just prior to being longitudinally compacted in accordance with the invention) of 2 cm, which is subsequently longitudinally compacted to a length of 1 cm, has a longitudinal compaction of 50% (i.e., (2 cm−1 cm)÷2 cm).
In a presently preferred embodiment, the medical device tubular component is an inflatable balloon for a catheter. A balloon which embodies features of the invention can be used on a variety of suitable balloon catheters including coronary and peripheral dilatation catheters, stent delivery catheters, drug delivery catheters and the like. A balloon catheter of the invention generally comprises an elongated shaft having a proximal shaft section, a distal shaft section, at least a first lumen, and a balloon on a distal shaft section with an interior in fluid communication with the first lumen of the shaft. Although discussed below primarily in terms of the embodiment in which the medical device component is an inflatable member such as a balloon for a catheter, it should be understood that other expandable medical devices and components are included within the scope of the invention, including stent covers and vascular grafts.
The catheter balloon typically has a proximal and a distal skirt section secured to the shaft, an inflatable working length section, an inflatable proximal section between the proximal skirt section and the working length which inflates to a tapered configuration (“the proximal tapered section”), and an inflatable distal section between the distal skirt section and the working length which inflates to a tapered configuration (“the distal tapered section”). It should be understood that in at least in one embodiment the balloon is not blow molded or otherwise preexpanded into the inflated configuration prior to use. Thus, the “proximal and distal tapered sections” typically do not have a tapered configuration prior to the balloon being inflated during use of the balloon catheter.
In a presently preferred embodiment, the section of the first layer located at the central working length section of the balloon (hereafter the first layer working section) has a longitudinal compaction greater than the sections of the first layer located at the proximal and distal tapered sections of the balloon (hereafter the first layer tapered sections). The inflated tapered sections of the balloon form the transition between the skirt sections bonded to the shaft and the inflated working length of the balloon. Thus, depending on the length and the diameter of the inflated tapered sections, this transition can range from a gradual shallow taper to a short sharp transition. In one embodiment, the first layer tapered sections have a longitudinal compaction such that, when inflated, the tapered sections have a desired length and a desired inflated outer diameter tapering between the inflated working length to the skirt section, to decrease the hoop stress and stress concentration at the end of the skirt section. Thus, the rupture pressure of the balloon is increased by increasing the rupture pressure of the bond between the skirt section and the shaft. Specifically, in one embodiment, in the inflated configuration, the tapered sections taper at an angle of about 5° to about 45°, and with an inflated length of about 1 to about 5 mm. Thus, in one embodiment, the longitudinal compaction percentages of the sections are selected to provide a balloon having a desired inflated dimension and shape.
Additionally, in one embodiment, axial shrinkage of the balloon sections which would otherwise occur during inflation of the balloon is decreased by the longitudinal compaction of the first layer sections. Specifically, for no axial shrinkage or a minimal amount of axial shrinkage (i.e., less than 5%), the presently preferred the inflatable length of a porous polymeric layer of a balloon has a longitudinal compaction of about 10% to about 60%, preferably about 20% to about 50%. When not compacted, the axial shrinkage is believed to be about 10% to about 30% of the length of the balloon.
In one embodiment, the sections of the first layer located at the skirt sections of the balloon (hereafter the first layer skirt sections) have a longitudinal compaction which is less than or at least not greater than the longitudinal compaction of the first layer tapered sections. In one embodiment the first layer skirt sections are not longitudinally compacted, and thus have a longitudinal compaction of 0%. The first layer skirt section preferably has improved flexibility and low profile due to the low or zero percent longitudinal compaction of the skirt section. The skirt sections of the first layer extend along the section of the balloon secured to the shaft. However, the skirt sections of the first layer are not necessarily directly secured to the shaft, and may instead have at least a portion secured to a section of a second (inner) layer of the balloon which is directly secured to the shaft. The terminology “directly secured” to the shaft should be understood to include a variety of bonding methods including fusion and adhesive bonding.
In a presently preferred embodiment, the polymeric material of the first layer of the balloon comprises a polymer having a porous structure, which in one embodiment is selected from the group consisting of expanded polytetrafluoroethylene (ePTFE), ultra high molecular weight polyolefin such as ultra high molecular weight polyethylene, and porous polyolefins such as polyethylene and polypropylene, and porous polyurethane. In one embodiment, the porous material has a node and fibril microstructure. For example, ePTFE and ultra high molecular weight polyethylene typically have a node and fibril microstructure, and are not melt extrudable. The node and fibril microstructure, when present, is produced in the material using conventional methods. However, a variety of suitable polymeric materials can be used in the method of the invention including conventional catheter balloon materials which are melt extrudable. In one presently preferred embodiment, the polymeric material is typically not formed into a balloon by conventional balloon blow molding, and is instead formed into a balloon by heat fusing wrapped layers of the polymeric material together to form a tubular member. Porous materials such as ePTFE and ultrahigh molecular weight polyethylene typically require a nonporous second layer or liner when used to form an inflatable balloon. Thus, the balloon or other tubular medical device or component having a first layer with longitudinally compacted sections, should be understood to include an embodiment where the first layer forms at least a layer of a multilayered catheter balloon. The balloon first layer is typically longitudinally compacted before being secured to the balloon second layer, and the second layer is typically not longitudinally compacted.
A method of making a first layer of an expandable tubular medical device or component having at least one layer, generally comprises individually compacting incremental segments of a porous polymeric tube to incrementally compact the porous polymeric tube. In one embodiment, the method comprises placing a porous polymeric tube having a length and an outer diameter on a mandrel, with a diameter limiting member around at least a portion of the tube. In one embodiment, the diameter limiting device is selected from the group consisting of a die, a polymeric tube, and a sheet of polymeric material wrapped around the porous polymeric tube. A mold may also be used as the diameter limiting device, although preferably such that the mold uniformly limits the diameter of the segment of the tube during compaction, to thereby produce uniform compaction along the length of the individual segment avoiding buckling of the segment into an accordion-like configuration during compaction. At least a first compactor member is releasably secured to the tube at a first location on the tube, the secured compactor member being slidably disposed relative to the mandrel, and the compactor member is moved a distance, to thereby compact a first segment of the tube which is within the diameter limiting member and which has a length less than the entire length of the tube. The compactor member is released and repositioned on the tube at a second location on the tube, and the compactor member is releasably secured to the tube at the second location. The compactor member is then moved a distance, to compact a second segment of the tube which is adjacent to the first segment and which is within the diameter limiting member and which has a length less than the entire length of the tube. In one embodiment, the incremental compaction method of the invention produces uniform compaction along the length of the porous polymeric tube. In an alternative embodiment, one or more segments are compacted by an amount different than the first segment, so that the incremental compaction method of the invention produces variable compaction along the length of the porous polymeric tube.
The invention provides a catheter balloon or other expandable tubular medical device or component, having improved performance characteristics due to the longitudinal compaction of sections of the porous polymeric layer. In one embodiment, the compacted sections provide a balloon having an inflated configuration with a desired shape and dimensions, and with reduced stress at the skirt sections. An improved method of compacting porous polymeric material during formation of a tubular medical device or component, provides controllable incremental compaction of the porous polymeric material to produce uniform or variable compaction along the length of the tube. These and other advantages of the invention will become more apparent from the following detailed description of the invention and the accompanying exemplary drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an elevational view, partially in section, of a stent delivery balloon catheter embodying features of the invention.
FIG. 2 is a transverse cross sectional view of the balloon catheter shown in FIG. 1 , taken along line 2 — 2 .
FIG. 3 is a transverse cross sectional view of the balloon catheter shown in FIG. 1 , taken along line 3 — 3 .
FIG. 4 illustrates the balloon catheter of FIG. 1 , with the balloon inflated.
FIGS. 5 a-d illustrate an assembly of a tube of polymeric material on a mandrel, partially in section, during longitudinal compaction of portions of the tube to form a layer of the balloon of FIG. 1 , in a method which embodies features of the invention, in which a block is moved to compact a portion of the tube into a die.
FIGS. 6 a-c illustrate an assembly of a tube of polymeric material on a mandrel, partially in section, during longitudinal compaction of portions of the tube to form a layer of the balloon of FIG. 1 , in an alternative method which embodies features of the invention, in which two blocks are moved together to compact a portion of the tube in a diameter limiting member between the blocks.
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 illustrates an over-the-wire type stent delivery balloon catheter 10 embodying features of the invention. Catheter 10 generally comprises an elongated catheter shaft 12 having an outer tubular member 14 and an inner tubular member 16 . Inner tubular member 16 defines a guidewire lumen 18 configured to slidingly receive a guidewire 20 , and the coaxial relationship between outer tubular member 14 and inner tubular member 16 defines annular inflation lumen 22 , as best shown in FIG. 2 illustrating a transverse cross section view of the distal end of the catheter shown in FIG. 1 , taken along line 2 — 2 . An inflatable balloon 24 disposed on a distal section of catheter shaft 12 has a proximal skirt section 25 sealingly secured to the distal end of outer tubular member 14 and a distal skirt section 26 sealingly secured to the distal end of inner tubular member 16 , so that its interior is in fluid communication with inflation lumen 22 . An adapter 30 at the proximal end of catheter shaft 12 is configured to provide access to guidewire lumen 18 , and to direct inflation fluid through arm 31 into inflation lumen 22 . FIG. 1 illustrates the balloon 24 in a low profile tubular configuration prior to complete inflation, with an expandable stent 32 having a stent cover 35 , mounted on the balloon 24 for delivery within a patient's body lumen 27 . The distal end of catheter 10 may be advanced to a desired region of the patient's body lumen 27 in a conventional manner, and balloon 24 inflated to expand covered stent 32 , and the balloon deflated, leaving covered stent 32 implanted in the body lumen 27 . FIG. 3 illustrates a transverse cross section view of the distal end of the catheter shown in FIG. 1 , taken along line 3 — 3 .
In the embodiment illustrated in FIG. 1 , balloon 24 has a first layer 33 and a second layer 34 . In a presently preferred embodiment, the balloon 24 first layer 33 comprises a microporous polymeric material, and preferably a microporous polymeric material having a node and fibril microstructure, such as ePTFE. In the embodiment illustrated in FIG. 1 , first layer 33 is formed of ePTFE, and the second layer 34 is formed of a polymeric material preferably different from the polymeric material of the first layer 33 . Although discussed below in terms of one embodiment in which the first layer 33 is formed of ePTFE, it should be understood that the first layer may comprise other materials, including ultrahigh molecular weight polyethylene. The second layer 34 is preferably formed of an elastomeric material, such as polyurethane elastomers, silicone rubbers, styrene-butadiene-styrene block copolymers, polyamide block copolymers, and the like. In a preferred embodiment, layer 34 is an inner layer relative to layer 33 , although in other embodiments it may be an outer layer. Layer 34 formed of an elastomeric material limits or prevents leakage of inflation fluid through the microporous ePTFE to allow for inflation of the balloon 24 , and expands elastically to facilitate deflation of the balloon 24 to a low profile deflated configuration. The elastomeric material forming layer 34 may consist of a separate layer which neither fills the pores nor disturbs the node and fibril structure of the ePTFE layer 33 , or it may at least partially fill the pores of the ePTFE layer.
FIG. 4 illustrates the balloon catheter 10 of FIG. 1 , with the balloon in an inflated configuration. The inflated balloon 24 has a central working section with covered stent 32 thereon, a proximal tapered section between the working section and the proximal skirt section 25 , and a distal tapered section between the distal skirt section 26 and the working section. The section of the first layer 33 extending along the working section of the balloon is hereafter referred to as the first layer working section. Similarly, the first layer proximal and distal tapered sections refer to the sections of the first layer 33 extending along the proximal and distal tapered sections of the balloon, and the first layer skirt sections refer to the sections of the first layer 33 extending along the balloon skirt sections 25 , 26 . In one presently preferred embodiment, the inflated first layer working section has a length of about 8 to about 80 mm, the inflated first layer proximal tapered section has a length of about 1 to about 5 mm, and the inflated first layer distal tapered section has a length of about 1 to about 5 mm. Although the balloon 24 is illustrated in FIG. 4 with a conventional inflated configuration having a cental working length between two tapered inflatable sections, it should be understood that the inflated balloon may have a variety of suitable configurations including balloon configurations specially shaped for a particular anatomy such as a focal balloon configuration, a conical balloon configuration, and the like, as are conventionally known to one of skill in the art.
The first and second layers 33 , 34 of balloon 24 each extend from the proximal skirt section 25 of the balloon to the distal skirt section 26 of the balloon. The first layer 33 can have a length which is the same as or a shorter than the length of the second layer 34 , or alternatively, can have end sections which extend beyond the end sections of the second layer 34 and directly onto the shaft. FIG. 4 illustrates one embodiment in which the layers 33 , 34 of the balloon have the same length, so that the skirt sections 25 , 26 consist of end sections of the second (inner) layer 34 having an inner surface bonded to the shaft, and end sections of the first (outer) layer 33 having an inner surface bonded to the end sections of the second layer 34 . However, in an alternative embodiment (not shown), the ends of the first layer 33 extend beyond the end sections of the second layer 34 and onto the shaft 12 , so that the skirt sections 25 , 26 are also formed in part by end sections of the first layer 33 which extend beyond the end sections of the second layer 34 and bond directly to the shaft 12 without the second layer 34 therebetween. The lengths of the first layer skirt sections will vary depending on a variety of factors including the method of bonding the balloon to the shaft. In one presently preferred embodiment, the first layer proximal skirt section has a length of about 1 to about 5 mm, and the first layer distal skirt section has a length of about 1 to about 5 mm.
The ePTFE layer 33 of balloon 24 has sections with different amounts of longitudinal compaction, at least prior to being inflated. Preferably, the ePTFE layer 33 has a first section, and a second section longitudinally adjacent to the first section and extending at least in part along a central portion of the length of the balloon, the second section being longitudinally compacted by more than the first section. In a presently preferred embodiment, the second section of the ePTFE layer 33 is located at the first layer working length (i.e., the section of the first layer 33 extending along the working section of the inflated balloon), and the first section of the ePTFE layer 33 is located at the first layer proximal or distal tapered sections. In a presently preferred embodiment, the section of the first layer located at the working length of the balloon has a greater longitudinal compaction than the sections of the first layer located at both the proximal and the distal inflated tapered sections of the balloon. Thus, in one embodiment, the central working section of the ePTFE layer 33 has a greater longitudinal compaction than the remaining inflatable sections of the ePTFE layer 33 .
In one embodiment, the first layer working section has a longitudinal compaction of about 10% to about 60%, more specifically about 20% to about 50% of a prelongitudinal compaction length of the section, and the first layer tapered sections have a longitudinal compaction of about 10% to about 40%, more specifically about 15% to about 30% of a prelongitudinal compaction length of the sections. The percent longitudinal compaction values should be understood to refer to values existing prior to inflation of the balloon, and defined as a percentage length reduction from before to after the compaction process. For example, longitudinally compacting an ePTFE tube so that a section corresponding to the first layer working section is compacted from a prelongitudinal compaction length of about 2.86 cm to a compacted length of about 2 cm, produces a 2 cm first layer working section having a longitudinal compaction of about 30% prior to inflation of the balloon.
The first layer skirt sections have a longitudinal compaction which is not greater than, and preferably is less than the longitudinal compaction of the first layer tapered sections. In a presently preferred embodiment, the first layer skirt sections are not longitudinally compacted, and thus have a longitudinal compaction of 0%. In one embodiment, the first layer skirt sections have a longitudinal compaction of about 0% to about 30%, more specifically about 10% to about 20%.
Thus, in one presently preferred embodiment, a balloon ePTFE layer having a post-compaction working length of 20 mm compacted by 40%, post-compaction tapered sections of 2 mm compacted by 30%, and post-compaction skirt sections of 2 mm compacted by 20%, had an original pre-compaction working length of 33.3 mm, original pre-compaction tapered sections of 2.86 mm, and original pre-compaction skirt sections of 2.5 mm.
The first layer working section, prior to inflation of the balloon 24 , preferably has a lower porosity than the first layer proximal and distal tapered sections and lower than the first layer proximal and distal skirt sections. Specifically, in one embodiment, the first layer working section (prior to inflation) has a porosity about 0 to about 40% lower than a porosity of the first layer proximal and distal tapered sections, and about 10 to about 60% lower than a porosity of the first layer proximal and distal skirt sections. In one embodiment, the first layer proximal and distal tapered sections have a porosity lower than the first layer proximal and distal skirt sections, and specifically about 10 to about 30% lower than the porosity of the first layer proximal and distal skirt sections.
The ePTFE layer 33 is preferably formed according to a method in which an ePTFE tube used to form layer 33 is incrementally compacted. Specifically, the individual sections of layer 33 having specific longitudinal compaction values (e.g., the working length, and the proximal and distal tapered sections) are each produced by compacting multiple smaller length portions of the tube. For example, to produce a working length having a length of 2 cm and a longitudinal compaction of about 30%, 3 portions each having an initial precompaction length of 0.95 cm would each be successively compacted to a compacted length of 0.65 cm (i.e., (0.7)(0.95 cm)), to collectively produce the 2 cm working length having a 30% longitudinal compaction.
FIGS. 5 a-d illustrate an assembly with a polymeric tube 40 during incremental longitudinal compaction of the tube 40 in a method which embodies features of the invention. The polymeric material of the tube 40 is ePTFE in the embodiment in which the tube forms ePTFE layer 33 of the balloon 24 of FIG. 1 . The tube 40 may be provided with uniform longitudinal compaction such that each incremental segment is compacted by the same amount, or alternatively, it may be provided with variable compaction in which one or more incremental segments are longitudinally compacted by different amounts.
In the embodiment illustrated in FIG. 5 , the tube 40 is on a mandrel 41 with a portion of the tube 40 in a die 42 having an inlet 43 and an outlet 44 . The mandrel 41 may optionally have a polymeric jacket (not shown) on an outer surface of the metallic body. A compactor member 45 is releasably secured to the tube at a location on the tube 40 spaced from the die inlet 43 by a distance “d”. In the embodiment of FIG. 5 , the compactor member 45 comprises a block with a bore configured to surround and clamp onto the tube 40 with the mandrel 41 therein, such as with a collet-type clamping mechanism. However, a variety of suitable compactor members may be used including a hydraulic clamp. The compactor member 45 is secured to the tube 40 such that it is slidably disposed relative to the mandrel within the tube. A fixing member 46 releasably secures the tube 40 to the mandrel at a location on the tube adjacent the outlet 44 of the die 42 . In the embodiment of FIG. 5 , the fixing member 46 comprises a block similar to the compactor member 45 , with a bore configured to surround and clamp onto the tube 40 and mandrel 41 therein. With the compactor member 45 releasably secured to the tube 40 the distance “d” from the inlet 43 of the die 42 , a first segment “S 1 ” of the tube 40 is located between the compactor member 45 and the outlet 44 of the die 42 , as illustrated in FIG. 5 a . The compactor member 45 is then moved toward the inlet 43 of the die 42 to compact the first segment “S 1 ” of the tube 40 into the die, thereby forming compacted segment “CS 1 ”, as illustrated in FIG. 5 b . The compacted segment is illustrated in the figures by closer-spaced cross hatching. The difference between the original precompacted length “S 1 ” of the segment, and the compacted length “CS 1 ” of the segment, expressed as a percentage of the original precompacted length “S 1 ”, is the percent longitudinal compaction of the segment. The die may be heated to thereby heat the compacted segment “CS 1 ” in the die, to heat stabilize the compacted segment in the compacted configuration. In a presently preferred embodiment, the die is heated to an elevated temperature of about 320° C. to about 400° C., preferably about 350° C. to about 370° C. to heat stabilize the compacted segment. The compactor member 45 and fixing member 46 are then released, and the tube 40 is repositioned by sliding the compacted segment “CS 1 ” through the die outlet 44 to place another noncompacted portion of the tube 40 in the die lumen, as illustrated in FIG. 5 c . With the compactor member 45 and fixing member 46 again releasably secured to the tube 40 , the second segment “S 2 ” is compacted as outlined above. The second segment “S 2 ” may be compacted by the same amount as the first segment “S 1 ” in order to continue formation of a first compacted section, or it may be compacted by a different amount in order to provide for a second compacted section. FIG. 5 d illustrates the assembly after the compaction of the second segment “S 2 ” to produce compacted segment “CS 2 ”, with the compactor member and fixing member again repositioned and secured on the tube 40 , ready for compaction of a third segment “S 3 ”.
During compaction, the compactor member 45 may be moved the entire distance “d” to the die inlet 43 , or alternatively, it may be moved a distance less than “d” depending on the amount of longitudinal compaction desired for the segment being compacted. For example, in the embodiments illustrated in FIGS. 5 a-d , the length of the first, second, and third segments S 1 , S 2 , S 3 are approximately equal, so that the compactor member 45 could be moved the entire distance “d” to produce an amount of compaction in one of the segments, and moved a distance less than “d” in order to produce a smaller amount of compaction in one or more of the remaining segments.
The inner diameter of the inner chamber of die 42 is sized so that the tube 40 compacts without the outer diameter of the tube increasing. The inner diameter of the inner chamber of die 42 is typically about equal to the outer diameter of the tube 40 on the mandrel 41 . Alternatively, the inner diameter of the inner chamber of die 42 may be smaller than the outer diameter of the tube 40 on the mandrel 41 , so that it provides resistance to movement of the tube 40 therein to increase the percent compaction of the tube 40 . The length of the inner chamber of the die 42 in which tube is compacted is typically about 1 to about 5 cm, preferably about 2 to about 3 cm. The length of the tube 40 is typically about 4 to about 20 cm to produce a layer 33 of a balloon having a length of about 2 to about 10 cm. The length of the segments S 1 , S 2 , S 3 is typically about 5 to about 25 mm, preferably about 5 to about 15 mm. The length of the segment is preferably sufficiently short such that the segment compacts uniformly along the length of the segment and without buckling.
After being longitudinally compacted, the tube 40 may be heat treated or otherwise further processed and secured to the second layer 34 , to complete formation of the balloon 24 . The tube 40 is typically longitudinally stretched prior to being longitudinally compacted, as for example by being placed on a mandrel and pulled at either end to stretch down on to the mandrel, although it can be longitudinally stretched using a variety of suitable methods. With the tube 40 restrained in the longitudinally stretched configuration, the tube 40 is typically heated, to stabilize the tube in the stretched configuration prior to being longitudinally compacted.
FIGS. 6 a-c illustrate an assembly of polymeric tube 40 on mandrel 41 during incremental longitudinal compaction of the tube in an alternative method which embodies features of the invention. Similar to the embodiment of FIG. 5 , the tube 40 may be provided with uniform longitudinal compaction, or alternatively, with variable compaction.
In the embodiment illustrated in FIG. 6 , the tube 40 is on a mandrel 41 with a portion of the tube 40 in a diameter limiting member 52 comprising a sheet of polymeric material wrapped around the tube 40 . In a presently preferred embodiment, the sheet of polymeric material forming the diameter limiting member 52 is ePTFE, although other polymeric materials may be used, including Teflon, and polyolefins such as high density polyethylene (HDPE), low density polyethylene (LDPE), and linear low density polyethylene (LLDPE). At least a first compactor member 53 is releasably secured at a first location to the tube and slidably disposed relative to the mandrel 41 . In the embodiment of FIG. 6 , the first compactor member 53 is a block similar to the block of the embodiment of FIG. 5 . In the embodiment of FIG. 6 , a second compactor member 54 is releasably secured at a second location to the tube 40 longitudinally spaced apart from the first compactor member 53 with at least a portion of the diameter limiting device 52 therebetween. With the first and second compactor members 53 , 54 releasably secured to the tube 40 a distance apart, a first segment “S 1 ” of the tube 40 is located between the compactor members, as illustrated in FIG. 5 a . The first and second compactor members 53 , 54 are then moved toward one another toward a center of the length of the tube 40 to longitudinally compact the first segment “S 1 ” therebetween in the diameter limiting device 52 , thereby forming compacted segment “CS 1 ”, as illustrated in FIG. 6 b . The compactor members 53 , 54 are then released and resecured to the tube. In a presently preferred embodiment, the compactor members 53 , 54 are resecured to the tube 40 at locations further apart on the tube and closer to the ends of the tube 40 , with second segment “S 2 ” greater than “S 1 ” therebetween, as illustrated in FIG. 6 c . The compactor members 53 , 54 are illustrated in FIG. 6 c ready to be moved toward one another toward a center of the length of the tube 40 , to longitudinally compact the second segment “S 2 ” therebetween in the diameter limiting device 52 . The ePTFE tape 52 typically has a length sufficient to cover the entire length of the tube 40 to be compacted, so that the ePTFE tape 52 does not have to be removed and replaced with a longer length of ePTFE tape 52 between the compaction of each individual segment of tube 40 . In alternative embodiments (not shown) using alternative diameter limiting members such as a tube, die, or mold, the diameter limiting device is typically released from the compacted segment and a longer diameter limiting device is secured to the tube to accommodate the longer length of the next segment to be compacted. The compacted tube 40 may be heated to heat stabilize the compacted segments, as discussed above in relation to the embodiment of FIG. 5 , as for example by traversing a heating nozzle along the length of the compacted segments. In the illustrated embodiment in which the diameter limiting member 52 is ePTFE tape wound around the tube 40 , the tube may be heated at the end of the compaction of the final segment rather than after each individual compaction, because the ePTFE tape 52 is typically not removed between the compaction of each individual segment of the tube 40 . The length of the segments being compacted is typically about 10 to about 50 mm, preferably about 20 to about 30 mm.
In an alternative embodiment (not shown), a fixing member is used in place of the second compaction member 54 , which is releasably secures the tube 40 to the mandrel 41 , so that compacting the segment therebetween comprises moving the first compactor member 53 toward the fixing member. The distance between the first compaction member 53 and the fixing member is then increased, for compacting another segment of the tube as described above.
To the extent not previously discussed herein, the various catheter components may be formed and joined by conventional materials and methods. For example, the outer and inner tubular members 14 , 16 can be formed by conventional techniques, such as by extruding and necking materials found useful in intravascular catheters such a polyethylene, polyvinyl chloride, polyesters, polyamides, polyimides, polyurethanes, and composite materials.
The length of the balloon catheter 10 is generally about 108 to about 200 centimeters, preferably about 137 to about 145 centimeters, and typically about 140 centimeters for PTCA. The outer tubular member 14 has an outer diameter (OD) of about 0.017 to about 0.036 inch (0.43-0.91 mm), and an inner diameter (ID) of about 0.012 to about 0.035 inch (0.30-0.89 mm). The inner tubular member 14 has an OD of about 0.017 to about 0.026 inch (0.43-0.66 mm), and an ID of about 0.015 to about 0.018 inch (0.38-0.46 mm) depending on the diameter of the guidewire to be used with the catheter. The balloon 24 is has a length of about 8 mm to about 80 mm, typically about 8 mm to about 38 mm, and an inflated working diameter of about 1.5 mm to about 20 mm, typically about 2 mm to about 10 mm.
While the present invention has been described herein in terms of certain preferred embodiments, those skilled in the art will recognize that modifications and improvements may be made without departing from the scope of the invention. For example, although the embodiment illustrated in FIG. 1 is an over-the-wire stent delivery catheter, balloons of this invention may also be used with other types of intravascular catheters, such as rapid exchange balloon catheters. Rapid exchange catheters generally comprise a distal guidewire port in a distal end of the catheter, a proximal guidewire port in a distal shaft section distal of the proximal end of the shaft and typically spaced a substantial distance from the proximal end of the catheter, and a short guidewire lumen extending between the proximal and distal guidewire ports in the distal section of the catheter. While individual features of one embodiment of the invention may be discussed or shown in the drawings of the one embodiment and not in other embodiments, it should be apparent that individual features of one embodiment may be combined with one or more features of another embodiment or features from a plurality of embodiments.
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A catheter balloon or other expandable tubular medical device or component, having at least a first layer with a first section and a second section longitudinally compacted by more than the first section. In a presently preferred embodiment, the second section of the first layer extends at least in part along a central portion of the length of the first layer. The longitudinal compaction of the material of the first layer preferably results in a balloon or other expandable tubular medical device or component having improved performance characteristics such as compliance and dimensional stability. One aspect of the invention is directed to a method of longitudinally compacting a porous polymeric tube incrementally along the length of the tube, to compact sections of the tube.
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BACKGROUND
[0001] A lipoprotein can be categorized as a high-density lipoprotein (HDL), a low-density lipoprotein (LDL), a very low-density lipoprotein (VLDL), or a chylomicron (CM). It is well known that HDL serves a protective function by removing cholesterol accumulated in tissues, including the arterial walls, and then returning it to the liver. Therefore, the cholesterol in HDL, also known as high-density lipoprotein cholesterol (HDLC), is a negative risk factor for various types of arteriosclerosis, such as coronary arteriosclerosis, and the HDLC level in blood is a useful index for the precognition of arteriosclerosis.
[0002] Solution assays are conventional methods used for determining the amount of HDLC in the blood. These methods consist of two steps, a fractionation step and a detection step. The fractionation step separates HDL from other lipoproteins whereas the detection step quantifies the cholesterol in the HDL. Examples of fractionation methods include an ultracentrifugation method, an immunochemical method, an electrophoretic method, and a precipitation method. An alternative approach to conventional solution methods is to perform the HDLC assay in a single step dry slide analytical element, also known as a dry slide, on a polyester support.
[0003] The development of the direct HDLC dry slide requires a preferential selectivity for HDLC over low-density lipoprotein cholesterol (LDLC), very low-density lipoprotein cholesterol (VLDLC), and cholesterol in CM. Many well-known solution methods have been shown to improve HDLC specificity by including non-high density lipoprotein precipitation methods ( 1 , 2 , 3 , 4 , 5 ), immuno-inhibition ( 6 , 7 ), selective surfactants ( 8 , 9 , 10 , 11 ), Catalase elimination ( 12 ), and polyethylene glycol (PEG) cross-linked cholesterol esterase ( 13 ). However, the precipitation methods and selective surfactant methods cannot be done as single-step dry slide assays because they do not yield the sufficient HDLC selectivity needed for such a method. Other methods use multiple reagent additions or separation steps that also are not amenable to the all inclusive single-step dry slide technology. Because none of the well-known HDLC methods yield sufficient selectivity in the dry slide assay, additional methods to improve HDLC specificity were pursued.
[0004] Surfactants are surface active agents that can alter the properties of fluid interfaces between polar and non-polar moieties. It has been well known among persons skilled in the art that surfactants can be used to selectively disrupt protein membranes or selectively solubilize their components. Surfactant solubilization methods have been used for several decades to purify proteins from a wide class of human, animal, and bacterial sources ( 14 , 15 , 16 , 17 ). A surfactant's hydrophile-lipophile balance (HLB) number indicates the relative strength of the hydrophilic and hydrophobic areas of the surfactant molecule and characterizes the surfactant's relative affinity for aqueous (polar) and organic phases (non-polar). For instance, it is well documented that polyethylene oxide chain surfactants with HLB numbers ranging from 12.5 to 13.5 are effective in selectively solubilizing high-density lipoproteins in solution ( 18 , 19 ). In addition, non-ionic surfactants with LB values less than 14.6 ( 20 ) have been found to preferentially solubilize LDL in solution. These general surfactant properties have been used in the development of several HDLC solution assays for automated analyzers ( 10 , 11 ). For example, Matsui et. al. use non-ionic polyalkylene oxide surfactants with HLB numbers of 13 to 14 (lines 2-4 on page 4, ref 10) to selectively solubilize LDL in their assay. After LDL is solubilized, Matsui et al. use Catalase to eliminate the LDLC-derived peroxide produced. The addition of the second reagent then inhibits the Catalase in the first reaction cascade and begins a second reaction sequence by solubilizing the remaining HDL. Use of this two-step elimination method in Matsui et al. is not possible in the single-step dry slide assay (Scheme 1). In addition, the surfactant concentration range claimed by Matsui et al. (0.05-3%, lines 14-15 on page 4, ref 10) is insufficient to selectively solubilize HDL in the approximate 5.5 minute time frame amenable to the dry slide format.
[0005] Hino et al. also disclose the use of non-ionic polyalkylene oxide surfactants with a HLB number of about 13. Like Matsui et al., Hino et al.'s use of surfactants is also not compatible with a dry slide assay ( 11 ). Hino et al. use a low concentration of surfactant in the range of 0.01 to 1% by weight that preferably does not dissolve lipoproteins. Along with the surfactant, a reagent is added which forms a complex with the non-high density lipoproteins. The complexed non-high density lipoproteins are then prevented from reacting with the enzymes used in the detection step of the assay. The enzymes used in the detection step are then added in the second reagent, which also contains the surfactant TRITON X-100 (a detergent produced by Dow Chemical) (lines 60-64 in column 2, ref 11). TRITON X-100 (TX-100) presumably solubilizes the uncomplexed HDL so that HDLC can react with the cholesterol detecting enzyme cascade (Scheme 2). Thus, the HDL selective surfactant in this solution assay has an inhibitory effect on the interaction between the detection enzymes and non-HDLs. The structure of the dry slide is not compatible with the above-described two-reagent addition method since all the reagents are contained together in the dry slide element and the TX-100 resolubilizes the precipitated non-HDL complexes in the dry slide format.
[0006] Since neither of the reaction mechanisms nor multiple reagent additions described in the above references are amenable to the all inclusive dry slide, new methods to employ the general property of selective surfactants need to be developed for the direct HDLC assay in a dry slide.
[0007] The effect of various surfactants on HDLC selectivity of the dry slide was assessed in combination with the other known effectors of HDLC selectivity. Reagents coated with selective surfactant in the dry slide to improve the HDLC selectivity include combinations of different non-HDL precipitating reagents (e.g., phosphotungstic acid, dextran sulfate, polyethylene glycol), ion exchange resins, LDL complex formers (calix[8]arene ( 21 )), magnetic particles ( 22 ), and HDL selective cholesterol esterase (CEH) enzyme sources.
[0008] The present inventors have found two surfactants useful in conferring HDLC selectivity in a single-step HDLC dry slide assay. These surfactants confer HDLC selectivity in the reaction of cholesterol esterase with lipoproteins. The HDL selectivity observed with these surfactants in the direct HDLC dry slide is unusual compared to the lack of selectivity shown by other screened surfactant sources because the two surfactants disclosed retain their selectivity in the presence of polyanion-non-HDL-lipoprotein complexes. These surfactants with HDL selectivity, when used in conjunction with polyanion precipitation and an HDL selective cholesterol esterase, adds a third selectivity mechanism to the assay. The additional selectivity from these surfactants greatly improves the overall selectivity of the assay and makes it possible to have a functional single-step direct HDLC dry slide assay. Without this enhancement, the overall selectivity and accuracy of the assay is insufficient due to the interference from non-high density lipoprotein cholesterol.
SUMMARY OF THE INVENTION
[0009] An object of the present invention is a method to provide for quantifying cholesterol in high-density lipoproteins using a single-step assay. A multi-layer analytical element is used wherein at least one layer contains phosphotungstic acid and another contains a surfactant that selectively acts on high-density lipoproteins and does not solubilize polyanion-non-high density lipoprotein complexes. In the present invention, the multi-layer analytical element is contacted with a sample that may contain high-density lipoprotein cholesterol. The non-HDL is precipitated and the HDL is solubilized in the spreading layer. Then the CEH reacts with the solubilized HDL cholesterol esters to form cholesterol. Finally the cholesterol in the high-density lipoprotein is detected and quantified.
[0010] In another embodiment, the surfactant is selected from EMULGEN B-66, EMULGEN A-90, or a mixture thereof.
[0011] In another embodiment, the amount of phosphotungstic acid present in a layer of the dry slide is preferably in an amount of 1 to 5 g/m 2 .
[0012] In another embodiment, the amount of surfactant present in a layer of the dry slide is preferably in an amount of 3 to 8 g/m 2 .
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a kinetic response for human HDL and LDL containing test fluids with the non-specific EMULGEN 109P surfactant without the presence of phosphotungstic acid.
[0014] FIG. 2 shows a kinetic response for human HDL and LDL containing test fluids with the non-specific EMULGEN 109P surfactant in the presence of phosphotungstic acid.
[0015] FIG. 3 shows a kinetic response for human HDL and LDL containing test fluids with HDL specific EMULGEN B-66 without the presence of phosphotungstic acid.
[0016] FIG. 4 shows a kinetic response for human HDL and LDL containing test fluids with HDL specific EMULGEN B-66 in the presence of phosphotungstic acid.
[0017] FIG. 5 shows a patient accuracy plot with the use of the non-selective surfactant EMULGEN 109P in the direct HDLC dry slide.
[0018] FIG. 6 shows a patient accuracy plot with the use of the non-selective surfactant EMULGEN 220 in the direct HDLC dry slide.
[0019] FIG. 7 shows a patient accuracy plot with the use of the HDL selective surfactant EMULGEN B-66 in the direct HDLC dry slide.
[0020] FIG. 8 shows a patient accuracy plot with the use of the HDL selective surfactant EMULGEN A-90 in the direct HDLC dry slide.
[0021] FIG. 9 shows a patient accuracy plot with the use of the non-selective surfactant EMULGEN 109P in the presence of phosphotungstic acid in the direct HDLC dry slide.
[0022] FIG. 10 shows a patient accuracy plot with the use of the non-selective surfactant EMULGEN 220 in the presence of phosphotungstic acid in the direct HDLC dry slide.
[0023] FIG. 11 shows a patient accuracy plot with the use of the HDL selective surfactant EMULGEN B-66 in the presence of phosphotungstic acid in the direct HDLC dry slide.
[0024] FIG. 12 shows a patient accuracy plot with the use of the HDL selective surfactant EMULGEN A-90 in the presence of phosphotungstic acid in the direct HDLC dry slide.
[0025] FIG. 13 shows a kinetic response for non-HDL precipitated with phosphotungstic acid and MgCl 2 , resuspended in NaCl, TRITON X-100, and EMULGEN B-66, and tested on a coating containing EMULGEN B-66 surfactant.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In the direct HDLC dry slide, two HDL selective surfactants were discovered which showed superior selectivity for HDL. Several surfactants were screened for HDLC selectivity (Table 1), but only two surfactants, EMULGEN B-66 (a polyoxyethylene derivative produced by KAO Corp.,) and EMULGEN A-90 (a polyoxyethylene derivative produced by KAO Corp.,) possessed the level of HDLC selectivity needed for a single-step direct HDLC dry slide assay. The superior HDL specificity of EMULGEN B-66 and EMULGEN A-90 is crucial to the direct HDLC dry slide because the currently known HDL specific methods do not provide adequate HDLC specificity needed for the dry slide assay. One example of the dry slide used in evaluating the various surfactants is shown in example 1, however, the location of the enzymes and other reactive ingredients may be placed in a variety of positions within the dry slide and a variety of different materials may be used for the various layers.
[0027] The HDL selectivity of EMULGEN B-66 and EMULGEN A-90 was demonstrated by comparing their performance against that of other surfactants using two different means: comparison of the kinetic response from pure human HDL and LDL in serum-based test fluids and accuracy comparisons with patient samples. The kinetics of the human LDL fluid at 75 mg/dL compared to the human HDL fluid at 75 mg/dL in FIGS. 1 through 4 show a much larger response for a non-selective surfactant EMULGEN 109P (a polyoxyethylene lauryl ether produced by KAO Corp.,) than the response for the HDL selective surfactant EMULGEN B-66. Similar non-selectivity or partial selectivity was observed with the other surfactants and is summarized in Table 2.
[0028] Dry analytical elements, or dry slides, and their use are described in numerous publications, including U.S. Pat. Nos. 4,123,528; 4,786,605; 3,992,158; 4,258,001; 4,670,381; and European Patent Application Nos. 051 183; 066 648. The layers of the element of the present invention can be self-supporting, but preferably, these layers are disposed on a suitable dimensionally stable, chemically inert support. A support choice should be compatible with the intended mode of detection. Useful support material include but are not limited to paper, metal, foils, polystyrenes, polyesters, polycarbonates, and cellulose esters.
[0029] In the direct HDLC dry slide, the level of HDLC selectivity achieved merely using the superior HDL selective surfactants was not sufficient to achieve the accuracy targets for a single step assay. It was found that in order to achieve further improvement in HDLC selectivity, it was desirable to couple the HDL selective surfactant with phosphotungstic acid (PTA) precipitation. PTA, a classical method for achieving HDLC selectivity, facilitates HDLC selectivity by precipitating non-HDL. With the addition of PTA, both EMULGEN B-66 and EMULGEN A-90 showed similar HDLC selectivity while the other surfactants showed little or no HDLC selectivity. (Table 4) For example in Table 4, the HDLC selectivity of EMULGEN A-60 (a polyoxyethylene derivative produced by KAO Corp.,) and EMULGEN 109P with PTA is similar to that of non-specific surfactant TX-100, suggesting that both EMULGEN A-60 and EMULGEN 109P have no HDLC selectivity. In contrast, EMULGEN 220 (a polyoxyethylene cetyl ether produced by KAO Corp.,) shows intermediate HDLC selectivity in the presence of PTA.
[0030] An additional unique feature of EMULGEN B-66 and EMULGEN A-90 is their compatibility with PTA precipitated non-HDL. Both surfactants do not resolubilize the PTA-MgCl 2 -non-HDL complexes. Although it has been documented that a surfactant's HLB number in solution methods is a good indicator of the its ability to solubilize certain proteins, in a direct HDLC dry slide the HLB number is a poor indicator. it has been shown that in the direct HDLC dry slide there is no correlation between the surfactant's HLB number (Table 1) and its ability to selectively solubilize HDL while not disrupting the PTA-MgCl 2 -non-HDL complexes (correlation coefficient=0.017). In contrast, EMULGEN B-66 and EMULGEN A-90's inherent HDL selectivity and the lack of solubilization of PTA precipitated non-HDL complexes are the essential properties responsible for the possibility of a single-step direct HDLC dry slide assay.
[0031] A “sample” as used herein, refers to any substance that may contain the analyte of interest. A sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, urine, cerebrospinal fluid, and other constituents of the body which may contain the analyte of interest. Optionally, samples may be obtained from water, soil, and vegetation.
[0032] In at least one of the layers of the element of this invention is a dye which is capable of reacting with an enzyme to form a color. The dye functions as an indicator of the presence and the amount of HDLC present in a given sample. In a preferred embodiment of this invention, a leuco dye is used that can react with hydrogen peroxide and peroxidase to form a color. The color can be detected optically by the naked eye, by a photodiode selected to respond to a particular wavelength of light, or by other optical detection systems known by those skilled in the art using absorption, reflectance, or fluorescence spectroscopy. In a preferred embodiment of this invention, a reflectometer is used to detect and quantitate the dye color.
[0033] The element of this invention can include a wide variety of additives in appropriate layers as are known in the art to aid in manufacture, fluid spreading, and absorption and unwanted radiation.
[0034] The element of the present invention can be prepared using conventional coating procedures and equipment as are described in the art including gravure, curtain, hopper, and other coating techniques. The element can be configured in a variety of forms, including elongated tapes of any desired width, sheets, slides or chips. The process can be manual or automated.
[0035] The following examples are intended to illustrate, not limit the scope of the present invention.
EXAMPLE 1
[0036] Several surfactants were evaluated in a dry slide in order to evaluate their HDL selectivity. Below is an example of a multilayer analytical element, or dry slide, used in the evaluative process.
MgCl 2 /Surfactant BaSO 4 Spreadlayer/Surfactant I-100 Adhesion Gel/COD/CEH Gel/Dye/POD
EXAMPLE 2
[0037] Table 1 details the surfactants screened in the direct HDLC dry slide.
TABLE 1 Vendor Surfactant HLB Number Kao Corp EMULGEN A-60 12.8 Kao Corp EMULGEN B-66 13.2 Kao Corp EMULGEN A-90 14.5 Kao Corp EMULGEN 109P 13.6 Kao Corp EMULGEN 220 14.2 Dow Chemicals TRITON X-100 13.5
EXAMPLE 3
[0038] Serum based test fluids containing pure human HDL and LDL were reacted with HDL specific EMULGEN B-66 surfactant and non-HDL specific EMULGEN 109P surfactant in a dry slide assay. The kinetic response for each reaction was recorded and shown in FIGS. 1 through 4 .
EXAMPLE 4
[0039] The HDL selectivity of each surfactant in Table 1 was screened by measuring and comparing each surfactant's kinetic response to serum based test fluids containing pure human HDL and LDL. The results were then normalized to EMULGEN B-66 with a lower normalized number signifying decreased HDLC selectivity.
[0040] As shown in Table 2, surfactants, EMULGEN B-66 and EMULGEN A-90, screened in the HDLC dry slide have less LDL reactivity compared to HDL reactivity than other screened surfactants. Furthermore, when evaluated with human HDL and LDL test fluids, EMULGEN B-66 and EMULGEN A-90 are shown to be considerably more selective for HDL than LDL. In addition, these surfactants have unique specificity characteristics not seen with other surfactants used in the well-known cholesterol enzymatic cascade.
TABLE 2 Normalized Surfactant LDL(75)/HDL(75) HDLC Selectivity EMULGEN A-60 0.881 0.36 EMULGEN B-66 0.321 1.00 EMULGEN A-90 0.365 0.88 EMULGEN 109P 0.976 0.33 EMULGEN 220 0.991 0.32
EXAMPLE 5
[0041] Further tests of HDLC selectivity were done using a split sample comparison with serum patient samples with varying concentrations of HDLC, also known as patient accuracy testing. The HDL selectivity of EMULGEN 109 P, EMULGEN 220, EMULGEN B-66, and EMULGEN A-90 in direct HDLC dry slide assays were evaluated. As shown in FIGS. 5 through 8 , the results from the dry slide assays where then compared to those obtained through the VITROS Magnetic HDLC precipitation method. The results from the direct HDLC slide assay were then correlated with the results from the VITROS Magnetic HDLC precipitation method and recorded in Table 3. The correlation results for the surfactants confirm the conclusions reached from the human HDL and LDL test fluids that EMULGEN B-66 and EMULGEN A-90 show HDL specificity that is not observed with other surfactants.
TABLE 3 Surfactant Source Slope Intercept Sy.x R 2 EMULGEN A-60 0.14 47.96 6.55 0.14 EMULGEN B-66 0.50 28.21 9.40 0.50 EMULGEN A-90 0.66 18.85 8.87 0.67 EMULGEN 109P 0.14 47.66 6.57 0.14 EMULGEN 220 0.05 53.04 4.18 0.05
EXAMPLE 6
[0042] To test the effect of PTA on each surfactant's HDLC selectivity in a direct HDLC dry slide, PTA was added to the MgCl 2 and surfactant layer of the slide. Serum based test fluids containing pure human HDL and LDL were than used to test the HDLC selectivity of the surfactants. Each surfactant's HDLC selectivity resulting from these PTA added coatings are found in Table 4. TX-100, a general surfactant known to completely dissociate lipoproteins, was added as a control. The HDL selectivity results were then normalized to the EMULGEN B-66 data. ( 24 ). A lower normalized number signifies decreased selectivity to HDLC.
TABLE 4 LDL(75)/ Normalized HDLC HDLC Selectivity Surfactant HDL(75) Selectivity +PTA/−PTA EMULGEN A-60 0.941 0.32 1.07 EMULGEN B-66 0.297 1.00 0.92 EMULGEN A-90 0.222 1.34 0.61 EMULGEN 109P 0.992 0.30 1.02 EMULGEN 220 0.621 0.48 0.63 Triton X-100 0.994 0.30 ND
The formulas containing EMULGEN B-66 and EMULGEN A-90 showed the same HDLC selectivity with PTA while the other surfactants tested showed little or no HDLC selectivity with addition of PTA.
EXAMPLE 7
[0044] Table 4 also compares each surfactant's HDLC selectivity with and without the PTA coating using the human HDL and LDL linearity series. The dry slides containing EMULGEN B-66, EMULGEN A-90, and EMULGEN 220 all showed some enhancement of selectivity with the addition of PTA. In contrast, EMULGEN A-60 and EMULGEN 109P showed no enhancement of selectivity with the addition of PTA.
EXAMPLE 8
[0045] A similar comparison to the one in Example 7, comparing each surfactant's HDLC selectivity with and without the PTA coating was also performed using patient samples. Patient accuracy plots comparing the use of non-selective surfactants, EMULGEN 109P and EMULGEN 220, with the HDL selective surfactants, EMULGEN B-66 and EMULGEN A-90, in the dry slide with the PTA addition versus the VITROS Magnetic HDLC precipitation method ( FIGS. 9-12 ). Table 5 depicts the correlation results between the direct HDLC slide and the VITROS Magnetic HDLC precipitation method for coatings containing PTA. Both EMULGEN B-66 and EMULGEN A-90 show HDLC selectivity while the other surfactants show little or no HDLC selectivity. The results using patient samples is also similar to those found with human HDL and LDL test fluids (Example 7) because EMULGEN B-66, EMULGEN A-90, and EMULGEN 220 all showed some enhancement of HDLC selectivity with the addition of PTA.
TABLE 5 Surfactant Source Slope Intercept Sy.x r 2 EMULGEN A-60 0.08 50.91 4.98 0.08 EMULGEN B-66 0.58 23.50 9.29 0.58 EMULGEN A-90 0.74 14.78 8.30 0.74 EMULGEN 109P 0.13 48.98 6.24 0.13 EMULGEN 220 0.19 45.50 7.34 0.19 Triton X-100 0.11 50.13 5.83 0.11
EXAMPLE 9
[0046] To demonstrate that these surfactants selectively solubilize HDL while leaving the complexes of non-HDL lipoprotein with PTA and MgCl 2 intact in solution as well as in the dry slide assay, classical techniques where used to precipitate and isolate the PTA-MgCl 2 -non HDL precipitated complex ( 4 ). The precipitate was collected by centrifugation and washed, then resuspended in NaCl, 1% EMULGEN B-66, or 1% TX-100. The NaCl treatment remained a cloudy suspension while the TX-100 treatment cleared completely. However, the EMULGEN B-66 solution cleared slightly, but remained predominantly cloudy. These three treatments were analyzed on a coating similar to the PTA/EMULGEN B-66 coating described above. The kinetics are shown in FIG. 13 . The kinetics of the TX-100-treated non-HDL precipitate are very rapid, confirming that the LDL has been resolubilized prior to application to the slide. Both the EMULGEN B-66 and NaCl-treated samples show much slower, but very similar kinetics. This confirms that the non-HDL has remained precipitated and is only slowly resolubilized. The kinetics of the EMULGEN B-66 sample are slightly faster than the NaCl sample, confirming the hypothesis that very little resolubilization of non-HDL precipitate occurs in the presence of the EMULGEN B-66 surfactant in solution or in the direct HDLC dry slide.
EXAMPLE 10
[0047] In a preferred embodiment of the invention, shown below, the surfactant used in the dry slide format is either HDL selective surfactant, EMULGEN B-66 or EMULGEN A-90.
PTA/MgCl 2 BaSO 4 Spreadlayer/Surfactant I-100 Adhesion PEG/COD/CEH Gel/Dye Gel/Dye/POD
[0048] Table 6 shows the correlation results between patient samples on the direct HDLC dry slide in the preferred embodiment and the VITROS Magnetic HDLC precipitation method. Results of patient accuracy tests (Table 6) indicate that EMULGEN B-66 confers more selectivity to the formula than EMULGEN A-90 in the preferred embodiment shown above. The selectivity observed for both EMULGEN B-66 and EMULGEN A-90 is better in this format than demonstrated previously ( FIGS. 9 through 12 and Table 5) due to the enhancements in the slide structure and optimization of the active reagents.
TABLE 6 Enzyme Source Slope Intercept Sy.x R 2 EMULGEN B-66 0.82 11.03 7.11 0.78 EMULGEN A-90 0.76 14.10 5.83 0.82 EMULGEN B-66* 0.89 6.53 4.89 0.90 EMULGEN A-90* 0.81 11.20 4.87 0.88 *One discrepant patient sample excluded from the data.
EXAMPLE 11
[0049] By incorporation of the above discoveries in the dry slide, an accurate, precise, and rapid HDLC assay has been developed. In another preferred embodiment of the invention, shown below, the surfactant used in the dry slide format is the HDL selective surfactant EMULGEN B-66.
PTA/MgCl 2 BaSO 4 Spreadlayer/Surfactant I-100 Adhesion PEG/COD/CEH PEG/Dye Gel/POD
[0050] Direct HDLC dry slides were made using the formula and format described as the preferred slide structure above, using 7 g/m 2 EMULGEN B-66 as the HDL selective surfactant, Candida rugosa lipase or Denka CEH as the HDL selective cholesterol ester hydrolase, and IMnAg or PEG as the −02 matrix. Accuracy versus the VITROS Magnetic HDLC precipitation method and pooled precision were evaluated using 30 patient samples. Results can found in Table 7. The results show that this assay has acceptable accuracy and precision and is free from significant interference from hemolysed patient samples.
TABLE 7 Precision, Accuracy, and Hemolysate Interference Results from a Dry Analytical Element Assay for HDLC Using the Preferred Format. −02 CEH Patient Accuracy Precision Interference Matrix Source Slope Intercept Sy.x Pooled SD Hemolysate PEG Denka 0.99 0.50 2.2 1.30 >500 mg/dL Hb IMnAg Candida 0.99 0.52 2.46 3.89 >500 rugosa mg/dL Hb
LITERATURE CITED
[0051]
1 Burstein, M., Scholnick, H. R., & Morfin, R. J Lipid Res, 1970, 11, 583-595.
2 Fredrickson, D. S., Levy, R. I., & Lindgren, F. T. J Clin Invest 1968, 47, 2446-2457.
3 Warnick, G., Benderson, J., & Albers, J. J. Clin Chem 1979, 25, 1309-1313.
4 Burstein, M., & Scholnick, H. R. Adv Lipid Res 1973, 11, 67-108.
5 Briggs, C., Anderson, D., Johnson, P., & Deegan, T. Ann Clin Biochem 1981, 18, 177-181.
6 Kakuyama, T., Kimura, S., & Hasiguchi, Y. Clin Chem 1994, 40, Al 104.
7 Nauck, M., Marz, W., & Wieland, H. Clin Chem 1998, 44, 1443-1451.
8 Arranz-Pena, M., Tasende-Mata, J., & Martin-Gil, F. J. Clin Chem 1998, 44, 2499-2505.
9 Yamamoto, M., Nakamura, M., Hino, K., Saito, K., & Manabe, M. Clin Chem 2000, 46, A98
10 Matsui, H., Ito, Y., Ohara, S., & Fujiwara, A. European Patent EP 0887422A1, Priority Date Sep. 12, 1996.
11 Hino, K., Nakamura, M., & Manabe, M. U.S. Pat. No. 5,773,304, Priority Date Jan. 31, 1995.
12 Izawa, S., Okada, M., Matsui, H., & Horita, Y. J Med Pharm Sci 1997, 37, 1385-1388.
13 Sugiuchi, H., Uji, Y., Okabe, H., Irie, T., Uekama, K., Kayahara, N., & Miyauchi, K. Clin Chem 1995, 41, 717-723.
14 Neugebauer, J. M. Methods Enzymol. 1990, 182, 239-253.
15 Hjelmeland, L. M. Methods Enzymol. 1990, 182, 253-264.
16 Marston, F. A. O., & Hartley, D. L. Methods Enzymol. 1990, 182, 264-276.
17 Hjelmeland, L. M. Methods Enzymol. 1990, 182, 277-282.
18 Egan, R. W. J Biol Chem 1976, 251, 4442-4447.
19 Slinde, E., & Flatmark, T. Biochim Biophys Acta 1976, 455, 796-805.
20 Tucker, I. G., & Florence, A. T. J Pharm Pharmocol 1983, 35, 705-711.
21 Kishi, K., Ochiai, K., Ohta, Y., Uemura, Y., Kanatani, K., Nakajima, K., Wang, T., & Nakamura, M. 69 th AACC 2001, Poster, Chicago, Ill.
22 Lawlor, J. F., & Musto, J. D. United State Patent 5,242,833, Sep. 7, 1993.
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Disclosed is a method for quantifying cholesterol in high-density lipoproteins in a single step assay using a dry slide. The method for quantifying cholesterol in high-density lipoprotein comprises a first step of adding a sample onto a multi-layered dry slide wherein at least one of the layers contains phosphotungstic acid and another contains a high-density lipoprotein selective surfactant. The phosphotungstic acid precipitates non-high-density lipoproteins while the high-density lipoprotein selective surfactant only solubilizes high-density lipoproteins and does not solubilize non-HDL precipitated complexes. The cholesterol esterase then reacts with the solubilized HDL cholesterol esters to form cholesterol. Finally the cholesterol in the high-density lipoprotein is detected and quantified.
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BACKGROUND OF THE INVENTION
This invention relates generally to railway wheels, axles and rails and has particular reference to an improved railway car wheelset having a novel wheel configuration.
For many years the track engaging surface of railway car wheels has been frusto-conical, the profile of the engaging surface being a straight line oblique to the horizontal in tangency with a convex rail radius. Wheels of this configuration, even when unworn, have a rolling speed upper limit that is considerably below that wanted and needed to make railroads competitive with trucking and flying. With worn wheels, rail misalignment and tight track curvatures, the upper speed limit is lowered further. Since correcting for rail misalignment and tight curvatures is a costly major undertaking, this means that the track engaging surfaces of the wheels must be machined frequently to compensate for wear and thereby attempt to operate as efficiently as possible.
The applicant's invention is a substantial departure from conventional frusto-conical car wheels, and the applicant is not aware of any prior art having a wheel configuration similar to his. U.S. Pat. Nos. 86,631 (Feb. 9, 1869) and 638,827 (Dec. 12, 1899) disclose substantially conventional frusto-conical car wheels. U.S. Pat. No. 10,714 (Mar. 28, 1854) to Wilder is the closest prior art known to the applicant in that it discloses a railway car wheelset wherein the track engaging surfaces of the wheels are curved. However, the wheels of U.S. Pat. No. 10,714 do not have a configuration like that of applicant's wheels nor can the patented wheelset function in the manner of applicant's wheelset, which manner of functioning is described hereinafter. In the Wilder patent, each wheel bears directly on the top of its rail and the contact point is at the point of the wheel's maximum convex radius, neither of which conditions exist in the present invention as will be explained.
SUMMARY OF THE INVENTION
The improved railway car wheelset of the invention comprises a pair of variable diametered wheels and an axle, the wheels being respectively secured at their inner sides to the opposite ends of the axle for rolling in unison therewith. Each wheel can be provided at its inner side with a radially outwardly projecting annular flange, and these flanges are spaced inwardly from the inside edges of their respective rails when the wheelset is in centered position on the track. The wheelset is thus able to have limited lateral movement with respect to the track before one or the other of the flanges contacts its rail. In this connection, it should be noted that the wheel flanges can be omitted without departing from the inventive concept.
Each wheel is contoured from the flange at its inner side to its outer side in a specific manner that determines how the wheelset will function when rolling on the track. Thus, adjacent the flange the wheel has a concave annular portion and then, moving outwardly, a convex annular portion and then either a frusto-conical portion or a second concave annular portion, the successive portions merging together in a smooth curve.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an elevation of a railway car wheel that exemplifies the prior art, the wheel being shown in engagement with a track rail;
FIG. 2 is an elevation of a railway car wheelset embodying the present invention, the wheelset being shown in centered position on the track rails;
FIG. 3 is an enlarged elevation of a portion of the left hand wheel and rail of FIG. 2;
FIG. 4 is an elevation corresponding to FIG. 2 but showing the wheelset laterally displaced from the track centerline;
FIG. 5 is a diagrammatic perspective view to illustrate the self steering action of the wheelset of the invention on a curved track;
FIG. 6 is an enlarged, fragmentary elevation showing the relationship between a car wheel of the invention and a rail in the extreme positions of the wheel on the rail; and
FIG. 7 is an enlarged, fragmentary elevation showing the relationship between a car wheel of the invention and an improved rail.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Having reference now to the drawings, FIG. 1 illustrates a railway car wheel that is typical of the prior art. In this wheel the portion 10 that rolls on the rail 11 is frusto-conical, the wheel being provided on its inner side, or side toward the centerline of the track, with the usual flange 12.
FIGS. 2-4 illustrate a railway car wheelset embodying the present invention on a convex radius or crowned rail of substantially conventional configuration. The wheelset comprises a pair of variable diametered wheels 14A and 14B that are fixed at their inner sides to the opposite ends of an axle 15. The two wheels and axle thus move in unison, with the wheelset being pivotally connected to the railway car (not shown) in a conventional manner. The wheels 14A and 14B engage and roll on the upper surfaces of rails 16A and 16B respectively, which rails are supported in the usual manner by ties (not shown) and together comprise the track.
Wheels 14A and 14B have identical configurations and each wheel has a flange 17 at its inner side, the inner sides of the wheels and rails being defined herein as the sides thereof towards the centerline 18 of the track. From its flange 17 to its outer side 20, the surface of each wheel has a concave annular portion 21, then a convex annular portion 22 and then a frusto-conical portion 24. The portion 24 could, alternatively, be a second concave annular portion. Stated generally, the diameter of each wheel decreases from its flange to its outer side, as shown by phantom line 2A in FIG. 2; except for the "bulge" or protrusion caused by the convex annular portion 22. The radius of the convex portion 22, which may vary under various conditions to be described hereinafter, is indicated by the arrows rW in FIG. 2.
In FIG. 2, the wheelset of the invention is shown in centered position on the track, i.e. with the wheelset centerline coincident with the centerline 18 of the track. Assuming negligible wear on wheels or rails, the wheel 14A engages its rail 16A at a point P and wheel 14B engages its rail 16B at a point Q, the distance between point P and point Q being the gage of the wheels. These points are displaced inwardly an equal amount from the centerlines 25A and 25B of the two rails.
The locations of points P and Q on the rails also determine the contact angle and the rolling radius for each wheel. The contact angle for wheel 14A is shown as φP and is defined as the angle between the centerline 25A of rail 16A and a line 26 passing through a point 27 on the rail centerline, the wheel contact point P and a point 27A which is the center of radius rW. The point 27 is the center of the arc that defines the upper surface of the rail 16A, the radius for this arc being shown in FIGS. 2 and 3 as rR. The contact angle for wheel 14B is shown as φQ and is, like φP, the angle between the centerline 25B of rail 16B and a line 28 passing through a point 30 on the centerline, the wheel contact point Q and a point 30A which is the center of radius rW. Again, point 30 is the center of the arc that defines the upper surface of the rail, the distance between point 27 and point 30 being the gage of the rails.
The rolling radius for wheel 14A is shown as rP and is the radius of the wheel rolling surface at the contact point P. Similarly, the rolling radius rQ for wheel 14B is the radius of the wheel rolling surface at the contact point Q. When the wheelset is in centered position on the track as indicated in FIGS. 2 and 3, φP=φQ and rP=rQ. In this position, in accord with the invention the point at which the wheel diameter (and hence its radius) is at a maximum is inwardly of, or towards the track centerline from, the contact points P and Q. The point of maximum diameter for wheel 14A is best shown in the enlarged fragmentary view of FIG. 3 where it is indicated at M. The wheel 14B, of course, has a corresponding maximum diameter point inwardly of its contact point Q.
In FIG. 7, which corresponds to FIG. 3, a wheel 34 as described above is shown in engagement with an improved rail 35 which also embodies the invention. Rail 35, rather than having a conventional convex crowned upper surface, has a concave or inverted crowned upper surface 36 with radius rR as shown. With such a rail configuration, the basic wheel/rail relationship is essentially the reverse of that described above in connection with FIGS. 2-4. Thus, the contact point P of the wheel with the rail is located inwardly of the maximum diameter of the wheel, indicated at M, rather than outwardly and this makes the contact angle θP a negative angle if contact angle φP is considered to be positive.
The centered wheelset position shown in FIG. 2 is the optimum condition for railway car movement but it is a condition that hardly ever continues for more than a fraction of a second at a time. Thus, the wheelset is normally displaced to the left or right of centered position, this displacement being caused by such things as rail misalignment, by suspension forces imposed on the wheelset by the movement of supported loads and/or by inertia forces created within the wheelset as it rolls in a wobble or hunting fashion.
Self Centering
The wheelset of the invention has a self centering capability, while rolling on a straight track, that will now be described. Reference is made to FIG. 4 where the wheelset is shown with a lateral displacement to the left of the track centerline 18, the displacement being indicated at Y and being the distance between the track centerline and the centerline 31 of the wheelset. In this connection, it will be understood that the displacements and the changes in contact angles and rolling radii referred to hereinafter are very small, being exaggerated in the drawings for illustration.
With a displacement Y to the left and a slight axle centerline roll, rolling radius rP increases and contact angle φP decreases for the wheel 14A which is on the same side of the track centerline as the wheelset centerline. The radius rP increases because the wheel contact point P moves towards the maximum radius point M, FIG. 3. At the same time, the rolling radius for wheel 14B decreases and its contact angle φQ increases.
As the wheelset moves forward on the track in this displaced condition, the distance on its rail that wheel 14A travels will be greater than the distance on its rail that wheel 14B travels and therefore the wheelset will turn inward and its centerline 31 will move toward the track centerline 18. This will cause rP to decrease and rQ to increase while increasing φP and decreasing φQ. This response to the disturbance, or cause of the displacement, tends to return the wheelset towards centered position on the track. As rolling continues, the wheelset will roll past the track centerline to the opposite side of the track causing the relationship of the rolling radii and contact angles to reverse which again turns the wheelset back towards the track centerline. The wheelset will thus continue to roll with a wobble or in a hunting fashion about the track centerline and this is the self centering motion. Friction forces at the contact points P and Q tend to reduce the wobble amplitude in time, other conditions remaining the same. A full self centering effect will be maintained as long as the lateral displacement in either direction doesn't cause either contact angle to decrease to zero.
Self-Steering
Reference is now made to FIG. 5 which shows diagrammatically and in exaggerated fashion the wheels 14A and 14B of the wheelset rolling on the rails 16A and 16B of a curved track. As indicated by the wheelset centerline 31, the wheelset has a lateral displacement Y to the left of the track centerline 18. The solid axle feature of the wheelset permits it to position itself outwardly and radially on the curved track such that it rolls steadily forward as a cone whose apex is at the center 32 of the track curvature.
The curved track displacement Y has the same effect as a disturbance caused displacement Y on a straight track discussed above. Thus, rolling radius rP increases and rQ decreases while φP decreases and φQ increases. This, as previously explained, causes wheel 14A to travel a greater distance on its track than wheel 14B travels on its track. For any given track radius, there is a particular self centering outward displacement Y that occurs from the rP/rQ relationship that is required to permit the wheelset to self steer around a curved track and roll steadily without wobble.
As with straight track disturbances, the outward displacement Y on a curved track is limited by not having either contact angle to decrease to zero. However, given any additional disturbance due to rail misalignment or load variations, the wheelset wobble now permissible will be further reduced by the amount of self steering displacement required for the curve.
Stability
At any rolling speed, the wheelset of the invention will wobble in response to a disturbance that moves it from a centered position on the track. The maximum displacement Y of the wheelset wobble due to a disturbance will steadily decrease when rolling is stable and steadily increase when unstable. Given any specific centered position values for rW,rR, the rolling radii and the contact angles, FIG. 2, there will be a rolling speed above which stability will end and instability will begin.
For a particular displacement Y and rolling speed of a wheelset, the rP/rQ relationship determines the angular speed at which the wheelset will turn inward and therefore the frequency with which a wheelset will wobble. The greater the difference between rP and rQ, the higher will be the wobble frequency and the greater will be the inertia forces developed within the wheelset itself, and therefore the lower will be the rolling speed at which instability begins. The centered position value of the contact angles φP and φQ determines the amplitude of wobble and/or the tightness of curve that can be negotiated and/or the wobble permissible on a particular curve.
The upper limit of rolling speed with prior wheelsets has been reached with unworn coned wheels, FIG. 1, and with a minimized contact angle with the wheelset in centered position on the track. In a coned wheel, the possible rP/rQ difference is reduced when the value of the contact angles in centered position is reduced. To achieve the current rolling speed upper limit, it has been necessary to remachine wheel cones frequently to compensate for increased rP/rQ differences coming from wheel contour wear, or to reduce the magnitude of disturbances by more perfectly aligning rails, or to increase track curvature which limits field use. The second and third alternatives are, of course, very costly.
Considerably higher rolling speeds (around 200 mph) are wanted and needed to maximize the social and economic value of railways in competition with trucking and flying. The rolling speed has been maximized in the above way for at least the last ten years. Any new approach to increasing stable rolling speeds on existing track requires a greater reduction in the magnitude of rP/rQ differences during wheelset displacements without reducing the centered position value of the contact angles.
In the present invention, the convex annular portions or protrusions 22, FIG. 2, of the wheels reduce rP/rQ differences on existing track considerably further than that now possible with the coned wheel. These convex annular wheel portions permit a much greater opportunity for higher rolling speed with stability, self steering, and self centering. Less restriction on rail alignment and track curvature is also obtainable because the centered position value of the contact angles can be increased. The exact values of rW and the contact angles for a particular application are dependent upon the ordering of priorities and chosen limits among rolling speed, track curvature, wear life, and rail misalignment.
Referring generally to FIG. 6, which illustrates wheel/rail relationships in extreme positions, there is a whole family of design options that are stable during rolling speeds near 200 mph. For example:
1. if a maximized rolling speed is most important, a wheel with a convex annular portion 22 having a small radius rW (less than one inch) rolling upon existing track for a specific displacement Y develops less than 1/7 the maximum rP/rQ difference that a 3° cone does. The value of the contact angles in centered position, the track curvature, and rail misalignment can be comparable to current practice. A wheel with a one inch convex radius rW then will be stable at more than twice the current maximum rolling speed (close to 300 mph);
2. if maximized rail misalignment is most important (about ±1/4 inch) the centered position contact angles can be increased (approx. 4°) and an average convex radius rW (about 3 inches) chosen with current limits maintained on track curvature;
3. if minimum wheel wear is most important, contact point pressure (therefore wear) can be reduced by maximizing the convex radius rW (approx. 7 inches) and by reducing the contact angles in centered position (approx. 11/2°) while the limits on track curvature and rail misalignment are still about typical of current practice;
4. if minimum track curvature is most important (about one mile), the contact angles in centered position can be increased (approx. 5°) and an average convex radius rW (about 3 inches) chosen with current limits being kept on rail misalignment.
If the existing convex radius rR of the rail (approx. 6 inches) is increased, then even higher rolling speeds with self centering, tighter curves and more track misalignment are obtainable. This leads to two additional, new possibilities:
1. A whole family of adjustable options emerges as the rail radius rR is progressively increased from 6 inches to near infinity and the convex wheel radius rW and value of the contact angle in centered position are both progressively increased. This can lead to obtaining rolling speeds of up to 300 mph on a track curve radius under three miles with track misalignment of ±1/4 inch.
2. Another whole family of duplicate adjustable options emerges as the radius rR of a rail having a concave upper surface as in FIG. 7 is progressively increased from 6 inches to near infinity and the convex wheel radius rW is progressively increased from one to seven inches and the contact angle in centered position is progressively decreased from minus 4° to minus 11/2°. A positive contact angle is not possible with a concave upper rail upper surface.
From the foregoing description it will be apparent that the invention provides an improved railway car wheelset having a novel wheel configuration that permits greatly improved performance on existing track. As will be understood by those familiar with the art, the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof.
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An improved railway car wheelset having a novel wheel configuration that permits greatly improved performance on existing and improved track. This means that substantially higher rolling speeds can be achieved without loss of stability and while maintaining the ability of the wheelset to self center and self steer. The improved performance is enabled primarily by forming the track engaging surface of each wheel of the wheelset with an outward arcuate projection also referred to herein as the convex annular portion of the wheel. This wheel configuration operates to reduce the magnitude of the effects that occur when disturbances cause the wheelset to deviate from centered position on the track.
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BACKGROUND OF THE INVENTION
The present invention relates to the diagnostic imaging arts. It finds particular application in conjunction with the real-time display of medical diagnostic images and will be described with particular reference thereto. However, it is to be appreciated that the invention may find application in conjunction with non-medical imaging, volumetric imaging, and the like.
Heretofore, x-rays have been projected through a patient onto a flat film box on the other side of the patient. X-ray film mounted in the film box was exposed with a projection of the radiation opacity of the tissue or other internal structure of an examined subject. Because all of the internal structure was projected into a common plane, such images were difficult to read.
Conventional x-ray tomo systems have a similar construction, but include structure for moving the x-ray tube and the film box counter-cyclically in planes parallel to the x-ray film. More specifically, a center ray of the x-ray beam was projected through the region of interest to the detector. The x-ray source and the detector were then moved such that the central ray pivots about a fixed point in the plane of interest. With this process, not only does the central ray pivot about the plane or slice of interest, the other rays from the x-ray source to the film box do as well. In this manner, the x-ray attenuation contribution to the final image from volumetric elements within the selected plane remains constant during the imaging procedure. However, outside of the selected slice, each of the rays pass through different surrounding tissue or structures as the source and detector move. In this manner, the contributions to the final image from structures outside of the plane of interest become blurred and averaged. With a sufficiently long exposure and motion through a relatively wide range, the out-of-slice structures can be reduced to background noise while the in-slice structures are displayed crisp and clear. Such systems required a significant time lag before the diagnostic image could be viewed. First, there was a delay while the x-ray source and the film box and the film box were moved back and forth to expose the film. This was followed by a further delay as the film was developed.
Real-time images were available from fluoroscopy systems. In a fluoroscopy system, the x-rays are projected through the patient onto an image intensifier, i.e., a fluorescent screen and electronics to make the resultant image brighter. A video camera was mounted to view the image generated by the image intensifier. The video camera was connected by a closed-circuit TV system with a monitor for viewing the fluoroscopic images. Although these images were real-time, they were again projection images which superimposed all the structure in the field-of-view onto a common plane. Moreover, image intensifiers were subject to non-uniform brightness across the field-of-view and significant image distortions. Fluoroscopic images typically had much less resolution than projection x-ray.
CT scanners have been utilized to generate images of internal structures quickly. However, CT scanners typically view the patient in slices which are orthogonal to those of the tomographic x-ray systems. That is, with the patient positioned prone on his back in the scanner, the tomographic x-ray systems generated an image of a horizontal slice. With the same orientation of the patient, CT scanners generate a vertical slice. Of course, CT scanners can be utilized to generate a large multiplicity of slices to define a volume from which a horizontal slice can be extracted. However, taking a large number of slices again introduces a time delay. Moreover, CT scanners are expensive and capable of performing only a limited number of diagnostic tasks.
The present invention contemplates a new and improved imaging technique which overcomes the above-referenced problems and others.
SUMMARY OF THE INVENTION
In accordance with the present invention, an apparatus for real-time diagnostic imaging of an object is provided. A support supports a region of interest of an object to be examined. A radiation source projects a beam of penetrating radiation through the region of interest. A movable gantry causes relative motion between the radiation source and the support. A radiation detector detects the beam of penetrating radiation and converted the detected radiation into electronic data. The radiation detector is repeatedly sampled as the radiation source moves to generate a plurality of electronic views. An image processing circuit processes the plurality of electronic views to generate an electronic image representation of a selected slice through the region of interest, which image representation is continuously updated as the radiation source moves.
In accordance with a more limited aspect of the invention, the moveable gantry rotates the radiation source in an annular trajectory of different radii so that different horizontal slice thicknesses of the object can be monitored in real-time and controlled in real-time by the user.
In accordance with a more limited aspect of the invention, the radius is controlled through a user input device which takes a selected thickness for a region of interest to be depicted in a slice image representation, converts the selected thickness into a corresponding radius value, and adjusts the radiation source to a radius corresponding to the selected radius value.
In accordance with a more limited aspect of the invention, a focal plane selection input device allows an operator to designate a focal plane corresponding to the selected slice, through use of a look-up table, to generate an appropriate section from the plurality of views for display.
A first advantage of the present invention is that it provides a new imaging modality for diagnostic imaging.
Another advantage of the present invention is that it provides a real-time display of slices taken longitudinally through a patient.
Other advantages of the present invention reside in the ready adjustability of the slice thickness and position.
The present system is amenable to imaging in other modes including tomographic, fluoroscopic, and projection x-ray modes.
Other advantages of the present invention reside in the ability to enhance and manipulate images easily.
Still further advantages of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention may take form in various components and arrangements of components, and in various steps and arrangements of steps. The drawings are only for purposes of illustrating a preferred embodiment and are not to be construed as limiting the invention.
FIG. 1 is a diagrammatic illustration of an imaging system in accordance with the present invention;
FIG. 2 is a detailed view of an image processor of FIG. 1;
FIG. 3 is a diagrammatic illustration of the geometric principles responsible for structures in the focal plane contributing coherently to the resultant image while structures outside of the focal plane contribute non-coherently and blur;
FIG. 4 is a diagrammatic illustration illustrating x-ray tube placement and principles behind generating thin slices; and,
FIG. 5 is a diagrammatic illustration illustrating the geometry and principles behind thick slice image generation.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
With reference to FIG. 1, a radiation source 10 , such as an x-ray tube, projects a beam of x-rays or other penetrating radiation through a region of interest 12 of a subject 14 , such as a patient or an object in a manufacturing environment, supported on a support 16 . Radiation which has passed through the region of interest impinges upon a flat panel radiation detector 18 . In the preferred embodiment, the radiation detector is a grid of amorphous silicon elements on the order of a millimeter square, with the overall detector being on the order of 45 cm x 45 cm. Each element of the amorphous silicon detector integrates the intensity of received radiation over a sampling period and generates an electronic data value indicative of the intensity of received radiation. Typically, all of the elements are read out concurrently or in close temporal proximity to generate a view representative of x-ray intensity variation, which, in turn, is indicative of a projection of radiation opacity of the region of interest taken in the direction of the x-ray beam.
The radiation source 10 is mounted on a rotational gantry 20 which rotates the radiation source 10 in an annular, preferably circular, trajectory 22 of adjustable radius. For clarity of illustration, the gantry 20 is illustrated as a concave dish of constant radius relative to a center point of the detector 18 . It is to be appreciated that other, more complex mechanical arrangements can be provided for rotating the x-ray source 10 in a circular trajectory 22 of selectable radius along the surface of the dish. For example, the x-ray source 10 can be mounted to a dish segment supported by rollers or bearings with an adjustment drive (not shown) for adjusting the position of the radiation source 10 radially. Once the radius of the circular trajectory 22 is selected, another drive motor 24 rotates the x-ray source 10 around the selected trajectory. A position encoder 26 measures the radius r of the trajectory 22 and the angular position φ of the x-ray source 10 around the trajectory 22 .
With reference to FIG. 3, the region of interest 12 is divisible into a series of focal planes F 1 , F 2 , . . . , F n . With the x-ray source 10 starting at an arbitrary initial position (x 0 , y 0 , z 0 ) on the trajectory 22 , the image of incremental element 32 on a first focal plane F 1 is located at an initial position (X 0 , Y 0 , Z 0 ) on the detector plane, where the z-coordinate of the radiation detector has been arbitrarily selected as z 0 =0 for simplicity of calculation. As the x-ray source 10 moves around the trajectory 22 to other positions (X i , Y i , Z 0 ), the image of the incremental element 32 now impinges on a point (x i , y i , z 0 ) on the radiation detector 18 . In fact, the image of the incremental elements 32 traverses a complementary trajectory 22 ′ on the detector 18 .
It will be seen that for any point outside of the focal plane F 1 , such as a point 34 on focal plane F 2 , different rays will pass through element 34 and the trajectory of the projection of element 34 onto the detector 18 will follow a different trajectory from trajectory 22 ′. More specifically, by simple geometry, it can be seen that when the image read out on the detector 18 is translated or shifted such that the point on the trajectory 22 ′ that corresponds to the projection of element 32 are aligned, the images can be enhanced with elements 32 adding coherently. More specifically, all of the points on plane F 1 will integrate substantially coherently such that incremental elements of the region of interest lying in plane F 1 are integrated coherently and will be strong and clear in the resultant image. On the other hand, the projection of point 34 and other incremental elements of the region of interest that are off the focal plane F 1 will sometimes contribute to one pixel of the integrated image and sometimes to others, thus blurring and becoming de-emphasized. With sufficient variation, the out of plane contribution to the image can be reduced to background noise. For any radius r of the circle, and any angular position φ along the circular trajectory 22 , the trajectory 22 ′ can be precalculated. In this manner, for any given position (r, φ) of the x-ray source around the trajectory 22 , the offset (x 0 −x i , y 0 −y i , z o −z i ) can be determined geometrically and stored. It is further to be appreciated that the same principle holds true for elements on focal plane F 2 and the other focal planes through F n .
With reference again to FIG. 1, as the motor 24 rotates the radiation source 10 along the trajectory 22 , the encoder 26 monitors the radius r of the trajectory and the angular position φ of the radiation source 10 around it. The coordinates (r, φ) are used to address a look-up table 40 which stores the offset or shift S i =(x 0 −x i , y 0 −y i , z o −z i ). The look-up table 40 is also addressed with the location of the focal plane to be imaged as input by an operator on a slice or focal plane selection input device 42 .
With reference to FIG. 2, and continuing reference to FIG. 1, each time a view is read out of the detector 18 , it passes to a view memory or buffer 44 . An image translation or shift circuit 46 shifts and interpolates the resultant image by the amount S to create a shifted view which is stored in a shifted view memory or buffer 48 . Although buffers 44 and 48 are shown separately for simplicity of illustration, it is to be appreciated that they may be the same element of hardware as may other memories described hereinbelow.
Each shifted image is integrated in summation circuit 50 with precedingly taken images and stored in a first slice memory 52 . In the preferred embodiment, the first slice memory 52 is used to accumulate the sum of the views taken over 180° of rotation about the trajectory 22 . Once 180° of views are integrated, the first slice memory 52 is interconnected with a video processor 54 which converts the slice image representation from the memory 52 into appropriate format for display on a video monitor 56 . While the first slice is being displayed, the x-ray source 10 is rotating through the next 180° and another image is being built in a second slice memory 58 . Once the next 180° slice is completed, the second slice 58 memory is connected with the video processor 54 and the first slice memory 52 is erased and commences building the next view of the slice.
Preferably, the x-ray source 10 is rotated at 15 rotations per second such that there is a frame rate of 30 images of the slice generated per second to match a standard video image frame rate. Of course, other frame rates may be selected such as 4-8 images per second. In that case, the x-ray source 10 is rotated at 2-4 rotations per second. Analogously, images may be built based on shorter arc segments such as 120°. As another option, the image may be built in a single memory with the oldest view being subtracted back out as the newest view is added in. As yet another alternative, the views can be continuously accumulated in the slice image memory with no deletions, possibly with the accumulated image and the new view being weighted that more recent views have greater prominence than older views.
The angular position of the x-ray source 10 is monitored by a frame rate controller 60 which changes the position of a switch 62 after each 180° or other selected distance along the trajectory 22 . The switch 62 switches which of the memories 52 , 58 is connected with the video processor 54 and which is receiving the additional views from the summation circuit 50 .
As another alternative, a plurality of planes or slices can be reconstructed. For simultaneous reconstruction of multiple planes, the look-up table 40 is addressed with each of the focal planes selected with the slice selection control 42 . The appropriate displacements for each of the focal planes S 1 , S 2 , . . . , S n are outputted to a respective image shifting circuit 46 2 , . . . , 46 n . The shifted views are conveyed to shifted view memories or buffers 48 2 , . . . , 48 n . The shifted views are summed 50 2 , . . . , 50 n . into the accumulating image in buffer 52 2 , . . . , 52 n. or 58 2 , . . . , 58 n . The switch circuits 62 2 , . . . , 62 n convey the most recently completed slice images to a volumetric image memory 64 . The volumetric image memory 64 then conveys the completed slice images through a digital enhancer 66 after the video processor 54 accesses the volumetric image memory 64 to retrieve operator selected slices, an oblique slice, volume renderings, volume images, or the like.
With continuing reference to FIGS. 1 and 2, there are shown a plurality of image processing systems 68 1 , 68 2 , . . . , 68 n . The image processing systems 68 1 , 68 2 , . . . , 68 n receive inputs from the view memory 44 and Look-Up-Table 40 , which are sent to the image transition/shift circuit 46 , and they receive inputs the frame rate controller 60 which is sent to the switch 62 . The image processing systems 68 1 , 68 2 , . . . , 68 n then outputs the volume image generated to the volume image memory 64 .
With references to FIGS. 4-5, the operator can select a desired slice thickness with a slice thickness selection input device 70 . The selected slice thickness is used as an input into a thickness/radius look-up table 72 . Based on the selected slice, the look-up table 72 retrieves the corresponding trajectory radius r. A radius adjustment driver 74 moves the radial position of the radiation source 10 inward or outward in accordance with the selected slice thickness and adjusts the direction of a collimator 76 such that a central ray of the radiation beam is aligned with the center of the detector 18 .
As illustrated in FIG. 4, when the radius of the trajectory 22 is relatively large, the radiation beam crosses the point 32 on the selected focal plane at a sharp angle. Due to the sharp angle, incremental elements only a relatively short distance off the focal plane are not held focused with the point 32 as the radiation source moves. In this manner, only data which spans a relatively narrow region to either side of the focal plane remains coherent, hence the resultant image is of a relatively thin slice. As illustrated in FIG. 5, when the radius of the trajectory 22 is very small, the central rays intersect at point 32 at very close angles. When the central ray oscillates about a very narrow range, structures that lie over a more significant distance to either side of the focal plane remain coherently focused on the detector as the radiation source 10 rotates. Hence, the resultant image represents a relatively thick slice. The exact thickness of the slice versus the radius is a relatively straightforward geometric calculation based on the size of the detector elements, the displacement between the detector and the focal plane, the distance between the focal plane and the radiation source, and the radius of the trajectory 22 .
Although the radiation source 10 is below the subject 14 in the preferred embodiment, for mechanical simplicity it is contemplated that the radiation source 10 may be disposed above the patient 14 with the detector 18 disposed below or built into the patient support 16 .
Although the trajectory 22 is defined along a spherical surface segment, for mathematical simplicity in operating on the resultant data, it is to be appreciated that the trajectories of various sizes could be defined on a flat surface or other surface. Also for mathematical simplicity, each view is subject to the same shift vector S. In some circumstances, it may be advantageous to shift different portions of each view differently. Such differential shifting may reduce edge distortion, may be used to generate oblique plane views, and the like.
It is also to be appreciated that the slice images built in slice image memories 52 and 54 are digital images which can be enhanced using any of a variety of image enhancement techniques. Such techniques include edge enhancement, smoothing, background noise suppression, and the like.
The invention has been described with reference to the preferred embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the invention be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
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An object ( 14 ) is positioned on an object support ( 16 ). A radiation source ( 10 ) projects a beam of radiation through a region of interest ( 12 ) of the object. A plurality of focal planes (F 1 , F 2 , . . . , F n ) mark the center of selected slice images through the region of interest. A gantry ( 20 ) rotates the radiation source ( 10 ) around a circular trajectory ( 22 ) as an encoder ( 26 ) monitors a radius (r) of the trajectory and an angular position (φ) of the radiation source ( 10 ) around the trajectory ( 22 ). A look-up table ( 40 ) is addressed with the selected focal plane(s) (F 1 , F 2 , . . . , F n ) and (r, φ) to generate a correction or shift value (S 1 , . . . , S n .) for each selected focal plane (F 1 , F 2 , . . . , F n ). A flat panel detector ( 18 ) is read out a plurality of times to generate a plurality of electronic data views as the radiation source rotates. Each view is corrected with a corresponding correction or shift value and integrated in a summation circuit ( 50 ) with preceding views to generate an image representation of the slice(s) through the selected focal plane(s) (F 1 , F 2 , . . . , F n ) or a 3D volume. The slice image or 3D volume image representation is converted into a human-readable display ( 56 ) substantially in real-time, e.g., each time a preselected number of views has been summed.
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FIELD OF THE INVENTION
The object of the present invention is the creation of offset printing presses which are designed on the basis of sheet-fed presses, modified, and provided with a set of complementary means making it possible for them to carry out simultaneous or successive sheet-fed or web-fed printing.
DESCRIPTION OF THE PRIOR ART
At present, web-fed printing is carried out on very large printing units which are well adapted, with their high speeds, to large runs, but whose means of implementation are lengthy and disproportionate to the printing of short runs.
Moreover, the rapid development in microcomputing as well as the fall in its production costs mean that mary average-sized firms are becoming computerized. This has created new requirements for web-fed printed matter in short and medium runs, to which requirements the traditional printers, equipped for the most part with sheet-fed presses, cannot respond.
The manufacturers of offset presses, aware of this new market, are beginning to produce, in relatively small quantities, appliances better adapted to this requirement. However, their lack of versatility added to the fact that their prices, compared to similar sheet-fed equipment, are approximately threefold makes it difficult for them to recoup their costs at the present stage of development of this new process. Moreover, these presses are designed solely for printing forms or labels intended for computing and cannot process web-fed printed matter not equipped with "caroll" punches, since their system for driving the web and for marking the margin are connected to the holes of these punches.
Now, in the field of printing from a non-punched web with short or medium runs, such as for example labels, a huge market exists which completely escapes the traditional printer possessing only sheet-fed offset presses.
Moreover, these presses are not easy for the user to employ, since their design derives directly from sheet-fed presses and, for this reason, they are poorly adapted to web-fed printing. As is shown in FIG. 1, the operator of these presses is forced to feed and to introduce the continuous web 1, prefolded into pages, into the drives 2, then to pass it through the blanket 3 and impression 4 cylinders, to take it out in order to introduce it into the outlet drives 2A and to forward it into the folder 5 as far as the receiving stack 6.
The start-up and operation of the machine, in this design, is not convenient for the operator, and this is all the more difficult when prefolded and transversely preperforated webs of light paper are being processed, in which case the risks of tearing are quite frequent and, in this case, make it necessary to carry out the same advance of the web again.
Another major shortcoming of these presses lies in the fact that they do not make it possible to carry out, when required and simultaneously, numbering on printed matter intended for the production of bundles of a large number of numbered pages.
SUMMARY OF THE INVENTION
The object of the present invention, as described in its various claims, is to overcome all these disadvantages by providing the sheet-fed presses with complementary means working together with their main components, so as to make it possible to carry out, simultaneously or successively, both sheet-fed or web-fed printing from webs with or without "caroll" punches.
The numbering unit fitted on these presses serves two purposes and makes it possible for numbering to be carried out equally well either sheet-fed or web-fed. The advantages obtained by means of this invention consist in providing the traditional printers with a substantially versatile printing press which is convenient to use and whose series-production cost will be appreciably higher than that of a sheet-fed press of the same capacity. The basic machine will be supplied in the version chosen by the customer and will comprise the set of components indispensable for driving the removable complementary components which it will be possible to add as and when needed. In order to allow the printer to change quickly from one version to another, each device is grouped together on a support taking the form of a sliding frame.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The invention is described in detail hereinafter with reference to the drawings attached to the text and showing one of the preferred embodiments of the different versions.
FIG. 2 shows a front view of a sheet-fed press, in which the arrangement of the different components has been chosen in such a way that it is possible to obtain, in each printing cycle, successively sheet-fed or web-fed, printed matter which may comprise two off-set colors plus a numbering in a third typographic color.
In the sheet-fed version, the sheet of paper is removed from the stack 7 (FIG. 2) and forwarded in the direction of the arrows towards the traditional feeding device 8, as far as the impression cylinder 4 where the grips catch it and press it successively against the blanket covering the blanket cylinder 3 and the numbering units arranged on their circular support 9 at the outlet from the impression cylinder 4. The grips open and the sheet is taken up by the grips of the outlet chain conveyor 10 which forward it and set it down on the receiving stack 11. The plate cylinders 12 of the main printing unit and of the movable support unit 13 print, before and during each printing cycle, their image on the blanket of the blanket cylinder 3. The inking of the offset plates comprising the image to be reproduced is carried out by the set of inking and damping rollers constituting their respective inking units 14 and 15.
In the version of web-fed printing, the advance of the web has been preferably modified in order to allow the operator of the press to work only on one side of it. This arrangement makes it possible, by using all the components in the press, including the numbering unit, to obtain a printed matter having, in each printing cycle, two colors and a numbering, in the same way as with sheet-fed printing (FIG. 2).
Before printing, the sheet-feeding is put out of operation by means of a pneumatic valve which closes the inlet of air caused by the absence of sheets under the air inlets of the feeder. The stack of paper pre-folded in the form of a blank page 11A is placed on its pallet on the floor. The top of the web 1 is unrolled, guided and brought under the bottom of the group of pin drives 2 which secure it by means of swinging flaps. It then passes around the complementary and removable impression cylinder 17 which will allow it to be pressed against the numbering unit 9 and the blanket cylinder 3 which will have received, as in sheet-fed printing, its print in one or two colors from the plate cylinder or cylinders 12 and 13. In this specification, the removable impression cylinder 17 is also referred to in an abbreviated fashion as the "press cylinder 17", and "impression cylinder 17".
The printed web is slightly tightened, guided and driven by the top of the pin drives 2 whose spacing corresponds to that of the "caroll" punches (1/2 inch).
In each of the examples which follow, with the exception of FIG. 8 which shows an accessory device to be fitted on a conventional sheet-fed press, the movement pieces for rotating the impression cylinder 17 and the folder 5 are included in the general mechanism of the press. Depending on the work to be carried out, either on a web with "caroll" punches or on a web with marks to be printed, the removable components are arranged on a sliding frame 64 (FIG. 4) sliding and locked on slide rails 65. The punched web of paper 1 (FIG. 3) to be printed is wound around the movable press-cylinder 17 and can be pressed against the blanket cylinder 3A by the action of the two eccentric bearings 18 (FIG. 4) appropriately adjusted.
FIG. 3 shows a front view of the printing press working simultaneously, on the one hand with sheet-fed printing from the pallet for feeding blank sheets 7 and, on the other hand, with web-fed printing from the pallet of prefolded blank paper 11A.
FIG. 4 shows a mechanical arrangement, preferably retained, making it possible to carry out simultaneous sheet-fed and web-fed printing. Sheet-fed printing involves the inking unit 15 (FIG. 3), the plate cylinder 12, the blanket cylinder 3, the impression cylinder 4, the numbering cylinder 9, the chain conveyor 10, the sheet-feeding stack 7 and the stack for receiving the printed sheets 11. The sheet-fed section operates as a conventional machine.
Web-fed printing involves the inking unit 14 (FIG. 3), the plate cylinder 13, the removable blanket cylinder 3A, the impression cylinder 17 the stack 11A for feeding prefolded paper and the stack 6 for receiving the printed and refolded paper. The displacement towards gear 27A (FIG. 4) fitted on the shaft of the blanket cylinder 3A, of the sliding gear 13A, activated by a hand-lever (not shown for reasons of clarity) uncouples the plate cylinder 13 from the cylinders 3 and 12 used for sheet-fed printing. In contrast, this movement of the gear 13A permits use of the inking unit 14 (FIG. 3) and of the plate cylinder 13 (FIG. 4) being moved by the rotation of its eccentric bearings 19A in order to carry out printing together with the blanket cylinder 3A and the press-cylinder 17 which are removable and appropriately positioned. The rotational movement originating from the common motor of the machine drives the gear 27 (FIG. 4) fastened on the shaft of the blanket cylinder 3 and the free gear 27B in rotation on the shaft of the blanket cylinder 3A by means of intermediate gears (not shown in order to improve understanding of the drawing). When it is supplied with electrical current, the electromechanical clutch 63 drives the blanket cylinder 3A and the impression cylinder 17 by means of the common motor of the printing press. The drive chains with pins 2 are driven by a grooved shaft 31 at the end of which an indexed pinion 45 is fastened. A step motor 46 drives the indexed pinion 45 by means of the indexed pinion 47 and the indexed belt 48.
The step motor 46 receives its pulses from an electronic casing 16 (FIG. 3) comprising a key unit or coding wheels capable of programming the length of the format to be printed. The pulse generator 49 (FIG. 4) enables the electronic casing 16 (FIG. 3) to calculate the speed of the step motor 46 as a function of the speed of the plate cylinder 13. Moreover, the pulse generator 49 (FIG. 4) supplies the electronic casing 16 (FIG. 3) with a synchronizing signal at each turn of the plate cylinder 13. At this signal, the electronic casing 16 (FIG. 3) supplies current to the electromagnet 50 (FIG. 4). The lever 24 drives the shaft 25 and the gears 26 which are fastened to it. The eccentric bearings 18 begin to rotate and bring about the "pressure" position of the press-cylinder 17 while it is still facing the hollow of the blanket cylinder 3A. At the same time the step motor 46 starts and moves the web of punched paper at the same speed as the circumferential speed of the blanket cylinder 3A. When the hollow of the blanket cylinder 3A has unwound, the press-cylinder 17 exerts pressure. The printing of the programmed format is achieved. At this moment, the programmed electronic casing 16 (FIG. 3) sends a signal for ending pressure. The electromagnet 50 (FIG. 4) is released, and the spring 51 provides the movement for ending pressure by turning the eccentric bearings 18 of the impression cylinder 17. When this movement has been completed, the electronic casing 16 (FIG. 3) stops the step motor 46, then reverses its direction of rotation by a length programmed as a function of the format to be produced which will then be obtained by the difference between the run of the web in the direction of the printing and the reverse run. It should be noted that the step motor 46 could be replaced by a direct-current motor and an associated electronic assembly. This is a known process for controlled displacement by an electronic monitoring device. A balance folder 5 ensures refolding of the printed web. An indexed pinion 35 is fastened on the shaft of the plate cylinder 13. The indexed belt 36 drives the indexed pinion 37 which is integral with the gear 38. The latter sets the crank gear 39 in rotation at half speed. The connecting rod 40 retransmits the movement to the folder 5 by means of the lever 41 integral with the gear 42 which oscillates while driving the gear 44 fixed on the shaft 43 of the folder 5. Therefore, to each turn of the plate cylinder 13, thus to each printed format, there corresponds a forward or return movement of the folder 5 ensuring refolding.
FIG. 5 shows a plan view with the drives 2 being controlled in a completely mechanical manner. The shaft of the blanket cylinder 3 carries, at one end, a set of adjustable cams 20 and 20A which swing the lever 21 either in one direction or in the other by means of the cam rollers 22. The connecting rod 23 in turn activates the lever 24 which, fastened on the shaft 25, drives it in its oscillation as well as the two gears 26 in gear with the two eccentric bearings 18 in which the shaft of the press-cylinder 17 turns. There are thus obtained, at each turn of the blanket cylinder 3, a pressure position followed by a position ending pressure controlled by the maneuvering cams 20 and 20A, and this at each rotation of the blanket cylinder 3, thus at each printing cycle. The gear 27 fastened on the shaft of the blanket cylinder 3 is in gear with the gear 28 fastened on the shaft of the impression cylinder 17, thus ensuring rotation of the latter, even during the part of the cycle when there is no pressure. The cam for causing pressure 20 always triggers pressure in the hollow of the blanket cylinder. The cam for ending pressure 20A triggers the end of pressure at will, depending on the chosen format to be printed, engraved on the vernier 29. The web of paper 1, printed by the pressure between the blanket cylinder 3 and the press-cylinder 17, drives the chain drives with pins 2 supported by the adjustment shaft 30 and the grooved shaft 31 one end of which comprises a fastened ratchet wheel 32. A catch 33 articulated on the lever 34 set in motion by the shaft for controlling the pressure 25 drives, in a small backward movement, the web of paper 1 each time the pressure is ended, thus smoothing the web in order to obtain the appropriate format after each printing cycle. The format printed will thus be defined by the difference between the length of web printed by the impression cylinder 17 adjusted by the cam 20A, chosen on the vernier 29, and the return movement due to the catch 33 which will be variable as a function of the number of teeth cut on the wheel 32. Thus, it will be possible to choose a format to be printed with a spacing of half an inch, a sixth of an inch or an eighth or an inch. During pressure, the catch 33 comes out from the toothed wheel 32, thus ensuring the free rotation of the drives 2 driven by the punched web of paper 1.
In the case where printing is carried out from a web of light paper, it is preferable, in order to avoid the risks of tearing the web, to drive the drives mechanically in phase with the machine.
A folder 5 ensures refolding of the printed web. An indexed pinion 35 is fastened on the shaft of the blanket cylinder 3. The indexed belt 36 drives the indexed pinion 37 which is integral with the gear 38. The latter ensures rotation of the crank gear 39 at half speed. The connecting rod 40 retransmits the movement to the folder 5 by means of the lever 41 which is integral with the gear 42 which sets the shaft 43 of the folder in oscillation, on which shaft the gear 44 is fastened.
FIGS. 6 and 7 show, in front and plan view, an example of the machine set up for web-fed printing from reel to reel. The latter are carried on a run-off reel 52 (FIG. 6). The motor 53 drives the shaft of the reels by means of clutches for the control loops 54 and 55. The rolls 56 tighten and guide the web 1A. The press-cylinder 17 (FIG. 7) ensures the pressing of the web of paper against the blanket cylinder 3 by means of its eccentric bearings 18 (FIG. 7) set in rotation by the electromagnet 50 as in the previous case. A step motor 46 drives the roller 57 by means of a free wheel gear 47A. The pressure rollers 58 ensure driving of the web of paper 1A. At the starting signal of the printing cycle given by the pulse generator 49, the electronic casing 16 (FIG. 6) activates the electromagnet 50 (FIG. 7) and ensures pressure of the impression cylinder 17 when it is facing the hollow of the blanket cylinder 3. The step motor 46 turns in the direction of unwinding of the web, but at a speed substantially greater than that corresponding to the turning of the blanket cylinder 3. The step motor 46 starts up in the same free direction of the free wheel gear 47A and, for this reason, has no effect on the drive of the web of paper 1A. At each printing cycle, when the hollow of the blanket cylinder 3 is at the end of its unwinding and comes into contact with the press-cylinder 17, the web of paper 1A, gripped between the two, is drawn and printed. At the end of printing, with the programmed format, a set of orders are emitted by the electronic control casing 16 and follow each other at very short intervals set by retarders acting on:
(a) the ending of pressure of the press-cylinder 17 (FIG. 7). The electromagnet 50 is released and the web of paper 1A moves away from the blanket cylinder 5 which no longer drives it,
(b) the stoppage of the step motor 46,
(c) the rotation of the step motor 46 at a very slow speed, in the reverse direction. The free wheel gear 47A becomes active, the roller 57 turns and moves the paper back,
(d) the stoppage of the step motor 46, obtained by the passage under the optical detector 59 of a line or a point serving as a mark and produced on the web during printing. The position of the optical detector 59 along the web of paper defining the printed format. Important variants of known means concerning the driving and feeding of the web can be applied without going beyond the scope of the present invention.
In the configuration in FIGS. 6 and 7, the machine makes it possible to successively carry out either sheet-fed printing or web-fed printing from reel to reel in 2 colors plus numbering. The configuration in FIGS. 3 and 4 makes it possible to simultaneously carry out sheet-fed and web-fed printing from reel to reel. In another form of the invention, an example of a removable device which can be fitted on existing sheet-fed presses to change them to mixed sheet-fed and web-fed presses, from the same basic components described in the present invention. According to the arrangement of the various components constituting the sheet-fed press to which it is attached, the results obtained will combine all or some of those achieved with the presses designed for this purpose and coming within the scope of the invention.
FIG. 8 shows a plan view of an example of a complete device fitted on a conventional sheet-fed offset press. The frame 60 fixed on the press by the adaptors 61 supports all the components necessary for operation of the device. The blanket cylinder 3 directly drives the impression cylinder 17. The eccentric shaft 62 makes it possible, by means of the rotation of the lever 24 activated by the electromagnet 50, to subject the web of paper 1 to pressure and print it. As in the previous example in FIG. 6, the combination of electronic casing 16, pulse generator 49 and step motor 46 permits displacement of the web of paper 1 and the appropriate positioning of the printing by means of the chain drives with pins 2. As in the examples in FIGS. 3 and 6, the web of paper 1 is returned into its folds by the folder 5 activated by the connecting rod 40 and the crank gear 39. Moreover, as in the example in FIG. 5, the system for driving the web of paper 1 can be achieved in a completely mechanical manner with cams and ratchet wheel.
These versatile presses, in their different versions, permit the following combinations:
(a) simultaneous sheet-fed and web-fed printing
1: sheet-fed printing in one color plus web-fed printing on one colors
2: sheet-fed printing in one color plus numbering plus web-fed printing in one color
3: sheet-fed printing in one color plus one support color plus web-fed printing in one color
4: sheet-fed printing in one color plus web-fed printing in one color plus one support color
5: sheet-fed printing in one color plus web-fed printing in one color plus numbering
(b) web-fed printing
6: web-fed printing, fold to fold, in one color
7: web-fed printing, reel to reel, in one color
8: web-fed printing, fold to fold, in one color plus numbering
9: web-fed printing, reel to reel, in one color plus numbering
10: web-fed printing, fold to fold, in two colors
11: web-fed printing, reel to reel, in two colors
12: web-fed printing, fold to fold, in two colors plus numbering
13: web-fed printing, reel to reel, in two colors plus numbering
14: web-fed printing, fold to fold, in two colors plus one support color
15: web-fed printing, reel to reel, in two colors plus one support color
(c) sheet-fed printing
16: sheet-fed printing in one color
17: sheet-fed printing in one color plus numbering
18: sheet-fed printing in two colors
19: sheet-fed printing in two colors plus numbering
20: sheet-fed printing in two colors plus one support color.
The advantages obtained from these presses are evident and, by virtue of the fact that they eliminate constraints placed on traditional printers, they will allow considerable development in all web-fed printed matter of small and medium runs.
In parallel to the services which these versatile presses will offer traditional printers, it is envisaged that they will be used by specialized printers in web-fed printing, since, at present, computer manufacturers are developing appliances equipped with laser-printers, sheet-fed or web-fed depending on the intended use of the printed matter.
The device which can be fitted on the existing sheet-fed presses is not unimportant since, for a small investment, the printer can develop, within his company, these new markets before equipping himself with better adapted presses.
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Offset printing press which comprises, in combination, a set of means making it possible for it to carry out, either independently or simultaneously, sheet-fed or web-fed printing. These webs are presented equally well either as perforated and prefolded pages or as reels. A set of mechanical movement pieces, working together with the removable components, makes it possible, depending on the types of printing to be carried out, to provide the press with twenty possibilities of different printing modes. A movable press-cylinder 17 (FIG. 3), working together with the blanket of the removable blanket cylinder 3A, makes it possible to prevent the web-fed printed matter, during its printing, from passing through the press and, by so doing, to make these two modes of feeding and printing, sheet-fed and web-fed, totally independent of each other. The same type of removable device as that fitted on the mixed press, also mounted on a sliding frame 64 (FIG. 4), can be fitted on a set of existing sheet-fed offset presses, in order to make it possible for them to carry out successive sheet-fed and web-fed printing.
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BACKGROUND
[0001] Certain devices that move or otherwise handle liquid(s) may produce froth. Froth, for example, can occur when the liquid(s) mix with gas to form bubbles. A build-up of such bubbles can lead to a layer of froth on top of the liquid. In certain instances gas maybe drawn into the liquid resulting in froth. In other instances gas may be drawn or otherwise released from within the liquid resulting in froth.
[0002] Froth will usually return to separate liquid and gas components, but this can take a significant amount of time and possibly also space to hold the froth as it slowly separates. Such time and or space are often unacceptable for certain devices or processes. Thus, to avoid froth or otherwise reduce the volume of froth produced, special chemicals or compounds are often added to the liquid that tend to reduce or eliminate unwanted froth.
[0003] However, there are some devices and processes that simply cannot accommodate such special chemicals or compounds. In other situations, the additional cost of such special chemicals or compounds may be prohibitive.
[0004] Consequently, there is a need for methods and apparatuses for handling froth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The following detailed description refers to the accompanying figures.
[0006] FIG. 1A is an illustrative diagram depicting an exemplary fluid handling device having a container configured to handle froth in accordance with certain implementations of the present invention.
[0007] FIG. 1B is an illustrative diagram depicting an exemplary fluid handling device having a container configured to handle froth as in FIG. 1A further illustrating froth, and froth that has been separated into liquid and gas portions, in accordance with certain implementations of the present invention.
[0008] FIG. 2 is an illustrative diagram depicting an exemplary printing device having a container configured to handle froth, in accordance with certain implementations of the present invention.
[0009] FIG. 3 is flow diagram depicting a method for use with devices, for example, such as those illustrated in FIGS. 1 A-B, and 2 , for handling froth, in accordance with certain implementations of the present invention.
[0010] FIG. 4 is a diagram depicting exemplary circuitry for applying an electrostatic charge, in accordance with certain implementations of the present invention.
DETAILED DESCRIPTION
[0011] Attention is drawn to FIG. 1A , which is an illustrative diagram depicting an exemplary fluid handling device 100 having a container 102 configured to handle froth in accordance with certain implementations of the present invention.
[0012] As shown, fluid handling device 100 includes container 102 having, in this example, a housing 104 forming therein an opening 106 which is suitable for holding froth. Here, froth is introduced into opening 106 through a froth port 108 . Opening 106 further includes a liquid port 110 that allows liquid separated from the froth within opening 106 to exit container 102 . Opening 106 also includes a gas port 112 that allows gas separated from the froth within opening 106 to exit container 102 .
[0013] In this embodiment, froth port 108 is fluidically coupled to a froth conduit 114 which is further fluidically coupled to a froth source 116 . Similarly, liquid port 110 is fluidically coupled to a liquid conduit 118 which is further fluidically coupled to a liquid destination 120 .
[0014] In certain other implementations, all or some of the froth and liquid components may be combined. For example, froth port 108 and liquid port 110 may be combined into a single port that allows froth to enter into opening 106 and liquid to exit from within opening 106 . Froth conduit 114 and liquid conduit 118 may be similarly combined into one conduit that carries froth towards container 102 and liquid away from container 102 . In such examples and/or other implementations, froth source 116 and liquid destination 120 may also be combined as a single container or vessel that is configured to hold both liquid and froth. Such combinations are represented by connector 132 shown in dashed line format.
[0015] With regard to the exemplary device in FIG. 1A , a gas conduit 122 is fluidically coupled to gas port 112 . Here, gas may exit opening 106 and be released (e.g., vented) into the atmosphere as illustrated as gas destination 124 a and/or collected or otherwise handled using a gas destination 124 b fluidically coupled to gas port 112 . In certain implementations, gas port 112 may directly vent gas into the atmosphere without requiring gas conduit 122 . Gas port 112 , gas conduit 122 and/or gas destination 124 b may be configured to reduce the chance for liquid or froth from escaping therethrough by including one or more controlling mechanisms as are well known in the art for reducing fluid leaks and the like. For example, in certain implementations, a gas-permeable filter (not shown) and/or a serpentine conduit shape (not shown) may be employed to hinder liquid movement.
[0016] Circuitry 126 is shown as being connected to at least two electrodes that are at least partially arranged within opening 106 . In this example, circuitry 126 is configured to generate a voltage potential between an upper electrode 128 a and a lower electrode 128 b, which are separated by a gap space 130 within opening 106 . When applied by circuitry 126 , the voltage potential creates an electrostatic charge between the electrodes. This electrostatic charge is discharged through the froth located within opening 106 . The electrostatic discharge tends to reduce the amount of froth.
[0017] The reduction of froth is believed to be caused by the electrostatic discharge creating localized heating of the bubble lamella, disrupting the surface tension and causing the bubble to rupture. The high temperature of the spark vaporizes the liquid faster than the surface tension can recover destabilizing the lamella.
[0018] Those skilled in the art will recognize that circuitry 126 may take on several forms, as there are many well known circuits that may be employed to generate the voltage potential.
[0019] By way of example, a simple charging/discharging circuit 400 is illustrated in FIG. 4 . Circuit 400 may be included, for example, in circuitry 126 . Circuit 400 includes a DC voltage source 402 coupled to a charging resistor 404 . Charging resistor 404 is further coupled to a relay 406 . When relay 406 is in a first position the voltage potential from source 402 is applied to charge storage capacitor 408 . Capacitor 408 is then charged. Subsequently, when relay 406 is in a second position the capacitor 408 is allowed to discharge through a current limiting resistor 410 and through froth between the electrodes in container 102 . In one exemplary implementation, DC voltage source 402 outputs 8,000 volts, charging resistor 404 is a 1 MΩ resistor, charge storage capacitor 408 is a 100 pF capacitor, current limiting resistor is a 1 kΩ resistor, and the resulting electrostatic discharge is about 8,000 volts.
[0020] Furthermore, those skilled in the art will recognize that the voltage potential will likely be different depending upon various design characteristics and the like. For example, the voltage potential may correspond in some manner to the arranged opening 106 , electrodes 128 , the gap space 130 (or gap spaces if more than two electrodes are used), certain properties or characteristics of the liquid and/or the gas, the amount of froth present or expected, etc. By way of example, in certain implementations a voltage potential of at least about 1,000 volts may be required, while in other implementations the requisite voltage potential may be lower or greater. In certain exemplary implementations such as that depicted in FIG. 2 , for example, the voltage potential is typically between about 8,000 and about 12,000 volts.
[0021] In certain implementations, circuitry 126 is configured to selectively apply the voltage potential when the volume of froth within opening 106 reaches or possibly exceeds a defined threshold froth volume level. Hence, circuitry 126 may include a monitoring mechanism 127 that senses the froth volume level or otherwise identifies the froth volume level in a manner that causes circuitry 126 to apply the voltage potential. Monitoring mechanism 127 may include, for example, electrical, mechanical, and/or optical based sensors or other like devices. Circuitry 126 may include logic and/or other mechanisms to respond to monitoring mechanism 127 . In certain implementations, circuitry 126 may be programmably configured and the threshold froth volume level(s) established.
[0022] In certain implementations, circuitry 126 may be configured to apply the voltage potential periodically, perhaps in accordance with a desired schedule. For example, the voltage potential may be applied every ten seconds.
[0023] Circuitry 126 may be configured to apply the voltage potential a plurality of times during a set period of time. For example, the voltage potential may be applied at a rate of once per second (i.e., 1 Hz). Such a rate may be higher or lower in other implementations. For example, a rate of about 20 Hz was found to be effective in certain implementations as for example in FIG. 2 .
[0024] Those skilled in the art will recognize also that circuitry 126 may be configured to apply different voltages at certain times, or upon different levels of froth, or through different electrodes, etc.
[0025] Attention is now drawn to FIG. 1B . Here, froth 134 is urged or otherwise allowed in some manner to travel from froth source 116 through froth conduit 114 and into opening 106 . An electrostatic discharge is illustrated by conductive path(s) 140 as passing between electrodes 128 a and 128 b through portions of froth 134 . The electrostatic discharge tends to separate at least some of froth 134 into liquid 136 and gas 138 portions. In this example, the separated liquid 136 descends within opening 106 following the electrostatic discharge where it may then be urged or otherwise allowed in some manner to travel from opening 106 through liquid conduit 118 and into liquid destination 120 . The separated gas 138 ascends within opening 106 , above any remaining froth 134 and/or liquid 136 , where it may then be urged or otherwise allowed in some manner to travel from opening 106 through gas conduit 122 and into a liquid destination 124 a and/or 124 b.
[0026] A threshold froth volume level 142 is illustrated in FIG. 1B . As described above, in certain implementations, circuitry 126 may be configured to selectively apply the voltage potential provided that the froth volume level is at or above threshold froth volume level 142 . In other implementations, threshold froth volume level 142 may reflect the level at which there is simply enough froth 134 between electrodes 128 a - b to cause the discharge via conductive path 140 .
[0027] FIG. 2 is an illustrative diagram depicting an exemplary printing device 200 having a container 216 configured to handle froth, in accordance with certain further implementations of the present invention.
[0028] Printing device 200 is a representative inkjet printing device. Printing device 200 includes a printhead 202 having one or more nozzles 204 configured to selectively eject droplets of fluid, such as for example, ink 214 . Printhead 202 is fluidically coupled to a printhead reservoir 206 that holds and supplies ink 214 to printhead 202 . Printhead reservoir 206 is further fluidically coupled through a conduit 208 a to a pump 210 . In this example pump 210 is a bidirectional pump and is further fluidically coupled to an ink cartridge 212 through a conduit 208 b. Ink cartridge 212 stores ink 214 . Pump 210 may be operated to selectively pump ink 214 from ink cartridge 212 to printhead reservoir 206 , or from printhead reservoir 206 to ink cartridge 212 . Froth may be created due to this pumping action and/or as a result of some other process or property. Thus, froth may accumulate in ink cartridge 212 .
[0029] The froth in ink cartridge 212 is allowed to enter into container 216 via conduit 208 c. Froth 134 within container 216 is then subjected to an electrostatic discharge and the separated ink is allowed to return to container 216 via conduit 208 c. The separated gas is allowed to exit container 216 via gas port 112 .
[0030] Although shown separately, in certain other implementations, ink cartridge 212 and container 216 may be combined to form a single vessel. Similarly, in still other implementations, ink cartridge 212 , container 216 and printhead reservoir 206 may be combined to form a single vessel.
[0031] FIG. 3 is flow diagram depicting a method 300 for use with fluid handling devices, for example, such as those illustrated in FIGS. 1 and 2 , for handling froth, in accordance with certain implementations of the present invention.
[0032] In act 302 a threshold froth volume level 142 is established, for example, as described in the examples above or in other ways. In act 304 an electrostatic charge is applied by circuitry 126 to electrodes 128 . In act 306 the froth discharges the electrostatic charge when the froth reaches the threshold froth volume level 142 . Acts 304 and 306 may then be repeated.
[0033] In a second exemplary method, as depicted with dashed lines in FIG. 3 , in act 308 the froth volume level may be measured. In act 310 , when the measured froth volume level reaches the threshold froth volume level 142 , circuitry 126 applies the electrostatic charge that then discharges through froth 134 . Acts 308 and 310 may then be repeated.
[0034] Although the above disclosure has been described in language specific to structural/functional features and/or methodological acts, it is to be understood that the appended claims are not limited to the specific features or acts described. Rather, the specific features and acts are exemplary forms of implementing this disclosure.
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Methods and apparatuses are provided for separating froth into liquid and gas components. One apparatus includes a container that is configured to hold froth therein and change at least a portion of the froth into substantially separate liquid and gas portions when an electrostatic charge is discharged through at least a portion of the froth between at least two electrodes at least partially arranged within the container.
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FIELD OF THE INVENTION
[0001] The present invention relates to a method for accumulating foil prior to welding. The foil is used for packaging and wrapping compressible and incompressible objects.
BACKGROUND
[0002] Packaging and wrapping of products using foil is widely used in many industries. Such packed or wrapped products could be for the building industry and range from building materials such as plaster plates to glass or mineral wool. One of the primary requirements for the packaging of such products is naturally that the material wrapped around the products serves to protect the products until it is purposely broken.
[0003] When wrapping insulation materials, such as glass or mineral wool, the foil is often used as a mean for maintaining a compression of normally a stack of insulation objects. The insulation objects are compressed and then wrapped in un-stretched or stretched foil, which is joined by welding. The welding is made by a welding assembly comprising two pairs of resilient jaws, between which a knife for cutting the foil is placed. Further, between the knife and the resilient jaws two pairs of welding beams for welding the foil are positioned. When the objects to be packed have been wrapped in the foil, the foil is stretched and held between the resilient jaws and at the same time clamped between the welding beams. Hereafter the foil is cut and welded at the same time.
[0004] To reduce the production costs, it is desired to reduce the amount of foil used per package. This is done by using thinner foils in the existing production facilities. However, using thinner foil is encumbered with one major problem. Regardless of the foil thickness, the thermal stress applied to the stretched foil during welding will release the tension present in the foil. This will lead the foil to creep and consequently lead to a thinning of the foil between the resilient jaws and the welding beam. When using thin foils, the thinning can reduce the yield strength of the foil below the required level or be so severe that the foil is reduced to only thin threads. There is therefore a great risk that the packaging process fails or that the finished package is torn or breaks open unintentionally.
SUMMARY OF THE INVENTION
[0005] The present invention relates to a method for welding at least one foil in a packaging process using a welding assembly comprising the steps of directing the foil into the welding assembly, holding the foil in at least one holding point of the welding assembly, welding the foil in at least one welding point of the welding assembly, and where a part of the foil is accumulated between the at least one holding point and the at least one welding point prior to welding the foil in the welding point. When the foil is welded the foil is drawn or crept towards the welding point, due to the heat in this point. Since a part of the foil has been accumulated between the welding point and the holding point, only the excess foil here is drawn towards the welding. Hereby it is avoided that the foil is over-stretched or that the welding will be too weak. Thinning of the foil between the welding point and the holding point will therefore not occur. Thus, the risk of breakage of the foil due to weaknesses in the foil and/or welding is reduced or eliminated. A packaging process using this method will therefore not fail nor will the finished package be torn or break open unintentionally.
[0006] In another embodiment the method further comprises the step of cutting the foil during welding of the foil. When using continuous foil this is particular advantageous, as this enables a continuous packaging process.
[0007] In a further embodiment the method comprises the step of clamping the foil in the at least one welding point prior to holding the foil in the at least one holding point. This is advantageous as it makes it possible to direct foil towards the welding point prior to holding the foil in the holding point, whereby foil will be accumulated between the welding point and the holding point.
[0008] In yet another embodiment the method comprises the step of clamping the foil in the welding point offset from the holding point of the foil ( 112 ), and then aligning the at least one welding point with the at least one holding point. By having the welding point and the holding point offset from each other and subsequently aligning these two points, foil can be accumulated between these two points. The length of the accumulated foil can be max. the distance between the welding point and the holding point. This distance can advantageously be adjusted according to e.g. the welding and/or foil properties.
[0009] The present invention further relates to a welding assembly for welding at least one foil comprising welding means for welding the foil, and where the welding assembly further comprises holding means for holding the foil while welding, and where the welding assembly comprises accumulation means for accumulating a part of the foil between at least one welding point of the welding means and the holding point of the holding means, prior to welding the foil. The welding assembly is advantageous in that the accumulation means ensures that a part of the foil can be accumulated between the welding point and the holding point. While welding, the foil will be drawn or crept towards the welding point of the welding means, but without weakening or breaking the foil both during and after the welding process.
[0010] In another embodiment the welding assembly comprises cutting means for cutting the foil. This is advantageous in that the welding assembly can be used in e.g. a continuous packaging process using continuous foils.
[0011] In a further embodiment the welding means of the welding assembly comprises a welding beam, at least one welding bar and a welding support. This is advantageous in that the foil can be clamped in a welding point by the welding beam and the welding, support, and further that the foil can be welded in the same welding point. In addition, the welding point can be moved independently of the holding means.
[0012] In yet another embodiment the holding means of the welding assembly comprises an upper jaw, a lower jaw and at least one resilient member. This is advantageous in that the foil can be held firmly prior to and during the welding process. By having at least one resilient member it is ensured that the foil maintains its position, thus the foil cannot slide in the holding point.
[0013] In another embodiment the accumulation means of the welding assembly is a pneumatic cylinder. Here the advantages are as mentioned above.
[0014] The present invention further relates to the use of a welding assembly as mentioned above employing the method for welding at least one foil in a packaging process also mentioned above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In the following, preferred embodiments of the invention will be described referring to the figures, where FIGS. 1 a - f illustrate a number of method steps of accumulating foil prior to welding the foil.
DESCRIPTION OF EMBODIMENTS
[0016] The welding assembly 100 comprises holding means, welding means, cutting means and accumulation means. The holding means comprises an upper jaw 102 and a lower jaw 104 . The lower jaw 104 comprises a resilient member 105 , in which the two protrusions of the upper jaw 102 can form a recess or groove. The jaws 102 , 104 can be moved vertically up and down and can hold the foil 112 in the holding point 124 , 125 . The welding means of the welding assembly 100 comprises a welding beam 106 and a welding support 108 . The welding beam 106 and the welding support 108 are placed in between the jaws of the upper jaw 102 and the lower jaw 104 , respectively. The welding support 108 can be moved vertically up and down, independently of the lower jaw 104 . This movement can be enabled by accumulation means (not shown), such as a pneumatic cylinder. The welding beam 106 can be moved vertically up and down relative to the upper jaw 102 . This relative movement is limited to the clearance 118 of the upper jaw 102 . For illustrative purposes, the clearance 118 is only depicted in FIG. 1 a . The welding beam 106 comprises two welding bars 107 , 109 , which can be heated e.g. by directing a current there through. When the welding beam 106 and the welding support 108 are brought together they clamp the foil 112 in the welding point 122 , 123 . The welding assembly 100 further comprises cutting means comprising a knife 110 for cutting the foil 112 . The knife 110 can be moved vertically up and down independently of the welding support 108 . The width of both the jaws 102 , 104 , the welding beam 106 , and the welding support 108 span at least the width of the foil, but does not have to be continuous. The knife 110 also spans at least the width of the foil.
[0017] In one embodiment the foil is welded using the welding assembly 100 according to the following method. The method relates to the joining of at least two foils. The at least two foils can originate from the same roll of foil but also be from two different rolls of foil. For the sake of simplicity the at least two foils are depicted and referenced as one foil 112 . The foil 112 used can be un-stretched or pre-stretched. The latter concerns a foil that has been stretched to such an extent that it is plastically deformed. This means that the foil cannot be stretched any further without breaking it.
[0018] As illustrated in FIG. 1 a , the foil 112 is directed or led in between the upper and lower jaws 102 , 104 and the welding beam 106 and the welding support 108 , which have been positioned so that they form a space there between. As depicted in FIG. 1 a , the welding beam 106 is positioned vertically higher than the jaws of the upper jaw 102 . The welding support 108 is then directed upwards towards the welding bars 107 , 109 , whereby the foil 112 is clamped in the welding point 122 , 123 , see FIG. 1 b . This upward movement of the welding support 108 could be enabled by using accumulation means (not shown), such as actuating a pneumatic cylinder. The upper jaw 102 and thereby also the welding beam 106 are then moved downwards towards the lower jaw 104 and the resilient member 105 . The downwards movement of the welding beam 106 can take place as the pressure in the pneumatic cylinder (accumulation mean) in the welding support 108 is reduced or removed. The foil 112 is now held between both upper and lower jaw 102 , 104 in the holding point 124 , 125 and clamped between the welding beam 106 and the welding support 108 in the welding point 122 , 123 . In the space between the holding point 124 , 125 and the welding point 122 , 123 , accumulated foil 112 is now placed. The foil 112 in this confined space will typically fold in e.g. an S- or Z-shape. As illustrated in FIG. 1 d , the foil 112 is hereafter cut by moving the knife 110 (cutting means) upwards and at the same time welded in the welding point 122 , 123 . Due to the heat, the accumulated foil 112 on both sides of the welding point 122 , 123 is drawn or crept towards the welding point 122 , 123 . If the foil 112 is pre-stretched (plastically deformed or stretched), the foil 112 will during welding creep or be drawn more towards the welding point 122 , 123 , than an un-stretched foil 112 . In order to provide the best possible welding, the welding support 108 can be pressed against the welding bars 107 , 109 during and after welding.
[0019] This is done by activating the pneumatic cylinder of the welding support 108 . Hereby it can be ensured that e.g. the transition from the performed welding and the regular foil 112 is such that the foil 112 does not break if the foil 112 is exposed to e.g. a force across the foil 112 .
[0020] The steps of the method described can vary both in terms of how they are performed and the sequence of them. Thus the foil can be accumulated in many other ways between the holding point 124 , 125 and the welding point 122 , 123 , using different accumulation means. This could e.g. be by moving the holding means and the welding means according to another method or sequence of steps. The foil could also be pre-accumulated before it is led in between the holding and the welding point. The accumulation means could also be an element or apparatus that conveys foil towards the welding point, e.g. when the welding means clamps the foil and before the foil is held in the holding point by the holding means.
[0021] The knife 110 which can be moved vertically up and down independently of the welding support 108 makes it possible to actually weld the foil 112 before cutting the foil 112 ensuring that the foil 112 has the time to be welded before cutting and further that the foil is not stressed while cutting. The possibility of independently moving the knife 110 makes it possible to ensure the correct timing between welding and cutting. The timing could depend on the amount of foil to be welded as well as the type of foil to be welded. Further, the independent movement up and down of the knife 112 as well as the vertical space within said welding support 108 makes it possible to control the extent of up and downwards movement to ensure that the foil 112 gets cut, which again could depend on the type of foil.
REFERENCES
[0000]
100 welding assembly
102 upper jaw
104 lower jaw
105 resilient member
106 upper welding beam
107 welding bar
108 welding support
109 welding bar
110 knife
112 foil
118 clearance for moving the welding beam 106 relative to the upper jaw 102
120 direction of movement of the foil 112
122 , 123 welding point of the welding means ( 106 , 107 , 108 , 109 )
124 , 125 holding point of the holding means ( 102 , 104 , 105 )
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The present invention relates to a method for welding at least one foil in a packaging process using a welding assembly, comprising the steps of directing the foil into the welding assembly, holding the foil in at least one holding point of the welding assembly, welding the foil in at least one welding point of the welding assembly, and where a part of the foil is accumulated between the at least one holding point and the at least one welding point prior to welding the foil in the welding point. The present invention further relates to a welding assembly employing this method.
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CROSS-REFERENCE TO RELATED U.S. APPLICATIONS
[0001] Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT
[0003] Not applicable.
REFERENCE TO AN APPENDIX SUBMITTED ON COMPACT DISC
[0004] Not applicable.
BACKGROUND OF THE INVENTION
[0005] 1. Field of the Invention
[0006] The present invention relates to cover embodiment, which is used in aseptic liquid cardboard packages, forms a flowing hole on the top part of the package so that the liquid inside the cardboard package can flow through, and can be screwed once more.
[0007] 2. Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98
[0008] In the currently used aseptic liquid cardboard package covers, various shapes of configurations have been applied. Some of these can be opened with the help of hinges and the aluminum foil that is previously attached on the package is broken off by hand after opening the upper cover, and thus they are made functional. In some embodiments, on the other hand, a hole is created, which enables the liquid to flow into the cover by piercing the foil part of the package as a result of pressing a plastic piece having incisors for piercing the foil located inside the cover, in the downward direction.
[0009] In some cover embodiments, on the other hand, the covers are provided to operate with a screwed system which is made of two- or three-piece plastic. On the part of the package under the cover, which has been previously prepared on the cardboard, the foil and the polyethylene layer are left in a one-piece structure so that they can pass through the cardboard in one-piece form while the cardboard laminations (layers) are being made, and this system is an embodiment which is safer compared to the other embodiments, and provides asepticity. Moreover, as these types of screwable covers are covered by being screwed again, it has become much easier to carry the products, and impermeability is provided.
[0010] The working system of the screwed covers which consist of three parts, is that the bottom and upper part are screwed in nested form and a third part is located in the center, and when the cylindrical cover having incisors at the bottom part is rotated in the opening direction, it progresses in the downward direction, and cuts the foil on the cardboard by rotating and therefore it pierces the part inside the cover of the package. In such types of cover embodiments, the applications numbered PCT/JP2004/014333, EP1820742 A1, PCT/EP03/50614 can be considered as example. In these two-piece covers, on the other hand, the cylindrical part located at the center is rotated; the plate or plates connected to the outer cover having flat incisors located on the lower part of the cover is/are pushed in the downward direction, the foil part of the package is made to be broken into pieces as much as the number of plates, and thus it is opened.
[0011] In that kinds of screwed covers consisting of two pieces, on the other hand, the cover embodiment presses the flat plate having incisors in the downward direction and provided pierce of the aluminum layer on the package when the cover embodiment is rotated in the opening direction thanks to foil protrusion provided at the upper section of flat plate having incisors, which is connected to the sides within the section attached to the lower package and protrusion being suitable with foil protrusion designed in the upper screwed cover; however, a completely homogeneous flowing hole cannot be opened as the opening process is not completed.
[0012] This case has restricted the product flow and made the usage difficult.
[0013] In the above-mentioned cover embodiments, on the other hand, after the foil available on the flowing mouth is cut, the part of the foil which is broken off stuffs up the flow net because of the pressure inside the box, and sometimes the foil piece falls into the liquid product, which causes undesired results.
[0014] As the cover height should be higher because of the rotation process of the parts having cylindrical incisors in the inner part of such type of screwable covers, it becomes quite difficult for them to be stored, transported and ordered on the shelves. Moreover, because of huge cover sizes and more number of parts, the amount of raw material used increases, which makes it economically non-efficient and thus, it is not that economic in efficient use of the resources.
OBJECT OF THE INVENTION
[0015] The object of the invention is to provide a much more apparent flowing ease compared to the other embodiments during the product flow by taking the foil totally away from the flowing mouth, and by opening at least 90% of the inner wall in a homogeneous structure.
BRIEF SUMMARY OF THE INVENTION
[0016] In order to achieve the above-mentioned object, the present invention relates to a screwed cover embodiment having apparatus of foil-cutting and folding to the inner part in the aseptic liquid cardboard packages, comprising lower cover, cylindrical ridge and flat plate located on the inner part of the lower cover; axial hinge and tension spring connected to the cylindrical wall in the lower cover; upper cover and spiral friction ramp located on the inner part of the upper cover.
[0017] In order to achieve said objects, there is a cylindrical ridge located on the inner part of the lower cover which is exposed to the pressure that the spiral friction ramp will apply in the downward direction after it is rotated in the cover-opening direction. Moreover, the left and right flat plates on which the cylindrical ridge on the inner part of the lower cover is located are connected to the cylindrical wall inside the lower cover by means of axial hinge. The connection of said flat plates to the cylindrical inner wall with an asymmetrical parallel angle via axial hinge is provided by means of the tension spring.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is the view of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention from the side profile.
[0019] FIG. 2 is the top perspective view of lower cover ( 01 ) of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention.
[0020] FIG. 3 is the bottom perspective view of the lower cover ( 01 ) of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention.
[0021] FIG. 4 is the bottom perspective view of the lower cover ( 01 ) and the upper cover ( 02 ) of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention.
[0022] FIG. 5 is the inner part profile view of the upper cover ( 02 ) of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention.
[0023] FIG. 6 is the perspective view of the lower cover ( 01 ) installed on the cardboard box ( 15 ) of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention.
[0024] FIG. 7 is the perspective view of the inner mechanism ( 03 , 04 , 05 , 13 ) of the lower cover ( 01 ) which is installed on the cardboard box ( 15 ) of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention, when it is semi-open together.
[0025] FIG. 8 is the top perspective view of the lower cover ( 01 ) of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention.
[0026] FIG. 9 is the perspective sectional view of the upper ( 02 ) and lower ( 01 ) covers of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention.
[0027] FIG. 10 is the perspective sectional view showing how the inner mechanism of the upper ( 02 ) and lower ( 01 ) covers of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention operate.
[0028] FIG. 11 is the perspective sectional view showing how the inner mechanism of the upper ( 02 ) and lower ( 01 ) covers of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention operate.
[0029] FIG. 12 is the perspective sectional view showing how the inner mechanism of the upper ( 02 ) and lower ( 01 ) covers of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention operate, as a continuation of FIG. 11 .
[0030] FIG. 13 is the perspective sectional view showing how the inner mechanism of the upper ( 02 ) and lower ( 01 ) covers of the preferred embodiment of the cover embodiment ( 101 ) according to the present invention operate, as a continuation of FIG. 12 .
DESCRIPTION OF PART REFERENCES
[0031] 101 . Cover embodiment
[0032] 01 : Lower cover
[0033] 02 : Upper cover
[0034] 03 : Cylindrical ridge
[0035] 04 : Tension springs
[0036] 05 : Axial hinges
[0037] 06 : Drainage protrusion 1
[0038] 07 . Drainage protrusion 2
[0039] 08 : Spiral friction ramp
[0040] 09 : Screwing threads (upper)
[0041] 10 : Cylindrical body
[0042] 11 : Screw threads (lower)
[0043] 12 : Incisors
[0044] 13 : Flat plates
[0045] 14 : Cylindrical wall
[0046] 15 : Aseptic cardboard box
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention relates to a screwed cover embodiment ( 101 ) having apparatus of foil-cutting and folding to the inner part in the aseptic liquid cardboard packages, comprising lower cover ( 01 ), cylindrical ridge ( 03 ) and flat plate ( 13 ) located on the inner part of the lower cover ( 01 ); axial hinge ( 05 ) and tension spring ( 04 ) connected to the cylindrical wall ( 14 ) in the lower cover ( 01 ); upper cover ( 02 ) and spiral friction ramp ( 08 ) located on the inner part of the upper cover ( 02 ).
[0048] When said spiral friction ramp ( 08 ) rotated in the cover-opening direction, it applies a pressure in the downward direction. Cylindrical ridge ( 03 ) located on the inner part of the lower cover ( 01 ) is exposed to this pressure.
[0049] The connection between the right and left flat plates ( 13 ) on which the cylindrical ridge ( 03 ) provided on the inner part of lower cover ( 01 ) is located and the cylindrical wall ( 14 ) inside the lower cover ( 01 ) is provided by means of the axial hinge ( 05 ). Moreover, a tension spring ( 04 ) which provides the connection of said flat plates ( 13 ) to the inner wall ( 14 ) with an asymmetrical parallel angle by means of the axial hinge ( 05 ), is used.
[0050] The cover embodiment ( 101 ) according to the present invention comprises a surface which will be attached on the surface of the cardboard package ( 15 ) forming the lower cover ( 01 ); a cylindrical body ( 10 ) to make the upper cover ( 02 ) screwed; cylindrical ridge ( 03 ) located in the middle of this cylindrical body ( 10 ) and flat plates ( 13 ) adjacent to the protrusions on both sides of this cylindrical ridge ( 03 ); incisors ( 12 ) ordered all around the lower part of the flat plates ( 13 ); axial hinges ( 05 ) connecting all these channel, plates and incisors to the cylindrical inner wall ( 14 ) from two points and tension springs ( 04 ) providing pushing and drawing force in a form inclined 90 degree located asymmetrically with different angles from different points by means of the axial hinges ( 05 ) connected to the inner wall ( 14 ) from two sides, which can rotate the embodiment 90 degree, to which these axial hinges ( 05 ) are connected; drainage protrusions ( 06 - 07 ) located at the front and back part on the cylindrical ridge ( 03 ) in the middle of the flat plates ( 3 ); screwable upper cover ( 02 ) and spiral friction ramp ( 08 ) on the inner part of this upper cover ( 02 ) embodiment; screwing threads ( 09 - 11 ) parallel to each other on the lower cover ( 01 ) and upper screwable cover, which provide this spiral friction ramp ( 08 ) to rotate. As a result of the calculations conducted, it has been found out that this embodiment provides an ease of flowing which is much more evident compared to the other embodiments while the product is flowing after the opening and homogeneous opening of at least 90% of the inner wall of the lower cover by taking the foil away from the flowing mouth.
[0051] When the upper cover ( 02 ) and the lower cover ( 01 ) are in a closed position, when the upper cover ( 02 ) is rotated in the opening direction, the spiral friction ramp ( 08 ) on the inner part of the upper cover ( 02 ) applies pressure on the lower flat plates ( 13 ) in the downward direction thanks to the spiral height of the spiral friction ramp ( 08 ) on the inner part of the upper cover ( 02 ) by rubbing onto the upper part of the cylindrical ridge ( 03 ) located in the middle of the inner wall ( 14 ) of the lower cover ( 01 ). Because of this pressure, the incisors ( 12 ) ordered all around the flat plates ( 13 ) create holes on the foil. On the foil, a hole is created in the form of the inner wall ( 14 ) of the lower cover ( 01 ), and a pressure is applied until it will be curved downwards with 30 degree. The movement of the spiral friction ramp ( 08 ) to the left and right because of the pressure applied is prevented by the drainage protrusion ( 06 ) located on the cylindrical ridge ( 03 ) and on the front part, and it is provided to progress on the same line. Following the 180-degree rotation in the opening direction, the spiral friction ramp ( 08 ) on the inner part of the upper cover ( 02 ) rests on the second drainage protrusion ( 07 ) located on the upper part of the cylindrical ridge ( 03 ) in the middle of the lower cover ( 01 ) and on the back part, pushes the flat plates ( 13 ) opening the foil in the downward direction, and enables it to be dragged until it reaches the 90-degree position. The flat plates ( 13 ) and the lower cover ( 01 ) are provided to rotate in the same axis thanks to the axial hinges ( 05 ) located onto the inner wall ( 14 ). Thanks to the tension springs ( 04 ) connected to the flat plates ( 13 ) located in the middle of of the lower cover ( 01 ) with in the inner wall ( 14 ) of the lower cover ( 01 ), the flat plates ( 13 ) which are provided to progress 90 degree in the backward direction are totally taken away from the flowing mouth, and therefore a homogeneous flowing mouth is obtained.
[0052] After the 360-degree rotation of the upper cover ( 02 ) on the cardboard package ( 15 ) is completed, the inner mechanism on the lower cover ( 01 ) is folded into the cardboard package ( 15 ) with 180 degrees, and therefore, makes the 90% of the flowing mouth open by compressing the foil between the mechanism and the cardboard package ( 15 ). Thus, both an ease of flowing is obtained, and the flowing mouth of the foil is prevented from getting closed.
[0053] The embodiment within the cover is an embodiment enabling cylindrical ridge ( 03 ) mechanism, the lower part of which is hollow, to be rotated 180 degrees.
[0054] It is illustrated with the drawings ( FIG. 1-13 ) that the operation of the mechanism is designed specifically and the items in the current embodiment constitute a whole. It is provided that the cover embodiment ( 101 ) consists of two pieces, and the inner mechanism rotates 180 degree with this configuration. Therefore the resources are used more efficiently, and the product is used in a more functional manner.
Adaptation of the Invention to the Industry
[0055] It is a cover embodiment which can be produced with the plastic injection molding method so as to be used in aseptic cardboard packages, attached onto the cardboard package with the help of an installation machine as in the other cardboard package covers, minimizing the raw material usage as its number of parts is quite low thanks to the functional characteristics of its design.
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A cover embodiment is provided to be used especially in aseptic cardboard liquid packages and to provide ease of use and it provides the flowing mouth to open in a maximum level by creating a homogeneous structure on the liquid flowing mouth thanks to the items located inside and enabling the aluminum foil part left inside the cardboard to be compressed between the package and the cover embodiment.
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CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is the U.S. National Phase of PCT Appln. No. PCT/EP2012/050202 filed Jan. 9, 2012 which claims priority to German Application No. 10 2011 002 668.1 filed Jan. 13, 2011, the disclosures of which are incorporated in their entirety by reference herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to aqueous silicone emulsions which comprise high-viscosity polyorganosiloxanes and have a particularly low content of cyclic siloxanes, to processes for preparation thereof and to the use thereof.
[0004] 2. Description of the Related Art
[0005] Silicones have a variety of uses. In order to facilitate application and metering, particularly in the case of viscous products, it is desirable for many applications that the organosilicon compounds are in dilute form. The use of organic solvents such as benzene or hydrochlorocarbons is possible for this purpose, but disadvantageous from an ecological and occupational health point of view. Therefore, use is usually effected in the form of aqueous emulsions or dispersions, typically in the form of oil-in-water emulsions (O/W emulsions) which can be diluted with water. The oil phase here is understood to mean the water-immiscible organosilicon compounds, optionally dissolved in organic solvents. For many uses, it is advantageous when the silicone has a high molecular weight and hence a high viscosity. A known way of arriving at emulsions comprising a high molecular weight silicone is the emulsion polymerization of low molecular weight, especially cyclic, organosiloxanes with arylalkylsulfonic acids (DE-A 14 95 512). In this context, through vigorous stirring or homogenization with a high-pressure homogenizer, exceptionally low particle sizes are achieved, these no longer being perceptible with an optical microscope. A disadvantage of this process is the fact that, because of the equilibrium character of this reaction, more than 10% volatile cyclic siloxanes are present based on siloxane, but are undesirable. It has therefore been proposed that these be distilled off subsequently (e.g. U.S. Pat. No. 4,600,436) or removed by a membrane process (EP-A 1 368 109). Both processes mean additional technical complexity and can impair the stability of the emulsion.
[0006] Alternatively, rather than cyclic siloxanes, linear oligomers having terminal silanol groups can be used. These oligomers, in the presence of emulsifiers, condensation catalysts and a very small amount of water, form a paste in which the polycondensation takes place. Subsequently, this paste is diluted to the desired concentration (EP 93 310 B2). In general, the proportions of cyclic volatile siloxanes are lower than in the case of emulsion polymerization of cyclic siloxanes. The proportion of these volatile siloxanes can be reduced, for example, by first producing an emulsion from the salt form of the anionic emulsifier/catalyst, then activating this emulsion by adding acid (EP-A 1 072 629). This ultimately increases the salt content in the emulsion, which is disadvantageous for stability. In the case of use of alkoxy-terminated siloxane oligomers, it is said that less cyclic siloxanes are likewise formed (JP-A2001288269). However, the production of these oligomers is more complex and hence more costly.
[0007] Specific emulsifiers based on taurocholates likewise make a contribution to the reduction in the amount of cyclic products which are formed in the emulsion condensation of siloxane oligomers (WO 2006102010). Here too, as the working examples clearly show, more than 1% octamethylcyclotetrasiloxane is formed.
[0008] There have also been suggestions of emulsifying dimethylpolysiloxanes, especially polysiloxanes terminated with trimethylsiloxy groups, having viscosities of up to 5,000,000 cSt, by mixing and heating them with 10-30% of a phosphoric partial ester based on siloxane until a clear solution has formed, which, after neutralization, is diluted with water (DE-A 27 30 923). However, this process has the disadvantage that the polydimethylsiloxane is usually depolymerized in the process, and so the emulsion obtained contains a low-viscosity siloxane and a high proportion of volatile cyclic siloxanes, e.g. octamethylcyclotetrasiloxane.
[0009] JP2002020490 proposes using, as emulsifiers, at least one two-substance combination of polyoxyethylene alkyl sulfates, polyoxyethylene alkyl phosphates and alkylsulfonates, or the corresponding acids, it being preferable that the acid is released in the emulsion only through addition of mineral acids such as sulfuric acid. Sole use of polyoxyethylene alkyl phosphates is said to lead only to low molecular weight polyorganosiloxanes, since the catalytic activity thereof is too low. Therefore, combinations with sulfates or sulfonates and activation of sulfuric acid are necessary. This ultimately leads in turn to more than 1% cyclic siloxane oligomers, unless the reaction time is extremely short, in which case, however, viscosities of >1,000,000 mm 2 /s are not achieved.
[0010] On the other hand, such emulsions are often produced practically in such a way that either several batches are produced batchwise and transferred into a maturing tank, or a continuous campaign is produced over a particular period in a maturing tank, where the reaction is stopped by neutralization after attainment of the desired viscosity. In this case, it is unavoidable that a considerable proportion of the emulsion resides in the tank for longer than required, as a result of which the proportion of cyclic oligomers exceeds the tolerable extent.
SUMMARY OF THE INVENTION
[0011] The invention provides emulsions of polyorganosiloxanes comprising
[0012] (A) polyorganosiloxanes having a viscosity greater than 10,000 mm 2 /s, measured at 25° C.,
[0013] (B) at least one emulsifier of the formula
[0000] (RO) n P(O)(OH) (3-n) (I),
[0000] in which
R may be the same or different and denotes monovalent hydrocarbyl radicals having 4 to 30 carbon atoms,
n is 1 or 2,
and/or salts thereof,
(C) at least one second emulsifier selected from the group consisting of
(C1) ethoxylated triglycerides having 40 to 400 ethylene glycol groups,
(C2) ethoxylated sorbitan esters of fatty acids having 12 to 18 carbon atoms and 10 to 40 ethylene glycol groups,
(C3) compounds of the formula
[0000] R 1 —O—(CH 2 CH 2 O) m —H (II)
[0000] and
(C4) compounds of the formula
[0000] R 2 CH 2 C(O)—O—(CH 2 CH 2 O) p —H (III),
[0000] in which
R 1 is an alkyl radical having 10 to 30 carbon atoms,
R 2 is an alkyl radical having 10 to 30 carbon atoms,
m is from 15 to 100 and
p from 15 to 100,
and
(D) water,
with the proviso that the emulsions contain less than 2% by weight of octaorganylcyclotetrasiloxane (D 4 ), based on component (A).
[0014] The invention further provides a process for producing the inventive emulsions, characterized in that (a) polyorganosiloxanes containing units of the general formula
[0000] R 4 a (R 3 O) b SiO (4-a-b)/2 (IV),
[0000] in which
R 4 may be the same or different and is a monovalent, optionally substituted hydrocarbyl radical having 1 to 30 carbon atoms or hydrogen atom,
R 3 may be the same or different and is a hydrogen atom or a monovalent, optionally substituted hydrocarbyl radical,
a is 0, 1, 2 or 3 and
b is 0, 1, 2 or 3,
with the proviso that the sum of a+b is less than or equal to 3,
and the polyorganosiloxanes contain 5 to 500 units of the formula (IV), where b is not 0 in at least one unit,
(b) emulsifier of the formula (I), wherein the OH groups may optionally be partly neutralized,
(c) emulsifier selected from the group consisting of ethoxylated triglycerides having 40 to 400 ethylene glycol groups, ethoxylated sorbitan esters of fatty acids having 12 to 18 carbon atoms and 10 to 40 ethylene glycol groups, compounds of the formula (II) and compounds of the formula (III),
(d) water and
optionally
(e) further substances
are mixed by stirring and/or homogenizing, and the organopolysiloxanes (a) containing units of the formula (IV) are allowed to condense at temperatures of 0 to 50° C. until the desired viscosity has been attained and then the emulsifier of the formula (I) is optionally neutralized with bases, such that the pH of the emulsion is greater than 5, and optionally further water (d) and/or further substances (e) are added.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The inventive emulsions can be produced by processes known to those skilled in the art.
[0016] Mixing and homogenizing tools used may be all emulsifying units known to those skilled in the art, for example high-speed stirrers, dissolver disks, rotor-stator homogenizers, ultrasound homogenizers and high-pressure homogenizers of various designs. The process according to the invention can be operated continuously, semicontinuously or batchwise.
[0017] A preferred embodiment of the process according to the invention is characterized in that
[0000] in a 1st step
(a) 100 parts by weight of polyorganosiloxanes containing units of the general formula
[0000] R 4 a (R 3 O) b SiO (r-a-b)/2 (IV),
[0000] in which
R 4 may be the same or different and is a monovalent, optionally substituted hydrocarbyl radical having 1 to 30 carbon atoms or hydrogen atom,
R 3 may be the same or different and is a hydrogen atom or a monovalent, optionally substituted hydrocarbyl radical,
a is 0, 1, 2 or 3 and
b is 0, 1, 2 or 3,
with the proviso that the sum of a+b is less than or equal to 3,
and the organopolysiloxanes contain 5 to 500 units of the formula (IV), where b is not 0 in at least one unit,
(b) 1 to 30 parts by weight of an emulsifier of the formula (I), wherein the OH groups may optionally be partly neutralized,
(c) 1 to 30 parts by weight of an emulsifier selected from the group consisting of ethoxylated triglycerides having 40 to 400 ethylene glycol groups, ethoxylated sorbitan esters of fatty acids having 12 to 18 carbon atoms and 10 to 40 ethylene glycol groups, compounds of the formula (II) and compounds of the formula (III),
(d) 1 to 50 parts by weight of water and
optionally
(e) further substances
are mixed by stirring and/or homogenizing;
in an optional 2nd step
further water (d) is added;
in a 3rd step
the organopolysiloxanes (a) containing units of the formula
[0018] (IV) are allowed to condense at temperatures of 0 to 50° C. until the desired viscosity has been attained;
[0000] in an optional 4th step
the emulsifier of the formula (I) is neutralized with bases, such that the pH of the emulsion is greater than 5; and
in an optional 5th step
the emulsion obtained in the 4th step is mixed with further water (d) and/or further substances (e).
[0019] The polyorganosiloxanes (A) present in the inventive emulsions are preferably those containing units of the formula (IV), more preferably those composed of units of the formula (IV) with an average value of a of 1.990 to 2.005 and an average value of b of 0.001 to 0.004, especially those composed of units of the formula (IV) where R 3 is a hydrogen atom, R 4 is a methyl radical and an average value of a is 1.990 to 2.005 and an average value of b is 0.001 to 0.004. Most preferably, the polyorganosiloxanes (A) are dimethylpolysiloxanes bearing trimethylsiloxy and/or dimethylhydroxysiloxy end groups.
[0020] Polyorganosiloxanes (A) present in the inventive emulsions preferably have a viscosity of greater than 100,000 mm 2 /s, more preferably greater than 1,000,000 mm 2 /s, in each case at 25° C.
[0021] Examples of R radicals are branched or unbranched alkyl radicals having 4 to 30 carbon atoms, such as butyl, hexyl, 2-ethylhexyl, octyl, isononyl, n-decyl, dodecyl, isotridecyl and n-tetradecyl radicals, unsaturated aliphatic radicals such as oleyl radicals, and aromatic radicals such as phenyl, toluyl, xylyl, nonylphenyl, naphthyl, anthracyl, tristyrylphenyl or benzyl radicals.
[0022] Preferably, the R radical comprises alkyl radicals having 4 to 18 carbon atoms, more preferably n-butyl, n-octyl, 2-ethylhexyl, n-decyl, n-dodecyl or n-tetradecyl radicals, especially n-octyl and n-decyl radicals.
[0023] Examples of compounds of the formula (I) used in accordance with the invention are di-n-butyl phosphate, di-n-hexyl phosphate, mono-n-octyl phosphate, di-n-octyl phosphate, mono-2-ethylhexyl phosphate, di-2-ethylhexyl phosphate, mono-i-nonyl phosphate, di-i-nonyl phosphate, mono-n-decyl phosphate, n-octyl n-decyl phosphate, di-n-decyl phosphate, monoisotridecyl phosphate, di-n-nonylphenyl phosphate, monooleyl phosphate and distearyl phosphate.
[0024] Preferably, the compounds of the formula (I) used in accordance with the invention are mono-n-octyl phosphate, di-n-octyl phosphate, mono-n-decyl phosphate, n-octyl-n-decyl phosphate and di-n-decyl phosphate.
[0025] Preferably, the compounds of the formula (I) used in accordance with the invention are mixtures of diesters and monoesters.
[0026] The inventive emulsions may comprise, as component (B), compounds of the formula (I) as such or salts thereof, preferably with alkali metal or alkaline earth metal hydroxides, ammonia or amines, or mixtures of acids of the formula (I) and salts thereof.
[0027] Component (B) of the inventive emulsions preferably comprises salts of the compounds of the general formula (I), especially alkali metal salts or triethanolamine salts.
[0028] The acid number of component (B) present in the inventive emulsion is determined by the number of free OH groups therein and the molar mass thereof, i.e. the amount of KOH in mg which is required for neutralization of 1 g of component (B). The acid number of component (B) is preferably in the range from 0 to 200, more preferably in the range from 0 to 20, especially 0, i.e. the inventive emulsions in this case contain, as component (B), fully neutralized compounds of the formula (I).
[0029] Compounds of the formula (I) are commercially available or preparable by commonly known chemical methods.
[0030] The inventive emulsions comprise, as a further emulsifier (C), at least one nonionic emulsifier selected from compounds of the formulae (II) and (III), ethoxylated triglycerides or ethoxylated sorbitan esters.
[0031] Examples of R 1 and R 2 radicals are each independently the n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl and the n-octadecenyl radical.
[0032] The R 1 radical preferably comprises alkyl radicals having 12 to 20 carbon atoms, more preferably linear alkyl radicals. The alkyl radical R 1 especially has an even number of carbon atoms.
[0033] The R 2 radical preferably comprises alkyl radicals having 12 to 20 carbon atoms, more preferably linear alkyl radicals. The alkyl radical R 2 especially has an even number of carbon atoms.
[0034] Preferably, m has a value of 20 to 40.
[0035] Preferably, p has a value of 20 to 50.
[0036] Examples of ethoxylated triglycerides having 40 to 400 ethylene glycol groups (C1) are ethoxylated castor oil having 200 ethylene glycol units, ethoxylated castor oil having 40 ethylene glycol units and ethoxylated hydrogenated castor oil having 200 ethylene glycol units.
[0037] Examples of ethoxylated sorbitan esters of fatty acids having 12 to 18 carbon atoms and 10 to 40 ethylene glycol groups (C2) are
[0000] polyoxyethylene(20) sorbitan stearate (Polysorbate® 60), polyoxyethylene(20) sorbitan tristearate (Polysorbate® 65), polyoxyethylene(20) sorbitan oleate (Polysorbate® 80) and polyoxyethylene(20) sorbitan laurate (Polysorbate® 20).
[0038] Examples of compounds (C3) of the formula (II) are
C 18 H 37 —O—(CH 2 CH 2 O) 20 —H,
C 18 H 35 —O—(CH 2 CH 2 O) 20 —H and
C 12 H 23 —O—(CH 2 CH 2 O) 23 —H.
[0039] Examples of compounds (C4) of the formula (III) are C 16 H 33— CH 2 —C(O)—O—(CH 2 CH 2 O) 20 —H, C 16 H 33— CH 2- C(O)—O—(CH 2 CH 2 O) 30 —H, C 16 H 33— CH 2— C(O)—O—(CH 2 CH 2 O) 40 —H and C 16 H 33— CH 2— C(O)—O—(CH 2 CH 2 O) 100 —H.
[0040] Preferably, the emulsifier (C) present in the inventive emulsion has an HLB value greater than 14, more preferably greater than 15.5, especially 16.5 to 20. The HLB value is the expression of the equilibrium between hydrophilic and hydrophobic groups of an emulsifier. The definition of the HLB value and processes for determination thereof are known to those skilled in the art and are described, for example, in Journal of Colloid and Interface Science 298 (2006) 441-450 and the literature cited therein, especially citation [23].
[0041] Preferably, emulsifier (C) is a compound of the formula (II).
[0042] In addition to components (A), (B), (C) and (D), the inventive emulsions may comprise all further substances which are typically added to silicone emulsions, for example further siloxanes different than component (a), silanes, especially alkoxysilanes, further emulsifiers different than components (b) and (c), thickeners and/or protective colloids, and also additives, for example preservatives, disinfectants, wetting agents, corrosion inhibitors, dyes and fragrances.
[0043] The inventive emulsions are preferably those which comprise component (A) to an extent of preferably 1 to 80% by weight, more preferably to an extent of 20 to 70% by weight,
[0000] component (B) to an extent of preferably 0.2 to 20% by weight, more preferably to an extent of 1 to 10% by weight, and
component (C) to an extent of preferably 0.2 to 20% by weight, more preferably to an extent of 1 to 10% by weight.
[0044] The inventive emulsions advantageously comprise only a very low proportion, if any, of cyclic siloxanes, especially of octaorganylcyclotetrasiloxanes (D 4 ). The organyl groups in the cyclosiloxanes correspond to the organyl groups in the organopolysiloxane used and are preferably methyl groups.
[0045] The inventive emulsion preferably contains preferably less than 1% by weight, more preferably less than 0.5% by weight, and especially less than 0.1% by weight, of octaorganylcyclotetrasiloxanes, especially octamethylcyclotetrasiloxane (D 4 ), based in each case on component (A).
[0046] The inventive emulsion preferably has a particle diameter of preferably 50 to 1000 nm, more preferably from 100 to 500 nm, especially from 100 to 200 nm, these figures being based on the mean of the volume distribution measured by the principle of Fraunhofer diffraction (according to ISO 13320).
[0047] The inventive emulsions have a content of nonvolatile components measured according to DIN EN ISO 3251 of preferably 1 to 80% by weight, more preferably 10 to 65% by weight, especially 30 to 60% by weight.
[0048] The pH of the inventive emulsion is preferably 5 to 10, more preferably 6 to 8, and especially about 7.
[0049] Examples of hydrocarbyl radicals R 4 are alkyl radicals such as the methyl, ethyl, n-propyl, isopropyl, 1-n-butyl, 2-n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and tert-pentyl radicals; hexyl radicals such as the n-hexyl radical; heptyl radicals such as the n-heptyl radical; octyl radicals such as the n-octyl radical and isooctyl radicals such as the 2,2,4-trimethylpentyl radical; nonyl radicals such as the n-nonyl radical; decyl radicals such as the n-decyl radical; dodecyl radicals such as the n-dodecyl radical; octadecyl radicals such as the n-octadecyl radical; cycloalkyl radicals such as the cyclopentyl, cyclohexyl, cycloheptyl radical and methylcyclohexyl radicals; alkenyl radicals such as the vinyl, 1-propenyl and 2-propenyl radical; aryl radicals such as the phenyl, naphthyl, anthryl and phenanthryl radicals; alkaryl radicals such as the o-, m-, p-tolyl radicals, xylyl radicals and ethylphenyl radicals; and aralkyl radicals such as the benzyl radical, and the α- and β-phenylethyl radicals.
[0050] Examples of substituted R 4 radicals are radicals substituted by halogen, cyano, glycidoxy, polyalkylene glycol or amino groups, for example trifluoropropyl, cyanoethyl, glycidoxypropyl, polyalkylene glycol propyl, aminopropyl or aminoethylaminopropyl radicals.
[0051] Preferably, not more than one R 4 radical in the units of the formula (IV) is a hydrogen atom.
[0052] Preferably, the R 4 radical comprises hydrocarbyl radicals having 1 to 18 carbon atoms, more preferably the methyl or phenyl radicals, and especially more than 80 mol % of the R 4 radicals in the siloxane (a) are methyl radicals.
[0053] Examples of R 3 radicals are the examples given for R 4 radicals. Preferably, the R 3 radical is a hydrogen atom or a hydrocarbyl radical having 1 to 4 carbon atoms, more preferably a hydrogen atom.
[0054] In formula (IV), the sum of a+b preferably has a value of on average 1.5 to 2.4, more preferably on average 1.8 to 2.3, and especially 1.9 to 2.1.
[0055] The siloxanes (a) used in the process of the invention consist preferably of 5 to 500, more preferably 10 to 200, and especially 20 to 100 units of the formula (IV).
[0056] In preferably 0.4 to 40%, more preferably 2 to 10%, of the units of the formula (IV) in the siloxanes (a) used in the process according to the invention, b is not 0.
[0057] Examples of siloxanes (a) used in accordance with the invention are polydiorganosiloxanes terminated with alkoxy or hydroxyl groups, especially polydiethyl- and polydimethylsiloxanes.
[0058] The siloxanes (a) used in the process according to the invention preferably have a viscosity of 5 to 10 000 mm 2 /s, more preferably 10 to 500 mm 2 /s, and especially 30 to 100 mm 2 /s, in each case at 25° C.
[0059] Preferably, the siloxanes (a) are those of the formula
[0000] HO[SiR 4 2 O] c —H (V)
[0000] where R 4 has one of the abovementioned meanings, especially a methyl radical, and c has a value of 5 to 500, preferably 10 to 200, and more preferably from 20 to 100.
[0060] The polysiloxanes (a) containing units of the formula (IV) are commercial products or can be prepared by known processes.
[0061] Examples of component (b) are the abovementioned examples for the compounds of the formula (I), optionally in a mixture with salts thereof.
[0062] The acid number of the compound of the formula (I) used in the process according to the invention is determined by the average value of n and the molar mass thereof, i.e. the amount of KOH in mg which is required for neutralization of 1 g of compound of the formula (I). The acid number of the compound of the formula (I) used in accordance with the invention is preferably in the range from 100 to 600, more preferably in the range from 200 to 500, and especially in the range from 250 to 450.
[0063] Component (b) is preferably used in the process according to the invention in amounts of 1 to 25 parts by weight, especially 2 to 10 parts by weight, based in each case on 100 parts by weight of polyorganosiloxane (a).
[0064] Examples of component (c) are the abovementioned examples for the compounds (C).
[0065] Component (c) is preferably used in the process according to the invention in amounts of 1 to 25 parts by weight, especially 5 to 20 parts by weight, based in each case on 100 parts by weight of polyorganosiloxane (a).
[0066] The water (d) may be any of water which is useful for production of dispersions.
[0067] The water (d) is preferably partly or fully demineralized water, distilled or (repeatedly) redistilled water, water for medical or pharmaceutical purposes, for example purified water (aqua purificata according to Pharm. Eur.).
[0068] The water (d) used in accordance with the invention preferably has a conductivity of less than 50 μS/cm, more preferably less than 10 μS/cm, and especially less than 1.3 μS/cm, in each case at 25° C. and 1010 hPa.
[0069] Water (d) is preferably used in the process according to the invention in amounts of 20 to 1000 parts by weight, especially 30 to 400 parts by weight, based in each case on 100 parts by weight of polyorganosiloxane (a). In the preferred process variant, water is added in the first step, in the optional second step and in the optional fifth step, preferably using water in the first step of the process in amounts of 1 to 30 parts by weight, especially 5 to 20 parts by weight, based in each case on 100 parts by weight of polyorganosiloxane (a).
[0070] In addition to components (a), (b), (c) and (d), all further substances (e) which are typically added to silicone emulsions can be used in the process according to the invention, for example further siloxanes different than component (a), silanes, especially alkoxysilanes, further emulsifiers different than components (b) and (c), thickeners and/or protective colloids, and also additives, for example preservatives, disinfectants, wetting agents, corrosion inhibitors, dyes and fragrances. The addition of these components (e) can be effected in the first step of the process according to the invention and/or else in a later process step, for example after the 5th step.
[0071] Examples of further siloxanes (e) which can be used in accordance with the invention are those of the formula (IV) where b is 0, for example trimethylsiloxy-terminated polydimethylsiloxanes. Such siloxanes (e) are advantageously used to control the viscosity of the polysiloxane obtained after the condensation reaction in the emulsion.
[0072] If further siloxanes (e) are used, the amounts are preferably 0.01 to 10 parts by weight, based on 100 parts by weight of component (a). In the process according to the invention, preferably no further siloxanes (e) are used.
[0073] Examples of silanes (e) which can be used in accordance with the invention are methyltrimethoxysilane, tetraethoxysilane, vinyltriethoxysilane, morpholinomethyltriethoxysilane, cyclohexylaminomethylmethyldiethoxysilane, the examples cited in DE-A 102005022099, paragraph [0052] and in DE-A 102004038148, paragraph [0033], or the hydrolysis/condensation products thereof. Such silanes (e) are advantageously used to obtain branched or crosslinked siloxanes, for example those which form elastic films after the drying of the emulsion. In the preferred process variant, these silanes (e) can be added in the 1st step, or else after the 3rd step.
[0074] If silanes (e) are used, the amounts are preferably 0.01 to 10 parts by weight, based on 100 parts by weight component (a). In the process according to the invention, preferably no silanes (e) are used.
[0075] Examples of further emulsifiers (e) which can be used in accordance with the invention are all emulsifiers known to date, such as anionic or nonionic emulsifiers, for example alkyl sulfates, ethoxylated alkyl sulfates, and polyethylene glycol ethers of alkylphenols and alkyl polyglycosides.
[0076] In the process according to the invention, preferably no cationic and no amphoteric emulsifiers are used.
[0077] In the process according to the invention, preferably no further anionic emulsifiers, more particularly no alkyl- or alkylarylbenzenesulfonic acids or salts thereof, are used as component (e).
[0078] If further emulsifiers (e) are used, the amounts are preferably 1 to 20 parts by weight, based on 100 parts by weight component (a).
[0079] Preferably, no further emulsifiers are used as component (e).
[0080] If thickeners or protective colloids are used as component (e) in the process according to the invention, these are preferably acrylic acid copolymers.
[0081] If thickeners and/or protective colloids (e) are used, the amounts are preferably 0.01 to 2 parts by weight, based on 100 parts by weight component (a). In the process according to the invention, preferably no thickener and/or protective colloid (e) is used.
[0082] Examples of additives (e) which can be used in accordance with the invention are, for example, preservatives, dyes or fragrances known to those skilled in the art, especially preservatives such as methylisothiazolinone, chloromethylisothiazolinone, benzylisothiazolinone, phenoxyethanol, methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, alkali metal benzoates, alkali metal sorbates, iodopropinyl butylcarbamate, benzyl alcohol and 2-bromo-2-nitropropane-1,3-diol.
[0083] If additives (e) are used, the amounts are preferably 0.0005 to 2 parts by weight, based on 100 parts by weight of component (a). In the process according to the invention, preference is given to using additives (e).
[0084] In the first step of the preferred embodiment of the process according to the invention, all components can be mixed with one another by stirring and/or homogenizing, for example in any sequence, the peripheral speed of the stirrer and/or rotor-stator homogenizer preferably being greater than 5 m/s, more preferably greater than 10 m/s, and especially 5 to 50 m/s.
[0085] Compounds of the formula (I) as component (b) can, if desired, be partly neutralized as early as in the first step of the process according to the invention with bases, for example alkali metal hydroxides or amines, but this is not preferred.
[0086] The mixture according to a first step of the process according to the invention has a pH of less than 6, preferably less than 5, more preferably less than 4, and especially 1 to 3.
[0087] Preferably, the emulsion composed of components (a), (b), (c), (d) and optionally (e) obtained in the first step is highly viscous and not free-flowing. It is especially preferable when the yield point (according to DIN 53019-1 and cited standards) of the emulsion obtained in the first step is greater than 100 Pa, especially greater than 1000 Pa.
[0088] The first step of the process according to the invention is preferably performed at temperatures of preferably 5 to 80° C., especially 10 to 50° C., and at the pressure of the surrounding atmosphere, i.e. between 900 and 1100 hPa, or at an elevated pressure of up to 20,000 hPa, especially up to 10,000 hPa.
[0089] Preferably, the duration of the first step of the invention is less than 4 hours, more preferably less than 2 hours, and especially 5 to 60 minutes.
[0090] The mixture obtained in the first step of the process according to the invention preferably has a particle size (mean of the volume distribution) of less than 1 pm, more preferably 100 to 500 nm, and especially 100 to 200 nm.
[0091] In the optional second step of the process according to the invention, the emulsion obtained in the first step, especially when it is highly viscous to firm, is diluted with water with stirring and/or homogenization, so as to form a free-flowing emulsion which preferably contains more than 50 parts water per 100 parts component (a).
[0092] The stirring or homogenizing can be effected under the same conditions as described for the first step.
[0093] The second step of the process according to the invention is preferably performed at temperatures of preferably 5 to 50° C., especially 10 to 30° C., and at the pressure of the surrounding atmosphere, i.e. between 900 and 1100 hPa, or at an elevated pressure of up to 20,000 hPa, especially of up to 10,000 hPa.
[0094] The second step can be effected in the same vessel as the first process step.
[0095] Preferably, the duration of the second step in accordance with the invention, which is optional, is less than 4 hours, more preferably less than 2 hours, and especially 5 to 60 minutes.
[0096] In the process according to the invention, the second step is preferably performed.
[0097] In the third step of the process according to the invention, the organopolysiloxanes (a) are allowed to condense until the viscosity as desired for siloxane (A) in the inventive emulsion has been attained, i.e. a viscosity of greater than 10,000 mm 2 /s, preferably greater than 100,000 mm 2 /s, more preferably greater than 1,000,000 mm 2 /s, in each case at 25° C.
[0098] Preferably, the duration of the third step of the invention is 1 to 200 hours, more preferably 8 to 96 hours, especially 12 to 72 hours. The third step can be effected in the same vessel as the first and second steps. However, the emulsion can also be transferred to a special vessel where, if appropriate, several batches produced in succession are mixed for the third step. However, it is also possible to perform the first and second steps continuously and the third step in a maturing tank.
[0099] The third step of the process according to the invention is preferably performed at temperatures of 2 to 50° C., more preferably 5 to 30° C., especially at 5 to 20° C., and a pressure of the surrounding atmosphere, i.e. between 900 and 1100 hPa.
[0100] Any alcohols obtained as condensation by-products in the process according to the invention, for example when R 3 in formula (IV) is not a hydrogen atom, may remain in the emulsion or else be removed, for example by distillation under reduced pressure or by extraction.
[0101] Examples of the bases used in the optional fourth step of the process according to the invention are alkali metal hydroxides such as NaOH and KOH, and amines, for example monoethanolamine and triethanolamine. The pH can in principle also be adjusted by the addition of alkali metal salts of weak acids, for example sodium citrate, sodium silicate, potassium acetate or potassium phosphate.
[0102] Preferably, the bases which can be used in the fourth step of the process according to the invention are alkali metal or alkaline earth metal hydroxides, ammonia and amines, more preferably NaOH, KOH, monoethanolamine and triethanolamine.
[0103] The pH of the emulsion after the inventive neutralization is preferably 5 to 10, more preferably 6 to 8, and especially about 7.
[0104] The optional fourth step of the process according to the invention is preferably performed at temperatures of 5 to 50° C., more preferably 15 to 30° C., and a pressure of the surrounding atmosphere, i.e. between 900 and 1100 hPa.
[0105] In the process according to the invention, the fourth step is preferably performed.
[0106] The emulsions obtained in accordance with the invention can then be mixed in an optional 5th step with further water (d) and/or further substances (e) as desired.
[0107] Preferably, no further components are used in addition to components (a), (b), (c), (d) and optionally (e) and bases in the process according to the invention.
[0108] The components used in the process according to the invention may each be one kind of such a component, or else a mixture of at least two kinds of a particular component.
[0109] The inventive emulsions, or those produced in accordance with the invention, have the advantage that they comprise high-viscosity polydiorganosiloxanes and have a low content of cyclic siloxanes.
[0110] In addition, the inventive emulsions, or those produced in accordance with the invention, have the advantage that they are very stable and thus have a long shelf life.
[0111] The inventive emulsions, or those produced in accordance with the invention, have the advantage that they are storage-stable and have excellent performance properties, for example very good action as separating agents and lubricants, good wetting capacity in different substrates, good conditioning action in haircare products, i.e. distinct reduction in wet and dry combing force.
[0112] The process according to the invention has the advantage that it is possible to produce emulsions comprising high molecular weight siloxanes in a simple and inexpensive manner.
[0113] The process according to the invention also has the advantage that, even after a relatively long duration of the third step, the proportion of cyclic siloxanes remains low, which is particularly favorable, for example, in the case of continuous production with a relatively broad residence time range.
[0114] The process according to the invention has the advantage that the viscosity of the oil can be varied and adjusted in a simple manner within a wide range without forming an elevated proportion of cyclic siloxanes.
[0115] The inventive emulsions, or those produced in accordance with the invention are usable for all purposes for which emulsions comprising high-viscosity siloxanes are useful, for example as separating agents, lubricants, hydrophobizing agents, and for textile impregnation, in the processing of rubber and plastics or in metalworking, hydrophobizing agents for glass and mineral building materials, or as a constituent of personal care products.
[0116] In the case of use as a lubricant for sewing threads, the inventive emulsions can be combined, for example, with wax emulsions. The inventive emulsions can be used to produce separating agent formulations, for example for the tire industry, these comprising, as well as the inventive emulsion, further components such as thickeners, for example xantham gum or polyacrylates, fillers such as talc or mica, waxes and further components known to those skilled in the art. The high viscosity and the low content of volatile siloxanes is particularly advantageous in these applications.
[0117] The invention further provides personal care compositions comprising inventive emulsions in amounts of 0.05 to 10% by weight, more preferably 0.5 to 5% by weight.
[0118] The inventive personal care compositions are preferably haircare compositions.
[0119] These haircare compositions comprise, as well as the inventive emulsions, or those produced in accordance with the invention, preferably one or more conditioners selected, for example, from the group of quaternary ammonium compounds, natural or synthetic waxes, vegetable oils, mineral oils, fluorinated oils, silicone oils, especially aminosilicone oils, organic polymers and copolymers, which may be nonionic, anionic, cationic or amphoteric, cationic proteins and cationic surfactants.
[0120] Further constituents of these haircare compositions are, for example, water, surfactants, fatty alcohols, rheology modifiers, pearlizers, organic acids, fragrances, preservatives, vitamins, sunscreens, salts, dyes, and further components of haircare compositions known to those skilled in the art.
[0121] The haircare compositions comprising the inventive emulsions, or those produced in accordance with the invention, may, for example, be shampoos, hair masks, hair rinses, hair waxes, hair creams, hair gels, hair foams, hairsprays and hair colorants. These care compositions improve both the dry and wet combability, and also the feel of the wet and dry hair.
[0122] Application can be effected, for example, in the course of washing, after washing, as a pre- or aftertreatment in the course of bleaching or in the course of coloring with direct or oxidation dyes, and in the course of permanent shaping of the hair (e.g. permanent wave).
[0123] The invention further provides haircare compositions comprising inventive emulsions and at least one conditioner.
[0124] In the examples which follow, all figures for parts and percentages, unless stated otherwise, are based on weight.
[0125] Unless stated otherwise, the examples which follow are performed at a pressure of the surrounding atmosphere, i.e. at about 1010 hPa, and at room temperature, i.e. about 25° C., or a temperature which is established on combination of the reactants at room temperature without additional heating or cooling. All viscosity figures given in the examples are based on a temperature of 25° C.
[0126] The emulsions produced in the examples which follow were tested as follows:
[0127] The particle size was determined with a Malvern Zetasizer ZEN1600/Nano-S particle size analyzer, Software Version 6.01, by means of dynamic light scattering. For this purpose, the emulsions were diluted to 0.5% with filtered and degassed water. The values reported are always based on the D(50) value.
[0128] To determine the oil viscosity, 20 g of emulsion were admixed with 30 g of acetone, and the emulsion separated. The acetone/water phase was removed and the operation was repeated once more. Subsequently, the polymer was washed three times with water and dried at 110° C. while stirring until no water droplets were visible any longer, and then aftertreated at 110° C. in a drying cabinet for another 8 h. The viscosity was determined with an MCR 300 cone-plate viscometer (Paar-Physika) at 25° C. and a shear gradient of 1/s.
[0129] To determine the content of octamethylcyclotetrasiloxane (D 4 ), a 29 Si NMR spectrum of the emulsion was recorded (Bruker Avance 400, 10 mm selective 29 Si NMR sample head, addition of 15% D 2 O to the original emulsion, pulse angle 30°, wait time 30 s, 400 scans).
[0000] The integrals of the signals between −19.75 and −20 ppm (D 4 ) and −21.5 to −23.25 (remaining D units) were used to determine the D 4 content in mol % of Si, and this, because of the equal molar mass of the individual siloxane unit (74 g/mol), is virtually equal to the proportion of D 4 in % by weight based on polydimethylsiloxanes.
EXAMPLE 1
[0130] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 10 parts of a 2-ethylhexyl phosphate having an acid number of 295 mg KOH/g (obtainable under the “Servoxyl VPTZ 100” name from Elementis Specialties Netherlands B.V. Delden), 14 parts of an ethoxylated stearyl alcohol of the formula C 18 H 37 —O—(CH 2 CH 2 O) 20 —H obtainable under the “Arlypon SA 20” name from Cognis AG, Düsseldorf) and 10 parts of water are added and homogenized for 10 min. The gel-like phase formed (yield point 920 Pa) having a particle size of less than 250 nm is diluted with 100 parts of water within 10 min and stored at 10° C. This emulsion had a pH of 2.6. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine, and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) is added.
[0131] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 2
[0132] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 10 parts of an isononyl phosphate having an acid number of 300 mg KOH/g (obtainable under the “Servoxyl VPXZ 100” name from Elementis Specialties Netherlands B.V. Delden), 10 parts of an ethoxylated lauryl alcohol of the formula C 12 H 23 —O—(CH 2 CH 2 O) 23 —H (obtainable under the “Brij 35” name from Croda GmbH, D-Nettetal) and 10 parts of water are homogenized for 5 min. The gel-like phase formed having a yield point of 1730 Pa and a particle size of less than 200 nm is diluted with 100 parts of water within 10 min and stored at 15° C. This emulsion had a pH of 2.3. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) is added.
[0133] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 3
[0134] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 10 parts of a 2-ethylhexyl phosphate having an acid number of 295 mg KOH/g (obtainable under the “Servoxyl VPTZ 100” name from Elementis Specialties Netherlands B.V. Delden), 10 parts of an ethoxylated lauryl alcohol of the formula C 12 H 23 —O—(CH 2 CH 2 O) 23 —H (obtainable under the “Brij 35” name from Croda GmbH, D-Nettetal) and 10 parts of water are added and homogenized for 10 min. The gel-like phase formed (yield point 1340 Pa) having a particle size of less than 200 nm is diluted with 100 parts of water within 10 min and stored at 10° C. This emulsion had a pH of 2.2. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical
[0135] Industries Ltd., CH-9471 Buchs/SG) is added.
[0136] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 4
[0137] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 6 parts of a 2-ethylhexyl phosphate having an acid number of 225 mg KOH/g (obtainable under the “Servoxyl VPDZ 100” name from Elementis Specialties Netherlands B.V. Delden), 10 parts of an ethoxylated lauryl alcohol of the formula C 12 H 23 —O—(CH 2 CH 2 O) 23 —H (obtainable under the “Brij 35” name from
[0138] Croda GmbH, D-Nettetal) and 10 parts of water are added and homogenized for 15 min. The gel-like phase formed (yield point 990 Pa) having a particle size of less than 200 nm is diluted with 100 parts of water within 10 min and stored at 10° C. This emulsion had a pH of 2.5. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) is added.
[0139] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 5
[0140] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 4 parts of a n-butyl phosphate having an acid number of 465 mg KOH/g (obtainable under the “Servoxyl VPIZ 100” name from Elementis Specialties Netherlands B.V. Delden), 10 parts of an ethoxylated stearic acid of the formula C 16 H 33 CH 2 C(O)—O—(CH 2 CH 2 O) 40 —H (obtainable under the “Myrj 52S” name from Croda GmbH, D-Nettetal) and 10 parts of water are added and homogenized for 15 min. The gel-like phase formed (yield point 720 Pa) having a particle size of less than 200 nm is diluted with 100 parts of water within 10 min and stored at 10° C. This emulsion had a pH of 1.8. After 96 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) is added.
[0141] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 6
[0142] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 10 parts of an octyl decyl phosphate having an acid number of 295 mg K0H/g (obtainable under the “Crodafos 810 A” name from Croda GmbH, D-Nettetal), 14 parts of an ethoxylated lauryl alcohol of the formula C 12 H 23 —O—(CH 2 CH 2 O) 23 —H (obtainable under the “Brij 35” name from Croda GmbH, D-Nettetal) and 10 parts of water are added and homogenized for 10 min. The gel-like phase formed (yield point 1940 Pa) having a particle size of less than 200 nm is diluted with 100 parts of water within 10 min and stored at 10° C. This emulsion had a pH of 2.0. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) is added.
[0143] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 7
[0144] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 6 parts of an octyl decyl phosphate having an acid number of 295 mg KOH/g (obtainable under the “Crodafos 810 A” name from Croda GmbH, D-Nettetal), 10 parts of an ethoxylated lauryl alcohol of the formula C 12 H 23 O—(CH 2 CH 2 O) 23 —H (obtainable under the “Brij 35” name from Croda GmbH, D-Nettetal) and 10 parts of water are added and homogenized for 10 min. The gel-like phase formed (yield point 1110 Pa) having a particle size of less than 200 nm is diluted with 100 parts of water within 10 min and stored at 15° C. This emulsion had a pH of 2.1. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) is added.
[0145] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 8
[0146] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 6 parts of an octyl decyl phosphate having an acid number of 295 mg KOH/g (obtainable under the “Crodafos 810 A” name from Croda GmbH, D-Nettetal), 10 parts of an ethoxylated sorbitan laurate (obtainable under the “Tween 20” name from
[0147] Croda GmbH, D-Nettetal) and 10 parts of water are added and homogenized for 10 min. The gel-like phase formed (yield point 820 Pa) having a particle size of less than 300 nm is diluted with 100 parts of water within 10 min and stored at 15° C. This emulsion had a pH of 2.0. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.18 part of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) is added.
[0148] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 9
[0149] 100 parts of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a beaker. With a rotor-stator homogenizer (Ultra-Turrax, peripheral speed 16 m/s), 6 parts of an octyl decyl phosphate having an acid number of 295 mg KOH/g (obtainable under the “Crodafos 810 A” name from Croda GmbH, D-Nettetal), 10 parts of an ethoxylated castor oil (obtainable under the “Atlas G1300” name from Croda GmbH, D-Nettetal) and 10 parts of water are added and homogenized for 10 min. The gel-like phase formed (yield point 900 Pa) having a particle size of less than 200 nm is diluted with 100 parts of water within 10 min and stored at 15° C. This emulsion had a pH of 2.1. After 24 h, the emulsion is adjusted to a pH of 7 with triethanolamine and 0.24 part of preservative based on methylisothiazolinones and ethylhexylglycerol (obtainable under the “Euxyl K220” name from Schülke & Mayr GmbH, Norderstedt) is added.
[0150] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
EXAMPLE 10
[0151] 950 kg of an α,ω-hydroxyl-terminated polydimethylsiloxane having a viscosity of 60 mPas are initially charged in a mixing agitator having a capacity of 2000 l (Becomix RW 2000). The homogenizer is switched on and set to a peripheral speed of 24 m/s. 60 kg of an octyl-decyl phosphate having an acid number of 295 mg KOH/g (obtainable under the “Crodafos 810 A” name from Croda GmbH, D-Nettetal), 100 kg of an ethoxylated lauryl alcohol of the formula C 12 H 23 —O—(CH 2 CH 2 O) 23 —H (obtainable under the “Brij 35” name from Croda GmbH, D-Nettetal) and 100 kg of water are added and homogenized for 15 min. A firm gel-like phase was formed, which had a yield point of 1050 Pa. This phase was homogenized for a further 45 min until a particle size of less than 500 nm had been attained. Subsequently, the emulsion was diluted with 900 kg of water within 10 min and stored at 15° C. This emulsion had a pH of 1.3. After 48 h, the emulsion is adjusted to a pH of 7 with triethanolamine. Subsequently, 1.8 kg of preservative based on isothiazolinones (obtainable under the “Kathon CG” name from Acima Chemical Industries Ltd., CH-9471 Buchs/SG) were added.
[0152] The emulsion thus obtained is then analyzed for particle size, oil viscosity and the content of octamethylcyclotetrasiloxane D 4 . The results can be found in Table 1.
[0000]
TABLE 1
Particle size
Oil viscosity
D 4 in % by
Example
D(50) in nm
in Pas
weight
1
205
1790
<0.05
2
153
762
0.05
3
143
1430
0.1
4
175
1050
0.05
5
162
1630
0.15
6
116
1220
0.05
7
154
2630
0.05
8
214
1730
0.1
9
174
1420
0.05
10
156
1560
0.08
[0153] All inventive emulsions were very stable; they did not exhibit deposition either in the course of centrifuging (1 h at 2500 g) or in the course of storage over 6 months. The particle size was also unchanged after 28 d at 50° C.
EXAMPLE 11
[0154] A shampoo 19:3 is formulated as follows, the individual components being designated according to INCI nomenclature: 0.2 part of guar hydroxypropyltrimonium chloride (obtainable under the N-Hance® 3000 name from Hercules Inc.) is dispersed in 11.98 parts of water. 71.7 parts of sodium laureth sulfate (obtainable under the Genapol® LRO 26.5% name from Clariant GmbH) are stirred in gradually and the mixture is heated to 75° C. In the course of this, 0.3 part of PEG-150 distearate (obtainable under the Emulgin® EO 33 name from Cognis Deutschland GmbH) is added on attainment of 50° C. and, when 65° C. has been attained, 1.2 parts of glycol distearate (obtainable under the Genapol® PMS name from Clariant GmbH). The mixture is mixed until 75° C. has been attained. Then the mixture is cooled. When 35° C. has been attained, 0.06 part of methylchloroisothiazolinone/methylisothiazolinone preservative (obtainable under the Kathon™ CG name from Rohm & Haas Company, Inc.) and 4 parts of the emulsion of example 6 are added and the mixture is stirred for 5 minutes. Finally, 10.06 parts of cocamidopropyl betaine (obtainable under the Genagen® CAB 30% name from Clariant GmbH) and 0.5 part of sodium chloride 25% are added and the mixture is stirred for 10 minutes in each case.
[0155] The shampoo thus obtained improves both wet and dry combability, and also the feel of the wet and dry hair.
EXAMPLE 12
[0156] A shampoo 11:4 is formulated as follows, the individual components being designated according to INCI nomenclature: 0.1 parts of Polyquaternium-10 (obtainable under the Ucare™ Polymer JR-400 name from Amerchol Corporation) are dispersed in 39.04 parts of water. 41.5 parts of sodium laureth sulfate (obtainable under the Genapol® LRO 26.5% name from Clariant GmbH) are stirred in gradually and the mixture is heated to 75° C. In the course of this, 0.2 part of hydroxyethyl cellulose (obtainable under the Tylose® H 4000 P2 name from Shin-Etsu Chemical Co.) is added on attainment of 50° C. and, when 65° C. has been attained, 1.2 parts of glycol distearate (obtainable under the Genapol® PMS name from Clariant GmbH). The mixture is mixed until 75° C. has been attained. Then the mixture is cooled. When 35° C. has been attained, 0.06 part of methylchloroisothiazolinone/methylisothiazolinone preservative (obtainable under the Kathon™ CG name from Rohm & Haas Company, Inc.) and 4 parts of the emulsion of example 6 are added and the mixture is stirred for 5 minutes. Finally, 13.4 parts of cocamidopropyl betaine (obtainable under the Genagen® CAB 30% name from Clariant GmbH) and 0.5 part of sodium chloride 25% are added and the mixture is stirred for 10 minutes in each case.
[0157] The shampoo thus obtained improves both dry and wet combability, and also the feel of wet and dry hair.
EXAMPLE 13
[0158] A conditioner is formulated as follows, the individual components being designated according to INCI nomenclature: 87.04 parts of water are initially charged and heated to 75° C. while stirring. In the course of this, 1.2 parts of hydroxyethyl cellulose (obtainable under the Tylose® H 4000 P2 name from Shin-Etsu Chemical Co.) are added. When 65° C. has been attained, 0.5 part of stearamidopropyl dimethylamine (obtainable under the Incromine™ SB name from Croda GmbH), 1 part of Polysorbate 80 (obtainable under the Tween™ 80 name from Croda GmbH), 3 parts of stearyl alcohol (obtainable under the stearyl alcohol name from Merck-Schuchardt), 1 part of cetyl alcohol (obtainable under the cetyl alcohol name from Merck KGaA) and 1.76 parts of behentrimonium chloride (obtainable under the Genamin® KDMP name from Clariant GmbH) are added. The mixture is mixed until 75° C. has been attained. Then the mixture is cooled. During the cooling, 0.2 part of citric acid (obtainable under the citric acid name from Sigma) and 0.2 part of tetrasodium EDTA (obtainable under the EDETA® B powder name from BASF Corporation) are added. When 35° C. has been attained, 0.1 part of methylchloroisothiazolinone/methylisothiazolinone preservative (obtainable under the Kathon™ CG name from Rohm & Haas Company, Inc.) and 4 parts of the emulsion of example 6 are added and the mixture is stirred for 5 minutes. Finally, the mixture is homogenized using the Turrax for 1 minute.
[0159] The conditioner thus obtained improves both wet and dry combability, and also the feel of dry and wet hair.
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Aqueous emulsions of high viscosity organopolysiloxanes can be prepared having a very low content of cyclic siloxanes by condensing low molecular weight alkoxy-functional organopolysiloxanes in aqueous emulsion with an alkylphosphate emulsifier and at least one selected polyoxyethylene non-ionic surfactant.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to a vise for clamping a workpiece and more particularly to a vise capable of clamping a workpiece having an angularly disposed member wherein the angled portion of the workpiece is clamped between a curvilinear surface and a pivotable surface.
2. Description of the Related Art
There are a variety of clamping devices available in the art for clamping a workpiece during machining or other manufacturing operations. Most of these devices secure the workpiece by clamping it between two or more jaws which are capable of movement relative to one another. The jaws may have fixed or moveable bearing surfaces which contact the workpiece. Where moveable bearing surfaces are utilized, they are typically designed to pivot in some fashion to conform to irregularly shaped workpieces.
Angled extrusions, such as those frequently seen in aircraft structural parts, present a particularly difficult clamping task to the machinist. If the base of an angled extrusion is to be machined, the angled portion of the extrusion must be clamped. For accurate machining, the base of the angled extrusion must remain parallel to and flush with the sacrificial material that is typically inserted between the vise jaws and the angled extrusion base. Ordinary vises tend to impart clamping forces at different locations on the angled portion of the angled extrusion, thereby creating a net moment which will tend to move some portion of the base out of parallel with the sacrificial material. Angled extrusions also typically require the machinist to manually adjust the vise jaws to approximately conform to the angle of the angled portion of the extrusion. This is a tedious time consuming process which, depending upon the size of the workpiece and vises involved, may involve a blind operation since the machinist may not be able to visually verify that the jaws have closely matched the angle of the extrusion. Even if the operator carefully manually positions the vise jaws to secure the angled extrusion in a vise which has pivoting bearing surfaces, the pivoting bearing surfaces may have a tendency to rotate during machining which will cause misalignment of the workpiece.
SUMMARY OF THE INVENTION
The present invention includes a new device for quickly and accurately clamping a workpiece, such as, for example, an angled extrusion. The invention is adapted to rapidly engage and automatically adjust to workpieces of various angularities without the need for lengthy manual adjustments. In a preferred embodiment, the invention includes a fixed jaw which has an external radius for contacting one surface of the workpiece, a second moveable jaw which is capable of movement relative to the fixed jaw, and which has a generally cylindrical recess, and a semi-cylindrical jaw insert which is pivotally mounted in the recess in the moveable jaw. The jaw insert may be mounted to freely pivot in the recess or it may be spring biased so that it returns to a set position when a workpiece is removed. The radius of the cylindrical portion of the jaw insert should be greater than the external radius on the fixed jaw to minimize the moment induced when severely angled workpieces are clampled. The radius of the cylindrical portion of the jaw insert should be slightly larger than the radius of the cylindrical recess in the moveable jaw such that when the jaw insert engages a workpiece under load, the cylindrical surface of the jaw insert will form a press fit with the cylindrical recess on the movable jaw, thereby locking the jaw insert, and thus the workpiece, into position.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the self-indexing vise, illustrated in section.
FIG. 2 depicts the self-indexing vise, illustrated in section with an angled workpiece in position.
FIG. 3 depicts a magnified view of the contacting portion of the self-indexing vise, illustrated in section.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to FIG. 1, self-indexing vise 10 includes a substantially rectangular base 12 which is generally configured to set on top of a machine tool. Base 12 may be of variable geometry depending upon the particular router or mill or other machine tool upon which it is placed.
A fixed jaw post 14 extends up from and is coupled to base 12. Post 14 includes bore 16 adapted to receive bolt 18.
Fixed jaw 20 rests upon spacer 22 which is placed between fixed jaw 20 and post 14. Spacer 22 is sized and designed to place fixed jaw 20 a certain height above base 12 and may be obviated depending upon the thickness of fixed jaw 20. Fixed jaw 20 and spacer 22 are fixed to post 14 by bolt 18. The number and spacing of bolts 18 will depend on the length of vise 10.
Fixed jaw 20 has at one terminus a full external radius 24. In FIG. 1, external radius 24 is shown sized smaller than the thickness of fixed jaw 20. However, the dimension of external radius may coincide with the thickness of fixed jaw 20, depending upon the thickness of fixed jaw 20.
A moving jaw post 26 is fixedly attached to and emanates up from base 12. Post 26 includes a bore 28 which is adapted to receive bolt 30. Moveable jaw 32 slidably rests on moveable jaw post 26. Alignment of moveable jaw 32 when moveable jaw 32 is translated, it is partially maintained by bolt 30 which is preferably a shoulder bolt. Moveable jaw 32 is prevented from upward translation during horizontal translation by bolt 30 which bears upon shoulders 36 and 38 of counterbore 40. Moveable jaw 32 is also fixedly attached to piston block 42. Piston block 42 includes a bore 44 adapted to receive bolt 46 which fixes moveable jaw 32 to piston block 42. The number and spacing of bolts 30, 46 will depend on the length of vise 10. Moveable jaw 32 may be alternatively fixed to piston block 42 by other fastening methods such as welding or riveting, etc.
Referring now also to FIG. 2 which shows vise 10 engaging a workpiece 47, moveable jaw 32 is translated horizontally by piston rod 48 which is attached to piston block 42 and cylinder 50. Cylinder 50 may be either hydraulically or pneumatically operated, or alternatively actuated by some other mechanical mechanism such as a rack and pinion gear system.
Moveable jaw 32 also includes a recess 52 which is positioned in opposition to the external radius 24 on fixed jaw 20. Recess 52 extends longitudinally along the entire length of moveable jaw 32. Recess is adapted to pivotally receive pivoting member 54.
Referring now also to FIG. 3, pivoting member 54 includes a substantially flat bearing surface 56 and a semi-cylindrical surface 58. Pivoting member 54 should be fabricated such that the center 60 of semi-cylindrical surface 58 of pivoting member 54 lies as close as possible to bearing surface 56. This will reduce the distance between the point where workpiece 47 contacts the external radius 24 of fixed jaw 20 and the center 60 of semi-cylindrical surface 58, and thereby reduce the moment induced by the force imparted by moveable jaw 32 and the opposing force exerted by fixed jaw 20. Pivoting member 54 should also be fabricated such that the radius of semi-cylindrical surface 58 is slightly larger than the radius of recess 52 in moveable jaw 32. The difference in radii will facilitate the locking feature to be discussed more fully below.
Pivoting member 54 also includes a passage 62 which is adapted to receive one anchor 64 of a spring 66. The second anchor 68 of spring 66 is connected to a pin 70 which protrudes from web 72 in moveable jaw 32. Spring 66 is adapted to bias pivoting member 54 such that the bearing surface 56 of pivoting member 54 returns to a nearly vertical position when pivoting member 54 is not in physical contact with workpiece 47. The number of springs 66 and thus the number of passages 62, pins 70 and webs 72 may be varied dependent upon the length of the vise 10.
Referring to FIGS. 2 and 3, vise 10 operates in the following manner. Workpiece 47 is placed in vise 10 such that the base 76 of workpiece 47 is placed on top of sacrificial material 78 and the angled portion 80 is sandwiched between fixed jaw 20 and moveable jaw 32. Moveable jaw 32 is then translated horizontally by piston rod 48 until the angled portion 80 of workpiece 47 is firmly clamped between external radius 24 and bearing surface 56. As bearing surface 56 begins to contact the angled portion 80, it pivots to automatically conform to the angle of the workpiece 47. When sufficient clamping force has been exerted by piston 48, a press fit is formed between the semi-cylindrical surface 58 of pivoting member 54 and the recess 52 in moveable jaw 32, thereby locking pivoting member 54 into position. This locking feature restricts the workpiece 47 from either rotating or translating upward which would cause misalignment during machining. This locking feature is facilitated by the differential radii of the semi-cylindrical surface 58 and the recess 52.
While the semi-cylindrical surface 58 and the recess 52 may be fabricated with the same radius, it is preferred that the radius of semi-cylindrical surface 58 exceed that of recess 52. The required differential between the radii of the semi-cylindrical surface 58 and the recess 52 may increase depending upon the sizes of the semi-cylindrical surface 58 and the recess 52. For example, experiment has shown that when the radius of the recess 52 is approximately 1/2 inch (approximately 0.001 m), the radius of the semi-cylindrical surface 58 should exceed the radius of the recess 52 by approximately 0.007 to 0.010 inches (approximately 0.0002 m to 0.0003 m). However, for larger sized embodiments of the present invention, the required radius differential may increase. For example, if the radius of the recess 52 is approximately 1 inch (approximately 0.03 m), the radius of the semi-cylindrical surface should exceed the radius of recess 52 by approximately 0.0014 to 0.02 inch (approximately 0.0004 m to 0.0005 m).
Experiment has also shown that when the radius of semi-cylindrical surface 58 and the recess 52 are approximately 1/2 inch (approximately 0.001 m), piston 48 should exert a force of approximately 200 pounds per linear inch (approximately 35,000N/m) of pivoting member 54 length in order to sufficiently lock semi-cylindrical surface 58 in recess 52.
It is also preferred that the radius of external radius 24 be smaller than the radius of the semi-cylindrical surface 58. This will assist in reducing the distance between the point on the external radius 24 that contacts the angled portion 80 of workpiece 47 and the point on the bearing surface 56 through which the clamping force of moveable jaw 32 acts, thereby reducing the moment that is induced by the clamping force and the resisting force imparted by the external radius 24. External radius 24 is preferably fabricated with a radius that is as small as possible, yet not so small, that contact between the angled portion 80 of workpiece 47 and external radius 24 will mar the surface of the angled portion 80 of workpiece 47, or cause external radius 24 to fail.
Once the workpiece 47 has been locked into position, machining can proceed. When machining is concluded the piston 48 is retracted and the moveable jaw 32 translated horizontally away from the workpiece 47. The pivoting member 54 biases back to its original position and the workpiece 47 may be removed.
The moveable jaw 32, including the external radius 24, the pivoting member 54, and the recess 52, and the fixed jaw 20 including the external radius 24, are preferably manufactured from 4140, 4142 or similar steel.
Many modifications and variations may be made in the techniques and structures described and illustrated herein without departing from the spirit and scope of the present invention. For example, both fixed jaw 20 and moveable jaw 32 may be capable of movement. Accordingly, the techniques and structures described and illustrated herein should be understood to be illustrative only and not limiting upon the scope of the present invention.
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A self-indexing vise 10 for clamping an angled workpiece 47 includes a pair of jaws 20, 32 capable of relative movement, a semi-cylindrical jaw insert 54 received in a recess 52 in one of the jaws 20, 32 and capable of pivoting to automatically conform to the contour of an angled workpiece 47. The jaw insert 54 is sized slightly larger than the recess 52 in which it pivots such that when the vise 10 is loaded, a press fit forms between recess 52 and jaw insert 54 which locks the insert 54 into position.
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This patent application claims priority based on related Provisional Patent Application No. 61/336,402 filed on date Jan. 20, 2010, Confirmation No. 1099 by J. Kellogg Burnham.
BRIEF DESCRIPTION OF THE INVENTION
This application describes a novel aircraft, herein called Sky Rover, which is capable of vertical take off and landing, and hover, as well as level flight and maneuver. Sky Rover produces its own airflows within its structures and applies them to its external airfoils mounted on both sides of its structure, thereby generating lift on the airfoils, without needing forward movement of the aircraft, and finally discharging the airflows outboard to both sides; because of its vertical and horizontal flight capacity it possesses unequaled free access to the natural environment.
BACKGROUND AND FIELD OF THE INVENTION
Atmospheric flight is currently achieved by different machines which use any of a great variety of flight means. Today airplanes and helicopters carry passengers and cargo in massive volume.
A century of development has taken the Wright Brothers' stroke of genius in aerodynamics to remarkable advances: a wing so light the craft can fly on sunlight; a far-ranging killer drone; the massive industrial transport institution.
Airplanes obtain their support, when airborne, from their wings which traverse relatively stationary true air while being tilted up at their respective angle of attack, such as 15°, and the curved aerodynamic shape of their wings at that slant produces the needed upward lift. The steeper the upward tilt of the wing leading edge, the greater the lift, for a given airspeed; but at less than obligatory airspeed, different for each case, the airplane stalls, falls. With increasing angle of attack, along with increased lift there comes increasing drag, which is undesirable for airplanes, lift-drag is of no effect on stationary airfoils, specifically for Sky Rover; and Sky Rover employs steep angles of attack of 60° to 100° to get high lift, which light airplanes cannot do.
It is a safe, easy-flying, responsive talented new aircraft essentially independent of its surroundings and of atmospheric changes, an aircraft that is airborne and airworthy from the first instant of flight, an aircraft able to soar freely like a bird. Research began with conventional airfoils which gave a mild response to increasingly powerful airflows, which were sometimes combed and retarded with straighteners. Steeper angles of attack, guides, scoops, panels, slotted airfoils were used in different experiments, obtaining encouraging results.
Also developed were propellers with long reverse nose cones and a matching conical shell giving concentrated airflow, which are indeed promising, being lighter and smaller than centrifugal blowers. Effective aerodynamic lift of over 40 lbs. per sq. ft. of airfoil has been reached, an exceptional ratio, believed to be adequate to put to work. Official record of the whole process must be made so it can be of general use, on its merits.
The airplane wing is problematical. Although supported at one end, the unsupported distal end may be of structural concern. In contrast, Sky Rover uses short robust airfoils built above roof level close to the fuselage and swept by a precise airflow. Likewise, Sky Rover needs no preliminary runway dash to get into supporting air; Sky Rover is fully airborne from the first second of lift-off until it touches down at its exact landing place.
Sky Rover is a part of Aviation in general, and more particularly of heavier-than-air craft; specifically Sky Rover relates in the first instance to an air vehicle for one or more persons, and more generally to the expanded performance possibilities it presents. For Sky Rover provides new close-up access to the environment of the air; the contributions include:
a) Sky Rover becomes airborne when vertically lifted by its airfoils, with or without horizontal travel; thus VTOL; b) Sky Rover when airborne possesses inherent upright stability, independent of movement, c) Sky Rover, having an efficient oblong shape, and being unencumbered by the traditional transverse airplane wing, possesses a reduced beam dimension which is advantageous for its broad scope of activity. d) Sky Rover, having self-powered flight, can go where conventional aircraft, captive to an obligatory airspeed and to a restricted horizon, may not; Sky Rover can stop in mid-flight, turn, reconsider, land or not land, depart or arrive vertically; explore; go almost everywhere; serve new functions.
GENERAL DESCRIPTION OF THE INVENTION
The invention, VTOL wingless Aircraft “Sky Rover”, comprises the patentable characteristics thereof and of the components which contribute to its ability to take off vertically, fly, hover, land vertically, and perform forward flight and maneuver. Sky Rover is “wingless” to avoid confusion, because its airfoils perform functions distinct from those of the airplane wing, but the airfoils do provide support in air for Sky Rover.
When the throttle-regulated lift, generated by airflow sweeping over and under its airfoils, exceeds the weight of Sky Rover, it rises, and when lift is less than aircraft weight, it descends. Since the lift is generated upon two opposed airfoils, or two opposed banks of airfoils, which airfoils stand higher than the center of gravity of the aircraft, the lift provides a stable upright position. Sky Rover can go where other aircraft can: but, having no need of forward travel to remain airborne, Sky Rover has unique access to the very ample environment reachable from the air. Being disencumbered of the usual transverse wing of airplanes, Sky Rover has a favorable overall beam dimension, for forward flight and varied activities.
Now a new dimension is added by Sky Rover which is the subject of the present patent application, an intensifier that multiplies and simplifies flight, the Sky Rover Flight System, which provides smaller and protected airfoils, able to generate greater lift. And lift is the essence of aviation.
The two-seater class of light airplanes today can generate five to ten lbs of lift for each square foot of its wing area but my present prototype Sky Rover, in the same weight class, lifts over four times as much—40: lbs per square foot of airfoil, about the same proportionate lift as the F22. And Sky Rover lifts 500 lbs. and flies on 20 HP per unit: my next prototype with 4 airfoils will lift 2000 lbs. and only weigh 700 lbs, dry weight.
There is more. Sky Rover can fly straight up, from the ground, a standing start. Sky Rover provides its own wind and serves its own airfoils, so it does not depend so much on flying weather. Sky Rover can take off, fly, and land vertically in near-zero visibility. And vertical access enables Sky Rover to go anywhere, hover under bridges to repair them, do rescue over floods, Sky Rover is the first aircraft to produce its own airflow and serve the airflow to its own high-lift airfoil. Sky Rover can fly standing still or go anywhere; it is not subject to a compulsory airspeed under threat of stall. Sky Rover can land on water or muskeg, it is able to take on tough jobs not doable before.
Sky Rover the prototype aircraft is mostly fuselage, having cockpit forward, tail structure aft, optionally with pusher-prop; amidships the structure encloses the power source driving the blowing means, consisting of powered centrifugal blowers, which produce the airflow that enables Sky Rover to fly; the airflow is delivered to opposed pairs of airflows installed on opposite sides of the aircraft structure; the shape and dimensions of Sky Rover may vary according to the particular work it does, based on this outline.
A controlled airstream going out through each wall opening, and sweeping over the respective airfoil from leading edge to trailing edge, and to its flap if any, passing above and below the airfoil, generates a lift effect upon the airfoil. The intensity of each airstream can be varied to increase or decrease the amount of lift, as control operation may require.
IN THE DRAWINGS
FIG. 1 is a perspective view of the aircraft.
FIG. 2 is a schematic angular view of the aircraft.
FIG. 3 a is a schematic view of assembly of airfoils and surfaces.
FIG. 3 b is a schematic view of assembly showing routes of passage of Airflows.
FIG. 4 is a vertical schematic cross section of Sky Rover showing assembly and function of parts.
FIG. 5 is an angular side view sketch of location of airfoils and panels.
DETAILED DESCRIPTION OF THE INVENTION
Sky Rover is a novel aircraft because it rises and descends vertically, and hovers, in addition to forward flight and maneuver; also because it does not have outstretched wings but only modest airfoils at its midriff, which supply its support in air; it is novel also because it provide its own airflows which it delivers to its own airfoils to generate lift; and which, when in the clutch of moderate air turbulence, will continue to generate its own lift; because it draws in atmospheric air to produce its airflows: no atmospheric disturbance reaches the airfoils; it generates its own lift, and flies; and because it can land on water or any moderately coherent surface; and perhaps most important of all its novelties, it can perform under many flying conditions and locations, a random or regular services of inspection, repair, delivery, search, lift-saving, and emergency.
The Airfoil
Numbered reference to FIG. 3 a and FIG. 5 , following.
The airfoil of Sky Rover consists of two members, as shown in FIG. 3 . The main body of the airfoil is Fatwing 14 ; but the leading edge and the whole front portion of the airfoil is severed from Fatwing 14 to provide an intervening airflow channel, called Slot Passage 13 . which extends across the full span of Fatwing 14 . This leading portion is separately identified as Scion 9 . The airflows pass over them as if they were a single composite airfoil; but the two separated airfoils also have separate respective functions, and the Slot Passage 13 plays its own role between the two airfoils, as illustrated in part in FIG. 3 . Fatwing 14 has thickness equal to 30% of its length; and when grouped with Scion 9 , as composite airfoil, its thickness is greater. The overall chord times span gives the basic airfoil area. Further information about and description of the composite split airfoil Fatwing 14 /Scion 9 is provided below, and in Claims.
The following discussion concerns the route that airflows take to pass through Sky Rover flight system.
Route of the Airflow Sequence
See FIG. 3
1 . BLOWER INLET
2 . BLOWER
3 . BLOWER OUTLET
4 . BLUE SKY
5 . BACKBOARD
6 . JACK-KNIFE-BEND
7 . TECHO
8 . Orifice at TECHO END
9 . Upper Camber of Airfoil SCION
10 . Orifice at end of Airfoil SCION
11 . Concave Face of Airfoil SCION in SLOT PASSAGE
12 . Leading Edge of Airfoil SCION (Nose)
13 . Entry into SLOT PASSAGE
14 . Upper Camber of Airfoil FATWING
15 . Trailing Edge of Airfoil FATWING
16 . Lower Camber of Airfoil FATWING
17 . Airfoil tail, to attach aileron to Airfoil FATWING
PRIME AIRFLOW (a)
SECONDARY AIRFLOW (b)
TERTIARY AIRFLOW (c)
Airflow
Numbered reference to FIG. 3 b and FIG. 3 a , following.
Sky Rover is able to fly by virtue of its airfoil and airflow system, which is an object of claims of the present patent application. The airflow can be produced by any suitable air-moving means, and is here preferably provided by centrifugal blower having its rotor composed of forward-curved blades, drawing in air at both its ends and accumulating the accelerated air in its spiral volute. The air delivered by the blower has been uniformly accelerated, but is not uniform because air proceeding from the volute is retarded, and because the middle of the rotor accelerates air more; the air discharged from the Blower 3 volute spreads and scatters, and varies widely in velocity, unlike the “true” placid air traversed by airplane wings, thus precluding close equivalence between Sky Rover and airplanes in considering respective Angles of Attack; however, the airflow above described is suitable feed for the Sky Rover Lift System, herein set forth with reference to FIG. 3 .
Numbered references are to FIG. 3 a , and to “Route of the airflow,” following.
The mixed airflow tumbles upward from Blower Outlet 3 ; and out of this air supply Sky Rover system produces three currents: the Prime Airflow (a), which is the main airflow; the Secondary Airflow (b); and the Tertiary Airflow (c). The total airflow is discharged upward from Blower Outlet 3 at angles between about 100° and 75° above the horizontal. The rising Prime Airflow (a) sweeps up Backboard 5 ; Backboard 5 has sideboards—not shown—to enclose all the airflow on 3 sides. Vertical Panel Backboard 5 at its upper end engages the adjoining end of horizontal panel Techo 7 . Prime Airflow (a) races up vertical Backboard 5 and strikes horizontal Techo 7 and is trapped; Prime Airflow (a) immediately goes through a right-angle change of direction, presumptively the area Jack-Knife-Bend 6 ; Prime Airflow (a) then flows horizontally under panel Techo 7 to the Orifice 8 located at the distal end of Techo 7 , and passes forward bursting into the area of Blue Sky 4 . There is no further cover over Prime Airflow (a) all the way to the Trailing Edge 15 of Fatwing 14 .
As Prime Airflow (a) moves forward under Blue Sky 4 , it is impacted from below by Secondary Airflow (b) riding up the upper Camber 9 of Scion 9 at Orifice 8 . And shortly beyond, Prime Airflow (a) is again impacted underneath by Tertiary Airflow (c) rising through the narrow orifice at the top of Slot Passage 10 . Prime Airflow (a) is pummeled and enriched by each of these impacting airflows.
We have seen that Prime Airflow (a), the strongest airflow, breaks direct from the top of Blower Outlet 3 . Secondary Airflow (b), issuing from Blower Outlet 3 , spreads out embracing approximately from 95° to 85° above the horizontal, striking toward airfoil Scion 9 ; the Secondary Airflow (b) rises up along the Scion 9 upper camber; on reaching the top of Scion 9 it impacts upon Prime Airflow (a). Since Tertiary Airflow (c) is the lower third of the airflow coming out of Blower Outlet 3 ; it flows toward the nose of airfoil Scion 9 , and Tertiary Airflow (c) separates from Secondary Airflow (b) on striking Scion 9 , Tertiary Airflow (c) passes below the nose of Scion 9 ; it is then exposed to entry to Slot Passage 13 ; and part of Tertiary Airflow (c) penetrates Slot Passage 13 and is accelerated as it rises in the diminishing space, and comes out at Orifice 10 to impact Prime Airflow (a). The part of Tertiary Airflow (c) that does not enter Slot Passage 13 continues along the lower Camber 16 of Fatwing 14 to Trailing Edge 15 where it mingles again with Prime Airfoil (a) in the atmosphere. The still energetic flow of Prime Airflow (a) as it reaches Trailing Edge 15 , and the remaining Tertiary Airflow (c), can be used to advantage with a flap or elevator: this potential is not shown in FIG. 3 .
Prototype
The prototype Sky Rover fuselage is six feet wide, and accommodate in its interior space a side-by-side pair of Blowers 3 which are nearly 3 feet in diameter, each Blower 3 serving its corresponding airfoil mounted on the exterior surface of the fuselage; hence the cockpit admits convenient side-by-side seating for two, with amplitude for work.
The wide fuselage slows airspeed but the absence of customary broad wingspread compensates in reduced air resistance. See FIG. 4
Balance
Sky Rover when flying is characterized by inherent and self-correcting balance. Support for the aircraft when airborne is provided by the two parallel lengthwise batteries of airfoils, which hold and carry the central structure below and between them. Much of the total weight of the aircraft is located in the lowest part of the central structure hangs below and is locked to the airfoils, like a frozen pendulum, and returns to the mid-point position of balance. Even if some phenomenon of weather or maneuver were to swing the aircraft out 90°, automatically and by gravity it will seek out and regain its balanced middle position.
The foregoing description refers preferentially to right and left, or roll, departures from and recovery of medial balance; however the same factors equally provide balance in pitch, or fore-and-aft variations, and for the same reason: that Sky Rover is balanced at center.
In fact, however, Sky Rover may have a slight but significant pitch preference, to be nose-heavy, so that it tends always to be very slightly head-down and to move forward, which is helpful for easy handling, but consistent, easily compensated for, and allowed for.
Balance Control
The automatic balancing instrument carried by Sky Rover consists of a rod suspended within a case and fitted with hair-thin springs urging it toward its vertical suspension position, having at its lower end six or other number of radially protruding wired electrodes closely surrounded by a grounded metallic ring which is fixed to the structure of Sky Rover.
Any slight change in the level position of Sky Rover actuates the suspended rod and causes the corresponding one of the electrodes (depending on the direction involved) to make contact with the grounded metal ring, closing the circuit associated with that particular electrode and causing it to actuate the corresponding element, compressed air valve or otherwise powered control assembly, and thereby to initiate the compensating operation. As the level position of Sky Rover is brought again to neutral, de-contacting the electrode involved, the power system returns to neutral.
The pilot's joystick, when initially moved from neutral position, makes selective contact with the circuits operating with the automatic balance control mechanism, independently of any imbalance detected by the apparatus; and a further movement of the joystick provides additional and stronger action for maneuver.
Power for operation of the aircraft is provided by a gasoline engine (or other suitable source). In one version of this invention the engine powers a hydraulic pump, and the pump provides oil under pressure to hydraulic motors; each hydraulic motor is coupled, directly or through gearing, propeller, turbine, centrifugal blower or other air mover; a return line from each hydraulic motor completes the circuit; thus uniform power and rotation are supplied to produce the required airflows. The hydraulic oil flow from the hydraulic pump is under governance, thus providing selective management of flight to the automatic balance mechanism above described, and to the pilot.
In other versions of this invention power is transmitted from the motor to the fans or propellers or blowers by means suited to the position, load, speed range and other factors related to the overall function and the vehicle, including provision of intermediate planetary gearing to accelerate rotation. Power transmission from engine to propeller or blowers is also feasible using sheave and belt, and by toothed belts. Similar convenience is afforded by sprocket and chain transmission. These drives require guard protection, and provision for tensioning.
ADDITIONAL COMMENTS ON DRAWINGS
The description of airfoils and airflows refers to the numbered points in the diagram of FIG. 3 . Air intake on top of rear fuselage in FIG. 1 is for an intended prototype Sky Rover aircraft having four units of the same capacity shown in FIG. 3 , with 80 HP, total estimated dry weight 700 lbs, total lift 2000 lb, two-seater side by side with cargo capacity fore and aft, suitable for diverse use, including lifesaving.
FIG. 4 shows a possible distribution of components for the suggested aircraft.
FIG. 5 shows the bare airfoil components:
Backboard, Techo, Scion, Fatwing
Since every patent should furnish information sufficient for the reader to reproduce the results cited, the following specifics as to successful trials are offered: FIG. 3 is diagram of one such tested lift-producing prototype having a powered blower with rotor 18⅛″ø×18⅛″; and outlet 30″×30″ revolving about at 1100 rpm, consuming 16 to 20 HP, having airfoils: Scion 12″×36″ and Fatwing 36″×36″, shaped and spaced as shown, and having Backboard 30″×30″ and Techo 35″×30″; this prototype produces 500 lbs avdp. lift at the forward third of Fatwing, or 42 lbs lift per sq. ft. of airfoil; these results being still less than the potential.
FIG. 1 depicts prototype Sky Rover 4 equipped with four units of Sky Rover flight assembly, each unit having powered 18⅛″ø blower with airflow direction and airfoils Fatwing and Scion, for total lift of 2000 lbs, showing the optional side-by-side seating cockpit, cargo or lab space, firewall, the four units of Sky Rover airfoils installed in opposed pairs, the rear fuselage air intake, and then rudders and elevators; reference FIG. 1 .
FIG. 2 depicts prototype Sky Rover 4 flight assembly as in to FIG. 1 with all four Techos removed and airfoils on right side removed, to illustrate one mode of compact grouping, the Sky Rover flight assemblies at mid-fuselage with power plant and drive partly balancing out cockpit and forward cargo load. These arrangements are optional. FIG. 4 is a vertical cross sectional view of the Sky Rover Prototype 4 of FIGS. 1 and 2 . Showing 2 units mounted side by side within the fuselage reverse rotation and airflows directed to opposite sides, thus counterbalanced.
The air passage area enclosed by the fuselage sides and bottom permits adequate air movement for the possible 60,000 cfm total volume which passes into end entries of the blowers; each blower outlet delivers its airflow to its respective airflow system. FIG. 5 is a sketch showing Scion 9 and Fatwing 14 in approximate position with horizontal panel Techo 7 and vertical panel Backboard 5 ; the terms “horizontal” and “vertical” are suggestive only.
|
Novel aircraft for vertical and horizontal flight having powered air acceleration means installed within its structure drawing air thereinto and delivering the resulting airflow upon airfoils installed in opposed pairs on opposite sides of its exterior structure thereby producing lift without forward travel of the aircraft.
| 1 |
FIELD OF THE INVENTION
[0001] This invention relates to prostaglandin analogs and their synthesis. More particularly, it relates to a novel, simplified synthesis of prostaglandin analogs, and novel chemical compounds useful as intermediates in such synthesis.
BACKGROUND OF THE INVENTION AND PRIOR ART
[0002] Prostaglandins (PGs) are organic carboxylic acids, namely cyclopentanes carrying two side chain substituents, typically linear C6-C8 side chains, bonded to adjacent positions on the cyclopentane nucleus. One of the side chains, the α-side chain, carries a terminal carboxylic acid group. Many are natural products found in mammalian organs and tissues (primary PGs), and exhibit a variety of physiological activities. Primary PGs generally have a prostanoic acid skeleton, which forms the basis of the nomenclature:
[0000]
[0003] A significant number of synthetic PG analogs have been made and found to have useful pharmacological properties. These may have modified skeletons, and substituted and unsaturated side chains. PGs are characterized by a hydroxyl (or ketone) substituent on the cyclopentane nucleus, position 9.
[0004] Prostaglandin analogs are difficult to synthesize. Complications arise because of the requirements of the end products to have several functional groups and two side chains of significant size and complexity. Stereospecificity is commonly required, for substituent groups and for bonds in the core. Since the products are intended for pharmaceutical use, the range of industrially acceptable reagents, solvents, catalysts, etc. which can be used in their synthesis is limited to those having pharmaceutical industry acceptability.
[0005] A common starting material for PG analog synthesis is the commercially available Corey alcohol benzoate, of formula:
[0000]
[0006] To convert this to a synthetic PG analog, many protection, functionalization, de-protection, etc. steps are required to form the desired side chains. U.S. Pat. No. 5,252,605 Ueno, issued Oct. 12, 1993, reports several PG syntheses starting from Corey alcohol which involve approximately fifteen steps. Inevitably, such a multi-step process is time consuming and expensive to conduct, and results in relatively low overall yield of final product.
[0007] An example of a synthetic prostaglandin analog of specific interest is lubiprostone, of formula:
[0000]
[0008] This compound is marketed as “Amitiza”, for use in treatment of chronic idiopathic constipation, irritable bowel syndrome and post-operative ileus. Its synthesis presents significant technical challenge because of the chemical complexity of the fluorine containing substituent chain at the 12-position. Known methods for its synthesis suffer from the aforementioned disadvantages, namely a multi-step (typically 15-step) synthesis from Corey alcohol with consequent low yields of final product and time consuming nature of the process.
[0009] It is an object of the present invention to provide a novel synthetic method for preparing PG analogs, in fewer steps and in improved overall yield.
[0010] It is a further object to provide novel chemical compounds useful in the synthesis of PG analogs.
[0011] It is a specific object of the present invention to provide a novel synthesis of lubiprostone, starting from commercially available Corey alcohol.
SUMMARY OF THE INVENTION
[0012] One significant aspect of the present invention is a small class of novel chemical compounds comprising a cyclopentane nucleus fused at its 4,5 position with a 4-substituted 3,5-dioxalane ring, and fused at its 3a,6a-position with a lactone ring. The compounds have the formula:
[0000]
[0013] where R represents an aryl group, preferably a substituted phenyl group such as p-methoxyphenyl (PMP). Subsequent reaction of a compound of formula A with a lower alkyl-aluminum compound such as di-isobutyl aluminum hydride (DIBAL) under properly selected conditions causes ring opening of the dioxalane at a specific position, as well as reduction of the lactone to lactol without over-reducing the lactol ring structure to a diol. The product of the ring opening reaction has a hydroxymethyl group at position 1 on the cyclopentane nucleus, ready for chemical expansion to provide the ω-chain of the selected target PG analog, and a protected hydroxyl group at position 2. In subsequent steps, the lactol ring can be opened chemically, and expanded to form the β-chain of the target compound, with the residue of the lactol ring forming the basis for the eventual 9-hydroxy or 9-keto group of the target PG analog. The formation of this ring-opened product B may be represented as follows:
[0000]
[0014] It is totally unexpected that this reaction should take place without over-reducing the lactone ring. One would have predicted formation of a complex mixture of different reduction products, with such a plurality of potentially reducible groups and sites being subject to such a powerful reducing agent as DIBAL. Instead, by selection of appropriate reaction conditions, a high degree of selectivity to from product B is achieved. These conditions include selection of a reaction solvent which is a good solvent for the cyclopentane compound, and which is a polar, non-co-ordinating solvent that permits, and does not interfere with, co-ordination of the aluminum complex with the available oxygen of the ring structure, to the substantial exclusion of co-ordination of the aluminum to the solvent itself; and temperatures appropriate to maintain the stability of the organo-aluminum compound. Suitable such solvents include methylene chloride, dichloroethane, chlorobenzene, chloroform, toluene and mixtures thereof, and similar polar hydrocarbons, with methylene chloride being most preferred. Preferably low temperatures, below 0° C. and most preferably in the −40-50° C. range.
[0015] Thus according to a first aspect of the present invention, there is provided in one embodiment a fused cyclopentane—4-substituted 3,5-dioxalane lactone compound useful as an intermediate in the synthesis of prostaglandin analogs, the compound having the formula A:
[0000]
[0000] wherein R represents a lower alkoxy substituted phenyl group.
[0016] According to a second aspect, there is provided a process of preparing a substituted cyclopentane lactone compound of formula B, which comprises subjecting a compound of formula A as defined above to selective ring opening reduction with a lower alkyl-aluminum reducing agent in solution in a polar, non-coordinating solvent at a temperature at which the reducing agent is stable.
BRIEF REFERENCE TO THE DRAWING
[0017] The single FIG. 1 of accompanying drawings illustrates the overall reaction scheme embodying the present invention, in the preparation of lubiprostone, a preferred embodiment thereof.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] Referring to the accompanying drawing FIG. 1 , the preferred synthesis according to the invention starts with Corey alcohol benzoate 10, which is commercially available. Reaction of this with sodium methoxide in methanol (room temperature, 1.5 hours) produces Corey lactone diol 12 in high yield (e.g. 97%) ready for further reaction.
[0019] A cyclopentane-lactone-dioxalane fused tricyclic compound 16, member of the class of compounds A of the present invention, is prepared by reacting Corey lactone diol 12 with anisaldehyde dimethyl acetal, compound 14, in the presence of trace amounts of acid. This reaction suitably takes place under reflux, over a period of, for example 3 hours. A dioxalane ring substituted at ring position 2 with p-methoxyphenyl (PMP) forms in high (85-90%) yield. In the next step, according to this preferred embodiment of the process, compound 16 is reacted with DIBAL, in solution in methylene chloride and toluene, and at a low temperature (e.g. −45° C.) at which DIBAL is stable. Ring opening of the dioxalane at a specific position occurs, without over-reduction the lactone structure to diol, thereby producing compound 18, a representative of class B referred to above, in a yield in excess of 80%. Compound 18 has a hydroxymethyl group at position 1 on the cyclopentane nucleus, ready for chemical expansion to provide the α-chain of the selected target PG analog, and a hydroxyl group protected with p-methoxy benzyl at position 2.
[0020] Side chain expansion and derivatization can now take place using compound 18, advantageously expanding one side chain to that required in the target prostaglandin analog first, and subsequently expanding the second one to the target. Thus in the preferred embodiment where lubiprostone is the target compound, the α-chain is formed first. This is a linear heptanoic acid chain, which after formation merely needs simple protection of its terminal carboxylic acid group to confer stability and prevent its interference with other reactions. The ω-chain of lubiprostone is more chemically complex, involving a hemi-acetal and a di-fluorinated side chain. Introducing this chain second reduces the chances of fluorinated side chain losses in subsequent reactions, as the number of such reactions is reduced, the α-chain being already formed.
[0021] The first step in the α-chain expansion is reaction of compound 18 with (4-carboxybutyl)triphenylphosphonium bromide and sodium hexamethyl disilazane to cause opening of the lactone ring and condensation thereof to form a compound 20. This reaction suitably takes place in toluene solvent and at a temperature of −20 to −30° C., over a period of 2-3 hours. A double bond forms at position 5,6 of the side chain. Stereospecificity of the original Corey alcohol is retained. This reaction is analogous to that conducted in known prostaglandin synthesis process, although according to the invention it is applied to novel reagents and produces novel intermediates. The next step is the protection of the terminal carboxylic acid group, and this is done in known manner, by reaction of compound 20 with benzyl bromide (BnBr) in the presence of potassium carbonate at room temperature in acetone solvent, in two steps, over 18 hours, producing protected acid compound 22. A 55-65% yield is typically obtained in this step.
[0022] Next, a double oxidation of hydroxyl groups to keto and aldehyde groups is conducted. Protected compound 22 is oxidized with pyridine-sulfur trioxide in the presence of diisopropylethylamine and DMSO and methylene chloride solvent. The result is oxidation of the primary alcohol side chain group to aldehyde, and oxidation of the secondary, nuclear alcohol group to a keto functionality, producing compound 24.
[0023] Now the fluorinated side chain required in lubiprostone can start to be introduced. Thus the next step in the process is the reaction of compound 24 with dimethyl-(2-oxo-3,3-difluoroheptyl)phosphonate (compound 26), in the presence of sodium hydride and dimethoxyethane (DME), for example at 50 to 70° over 18 hours. The result is compound 28, in 60-70% yield.
[0024] The final reactions in lubiprostone synthesis are the hydrogenation of the double bonds in compound 28, ((Z)-benzyl 7-((1R,2R,3R)-2-((E)-4,4-difluoro-3-oxooct-1-enyl)-3-(4-methoxybenzyloxy)-5-oxocyclopentyl)hept-5-enoate) which is itself a novel, inventive compound and a feature of the present invention, and the deprotection thereof to remove the carboxylic acid protectant from the α-chain terminus, and the removal of the p-methoxybenzyl (OPMB) protectant to form the desired hemi-acetal ring. This is done in a single step, by hydrogenation using hydrogen over palladium/carbon catalyst in isopropanol medium, at room temperature over, e.g., 2 hours. This process is another significant feature of the present invention. The product is lubiprostone, compound 30, in a 75-80% yield for this step.
[0025] The illustrated process is capable of producing lubiprostone from Corey alcohol in eight steps at an overall yield in excess of 15%, which is most acceptable in syntheses of this type and is significantly higher than that achieved with prior art processes. Most of the reagents used are relatively inexpensive, with the possible exception of dimethyl-(2-oxo-3,3-difluoroheptyl)phosphonate (compound 26). This is a known compound, preparable from ethyl 2-oxo-hexanoate by reaction with ethyl 2,2-difluorohexanoate he following reaction scheme:
[0000]
[0026] The specific preferred embodiment of the present invention is further described, for illustrative purposes, in the following specific experimental examples.
Experimental Procedure
[0027]
[0028] Corey Lactone Diol 12. To a suspension of 12 (15 g, 54 mmol, 1 equiv) in methanol (75 mL) was added sodium methoxide (25% wt in methanol, 1.2 mL, 5.4 mmol, 0.1 equiv). The mixture was stirred at room temperature for 1.5 h and then hydrochloric acid solution (4 M in dioxane, approximately 1 mL) was added until the pH was 3-4. The solution was stirred at room temperature for 10 min and then concentrated to dryness under vacuum on a rotary evaporator. The resulting white solid was suspended in methyl tert-butyl ether (150 mL) and stirred at room temperature for 1 h. The solid was filtered, washed with methyl tert-butyl ether, and dried under vacuum for 10 min to afford 9.1 g of 12 (97%) as a white solid.
[0000]
[0029] Protected Diol 16. To a suspension of 12 (5.0 g, 29 mmol, 1 equiv) in toluene (100 mL) was added anisaldehyde dimethyl acetal (14) (7.4 mL, 44 mmol, 1.5 equiv) and p-methoxy benzoic acid (44 mg, 0.29 mmol, 0.01 equiv). A condenser and a Dean-Stark apparatus were attached and the mixture was heated at 120° C. for 3 h while removing methanol by the Dean-Stark apparatus (approximately 2 mL). The reaction mixture was removed from the oil bath and stirred at room temperature for 15 min. Methyl tert-butyl ether (100 mL) was added and the mixture was cooled in an ice bath for 45 min. The resulting suspension was filtered, washed with methyl tert-butyl ether, and dried under vacuum for 10 min to afford 7.3 g of 16 (87%) as a white solid.
[0000]
[0030] Lactol 18. A solution of 16 (14 g, 50 mmol, 1 equiv) in dichloromethane (500 mL) in a round-bottom flask containing a dropping funnel was flushed with N 2 for 5 min. The solution was cooled to −45° C. and diisobutylaluminum hydride (1M in toluene, 150 mL, 150 mmol, 3 equiv) was added dropwise. The mixture was stirred for 1 hour and 20 min at −45° C. Buffer solution pH 7 (21 mL) was added dropwise and the solution was warmed to room temperature over 2 h. The suspension was filtered and washed with dichloromethane. The filtrate was concentrated to dryness under vacuum on a rotary evaporator to afford 13 g of 18 as a yellow oil (88% yield) which was used directly in the next step.
[0000]
[0031] Diol 20. To a suspension of (4-carboxybutyl)triphenylphosphonium bromide (33 g, 75 mmol, 2 equiv) in toluene (220 mL) was added sodium hexamethyl disilazane (1 M in tetrahydrofuran, 262 mL, 262 mmol, 7 equiv). The mixture was stirred at room temperature for 1 h and then cooled to −25° C. Compound 18 in tetrahydrofuran (60 mL) was added dropwise and then warmed to room temperature over 4 h. Water (200 mL) was added and the organic layer was separated and extracted with water (2×50 mL). The aqueous washings were combined and 20% aqueous citric acid solution (125 mL) was added. The suspension was extracted with dichloromethane (4×100 mL). The organics were combined, dried over sodium sulfate, filtered, and concentrated to dryness under vacuum on a rotary evaporator to afford a yellow oil containing 20. The oil was dissolved in acetone (433 mL) and potassium carbonate (11 g, 77 mmol, 2 equiv) and benzyl bromide (9.1 mL, 77 mmol, 2 equiv) were added. The mixture was stirred at room temperature for 18 h, filtered, and concentrated to dryness under vacuum on a rotary evaporator. The crude oil was purified by column chromatography using 50% ethyl acetate/hexanes as eluant to afford 11 g of 22 as a yellow oil (64%).
[0000]
[0032] Aldehyde 24. A solution of 22 (3.5 g, 7.4 mmol, 1 equiv) and dimethyl sulfoxide (10.5 mL) in dichloromethane (70 mL) was cooled to −15° C. Diisopropyl ethylamine (4.3 mL, 45 mmol, 6 equiv) was added followed by the addition of a solution of sulfur trioxide pyridine complex (7.1 g, 45 mmol, 6 equiv) in dimethyl sulfoxide (21 mL). The mixture was stirred at −15° C. for 1 h and was then diluted with 20% aqueous citric acid solution (20 mL). The aqueous layer was extracted with dichloromethane (3×20 mL) and the organics were combined, dried over sodium sulfate, filtered, and concentrated to dryness under vacuum on a rotary evaporator. The crude oil was purified by column chromatography using 20-40% ethyl acetate/hexanes as a gradient eluant to afford 3.1 g of 24 as a yellow oil (90%).
[0000]
[0033] Protected Unsaturated Lubiprostone 28. A suspension of sodium hydride (60% dispersion in oil, 2.1 g, 53 mmol, 2.5 equiv) in tetrahydrofuran (500 mL) was added dropwise a solution of 26 (14 g, 53 mmol, 2.5 equiv) in tetrahydrofuran (165 mL). The mixture was stirred for 1 h at room temperature. A solution of 24 (9.9 g, 21 mmol, 1 equiv) in tetrahydrofuran (165 mL) was added dropwise. The mixture was then heated with stirring at 58° C. for 2 days. The mixture was cooled to room temperature and saturated aqueous ammonium chloride (200 mL) was added followed by water (200 mL). The aqueous layer was separated and extracted with ethyl acetate (3×150 mL). The organics were combined, dried over sodium sulfate, filtered, and concentrated to dryness under vacuum on a rotary evaporator. The crude oil was purified by column chromatography using 10-25% ethyl acetate/hexanes as a gradient eluant followed by a second column chromatography using 20% ethyl acetate/hexanes as to afford 7.8 g of 28 as a yellow oil (61%).
[0000]
[0034] Lubiprostone. A mixture of 28 (8.0 g, 13 mmol, 1 equiv) and 5% palladium on carbon (containing 54.02% water, 5.1 g, 1.3 mmol, 0.1 equiv) in isopropanol (300 mL) was stirred under an atmosphere of H 2 (g) in a Parr hydrogenator at 40 psi for 2 h. The solution was then filtered through Celite™ and washed with methyl tert-butyl ether. The filtrate was concentrated to dryness under vacuum on a rotary evaporator and the resulting yellow oil was purified by a silica plug by first eluting with dichloromethane to remove impurities and then with methyl tert-butyl ether to remove the product. The methyl tert-butyl ether filtrate was concentrated to dryness under vacuum on a rotary evaporator to afford a yellow oil that was dried under vacuum for 3 h. The resulting oil was dissolved in dichloromethane (5 mL) with heating and a 1:1 solution of hexanes:petroleum ether (50 mL) was added. The solution was placed in an ice bath and stirred vigorously. Methyl tert-butyl ether (1 mL) was added and the product began precipitating out of solution. The mixture was stirred for 2 h, filtered, and washed with a solution of 2% dichloromethane in 1:1 mixture hexanes:petroleum ether to afford 4.1 g of Lubiprostone (78%) as a white solid.
[0000]
[0035] Ethyl 2,2-Difluorohexanoate. To a 0° C. solution of ethyl 2-oxohexanoate (6.3 g, 40 mmol, 1 equiv) in dichloromethane (125 mL) was added dropwise (diethylamino)sulfur trifluoride (6.3 mL, 48 mmol, 1.2 equiv). The solution was warmed to room temperature over 4 h. Saturated aqueous sodium bicarbonate (100 mL) was slowly added. The aqueous layer was separated and extracted with dichloromethane (3×50 mL). The organics were combined, dried over sodium sulfate, filtered, and concentrated to dryness under vacuum on a rotary evaporator to afford 6.5 g of ethyl 2,2-difluorohexanoate (91%) as a yellow oil.
[0000]
[0036] Dimethyl-(2-oxo-3,3-difluoroheptyl)phosphonate 26. A solution of dimethyl methylphosphonoate (6.5 g, 80 mmol, 2.2 equiv) in tetrahydrofuran (100 mL) was cooled to −78° C. and n-butyllithium (2.5 M in hexanes, 14 mL, 36 mmol, 1 equiv) was added dropwise. The solution was stirred at −78° C. for 30 min and ethyl 2,2-difluorohexanoate (6.5 g, 36 mmol, 1 equiv) was added dropwise. The solution was stirred at −78° C. for 1 h and warmed to 0° C. over 1 h. Pentane (100 mL) was added followed by the dropwise addition of 2M H 2 SO 4 to pH=6. The aqueous layer was separated and extracted with pentane (3×15 mL). The organics were combined, dried over sodium sulfate, filtered, and concentrated to dryness under vacuum on a rotary evaporator. The crude oil was purified by column chromatography using methyl tert-butyl ether as eluant to afford 4.1 g of 26 as a yellow oil (44%).
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Fused cyclopentane—4-substituted 3,5-dioxalane lactone compounds useful as an intermediate in the synthesis of prostaglandin analogs are provided. The compounds have the formula A:
wherein R represents an aryl group such as p-methoxyphenyl.
This compound can be reacted with a lower alkyl aluminum compound to open the dioxalane ring and reduce the lactone to lactol, without over-reducing to diol. The resulting compound can be functionalized to insert chemical side groups of target prostaglandins, adding the required α-side chain and then the required ω-side chain sequentially and independently of each other. The compounds and process are particularly suitable for preparing lubiprostone.
| 2 |
FIELD OF THE INVENTION
The present invention relates to a process for the separation of racemic mixtures comprising development of a denser molecular imprint on silica with a desired enantiomer by sol-gel protocol. More particularly, nanoporous silica with a defined shape and size is developed by molecular imprinting of the desired enantiomer for the resolution of corresponding racemic mixture. The present invention relates to the functionalized imprinted materials. More particularly, the present invention relates to amorphous silicas having discrete pores of controlled size and shape with one spatially organized functional groups formed therein.
BACKGROUND OF THE INVENTION
Reference is made to Wulf Angew, Chem.Int.Ed ., 1812, 34, 1995, wherein separation of aminoacid derivatives and sugar compounds is carried out by using bulk polymer net work using functional monomers through covalent interactions. The major disadvantages are the amount of imprint used in the polymer preparation is only up to 3% of the total polymer and after the porogens (solvents) are removed, some of the structural integrity of the polymer is lost, leading to deformation of the sites.
Reference is made to Davis et al; Nature , 283, 403, 2000, wherein molecular imprinting of bulk microporous silica is carried out by covalent bonding approach using tetraethylorthosilicate as a silicon source. The major disadvantage in this process is the amount of imprint used in the sol-gel synthesis correspond to only 2 mol % of the imprint silicon relative to TEOS silicon.
Reference is made to Mosbach et al; Biotechnology 163, 1996 ; Journal of Chromatography 391, 470, 1989; wherein enantiomeric resolution of amino acid derivatives was carried out using polymerization of monomer with imprint molecule in the presence of crosslinking agent. The major drawback in this process is some of the structural integrity of the polymer is lost, leading to deformation of the sites. Reference is also made to Pinel et al., Advanced Materials .1997, 9, 582, wherein (−)-menthol was used as a imprint molecule using silica. The major drawbacks in this process is poor enantioselectivity.
Reference is made to U.S. Pat. No. 5,587,273 wherein molecular imprinting method is used for organic polymers, particularly allowing the manufacture of thin films on surfaces such as silicon wafers. This method has shown that some highly selective receptor sites can be built for complex molecules such as sugars, amino acids. The major drawbacks in this process is imprinted polymers lose their affinity for substrates in aqueous solutions.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a process for the separation of racemic mixtures comprising development of a denser molecular imprint on silica with a desired enantiomer by sol-gel protocol.
It is another object of the invention to develop an nanoporous silica with a defined shape and size by molecular imprinting of the desired enantiomer for the resolution of corresponding racemic mixture.
It is yet another object of the invention to provide a process for the separation of racemic mixtures comprising development of a denser molecular imprint on silica with a desired enantiomer by sol-gel protocol where the selectivity is good.
It is yet another object of the invention to provide a process for the separation of racemic mixtures wherein the use of expensive chemicals for resolution is avoided.
It is yet another object of the invention to provide a process for the separation of racemic mixtures which is environmentally safe, simple and economical.
It is a further object of the invention to provide a process for the separation of racemic mixtures where imprinted silica can be used for number of cycles with consistent selectivity.
SUMMARY OF THE INVENTION
The novelty of the present invention lies in the design and development of nanoporous silica with a defined shape and size to suit the desired enantiomer for resolution of the corresponding racemic mixture with high enantiomeric purity for the first time. The nanoporous silica is developed by sol-gel protocol, involving the hydrolytic polymerization of tetraethylorthosilicate using as a monomer and amino alkylsilane as a functional monomer in presence of desired enantiomer, capping of the surface OH groups and finally desorption of the encapsulated enantiomer, for the selective adsorption of the said enantiomer from the racemic mixture to effect resolution with high optical purity.
During the hydrolytic polymerization, the monomer of the functional silica of 3-amino alkanes form strong hydrogen bonding interactions with the —COOH group of protected amino acid and also forms hydrophobic and dipole-dipole interactions between two functional monomers or functional monomer and imprint molecule. Higher enantioselectivities are obtained when silica as synthesized is used in the resolution of amino acid derivatives and mandelic acid. The nanoporous silica thus developed possess high loading capacity to enable to adsorb desired enantiomer 3-12%. Thus the nanoporous silica gives higher through put in the resolution of racemic mixture for the first time, which is not possible with polymers. Thus earlier patents fell short of expectations for commercial reality and economics of the process. Therefore, silica as synthesized is better option in particular for the resolution of racemic compounds having functional groups. Thus, this invention offers the best techno-economic route for resolution of amino acid derivatives and for mandelic acid.
Accordingly the present invention provides a process for the separation of racemic mixtures comprising developing a denser molecular imprint on silica with a desired enantiomer by sol-gel protocol, said sol-gel protocol comprising hydrolytic control polymerization of a silica source as the monomer and amino alkylsilane as a functional monomer in the presence of the desired enantiomer, capping of surface OH groups and desorption of encapsulated enantiomer from the silica, for the selective adsorption of 3-12% of the said enantiomer from the racemic mixture to effect resolution with high optical purity.
In one embodiment of the invention, the alkyl group in the amino alkyl silane is selected from the group consisting of ethyl, propyl and butyl.
In another embodiment of the invention the silica used is a nanoporous silica designed and developed with the defined shape and size to suit the desired enantiomer for resolution of the corresponding racemic mixture.
In another embodiment of the invention, the silica source used is tetraethylorthosilicate (TEOS).
In another embodiment of the invention, the functional monomer comprises 3-amino alkyl triethoxysilane.
In yet another embodiment of the invention, the alkyl is selected from the group consisting of ethyl, propyl, and butyl.
In another embodiment of the invention the imprinted silica used for resolution is recycled for number of times.
In another embodiment of the invention the ratio of imprint to the functional monomer molecule is from 1:2 to 1:5.
In an embodiment of the invention the enantiomeric imprints used comprises Cbz protected (L)-alanine, (L)-phenylalanine and (L)-glutamic acid, and 1-mandelic acid.
In another embodiment of the invention the percentage of imprint molecule to TEOS used is from 2% to 10%.
In yet another embodiment of the invention, the solvent used for controlled hydrolysis is distilled water.
In still another embodiment of the invention the capping of surface-OH group is carried out with an equimolar mixture of 1,1,1,3,3,3-hexamethyldisilazane arid chlorotrimethyl silane.
DETAILED DESCRIPTION OF THE INVENTION
The present invention applies the principles of enzyme specificity and catalysis in a non-biological context. In the most general terms, the present invention relates to the development of amorphous inorganic materials having discrete voids of controlled sized and shape that are akin to enzymatic active sites. The size and shape of the voids are readily varied and are typically complementary to the desired substrate (depend on desired enantiomer molecule). One or more spatially organized functional groups are positioned in a defined three dimensional relationship within each void and with respect to each other such that the imprinted material contains a plurality of substantially similar functionalized void spaces. By varying both the positions and identities of the one or more functional groups, diverse sets of substrate specific adsorbents and non-biologically-based catalysts are created.
The novelty of the present invention lies in the design and development of nanoporous silica with the defined shape and size to suit the desired enantiomer for resolution of the corresponding racemic mixture with high enantiomeric purity for the first time. The nanoporous silica is developed by sol-gel protocol, involving the hydrolytic polymerization of tetraethylorthosilicate using as a monomer and amino alkylsilane as a functional monomer in presence of desired enantiomer, capping of the surface OH groups and finally desorption of the encapsulated enantiomer, for the selective adsorption of the said enantiomer from the racemic mixture to effect resolution with high optical purity.
During the hydrolytic polymerization, the monomer of the functional silica of 3-amino alkanes form strong hydrogen bonding interactions with the —COOH group of protected amino acid and also forms hydrophobic and dipole-dipole interactions between two functional monomers or functional monomer and imprint molecule. Higher enantioselectivities are obtained when silica as synthesized is used in the resolution of amino acid derivatives and mandelic acid. The nanoporous silica thus developed possess high loading capacity to enable to adsorb desired enantiomer 3-12%. Thus the nanoporous silica gives higher through put in the resolution of racemic mixture for the first time, which is not possible with polymers. Thus earlier patents fell short of expectations for commercial reality and economics of the process. Therefore, silica as synthesized is better option in particular for the resolution of racemic compounds having functional groups. Thus, this invention offers the best techno-economic route for resolution of amino acid derivatives and for mandelic acid.
As explained above, the source of silica used is preferably tetraethylorthosilicate (“TEOS”) monomer and the functional monomer is preferably 3-amino alkyl triethoxysilane, where the alkyl is selected from ethyl, propyl and butyl. Preferably, the ratio of imprint to the functional monomer molecule is from 1:2 to 1:5. The imprinted silica used for resolution of racemic mixture can be recycled for number of cycles.
The enantiomeric imprints used are Cbz protected (L)-alanine, (L)-phenylalanine and (L)-glutamic acid, 1-mandelic acid etc. The percentage of imprint molecule to TEOS used is from 2% to 10%. The solvent used for controlled hydrolysis is preferably distilled water while the capping of the surface —OH group is with equimolar mixture of 1,1,1,3,3,3-hexamethyldisilazane and chlorotrimethyl silane.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the invention.
EXAMPLE 1
a) Cbz-protected (L)-Alanine as a Print Molecule:
3.77 g of Cbz-protected (L)-alanine and 6.558 g of 3-aminopropyltriethoxysilane were taken in a 500 ml beaker containing 100 nm of absolute ethanol and stirred at room temperature for 48 h. to allow strong acid-amine hydrogen bonding interactions.
b) Synthesis of Imprinted Silica Gel:
All imprinted materials were synthesized according to the following protocol. The amount of the imprint compound used in the synthesis corresponds to about 6 mol % of imprint Si relative to the source of silica, Tetraethylorthosilicate (“TEOS”).
In a typical procedure, To the 1.0 lit beaker containing acid-amine mixture (1.31 g in 100 ml ethanol) 70 ml of dry ethanol (Total ethanol quantity is 170 ml), 23.0 ml of TEOS was added to this mixture and 3.0 ml of 2-propanol was added to this mixture. Finally 61 ml of pH 2.0 aqueous HCl were added to the gel mixture. The mixture was covered loosely with a jar cap and stirred for 12 hours at 0° C. It was then covered with wax paper and stirred for 6 hours at 8° C. and for 6 hours at 15° C. and five days at room temperature. With approximately ¾ of an inch liquid head remaining in the jar after this period, the mixture was transferred to a 40° C. oven and covered loosely with a jar cap. The mixture was aged in the oven for four days and at 80° C. for two days after which time gelation had occurred and the caps were removed. The resulting glass monoliths were further aged in the oven for a period of two days. The obtained silica gel was washed thoroughly with methanol. The as-made imprinted silica monoliths were ground into a powder of, a mortar and pestle. The resulting powder was dried under ambient conditions and Soxhlet extracted with acetonitrile refluxing in calcium hydride for a period of 24 hours to remove water and ethanol from the pores of amorphous silica. The amorphous silica was then separately washed with 25 ml/g of silica with chloroform and pentane and allowed to dry.
c) Capping the Surface of Silica:
Capping OH-defect sites on the surface with an equimolar mixture of 1,1,1,3,3,3-hexamethyldisilazane and chlorotrimethylsilane at room temperature for 24 hours (“capped material”) further processed the extracted material.
d) Removal of the Imprint Molecule:
Typical weight increase before/after the capping procedure is approximately 3-5 weight %. Subsequent to the capping procedure, the silica was washed with 50 ml/g of silica with anhydrous THF, anhydrous acetonitrile, chloroform, and pentane, and allowed to dry in a desiccator under ambient conditions. The washed and capped material is then ready for imprint removal.
The capped silica (2.00 g) was taken in 100 ml round-bottomed flask containing methanol (50 ml). To this 1M solution of 6 ml of NaHCO 3 solution was added and refluxed for 24 h. The reaction mixture was filtered off to separate the silica and washed with water and methanol respectively. The resultant silica gel was Soxhlet extracted with methanol for 36 h to get the complete removal of print molecule from silica. After that it was washed with chloroform, acetonitrile and dry THF.
e) 2.0 g of silica was taken in 50 ml of round-bottomed flask containing 20 ml-of ethanol. To this 0.12 gm of CBZ-protected D, L-alanine was added and stirring continued. After 12 h 90% of (L)-alanine derivative was in equilibrium with the imprinted silica and analyzed by chiral HPLC.
EXAMPLE 2
Cbz-protected (L)-phenylalanine as a print molecule:
The imprinted silica was prepared in the same manner as in example 1 using Cbz-protected (L)-phenylalanine as a print molecule. 2.0 g of silica was taken in 50 ml of round-bottomed flask containing 20 ml of ethanol. To this 0.12 gm of Cbz-protected D, L-phenylalanine was added and stirring continued. After 12 h 70% of (L)-Phenyl alanine derivative was in equilibrium with the imprinted silica and analyzed by chiral HPLC.
EXAMPLE 3
Cbz-protected L-Glutamic acid as a print molecule:
The imprinting silica was prepared in the same manner as in example 1, using Cbz-protected (L)-Glutamic acid as a print molecule. 2.0 g of silica was taken in 50 ml of round-bottomed flask containing 20 ml of ethanol. To this 0.12 gm of Cbz-protected D, L-Glutamic acid was added and stirring continued. After 12 h 63% of (L)-Glutamic acid derivative was in equilibrium with the imprinted silica and analyzed by chiral HPLC.
EXAMPLE 4
1-mandelic acid as a print molecule:
The imprinting silica was prepared in the same manner as in example 1, using 1-mandelic acid as a print molecule. 2.0 g of silica was taken in 50 ml of round-bottomed flask containing 20 ml of ethanol. To this 0.12 gm of d,l mandelic acid was added and stirring continued. After 12 h 90% of 1-mandelic acid was in equilibrium with the imprinted silica and analyzed by chiral HPLC.
The Main Advantages of the Present Invention are:
1. A novel process for the resolution of racemic compounds using nanoporous imprinted silica.
2. Cheaply and readily available tetraethylorthosilicate is used as the silicon source for the preparation of imprinted silica.
3. The selectivities are good and comparable with conventional methods
4. The present process dispenses the use of expensive chemicals for resolution
5. The present process is environmentally safe since there is no disposal problem.
6. The imprinted silica can be used for number of cycles with consistent selectivity.
7. The process is simple, clean and neat
8. The process is economical since it is having high imprint loading.
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The present invention relates to a process for the separation of racemic mixtures comprising development of a denser molecular imprint on silica with a desired enantiomer by sol-gel protocol.
| 1 |
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates in general to an improved ultrafine finish for workpieces having various elements with different hardnesses and, in particular, to an improved system, method, and apparatus for nanogrinding and chemical mechanical nanogrinding of workpieces with both chemical and mechanical processes.
2. Description of the Related Art
Data access and storage systems generally comprise one or more storage devices that store data on magnetic or optical storage media. For example, a magnetic storage device is known as a direct access storage device (DASD) or a hard disk drive (HDD) and includes one or more disks and a disk controller to manage local operations concerning the disks. The hard disks themselves are usually made of aluminum alloy or a mixture of glass and ceramic, and are covered with a magnetic coating. Typically, one to five disks are stacked vertically on a common spindle that is turned by a disk drive motor at several thousand revolutions per minute (rpm). Hard disk drives have several different typical standard sizes or formats, including server, desktop, mobile (2.5 and 1.8 inches) and microdrive.
SUMMARY OF THE INVENTION
A typical HDD uses an actuator assembly to move magnetic read/write heads to the desired location on the rotating disk so as to write information to or read data from that location. The magnetic read/write devices are mounted on a slider. A slider generally serves to mechanically support the head and any electrical connections between the head and the rest of the disk drive system. The slider is aerodynamically shaped to glide over moving air in order to maintain a uniform distance from the surface of the rotating disk, thereby preventing the head from undesirably contacting the disk.
A slider is typically formed with an aerodynamic pattern of protrusions on its air bearing surface (ABS) that enables the slider to fly at a constant height close to the disk during operation of the disk drive. A slider is associated with each side of each disk and flies just over the disk's surface. Each slider is mounted on a suspension to form a head gimbal assembly (HGA). The HGA is then attached to a semi-rigid actuator arm that supports the entire head flying unit. Several semi-rigid arms may be combined to form a single movable unit having either a linear bearing or a rotary pivotal bearing system.
The head and arm assembly is linearly or pivotally moved utilizing a magnet/coil structure that is often called a voice coil motor (VCM). The stator of a VCM is mounted to a base plate or casting on which the spindle is also mounted. The base casting with its spindle, actuator VCM, and internal filtration system is then enclosed with a cover and seal assembly to ensure that no contaminants can enter and adversely affect the reliability of the slider flying over the disk. When current is fed to the motor, the VCM develops force or torque that is substantially proportional to the applied current. The arm acceleration is therefore substantially proportional to the magnitude of the current. As the read/write head approaches a desired track, a reverse polarity signal is applied to the actuator, causing the signal to act as a brake, and ideally causing the read/write head to stop and settle directly over the desired track.
The motor used to rotate the disk is typically a brushless DC motor. The disk is mounted and clamped to a hub of the motor. The hub provides a disk mounting surface and a means to attach an additional part or parts to clamp the disk to the hub. In most typical motor configurations of HDDs, the rotating part of the motor (the rotor) is attached to or is an integral part of the hub. The rotor includes a ring-shaped magnet with alternating north/south poles arranged radially and a ferrous metal backing. The magnet interacts with the motor's stator by means of magnetic forces. Magnetic fields and resulting magnetic forces are induced via the electric current in the coiled wire of the motor stator. The ferrous metal backing of the rotor acts as a magnetic return path. For smooth and proper operation of the motor, the rotor magnet magnetic pole pattern should not be substantially altered after it is magnetically charged during the motor's manufacturing process.
As mentioned above the read/write head comprises an electromagnetic coil writer, a GMR or TMR reader, and a slider body. It flies over the magnetic disk to perform the read and write functions. To achieve optimum performance, the spacing between the transducer and the disk, called the magnetic space 19 ( FIG. 1 ), must be consistently maintained and has become consistently smaller over time with the increasing of recording areal density. The magnetic space 19 is defined as the fly height 21 plus the pole tip recession (PTR) 11 .
The PTR 11 has been a major contributor to the magnetic space loss for high areal density products. As shown in FIG. 1 , the PTR is the height difference between the pole tips 13 and a plane 15 fitted to the ABS 17 . It is caused by the differences in the removal rates of metal poles, alumina, and AlTiC in the slider abrasive finishing process. The slider abrasive finishing process critically affects the magnetic, electrical, and mechanical performances, as well as the stability of the recording heads. Therefore, ultraprecision abrasive finishing is a key technology in the final finishing of thin film magnetic recording heads.
SUMMARY OF THE INVENTION
One embodiment of a system, method, and apparatus for nanogrinding and chemical-mechanical nanogrinding is disclosed. The present invention achieves near-zero pole tip recession (PTR) to minimize magnetic space loss of the head transducer to media spacing loss, alumina recession (AluR)/and trailing edge profile variation, and smooth surface finish (sub-nm Ra) and minimal smearing across multi-layers of thin films and the hard substrate to meet the requirements of high areal density thin film magnetic head for hard disk drive (HDD).
Lapping is a material removal process for the production of flat surfaces by free-abrasive three-body abrasion. A loose abrasive and a hard lapping plate are used for this purpose. During lapping, besides three-body abrasive abrasion (i.e., rolling), some abrasives also temporarily embed in the lapping plate to cause some temporal two-body abrasion. High material removal rate can be achieved by free-abrasive lapping.
Nanogrinding is a fixed abrasive two-body abrasion process that uses fixed-abrasive embedded in a soft plate as a finishing process for producing flat and good surface finish. The material removal rate from fixed-abrasive nanogrinding is lower than from free-abrasive lapping, but it can produce superior surface planarity (e.g., less recession). The recording heads are finished by free-abrasive lapping followed by nanogrinding. High material removal is achieved by free-abrasive lapping, and good surface finish and planarity are obtained by nanogrinding. Appropriate chemical-mechanical interactions in nanogrinding, called chemical-mechanical nanogrinding, result in further improvements in achieving good surface finish and planarization.
The planarity and surface finish from nanogrinding are superior to those from free-abrasive lapping. The PTR can be improved to about 8 nm by nanogrinding process versus about 30 nm by free-abrasive lapping process. With a fine chemical mechanical nanogrinding process, PTR can be improved to a mean of about less than 1.0 nm. In addition, nanogrinding is virtually scratch-free in contrast to the significant scratching of free-abrasive lapping.
Process integration and throughput issues are considered for free-abrasive and fixed-abrasive processes. Free-abrasive lapping process is recommended for high material removal rates followed by the fixed-abrasive nanogrinding process for achieving excellent finish. Further planarity and surface finish improvements are achieved by adjusting mechanical and chemical interaction in fixed-abrasive nanogrinding and chemical-mechanical nanogrinding.
A metal plate (e.g., zinc lapping plate) may be used for free-abrasive rough lapping, and a tin lapping plate is used for nanogrinding. Monocrystalline diamond slurry is used for high material removal free-abrasive lapping, and polycrystalline diamond slurry for nanogrinding. The polycrystalline diamond abrasive in ethylene glycol is dispensed on the plate surface and then the diamond abrasive is embedded or charged onto the lapping plate with a ceramic conditioning ring to form the nanogrinding plate.
The reactive solution plays an important role in chemical-mechanical nanogrinding. The chemistry of the reactive solution facilitates selective removal of the ceramic layers, namely, AlTiC and Al 2 O 3 to metal, namely, NiFe and, hence, compensate for the preferential mechanical removal of the softer metal over the harder ceramic. The specific choice of the solution (e.g., viscosity, suspension, surfactant) and its chemical interaction (e.g., oxidizer, corrosion inhibitor, pH, and complex chelating agent) with the work material help achieve good surface and subsurface integrity, machining accuracy (e.g., less PTR), high material removal, final cleaning (e.g., rinseability), and abrasive and plate life.
The foregoing and other objects and advantages of the present invention will be apparent to those skilled in the art, in view of the following detailed description of the present invention, taken in conjunction with the appended claims and the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
So that the manner in which the features and advantages of the invention, as well as others which will become apparent are attained and can be understood in more detail, more particular description of the invention briefly summarized above may be had by reference to the embodiment thereof which is illustrated in the appended drawings, which drawings form a part of this specification. It is to be noted, however, that the drawings illustrate only an embodiment of the invention and therefore are not to be considered limiting of its scope as the invention may admit to other equally effective embodiments.
FIG. 1 is a schematic side view of a conventional slider flying over a surface of a disk media;
FIGS. 2 and 3 are sectional side views of the topography results of free-abrasive lap and nanogrinding, respectively; and
FIG. 4 is a high level flowchart depicted one embodiment of a method constructed in accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Referring to FIGS. 2-4 , one embodiment of the present invention comprises a system, apparatus, and method of treating a surface of a workpiece 31 in order to improve a surface finish thereof. The workpiece 31 may comprise many different objects, but is well suited for a hard disk drive slider that is formed from a variety of different materials, such as metallic and ceramic materials.
One embodiment of a method of the present invention first comprises providing a workpiece 31 having a first material 33 (e.g., pole material) and a second material 35 (e.g., Al 2 O 3 ) that differs from the first material 33 . As depicted at block 61 ( FIG. 4 ), the method further comprises lapping the workpiece 31 with a lapping substrate 37 and an abrasive slurry 39 between the workpiece 31 and the lapping substrate 37 such that portions of the first material 33 and the second material 35 are mechanically removed from the workpiece 31 .
The method additional comprises nanogrinding the workpiece 31 with a nanogrinding substrate 41 and a nonabrasive solution 45 , the nanogrinding substrate 41 having an abrasive 43 embedded in a surface thereof that mechanically removes additional portions of the first material 33 and the second material 35 from the workpiece 31 , as illustrated at block 63 . In addition, the method comprises selectively chemical mechanical removing (block 67 ) an additional portion of the second material 35 from the workpiece 31 with the nonabrasive solution 45 . In one embodiment, both the mechanical and the chemical removal of material occur simultaneously to provide a very efficient and effective process.
The workpiece 31 defines a plane 47 ( FIG. 3 ), and both the first and second materials 33 , are removed from the workpiece 31 to within about one nanometer of the plane 47 , and a surface roughness of approximately 0.5 nm rms. In one embodiment, nanogrinding removes more of the first material 33 than the second material 35 , and the chemical removal step removes more of the second material 35 than the first material 33 .
The method optionally further comprises providing the workpiece 31 with a third material 49 (such as AlTiC), and the chemical removal step comprises adding a nonabrasive substance 51 to the nonabrasive solution 45 for selectively chemically mechanical removing a portion of the third material 49 from the workpiece 31 . The method also optionally comprises providing the nonabrasive solution 45 with desired properties (block 65 ) selected for viscosity, suspension, surfactant, and chemical interaction with the workpiece, including oxidizer, corrosion inhibitor, pH, complex chelating agent, and a selected conductivity that avoids corrosion of the workpiece 31 and reduces electrostatic discharge.
The nonabrasive solution 45 may include a water-soluble hydrocarbon chain of a hydroxyl (OH) group ethylene glycol solution, and may further comprise colloidal silica solution of having an average size of approximately 10 nm. Furthermore, the pH of the nonabrasive solution may be adjusted with organic additives, and the viscosity of the nonabrasive solution may be altered (e.g., increased) by partially replacing ethylene glycol with diethylene glycol, triethylene glycol, or propylene glycol and dipropylene glycol. The method may further comprise adding a corrosion inhibitor, such as BTA, Triton, Standapol, or Texapon, for example.
The slider abrasive finishing process critically affects the magnetic, electrical, and mechanical performance of the recording heads. Therefore, ultraprecision abrasive finishing is a key technology for final finishing of the thin film magnetic recording heads. The reactive solution plays an important role in chemical-mechanical nanogrinding. The chemistry of the reactive solution can facilitate selective removal of the ceramic layers, such as AlTiC and Al 2 O 3 to metal, e.g., NiFe, and hence compensate for the preferential mechanical removal of the softer metal over the harder ceramic.
The specific choice of the solution (viscosity, suspension, surfactant) and its chemical interaction (oxidizer, corrosion inhibitor, pH, and complex chelating agent) with the workpiece or work material are critical in achieving good surface and subsurface integrity, machining accuracy (less PTR), high material removal, final cleaning (rinseability), and abrasive and plate life. Proper conductivity of the reactive solution is also required, e.g., low conductivity to avoid GMR/TMR stack corrosion but some conductivity to eliminate ESD damage issue during lapping and nanogrinding.
The pH of reactive solutions for chemical-mechanical nanogrinding may be adjusted by organic additives. PTR generally decreases with increasing pH and is smallest around pH 10. However, if the pH level becomes too high (e.g., pH 11), it may contribute to sensor corrosion, especially for copper layer in sensors.
Viscosity may be increased by partially replacing ethylene glycol (C 2 H 6 O 2 ) with either diethylene glycol, triethylene glycol, or propylene glycol and dipropylene glycol (viscosity increasing). The viscosity of an oil-soluble solution (e.g. petroleum-base) is lower than the water-soluble solution (e.g. ethylene glycol). Corrosion inhibitors such as BTA, Triton, Standapol, or Texapon may be used.
There are also oil-soluble nanogrinding solutions. By adding carboxyl (COOH) polar functional groups, such as C 17 H 31 COOH, C 17 H 33 COOH, and C 17 H 33 COOH to oil-soluble lapping/nanogrinding solutions, such as petroleum, can achieve better metal surface finish.
The planarity and surface finish from nanogrinding are found to be much better than those from free-abrasive lapping. The planarity and surface finish improvement can be achieved by adjusting mechanical and chemical interaction in chemical mechanical nanogrinding. PTR can be improved to a mean of about 0.5 nm. The surface roughness of AITiC/NiFe/Al 2 O 3 improves to about 0.5 nm rms. Process integration and throughput issues are considered for free- and fixed-abrasive processes. Free-abrasive process is recommended for high material removal rate followed by chemical-mechanical nanogrinding for achieving excellent finish.
The present invention has several advantages, including the ability to achieve near-zero PTR. A high material removal is achieved by free-abrasive lapping, and good surface finish and planarity are obtained by fixed-abrasive nanogrinding. Chemical-mechanical interactions in chemical mechanical nanogrinding result in further improvements in achieving good surface finish and planarization.
The planarity and surface finish from fixed-abrasive nanogrinding are superior to those from free-abrasive lapping. In addition, fixed-abrasive nanogrinding is virtually scratch-free in contrast to the significant scratching of free-abrasive lapping. Further planarity and surface finish improvements are achieved by adjusting mechanical and chemical chemical-mechanical nanogrinding. The chemistry of the reactive solution facilitates selective removal of the ceramic layers and compensate for the preferential mechanical removal of the softer metal over the harder ceramic. The solution and its chemical interaction with the work material help achieve good surface and subsurface integrity, machining accuracy, high material removal, final cleaning, and abrasive and plate life.
While the invention has been shown or described in only some of its forms, it should be apparent to those skilled in the art that it is not so limited, but is susceptible to various changes without departing from the scope of the invention.
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A chemical-mechanical nanogrinding process achieves near-zero pole tip recession (PTR) to minimize magnetic space loss of the head transducer to media spacing loss, alumina recession and trailing edge profile variation, and smooth surface finish with minimal smearing across multi-layers of thin films and the hard substrate to meet the requirements of high areal density thin film magnetic heads for hard disk drives (HDD). With a fine chemical mechanical nanogrinding process, PTR can be improved to a mean of about 0.5 nm.
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TECHNICAL FIELD
The present invention relates to paperboard cartons. More particularly, it relates to pouring spouts for paperboard cartons wherein the spout is integrated with the carton, is useful with cartons having a relatively small or narrow width or thickness, is "sift proof" to protect the contents and prevent leakage, and provides evidence of tampering.
BACKGROUND OF THE INVENTION
U.S. Pat. Nos. 4,569,443 and 5,215,250 address many of the problems associated with pouring spout structures for paperboard cartons. However, one problem unsolved by the cartons of the two patents is how to provide optimal barrier protection for the material contained in a carton with a pouring spout, while at the same time providing a pouring spout that is easy for a consumer to open.
Typically, cartons with pouring spouts for containing hygroscopic material or particulate materials requiring a degree of barrier protection are adapted to prevent the absorption of moisture or other contaminants by having, for example, films or other coatings applied to or integrated with the carton walls. Sifting or leakage from pouring spout containers can be a particular problem, because the spout structure generally includes aligned or overlaid seams, edges, cuts or perforated lines. Cuts that penetrate completely through the carton walls are a particular problem, especially if the contents are in direct contact with the cuts and the cuts provide a pathway directly to the exterior of the carton.
U.S. Pat. Nos. 4,732,315; 4,718,557; 3,346,165; and 2,819,832 are representative of attempts to solve the barrier protection and leakage problems. The latter patent discloses a leak-proof carton with superimposed inside and outside spout openings. However, when such double perforated flap structures are used, the material contained in the carton may still leak out and moisture may still easily penetrate the carton, particularly when perforations and throughcuts in separate layers are adjacent or superimposed along their length.
U.S. Pat. No. 4,909,395 discloses a carton having a double panel end closure with an opening flap in the outer panel providing access to a dispensing aperture in an inner panel. The opening structure includes a partially pre-cut bridge in an adhesive area for securing the outer opening flap to the inner panel, but again, aligned cuts or cuts that run from the carton exterior to the inner opening can provide a path for moisture entry.
It is well recognized that it is desirable to have an opening structure that can provide evidence of attempted or actual tampering. In this regard, U.S. Pat. No. 4,569,443 discloses a carton with a pouring spout and a removable access tab that provides a means for determining whether tampering has occurred. U.S. Pat. Nos. 3,395,848; 4,706,875 and 4,799,594 all disclose recloseable opening and dispensing structures for cartons in which perforations or other lines of weakness are used to form a portion of the structure to help provide evidence of tampering if they appear to have been torn.
Another problem in cartons with pouring spouts is that the spout or carton may be damaged or rendered useless by pulling the spout too far out while opening it or forcing it inwardly too far into the carton interior when reclosing it. This problem is partially addressed in U.S. Pat. No. 5,215,250, wherein a spout structure includes wings for forming the sides of the spout and for guiding the outward travel of the spout. Another portion of the spout acts as a stop to prevent the spout from being pushed inadvertently into the carton interior. However, as mentioned above, the disclosed spout does not adequately address the problem of barrier protection and resistance to leaking, nor does it include positive stop features for controlling the inward and outward movement of the spout.
Clearly, despite the improvements described in the above-cited patents, there is a need for a carton including a durable, integral dispensing spout structure for powdered or granular materials that prevents leakage and provides barrier protection for the contents, while at the same time providing tamper evidence and easy opening.
SUMMARY OF THE INVENTION
The present invention overcomes the problems unaddressed by known cartons with pouring spout structures and provides a sift proof carton with a pouring spout with complete side walls, a tamper evidence feature and control of the inward and outward movement of the spout structure during opening and closing. It further provides a carton that can be produced in a rapid, efficient and economical manner.
A substantially sift proof pouring spout carton is provided by the carton body and cooperating interior and exterior pouring spout components formed in part by cuts partially penetrating the thickness of the material from which the carton is formed, free edges or lines of perforations, none of which are aligned and overlaid along their length. The exterior spout component is split to form an automatic supplemental side wing, includes an integral inward travel stop tab, and is releasably connected to a tamper evidence tab formed in the carton body. The inward travel stop tab and tamper evidence tab are defined at least in part by cuts partially penetrating the carton body and resultant delamination areas.
The invention encompasses a single-piece fiat blank for forming the spout and carton with which it is integrated. The carton includes first and second side wall panels, first and second end wall panels hingedly connected to the first and second side wall panels, and top and bottom closure panels attached to the side wall panels to form a tubular carton having a rectangular cross-section. The pouring spout is formed integrally in one of the wall panels of the carton by the cooperating interior and exterior pouring spout components formed by die-cutting the blank. The carton and interior and exterior pouring spout components have no throughcuts, free edges or lines of perforations which are aligned and overlaid along their length and is, therefore, substantially sift proof.
It is an object of the present invention to provide a substantially sift proof paperboard carton, formed from a single-piece blank, having an integral pouring spout.
Another object of the present invention is to provide a pouring spout for a carton wherein the spout has full spout-forming side walls when deployed to its open, dispensing position, and includes an outward travel stop feature to control the outward movement of the spout.
Still another object of the present invention is to provide an integral pouring spout for a carton wherein the spout is recloseable and includes an inward travel stop to prevent a user from pushing it too far into the carton.
Yet another object of the present invention is to provide a carton with an integral pouring spout wherein the carton includes a tamper evidence feature adjacent to the spout to provide evidence of attempted or actual tampering.
An advantage of the pouring spout of the present invention is that it provides a recloseable pouring spout with complete spout sidewalls and inward and outward travel stops and a tamper evidence tab, yet is also substantially leak proof and provides significant barrier protection for the contents.
A feature of the present invention which accomplishes the advantage of significant barrier protection and leakage prevention are delamination regions formed in the carton adjacent to the pouring spout structure to provide the stops for controlling movement of the spout and the tamper evidence tab. A first delamination region is formed in the body of the carton adjacent to the spout and provides the inward travel stop associated with the spout, and a second delamination region is formed in the carton body adjacent to the spout to provide the tamper evidence tab of the present invention.
These and other objects, features and advantages of the present invention will become more fully apparent and understood with reference to the following specification, and to the appended drawings and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plan view of the inside of the blank from which the carton of the present invention is formed and shows the die cut profile thereof;
FIG. 1a is a cross sectional view taken along line 1a--1a in FIG. 1 and shows the partial cut bridge incisions defining, in part, the tamper evidencing tab of the present invention;
FIG. 1b is a cross sectional view taken along line 1b--1b in FIG. 1 and shows the partial cut gate incisions for forming, in part, an inward travel stop feature associated with the spout of the present invention;
FIG. 2 is a perspective view showing an initial fold in the forming of the carton of the present invention;
FIG. 3 is a perspective view showing the initial fold completed;
FIG. 4 is a perspective view showing a second fold in the forming of the carton;
FIG. 5 is a perspective view showing the tubed blank of the carton of the present invention;
FIG. 6 is a perspective view showing the carton of the present invention folded up and closed, generally as it would appear when filled, sealed and ready for purchase by a consumer;
FIG. 7 is a perspective view depicting an initial step in opening-the carton of the present invention, namely, the removal of the tamper evidencing tab;
FIG. 8 is a perspective view depicting a subsequent step in opening the spout of the carton of the present invention;
FIG. 9 is a perspective view depicting the spout open and ready to dispense contents, and also revealing the inward movement stop tab feature of the present invention;
FIG. 10 is a perspective view depicting the outward movement stop feature of the carton of the present invention, and the cooperation of the internal and external spout components of the present invention; and
FIG. 11 is a perspective view depicting the carton of the present invention reclosed, and showing the inward movement stop tab abutting the carton body to control the inward movement of the spout.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
FIG. 1 depicts a flat, single-piece paperboard blank 2 for forming a pouring spout carton (depicted fully erected in FIG. 6) in accordance with the present invention. The blank 2 may be formed from conventional paperboard or box board of any desired caliper, and is die-cut and scored as depicted by well-known, conventional methods. In all the Figures, double lines indicate fold score lines and single solid or dashed lines indicate cuts, partial cuts, or free edges.
The blank 2 is cut and scored to define first and second main generally rectangular side wall panels 4, 6, respectively, a first end wall panel 8 between and hingedly connected to the side wall panels 4, 6 at parallel fold lines 10, 12, respectively. A glue flap 14 is hingedly connected to the remaining edge of the first side wall panel 4 at a fold line 16, and a second end wall panel 18 is hingedly connected at a fold line 20 to the remaining edge of the second side wall panel 6. Major top and bottom closure flap panels 22, 24, respectively, are hingedly attached to the side walls 4, 6 at fold lines 26, 28, respectively. Minor top closure flap panels 30 are hingedly connected to the end wall 8 and glue panel 14 at fold lines 32 generally collinear with fold line 26. Similarly, minor bottom closure flap panels 34 are foldably connected to the end wall panels 8, 18 at fold lines 36 collinear with the fold lines 28. A small glue receiving bottom closure tab 34' is foldably connected to the glue flap 14. The fold lines 26, 32 at the top of the blank 2 are generally collinear, perpendicular to the fold lines 10, 12, 16, 20 at the side edges of the main side and end wall panels and will form the top edge 37 of the carton (FIG. 6). Similarly, the fold lines 28, 36 at the bottom of the blank 2 are generally collinear, perpendicular to the fold lines 10, 12, 16, 20 and will form the bottom edge 39 of the carton (FIG. 6). Thus, the blank 2 will form a generally tubular carton of rectangular cross-section with closed ends when foldably erected.
A glue flap extension panel 40 is hingedly connected to the glue flap 14 at a fold line 42. An interior pouring spout component, indicated generally at 44, is formed in part in glue flap 14 and in part in the extension panel 40. The interior or inside pouring spout component 44 includes a generally rectangular center part 46 with a base hinge line 48, parallel side edges 50, 52 and a top free edge 54. One of the side edges, the edge 52, is collinear with and lies along the fold line 42 between the glue panel 14 and the extension panel 40. A first, generally rectangular, small spout side wall wing 56 is hingedly attached at one side 50 of the center part 46 and extends laterally away therefrom. The small wing 56 has upper and lower edges 58, 60, rounded corners 62 and a free side edge 64 parallel to the side edge 50. A second, larger spout side wall or wing 66 is hingedly attached to the other side 52 of the center part 46, and extends generally outwardly away therefrom in the plane of the blank 2. The larger spout wing 66 has a bottom edge 68 generally collinear with the base hinge edge 48 of the center portion 46, a rounded corner 70, a free side edge 72 generally parallel with the edge 52 of the center part 46 and an irregular upper edge 74. Immediately adjacent to the fold line 42, the upper edge 74 is collinear with the upper edge 54 of the center part, but then it curves sharply toward the edge bottom 68 to a V-shaped point 76. The upper edge 74 includes a straight portion adjacent to the edge 72 of the large wing 66 that extends at an angle from the point 76 to the outer edge 72 thus forming an outward travel stop lobe 82 for controlling the outward movement of the pouring spout of-the present invention. A stripped out area 80 is formed adjacent to the larger wing 66.
With continued reference to FIG. 1, the blank 2 includes an external or exterior pouring spout component, indicated generally at 86. The exterior pouring spout component is formed substantially in the second end wall 18 in the upper region thereof. The exterior pouring spout component 86 is split into at least two portions, including a generally rectangular exterior pouring spout center tab 88 having a hinge base 90, two parallel side edges 92, 94, and a top free edge 96. Although the center tab 88 will be generally rectangular in most cartons, other shapes (e.g., parallelogram, truncated pyramidal, trapezoidal) may be used as well. One of the side edges, edge 92, is collinear with the fold line 20 between the side wall 6 and the end wall 18. The external pouring spout component 86, particularly the center tab 88, is hingedly connected at its base 90 to the side wall 18.
The split or divided external pouring spout component of 86 includes a generally triangularly shaped exterior supplemental wing 98. The exterior wing 98 and the center tab 88 have a common side edge 100 formed by a single continuous incision cut entirely through the thickness of the blank 2 for forming the carton body. The wing 98 is hingedly connected to the end wall 18 along a hypotenuse edge formed by a perforated line 102. The remaining, uppermost edge of the wing 98 is defined by a single, continuous cut line 104 slightly offset with respect to the top free edge 96 of the center tab 88. The exterior pouring spout component 86 is held in place in the plane of the blank 2 (the side wall 18) by slight interruptions or nicks 106 in the cut line defining the side edge 100 of the center tab 88.
With continued reference to FIG. 1, and referring to FIG. 1b, an inward travel stop tab 110 is associated with and carried by the external pouring spout component 86, specifically the center tab 88 thereof. The stop tab 110 is defined by a first, straight cut 112 cut partially through the thickness of the side wall 6 on the inside surface of the blank 2 and parallel to the fold line 20. Upper and lower short angled cuts 114, 116, respectively, connect the ends of the straight partial cut 112 and the fold line 20. The term "partial" is intended to describe a cut which only partially penetrates the thickness of blank 2 (and, thus, the walls forming the carton body). Cut lines 112, 114, 116 are partial cuts, penetrating part way into the paperboard forming the blank 2 (side wall 6) to a depth of from 50 to 60 percent of the thickness of the blank 2. As will be explained below, the partial cut lines, 112, 114, 116 create a delaminating region basically congruent or co-existent with the stop tab 110 which, upon opening of the spout as explained below, delaminates or separates from the inside of the side wall 6 of the carton to form the tab 110.
Referring to FIG. 1 and FIG. 1a, the tamper evidence tab 120 of the present invention (see also FIG. 6) is defined in part by cuts on the inside surface of the blank 2 and partially through the end wall 18. The partial cuts include a lower cut 122 generally parallel with the free edge 96 of the center tab 88 and two angled end cuts 124. The upper portion 123 of the tamper evident tab 120 includes a free edge 126 and a parallel partial cut 128 in the inside surface of the blank 2. The free edge 126 and the partial cut 128 are connected by angled free edges 130 and angled partial cuts 132. The partial cuts 122, 124 and 128, 132 associated with the tamper evident tab 120 should penetrate the paperboard forming the blank 2 to a lesser extent than the partial cuts associated with the stop tab 110, penetrating the blank material from about 30 to 40 percent of the thickness of the blank 2.
Referring to FIG. 4, wherein the opposite or exterior side of the blank 2 is visible, the tamper evident tab 120 is completed by two parallel partial cuts 136, 138. These two cuts 136, 138 penetrate the thickness of the blank 2 from about 50 to 60 percent and at one end, are connected to the cut edge 96 forming the top of the central tab 88. At their upper end, the partial cut lines 136, 138 are connected to the ends of the angled free edges 130, terminating at the free edge 19 of the panel 18. With continued reference to FIG. 4, the partial cut line 138 defining one side edge of the tamper evidencing tab 120 continues downwardly continuously along the fold line 20 to the hinge base 90 to assist in creating the delaminating stop tab 110 on the inside of the blank 2.
FIGS. 2-5 depict selected steps in the erection or fold up sequence of blank 2 to form the carton 150 of the present invention (depicted fully formed in FIG. 6). As depicted in FIG. 2, the glue panel 14 and extension panel 40 are first folded about the fold line 16 into the close, parallel, overlapping relationship with the side wall panel 4 as depicted in FIG. 3. Next, as shown in FIG. 4, the blank 2 is folded about the fold line 12 so that the main side wall panels 4, 6 are in close parallel overlapping relation and so that the end wall panel 18 is in contact with the glue flap 14. In this position, the extension panel 40 is in contact with the second main side wall panel 6. Also, in this position, the interior pouring spout component 44, particularly the center portion 46 thereof, is aligned with and generally under the exterior pouring spout component 86, particularly the center tab 88. The blank 2 is secured or held in this position by previously applied glue or other adhesive means applied in the adhesive strip areas 152 depicted in FIG. 3.
In the condition depicted in FIG. 4, the partially folded blank can be shipped to a user who will erect it by "tubing" it, as shown in FIG. 5. The major and minor top and bottom closure panels, indicated generally at 154, 156 in FIG. 5, are folded inwardly one after the other and secured by appropriate means (adhesive, slot and tab arrangements, or the like), whereby a fully erected, sealed carton 150 of substantially rectangular shape is formed as depicted in FIG. 6. The dispensing spout, indicated generally at 158 in FIG. 6, is held closed by the tamper evident tab 120 and the nicks 106.
FIGS. 7 and 8 are fragmentary perspective views illustrating the opening of the carton 150, particularly the opening of the pouring spout 158. With reference to FIG. 7, the tamper evidencing tab 120, particularly the uppermost portion 123 thereof, forms an upstanding, graspable pull tab 160 defined by the free edge 126 which allows the tab 160 to stand up when the carton top closure flaps 154 are folded, is grasped and pulled away from the end wall panel 18 in the direction of arrow "A". As is visible in FIGS. 9-11, removal of the tab 120 and the resultant delamination of the delaminating region of the end wall 18 formed by the partial cut lines identified above, creates a bridge structure 162. The bridge structure 162 is formed by the region of the side wall 18 left when the tamper evidence tab 120 is removed, specifically the area between the upper and lower partial cut lines 128, 122. The bridge structure 162 serves to maintain the integrity and structure of the upper region of the carton 150 after the tamper evident tab 120 is removed.
Referring to FIG. 8, removing the tab 120 creates a space between the bottom of the bridge 162 and the upper edge of the pouring spout 158, specifically the upper free edge 54 of the central part 46. A finger may be used to contact or grasp the top of the pouring spout 158, and the top of the spout 158 is pulled outwardly in the direction of arrow "B", causing the spout 158 to pivot about the superimposed hinge lines 48, 90. The spout 158 is thus extended to the open position shown in FIGS. 9 and 10.
With continued reference to FIG. 8, the function of the inward travel stop tab 110 can be appreciated. As the spout 158 is being opened, the partial cut lines on the inside of the blank 2 defining the inward travel stop tab 110 cause the delamination of delaminating region comprising the tab 110. As the spout 158 is moved to the position depicted in FIG. 9, the stop 110 moves out from under the main side wall 6 with the spout 158 until the bottom edge of the tab 110 clears the fold line 20. The natural resiliency of the material from which the carton of the present invention is formed, and the resiliency created by the tension in that material along the fold line 20, cause the stop 110 to extend outwardly from the spout 158 becomes coplanar or nearly coplanar therewith.
As the spout 158 is being pulled out to its fully opened position, the wings, including the smaller wing 56 and the large wing 66, fold about fold lines 50, 52, respectively, and enable a complete pouring spout 158. One spout side wall is formed by the larger wing 66 and the other by the cooperation of the combined small wing 56 and the exterior supplemental wing 98. As the spout 158 is opened, the irregular upper edge 74 of the large wing 66 follows the edge formed by cut line 54 which defines the free edge of the center part 46 and, thus, the top of the dispensing opening, until reaching the point 76 wherein the angled, catch V-shaped portion of the stop lobe 72 prevents further outward movement of the spout 158.
After a sufficient amount of contents has been dispensed, the user merely pushes (in the direction of arrow "C" of FIG. 11) the upper, free edge of the pouring spout 158 inwardly toward the carton interior. The stop tab 110 delaminated from the inside of the main side wall 6 will abut the corner of the carton formed along the fold line 20 to prevent inward movement past the point at which the spout 158 is generally coplanar with the end wall 18 of the carton 150, thereby ensuring that the spout 158 can be re-opened easily and is not damaged by being pushed too far into the carton. The space between edge 96 and the cut line 122 forming the lower edge of the bridge 162, although closed by the upper region of the center part 46 when the spout 158 is closed (thereby preventing leakage), facilitates re-opening the spout 158.
A number of variations of the present invention can be made. For example, the size of the carton 150 may be varied as may the size of the pouring spout 158. The carton including the pouring spout 158 of the present invention could be produced in cylindrical or other shapes. The preferred material for the carton 150 of the present invention is paperboard, but other suitable material may be used for all or a portion of the invention. The carton 150 may carry indicia, graphics or printing including instructions for opening and reclosing the spout, and it may be overwrapped with appropriate material.
It is contemplated that additional changes, including those mentioned above, can be made without deviating from the spirit of the present invention. Therefore, it is desired that the foregoing description be considered as illustrative, not restrictive, and that reference be made to the appended claims to indicate the scope of the invention.
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A substantially sift proof pouring spout is provided by cooperating interior and exterior pouring spout components formed by partial throughcuts, edges or lines of perforations. The exterior spout component is split to form an automatic supplemental side wing, includes an integral inward travel stop tab, and is releasably connected to a tamper evidence tab. The invention encompasses a single-piece flat blank for forming the spout and a carton with which it is integrated. The inward travel stop tab and tamper evidence tab are defined at least in part by the partial cuts and resultant delamination areas.
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This is a continuation, of application Ser. No. 563,830, filed Dec. 21, 1983, now abandoned.
BACKGROUND OF THE INVENTION
In a molder, for example, a molder disclosed in U.S. Pat. No. 4,105,391, wherein a plurality of neck molds are mounted at given intervals at the undersurface of a transferring platen the neck molds and injection molds are closed, injection cores are inserted, molten resin is injected towards the inside of the neck molds to mold a preform with a bottom around the injection cores, and thereafter the injection cores are removed and transferred together with the neck molds to the succeeding step after the preform has been opened. If the injection molding stage is expanded into the range including stopping positions of two neck molds and the injection molds for molding an inner layer and an outer layer of the preform with a bottom are disposed on the neck molds, as described in U.S. Pat. No. 4,321,029, the inner layer preform and the outer layer preform can be injection molded continuously, and the double-layered preform can be transported to the succeeding step in the state without being modified.
However, in the molder which transfers the preform while being held by the neck mold, the molten resin is poured to the inside of the neck mold to mold the inner layer preform first. Therefore, in the next step of molding the outer layer preform, a space for receiving the poured molten resin poured cannot be formed between the neck mold and the inner layer preform without modification, and the outer layer preform molded therein is in the range molded by the injection mold. Thus, another means has to be used to mold a double-layered preform 1, that is, a preform in which an inner layer 2 and an outer layer 3 are formed up to a neck portion, as shown in FIG. 1.
The necessity of double-layered containers such as bottles for beverages is increasing with the wide use of synthetic resin containers. Particularly, in double-layered containers with an inner layer formed of polyethylene-terephtalate which is liable to give rise to thermal deformation when a filling temperature of content exceeds 80° C., if the neck portion is formed by the inner layer alone even if the outer layer is formed of polycarbonate of excellent heat resistance, the neck portion undergoes thermal deformation at the time of filling to break a seal of a lip portion, failing to serve as a container. However, if the neck portion is formed on both inner and outer layers, deformation of the neck portion can be prevented by polycarbonate of the outer layer.
SUMMARY OF THE INVENTION
The present invention provides an apparatus which can injection mold a double-layered preform having the above-described construction shown in FIG. 1 by adding to the injection molding stage a mold for molding an inner layer preform without changing the structure of the conventional molding machine.
The molder in accordance with the present invention is characterized in that clamping devices are provided internally and externally of a transferring platen on the side of a machine base. The transferring platen includes a plurality of neck molds which rotates and each neck mold stops at a position within the injection molding stage. A first mold closed with an injection core externally of the transferring platen to mold an inner layer preform in the circumference of the injection core and a second mold closed with the injection core having a neck mold and an inner layer preform to mold an outer layer preform in the whole circumference of the inner layer preform are provided, parallel to each other on a clamping plate of the clamping device, and a hydraulically-operated core mold clamping device comprising a rotary plate and a lifting plate for alternately inserting the injection cores into both the molds of from the top.
In the present invention, the clamping devices provided with the two sets of molds and the core mold clamping device are opened and closed in the state where the transferring plate stops and during the time the neck molds are positioned at the injection molding stage, and replacement of the injection cores relative to the two sets of molds is effected in synchronism with the rotation of the transferring platen. Thus, in order to mold a double-layered preform in association with intermittent movement, it is necessary to simultaneously mold an inner layer parison and an outer layer parison, and therefore, at least a pair of injection cores of the same shape are mounted on the core mold clamping device. The injection core has a length sufficient to mold both inner and outer layers up to a lip portion of the preform and is projectingly provided in the center of the lower surface of a member for closing the first mold and neck mold.
In the present invention, the double-layered preform can be molded up to the lip portion similarly to the case where a single layer preform is injection molded. In addition, the molder need not be modified in portions other than the injection molding stage and no considerable delay occurs even during the molding cycle. Moreover, as the case may be, a single layer preform having a wall-thickness of a double layer portion can be molded merely by stopping replacement of the injection core.
Furthermore, in the molder, the molds for molding the inner layer can be provided parallel to each other externally of the injection mold closed with the neck mold, and a core clamping device provided with replacing means can be merely disposed above the molds. Therefore, the molder has features in that construction and operation are not particularly complicated and the basic construction of the existing molding machine need not be changed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a longitudinal sectional front view of a double-layered preform which can be injection molded by a molder for molding double-layered preforms in accordance with the present invention.
FIG. 2 is a side view of a molding machine with an injection molding stage shown in longitudinal section.
FIG. 3 is a plan view of the molding machine.
FIG. 4 is a front view of an injection molding stage of the molding machine.
FIG. 5 is a longitudinal sectional side view of the injection molding stage with molds closed.
FIG. 6 is a longitudinal sectional side view of the injection molding stage wherein an inner layer preform and a double-layered preform are simultaneously injection molded.
DETAILED DESCRIPTION OF THE INVENTION
Specific embodiments of the present invention will now be described in detail with reference to FIGS. 2 to 6.
Referring to FIG. 2 and thereafter, a molder has a lower base platen 12 on a machine base 11 and an upper base platen 13 provided thereabove at a predetermined distance. The space between these base platens is the molding space.
A transferring platen 16, disposed at the undersurface of the upper base platen 13, is intermittently driven through an angle of 90° around a central support shaft 14 by means of a hydraulically or electrically-operated motor 15. Five neck molds 17 closed by springs 17a are disposed at the undersurface of the transferring platen 16 and are equidistant from one another so that they can be opened and closed when moved radially. Each neck mold 17, stops at a preform injection molding stage A, a heating stage B, a stretching and blow molding stage C, and a mold releasing stage D in said order.
In the injection molding stage A, a hydraulically-operated mold clamping device 18 and a core mold clamping device 19 are vertically arranged, and in the heating stage B, a device 20 is provided to heat or cool the preform to control a preform temperature to the stretching and blow temperature.
In the stretching and blow molding stage C, a blowing mold 21, opened and closed radially by oil pressure, as well as a hydraulically operated opening and closing device 22, are provided on the lower base platen 12, and a lifting device 23 for a stretching and blowing rod is provided on the upper base platen 13. In the mold releasing stage D, a device 24 is provided for pushing open the neck mold 17 against the force of 17a.
The above-described devices are opened and closed or moved up and down when the transferring platen 16 stops and each of the neck molds 17 stops at a predetermined position.
The mold clamping devices 18 are provided internally and externally of the transferring platen 16 on the line l--l of FIG. 3 which connects the center of rotation of the transferring platen 16 and the center of the neck mold 17.
Connected to a fixed plate 26 on the side of the machine base on which the mold clamping device 18 is mounted are a pair of tie-rods 27, which serve for holding the upper base platen 13 and for supporting the upper core mold clamping device 19. On the mold clamping plate 28 provided with the mold clamping device 18 is a mold place bed 28a interiorly having two sets of hot runner blocks 29a, 30a, and two sets of molds 29, 30 are mounted parallel to one another on the mold place bed 28a. One (referred to as a first mold) 29 of said molds is positioned externally of the transferring platen 16 and the other (referred to as a second mold) 30 is mounted at a position which is close to said neck mold 17. These molds have cavities in the same number as that of the neck molds 17. The size of the cavity in the first mold 29 sufficient to mold the inner layer preform 2 between it and the injection core 31 inserted into the mold. The size of the cavity in the second mold 30 is sufficient enough to form a space for receiving the injection core 31 along with the inner layer preform 2 adhered to the periphery of the core to mold the outer layer preform 3 between it and the inner layer preform 2, said space being connected to the space formed between the neck mold 17 and the inner layer preform 2.
Replacement of the injection cores 31 relative to the two sets of molds 29, 30 is effected by a rotating plate 33 at the underside of the lifting plate 32 disposed on the tie-rods 27. A rotating device 34 such as a rotary actuator on the molding clamping plate is also used. for operating the rotating plate 33. The injection cores 31 are projectingly provided at the lower ends of mold beds 36, 36 at both ends of the lower surface of the rotating plate 33 connected to the rotating shaft 35, and the first mold 29 and the neck mold 17 are closed by the mold beds 36, 36. The movement of the lifting plate 32 is effected by connection of plungers 38, 38 of hydraulic cylinders 37, 37 mounted on the side of the tie-rods 27, 27.
The molding of the double-layered preform 1 by the above-described molder can be performed without much difference from the case where the single layer preform is molded. When the transferring platen 16 stops and one of the neck molds 17 is positioned at the injection molding stage A, the first mold 29 and second mold 30 are moved up by the operation of the mold clamping device 18, and the neck mold 17 and second mold 30 are closed.
Next, the injection cores 31, 31 along with the mold clamping plate 32 are moved down by the operation of the core mold clamping device 19 and inserted into the the second mold is second mold 30, and the first mold 29 and closed by the mold beds 36, 36, respectively. At this time, the inner layer preform 2, which is premolded in the first mold 29 by the previous step, is adhered to the periphery of one of the injection cores, and the inner layer preform 2 is positioned within the second mold extending through the neck mold 17, as shown in FIG. 5.
After powerful mold clamping has been performed following the above-described closure of molds, molten resin is poured into both molds 29, 30 from an injection device (not shown in the figure) in nozzle touch with the above-described hot runner blocks 29a, 30a.
Thereby, in the first mold 29, the aforesaid preform 2 is molded on the periphery of the injection core 31, and the outer layer preform 3 of the length reaching the inside of the neck mold 17 is formed on the periphery of the inner layer preform 2, as shown in FIG. 6.
After the preform has been injection molded, the mold clamping device 18 and the core mold clamping device 19 are operated to open both of the molds 29, 30 and to raise the injection cores 31, 31. On the first mold side, the inner layer preform 2 is adhered to the periphery of the injection core 31 and removed from the first mold 29, and on the second mold side, only the injection core 31 is removed, and the molded double-layered preform 1 is held in the neck mold 17 in the hollow state and remains.
When both of the injection cores are returned to their original position, the transferring platen 16 is rotated, and the double-layered preform 1 is held by the neck mold 17 and sent to the position of the heating stage B. The rotating and driving device 34 is operated at substantially the same time as the rotation of the transferring platen 16, and the rotating plate 33 is rotated through an angle of 80° to perform the replacement of the injection cores 31, 31 to assume the state as shown in FIG. 2. When the transferring platen 16 again stops and the neck mold 17 is positioned, the molding of the inner layer preform 2 and the molding of the double-layered preform 1 are simultaneously performed. The inner layer preform 2 is not completely cooled before it is rotated into position above molding station 30 and inserted into mold 30.
While in the above-described embodiment, the case of the molding machine provided with the heating stage B has been described, it will be noted that where the stretching and blow molding can be performed without requiring the heating, the molding machine can be used without the heating stage. The inner layer preform 2 and the outer layer preform 3 can be molded of the same or different resin, and no limitation is made particularly to such a thing in the preform molding apparatus.
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A molder for molding double-layered preforms in an injection, stretching and blow molding machine wherein a plurality of neck molds which also serve as a member for holding preforms and molded articles are mounted at given intervals on the undersurface of a transferring platen provided above a machine base and which is intermittently rotated so that the neck molds can be opened and closed, and devices required from injection molding of preforms to stretching and blow molding are disposed at stopping positions of said neck molds.
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FIELD OF THE INVENTION
The present invention relates generally to the field of replacing portions of the human structural anatomy with medical implants, and more particularly relates to an expandable implant and method for replacing bone structures such as one or more vertebrae or long bones.
BACKGROUND
It is sometimes necessary to remove one or more vertebrae, or a portion of the vertebrae, from the human spine in response to various pathologies. For example, one or more of the vertebrae may become damaged as a result of tumor growth, or may become damaged by a traumatic or other event. Excision of at least a generally anterior portion, or vertebral body, of the vertebra may be referred to as a corpectomy. An implant is usually placed between the remaining vertebrae to provide structural support for the spine as a part of a corpectomy. FIG. 1 illustrates four vertebrae, V 1 -V 4 of a typical lumbar spine and three spinal discs, D 1 -D 3 . As illustrated, V 3 is a damaged vertebra and all or a part of V 3 could be removed to help stabilize the spine. If removed along with spinal discs D 2 and D 3 , an implant may be placed between vertebrae V 2 and V 4 . Most commonly, the implant inserted between the vertebrae is designed to facilitate fusion between remaining vertebrae. Sometimes the implant is designed to replace the function of the excised vertebra and discs. All or part of more than one vertebrae may be damaged and require removal and replacement in some circumstances.
Many implants are known in the art for use in a corpectomy procedure. One class of implants is sized to directly replace the vertebra or vertebrae that are being replaced. Another class of implants is inserted into the body in a collapsed state and then expanded once properly positioned. Expandable implants may be advantageous because they allow for a smaller incision when properly positioning an implant. Additionally, expandable implants may assist with restoring proper loading to the anatomy and achieving more secure fixation of the implant. Implants that include insertion and expansion members that are narrowly configured may also provide clinical advantages. In some circumstances, it is desirable to have vertebral endplate contacting surfaces that effectively spread loading across the vertebral endplates. Effective implants should also include a member for maintaining the desired positions, and in some situations, being capable of collapsing. Fusion implants with an opening may also be advantageous because they allow for vascularization and bone growth through all or a portion of the entire implant.
Expandable implants may also be useful in replacing long bones or portions of appendages such as the legs and arms, or a rib or other bone that is generally longer than it is wide. Examples include, but are not limited to, a femur, tibia, fibula, humerus, radius, ulna, phalanges, clavicle, and any of the ribs.
SUMMARY
In one exemplary aspect, an expandable medical implant for supporting bone structures is disclosed. The implant has an overall implant height adjustable along a longitudinal axis. The implant may include an outer member configured to cooperatively engage a first bone structure and an inner member receivable in the outer member. The inner member may be movable relative to the outer member to increase and decrease the overall implant height. The inner member may be configured to cooperatively engage a second bone structure. One of the outer and inner members includes a tapered surface and the other of the outer and inner members includes a scalloped surface. The implant may also include a locking element disposed between the tapered surface and the scalloped surface. The tapered surface may be movable relative to the locking element to transversely shift the locking element into engagement with the scalloped surface to inhibit a decrease in the overall implant height.
In another exemplary aspect, a locker member may be disposed between the inner and outer member. The locker member may include a receiving aperture containing the locking element, and may be configured to act on the locking element to affect the position of the locking element relative to the outer member. The tapered surface of the outer member may be configured to affect the position of the locking element relative to the scalloped surface of the inner member.
In another exemplary aspect, an expandable medical implant for supporting bone structures may include an outer member having an inner surface configured to cooperatively engage a first bone structure. The implant also may include an inner member receivable in the outer member and movable relative to the outer member to increase and decrease the overall implant height. The inner member may have a scalloped surface and may be configured to cooperatively engage a second bone structure. A locking element may be disposed between the inner surface of the outer member and the scalloped surface of the inner member. The locking element may be movable between a locked condition and an unlocked condition and may be biased toward the locked condition. The locking element may be disposed to selectively engage the scalloped surfaces to inhibit a decrease in the overall implant height.
In another exemplary aspect, the implant may include a locker member disposed between the inner and outer member, the locker member including a receiving aperture containing the locking element.
In yet another exemplary aspect, an expandable medical implant for supporting bone structures may include an outer member having a tapered inner surface and being configured to cooperatively engage a first bone structure. The implant also may include a locker member receivable in the outer member and movable relative to the outer member. The locker member may include a receiving aperture. An inner member may be receivable in the locker member and movable relative to the locker member and the outer member to increase and decrease the overall implant height. The inner member may have a scalloped surface and may be configured to cooperatively engage a second bone structure. A locking element may be disposed within the receiving aperture of the locker member. The locking element may be associated with the tapered inner surface of the outer member and the scalloped surface of the inner member. The tapered surface may be movable relative to the locking element to transversely shift the locking element into engagement with the scalloped surface to inhibit a decrease in the overall implant height.
In yet another exemplary aspect, a method of supporting bone structures with an expandable medical implant is disclosed. The implant may have an overall implant height adjustable along a longitudinal axis. The method may include placing the implant between bone structures to be supported and displacing an inner member having a scalloped surface relative to an outer member having a tapered inner surface in order to increase the overall implant height. The outer member may be configured to cooperatively engage a first bone structure and the inner member may be configured to cooperatively engage a second bone structure. Displacing the inner member may allow a locking element to disengage the scalloped surface. A compressive load may be supported from the bone structures on the inner and outer members, and the compressive load may cause the tapered surface to shift the locking element into engagement with the scalloped surface and to inhibit a decrease in the overall implant height.
In yet another exemplary aspect, an expandable medical implant for supporting bone structures includes an outer member being configured to cooperatively engage a first bone structure and an inner member receivable in the outer member. The inner member may be movable relative to the outer member to increase and decrease the overall implant height and may be configured to cooperatively engage a second bone structure. At least one of the inner and outer members includes vascularization openings formed on first and second opposing sides of the implant. The vascularization openings on the first opposing side may be larger than the vascularization openings on the second opposing side.
Further aspects, forms, embodiments, objects, features, benefits, and advantages of the present invention shall become apparent from the detailed drawings and descriptions provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an elevation view of a segment of a lumbar spine.
FIG. 2 is a pictorial illustration of an exemplary expandable implant according to one embodiment of the present invention.
FIGS. 3 a - 3 c are pictorial illustrations of exploded views of the implant of FIG. 2 .
FIG. 4 is an isometric pictorial illustration of a base component of the implant of FIG. 2 .
FIG. 5 is a top pictorial illustration of the base component of FIG. 4 .
FIG. 6 is a sectional pictorial illustration of the base component of FIG. 5 , taken along line 6 - 6 .
FIG. 7 is an isometric pictorial illustration of a locker component of the implant of FIG. 2 .
FIG. 8 is an isometric pictorial illustration of a post component of the implant of FIG. 2 .
FIG. 9 is a side pictorial illustration of the implant of FIG. 2 .
FIG. 10 is a sectional pictorial illustration taken along line 10 - 10 in FIG. 9 .
FIG. 11 is a sectional pictorial illustration taken along line 11 - 11 in FIG. 9 .
FIG. 12 is a sectional pictorial illustration of an exemplary locking arrangement usable with the implant of FIG. 2 .
FIG. 13 is an elevation view of another exemplary embodiment of the present invention.
FIG. 14 is another elevation view of the exemplary embodiment of FIG. 13 .
FIG. 15 is an elevation view of another exemplary embodiment of the present invention.
FIG. 16 is another elevation view of the exemplary embodiment of FIG. 15 .
DETAILED DESCRIPTION
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates.
FIGS. 2 and 3 a - 3 c show an exemplary expandable implant 100 usable to secure and space adjacent bone structures. In FIG. 2 , the implant 100 is shown fully assembled, while FIGS. 3 a - 3 c show the implant 100 in an exploded condition, along a longitudinal axis L. Referring to these figures, the implant 100 includes three main components, including a base 102 , a locker 104 , and a post 106 . These main components operate together to provide the support and spacing between the adjacent bone structures. In addition to these components, the exemplary implant 100 includes locking elements 108 , pegs 110 , and biasing elements 112 .
In the exemplary embodiment shown in FIGS. 2 and 3 a - 3 c , the base 102 is configured and shaped to receive and house the locker 104 , which, in turn, is configured and shaped to receive and house the post 106 . The locking elements 108 cooperate with the locker 104 to control displacement of the post 106 relative to the base 102 , thereby controlling the overall height of the implant 100 . In this embodiment, the pegs 110 connect the base 102 , the locker 104 , and the post 106 into a unitary mechanism. The biasing elements 112 cooperate with the base 102 and the locker 104 to bias the locker 104 , and likewise the locking element 108 , into a position that selectively locks or secures the post 106 relative to the base 102 , thereby hindering the ability of the implant 100 to collapse after implantation. In the embodiments shown, the biasing element 112 is a leaf spring. However, the biasing element could be any type of spring, including a coil spring, or a material, such as a silicone or elastomeric bumper, or an elastic member, such as a stretchable band that may act in compression or tension.
The components of the exemplary implant 100 will be described in further detail with reference to FIGS. 4-12 . The base 102 will be described first, with reference to FIGS. 4-6 , as well as FIGS. 3 a - 3 c . FIG. 4 shows an isometric view of the base 102 ; FIG. 5 shows a top view; and FIG. 6 shows a cross-sectional view.
The base 102 includes a top surface 114 , a bottom surface 116 , an outer wall 118 , and an inner wall 120 , defining a bore 122 . The top and bottom surfaces 114 , 116 may be relatively flat surfaces having the bore 122 formed therein. The top surface 114 may be configured to cooperate with locker 104 , and the bottom surface 116 may be configured to cooperatively engage a bone structure, either directly or through additional components, such as endplates. The bottom surface 116 may include features for attachment to endplates or other components. Some exemplary features are described below with respect to the post 106 .
In the exemplary embodiment shown, the outer wall 118 may include instrument receiving features 124 that cooperate with surgical instruments for placement of the implant 100 between desired bone structures. In the embodiment shown, the instrument receiving features 124 are indentations on opposite sides of the base outer wall 118 , however, it is contemplated that many other features could be used to cooperate with instruments that would allow the instruments to grip, support, or otherwise place the implant 100 in a desired location. Further, some embodiments lack any instrument receiving features.
In the exemplary embodiment shown, in addition to the instrument receiving features 124 , the outer wall 118 includes additional cut outs and features that function to reduce the mass of the implant 100 while maintaining sufficient strength to properly support the bone structures and weight of a patient. In addition, these additional cutouts and features may simplify additional processing, such as, for example, when using a wire EDM to cut features at the bottom surface 116 .
Referring now to FIGS. 5 and 6 , the base 102 includes the inner wall 120 , forming the bore 122 . In this exemplary embodiment, the bore 122 extends longitudinally from the top surface 114 through the base 102 , to the bottom surface 116 , as best seen in FIG. 6 . As best seen in FIG. 5 , the bore 122 in this exemplary embodiment is substantially rectangular. Accordingly, the inner wall 120 may be formed of substantially planar surfaces that form the rectangular shape. It should be noted that in other embodiments, the bore 122 is formed of other polygon shapes, such as, for example, triangular, square, or pentagon. Still other embodiments have bores that are oval or circular shaped. As illustrated in FIG. 2 , the bore 122 is configured to receive the locker 104 of the implant 100 .
The inner wall 120 has a tapered section 128 and a non-tapered section 130 . In the exemplary embodiment shown, the tapered section 128 is adjacent the top surface 114 of the base 102 , while the non-tapered section 130 is adjacent the bottom surface 116 of the base 102 . However, the tapered section 128 may be otherwise arranged or placed. As discussed further below, the tapered section 128 cooperates with the locking element 108 to secure the height of the implant 100 at a desired level. Also, in the exemplary embodiment shown, the inner wall 120 includes two tapered sections 128 , disposed on opposite sides of the bore 122 . Other embodiments include one or more than two tapered sections, and for the reasons described below, symmetry may provide advantages when expanding the implant 100 .
In addition to the elements described, the base 102 also includes a peg aperture 132 , a biasing member aperture 134 , and a vascularization aperture 136 . During assembly, the peg 110 may be inserted into the peg aperture 132 , and the biasing element 112 may be inserted through the biasing member aperture 134 . The vascularization aperture 136 provides access to the bore 122 and may be used to introduce bone graft, tissue, or other material into the bore 122 after implantation. In addition, it allows fluid into the interior of the base 102 thereby, encouraging bone growth. Because of the cutouts, the outer wall 118 of the base 102 also forms a flange 138 , as best seen in FIGS. 2 , 3 a , and 4 .
As shown in FIG. 2 , the base 102 receives the locker 104 , which is described with reference to FIGS. 2 , 3 a - 3 c , and FIG. 7 . The locker 104 includes an upper end 150 , a lower end 152 , an inner surface 154 , an outer surface 156 , and a flange 157 .
In this exemplary embodiment and as shown in FIG. 2 in an assembled condition, the upper end 150 is disposed outside the base 102 , and the lower end 152 is disposed within the bore 122 of the base 102 . At the upper end 150 , the flange 157 radially extends to have an outer perimeter substantially matching that of the base 102 . As described below, the flange 157 may be used to displace the locker 104 relative to the base 102 in order to change the overall height of the implant 100 .
In the embodiment shown, the outer surface 156 of the locker 104 is sized and formed to be received within the bore 122 of the base 102 . In this embodiment, like the bore 122 of the base 102 , the outer surface 156 is substantially rectangular. However, the outer surface may be in the form of other shapes, as described above with reference to the base 102 .
The outer surface 156 includes a locking element receiver 158 , which in this embodiment is an aperture from the outer surface 156 to the inner surface 154 . In addition, the outer surface includes a biasing member support 160 , a vascularization aperture 162 , and a peg slot 164 .
As described further below, the locking element 108 fits within and extends through the locking element receiver 158 to engage and disengage the base 102 and the post 106 , restricting movement of the post 106 relative to the base 102 . The biasing member support 160 cooperates with the biasing member 112 to provide a biasing force on the locker 104 to maintain it within the base 102 . The peg slot 164 receives the peg 110 , which also extends through the base 102 . This allows the locker 104 to move relative to the peg 110 , but the peg 110 blocks removal of the locker 104 from the base 102 . Accordingly, the peg slot 164 cooperates with the peg 110 to slidably maintain the locker 104 within the base 102 .
The inner surface 154 of the locker 104 forms a locker bore 166 . The locker bore 166 in this exemplary embodiment is rectangular, as is the outer surface 156 . However, the locker bore 166 need not be rectangular but could be formed into some other shape. As will be described below, the locker bore 166 is configured and sized to receive the post 106 .
The post 106 will be described with reference to FIGS. 8 and 3 a - 3 c . The post 106 includes a top end 180 , a bottom end 182 , and a main body 184 extending therebetween. The top end 180 includes a top surface 181 having end plate connectors 186 formed therein. In the embodiment shown, the end plate connectors 186 are configured for attachment to an end plate (not shown). In the embodiment shown, the end plate connectors 186 are a series of holes configured to attach to endplates. In some embodiments, instead of attaching to separate endplates, the post 106 is configured to cooperatively attach directly to bone structure. In this exemplary embodiment, one end plate connector 186 may include an attachment aid 188 that cooperates with an end plate to secure the end plate onto the top surface 180 of the post 106 . In this embodiment, the attachment aid 188 is a spring feature that is deformable to receive an endplate post and frictionally grip it to hold the endplate in place during implantation. In addition, the bottom surface 116 of the base 102 may include similar features, including the end plate connectors and the attachment aid, such as the spring feature. In some embodiments, the end plate connectors are cylindrical posts that extend from an endplate and are configured to be received by the end plate connectors 186 . The end plates could be at any angle or of various types. Alternatively, the end plate connectors may be used with an intermediate spacer to connect and stack two implants.
The bottom end 182 of the post 106 includes a bottom surface 183 having a vascularization aperture 190 formed therein. The bottom end 182 fits within the locker bore 166 , and is slidable relative to the locker 104 and the base 102 to increase and decrease the overall height of the implant 100 . In the embodiment shown, the bottom end 182 of the post 106 is sized and formed to be received within the locker bore 166 . In this embodiment, like the locker bore 166 , the bottom end 182 is substantially rectangular. However, the bottom end may be in the form of other shapes, as described above with reference to the base 102 .
The main body 184 includes a peg slot 192 , additional vascularization holes 194 , and a locking surface 196 . The peg slot 192 is configured to receive the peg 110 , which also extends through the base 102 and locker 104 . Because of the length of the peg slot 192 , the post 106 may be raised or lowered relative to the peg 110 to increase or decrease the overall height of the implant 100 . The vascularization holes 194 and the vascularization aperture 190 provides access for placement of bone graft or other material and allows fluid flow to promote bone growth and attachment to the bone structures.
The locking surface 196 is the area configured to contact the locking element 108 , and in this exemplary embodiment, may include roughened features, such as, for example, a series of roughening scallops aligned transverse to a longitudinal axis of the implant 100 , as shown in the figures. As will be described below, the roughened features, such as the scallops cooperate with the locking element 108 to secure the post at a desired height relative to the locker 104 and the base 102 . In this embodiment, the scallops of the locking surface 196 are spaced less than 1 mm apart and enable an incremental increase and decrease in the height of the implant 100 . A scallop radius may substantially correspond with a radius of the locking element 108 , providing a relatively tight fit when the locking element is engaged with the locking surface 196 . In some embodiments, the locking surface 196 is not scalloped, but includes other roughening features. For example, in some embodiments the roughened features of the locking surface includes protruding triangular features or block-like features forming teeth. Still other surface features may be simply rough surfaces, such as those formed by shot peening, blasting, etching, or machining to increase the frictional properties of the locking surface 196 . Still other roughened surface features are contemplated. In yet other embodiments, the locking surface 196 is relatively smooth, thereby allowing for an infinite number of expansion increments.
In addition to the features described above, the post 106 includes a flange 198 . In the exemplary embodiment shown, the flange 198 includes instrument receiving grips 200 along its outer edges, formed to fit instruments during implantation or expansion.
Operability of the implant 100 will be described with reference to FIGS. 9 through 12 . FIG. 9 shows the implant 100 in an assembled condition. FIGS. 10 and 11 show cross-sectional views of the implant 100 . FIG. 12 shows one model of components used to illustrate the functionality of the locking mechanism of the implant 100 .
Referring now to the cross-sectional view in FIG. 10 , the peg 110 is shown extending through the base 102 , the locker 104 , and into the post 106 . As can be seen, the peg slot 164 in the locker 104 allows the locker 104 to move along the longitudinal axis L of the implant 100 relative to the base 102 . Likewise, the peg slot 192 in the post 106 allows additional movement of the post 106 relative to the base 102 . In this manner, the peg 110 may maintain the components of the implant 100 together, while at the same time allowing them to expand longitudinally to increase and decrease the overall implant height.
FIG. 11 is a cross-sectional view through the biasing element 112 and through the locking element 108 . The locking element 108 is maintained in its location by the locking element receiver 158 of the locker 104 . As shown in FIG. 11 , the biasing element 112 cooperates with the base 102 and the biasing member support 160 of the locker 104 to limit the axial movement of the locker 104 relative to the base 102 . The biasing element 112 provides a continuous biasing force against the locker 104 to maintain the locker 104 in a position that locks the height of the implant.
FIG. 12 shows the relationship of the locking element 108 with the base 102 , the locker 104 , and the post 106 , according to one embodiment of the implant. The locking element 108 is disposed in the locking element receiver 158 , and protrudes through the receiver 158 such that the locking element 108 is selectively in contact with both the base 102 and the post 106 .
In use, when the locker 104 is raised relative to the base 102 , the locking element 108 also raised relative to the base 102 . Because the base 102 has a tapered section 128 , upward movement of the locking element 108 relative to the base may provide free space for the locking element 108 to move away from the post 214 . This may be referred to as an unlocked condition, allowing the post 214 to slide freely to either increase or decrease the overall height of the implant 100 . Once the desired height is achieved, the locker 104 may be moved downward relative to the base 102 , wedging the locking element 108 between the tapered surface 128 of the base 102 and the post 106 . So doing locks the overall height of the implant at its desired height. This may be referred to as a locked condition. The roughened surface features, such as the scallops, of the post 106 may provide a locking location for the locking element 108 and may reduce slippage between the locking element 108 and the post 106 .
In the embodiment shown, the overall height of the implant 100 can be increased simply by raising the post 106 relative to the base 104 . So doing may force the locking element 108 to move upwardly along the tapered section 128 to the unlocked condition, thereby allowing the implant height to be increased without requiring any separate attention to the locker 104 . This also allows the locking element 108 to freely engage and disengage the roughened features of the locking surface 196 . Accordingly, in some embodiments such as those shown having the scalloped surface features, during expansion, an audible clicking may be generated as the locking element 108 moves past and falls into each scalloped feature of the locking surface 196 . In some embodiments, the locker 104 and the locking element 108 are configured to require manual or separate displacement of the locker 104 and the locking element 108 to reduce the overall height of the implant 100 .
In the embodiment shown, the locking element 108 is a cylindrical rod that distributes its locking force over a wide surface area and in the embodiment shown over the entire width of the post 106 . Accordingly, the locking element 108 contacts the post 106 along a contact line transverse to the longitudinal axis L of the implant 100 , rather than at a single point. Because of this, the implant is less conducive to undesired slipping. It should be noted that the scalloped surface on the post 106 is optional and the post 106 may include other roughened features, indentations or elements that increase the friction between the locking element and the post.
In the embodiment shown, the implant 100 includes symmetrically locking features, including opposed tapered surfaces on the base 102 , two locking elements 108 in two opposed receiving apertures 158 , and two opposite locking surfaces 196 . This symmetry may aid expansion and collapse of the implant by substantially equalizing the forces required at each side of the implant to expand or collapse it, providing a better level of control to the physician placing or removing the implant.
During implantation, the implant 100 may be gripped with a surgical instrument at instrument receiving features of the base 102 and at the instrument receiving grips 200 of the post 106 . In its smallest condition, the implant may be introduced to a patient through the smallest possible incision. In one exemplary embodiment, the implant 100 may be introduced between two bone structures, such as adjacent vertebral bodies, such as the vertebral bodies V 2 and V 4 in FIG. 1 , replacing the vertebral body V 3 along with the discs D 2 and D 3 . Once positioned between the adjacent bone structures, the implant 100 may be distracted to increase the overall implant height. Using the instruments, the post 106 is longitudinally displaced relative to the base 102 . In so doing, the post 106 frictionally acts on the locking element 108 to raise it relative to the base 102 , along the tapered section 128 . Once a desired height is achieved, the base 102 and post 106 are released. The continuous biasing force of the biasing member 112 acting on the locker 104 draws the locker 104 and the locking element 108 into a locking condition, where the locking element is wedged between the tapered section 128 and the locking surface 196 of the post 106 . This compressive force locks the implant 100 against further decreases in the overall height. Once expanded, an implanting physician may introduce optional bone growth promoters into the base 102 or post 106 through the vascularization aperture 136 and the vascularization holes 194 , respectively.
If it later becomes necessary to remove the implant, the locker flange 157 may be raised relative to the base 102 to remove the locking element 108 from its wedged position. Once the locking element 108 is free to disengage the locking surface 196 of the post 106 , the post 106 may collapse into the locker 104 , and the implant 100 may be removed from the patient. Again, although described with reference to one locking element, it is understood that two or more locking elements may be includes to provide symmetry.
In the implantation method described above, some amount of the distraction force is used to overcome the biasing force of the biasing member 112 . In some embodiments, the biasing member may be adjusted to provide a stronger biasing force to resist undesirable actuation of the implant once released. The stronger the biasing member, the greater the force required to deploy the device. However, in other implantation methods, the locker 104 may be separately raised relative to the base 102 to release the locking element prior to distracting the post 106 from the base 102 . In this way, the complete distraction force may be used for distraction, rather than a portion being used to overcome the biasing force acting on the locker 104 .
In yet other implantation methods, the implant may also be deployed by raising the center post 106 relative to the base 102 from the bottom end 182 . In these embodiments, deploying instruments may attach to the post bottom end 182 , such as at the bottom surface 183 , or to features on the post 106 such as the vascularization apertures 194 , and in addition, attach to the instrument receiving features 124 on the base 102 . By moving the post 106 from the bottom end 182 (or the vascularization apertures 194 ) relative to the instrument receiving features 124 , the distance between the bottom end 182 (or the vascularization apertures 194 ) and the instrument receiving features 124 decreases, while the overall height of the implant increases. Accordingly, during deployment, the gripping portions of the instrument move closer together (decreasing the instrument size), while the height of the implant increases. Because in this embodiment, the instrument does not grip at the ends of the implant, the implant can be deployed into a space or cavity where both ends of the implant are not directly accessible at the same time.
Although the implant 100 is described as being somewhat porous with vascularization apertures 136 , 162 , 190 , 194 , other embodiments include either more or less vascularization apertures. In some embodiments, the post is substantially solid such that while it is telescopically received within the locker and base, no material may be received within the post, or alternatively, with in the base.
FIGS. 13 and 14 show an embodiment of another exemplary expandable implant 300 having additional vascularization openings. FIG. 13 shows a back side and FIG. 14 shows a front side. The implant 300 is similar to the implant 100 described above, including a base 302 , a locker 304 , and a post 306 . In this embodiment, the heights of the base 302 and the post 306 are greater than those of the base 102 and post 106 described above. To accommodate grafting, tissue, or other material, the implant 300 includes rear vascularization openings 308 , side vascularization openings 310 , and at least one front access window 312 . The rear and side openings 308 , 310 , as well as the access window 312 , increase the porosity of the implant, promoting breathability and bone growth. The access window 312 is larger than the rear and side openings 308 , 310 and provides access to the interior of the base 302 . Accordingly, during implantation, a physician may introduce grafting material through the access window 312 to pack grafting material, tissue, or other material into the base 302 . The larger size of the access window 312 simplifies the packing process, while the smaller size of the rear and side openings 308 , 310 help reduce the opportunity for the material being packed to extrude from the rear or side openings. This may become important when the implant 300 is placed in a spine and the rear of the implant 100 is facing or located adjacent the spinal cord. Similarly, the larger size of the access window 312 may allow placement of large segments of grafting, tissue, or other material, while the smaller rear and side openings 308 , 310 help contain the large segments within the base 302 . The post 306 of the implant 300 also includes vascularization holes 314 similar to the vascularization holes 194 described above.
FIGS. 15 and 16 show another embodiment of an exemplary expandable implant 400 . FIG. 15 shows a back side and FIG. 16 shows a front side. Again, the implant 400 is similar to the implant 100 described above, including a base 402 , a locker 404 , and a post 406 . To accommodate grafting, tissue, or other material, the implant 400 includes rear vascularization openings 408 , side vascularization openings 410 , and front access windows 412 that increase the porosity of the implant, promoting breathability and bone growth. As described above, the access window 412 is larger than the rear and side openings 408 , 410 and provides access to the interior of the base 402 . In this embodiment, the rear openings 408 are larger than the side openings 410 . Nevertheless, in this embodiment, the rear openings 408 are smaller than the access window 412 . As can be seen, in this embodiment, the base 402 includes three rear openings 408 .
The post 406 of the implant 400 also includes vascularization holes 414 similar to the vascularization holes 194 described above. In addition, the post 406 includes post openings 416 in a locking surface 418 . The locking surface 418 may be similar to the locking surface 196 described above. The post openings 416 provide additional vascularization to the implant 400 .
In the embodiments shown in FIGS. 13-16 , the implants include only one access window. However, in other embodiments, the implants include more than one access window on the front side, while the rear and side openings are still maintained smaller than the front access windows. In other embodiments, the rear openings are smaller than the side openings. It also should be noted that the implant may include more or less than three rear openings, and the size of the openings may be determined in part based upon the size of the implant and based upon the size or amount of packing material anticipated.
While the post has been shown as telescopically received within the locker and the base, it will be appreciated that in a further embodiment the respective configuration is inverted such that a portion of the base is received within the post. Moreover, while a substantially cylindrical structure having rectangular bores has been shown for the purposes of illustration, in alternative embodiments the tubular and rectangular shapes may take the form of a rectangle, square, ellipse, diamond, oval, D-shape or any shape desired to conform and substantially match the adjacent bone or the bone structure that is being replaced. As a result, the definition of tubular is not intended to be limited to cylindrical but is instead intended to cover all components that may be utilized to reduce the present invention.
While the present device has been described with respect to insertion between two vertebrae after removal of the intervening vertebrae and intervertebral disc, it is contemplated that the length of the device may be sized appropriate to span multiple vertebrae. Additionally, the device may find application in other orthopedic areas and the size and shape of the device may be made to substantially match the implantation site. For example, while the present embodiment has been illustrated as a substantially cylindrical device, it is contemplated that in certain spinal applications it is desirable that the device have a substantially D shaped cross-section as viewed from top to bottom such that the anterior portion of the device has an exterior convexly curved surface matching the anterior of the vertebral body while the posterior portion of the device is substantially flat or concave allowing it to be positioned closer to the spinal canal without protruding into the spinal canal.
Embodiments of the implant in whole or in part may be constructed of biocompatible materials of various types. Examples of implant materials include, but are not limited to, non-reinforced polymers, carbon-reinforced polymer composites, PEEK and PEEK composites, shape-memory alloys, titanium, titanium alloys, cobalt chrome alloys, stainless steel, ceramics and combinations thereof. In some embodiments, the locking elements 108 are formed or cobalt chrome and the base 102 , locker 104 , and post 106 are formed of titanium.
If the implant is made from radiolucent material, radiographic markers can be located on the implant to provide the ability to monitor and determine radiographically or fluoroscopically the location of the implant in the spinal disc space. In some embodiments, radiographic markers are placed to show the location of the locking elements relative to the post and base.
In some embodiments, the implant or individual components of the implant are constructed of solid sections of bone or other tissues. In other embodiments, the implant is constructed of planks of bone that are assembled into a final configuration. The implant may be constructed of planks of bone that are assembled along horizontal or vertical planes through one or more longitudinal axes of the implant. In some embodiments, a cavity is cut or constructed through the implant. The cavity may be useful to contain grafting materials. Tissue materials include, but are not limited to, synthetic or natural autograft, allograft or xenograft, and may be resorbable or non-resorbable in nature. Examples of other tissue materials include, but are not limited to, hard tissues, connective tissues, demineralized bone matrix and combinations thereof. Examples of resorbable materials that may be used include, but are not limited to, polylactide, polyglycolide, tyrosine-derived polycarbonate, polyanhydride, polyorthoester, polyphosphazene, calcium phosphate, hydroxyapatite, bioactive glass, and combinations thereof. Implant may be solid, porous, spongy, perforated, drilled, and/or open.
In some circumstances, it is advantageous to pack all or a portion of the interior and/or periphery of the implant with a suitable osteogenetic material or therapeutic composition. Osteogenic materials include, without limitation, autograft, allograft, xenograft, demineralized bone, synthetic and natural bone graft substitutes, such as bioceramics and polymers, and osteoinductive factors. A separate carrier to hold materials within the device can also be used. These carriers can include collagen-based carriers, bioceramic materials, such as BIOGLASS®, hydroxyapatite and calcium phosphate compositions. The carrier material may be provided in the form of a sponge, a block, folded sheet, putty, paste, graft material or other suitable form. The osteogenetic compositions may include an effective amount of a bone morphogenetic protein, transforming growth factor β1, insulin-like growth factor 1, platelet-derived growth factor, fibroblast growth factor, LIM mineralization protein (LMP), and combinations thereof or other therapeutic or infection resistant agents, separately or held within a suitable carrier material. A technique of an embodiment of the invention is to first pack the interior of an unexpanded implant with material and then place one or both end members if desired.
Access to the surgical site may be through any surgical approach that will allow adequate visualization and/or manipulation of the bone structures. Example surgical approaches include, but are not limited to, any one or combination of anterior, antero-lateral, posterior, postero-lateral, transforaminal, and/or far lateral approaches. Implant insertion can occur through a single pathway or through multiple pathways, or through multiple pathways to multiple levels of the spinal column. Minimally invasive techniques employing instruments and implants are also contemplated.
It is understood that all spatial references, such as “top,” “inner,” “outer,” “bottom,” “left,” “right,” “anterior,” “posterior,” “superior,” “inferior,” “medial,” “lateral,” “upper,” and “lower” are for illustrative purposes only and can be varied within the scope of the disclosure.
FIG. 1 illustrates four vertebrae, V 1 -V 4 , of a typical lumbar spine and three spinal discs, D 1 -D 3 . While embodiments of the invention may be applied to the lumbar spinal region, embodiments may also be applied to the cervical or thoracic spine or between other bone structures.
While embodiments of the invention have been illustrated and described in detail in the disclosure, the disclosure is to be considered as illustrative and not restrictive in character. All changes and modifications that come within the spirit of the invention are to be considered within the scope of the disclosure.
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An expandable medical implant for supporting bone structures is disclosed. The implant may include an outer member and an inner member receivable in the outer member. One of the outer and inner members includes a tapered surface and the other of the outer and inner members includes a scalloped surface. The implant may also include a locking element disposed between the tapered surface and the scalloped surface. The tapered surface may be movable relative to the locking element to transversely shift the locking element into engagement with the scalloped surface to inhibit a decrease in the overall implant height.
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FIELD OF THE INVENTION
The present invention relates to a ceramic Composite material, for example, a ceramic molded body or a layer, as well as a use of the ceramic molded body or a layer.
BACKGROUND INFORMATION
European Published Patent Application No. 0 412 428 refers to a ceramic composite body and a method of producing same, in which an organosilicon polymer, as a precursor material, together with incorporated particles of hard material and/or other reinforcing components, as well as one or more metallic fillers, is subjected to pyrolysis. In pyrolysis, the decomposition products formed from the polymer compounds react with the metallic filler, which may result in a ceramic composite body having a matrix with particles of hard material and/or reenforce embedded components.
For example, carbides or nitrides of titanium, zirconium or other transition metals may be used as the hard material particles or reinforcing components as referred to, for example, in European Published Patent Application No. 0 412 428, in which the particle sizes of the powder particles are in the range of approximately 1 μm to approximately 300 μm.
The matrix formed from the organosilicon polymer after pyrolysis is a monophase or polyphase, amorphous, partially crystalline or crystalline matrix of silicon carbide, silicon nitride, silicon dioxide or mixtures thereof.
In addition to microscale powder materials, nanoscale powder materials may be single-phase or polyphase powders having particle sizes in the nanometer range. Due to their small particle dimensions, they are characterized by a very high proportion of particle boundaries or phase boundaries per volume. In addition, the physical, chemical and mechanical properties of such nanoscale powders may differ from those of conventional coarse-grained materials having the same chemical composition. For example, such nanoscale powders may have greater hardness, increased diffusivity and increased specific heat.
Nanoscale powdered materials may be produced by flame pyrolysis, gas condensation, spray conversion or crystallization of amorphous substances. Industrial production has advanced in the case of zirconium dioxide, silicon dioxide, titanium dioxide and aluminum oxide.
It is believed that the properties of ceramic composite materials having microscale fillers are determined largely by the properties of the fillers. Thus, local stress peaks or cracks may occur in the composite material when the properties of the matrix and fillers differ, e.g., different coefficients of thermal expansion. This may result in an increased failure rate of such components.
When using reactive microscale fillers as referred to, for example, in European Published Patent Application No. 0 412 428, the effect of which is based on reaction of the fillers with the ambient matrix, only an incomplete reactive conversion of filler may be achieved in the edge area of the filler grains.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a ceramic composite material, which may be suitable, for example, for producing ceramic molded bodies or layers and with which the profile of electrical and physical properties may be easily and reliably adjusted.
Another object of the present invention is to provide a ceramic composite material, the electric properties of which, porosity, high-temperature stability, mechanical strength, i.e., fracture toughness and homogeneity are improved in comparison with the related art.
It is believed that an exemplary ceramic composite material according to the present invention has the advantage in that the profile of electrical and physical properties of the ceramic composite material obtained after pyrolysis may be adapted to a profile of properties predetermined for the respective application, i.e., the composition of the composite material may be tailored to this profile of properties. For example, the large selection of fillers may permit the properties of the resulting ceramic composite materials to be varied or adjusted over a wide spectrum.
In addition, it is believed that an exemplary ceramic composite material according to the present invention has the advantage in that, due to the small particle size of the reactive filler, the process temperatures may be lowered and the process times required for a complete reaction may be shortened in comparison with the related art, so that with the process temperatures required in the past, liquid or volatile fillers may still be solid and thus may be used at the pyrolysis and sintering temperatures. Furthermore, unwanted phase reactions, which may occur at higher temperatures, i.e., reactions between the matrix and filler, may be avoided by using reduced process temperatures.
It is believed that one advantage of the composite material according to the present invention is that the porosity of the composite material may be adjusted in a defined manner using the fillers, the combination of a suitable nanoscale filler with defined pyrolysis conditions thus allowing the production of both highly porous composite materials and dense composite materials by varying the pyrolysis conditions, while otherwise using the same polymer precursor material, i.e., the same starting mixture.
Exemplary porous ceramic composite materials according to the present invention may also have a very good spalling resistance and may be applied to various applications, for example, as lightweight structural materials, as porous protective shells for sensors, as filters, as catalyst support materials or as a matrix for infiltrated reactive composite materials, while exemplary high-density ceramic composite materials according to the present invention have an increased mechanical strength, improved fracture toughness and improved corrosion resistance.
In production of a n exemplary ceramic composite material according to the present invention, shaping and production methods may be used, so that even ceramic fibers, layers and molded bodies of different sizes or having a complex geometry are readily obtainable, which may permit an exemplary composite material to be applied to a broad spectrum of applications. For example, shaping methods, such as compression molding, injection molding, joining and fiber extrusion may be used. With regard to the production method used, pyrolysis, under a protective gas and laser pyrolysis may be employed.
In this regard, a simple and reliable control or adjustability of the flow properties and pourability of the starting mixture may be achieved through the type and quantity of the nanoscale filler. This may also be true of the process parameters in powder transport, in cold molding, in injection molding, in spin coating or in dip coating.
Moreover, due to the small size of the filler, detailed replicas of embossed, cast or injection molded shapes may also be produced by pouring the starting mixture into a mold and then performing pyrolysis. In addition to the fidelity in detail, these replicas may have a high surface quality, allowing details having dimensions of less than 1 μm to be molded.
It is also believed that an exemplary ceramic composite material according to the present invention has the advantage in that, due to the use of highly dispersed insulating fillers, the electric resistance of the composite material is increased significantly and the long-term stability of this electric resistance may be improved. In addition, due to the improved homogeneity and stability of the thermal and electrical properties of the resulting composite material, reliability may also increase.
It is also believed that another advantage of an exemplary ceramic composite material according to the present invention is that it may permit high degrees of filling and short pyrolysis times, and the flow properties of the polymer precursor materials used may be regulated through the addition of suitably selected fillers. Thus, for example, suspensions of starting mixtures that remain stable and processable over long periods of time may-be produced.
The polymer precursor material may be an oxygen-containing polysiloxane precursor or a polysilazane precursor that is stable in air, since these materials allow processing in air and thus may allow the production of inexpensive composite materials. In addition, the resulting pyrolysis product may be chemically stable with regard to oxidation and corrosion and at the same time may be unobjectionable from a health standpoint.
In addition to the nanoscale fillers having an average particle size of less 200 nm, other fillers, such as a powdered aluminum oxide (Al 2 O 3 ) having a larger particle size of 500 nm to 10 μm, for example, 500 nm to 3 μm, may also be used. This may broaden the spectrum of achievable electrical and physical properties and thus may broaden the spectrum of applications of the resulting composite materials. For example, the electric resistance of the resulting ceramic composite material may increase by several orders of magnitude at room temperature and also at temperatures greater than 1200° C. Also, when conventional microscale aluminum oxide fillers are replaced largely or completely by nanoscale silicon dioxide, for example, amorphous silicon dioxide or corresponding highly dispersed silicic acid, for example, pyrogenic silicic acid, the long-term stability of the mechanical and electrical properties of the ceramic composite material obtained may be improved at temperatures above 1200° C. Simultaneously, an increase in the allowed heating rates in pyrolysis and a shortening of the time required for shaping by compression molding may be achieved.
With regard to the highest possible specific electric resistance of the composite material, it is believed to be advantageous if, in addition to the polymer precursor material and instead of or in addition to a conventional, microscale aluminum oxide filler, the starting mixture also contains nanoscale silicon dioxide, for example, amorphous silicon dioxide, nanoscale silicon dioxide provided having a carbonaceous and/or hydrophilic surface modification, pyrogenic silicic acid or silicic acid provided with a carbonaceous and/or hydrophilic surface modification to which may be added a boron compound in the amount of 10 wt % to 30 wt %, for example, a boron oxide such as B 2 O 3 .
In this connection, the specific electric resistance of the resulting composite material depends not only on the particle size of the filler but also on the BET surface area of the filler, so that the resistance may be easily adjustable to unexpectedly high values. The surface properties of the filler are additional variables, which effect the resulting specific electric resistance of the composite material, for example, in conjunction with a change in the BET surface area. Thus, the transition from a hydrophobic surface to a hydrophilic surface of the filler particles, for example, may result in an increase in the specific electric resistance obtained.
Especially high values for the specific electric resistance may also be achieved, for example, when the filler, for example, SiO 2 or silicic acid, is used in an amount of at least 9 vol % in the starting mixture, whereby at the same time, another filler such as Al 2 O 3 , which may optionally be used in the starting mixture, should amount to less than 7 vol %, for example, less than 3 vol %.
Due to the small particle size of the filler, the surface quality of coatings produced with this ceramic composite material may be improved because the starting mixture applied before pyrolysis to the surface of a substrate to be coated penetrates into all the surface detects and irregularities in this substrate, thereby increasing adhesion of the coating, as well as equalizing irregularities and defects in the substrate-layer interface.
With respect to nanoscale fillers, at least approximately complete conversion of these fillers with the surrounding matrix in pyrolysis may be achieved in the ceramic composite material. This may result in, for example, a definite shortening of pyrolysis cycles. Furthermore, chemical reaction of the nanoscale filler with the polymer precursor material may proceed more rapidly in comparison with microscale fillers.
Also, by adding a suitable stabilizer to the starting mixture, for example, production of a stable suspension of the polymer precursor material with the filler in an organic solvent may be produced. For example, the stability of such a suspension with respect to sedimentation may increases in comparison with similar starting mixtures having microscale fillers, so that coating methods performed with such suspensions by dip coating or spin coating may be facilitated. Furthermore, the nanoscale filler may be suitable as a dispersant for a microscale filler used concurrently.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing specific electric resistance R of the composite ceramic as a function of the BET surface area of highly dispersed silicic acid having a hydrophobic surface as the filler or highly dispersed silicic acid having a hydrophilic surface as the filler.
DETAILED DESCRIPTION
Initially placed in a milling pot are 64.4 g powdered polymethylsiloxane, 0.6 g of a catalyst and 35.6 g nanoscale silicon dioxide powder, for example, amorphous silicon dioxide powder or highly dispersed nanoscale pyrogenic silicic acid having a BET surface area of 140 m 2 /g (also referred to as highly dispersed silicic acid) on 1000 g iron milling balls. This corresponds to a degree of filling of 20 vol % silicon dioxide or silicic acid, based on the starting mixture of the polymer precursor material polymethylsiloxane and SiO 2 as the filler. The filler silicon dioxide or the starting pyrogenic silicic acid also has an average particle size of less 200 nm. For example, the powder particles in this exemplary embodiment are primary particles having an average particle size of, for example, 5 nm to 80 nm or aggregates of such primary particles, but the average particle size of the aggregates is less than 200 nm. An SiO 2 powder or a corresponding highly dispersed silicic acid may be used as the nanoscale filler, which contains the powder particles as primary particles having an average particle size of 5 nm to 30 nm.
The added catalyst initiates or accelerates the crosslinking of the polymer precursor material in compression molding or other suitable shaping operations. For example, catalysts, such as aluminum acetylacetonate or zirconium acetylacetonate may be used for this purpose. In addition, a catalytically active surface may be provided on the nanoscale filler, so that the filler may assume the role of or replace the catalyst or additionally act as an alternative to the catalyst.
After a milling time of a few minutes for the starting mixture, the resulting powder mixture, having the precursor material and the filler, is separated from the iron balls and screened through a 150 μm screen. Then, the screened powder mixture is poured into a mold, where molding is performed at a pressure of 100 MPa to 200 MPa. Then, the molded powder mixture is crosslinked at a compression molding temperature of 160° C. to 200° C. and a pressure of 3 MPa to 6 MPa. Then, the resulting molded body or the molded starting mixture is pyrolyzed in an argon atmosphere at a temperature of 1050° C. to 1350° C., for example, approximately 1300° C., resulting in the a ceramic composite body.
The following table shows comparative experiments between a ceramic composite material having an aluminum oxide filler having an average particle size of approximately 1 μm and a specific electric resistance of approximately 10 15 Ωcm and a comparable ceramic composite material in which, however, the microscale aluminum oxide powder has been replaced by nanoscale silicon dioxides having an average particle size of less than 200 nm, a specific electric resistance of approximately 10 9 Ωcm and a BET surface area of approximately 140 m 2 /g. The starting mixture of the composite materials according to the following table also contains molybdenum disilicide powder and silicon carbide powder as fillers in addition to the polymer precursor material polysiloxane. However, these fillers have been kept constant with regard to particle size and amount.
TABLE 1
Specific electric
Polysiloxane
SiC
MoSi 2
Al 2 O 3
SiO 2
resistance R
(vol %)
(vol %)
(vol %)
(vol %)
(vol %)
(Ωcm)
65%
8%
13%
14%
0%
<10 3
60%
8%
13%
9%
10%
<10 3
57%
8%
13%
5%
17%
approximately 10 3
65%
8%
13%
0%
14%
>10 3
The increased specific electric resistances R achieved according to the preceding table by using nanoscale pyrogenic silicic acid or SiO 2 particles remain largely stable, even after prolonged storage times at a temperature of 1300° C., and are significantly higher than the value observed in corresponding composite materials, which do not contain a nanoscale filler. However, mixtures, in which SiO 2 or silicic acid has been added in the form of powders having an average particle size of more than 1 μm have a high porosity, a low electric resistance and an inadequate high-temperature stability.
A second embodiment begins with the same starting mixture, which has already been described with respect to the first exemplary embodiment according to the present invention described above. In this exemplary embodiment, however, the open porosity of the resulting ceramic composite material is adjusted by varying the pyrolysis time and the final temperature in pyrolysis. In this way, an open porosity amounting to between approximately 1% and more than 30% may be obtained by using the same starting mixture composition, i.e., using polysiloxane as the polymer precursor material and adding nanoscale SiO 2 , by varying the pyrolysis time and the final temperature in pyrolysis, as illustrated in the following table.
TABLE 2
Pyrolysis
Final
Relative
Relative
Open
time
temperature
weight loss
shrinkage
porosity
22.5 h
1100° C.
25.5%
14.8%
30%
34.0 h
1100° C.
24.4%
14.2%
30%
37.3 h
1300° C.
24.8%
18.1%
13%
60.0 h
1300° C.
25.3%
20.1%
5%
102.5 h
1300° C.
24.8%
20.3%
<1%
In addition to dry milling of the powder mixture of the precursor material and filler in a ball mill, a conventional wet processing method may be used for producing the starting mixture described above. In this case, the polymer precursor material, e.g., polysiloxane, is first dissolved in acetone with the catalyst to homogenize the filler, and then the nanoscale filler is incorporated into this mixture. Next, this suspension is mixed, for example, for two hours using a magnetic stirrer and finally is vacuum dried. In this procedure, the starting mixture is not heated, so that there is no thermal crosslinking of the polymer precursor material before compression molding or shaping.
Mixing the precursor material and the filler in a mixer, for example, a heated mixer, and then kneading the resulting granulated mass, offers an other manner of incorporating the nanoscale filler into the precursor material . As in dry milling, no additional solvent may be necessary.
By using a vibrating screen, the resulting or used powder or powder mixtures may be first freed of uncrushed, i.e., unmilled, agglomerates before pyrolysis or before a molding step. The mesh of this screen may be, for example, 150 μm.
In addition to compression molding, injection molding may be used to shape the starting mixture before pyrolysis.
Moreover, pyrolysis of the prepared starting mixture to form the ceramic composite material may be performed in an inert gas atmosphere, using final temperatures of 600° C. to 1400° C., depending on the precursor material and the filler.
After pyrolysis, a ceramic composite material is obtained, in which the filler either forms at least partially nanoscale inclusions in a matrix formed essentially by the polymer precursor material or in which the filler has reacted with the matrix material so that there is little or no differentiation between the filler and matrix, due to diffusion processes. In this case, a largely homogeneous ceramic composite material, in which the filler has reacted with gases released during pyrolysis, for example, may be formed from the starting mixture with the filler in pyrolysis.
The filler first used in the starting mixture may also undergo thermal decomposition in pyrolysis and/or it may react with the precursor material so that, for example, nanoscale pores formed in the matrix may be at least partially attributable to pyrolysis of the filler in the matrix. The average resulting pore size amounts to less than 200 nm, for example, 5 nm to 100 nm, depending on the average particle size of the filler used.
In addition to the silicon dioxide described above, other oxides, nitrides or carbides of silicon, aluminum, titanium, zirconium, boron, tungsten, vanadium, hafnium, niobium, tantalum or molybdenum or a mixture thereof, e.g., in the form of oxycarbides, oxynitrides, carbonitrides or oxycarbonitrides may also be used as nanoscale fillers.
In addition, the nanoscale filler may be a metallic powdered filler and/or a filler containing gold, palladium, platinum, rhodium or iridium, e.g., in the form of a sol with nanoscale colloids contained therein or a suspension containing this metallic filler.
If the nanoscale filler at first is to be at least mostly decomposed in pyrolysis to form pores in the composite material, an organic filler, such as nanoscale carbon particles or nanoscale carbon black or nanoscale organic polymers may be suitable as the filler. Depending on the amount of the filler, the pyrolysis temperature and the duration of pyrolysis, the open porosity of the ceramic composite material obtained with these fillers may be adjusted to levels between 1% and 50%, the pyrolysis, for example, being followed by aging in an oxygen-containing atmosphere, which allows carbon to burn-off as thoroughly as possible.
Suitable polymer precursor materials include a variety of known precursor materials, such as organosilicon polymer compound, for example, polysiloxanes, polysilanes, polycarbosilanes or polysilazanes, organozirconium polymer compounds, organoaluminum polymer compounds, organotitanium polymer compounds, boron-containing polymer precursor materials or mixtures or intermediates of these precursor materials.
In addition, a stabilizer and a solvent, e.g., an organic solvent, such as acetone or an alcohol or water may be added to the starting mixture, depending on the individual case. In any case, the amount of nanoscale filler in the starting mixture should be between 2 vol % and 50 vol %.
With processing properties otherwise being the same, the specific electric resistance of the resulting ceramic composite material may be increased by several orders of magnitude at both room temperature and temperatures >1200° C. when a conventional coarse filler, such as Al 2 O 3 , is replaced at least largely or completely by nanodisperse, for example, amorphous silicon dioxide (also referred to as standard SiO 2 ) or highly disperse, for example, pyrogenic silicic acid (HDS).
Also, the filler permits the coefficient of thermal expansion of a conductive ceramic composite material to be adapted, for example, to that of an adjacent nonconducting ceramic composite material bonded to it through the comparatively low coefficient of thermal expansion of silicon dioxide.
Also, if only a small amount of nanodispersed SiO 2 is added to the composite material for adjusting the coefficient of thermal expansion and increasing the specific electric resistance of the composite material, and if working with a large amount of comparatively coarse Al 2 O 3 , i.e., an amount of more than 7 vol %, in many cases, more than 3 vol %, no significant desired increase in the specific electric resistance may be observed.
The use of a microscale additional filler, such as Al 2 O 3 together with the nanoscale filler has positively effects a desired surface vitrification of the ceramic composite material, which may be used as a ceramic heater, for example, which may result in an improved high-temperature stability, for example, when the amount of nanoscale filler is greater than 9 vol %, for example, significantly greater than 10 vol %.
In another exemplary embodiment according to the present invention, as an additional parameter, the BET surface area of the filler used, which may be highly dispersed silicic acid (HDS) or powdered, for example, amorphous SiO 2 , is altered with otherwise the same composition of the starting mixture according to Table 1. As part of these experiments, the properties of the surface of the nanoscale filler have a considerable effect on the specific electric resistance ultimately achieved of the composite material.
For example, in the case of a high BET surface area of the filler, a definitely increased specific electric resistance and thus a very good insulating ceramic composite material may be obtained, while the amount of another microscale filler, such as Al 2 O 3 in the starting mixture is greater than 2%.
Through the targeted adjustment of the BET surface area of the filler in the starting mixture to largely or completely replace the microscale filler Al 2 O 3 , which would otherwise be conventional in the starting mixture by highly dispersed nanoscale, for example, amorphous SiO 2 or highly dispersed silicic acid, without causing a relevant reduction in the specific electric resistance of the resulting ceramic composite material.
Therefore, the differentiated use of, for example, highly dispersed silicic acids, i.e., nanoscale, for example, amorphous, SiO 2 powder particles having various BET surface areas and various surface modifications as fillers may thereby influence, in a controlled manner, the electric properties of the ceramic composite material produced by pyrolysis.
By using highly dispersed silicic acid (HDS), e.g., corresponding nanoscale, for example, amorphous, SiO 2 powder particles having a BET surface area of at least 50 m 2 /g, for example, 90 m 2 /g, up to 450 m 2 /g with otherwise the same composition of the starting mixture, the specific electric resistance of the composite material may be increased. The increase in specific resistance is greater, because, at the same BET surface area, instead of a highly dispersed silicic acid having a hydrophobicized surface, i.e., a pyrogenic nanoscale silicic acid, for example, a highly dispersed silicic acid without surface hydrophobicization or with a hydrophilic surface is used.
Two additional parameters as the BET surface area and the properties of the surface of the tiller (hydrophilic/hydrophobic) may allow the mechanical properties of the composite material, such as the coefficient of thermal expansion or the surface vitrification properties, to be adjusted to the respective individual case in a wide range, in addition to adjusting the specific electric resistance. In addition, varying the BET surface area of the nanoscale filler also influences the pyrolysis rate, sintering properties, porosity and viscosity of the starting mixture and the resulting composite material.
FIG. 1 illustrates a starting mixture containing 60 vol % polysiloxane, 10 vol % silicon carbide, 13 vol % molybdenum disilicide and 17 vol % SiO 2 particles or highly dispersed silicic acid having an average particle size of less than 200 nm as nanoscale filler. Then, a ceramic composite material was produced from this starting mixture, and the specific electric resistance R (Ωcm) was determined as a function of the BET surface area (m 2 /g) of the nanoscale filler.
The circular measurement points in FIG. 1 show measurements using a nanoscale, highly dispersed silicic acid having the BET surface area indicated, in which the powder particles have a hydrophobic surface area obtained by a carbonaceous surface modification (pyrolysis). The squares at a corresponding particle size or particle size distribution of the powder particles indicate measurement points obtained by using highly dispersed silicic acid of the stated BET surface area having a hydrophilic surface.
The increase in specific electric resistance R as a function of the BET surface area may be greater when using hydrophilic highly dispersed silicic acid than when using hydrophobic highly dispersed silicic acid. Furthermore, the BET surface area of the nanoscale filler may have a significant influence on resulting specific electric resistance R of the composite material. Specific electric resistance R, as shown in FIG. 1, are presented on the y axis in a logarithmic scale.
Starting with a composition according to Table 1 having an Al 2 O 3 content of 5 vol %, replacing the highly dispersed pyrogenic silicic acid having a BET surface area of 140 m 2 /g by a corresponding highly dispersed pyrogenic silicic acid having a BET surface area of 225 m 2 /g may permit the amount of highly dispersed silicic acid to be reduced in this starting mixture and through a correspondingly greater amount of Al 2 O 3 having an average particle size of approximately 1 μm, polysiloxane and conductive molydenum disilicide, i.e., a starting mixture having 60 vol % polysiloxane, 8 vol % silicon carbide, 13 vol % molydenum disilicide, 6 vol % Al 2 O 3 and 13 vol % SiO 2 (highly dispersed pyrogenic silicic acid) may be replaced, while achieving an increase in specific electric resistance R of the composite material from approximately 10 3 Ωcm to more than 10 4 Ωcm.
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A ceramic composite material, for example, a ceramic molded body or a layer obtained by pyrolysis of a starting mixture, containing at least one polymer precursor material and at least one filler, which has an average particle size of less than 200 nm. Such a composite material may be used, for example, for producing fibers, filters, catalyst support materials, ceramic sheathed-element glow plugs, metal-containing reactive composite materials, porous protective shells for sensors, ceramic or partially ceramic coatings or microstructured ceramic components.
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CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional appl. No. 61/484,558 filed 10 May 2011, the entire disclosure of which is incorporated herein by reference.
BACKGROUND INFORMATION
[0002] Bacteria is found virtually everywhere and is responsible for a significant amount of disease and infection. Killing and/or eliminating these microorganisms is desirable to reduce exposure and risk of disease.
[0003] Bacteria in many environments are present in high concentrations and have developed self preservation mechanisms and, therefore, are extremely difficult to remove and/or eradicate. They can exist in planktonic, spore and biofilm forms.
[0004] In a biofilm, bacteria interact with surfaces and form surface colonies which adhere to a surface and continue to grow. The bacteria produce exopolysaccharide (EPS) and/or extracellularpolysaccharide (ECPS) macromolecules that keep them attached to the surface and form a protective barrier effective against many forms of attack. Protection most likely can be attributed to the small diameter of the flow channels in the matrix, which restricts the size of molecules that can transport to the underlying bacteria, and consumption of biocides through interactions with portions of the EPS/ECPS macromolecular matrix.
[0005] Bacteria often form spores, which provide additional resistance to eradication efforts. In this form, the bacteria create a hard, non-permeable protein/polysaccharide shell around themselves which prevents attack by materials that are harmful to the bacteria.
[0006] Additionally, bacteria in biofilm- or spore forms are down-regulated (sessile) and not actively dividing. This makes them resistant to attack by a large group of antibiotics and antimicrobials, which attack the bacteria during the active parts of their lifecycle, e.g., cell division.
[0007] Due to the protection afforded by a macromolecular matrix (biofilm) or shell (spore) and their down-regulated state, bacteria in biofilm- and spore states are very difficult to treat. The types of biocides and antimicrobials effective in treating bacteria in this form are strongly acidic, oxidizing, and toxic, often involving halogen atoms, oxygen atoms, or both. Common examples include concentrated bleach, phenolics, strong mineral acids (e.g., HCl), hydrogen peroxide and the like. Commonly, large dosages of such chemicals are allowed to contact the biofilm or spore for extended amounts of time (up to 24 hours in some circumstances), which makes them impractical for many applications.
[0008] Recently developed formulations intended for use in connection with compromised animal/human tissue can solvate a biofilm matrix so that still-living bacteria can be rinsed or otherwise removed from infected tissue; the concentrations of active ingredients in these formulations are too low to effectively kill the bacteria, thus making them ill suited for use as disinfecting agents. More recently, solutions that can disrupt the macromolecular matrix, or bypass and/or disable the defenses inherent in these matrices, allowing lethal doses of antimicrobial ingredients in the solutions to access and kill the bacteria in their biofilm and sessile states have been described; unlike the aforementioned formulations, these solutions can be used as disinfectants.
[0009] Most water filtration is accomplished using filters made of materials such as paper, fiber, and synthetic fibers. Unclean, bacteria-laden water is passed through a membrane having a controlled pore size, typically on the order of ˜0.20 to ˜0.45 μm. These membranes are effective at keeping bacteria from passing through them into a clean water reservoir, but they do not weaken, disable or kill the bacteria. This latter characteristics make such membranes susceptible to bacterial growth, thereby increasing the risks of contamination with biofilms and spore-forming bacteria and reduced flow rates due to clogging.
[0010] Silver-loaded ceramic filters use the antimicrobial properties of silver to kill bacteria as they pass through a porous ceramic substrate. To achieve high efficacy, flow rates must be kept low. Further, these filters have a high propensity for clogging. Finally, silver ions are not particularly efficacious in debilitating and killing bacteria in biofilm- and spore forms.
[0011] Devices and articles can be provided with coatings that include antimicrobials such as cationic compounds (e.g., quaternary ammonia compounds), silver and copper compounds, and peptides. These coatings are limited in their efficacy against resistant forms of bacteria and have very thin regions of effective antimicrobial effect. These types of coatings are generally designed to prevent surface attachment of bacteria rather than to disinfect.
[0012] Certain eluting devices and articles are designed to slowly release anti-bacterial compounds when exposed to moisture. These solids typically been impregnated by antimicrobial agents which, over time, work their way to the surface and are released. The concentrations of solutions eluted from these devices and articles, as well as the efficacy of the employed antimicrobial agents against resistant forms of microbes, are low. The utility of such devices and articles is further reduced in situations where a liquid is to pass through the device due to more rapid depletion of the antimicrobial agent(s).
[0013] A solid material capable of preventing bacterial growth, and preferably killing bacteria coming into contact with or close proximity to the solid material, remains desirable. Such a solid preferably can be useful in a variety of forms including, but not limited to, filters, eluting devices, and coatings.
SUMMARY
[0014] Liquid compositions effective for disinfection purposes are described in U.S. Pat. Publ. No. 2010/0086576 A1. Those compositions display both moderately high tonicity (i.e., large amounts of osmotically active solutes) and relatively low pH (about 4≦pH≦6) which work with surfactants to induce membrane leakage in bacteria, leading to cell lysis. The composition acts at least in part to interrupt or break ionic crosslinks in the macromolecular matrix of a biofilm, facilitating the passage of solutes and surfactant through the matrix to bacteria entrained therein and/or protected thereby. In addition to being lethal toward a wide spectrum of gram positive and gram negative bacteria, these liquid compositions also exhibit lethality toward other microbes such as viruses, fungi, molds, and yeasts.
[0015] However, some end-use applications are not conducive to the relatively high concentrations that provide the liquid compositions with their efficacy. These include, but are not limited to, applications where a high concentration of free (unbound) species of these ingredients is unacceptable, applications where an extremely large volume of liquid needs to be disinfected, and applications where such ingredients will be consumed.
[0016] The solid materials of the present invention are designed and intended to achieve, in a non-liquid form, a set characteristics similar to those displayed by the aforementioned liquid compositions: high tonicity and surfactant availability.
[0017] These solid materials, adapted to kill bacteria in planktonic, spore and biofilm states, include a crosslinked version of a water soluble polyelectrolyte and entrained surfactant. This combination of components permits the local chemistry within the solid material and in its immediate vicinity, when in use in an aqueous environment, to mimic that of the previously described liquid disinfecting composition: high tonicity and high surfactant concentration. In at least some embodiments, the solid material includes no biocidal additives, particularly active antimicrobial agents.
[0018] In certain aspects, the solid material can be prepared by crosslinking a liquid or flowable polyelectrolyte in the presence of the surfactant(s).
[0019] Also provided are methods of using the foregoing composition. When a liquid is passed through or in proximity to the solid material, any bacteria or other microorganism is exposed to the local chemistry conditions discussed above: high tonicity, relatively low pH, and available surfactant, a combination that can induce membrane leakage in bacteria leading to cell lysis. These characteristics permit the solid material to be very effective at bypassing and disabling bacterial biofilm and spore defenses, allowing the solid material to kill bacteria in any of its several states.
[0020] The solid material can be used to disinfect liquids, in either filter or insert form, and as surface coating that prevents bacterial contamination by killing any bacteria that come into contact therewith. That it can perform these tasks while losing or transmitting very little of its chemical components into the environment being treated is both surprising and advantageous. Further, any chemical components that do enter the environment are relatively benign.
[0021] To assist in understanding the following description of various embodiments, certain definitions are provided immediately below. These are intended to apply throughout unless the surrounding text explicitly indicates a contrary intention:
“microbe” means any type of microorganism including, but not limited to, bacteria, viruses, fungi, viroids, prions, and the like; “antimicrobial agent” means a substance having the ability to cause greater than a 90% (1 log) reduction in the number of one or more of microbes; “active antimicrobial agent” means an antimicrobial agent that is effective only or primarily during the active parts of the lifecycle, e.g., cell division, of a microbe; “biofilm” means a community of microbes, particularly bacteria and fungi, attached to a surface with the community members being contained in and/or protected by a self-generated macromolecular matrix; “residence time” means the amount of time that an antimicrobial agent is allowed to contact a bacterial biofilm; “biocompatible” means presenting no significant, long-term deleterious effects on or in a mammalian species; “biodegradation” means transformation, via enzymatic, chemical or physical in vivo processes, of a chemical into smaller chemical species; “polyelectrolyte” means a polymer with multiple mer that include an electrolyte group capable of dissociation in water; “strong polyelectrolyte” is a polyelectrolyte whose electrolyte groups completely dissociate in water at 3 pH 9; “weak polyelectrolyte” is a polyelectrolyte having a dissociation constant of from ˜2 to ˜10, i.e., partially dissociated at a pH in the range where a strong polyelectrolyte's groups are completely dissociated; and “polyampholyte” is a polyelectrolyte with some mer including cationic electrolyte groups and other mer including anionic electrolyte groups.
[0033] Hereinthroughout, pH values are those which can be obtained from any of a variety of potentiometric techniques employing a properly calibrated electrode.
[0034] The relevant portions of any specifically referenced patent and/or published patent application are incorporated herein by reference.
DETAILED DESCRIPTION
[0035] The antimicrobial solid material can contain as few as two components: a crosslinked polymer network and at least one entrained surfactant, each of which generally is considered to be biocompatible. Certain embodiments of the composition employ no active biocides. In these and other embodiments, the identity of the polymers and surfactants, as well as the concentrations in which each is discharged from the solid material, can be such that recognized toxicity limits are not exceeded during normal use.
[0036] The solid material is lethal to planktonic and bacterial cells with high efficacy, is not readily consumed, provides a significant amount of surface area for microbial interactions, and does not create toxicity in solutions being treated. The solid material is not particularly soluble in water under most conditions (e.g., moderate temperatures and solute concentrations), but the polyelectrolyte chains are at least hydrophilic and, where the solid material is to be used in a setting where it might not be immersed in an aqueous medium, preferably hygroscopic, thereby permitting the solid material to swell somewhat when in the presence of moisture, particularly water.
[0037] The solid material of the present invention requires some level of water or humidity to function appropriately. This can determined or defined in a variety of ways. The polyelectrolytes must be capable of localized liquid charge interaction (meaning at least two water molecules are contacting or very near an electrolyte group); alternatively, sufficient water must be present to activate the charge of the electrolyte; and/or sufficient water to permit bacterial growth. As non-limiting examples, gaseous or liquid water can be applied directly to the solid material or can result from other, indirect means, e.g., water vapor contained in breath or ambient air, condensates, etc.
[0038] Because the antimicrobial material is solid, it does not itself have a true pH; in use, however, the local pH of any aqueous composition in which it is deployed preferably is lower than ˜7 to ensure proper antimicrobial activity. Reduced pH values (e.g., less than ˜6.5, ˜6.0, ˜5.5, ˜5.0, ˜4.5 and even ˜4.0) generally are believed to correlate with increases in efficacy of the solid material, although this effect might not be linear, i.e., the enhancement in efficacy may be asymptotic past a certain hydronium ion concentration. Without wishing to be bound by theory, acidic protons (i.e., hydronium ions) might be involved in breaking ionic crosslinks in the macromolecular matrix of a biofilm.
[0039] In addition to more strongly acidic local environments, high local osmolarity conditions also are believed to increase efficacy. Accordingly, larger concentrations of polyelectrolytes, larger concentrations of surfactant, surfactants with shorter chain lengths (e.g., no more than C 10 , typically no more than C 8 , commonly no more than C 6 ), and surfactants with smaller side groups around the polar group each are more desirable.
[0040] The lethality of the surfactant component(s) is increased and/or enhanced when the solid material can provide to the local environment in which it is deployed at least moderate effective solute concentrations (tonicity). (In biological applications, a 0.9% (by wt.) saline solution, which is ˜0.3 Osm, typically is considered to be have moderate tonicity, while a 3% (by wt.) saline solution, which is ˜0.9 Osm, generally is considered to be hypertonic.) Without wishing to be bound by theory, higher tonicities may exert higher osmotic pressure to the bacterial cell wall, which increases its susceptibility to interruption by surfactant. Local osmolarity (tonicity) generally increases in proportion to the number and type of electrolytes present in the polymeric network. (By local osmolarity is meant that of a liquid contained in the solid material. While this might vary from place to place throughout the article, preference is given to those solid materials capable of providing high local osmolarities throughtout.)
[0041] The polyelectrolyte(s) that form the bulk of the solid material preferably are at least somewhat water soluble but also essentially water insoluble after being cross-linked. A partial list of polyelectrolytes having this combination of characteristics included, but are not limited to, strong polyelectrolytes such as polysodium styrene sulfonate and weak polyelectrolytes such as polyacrylic acid, pectin, carrageenan, any of a variety of alginates, polyvinylpyrrolidone, carboxymethylchitosan, and carboxymethylcellulose. Included in potentially useful polyamphyolytes are amino acids and betaine-type cross-linked networks; examples would be hydrogels based on sodium acrylate and trimethyl-methacryloyloxyethylammonium iodide, 2-hydroxyethylmethacrylate, or 1-vinyl-3(3-sulfopropyl)imidazolium betaine. Those polymeric materials having electrolyte groups that completely (or nearly completely) dissociate in water and/or provide relatively low local pH values are desired for efficacy are preferred.
[0042] Also preferred are those polyelectrolytes having a high density of mer with electrolyte-containing side groups. Without wishing to be bound by theory, the large number of acidic or polar side groups on the polyelectrolyte are believed to function equivalently to the high tonicity solution of the previously described liquid composition.
[0043] Several crosslinking mechanisms including but not limited to chemical, high temperature self-crosslinking (i.e., dehydrothermal crosslinking), and irradiation (e.g., e-beam or gamma rays) can be employed. The ordinarily skilled artisan can discern and select an appropriate crosslinking mechanism once a polyelectrolyte is selected.
[0044] Another option is to create crosslinks during the polymerization process itself, such as by condensing adjacent sulfonic acid groups to yield sulfonyl crosslinks.
[0045] Independent of crosslinking method, the solid material can be formed by crosslinking polymers (or polymerizable monomers) in an aqueous solution contained in a heat conductive mold, followed by rapid freezing and subsequent lyophilizing. The resulting sponge-like material generally takes the shape of the mold in which it was formed. A potential advantage of this process is that it can provide a ready means for removing any hazardous or undesirable precursor chemicals used in the polymerization and/or cross-linking steps. Solids resulting from this type of process often have a spongy appearance, with relatively large pores connected by tortuous paths. Often, pores less than ˜0.22 μm, less than ˜0.45 μm, less than ˜0.80 μm, and less than ˜0.85 μm are desirable (based on the diameters of endotoxins, bacteria, and spores); for these and other applications, a solid material with at least some larger pores (e.g., less than ˜1, 2, 5, 10, 50, or 100 μm) can be used.
[0046] The crosslink density in the solid material plays an important role, specifically, those solid materials with higher crosslink densities tend to maintain higher surfactant concentrations for a longer period of time due to, presumably, longer mean free paths in the polymeric network.
[0047] The solid material contains a sufficient amount of surfactant to interrupt or rupture cell walls of bacteria contacting or coming into the vicinity of the solid material. This amount can vary widely based on a variety of factors including, for example, whether a biofilm already exists in the area to be treated (and whether that biofilm is entrenched, a factor which relates to the type of proteins and mass of the macromolecular matrix), the species of bacteria, whether more than one type of bacteria is present, the solubility of the surfactant(s) in the local environment, and the like. The surfactant component(s) generally constitute as low as ˜0.03% and as high as ˜10%, ˜15% or even ˜17.5% (all by wt.) of the solid material.
[0048] Essentially any material having surface active properties in water can be employed, although those that bear some type of ionic charge are expected to have enhanced antimicrobial efficacy because such charges, when brought into contact with a bacteria, are believed to lead to more effective cell membrane disruption and, ultimately, to cell leakage and lysis. This type of antimicrobial process can kill even sessile bacteria because it does not involve or entail disruption of a cellular process. Cationic surfactants are most desirable followed by, in order, zwitterionic, anionic and non-ionic.
[0049] Potentially useful anionic surfactants include, but are not limited to, sodium chenodeoxycholate, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octane-sulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, sodium dodecyl sulfate, sodium glycodeoxycholate, sodium lauryl sulfate, and the alkyl phosphates set forth in U.S. Pat. No. 6,610,314. Potentially useful cationic surfactants include, but are not limited to, hexadecylpyridinium chloride monohydrate and hexadecyltrimethylammonium bromide, with the latter being a preferred material. Potentially useful nonionic surfactants include, but are not limited to, polyoxyethyleneglycol dodecyl ether, N-decanoyl-N-methyl-glucamine, digitonin, n-dodecyl B-D-maltoside, octyl B-D-glucopyranoside, octylphenol ethoxylate, polyoxyethylene (8) isooctyl phenyl ether, polyoxyethylene sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate. Potentially useful zwitterionic surfactants include, but are not limited to, 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propane sulfonate, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate, 3-(decyldimethylammonio) propanesulfonate inner salt, and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. For other potentially useful materials, the interested reader is directed to any of a variety of other sources including, for example, U.S. Pat. Nos. 4,107,328 and 6,953,772 as well as U.S. Pat. Publ. No. 2007/0264310.
[0050] The surfactant preferably is present in the polymer network at the time that crosslinking occurs (or the time of polymerization in the case of the type of simultaneous polymerization and condensation discussed above). If it is not, a crosslinked polymer article or film must be post-treated to ensure proper entrainment of the surfactant. A possible method for accomplishing this is immersion of the article or film in an aqueous solution that contains one or more surfactants, followed by removal of excess water via a drying (e.g., thermal or freeze) or evacuation process.
[0051] In certain embodiments, the surfactant(s) can be the only antimicrobial agents in the composition, specifically, the composition can be free of active antimicrobial agents.
[0052] In addition to the surfactant(s), one or more ionic compounds (salts) can be incorporated into the solid material so as to enhance its ability to create localized regions of high tonicity.
[0053] Regardless of how achieved, the local tonicity around the solid material is at least moderately high, with an osmolarity of at least about 0.1 Osm being preferred for most applications. Solid materials that create local osmolarities greater than about 0.1 Osm will have enhanced bactericidal activity; increases in the osmotic pressure applied to the bacteria enhance antimicrobial efficacy.
[0054] A variety of additives and adjuvants can be included to make a solid material more amenable for use in a particular end-use application without negatively affecting its efficacy in a substantial manner. Examples include, but are not limited to, emollients, fungicides, fragrances, pigments, dyes, abrasives, bleaching agents, preservatives (e.g., anti-oxidants) and the like. Depending on the identity and nature of a particular additive, it can be introduced at any of a variety of times during production of the solid material.
[0055] The solid material does not require inclusion of an active antimicrobial agent for efficacy, but such materials can be included in certain embodiments. For example, one or more of bleach, any of a variety of phenols, aldehydes, quaternary ammonium compounds, etc., can be added.
[0056] As previously stated, bacteria present in a biofilm derive some inherent protection offered by the EPS/ECPS macromolecular matrix. Without wishing to be bound by theory, the high tonicity and slightly acidic nature of the solid material (as well as the region immediately surrounding it when it is in use) are believed to interfere with and break the ionic crosslinks in the macromolecular matrix of any biofilm passing near or through the material, thus permitting better access to the previously protected bacteria. Additionally, the high tonicity provided in and around the solid material means that an abundance of ions are available, even though some are consumed in the EPS. These ions can assist in killing the bacteria while they remain in the biofilm and after they are freed therefrom, perhaps by making the bacterial cell walls susceptible to being ruptured by the surfactant component(s).
[0057] Thus, the solid material that includes one or more surfactants entrained in a polymer network possesses a combination of characteristics and attributes that allow it to be a highly effective yet non-toxic antimicrobial:
1) a capability to provide an aqueous liquid contacting it a local pH (in and/or very near it) of less than 7, preferably less than 6; 2) the polymeric network is hydrophilic (and, where the solid material is intended for use at least some of the time in a non-immersed state, perhaps even hygroscopic); 3) a capability to provide an aqueous liquid contacting it an effective local solution osmolarity (in and/or very near the solid material) of at least ˜0.1 Osm; 4) a sufficient concentration of one or more surfactants to rupture cell walls of bacteria contacting or coming near to the solid material; and 5) a crosslink density of the polymeric network is great enough to greatly slow the rate of surfactant loss from the material.
[0063] This solid material is actively antimicrobial, has greater antimicrobial efficacy against bacteria in resistant forms, is not rapidly consumed, and does not create toxicity in the medium being treated.
[0064] The solid material can take any of a variety of intermediate and final shapes or forms including, but are not limited to, a spongy solid that is permeable to vapor and or liquids; a molded, extruded or deposited sheet; and an extruded fiber or thread. Once in a particular shape, the material then can be further processed or manipulated so as to provide a desired shape, e.g., a sheet good can be rolled or folded so as to provide a membrane of a particular geometry. Thus whether the material is used in its manufactured form or it is post processed by thermal forming, mechanical shaping, lamination, granulation, pulverization, etc., it is considered to be within the present disclosure.
[0065] A single, non-limiting example of a potential use for a solid antimicrobial material is as a filter (or part of a filtration device) to be placed in the flow path of a vapor or liquid passing there-through, -over or -by. Such a material can be housed, sealed, or adhered in a variety of ways so as to permit fluid flow to be directed through, around, or over it.
[0066] A filter can be provided by making a spongy solid (via, for example, a lyophilization process such as the one described above) with a surfactant trapped therein. Water can be passed through or past the spongy solid, which will work as a filter device, which is actively antimicrobial and kills any bacteria passing through the element.
[0067] Such a filter can have high flow rates because of its active antimicrobial nature and, therefore, can have larger pore sizes than current sterile filters which rely on extremely small pores to prevent passage of bacteria through the filter. Larger pores also mean that such a filter will be less susceptible to clogging, thus increasing its viable lifecycle. Thus, the resulting filtration device has high bactericidal activity toward planktonic and bacterial cells, permits high fluid flow rates, is less susceptible to clogging, and produces disinfected water which is non-toxic when ingested.
[0068] As an alternative to a spongy, amorphous mass, a much more structured form, e.g., a fabric (woven or nonwoven) made from or incorporating threads provided from a solid antimicrobial material of the present invention, also can be employed for such filtration applications.
[0069] In addition to water filtration, other potential uses for solid materials of the present invention include, but are not limited to, air filters, odor controlling articles (e.g., clothing such as socks, shoe inserts, etc.), pool water treatment articles, disinfecting wipes, mine waste pool barriers (to prevent acidic leakage due to bacterial activity), bandages, humidifier wicking elements, layers in personal protection articles such as diapers and feminine hygiene products, and the like.
[0070] The solid material of the present invention also can be used as an antimicrobial surface coating or external surface layer for the prevention of bacterial contamination of the protected surface. In this manner, the material will kill bacteria, in any form, coming into contact with the surface of the material. Potential end use applications for such coatings include, but are not limited to, cooler surfaces, refrigerator interiors, drip pans (e.g., refrigerators, dehumidifiers, etc.), food storage containers, tracheotomy tubes, external surfaces of temporarily or permanently implanted medical devices, contact surfaces in medical equipment (e.g., fluid lines, fittings, joints, reservoirs, covers, etc.), reagent bottles, telephone and remote control surfaces (e.g., buttons), medical devices intended to contact more than one patient (e.g., blood pressure cuffs, stethoscopes, wheelchairs, gurneys, etc.), plumbing fixtures, pipes and traps, recreational vehicle cisterns and tanks, shower walls and components, canteens, beverage dispensers and transfer lines, baby feeding equipment (e.g., bottles, nipples, etc.), pacifiers, teething rings, toys, playground and exercise equipment, outdoor equipment (e.g., tents, boat covers, sleeping bags, etc.), and the like.
[0071] As is clear from the foregoing description, the solid material may take many different physical forms and find use in a variety of devices. Its components can be provided from a wide variety of materials, and its polymer network can be crosslinked in a variety of ways. Thus, the ordinarily skilled artisan understands that the functionality of the components and not their specific identity or manner of processing is that which is most important; the ever evolving fields of chemistry and polymer science are anticipated to provide additional options not known at the time of this writing that provide similar functionality. (By way of non-limiting example, surfactants are described here as a key component for providing bactericidal activity; however, newly developed compounds that do not fit entirely within the definition of “surfactant” yet still possess the types of charged or polar side groups that provide the same functional mechanism are quite reasonably expected to be useful in solid material.)
[0072] While various embodiments of the present invention have been provided, they are presented by way of example and not limitation. The following claims and their equivalents define the breadth and scope of the inventive methods and compositions, and the same are not to be limited by or to any of the foregoing exemplary embodiments.
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A solid material adapted to kill bacteria in planktonic, spore and biofilm states is lethal toward a wide spectrum of gram positive and gram negative bacteria as well as other microbes. The solid material includes a significant amount of one or more surfactants entrained in a crosslinked polymeric network.
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FIELD OF THE INVENTION
The invention relates generally to fold out tables. In particular, the invention relates to a fold out table stored in a cabinet-like or desk-like structure.
BACKGROUND
One conventional method to improve space is the use of a fold out table stored in a cabinet-like or desk-like structure. Such a table typically includes table leafs which can open and fold out into a table position and then fold in and close to revert to the cabinet-like or desk-like structure. Examples of prior fold out tables can be found in U.S. Pat. Nos. 3,999,822, 2,672,384, and 2,059,994.
BRIEF DESCRIPTION OF THE DRAWINGS
A complete understanding of the preferred embodiments will be obtained from the following description when taken in connection with the accompanying drawing figures, wherein like reference numerals identify the same parts throughout.
FIG. 1 is an elevated front view of a cabinet made in accordance with a preferred embodiment of the present invention with the front face thereof in a closed position.
FIG. 2 is an elevated front view of the cabinet table in accordance with the preferred embodiment in the open position and an upper balance plate piece in a folded, storage position.
FIG. 3A is a side view of the cabinet table in FIG. 2 illustrating the upper balance plate piece moving from a folded, storage position to an unfolded, vertical position.
FIG. 3B is a side view of the cabinet table in FIG. 2 illustrating the upper balance plate piece moving downward from the unfolded, vertical position.
FIG. 3C is a side view of the cabinet table in FIG. 2 illustrating the upper balance plate piece moving to an unfolded, deployed position.
FIG. 4 is a side view of the cabinet table shown in FIG. 3C .
FIG. 5 is an elevated front view of the cabinet table in FIG. 2 illustrating the interior box in the open position and the upper balance plate piece in the unfolded, deployed position.
FIG. 6 is an elevated front view of the cabinet table in FIG. 2 illustrating certain table leafs in an external, vertical, folded, raised, and unlocked position.
FIG. 7 is an elevated front view of the cabinet table in FIG. 2 illustrating certain table leafs in an external, horizontal, folded, and unlocked position.
FIG. 8 is a sectional view of the posterior of the cabinet table in FIG. 2 from behind the cabinet table illustrating certain slots and tracking mechanisms.
FIG. 9 is an elevated front view of the cabinet table shown in FIG. 7 illustrating two table leafs in an external, horizontal, folded, and unlocked position and showing the lock mechanism.
FIG. 10A is a sectional view of a key in relation to the two table leafs shown in FIG. 9 in an external, horizontal, folded, and unlocked position.
FIG. 10B is a sectional view of the key in relation to the two table leafs in an external, horizontal, folded, and locked position.
FIG. 11 is an elevated front view of the cabinet table shown in FIG. 9 illustrating the two table leafs in an external, horizontal, folded, and locked position.
FIG. 12 is an elevated front view of the cabinet table shown in FIG. 9 illustrating four table leafs in an external, horizontal, unfolded, and locked position.
FIG. 13 is a front view of the cabinet table when in the open position shown in FIG. 12 .
FIG. 14 is an elevated front view illustrating multiple tables according to the preferred embodiment oriented to form one continuous table.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
10
cabinet containing a fold out table
11
top plate
12
front face
14
top front face
16
bottom front face
18
cabinet front frame
20
back plate
22
receptacle
24
slot
25
tracking mechanism
26
side plate
28
front plate
30
hinge
31
hinge mechanism
32
support wall
34
key
35
key notch
36
guide pin
37
intermediate plate
38
stopper
39
balance plate
40
upper balance plate piece
41
lower balance plate piece
42
large table leaf
44
small table leaf
46
hand grip
48
leaf hinge
50
lock receiver
52
lock prong
56
balance plate foot
60
side wall
62
interior box
64
cabinet body
66
holding mechanism
67
balance plate stop
68
balance plate guide track
69
balance plate clearance
70
toe space
Referring now to the drawings, as shown in FIG. 1 , a cabinet table includes a cabinet containing a fold out table 10 . The cabinet containing a fold out table 10 is partially slid out and away from the cabinet body 64 to an open position as shown in FIG. 2 by a hinge 30 which attaches the top front face 14 to the intermediate plate 37 so that the top front face 14 can rotate 90 degrees away from the top plate 11 from a vertical position to a horizontal position and rest in a horizontal position that is substantially perpendicular to the bottom front face 16 when the cabinet is open. Preferably, there is at least one stopper 38 attached to the top surface of the intermediate plate 37 . Due to this stopper 38 , the exterior of the cabinet front frame 18 lies substantially flush against the interior of the top front face 14 when the cabinet containing a fold out table 10 is in a closed position so that it is not possible to distinguish the cabinet containing the fold out table 10 from a cabinet that does not contain a fold out table.
When the cabinet containing a fold out table 10 is slid out, there is an interior box 62 formed by a back plate 20 , side plates 26 , and a front plate 28 . The interior box 62 is attached to the cabinet body 64 in a manner that allows the interior box 62 to slide out and away from the cabinet body 64 to an open position. An upper balance plate piece 40 is in a horizontal position over part of the interior box 62 as shown in FIG. 2 . The upper balance plate piece 40 and lower balance plate piece 41 are connected via a hinge mechanism 31 so that the upper balance plate piece 40 can rotate toward the interior box 62 . A balance plate clearance 69 allows the upper balance plate piece 40 to rest on the support wall 32 in a folded, horizontal, internal storage position.
As shown in FIG. 3A , to deploy the fold out table, the upper balance plate piece 40 first rotates 90 degrees from a folded, horizontal, internal storage position in which the upper balance plate piece 40 is perpendicular to the lower balance plate piece 41 to a vertical deployed position in which the upper balance plate piece 40 is vertically aligned with the lower balance plate piece 41 . As shown in FIG. 3B , after the upper balance plate piece 40 rotates to the vertical deployed position so that the upper balance plate piece 40 is vertically aligned with the lower balance plate piece 41 , a balance plate guide track 68 guides the downward vertical movement of balance plate pieces 40 and 41 as gravity pulls balance plate pieces 40 and 41 toward the floor. As shown in FIG. 3C , after the balance plate guide track 68 guides the downward vertical movement of balance plate pieces 40 and 41 toward the floor, the lower balance plate piece 41 rests on the floor. The preferred embodiment includes at least one balance plate foot 56 . Preferably, this balance plate foot 56 is adjustable. Due to an adjustable balance plate foot 56 , it is unnecessary to have wheels which can easily damage floors and which cannot be adjusted to provide a level table surface on an unlevel floor. The preferred embodiment includes at least one finger grip located on the part of the upper balance plate piece 40 which faces the interior box. The lower balance plate piece 41 contains at least one balance plate stop 67 to stop the vertical upward motion of balance plate pieces 40 and 41 at the proper height when the upper balance plate piece 40 is pulled in a vertical upward motion to return the upper balance plate piece 40 to a storage position.
As shown in FIG. 4 , the cabinet containing a fold out table 10 has a cabinet body 64 including a top plate 11 , two substantially vertical side walls 60 , toe space 70 , and a cabinet front frame 18 . The top plate 11 is mounted to the two substantially vertical side walls 60 . The top plate 11 and side walls 60 are each attached to the cabinet front frame 18 . A front face 12 contains a top front face 14 and a bottom front face 16 . The top front face 14 is located above and is in vertical alignment with the bottom front face 16 .
FIG. 5 shows the cabinet containing a fold out table 10 in a fully open position with a support wall 32 , a key 34 in the support wall 32 , two large table leafs 42 , and two small table leafs 44 . Each side plate 26 is attached to the front plate 28 and to the back plate 20 . The support wall 32 is attached to the front plate 28 and the back plate 20 , preferably at a location that is equidistant from each side plate 26 .
As shown in FIG. 5 , the back plate 20 includes a receptacle 22 , two slots 24 , and two tracking mechanisms 25 . While the only depiction of the receptacle 22 in the drawings is that of an electrical receptacle, other embodiments could include other types of receptacles, including, but not limited to, receptacles for telephone and internet connections. Each tracking mechanism 25 is attached to the side of a large table leaf 42 . Each large table leaf 42 is attached to a small table leaf 44 by a leaf hinge 48 , which allows the small table leafs 44 to remain in a folded position as shown in FIG. 11 or an unfolded position as shown in FIG. 12 . Each large table leaf 42 and each small table leaf 44 contain a hand grip 46 .
Each large table leaf 42 is pivotably slidable to various positions without difficulty via the slot 24 and tracking mechanism 25 . Each large table leaf 42 and small table leaf 44 is raised from the internal, folded position as shown in FIG. 5 by a slot 24 and tracking mechanism 25 to the folded, raised position as shown in FIG. 6 . Each large table leaf 42 and small table leaf 44 is moveable from the folded, raised position as shown in FIG. 6 by the slot 24 and tracking mechanism 25 to the horizontal, folded, unlocked position as shown in FIG. 7 .
As shown in FIG. 7 , each large table leaf 42 contains a holding mechanism 66 which prevents the large table leaf 42 and small table leaf 44 from separating or flapping open as the large table leaf 42 and small table leaf 44 are moved from position to position. Preferably, this holding mechanism 66 is a rare earth magnet located near the surface of the large table leaf 42 .
FIG. 8 illustrates the cabinet containing a fold out table 10 shown in FIG. 7 from behind with one tracking mechanism 25 shown in the position it would have when one large table leaf 42 and one small table leaf 44 are in the vertical position and one tracking mechanism 25 shown in the position it would have when one large table leaf 42 and one small table leaf 44 are in the horizontal position.
One small table leaf 44 contains a lock prong 52 , while the other small table leaf 44 contains a lock receiver 50 . Both the lock prong 52 and the lock receiver 50 are preferably fixed to the center of the respective small table leaf 44 . One small table leaf 44 contains at least one guide pin 36 and the other small table leaf 44 contains at least one corresponding guide pin hole on the respective interior surface.
As shown in FIG. 9 , each large table leaf 42 and small table leaf 44 in a horizontal, folded, unlocked position is moved horizontally and inwardly and then locked in the position shown in FIG. 11 . Each large table leaf 42 contains a key notch 35 which allows the large table leafs 42 to slide together and lock via the key 34 as shown in FIG. 10A and FIG. 10B . The key notch 35 aids in the ability of the unfolded table to support substantial amounts of weight and downward force while maintaining a level surface.
The small table leafs 44 are unfolded to create a table shown in FIG. 12 which is large enough to comfortably seat at least three adults. As shown in FIG. 13 , the unfolded table top rests in a flat position with no interference from the upper balance plate piece 40 or other elements of the interior box 62 .
The cabinet containing a fold out table 10 is retrofittable to various cabinet sizes and arrangements. As shown in FIG. 14 , one preferred embodiment has a horizontal arrangement of multiple cabinets containing a fold out table 10 so that when the cabinet containing a fold out table 10 is in the open position, and the large table leafs 42 and small table leafs 44 are in the horizontal, unfolded position, a continuous table top surface can be created.
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A cabinet table has a cabinet body, a front face, an interior box, and a sliding mechanism permitting the interior box to slide out of the cabinet body. The front face is connected to the interior box and has a top front face and a bottom front face. The interior box has a plurality of table components which are moveable into a table surface.
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FIELD OF THE INVENTION
The present invention is directed to pharmaceutical formulations and their manufacture. One or more pharmacologically active substances are incorporated into the new formulations in order to be released over a desired period of time and, at the same time the dependence of the release rate on the fraction of substance remaining in the formulation, is minimized.
BACKGROUND OF THE INVENTION
Pharmaceutical preparations based on eroding, hydrophilic matrices, showing extended release properties, have been described for pharmacologically active substances of low and high water solubility. The release may be described by a simple exponential function,
M(t)/M(∞)=k·t.sup.n ( 1)
where n reflects the basic kinetics of the release (Ritger and Peppas, J.Contr.Rel. 5 (1987) 23-26). The most beneficial situation is when the release rate is totally independent of the fraction of substance remaining in the formulation that is n=1.
Active substances showing low water solubility have successfully been formulated into hydrophilic, eroding matrices. This has been described in U.S. Pat. No. 4,803,081, which shows favourable release kinetics. The same technique applied on substances of higher water solubility, such as metoprolol succinate, do not give the same beneficial release kinetics. This has limited the medical usefulness of this pharmaceutical principle.
Attempts have been made to improve the release kinetics of the hydrophilic eroding matrix, by using special geometrical arrangements, or introducing a gradient in drug concentration, of the formulations (P. I.Lee, Proc. Int. Symp. Contr. Rel. Bioact. Matr., 15 (1988) 97-98). It has also been proposed to restrict the access of water to the eroding matrix by applying coatings on selected surfaces, which raised the kinetic exponent n in Equation 1 (P. Colombo et al Int J Pharm., 63 (1990) 43-48). Probably none of these concepts has reached the open market, as the complicated manufacturing processes will make the products comparably expensive.
The technique to complex pharmacologically active substances to ionizable, crosslinked polymer particles (ion-exchange resins) is well known (A. T. Florence and D. Attwood, Physiochemical Principles of Pharmacy, Macmillan Press, London, 1982, 297-300, GB Pat 907,021 (1962)). The release of active substance can be controlled by varying the crosslinking density and particle size of the resin. The release rate is, however, depending on the fraction of substance remaining in the particles. The complex has also been coated to further reduce the release rate (U.S. Pat. No. 4,221,778 (1980)). To reach an improvement in the overall release kinetics pellets with different coatings have to be mixed.
It has been suggested to use ion-exchange resins to reduce the release rate from hydrophilic matrices (L. C. Feely and S. S. Davis, Int. J. Pharm. 44 (1988) 131-139). The pure resins were mixed with a pharmacologically active substance as a salt and a gel-forming polymer, a high viscosity hydroxypropyl methylcellulose (HPMC). No complex was, however, formed per se and the effect of the ion-exchange resin was only a reduction in the release rate.
GB 2 218 333 describes a preparation containing one active ingredient, namely ranitidine together with a synthetic cation exchange resin. Hydroxylpropyl methylcellulose may be added and is in that case used as granulating additive and does not control the release rate.
EP 241 178 describes a pharmaceutical composition comprising one or more therapeutically active ingredients dispersed in a carrier. In this case no complex is formed.
EP 338 444 describes a composition containing azelastin which may be bound to a cation exchange resin. It has however not been proposed that a hydrophilic eroding matrix should be added.
EP 195 643 describes release by diffusion through a gel-forming layer in a transdermal preparation. Also a salt must be added to the composition in order to make the composition suitable for use.
BRIEF DESCRIPTION OF THE INVENTION
Active substances, available as dissociated ions, are complexed to insoluble, oppositely charged polymers, such as an ion-exchange resin. The particles formed, the complex, are embedded into a hydrophilic eroding matrix. Surprisingly, the release kinetics obtained were more beneficial, showing a higher value of the exponent n (Equation 1) than for the ordinary salt, base or acid.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 compares the release kinetics of metoprolol in the formulation of Example 1 with that in the formulation of Reference Example 1.
FIG. 2 compares the release kinetics of metoprolol in the formulation of Example 2 with that in the formulation of Reference Example 1.
FIG. 3 compares the release kinetics of metoprolol in the formulation of Example 4 with that in the formulation of Reference Example 4.
FIG. 4 compares the release kinetics of terbutaline in the formulation of Example 6 with that in the formulation of Reference Example 6.
DESCRIPTION OF THE INVENTION
The new preparations defined above give an even release of the active substance with high solubility in water. The different ingredients in the preparation are defined more in detail in the following:
Active substances are defined as compounds, which give a pharmacological effect when administered to humans or animals. To be useful in the present invention the substance must be available as dissociated ions. Therefore substances like glucose cannot be used. Instead bases, acids or amphoteric substances can be used.
It is preferable to use an active substance, which has a solubility greater than 10 mg/ml in water.
The ion-exchange resin has to be matched to the active substance and its physicochemical properties. Weak bases are best complexed with strong acid exchangers like sulphonic acids. These are often based on polystyrene crosslinked with divinylbenzene, and marketed under trademarks Resonium, Amberlite and Dowex.
The active substances may be used in the process as a salt or free base. The resin may be used in the acid form or as a salt of a suitable cation, such as sodium.
Stronger bases can be complexed to ion-exchange resins of lower acidity, such as crosslinked poly (acrylic acid) or styrene-divinylbenzene modified to contain carboxylic groups. It is also possible to use the mentioned sulphonic acid ion-exchangers.
Acids may be complexed with crosslinked polystyrene with quarternary amines, or other basic anion-exchangers. The acids may be used as free acids or suitable salts. The anion-exchanger may be used as base, with a hydroxylic ion on every amine, or a salt of a suitable anion, such as chloride.
The hydrophilic eroding matrix may consist of a polysaccharide. Especially useful are derivatised celluloses such as methylcellulose (MC), hydroxypropyl metylcellulose (HPMC), both marketed under the tradenames Metolose and Methocel, and ethylhydroxy ethylcellulose (EHEC). We have found a grade HPMC, Metolose 60SH50 (viscosity 2% solution in water at 20° C. of approx. 50 mPas, 27.0-30.0% w/w methoxy groups and 7.0-12.0% w/w hydroxypropoxy groups) especially useful. Also a mixture of low and high molecular weight HPMC can be used. The use of different mixtures of HPMC gives according to known technique different release rates of the active ingredient. Cf J. Contr. Rel. 5 (1987) p. 159-172. The eroding matrix may also consist of synthetic hydrophilic polymers, such as polyvinylalcohol or polyvinylpyrrolidone.
Other useful materials are bioeroding polymers such as polyorthoesters and polyanhydrides, such as those described by Nguyen et al (J. Contr. Rel. 4 (1986) 9-16) and polyanhydrides (R. Langer et al, Proc. Int. Symp. Control. Rel. Bioact. Mater., 16 (1989) 119-120, 161-162, 338-339).
PROCESSES FOR THE PREPARATION OF TABLETS
Tablets are preferably prepared by embedding the complex into a hydrophilic eroding matrix by compression in an ordinary tablet press. Processes including solvent evaporation (casting), precipitation or polymerisation may also be used.
EXAMPLES
Example 1
1 kg Dowex 50W-X4, 200-400 mesh, was washed with 2 L 1M NaOH, 8 L deionized water, 2 L 0.1M NaOH, 8 L deionized water, 0.8 L methanol, 4 L water, 1.6 L 10% HCl and 12 L deionized water. The resin was dried overnight at 80° C., yielding 352 g resin with 8.5% moisture and 4.86 milliequivalents/g dry resin. 30.15 g resin was slurried in deionized water and a solution containing 44.06 g metoprolol succinate was added. After 10 minutes stirring, the resin was filtered on a sintered glass funnel. Another 8.01 g metoprolol succinate in water was added to the resin, and filtered off. The resin was rinsed with 2L deionized water and dried overnight at 80° C., giving 64.44 g complex with a metoprolol content, determined spectrophotometrically at 274 nm, of 1.98 mmol/g. 1 g of the complex was carefully mixed with 3 g Metolose 60SH50 (viscosity 49 mPas in 2% water solution, 28.2% methoxy groups and 8.2% hydroxypropoxy groups) with a mortar and pestle. 400 mg of the mixture was filled by hand into 20 mm flat punches and compressed into tablets. The release of metoprolol was measured in a USP apparatus no 2 (paddle) at 50 rpm, with the tablets mounted in a stationary basket, in 1 L phosphate buffer at pH 7.5 and 37° C. The amount drug released was measured spectrophotometrically, for metoprolol at 274 nm.
Reference Example 1
1 g metoprolol succinate was mixed with 3 g Metolose 60SH50 (same lot as above) with a mortar and pestle. 400 mg of the mixture was filled by hand into 20 mm flat punches and compressed into tablets.
The fraction drug released is plotted versus time in FIG. 1. The exponent describing the release kinetics, defined in Equation 1, is evaluated using non-linear least square fitting available in the software package RS/1 (RTM). The exponent was found to be 0.92 for the tablet containing the complexed drug and 0.61 for the low molecular weight salt, the succinate.
Example 2
0.9 kg Dowex 50W-X8 200-400 mesh was treated similarly as in Example 1. The resin contained 5.10 mekv/g dry resin and 7.3% moisture. 30.02 g resin was treated with 44.06 g and 8.00 g metoprolol succinate in a similarly way as in Example 1. 57.76 g complex with 1.80 mmol/g was obtained. The tablets were manufactured and analyzed similarly as in Example 1 and the same reference was used. The release of the tablets is shown in FIG. 2. The release-describing exponent of Equation 1 was 0.97 for the tablets made according to this invention, compared to 0.61 for the reference tablet.
Example 3
1 g of the complex of Example 1 was mixed with 3 g Metolose 65SH50 (viscosity 47 mPas of 2% water solution, 27.3% methoxy groups and 4.2% hydroxypropoxy groups), compressed into tablets and analyzed similarly.
Reference Example 3
1 g metoprolol succinate was mixed with 3 g Metolose 65SH50 (same lot as above), compressed into tablets and analyzed with the method described in Example 1
The kinetic exponent, defined in Equation 1, increased from 0.44 for the succinate salt to 0.68 for the complex.
Example 4
1 g of the complex of Example 1 was mixed with 3 g Methocel E4MCR (viscosity 4077 of 2% water solution, 30.0% methoxy groups and 8.6% hydroxypropoxy groups), compressed into tablets and analyzed similarly.
Reference Example 4
1 g metoprolol succinate was mixed with 3 g Methocel E4MCR (same lot as above), compressed into tablets and analyzed with the method described in Example 1
The release kinetics of the inventive and reference formulations are shown in FIG. 3.
The exponent describing the kinetics of release increased from 0.46 (low molecular weight salt) to 0.66 (ion exchange resin complex).
Example 5
14.67 g Dowex 50W-X4 (from Example 1) was slurried in water. A water solution of 20.25 g lidocaine HCl. H 2 O was added. After 10 minutes stirring the complex was filtered and washed with 4 L deionized water. After drying, the complex (24.84 g) contained 1.86 mmol/g, determined spectrophotometrically at 262 nm. Tablets were made according to Example 1 with the same lot of polymer and analyzed.
Reference Example 5
Tablets were also made from lidocaine-HCl. H 2 O and Metolose 60SH50.
The kinetic exponent of Equation 1 was 0.95 for the tablet containing the complex, and only 0.58 for the low molecular weight salt.
Example 6
14.67 g Dowex 50W-X4 (from Example 1) was slurried in water. A water solution of 19.20 g terbutaline sulphate was added. After 10 minutes stirring the complex was filtered and washed with 4 L deionized water. After drying, the complex (25.57 g) contained 1.91 mmol/g, determined spectrophotometrically at 278 nm. Tablets were made according to Example 1 and analyzed.
Reference Example 6
Tablets were also made from terbutaline sulphate.
The release profiles of FIG. 4 demonstrate that the kinetic exponent was improved to 1.00 from 0.60 for the corresponding sulphate salt.
Example 7
13.70 g Dowex 50W-X4 (from Example 1) was slurried in water and filtered on a sintered glass funnel. The resin was washed with 1 L water containing 5% NaCl. The resin was further washed with 2 L deionized water. The resin was slurried in 100mL water containing 20.05 g alprenolol HCl. After 10 minutes stirring the complex was filtered and washed with 4 L deionized water. After drying, the complex (27.15 g) contained 1.97 mmol/g, determined spectrophotometrically at 270 nm. Tablets were made according to Example 1 and analyzed.
Reference Example 7
Tablets were also made from alprenolol HCl.
The hydrochloric salt had an exponent of 0.63, significantly lower than the complex, 1.16.
Example 8
100 g Dowex 1X-2 was washed with 0.5 L 0.1M HCl, 1 L water, 200 mL methanol, 0.5 L water, 0.5 L 0.5M NaOH, 200 mL methanol, 0.5 L water, 1 L 5% NaCl followed by 2 L deionized water. The resin was dried at 80° C. overnight yielding approx. 60 g resin containing 11.5% water and 4.49 mekv/g dry resin. 6.68 g resin was treated with 100 mL 1M NaOH, filtered and washed with 2 L water and 2 lots of 200 mL ethanol 95% and slurried in 200 mL ethanol. 3.46 g salicylic acid was added and the slurry was agitated for 9 hours. The complex was filtered and washed with two lots of 200 mL ethanol and 2 L water. 6.25 g complex containing 19.5% salicylic acid, measured spectrophotometrically at 296 nm, was obtained after drying overnight. 1 g complex was mixed with 3 g Metolose 60SH50 and tablets were prepared according to Ex. 1.
Reference Example 8
1 g salicylic acid was mixed with 3 g Metolose 60SH50 and compressed to tablets by the method described in Ex. 1.
The release curves were fitted to Equation 1, giving an exponent of 0.56 for the acid and 0.96 for the complex.
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A preparation of a pharmacologically active ionizable substance, wherein active substance is ionically complexed to an ion-exchanger resin, which is embedded in a hydrophilic eroding matrix as well as a process for the manufacture thereof.
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CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a divisional application of and claims priority to pending U.S. application Ser. No. 11/812,300 filed on Jun. 18, 2007, and entitled RADIO FREQUENCY SHIELDING APPARATUS SYSTEM AND METHOD, the entire contents of which is expressly incorporated by reference herein.
BACKGROUND
The present disclosure generally relates to radio frequency shielding for a commercial aircraft. More particularly, the disclosure pertains to a method and system that assists in attenuating electromagnetic propagation through commercial aircraft passenger windows, aircraft doors or the like.
BACKGROUND
Generally, the fuselage of commercial aircraft are extremely efficient at attenuating electromagnetic radiation or energy such as radio frequency (RF) energy. Conventional aircraft typically include an outer skin of aluminum or include a conductive mesh or coating to dissipate lightning strikes. This conductive skin reflects and attenuates RF energy to a high degree. However, commercial aircraft generally also include a number of electromagnetic apertures. Aircraft windows and doors are two of the most common electromagnetic apertures inherent to most commercial aircraft designs. During operation of commercial aircraft, these apertures allow RF energy to enter and exit the aircraft.
This property of aircraft windows and doors is undesirable for several reasons. For example, externally generated RF transmissions may interfere with on-board systems. In another example, internally generated RF transmissions may interfere with on-board systems and/or may violate the rules of the United States Federal Communications Commission (FCC) and other such regulatory institutions.
Accordingly, it is desirable to provide a cost effective method and apparatus for attenuating electromagnetic propagation through aircraft passenger windows or the like at least to some extent.
SUMMARY
The foregoing needs are met, at least to some extent, by the present disclosure, wherein in one respect a system, assembly, and method is provided that in some embodiments attenuates electromagnetic propagation through an aperture in an aircraft.
An embodiment relates to a system for shielding an aircraft from electromagnetic energy. The system includes a fuselage, aperture, window mounting, and window plug. The fuselage provides an electrically conductive envelope. The aperture is disposed in the fuselage. The window mounting spans the aperture. The window plug spans the aperture. The window mounting and the window plug are electrically coupled to the fuselage and provide an electrical path spanning the aperture.
Another embodiment pertains to an assembly for shielding an aperture in a fuselage of an aircraft from electromagnetic energy. The assembly includes a window mounting and a window plug. The window mounting spans the aperture. The window plug spans the aperture. The window mounting and the window plug are electrically coupled to the fuselage and provide an electrical path spanning the aperture.
Yet another embodiment relates to a method of shielding an aperture in a fuselage of an aircraft from electromagnetic energy. In this method, a window mounting is conductively connected to the fuselage and a window plug is conductively connected to the window mounting.
There has thus been outlined, rather broadly, certain embodiments that the detailed description thereof herein may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional embodiments that will be described below and which will form the subject matter of the claims appended hereto.
In this respect, before explaining at least one embodiment in detail, it is to be understood that embodiments are not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. In addition to the embodiments described, the various embodiments are capable of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the disclosure. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the various embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an exploded view of a system for shielding an aperture according to an embodiment.
FIG. 2 is a cross-sectional perspective view of a window mounting suitable for use with the system according to FIG. 1 .
FIG. 3 is a cross-sectional view of a capacitive gasket suitable for use with the window mounting according to FIG. 2 .
FIG. 4 is an example of a graph showing frequency in MHz (abscissa) as it affects the shielding effectiveness in dB (ordinate) of coated windows suitable for use with the system according to FIG. 1 .
FIG. 5 is an example of a graph showing frequency in MHz (abscissa) as it affects the shielding effectiveness in dB (ordinate) of coated windows and electronically dimmable windows suitable for use with the system according to FIG. 1 .
FIG. 6 is an example of a graph showing frequency in MHz (abscissa) as it affects the shielding effectiveness in dB (ordinate) of coated windows and grounded electronically dimmable windows suitable for use with the system according to FIG. 1 .
FIG. 7 is an example of a graph showing frequency in MHz (abscissa) as it affects the shielding effectiveness in dB (ordinate) of coated windows and circumferentially bonded electronically dimmable windows suitable for use with the system according to FIG. 1 .
FIG. 8 is an example of a graph showing frequency in MHz (abscissa) as it affects the shielding effectiveness in dB (ordinate) of a coated bellows suitable for use with the system according to FIG. 1 .
DETAILED DESCRIPTION
Various embodiments will now be described with reference to the drawing figures, in which like reference numerals refer to like parts throughout. An embodiment in accordance with the present disclosure provides a method and system that assists in attenuating electromagnetic propagation, for example RF energy, through commercial aircraft apertures such as passenger windows, aircraft doors or the like. More particularly, an embodiment provides an aircraft aperture assembly or system having a plurality of components that, when assembled in an aircraft frame or fuselage, assists in the attenuation of the transmission of RF energy therethrough.
Referring now to FIG. 1 , a window system 10 includes a window mounting 14 and window plug 16 . The window mounting 14 is configured to be mounted in or mated with a window opening 18 in an outer skin 20 of an aircraft (not shown). The window plug 16 is configured to be mounted in or mated with a plug opening 22 in an inner skin 24 of the aircraft. The window mounting 14 includes a capacitive gasket 28 , outer window 30 , inner window 32 , and window forging 34 . The window mounting 14 is further described in FIGS. 2 and 3 . The window plug 16 includes a bellows seal 40 , outer reveal 42 , electronically dimmable window (EDW) 44 , inner reveal 46 , dust cover 48 , and window plug molding 50 .
In general, some or all of the various components of the window system 10 are configured to conduct electricity sufficiently well enough to reflect and/or attenuate electromagnetic energy such as RF energy. More particularly, when installed in an electrically conductive envelope such as a fuselage of an aircraft, the assembled components of the window system 10 provide a conductive path spanning the window opening 18 in the outer skin 20 of the fuselage. In this manner, electromagnetic energy such as RF energy generated within the fuselage may be attenuated or essentially prevented, to a large extent, from entering or exiting the fuselage. It is an advantage of various embodiments that RF energy may be attenuated to such an extent that signals emanating from within the fuselage can not reasonably be detected outside the fuselage. It is another advantage of various embodiments that, for the purposes of the United States Federal Communications Commission (FCC) and other such regulatory institutions, the interior of an aircraft outfitted with the window system 10 may be classified an indoor environment due to the attenuation of RF energy provided by the window system 10 .
In FIG. 2 , a particular embodiment of the commercial aircraft window mounting, generally designated 14 , is illustrated. The commercial aircraft window mounting 14 includes the capacitive gasket 28 positioned between and/or partially surrounding the outer window 30 and the inner window 32 . The commercial aircraft mounting 14 additionally includes the window forging 34 that is configured to mate with the airframe or outer skin 20 of the aircraft. The window forging 34 includes a radial flange 56 and an axial flange 58 . The window forging 18 also includes a base portion 60 that extends in opposing relationship to the radial flange 56 . That is, the base portion 60 extends generally inwardly or opposite the radial flange 56 as previously discussed, and provides an inwardly and downwardly sloping surface 62 .
As illustrated in FIG. 2 , the commercial aircraft window mounting 14 further includes a series of spring clips 64 positioned about the periphery of the window forging 34 . The commercial aircraft window mounting 14 also has a series of mounting flanges 66 and a series of bolts 68 , or other such mechanical attachments or fasteners, also positioned about the periphery of the forging 34 . The mounting flanges 66 are connected to, and extend from, the axial flange 58 of the window forging 34 . The mounting flanges 66 are positioned about the periphery of the window forging 34 as illustrated in FIG. 1 , and combine with the spring clips 64 and the bolts 34 to mount the gasket 28 and outer and inner windows 30 , 32 to the window forging 34 .
Referring now to FIGS. 2 and 3 , a cross-sectional view of the gasket 28 is illustrated. As depicted in FIGS. 2 and 3 , the gasket 28 encircles the outer window 30 and inner window 32 and provides a circumferential bond between the outer and inner windows 30 , 32 and the window forging 34 . The gasket 28 is a capacitive gasket that provides a capacitive bond between the windows 30 , 32 and the window forging 34 . The gasket 28 includes a lower portion or section 70 , a mid-section or portion 72 and an upper portion or section 74 .
As illustrated in FIGS. 2 and 3 , the lower section 70 of the gasket 28 extends from the mid-section 72 of the gasket 28 at an angle in a downwardly direction, away for the window forging 34 . The aforementioned geometry of the lower section 70 of the gasket 28 generally mirrors or compliments the downwardly sloping surface 62 of the base portion 60 . The lower section 70 includes a series of ridges, generally designated 78 , that extend outwardly from the lower section 70 . As depicted in FIGS. 2 and 3 , the mid-section 72 , as the name suggests, occupies the middle portion of the gasket 28 and functions as a spacer between the outer window 30 and inner window 32 . The upper portion 74 extends upwardly from the mid-section 72 , generally parallel to the axial flange 58 of the window forging 34 .
In various embodiments, the gasket 28 includes a conductive media that is bound by an elastomeric matrix. The conductive media includes any suitable strongly, weakly, and/or semi-conductive materials. Specific examples of conductive materials include conductive carbon black, aluminum, silver, and the like. The elastomeric matrix includes ethylene propylene diene monomer (EPDM) and the like. In one embodiment, the capacitive gasket 28 includes a carbon black media in an EPDM or other such elastomeric matrix. Alternatively, the gasket 28 may include silver and/or aluminum flakes in an EPDM or other such elastomeric matrix. The carbon black media provides greater than 20 dB to about 45 dB of RF energy shielding in the range of from about 80 MHz to approximately 18 GHz of the electromagnetic spectrum. The silver and/or aluminum flake media provides approximately 10 dB to about 47 dB of RF energy shielding in the range of from about 80 MHz to approximately 18 GHz of the electromagnetic spectrum.
As previously discussed, during operation of commercial aircraft for example, the aircraft encounters electromagnetic energy in the form of RF radiation from external sources. This RF radiation can interfere with the operation of the commercial aircraft systems such as the communication system and the navigation system. Accordingly, in order to attenuate the propagation of RF radiation through the commercial aircraft passenger windows, techniques such as shielding are implemented to reduce electromagnetic propagation. During the shielding process and, prior to assembly of the window system 10 the windows are treated with a film or material that reflects electromagnetic energy. As illustrated in FIG. 1 , the inner window 32 has been shielded or treated, as generally designated by reference numeral 76 , with a film or other material that reduces or attenuates the propagation of electromagnetic radiation. The shielding 76 includes any suitable film, layer, and/or treatment operable to reflect, attenuate, or otherwise reduce the propagation of electromagnetic energy. Suitable examples of the shielding 76 include conductive films, meshes, and the like.
The shielded inner window 32 combines with the gasket 28 to reduce electromagnetic propagation through the passenger windows of a commercial aircraft. As previously discussed, the shielded window 32 reflects electromagnetic radiation, however as the frequency of electromagnetic energy increases, for example, to approximately 1 GHz to approximately 2 GHz, the window may begin to lose its attenuation characteristics and begin to resonate and retransmit the electromagnetic energy. To avoid such instances, the gasket 28 provides a capacitive coupling between the inner window 32 and the commercial aircraft frame, dissipating the electromagnetic energy onto the aircraft frame or outer skin 20 . In this regard, the gasket 28 includes a material having a dielectric constant, permittivity, and/or resistance such that the gasket 28 is configured to discharge electromagnetic energy from the window 32 to the window forging 34 prior to resonance of the window 32 . That is, the window 32 is configured to reflect electromagnetic energy until the energy exceeds a predetermined maximum amount of energy. If the window 32 were to remain electrically isolated past this predetermined maximum amount of energy, the window 32 may transmit RF energy. The gasket 28 is configured to conduct electromagnetic energy or electricity from the window 32 to the window forging 34 prior to the amount of energy in the window 32 exceeding the predetermined maximum. The gasket 28 further assists the attenuation electromagnetic radiation by absorbing some of the electromagnetic energy as heat.
FIGS. 4-7 are examples of graphs showing frequency in MHz (abscissa) as it affects the shielding effectiveness in dB (ordinate) of components suitable for use with the system according to FIG. 1 . As shown in FIG. 4 , the window 30 and/or 32 , when coated with a thin, essentially transparent, coating of gold, attenuates approximately 20 decibels (dB) of electromagnetic (EM) energy within a frequency range of about 300 megahertz (MHz) to about 11,000 MHz. As shown in FIG. 5 , when the coated window 30 and/or 32 is combined with the EDW 44 , approximately 25 dB of EM energy is attenuated within a frequency range of about 300 MHz to about 11,000 MHz. That is, assembling these two components increases the attenuation. Similarly, as shown in FIG. 6 , by grounding the EDW 44 , approximately 35 dB of EM energy is attenuated within a frequency range of about 300 MHz to about 11,000 MHz. The attenuation is further again increased by circumferentially bonding the EDW 44 within the window system 10 . In a particular embodiment, the EDW 44 is circumferentially bonded to the window system 10 via the bellows seal 40 . For example the bellows seal 40 is conductively coated or otherwise configured to conduct EM energy. In a particular example, the bellows seal 40 is coated with an electrically conductive silicone-based ink. This ink may include any suitable conductive material such as, for example, aluminum, silver, gold, carbon, and the like. While in general, any suitable coating material that exhibits good adhesion to the bellows seal 40 , flexibility, and conductivity may be utilized in various embodiments, specific examples of coating materials may be manufactured by Creative Materials, Inc. of Tyngsboro, Mass. 01879, U.S.A. In particular, product number 115-08, electrically conductive silicone ink with 87% silver (cured) is suitable for use with various embodiments. It is to be understood that the graphs illustrated in FIGS. 4-7 are for illustrative purposes only, and thus, the respective curvatures, slopes and y-intercepts may be the same or different depending on the response of the various EM energy attenuators.
FIG. 8 is an example of a graph showing frequency in MHz (abscissa) as it affects the shielding effectiveness in dB (ordinate) of a coated bellows suitable for use with the system according to FIG. 1 . As shown in FIG. 8 , when coated with electrically conductive silicone ink with 87% silver (cured), the bellows seal 40 attenuates approximately 20 dB. It is to be understood that the graph illustrated in FIG. 8 is for illustrative purposes only, and thus, the respective curvatures, slopes and y-intercepts may be the same or different depending on the response of the various EM energy attenuators.
The many features and advantages of the various embodiments are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages that fall within the true spirit and scope of the embodiments. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the embodiments to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the various embodiments.
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An assembly for shielding an aircraft from electromagnetic energy may include a window mounting configured to be conductively coupled to an aperture in a fuselage of an aircraft. The window mounting may include a window pane having an electromagnetically-reflective coating for reflecting electromagnetic energy. The window pane may remain electrically isolated from the fuselage prior to the electromagnetic energy exceeding a frequency of approximately 1 GHz. The window mounting may further include a capacitive gasket capacitively coupling the window pane to the fuselage after the frequency of the electromagnetic energy reflected by the window pane exceeds approximately 1 GHz.
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BACKGROUND OF THE INVENTION
Water-in-oil emulsions (oil-mud) are often used in circulating fluids required in the rotary drilling of formations containing hydrocarbons. These circulating fluids are referred to as drilling muds. These oil-mud circulating fluids are pumped down the drill pipe and out into the wellbore through holes in the drill bit and back up the well in the annular space between the drill pipe and walls of the wellbore, carrying with it drill cuttings and the like that are then removed before recirculation. This mud performs a number of functions, including removing drill cuttings, lubricating and keeping the bit cool, providing flotation to help support the weight of the drill pipe and casing, coating the wellbore surface to prevent caving in and undesirable flow of fluids in or out of the wellbore, including drilling fluids, brine, and the like.
Obviously, the properties of and the composition of these drilling mud formulations are complex and variable, depending on the conditions involved and the results desired or required including reuse and recycling of mud formulations. One of the most important properties of these drilling muds and other drilling fluids is that they be thermally stable and do not present rheological and thixotropic problems under the conditions of drilling.
In these oil mud drilling fluids, the oil is the continuous phase and the water is present in a dispersed phase. This is necessary to maintain the required rheology of the mud for drilling and completion, including a balance between gel strength and viscosity, i.e., the balance for example between pumpability of the mud and its hole cleaning capability. In an oil mud, the surface of the solid materials in the mud are essentially oil wet. If, because of a number of possible factors, the solid particles begin to be water wet, handling and other problems begin. When the suspended particle surfaces become water wet and the water in oil emulsion is converted to an oil in water emulsion, which condition is referred to as "flipped", the properties of the mud are extensively affected to a degree that the mud is no longer useful. At this point, these expensive mud formulations normally have to be discarded. Usually, an oil mud in a flipped state is too thick and viscous to handle with the normal well equipment.
The undesired conversion from water in oil to oil in water emulsions includes conversion of the solid particles from an oil wet to water wet stage. This conversion or flipping can be caused by a number of factors and conditions. One source of excess water contributing to this undesirable state may come from accidental addition of water to the mud during makeup or during recycling, and cognate water introduced into the mud from formations during drilling operations. High temperature drilling conditions can cause the conversion of the water in oil to oil in water conditions. Drilled cuttings and other materials getting into the mud during drilling operations such as shale, water sensitive and water swellable clays, drilled cement, water wet solid materials, and the like can contribute to a change in the emulsion state of the oil mud. Inexpensive and incomplex techniques and methods for preventing, or more importantly, for reversing this change in emulsion state, to reverse the flipped mud, are among the objectives of this invention.
SUMMARY OF THE INVENTION
In oil base drilling muds, the solid materials have oil wet surfaces because of the water in oil emulsion and the resulting oil base muds have the desired rheology for drilling under a variety of conditions. On occasion, during drilling operations in the field, conditions occur that cause the surface of the solid materials in the mud to be water wet, changing the rheology of the drilling mud such that it is unusable and has to be discarded. In accordance with this invention, a novel method is provided to prevent or reverse this condition so that the water wet surfaces of the solid particles become oil wet again, restoring the drilling mud to substantially its original condition or level or to such a condition that it can continue to be used in drilling operations. This is accomplished by adding to the drilling mud containing water wet particles at least one of a specific and critical group of imidazolines having the general formula ##STR1## wherein Y is phosphate, hydroxyl, amine, amide or an ester, R 1 is hydrogen, alkyl, or carboxylic acid salt, R 2 is alkyl carboxylic acid salt, and R 3 is an alkyl radical containing 1 to 14 carbon atoms, all as hereinafter defined.
DETAILED DESCRIPTION
The imidazolines useful in the practice of the invention have the general formula ##STR2## wherein (1) Y is a phosphate group having the formula ##STR3## wherein X is hydrogen, --NH 3 , or an alkaline metal cation; --OH; --NH 2 , an amide of the formula ##STR4## and esters of the formula ##STR5## wherein R is an alkyl radical containing 1 to 12 carbon atoms; (2) R 1 is --H, an alkyl radical containing 1 to 4 carbon atoms, or an alkyl carboxylate radical ##STR6## wherein n is an integer from 1 to 4, X is --H, --NH 3 , or an alkali metal cation; (3) R 2 is an alkyl carboxylate radical having the formula ##STR7## wherein n is an integer from 1 to 4, and X is --H, --NH 3 , or an alkali metal cation; and (4) R 3 is an alkyl radical containing 1 to 14 carbon atoms, and may be a mixture of alkyl radicals within the range of C 6 -C 14 , i.e., obtained from coco fatty acid, for example. More preferably, Y is --OH or ##STR8## R 1 is H or ##STR9## R 2 is ##STR10## and R 3 is C 6 to C 8 .
The preparation of imidazolines related to some of those defined herein is found in U.S. Pat. No. 4,490,536.
To be successful in the practice of this invention, it has been found that the imidazolines must have some degree of water solubility. The imidazolines that are either completely water insoluble or completely oil soluble are not effective in the practice of the invention. Water soluble imidazolines contain phosphates and/or 1 or 2 carboxylic acid groups.
It has been found that the length of the carbon chain R 3 is critical to the practice of the invention. When R 3 is an alkyl group containing 16 or more carbon atoms, the imidazoline is not effective in the practice of the invention. However, when R 3 alkyl groups contain less than 14, preferably an average of about C 4 and C 12 , good results are obtained. Mixtures of alkyl groups may be used so long as the average carbon content of the alkyl groups in the molecule is about 12 or less.
Oil base drilling muds are prepared by a great variety of formulations and with a large number of ingredients, as is well known to those skilled in the art. Specific formulations depend on the state of drilling a well at any particular time, for instance, depending on the depth, the nature of the strata encountered, and the like. The process of this invention is directed to and particularly adapted to provide improved oil base drilling muds useful under conditions of high temperature and pressure, such as those encountered in deep wells, where many previously and under conditions such that water or water sensitive materials are introduced into the oil mud drilling fluids.
Oil base mud formulations including those intended for use under high temperature (up to about 500° F.) and high pressure (up to about 25,000 psi) conditions may contain a petroleum oil, a weighting agent, an emulsifier, a gelling or thixotropic agent, salts and a fluid loss control agent as the ingredients, if desired. Water is often added.
The oil (continuous phase) used in a petroleum oil, generally diesel oil or mineral seal oil, although lighter oils such as kerosene, or heavier oils such as fuel oil, white oil, crude oil, and the like may also be used. The invention is particularly useful with low viscosity, low aromatic oils, No. 2 diesel oil and mineral oils.
If water is used, the amount normally is small, and while usually is less than about 10 weight percent, amounts as high as about 60 volume percent may be present under some conditions.
Emulsifiers, both invert and wetting agents, include those normally used, including alkali and alkaline earth metal salts of fatty acids, rosin acids, tall oil acids, the synthetic emulsifiers such as alkyl aromatic sulfonates, aromatic alkyl sulfonates, long chain sulfates, oxidized tall oils, carboxylated 2-alkyl imidazolines, imidazoline salts, amido amines, alkoxy phenols, polyalkoxy alcohols, alkyl phenols, high molecular weight alcohols, and the like.
Water soluble salts often are added to the formulations normally are the brine salts such as sodium chloride, potassium chloride, sodium bromide, calcium chloride, more preferably, and the like, usually in a water solution. Formation brines and seawater may be used. These salts are added to control the osmotic pressure of the formulations as needed, according to drilling conditions.
Weighting materials, if used, include such materials as calcium carbonate, silicates, clays, and the like, but more preferably are the heavier materials such as the barites, specular hematite, iron ores, siderite, ilmenite, galena, and the like.
The oil-muds normally will be formulated to weigh from greater than about 7 (no weighting agent) to about 22 pounds per gallon of mud. Usually the range is from about 10 to 18 pounds per gallon. The water content will normally be from 0 to 60 percent by volume.
The thixotropic thickening and gelling agents normally used in many oil-mud formulations are organophilic clays. The clays used may be any of those that have substantial base-exchange capacity. A variety of such materials are known to those skilled in the art, including Wyoming bentonite, montmorillonite, hectorite, attapulgite, illite, fullers earth, beidellite, saponite, vermiculite, zeolites, and the like. Wyoming swelling bentonite and hectorite are normally utilized.
To obtain the desired organophilic clays, the swelling bentonites and hectorites are reacted with functional organic compounds, as is well known to those skilled in the art. The amount of organic compound used will be dependent on the reactivity of the clays used, but usually is from about 50 to 300 milliequivalents of an organic ammonium salt, for example, per 100 grams of clay. The reactions are normally conducted in water and the treated clay is separated and dried. Normally used are onium compounds, such as organic ammonium salts such as quaternary ammonium salts having the structural formula ##STR11## wherein R 1 are alkyl groups containing 1 to 20 carbon atoms, R 2 are alkyl groups containing 1 to 20 carbon atoms; R 3 are alkyl groups containing 1 to 20 carbon atoms; R 4 are alkyl groups containing 1 to 20 carbon atoms; and at least one of R 1 , R 2 , R 3 or R 4 contains at least 12 carbon atoms, and M is Cl, Br, I, OH or SO 4 . Typical reactants include those containing quaternary ammonium cations selected from the group consisting of trimethyl octadecyl ammonium, dimethyl dihydrogenated tallow ammonium, methyl benzyl dicoco ammonium, methyl trihydrogenated tallow ammonium, methyl benzyl dihydrogenated tallow ammonium chloride, and the like. Descriptions of preparation of typical organophilic clays can be found in U.S. Pat. Nos. 2,966,506; 4,105,578; 4,382,868; and 4,425,244.
The organophilic clay content of the oil-mud formulation will usually vary inversely as the density of the oil-mud. The organoclay content may range from about 25 to 30 pounds per barrel (ppb) in low densities, to almost 0 in high densities. Normally an amount from about 2 to about 15 pounds of clay per barrel of mud will be used. The degree of suspension or hole cleaning required or requested will have an impact on the clay concentration as is well known to those skilled in the art.
Useful fluid loss control agents, if used, include for example lignite and its derivatives, humic acid, quebracho and derivatives thereof, asphalts, oxidized pitch, polymeric or natural rubber, latex materials, and the like.
In a preferred practice of the invention, the defined imidazolines are added to a flipped mud, i.e., an oil base mud wherein the particles have changed from an oil wet condition to a water wet condition. The imidazoline is usually dissolved in water, preferably in a minimum amount of water. The imidazolines may be added as such and thoroughly mixed in the oil mud. The amounts used will be such that the particles are converted from the water wet to oil wet stage. This effective amount is readily determined by a simple test on a portion of the mud, as is shown in the Examples, and is normally in an amount from about 0.1 to about 5 pounds per barrel of mud. While it may be more expensive, the imidazolines may be added to a stable oil mud drilling fluid as a precaution to prevent water wetting of particles of the mud if conditions occur such as might cause water wetting in the absence of the defined imidazoline. However, the greater value of the process of the invention is to convert water wet particles in an original oil base mud to an oil wet state so that a flipped mud is made usable again.
EXAMPLES 1-10
To demonstrate the practice of the invention and the advantages thereof, a standard oil mud was prepared and converted to a water wet mud state. A variety of imidazolines were then added to demonstrate the criticality and necessity for a particular class of imidazoline required to reverse the water wet mud to an oil wet mud, an invert emulsion.
A 15.0 pound per gallon (ppg) oil mud was prepared according to the following formulation:
147.5 weight parts mineral oil (Shell D-70)
2.0 weight parts an amido-amine emulsifier 1
63.0 weight parts an aqueous 25% CaCl 2
10.0 weight parts organophilic clay 2
5.0 weight parts lime
396.5 weight parts Barite
1.0 weight part oxidized tall oil emulsifier
This mud had an electrical stability of 180 volts and was grainy and water wet.
The following indicated materials in amounts of one weight part were mixed into portions of this mud, and the emulsion stability of the samples was determined in accordance with API RP 13B, Eighth Edition, 1980, STANDARD PROCEDURE FOR TESTING DRILLING FLUIDS, Section 8. Refer to Table 1 for the data. The electrical stability is reported in volts, and the higher the value, the more stable the water in oil emulsion. A value of greater than about 225 volts is preferred. Observations of the treated muds are recorded below as to appearance and oil or water wet state are noted. A flipped mud is rough and grainy in appearance. A useful oil wet mud is smooth and shiny in appearance.
TABLE 1__________________________________________________________________________ Electrical StabilityAdditive Volts Observation__________________________________________________________________________ ##STR12## 180 Grainy, water wet ##STR13## 180 Grainy, water wet ##STR14## 140 Grainy, water wet ##STR15## 220 Grainy, water wet ##STR16## 180 Grainy, water wet ##STR17## 380 Smooth shiny slurry, oil wet ##STR18## 480 Smooth shiny slurry, oil wet ##STR19## 520 Smooth shiny slurry, oil wet ##STR20## 988 Smooth shiny slurry, oil wet ##STR21## 998 Smooth shiny slurry, oil__________________________________________________________________________ wet
The first 5 imidazolines used were completely ineffective in reversing the water wet mud back to the oil wet stage. The criticality of the defined and useful imidazolines is obvious when one compares Example (4) that was completely unsatisfactory and ineffective to Example (8) which was very effective in reversing the water wet to oil wet state, as shown by the electrical stability values and the oil wet condition of the treated mud.
EXAMPLE 11
This Example demonstrates how exposure to high temperature can convert a satisfactory oil mud into an unsatisfactory mud wherein the oil wet particles become water wet and the mud is useless, and how this useless mud can be converted to a useful state in accordance with this invention.
A test mud was prepared according to the following recipe:
142.0 weight parts low aromatic mineral oil (Conoco LVT)
9.0 weight parts quaternary ammonium bentonite clay
4.0 weight parts amido-amine emulsifier (Example 1)
2.0 weight parts oxidized crude tall oil wetting agent
26.6 weight parts 25% CaCl 2 solution
556.0 weight parts Barite
This mud as prepared had the following physical properties (API RP 138, Eighth Edition, 1980): Plastic Viscosity-38, Yield Point-11, 10 second gel-8, 10 minute gel-8, stability-1300 volts, and the mud was shiny, smooth and oil wet. After aging, this mud formulation for 16 hours at 400° F., the mud became unusable and too thick to pump because the formulation particles became water wet, as shown by the following data: Plastic Viscosity-86, Yield Point-48, 10 second gel-8, 10 minute gel-8, and emulsion stability-460 volts.
To this useless heat aged mud there was added one weight part of a 40 weight percent solution in water of ##STR22## On addition of the above imidazoline, the mud became shiny and smooth, having oil wet particles. The mud had the following physical properties, Plastic Viscosity-61, Yield Point-8, 10 second gel-6, 10 minute gel-8, and an emulsion stability of 880 volts.
EXAMPLE 12
This Example demonstrates that the addition of one of the defined imidazolines in accordance with this invention to a mud before exposure to destabilizing conditions, will prevent the conversion of the mud from an oil wet to a water wet condition, thus maintaining the desired rheology of the mud under conditions that would normally change the mud to a useless state so that it would have to be discarded.
To a sample of the oil mud prepared as described in Example 11, one weight part of a 40 weight percent solution in water of the imidazoline of Example 11 was added to the oil mud. This material had the following physical properties. Plastic Viscosity 43, Yield Point 11, 10-second gel 8, 10-minute gel 8, volt stability 1400, and the mud had a shiny, smooth appearance and the particles were oil wet. After aging the mud at 400° F. for 16 hours, the mud was still shiny and smooth, as well as oil wet, had a Plastic Viscosity value of 49, a Yield Point of 12, a 10-second yield point of 8 and a 10-minute gel of 10, and an emulsion stability of 1060 volts.
EXAMPLE 13
This Example demonstrates the value of the process of this invention in reversing the adverse effects of the invasion into the oil mud of water wet solids during drilling operations. A test mud was prepared according to the following formulation:
149.0 weight parts low aromatic mineral oil
4.0 weight parts oxidized tall oil wetting agent
2.0 weight parts amido-amine (Example 1)
8.5 weight parts quaternary ammonium bentonite
3.0 weight parts lime
60.6 weight parts 25% aqueous CaCl 2 solution
349.4 weight parts Barite
This oil base mud as mixed had the following properties: plastic viscosity-39, Yield Point-22, 10 second gel-12, 10 minute gel-28, emulsion stability-1080 volts, and was shiny, smooth and oil wet.
This satisfactory and useful oil mud was then contaminated with 1/4 barrel equivalent of a 12 pound per gallon water base mud per barrel equivalent of the above oil base mud. This mixture immediately became a semi-solid, could not be pumped, and was so thick that no physical properties other than stability could be measured. This value was 140 volts, to be compared to a value of 1080 volts obtained on the original oil base mud.
This contaminated and useless mud was then mixed with one pound per barrel of a 40 weight percent solution in water of the imidazoline of Example 11. This useless mud became shiny and smooth, in an oil wet condition, and the physical properties of the treated mud were, Plastic Viscosity-70, Yield Point-25, 10 Second gel-10, 10 minute gel-31, and the emulsion stability was 770 volts, all clearly demonstrating the conversion of the contaminated mud to a useful oil mud.
When Examples 11 and 12 were repeated with the imidazolines of Examples 9 and 10, similar excellent results were obtained.
In another application of the invention, a sample of mud with undesirable properties as to water wetting of solids was treated by adding 1 weight part of the above imidazoline derivative. The resulting formulation was converted to a useful oil wet product having excellent properties, 1240 volt stability, and the product had a shiny, smooth appearance.
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On occasion, during drilling operations employing oil base drilling muds, conditions occur that cause the surface of the solid materials in the mud to be water wet, changing the rheology of the drilling mud such that it is unusable and has to be discarded. In accordance with this invention, a novel method is provided to reverse this condition so that the water wet surfaces of the solid particles become oil wet again, restoring the drilling mud to substantially its original condition or level or to such a condition that it can continue to be used in drilling operations. This is accomplished by adding to the drilling mud containing water wet particles at least one of a specific and critical group of imidazolines.
The imidazolines may be added to stable oil mud drilling fluids to decrease possible water wetting of the particle surfaces therein.
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CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation application of PCT/US2011/040102, filed Jun. 10, 2011, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/354,129 filed Jun. 11, 2010, the contents of which are hereby incorporated by reference in their entirety.
FIELD
[0002] The present disclosure relates to recombinant cells containing improved pathways for biofuel synthesis. In particular, recombinant cells and methods for the synthesis of n-butanol are provided.
BACKGROUND
[0003] Liquid fuels derived from plant biomass are renewable energy sources and the global demand for such biofuels is rising. Ethanol is the most widely used biofuel today, but its low energy return, high vaporizability and miscibility with water present major technical challenges. Alternative biofuels, such as n-butanol, more closely resemble gasoline and have the potential to replace ethanol as the predominant biofuel in the future.
[0004] While several microorganisms can produce ethanol as a fermentation product, only few natural micoorganisms can produce n-butanol. Natural n-butanol producers, such as Clostridium acetobutylicum ( C. acetobutylicum ), can be used for industrial applications but are not as genetically tractable or robust fermentation hosts as, for example, Escherichia coli ( E. coli ) or Saccharomyces cerevisiae ( S. cerevisiae ). It is therefore attractive to engineer a recombinant pathway for biofuel production in such host as E. coli or S. cerevisiae.
[0005] n-Butanol biosynthesis typically includes several enzymatic steps, whereby different n-butanol synthesizing organisms can utilize different classes and combinations of enzymes to mediate the conversion from pyruvate to n-butanol. Generally, the startpoint of n-butanol synthesis, pyruvate, can be derived through the metabolism of various sugar substrates, including glucose and xylose, but also starches and lignocellulosics. Pyruvate is then converted to acetyl-CoA. Acetyl-CoA is subsequently converted to acetoacetyl-CoA, which is itself converted to 3-hydroxybutyryl-CoA. 3-Hydroxybutyryl-CoA is converted to crotonyl-CoA. Crotonyl-CoA is converted to butyryl-CoA. Finally, butyryl-CoA is converted to n-butanol.
[0006] The n-butanol biosynthesis pathway of C. acetobutylicum converting acetyl-CoA to n-butanol can be lifted out and inserted into E. coli , thereby generating a recombinant cell that produces n-butanol (Inui, et al., 2008, Appl. Microbiol. Biotechnol. 77, 1305-16; Atsumi et al., 2008, Metab. Eng. 10, 305-11; Nielsen et al., 2009, Metab. Eng. 11, 262-73). However, the C. acetobutylicum derived n-butanol biosynthesis pathway contains multiple bottlenecks that limit the yields of biofuel production.
[0007] In view of these facts and the growing global demand in biofuels, a significant need exists for more productive recombinant cells and improved methods for biofuel synthesis. Specifically, new recombinant cells are needed providing for robust and high-yielding n-butanol synthesis pathways.
BRIEF SUMMARY
[0008] Provided herein are recombinant cells for the production of n-butanol. Also provided are methods for producing n-butanol using the recombinant cells described herein.
[0009] Particularly, recombinant cells are provided including recombinant sequences encoding enzymes that constitute a synthetic pathway for n-butanol production. In one embodiment of the invention the enzymes include an acylating aldehyde dehydrogenase catalyzing the conversion of acetaldehyde to acetyl-CoA. In another embodiment the enzymes include a pyruvate:flavodoxin/ferredoxin-oxidoreductase catalyzing the conversion of pyruvate to acetyl-CoA. The acylating aldehyde dehydrogenase or pyruvate:flavodoxin/ferredoxin-oxidoreductase are combined with a keto-thiolase or acetyl-CoA acetyltransferase catalyzing the conversion of acetyl-CoA to acetoacetyl-CoA, an acetoacetyl-CoA reductase or hydroxybutyryl-CoA dehydrogenase catalyzing the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA, a crotonase catalyzing the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA a crotonyl-CoA reductase, butyryl-CoA dehydrogenase or trans-enoyl-CoA reductase catalyzing the conversion of crotonyl-CoA to butyryl-CoA, and a butyraldehyde/butanol dehydrogenase catalyzing the conversion of butyryl-CoA to n-butanol.
[0010] Furthermore, methods for n-butanol production are provided. The methods include the step of growing a recombinant cell of the invention in the presence of a suitable carbon source.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the biosynthesis pathways for n-butanol. Enzyme 1 is a pyruvate dehydrogenase or a pyruvate dehydrogenase bypass consisting of a pyruvate decarboxyase and a acylating aldehyde dehydrogenase, or a pyruvate dehydrogenase bypass consisting of a pyruvate decarboxyase, a non-acylating aldehyde dehydrogenase, and an acetyl-CoA synthetase, or a pyruvate:flavodoxin/ferredoxin-oxidoreductase, or a pyruvate formate lyase and formate dehydrogenase; Enzyme 2 is a keto-thiolase or acetyl-CoA acetyltransferase; Enzyme 3 is an acetoacetyl-CoA reductase or hydroxybutyryl-CoA dehydrogenase; Enzyme 4 a crotonase; Enzyme 5 is a trans-2-enoyl-CoA reductase; Enzyme 6 is a butyraldehyde/butanol dehydrogenase. Subclasses of Enzymes 3 and 4, such Enzyme 3.1 and 3.2, may feature different stereoselectivities and produce different chiral intermediates. For example, the acetoacetyl-CoA reductase Hbd produces (S)-hydroxybutyryl-CoA while PhaB produces (R)-hydroxybutyryl-CoA.
[0012] FIG. 2 shows the production of n-butanol using different strains (1-6), promoters (6-12), enoyl-CoA reductase and ketoreductase/enoyl-CoA hydratase selection (10, 13-18) and overexpression of a pyruvate dehydrogenase (PDH) (19-20).
[0013] FIGS. 3A and 3B show the promoter optimization for Ccr expression and the S-tag analysis of Ccr solubility. FIG. 3A shows butanol production using pBT33-phaA.phaB-crt in combination with ccr-adhE2 and using promoters with variable strengths to transcribe the ccr-adhE2 operon. The plasmids used for expression of the ccr-adhE2 operon were pBAD33-ccr.adhE2, pTrc99a-ccr.adhE2, pCWOri-ccr.adhE2, and pET29a-ccr.adhE2. FIG. 3B shows the relationship between n-butanol production (quantified by GC-MS) and soluble Ccr-Stag protein (quantified by S-Tag Rapid Assay Kit, Novagen).
[0014] FIGS. 4A and 4B show the trapping of pathway intermediates by Ter. FIG. 4A shows the reaction catalyzed by Crt is reversible in cell lysate after 2 hours. FIG. 4B shows that Ter is effectively irreversible in cell lysate with no observable reaction occurring within 2 hours.
[0015] FIG. 5 shows a Neighbor Net graph of Ter from T. denticola (Tucci and Martin, 2007, FEBS Lett. 581 (2007) 1561-66). The scale bar at the lower right indicates estimated substitutions per site. Abbreviations are as follows: β and γ, proteobacteria; bactero, bacteroides; entero, enterobacteria; spiro, spirochete.
[0016] FIG. 6 shows the impact of replacing Ccr for Ter on n-butanol yields in recombinant E. coli . Elevating E. coli PDH levels in the presence of Ter results in further increases in n-butanol yields.
[0017] FIG. 7 shows n-butanol production in E. coli cell genetically modified to express the butanol biosynthetic pathway of FIG. 1 . The product retention time was compared to an authentic n-butanol standard in a chromatograph (left), and a product mass spectrum was compared to an authentic n-butanol standard (right) to confirm the identity of the fermentation product.
[0018] FIG. 8 compares the n-butanol production and the ethanol to butanol ratio in E. coli in the presence of basal levels of acetyl-CoA versus and after overexpression of the variants of E. coli PDH (pyruvate dehydrogenase complex), PFOR complex (pyruvate:flavodoxin/ferredoxin-oxidoreductase (YdbK), a flavodoxin-NADP reductase (Fpr), a ferredoxin (Fdx), and one of two flavodoxins (FldA or FldB) all of which are from E. coli ), pyruvate formate lyase (Pfl) and formate dehydrogenase (Fdh), and PDH bypass (a pyruvate decarboxylase from Z. mobilis , an acylating aldehyde dehydrogenase from E. coli , and a pantothenate kinase from E. coli ).
[0019] FIG. 9 shows total fuel (butanol and ethanol) titer in E. coli DH1 and a knockout strain in the presence of basal levels of acetyl-CoA versus expression of the PDHc bypass (a pyruvate decarboxylase from Z. mobilis , an acetylating aldehyde dehydrogenase from E. coli , and a pantetheinate kinase from E. coli ).
[0020] FIG. 10 shows the four general pathways for the conversion of pyruvate to acetyl-CoA consisting of a pyruvate dehydrogenase complex, or a pyruvate:flavodoxin/ferrodoxin-oxidoreductase, or a pyruvate dehydrogenase bypass consisting of a pyruvate decarboxyase and acylating aldehyde dehydrogenase, or a pyruvate dehydrogenase bypass consisting of a pyruvate decarboxyase and a non-acylating aldehyde dehydrogenase and an acetyl-CoA synthetase, or pyruvate formate lyase and formate dehydrogenase.
[0021] FIG. 11 shows native fermentation pathways in E. coli that compete with fuel production under anaerobic and microaerobic conditions.
[0022] FIGS. 12A and 12B show n-butanol production in S. cerevisiae . FIG. 12A shows the recombinant pathway for n-butanol production in recombinant S. cerevisiae cells. FIG. 12B shows butanol production in S. cerevisiae BY4741Δadh. Column 1 shows background level production of butanol. Column 2 shows butanol titer by Saccharomyces cerevisiae BY4741Δadh strain harboring butanol production pathway and PDH bypass.
[0023] FIG. 13 shows the pentose phosphate pathway. The pentose phosphate pathway takes in C5 sugars, including xylose and arabinose, and using NADP + /H as a cofactor converts the sugars into molecules that can enter into glycolysis and then into the n-butanol producing pathway.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0024] The present disclosure relates to recombinant cells producing n-butanol and to methods of using these recombinant cells for the production of n-butanol from fermentable carbon sources.
[0000] n-Butanol Synthesis Pathway
[0025] n-Butanol can be produced by a recombinant cell containing recombinant sequences of at least six enzymes catalyzing the generation of acetyl-CoA and its stepwise conversion to n-butanol ( FIGS. 1 and 10 ). Acetyl-CoA can be generated from the glycolysis product pyruvate by means of a pyruvate dehydrogenase complex (PDHc), a pyruvate formate oxidoreductase (PFOR), the combined activities of a pyruvate formate lyase and a formate dehydrogenase (PFL-FDH), or a pyruvate dehydrogenase bypass pathway (PDH bypass). PDH bypass pathways can include a pyruvate dehydrogenase (PDC) in combination with an acylating aldehyde dehydrogenase (AlDH) or a non-acylating aldehyde dehydrogenase and an acetyl-CoA synthetase. The conversion of acetyl-CoA to n-butanol may proceed through the intermediates acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA. The recombinant cells of this invention are engineered to contain efficient heterologous pathways for n-butanol production.
[0026] In one embodiment of the invention the recombinant cell contains recombinant sequences encoding i) an acylating aldehyde dehydrogenase catalyzing the conversion of acetaldehyde to acetyl-CoA ( FIG. 1 , Enzyme 1), ii) a keto-thiolase or acetyl-CoA acetyltransferase catalyzing the conversion of acetyl-CoA to acetoacetyl-CoA ( FIG. 1 , Enzyme 2), iii) an acetoacetyl-CoA reductase or hydroxybutyryl-CoA dehydrogenase catalyzing the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA ( FIG. 1 , Enzyme 3), iv) a crotonase catalyzing the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA ( FIG. 1 , Enzyme 4), v) a crotonyl-CoA reductase, butyryl-CoA dehydrogenase or trans-enoyl-CoA reductase catalyzing the conversion of crotonyl-CoA to butyryl-CoA ( FIG. 1 , Enzyme 5), and vi) a butyraldehyde/butanol dehydrogenase catalyzing the conversion of butyryl-CoA to n-butanol ( FIG. 1 , Enzyme 6).
[0027] In one specific embodiment the sequences encoding the acylating aldehyde dehydrogenase, the keto-thiolase or acetyl-CoA acetyltransferase, the acetoacetyl-CoA reductase or hydroxybutyryl-CoA dehydrogenase, the crotonase, the crotonyl-CoA reductase, butyryl-CoA dehydrogenase or trans-enoyl-CoA reductase, and the butyraldehyde/butanol dehydrogenase are linked. In another specific embodiment the sequences are not linked.
[0028] Some organisms may not express an endogenous pyruvate decarboxylase or may express only low levels of pyruvate decarboxylase activity that limit the availability of acetaldehyde, the activity of the acylating aldehyde dehydrogenase, and the overall n-butanol yields of the recombinant biosynthesis pathway. Therefore, in some embodiments the recombinant cell further contains a recombinant sequence encoding a pyruvate decarboxylase catalyzing the conversion of pyruvate to acetaldehyde. In another specific embodiment the pyruvate decarboxylase is derived from Z. mobilis or S. cerevisiae.
[0029] In one embodiment of the invention the recombinant cell contains recombinant sequences encoding i) a pyruvate:flavodoxin/ferredoxin-oxidoreductase catalyzing the conversion of pyruvate to acetyl-CoA ( FIG. 1 , Enzyme 1), ii) a keto-thiolase or acetyl-CoA acetyltransferase catalyzing the conversion of acetyl-CoA to acetoacetyl-CoA ( FIG. 1 , Enzyme 2), iii) an acetoacetyl-CoA reductase or hydroxybutyryl-CoA dehydrogenase catalyzing the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA ( FIG. 1 , Enzyme 3), iv) a crotonase catalyzing the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA ( FIG. 1 , Enzyme 4), v) a crotonyl-CoA reductase, butyryl-CoA dehydrogenase or trans-enoyl-CoA reductase catalyzing the conversion of crotonyl-CoA to butyryl-CoA ( FIG. 1 , Enzyme 5), and vi) a butyraldehyde/butanol dehydrogenase catalyzing the conversion of butyryl-CoA to n-butanol ( FIG. 1 , Enzyme 6).
[0030] In one specific embodiment the sequences encoding the pyruvate:flavodoxin/ferredoxin-oxidoreductase, the keto-thiolase or acetyl-CoA acetyltransferase, the acetoacetyl-CoA reductase or hydroxybutyryl-CoA dehydrogenase, the crotonase, the crotonyl-CoA reductase, butyryl-CoA dehydrogenase or trans-enoyl-CoA reductase, and the butyraldehyde/butanol dehydrogenase are linked. In another specific embodiment the sequences are not linked.
[0031] In one specific embodiment the recombinant cell further comprising recombinant sequences encoding the ferredoxin-NADP reductase from E. coli , the ferredoxin FdC from E. coli , and the flavodoxins FldA and FldB from E. coli.
[0032] In one embodiment of the invention the recombinant cell produces n-butanol under aerobic conditions. In one embodiment of the invention the recombinant cell produces n-butanol under microaerobic conditions. Microaerobic conditions refer to an environment where the concentration of oxygen is less than that in the air. In one embodiment of the invention the recombinant cell produces n-butanol under anaerobic conditions. In one specific embodiment the recombinant cell produces more n-butanol under anaerobic conditions than under aerobic or microaerobic conditions. In another specific embodiment the recombinant cell produces near quantitative yields of n-butanol under anaerobic conditions.
[0033] In one embodiment of the invention the recombinant cell produces n-butanol and ethanol under aerobic conditions. In one embodiment of the invention the recombinant cell produces n-butanol and ethanol under microaerobic conditions. In one embodiment of the invention the recombinant cell produces n-butanol and ethanol under anaerobic conditions. In one specific embodiment the recombinant cell produces more total levels of n-butanol and ethanol under anaerobic conditions than under aerobic or microaerobic conditions. In another specific embodiment the recombinant cell produces near quantitative yields of n-butanol and ethanol under anaerobic conditions.
[0034] In one embodiment of the invention the recombinant cell produces elevated levels of n-butanol compared to a wild-type cell under aerobic conditions. Elevated levels of n-butanol produced by the recombinant cell under aerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 1,000,000-fold compared to the n-butanol levels produced by a wild-type cell under aerobic conditions. In specific embodiments the recombinant cell produces at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L n-butanol under aerobic conditions.
[0035] In one embodiment of the invention the recombinant cell produces elevated total levels of n-butanol and ethanol compared to a wild-type cell under aerobic conditions. Elevated total levels of n-butanol and ethanol produced by the recombinant cell under aerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 1,000,000-fold compared to the total levels of n-butanol and ethanol produced by a wild-type cell under aerobic conditions. In specific embodiments the recombinant cell produces under aerobic conditions total levels of n-butanol and ethanol of at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L.
[0036] In one embodiment of the invention the recombinant cell produces elevated levels of n-butanol compared to a wild-type cell under anaerobic conditions. Elevated levels of n-butanol produced by the recombinant cell under anaerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 1,000,000-fold compared to the n-butanol levels produced by a wild-type cell under anaerobic conditions. In specific embodiments the recombinant cell produces at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L n-butanol under anaerobic conditions.
[0037] In one embodiment of the invention the recombinant cell produces elevated total levels of n-butanol and ethanol compared to a wild-type cell under anaerobic conditions. Elevated total levels of n-butanol and ethanol produced by the recombinant cell under anaerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 1,000,000-fold compared to the total levels of n-butanol and ethanol produced by a wild-type cell under anaerobic conditions. In specific embodiments the recombinant cell produces under anaerobic conditions total levels of n-butanol and ethanol of at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L.
Enzyme 1: Acetyl-CoA Generation
[0038] Recombinant cells of this invention contain at least one recombinant pathway for the production of acetyl-CoA ( FIG. 10 ). In one embodiment of the invention the recombinant cell contains recombinant sequences encoding a pyruvate dehydrogenase complex (PDH). In a specific embodiment the PDH is Pdh from E. coli . In another embodiment the recombinant cell contains recombinant sequences encoding a pyruvate formate lyase (PFL) and a formate dehydrogenase (FDH).
[0039] In another embodiment the recombinant cell contains recombinant sequences encoding a pyruvate formate oxidoreductase complex (PFOR). In one specific embodiment PFOR includes a pyruvate:flavodoxin/ferredoxin-oxidoreductase, a flavodoxin-NADP reductase, a ferredoxin, and at least one flavodoxins. In another specific embodiment the recombinant sequences encoding PFOR includes YdbK (SEQ ID NOs: 472, 473), Fpr (SEQ ID NOs: 464, 465), Fdx (SEQ ID NOs: 466, 467), and FldA (SEQ ID NOs: 468, 469), or FldB (SEQ ID NOs: 470, 471) from E. coli.
[0040] In another embodiment the recombinant cell contains recombinant sequences encoding a pyruvate dehydrogenase bypass (PDH bypass). In one specific embodiment the PDHc bypass includes recombinant sequences encoding a pyruvate decarboxylase (PDC). In another specific embodiment the PDHc bypass includes recombinant sequences encoding a non-acylating aldehyde dehydrogenase (AlDH). In another specific embodiment the PDH bypass includes recombinant sequences encoding an acetyl-CoA synthetase (ACS). In another specific embodiment the PDHc bypass includes recombinant sequences encoding a PDC, a non-acylating AlDH, and an ACS. In another specific embodiment the PDHc bypass includes recombinant sequences encoding an acetylating AlDH. In a preferred embodiment the PDHc bypass includes recombinant sequences encoding a PDC and an acylating AlDH. In another preferred embodiment the PDHc bypass includes recombinant sequences encoding a PDC from Z. mobitilis and an acylating aldehyde dehydrogenase from E. coli . In another preferred embodiment the PDHc bypass contains recombinant sequences encoding Pdc from Z. mobitilis and EutEA from E. coli.
[0041] Recombinant sequences encoding PDHc, PFOR, PFL, FDH, acylating AlDH and non-acylating AlDH enzymes may be derived from all prokaryotic organisms, including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal organisms, and all eukaryotic organisms, including mammalian, insect, fungal and yeast organisms. Preferred examples include, but are not limited to: E. coli Pdh, which is composed of the three genes aceE (SEQ ID NOs: 1, 2), aceF (SEQ ID NOs: 3, 4), and lpdA (SEQ ID NOs: 5, 6), the E. faecalis Pdh, which is composed of the four genes pdhA (SEQ ID NOs: 7, 8), pdhB (SEQ ID NOs: 9, 10), aceF (SEQ ID NOs: 11, 12), and lpdA (SEQ ID NOs: 13, 14), the E. coli Pfor genes ydbK (SEQ ID NOs: 35, 36), fpr (SEQ ID NOs: 37, 38), fdx (SEQ ID NOs: 39, 40), fldA (SEQ ID NOs: 41, 42), and fldB (SEQ ID NOs: 43, 44), the Z. mobiilis pdc gene (SEQ ID NOs: 474, 475), and the E. coli acetylating aldehyde dehydrogenase gene eutE (SEQ ID NOs: 476, 477).
Enzyme 2: Keto-Thiolase or Acetyl-CoA Acetyltransferase
[0042] Recombinant sequences encoding the keto-thiolase or acetyl-CoA acetyltransferase may be derived from all prokaryotic organisms, including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal organisms, and all eukaryotic organisms, including mammalian, insect, fungal and yeast organisms. Preferred examples include, but are not limited to: the Rastonia eutrophus acetoacetyl-CoA thiolase/synthase phaA (SEQ ID NOs: 15, 16) and related enzymes from cells that make polyhydroxyalkanoates, C. acetobutylicum acetoacetyl-CoA thiolase/synthase thI, and E. coli acetoacetyl-CoA thiolase/synthase atoB.
Enzyme 3: Acetoacetyl-CoA Reductase or Hydroxybutyryl-CoA Dehydrogenase
[0043] Recombinant sequences encoding acetoacetyl-CoA reductase or hydroxybutyryl-CoA dehydrogenase may be derived from all prokaryotic organisms, including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal organisms, and all eukaryotic organisms, including mammalian, insect, fungal and yeast organisms. Preferred examples include, but are not limited to: the R. eutrophus 3-hydroxybutyryl-CoA dehydrogenase phaB (SEQ ID NOs: 17, 18), the C. acetobutylicum acetoacetyl-CoA reductase hbd (SEQ ID NOs: 19, 20).
Enzyme 4: Crotonase
[0044] Recombinant sequences encoding crotonase may be derived from all prokaryotic organisms, including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal organisms, and eukaryotic organisms, including mammalian, insect, fungal and yeast organisms. Preferred examples include, but are not limited to: the C. acetobutylicum crotonase crt (SEQ ID NOs: 21, 22) or the A. cavaie crotonase phaJ (SEQ ID NOs: 478, 479).
Enzyme 5: Crotonyl-CoA Reductase or Trans-Enoyl-CoA Reductase
[0045] Recombinant sequences encoding crotonyl-CoA reductase or trans-enoyl-CoA reductase may be derived from all prokaryotic organisms, including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal organisms, and all eukaryotic organisms, including mammalian, insect, fungal and yeast organisms. Preferred examples include, but are not limited to: T. denticola (SEQ ID NOs: 29, 30), E. gracilis (SEQ ID NOs: 31, 32), Burkhoderia mallei, Burkhoderia pseudomallei, Burkhoderia cepacia, Methylobacillus flagellatus, Xylella fastidiosa, Xanthomonas campestris, Xanthomonas cryzae, Pseudomonas putida, Pseudomonas entomophila, Marinomonas sp., Psychromonas ingrahmii, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio splendidus, Vibrio sp., Shewanella frigidimarina, Oceanospirillum sp., Aeromonas hydrophila subsp., Serratiae proteamaculans, Saccharophagus degradans, Colwellia psychrerythraea, Reine kea sp., Idiomarina loihiensis, Streptomyces avermitilis, Coxiella burnetii Dugway, Polaribacter irgensii, Flavobacterium johnsoniae, Cytophaga hutchisonii, E. coli, R. eutrophus, A. caviae , or C. acetobutylicum.
[0046] The disclosure includes examples for the use of Ters from T. denticola and Euglena gracilis ( E. gracilis ), the polypeptide sequences of which are 48% homologous.
[0047] In a specific embodiment the recombinant sequence encoding the crotonyl-CoA reductase is derived from Streptomyces collinus ( S. collinus ). In another specific embodiment the recombinant sequence encoding the trans-enoyl-CoA reductase (TER) is derived from T. denticola . In another specific embodiment the crotonyl-CoA reductase is ccr from S. collinus . In another specific embodiment the trans-enoyl-CoA reductase is ter from T. denticola.
Enzyme 6: Butyraldehyde/Butanol Dehydrogenase
[0048] Recombinant sequences encoding the butyraldehyde/butanol dehydrogenase may be derived from all prokaryotic organisms, including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal organisms, and all eukaryotic organisms, including mammalian, insect, fungal and yeast organisms. Preferred examples include, but are not limited to: the C. acetobutylicum butyraldehyde/butanol dehydrogenases adhE2 (SEQ ID NOs: 33, 34) or aad (SEQ ID NOs: X, Y) and related sequences from Clostridia sp, including but not limited to adhE1, bdhA, bdhB from C. acetobutylicum ; and aldH from Clostridium perfringens, Clostridium botulinum A, Clostridium beijerinckii , and Clostridium difficile . In another specific embodiment the butyraldehyde/butanol dehydrogenase is the butyryl-CoA dehydrogenase bcd from C. acetobutylicum.
Cofactor Specificity
[0049] Biomass degradation, and especially the degradation of hemicellulose, yields both C6 sugars such as glucose and C5 sugars such as xylose. Whereas C6 sugars are typically metabolized through the NAMNADH-dependent Embden-Meyerhof-Parnas pathway (the most common glycolytic pathway), C5 sugars are typically metabolized through the Pentose Phosphate Pathway, which is NADP + /NADPH-dependent ( FIG. 13 ). NADP + /NADPH-dependent enzymes of the Pentose Phosphate Pathway include a glucose dehydrogenase, such as gcd of E. coli , and a 2-keto-D-gluconate reductase, such as tiaE of E. coli . Applicants do not wish to be bound by theory. However, when producing n-butanol from hemicellulose-derived carbon sources it is believed to be beneficial to integrate NADPH-specific enzymes, such as the 3-hydroxybutyryl-CoA dehydrogenase PhaB from R. eutrophus , in the n-butanol synthesis pathway to rebalance the NADP required for continued C5 sugar assimilation.
[0050] Because the metabolism of different carbon sources may differently affect cellular NAD + /NADH- and NADP + /NADPH-redox systems, without wishing to be bound by theory, it is further believed that it is beneficial to tailor recombinant n-butanol synthesis pathways to contain an optimized number of either NAD + /NADH-dependent or NADP + /NADPH-dependent enzymes. This tailoring allows for an optimal rebalancing of the respective redox systems and ultimately leads to optimized carbon source utilization and n-butanol yields. For example, when metabolizing a hexose-rich carbon source, recombinant cells containing a greater number of NAD + /NADH-dependent enzymes are preferred. On the contrary, when metabolizing a pentose-rich carbon source recombinant cells containing a greater number of NADP + /NADPH-dependent enzymes are preferred. When metabolizing a carbon source yielding a mix of hexoses and pentoses, such as hemicellulose, recombinant cells containing a mix of NAD + /NADH-dependent and NADP + /NADPH-dependent enzymes within the recombinant n-butanol pathway are preferred.
[0051] In one embodiment of the invention the recombinant n-butanol synthesis pathway uses NADH, but no NADPH. In one specific embodiment, the recombinant n-butanol synthesis pathway ( FIG. 1 , Enzymes 1-6) uses 4 moles of NADH for the production of one mole of n-butanol. Such a recombinant n-butanol synthesis pathway includes the C. acetobutylicum acetoacetyl-CoA reductase Hbd and the C. acetobutylicum crotonase Crt. In another embodiment of the invention the recombinant n-butanol synthesis pathway uses both NADH and NADPH. In one specific embodiment, the recombinant n-butanol synthesis pathway uses 3 moles of NADH and 1 mole of NADPH for the production of one mole of n-butanol. Such a recombinant n-butanol synthesis pathway includes the R. eutrophus 3-hydroxybutyryl-CoA dehydrogenase PhaB and the A. cavaie crotonase PhaJ. In a preferred embodiment the recombinant n-butanol synthesis pathway using 3 moles of NADH and 1 mole of NADPH includes the acetyl-CoA acetyltransferase PhaA, the R. eutrophus 3-hydroxybutyryl-CoA dehydrogenase PhaB, the A. cavaie crotonase PhaJ and the trans-enoyl-coA reductase Ter from T. denticola.
Coenzyme A Synthesis
[0052] In one embodiment the recombinant cell further contains recombinant sequences encoding one or more enzymes of the coenzyme A biosynthesis pathway.
[0053] In one embodiment the recombinant cell further contains a recombinant sequence encoding a pantothenate kinase catalyzing the conversion of pantothenate to 4′-phosphopantothenate. In one specific embodiment the pantothenate kinase is derived from E. coli . In another specific embodiment the pantothenate kinase is PanK/CoaA (SEQ ID NOs: 455, 456), or CoaX SEQ ID NOs: 457, 458).
[0054] In another embodiment the recombinant cell further contains a recombinant sequence encoding a phosphopantothenoylcysteine synthetase catalyzing the conversion of 4′-phosphopantothenate to 4′-phosphopantothenoylcysteine. In a specific embodiment the phosphopantothenoylcysteine synthetase is derived from E. coli . In another specific embodiment the phosphopantothenoylcysteine synthetase is Ppcs or CoaB (SEQ ID NOs: 459, 460).
[0055] In another embodiment the recombinant cell further contains a recombinant sequence encoding phosphopantothenonylcysteine decarboxylase catalyzing the conversion of 4′-phosphopantothenoylcysteine to 4′-phosphopantetheine. In a specific embodiment the phosphopantothenonylcysteine decarboxylase is derived from E. coli . In another specific embodiment the phosphopantothenonylcysteine decarboxylase is Ppcdc or CoaC (SEQ ID NOs: 459, 460).
[0056] In another embodiment the recombinant cell further contains a recombinant sequence encoding phosphopantetheine adenylyl transferase catalyzing the transfer of an adenylyl group from ATP to 4′-phosphopantetheine. In a specific embodiment the phosphopantetheine adenylyl transferase is derived from E. coli . In another specific embodiment the phosphopantetheine adenylyl transferase is Ppat or CoaD (SEQ ID NOs: 461, 462).
[0057] In another embodiment the recombinant cell further contains a recombinant sequence encoding dephosphocoenzyme A kinase catalyzing the phosphorylation of dephospho-CoA. In a specific embodiment the dephosphocoenzyme A kinase is derived from E. coli . In another specific embodiment the dephosphocoenzyme A kinase is CoaE (SEQ ID NOs: 463, 464).
[0058] Recombinant sequences encoding pantothenate kinase, phosphopantothenoylcysteine synthetase, phosphopantothenonylcysteine decarboxylase, phosphopantetheine adenylyl transferase, or dephosphocoenzyme A kinase may be derived from all prokaryotic organisms, including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal organisms, and all eukaryotic organisms, including mammalian, insect, fungal and yeast organisms.
Competing Pathways
[0059] In one embodiment of the invention the recombinant cell further contains mutations reducing or eliminating the activity of enzymes in pathways that utilize pyruvate or acetyl-CoA to synthesize products other than n-butanol ( FIG. 11 ). In one specific embodiment enzyme activities are reduced or eliminated in a pathway synthesizing lactate from pyruvate. In another specific embodiment enzyme activities are reduced or elimimanted in a pathway synthesizing acetate from pyruvate. In another specific embodiment enzyme activities are reduced or eliminated in a pathway synthesizing acetate from acetyl-CoA. In another specific embodiment enzyme activities are reduced or eliminated in a pathway synthesizing ethanol from acetyl-CoA.
[0060] In one embodiment the recombinant cell contains a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate with reduced or eliminated activity. In a specific embodiment the lactate dehydrogenase is ldhA from E. coli . In another embodiment the recombinant cell contains a pyruvate oxidase that catalyzes the conversion of pyruvate to acetate with reduced or eliminated activity. In a specific embodiment the pyruvate oxidase is poxB from E. coli . In another embodiment the recombinant cell contains an alcohol dehydrogenase that catalyzes the conversion of acetyl-CoA to ethanol with reduced or eliminated activity. In a specific embodiment the alcohol dehydrogenase is adhE from E. coli . In another embodiment the recombinant cell contains an acetate kinase that catalyzes the conversion of acetyl-CoA to acetate with reduced or eliminated activity. In a specific embodiment the acetate kinase is ackA. In another embodiment the recombinant cell contains a phosphotransacetylase that catalyzes the conversion of acetyl-CoA to acetate with reduced or eliminated activity. In a specific embodiment the phosphotransacetylase is pta. In another embodiment the recombinant cell contains a fumarate dehydrogenase that catalyzes the conversion of succinate to fumarate with reduced or eliminated activity. In a specific embodiment the phosphotransacetylase is frd from E. coli.
[0061] The activity of an enzyme having reduced or eliminated activity may be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to a wild type enzyme, the activity of which is not reduced. Mutatations reducing or eliminating the activity of enzymes may include point mutations that cause amino acid changes in the enzymes, deletion mutations, nonsense mutations, frameshift mutations, sequence duplications or inversions and insertions. Mutations may be introduced in a targeted or non-targeted manner. Mutations may be introduced by molecular biology means, such as homologous recombinations, antisense technologies or RNA interference, or by chemical means, such as treatments with DNA intercalators or DNA methylating agents.
[0062] In one embodiment the recombinant cell is a yeast cell. In a specific embodiment the yeast cell further contains mutations reducing or eliminating the activity of enzymes in pathways that utilize pyruvate or acetyl-CoA to synthesize products other than n-butanol. In another specific embodiment the enzymes may include the alcohol dehydrogenase adh1, the NAD-dependent glycerol-3-phosphate dehydrogenases gpd1 or gpd2, the NADP-dependent glutamate dehydrogenase gdh1, the aquaglyceroporin fps1, the pyruvate decarboxylases pdc1, pdc2, pdc3, pdc4, and pdc5, the acetyl-CoA synthetases acs1 and acs2, and the acetaldehyde dehydrogenases ALDH1, ADLH2, ALDH3, ALDH4, ALDH5, ALDH6.
[0063] In another specific embodiment the recombinant cell further contains recombinant sequences encoding the glutamate synthase glt1 or the glutamine synthetase gln1.
Cells
[0064] Recombinant cells of the invention may include all prokaryotic-including proteobacterial, archaebacterial, bacteroidal, enterobacterial, spirochetal- and eukaryotic-including mammalian, insect, fungal and yeast-cell types. Preferred embodiments of the invention include, but are not limited to E. coli cells, Zymomonas mobilis ( Z. mobilis ) cells, Bacillus subtilis ( B. subtilis ) cells, yeast cells including S. cerevisiae cells and S. pombe cells, cyanobacterial cells such as Synechocystis sp. and Synechococcus sp., photosynthetic cells such as Rhodospirillum sp., solvent producing cells such as Clostridium sp. (including but not limited to Clostridium acetobutylicum and Clostridium beijerinckii ), chemoautotrophic cells such as Ralstonia sp., in general and Ralstonia eutrophus in particular, aromatic-degrading cells such as Pseudomonas sp. and Rhodococcus sp., thermophilic cells such as Thermoanaerobacterium saccharolyticum ( T. saccharolyticum ) and Thermotoga sp., cellulytic cells such as Trichoderma reesei ( T. reesei ) cells, and Aspergillus niger ( A. niger ) cells, and lignocellulytic cells such as Phanerochaete chrysosporium ( P. chrysosporium ), CHO cells, SF9 cells.
General Methods
[0065] Metabolites and products formed as part of the recombinant biofuel pathway can be identified and quantified using standard HPLC chromatography and mass spectrometry techniques. Enzymatic activities can be determined using traditional spectrophotometric activity assays relying on the detection of NAD(P)H cofactor consumption.
[0066] The nucleic acids may be synthesized, isolated, or manipulated using standard molecular biology techniques such as those described in Sambrook, J. et al. 2000. Molecular Cloning: A Laboratory Manual (Third Edition). Techniques may include cloning, expression of cDNA libraries, and amplification of mRNA or genomic DNA.
[0067] The nucleic acids of the present disclosure, or subsequences thereof, may be incorporated into a cloning vehicle comprising an expression cassette or vector. The cloning vehicle can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, or an artificial chromosome. The viral vector can comprise an adenovirus vector, a retroviral vector, or an adeno-associated viral vector. The cloning vehicle can comprise a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
[0068] The nucleic acids may be operably linked to a promoter. The promoter can be a viral, prokaryotic, or eukaryotic promoter. The promoter can be a constitutive promoter, an inducible promoter, a tissue-specific promoter, or an environmentally regulated or a developmentally regulated promoter.
[0000] Methods for Producing n-Butanol
[0069] In one embodiment of the invention the method for the production of n-butanol includes the step of growing a recombinant cell of the invention in the presence of a suitable carbon source.
[0070] Suitable carbon sources may include, but are not limited to glucose, glycerol, sugars, starches, and lignocellulosics, including but not limited to glucose derived from cellulose and C 5 sugars derived from hemicellulose, such as xylose.
[0071] In one specific embodiment the recombinant cell of the invention is grown under aerobic conditions. In another specific embodiment the recombinant cell of the invention is grown under microaerobic conditions. In another specific embodiment the recombinant cell of the invention is grown under anaerobic conditions. In another specific embodiment the recombinant cell of the invention is grown under conditions wherein it produces more n-butanol under anaerobic conditions than under aerobic or microaerobic conditions. In another specific embodiment the recombinant cell of the invention is grown under conditions wherein it produces more total levels of n-butanol and ethanol under anaerobic conditions than under aerobic or microaerobic conditions. In another specific embodiment the recombinant cell of the invention is grown under anaerobic conditions wherein it produces near quantitative yields of n-butanol. In another specific embodiment the recombinant cell of the invention is grown under anaerobic conditions wherein it produces near quantitative yields of n-butanol and ethanol.
[0072] In one specific embodiment the recombinant cell of the invention is grown under aerobic conditions wherein it produces elevated levels of n-butanol compared to a wild-type cell grown under aerobic conditions. Total levels of n-butanol produced by the recombinant cell of the invention under aerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 100,000-fold compared to the n-butanol levels produced by a wild-type cell under aerobic conditions. In specific embodiments the recombinant cell of the invention is grown under aerobic conditions wherein it produces at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L n-butanol.
[0073] In one specific embodiment the recombinant cell of the invention is grown under aerobic conditions wherein it produces elevated total levels of n-butanol and ethanol compared to a wild-type cell grown under aerobic conditions. Total levels of n-butanol and ethanol produced by the recombinant cell of the invention under aerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 100,000-fold compared to the total levels of n-butanol and ethanol produced by a wild-type cell under aerobic conditions. In specific embodiments the recombinant cell of the invention is grown under aerobic conditions wherein it produces total levels of n-butanol and ethanol of at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L.
[0074] In one specific embodiment the recombinant cell of the invention is grown under anaerobic conditions wherein it produces elevated levels of n-butanol compared to a wild-type cell grown under anaerobic conditions. Total levels of n-butanol produced by the recombinant cell of the invention under anaerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 100,000-fold compared to the n-butanol levels produced by a wild-type cell under anaerobic conditions. In specific embodiments the recombinant cell of the invention is grown under anaerobic conditions wherein it produces at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L n-butanol.
[0075] In one specific embodiment the recombinant cell of the invention is grown under anaerobic conditions wherein it produces elevated total levels of n-butanol and ethanol compared to a wild-type cell grown under anaerobic conditions. Total levels of n-butanol and ethanol produced by the recombinant cell of the invention under anaerobic conditions may be elevated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, 1,000-fold, 3,000-fold, 10,000-fold, 30,000-fold, 100,000-fold, 300,000-fold or 100,000-fold compared to the total levels of n-butanol and ethanol produced by a wild-type cell under anaerobic conditions. In specific embodiments the recombinant cell of the invention is grown under anaerobic conditions wherein it produces total levels of n-butanol and ethanol of at least 0.01 g/L, at least 0.03 g/L, at least 0.1 g/L, at least 0.3 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least 4.0 g/L, at least 4.5 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10.0 g/L, at least 15.0 g/L, at least 20.0 g/L, at least 30.0 g/L, at least 50.0 g/L, or at least 75.0 g/L.
[0076] The methods described herein can be practiced in combination with other methods useful for the production of n-butanol, such as methods for the conversion of lignocellulosic materials into biofuels.
[0077] For example, plant material may be subjected to pretreatment including ammonia fiber expansion (AFEX), steam explosion, treatment with alkaline aqueous solutions, acidic solutions, organic solvents, ionic liquids (IL), electrolyzed water, phosphoric acid, and combinations thereof. Pretreatments that remove lignin from the plant material may increase the overall amount of sugar released from the hemicellulose.
[0078] Because hemicellulose degradation yields both C6 sugars (e.g., glucose) and C5 sugars (e.g., xylose) a combination of recombinant n-butanol biosynthesis pathways with optimized recombinant glycolysis pathways (for C6 sugar assimilation) or optimized recombinant pentose phosphate pathways (for C5 sugar assimilation) may be useful for the achievement of optimal biomass utilization and n-butanol yields.
Preferred Embodiments
[0079] In one preferred embodiment of the invention the recombinant cell contains recombinant sequences encoding the pyruvate decarboxylase Pdc from Z. mobilis , the acylating aldehyde dehydrogenase EutE from E. coli , the keto-thiolase PhaA from R. eutrophus , the hydroxybutyryl-CoA dehydrogenase Hbd from C. acetobutylicum , the crotonase Crt from C. acetobutylicum , the crotonyl-CoA reductase Ter from T. denticola , and the alcohol dehydrogenase AdhE2 from C. acetobutylicum . In another preferred embodiment the recombinant cell contains recombinant sequences encoding the pyruvate:flavodoxin/ferredoxin-oxidoreductase YdbK from E. coli , the keto-thiolase PhaA from R. eutrophus , the hydroxybutyryl-CoA dehydrogenase Hbd from C. acetobutylicum , the crotonase Crt from C. acetobutylicum , the crotonyl-CoA reductase Ter from T. denticola , and the alcohol dehydrogenase AdhE2 from C. acetobutylicum . In another preferred embodiment the recombinant cell is a S. cerevisiae cell, an E. coli cell, a C. acetobutylicum cell, or a C. beijerinckii cell.
[0080] In another preferred embodiment the recombinant cell further contains a recombinant sequence encoding a component of an acetyl-CoA synthesis pathway, including pantothenate kinase (PanK, CoaA, CoaX), phosphopantothenoylcysteine synthetase (Ppcs, CoaB), phosphopantothenonylcysteine decarboxylase (Ppcdc, CoaC), and phosphopantetheine adenylyl transferase (Ppat, CoaD), and dephosphocoenzyme A kinase (CoaE).
[0081] In another preferred embodiment the recombinant cell further contains reduced or eliminated activities of at least one enzyme of a biosynthesis pathways utilizing pyruvate or acetyl-CoA for other purposes than n-butanol biosynthesis, such as lactate dehydrogenase, pyruvate oxidase, alcohol dehydrogenase, acetate kinase, or phosphotransacetylase.
[0082] In another preferred embodiment a preferred recombinant cell of the invention is grown in the presence of a suitable carbon source. In another preferred embodiment the preferred cell of the invention is grown under anaerobic conditions. In another preferred embodiment the preferred cell of the invention is grown under conditions wherein the cell produces total levels of n-butanol and ethanol of at least 5.0 g/L.
EXAMPLES
[0083] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.
Summary of Examples
[0084] Example 1:Production of n-butanol in recombinant E. coli
[0085] Example 2: Identification of bottleneck in recombinant n-butanol synthesis pathway
[0086] Example 3: Ter increases n-butanol production in recombinant cells
[0087] Example 4: Elevation of PDH and PFOR activities further increase n-butanol yields
[0088] Example 5: Efficient production of n-butanol in a recombinant cell
[0089] Example 6: Construction of a recombinant S. cerevisiae cell for n-butanol production
Materials and Methods.
[0090] Terrific Broth (TB), LB Broth Miller (LB), LB Agar Miller, sulfuric acid and glycerol were purchased from EMD Biosciences (Darmstadt, Germany). Isopropyl β-D-1-thiogalactopyranoside (IPTG) D-glucose, Dithiothreitol (DTT), Tris-HCl, phenylmethanesulfonyl fluoride (PMSF), carbenicillin (Cb), ammonium acetate, streptomycin sulfate and HPLC-grade acetonitrile were purchased from Fisher Scientific (Pittsburgh, Pa.). L-arabinose, chloramphenicol (Cm), kanamycin (Km), coenzyme A (CoASH), acetyl-CoA, acetoacetyl-CoA, crotonyl-CoA, butyryl-CoA, butyraldehyde, N,N,N′,N′-Tetramethylethylenediamine (TEMED), NADH, NADPH, and NAD were purchased from Sigma-Aldrich (St. Louis, Mo.). Polyacrylamide, Protein Assay reagent, electrophoresis grade sodium dodecyl sulfate (SDS), and ammonium persulfate were purchased from Bio-Rad Laborabories (Hercules, Calif.). All PCR amplifications were carried out with Phusion polymerase (New England BioLabs; Ipswich, Mass.), unless otherwise noted. Deoxynucleotides (dNTPs) and Platinum Taq High-Fidelity polymerase (Pt Taq HF) were purchased from Invitrogen (Carlsbad, Calif.). All restriction enzymes, antarctic phosphatase, polynucleotide kinase, T4 Polymerase and T4 DNA ligase were purchased from New England Biolabs (Ipswich, Mass.). DNA was isolated using the QIAprep Spin Miniprep Kit, QIAquick PCR Purification Kit, and QIAquick Gel Extraction Kit (QIAGEN; Valencia, Calif.) as appropriate. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, Iowa) and resuspended at a stock concentration of 100 μM in 10 mM Tris-HCl, pH 8.5. Codon optimization and back-translation were carried out using Gene Designer 2.0 (DNA 2.0; Menlo Park, Calif.). All synthetic genes and inserts were sequenced using the sequencing primers for the appropriate gene(s) following plasmid construction by the UC Berkeley Sequencing Facility, Sequetech (Mountain View, Calif.), or Quintara Biosciences (Berkeley, Calif.). All absorbance readings were taken on a DU-800 spectrometer (Beckman-Coulter; Fullerton, Calif.) or a SpectraMax M2 plate reader (Molecular Devices; Toronto, Canada).
Bacterial Strains.
[0091] E. coli DH10B-T1R, DH10B-T1R(de3), DH1, DH1(de3), and BL21(de3), and were used for protein and n-butanol production studies. DH10B-T1R and DH1 were lysogenized using λDE3 Lysogenization Kit from Novagen (San Diego, Calif.). Additional strain optimization in E. coli DH1 was achieved by knocking out metabolic genes to divert carbon flux from organic acid metabolites to the synthetic butanol pathway (Table 1, FIG. 9 ).
Cell Culture.
[0092] E. coli strains were transformed by electroporation using the appropriate plasmids. A single colony from a fresh transformation was then used to seed an overnight culture grown in Terrific Broth (TB) supplemented with 0.5% glucose and appropriate antibiotics at 37° C. in a rotary shaker (200 rpm). Antibiotics were used at a concentration of 50 μg/mL for strains with a single resistance marker. For strains with multiple resistance markers, kanamycin (Km) and chloramphenicol (Cm) were used at 25 μg/mL and carbenicillin (Cb) was used at 50 μg/mL.
Example 1
Production of n-Butanol in E. coli
[0093] A recombinant pathway for n-butanol synthesis in E. coli was constructed in the form of a two plasmid system in E. coli BL21(de3) cells comprising the R. eutrophus genes phaA and phaB, the C. acetobutylicum genes crt and adh2 and the S. cinnamonensis gene ccr ( FIG. 2 ). Although n-butanol formation could be observed by gas chromatography-mass spectometry, the titer achieved in E. coli BL21(de3) cells was low (˜2 mg/L).
Gene Synthesis
[0094] Synthetic genes encoding PhaA (SEQ ID NO 15), PhaB (SEQ ID NO 16), Crt (SEQ ID NO 21), Ccr (SEQ ID NO 23), and AdhE2 (SEQ ID NO 33) were optimized for E. coli class II codon usage and obtained from Epoch Biosciences (Sugar Land, Tex.). Gene2Oligo (http://berry.engin.umich.edu/gene2oligo) was used to convert the gene sequence into primer sets using default optimization settings (Gene Construction Primers: Ter ( E. gracilis )—SEQ ID NOs 45-112; Ter ( T. denticola )—SEQ ID NOs 113-184; Ccr ( S. cinnamonensis )—SEQ ID NOs 185-260; Hbd ( C. acetobutylicum )—SEQ ID NOs 261-314). To assemble the synthetic gene, each primer was added at a final concentration of 1 μM to the first PCR reaction (50 μL) containing 1×Pl Taq HF buffer (20 mM Tris-HCl, 50 mM KCl, pH 8.4), MgSO 4 (1.5 mM), dNTPs (250 μM each), and Pt Taq HF (5 U). The following thermocycler program was used for the first assembly reaction: 95° C. for 5 min; 95° C. for 30 s; 55° C. for 2 min; 72° C. for 10 s; 40 cycles of 95° C. for 15 s, 55° C. for 30 s, 72° C. for 20 s plus 3 s/cycle; these cycles were followed by a final incubation at 72° C. for 5 min. The second assembly reaction (50 μL) contained 16 μL of the unpurified first PCR reaction with standard reagents for Pt Taq HF. The thermocycler program for the second PCR was: 95° C. for 30 s; 55° C. for 2 min; 72° C. for 10 s; 40 cycles of 95° C. for 15 s, 55° C. for 30 s, 72° C. for 80 s; these cycles were followed by a final incubation at 72° C. for 5 min. The second PCR reaction (16 μL) was transferred again into fresh reagents and run using the same program. Following gene construction, the DNA smear at the appropriate size was gel purified and used as a template for the rescue PCR (50 μL) with Pt Taq HF and rescue primers (TdTer F1 and R1) under standard conditions. The resulting rescue product was either inserted directly in the appropriate vector or first cloned into pCR2.1-TOPO using a TOPO TA Cloning Kit from Invitrogen.
Construction of Plasmids
[0095] Standard molecular biology techniques were used to carry out plasmid construction using E. coli DH10B-T1R as the cloning host. Primers are listed in SEQ ID NOs 315-334. Annealed inserts were generated by phosphorylating each primer (1.5 pmol) individually with polynucleotide kinase in T4 DNA ligase buffer followed by incubation at 37° C. for 30 min and heat inactivation at 65° C. for 20 min. The phosphorylated primers were then mixed in 1× annealing buffer (100 mM NaCl, 50 mM HEPES, pH 7.4) and annealed using the following program and used immediately once the reaction reached 25° C.: 90° C. for 4 min, 70° C. for 10 min, ramped to 37° C. at 0.5° C./s, 37° C. for 15 min, ramped to 25° C. at 0.5° C./s.
[0096] pBT33-phaAB-crt. The phaAB operon was amplified from pCR2.1-phaA2.phaB using the phaA2 F2 and phaB R2 primers and inserted into the SacI-XbaI restriction sites of pBAD33 to generate pBAD33-phaAB. The pTrc99a-crt cloning intermediate was made by inserting the synthetic crt gene into the NcoI-XmaI restriction sites of pTrc99a using the crt F2 and crt R2 primers to amplify the insert. The resulting PTrc.crt.rrnB cassette was amplified from pTrc99a-crt using the pTrc99a F4 and pTrc99a R4 primers and inserted non-directionally into the BglI site of pBAD33-phaAB to produce pBT33-phaABcrt. Sequencing showed the coding strand of the phaAB operon was on the same strand as the crt gene. pBT33-phaB-hbd. The pCR2.1-phaA.hbd cloning intermediate was constructed by amplification of the synthetic hbd gene from pCR2.1-hbd with the hbd F1 and hbd R1 primers and insertion into the EcoRIHindIII restriction sites of pCR2.1-phaA2.phaB. The phaAB operon of pBT33-phaAB-crt was then replaced with a new multiple cloning site by digestion with NdeI and XhoI and insertion of a linker using sequence and ligation independent cloning (SLIC) (Li and Elledge, 2007, Nature Methods. 4, 251-56). The insert was made by amplifying the rrnB terminator from pBAD33 using primers rrnB SLIC F1 and rrnB SLIC R1. The amplified fragment and digested vector were independently treated with 0.5 U T4 polymerase for 30 min and the reaction was quenched with the addition of dATP. The insert and vector were incubated in 1× ligation buffer for 30 min at 37° C. and transformed immediately.
[0097] pCWOri-ccr.adhE2. pCWOri-ccr.adhE2 was made by inserting the ccr-adhE2 operon from pET29accr. adhE2 into the NdeI-HindIII sites of pCWOri. The primers used to amplify the operon were ccr F1 and adhE2 R1.
[0000] In Vivo Production of n-Butanol
[0098] For production of n-butanol production in baffled flasks, the overnight cultures were grown for 12-16 h and used to inoculate TB (50 mL) with either 2% glucose or 2% glycerol replacing the standard glycerol supplement and appropriate antibiotics in a 250 mL-baffled flask to a starting OD600=0.05. The cultures were grown at 37° C. in a rotary shaker (200 rpm) and induced with IPTG (1.0 mM) and L-arabinose (0.2%) when appropriate at OD600=0.35-0.45. At this time the growth temperature was reduced to 30° C. Upon induction and following all daily samplings, flasks were sealed with Parafilm M (Pechiney Plastic Packaging, Chicago, Ill.). For production of n-butanol production in culture tubes, the overnight cultures were grown for 22-26 h and used to inoculate (1%, 50 μL) precultures in TB with 0.5% glucose (5 mL). After incubation at 37° C. in rotary shaker (250 rpm) for 16 h, precultures were back-diluted 8 to OD600=0.4 in TB with 2.5% glucose replacing the standard glycerol supplement (5 mL) in anaerobic tubes (20 mm; Bellco Glass; Vineland, N.J.) and induced with IPTG (1.0 mM) and L-arabinose (0.2%). The growth temperature was then reduced to 30° C. and the culture tubes sealed with aluminum seals using butyl rubber septa (Bellco Glass) unless otherwise noted. For anaerobic growth, the headspace of the cultures was deoxygenated with Ar gas after backdilution and induction. Semi-anaerobic growth was performed with cultures in sealed tubes without degassing with Ar and aerobic growth was performed in unsealed tubes. Extraction and quantification of n-butanol. Samples (2 mL) were removed from cell culture and cleared of biomass by centrifugation at 20817×g for 2 min using an Eppendorf 5417R centrifuge (Hamburg, Germany). The supernatant or cleared media sample was then mixed 1:1 with an aqueous solution containing the isobutanol internal standard (1000 mg/L). These samples were then analyzed on a Trace GC Ultra (Thermo Scientific; Waltham, Mass.) using an HP-5MS column (0.25 mm×30 m, 0.25 μM film thickness, J & W Scientific). The oven program was as follows: 75° C. for 3 min, ramp to 300° C. at 45° C./min, 300° C. for 1 min. n-Butanol was quantified using by flame ionization detection (FID) (using flow of 350 ml/min air, 35 ml/min H 2 , and 30 ml/min He). Samples containing n-butanol levels below 500 mg/L were then re-quantified with a DSQII single-quadrupole mass spectrometer (Thermo Scientific; Waltham, Mass.) using single ion monitoring (m/z 41 and 56) concurrent with full scan mode (m/z 35-80) for samples with n-butanol levels lower than 500 mg/L. Samples were quantified relative to a standard curve of 2, 5, 10, 25, 50, and 100 mg/L n-butanol for MS detection or 62.5, 125, 250, 500, 1000, 2000, 4000 mg/L n-butanol for FID detection. Standard curves were prepared freshly during each run and normalized for injection volume using the internal isobutanol standard
Example 2
Identification of Bottleneck in Recombinant n-Butanol Synthesis Pathway
[0099] The initial n-butanol yields obtained with the recombinant cellular system of Example 1 were subsequently improved ˜60-fold by promoter and host cell optimization ( FIGS. 2 and 3A ).
[0100] A correlation was observed between n-butanol yields and solubility of the Ccr protein, which pointed to a bottleneck in the n-butanol biosynthesis pathway at the conversion step of crotonyl-CoA to butyryl-CoA ( FIG. 3B ).
Construction of Plasmids
[0101] pBAD33-ccr.adhE2. The ccr-adhE2 operon was amplified from pET29a-ccr.adhE2 using the ccr F1 and adhE2 R17 primers and inserted into the NdeI-SalI sites of pBAD33-phaAB, the insert was digested using NdeI and XhoI.
[0102] pTrc99a-ccr.adhE2. pTrc99a-ccr.adhE2 was made by inserting the ccr-adhE2 operon from pET29accr.adhe2 into the NcoI-SacI sites. The primers used to amplify the operon were ccr F15 and adhE2 R2.
[0103] pCWOri-ter.adhE2. The ter gene was amplified from pET16b-His-ter with TdTer F1 and TdTer R102 and inserted directly into the NdeI-EcoRI restriction sites of pCWOri-ccr. adhE2.
[0104] pET29a-ccr.adhE2. The ccr gene was amplified using the ccr F1 and ccr R2 primers and inserted into the NdeI-EcoRI sites of pET29a. pET29-ccr.adhE2 was constructed by insertion of the adhE2 gene into the EcoRI-SacI restriction sites of pET29a-ccr after amplification using the adhE2 μl and adhE2 R2 primers.
Example 3
Ter Increases n-Butanol Production in Recombinant Cells
[0105] In an experiment similar to Example 1, the replacement of the S. cinnamonensis gene ccr for ter genes from E. gracilis and T. denticola resulted in significantly increased n-butanol yields, where the recombinant biosynthesis pathway further comprised the R. eutrophus gene phaA, and the C. acetobutylicum genes hbd, crt and adh2 ( FIG. 5 ). This experiment thus demonstrates that the incorporation of Ter enzymes into the recombinant biosynthesis pathway for n-butanol relieves a bottleneck at the stage of crotonyl-CoA to butyryl-CoA conversion.
Example 4
Elevation of PDH and PDHc Bypass Activities Further Increase n-Butanol Yields
[0106] Acetyl-CoA is the building block for the production of advanced fuels ranging from short-, medium-, and long-chain length fatty alcohols, fatty acids, fatty acid esters, and alkanes. A major challenge in the production of these molecules is the bottleneck from the endpoint of glycolysis, the conversion of pyruvate to acetyl-CoA. Four classes of enzymes were identified that can relieve this bottleneck: pyruvate dehydrogenase PDH, PDHc bypass comprised of two enzymes (pdc and eutE), E. coli pyruvate formate oxido-reductace (PFOR), and E. coli pyruvate formate lyase with C. boidinii formate dehydrogenase (pfl and fdh).
[0107] In an experiment similar to Example 4, the elevation of PDH activity further increased n-butanol yields beyond the yields observed in the presence of Ter alone ( FIGS. 5 and 6 ). This finding demonstrates that a second bottleneck existed in the n-butanol biosynthesis pathway at the initial conversion of pyruvate to acetyl-CoA. Increasing the concentration of acetyl-CoA by increasing the turnover of pyruvate relieved this second bottleneck and resulted in higher n-butanol yields.
[0108] The third route to generate acetyl-CoA from pyruate is catalyzed by PDHc bypass that is composed of two enzymes, pyruvate decaroboxylase and acetylating aldehyde dehydrogenase. Acetaldehyde is generated by pyruvate decarboxylase from pyruvate and then oxidized to acetyl-CoA, coupled with the reduction of NAD+ to balance the reducing equivalent required for butanol synthesis. In the presence of these enzymes, and under anaerobic conditions, n-butanol yield can increase by 50% ( FIG. 8 ).
Example 5
Efficient Production of n-Butanol in a Recombinant Cell
[0109] Through the use of Ter from T. denticola and overexpression of the E. coli pyruvate dehydrogenase complex or the pyruvate decarboxylase of Z. mobilis and the acetylating aldehyde dehydrogenase of E. coli in a pathway otherwise comprising the R. eutrophus gene phaA, and the C. acetobutylicum genes hbd, crt and adh2 it was possible to engineer a highly efficient recombinant cell for the production of n-butanol.
[0000]
TABLE 1
Knockout E. coli DH1 host strains for the
production of n-butanol.
Strain
Genotype
E. coli
endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(rK− mK+) λ−
DH1
MC001
E. coli DH1 ΔadhE
MC002
E. coli DH1 ΔadhE, ΔldhA
MC003
E. coli DH1 ΔadhE, ΔldhA, ΔackA-pta
MC004
E. coli DH1 ΔadhE, ΔldhA, ΔpoxB
MC005
E. coli DH1 ΔadhE, ΔldhA, ΔackA-pta, ΔpoxB
MC006
E. coli DH1 ΔadhE, ΔldhA, ΔackA-pta, ΔpoxB, ΔfrdBC
Example 6
n-Butanol Production in a Recombinant S. Cerevisiae Cell
[0110] S. cerevisiae is another preferred host for a recombinant n-butanol production pathway and well suited to support industrial fuel production. The preferred recombinant n-butanol synthesis pathway was inserted into S. cerevisiae ( FIG. 12A ). The recombinant pathway includes the pyruvate decarboxylase Pdc from Z. mobilis , the acylating aldehyde dehydrogenase EutE from E. coli , the keto-thiolase PhaA from R. eutrophus , the hydroxybutyryl-CoA dehydrogenase Hbd from C. acetobutylicum , the crotonase Crt from C. acetobutylicum , the crotonyl-CoA reductase Ter from T. denticola , and the alcohol dehydrogenase AdhE2 from C. acetobutylicum ( FIG. 12A ). The DNA constructs shown in FIG. 12A for both plasmid-based and chromosomal gene expression were made using standard methods described above and one-step isothermal DNA assembly as described by Gibson, et al., Nat. Methods . (2009) 6, p. 343.
[0111] To optimize production of n-butanol, pyruvate decarboxylase pdc (mutant cell: Δpdc) and the alcohol dehydrogenase adh1 (mutant cell: Δadh1) were targeted for deletion in S. cerevisiae because these enzymes are involved in competing, acetyl-CoA consuming pathways other than n-butanol production. (See also FIG. 11 for analogous E. coli pathways). Wild-type S. cerevisiae as well as Δpdc and Δadh1 strains bearing a plasmid-based n-butanol genetic system were prepared using standard molecular biology techniques. Recombinant S. cerevisiae cells with the preferred n-butanol pathway were shown to produce at least 10 mg/L n-butanol. For example, a Δadh1 mutant cell, S. cerevisiae BY4741Δadh, containing the n-butanol production pathway ( FIG. 12A ) was shown to produce greater than 12 mg/L n-butanol ( FIG. 12B , column 2), whereas the background level of n-butanol production of S. cerevisiae BY4741Δadh was only about 2 mg/L ( FIG. 12B , column 1).
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The present disclosure provides optimized recombinant cells for the production of n-butanol. Methods for the use of these cells are also provided. Specifically, the utility of acylating aldehyde dehydrogenases and pyruvate:flavodoxin/ferredoxin-oxidoreductase for the improvement of n-butanol yields from recombinant cells is disclosed.
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CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. §119 of: U.S. Provisional Application Ser. No. 62/243,938 filed on Oct. 20, 2015 the content of which is relied upon and incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The disclosure relates generally to a composition for gluing laminates and a process for making the same, and more particularly resin glue sheets for gluing laminates and a process for making the same.
BACKGROUND
[0003] The disclosure relates generally to an adhesive compound in the form of a resin sheet for fabricating laminated wood products and method for making the resin sheets. Laminated wood structures have been made for centuries. Laminated wood structures can range from furniture to plywood for construction. One of the problems with the fabrication process of a laminate is the glue. It typically is a highly viscous liquid. It is messy to apply; the glue has a tendency to squeeze and ooze out and on to the machinery and tooling used to make the laminated product. This leaves the tooling and machinery gluey and messy requiring it to be cleaned before it is used again. The cleanup is time consuming and dangerous.
[0004] The gluing process, once started, requires the glue to be quickly applied and pressed in a short period of time. Water from the glue can cause delamination or steam bubbles in the final pressing step. Extra water is typically needed to produce the right glue viscosity resulting in lower solids content. The water in the glue adds additional time to the curing cycle and requires the expending of additional energy to complete it.
[0005] No admission is made that any reference cited herein constitutes prior art. Applicant expressly reserves the right to challenge the accuracy and pertinence of any cited documents.
SUMMARY
[0006] In one variation of the present invention it provides a process for making resin glue sheets with the following steps of: a) reducing to a dry powdered form any dry ingredients that will make up a composition that will be used to form the glue sheets; b) mixing into a homogenous mixture all of the dry ingredients that will make up the composition; c) mixing into the composition any liquid ingredients including water to achieve a predetermined moisture content; d) mixing into the composition glycerin in a preset amount; e) mixing the composition to achieve a semidry doughy composition that is highly viscous; f) shaping the composition into a film; and g) drying the composition in the film form.
[0007] In a variation of this process the ingredients are: soy, sorbitol, lime, SMBS, steric acid and zinc omedine. In another variation of this process the step of drying the semi-dry doughy composition into a film is done by drying it with a rotary drum dryer. In another variation of this process drying the composition into a film with a rotary drum dryer consists of passing it through a gap between two counter rotating drum dryers. In another variation the doughy composition is passed downward through a gap and a first portion of the composition adheres to a first drum of the counter rotating drums and a second portion of the composition adheres to a second drum of the counter rotating drums and the first portion of the composition is peeled off of the first counter rotating drum to form a thin film and the second portion is peeled off of the second drum to form a thin film. In another variation of the process heat is added to achieve an optimal temperature for mixing of the ingredients. In a further variation of the process the optimal temperature sought for mixing is 120°.
[0008] In another variation of the process reinforcing material is added to the film. In a further aspect of the process adding the reinforcing material includes the step of selecting a material from the group consisting of natural fibers, artificial fibers, synthetic fibers, and recycled fibers. In yet another aspect of this process it includes the additional steps of forming a fiber into a matt; passing the fiber matt down through the gap between the two counter rotating drums with the ingredients so that the ingredients suffuse the matt.
[0009] The invention also provides a glue sheet with: a) core ingredients; b) resin additives; c) glycerin of a predetermined amount; d) moisture of a predetermined amount; wherein the sheet is formed into a membrane that can be placed between structural sheets of another substance to form a composite material upon the application of suitable pressure and temperature. In a variation of the invention the core ingredients are: soy, sorbitol, lime and SMBS. The resin additives are zinc omedine and steric acid. The structural material is wood.
[0010] Another variation of the invention provides a glue sheet made of a) core ingredients; b) resin ingredients; c) at least one plasticizer, wherein the core ingredients, the resin ingredients, the plasticizer are mixed to a semi-dry doughy mixture then under suitable pressure and temperature formed into a membrane which is dried to form the glue sheet. In an aspect of this variation of the invention forming the semi-dry doughy mixture into a membrane to create the film can be achieved by passing the semi-dry doughy mixture between two counter rotating heated drums set at a predetermined distance to create the glue sheets of a predetermined thickness. In this variation of the invention the core ingredients are soy, sorbitol, lime and SMBS. The resin ingredients are the following; zinc omedine, and steric acid. The plasticizer is glycerin.
[0011] In another variation of the glue sheets material is added to reinforce said glue sheets. In additional aspect the material is selected from a group of fibers consisting of cotton, kenaf, flax, Hemp, Jute (burlap), wood fiber, wool, cellulose, carbon, glass, basalt and plastic. In yet another aspect the material is selected from a group of consisting of natural fibers, synthetic fibers, artificial fibers, staple fibers and waste fibers. In yet another aspect the fibers are formed into a matt selected from a group consisting of woven matts and non-woven matts.
[0012] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the written description and claims hereof, as well as the appended drawings.
[0013] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary, and are intended to provide an overview or framework to understand the nature and character of the claims.
[0014] The accompanying drawings are included to provide a further understanding, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a matrix chart showing the various ingredients that make up the composition that forms the resin sheets and various embodiments of possible combinations of those ingredients;
[0016] FIG. 2 is a flow chart setting forth the basic steps of the process used to make the resin sheets;
[0017] FIG. 3 is a schematic view of the counter rotating drums that make up the preferred apparatus for forming the composition into a film and drying it;
[0018] FIG. 4A depicts resin glue sheets of the present invention positioned between wood to be formed into a laminate structure;
[0019] FIG. 4B shows the wood and glue sheets of FIG. 4A being compressed in a press to form the laminate structure;
[0020] FIG. 4C shows the finished laminate product after it has come out of the press process depicted in FIG. 4B ; and
[0021] FIG. 5 is a schematic view of the counter rotating drums that make up the preferred apparatus for forming the composition into a film and drying it, which includes the addition of a reinforcing matt.
DETAILED DESCRIPTION
[0022] The present invention provides a resin adhesive composition that can be fabricated into sheets of resin for use in a lamination process to make products varying from furniture to plywood. The composition has three main constituents: core ingredients that provide the body and adhesive qualities, resin additives and water. Additionally, reinforcing material can be added as a fourth constituent when needed or desired.
[0023] The core material in a preferred embodiment includes soy, sorbitol, lime, sodium metabisulfite (SMBS) and steric acid. Soy is the main adhesive. As used in this application “Soy” refers to several different products, all involving soy protein. Specifically, any of the following forms of soy protein may be used: soy flour (SF), soy protein concentrate (SPC) and soy protein isolate (SPI). The difference between these versions is that the protein content increases from soy flour (about 50% protein) to SPC (about 65% protein) to SPI (about 90% protein). In this document anywhere we say “soy”, any of these versions can be used interchangeably. The use of the different variations of the soy discussed above depends on technical needs and cost. For example SPI provides better wet performance but entails a higher cost. Likewise, using soy flour results in a better cost position, but entails worse wet performance. When soy flour is used the moisture or water content typically is up to 40% for optimal mixing viscosity. When SPI is used the moisture content needs to be as high as 65% to reach an optimal mixing.
[0024] Sodium metabisulfite, AKA sodium pyrosulfite, is an inorganic compound that is obtained from the evaporation of sodium bisulfite saturated with sulfur dioxide. When added to a water based resin this compound helps to disperse the large protein molecules in solution and results in a decreased viscosity resin system that is easier to spray. This may also have an advantageous effect on the antimicrobial properties of the composition. In the presence of water, SMBS facilitates the cross linking of soy protein.
[0025] The lime is added to adjust the pH of the composition to help the soy proteins unravel and the sorbitol is a plasticizer to help reduce brittleness. It should be noted that these are only the primary intended purposes of the listed ingredients; the ingredients may also provide other benefits.
[0026] In one embodiment the resin additives can include zinc omedine, steric acid and glycerin. The zinc omedine is added for microbial resistance. Steric acid provides moisture resistance. The glycerin reduces the fragility of the sheets that are made of the composition making them pliable and less brittle. Other substances can be added, such as fire retardants, including aluminum trihydrate, ammonium polyphosphate and boric acid.
[0027] FIG. 1 provides a spreadsheet 20 of different embodiments in which the core, resin and water components can be combined depending on the use it will be put to. (Spreadsheet 20 is incorporated into this text.) However, this is not an exhaustive list just an illustrative list of the various proportions the ingredients can be combined. In FIG. 1 in its top row 21 provides the overall percentage of glycerin in the composition. The first column 23 provides the major category of ingredients: the core ingredients, the resin additives and moisture content. The second column provides a breakdown of the preferred ingredients. Those that make up the core: soy, lime sorbitol, lime and SMBS. Those that make up the resin additives: zinc omedine, steric acid and glycerin. The moisture component being water.
[0028] Columns 25 , 26 , 27 , 28 , 29 , 30 and 31 providing various percentages of combinations of the ingredients. As you move from column 25 on the left to column 31 on the right the glycerin content of the combination increases which in turn increases the flexibility and decreases the brittleness of the sheets that will be formed out of the mixture of ingredients. The percentages of each column add up to approximately 100% before adding the moisture.
[0029] FIG. 2 is a flow chart of the basic steps used to form the resin sheets from the ingredients identified above or which are similar to those identified above. The first step 41 is to break all of the dry components down into a powder. Once the dry ingredients have been broken down into a powder they are mixed to form a homogenous mixture 43 . In the next step the liquid components are mixed in to form a resin paste 45 . After the resin paste is formed, glycerin of a predetermined amount is mixed in to create a semi-dry highly viscous doughy composition 47 . In the next step the semi-dry doughy composition is formed into a film 49 and then dried 51 . The final step is cutting the film formed into sheets or rolling it into a roll 53 .
[0030] FIG. 3 is a schematic diagram of a dual counter rotating drum dryer use in an embodiment of the invention to form the semi-dry doughy composition into a thin film and dry it. The dryer has two counter rotating drums 61 and 63 . Drum 61 rotates in a clockwise direction as indicated by arrow 65 , and drum 63 rotates in a counter clockwise direction as indicated by arrow 67 . A small gap or nip 64 exists between drum 61 and 63 . The drums are heated. In the embodiment shown the drums have a hollow interior portion that is heated by steam. In the embodiment of the invention discussed herein the temperature is generally kept between 100° C. to 150° C., with the target temperature being 120° C. The steam pressure is kept at about 80 psi and the rate of rotation is 8.87 rpm. It should be noted that the temperature, rpm's and steam pressure are cited here only as examples. In practice the invention can be practiced with a broad range of temperatures, pressures and rpm's and the desired results achieved. The gap between the rollers depends on the thickness of film desired. In the embodiment of the process discussed it is between 0.005 inches to 0.03 inches with the average being 0.01 inches. If for some reason the target temperature is not achieved to obtain the optimal mixing of the resin mixture additional heat can be added to the system.
[0031] As can be seen in FIG. 3 the semi-dry doughy composition of the resin mixture 72 is placed between the upper parts of drum 61 and 63 just above gap 64 . As the semi-dry doughy composition 72 passes downward through gap 64 it is compressed by the force of the rollers to the thickness of the gap. After the semi-dry doughy composition passes down beyond gap 64 a portion adheres to drum 61 to form thin film 73 and a portion adheres to drum 63 to form thin film 79 . As thin film 73 moves up with drum 61 adjustable knife 69 peels the film off of drum 61 . After being peeled off of drum 61 thin film 73 is cut into sheets by cutter 75 . Likewise as thin film 79 moves up with drum 63 adjustable knife 71 peels off thin film 79 . Thin film 79 is then rolled into a roll 81 .
[0032] Depending on the final moisture content needed the thin films created can be used as they are or dried further to reduce moisture content. Optimal moisture content can vary depending on use. For use in making plywood or veneers it will typically be in the 6% to 10% range.
[0033] FIGS. 4A, 4B and 4C depict the fabrication of a laminated piece using the film produced with the thin film of resin glue sheets of the present invention. In FIG. 4A three sheets of wood 101 have sandwiched between them two of the resin glue sheets 103 of the present invention. In FIG. 4B the combination of the two resin sheets 103 and three wood sheets are compressed in a press 105 used to make laminated wood pieces. The temperature of such a press would be between 100° C. and 150° C. with the target temperature being 120° C. The pressure would be between 30 psi to 700 psi with the target pressure being 300 psi. The time the combination would stay in the press depends on the thickness of the combination of wood pieces 101 and glue sheets 103 . This generally would be 11 seconds for every millimeter of thickness of the combination of wood pieces and resin glue sheets of the present invention.
[0034] In another variation of the present invention the glue sheets of the present invention have reinforcing material added to them to make them less likely to break during handling. The reinforcing material gives the glue sheets greater tensile strength. Fibers constitute the primarily reinforcing material. This is not to be confused with the fibers (structural material) that are bonded together for the overall composite or laminate material glue sheets.
[0035] In the preferred embodiment the reinforcing fiber is typically configured in a loosely woven matt or non-woven matt. Fibers that can be used as the reinforcing material are natural plant fiber materials, artificial/synthetic and recycled fibers.
[0036] Natural fibers that can be used as the reinforcing materials include cotton, kenaf, flax, Hemp, Jute (burlap), wood fiber, wool, cellulose or other natural fibers. The physical structure of the fiber includes short or long fibers. Additionally, the fiber can be staple fiber or waste fiber. Textile waste, short fibers or mill filings can be used. Also staple fibers up to and greater that inch in length can be used as the reinforcing fiber.
[0037] Artificial or synthetic fibers can be used; among some of the materials that can be used carbon, glass, basalt and plastic fibers. Both waste and staple fiber can be used. Synthetic fibers made of cellulose such as rayon, viscose, lyocell, and acetate can be used. Both waste and staple fiber can be used.
[0038] Other miscellaneous fibers that can be used are Wollastonite, recycled fibers: paper, old corrugated cardboard (OCC), Nano clays, feather waste, animal based fiber waste such as wool, hair, etc. Additionally, thin paper, plastic, or scrim made from any of the aforementioned materials that can form the reinforcing materials.
[0039] In a preferred embodiment the resin adhesive material formed as described above is suffused or embedded in reinforcing fiber material. Referring to FIG. 5 one of the preferred methods of suffusing of embedding the resin glue in the reinforcing fiber consists of forming the fiber in a woven or non-woven matt 111 A and 111 B from one of the materials listed above. In the example given it is formed into spools 113 A and 113 B for integration into the manufacturing process. The fiber matts 111 A and 111 B are threaded down between drum 61 and 63 and as the doughy resin material 72 moves down between the drums matts 111 A and 111 B are also drawn through. As matts 111 A and 111 B pass down through the resin composition is suffused or embedded in matts 111 A and 111 B by a combination of pressure exerted by rotating drums 61 and 63 and the heat generated by the process and added if such added heat is necessary to achieve the right viscosity for suffusing or embedding resin composition in matts 111 A and 111 B.
[0040] The combined matt 111 A and resin composition 72 can be rolled up into a spool 123 for storage and shipment. Alternatively, as depicted in FIG. 5 the combined resin composition 72 and matt 111 B can be cut into sheets 121 .
[0041] Although the preferred embodiment discussed above shows two thin films coming off of drums 61 and 63 in FIG. 3 , namely thin films 73 and 79 , the process depicted could just as easily produce only one thin film. Likewise in the process depicted in FIG. 5 only one matt such as 113 A could be used and only one thin film would be produced 119 .
[0042] The resin films of the present invention offer a number of advantages; they can be stored for a long time without degradation for up to at least one year prior to use. The films can be stored in the roll or sheet form. Another alternative is to store the resin sheets with the wood sheets it is to be combined with alternating film adhesive with wood sheets to bring the material to the same moisture content.
[0043] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the invention. Since modifications combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and their equivalents.
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Various ways for formulating resin glue sheets or film for gluing and laminating items together are disclosed. Also, methods for fabrication of the resin glue sheets or film are disclosed.
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CROSS-REFERENCE(S) TO RELATED APPLICATION(S)
[0001] This application claims priority to Chinese Patent Application No. 201020612014.6, filed Nov. 9, 2010, and to Chinese Patent Application No. 201120018102.8, filed Jan. 20, 2011, each of which is hereby incorporated by reference in the present disclosure in its entirety.
BACKGROUND
[0002] 1. Field
[0003] The present disclosure relates generally to capstans, and more specifically to electric capstans.
[0004] 2. Description of Related Art
[0005] At present, electric capstan in the related art includes a motor, a reduction gear and a winching roller, wherein the output shaft of the motor is connected to the reduction gear, and then the reduction gear is connected to the winching roller. Electric capstans are mainly used in off-road vehicle, agricultural vehicle, barge and other special vehicles, and are used as a self-protection device and a traction device for a vehicle or a ship. In the case of driving vehicles or ships for purpose of traction operation and rescue operation (including self-rescue operation), electric capstans act an important part.
[0006] However, in the practical purposes, in addition to the traction function, sometimes there is a need for being supplied with water. For example, especially, the off-road vehicle, the agricultural vehicle and the barge can be easily stained with feculency such as mud and thus pollute the vehicle body or ship body thereof, when they are operated in the wild or outdoors. Here, water is needed to flush the stained vehicle or ship. In order to solve above problem, a typical solution, at present, is to equip the vehicle or ship with a separate water pump. The equipment with a separated water pump may not only occupy the space inside the vehicles or ships, but also increase the weight and cost.
[0007] Similarly, in order to accomplish further functions, the vehicles or ships are required to be equipped with devices with respective functions. Therefore, much more space inside the vehicles or ships may be occupied to mount the devices with respective functions, thus causing a great increment in cost.
BRIEF SUMMARY
[0008] One object of the present disclosure is to provide an electric capstan which is able to easily and accessibly connect external functional models to further accomplish multiple functions and which has a good reliability.
[0009] The object of the present disclosure may be accomplished by following configuration. An electric capstan of the disclosure includes a motor, a reduction gear and a winching roller, and further includes an engagement-disengagement gear and functional module, wherein the input end of the engagement-disengagement gear is connected to the output shaft of the motor and the output end of the engagement-disengagement gear is connected to the input end of the functional module.
[0010] Compared with the related art, the present disclosure, with such above configuration, has following advantages. The electric capstan according to the present disclosure includes an engagement-disengagement gear and functional module, wherein the input end of the engagement-disengagement gear is connected to the output shaft of the motor and the output end of the engagement-disengagement gear is connected to the input end of the functional module. As a result, the functional module is integrated with the electric capstan, and thus the electric capstan can accomplish multiple functions through the variable attached external functional modules and have a good reliability.
[0011] As a modification, the engagement-disengagement gear and the functional module both have accessible interfaces for facilitating the removable connection therebetween, wherein a first connecting member is arranged at the engagement-disengagement gear, and a second connecting member is arranged at the functional module. As a result, this can facilitate the accessible assembly between the engagement-disengagement gear and the functional module and allow the present disclosure to be conveniently used.
[0012] As a further modification, the engagement-disengagement gear includes a clutch and a lever that is able to engage or disengage the clutch, wherein the clutch has a groove, the lever at the end thereof has an eccentric and axial protrusion which can be embedded in the groove of the clutch, and the lever is rotatably attached to the housing of the clutch. Although the engagement-disengagement gear can be only mounted in the axial direction of motor, this engagement-disengagement gear has a simple and reliable structure, high transmission efficiency and easy manipulation, which is advantageous to implement the electric capstan according to the present disclosure and improve the performance thereof.
[0013] As a further modification, the output end of the engagement-disengagement gear is located on the sideface of the motor. Since the output end of the engagement-disengagement gear is connected to the input end of the functional module, when the output end of the engagement-disengagement gear is located on the sideface of the motor, the functional module is located on the sideface of the motor. As a result, the dimension of the electric capstan in the axial direction thereof can greatly reduce, that is the dimension of the electric capstan in the lengthwise direction thereof can greatly reduce, and therefore, the electric capstan according to the present disclosure has an advantage that it can be mounted on an object which provides a narrow mounting place.
[0014] As a further modification, the functional module is located above the sideface of the motor. As a result, this can make the whole structure of the electric capstan more compact and mounting of the functional module more convenient.
[0015] As a further modification, the engagement-disengagement gear includes a bracket, a first gear, a second gear, a third gear and an engagement-disengagement handle that can be operated so as to move the second gear in the axial direction of the revolution shaft thereof and further engaged with or disengaged from the first gear, wherein the engagement-disengagement handle is mounted on the bracket, the movable end of the engagement-disengagement handle is located on the side of the second gear, the bracket is connected to the housing of the motor, the first connecting member is mounted on the bracket; the connection of the input end of the engagement-disengagement gear and the output shaft of motor means the connection of the first gear and the output shaft of motor; the second gear is mounted on the bracket; the connection of the output end of the engagement-disengagement gear and the input end of the functional module means the connection of the third gear and the input end of the functional module; the first gear, the second gear and the third gear are engaged successively. As a result, since the three gears constitute a gear transmission mechanism, the engagement-disengagement gear has a simple and reliable structure and a light weight, and therefore, it is advantageous for the electric capstan to be mounted on transportation such as a vehicle.
[0016] As a further modification, the first and second connecting members have chuck structures that can be matched each other. The chucks that can be matched each other have a simple and reliable structure. Such a structure not only allows the electric capstan to easily and accessibly connect external functional models, but also improves reliability of the connection between engagement-disengagement gear and functional module. Therefore, this makes the electric capstan according to the present disclosure more compact, simple and convenient for bringing into produce.
DESCRIPTION OF THE FIGURES
[0017] FIG. 1 is an exemplary structural view of the electric capstan according to the first embodiment of the present disclosure;
[0018] FIG. 2 is an exemplary cross-sectional view, in enlarged manner, showing the engagement-disengagement gear of the electric capstan according the first embodiment of to the present disclosure;
[0019] FIG. 3 is an exemplary structural view of the electric capstan according to the second embodiment of the present disclosure; and
[0020] FIG. 4 is another exemplary structural view of the electric capstan according to the second embodiment of the present disclosure.
[0021] In the figures, reference numeral 1 indicates a motor, reference numeral 2 indicates a winching roller, reference numeral 3 indicates functional module, reference numeral 4 indicates a first connecting member, reference numeral 5 indicates a second connecting member, reference numeral 6 indicates a clutch, reference numeral 7 indicates a lever, reference numeral 8 indicates a bracket, reference numeral 9 indicates a first gear, reference numeral 10 indicates a second gear, reference numeral 11 indicates a third gear and reference numeral 12 indicates an engagement-disengagement handle.
DETAILED DESCRIPTION
[0022] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.
[0023] The details of the present disclosure are set forth in the accompanying drawings and the embodiments below.
First Embodiment
[0024] In the first embodiment of the present disclosure, as shown in FIGS. 1 and 2 , an electric capstan includes a motor 1 , a reduction gear and a winching roller 2 , and further includes an engagement-disengagement gear and functional module 3 . The input end of the engagement-disengagement gear is connected to the output shaft of the motor 1 and the output end of the engagement-disengagement gear is connected to the input end of the functional module 3 . Also, the output shaft of the motor 1 is connected to the reduction gear, and the reduction gear is connected to the winching roller 2 .
[0025] The engagement-disengagement gear and the functional module 3 both have accessible interfaces for facilitating the removable connection therebetween. A first connecting member 4 is arranged at the engagement-disengagement gear, and a second connecting member 5 is arranged at the functional module 3 .
[0026] The engagement-disengagement gear includes a clutch 6 and a lever 7 that is able to engage or disengage the clutch 6 . The clutch 6 has a groove, and the lever 7 at the end thereof has an eccentric and axial protrusion which can be embedded in the groove of the clutch. The lever 7 is rotatably attached to the housing of the clutch 6 .
[0027] Such above configuration is shown in FIG. 1 or 2 . FIG. 1 schematically depicts two functional modules 3 (the upper one is a water pump, and the lower one is an air pump), and the two functional modules 3 are in a condition that they are removed from the electric capstan.
Second Embodiment
[0028] In the second embodiment of the present disclosure, as shown in FIGS. 3 and 4 , an electric capstan includes a motor 1 , a reduction gear and a winching roller 2 , and further includes an engagement-disengagement gear and functional module 3 . The input end of the engagement-disengagement gear is connected to the output shaft of the motor 1 , and the output end of the engagement-disengagement gear is connected to the input end of the functional module 3 . Also, the output shaft of the motor 1 is connected to the reduction gear, and the reduction gear is connected to the winching roller 2 .
[0029] The engagement-disengagement gear and the functional module 3 have accessible interfaces for facilitating the removable connection therebetween. A first connecting member 4 is arranged at the engagement-disengagement gear, and a second connecting member 5 is arranged at the functional module 3 .
[0030] The output end of the engagement-disengagement gear is located on the sideface of the motor 1 , the functional module 3 is located above the sideface of the motor 1 , and the axis of the input shaft of the functional module 3 is in parallel with the axis of the output shaft of the motor 1 .
[0031] The engagement-disengagement gear includes a bracket 8 , a first gear 9 , a second gear 10 , a third gear 11 and an engagement-disengagement handle 12 that can be operated so as to move the second gear 10 in the axial direction of the revolution shaft thereof and further engaged with or disengaged from the first gear 9 . The engagement-disengagement handle 12 is mounted on the bracket 8 . The movable end of the engagement-disengagement handle 12 is located on the side of the second gear 10 . The bracket 8 is connected to the housing of the motor 1 . The first connecting member 4 is mounted on the bracket 8 . The connection of the input end of the engagement-disengagement gear and the output shaft of motor 1 means the connection of the first gear 9 and the output shaft of motor 1 . The second gear 10 is mounted on the bracket 8 . The connection of the output end of the engagement-disengagement gear and the input end of the functional module 3 means the connection of the third gear 11 and the input end of the functional module 3 . The first gear 9 , the second gear 10 and the third gear 11 are engaged successively.
[0032] Such above configuration is shown in FIGS. 3 and 4 . FIG. 3 schematically depicts two functional modules 3 (the upper one is a water pump, and the lower one is an air pump), and the two functional modules 3 are in a condition that they are removed from the electric capstan.
[0033] In this embodiment, the third gear 11 may be attached on the functional module 3 and removed along with the functional module 3 from the electric capstan. Therefore, each of the functional modules 3 is provided with a third gear 11 .
[0034] The preferable first and second embodiments according to the present disclosure have been described above. In the embodiments, the first and second connecting members 4 and 5 both have chuck structures that can be matched each other. In addition, the functional module 3 may be a water pump, an oil pump, an air pump, a vacuum cleaner, a reduction gear built-in air pump or an acceleration gear built-in vacuum cleaner.
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An electric capstan that is able to easily and accessibly connect external functional models and accomplish multiple functions with good reliability. This electric capstan includes a motor, a reduction gear and a winching roller, and further includes an engagement-disengagement gear and functional module, wherein the input end of the engagement-disengagement gear is connected to the output shaft of the motor and the output end of the engagement-disengagement gear is connected to the input end of the functional module.
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SUMMARY OF THE INVENTION
This invention relates to a rotative inductive coupling for the transmission of electrical power from a stationary part to a rotating part. More particularly, it relates to a power coupling combined with means for transmitting informational signals between the stationary part and the rotating part.
Briefly and in summary, this invention is a rotative, inductive coupling transferring electrical power and information between a rotating part and a stationary part comprising: (a) a stationary core means mounted on the stationary part; (b) an annular stationary coil mounted on the stationary part and encircling a hollow, rotating core means of magnetic material constructed to rotate in the stationary core for establishing a flux path; (c) an annular, rotative coil mounted on the rotating part, encircling the hollow, rotative core means, (d) means for connecting one of the coils to a source of alternating electrical potential and means for connecting the other coil to a use of electrical potential; and (e) a plurality of optical transceivers means mounted at opposing positions on the axis of the hollow, rotating core means, transmitting and receiving information to and from the stationary and rotating parts on a modulated beam of light passing through and along the axis of the hollow, rotative core means.
In the description which follows, the terms stationary and rotating are relative and are used to describe the relationship among the parts of the apparatus in the circumstances being described. In other instances, the stationary part could be rotating and the rotating part could be stationary relative to each other or to the earth or to other objects.
It has often been necessary in the design and construction of apparatus in the past, to provide a way of transmitting electrical power from a stationary part to a rotating part. One means of accomplishing this has been the use of slip rings. In these devices, a conductor terminal on the rotating part slides on a conductor terminal of the stationary part. While still used in some instances, this technique has disadvantages such as friction, and noise both mechanical and electrical.
The alternative use of rotating transformer-type couplings is well developed as exemplified by U.S. Pat. No. 3,535,618. In this type of coupling, a coil is mounted on a stationary member and another coil is mounted on a rotating member. A magnetic core is associated with each coil and the magnetic flux from one coil is linked to the other coil by passing through an air gap separating the two coils. As the air gap increases in width, the mutual degree of flux linking becomes less which is sometimes a problem.
U.S. Pat. No. 3,317,873 is another example of a rotary transformer in which power is transmitted from a stationary part to a rotating part.
It is also often necessary in the design and construction of apparatus that electrical signals be transmitted from a rotating part to a stationary part. These electrical signals may be used in measurement of certain quantities on the rotating elements or they may be used to control equipment on the rotating parts. For example, the temperature and stress in a rotating element such as the rotor of an electric motor have been measured using systems which transfer informational signals from the rotor apparatus to the stationary recording apparatus. The well known way of accomplishing this is by the use of slip rings, but again, in this situation, the slip rings frequently introduce electrical contact noise, friction and heating problems.
In present state-of-the-art communication systems in which information is transmitted at rates of of 100K to 50 M bits per second, the use of slip rings is not a satisfactory means.
Examples of signal coupling devices between stationary and rotating parts are to be found in U.S. Pat. Nos. 2,894,231 and 3,268,880.
In the field of Robotics, the situation is frequently encountered where it is necessary to transmit power and informational signal through a "joint" where one part is stationary and the other is rotating relative thereto. All of the parameters are critically limited in this situation, and a rotative, conductive coupling with electrical signal transmitting capability is needed which incorporates the maximum electrical power transmission in the smallest possible size package, combined with maximum informational signal transmission capability in the smallest size package. The signal must be transmitted free of distortion and at maximum data rates.
The rotative inductive and optical coupling of this invention combines the desired features of large power transmission capability, large signal communication capability, with compact size and weight.
It is therefore an object of the present invention to couple high current flows with efficiency. It is another object to accomplish this with lightweight and small size apparatus.
It is still another object of this invention to communicate signals at a high rate, clear of noise and interference, between a rotative and a stationary part. It is a further object to accomplish this with lightweight and small size apparatus.
To achieve the efficiency objective in the transmission of power in this invention, a flux path of low reluctance is provided. This is accomplished by the provision of a small gap between one core element or means relative to the other. Another object is to improve the size and weight characteristics of the power transmission portion of the apparatus by providing for a high frequency current.
The foreging and other advantages of the invention will become apparent from the following disclosure in which a preferred embodiment of the invention is described in detail and illustrated in the accompanying drawing. It is contemplated that variations in structural features and arrangement of parts may appear to the person skilled in the art, without departing from the scope or sacrificing any of the advantages of the invention.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is an elevational, section view of the electrical coupling of the present invention shown with respect to the rotating and stationary parts.
FIG. 2 is a sectional, plan view of the coupling taken on the line 2 of FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
Referring to FIGS. 1 and 2, a coupling 20 comprises a rotating part 21 and a stationary part 22. Rotation between parts 21 and 22 is provided by an upper bearing 23 and a lower bearing 24, shown as ball bearings. The parts 21 and 22 are generally circular in cross-section about a longitudinal axis of rotation 19.
The rotating part 21 includes a frame member 35 which supports and surrounds a hollow, rotative core means 36 which also serves as a shaft of rotation for the stationary part 22. An optical transceiver means 37 is mounted in the end of the frame member 35. The end may be enclosed in a cover 38. The transceiver means 37 is provided with one or more signal output leads 39.
An annular, rotative coil 40 is mounted on and in contact with the rotative core means 36. AC voltage power output leads 41 are connected from the rotative coil 40 through an aperture to the inside of the hollow, rotative core means 36 where they may pass through grooves 42 and out through the end of the frame member 35, and the cover 38.
Stationary part 22 includes a main frame 25. The main frame 25 supports an outer stationary core 26 as by bracket means 27 at each end. An optical transceiver means 28 is provided in a housing 29. The optical transceiver means 28 is connected to one or more signal input leads 30.
An annular, stationary coil 45 encircles the rotative core means 36 in close proximity thereto. The stationary core 45 may be supported within the outer core 26 by a bracket means 46. AC voltage power input leads 47 are connected to the ends of coil 45. The hollow, rotative core means 36 passes through a closely separated matching hole in the outer stationary core 26 at an upper end 50 and a lower end 51. Only a very small gap 49 is provided between the core means 26 and 36. A magnetically susceptible lubricant 52 may be interposed in the gap 49.
By way of example, rotative motion may be imparted to part 21 by means of a V-belt 55 running in a groove of a pulley 56 (shown in cross-section). The means of imparting rotation to the rotating part 21 is not an important part of this invention and this may be accomplished by various conventional means.
In the operation and use of this invention, rotative part 21 rotates in and relative to the part 22 by means of the bearings 23 and 24. While rotation takes place, and independently thereof, electrical power in the form of an alternating voltage (a potential) is applied to the coil 45 through the leads 47. This creates an inductive, magnetic field passing through the rotating core means 36 and the outer stationary core 26, generally as indicated by the dashed lines of flux, shown in FIG. 1. The magnetic field induces an alternating current input through the leads 41.
Since the power coupling feature of this invention is in many ways a rotary transformer, many of the design techniques for the increased efficiency of transformers may be applied to this invention. High current flows such as 10 amperes may be coupled with reasonable efficiency. This can be accomplished in a very small size unit with relatively light weight. Because of the manner and configuration of construction, a flux path of low reluctance couples the stationary coil 45 (primary) and the rotative coil 40 (secondary). The gap in the flux path in the construction of the rotative transformer is reduced to an absolute minimum and may be eliminated with the proper selection of a magnetically susceptible lubricant 52.
As a further factor in the reduced size and weight characteristics of the transformer, the frequency of the power signal may be optimized at a much higher rate than the usual 60 Hz in common use. At the higher frequencies, the need for elimination of power losses due to eddy currents may be met by the selection of laminated or powdered ferrite cores which provide low reluctance to magnetic flux. Because of the compactness of the construction of this invention, the inner rotative core means 36 and the outer stationary core 26 may be readily fabricated of powdered ferrites or amorphous silicon steel. These materials may be easily manufactured using powder metallurgy techniques to very close tolerances, and a desirable gap between the ends 50, 51 and the rotating core 36 of 0.001 inches can be maintained with little or no machining.
During rotation of the part 21, signals in the form of a modulated light beam are transmitted and received between the optical transceivers 28 and 37. The transceivers 28 and 37 are positioned so that the center of the beam of light coincides with the axis of rotation 19 of the part 21 in the stationary part 22. This positioning essentially eliminates any need for synchronization between the frequencies or rate of rotation of the rotating part 21 with the frequencies in the signal communication system. The frequency of the information transmission is unlimited. There is no noise or interference in the signal as a result of the mechanics of the rotational aspects of the apparatus.
This is very helpful and a significant improvement in rotative coupling apparatus. In telemetry and robotics where signal frequencies are desirably in the range of 5×10 6 Hz and information communication rates are desirably in the range of 10×10 5 bits per sec, this separation of the electronics from interrelation with the mechanics of the system brings about a new freedom of choices.
Prior rotative couplings using some form of mechanical signal connectors, such as slip rings could not be used in signal communications at the above described rates.
The art of transmitting information signals on light beams is well developed and the apparatus and systems necessary to carry out transmission along the centerline of the coupling are well within understanding of those skilled in art.
In a typical use of the invention a rotative part 21 would turn at <3,000 rpm relative to the stationary part 22 driven by belt 55 through pulley 56. A VAC potential of 220 volts at a frequency of 60 Hz is applied at the VAC INPUT.
A stationary coil 45 of N turns is supported in the stationary core 26 which is constructed of pressed powdered ferrite. A gap 49 of 0.001 inch separates the stationary core 26 from the rotative core 36. The gap 49 is filled with a magnetically susceptible lubricant 52. A rotative coil 40 of N turns is wound on the rotative core 36. A VAC potential of 220 volts is available at the VAC OUTPUT.
On the stationary part 22 an optical transceiver 28 in transmitting mode provides signals on a light beam of 0.001 watts modulating at 5×10 6 Hz to an optical transceiver 37 in receiving mode on the rotating part 21.
It is herein understood that although the present invention has been specifically disclosed with the preferred embodiments and examples, modification and variations of the concepts herein disclosed may be resorted to by those skilled in the art. Such modifications and variations are considered to be within the scope of the invention and the appended claims.
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A rotative coupling allows the inductive transmission of power and the transmission of informational signals between a stationary part and a hollow, rotating part. The informational signals are transmitted on a modulated beam of light passing through and along the axis of rotation of the hollow, rotative part. The power is transmitted by induction from a coil on the stationary part to a coil on the rotative part, both coils being positioned within stationary core means and the rotative core means, which combine to establish a continuous flux path around the coils.
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CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. patent application Ser. No. 10/330,853 filed Dec. 26, 2002, the disclosure of which is incorporated by reference in its entirety.
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
[[0002]] This invention was made in part with Government support under a grant from the National Science Foundation (Cooperative Agreement No. DMR-980677). Accordingly, the Government may have certain rights to this invention.
TECHNICAL FIELD
[0003] This invention relates generally to the depolymerization of polymers, and, more particularly relates to an organocatalytic method for depolymerizing polymers using nucleophilic reagents. The invention is applicable in numerous fields, including industrial chemistry and chemical waste disposal, plastics recycling, and manufacturing processes requiring a simple and convenient method for the degradation of polymers.
BACKGROUND OF THE INVENTION
[0004] Technological advances of all kinds continue to present many complex ecological issues. Consequently, waste management and pollution prevention are two very significant challenges of the 21 st century. The overwhelming quantity of plastic refuse has significantly contributed to the critical shortage of landfill space faced by many communities. For example, poly(ethylene terephthalate) (poly(oxy-1,2-ethanediyl-oxycarbonyl-1,4-diphenylenecarbonyl); “PET”), a widely used engineering thermoplastic for carpeting, clothing, tire cords, soda bottles and other containers, film, automotive applications, electronics, displays, etc. will contribute more than 1 billion pounds of waste to land-fills in 2002 alone. The worldwide production of PET has been growing at an annual rate of 10% per year, and with the increase in use in electronic and automotive applications, this rate is expected to increase significantly to 15% per year. Interestingly, the precursor monomers represent only about 2% of the petrochemical stream. Moreover, the proliferation of the use of organic solvents, halogenated solvents, water, and energy consumption in addressing the need to recycle commodity polymers such as PET and other polyesters has created the need for environmentally responsible and energy efficient recycling processes. See Nadkarni (1999) International Fiber Journal 14(3).
[0005] Significant effort has been invested in researching recycling strategies for PET, and these efforts have produced three commercial options; mechanical, chemical and energy recycling. Energy recycling simply burns the plastic for its calorific content. Mechanical recycling, the most widespread approach, involves grinding the polymer to powder, which is then mixed with “virgin” PET. See Mancini et al. (1999) Materials Research 2(1):33-38. Many chemical companies use this process in order to recycle PET at the rate of approximately 50,000 tons/year per plant. In Europe, all new packaging materials as of 2002 must contain 15% recycled material. However, it has been demonstrated that successive recycling steps cause significant polymer degradation, in turn resulting in a loss of desirable mechanical properties. Recycling using chemical degradation involves a process that depolymerizes a polymer to starting material, or at least to relative short oligomeric components. Clearly, this process is most desirable, but is the most difficult to control since elevated temperature and pressure are required along with a catalyst composed of a strong base, or an organometallic complex such as an organic titanate. See Sako et al. (1997) Proc. of the 4 th Int'l Symposium on Supercritical Fluids , pp. 107-110. The use of such a catalyst results in significant quantities of undesirable byproducts, and materials processed by these methods are thus generally unsuitable for use in medical materials or food packaging, limiting their utility. Moreover, the energy required to effect depolymerization essentially eliminates sustainability arguments.
[0006] Accordingly, there is a need in the art for an improved depolymerization method. Ideally, such a method would not involve extreme reaction conditions, use of metallic catalysts, or a process that results in significant quantities of potentially problematic by-products.
SUMMARY OF THE INVENTION
[0007] The invention is directed to the aforementioned need in the art, and, as such, provides an efficient catalytic depolymerization reaction that employs mild conditions, wherein production of undesirable byproducts resulting from polymer degradation is minimized. The reaction can be carried out at temperatures of at most 80° C., and, because a nonmetallic catalyst is preferably employed, the depolymerization products, in a preferred embodiment, are substantially free of metal contaminants. With many of the carbene catalysts disclosed herein, the depolymerization reaction can be carried out at a temperature of at most 60° C. or even 30° C. or lower, i.e., at room temperature.
[0008] More specifically, in one aspect of the invention, a method is provided for depolymerizing a polymer having a backbone containing electrophilic linkages, wherein the method involves contacting the polymer with a nucleophilic reagent and a catalyst at a temperature of less than 80° C. An important application of this method is in the depolymerization of polyesters, including homopolymeric polyesters (in which all of the electrophilic linkages are ester linkages) and polyester copolymers (in which a fraction of the electrophilic linkages are ester linkages and the remainder of the electrophilic linkages are other than ester linkages).
[0009] In a related aspect of the invention, a method is provided for depolymerizing a polymer having a backbone containing electrophilic linkages, wherein the method involves contacting the polymer with a nucleophilic reagent and a catalyst that yields depolymerization products that are substantially free of metal contaminants. The polymer may be, for example, a polyester, a polycarbonate, a polyurethane, or a related polymer, in either homopolymeric or copolymeric form, as indicated above. In this embodiment, in order to provide reaction products that are substantially free of contamination by metals and metal-containing compounds, the catalyst used is a purely organic, nonmetallic catalyst. Preferred catalysts herein are carbene compounds, which act as nucleophilic catalysts, as well as precursors to carbene compounds, as will be discussed infra. As is well understood in the art, carbenes are electronically neutral compounds containing a divalent carbon atom with only six electrons in its valence shell. Carbenes include, by way of example, cyclic diaminocarbenes, imidazol-2-ylidenes (e.g., 1,3-dimesityl-imidazol-2-ylidene and 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene), 1,2,4-triazol-3-ylidenes, and 1,3-thiazol-2-ylidenes; see Bourissou et al. (2000) Chem. Rev. 100:39-91.
[0010] Since the initial description of the synthesis, isolation, and characterization of stable carbenes by Arduengo (Arduengo et al. (1991) J. Am. Chem. Soc. 113:361; Arduengo et al. (1992) J. Am. Chem. Soc. 114:5530), the exploration of their chemical reactivity has become a major area of research. See, e.g., Arduengo et al. (1999) Acc. Chem. Res. 32:913; Bourissou et al. (2000), supra; and Brode (1995) Angew. Chem. Int. Ed. Eng. 34:1021. Although carbenes have now been extensively investigated, and have in fact been established as useful in many synthetically important reactions, there has been no disclosure or suggestion to use carbenes as catalysts in nucleophilic depolymerization reactions, i.e., reactions in which a polymer containing electrophilic linkages is depolymerized with a nucleophilic reagent in the presence of a carbene catalyst.
[0011] Suitable catalysts for use herein thus include heteroatom-stabilized carbenes or precursors to such carbenes. The heteroatom-stabilized carbenes have the structure of formula (I)
wherein:
E 1 and E 2 are independently selected from N, NR E , O, P, PR E , and S, R E is hydrogen, heteroalkyl, or heteroaryl, x and y are independently zero, 1, or 2, and are selected to correspond to the valence state of E 1 and E 2 , respectively, and wherein when E 1 and E 2 are other than O or S, then E 1 and E 2 may be linked through a linking moiety that provides a heterocyclic ring in which E 1 and E 2 are incorporated as heteroatoms; R 1 and R 2 are independently selected from branched C 3 -C 30 hydrocarbyl, substituted branched C 3 -C 30 hydrocarbyl, heteroatom-containing branched C 4 -C 30 hydrocarbyl, substituted heteroatom-containing branched C 4 -C 30 hydrocarbyl, cyclic C 5 -C 30 hydrocarbyl, substituted cyclic C 5 -C 30 hydrocarbyl, heteroatom-containing cyclic C 1 -C 30 hydrocarbyl, and substituted heteroatom-containing cyclic C 1 -C 30 hydrocarbyl; L 1 and L 2 are linkers containing 1 to 6 spacer atoms, and are independently selected from heteroatoms, substituted heteroatoms, hydrocarbylene, substituted hydrocarbylene, heteroatom-containing hydrocarbylene, and substituted heteroatom-containing hydrocarbylene; and m and n are independently zero or I, such that L 1 and L 2 are optional.
[0017] Certain carbene catalysts of formula (I) are novel chemical compounds and are claimed as such herein. These novel carbenes are those wherein a heteroatom is directly bound to E 1 and/or E 2 , and include, solely by way of example, carbenes of formula (I) wherein E 1 is NR E and R E is a heteroalkyl or heteroaryl group such as an alkoxy, alkylthio, aryloxy, arylthio, aralkoxy, or aralkylthio moiety.
[0018] Carbene precursors suitable as catalysts herein include tri-substituted methanes having the structure of formula (PI), metal adducts having the structure of formula (PII), and tetrasubstituted olefins having the structure (PIII)
wherein, in formulae (PI) and (PII):
E 1 and E 2 are independently selected from N, NR E , O, P, PR E , and S, R E is hydrogen, heteroalkyl, or heteroaryl, x and y are independently zero, 1, or 2, and are selected to correspond to the valence state of E 1 and E 2 , respectively, and wherein when E 1 and E 2 are other than O or S, then E 1 and E 2 may be linked through a linking moiety that provides a heterocyclic ring in which E 1 and E 2 are incorporated as heteroatoms; R 1 and R 2 are independently selected from branched C 3 -C 30 hydrocarbyl, substituted branched C 3 -C 30 hydrocarbyl, heteroatom-containing branched C 4 -C 30 hydrocarbyl, substituted heteroatom-containing branched C 4 -C 30 hydrocarbyl, cyclic C 5 -C 30 hydrocarbyl, substituted cyclic C 5 -C 30 hydrocarbyl, heteroatom-containing cyclic C 1 -C 30 hydrocarbyl, and substituted heteroatom-containing cyclic C 1 -C 30 hydrocarbyl; L 1 and L 2 are linkers containing 1 to 6 spacer atoms, and are independently selected from heteroatoms, substituted heteroatoms, hydrocarbylene, substituted hydrocarbylene, heteroatom-containing hydrocarbylene, and substituted heteroatom-containing hydrocarbylene; m and n are independently zero or 1; R 7 is selected from alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, substituted with at least one electron-withdrawing substituent; M is a metal; Ln is a ligand; and j is the number of ligands bound to M.
[0028] In compounds of formula (PIII), the substituents are as follows:
E 3 and E 4 are defined as for E 1 and E 2 ; v and w are defined as for x and y; R 8 and R 9 are defined as for R 1 and R 2 ; L 3 and L 4 are defined as for L 1 and L 2 ; and h and k are defined as for m and n.
[0034] The carbene precursors may be in the form of a salt, in which case the precursor is positively charged and associated with an anionic counterion, such as a halide ion (I, Br, Cl), a hexafluorophosphate anion, or the like.
[0035] Novel carbene precursors herein include compounds of formula (PI), those compounds of formula (PII) in which a heteroatom is directly bound to E 1 and/or E 2 , and those compounds of formula (PIII) in which a heteroatom is directly bound to at least one of E 1 , E 2 , E 3 , and E 4 , and may be in the form of a salt as noted above.
[0036] Ideally, the carbene catalyst used in conjunction with the present depolymerization reaction is an N-heterocyclic carbene having the structure of formula (II)
wherein:
R 1 , R 2 , L 1 , L 2 , m, and n are as defined above; and L is a hydrocarbylene, substituted hydrocarbylene, heteroatom-containing hydrocarbylene, or substituted heteroatom-containing hydrocarbylene linker, wherein two or more substituents on adjacent atoms within L may be linked to form an additional cyclic group.
[0040] As alluded to above, one important application of the present invention is in the recycling of polyesters, including, by way of illustration and not limitation: PET; poly (butylene terephthalate) (PBT); poly(alkylene adipate)s and their copolymers; and poly(ε-caprolactone). The methodology of the invention provides an efficient means to degrade such polymers into their component monomers and/or relatively short oligomeric fragments without need for extreme reaction conditions or metallic catalysts.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 illustrates the organocatalytic depolymerization of PET in the presence of excess methanol using N-heterocyclic carbene catalyst, as evaluated in Example 7.
[0042] FIG. 2 illustrates the organocatalytic depolymerization of PET in the presence of ethylene glycol using N-heterocyclic carbene catalyst, as evaluated in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Unless otherwise indicated, this invention is not limited to specific polymers, carbene catalysts, nucleophilic reagents, or depolymerization conditions. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
[0044] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polymer” encompasses a combination or mixture of different polymers as well as a single polymer, reference to “a catalyst” encompasses both a single catalyst as well as two or more catalysts used in combination, and the like.
[0045] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:
[0046] As used herein, the phrase “having the formula” or “having the structure” is not intended to be limiting and is used in the same way that the term “comprising” is commonly used.
[0047] The term “alkyl” as used herein refers to a linear, branched, or cyclic saturated hydrocarbon group typically although not necessarily containing 1 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although again not necessarily, alkyl groups herein contain 1 to about 12 carbon atoms. The term “lower alkyl” intends an alkyl group of 1 to 6 carbon atoms, and the specific term “cycloalkyl” intends a cyclic alkyl group, typically having 4 to 8, preferably 5 to 7, carbon atoms. The term “substituted alkyl” refers to alkyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl and lower alkyl, respectively.
[0048] The term “alkylene” as used herein refers to a difunctional linear, branched, or cyclic alkyl group, where “alkyl” is as defined above.
[0049] The term “alkenyl” as used herein refers to a linear, branched, or cyclic hydrocarbon group of 2 to about 20 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, cicosenyl, tetracosenyl, and the like. Preferred alkenyl groups herein contain 2 to about 12 carbon atoms. The term “lower alkenyl” intends an alkenyl group of 2 to 6 carbon atoms, and the specific term “cycloalkenyl” intends a cyclic alkenyl group, preferably having 5 to 8 carbon atoms. The term “substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkenyl” and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
[0050] The term “alkenylene” as used herein refers to a difunctional linear, branched, or cyclic alkenyl group, where “alkenyl” is as defined above.
[0051] The term “alkoxy” as used herein refers to a group —O-alkyl wherein “alkyl” is as defined above, and the term “alkylthio” as used herein refers to a group —S-alkyl wherein “alkyl is as defined above.
[0052] The term “aryl” as used herein, and unless otherwise specified, refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Preferred aryl groups contain 5 to 20 carbon atoms and either one aromatic ring or 2 to 4 fused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl, and the like, with more preferred aryl groups containing 1 to 3 aromatic rings, and particularly preferred aryl groups containing 1 or 2 aromatic rings and 5 to 14 carbon atoms. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the terms “heteroatom-containing aryl” and “heteroaryl” refer to aryl in which at least one carbon atom is replaced with a heteroatom. Unless otherwise indicated, the terms “aromatic,” “aryl,” and “arylene” include heteroaromatic, substituted aromatic, and substituted heteroaromatic species.
[0053] The term “aryloxy” refers to a group —O-aryl wherein “aryl” is as defined above.
[0054] The term “alkaryl” refers to an aryl group with at least one and typically 1 to 6 alkyl, preferably 1 to 3, alkyl substituents, and the term “aralkyl” refers to an alkyl group with an aryl substituent, wherein “aryl” and “alkyl” are as defined above. Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl, 2,4,6-trimethylphenyl, and the like. The term “aralkyl” refers to an alkyl group substituted with an aryl moiety, wherein “alkyl” and “aryl” are as defined above.
[0055] The term “alkaryloxy” refers to a group —O—R wherein R is alkaryl, the term “alkarylthio” refers to a group —S—R wherein R is alkaryl, the term aralkoxy refers to a group —O—R wherein R is aralkyl, the term “aralkylthio” refers to a group —S—R wherein R is aralkyl.
[0056] The terms “halo,” “halide,” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro, or iodo substituent. The terms “haloalkyl,” “haloalkenyl,” and “haloalkynyl” (or “halogenated alkyl,” “halogenated alkenyl,” and “halogenated alkynyl”) refer to an alkyl, alkenyl, or alkynyl group, respectively, in which at least one of the hydrogen atoms in the group has been replaced with a halogen atom.
[0057] “Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, preferably 1 to about 20 carbon atoms, more preferably 1 to about 12 carbon atoms, including linear, branched, cyclic, saturated, and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, alkaryl groups, and the like. The term “lower hydrocarbyl” intends a hydrocarbyl group of 1 to 6 carbon atoms, and the term “hydrocarbylene” intends a divalent hydrocarbyl moiety containing 1 to about 30 carbon atoms, preferably 1 to about 20 carbon atoms, most preferably 1 to about 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species. The term “lower hydrocarbylene” intends a hydrocarbylene group of 1 to 6 carbon atoms. Unless otherwise indicated, the terms “hydrocarbyl” and “hydrocarbylene” are to be interpreted as including substituted and/or heteroatom-containing hydrocarbyl and hydrocarbylene moieties, respectively.
[0058] The term “heteroatom-containing” as in a “heteroatom-containing alkyl group” (also termed a “heteroalkyl” group) or a “heteroatom-containing aryl group” (also termed a “heteroaryl” group) refers to a molecule, linkage, or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly, the term “heteroalkyl” refers to an alkyl substituent that is heteroatom-containing, the term “heterocyclic” refers to a cyclic substituent that is heteroatom-containing, the terms “heteroaryl” and heteroaromatic” respectively refer to “aryl” and “aromatic” substituents that are heteroatom-containing, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, etc. It should be noted that a “heterocyclic” group or compound may or may not be aromatic, and further that “heterocycles” may be monocyclic, bicyclic, or polycyclic as described above with respect to the term “aryl.”
[0059] By “substituted” as in “substituted hydrocarbyl,” “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with a non-hydrogen substituent. Examples of such substituents include, without limitation, functional groups such as halide, hydroxyl, sulfhydryl, C 1 -C 20 alkoxy, C 5 -C 20 aryloxy, C 2 -C 20 acyl (including C 2 -C 20 alkylcarbonyl (—CO-alkyl) and C 6 -C 20 arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C 2 -C 20 alkoxycarbonyl (—(CO)-O-alkyl), C 6 -C 20 aryloxycarbonyl (—(CO)-O-aryl), halocarbonyl (—CO)—X where X is halo), C 2 -C 20 alkyl-carbonato (—O—(CO)—O-alkyl), C 6 -C 20 arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO − ), carbamoyl (—(CO)—NH 2 ), mono-(C 1 -C 20 alkyl)-substituted carbamoyl (—(CO)—NH(C 1 -C 20 alkyl)), di-(C 1 -C 20 alkyl)-substituted carbamoyl —(CO)—N(C 1 -C 20 alkyl) 2 ), mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH 2 ), carbamido (—NH—(CO)—NH 2 ), cyano(—C≡N), cyanato (—O—C≡N), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH 2 ), mono- and di-(C 1 -C 20 alkyl)-substituted amino, mono- and di-(C 5 -C 20 aryl)-substituted amino, C 2 -C 20 alkylamido (—NH—(CO)-alkyl), C 6 -C 20 arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C 1 -C 20 alkyl, C 5 -C 20 aryl, C 6 -C 24 alkaryl, C 6 -C 24 aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO 2 ), nitroso (—NO), sulfo (—SO 2 —OH), sulfonato (—SO 2 —O − ), C 1 -C 20 alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”), C 1 -C 20 alkylsulfinyl (—(SO)-alkyl), C 5 -C 20 arylsulfinyl (—(SO)-aryl), C 1 -C 20 alkylsulfonyl (—SO 2 -alkyl), C 5 -C 20 arylsulfonyl (—SO 2 -aryl), and thiocarbonyl (═S); and the hydrocarbyl moieties C 1 -C 20 alkyl (preferably C 1 -C 18 alkyl, more preferably C 1 -C 12 alkyl, most preferably C 1 -C 6 alkyl), C 2 -C 20 alkenyl (preferably C 2 -C 18 alkenyl, more preferably C 2 -C 12 alkenyl, most preferably C 2 -C 6 alkenyl), C 2 -C 20 alkynyl (preferably C 2 -C 18 alkynyl, more preferably C 2 -C 12 alkynyl, most preferably C 2 -C 6 alkynyl), C 5 -C 20 aryl (preferably C 5 -C 14 aryl), C 6 -C 24 alkaryl (preferably C 6 -C 18 alkaryl), and C 6 -C 24 aralkyl (preferably C 6 -C 18 aralkyl).
[0060] In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.
[0061] By “substantially free of” a particular type of chemical compound is meant that a composition or product contains less 10 wt. % of that chemical compound, preferably less than 5 wt. %, more preferably less than 1 wt. %, and most preferably less than 0.1 wt. %. For instance, the depolymerization product herein is “substantially free of” metal contaminants, including metals per se, metal salts, metallic complexes, metal alloys, and organometallic compounds.
[0062] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. For example, the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
[0063] Accordingly, the invention features a method for depolymerizing a polymer having a backbone containing electrophilic linkages. The electrophilic linkages may be, for example, ester linkages (—(CO)—O—), carbonate linkages (—O—(CO)—O)—, urethane linkages (—O—(CO)—NH), substituted urethane linkages (—O—(CO)—NR—, where R is a nonhydrogen substituent such as alkyl, aryl, alkaryl, or the like), amido linkages (—(CO)—NH—), substituted amido linkages (—(CO)—NR— where R is as defined previously, thioester linkages (—(CO)—S—), sulfonic ester linkages (—S(O) 2 —O—), and the like. Other electrophilic linkages that can be cleaved using nucleophilic reagents will be known to those of ordinary skill in the art of organic chemistry and polymer science and/or can be readily found by reference to the pertinent texts and literature. The polymer undergoing depolymerization may be linear or branched, and may be a homopolymer or copolymer, the latter including random, block, multiblock, and alternating copolymers, terpolymers, and the like. Examples of polymers that can be depolymerized using the methodology of the invention include, without limitation:
poly(alkylene terephthalates) such as fiber-grade PET (a homopolymer made from monoethylene glycol and terephthalic acid), bottle-grade PET (a copolymer made based on monoethylene glycol, terephthalic acid, and other comonomers such as isophthalic acid, cyclohexene dimethanol, etc.), poly (butylene terephthalate) (PBT), and poly(hexamethylene terephthalate); poly(alkylene adipates) such as poly(ethylene adipate), poly(1,4-butylene adipate), and poly(hexamethylene adipate); poly(alkylene suberates) such as poly(ethylene suberate); poly(alkylene sebacates) such as poly(ethylene sebacate); poly(c-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalates) such as poly(ethylene isophthalate); poly(alkylene 2,6-naphthalene-dicarboxylates) such as poly(ethylene 2,6-naphthalene-dicarboxylate); poly(alkylene sulfonyl-4,4′-dibenzoates) such as poly(ethylene sulfonyl-4,4′-dibenzoate); poly(p-phenylene alkylene dicarboxylates) such as poly(p-phenylene ethylene dicarboxylates); poly(trans-1,4-cyclohexanediyl alkylene dicarboxylates) such as poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate); poly(1,4-cyclohexane-dimethylene alkylene dicarboxylates) such as poly(1,4-cyclohexane-dimethylene ethylene dicarboxylate); poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylates) such as poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate); lactic acid polymers and copolymers such as (S)-polylactide, (R,S)-polylactide, poly(tetramethylglycolide), and poly(lactide-co-glycolide); and polycarbonates of bisphenol A, 3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A, 3,3′,5,5′-tetramethylbisphenol A; polyamides such as poly(p-phenylene terephthalamide) (Kevlaro); poly(alkylene carbonates) such as poly(propylene carbonate); polyurethanes such as those available under the tradenames Baytece and Bayfil®, from Bayer Corporation; and polyurethane/polyester copolymers such as that available under the tradename Bayda®, from Bayer Corporation.
[0082] Depolymerization of the polymer is carried out, as indicated above, in the presence of a nucleophilic reagent and a catalyst. Nucleophilic reagents, as will be appreciated by those of ordinary skill in the art, include monohydric alcohols, diols, polyols, thiols, primary amines, and the like, and may contain a single nucleophilic moiety or two or more nucleophilic moieties, e.g., hydroxyl, sulfhydryl, and/or amino groups. The nucleophilic reagent is selected to correspond to the particular electrophilic linkages in the polymer backbone, such that nucleophilic attack at the electrophilic linkage results in cleavage of the linkage. For example, a polyester can be cleaved at the ester linkages within the polymer backbone using an alcohol, preferably a primary alcohol, most preferably a C 2 -C 4 monohydric alcohol such as ethanol, isopropanol, and t-butyl alcohol. It will be appreciated that such a reaction cleaves the ester linkages via a transesterification reaction, as will be illustrated infra.
[0083] The preferred catalysts for the depolymerization reaction are carbenes and carbene precursors. Carbenes include, for instance, diarylcarbenes, cyclic diaminocarbenes, imidazol-2-ylidenes, 1,2,4-triazol-3-ylidenes, 1,3-thiazol-2-ylidenes, acyclic diaminocarbenes, acyclic aminooxycarbenes, acyclic aminothiocarbenes, cyclic diborylcarbenes, acyclic diborylcarbenes, phosphinosilylcarbenes, phosphinophosphoniocarbenes, sulfenyl-trifluoromethylcarbene, and sulfenyl-pentafluorothiocarbene. See Bourissou et al. (2000), cited supra. Preferred carbenes are heteroatom-stabilized carbenes and preferred carbene precursors are precursors to heteroatom-stabilized carbenes. nitrogen-containing carbenes, with N-heterocyclic carbenes most preferred.
[0084] In one embodiment, heteroatom-stabilized carbenes suitable as depolymerization catalysts herein have the structure of formula (I)
wherein the various substituents are as follows:
[0086] E 1 and E 2 are independently selected from N, NR E , O, P, PR E , and S, R E is hydrogen, heteroalkyl, or heteroaryl, and x and y are independently zero, 1, or 2, and are selected to correspond to the valence state of E 1 and E 2 , respectively. When E 1 and E 2 are other than O or S, then E 1 and E 2 may be linked through a linking moiety that provides a heterocyclic ring in which E 1 and E 2 are incorporated as heteroatoms. In the latter case, the heterocyclic ring may be aliphatic or aromatic, and may contain substituents and/or heteroatoms. Generally, such a cyclic group will contain 5 or 6 ring atoms.
[0087] For example, in representative compounds of formula (I):
(1) E 1 is O or S and x is 1; (2) E 1 is N, x is 1, and E 1 is linked to E 2 ; (3) E 1 is N, x is 2, and E 1 and E 2 are not linked; (4) E 1 is NR E , x is 1, and E 1 and E 2 are not linked; or (5) E 1 is NR E , x is zero, and E 1 is linked to E 2 .
[0093] R 1 and R 2 are independently selected from branched C 3 -C 30 hydrocarbyl, substituted branched C 3 -C 30 hydrocarbyl, heteroatom-containing branched C 4 -C 30 hydrocarbyl, substituted heteroatom-containing branched C 4 -C 30 hydrocarbyl, cyclic C 5 -C 30 hydrocarbyl, substituted cyclic C 5 -C 30 hydrocarbyl, heteroatom-containing cyclic C 1 -C 30 hydrocarbyl, and substituted heteroatom-containing cyclic C 1 -C 30 hydrocarbyl. Preferably, at least one of R 1 and R 2 , and more preferably both R 1 and R 2 , are relatively bulky groups, particularly branched alkyl (including substituted and/or heteroatom-containing alkyl), aryl (including substituted aryl, heteroaryl, and substituted heteroaryl), alkaryl (including substituted and/or heteroatom-containing aralkyl), and alicyclic. Using such sterically bulky groups to protect the highly reactive carbene center has been found to kinetically stabilize singlet carbenes, which are preferred reaction catalysts herein. Particular sterically bulky groups that are suitable as R 1 and R 2 are optionally substituted and/or heteroatom-containing C 3 -C 12 alkyl, tertiary C 4 -C 12 alkyl, C 5 -C 12 aryl, C 6 -C 18 alkaryl, and C 5 -C 12 alicyclic, with C 5 -C 12 aryl and C 6 -C 12 alkaryl particularly preferred. The latter substituents are exemplified by phenyl optionally substituted with 1 to 3 substituents selected from lower alkyl, lower alkoxy, and halogen, and thus include, for example, p-methylphenyl, 2,6-dimethylphenyl, and 2,4,6-trimethylphenyl (mesityl).
[0094] L 1 and L 2 are linkers containing 1 to 6 spacer atoms, and are independently selected from heteroatoms, substituted heteroatoms, hydrocarbylene, substituted hydrocarbylene, heteroatom-containing hydrocarbylene, and substituted heteroatom-containing hydrocarbylene; and m and n are independently zero or 1, meaning that each of L 1 and L 2 is optional. Preferred L 1 and L 2 moieties include, by way of example, alkylene, alkenylene, arylene, aralkylene, any of which may be heteroatom-containing and/or substituted, or L 1 and/or L 2 may be a heteroatom such as O or S, or a substituted heteroatom such as NH, NR (where R is alkyl, aryl, other hydrocarbyl, etc.), or PR; and
[0095] In one preferred embodiment, E 1 and E 2 are independently N or NR E and are not linked, such that the carbene is an N-heteroacyclic carbene. In another preferred embodiment, E 1 and E 2 are N, x and y are 1, and E 1 and E 2 are linked through a linking moiety such that the carbene is an N-heterocyclic carbene. N-heterocyclic carbenes suitable herein include, without limitation, compounds having the structure of formula (II)
wherein R 1 , R 2 , L 1 , L 2 , m, and n are as defined above for carbenes of formula (I). In carbenes of structural formula (II), L is a hydrocarbylene, substituted hydrocarbylene, heteroatom-containing hydrocarbylene, or substituted heteroatom-containing hydrocarbylene linker, wherein two or more substituents on adjacent atoms within L may be linked to form an additional cyclic group. L is a hydrocarbylene, substituted hydrocarbylene, heteroatom-containing hydrocarbylene, or substituted heteroatom-containing hydrocarbylene linker, wherein two or more substituents on adjacent atoms within L may be linked to form an additional cyclic group. For example, L may be —CR 3 R 4 —CR 5 R 6 — or —CR 3 ═CR 5 —, wherein R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, halogen, C 1 -C 12 alkyl, or wherein any two of R 3 , R 4 , R 5 , and R 6 may be linked together to form a substituted or unsubstituted, saturated or unsaturated ring.
[0097] Accordingly, when L is —CR 3 R 4 —CR 5 R 6 — or —CR 3 ═CR 5 —, the carbene has the structure of formula (II)
in which q is an optional double bond, s is zero or 1, and t is zero or 1, with the proviso that when q is present, s and t are zero, and when q is absent, s and t are 1.
[0099] Certain carbenes are new chemical compounds and are claimed as such herein. These are compounds having the structure of formula (I) wherein a heteroatom is directly bound to E 1 and/or E 2 . e.g., with the proviso that a heteroatom is directly bound to E 1 , E 2 , or to both E 1 and E 2 , and wherein the carbene may be in the form of a salt (such that it is positively charged and associated with a negatively charged counterion). These novel carbenes are those wherein a heteroatom is directly bound to E 1 and/or E 2 , and include, solely by way of example, carbenes of formula (I) wherein E 1 and/or E 2 is NR E and R E is a heteroalkyl or heteroaryl group such as an alkoxy, alkylthio, aryloxy, arylthio, aralkoxy, or aralkylthio moiety. Other such carbenes are those wherein x and/or y is at least 1, and L 1 and/or L 2 is heteroalkyl, heteroaryl, or the like, wherein the heteroatom within L 1 and/or L 2 is directly bound to E 1 and/or E 2 , respectively.
[0100] Representative of such novel carbenes are compounds of formula (I) wherein E 1 is NR E , and R E is alkoxy, substituted alkoxy, aryloxy, substituted aryloxy, aralkoxy, or substituted aralkoxy. A preferred subset of such carbenes are those wherein E 2 is N, x is zero, y is 1, and E 1 and E 2 are linked through a substituted or unsubstituted lower alkylene or lower alkenylene linkage. A more preferred subset of such carbenes are those wherein R E is lower alkoxy or monocyclic aryl-substituted lower alkoxy, E 1 and E 2 are linked through a moiety —CR 3 R 4 —CR 5 R 6 — or —CR 3 ═CR 5 —, wherein R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, halogen, and C 1 -C 12 alkyl, n is 1, L 2 is lower alkylene, and R 2 is monocyclic aryl or substituted monocyclic aryl. Examples 8-11 describe syntheses of representative compounds within this group.
[0101] As indicated previously, suitable catalysts for the present depolymerization reaction are also precursors to carbenes, preferably precursors to N-heterocyclic and N-heteroacyclic carbenes. In one embodiment, the precursor is a tri-substituted methane compound having the structure of formula (PI)
wherein E 1 , E 2 , x, y, R 1 , R 2 , L 1 , L 2 , m, and n are as defined for carbenes of structural formula (I), and wherein R 7 is selected from alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, and is substituted with at least one electron-withdrawing substituent such as
[0103] fluoro, fluoroalkyl (including perfluoroalkyl), chloro, nitro, acytyl. It will be appreciated that the foregoing list is not exhaustive and that any electron-withdrawing group may serve as a substituent providing that the group does not cause unwanted interaction of the catalyst with other components of the depolymerization mixture or adversely affect the depolymerization reaction in any way. Specific examples of R 7 groups thus include p-nitrophenyl, 2,4-dinitrobenzyl, 1,1,2,2-tetrafluoroethyl, pentafluorophenyl, and the like.
[0104] Catalysts of formula (PI) are new chemical entities. Representative syntheses of such compounds are described in Examples 13 and 14 herein. As may be deduced from those examples, compounds of formula (PI) wherein E 1 and E 2 are N may be synthesized from the corresponding diamine and an appropriately substituted aldehyde.
[0105] Another carbene precursor useful as a catalyst in the present depolymerization reaction has the structure of formula (PII)
wherein E 1 , E 2 , x, y, R 1 , R 2 , L 1 , L 2 , m, and n are as defined for carbenes of structural formula (I), M is a metal, e.g., gold, silver, other main group metals, or transition metals, with Ag, Cu, Ni, Co, and Fe generally preferred, and Ln is a ligand, generally an anionic or neutral ligand that may or may not be the same as -E 1 -[(L 1 ) m -R 1 ] x or -E 2 -[(L 2 ) n -R 2 ] y . Generally, carbene precursors of formula (PII) can be synthesized from a carbene salt and a metal oxide; see, e.g., the synthesis described in detail in Example 12.
[0107] Still another carbene precursor suitable as a depolymerization catalyst herein is a tetrasubstituted olefin having the structure of formula (PIII)
wherein: E 1 , E 2 , x, y, R 1 , R 2 , L 1 , L 2 , m, and n are defined as for carbenes of structural formula (I); E 3 and E 4 are defined as for E 1 and E 2 ; v and w are defined as for x and y; R 8 and R 9 are defined as for R 1 and R 2 ; L 3 and L 4 are defined as for L 1 and L 2 ; and h and k are defined as for m and n. These olefins are readily formed from N,N-diaryl- and N,N-dialkyl-N-heterocyclic carbene salts and a strong base, typically an inorganic base such as a metal alkoxide.
[0109] As with the carbenes per se, those catalyst precursors having the structure of formula (PII) or (PIII) in which a heteroatom is directly bound to an “E” moiety, i.e., to E 1 , E 2 , E 3 , and/or E 4 , are new chemical entities. Preferred such precursors are those wherein the “E” moieties are NR E or linked N atoms, and the directly bound heteroatom within R E is oxygen or sulfur.
[0110] The depolymerization reaction may be carried out in an inert atmosphere by dissolving a catalytically effective amount of the selected catalyst in a solvent, combining the polymer and the catalyst solution, and then adding the nucleophilic reagent. In a particularly preferred embodiment, however, the polymer, the nucleophilic reagent, and the catalyst (e.g., a carbene or a carbene precursor) are combined and dissolved in a suitable solvent, and depolymerization thus occurs in a one-step reaction.
[0111] Preferably, the reaction mixture is agitated (e.g., stirred), and the progress of the reaction can be monitored by standard techniques, although visual inspection is generally sufficient, insofar as a transparent reaction mixture indicates that the polymer has degraded to an extent sufficient to allow all degradation products to go into solution. Examples of solvents that may be used in the polymerization reaction include organic, protic, or aqueous solvents that are inert under the depolymerization conditions, such as aromatic hydrocarbons, chlorinated hydrocarbons, ethers, aliphatic hydrocarbons, or mixtures thereof. Preferred solvents include toluene, methylene chloride, tetrahydrofuran, methyl t-butyl ether, Isopar, gasoline, and mixtures thereof. Supercritical fluids may also be used as solvents, with carbon dioxide representing one such solvent. Reaction temperatures are in the range of about 0° C. to about 100° C. , typically at most 80° C., preferably 60° C. or lower, and most preferably 30° C. or less, and the reaction time will generally be in the range of about 12 to 24 hours. Pressures range from atmospheric to pressures typically used in conjunction with supercritical fluids, with the preferred pressure being atmospheric.
[0112] It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
[0113] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.
[heading-0114] Experimental:
[0115] General Procedures. 1 H and 13 C NMR spectra were recorded on a Bruke-Avance (400 MHz for 1 H and 100 MHz for 13 C). All NMR spectra were recorded in CDCl 3 . Materials. Solvents were obtained from Sigma-Aldrich and purified by distillation. Other reagents were obtained commercially or synthesized as follows: poly(propylene carbonate), poly(bisphenol A carbonate), poly(1,4-butylene adipate), 1-ethyl-3-methyl-1-H-imidazolium chloride, ethylene glycol, butane-2,3-dione monooxime, ammonium hexafluorophosphate, pentafluorobenzaldehyde, and mesityl diamine, obtained from Sigma-Aldrich; 1,3-(2,4,6-trimethylphenyl)imidazol-2-ylidene, synthesized according to the method of Arduengo et al. (1999) Tetrahedron 55:14523; N,N-diphenyl imidazoline, chloride salt, synthesized according to the method of Wanzlick et al. (1961) Angew. Chem. 73:493 and Wanzlick et al. (1962) Angew. Chem. 74:128, and Wanzlick et al. (1963) Chem. Ber. 96:3024; 1,3,5-tribenzyl-[1,3,5]triazinane, synthesized according to the method of Arduengo et al. (1992) J. Am. Chem. Soc. 114:5530, cited supra.
EXAMPLE 1
[0116] Depolymerization of Poly(propylene carbonate) (M w =50,000) with isolated carbene: 7 mg (0.02 mmol) of 1,3-(2,4,6-trimethylphenyl)imidazol-2-ylidene dissolved in toluene (0.6 mL), was added to a stirred mixture of 0.5 g of poly(propylene carbonate) in toluene (10 mL), under N 2 . After stirring for 5 minutes at room temperature, 2 mL of methanol were added to the reaction mixture and the temperature was brought to 80° C. Stirring was continued for 3 hours followed by the evaporation of the solvent in vacuo. The 1 H and 13 C NMR spectra showed the presence of a single monomer, 4-methyl-[1,3]-dioxolan-2-one. However, there were 4 peaks in the GC-MS.
[heading-0117] GC-MS:
[0118] a) m/z (5%) 5.099 min=106 (42), 103 (5), 91 (100), 77 (8), 65 (8)
[0119] b) m/z (5%) 5.219 min=106 (60), 105 (30), 103 (8), 91 (100), 77 (8), 65 (5), 51 (5)
[0120] c) m/z (85%) 6.750 min=102 (15), 87 (40), 58 (20), 57 (100). Major product.
[0121] d) m/z (5%) 9.030 min=136(10), 135 (100), 134 (70), 120 (85), 117 (8), 103 (5), 91 (14), 77 (10), 65 (5).
[0122] 1 H NMR :1.4 (d, 3H), 3.9 (t, 1H), 4.5 (t, 1H), 4.8 (m, 1H).
[0123] 13 C NMR: 18.96, 70.42, 73.43, 154.88
EXAMPLE 2
[0124] Depolymerization of Poly(Bisphenol A carbonate) (M w =65,000) with isolated carbene: 7 mg (0.02 mmol) of 1,3-(2,4,6-trimethylphenyl)imidazol-2-ylidine dissolved in toluene (1 mL), was added to a stirred mixture of 0.5 g of poly(bisphenol A carbonate) in toluene (10 mL), under N 2 . After stirring for 5 minutes at room temperature, 2 mL of methanol were added to the reaction mixture. The temperature was brought to 80° C. and stirring was continued for 18 hours followed by the evaporation of the solvent in vacuo. The 1 H and 13 C NMR spectra showed the presence of two compounds identified as, bisphenol A and carbonic acid 4-[1-hydroxy-phenyl)-1-methyl-ethyl]-phenyl ester 4-[1-(4-methoxy-phenyl)-1-methyl-ethyl] phenyl ester. However, GC-MS indicated 4 peaks.
[heading-0125] GC-MS:
[0126] a) m/z (5%) 5.107 min=106 (40), 103 (5), 91 (100), 77 (8), 65 (8), 51 (8)
[0127] b) m/z (5%) 5.210 min=106 (60), 105 (30), 103 (8), 91 (100), 77 (8), 65 (5), 51 (5)
[0128] c) m/z (60%) 14.301 min=228 (30), 213 (100), 119 (15), 91 (10). Major product
[0129] d) m/z (30%) 16.016 min=495 (30), 333 (10), 319 (20), 299 (5), 281 (5), 259 (25), 239 (38), 197 (40), 181 (12), 151 (12), 135 (100), 119 (10), 91 (10).
[0130] 1 H NMR: 1.6-1.8 (m), 2,4 (s), 3.96 (s), 6.7-6.8 (t), 7.0-7.3 (m).
EXAMPLE 3
[0131] Depolymerization of Poly(1,4-butylene adipate) (M n =12,000) with isolated carbene: 0.006 g (0.02 mmol) of 1,3-(2,4,6-trimethylphenyl)imidazol-2-ylidine dissolved in toluene (1 mL), was added to a stirred mixture of 1.0 g of poly(1,4-butylene adipate) in toluene (10 mL), under N 2 . After stirring for 5 minutes at room temperature, 2 mL of methanol were added to the reaction mixture. The temperature was brought to 80° C. and stirring was continued for 6 hours followed by the evaporation of the solvent in vacuo. The
[0132] 1 H and 13 C NMR showed the presence of a single product, and the GC-MS showed two products.
[heading-0133] GC-MS:
[0134] a) m/z (95%) 5.099 min=143 (80), 142 (20), 115 (20), 114 (100), 111 (70), 101 (65), 87 (12), 83 (25), 82 (12), 74 (36), 73 (26), 69 (10), 59 (72), 55 (60). Major product.
[0135] b) m/z (5%) 12.199 min=201 (4), 161 (6), 143 (100), 129 (32), 116 (12), 115 (25), 111 (70), 101 (12), 87 (10), 83 (15), 73 (34), 71 (12), 59 (14), 55 (42).
[0136] 1 H NMR :1.67 (m), 2.32 (s), 4.08 (s).
[0137] 13 C NMR: 24.26, 25.18, 33.74, 63.75, 173.23
EXAMPLE 4
[0138] Depolymerization of Poly(propylene carbonate) (M w =50,000) with in-situ carbene: To a mixture of 7 mg (0.047 mmol) of 1-ethyl-3-methyl-1-H-imidazolium chloride in tetrahydrofuran (THF) was added 4 mg (0.038 mmol) of potassium t-butoxide (t-BOK), under N 2 . After 30 min stirring, 0.1 mL of the reaction mixture was transferred to a flask that was charged with 0.5 g of poly(propylene carbonate) in 10 mL of THF. The reaction mixture was stirred for 10 min at room temperature followed by the addition of 2 mL of methanol. Stirring was continued at room temperature for 3 hours. Solvent was removed and the 1 H and 13 C NMR spectra showed the presence of a single product, 4-methyl-[1,3]-dioxolan-2-one. However, before the removal of the solvent the GC-MS of the crude reaction mixture showed 6 different compounds.
[heading-0139] GC-MS:
[0140] a) m/z (15%) 6.268 min=119 (4), 90 (100), 75 (4), 59 (25).
[0141] b) m/z (5%) 6.451 min=104 (40), 103 (30), 90 (5), 77 (5), 59 (100), 58 (10), 57 (10).
[0142] c) m/z (70%) 6.879 min=102 (10), 87 (25), 58 (14), 57 (100). Major product.
[0143] d) m/z (1%) 7.565 min=103 (40), 89 (5), 59 (100), 58 (5), 57 (8).
[0144] e) m/z (4%) 8.502 min=207 (14), 133 (10), 103 (35), 90 (10), 89 (10), 59 (100) 58 (12), 57 (14).
[0145] f) m/z (5%) 8.936 min=148 (8), 118 (8), 117 (15), 103 (20), 77 (60), 72 (8), 59 (100), 58 (5), 57 (5).
[0146] 1 H NMR :1.4 (d, 3H), 3.9 (t, 1H), 4.5 (t, 1H), 4.8 (m, 1H).
[0147] 13 C NMR: 18.96, 70.42, 73.43, 154.88
EXAMPLE 5
[0148] Depolymerization of Poly(bisphenol A carbonate) (M w =65,000) with in situ carbene: To a mixture of 7 mg (0.047 mmol) of 1-ethyl-3-methyl-1-H-imidazolium chloride in THF (1 mL) was added 4 mg (0.038 mmol) of t-BOK, under N 2 . After 30 min, stirring 0.1 mL of the reaction mixture was transferred to a flask that was charged with 0.5 g of poly(bisphenol A carbonate) in 10 mL of THF. The reaction mixture was stirred for 10 min at room temperature followed by the addition of 2 mL of methanol. Stirring was continued at room temperature for 3 hours. The solvent was removed in vacuo and the 1 H, 13 C NMR and GC-MS spectra showed a mixture of monomer and oligomers, where the major product was bisphenol A.
[heading-0149] GC-MS:
[0150] a) m/z (10%) 12.754 min=212 (30), 198 (20), 197 (100), 182 (10), 181 (10), 179 (10), 178 (10), 165 (8), 152 (8), 135 (10), 119 (12), 103 (15), 91 (12), 77 (10), 65 (5).
[0151] b) m/z (5%) 13.674 min=282 (5), 281 (10), 255 (8), 229 (10), 228 (40), 214 (20), 213 (100), 208 (30), 197 (30), 191 (5), 181 (5), 179 (5), 165 (10), 152 (8), 135 (25), 134 (25), 133 (5), 120 (5), 119 (50), 115 (10), 103 (10), 99 (5), 97 (5), 96 (5), 91 (30), 79 (5), 77 (10), 65 (8).
[0152] c) m/z (35%) 14.286 min=228 (34), 214 (20), 213 (100), 197 (5), 165 (5), 135 (5), 119 (20), 107 (5), 91 (10), 77 (5), 65 (5). Major Product.
[0153] d) m/z (35%) 15.189 min=286 (20), 272 (15), 271 (100), 227 (5), 212 (5), 197 (3), 183 (2), 169 (3), 133 (3), 119 (5).
[0154] e) m/z (10%) 15.983 min=344 (20), 330 (20), 329 (100), 285 (5), 269 (3), 226 (3), 211 (2), 183 (3), 165 (3), 153 (2), 133 (6), 121 (2), 91 (2), 77 (1), 59 (3).
[0155] Depolymerization of PET according to the above scheme: 20 mg of t-BOK and 45 mg of N,N-diphenyl imidazoline, chloride salt, were placed in a vial with 2 mL THF and stirred for 15 minutes. Ethylene glycol (2.3 g) and PET (0.25 g) (pellets obtained from Aldrich dissolved in CHCl 3 and trifluoroacetic acid and precipitated with methanol to form a white powder) were combined to form a PET slurry. The catalyst was added to the slurry with approximately 5 additional mL THF. After 2 hours, the solution became more transparent, indicating dissolution of the components of the depolymerization mixture. The admixture was stirred overnight, yielding a completely clear solution the following day. the THF was removed, yielding 225 mg of white solid. 1 H NMR 13 C NMR, and GC-MS were all consistent with bis(hydroxy ethylene) terephthalate.
[0156] Depolymerization of PET according to the above scheme: 25 mg of 1,3-dimethyl imidazole, iodide salt, and 11 mg of t-BOK were placed in a vial with 2 mL of THF and stirred for 15 min. Methanol (3.11 g) and PET (308 mg, as in Example 6) were combined with 5 mL of THF to form an insoluble mixture. The catalyst mixture was filtered into the PET/methanol mixture. After 1 hour, there was a noticeable increase in transparency. After 14 hours, the solution was completely homogeneous and clear. The solvent was removed by rotary evaporation to yield a white crystalline product (250 mg). 1 H NMR indicated complete conversion to dimethyl terephthalate.
[0157] Examples 6 and 7 may be better understood by reference to the synthetic route used to prepare the PET and the possible depolymerization products obtained therefrom. The PET obtained in each example was prepared by synthesis according to a two-step transesterification process from dimethyl teraphthalate (DMT) and excess ethylene glycol (EO) in the presence of a metal alkanoate or acetate of calcium, zinc, manganese, titanium etc. The first step generates bis(hydroxy ethylene) teraphthalate (BHET) with the elimination of methanol and the excess EO. The BHET is heated, generally in the presence of a transesterification catalyst, to generate high polymer. This process is generally accomplished in a vented extruder to remove the polycondensate (EO) and generate the desired thermoformed object from a low viscosity precursor. The reaction takes place according to the following scheme:
[0158] The different options for chemical recycling are regeneration of the base monomers (DMT and EG), glycolysis of PET back to BHET, decomposition of PET with propylene glycol and reaction of the degradation product with maleic anhydride to form “unsaturated polyesters” for fiber reinforced composites and decomposition with glycols, followed by reaction with dicarboxylic acids to produce polyols for urethane foam and elastomers.
[0159] In Example 7, PET powder was slurried in a THF/methanol solvent mixture. N-heterocyclic carbene (3-5 mol %), generated in situ, was added and within approximately 3 hours the PET went into solution. Anaylsis of the degradation product indicated quantitative consumption of PET and depolymerization via transesterification to EO and DMT. The DMT is readily recovered by recrystallization, while EO can be recovered by distillation ( FIG. 1 ). Alternatively, and as established in Example 6, if EO is used as the alcohol (˜50 to 200 mol % excess) in the THF slurry, the depolymerization product is BHET, which is the most desirable and can be directly recycled via conventional methods to PET ( FIG. 2 ). The N-heterocyclic carbene catalyst platform is extremely powerful, as the nature of the substituents has a pronounced effect on catalyst stability and activity towards different substrates.
[0160] The PET depolymerization reactions of Examples 6 and 7 are illustrated schematically below.
[0161] The following Examples 8-11 describe synthesis of new carbcne precursors as illustrated in the following scheme:
EXAMPLE 8
[0162] 3-Benzyl-1-methoxy-4,5-dimethylimidazolium iodide (2): Methyl iodide (0.5 mL, 7.8 mmol) was added via syringe to a solution of imidazole-N-oxide 1 (1.0 g, 4.9 mmol) in ca. 20 mL of CHCl 3 (compound 1 was prepared from 1,3,5-tribenzyl-[1,3,5]triazinane and butane-2,3-dione monooxime using the procedure of Arduengo et al. (1992), supra.) The resulting mixture was stirred at room temperature overnight. Removal of the volatiles in vacuo afforded a thick yellow oil of suitable purity in an undetermined yield. 1 H-NMR (δ, CDCl 3 ): 10.32 (s, 1H, N—CH—N); 7.39 (m, 5H, C 6 H 5 ); 5.56 (s, 2H, NCH 2 ); 4.38 (s, 3H, OCH 3 ); 2.27 (s, 3H, CH 3 ); 2.20 (s, 3H, CH 3 ).
EXAMPLE 9
[0163] 3-Benzyl-1-methoxy-4,5-dimethylimidazolium hexafluorophosphate (3): Crude iodide 2 was taken up in deionized (DI) water, which separated the product from small amounts of a dark, insoluble residue. The water solution was decanted to a second flask and a solution of ammonium hexafluorophosphate (950 mg, ca. 5.8 mmol) in 10 mL of DI water was added in portions. An oil separated during the addition, and the supernatant solution was decanted out. The oil was crushed in cold (0° C.), and subsequently recrystallized in methanol. Yield: 1.3 g (73% from 1). 1 H-NMR (δ, CDCl 3 ): 8.67 (s, 1H, N—CH—N); 7.39 (m, 3H, C 6 H 5 ); 7.29 (d, 2H, C 6 H 5 ); 5.24 (s, 2H, NCH 2 ); 4.21 (s, 3H, OCH 3 ); 2.27 (s, 3H, CH 3 ); 2.17 (s, 3H, CH 3 ).
EXAMPLE 10
[0164] 1-Benzyloxy-3-benzyl-4,5-dimethylimidazolium bromide (4): Benzyl bromide (1.2 mL, ca. 10 mmol) was added via syringe to a refluxing suspension of 1 (1.0 g, 5.0 mmol) in dry benzene. A dark orange oil separated after refluxing for 6 h, and cooling to room temperature. The supernatant was decanted and the remaining oil was dried under vacuum overnight, which caused the product to solidify. The solid mass was crushed in pentane, filtered and dried under vacuum. Yield: 1.34 g (63%). 1 H-NMR (δ, CDCl 3 ): 11.04 (s, 1H, N—CH—N); 7.6-7.2 (ov. m, 10H, 2×C 6 H 5 ); 5.59, 5.58 (s+s, N—CH 2 , O—CH 2 ); 2.09, 1.94 (s, 3H, CH 3 , CH 3 ). 13 C-NMR (δ, CDCl 3 ): 132.8 (OCH 2 —C 6 H 5 ); 132.5 (NCN); 131.5 (NCH 2 - i C 6 H 5 ); 130.6, 130.3, 129.2, 129.0, 129.0, 128.9, 128.0 ( omp C 6 H 5 ); 124.8; 124.1 (NCCN); 83.9 (OCH 2 ); 51.2 (NCH 2 ); 8.89 (CH 3 ); 7.11 (CH 3 ).
EXAMPLE 11
[0165] 3-Benzyl-1-benzyloxy-4,5-dimethylimidazolium hexafluorophosphate (5): A batch of crude bromide 4 (still as an oil before drying under vacuum) was dissolved in DI water and extracted with hexanes. The aqueous layer was separated and a solution of ammonium hexafluorophosphate (ca. 1.3 equiv.) was added dropwise with constant stirring. The yellow oil deposited on the walls of the flask was dissolved in warm methanol and a few drops of hexanes were added. Cooling to room temperature afforded off-white crystals of pure 5, which were rinsed with pentane and dried under vacuum. Yield: (82% from 1). 1 H-NMR (δ, CDCl 3 ): 8.42 (s, 1H, N—CH—N); 7.45-7.35, 7.18 (ov. m, C 6 H 5 ); 5.31, 5.20 (s+s, N-CH 2 , O—CH 2 ); 2.13 (s, 3H, CH 3 ); 2.05 (s, 3H, CH 3 ).
[0166] Bis(1-Benzyloxy-3-benzyl-4,5-dimethylimidazolylidene)silver(I) dibromoargentate (6). The carbene precursor 6 was prepared as follows: A mixture of silver oxide (128 mg, 0.55 mmol) and imidazolium bromide 4 (396 mg, 1.06 mmol) was taken up in dry CH 2 Cl 2 and stirred at room temperature for 90 minutes. The dark orange suspension was filtered through a pad of celite and evaporated to dryness, yielding an orange powder. Crystallization from THF afforded a white powder (2 crops). Yield: 291 mg (57%). 1 H-NMR (δ, CD 2 Cl 2 ): 7.47-7.32 (ov. m, 10H, 2×CrH 5 ); 5.23, 5.22 (s+s, NCH 2 , OCH 2 ); 2.01, 1.95 (s, 3H+3H, CH 3 , CH 3 ). 13 C-NMR (δ, CD 2 Cl 2 ): 136.2 (NCN); 133.3 (OCH 2 — i C 6 H 5 ); 130.8 (NCH 2 — i C 6 H 5 ); 130.7, 130.0; 129.3, 129.3, 128.5, 127.1, 123.9 ( omp C 6 H 5 +NCCN); 82.6 (OCH 2 ); 54.0 (NCH 2 ); 9.4 (CH 3 ); 7.8 (CH 3 ). Anal. Found: C, 47.56; H, 4.26; N, 5.79%. Calc. for C 38 H 40 Ag 2 Br 2 N 4 O 2 : C, 47.53; H, 4.20;; N, 5.83%.
[0167] Examples 13 and 14 describe preparation of additional carbene precursors from N,N-diaryl-substituted diamines as illustrated in the schemes below.
[0168] Synthesis of carbene precursor 7 (2-pentafluorophenyl-1,3-diphenyl-imidazolidine): 200 mg (0.94 mmol, FW=212.12) N,N′-diphenyl-ethane-1,2-diamine was placed in a vial and dissolved in 5mL CH 2 Cl 2 . A catalytic amount of p-toluenesulfonic acid and 50 mg of Na 2 SO 4 were added, followed by 230 mg (0.94 mmol, FW=196.07) of pentafluorobenzaldehyde. The mixture was stirred for 8 h. The Na 2 SO4 was filtered off and solvent was removed under reduced pressure to yield a light brown powder 395 mg (FW=436.2), 96% yield. 1 H NMR: (400 MHz, CDCl 3 , 25° C.)=3.7-3.9 (m, 2H), 3.9-4.1 (m, 2H), 6.5 (s, 1H), 6.7-6.8 (m, 2H), 6.8-6.9 (m, 1H), 7.2-7.5 (m, 2H). 19 F NMR: δ=−143.2 (s br, 2F), −153.7-−153.8 (m, 1F), 161.7-−161.8 (m, 2F).
[0169] Synthesis of carbene precursor 8 (2-pentafluorophenyl-1,3-bis-(2,4,6-trimethyl-phenyl)-imidazolidine): Mesityldiamine (512 mg, 1.7 mmol) was placed into a vial, equipped with a stirbar, with pentafluorobenzaldehyde (340 mg, 1.7 mmol). Glacial acetic acid (5 mL) was added and the reaction was stirred at room temperature for 24 h. The acetic acid was removed under reduced pressure and the product was washed several times with cold methanol to afford the product as a white crystalline solid (543 mg, 65%). 1 H NMR: (400 MHz, CDCl 3 , 25° C.) δ: 2.2 (s, 12H), 2.3 (s, 6H), 3.5-3.6 (m, 2H), 3.9-3.4 (m, 2H), 6.4 (s, 1H), 6.9 (s, 4H). 19 F NMR: −136.3-−136.4 (m, 1F), −148.6-−148.7 (m, 1F), −155.8-−155.9 (m, 1F), −163.0-−163.3 (m, 2F).
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A method is provided for carrying out depolymerization of a polymer containing electrophilic linkages in the presence of a catalyst and a nucleophilic reagent, wherein production of undesirable byproducts resulting from polymer degradation is minimized. The reaction can be carried out at a temperature of 80° C. or less, and generally involves the use of an organic, nonmetallic catalyst, thereby ensuring that the depolymerization product(s) are substantially free of metal contaminants. In an exemplary depolymerization method, the catalyst is a carbene compound such as an N-heterocyclic carbene, or is a precursor to a carbene compound. The method provides an important alternative to current recycling techniques such as those used in the degradation of polyesters, polyamides, and the like.
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BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to processes for preparing iodoaromatic compounds. This invention further relates to the use of iodoaromatic compounds in the formation of triarylamine hole transport small molecules, which may be used in electrophotography.
[0003] 2. Description of Related Art
[0004] Electrophotographic imaging members (i.e. photoreceptors) are well known. Electrophotographic imaging members are commonly used in electrophotographic (xerographic) processes and may comprise a photoconductive layer including a single layer or composite layers. These electrophotographic imaging members take many different forms. For example, layered photoresponsive imaging members, such as those described in U.S. Pat. No. 4,265,990 to Stolka et al., which is incorporated by reference in its entirety, are known in the art.
[0005] More advanced photoconductive photoreceptors containing highly specialized component layers are also known. For example, a multilayered photoreceptor employed in electrophotographic imaging systems sometimes includes one or more of a substrate, an undercoating layer, an intermediate layer, an optional hole or charge blocking layer, a charge generating layer over an undercoating layer and/or a blocking layer, and a charge transport layer (including a charge transport material in a binder). Additional layers such as one or more overcoating layer or layers are also sometimes included.
[0006] Various compounds are known for their uses as charge transporting materials, including hole transporting materials, in charge transporting layers of electrophotographic apparatuses, including pyrazoline compounds such as those disclosed in JP-B-37-10696, triarylamine compounds such as those disclosed in U.S. Pat. No. 3,180,730 to Klupfel et al., stilbene compounds as disclosed in published unexamined Japanese Patent Application JP-A-58-198043, hydrazone compounds as disclosed in JP-B-55-42380, oxadiazone compounds as disclosed in JP-B-34-5466, butadiene compounds as disclosed in published unexamined Japanese Patent Application JP-A-63-314554, and so on, are known. Among these compounds, triarylamine compounds are of particular importance, in view of high charge-transporting and hole-transporting ability (mobility), and various triarylamine compounds have been disclosed, e.g., in JP-A-1-280763, JP-A-2-178666, JP-A-2-178667, JP-A-2-178668, JP-A-2-178669, JP-A-2-178670, JP-A-2-190862, JP-A-2-190863, JP-A-2-230255, JP-A-3-78755, JP-A-3-78756, JP-A-3-78757, JP-A-3-114058, JP-A-4-133064, JP-A-4-193852, JP-A-4-312558, JP-A-5-19509, JP-A-5-80550, and JP-A-5-313386.
[0007] It is generally known that triarylamine compounds can be synthesized by coupling an arylamine compound with an aryl halide, usually an aryl bromide or an aryl iodide, using a copper catalyst. Aryl iodides are preferred due to the low reactivity of aryl bromides during tertiary amine formation.
[0008] Typically, large-scale production of triarylamine small molecules is accomplished by use of an Ullmann condensation reaction, such as those illustrated in reaction schemes (1) and (2):
[Insert Chemical Drawing (1)]
[Insert Chemical Drawing (2)]
[0009] The use of Ullmann condensation reactions in the production of tertiary amines, specifically triarylamines, is described in detail in, for example, U.S. Pat. No. 4,764,625 to Turner, et al., the entire disclosure of which is incorporated herein by reference.
[0010] In such Ullmann condensation reactions, aromatic halides, such as aryl iodides, are reacted with aromatic amine compounds in the presence of a base, a copper catalyst and, optionally, an inert solvent. Aromatic iodides possess a much higher reactivity than other aromatic halides in these reactions. Typically, aryl iodides have higher kinetic rates of product formation than other aromatic halides, as illustrated by the reduced reaction times necessary to produce higher yields of highly pure products than with other aromatic halides. Thus, aryl iodides are key substrates in the Ullmann condensation reactions traditionally used to manufacture triarylamine hole transport small molecules.
[0011] However, the benefits of using aryl iodides to form triarylamines by Ullmann condensation are not without cost. Aryl iodides are generally more expensive than aryl bromides or aryl chlorides.
[0012] The formation of aryliodides by iodinating an aromatic compound with a sulfuric acid catalyst in a mixed water/acetic acid solvent, using iodic acid and iodine, as shown in Ann., 634, 84 (1960) is known, as exemplified by the iodination of biphenyl in reaction scheme (3). Reaction scheme (3) represents a common, currently employed manufacturing method for 4-iodobiphenyl.
[Insert Chemical Drawing (3)]
[0013] In this reaction, biphenyl is iodinated by a hypervalent iodine species, prepared from elemental iodine and periodic acid in a strongly acidic solution. Preparation of monoiodo compound by this method is difficult, at least because over-iodination occurs. In the reaction scheme (3) above, the reaction mixture contains starting material, 4-iodobiphenyl, and 1,4-diiodobiphenyl. In order to prepare the mono-iodo compound for use in an Ullmann condensation, extensive purification by recrystallization is necessary, and yields of only about 60% can be obtained.
[0014] Iodination with periodic acid is carried out with ease. However the reaction selectivity is low. The reaction product is a mixture of monoiodo and diiodo compounds. When a subsequent amination reaction is carried out using this mixture, the reaction product after amination also comprises a mixture. Since impurities in the amination product have detrimental effects on the electrical characteristics of the charge transport material, purification of the product is required. The molecular weight of the impurities is large enough to render purification by distillation, etc., impractical. Thus, a very expensive purification method, such as a column purification, etc., must be used. In addition to the expense of iodine, a method in which many diiodo byproducts are formed is a costly process for preparing arylamine compounds.
[0015] Also, the solubility of diiodo compounds is very low, the diiodo compound cannot be removed from the product by recrystallization, which may be easily incorporated into industrial operations at low cost. Therefore, for product mixtures containing about 10% or more of diiodo compounds, purification by distillation is required.
[0016] The monoiodo compound, in contrast, has a high boiling point and a high melting point. For these reasons, high vacuum conditions are required in the distillation of monoiodo compound, and the product recovered by distillation is apt to solidify and become difficult to handle. When the mixture to be subjected to distillation contains a large amount of diiodo compounds, distillation must be carried out multiple times. The purity of the product subjected to a single distillation is lowered by “splashing” of the distillation mixture, resulting in contamination of the distillation product by diiodo compounds. Thus, fractional distillation is required to purify the monoiodo product from a reaction mixture containing a large amount of diiodo compounds. The more involved purification process complicates operation and increases manufacturing costs.
[0017] Also, Ann., 634, 84 (1960) describes carrying out the iodination reaction in a saturated solution of an aromatic compound to increase the selectivity of monoiodo compound formation. This method, however, does not provide a low-cost product having sufficient purity as a raw material used for a charge transporting material.
[0018] The present invention is provided to solve the problems described above. That is, the present invention provides a low cost route to iodoaromatic molecules, having high yields of highly pure monoiodo compounds, and thus a low cost route to triarylamine hole transport small molecules.
SUMMARY OF THE INVENTION
[0019] The present invention provides a process for preparing mono-iodinated aromatic compounds having high purity with high yield and at low cost.
[0020] Further, the present invention provides a process for preparing mono-iodinated aromatic compounds that are useful as intermediates for preparing charge transporting and hole transporting amino compounds.
[0021] Specifically, this invention provides process for preparing an aryl iodide compound by reacting an aryl halide compound with a metal iodide, a metal catalyst and a catalyst coordinating ligand in at least one solvent to form an aryl iodide; and purifying the aryl iodide.
[0022] This invention separably provides an aryl iodide compound prepared by reacting an aryl halide compound with a metal iodide, a metal catalyst and a catalyst coordinating ligand in at least one solvent to form an aryl iodide; and purifying the aryl iodide.
[0023] This invention separably provides a process for preparing a triarylamine compound by reacting an aryl halide compound with a metal iodide, a metal catalyst and a catalyst coordinating ligand in at least one solvent to form an aryl iodide; purifying the aryl iodide, reacting the aryl iodide with a diarylamine in the presence of potassium hydroxide and a copper catalyst in at least one solvent to form a triarylamine, and purifying the triarylamine.
[0024] This invention separably provides a triarylamine compound prepared by reacting an aryl halide compound with a metal iodide, a metal catalyst and a catalyst coordinating ligand in at least one solvent to form an aryl iodide; purifying the aryl iodide, reacting the aryl iodide with a diarylamine in the presence of potassium hydroxide and a copper catalyst in at least one solvent to form a triarylamine, and purifying the triarylamine.
[0025] This invention further separably provides a photoconductive imaging member comprising a charge transport layer that comprises at least one triarylamine compound prepared by reacting an aryl halide compound with a metal iodide, a metal catalyst and a catalyst coordinating ligand in at least one solvent to form an aryl iodide; purifying the aryl iodide, reacting the aryl iodide with a diarylamine in the presence of potassium hydroxide and a copper catalyst in at least one solvent to form a triarylamine, and purifying the triarylamine.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0026] The iodination reaction is carried out by a catalyzed halogen exchange reaction in which an aryl halide compound, a metal iodide, a catalyst and a catalyst coordinating ligand are added to a solvent, and thereafter, the reaction mixture is heated. After the reaction mixture is cooled, the reaction product is isolated and may be used in preparation of triarylamine small molecules.
[0027] The aryl portion of the aryl halide compound is not particularly limited. The aryl portion may include, but is not limited to, benzyl, cyclopentadienyl, biphenyl, terphenyl, tolyl, naphthyl, anthryl, phenanthryl, pyryl, fluorenyl and the like. The aryl portion may include one or more substituent group. Such substituents are not particularly limited, and may include but are not limited to alkyl groups, alkoxy groups, amide groups, sulfonamide groups, indole groups, nitrile groups, ester groups, fluoride atoms and the like. The halide portion of the aryl halide may be a bromine or chlorine atom.
[0028] The metal iodide used in the iodination reaction is not particularly limited. In particular, Group I and Group II metal iodides may be used, and Group I iodides, such as lithium iodide, sodium iodide and potassium iodide, are preferred. The metal iodide may be present in any effective amount, but the metal iodide is generally present in excess, with respect to the aryl halide reactant. For example, the metal iodide may be present in an amount such that the ratio of iodine atoms to aryl halide reactant molecules is in the range of from about 1:1 to about 10:1, preferably in the range of from about 2:1 to about 5:1.
[0029] As the catalyst in the iodination reaction, any suitable transition metal catalyst may be used. In particular, copper catalysts may be used as the catalyst for the iodination reaction. Examples of copper catalysts include but are not limited to copper powder, cuprous oxide and copper I and copper II halides, such as copper I and copper II chlorides, bromides and iodides. In fact, any copper catalyst heretofore commonly used in the halogen exchange reactions can be employed.
[0030] The catalyst may be present in any effective amount. The catalyst is preferably present in an amount in the range of from about 1 mole % to about 50 mole % with respect to the amount of aryl halide, more preferably in the range of from about 2 mole % to about 10 mole % with respect to the amount of aryl halide, and even more preferably in an amount that is about 5 mole % with respect to the amount of aryl halide.
[0031] Any ligand suitable for use with the chosen catalyst may be used as the catalyst coordinating ligand in the iodination reaction. Diamine ligands are particularly useful as catalyst coordinating ligands for preparing aryl iodides by this iodination reaction. In particular, 1,2-diamine ligands and 1,3-diamine ligands are preferred, and 1,3-propanediamine is most preferred. In addition, mono- and bidentate nitrogen-containing heterocycle ligands, including but not limited to 1,10-phenanthroline, may be used as the catalyst coordinating ligand.
[0032] The catalyst coordinating ligand may be present in any effective amount. The catalyst coordinating ligand may preferably be present in an amount in the range of from about 1 mole % to about 50 mole % with respect to the amount of aryl halide, more preferably in the range of from about 2 mole % to about 25 mole % with respect to the amount of aryl halide, and even more preferably in an amount that is about 10 mole % with respect to the amount of aryl halide.
[0033] The solvent to be used in the iodination reaction may be any polar organic solvent, such as tetrahydrofuran, dioxane, xylene, n-alcohols, and mixtures thereof. In particular, alcohols such as n-butanol, n-pentanol, n-hexanol, n-heptanol may be used. Preferably, in embodiments, low-cost, high-boiling alcohols, such as n-propanol, n-butanol and n-pentanol, and combinations thereof are used as the solvent. In some embodiments, the iodination reaction solvent has a boiling point of at least about 100° C. In some further embodiments, the iodination reaction is carried out under pressure greater than standard pressure, in a solvent that has a boiling point of less than 100° C. at standard pressure.
[0034] The reaction is carried out by stirring under heating. The reaction is preferably carried out under heating to a temperature of 100° C. or more, more preferably 125° C. or more, and most preferably 130° C. or more. In addition, it may be desirable to reflux the solvent vapor, since iodine has sublimating properties and may deposit on the upper portion of the reaction vessel.
[0035] The time necessary for the reaction to run to completion may vary with the identities of the reactants and solvent system. For example, a reaction to form 4-iodobiphenyl, in which the catalyst is CuI and the catalyst coordinating ligand is 1,3-propanediamine, may be complete after approximately four (4) hours in a solvent of 1-hexanol, which has a boiling point of 156° C. However, the same reaction in a solvent of 1-pentanol, which has a boiling point of 136° C., may require a significantly longer reaction time—about 18 hours.
[0036] After the conclusion of the reaction, the reaction mixture obtained is allowed to cool to 80° C., and the reaction mixture obtained is separated. Reaction products may be separated by any known or later developed method. Liquid products, for example, may be separated by extraction and/or distillation, and solid products may be separated by crystallization. For example, in some embodiments, a reaction mixture may be diluted with 2 volume equivalents of 30% ammonium hydroxide solution and allowed to cool further, resulting in product crystallization. The reaction product of the iodination reaction may be purified by any known method, such as chromatography or recrystallization. For example, recrystallization of 4-iodobiphenyl from 1-hexane results in a highly pure product.
[0037] The separated reaction product may be purified by recrystallization. If the reaction product is purified by recrystallization, the reaction product is dissolved in an organic solvent, such as methylene chloride, toluene, ethyl acetate, higher alcohols or the like. The organic phase formed by the reaction product and solvent may be washed with a dilute aqueous solution of a sodium salt, such as sodium thiosulfate or sodium carbonate. The organic phase may be then washed with water, dried, and the solvent may be removed by distillation. The residue may be recrystallized from any suitable organic solvent. The organic solvent for recrystallization may be chosen from ethyl acetate, toluene, ethanol, and mixtures thereof. In addition, liquid products may be purified by vacuum distillation.
[0038] The iodination reaction described herein may produce high yields of highly pure aryl iodides. Aryl iodide yields of at least about 75%, particularly in the range of from about 85% to about 95%, and more particularly of about 90%, may be obtained. In addition, the aryl iodide recovered may be highly pure. The aryl iodides obtained may be at least 90% pure; in particular, the aryl iodides may be at least 95% pure; more particularly, the aryl iodides may be at least 98% pure. The purity of the aryl iodides may be confirmed by any known or later developed analytical techniques, including but not limited to high performance liquid chromatography, gas-liquid chromatography and nuclear magnetic resonance spectroscopy.
[0039] A tertiary amine may be prepared by Ullmann condensation of a secondary amine and the aryl iodide compound prepared by the above-described process. Specifically, triarylamines such as those used as hole transport small molecules and charge transport small molecules, may be prepared by Ullmann condensation of a diarylamine and an aryl iodide compound obtained by the process described above. A process by which triarylamines may be prepared is described in detail in, for example, U.S. Pat. No. 4,764,625 to Turner, et al., and as set forth below.
[0040] The diarylamine has the general formula R 2 R 3 NH wherein R 2 and R 3 are the same or different members selected from the group consisting of substituted and unsubstituted aryl, alkaryl and aralkyl. Examples of these amines include but are not limited to diphenylamine, N,N′-3,4-(dimethyl)phenylamine, N,N′-bis(alkylphenyl)-(1,1′-biphenyl)-4,4′-diamine wherein the alkyl is, for example, methyl, ethyl, propyl, n-butyl, etc., N,N′-diphenyl-N,N′-bis(chlorophenyl)-(1,1′-biphenyl)-4,4′-diamine, N,N′-diphenyl-N,N′-bis(3-methylphenyl)-(1,1′-biphenyl)-4,4′-diamine, and N,N-diphenyl-N,N-bis(3-hydroxphenyl)-(1,1″-biphenyl)-4,4″-diamine.
[0041] The condensation reaction is conducted in the presence of potassium hydroxide and finely divided copper catalyst, in an inert atmosphere, such as argon, nitrogen, or methane. The condensation reaction may be conducted either in the absence of a solvent or with an inert saturated hydrocarbon solvent. The reaction mixture may be maintained at a temperature between about 120° C. and 190° C. for a period of time sufficient to substantially complete the reaction.
[0042] Any commercially available potassium hydroxide in flake or pellet form with a low water content can be employed. Examples of copper catalysts include but are not limited to, copper powder, cupric oxide, cuprous oxide, cuprous sulfate, cuprous sulfide, etc. In fact, any copper catalyst commonly used in the Ullmann condensation reaction may be used.
[0043] The ratio of base to amine should be such that the base is present as an excess in relation to the amine. This excess can range from about 1.5:1 to about 6:1 moles.
[0044] The temperature range for conducting the reaction can be from about 120° C. to about 190° C., with the preferred reaction temperature being from about 135° C. to about 165° C.
[0045] The Ullmann condensation reaction of the present invention may be carried out in the absence of a solvent when the intended product is very soluble at ambient temperature in inert, high-boiling hydrocarbon solvents. When the intended product is at least relatively insoluble at ambient temperature in an inert hydrocarbon solvent, the use of potassium hydroxide yields a relatively pure product which can be further highly purified by recrystallization from the same solvent.
[0046] The inert hydrocarbon solvent can be dodecane, tetradecane or any other hydrocarbon having an initial boiling point about above 170° C., or mixtures thereof. In particular, SOLTROL® 170 (initial b.p. 218° C.), which is a mixture of C 13 -C 15 aliphatic hydrocarbons, and SOLTROL® 130 (initial b.p. 176° C.), available from Phillips Chemical Company, are suitable solvent systems. The SOLTROL® hydrocarbons dramatically reduce work-up times, yielding a much purer product while being less expensive solvents.
[0047] Triarylamines formed by the above-described processes may be used as charge transporting molecules in charge transporting layers. Specifically, photoconductive imaging members for photoconductive photoreceptors and electrophotographic imaging members may comprise at least a charge transport layer comprising at least one triarylamine formed by the above-described processes.
[0048] Thus, this invention provides a simple, economical method for producing aryl iodides using fairly inexpensive reagents and mild reaction conditions to produce excellent yields of highly pure product.
[0049] The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the invention.
EXAMPLES
Example 1
Preparation of Iodobenzene
[0050] 15.7 g (0.1 mole) of bromobenzene is dissolved in 150 mL of n-pentanol. 2 equivalents of sodium iodide and 10 mole % of 1,3-propanediamine are added to the bromobenzene solution. The reaction mixture is refluxed at 130° C. for eighteen (18) hours to produce iodobenzene, as shown in reaction scheme (4).
[Insert Chemical Drawing (4)]
[0051] The reaction proceeds very rapidly. The reaction mixture is cooled to 80° C. and washed with a 30% ammonium hydroxide solution. The organic phase is washed with deionized water and brine. The organic phase is then dried over anhydrous sodium sulfate. The dried organic phase is subjected to rotary evaporation and/or vacuum distillation to afford a 92% yield of high purity iodobenzene.
Example 2
Preparation of 4-Iodobiphenyl
[0052] 30 g of 4-bromobiphenyl is dissolved in 40 mL of n-pentanol. 2 equivalents of sodium iodide and 10 mole % of 1,3-propanediamine are added to the 4-bromobiphenyl solution. The reaction mixture is refluxed at 130° C. for sixteen (16) hours to produce 4-iodobiphenyl, as shown in reaction scheme (5).
[Insert Chemical Drawing (5)]
[0053] The reaction proceeds very rapidly. The reaction mixture is cooled to 80° C. and washed with a 30% ammonium hydroxide solution. The organic phase is washed with deionized water and brine. The organic phase is then dried over anhydrous sodium sulfate. The dried organic phase is subjected to rotary evaporation and/or vacuum distillation to afford crude product. The reaction mixture is recrystallized one time from n-pentanol, and a 92% yield of high purity 4-iodobiphenyl is obtained.
Example 3
Preparation of N,N-bis(phenyl)-4-biphenylamine
[0054] 1143.5 g of recrystallized 4-iodobiphenyl, obtained in Example 2 is dissolved in 125 ml of ISOPAR M (trademark, Ashland Chemical), a commercially available mixture of high-boiling solvents having a boiling point of more than 200° C. 760 g of diphenylamine, 846 g of potassium carbonate, and 36.2 g of copper sulfate pentahydrate are added. The reaction mixture is placed under a nitrogen atmosphere and heated to 230° C. for two (2) hours. The reaction product is allowed to cool to 80° C. and 2000 ml of toluene is added. The reaction mixture is then slurry treated with 350 g of ENGELHARD F-20 activated clay (trademark, Engelhard Corporation). The clay is removed by filtration, and the solvent is removed by distillation under reduced pressure.
[0055] The residue is recrystallized in octane to provide 1127.8 g of N,N-bis(phenyl)-4-biphenylamine. The product is characterized as follows: yield: 86%, purity: 98.9, melting point: 117-118° C. The purity of the product is sufficient for use as a charge transport material.
[0056] While this invention has been described in conjunction with the exemplary embodiments outlined above, various alternatives, modifications, variations, improvements, and/or substantial equivalents, whether known or that are, or may be, presently unforeseen, may become apparent to those having at least ordinary skill in the art. Accordingly, the exemplary embodiments of the invention, as set forth above, are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention. Therefore, the systems, methods and devices according to this invention are intended to embrace all known or later-developed alternatives, modifications, variations, improvements, and/or substantial equivalents.
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The present invention provides a process for preparing monoiodinated aromatic compounds that are useful as intermediates for preparing charge transporting and hole transporting amino compounds and have high purity with high yield and at low cost. A process for preparing aryl iodide compounds comprises reacting an aryl halide compound with a metal iodide, a metal catalyst and a catalyst coordinating ligand in at least one solvent to form an aryl iodide; and purifying the aryl iodide. A triarylamine compound and a process for preparing a triarylamine compounds reacting the aryl iodide with a diarylamine is also provided. Further, a photoconductive imaging member comprising a charge transport layer that comprises at least one triarylamine compound prepared reacting the aryl iodide with a diarylamine is provided.
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CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The instant application is a U.S. non-provisional Application that is based on and claims the benefit of U.S. provisional application No. 61/498,133, filed Jun. 17, 2011, the disclosure of which is hereby expressly incorporated by reference thereto in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to devices used to collect fluid samples from and/or inject fluids into patients. More specifically, this invention relates to a device which utilizes a holder utilizing a double-ended needle that can be released, retracted or removed from the holder in a more safe and easy manner. The device can be a single-use device or multi-use device. The invention also relates to a method of collecting a fluid sample with the device as well as a method of making the device. The invention also relates to a blood sample collection device that is easier to use, is less costly to produce, is safer to use, and/or is easier to manufacture.
[0004] 2. Discussion of Background Information
[0005] Prevention of needle sticks is of paramount concern in the healthcare industry because of serious and deadly risk factors associated with AIDS and other serious communicable diseases. Typical blood collection devices utilize a needle inserted into a patient's vein so as to draw blood through the needle into an associated separate collection reservoir. Accidental needle sticks from previously used needles can occur during the fluid withdrawing process and subsequent handling and disposal operation. Until such used medical devices are destroyed, they remain a risk to those handling them.
[0006] Devices used for blood sampling are well know and include a collection device sold under the trademark Vacutainer® by Becton Dickinson Corporation. This device has a tubular syringe-like body with a needle in the front end, part of which extends back into a tubular syringe-like shell. Part of the needle extends externally for punching the skin. An evacuated collection tube with a rubber stopper is placed into the open back of the syringe-like shell with the rubber stopper against the internal end of the needle. After the skin is punctured, the collection tube is pushed forward to cause the needle to enter the evacuated tube. Vacuum helps draw blood into the collecting tube. When a sufficient sample has been obtained, the collecting tube and the stopper are simply withdrawn from the tubular shell and sent to the laboratory. This particular device has a permanently extended needle and an opening in the back for the collection tube which remains open after the collection tube is removed, leaving small quantities of blood and an internally exposed needle.
[0007] Medical devices which are used for collecting fluid samples from patients which have quick release needle systems are also known. Such devices include: U.S. Pat. No. 5,797,490 to FUJI et al; U.S. Pat. No. 5,755,673 to KINSEY; U.S. Pat. No. 4,822,343 to BEISER; U.S. Pat. No. 4,984,580 WANAMAKER; U.S. Re. 38,964 to SHILLINGTON; U.S. Pat. No. 5,616,136 to SHILLINGTON et al.; U.S. Pat. No. 5,637,101 to SHILLINGTON; U.S. Pat. No. 5,117,837 to WANAMAKER et al.; U.S. Pat. No. 4,907,600 to SPENCER; U.S. Pat. No. 4,993,426 to SPENCER; U.S. Pat. No. 4,904,244 to HARSH et al.; U.S. Pat. No. 4,490,142 to SILVERN. The disclosures of each of these documents is expressly incorporated by reference herein in their entireties.
[0008] Medical devices which are used for collecting fluid samples from patients which can benefit from the improvement offered by the instant invention include: U.S. 2010/0286558 to SCHRAGA; U.S. 2008/0262421 to SCHRAGA; U.S. Ser. No. 12/974,908 to SCHRAGA filed on Dec. 21, 2010; and U.S. 61/480,787 to SCHRAGA filed on Apr. 29, 2011. The disclosures of each of these documents is expressly incorporated by reference herein in their entireties.
[0009] The invention aims to improve devices of the type described above by making a needle assembly for a fluid collection device which includes a standard interface needle. The device is also believed to be as safe or safer to use and/or dispose-of than the above-noted devices.
SUMMARY OF THE INVENTION
[0010] According to one non-limiting aspect of the invention there is provided a fluid collection/injection device including one or more features shown herein in combination with one or more devices disclosed above. In embodiments, there is provided a fluid collection/injection device including one or more features shown herein in combination with one or more devices disclosed in U.S. 2010/0286558 to SCHRAGA; U.S. 2008/0262421 to SCHRAGA; U.S. Ser. No. 12/974,908 to SCHRAGA filed on Dec. 21, 2010; and U.S. 61/480,787 to SCHRAGA filed on Apr. 29, 2011. The disclosures of each of these documents is expressly incorporated by reference herein in their entireties.
[0011] The invention provide for a fluid collection device comprising a body having a front end and a back end. The back end that is at least one of open and sized and configured to receive therein a receptacle. A needle holding member is arranged in an area of the front end of the body. A needle holding member comprising a needle connecting interface adapted to mate with a needle interface.
[0012] The needle holding member may be a spring biased member that is movable from an initial position to a retracted position within the body.
[0013] The needle connecting interface may be a luer-lock interface, a standard interface and/or a universal interface.
[0014] The fluid collection device may further comprise a spring structured and arranged to move the needle holding member to a retracted position within the body when a movable member arranged with the body experiences at least one of: a front end of the movable member is caused to expand generally radially; a back end of the movable member is caused to contract generally radially; a front end of the movable member is caused to expand generally radially when a back end of the movable member is caused to contract generally radially; axial movement caused by a cap closing off the back end of the body, wherein, during the closing off of the back end of the body, a tapered surface of a back end of the movable member is contacted by a portion of the cap; circumferential portions of a front end of the movable member are caused to expand outwardly or generally radially so as to allow the needle holding member to disengage from the front end of the movable member; and portions of a front end of the movable member separated by slots are caused to expand outwardly or generally radially so as to allow the needle holding member to disengage from the front end of the movable member.
[0015] The invention also provides for a fluid collection/injection device comprising a body having a front end, a back end, and a main hollow section arranged between the front and back ends. A needle hub securing section is arranged on the front end and being structured and arranged to receive therein a needle member. The needle hub securing section comprises a fixed part and a movable part. A needle holding member is connectable to the needle hub securing section. The needle holding member comprising a needle connecting interface adapted to mate with a needle interface. The fixed part is integrally formed with the front end and the movable part is arranged on a member that has one end which is one of: fixed to a portion of the main hollow section; connected to a portion of the main hollow section via a living hinge; removably connected to a portion of the main hollow section; and integrally formed with the main hollow section.
[0016] The body may be one of generally cylindrical and generally tubular.
[0017] The fixed part and the movable part may form a generally cylindrical section which the movable part is in an initial position.
[0018] The fixed part and the movable part may each generally comprise one-half of an internal locking thread structured and arranged to engage with an external thread of the needle member.
[0019] The member may have one end which is fixed to the portion of the main hollow section.
[0020] The member may have one end which is removably connected to the portion of the main hollow section.
[0021] The invention also provides for a retractable medical device comprising a body having and a back end that is at least one of open and sized and configured to receive therein a receptacle, a movable member arranged within the body, the movable member having a back end and a front end, a movable holding member arranged in an area of the front end of the movable member, a needle connecting interface adapted to mate with a needle interface and being movable with the movable holding member, and a spring structured and arranged to move the movable holding member to a retracted position within the movable member when the movable member experiences at least one of: the front end of the movable member is caused to expand generally radially; the back end of the movable member is caused to contract generally radially; the front end of the movable member is caused to expand generally radially when the back end of the movable member is caused to contract generally radially; axial movement caused by a cap closing off the back end of the body, wherein, during the closing off of the back end of the body, a tapered surface of the back end of the movable member is contacted by a portion of the cap; circumferential portions of the front end of the movable member are caused to expand outwardly or generally radially so as to allow the needle holding member to disengage from the front end of the movable member; and portions of the front end of the movable member separated by slots are caused to expand outwardly or generally radially so as to allow the needle holding member to disengage from the front end of the movable member.
[0022] The needle connecting interface may be a luer-lock interface, a standard interface and/or a universal interface.
[0023] The invention also provides for a fluid collection/injection device comprising a body having a front end, a back end, and a main hollow section arranged between the front and back ends, a needle hub securing section arranged on the front end, a needle receiving member having a needle connecting interface, and the fluid collection/injection device is structured and arranged to utilize at least an operational mode, an operational mode, and a post-use mode, in the installation mode, the needle receiving member being coupled to the body via the needle hub securing section, in the operational mode, fluid passing through the needle receiving member and into or out of a receptacle inserted into the main hollow section; and in the post-use mode, a safety cover at least one of; prevents re-use of the fluid collection/injection device; prevents removal of the needle receiving member from the fluid collection/injection device; prevents removal of the needle receiving member from the needle hub securing section; locks in a covering position and prevents removal of a needle from the needle receiving member; activates release of the needle receiving member from the body such that when the safety cover is in a position covering a needle of the needle receiving member, the needle receiving member can fall out of the needle hub securing section; activates release of the needle receiving member from the body such that when the safety cover is in a position covering a needle of the needle receiving member, the needle receiving member is no longer securely retained to the needle hub securing section; releases a securing engagement between the needle receiving member and the needle hub securing section; unlocks a locking connection between the needle receiving member and the needle hub securing section; and moves a mechanism arranged in an area of the needle hub securing section which releases a connection between the needle receiving member and the needle hub securing section.
[0024] The needle connecting interface is a luer-lock interface, a standard interface and/or a universal interface.
[0025] The invention also provides for a method drawing fluid using the device of any of the types disclosed herein, wherein the method comprises installing the needle connecting interface and thereafter installing a needle having a standard interface on the needle connecting interface.
[0026] The invention also provides for a method drawing fluid using the device of any of the types disclosed herein, wherein the method comprises installing a needle having a standard interface on the needle connecting interface of the needle holding member so as to form a needle assembly and thereafter installing the needle assembly on the body.
[0027] The invention also provides for a method drawing fluid using the device of any of the types disclosed herein, wherein the method comprises installing a needle having a standard interface on the needle connecting interface and one of injecting fluid through the needle and withdrawing fluid into a container installed in the body via the needle.
[0028] The invention also provides for a needle assembly for a fluid collection device comprising a needle holding member installable on a fluid collection device, a needle connecting interface arranged on one end of the needle holding member, and a needle having a needle interface, wherein the needle is at least one of installable on the needle holding member such that the needle is in fluid communication with another needle arranged on the needle holding member and removably installable on the needle holding member.
[0029] The invention also provides for a double-ended needle assembly comprising a needle holding member installable on a fluid collection device and comprising a needle connecting interface arranged on one end of the needle holding member and a needle arranged on another end and a needle having a standard needle interface compatible with the needle connecting interface of the needle holding member.
[0030] The needle may be at least one of installable on the needle holding member such that the needle is in fluid communication with the needle arranged on the needle holding member and removably installable on the needle holding member.
[0031] Other exemplary embodiments and advantages of the present invention may be ascertained by reviewing the present disclosure and the accompanying drawing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The present invention is further described in the detailed description which follows, in reference to the noted plurality of drawings by way of non-limiting examples of exemplary embodiments of the present invention, in which like reference numerals represent similar parts throughout the several views of the drawings, and wherein:
[0033] FIG. 1 shows a needle holding member in accordance with one non-limiting embodiment of the invention. The member is shown in a prior-use configuration such that an interface cover is shown arranged on the interface portion of the member;
[0034] FIG. 2 shows a needle member in accordance with one non-limiting embodiment of the invention. The needle member is shown in a prior-use configuration such that a needle cover is shown arranged on the needle portion of the needle member;
[0035] FIG. 3 shows the needle holding member of FIG. 1 in a ready to use configuration such that an interface cover is shown removed so as to expose the interface portion of the member;
[0036] FIG. 4 shows the needle holding member of FIG. 3 in a configuration wherein needle cover is shown retracted. This typically occurs only when the member is installed on a fluid collection device and a collection vial is fully inserted therein;
[0037] FIG. 5 shows a needle member similar to that shown in FIG. 2 in a ready to use configuration;
[0038] FIGS. 6 and 7 show different needle members from that of FIG. 5 , but which utilize the same interface or connecting portion so as to be usable with the member of FIG. 1 . In FIG. 6 , the needle is different or shorter in length than that of FIG. 5 . In FIG. 7 , the needle is different or shorter in length and different or smaller in diameter than that of FIG. 5 ;
[0039] FIG. 8 shows the needle member of FIG. 5 as it is about to be connected to the needle holding member of FIG. 3 . This can occur before the needle holding member is installed on a fluid collection device or after;
[0040] FIG. 9 shows a needle assembly resulting from the connection of the components shown in FIG. 8 ;
[0041] FIG. 10 shows the needle holding member of FIG. 3 installed on a fluid collection device in accordance with one non-limiting embodiment of the invention;
[0042] FIG. 11 shows the needle assembly of FIG. 9 installed on a fluid collection device in accordance with one non-limiting embodiment of the invention;
[0043] FIG. 12 shows a needle holding member in accordance with another non-limiting embodiment of the invention. The member is shown in a ready to use configuration such that an interface cover has been removed from the interface portion of the member;
[0044] FIG. 13 shows the needle holding member of FIG. 12 installed on a fluid collection device in accordance with another non-limiting embodiment of the invention;
[0045] FIG. 14 shows a needle assembly installed on a fluid collection device in accordance with another non-limiting embodiment of the invention;
[0046] FIG. 15 shows the device of FIG. 14 before the needle member is connected to the needle holding member already installed on the fluid collection device in accordance with another non-limiting embodiment of the invention;
[0047] FIG. 16 shows a needle holding member in accordance with another non-limiting embodiment of the invention. The member is shown in a prior-use configuration such that an interface cover is shown arranged on the interface portion of the member;
[0048] FIG. 17 shows a needle member in accordance with another non-limiting embodiment of the invention. The needle member is shown in a prior-use configuration such that a needle cover is shown arranged on the needle portion of the needle member;
[0049] FIG. 18 shows a needle assembly in accordance with another non-limiting embodiment of the invention. In this embodiment, the needle member is already installed on the needle holding member, a needle cover is installed on the needle of the needle member, and a safety cover is pivotally connected to the needle holding member; and
[0050] FIG. 19 shows a needle holding member in accordance with another non-limiting embodiment of the invention. The member has two main components which are threadably connected to one another.
DETAILED DESCRIPTION OF THE INVENTION
[0051] FIGS. 1-4 show a first non-limiting embodiment of the invention. In FIG. 1 , a needle holding member 10 in accordance with one non-limiting embodiment of the invention has hub portion 11 , a connecting interface portion 12 , a retaining section 13 , an inner hollow needle 14 , and a retractable needle cover 15 . The configuration of the interface 12 , i.e., the receiving portion thereof, can be of any conventional standard type interface such as a luer-lok type interface. Similarly, the configuration of elements 13 , 14 , and 15 can be of any conventional type.
[0052] In FIG. 2 , a needle member 20 in accordance with one non-limiting embodiment of the invention is shown in a prior-use configuration. In this configuration, a removable needle cover 30 is shown mounted covering the hollow needle portion 21 of the needle member 20 . The cover 30 has a closed proximal end and an open distal end which is removably and releasably connected (via e.g., a snap and/or projection/recess connection) to the connecting interface section 23 of the member 20 .
[0053] In FIG. 3 , the needle holding member 10 of FIG. 1 is shown in a ready to use configuration such that an interface cover 40 (see FIG. 1 ) has been removed so as to expose the interface portion 12 of the member 10 .
[0054] FIG. 4 shows the needle holding member 10 of FIG. 3 in a configuration wherein needle cover 15 is retracted to expose the inner needle 14 . This typically occurs only when the member 10 is installed on a fluid collection device (see e.g., FIG. 10 ) and when a collection vial is fully inserted into the fluid collection device. A typical vacutainer-type vial has an insertion end whose self-sealing septum can be punctured by the needle 14 and can cause retraction of the flexible cover 15 during insertion of the vial.
[0055] FIGS. 5-7 show how different sized needle members 20 having a common standard connecting interface can be used with the needle holding member 10 of FIG. 1 . In FIG. 5 , the needle portion of the member 20 A has a diameter and an axial length that is larger than that of the member of FIG. 7 . In FIG. 6 , the needle portion of the member 20 B has a diameter that is larger than that of the member of FIG. 7 , but a length that is longer. In FIG. 7 , the needle portion of the member 20 C has a diameter that is smaller than that of the member 20 A of FIG. 5 , but a length that is between that of FIGS. 5 and 6 .
[0056] FIG. 8 shows the needle member of FIG. 5 as it is about to be connected to the needle holding member 10 of FIG. 3 . This can occur, by way of non-limiting example, before the needle holding member 10 is installed on a fluid collection device or after installed therein. FIG. 9 shows a needle assembly NA resulting from the connection of the components, i.e., needle member 20 and holding member 10 , shown in FIG. 8 .
[0057] According to one non-limiting example, a user can connect these components while the cover 30 is installed to make for a more safe assembly of the assembly NA. Assembly of these components can also occur in a factory setting so that the assembly NA shown in FIG. 9 (but optionally additionally including at least a safety cover 30 on the forward needle) is packaged in an assembled state. In this case, a user can remove the assembly NA from its packaging and install it on a fluid collection device. In other embodiments, the needle member 20 and holding member 10 are each packaged separately and in a non-attached manner. In other embodiments, the needle member 20 and holding member 10 are each packaged separately, but with both packages attached to one another. In still other embodiments, two or more needle members 20 of the same type are packaged together. In still other embodiments, two or more needle members 20 of different types, i.e., needle sizes, are packaged together. In still other embodiments, one holding member 10 and two or more, e.g., different, needle members 20 are placed in the same package in an unassembled state, e.g., in a factory setting. This allows a user to customize the needle assembly NA and/or tailor it to the circumstances. For example, if the package contains a single holding member 10 and, e.g., three needle members of the type as shown in FIGS. 5-7 , the user can place any one of the needle members 20 A- 20 C on the holding member 10 depending on which needle member 20 A- 20 C is called for and discard the other two. It is also possible to package a single holding member 10 along with, e.g., three individually packaged needle members of the type as shown in FIGS. 5-7 . The user can place any one of the needle members 20 A- 20 C on the holding member 10 depending on which needle member 20 A- 20 C is called for and store the packaged and unused other two.
[0058] FIG. 10 shows the needle holding member 10 of FIG. 3 installed on a fluid collection device 80 in accordance with one non-limiting embodiment of the invention. In embodiments, the fluid collection device 80 can be of any of the types disclosed in U.S. 2010/0286558 to SCHRAGA. In other embodiments, the invention can also be used with any one of the fluid collection systems disclosed in U.S. 2011/0160613 to SCHRAGA. The disclosure of each of these documents is expressly incorporated by reference herein in their entireties. As details of the fluid collection devices are described in these documents, additional details of such devices are not described herein. In still other embodiments, the fluid collection device 80 can be of any of the types conventionally known provided they function with a holing member 10 having a common or standard interface. FIG. 11 shows the needle assembly NA of FIG. 9 installed on a fluid collection device 80 in accordance with one non-limiting embodiment of the invention. Again, in embodiments, the fluid collection device 80 can be of any of the types disclosed in U.S. 2010/0286558 to SCHRAGA. In other embodiments, the invention can also be used with any one of the fluid collection systems disclosed in U.S. 2011/0160613 to SCHRAGA.
[0059] FIG. 12 shows another non-limiting embodiment of the invention. In FIG. 12 , a needle holding member 100 in accordance with one non-limiting embodiment of the invention has hub portion 111 , a connecting interface portion 112 , a retaining section 113 , an inner hollow needle, and a retractable needle cover 115 . The configuration of the interface 112 , i.e., the receiving portion thereof, can be of any conventional standard type interface such as a luer-lok type interface. Similarly, the configuration of elements 14 and 15 can be of any conventional type. In embodiments, the member 100 can be of any of the types disclosed in U.S. 2008/0262421 to SCHRAGA with the exception that it utilizes the interface 112 and a needle member 20 of the type described above such as that shown in FIGS. 5-7 . The disclosure of U.S. 2008/0262421 is expressly incorporated by reference herein in its entirety. FIG. 13 shows one way in which a fluid collection device 90 of the type disclosed in U.S. 2008/0262421 can be modified to utilize the holding member 100 of FIG. 12 . The normally compressed spring 50 is utilized to automatically cause the member 100 (typically with the needle member 20 attached) to retract into the body of the device 90 when activated after use.
[0060] FIG. 14 shows a needle assembly installed on a fluid collection device 90 in accordance with another non-limiting embodiment of the invention. The needle assembly includes the holder member 100 and a needle member 200 shown schematically. In embodiments, the needle member 200 can be of same as the member 20 shown and described above. The fluid collection device 90 can function in a manner similar to similar devices described in U.S. 2008/0262421. For example, the device 90 can function as follows: after the device 90 is used to obtain a fluid sample, the use can move the cap C to the closed position. Upon doing so, the cap C becomes non-releasably locked and the member MM is caused to move forward slightly. This movement, in turn, results in the proximal portion of the member MM expanding radially. This radial expansion causes the holder member 100 to disengage from the member MM. At this point, the spring 50 is free to expand axially and cause the member 100 (with the needle member 200 connected thereto) to retract fully into the device 90 and be safely contained therein so that the used device 90 can be handled safely and disposed of in a safer manner. The details of how such a device (but without the member 100 / 200 combination) functions are disclosed in U.S. 2008/0262421 and are therefore not described in more detail herein. FIG. 15 shows the device of FIG. 14 before the (schematically shown) needle member 200 is connected to the needle holding member 100 already installed on the fluid collection device 90 in accordance with another non-limiting embodiment of the invention.
[0061] FIGS. 16 and 17 show another non-limiting embodiment of the invention. In FIG. 16 , a needle holding member 100 ′ in accordance with another non-limiting embodiment of the invention has hub portion 111 ′, a connecting interface portion 112 ′, a retaining section 113 ′, an inner hollow needle, and a retractable needle cover 115 ′ normally covering the inner needle. The configuration of the interface 112 ′, i.e., the receiving portion thereof, can be of any conventional standard type interface such as a luer-lok type interface without the locking outer portion. Similarly, the configuration of elements 113 ′ and 115 ′ can be of any conventional type. An optional removable 40 can be used to protect the interface 112 ′.
[0062] In FIG. 17 , a needle member 200 ′ in accordance with another non-limiting embodiment of the invention is shown in a prior-use configuration. In this configuration, a removable needle cover 30 is shown mounted covering the hollow needle portion 210 ′ of the needle member 200 ′. The cover 30 has a closed proximal end and an open distal end which is removably and releasably connected (via e.g., a snap and/or projection/recess connection) to the connecting interface section 220 ′ of the member 200 ′.
[0063] FIG. 18 shows another non-limiting embodiment of the invention. In FIG. 18 , a needle holding member 100 ″ in accordance with another non-limiting embodiment of the invention has hub portion 111 ″, a connecting interface portion, a retaining section 113 ″, an inner hollow needle, and a retractable needle cover 115 ″ normally covering the inner needle. The configuration of the interface, i.e., the receiving portion thereof, can be similar to that of FIG. 1 and/or of any conventional standard type interface such as a luer-lok type interface. In addition, the member 100 ″ includes two oppositely arranged projections P arranged on the section 111 ″ to which is pivotally mounted a needle shield 60 . An optional safety cover 70 covers the needle 210 ″ of the removably mounted needle member 200 ″. The details of how such a device (but without the removable connection between members 100 ″/ 200 ″) functions are disclosed in U.S. Ser. No. 13/458,468 filed on Apr. 27, 2012 to SCHRAGA and are therefore not described in more detail herein. The disclosure of U.S. Ser. No. 13/458,468 filed on Apr. 27, 2012 is expressly incorporated by reference herein in its entirety. In embodiments, the assembly NA′ shown in FIG. 18 is packaged for use in the configuration shown. To use the assembly, a user need only install the assembly on a fluid collection device of the type shown in, e.g., FIG. 11 , and then remove the cover 70 . After use, the user can flip up the shield 60 so that it non-removably covers the needle 210 ″. In this position, the user is prevented from removing the needle member 200 ″ from the holding member 100 ″.
[0064] FIG. 19 shows another non-limiting embodiment of the invention. In FIG. 19 , a needle holding member 100 III in accordance with another non-limiting embodiment of the invention has hub portion 111 III , a connecting interface portion, a retaining section 113 III threadably and/or removably installable and/or installable only once on the section 100 a III , an inner hollow needle, and a retractable needle cover 115 III normally covering the inner needle. The configuration of the interface, i.e., the receiving portion thereof, can be similar to that of FIG. 1 and/or of any conventional standard type interface such as a luer-lok type interface. In embodiments, the assembly shown in FIG. 19 is packaged for use in the configuration shown. To use the assembly, a user need only install the assembly on a fluid collection device of the type shown in, e.g., FIG. 14 . The user can then install one of the needle members of the type shown in FIGS. 5-7 . After use, both the needle member and the assembly 100 III are safely arranged within the body shown I FIG. 14 and can be safely disposed of.
[0065] The devices described herein can also utilize one or more features disclosed in the documents expressly incorporated by reference herein. Furthermore, one or more of the various parts of the device can preferably be made as one-piece structures by e.g., injection molding, when doing so reduces costs of manufacture. Non-limiting materials for most of the parts include synthetic resins such as those approved for syringes, blood collection devices, or other medical devices. Furthermore, the invention also contemplates that any or all disclosed features of one embodiment may be used on other disclosed embodiments, to the extent such modifications function for their intended purpose.
[0066] It is noted that the foregoing examples have been provided merely for the purpose of explanation and are in no way to be construed as limiting of the present invention. While the present invention has been described with reference to an exemplary embodiment, it is understood that the words which have been used herein are words of description and illustration, rather than words of limitation. Changes may be made, within the purview of the appended claims, as presently stated and as amended, without departing from the scope and spirit of the present invention in its aspects. Although the present invention has been described herein with reference to particular means, materials and embodiments, the present invention is not intended to be limited to the particulars disclosed herein; rather, the present invention extends to all functionally equivalent structures, methods and uses, such as are within the scope of the appended claims.
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Fluid collection device includes a body having a front end and a back end. The back end is open and/or sized and configured to receive therein a receptacle. A needle holding member is arranged in an area of the front end of the body. A needle holding member comprising a needle connecting interface adapted to mate with a needle interface
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CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. §119 of European Patent Application EP 09 158 904.4 filed Apr. 28, 2009, the entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a slot valve for use in the pneumatic switching circuit of a respirator (also known as ventilator) and more particularly to a valve connected at a tube (e.g., an endotracheal tube or a tracheotomy cannula) coupled with the respirator.
BACKGROUND OF THE INVENTION
[0003] ARDS (acute respiratory deficiency syndrome) designates a sudden respiratory failure, which develops due to an acute inflammatory process of the lung tissue, in which the lungs extensively lose their ability to exchange gas. The permeability of the blood vessels in the pulmonary alveoli increases in ARDS and the pressure in the vessels drops, whereas it rises in other parts of the lung tissue. This leads to a life-threatening shortness of breath and to insufficient oxygen supply for the blood. The life-threatening hypoxia in the blood must be treated as quickly as possible by mechanically supporting breathing, i.e., artificial respiration with air enriched with oxygen. However, noninvasive respiration methods with merely increasing the oxygen concentration in the breathing air being supplied are often insufficient for the treatment of ARDS, because respirated ARDS patients have atelectatic (i.e., collapsed) lung areas, which can be opened (recruited) and made usable for gas exchange with a high respiration pressure only. However, the patient must be intubated for this, i.e., a tube (flexible tube) is pushed into the patient's trachea through the mouth or through the nose. The respiration is preferably carried out via an endotracheal tube or via a tracheotomy cannula. An endotracheal tube normally comprises a thin flexible tube, which is opened at both ends and whose lower end is pushed into the trachea. A cuff, which can be inflated via a thin flexible tube, which extends on the side of the tube, is located a short distance above the lower end. The trachea is sealed hereby. At the upper end, the endotracheal tube is equipped with a standardized connection piece, which makes possible connection to a respirator. A tracheotomy cannula is used in case of tracheotomy. The tracheotomy cannula also has an inflatable “block,” which makes respiration possible and at the same time prevents pharyngeal secretion from entering the lungs downwards.
[0004] A residual pressure (PEEP=positive end-expiratory pressure) is preferably maintained at the end of expiration during the artificial respiration of ARDS patients. The pressure in the pulmonary alveoli is increased by the PEEP respiration, as a result of which the pulmonary alveoli are expanded, which leads to an enlargement of the area for the gas exchange and thus to an improvement of oxygen uptake. Furthermore, the risk of collapse of the pulmonary alveoli during expiration is reduced. The end-expiratory pressure in PEEP respiration frequently equals 10 mbar or higher in ARDS patients in order to prevent the lung areas opened with difficulty from recollapsing. It is necessary in most cases to artificially respirate an ARDS patient over several days or even weeks. However, some steps are necessary in the course of respiration in routine clinical practice, for example, suction of fluids from the lungs, repositioning of the patient, changing of the tube system, of the filter or of the respirator. The necessary pressure in the lungs cannot be continuously maintained during the performance of these clinically necessary steps, so that the damaged lung areas must be recruited time and time again.
[0005] U.S. Pat. No. 4,351,328 describes an adapter, which is designed to connect a respirator and an endotracheal tube. The adapter is provided, furthermore, with an opening, which is closed by means of a valve. The valve is designed as a slot valve and can be pierced by the suction tube when the latter is introduced into the opening from the outside.
[0006] U.S. Pat. No. 4,416,273 discloses a connection adapter for an endotracheal tube. The adapter has a port provided with a lamellar valve in order to make it possible to insert a suction catheter into the tube from the outside.
[0007] DE 32 04 110 C2 pertains to a tracheal tube for artificial respiration. The lower part of the tracheal tube is surrounded by a balloon cuff, which can be inflated via an inflating cannula to the extent that it comes into contact with the tracheal wall. A breathing tube connected to a respirator and a pressure-measuring cannula are provided in the interior space of the tracheal tube in order to make it possible to measure the pressure drop in the breathing tube or the intratracheal pressure.
[0008] DE 198 38 370 C1 describes a device for removing sputum from a tracheal catheter. The device has three openings, wherein a first opening is connected to the end of the catheter projecting from the trachea, a second opening can be coupled with an air filter for cleaning and sterilizing the air to be breathed in, and a third opening is connected to a collecting bag into which the sputum can flow. A spring-tensioned piston for closing the third opening is controlled by the breathing air during inspiration and expiration.
[0009] DE 41 42 295 C2 pertains to a valve for generating a control pressure in a pneumatic switching circuit. The valve has the embodiment of a circular closing element and has incisions, so that eight circle segments are formed, which can be bent up around the circumferential line of the closing segment. The extent of bending up changes depending on the pressure of the fluid acting on one side of the valve.
[0010] DE 10 2005 014 650 B3 discloses a connection piece with a distal end and a proximal end for connecting a tracheal tube and a respirator as well as with a branch for inserting a catheter. A valve made of material deformable elastically at least in some areas is provided in the branch, and said valve forms a beak section with a slot, which is opened during the insertion of the catheter. Closed suction systems, as they are shown in this document, prevent only the pressure during suction. Changing of the device and changing of the closed suction system itself, which is necessary at 48-hour intervals for hygienic reasons, continue to lead to collapse of the lungs and to a subsequent stressful recruitment maneuver.
[0011] None of the above-mentioned documents pertains to the respiration of ARDS patients, and none of these documents discloses respirators or respirating means in the pneumatic switching circuit between a respirator and a patient, which are designed to maintain a certain air pressure in the lungs of the patient to be respirated even when, for example, the respirator is being replaced.
SUMMARY OF THE INVENTION
[0012] The object of the present invention is therefore to make available a valve for use in the pneumatic switching circuit of a respirator, by means of which the above-mentioned drawbacks are overcome. In particular, it is the object of the present invention to make available a valve for use in or at a tube (e.g., an endotracheal tube or a tracheotomy cannula) coupled with the respirator, by means of which a pressure drop in the lungs of a patient (especially of an ARDS patient) can be effectively prevented from occurring.
[0013] These and other objects are accomplished by means of a slot valve having bidirectionally acting means for responding at different pressure threshold values depending on the direction of flow of a fluid.
[0014] An essential advantage of the slot valve according to the present invention is that the valve acts bidirectionally and responds independently from the direction of flow or the direction of action of a fluid at different threshold pressures.
[0015] Another advantage is that the slot valve according to the present invention is a self-closing valve and passes over into the opened state only when a threshold pressure is overcome in order to make it possible for a fluid to flow through. The quantity of fluid flowing through depends on the pressure of the fluid. Flow through the valve according to the present invention is possible from both directions (i.e., bidirectionally), and the threshold pressure that brings about opening of the slot valve differs depending on the direction of flow. The threshold pressure is preferably in a range of about 0 mbar to about 5 mbar in a first direction and in a range of about 5 mbar to about 15 mbar in a second direction. However, the exact value of these threshold values depends on the field of application of the valve and may also be, for example, in the range from markedly above 10 mar to a few 100 mbar.
[0016] The slot valve according to the present invention is preferably used in the pneumatic switching circuit of a respirator and is consequently arranged in the flow path between the respirator and the patient to be respirated, especially an ARDS patient, who is preferably respirated in the PEEP mode. The pressure drop in the patient's lungs to below a predetermined pressure level of, for example, between about 5 and 15 mbar can be prevented from occurring by means of the slot valve according to the present invention during disconnection at the endotracheal tube or at the tracheotomy cannula (when, for example, the respirator is being replaced). Furthermore, the valve is designed to make inspiration possible without the patient having to generate a high suctioning pressure for this. The threshold value for opening the valve in the suction direction is therefore preferably below 5 mbar. The necessary threshold pressure to open the valve must be higher in the direction of expiration and equals more than 5 mbar, preferably more than 10 mbar or in exceptional cases more than 15 mbar, and these values may also vary in an especially advantageous embodiment depending on the patient to be respirated, depending on the intensity of the ARDS and depending on other factors. Furthermore, the valve is designed to make it possible to suction fluids from the lungs by means of a special cannula. The slot valve according to the present invention has a hygienic design, which permits use for more than 1 week. In a preferred embodiment the valve is designed in a special manner in order not to hinder the flow of breath during normal respiration. The slot valve according to the present invention is provided for this purpose with means that can be actuated manually in order to make a changeover between different modes of operation possible in a simple manner.
[0017] The slot valve according to the present invention is formed by a membrane made of an elastic plastic or rubber in a preferred embodiment. The membrane preferably has a round basic shape, i.e., a circular contour line. Other shapes, for example, oval, rectangular or square shapes, are, of course, also conceivable, but the round shape is preferred because of the symmetry. A plurality of mutually intersecting slots, which fully extend through the membrane and thus form a plurality of circle segment-shaped lamellae, are provided in the middle of the membrane. For example, a total of four lamellae are formed by two slots intersecting each other at right angles, and six lamellae are formed by three slots, etc. Embodiments with more than six lamellae are possible as well.
[0018] The lamellae are closed, i.e., the slot surfaces of adjacent lamellae sealing abut against one another in the resting state (i.e., when there is essentially no pressure difference between the opposite sides of the membrane. According to a first embodiment, the membrane lamellae are located in an arched surface in their closed position. This arched surface may have, for example, the shape of a dome or of a spherical surface segment or correspond to the outer surface of a flat cone or of a flat pyramid. Regardless of the selected design, the lamellae are designed such that they open more easily in a first direction of flow than in a second direction when pressure (which acts, for example, via a fluid on the membrane lamellae) is applied. Consequently, no or only a low fluid pressure (first threshold pressure) is necessary in a first direction of flow to bend the lamellae from their closed position into their opened position (i.e., to open the valve), whereas a higher pressure (second threshold pressure) is necessary in the opposite, second direction of flow.
[0019] According to a second embodiment, the membrane lamellae are in an essentially planar plane in this closed state and are designed to bring about different threshold values to open the valve or the lamellae when pressure is being applied in different directions. This can be achieved by the thickness of the material of the lamellae being greater in an axial direction than the thickness of the material of the annular edge area or supporting ring of the valve membrane. It can be achieved hereby that the contact area of the slot surfaces is relatively large in the axial direction. However, the pivot lines of the lamellae which are present in the transition between the supporting ring and the lamellae are slightly offset at the same time in relation between the axial center of the radially extending contact areas between the lamellae, so that a higher pressure must be built up in an axial direction to pivot or fold over the lamellae from the closed position into the opened position. As an alternative, the thickness of the material of the lamellae may increase starting from the pivot lines of the lamellae in the radial direction towards the center of the valve membrane, whereby a similar effect is brought about. It is also possible to provide an annular groove on one side of the valve membrane. The “film hinges” for the membrane lamellae are formed by this groove, but the pivot lines of the hinges are shifted at the same time in the axial direction due to the provision of the annular groove. Consequently the threshold pressure for opening the valve is higher in one direction than in the opposite direction in this embodiment as well. The magnitude of the particular threshold values can be determined by the thickness of the membrane material, depth of the groove and elasticity of the membrane material. Furthermore, it is possible to provide the membrane lamellae with axially extending projections on one side directly at the slot surfaces abutting against each other between adjacent lamellae in order to thus enlarge the contact areas of the slots in the axial direction, as a result of which a relative axial displacement of the pivot lines of the lamellae is brought about at the same time.
[0020] The slot valve according to the present invention is suitable, for example, for being used in the breathing circuit for positive pressure respiration (e.g., PEEP) between a respirator and the patient. However, other applications, in which a similar valve function is desired, are conceivable as well.
[0021] The slot valve according to the present invention preferably has an essentially tubular valve housing with two ports generally located opposite each other and breathing gas can flow through it in two opposite direction. As was explained above, the slot valve according to the present invention has, in the flow channel between the two ports, a slotted membrane, which is fixed at its annular edge directly or indirectly in the housing and has membrane lamellae separated by incisions essentially radially in its center. According to a preferred embodiment, the membrane lamellae are arched at least partly such that when the valve is closed, the convex side of the arch points in the direction of the patient. During expiration, a moderate overpressure on the patient side against the convex arch at first leads to pressing of the arch along the radial partition lines (slots) between the membrane lamellae and hence to blocking of the flow channel. It is only when a predetermined threshold pressure of, for example, about 10 mbar is exceeded that this blocking force is overcome, the lamellae are folded over in the opposite direction (i.e., opposite the direction of the arching) and the flow channel is released for breathing gas to flow through. When the pressure again drops below the threshold pressure, the membrane lamellae fold back into their original arched position because of their own restoring forces and the flow channel is again blocked. By contrast, an overpressure on the opposite side (i.e., on the side of the respirator) against the concave underside of the arch leads to an immediate, nearly forceless flow of fluid in the direction of the patient during inspiration, because only a very low threshold value is necessary to open the lamellae.
[0022] The valve according to the present invention may be designed, furthermore, as a “pop-up” valve. A depressible or foldable, annular intermediate area is provided for this between the inner, circular slot area and the outer supporting ring. This intermediate area is folded up or into one another in the closed resting state of the valve or in the state that prevails when the patient is breathing in and only a low threshold value (overpressure on the side of the respirator) is necessary for opening the membrane lamellae. When the patient is breathing out (overpressure on the patient side), the folded-up intermediate area is first unfolded. If the pressure rises further and exceeds said threshold value (e.g., 10 mbar or more), the membrane lamellae are folded over as well. It was found that the membrane lamellae are pressed better against each other when the intermediate area is folded up and overpressure prevails on the patient side. It is only when the intermediate area is unfolded that the transition section between the membrane lamellae and the intermediate area acquires the necessary flexibility to make it possible for the membrane lamellae to fold over easily when the patient-side pressure exceeds the predetermined threshold value during expiration. The foldable intermediate area thus acts as a kind of securing means against premature folding over of the membrane lamellae under the threshold pressure.
[0023] A releasing means, which folds the membrane lamellae out of the closed position on actuation, so that the flow channel in the interior of the valve is released, is additionally provided in another advantageous embodiment of the present invention. In a preferred embodiment of the slot valve according to the present invention, the valve is inserted into the breathing circuit between an endotracheal tube or a tracheotomy cannula on one side (patient side) and the filter, artificial nose, closed suction system or Y-piece on the other side (respirator side). The valve has a housing, a slot membrane with, for example, four or six radially extending slots, as a result of which cut membrane lamellae are formed, a rotary ring and a spreader. The spreader can be pushed into the range of action of the slot membrane such that the membrane lamellae of the slot membrane permanently release the flow center and do not represent a relevant flow resistance. The spreader has two oblique holding noses, which open through the housing into a groove of the rotary ring, which said groove is designed as an oblique path. The rotary ring additionally has an annular groove, into which snaps a bead of the housing. As a result, the rotary ring is fixed against axial displacement at the housing. When rotating the rotary ring, the spreader is displaced axially over the two oblique paths and can thus be brought optionally into a position close to the Y-piece, where the spreader does not mesh with the membrane and a pressure drop in the patient's lungs below, for example, 10 mbar is avoided. The spreader meshes with the membrane lamellae in the opposite position near the patient and pushes same out of the flow center, whereby unhindered, bidirectional flow of fluid through the valve is made possible.
[0024] The present invention will now be described on the basis of an example with reference to the drawings. The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and specific objects attained by its uses, reference is made to the accompanying drawings and descriptive matter in which preferred embodiments of the invention are illustrated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In the drawings:
[0026] FIG. 1 a is a schematic top view of an exemplary embodiment according to the present invention;
[0027] FIG. 1 b is a cross-sectional view through line A-A from FIG. 1 a , in which the arched lamella area is seen. The arrows indicate the direction of pressure in which a lower threshold value is necessary to open the valve;
[0028] FIG. 1 c is a cross-sectional view through line A-A from FIG. 1 a , in which an alternative embodiment to FIG. 1 b with varying material thickness of the membrane lamellae is seen;
[0029] FIG. 1 d is a cross-sectional view through line A-A from FIG. 1 a , in which another alternative embodiment to FIG. 1 c with constant material thickness of the membrane lamellae is seen, wherein the material thickness of the supporting ring is markedly smaller than that of the membrane lamellae;
[0030] FIG. 1 e is a cross-sectional view through line A-A from FIG. 1 a , in which another alternative embodiment to FIG. 1 d with constant material thickness of the entire membrane is seen, wherein an annular groove is provided between the supporting ring and the middle lamella area;
[0031] FIG. 2 a is an embodiment of the slot membrane according to the present invention from FIG. 1 b in the closed state;
[0032] FIG. 2 b shows the slot membrane from FIG. 2 a in an opened state, wherein the pressure acts in the direction of inspiration and only a very low pressure is necessary to press the membrane lamellae downwards;
[0033] FIG. 2 c shows the slot membrane from FIG. 2 a in an opened state, wherein the pressure acts in the direction of expiration and a rather high pressure threshold value is necessary to fold over the membrane lamellae upwards;
[0034] FIG. 3 a is another preferred embodiment of the slot valve from FIGS. 1 and 2 in the closed state;
[0035] FIG. 3 b shows the slot valve from FIG. 3 a shortly before the opened state is reached in the direction of expiration;
[0036] FIG. 3 c is a variant of the slot valve from FIG. 3 a in the closed state;
[0037] FIG. 3 d shows the slot valve from FIG. 3 c shortly before the opened state is reached in the direction of expiration;
[0038] FIG. 4 is another embodiment of the slot valve according to the present invention, which is provided with a releasing means that can be actuated manually.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] Referring to the drawings in particular, the figures will be described in detail below. FIG. 1 a shows a top view of an exemplary slot membrane according to the present invention in its simplest embodiment. The slot membrane 1 has essentially the shape of a disk and is made of rubber, silicone rubber or a suitable elastic plastic material. The slot membrane 1 comprises a circular lamella area 3 with lamellae 3 a through 3 d , which are formed by slots 4 a , 4 b that intersect each other, and an annular supporting area 2 , which surrounds the lamella area 3 and is used to fasten the slot membrane in a preferably annular valve housing. FIG. 1 a shows two slots that intersect each other, as a result of which four circle segment-shaped lamellae are formed. However, it is also possible to provide three or more slots that intersect each other, as a result of which the total number of lamellae increases correspondingly.
[0040] FIG. 1 b shows a cross-sectional view through line A-A from FIG. 1 a , in which the arched lamella area 3 is seen along slot 4 a . The membrane lamellae 3 a - 3 d are in their closed position in an arched surface in this embodiment, which surface has the shape of a dome or a spherical surface segment in this case. However, the lamellae may also form the outer surface of a flat cone or of a flat pyramid. The lamellae are closed in the resting state being shown, and the slot surfaces of adjacent lamellae sealingly abut against one another. Arrows 5 show the direction of pressure of a fluid when an overpressure prevails on the side of a respirator (not shown), as it happens during inspiration by the patient. Only a low pressure threshold value is necessary here to fold the lamellae down to open the valve. A slight overpressure, which presses the downwardly pointing, convex arch of the lamellae and leads to the membrane lamellae being pressed mutually and hence to blocking of the flow channel, is at first generated during expiration (cf. FIG. 2 c ) by the patient on the patient side. It is only when a predetermined threshold pressure between about 5 mbar and 15 mbar is exceeded that this blocking force is overcome, the lamellae 3 a - 3 d are folded upward and the flow channel is released. When the pressure again drops below the threshold pressure, the membrane lamellae fold back into their arched starting position because their own restoring forces and the flow channel is again blocked.
[0041] FIG. 1 c shows a cross-sectional view through line A-A from FIG. 1 a , in which an alternative embodiment of the lamellae 3 a - 3 d is seen. As is shown, the lamellae have a thickness increasing towards the center, as a result of which the contact surfaces between adjacent membrane lamellae rise in the axial direction in the area of the slots 4 a , 4 b . Since the pivot lines 6 of the lamellae are located above the center 7 of the contact surface in the axial direction in the figures, the lamellae can be pivoted downward with a low pressure, whereas a higher pressure is necessary to pivot the lamellae upward.
[0042] FIG. 1 d shows an alternative embodiment to FIG. 1 c , in which the membrane lamellae 3 a - 3 b have a constant material thickness, which is greater than the material thickness of the annular supporting area 2 . Pivot line 6 of the lamellae is above the center 7 of the contact surface between the lamellae in the axial direction in this case as well. As a result, the lamellae can be pivoted downwards with a low pressure during inspiration, whereas a higher threshold pressure is needed to pivot the lamellae upwards.
[0043] FIG. 1 e shows a cross-sectional view through line A-A from FIG. 1 a , in which an alternative embodiment to FIG. 1 d with a constant material thickness of the entire slot membrane 1 is seen. An annular groove 8 is provided between the supporting ring 2 and the middle lamella area 3 . The function of the different pressure threshold values in different directions of flow is embodied in this embodiment as well.
[0044] It is obvious that the slot membrane 1 from FIGS. 1 a through 1 e can be mounted into a preferably annular valve housing, wherein said supporting ring 2 can be inserted, for example, into an annular groove in the interior of the valve housing. Other modes of fastening, for example, bonding, welding, fusion, etc., are conceivable as well.
[0045] FIG. 2 a shows an embodiment of the slot membrane according to the present invention from FIG. 1 b in the closed state. No pressure prevails on either side in the closed state or the pressures are below the threshold values on both sides.
[0046] FIG. 2 b shows the slot membrane from FIG. 2 a in a downwardly opened state, wherein the pressure in the direction of inspiration is above a low predetermined threshold pressure of, for example, 0 to 5 mbar, which is necessary to push the membrane lamellae 3 a - 3 d downwards.
[0047] FIG. 2 c shows the slot membrane from FIG. 2 a in an opened state, wherein the pressure acts in the direction of expiration. This pressure is above the threshold pressure of, for example, 10 or 15 mbar, which is necessary to push the membrane lamellae 3 a - 3 d upwards.
[0048] FIG. 3 a shows a preferred embodiment of the slot valve from FIGS. 1 and 2 in the closed state or shortly before the opened state is reached in the direction of inspiration (overpressure on the side of the respirator). Valve 10 preferably has an essentially tubular valve housing 11 with two generally opposite ports and breathing gas can flow through it in two opposite directions. Valve housing 11 may be designed to enable an adapter or a tube to be connected. A slot membrane 3 , which is fastened to the valve housing at its annular supporting area 2 , is provided between the two ports 2 . The slot membrane 3 may have one of the embodiments shown in FIGS. 1 and 2 . The valve 10 shown in FIGS. 3 a through 3 d is designed as a “pop-up” valve. A depressible or foldable, annular intermediate area 12 , which may have different shapes, as this can best be seen in FIGS. 3 a and 3 d , is provided between the inner circular lamella area 3 and the outer supporting ring 2 . As is shown in FIG. 3 b , the intermediate area 12 is formed by an essentially conically tapering ring section, whereas the intermediate area 12 in FIG. 3 d is designed as a cylindrical ring section, which is joined by a radially outwardly curved wall section, which is in turn connected to the supporting ring 2 . FIG. 3 a shows the closed resting state of the valve or the state that prevails when the patient is breathing in and only a low threshold value of, for example, less than 5 mbar (overpressure on the side of the respirator) is necessary for opening the membrane lamellae 3 . When the pressure threshold value is exceeded, the lamellae 3 a - 3 d would open in the downward direction, as this is shown in FIG. 2 b . It is obvious that the lamella area 3 may have one of the configurations shown in FIGS. 1 b through 1 e.
[0049] As is shown in FIGS. 3 a and 3 c , the intermediate area 12 can be folded up or into one another. In this state the membrane lamellae 3 a - 3 d are closed or folded downwards when the pressure threshold value is exceeded (overpressure on the side of the respirator=inspiration by the patient). When the patient stops breathing in, the lamellae are again closed because of their restoring force. When the patient begins to breath out, the lamellae are at first pressed against each other, whereby the lamellae are prevented from being folded over. Folding over is prevented, furthermore, by the folded-up intermediate area 12 applying a radially inwardly directed force to the lamellae. If the pressure continues to rise on the patient side, the intermediate area 12 is first unfolded, as is shown in FIGS. 3 b and 3 d . The force exerted by the intermediate area on the lamellae decreases in this state because it is only when the intermediate area is unfolded that the section between the membrane lamellae and the intermediate area acquires the necessary flexibility to make it possible for the membrane lamellae to fold over in the direction of expiration. Consequently, the lamellae are folded upward when the pressure threshold value is subsequently exceeded (for example, 10 or 15 mbar) on the patient side, as is shown in FIG. 2 c . When the patient again stops breathing in, the pressure on the patient side will again drop below the above threshold value, and the lamellae will again return into their closed starting position because of their restoring force. The intermediate area is again folded up into the position shown in FIGS. 3 a and 3 c during the subsequent inspiration. The foldable intermediate area 12 thus acts as a kind of securing means against premature folding over of the lamellae below a defined pressure threshold value.
[0050] FIG. 4 shows another embodiment of the slot valve according to the present invention, which is additionally provided with a releasing means. The slot valve 20 has a lamella area 3 , in which a plurality of slotted lamellae 3 a - 3 d are formed. The lamellae have a configuration similar to that in FIGS. 1 b and 2 a - 2 c . Only the extent of arching of the lamellae is greater in the embodiment according to FIG. 4 . However, the lamellae may also have the configuration according to FIGS. 1 c - 1 e . Furthermore, supporting ring 2 is bent downwardly/inwardly in a C-shaped manner in the direction of the arch in this embodiment in order to mesh with a correspondingly shaped recess 21 of a female tube connector 22 . The female tube connector 22 is connected in one piece to a male tube connector 24 to form the valve housing. The male tube connector 24 is equipped to be connected to an opening of a Y-piece (not shown). A closed suction device, whose suction cannula can pass axially through the slot membrane, is arranged for suctioning secretion between the Y-piece and the slot valve in an advantageous embodiment. Besides the housing formed by the female connector 22 and the male connector 24 and the slot membrane 1 with, for example, four or six radially extending slots, valve 20 has a rotary ring 25 held rotatably on housing 22 , 24 and a spreader 26 mounted displaceably in the housing. The spreader 26 can be pushed into the range of action of the slot membrane 1 or of the lamella area 3 such that the membrane lamellae 3 a - 3 d of the slot membrane are permanently opened in the direction of the patient in order to thus release the flow center in the interior of the valve and not to represent a relevant flow resistance.
[0051] Spreader 26 has two holding noses 27 , which open through the housing (between the male and female tube connectors 22 , 24 ) into the oblique path 28 of the rotary ring 25 . The rotary ring 25 has, besides the oblique path 28 , an annular groove 29 , into which snaps a bead 30 of the housing. The rotary ring 25 is axially fixed at the housing hereby. When rotating the rotary ring, spreader 26 is axially displaced over the oblique bath 28 and can thus be brought optionally into a position close to the Y-piece (respirator side), where the spreader does not mesh with the membrane lamellae and a pressure drop in the patient's lungs below, for example, 10 mbar is thus avoided. In the opposite position near the patient the spreader meshes with the membrane lamellae and pushes these out of the flow center, as a result of which bidirectional flow of fluid through the slot valve 20 is made possible.
[0052] While specific embodiments of the invention have been described in detail to illustrate the application of the principles of the invention, it will be understood that the invention may be embodied otherwise without departing from such principles.
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A slot valve ( 20 ) for use in the pneumatic switching circuit of a respirator, wherein said slot valve is designed to act bidirectionally and to pass over into the opened state to make possible the flow of a fluid at different pressure threshold values depending on the direction of flow of a fluid.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority of Korean Application No. 10-2003-004 5717, filed on Jul. 7, 2003 reference.
FIELD OF THE INVENTION
Generally, the present invention relates to a powertrain system for a hybrid electric vehicle, and more specifically, to control of an automated shift gearbox in such a system.
BACKGROUND OF THE INVENTION
In general, a hybrid electric vehicle (HEV) uses two power sources, an internal combustion engine and electric motor. This is the feature of the HEV that differentiates it from a conventional vehicle using only an internal combustion engine. An appropriate powertrain system and shift control technique must therefore be provided for the HEV, so that power transmission of the two power sources may be smoothly maintained.
In conventional schemes for powertrain systems for such HEVs, power from the engine and power from the electric motor are not perfectly separated. Therefore, an optimum electric drive mode is very difficult to realize. In addition, inertial and/or frictional loads of the engine are active under regenerative braking, which may deteriorate efficiency of the regenerative braking.
In particular, because a clutch in a conventional HEV receives both the power of the engine and the power of the electric motor, the clutch is required to have a large torque capacity. Therefore, a hydraulic pump for controlling engagement of the clutch also must have a large capacity in order to ensure sufficient torque for engaging the clutch at a low speed (i.e., at a low shift-speed), which decreases overall efficiency of power of the vehicle.
Considering that a continuously variable transmission (CVT) is usually limited in its torque capacity, it is generally not appropriate for use as a transmission in an HEV, especially for a vehicle larger than a middle-sized sedan that has a relatively powerful engine. When a CVT is used for such a larger sedan, the CVT may experience slippage because of excessive torque.
As an alternative, an automated shift gearbox (ASG) has been proposed as a transmission for an HEV. In this case, an appropriate scheme of the powertrain and algorithm for controlling shift operation thereof must be newly designed in order to obviate shift-shock and/or a period of no power transmission.
FIG. 7 is a graph illustrating a shift-shock of an HEV having an ASG transmission that is conventionally controlled. As shown therein, a starting point of a shift operation, power from all power sources is separated from drive wheels, which causes output torque of the vehicle to abruptly become zero (0). In addition, a transient shift-shock may occur at a point of engagement of synchronizers, i.e., at a point of engagement of a shift-speed, (refer to circle 1), and also at a point of an engagement of the clutch. Since such a shift-shock is caused by a schematic structure of the powertrain system, the structure of a powertrain system and/or an algorithm for its shift operation should be newly designed to eliminate such a shift-shock.
The information disclosed in this Background of the Invention section is only for enhancement of understanding of the background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is already known to a person skilled in the art in this country.
SUMMARY OF THE INVENTION
Embodiments of the present invention provide a powertrain system for a hybrid electric vehicle that includes non-limiting advantages of enhanced shift performance. An exemplary powertrain system of a hybrid electric vehicle (HEV) according to an embodiment of the present invention includes an engine, a clutch, primary and secondary motors, an automated shift gearbox (ASG) connected to the engine and interposing the clutch therebetween, and a differential gearbox. The primary motor is disposed between an output shaft of the clutch and an input shaft of the ASG. The secondary motor is disposed between an output shaft of the ASG and an input shaft of the differential gearbox.
In a further embodiment, an exemplary powertrain system of an HEV according to an embodiment of the present invention includes a controller executing a shift operation by controlling the engine, the clutch, the primary and secondary motors, and the ASG. The controller adjusts outputs of the engine and the primary and secondary motors during the shift operation.
In a yet further embodiment, the controller executes a clutch disengagement mode during the shift operation. The clutch disengagement mode includes controlling an output torque of the engine during a disengagement of the clutch such that the output torque of the engine lies within a predetermined torque capacity of the clutch, controlling a rotation speed of the engine after the disengagement of the clutch such that the rotation speed of the engine is synchronized with a rotation speed of the output shaft of the clutch at a target shift-speed, and controlling the primary and secondary motor such that a required torque is output through the input shaft of the differential gearbox.
In a yet further embodiment, the controller executes a current shift-speed release mode during the shift operation, wherein the current shift-speed release mode. includes disengaging a current shift-speed of the ASG after the disengagement of the clutch, and controlling, when the current shift-speed is disengaged, a rotation speed of the input shaft of the ASG using the primary motor such that the rotation speed of the input shaft of the ASG is synchronized with one at the target shift-speed.
In a yet further embodiment, the controller executes a target shift-speed engagement mode during the shift operation, wherein the target shift-speed engagement mode includes engaging the target shift-speed after the rotation speed of the input shaft of the ASG is synchronized with the one at the target shift-speed, and controlling, after the engaging the target shift-speed, the primary and secondary motors such that the required torque is output through the input shaft of the differential gearbox.
In a yet further embodiment, the controller executes a shift finishing mode during the shift operation, wherein the shift finishing mode includes engaging the clutch after the target shift-speed is engaged and the rotation speed of the engine is synchronized with the rotation speed of the output shaft of the clutch at a target shift-speed.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, serve to explain the principles of the invention:
FIG. 1 is a schematic diagram of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention;
FIG. 2 illustrates a normal drive mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention;
FIG. 3 illustrates a clutch disengagement mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention;
FIG. 4 illustrates a current shift-speed release mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention;
FIG. 5 illustrates a target shift-speed engagement mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention;
FIG. 6 illustrates a shift finishing mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention; and
FIG. 7 is a graph for illustrating a shift-shock of an HEV having an ASG transmission that is conventionally controlled;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A preferred embodiment of the present invention will hereinafter be described in detail with reference to the accompanying drawings.
As shown in FIG. 1 , a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention includes an engine 310 , a clutch 320 , primary and secondary motors 330 and 350 , an automated shift gearbox (ASG) 340 , and a differential gearbox 360 . A controller 300 controls shift operation via control of the engine 310 , the clutch 320 , the primary and secondary motors 330 and 350 , and the ASG 340 . Engine 310 may be an internal combustion engine and primary and secondary motors 330 , 350 are preferably electric motors. The controller 300 can be realized by one or more processors activated by a predetermined program, and the predetermined program can be a set of instructions to form an algorithm for a shift operation according to a preferred embodiment of this invention.
The ASG 340 is a transmission that has a manual-transmission-based gear mechanism, and its gear engagement/disengagement is controlled by a controller. Such an ASG is well understood by persons of ordinary skill in the art. The ASG 340 is connected to the engine 310 via the clutch 320 . The primary motor 330 is interposed between an output shaft of the clutch 320 and an input shaft of the ASG 340 . The secondary motor 350 is interposed between an output shaft of the ASG 340 and an input shaft of the differential gearbox 360 .
Therefore, the primary motor 330 can apply its motor torque to the input shaft of the ASG 340 under the control of the controller 300 . In addition, the secondary motor 350 can apply its motor torque to the output shaft of the ASG 340 (i.e., the input shaft of the differential gearbox 360 ) under the control of the controller 300 .
An algorithm for shift operation adaptable for such a powertrain system of an HEV according to an embodiment is as follows.
A shift point is preferably determined on the basis of a conventional shift scheme. That is, according to a predetermined shift pattern, an upshift/downshift point is determined on the basis of a throttle valve opening controlled by an accelerator pedal operation of a driver, a current vehicle speed, and a current shift-speed.
A conventional shift pattern may be used as the shift pattern according to the present embodiment if capacities of the motors 330 and 350 are relatively small as in when the engine 310 is a diesel engine. When capacities of the motors 330 and 350 are relatively large or when the engine 310 is a gasoline engine, a shift pattern modified from a conventional one under consideration of load leveling may be used as the shift pattern according to the present embodiment.
A shift operation according to a present embodiment is hereinafter described in detail.
The controller 300 starts the shift operation when a shift point such as an upshift or a downshift point is determined. When the shift operation is started, the controller 300 disengages the clutch 320 , and at the same time, controls an output torque of the engine 310 such that the output torque lies within a predetermined torque capacity of the clutch 320 .
The controller 300 then respectively controls the primary and secondary motors 330 and 350 such that a power requested by a driver (e.g., by operation of an accelerator pedal) may be transmitted to the input shaft of the differential gearbox 360 .
When the clutch 320 is fully disengaged, the controller 300 controls rotation speed of the engine 310 such that it is synchronized with the rotation speed of the output shaft of the clutch 320 at a next shift-speed (i.e., a target shift-speed), i.e., such that a speed difference between both ends of the clutch 320 is reduced below a target difference.
When the clutch 320 is fully disengaged, the controller 300 disengages a current shift-speed. The disengagement of the current shift-speed may be realized by disengaging synchronizers for the current shift-speed e.g., by use of a shift actuator.
When the current shift-speed is fully disengaged, the controller 300 controls the rotation speed of the input shaft of the ASG 340 using the primary motor 330 such that the rotation speed of the input shaft of the ASG 340 is synchronized with one at the target shift-speed. The controller 300 then controls the secondary motor 350 such that the secondary motor 350 realizes its output power as close as possible to the required power requested by the driver. An output power of the vehicle is thereby prevented from becoming zero (0).
When the disengagement of the current shift-speed is completed and the rotation speed of the input shaft of the ASG 340 is synchronized with the rotation speed at the target shift-speed, the controller 300 engages the next shift-speed (i.e., the target shift-speed) of the ASG 340 . The engagement of the target shift-speed may be realized by engaging synchronizers for the target shift-speed e.g., by use of a shift actuator. During this operation, the controller 300 continues its controlling of the engine 310 regarding the revolution speed thereof.
When the target shift-speed is engaged and the rotation speed of the engine 310 is synchronized with the rotation speed of the output shaft of the clutch 320 at a target shift-speed, the controller 300 engages the clutch 320 , which finishes the shift operation.
The shift operation described above is described in further detail with respect to a 1→2 upshift with reference to FIGS. 2–6 .
Shift operations between different shift-speeds are the same as will be described with respect to the 1→2 upshift, as will be apparent to a person of ordinary skill in the art from the description below.
A normal drive mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention is hereinafter described with reference to FIG. 2 .
In the normal drive mode, power of the engine 310 and of the primary and secondary motors 330 and 350 is used. During the normal drive mode, the controller 300 starts the shift operation when a shift point such as an upshift or a downshift point is determined.
A clutch disengagement mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention is hereinafter described with reference to FIG. 3 .
When the shift operation is started, the controller 300 disengages the clutch 320 .
While the clutch 320 is being disengaged, the controller 300 controls an output torque of the engine 310 such that the output torque lies within a predetermined torque capacity of the clutch 320 .
When the clutch 320 is fully disengaged, the controller 300 controls a rotation speed of the engine 310 such that the rotation speed of the engine 310 is synchronized with a rotation speed of the output shaft of the clutch 320 at a target shift-speed.
The controller 300 then respectively controls the primary and secondary motors 330 and 350 such that power requested by a driver may be transmitted to the input shaft of the differential gearbox 360 .
A current shift-speed release mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention is hereinafter described with reference to FIG. 4 .
When the clutch 320 is fully disengaged, the controller 300 disengages a current shift-speed of the ASG 340 . Therefore, the power requested by the driver is realized by the secondary motor 350 only.
When the current shift-speed is fully disengaged, the controller 300 controls a rotation speed of the input shaft of the ASG 340 using the primary motor 330 such that the rotation speed of the input shaft of the ASG 340 is synchronized with a rotation speed at the target shift-speed.
During this operation, the controller 300 continues its controlling of the engine 310 regarding the revolution speed thereof.
A target shift-speed engagement mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention is hereinafter described with reference to FIG. 5 .
When the rotation speed of the input shaft of the ASG 340 is synchronized with the rotation speed at the target shift-speed, the controller 300 engages the target shift-speed of the ASG 340 .
The controller 300 then realizes the requested power using the primary and secondary motors 330 and 350 and outputs the power through the input shaft of the differential gearbox 360 .
During this operation, the controller 300 continues its controlling of the engine 310 regarding the revolution speed thereof.
A shift finishing mode of a powertrain system of a hybrid electric vehicle according to an embodiment of the present invention is hereinafter described with reference to FIG. 6 .
When the target shift-speed is engaged and rotation speeds of both ends of the clutch 320 are synchronized with each other, the controller 300 engages the clutch 320 . During the engagement of the clutch 320 , the controller 300 realizes the requested power using the primary and secondary motors 330 and 350 and outputs the power through the input shaft of the differential gearbox 360 .
By the engagement of the clutch 320 , the shift operation is finished, and after finishing the shifting operation, the controller 300 realizes the requested power using the engine 310 , and the primary and secondary motors 330 and 350 , and outputs the power through the input shaft of the differential gearbox 360 .
As described herein, a powertrain system of an HEV according to an embodiment of the present invention has the following merits.
A torque capacity required for the clutch is reduced since two motors are disposed after the clutch.
Shift-shock during engagement of a target shift-speed is reduced by controlling the two motors.
Power output is maintained by the primary and/or secondary motors in the case that the power of the engine is not transmitted through the clutch due to disengagement thereof.
Power output is maintained by the secondary motor in the case that the power is transmitted from neither the engine nor the primary motor.
While this invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
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Shift performance of a powertrain system of a hybrid electric vehicle is enhanced by a powertrain system including a clutch, a primary motor, an automated shift gearbox (ASG) connected to the engine interposing the clutch, a secondary motor; and a differential gearbox, wherein the primary motor is disposed between an output shaft of the clutch and an input shaft of the ASG, and the secondary motor is disposed between an output shaft of the ASG and an input shaft of the differential gearbox.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is in the field of organic chemistry. The invention relates to a process for the synthesis of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin from a dehydration product of (3R,3′R,6′R)-lutein, namely, (3R)-3′,4′-didehydro-β,β-caroten-3-ol [(3R)-3′,4′-anhydrolutein]. The process involves regioselective hydroboration of (3R)-3′,4′-anhydrolutein to a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin followed by separation of these carotenoids by enzyme-mediated acylation.
2. Related Art
(3R,3′R,6′R)-Lutein and (3R,3′R)-zeaxanthin are two dietary carotenoids that are present in most fruits and vegetables commonly consumed in the US. These carotenoids accumulate in the human plasma, major organs, and ocular tissues [macula, retinal pigment epithelium (RPE), ciliary body, iris, lens]. In the past decade, numerous epidemiological and experimental studies have shown that dietary lutein and zeaxanthin play an important role in the prevention of age-related macular degeneration (AMD) that is the leading cause of blindness in the U.S. and Western World. Among the 7 stereoisomers of dietary (3R,3′R,6′R)-lutein, only (3R,3′S,6′R)-lutein (3′-epilutein) has been detected in the human plasma and tissues. There are also two stereoisomers of (3R,3′R)-zeaxanthin, these are: (3R,3′S;meso)-zeaxanthin and (3S,3′S)-zeaxanthin; these carotenoids are not of dietary origin. However, meso-zeaxanthin that is absent from human plasma, has been found in nearly all human ocular tissues. This carotenoid is presumably formed in the human eye tissues as a consequence of metabolic transformation of dietary (3R,3′R,6′R)-lutein (Khachik F. et al. J. Invest. Ophthalmol. Vis. Sci. 43, 3383-92, 2002). The chemical structures of these carotenoids are shown in Scheme 1. In view of the potential therapeutic application of dietary lutein, (3R,3′R)-zeaxanthin, and (3R,3′S;meso)-zeaxanthin, the industrial production of these carotenoids has received considerable attention. Due to its challenging total synthesis, dietary (3R,3′R,6′R)-lutein is isolated from saponified extracts of marigold flowers ( Tagete erecta , variety orangade ) and is commercially available as a nutritional supplement (Khachik, F. U.S. Pat. No. 5,382,714, Jan. 17, 1995).
Although dietary (3R,3′R)-zeaxanthin is very widely distributed in Nature, its concentration in most readily available natural products is not sufficiently high for commercial production by extraction and isolation. Contrary to the situation with (3R,3′R,6′R)-lutein, numerous lengthy multistep processes have been developed for the total synthesis of (3R,3′R)-zeaxanthin (Mayer, H. Pure Appl. Chem. 1979, 51, 535-564; Saucy, G. U.S. Pat. No. 4,153,615, May 8, 1979; Rüttimann, A., Mayer, H. Helv. Chim. Acta 1980, 63, 1456-1462; Müller, R. K. et al. Food Chem. 1980, 5, 15-45; Widmer, E. et al. Helv. Chim. Acta 1990, 73, 861-867).
There are also several processes that convert the commercially available (3R,3′R,6′R)-lutein or crude saponified extracts of marigold flowers by base-catalyzed isomerization to optically inactive (3R,3′S;meso)-zeaxanthin (Torres-Cardona, M. D.; Quiroga, J. U.S. Pat. No. 5,523,494, Jun. 4, 1996; Bernhard, K, Giger, A. U.S. Pat. No. 5,780,693, Jul. 14, 1998; Rodriguez, G. A. International Patent to Prodemex, WO 99/03830, Jan. 28, 1999). In another process, (3R,3′R,6′R)-lutein is transformed into (3R,3′S;meso)-zeaxanthin similar to the above methods and the latter is oxidized to β,β-carotene-3,3′-dione followed by reduction with sodium or potassium borohydride to give a racemic mixture of (3R,3′R)-zeaxanthin, (3S,3′S)-zeaxanthin, and (3R,3′S;meso)-zeaxanthin (Virgili, S. et al. International Patent to Investigaciones Quimicas Y Farmaceuticas, WO 97/31894, Sep. 4, 1997). However, due to the low overall yield and the fact that the racemic mixture of (3RS,3′RS)-zeaxanthin was not resolved, this approach does not provide an attractive route for the industrial production of these carotenoids.
To circumvent the problems associated with the poor yield and the control of the stereochemistry in transformation of (3R,3′R,6′R)-lutein to (3R,3′R)-zeaxanthin, an efficient process has been reported by the author (Khachik, F. U.S. Pat. No. 6,818,798 B1, Nov. 16, 2004; Khachik, F. J. Nat. Prod. 2003, 66, 67-72). According to this process, lutein was first converted to a diastereomeric mixture of (3R,3′R,6′R)-lutein and (3R,3′S,6′R)-lutein (3′-epilutein) by acid-catalyzed epimerization followed by separation of these carotenoids by enzyme-mediated acylation. The resulting 3′-epilutein was then converted to (3R,3′R)-zeaxanthin by base-catalyzed isomerization. This process provided a convenient and alternative route to the total synthesis of dietary zeaxanthin.
The author now wishes to report yet another alternative method for transformation of dietary lutein to (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin. These carotenoids are initially prepared as a diastereomeric mixture that is subsequently separated into individual compounds with the aim to provide access to both of these important nutrients.
BRIEF SUMMARY OF THE INVENTION
This invention takes advantage of the readily accessible (3R)-3′,4′-didehydro-β,β-caroten-3-ol [(3R)-3′,4′-anhydrolutein] as the starting material for preparation of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin. (3R)-3′,4′-Anhydrolutein has been previously prepared as the major acid-catalyzed dehydration product of (3R,3′R,6′R)-lutein by the author as shown in Scheme 2 (Khachik, F. U.S. Pat. No. 7,115,786 B2, Oct. 3, 2006; Khachik, F. J. Nat. Prod. 2007, 70, 220-226).
The acid-catalyzed dehydration of (3R,3′R,6′R)-lutein, also results in the formation of two other minor dehydration products in addition to (3R)-3′,4′-anhydrolutein; these are: (3R,6′R)-3′,4′-didehydro-β,γ-caroten-3-ol [(3R,6′R)-anhydrolutein I] and (3R,6′R)-2′,3′-didehydro-β,ε-caroten-3-ol [(3R,6′R)-2′,3′-anhydrolutein II]. However, these carotenoids do not interfere with the conversion of (3R)-3′,4′-anhydrolutein to (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin. This transformation is accomplished by regioselective hydroboration of (3R)-3′,4′-anhydrolutein followed by crystallization as shown in Scheme 3.
The regioselective hydroboration of (3R)-3′,4′-anhydrolutein is carried out with a wide range of hydroborating reagents such as borane-tetrahydrofuran complex solution (BH 3 .THF), borane-dimethyl sulfide complex solution (BH 3 .SMe 2 ), borane-N,N-diethylaniline complex (BH 3 .NPhEt 2 ), borane-N-ethyl-N-isopropylaniline complex (BH 3 .NPhEtCHMe 2 ) (BACH-EI™), (−)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex [(R)-ALPINE-BORAMINE™], and (−)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex [(S)-ALPINE-BORAMINE™]. The reaction can be carried out in ether solvents such as tetrahydrofuran (THF), 1,2-dimethoxyethane (DME, ethylene glycol dimethyl ether), bis(2-methoxyethyl)ether (diglyme, diethylene glycol dimethyl ether), tert-butyl methyl ether (TBME)-diglyme, or in dichloromethane and 1,2-dimethoxyethane (CH 2 Cl 2 -DME). As shown in Scheme 3, the hydroboration of (3R)-3′,4′-anhydrolutein, in addition to the desired products also results in the formation of minor amounts of (3R,4′RS)-β,β-caroten-3,4′-diol and (3R,6′RS)-β,ε-caroten-3-ol (α-cryptoxanthin). However, these minor side products can be readily removed by crystallization and do not contaminate the product. Although the diastereomeric mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin can be directly used as a nutritional supplement, the present invention has developed a process for the separation of these carotenoids. This involves enzyme-mediated acylation of the mixture with immobilized lipase PS ( Pseudomonas cepacia ) or lipase AK ( Pseudomonas fluorescens ) in the presence of vinyl acetate which initially acylates these carotenoids into a mixture of (3R,3′R)-zeaxanthin-3-acetate and (3R,3′S)-zeaxanthin-3-acetate (Scheme 4). Further enzyme-mediate acylation of this mixture results in the formation of (3R,3′R)-zeaxanthin-3,3′-diacetate and (3R,3′S)-zeaxanthin-3-acetate. The separation of the zeaxanthin monoacylester and zeaxanthin diacylester is readily accomplished by column chromatography. After alkaline hydrolysis of zeaxanthin acyl esters, (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin are obtained in 92% and 94% diastereomeric excess (de), respectively.
In one embodiment of the present invention, (3R)-3′,4′-anhydrolutein is converted to a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin by regioselective hydroboration by reacting (3R)-3′,4′-anhydrolutein with a hydroborating reagent, in a solvent, at a temperature ranging from 0-30° C. to obtain an isomeric mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin as major products, in a ratio ranging from 1:1 to 3.4:1 and α-cryptoxanthin and (3R,4′RS)-β,β-caroten-3,4′-diol as two minor products. In some embodiments, the hydroborating reagent is selected from the group consisting of borane-tetrahydrofuran complex solution (BH 3 .THF), borane-dimethyl sulfide complex solution (BH 3 .SMe 2 ), borane-N,N-diethylaniline complex (BH 3 .NPhEt 2 ), borane-N-ethyl-N-isopropylaniline complex (BH 3 .NPhEtCHMe 2 )(BACH-EI™), (−)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex ((R)-ALPINE-BORAMINE™) and (+)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex ((S)-ALPINE-BORAMINE™). In some embodiments, the hydroborating reagent is prepared in situ and then allowed to react with (3R)-3′,4′-anhydrolutein. In one embodiment, the hydroborating agent is prepared by a method comprising adding a solution of methyl iodide (MeI) in an ether solvent to a solution of (3R)-3′,4′-anhydrolutein in an ether solvent or in dichloromethane, in which sodium borohydride (NaBH 4 ) is suspended. In some embodiments, the hydroborating agent is prepared in an ether solvent is selected from the group consisting of tetrahydrofuran (THF), 1,2-dimethoxyethane (DME), bis(2-methoxyethyl)ether (diglyme), tert-butyl methyl ether (TBME) and a combination thereof, and the ether solvent optionally contains dichloromethane.
In one embodiment, a hydroborating reagent is allowed to react with (3R)-3′,4′-anhydrolutein in an ether solvent or in dichloromethane at a temperature ranging from 0° C. to room temperature.
In one embodiment, a chiral hydroborating reagent is allowed to react with (3R)-3′,4′-anhydrolutein wherein (3R,3′R)-zeaxanthin is obtained as the major product and (3R,3′S;meso)-zeaxanthin is obtained as the minor product. In some embodiments, the chiral hydroborating reagent is (−)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex ((R)-ALPINE-BORAMINE™).
In one embodiment, a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin is purified by crystallization to give a purity of greater than 95%. In some embodiments, the crystallization is carried out by a process comprising dissolving a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin in a solvent selected from the group consisting of dichloromethane, tetrahydrofuran (THF), tert-butyl methyl ether (TBME) and ethyl acetate, and adding a hydrocarbon solvent selected from the group consisting of pentane, hexane, heptane, cyclohexane and petroleum ether, followed by cooling to induce crystallization. In some embodiments, a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin is obtained from the crystallization.
In some embodiments, a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin with a purity of greater than 95% is used a nutritional supplement or a food coloring additive.
In one embodiment, a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin is separated by enzyme-mediated acylation by acylating a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin with immobilized lipase PS ( Pseudomonas cepacia ) or lipase AK ( Pseudomonas fluorescens ) in the presence of an acyl donor such as vinyl acetate at a temperature ranging from ambient to 50° C. in a solvent to obtain a mixture of (3R,3′R)-zeaxanthin-3,3′-diacetate and (3R, 3S″)-zeaxanthin-3-acetate. In some embodiments, enzyme-mediated acylation is carried out in solvent such as ethyl acetate, or a ether solvent selected from the group consisting of ethyl ether, tetrahydrofuran (THF) and tert-butyl methyl ether (TBME).
In one embodiment, a mixture of (3R,3′R)-zeaxanthin-3,3′-diacetate and (3R, 3′S)-zeaxanthin-3-acetate obtained by enzyme-mediated acylation is separated by column chromatography using acetone, dichloromethane or ethyl acetate in combination with a hydrocarbon solvent selected from the group consisting of pentane, hexane, heptane, cyclohexane and petroleum ether to obtain (3R,3′R)-zeaxanthin-3,3′-diacetate and (3R,3′S)-zeaxanthin-3-acetate in 92% and 94% diastereomeric excess (de), respectively. In some embodiments, column chromatography is on silica gel.
In some embodiments, (3R,3″R)-zeaxanthin-3,3′-diacetate is saponified with alcoholic potassium hydroxide (KOH) or sodium hydroxide (NaOH) to obtain (3R,3′R)-zeaxanthin in 92% de or greater.
In some embodiments, (3R,3″S)-zeaxanthin-3-acetate is saponified with alcoholic potassium hydroxide (KOH) or sodium hydroxide (NaOH) to obtain (3R,3′S;meso)-zeaxanthin in 94% de or greater.
DETAILED DESCRIPTION OF THE INVENTION
Borane-ether (BH 3 .ether) complex solutions were prepared fresh from sodium borohydride (NaBH 4 ) and methyl iodide (MeI) in ether solvents or in dichloromethane and an ether by a slight modification of a published method (Bell H. M. et al. J. Org. Chem. 34, 3923-26, 1969). All other chemicals and reagents including borane-dimethyl sulfide (BH 3 .SMe 2 ), BH 3 .NPhEt 2 , BH 3 .NPhEtCHMe 2 , (R)-ALPINE-BORAMIN™, and (S)-ALPINE-BORAMINE™ were commercially available and obtained from Aldrich Chemical Co. (St. Louis, Mo.). Lipase PS ( Pseudomonas cepacia ) and lipase AK ( pseudomonas fluorescens ) were from Amano Enzyme USA (Lombard, Ill.); these enzymes were immobilized according to a known procedure. All carotenoids were fully characterized by 1 H and 13 C-NMR, MS, and UV-Vis, and circular dichroism (CD).
Hydroboration reactions were monitored by HPLC (eluent A) on a silica-based nitrile bonded (25-cm length×4.6 mm i.d.; 5-μm spherical particle) column (Waters Corporation, Milford, Mass.). The column was protected with a Brownlee nitrile bonded guard cartridge (3-cm length×4.6 mm ID; 5-μm particle size). Eluent A consisted of an isocratic mixture of hexane (75%), dichloromethane (25%), and methanol (0.6%). The column flow rate was 0.7 mL/min and the separations were monitored at 454 nm for zeaxanthin and 466 nm for (3R)-3′,4′-anhydrolutein. In the order of elution, the HPLC retention times were: α-cryptoxanthin (10.37 min), (3R)-3′,4′-anhydrolutein (10.74 min), (3R,4′RS)-β,β-caroten-3,4′-diol (28.71 min), and (3R,3′R)-zeaxanthin+(3R,3′S;meso)-zeaxanthin (coeluting peaks at 37.68 min).
The optical purity of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin was assessed by chiral HPLC (eluent B) on a CHIRALPAK® AD column (25-cm length×4.6 mm internal diameter) purchased from Chiral Technologies (Exton, Pa.). The column packing consisted of amylose tris-(3,5-dimethylphenylcarbamate) coated on 10 μm silica gel substrate and the column was protected with a silica gel guard cartridge (3-cm length×4.6 mm ID; 5 μm particle). For eluent B, a two pumps system with a combination of isocratic and gradient HPLC was employed that separated the stereoisomers of zeaxanthin at 450 nm. Pump One pumped a mixture of hexane (95%) and 2-propanol (5%) and pump Two pumped a mixture of hexane (85%), and 2-propanol (15%). At time zero, 95% solvents from pump One and 5% solvents from pump Two were pumped isocratically for 10 minutes. After 10 minutes, a linear gradient was run for 15 minutes during which the solvents from pump Two were linearly increased from 5% to 40% while that of pump One were reduced from 95% to 60%. At the end of each run, the column was re-equilibrated under the original isocratic conditions for 20 minutes. It should be noted that in addition to the separation of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin, this HPLC system can also separate (3S,3′S)-zeaxanthin. However, this stereoisomer of zeaxanthin is not formed according to the process described here.
The enzymatic acylation of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin were initially monitored by chiral HPLC employing the above eluent B until both zeaxanthins were monoacylated to (3R,3′R)-zeaxanthin-3-acetate and (3R,3′S)-zeaxanthin-3-acetate, respectively. To monitor the enzyme-mediate acylation of (3R,3′R)-zeaxanthin-3-acetate to (3R,3′R)-zeaxanthin diacetate, an isocratic mixture (eluent C) of hexane (95%) and isobutanol (5%) was used with the CHIRALPAK® AD column at a flow rate of 0.7 mL/min. Under these conditions, the monoacyl esters were separated and the course of the enzyme-mediated acylation could be easily monitored.
Purification of (3R)-3′,4′-Anhydrolutein by Crystallization. A crude mixture (200 g) of (3R)-3′,4′-anhydrolutein (84%, anhydrolutein III), (3R,6′R)-anhydrolutein I (10%), and (3R,6′R)-2′,3′-anhydrolutein II (6%) was crystallized from dichloromethane (1000 mL) and ethanol (2000 mL) by stirring at room temperature for 6 h. After filtration and drying, the dark red crystals (70 g) were shown by HPLC to consist of (3R)-3′,4′-anhydrolutein (92%), (3R,6′R)-anhydrolutein I (5%), and (3R,6′R)-2′,3′-anhydrolutein II (3%). A second crystallization was performed by stirring 10 g of this mixture in dichloromethane (50 mL) for 30 min at room temperature followed by addition of ethanol (100 mL) and stirring for 2 h. After filtration and drying under high vacuum, the dark red crystals (8.2 g) were shown by HPLC to consist of (3R)-3′,4′-anhydrolutein (96%), (3R,6′R)-anhydrolutein I (3%), and (3R,6′R)-2′,3′-anhydrolutein II (1%). Hydroboration reactions were carried out with both, the anhydroluteins crystallized once and the anhydroluteins crystallized twice and the results were nearly identical. Based on the proton and carbon NMR data, no major differences between the profiles of the anhydroluteins crystallized once and anhydrolutein crystallized twice was observed.
Hydroboration of (3R)-3′,4′-Anhydrolutein (Anhydrolutein III). In a preferred embodiment, a mixture of (3R)-3′,4′-anhydrolutein (1 equiv.) and NaBH 4 (2 equiv.) in an ether solvent [20-30 mL/g of (3R)-3′,4′-anhydrolutein] such as THF, DME, diglyme, TBME-diglyme or in CH 2 Cl 2 -DME is treated dropwise with a solution of methyl iodide (2 equiv.) in an ether solvent at ambient temperature under argon or nitrogen. The borane-ether complex generated in situ reacts with (3R)-3′,4′-anhydrolutein at 20-25° C. very slowly with evolution of methane. The reaction time varies from 3-5 h and depends on the solubility of (3R)-3′,4′-anhydrolutein in the solvent employed. During addition of MeI, occasional external cooling using a cold-water bath (≈10° C.) is necessary to maintain the temperature between 20-25° C. In addition to monitoring the reaction by HPLC (eluent A), the reaction can also be monitored by thin layer chromatography (TLC) employing hexane:acetone=7/3. The reaction mixture is then cooled down to −10° C. and the product is slowly treated with MeOH and stirred at this temperature for 30 min. The borane complex is then oxidized by dropwise addition of 3N NaOH followed by 30% H 2 O 2 while the temperature is maintained at −10° C. After the addition is completed, the mixture is allowed to warm up to room temperature and stirred for 1 h. The product is then filtered and extracted with a water-immiscible organic solvent. After, drying over sodium sulfate and solvent evaporation, the product is crystallized from dichloromethane and hexane and dried under high vacuum.
In an alternative embodiment, NaBH 4 (2 equiv.) in an ether solvent such as THF, DME, diglyme, TBME-diglyme or in CH 2 Cl 2 -DME is treated dropwise with a solution of methyl iodide (2 equiv.) in an ether solvent at ambient temperature under argon or nitrogen to generate borane-ether complex. The solution is then cooled down to 0° C. and (3R)-3′,4′-anhydrolutein (1 equiv.) in THF or CH 2 Cl 2 is added and after stirring for 2 h at 0° C. and 2 h at room temperature, the product is worked up as described above.
The hydroboration may be similarly carried out with borane-N,N-diethylaniline complex [BH 3 .NPhEt 2 ), borane-N-ethyl-N-isopropylaniline complex (BH 3 .NPhEtCHMe 2 )(BACH-EI™), (−)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex [(R)-ALPINE-BORAMINE™], and (+)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex [(S)-ALPINE-BORAMINE™]. The results of hydroboration of (3R)-3′,4′-anhydrolutein with various reagents in a wide range of solvents are shown in Table 1. Due to the excellent solubility of (3R)-3′,4′-anhydrolutein in THF, hydroboration in this solvent is preferred and gives the best results.
The reactions with BH 3 .Ether and BH 3 .SMe 2 in ether solvents and in CH 2 Cl 2 in all cases resulted in complete conversion of (3R)-3′,4′-anhydrolutein to the product. However, this was not found to be the case in hydroboration reactions with various borane-amine complexes in which 30-40% of (3R)-3′,4′-anhydrolutein remained unreacted. The hydroboration of (3R)-3′,4′-anhydrolutein in all cases also resulted in the formation of two minor side products that were isolated and identified from their NMR, UV-Vis, and MS as: α-cryptoxanthin and (3R, 4′RS)-β,β-caroten-3,4′-diol. These carotenoids were readily removed by crystallization of the crude product with CH 2 Cl 2 and hexane. Instead of CH 2 Cl 2 , other solvents such as ethyl acetate, or ethers such as THF or TBME can be employed. Similarly, hexane can be replaced with other hydrocarbon solvents such as pentane, heptane, or cyclohexane. Unless otherwise stated, the yields shown in Table 1 are based on the isolated yields of the purified mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin that were obtained after crystallization.
TABLE 1
Results of hydroboration of (3R)-3′,4′-anhydrolutein in different solvents.
Distribution of Carotenoids in the
Crude Products (%)
Zeaxanthin
Yield of
Hydroborating
(3R,3′R) +
α-Crypto-
(3R,4′RS)-β,β-
Zeaxanthin a
Reagent-Solvent
(3R,3′S; meso)
xanthin
caroten-3,4′-diol
(%)
BH 3 -THF
92
6
2
75
BH 3 -DME
85
10
5
57
BH 3 -Diglyme
84
12
4
45
BH 3 -TBME-Diglyme
87
9
4
55
BH 3 -DME-CH 2 Cl 2
85
10
5
58
BH 3 -Me 2 S—CH 2 Cl 2
59
35
6
40
H 3 B—NPhEt 2 -THF
46
0.0
6
46 b
H 3 B—NPhEtCHMe 2 -THF
78
10
12
56
(R)-ALPINE-BORAMINE ™-THF
68
25
7
40 c
(S)-ALPINE-BORAMINE ™-THF b
65
27
8
40 d
a If not specified, yield refers to crystallized zeaxanthin that does not contain any α-cryptoxanthin nor (3R,4′RS)-β,β-caroten-3,4′-diol;
b 48% of (3R)-3′,4′-anhydrolutein remained unreacted and the yield was determined by HPLC;
c (3R,3′R):(3R,3′S; meso) = 77:23 (54% diastereomeric excess, de);
d (3R,3′R):(3R,3′S; meso) = 43:57.
Particularly interesting were the hydroboration of (3R)-3′,4′-anhydrolutein with (±)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex [(R)- and (S)-ALPINE-BORAMINE™] that are well-established reagents for stereospecific hydroboration of alkenes (H. C. Brown, J. Organometallic Chem, 500, 1995, 1-19). With (−)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex [(R)-ALPINE-BORAMINE™] (3R,3′R)-zeaxanthin was obtained in 54% diastereomeric excess (de) while (+)-isopinocamphylborane-N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) complex [(S)-ALPINE-BORAMINE™] did not show any significant diastereoselectivity and the de of (3R,3′S;meso)-zeaxanthin was only 14%.
Separation of (3R,3′R)-Zeaxanthin and (3R,3′S;meso)-Zeaxanthin by Enzyme-Mediated Acylation. In a preferred embodiment, a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin in ethyl acetate or ether solvents such as ethyl ether, THF, or TBME is stirred with immobilized lipase PS ( Pseudomonas cepacia ) or lipase AK ( Pseudomonas fluorescens ) in the presence of an acyl donor such as vinyl acetate at ambient temperature. This initially acylates these carotenoids within the first 24 h into a mixture of (3R,3′R)-zeaxanthin-3-acetate and (3R,3′S)-zeaxanthin-3-acetate as shown in Scheme 4. The course of the reaction is monitored by chiral HPLC (eluent B) that show the gradual conversion of zeaxanthins to their corresponding monoacetates. When the reaction is allowed to proceed for another 24 h at room temperature, (3R,3′R)-zeaxanthin-3-acetate is acylated to (3R,3′R)-zeaxanthin-3,3′-diacetate while (3R,3′S)-zeaxanthin-3-acetate remains unchanged. The progress of this reaction can be best monitored by HPLC employing eluent C that separates these zeaxanthin monoacetates. The overall reaction time can be substantially reduced if the enzyme-mediated acylation is carried out at 40-45° C. After the removal of the enzyme, the crude product is separated by column chromatography (hexane:acetone, 95:5 to 90:10) on n-silica gel. Due to the difference in their solubility and chromatographic properties, (3R,3′R)-zeaxanthin-3,3′-diacetate is almost immediately eluted from the column whereas (3R,3′S)-zeaxanthin-3-acetate elutes later from the column. The alkaline hydrolysis of these individually pure mono- and diacetates of zeaxanthin is carried out in an alcoholic solution of KOH or NaOH to afford (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin in 92% and 94% diastereomeric excess (de), respectively.
It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.
Example 1
Hydroboration of (3R)-3′,4′-Anhydrolutein with BH 3 -THF at Ambient Temperature
(3R)-3′,4′-Anhydrolutein (5 g, 9.08 mmol) and NaBH 4 (0.72 g, 19.02 mmol) were transferred into a 250 mL flask equipped with an argon inlet, a thermometer, a mechanical stirrer, and an addition funnel. Dry THF (130 mL) was added and the mixture was stirred under argon. The flask was immersed in a cold-water bath (≈10-15° C.) and a solution of methyl iodide (1.2 mL, 2.736 g, 19.28 mmol) in dry THF (10 mL) was added dropwise at ambient temperature (20-25° C.) in 10 min during which gas evolution began and a thick dark red paste was formed. After 2 h, no detectable amount of (3R)-3′,4′-anhydrolutein was shown by HPLC (eluent A) to be present. The mixture was cooled down to −10° C., methanol (15 mL) was added drop wise, and the mixture was stirred until all the solids were dissolved. This was followed by sequential addition of 3N NaOH (15 mL) and 30% H 2 O 2 (15 mL) while maintaining the temperature at −10° C. The mixture was allowed to warm up to room temperature and stirred for 1 h; during this period the temperature rose to 29° C. and then dropped back to ambient temperature (25° C.). The solids were removed by filtration and the product was partitioned between water (150 mL) and CH 2 Cl 2 (150 mL). The organic layer was washed with water (2×100 mL), dried over sodium sulfate, and filtered. The HPLC analysis (eluent A) of the crude product showed the presence of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin as coeluting peaks (92%), α-cryptoxanthin (6%), and (3R, 4′RS)-β,β-caroten-3,4′-diol (2%). The solution of the crude product was concentrated under reduced pressure and the dark orange residue was crystallized from CH 2 Cl 2 (100 mL) and hexane (150 mL). After cooling at 0-5° C. for several hours, the orange crystals were collected by filtration and dried under high vacuum overnight to give a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (3.87 g, 6.80 mmol; 75%). The HPLC analysis of the product did not show the presence of any other carotenoids or impurities and the 1 H- and 13 C-NMR spectra of zeaxanthins were consistent with the reported literature data.
Example 2
Hydroboration of (3R)-3′,4′-Anhydrolutein with BH 3 -THF at 0° C.
Methyl iodide (1.2 mL, 2.736 g, 19.28 mmol) in dry THF (10 mL) was added dropwise to a suspension of NaBH 4 (0.72 g, 19.02 mmol) in THF (50 mL) under argon. After stirring for 40 min at room temperature (R.T.), the mixture was cooled down to 0° C. and a solution of (3R)-3′,4′-anhydrolutein (5 g, 9.08 mmol) in dry THF (80 mL) was added dropwise while maintaining the temperature at 0° C. After stirring for 2 h at 0° C. and 2 h at R.T., the mixture was cooled down to −10° C., methanol (15 mL) was added dropwise, and the mixture was stirred until all the solids were dissolved. This was followed by sequential addition of 3N NaOH (15 mL) and 30% H 2 O 2 (15 mL) while maintaining the temperature at −10° C. The mixture was allowed to warm up to room temperature and stirred for 1 h. The product was worked up as in example 1 and crystallized from CH 2 Cl 2 and hexane to give a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (3.98 g, 7.00 mmol; 77%).
Example 3
Hydroboration of (3R)-3′,4′-Anhydrolutein with BH 3 -DME
To a mixture of (3R)-3′,4′-anhydrolutein (1 g, 1.82 mmol) and NaBH 4 (0.145 g, 3.83 mmol) in dry 1,2-dimethoxyethane (DME, 30 mL) under argon was added a solution of methyl iodide (0.24 mL, 0.547 g, 3.86 mmol) in dry DME (2 mL) at room temperature (20-25° C.) in 5 min. Occasional cooling with a cold-water bath (10-15° C.) was necessary to maintain the temperature between 20-25° C. After 3 h, a thick dark red paste was formed and no detectable amount of (3R)-3′,4′-anhydrolutein was shown by HPLC (eluent A) to be present. The mixture was cooled down to −10° C., methanol (3 mL) was added, and the mixture was stirred until all the solids were dissolved. This was followed by sequential addition of 3N NaOH (3 mL) and 30% H 2 O 2 (3 mL) while maintaining the temperature at −10° C. The mixture was allowed to warm up to room temperature and stirred for 1 h. The solids were removed by filtration and the product was partitioned between water (40 mL) and ethyl acetate (40 mL). The organic layer was washed with water (2×100 mL), dried over sodium sulfate, and filtered. After solvent evaporation, the product was crystallized from ethyl acetate (30 mL) and hexane (45 mL) to give a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (0.59 g, 1.04 mmol; 57%).
Example 4
Hydroboration of (3R)-3′,4′-Anhydrolutein with BH 3 -DME in CH 2 Cl 2
To a mixture of (3R)-3′,4′-anhydrolutein (1 g, 1.82 mmol) and NaBH 4 (0.145 g, 3.83 mmol) in CH 2 Cl 2 (30 mL) under argon was added a solution of methyl iodide (0.24 mL, 0.547 g, 3.86 mmol) in dry 1,2-dimethoxyethane (DME, 3 mL) at room temperature (20-25° C.) in 5 min. After 4 h, a thick orange paste was formed and no detectable amount of (3R)-3′,4′-anhydrolutein was shown by HPLC to be present. The mixture was cooled down to −10° C. and was sequentially treated with methanol (3 mL), 3N NaOH (3 mL), 30% H 2 O 2 (3 mL), and worked up as described in Example 3. Crystallization from CH 2 Cl 2 (20 mL) and hexane (30 mL) gave a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (0.58 g, 1.02 mmol; 56%).
Example 5
Hydroboration of (3R)-3′,4′-Anhydrolutein with BH 3 —SMe 2 in CH 2 Cl 2
To a solution of (3R)-3′,4′-anhydrolutein (1 g, 1.82 mmol) in CH 2 Cl 2 (30 mL) under argon was added a 2M solution of borane-methyl sulfide (1.9 mL, 3.8 mmol) at room temperature (20-25° C.) in 5 min. After 3 h, a thick orange paste was formed and no detectable amount of (3R)-3′,4′-anhydrolutein was shown by HPLC to be present. The mixture was cooled down to −10° C. and was sequentially treated with methanol (3 mL), 3N NaOH (3 mL), 30% H 2 O 2 (3 mL), and worked up as described in Example 3. Crystallization from CH 2 Cl 2 (20 mL) and hexane (30 mL) gave a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (0.414 g, 0.728 mmol; 40%).
Example 6
Hydroboration of (3R)-3′,4′-Anhydrolutein with BH 3 —NPhEt 2 in THF
To a solution of (3R)-3′,4′-anhydrolutein (1 g, 1.82 mmol) in THF (30 mL) under argon was added a solution of BH 3 —NPhEt 2 (0.84 mL, 0.770 g, 4.72 mmol) at room temperature (20-25° C.). After 3 h, the mixture was cooled down to −10° C. and was treated with methanol (3 mL), 3N NaOH (3 mL), 30% H 2 O 2 (3 mL), and worked up with ethyl acetate as described in Example 3. The HPLC analysis (eluent A) of the crude product showed the presence of unreacted (3R)-3′,4′-anhydrolutein (48%), (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin as coeluting peaks (46%), and (3R, 4′RS)-β,β-caroten-3,4′-diol (6%).
Example 7
Hydroboration of (3R)-3′,4′-Anhydrolutein with BH 3 —NPhEtCHMe 2 in THF
To a solution of (3R)-3′,4′-anhydrolutein (1 g, 1.82 mmol) in THF (30 mL) under argon was added a 2.0 M solution of BH 3 —NPhEtCHMe 2 (2.4 mL, 4.80 mmol) in THF at room temperature (20-25° C.) in 5 min. After 3 h, the mixture was cooled down to −10° C. and was treated with methanol (3 mL), 3N NaOH (3 mL), 30% H 2 O 2 (3 mL), and worked up with ethyl acetate as described in Example 3. The HPLC analysis (eluent A) of the crude product showed the presence of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin as coeluting peaks (78%) as well as (3R, 4′RS)-β,β-caroten-3,4′-diol (12%) and α-cryptoxanthin (10%). Purification of the product by column chromatography (hexane:acetone, 95:5 to 70:30) gave a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (0.58 g, 1.02 mmol; 56%).
Example 8
Hydroboration of (3R)-3′,4′-Anhydrolutein with (−)-Isopinocamphylborane-N,N,N′-tetramethylethylenediamine (TMEDA) complex [(R)-ALPINE-BORAMINE™] in THF
To a solution of (R)-ALPINE-BORAMINE™ (0.33 g, 0.793 mmol) in THF (5 mL) was added a solution of boron trifluoride-diethyl etherate (0.2 mL, 0.224 g, 1.58 mmol) at room temperature under argon and the mixture was stirred for 1 h. The mixture was then cooled down to 0° C. and a solution of (3R)-3′,4′-anhydrolutein (0.25 g, 0.44 mmol) in THF (5 mL) was added. After 3 h, the mixture was sequentially treated with methanol (0.5 mL), 3N NaOH (0.5 mL), 30% H 2 O 2 (0.5 mL), and worked up with ethyl acetate as described in Example 3. The HPLC analysis (eluent A) of the crude product showed the presence of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin as coeluting peaks (68%) as well as (3R, 4′RS)-β,β-caroten-3,4′-diol (25%) and α-cryptoxanthin (7%). Purification of the product by column chromatography (hexane:acetone, 95:5 to 70:30) gave a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (0.100 g, 0.176 mmol; 40%). Chiral HPLC analysis (eluent B) of the product revealed the ratio of (3R,3′R)-zeaxanthin:(3R,3′S;meso)-zeaxanthin=77:23 corresponding to a de of 54% for the (3R,3′R)-isomer.
Example 9
Hydroboration of (3R)-3′,4′-Anhydrolutein with (+)-Isopinocamphylborane-N,N,N′,N′-tetramethylethylenediamine (TMEDA) complex [(S)-ALPINE-BORAMINE™] in THF
To a solution of (R)-ALPINE-BORAMINE™ (0.33 g, 0.793 mmol) in THF (5 mL) was added a solution of boron trifluoride-diethyl etherate (0.2 mL, 0.224 g, 1.58 mmol) at room temperature under argon and the mixture was stirred for 1 h. The mixture was then cooled down to 0° C. and a solution of (3R)-3′,4′-anhydrolutein (0.25 g, 0.44 mmol) in THF (5 mL) was added. After 3 h, the mixture was treated with methanol (0.5 mL), 3N NaOH (0.5 mL), 30% H 2 O 2 (0.5 mL), and worked up with ethyl acetate as described in Example 3. The HPLC analysis (eluent A) of the crude product showed the presence of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin as coeluting peaks (65%) as well as (3R,4′RS)-β,β-caroten-3,4′-diol (27%) and α-cryptoxanthin (8%). Purification of the product by column chromatography (hexane:acetone, 95:5 to 70:30) gave a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (0.100 g, 0.176 mmol; 40%). Chiral HPLC analysis (eluent B) of the product revealed the ratio of (3R,3′R)-zeaxanthin:(3R,3′S;meso)-zeaxanthin=43:57 corresponding to a de of 14% for the (3R,3′S;meso)-isomer.
Example 10
Separation of (3R,3′R)-Zeaxanthin and (3R,3′S;meso)-Zeaxanthin by Enzyme-Mediated Acylation with Lipase PS ( Pseudomonas cepacia )
Immobilization of Lipase PS ( Pseudomonas cepacia ). Lipase PS (3.0 g) and diatomaceous earth (HYFLO SUPER CEL®) (10 g) were mixed and 10 mL of phosphate buffer (pH=7.0) was added. After mixing for 15 min, the paste was spread out on a Petri dish and allowed to dry for 2 days in the presence of air. The same procedure was employed for the immobilization of Lipase AK ( Pseudomonas fluorescens ).
A mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin (0.6 g, 1.06 mmol) in THF (20 mL) was treated with immobilized lipase PS (0.6 g) and vinyl acetate (3 mL) and the mixture was stirred at room temperature under argon. The course of the reaction was monitored by chiral HPLC (eluent B). After 24, all of zeaxanthins were acylated to (3R,3′R)-zeaxanthin-3-acetate and (3R,3′S)-zeaxanthin-3-acetate. Stirring continued for another 24 h and the progress of the reaction was monitored by chiral HPLC employing eluent C. When all the (3R,3′R)-zeaxanthin-3-acetate was acylated to (3R,3′R)-zeaxanthin-3,3′-diacetate (total of 48 h), the enzyme was removed by filtration and the solvent was evaporated under reduced pressure to give 0.65 g of a crude product. Column chromatography of the residue on n-silica gel (hexane:acetone, 95:5 to 90:10) gave two main fractions that were identified from their mass and NMR spectra as (3R,3′R)-zeaxanthin-3,3′-diacetate (0.274 g, 0.42 mmol) and (3R,3′S)-zeaxanthin-3-acetate (0.244 g, 0.40 mmol).
Hydrolysis of (3R,3′R)-Zeaxanthin-3,3′-Diacetate. A solution of (3R,3′R)-zeaxanthin-3,3′-diacetate (0.274 g, 0.42 mmol) in THF (10 mL) was stirred with 10% methanolic KOH (2 mL) under argon. After 1 h, the product was partitioned between water (20 mL) and CH 2 Cl 2 (20 mL). The organic layer was removed and washed with water (2×20 mL), dried over Na 2 SO 4 , and evaporated to dryness to give (3R,3′R)-zeaxanthin (0.221 g, 0.39 mmol). The product was shown by chiral HPLC (eluent B) to consist of (3R,3′R)-zeaxanthin (96%) and (3R,3′S;meso)-zeaxanthin (4%) corresponding to a diasteromeric excess (de) of 92% for the (3R,3′R)-isomer.
Hydrolysis of (3R,3′S)-Zeaxanthin-3-Acetate. A solution of (3R,3′S)-zeaxanthin-3-acetate (0.20 g, 0.33 mmol). in THF (10 mL) was stirred with 10% methanolic KOH (2 mL) under argon. After 1 h, the product was partitioned between water (20 mL) and CH 2 Cl 2 (20 mL). The organic layer was removed and washed with water (2×20 mL), dried over Na 2 SO 4 , and evaporated to dryness to give (3R,3′S;meso)-zeaxanthin (0.176 g, 0.31 mmol). The product was shown by chiral HPLC (eluent B) to consist of (3R,3′S;meso)-zeaxanthin (97%) and (3R,3′R)-zeaxanthin (3%) corresponding to a diasteromeric excess (de) of 94% for the (3R,3′S;meso)-isomer.
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(3R,3′R,6′R)-Lutein and (3R,3′R)-zeaxanthin are two dietary carotenoids that are present in most fruits and vegetables commonly consumed in the US and accumulate in the human plasma, major organs, and ocular tissues. Another stereoisomer of (3R,3′R)-zeaxanthin that is not of dietary origin but is found in the human ocular tissues is (3R,3′S;meso)-zeaxanthin. There is growing evidence that these carotenoids play an important role in the prevention of age-related macular degeneration (AMD) that is the leading cause of blindness in the U.S. and the Western World. In view of the potential therapeutic application of dietary lutein, (3R,3′R)-zeaxanthin, and (3R,3′S;meso)-zeaxanthin, the industrial production of these carotenoids is of considerable importance. The present invention provides a process for the partial synthesis of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin from a readily accessible dehydration product of (3R,3′R,6′R)-lutein, namely, (3R)-3′,4′-didehydro-β,β-caroten-3-ol [(3R)-3′,4′-anhydrolutein]. The process involves regioselective hydroboration of (3R)-3′,4′-anhydrolutein to a mixture of (3R,3′R)-zeaxanthin and (3R,3′S;meso)-zeaxanthin followed by separation of these carotenoids by enzyme-mediated acylation.
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BACKGROUND OF THE INVENTION
The present invention is related bags, sacks and packs for storing clothing, gear or other types of articles and also preventing odors from either entering or escaping from the bag, sack or pack. The present invention also relates to camouflaged bags, sacks and packs for use by soldiers and hunters.
In situations where a person in the outdoors wishes to approach wild game, it is desirable that steps be taken to ensure that no odors or scents emanating from that person or that person's clothing and gear can be detected by the keen sense of smell of that wild animal. One solution to this problem is to provide articles of clothing that absorb odors emanating from covered or surrounded body portions. U.S. Pat. No. 6,134,718 discloses that such clothing articles (or a duffle bag or knapsack) may comprise inner and outer layers with “odor absorbing means” being enclosed between the inner and outer layers. The “odor absorbing means” may include “an odor absorbing agent” selected from the group consisting of activated charcoal, chlorophyll, baking soda, activated alumina, soda lime, zeolite, calcium oxide, potassium permanganate or a similar substance. In one example, the “odor absorbing means” takes the form of fibers treated with or having incorporated therein activated carbon or charcoal.
Activated charcoal is charcoal that has been treated with oxygen to open up millions of tiny pores between the carbon atoms and is widely used to “adsorb” odorous substances from gases or liquids. When a material “adsorbs” a molecule, the molecule is attached by chemical attraction. The highly porous activated charcoal provides countless bonding sites on its surface, where molecules attach and are trapped. As used herein, the term “adsorption” means the surface retention of solid, liquid or gas molecules, atoms or ions by a solid or liquid, as opposed to “absorption”, which means the penetration of substances into the bulk of the solid or liquid. As used herein, the term “odor-eliminating” means that at least some molecules of an odorous substance are adsorbed or absorbed by the material in question, not that all odor is eliminated.
In addition, special care is taken to remove odors normally associated with humans and other sources of odors not typically found in nature. For example, scent-free detergents are used to wash the clothing. In some cases, a natural scent that emits a desirable (non-human) odor is added to the clothing in order to cover up any remaining human and unnatural odors.
While these approaches have been used in the past to assist outdoorsman in odor control, steps must be taken to maintain the scent-free or scented state of the clothing during transport to the outdoor site. For this purpose special bags, packs and sacks have been designed which have means for blocking odors emanating from outside the bag from contaminating the scent-free or scented clothing inside the bag. More specifically, it is known to provide a bag, sack or pack comprising a layer of material designed to prevent odorous substances from entering the bag, sack or pack and being adsorbed by the clothing therein.
Conversely, in the case of sports accessory bags, it is desired that odors emanating from used athletic uniforms and footwear contained in the bag be prevented from escaping. Thus a properly constructed bag may serve either purpose, by blocking odors from entering or leaving the space enclosed by the bag.
There is a need for improvements in odor-eliminating storage bags. In the case of bags, sacks and packs used by outdoorsmen, such as hunters and soldiers, it is also desirable to provide camouflage that will blend in with a particular outdoor environment.
BRIEF DESCRIPTION OF THE INVENTION
The present invention is directed to bags, packs and sacks for storing clothing and gear of outdoorsmen, such as hunters and military personnel. These bags, packs and sacks may have one or both of two features: means for preventing odors from entering or leaving the interior volume of the bag, pack or sack; and a camouflage pattern visible from a vantage on the exterior of the bag.
One aspect of the invention is a bag comprising a receptacle having a mouth at an upper end, and a plastic zipper attached to the mouth, wherein the zipper comprises first and second zipper strips that extend across the mouth, the first zipper strip comprising a first closure profile and the second zipper strip comprising a second closure profile, the first and second closure profiles being mutually interlockable, the mouth being closed when the first and second closure profiles are interlocked and being open when the first and second closure profiles are disengaged, and wherein the receptacle is made of laminated material, the laminated material comprising first and second layers laminated to each other, the first layer comprising an odor-eliminating agent.
Another aspect of the invention is a bag comprising a receptacle having a mouth at an upper end, and a plastic zipper attached to the mouth, wherein the zipper comprises first and second zipper strips that extend across the mouth, the first zipper strip comprising a first closure profile and the second zipper strip comprising a second closure profile, the first and second closure profiles being mutually interlockable, the mouth being closed when the first and second closure profiles are interlocked and being open when the first and second closure profiles are disengaged, and wherein the receptacle is made of laminated material, the laminated material comprising first and second layers laminated to each other, opposing surfaces of the first and second layers forming an interface, at least one of the opposing surfaces of the first and second layers having a camouflage pattern printed thereon.
A further aspect of the invention is a method of manufacture comprising the following steps: (a) printing a camouflage pattern on a surface of a first web of film; (b) laminating the first web of film to a second web of film to form a laminated web having first and second mutually parallel edges, the second web of film being optically transparent and the printed surface of the first web of film being trapped between the first and second webs; (c) folding the laminated web along a fold line with the second web disposed on the outside of the fold, the fold line being generally parallel to the first and second edges; (d) joining a first zipper strip to a first portion of the first web of the laminated web along a first zone of joinder extending generally parallel to the first and second edges; (e) joining a second zipper strip to a second portion of the first web of the laminated web along a second zone of joinder extending generally parallel to the first and second edges; (f) joining a first transverse portion of the laminated web to a second transverse portion of the laminated web to form a first cross seal generally orthogonal to the first and second edges; and (g) joining a third transverse portion of the laminated web to a fourth transverse portion of the laminated web to form a second cross seal generally parallel to the first cross seal and separated therefrom by a predetermined distance, wherein the first and second cross seals extend from the fold to at least the first and second zipper strips.
Yet another aspect of the invention is a method of manufacture comprising the following steps: (a) blending resin containing odor-eliminating agent with resin for making bag making film; (b) extruding a first web of film using the blended resins; (c) laminating the first web of film to a second web of film to form a laminated web having first and second mutually parallel edges; (d) folding the laminated web along a fold line that lies generally parallel to the first and second edges; (e) joining a first zipper strip to a first portion of the first web of the laminated web along a first zone of joinder extending generally parallel to the first and second edges; (f) joining a second zipper strip to a second portion of the first web of the laminated web along a second zone of joinder extending generally parallel to the first and second edges; (g) joining a first transverse portion of the laminated web to a second transverse portion of the laminated web to form a first cross seal generally orthogonal to the first and second edges; and (h) joining a third transverse portion of the laminated web to a fourth transverse portion of the laminated web to form a second cross seal generally parallel to the first cross seal and separated therefrom by a predetermined distance, wherein the first and second cross seals extend from the fold to at least the first and second zipper strips.
A further aspect of the invention is a bag comprising a receptacle having a mouth at an upper end, and a plastic zipper attached to the mouth, wherein the zipper comprises first and second zipper strips that extend across the mouth, the first zipper strip comprising a first closure profile and the second zipper strip comprising a second closure profile, the first and second closure profiles being mutually interlockable, the mouth being closed when the first and second closure profiles are interlocked and being open when the first and second closure profiles are disengaged, and wherein the receptacle is made of laminated material, the laminated material comprising first and second layers laminated to each other, the first layer comprising a thermoplastic material and a corrosion-inhibiting agent.
Other aspects of the invention are disclosed and claimed below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing showing a camouflaged reclosable storage bag having an extruded plastic zipper in accordance with one embodiment of the invention.
FIG. 2 is a drawing showing a sectional view of the storage bag depicted in FIG. 1 .
FIG. 3 is a drawing showing a rear view of one wall of the storage bag depicted in FIG. 1 . The side of the wall visible in FIG. 3 is the side that faces the interior of the bag.
FIG. 4 is a drawing showing a sectional view of a reclosable storage bag having a slider-operated extruded plastic zipper in accordance with another embodiment of the invention.
Reference will now be made to the drawings, in which similar elements in different drawings bear the same reference numerals.
DETAILED DESCRIPTION OF THE INVENTION
A camouflaged reclosable storage bag 2 is shown in FIG. 1 with its mouth open. The bag is made from a web of laminated film material that has been folded, sealed and cut to form an individual bag. In general, the bag 2 comprises front and rear walls that have their side edges sealed together to form respective side seals 26 and 28 . The bottom of the bag has a gusset, which is not visible in FIG. 1 , but is designated by numeral 10 in FIG. 2 . One extruded flangeless zipper strip 12 having a substantially constant profile along its length is joined to the interior surface of the front wall of the bag, while another extruded flangeless zipper strip 14 having a substantially constant profile along its length is joined to the interior surface of the front wall of the bag. The flangeless zipper strips 12 and 14 extend from one side of the bag to the other side, with the terminal portions of the zipper strips at one end being sealed together and captured in the side seal 26 of the bag, while the terminal portions of the zipper strips at the other end are sealed together and captured in the side seal 28 of the bag. As best seen in FIG. 2 , the zipper strips 12 , 14 form a so-called “string” zipper 4 at an elevation in the bag that, for purposes of this disclosure, will be deemed the “mouth” of the bag. That mouth is closed by the string zipper 4 when the closure profiles of the zipper strips are interlocked with each other along their entire lengths, and is open when the closure profiles of the zipper strips are disengaged.
The portions of the bag at and below the elevation of the string zipper 4 form a receptacle in which articles of clothing or other items can be stored. As best seen in FIG. 2 , these portions include a receptacle front wall 6 , a receptacle rear wall 8 and the panels of the bottom gusset 10 , which together form the boundaries of an interior volume 24 of the receptacle. The bottom gusset 10 is formed by folding the laminated film material during the manufacturing process. In FIG. 2 , the mouth of the bag is shown closed by interlocking of respective mating closure profiles of the zipper strips 12 and 14 , described in more detail below. The receptacle walls 6 and 8 are respectively sealed to the backs of the bases of the interlockable zipper strips 12 and 14 by respective permanent or hard seals, formed by conduction heat sealing along respective lines or bands of joinder, respectively. Alternatively, the interlockable zipper strips can be attached to the walls by adhesive, application of ultrasonic energy, or other suitable bonding or sealing means.
The walls of the bag 2 extend beyond the zipper seals 12 and 14 , these upper portions of the walls being respectively designated by numerals 16 and 18 and will be referred to herein as the “header walls”. In the disclosed embodiment, the receptacle walls 6 , 8 and the header walls 16 , 18 are all portions of the same web of laminated film material. However, it should be appreciated that the header walls could be formed separately from and then joined to the receptacle walls in the area of the zipper seals 12 , 14 , and joined to each other to form side seals 26 , 28 .
As seen in FIG. 1 , each header wall 16 , 18 has a respective opening 30 , 32 . When the header walls 16 and 18 are brought together, the openings 30 and 32 overlap each other or are aligned. In this state, the portions of the header walls immediately above the openings 30 and 32 serve the function of a handle, with the openings allowing a person to pass his fingers through for grasping the handle. To prevent the material above openings 30 and 32 from being stretched by the weight of the contents of the storage bag, the laminated wall material of the entire bag may be made of sufficiently strong and thick material, or only the header wall material along the periphery of the openings 30 and 32 can be reinforced. In accordance with the latter embodiment, partly depicted in FIG. 3 , a reinforcement patch 34 can be seen attached or laminated to the header wall 18 along the periphery of the opening 32 . A similar reinforcement patch would be attached or laminated to the opposing header wall. In accordance with one method manufacture, a pair of rectangular patches could be sealed to each bag-width section of the continuous web of laminated film material at respective mirror-image positions on opposing sides of a centerline of the web. Each opening 30 and 32 would then be formed by cutting or punching through both the web and the respective reinforcement patch.
As seen in FIG. 1 , the laminated film material of the bag has a camouflage pattern printed thereon. In accordance with one embodiment of the invention, the laminated film material comprises a first layer of thermoplastic film material having a surface suitable for printing thereon, a camouflage pattern printed on that print-receptive surface, and a second layer of clear thermoplastic film material that is laminated to the printed side of the first layer, thereby trapping any emanations or odors from the printing in between the two thermoplastic layers. The first layer will form the inner layer of the bag, while the second layer forms the outer layer of the bag. This entrapment between layers of emanations from the printed matter protects the printed matter from being damaged or degraded by frictional contact with or scraping against external objects or the articles stored inside the bag, exposure to the elements of the weather, or other factors. In one non-limitative example, the print-receptive layer is made of low-density polyethylene (LDPE), while the covering layer is made of gas-impermeable thermoplastic material, such as nylon, polyester, polyvinyl dichloride, or ethylene vinyl alcohol.
The embodiments having printed camouflage are not limited to any particular camouflage pattern. Depending on the intended field of use, a camouflage pattern may be selected that mimics the visual presentation of a particular outdoor environment.
In accordance with a further aspect, the bag 2 shown in FIGS. 1 and 2 may also have the capability to prevent the diffusion of odors through the bag material. In accordance with one embodiment, this is accomplished by providing a laminated film material wherein the inner layer has odor-adsorbing or odor-absorbing properties, while the outer layer is made of a gas-impermeable thermoplastic material, such as nylon, polyester, polyvinyl dichloride, or ethylene vinyl alcohol, that acts as a barrier to gas passing through the bag walls.
More specifically, the inner layer is formed by blending an odor-eliminating chemical agent into the extrusion melt and then extruding a layer of film. Resin pellets containing a selected odor-eliminating chemical agent are commercially available and can be mixed with resin pellets not containing that chemical agent when the pellets are melted and mixed to form a homogeneously blend. The composition or formulation of the chemical agent will depend on the particular odor or odors sought to be eliminated. For example, the chemical agent may be a desiccant that absorbs atmospheric moisture, which contains odor molecules. One suitable resin for the inner layer is LDPE, which forms a breathable substrate. The additive will tend to exude to the surface of the polyethylene layer, so that the majority of the odor-eliminating chemical agent will reside on the surface of the polyethylene, where it is most effective. The outer barrier layer prevents contamination of the inner layer from outside the bag. For example, in the case of a chemical agent that adsorbs odorous substances, the odorous substances attach to bonding sites until the bonding sites are filled, at which point the odor-eliminating agent loses effectiveness. The gas barrier layer allows the inner layer to absorb or adsorb odors from the contents of the bag without absorbing or adsorbing odors from outside the bag, which might diminish its effectiveness.
In accordance with a further variation, the inner layer may further comprise a chemical agent that inhibits oxidation of metal inside the bag. One suitable corrosion-inhibiting chemical agent is triazole. This feature has useful application in a hunter's gun bag, with or without camouflage.
The string zipper depicted in FIG. 2 is commercially available from Minigrip/Zip-Pak, a division of Illinois Tool Works Inc. having offices in Glenview Ill. One zipper strip 12 has a profiled structure comprising two male members, while the other zipper strip 14 has a profiled structure comprising two female members which respectively mate with the male members during zipper closure. Each male member has a generally arrow-shaped rib-like male profile; each female member has a complementary, generally C-shaped female profile. Each zipper strip further comprises a respective base portion having substantially no flanges. Preferably, each base portion is a resiliently flexible self-supporting structure having a thickness greater than the thickness of the receptacle walls of the bag in which the zipper will be installed. FIG. 2 shows one male member engaged in one female member and the other male member engaged in the other female member.
In the particular zipper embodiment shown in FIG. 2 , the profile of each male member has a stem with a generally triangular head at the tip of the stem, the tip being the portion of the male member furthest away from the base of the profiled structure. The profile of each female member comprises a pair of hooks that grip the head of the male member and latch under it. These hooks extend from a base or root of the female member. The detents at the ends of the hooks are inclined and generally directed toward each other, the detents of the hooks defining a mouth that communicates with a groove defined by the walls of the hooks and root of the female member. The groove of each female member receives the head of the corresponding male member when the zipper is closed, as shown in FIG. 2 .
To open the closed zipper, the two sides of the zipper are pulled apart with sufficient force to pull the heads of the male members out of the female members. When the sides of the heads of the male members clear the detents of the hooks of the female member, the male and female members are no longer interlocked and the zipper is open.
The present invention does not require a zipper of the type shown in FIG. 2 . Other types of zippers can be employed. For example, instead of interlocking rib and groove closure elements having so-called male and female profiles, interlocking alternating hook-shaped closure elements can be used. In other words, hooks of the type shown in FIG. 2 as part of zipper strip 14 can be incorporated on both zipper strips.
Furthermore, zippers comprising flanged zipper strips can be employed in place of string zippers, as will now be described with reference to FIG. 4 . In this embodiment, a slider-zipper assembly 20 is installed in the mouth of the receptacle formed by walls 6 and 8 and bottom gusset 10 . The assembly 20 comprises an extruded plastic zipper and a molded plastic slider 48 mounted thereto. The zipper comprises first and second flanged zipper parts. The first flanged zipper part comprises a first profiled closure member 36 and a first flange 38 , and the second flanged zipper part comprises a second profiled closure member 40 and a second flange 42 . The profiled closure members 36 and 40 are mutually interlockable. Flange 38 has a band-shaped portion joined to an opposing portion of wall 8 by conduction heat sealing, while the flange 42 has a band-shaped portion joined to an opposing portion of wall 6 by conduction heat sealing, thereby forming respective so-called “permanent” seals 44 and 46 .
The slider 48 is mounted to the zipper and is configured to close portions of the zipper as the slider is moved in one direction along the zipper and to open portions of the zipper as the slider is moved in the opposite direction along the zipper in the manner disclosed in detail in U.S. Pat. No. 6,047,450. More precisely, the slider 48 is designed to cam the lower portions of the closure profiles toward each other to the closure profiles to rotate in opposite directions about a fulcrum point of contact. This causes the upper portions of the closure profiles to move apart and disengage. In this example, slider 48 does not have a plow or separating finger. However, slider-zipper assemblies of the type wherein the slider has a separating finger for separating the zipper halves during slider travel can be used in the reclosable camouflaged and/or scent-free bags, packs or sacks of the type disclosed herein.
In accordance with some embodiments of the invention, the bag, pack or sack incorporates both camouflage and odor elimination and/or corrosion inhibition means. In accordance with other embodiments, the bag, pack or sack incorporates camouflage, but not odor elimination or corrosion inhibition means. In accordance with further embodiments, the bag, pack or sack incorporates odor elimination means or corrosion inhibition means, but not camouflage. The method of manufacturing the first category of embodiments will now be described in detail, but it should be understood that the method steps for including an odor-eliminating chemical agent in the extrusion melt can be eliminated if a camouflaged bag without odor elimination is called for, whereas the method steps for including camouflage can be eliminated if a bag without camouflage is called for.
In accordance with one embodiment of the method of manufacture, pellets of resin containing a concentrate comprising an odor-eliminating chemical agent and/or a corrosion-inhibiting chemical agent are added to pellets of resin that do not contain that chemical agent. A suitable resin is LDPE. However, the invention is not limited to the use of polyethylene. Other thermoplastic materials can be used. These pellets are then melted and mixed to homogeneously blend the chemical agent throughout the molten resin. The molten resin is then pressed through a die orifice to extrude a continuous sheet of thermoplastic film that will be wound on a roll after cooling.
Alternatively, the odor-eliminating and/or corrosion-inhibiting chemical agent could be sprayed onto one or both surfaces of a web of thermoplastic film before lamination to another web.
In the next stage of manufacture, the polyethylene film with odor-eliminating chemical agent blended therein is unwound from the roll and fed to a printer that prints a camouflage pattern onto the surface on one side of the polyethylene film. When the print has dried, the polyethylene film can be rewound and transported to a laminator at a different site or can be fed directly to a laminator at the same site. The laminator continuously laminates the continuous web of printed film to another continuous web of thermoplastic film, preferably made of a gas-impermeable thermoplastic material. The two continuous webs are fed to the laminator in overlapping relationship, with the printed side of the first web in contact with the second web. The two webs can be laminated together, for example, by passing them through a pair of heated press rolls. The end result is a continuous web of laminated material, with the printed camouflage pattern trapped between the two layers at their interface.
The laminated web is then fed to a conventional bag making machine (or wound on a roll and transported to a bag making machine). An automated system (not shown) is used to apply a continuous length of zipper tape to the laminated web. The laminated web is advanced by pinch rollers that pull the web through the machine. Zipper sealing can be performed continuously using a drag sealer or intermittently using reciprocating sealing bars. If a slider is to be inserted at spaced intervals along the zipper tape, then zipper sealing to the laminated web should be performed intermittently to accommodate intermittent ultrasonic welding to form slider end stops on the zipper tape followed by intermittent slider insertion, both steps occurring upstream of the zipper sealing station.
One method for forming the laminated web into a reclosable bag having a string zipper and no slider will now be described. The laminated web is pulled over a V-shaped folding board or plow 40 , which folds the laminated web into a V shape with the odor-eliminating layer on the inside of the V. The zipper tape is paid out from a spool and pulled through a zipper guide that guides the zipper into a position inside the folded laminated web and oriented generally parallel to the fold in the web and at a predetermined elevation above the fold and a predetermined depth below the edges of the laminated web. In this example, it will be assumed that the zipper tape and the laminated web are moved intermittently with most operations being performed during dwell times.
At the sealing stations, a pair of opposing reciprocating horizontal heated sealing bars are extended at the elevation of the string zipper. The sealing bars apply sufficient heat and pressure to cause the portions of the laminated web in contact with the backs of the respective zipper strips of the string zipper to be sealed thereto. This section of the laminated web with attached string zipper section is then advanced to a cross sealing station, where the zipper/web assembly is cross sealed by a pair of opposing reciprocating vertical heated sealing bars, which are extended to form a cross seal that extends from the fold in the laminated web to the edges of the laminated web, which are disposed above the string zipper. After each cross sealing operation, the zipper/web assembly is advanced one bag width and the process is repeated. At the next station, openings are punch or cut in those portions of the laminated web that are disposed above the elevation of the string zipper. These are the openings 30 and 32 shown in FIG. 1 . This section of the zipper/web assembly is then advanced to the cutting station, where the zipper/web assembly is cut along a vertical line that generally bisects the cross seal, thereby severing an individual bag from the zipper/tape assembly.
If the openings in the top of the bag require reinforcement, the reinforcement patches can be attached to the laminated web before the latter is folded. It should also be appreciated that instead of folding the laminated web and then attaching both sides of the string zipper to the folded web, it is possible to first seal one of the interlocked zipper strips of the string zipper to the unfolded web, then fold the web over and seal the other zipper strip to the web. Alternatively, the zipper strips of the string zipper could be unwound from separate spools and sealed to the unfolded web separately, but in parallel with each other. After the zipper strips are sealed to the web, the web would be folded so that the zipper strips are aligned and then the aligned zipper strips would be pressed together by press rolls, causing the string zipper to close.
While the invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for members thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation to the teachings of the invention without departing from the essential scope thereof. Therefore it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
As used in the claims, the term “bag” includes bags, pouches, sacks, packs, and the like. As used in the claims, the verb “joined” means fused, bonded, sealed, adhered, etc., whether by application of heat and/or pressure, application of ultrasonic energy, application of a layer of adhesive material or bonding agent, interposition of an adhesive or bonding strip, etc. As used in the claims, the term “string zipper” means a zipper comprising two zipper strips that have mutually interlockable closure profiles and substantially no flange portions.
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A bag, pack or sack for storing clothing and gear of outdoorsmen, such as hunters and military personnel. The bag, pack or sack may have one or both of two features: means for preventing odors from entering or leaving the interior volume of the bag, pack or sack; and a camouflage pattern visible from a vantage on the exterior of the bag.
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BACKGROUND OF THE INVENTION
The invention relates to a process changing the relative postition of packs, especially of cuboidal cigarette packs of the hinge-lid type, the packs being successively fed to a turning station.
SUMMARY OF THE INVENTION
In packaging machines, it is a common necessity to change the relative position of the packs For this process, the often delicate wrappings of the packs have to be taken into account. It is known in the art to discharge the packs transverse to the original moving direction. Herewith, the position relative to the conveying direction also changes. This abrupt change in direction effects an application of extremely high forces on the pack contents and the pack itself. With non-rigid packs having soft contents, the risk of packs getting damaged can not be ruled out. This applies even more so to cigarette packing machines, since they work at particularly high operating speeds. The present invention has the object to create a process and an apparatus, with which the relative position of the packs can be changed in a gentle way so the packs are treated with care.
BRIEF DESCRIPTION OF THE DRAWINGS
This object is attained according to the invention by a process, in which the packs are turned in the region of a turning station during transport by at least two separate conveying means (turning conveyors) which engage opposite sides of the packs and which are driven by different (pulling) velocities V4 and V5. The packs are not diverted from their original conveying direction, but are only turned in a gentle way. Especially cuboidal cigarette packs with rectangular cross-section can be oriented from a position transverse to the conveying direction to a position parallel to the conveying direction in a relatively gentle but nevertheless fast way.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Further fundamental process features of the invention are disclosed in the (process) subclaims.
The object is further attained according to the invention by an apparatus comprising two separate conveying means (turning conveyors) in the region of the turning station which are drivable by different (pulling) velocities V4 and V5 and by means of which the packs are turnable by being pulled at opposite pack sides during transport. The conveying direction is not changed by the conveying means adjoining the feed track, so that a gentle turning movement of the individual packs is ensured.
Further fundamental apparatus features of the invention follow from the (apparatus) subclaims.
Both, the process and the apparatus according to the invention are not restricted to conveying and changing the relative position of exactly cuboidal packs, but can also handle packs of other designs like those having partially square cross-sections, cubic shape, rounded edges and the like.
Preferred embodiments of the invention are described in more detail below with reference to the drawings which show:
FIG. 1 a top plan view of a turning station with feed track and a discharge conveyor adjoining the feed track in the region of the turning station;
FIG. 2 a side view of the apparatus according to FIG. 1;
FIG. 3 a top plan view of a further embodiment of the invention with turning conveyors arranged differently to those of FIG. 1.
A feed track 10 delivers packs 11 to a conveying passage 14 formed by two separate conveying means, namely turning conveyors 12, 13 in the region of a turning station 14a. The feed track 10 has two feeder pairs 15, 16 being arranged in parallel planes above one another. Each feeder pair 15, 16 is formed by two conveying belts 17, 18 and 19, 20, respectively. The upper feeder pair 15 is therefore formed by the upper left conveying belt 17 in the conveying direction and the upper right conveying belt 18. Correspondingly, the lower feeder pair 16 is formed by the lower left conveying belt 19 and the lower right conveying belt 20. FIGS. 1 and 2 show that packs 11 are each held between the feeder pairs 15, 16 at four points of support.
A simpler embodiment is only provided with a lower feeder pair, so that the packs just have two points of support.
The conveyor belts 17 to 20 of the feed track 10 are led over deflecting rollers 21. 22 and 23, 24. The deflecting rollers for one feeder pair 15. 16 are axially aligned without being rigidly connected to one another.
The packs 11 are rectangular cigarette packs made of hard cardboard and contact the conveyor belts 17 to 20 with their side walls 25, 26. The end wall 27 and bottom wall 28 of each pack approximately point in a direction transverse to the conveying direction. Front wall 29 points in conveying direction and rear wall 30 in opposite direction of the conveying direction.
The turning conveyors 12, 13 are also designed as conveyor belts 35 and 36 being led over deflecting rollers 31. 32 and 33. 34. A pulling strand 37 or 38 being inside the conveying passage 14 comes to contact a certain portion of the pack 11. The conveyor belts 35, 36 conically taper in conveying direction, such that the conveying passage narrows in the conveying direction (see FIG. 1). The narrowest region of the conveying passage 14 is defined between the deflecting rollers 31 and 33 such that a pack 39 oriented parallel to the conveying direction is just able to pass this narrow region. The entering region into the conveying passage 14 between deflecting rollers 32 and 34 at the end of the feed track 10 is at least as wide as the diagonal line across one of the side walls 25, 26 is long.
For conveying the packs from the feed track 10 further and beyond the region of the conveying passage 14, two approximately parallel discharge conveyors 40, 41 contacting the side walls 25, 26 are provided. These discharge conveyors are also designed as belt conveyors 44. 45 being led over deflecting rollers 42 or 43. An interior strand 46 or 47 of the discharge conveyor 40, 41 comes to contact the side walls 25. 26 of the packs 11. 39. 48. The discharge conveyors thus form a conveying channel 49, in which the packs 11, 39, 48 are conveyed through the conveying passage 14.
The deflecting rollers 42, 43 for the conveyor belts 44, 45 of the discharge conveyors 40, 41 are axially aligned with the deflecting rollers 21, 22 and 23, 24 of the feed track 10, so that the packs 11, 39, 48 can enter the conveying passage 14 coming from the feed track without any vertical displacement or change of direction.
In the region adjoining the end of the conveying passage 14 in conveying direction, the conveying channel 49 is limited by lateral guiding means 50, 51, so that the packs 39 are not displaced by vibrations or the like. The guiding means 50, 51 are slightly warped to the outside (FIG. 1) near the deflecting rollers 30, 33, at the narrowest point of the conveying passage 14, in order to completely avoid the risk of the packs getting jammed. In the region of the upper discharge conveyor 40, there is a pressure means, namely a pressure roller 52, which rests on the strand 46 and thus applies pressure on the upper side wall 25 of each pack.
In the following, the process for changing the relative position of the packs will be described, the arrows A-F indicating the respective moving direction of the belt conveyors 17..20, 35, 36 and 44, 45. The packs 11, being spaced out from one another, are delivered on the feed track 10. The conveyor belts 17 and 19 being arranged on the left hand side in conveying direction run at a velocity of V2, which is higher than the velocity V1 of the conveyor belts 15, 16 arranged on the right hand side. This effects the packs 11, which are oriented transverse to the conveying direction at the beginning of the feed track 10, to be slightly turned at the end of the feed track 10, i e. when entering the conveying passage 14, thus being oriented in oblique position. It is of particular advantage, if the packs enter the conveying passage 14 in a relative position in which the diagonal line across the side walls 25 or 26 is at right angles to the conveying direction, as shown in FIG. 1. This relative position at the beginning of the conveying passage 14 ensures that the effective dimension being transverse to the conveying direction does not increase any more during the turning process but only decreases. The pack 11 then contacts the pulling strand 37 and 38 with a transverse edge 53 and an opposite transverse edge 54, respectively. While the pack 11 is now moved through the conveying passage 14 by the discharge conveyors 40, 41 at a velocity V3, said pack 11 is turned at the same time by the conveyor belts 35. 36 running at different velocities V4 and V5, as these conveyor belts pull the transverse edges 53, 54 relative to the movement of the discharge conveyors 40, 41 with them. In this manner the pack 11 is turned until it is oriented parallel to the conveying direction, just like pack 39 in FIG. 1. The velocity V3 of the conveyor belts 44, 45 of the discharge conveyors 40, 41 is higher than the velocity V2 of the faster conveyor belts 17, 19 of the feed track 10. The velocity V5 of the turning conveyor 13 (being on the right hand side in conveying direction) is smaller than the velocity V3 and the velocity V4 of the turning conveyor 12 (being on the left hand side in conveying direction). V5 is higher than V2 and V4 higher than V3.
If velocity V2 is taken as the base being 100% V1 approximately corresponds to 98%, V3 to 150%, V4 to 200% and V5 to 130%. In absolute terms, with a length of the feed track 10 of 1,000 mm and a pack width of 20 mm effective in conveying direction, the following velocities are particularly advantageous:
V1=49 m/min
V2=50 m/min
V3=77 m/min
V4=100 m/min
V5=65 m/min
Some of the figures given are rounded off. The velocity V3 of the discharge conveyors 40, 41 is chosen to be considerably higher than the velocities VI and V2 so that the packs 11, 39, 48 are spaced out more on their way through the conveying passage 14. In dependence on the already existing distances between the packs 11 on the feed track 10, this relation of velocities is necessary to avoid the risk of the successive packs wedging during the turning movement. If the packs 11 are already spaced out at sufficient distances on the feed track 10, there is no need for increasing the velocity in the conveying passage 14. FIG. 2 shows that the external diameter of the deflecting roller 42 for the conveyor belt 44 is smaller than the external diameter of the deflecting roller 21 for the conveyor belt 17. For this reason, the packs 11 do not contact the strand 46 with their upper side walls 25 immediately after entering the conveying passage 14. At this stage, the packs only rest on the strand 47 of the lower discharge conveyor and do not receive any counterpressure by the strand 46. This counterpressure does not effect the packs (pack 48 in FIGS. 1 and 2) until they have passed the pressure roller 52 in conveying direction. The pressure roller 52 effects a slight deflection of the strand 46 and at the same time regulates an exact distance between the two strands 46 and 47. The distance between the pressure roller 52 and the strands 31, 33 or 32, 34 is defined such that the pack 48 is not exposed to the pressure of the pressure roller 52, until a transverse edge 55 of the pack 48 which is the last one to pass the pressure roller in conveying direction, is between the strand 46 and the strand 47. This way, the risk of the edge 45 wedging with the strand 46 or 47 is avoided and a reliable turning process ensured.
In a further embodiment not shown in the drawings, the apparatus according to FIGS. 1 and 2 or the apparatus according to FIG. 3 which will be described below, is part of a packaging machine for cigarette packs, such as the one known from DE 34 00 650 A1. This prior art machine produces cuboidal (cigarette) packs from one blank by means of folding and adhesive bonding. In order to set the adhesive, the packs are taken over individually by a first drying turret and then fed to a second drying turret in groups, so that the adhesive can dry or harden completely. In the present embodiment of the invention, this second drying turret is replaced by the apparatus according to FIGS. 1 and 2 or 3. For this purpose, the cross-section of the conveying channel 49 formed by the discharge conveyors 40, 41 and the guiding means 50, 51 is dimensioned such that the packs receive pressure in a form-stabilizing manner, thus ensuring that the desired form of the packs is exactly preserved. Moreover, possible displacements in the region of the adhesive points in the packs can be corrected.
FIG. 3 shows a further embodiment of the apparatus according to the invention. Structure and function of this embodiment correspond to that of FIG. 1 unless it is explicitly stated to the contrary in the following description. In order to improve distinguishability, different reference numerals are used below. In contrast to the symmetrically arranged turning conveyors 12, 13 of FIG. 1, the turning conveyors 56, 57 in this embodiment are offset to one another in conveying direction. Correspondingly, the adjoining guide means 58, 59 are also offset to one another. The turning conveyor 57 being on the right hand side in conveying direction terminates approximately at a distance corresponding to half the length of a turning conveyor before the left-hand turning conveyor 56. Both conveying means 56, 57 are nevertheless of approximately the same length, just like the turning conveyors 12, 13 of FIG. 1.
A further difference is the structure of the feed track. The deflecting rollers 60 to 63 of FIG. 3 corresponding to the deflecting rollers 21 to 24 of FIG. 1 are in contrast to the latter not separately mounted in a rotatable manner. The deflecting rollers 60 and 61 for the conveyor belts 64, 65 contacting the side wall 70 (corresponding to side wall 25) are arranged on a common axis. The same applies to the deflecting rollers 62, 63 of the conveyor belts 66, 67
The turning process of the packs in the embodiment according to FIG. 3 can be described as follows:
Packs 69 delivered on the feed track 68 are spaced out from one another and oriented transverse to the conveying direction. When entering a conveying passage 71 starting at the end of the feed track 68, the packs 69 first of all contact the right hand turning conveyor 57. The packs 69 are led or held by the discharge conveyors 72, 73 bordering on the feed track 68. Due to the difference in velocities of the discharge conveyor 72, 73 and a conveyor belt 74 of the right-hand turning conveyor 57 and due to the transverse pack edge 75 lying ahead in conveying direction contacting the conveyor belt 74, the turning movement of the pack 69 commences. After the pack has turned approx. by 45° another transverse edge 76, which was originally pointing backwards contacts a conveyor belt 77 of the left-hand turning conveyor 56. This conveyor belt 77 runs faster than the discharge conveyors 72, 73 and therefore causes the turning process to continue. The individual phases from this moment until the end of the turning process are illustrated by the packs with reference numerals 78, 79, 80. Pack 78 contacts the conveyor belt 77 first. The effect of the conveyor belt 74 on the turning of the pack ends approximately at the position of pack 79. Pack 80 illustrates a position in which the turning process has already been completed. The embodiment according to FIG. 3 also comprises a pressure means in the form of a pressure roller 81, which takes effect in the region of the upper discharge conveyor 72 on the upper side wall 70 of each pack. Here too, the pressure roller 81 is arranged in the conveying direction such that the transverse edges 82, 83 of the pack 78 which are turning to come inbetween the discharge conveyors 72, 73 can not obstruct the turning process. The relative velocities of the individual conveyor belts approximately correspond to those of the embodiment according to FIG. 1, except that the velocities of the conveyor belts 64 to 67 of the feed track 68 do not differ, since the packs 69 are not partially turned on the feed track 68.
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Process and apparatus for changing the relative position of packs, especially of cuboidal cigarette packs of the hinge-lid type. Prior art processes and apparatuses work with forces having shock-like impacts on the packs, so that there is a risk of the packs getting damaged. The invention guarantees gentle changes of the relative position. Each pack is turned by way of being pulled by two conveyor belts running at different velocities and being arranged at two opposite regions of the pack.
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This is a division of application Ser. No. 07/376,289, filed Jul. 6, 1989 now U.S. Pat. No. 5,120,763, which is a continuation-in-part of U.S. application Ser. No. 07/120,830, filed Nov. 16, 1987 entitled "Physiologically Active and Nutritional Composition" now U.S. Pat. No. 4,851,431; of U.S. application Ser. No. 263,548, filed Oct. 27, 1988 entitled "Method for Treating Alzheimer's Disease and Related Dementias" now abandoned; and of U.S. application Ser. No. 07/359,562, filed Jun. 1, 1989 entitled "Method for Treating Epilepsy," now abandoned.
FIELD OF THE INVENTION
The present invention relates to a composition of matter having useful physiological and nutritional activity and applications, and to a method for inducing memory enhancement, analgesia, sleep regulation and inhibition of the symptoms of senility, for treating Alzheimer's disease and related dementias, and for treating epilepsy. The present application is filed as a continuation-in-part of U.S. application Ser. No. 07/120,830 filed Nov. 15, 1987 entitled "Physiologically Active and Nutritional Composition"; of U.S. application Ser. No. 263,548 filed Oct. 27, 1988 entitled "Method for Treating Alzheimer's Disease and Related Dementias"; and of U.S. application Ser. No. 07/359,562 filed Jun. 1, 1989 entitled "Method for Treating Epilepsy".
BACKGROUND OF THE INVENTION
It has been recognized for many years that the mammalian body requires for its nutrition relatively large amounts of fats, carbohydrates and proteins, and by contrast relatively small amounts of vitamins and minerals; lack of these latter classes of substances has been held to be accountable for the absence of general good health as well as the incidence of various specific bodily ailments. Vitamins and minerals are normally ingested or otherwise produced from the mammalian diet, but to a certain extent may also or alternatively be produced in the body. For various reasons which may be related to the source of supply or the manufacturing processes used, foods are sometimes lacking or dificient in vitamins and/or minerals, and even where vitamins are synthesized in the body, such a process may not produce the amount required. Over a period of time there has therefore grown up the use of food supplements, or nutritional compositions, to supply the ingredients of this nature required by the body, but which are either not produced therein in sufficient amounts, or are not supplied thereto by the regular diet of the subject in sufficient amounts.
Nutritional compositions are not at the present time, however, restricted merely to a content of vitamins and minerals, as the sole active ingredients. Other materias which are intermediate in metabolic processes and which it is though may not be produced in sufficient amounts (at least in subjects with abnormal metabolism) may also be present in nutritional compositions. Examples of such other materials are unsaturated fatty acids, such as linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid arachidonic and eicosapentaenoic aoids, as well as physiologically compatible derivatives thereof, such as salts, esters and amides of such acids, which may be metabolized in the body to prostaglandins. Prostaglandins are an important group of local hormones which act within the body tissues in which they are synthesized, in roles which are not entirely understood, though they may act at least to lower blood pressure, and to induce smooth muscle to contract.
Horrobin, in Med. Hypotheses 6: 469-486 (1980), has also proposed that a metabolic abnormality in the synthesis of certain prostaglandins is responsible for allowing an initial cancer cell to divide indefinitely, the abnormality being in particular, inhibition of the enzyme delta-6-desaturase which converts essential unsaturated fatty acids in normal cells to prostaglandins. He has also proposed pharmaceutical compositions (see e.g. EP 0037175 published Oct. 7, 1981 and prior patent applications referred to therein, the contents of which are to be regarded as incorporated herein by reference) comprising certain unsaturated fatty acids together with other ingredients which enhance formation in the body of essential prostaglandins and therefore bypass the metabolic abnormality referred to above.
In general, fatty acids in combined form are present in animal and vegetable fats and oils, but vegetable oils such as corn, cottonseed and soya oils contain in general a higher ratio of unsaturated to saturated acids, than do animal fats. A higher proportion of polyunsaturated fatty acids (such as linoleic and linolenic acids) in the diet apparently tends to reduce the incidence of heart disease, although whether this is due to a positive effect of the polyunsaturated compounds themselves, rather than to an intake of a correspondingly lower proportion of saturated compounds (and of cholesterol which is also present in animal fats), or to a lower fat intake overall, remains uncertain.
Senile dementia of Alzheimer's type (more commonly known as "Alzheimer's disease") appears to be the most widespread neuropsychiatrio disease and is becoming increasingly prevalent with the general aging of the population. It is not, however, confined to the elderly, as this term is generally understood. To the best of the inventor's knowledge, no satisfactory treatment for this type of dementia or for related dementias has yet been evolved.
The term "epilepsy" denotes a group of central nervous system disorders characterized by transitory episodes of abnormal motor, sensory, autonomic or psychic phenomena, and are almost always correlated with abnormal and excessive discharges in the encephalogram. Most known useful antiepileptic agents are either barbiturates or are structurally related thereto, or are benzodiazepines, dibenzoazepines, or lipide or lipid-like compounds containing phosphorus and/or nitrogen. Valproic acid (Pr 2 CHCOOH) and its derivatives are also useful in this respect. However, to the inventor's knowledge, it has not hitherto been proposed to use polyunsaturated unbranched long-chain carboxylic acids, containing only C, H and O, as antiepileptic compounds.
It has now been surprisingly found in accordance with the present invention, that a combination of two naturally occurring polyunsaturated acids within a certain range of proportions, produces certain beneficial effects in the human and animal body, including memory enhancement, analgesia, sleep regulation and inhibition of the symptoms of senility; and may also be used in a method of treatment for Alzheimer's disease and related dementias, and epilepsy, which method relieves at least certain symptoms of such diseases.
Experiments carried out by the inventor support the belief that it is the combination of these acids themselves in particular proportions which is the active factor in producing the beneficial and therapeutic effects just referred to; there is no evidence at present that such effects are connected with the metabolization of these acids to other substances.
It is particularly to be noted that the beneficial and therapeutic effects of the combination of the two specified acids (or their derivatives) may be adversely effected by the presence of an oily carrier or diluent which comprises at least one member of the group consisting of C 8-18 saturated fatty acids, oleic acid and derivatives of these acids, and the invention thus excludes the use of such diluents or carriers. As persons skilled in the art will be aware, the use of natural oily diluents and carriers of vegetable or animal origin is accordingly excluded from the scope of the invention.
SUMMARY OF THE INVENTION
The present invention accordingly provides a composition of matter which consists of (a) from about 13.0 to about 27.5% by weight of a compound selected from linolenic acid and derivatives thereof, calculated as the free acid, the derivatives of linolenic acid being both physiologically hydrolyzable and pharmacologically acceptable, and (b) about 87.0 to about 72.5% by weight of a compound selected from linoleic acid and derivatives thereof, calculated as the free acid, the derivatives of linoleic acid being both physiologically hydrolyzable and pharmacologically acceptable.
The present invention also provides, in another aspect, a pharmaceutical formulation which comprises the composition of matter as just defined, together with at least one pharmaceutically acceptable substance selected from diluents, carriers and adjuvants, excluding an oily carrier or diluent which comprises at least one member of the group consisting of C 8-18 saturated fatty acids, oleic acid and derivatives of these acids.
In yet another aspect, the invention provides a nutritional composition, adapted for consumption by mammals, characterized by the presence of (i) an orally ingestible diluent or carrier, excluding an oily carrier or diluent which comprises at least one member of the group consisting of C 8-18 saturated fatty acids, oleic acid and derivatives of these acids, (ii) at least one compound selected from linolenic acid and physiologically non-deleterious and hydrolyzable derivatives thereof and (iii) at least one compound selected from linoleic acid and physiologically non-deleterious and hydrolyzable derivatives thereof, such that the proportion of ingredient (ii) calculated as a percentage by weight of the combined ingredients (ii) and (iii) is from about 13.0 to about 27.5%, each of (ii) and (iii) being calculated as free acids.
In still another aspect, there is provided by the invention a method for treating a mammal for the purpose of inducing in the mammal a beneficial effect selected from memory enhancement, analgesia, sleep regulation and inhibition of the symptome of senility which comprises administering to the mammal a composition of matter as defined above in an amount effective to induce the at least one physiological effect, or a pharmaceutical formulation as defined above which contains an amount of the composition of matter effective to induce the at least one physiological effect. It will be appreciated that the compositions and formulations of the invention are intended in particular for administration to humans, or for human consumption, for the purpose of inducing in humans at least one of the aforementioned beneficial effects.
In a further aspect, the invention provides a method for treating Alzheimer's disease and related dementias, and epilepsy, which comprises administering to a person having the symptoms of Alzheimer's disease or related dementias, or to a person susceptible to epilepsy, a symptom-alleviating amount of a composition of matter or pharmaceutical formulation as defined above.
It will be apparent that the methods of the invention may utilize the nutritional compositions as described and claimed herein, which contain either an amount of the composition of matter effective to induce the at least one physiological effect, or a symptom-alleviating effective amount of the composition of matter (in the case of treating Alzheimer's disease or related dementias, or epilepsy), as the case may be.
With regard to the method of the invention as applied to the treatment of epilepsy, it should be noted that for purposes of definition, such terms as "method for the treatment of epilepsy", "alleviation of symptoms", and similar expressions, where used in the present specification and claims, are intended to include inter alia any such treatment which is effective to reduce the incidence and/or intensity of epileptic occurrences in subjects susceptible to such occurrences.
DETAILED DESCRIPTION OF THE INVENTION
The composition of matter according to the invention preferably consists of from about 14.3 to about 25.0% by weight of component (a) and about 85.7 to about 75.0% by weight of component (b), more preferably from about 16.3 to about 24.4% by weight of component (a) and about 83.7 to about 75.6% by weight of component (b).
In accordance with a particular embodiment of the invention, a special memory enhancement effect has been noted when the composition of matter consists of from about 20.0 to about 24.4% by weight of component (a) and about 80.0.to about 75.6% by weight of component (b), or from about 18.2 to about 22.2% by weight of component (a) and about 81.8 to about 77.8% by weight of component (b); and particularly when the composition consists of either about 22.2% by weight of component (a) and about 77.8% by weight of component (b), or about 20.0% by weight of component (a) and about 80.0% by weight of component (b).
As regards the method according to the invention treating Alzheimer's disease and related dementias, and epilepsy, the composition of matter useful therein preferably consists of from about 15.0 to about 24.5% by weight of component (a) and about 85.0 to about 75.5% by weight of component (b), more preferably from about 16.7 to about 22.2% by weight of component (a) and about 83.3.to about 77.6% by weight of component (b), and it is especially preferred to use such a composition of matter which consists of about 19.0% by weight of component (a) and about 81.0% by weight of component (b).
The preferred percentage proportions by weight are also of course applicable to the relationship between the at least one compound selected from linolenic acid and physiologically non-deleterious and hydrolyzable derivatives thereof, and the at least one compound selected from linoleic acid and physiologically non-deleterious and hydrolyzable derivatives thereof (calculated as the free acids), in the nutritional compositions of the invention.
Since, as has been intimated above, it is believed that the combination of linoleic and linolenic acids is the active principle per se which induces the effects mentioned, it will be appreciated by those skilled in the art that instead of the acids themselves, there may be utilized in the composition of the invention derivatives of these acids which are both physiologically hydrolyzable (to the corresponding acids) and pharmacologically acceptable. Such derivatives may for example be selected from salts, esters and amides of the respective acids.
Among suitable salts there may be mentioned the ammonium, sodium, potassium, calcium and magnesium salts as salts with substituted mono- and di-substituted amines and the analogous saturated heterocyclic compounds containing an NH group in the ring, so long as the amines and the analogues in question are physiologically acceptable. As suitable esters there may be mentioned, for example, the ethyl and glyceryl esters. Amides of the acids may also be utilized, e.g. amides of the acids with substituted mono- and di-substituted amines and with the analogous saturated heterocyclic compounds containing an NH group in the ring, so long as the amines and the analogues in question are physiologically acceptable. It will be appreciated that the latter stipulation is necessary (in the case of the amine salts, the amides and their heterocyclic analogues) since it is to be expected that such derivatives will metabolize in the body to the desired acids and the starting amines or heterocyclic compounds. It will of course be evident to a person skilled in the art how to select a particular salt, ester or amide, so as to comply with the requirements of physiologically hydrolyzing to the corresponding acids, and pharmacological acceptability.
The pharmaceutical formulation provided in accordance with the present invention may be adapted for oral, parenteral or rectal administration, and it may be in the form of dosage units. The diluents, carriers and adjuvants are those conventionally used in pharmaceutical and veterinary formulation.
For oral administration, the pharmaceutical formulations of the invention may be utilized as e.g. tablets, capsules, emulsions, solutions, syrups or suspensions. For parenteral administration, the formulations may be utilized as ampoules, or otherwise as suspensions, solutions or emulsions in aqueous or oily vehicles. The need for suspending, stabilizing and/or dispersing agents will of course take account of the fact of the solubility or otherwise of the linoleic and linolenic acids, or of their derivatives used in the formulations, in the vehicles which are used in particular embodiments. Thus, for example, where the acids themselves are used, account will be taken of the fact that these have a relatively low water solubility and in general a relatively high oil solubility. The formulations may additionally contain e.g. physiologically compatible preservatives and antioxidants.
The pharmaceutical formulations of the invention may also be utilized as suppositories with conventional suppository bases such as cocoa butter or other glycerides. As is well known in the pharmaceutical art, the formulations may also be made available in a depot form which will release the active composition slowly in the body, over a preselected time period.
The nutritional composition according to the invention includes as a necessary component an orally ingestible diluent or carrier; this may for example comprise a substance selected from sugar-based confectionery, a manufactured cereal, a fruit or vegetable product, a beverage or beverage concentrate, or any inert diluent, carrier or excipient known in the pharmaceutical art. It is intended generally that ingredients (ii) and (iii), as previously defined, may be used in nutritional compositions in any of the forms in which these are known and practiced in the art. Thus, the nutritional compositions may take the form of, e.g., sugar-based confectionery such as candies or chocalate, breakfast cereals, fruit or vegetable purees or beverages, other beverages (including those based on carbonated water), or beverage concentrates generally (including those in the form of e.g. powders, granules, flakes or crystals, which are intended to be mixed with hot or cold water and/or milk). The nutritional compositions may also generally be in the form of powders, tablets, capsules, solutions, concentrates, syrups, suspensions, gels or dispersions. It will be evident that when the nutritional compositions take the form of dispersions or suspensions, it will usually be necessary to use an acceptable (i.e. non-toxic and otherwise suitable) dispersing or suspending agent, as is well known in the nutritional and pharmaceutical arts. When these compositions are utilized in the form of capsules, it will be evident that gelatin or other known suitable ingestible materials may be used for encapsulation.
The present invention moreover includes the nutritional compositions described herein, which are adapted for consumption by non-human, as well as human mammals.
The present invention further includes nutritional compositions which also include any of the known vitamins. Thus for example, such compositions (which may be, but need not be, in the form of aqueous suspensions) may comprise at least one water-soluble vitamin selected from thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic acid, cobalamin and ascorbic acid. Alternatively or additionally, such compositions may comprise at least one oil-soluble vitamin selected from retinol, calciferol, tocopherol and menadione. The nutritional compositions of the present invention may also comprise in combined form at least one element selected from sodium, potassium, calcium, phosphorus, magnesium, chlorine and sulfur, and additionally or alternatively, at least one element selected from iron, copper, iodine, manganese, cobalt, zinc, molybdenum, fluorine, selenium and chromium. These compositions may also contain other natural or synthetic antioxidants.
The nutritional compositions of the present invention may also comprise other unsaturated fatty acids, such as for example those known to be metabolized in the body to prostaglandins, e.g. dihomo-gamma-linolenic acid, arachidonic and eicosapentaenoic acids, as well as physiologically compatible derivatives thereof, such as salts, esters and amides of such acids.
The invention will be illustrated by the following Examples.
EXAMPLE I
TREATMENT OF EXPERIMENTAL ANIMALS
METHOD
Subjects were male Long Evans (hooded) rats weighing initially 100 g. They were housed individually in hanging stainless steel, wire-mesh cages in a well-ventilated room at an ambient temperature of 20°-22° C. Light (Vita-Lite, Dura-Test, N.J.) was provided from 06.00 hrs. to 18.00 hrs. daily. A control group of rats (Group A) was fed a diet of linoleic acid 35 mg./kg. diet plus linolenic acid 0.15 mg./kg. diet. Other groups of rats received the same diet, plus a daily aqueous injection of a mixture of linoleic and linolenic acids (with polyethylene glycol emulsifier), each rat receiving by injection 25 mg. linolenic acid, the balance being linoleic acid. (It will be appreciated that the amounts of linoleic and linolenic acids ingested by the rats from the diet just described is insignificant compared with the amounts of these substances administered by injection.) The composition of the injected unsaturated fatty acid mixture was varied among different experimental groups; the percentage by weight of linolenic acid in the mixture was as follows (balance linoleic acid): 25.0; 22.2; 20.0; 18.2; 16.7; 15.4; and 14.3 (these groups were respectively labelled B, C, D, E, F, G and H. Otherwise expressed, the ratios by weight were respectively; 1:3; 1:3.5; 1:4; 1:4.5; 1:5; 1:5.5; and 1:6.0. Another route of administration (i.e., supplemented water or an enriched diet) was tested with closely similar results. Throughout the experiment the rate had free access to food and water. Handling of the rats was kept to a minimum, so as not to interfere with the learning.
Every week groups of rats, from each treatment regimen, were tested in the learning apparatus. The level of motor activity, pain threshold, colonic temperature and d-amphetamine-induced hypothemis were tested in different groups. The order of testing was as follows: first day, rotor activity; second day, pain threshold; third day, thermoregulation.
The learning apparatus is known from the scientific literature. Briefly, a circular tank (110 cm. in diameter) was filled with water (at the 40 cm. level), which was made opaque by the addition of powdered milk, so that rats swimming in the tank were unable to see an escape platform (7.5 cm. in diameter) submerged 2 cm. below the water level. Each animal was released facing the wall in one of four predetermined starting points located every 90° around the inner perimeter. A mass-learning technique was used, and each rat was tested 8 times per day in the tanks. The order of starting points was randomly predetermined. Each rat was allowed 120 seconds to find the platform, and intertrial interval was 20 seconds. The rate were tested during 3 consecutive days. During this period the platform was in the same location in the tank. After completion of the session on day 3, the platform was removed to another location in the tank, and the performance of rats in the new position was recorded. For each of the 24 trials (8 trials×3 days) the latency to reach the platform was recorded. A cut-off point criterion (i.e., the first trial to reach latency of 10 seconds, without increasing the latency in a later trial) was used to calculate the learning capacity of each diet group. To calculate the resistance to extinction, the time which the rats spent in the "old position" in the first trial was recorded.
The level of motor activity was assessed in an open field apparatus by recording the number of horizontal movements (infrared photobeam crossing) and rearing movements (determined from videotapings) made during the 15 minute sessions. The apparatus was very similar to the one previously described by Coscina et al. in 1986.
Pain threshold was measured with a hot plate (60×60 cm.) heated by a thermostatic bath (Hakke, Germany) to 58+/-0.20 C. The latency to lick the paw was recorded to the nearest 0.1 second. On the third day, the basic colonic temperature of each rat was measured (YSI telethermometer, model 43TA, Yellow Springs, Ohio). The rat was then injected with 15.0 mg./kg., i.p., d-amphetamine and placed immediately into a 4° C. cold room for 1 hour.
All tests were conducted between 10.00 and 14.00 hrs. There were 9 rats in each experimental group. At the end of each week of the experiment, the brain of the rate was removed for biochemical analysis for a different study. All experiments were conducted "double blind," i.e., the experimenter was unaware of the diet fed to the individual subjects or which treatment the rat received. Comparisons across diets and weeks of treatment were analyzed by analysis of variance with contrast tests.
RESULTS
TABLE 1______________________________________NUTRITIONAL FACTORSGROUP FOOD INTAKE (K Cal) WEIGHT GAIN______________________________________A 2565 +/- 39 237 +/- 4.7B 2575 +/- 80 230 +/- 7.0C 2545 +/- 75 235 +/- 2.8D 2534 +/- 68 237 +/- 4.6E 2543 +/- 72 239 +/- 6.1F 2562 +/- 57 235 +/- 3.3G 2586 +/- 48 238 +/- 3.9H 2533 +/- 61 234 +/- 5.5Data expressed as M +/- SEMP N.S. N.S.______________________________________
Unsaturated fatty acid treatment has no effect on either the amount of food intake (in kCal. units) nor on the rate of body weight gain.
LEARNING
TABLE 2__________________________________________________________________________NUMBER OF TRIALS TO REACH CRITERION (10 secs.) WEEKS OF TREATMENTGROUP (P) 0 1 2 3 4__________________________________________________________________________A (N.S.) 19.6 +/- 3.3 19.0 +/- 3.7 20.3 +/- 2.5 18.5 +/- 2.9 19.1 +/- 2.7B (N.S.) 20.1 +/- 4.1 18.0 +/- 4.0 19.9 +/- 4.5 17.1 +/- 4.0 17.0 +/- 3.2C (0.01) 17.1 +/- 3.3 12.5 +/- 2.1* 10.7 +/- 4.1* 7.9 +/- 3.9* 5.6 +/- 2.5*D (0.001) 18.5 +/- 2.0 9.3 +/- 2.6* 7.1 +/- 2.9* 6.1 +/- 2.8* 6.1 +/- 2.5*E (0.01) 19.1 +/- 2.3 14.2 +/- 3.7* 12.8 +/- 3.9* 9.6 +/- 3.0* 9.0 +/- 3.4*F (0.01) 19.5 +/- 3.5 16.1 +/- 2.6 11.2 +/- 1.1* 9.2 +/- 1.8* 7.9 +/- 1.0*G (N.S.) 19.7 +/- 3.8 18.1 +/- 3.3 18.4 +/- 2.9 17.9 +/- 4.1 18.6 +/- 2.6H (N.S.) 21.0 +/- 4.0 20.0 +/- 3.0 19.6 +/- 3.1 18.8 +/- 3.9 19.1 +/- 3.0P N.S. 0.01 0.01 0.01 0.01__________________________________________________________________________ *Statistically differs from Control (M +/- SEM)
Treatment with the ratios 1:3.5 to 1:5 (Groups C to F) has a significant effect on the rate of learning. The optimal ratio was 1:4 (Group D).
Comparative experiments were carried out by adminstering, in place of the inventive compositions, eleven similar non-inventive compositions comprising (i) 100% linolenic, linoleic or gamma-linolenic soids, respectively, or (ii) 95.2, 90.9, 83.3, 50.0, 9.1, 6.3, 4.8, or 3.8 linolenic acid (balance linoleic acid). After 4 weeks, the results in these cases generally showed either no significent change in learning ability, or a tendency to detract from learning ability; in two cases (9.1 and 6.3) an initial apparent small increase in learning ability had diminished by the end of 4 weeks.
TABLE 3__________________________________________________________________________TIME IN THE `WRONG' LOCATION; MEANS OF THE FIRST 2 TRIALS WEEKS OF TREATMENTGROUP (P) 0 1 2 3 4__________________________________________________________________________A (N.S.) 22.9 +/- 3 24.3 +/- 4 19.0 +/- 3 22.3 +/- .4 25.1 +/- 4B (N.S.) 18.5 +/- 3 19.4 +/- 4 20.6 +/- 6 20.6 +/- 4 20.1 +/- .5C (0.001) 20.3 +/- 4 30.9 +/- 2* 35.3 +/- 4* 39.2 +/- 4* 49.4 +/- 3*D (0.01) 19.5 +/- 3 24.1 +/- 3 29.3 +/- 4* 36.6 +/- 4* 39.1 +/- 4*E (0.01) 20.8 +/- 4 25.1 +/- 4 30.1 +/- 3* 33.1 +/- 4* 36.1 +/- 5*F (0.01) 19.4 +/- 3 22.1 +/- 3 29.1 +/- 5* 30.1 +/- 5* 32.2 +/- 5*G (N.S.) 22.8 +/- 4 19.4 +/- 3 19.0 +/- 3 19.6 +/- 4 18.1 +/- 4H (N.S.) 19.1 +/- 5 18.7 +/- 5 19.9 +/- 4 21.1 +/- 3 19.6 +/- 5P N.S. 0.01 0.001 0.001 0.001__________________________________________________________________________ *Statistically differs from Control (M +/- SEM).
Unsaturated fatty acid treatment with ratios of 1:3.5-1:5 (Groups C to F) has a significant effect on retention of the old learning. The most effective ratio was 1:3.5 (Group C).
MOTOR ACTIVITY
TABLE 4______________________________________AT THE END OF THE 4 WEEKS' TREATMENTGROUP LINE CROSSING REARING______________________________________A 76.0 +/- 27 75.0 +/- 5.0B 74.0 +/- 30 75.5 +/- 5.5C 70.7 +/- 25 76.0 +/- 4.5D 70.3 +/- 33 84.3 +/- 5.5E 72.1 +/- 29 77.7 +/- 6.6F 74.1 +/- 32 74.6 +/- 5.1G 72.5 +/- 25 76.9 +/- 6.1H 75.5 +/- 31 80.0 +/- 5.5P N.S. N.S.______________________________________
None of the treatment has any effect on horizontal or on vertical movement.
PAIN THRESHOLD
TABLE 5__________________________________________________________________________ WEEKS OF TREATMENTGROUP (P) 0 1 2 3 4__________________________________________________________________________A (N.S.) 7.9 +/- .9 7.8 +/- .8 8.0 +/- .6 7.9 +/- .9 8.1 +/- .9B (N.S.) 8.0 +/- .8 7.9 +/- .7 8.0 +/- .9 8.1 +/- .7 7.8 +/- .7C (0.01) 7.8 +/- .6 11.9 +/- .7 13.9 +/- .7* 16.5 +/- .6* 20.1 +/- 1.1*D (0.01) 8.1 +/- .8 12.1 +/- .6* 14.5 +/- .6* 18.2 +/- .7* 21.1 +/- .9*E (0.01) 7.8 +/- .6 9.0 +/- .9* 9.0 +/- .8* 14.1 +/- .7* 17.4 +/- .7*F (0.01) 8.1 +/- .9 9.9 +/- .9 11.5 +/- .7 14.1 +/- .7* 16.3 +/- .7*G (N.S.) 7.6 +/- .7 8.0 +/- .3 8.8 +/- .8 8.0 +/- .8 8.1 +/- .9H (N.S.) 8.0 +/- .9 8.0 +/- .4 8.5 +/- .5 8.3 +/- .7 8.3 +/- .7P N.S. 0.05 0.01 0.01 0.01__________________________________________________________________________ *Statistically differs from control (M +/- SEM).
Unsaturated fatty acid treatments with ratios of 1:3.5 to 1:4.5 (Groups C to E) cause analgesia among rats which were placed on a hot plate (58° C.). The most effective ratio seems to be 1:4 (Group D).
THERMAL RESPONSE TO D-AMPHETAMINE AT 4° C.
TABLE 6__________________________________________________________________________ WEEKS OF TREATMENTGROUP (P) 0 1 2 3 4__________________________________________________________________________A (N.S.) -1.9 +/- .7 -1.8 +/- .8 -1.7 +/- .6 -1.9 +/- .7 -2.0 +/- .9B (N.S.) -1.9 +/- .9 -1.9 +/- .9 -1.8 +/- .7 -2.0 +/- .9 -2.1 +/- .7C (0.001) -2.0 +/- .5 -.9 +/- .3* -.8 +/- .3* +.9 +/- .9* +1.1 +/- .7*D (0.001) -2.0 +/- .7 -.9 +/- .6* +.9 +/- .5* +1.1 +/- .7* +1.2 +/- .6*E (0.001) -1.9 +/- .8 -.6 +/- .9* +.6 +/- .3* +.9 +/- .9* +.9 +/- .9*F (0.01) -1.9 +/- .8 -.9 +/- .7* -.9 +/- .7* +.7 +/- .5* +1.1 +/- .9*G (N.S.) -1.9 +/- .9 -2.0 +/- .7 -1.8 +/- .7 -1.8 +/- 1.2 -1.9 +/- .7H (N.S.) -2.0 +/- .9 -1.9 +/- .7 -2.1 +/- .9 -2.0 +/- .9 -2.2 +/- 1.1P N.S. 0.05 0.01 0.01 0.01__________________________________________________________________________ *Statistically differs from Control (M +/- SEM)
Unsaturated fatty acid treatment protected rats from d-amphetamine induced hypothermia at ambient temperature of 4° C. A ratio of 1:4 (Group D) seems to be most effective.
SLEEP PARAMETERS
A small number of rats (n=6) received unsaturated fatty acids at a ratio of 1:3.5 (as in Group C) for 4 weeks. At the end of the treatment period the length of total sleeping hours and REM percentage was examined and compared with saline treated rats. A strong tendency (but not statistically significant) of longer sleeping hours (+30%) and an increase in REM periods (+18%) were found in treated rats.
THE EFFECT OF IRON DEFICIENCY ON LEARNING
Iron deficiency induced severe learning deficits both in water maze and in water tank learning. Similar deficits were obtained by brain lesions. While control rats needed 19.6+/-3.3 trial to reach the criterion of learning, iron-deficient rats needed 26.4+/-1.1 to reach the same performance. Iron-deficient rats treated for 3 weeks with 1:4 ratio (as in Group D) before training reached the criterion in 15.9+/-4.8 trial while saline treated rats had the same 27.0+/-1.2 trials to criterion.
THE EFFECT OF AGING ON LEARNING
Old male rats (20-22 months old) showed a strong deficit in learning. Among 7 non-treated rats none was able to learn the swim test. The group of old rats (n=6) was able to learn the swim test after 1:4 ratio treatment (as in Group D) of 6 weeks duration. They learned the task in 15.9+/-6.1 trials. However, due to the small number of rats, and because it is one trial without replication, it is difficult to draw positive conclusions on the effect of such treatment on old age learning deficit.
THE EFFECT OF OTHER FATTY ACID & NATURAL OIL CARRIERS ON LEARNING
When linolenic acid was added to corn oil, olive oil or sunflower oil, or when linoleic acid was added to linseed oil, in order that the linolenic:linoleic acid ratio therein (calculated as free acids) should be 1:4 by weight, such compositions had no effect on learning. Also, when a mixture of free linoleic and linolenic acids in this ratio was mixed with free palmic acid or free stearic acid, as carrier, such compositions had no effect on learning, while substitution of oleic acid for the palmic or stearic acid in these experiments gave inconclusive results.
It is concluded that palmic, stearic and (probably) C 8-18 saturated fatty acids generally and oleic acid, as well as derivatives thereof, when comprised in carriers or diluents, adversely affect the beneficial properties of the inventive compositions, and also that natural oils are unsuitable for use as carriers or diluents for the compositions of the invention. A person skilled in the art will be able to determine without undue experimentation whether or not smaller than carrier quantities of the thus excluded acids (and their derivatives), would adversely affect the advantageous biological activity of the inventive composition of matter. Insofar as it may be found that these smaller quantities would not unduly affect such activity, compositions including them as well as the inventive combination of linoleic and linolenic acids are deemed to be comprised within spirit and scope of the invention.
EXAMPLE II
STUDY OF HUMANS
225 mg. of the composition of one embodiment of the present invention which contained 22.2% by weight linolenic acid and the balance linoleio acid, was given twice daily to 12 demented geriatric patients (male and female, mean age 76.5+/-2). A comparable group of 12 geriatric patients (similar in age and severity of dementia) was given a placebo (lemonade syrup, the vehicle of the unsaturated fatty acid mixture). The treatment length was 28 days. The study was carried out in double-blind fashion; (however, there were some differences between the treatment and the lemonade, mainly in oclor). The medical staff, doctors and nurses, were instructed to follow the 24 subjects and to tell by the end of the period which one was "improved" on a subjective scale. The medical staff identified correctly 10 of the 12 treated patients as improved, and none of the 11 placebo group (one of the subjects in this group had to leave the experiment because of other medical problems). The patients seemed to be improved in the following aspects: they were more cooperative; they were in a better mood; appetites improved; they were able to remember their way around the hospital, and complained less about sleep disturbances. The food intake (amount and type of food) was not controlled. However, the rate of success in identifying treated subjects was highly significant.
EXAMPLE III
TESTS ON ALZHEIMER'S DISEASE PATIENTS
Example IIIa
Selection of Patients
Alzheimer's disease was defined according to the Diagnostic & Statistical Manual of the American Psychiatrists Association, 3rd Edn., March 1980. The criteria for inclusion are: complaints of discrientation in space and time, cognitive deficits and low scores in the Mini-Mental Test. Criteria for exclusion from the study are: multi infarotion dementia, depressive or post-depressive dementia, post-traumatic dementia, post-psychotic dementia, known endocrine disorder, normintensive hydrocephalus, very aggressive patient, very severe condition requiring constance assistance in the daily routine.
Criteria for Assessment of Results
The guardian was asked to assess the severity of the patient's condition in 12 areas of behaviour on a five-point scale, a score of 5 indicating a severe problem, a score of 1 indicating no problem. The 12 areas were as follows: 1. Space orientation: can the patient find the way to return alone without confusion and without losing sense of direction?2. Level of cooperation with family and doctors etc. 3. General mood of patient, especially whether aggressive. 4. The patient's appetite, attention to food, time of meals, interest in food. 5. Ability of patient to keep belongings in order and organize his life. 5. Short-term memory--ability to remember recent events. 7. Long-term memory--ability to remember remote events. 8. Sleep habits and sleep disturbances. 9. Whether patient is alert during daytime: periods of alertness and span of attention. 10. Does patient have auditory or visual hallucinations; if so, at what time of day? 11. Is patient capable of self-expression in clear speech and ideas? 12. Control of urination.
Method
Treatment in accordance with the present invention (or placebo) were carried out on 100 patients (79 male and 21 female; 24 of the 100 were hospitalized), of ages in the range 50-73 years. 71 of the patients had been definitely diagnosed as having Alzheimer's disease at least 4 years previously; in the past they had been treated with hydergin, various cholinergic drugs, piracetam or/and lecithin. The present treatment was administered to 60 patients, while 40 received placebo. The following table summarizes the patient sample.
______________________________________Classification of Alzheimer's Patients Hospitalized Non-hospitalized M F M F______________________________________Treatment 9 3 40 8Placebo 9 3 21 7Totals 18 6 61 15______________________________________
Patients received oral doses, 1 ml. morning and evening, of either medication or placebo, over a period of three weeks; the medication, which was kept in the cold or refrigerated, comprised per ml. 0.25 ml. of a mixture of linolenic and linoleic acids (Sigma) in a 1:4.25 weight ratio (19.05%: 80.95%), 0.73 ml. paraffin oil, 0.02 ml. alphatocopherol (Vitamin E, Sigma) and a few drops of flavoring (almond essence). At the end of the three-week period, each patient was medically reexamined, PEG recorded and samples of blood and urine were tested. A Mini Mental test was administered and the guardian was again asked to rate the severity of complaints on the basis of the above 12 questions.
Results
(1) The following table summarizes scores in the Mini Mental Test. The stated figures are average values for the number of patients (n).
______________________________________Treatment of Alzheimer's Patients - Mini Mental Test Scores Treatment (n = 60) responders non-respondersPlacebo (n = 40) (n = 49) (n = 11)______________________________________Before 7.3 ± 3.1 7.8 ± 2.9 7.6 ± 3.2After 7.5 ± 3.5 16.0 ± 3.7* 8.0 ± 2.5______________________________________ *P<0.01
(2) The following table summarizes the results of assessment of patients' condition in the 12 areas detailed above, on the 5-point scale, before and after treatment. A change of at least 1.4 units is considered significant. Each fraction denotes: ##EQU1##
______________________________________Treatment of Alzheimer's Patients - Ratings in 12 Areas Placebo (n = 40) Treatment (n = 60) fraction fractionAREA improved % improved %______________________________________1. Space orientation 3/33 9.0 37/50 74.02. Cooperativeness 2/31 5.1 28/49 57.13. Mood 5/27 18.5 27/44 61.44. Appetite 2/31 5.1 26/48 54.25. Organization 4/32 12.5 33/48 68.76. Short-term memory 1/3- 2.9 40/59 74.07. Long-term memory 0/38 0 34/58 58.78. Sleep problems -2/27 -7.4 21/29 74.49. Daytime alertness -2/33 -5.1 29/47 61.710. Hallucinations -2/10 -20.0 12/14 85.011. Self-expression 1/36 2.7 16/52 30.712. Bladder control 3/14 21.4 -2/27 -7.4______________________________________
(3) General physiological effects
No major side effects were found after the three week treatment period; one patient only suffered from severe stomach upset and diarrhhea. Body temperature and blood pressure (systolic and diastolic) were unchanged at the end of this period. Biochemical blood and urine laboratory tests did not show significant changes after treatment. There was no increase in total lipids in the blood. There was a tendency of the blood cholesterol level to decrease, but this was not necessarily of statistical significance.
Example IIIb
A further group of 13 Alzheimer's Disease out-patients, 5 male and 4 female, in the age range of 59-71 years, was treated with the same oral medication (no placebo), in a similar manner to Example IIIa.
Results
(1) The following table summarizes scores in the Mini Mental Test. The stated figures are average values for the number of patients (n).
______________________________________Treatment of Alzheimer's Patients - Mini Mental Test Scores Treatment (n = 13) responders (n = 10)______________________________________Before 7.0 ± 1.7After 15.6 ± 2.1*______________________________________ *P<0.01
(2) The following table summarizes the results of assessment of patients' condition in the 12 areas detailed above, on the 5-point scale, before and after treatment. A change of at least 1.4 units is considered significant. Each fraction denotes: ##EQU2##
______________________________________Treatment of Alzheimer's Patients - Ratings in 12 Areas Treatment (n = 13) fractionAREA improved %______________________________________1. Space orientation 8/12 662. Cooperativeness 5/12 423. Mood 5/9 554. Appetite 6/11 555. Organization 8/11 726. Short-term memory 10/12 837. Long-term memory 9/12 758. Sleep problems 4/6 669. Daytime alertness 5/11 4510. Hallucinations 5/6 8311. Self-expression 5/12 4212. Bladder control 0/7 0______________________________________
Unwanted side effects were not observed in the group of 13 patients. It is seen that the results in Example IIIb are in line with the noted improvement in the condition of Alzheimer's patients found in Example I, when the method according to the present invention is utilized.
EXAMPLE IV
TESTS ON ANIMAL MODELS OF EPILEPSY
Preliminary note
Tests on animal models enable possible antiepileptic drugs to be evaluated for potential therapeutic use in humans. Such tests measure the protection afforded by the drug against the convulsant effects of chemical or electrical stimulants. An important chemical stimulant utilized for these tests is pentylenetetrazol (PTZ, Metrazol-Knoll), which has been used in experimental animals to induce grand mal seizures.
EXAMPLE IVa
The inventive composition (labelled "SR-3") administered to animal models in accordance with the method of the invention comprised per ml. 0.40 ml. of a mixture of linolenic and linoleic acids (Sigma) in a 1:4 weight ratio (20%:80%), 0.59 rl. paraffin oil, and 0.01 ml. alpha-tocopherol (Vitamin E, Sigma).
In a first stage, the ED 50 for PTZ-induced grand mal seizures in SPD rats was investigated and found to be 76.5 mg./kg. The LD 50 is tightly closed at 81.0 mg./kg.
In a second stage, 80 (150 g.) male rats were divided into two groups. One group received 0.2 ml. i.p. saline treatment daily (4C rats) and the other group received treatment with 0.2 ml. i.p. SR-3 daily (40 rats), for a period of three weeks. After the three-week treatment, the rats were given one of two doses of PTZ, 50 mg./kg. or 100 mg./kg. The rats were observed by two independent observers, who were ignorant of which treatment each rat received. An EEG recording was not performed. The following variables were recorded in Table 7, below:
1. latency (seconds) to the first grand mal seizure (tonic-clonic contractions of the limbs, trunk and head, falling, saliva and blood discharge from the mouth);
2. number of rats responding with grand mal seizures;
3. mean duration (in seconds) of the grand mal seizures;
4. number of rats that died not exhibiting grand mal seizures but showing "infantile spasms" (sudden flexion of the forelegs, forward flexion of the trunk, or extension of the rear legs), the attack lasting only a few seconds, but it was repeated several times;
5. number of rats that died 15 minutes after PTZ injection.
TABLE 7______________________________________Study of the effect of SR-3 on animal modesl ofPTZ-induced epilepsy PTZ dose Variable studiedPretreatment (mg./kg.) 1 2 3 4 5______________________________________Saline* 50 28 ± 12 8 637 ± 31 8 9SR-3* 50 252 ± 11 1 24 1 0Saline* 100 7 ± 3 19 893 ± 14 1 20SR-3* 100 154 ± 25 3 27 ± 6 4 2______________________________________ *number of rats in each group = 20
The results clearly showed that pretreatment in accordance with the invention protected the rats from grand mal seizures induced by PTZ. Most of the rats (19/20) receiving the placebo that were challenged with 100 mg./kg. PTZ exhibited clear out grand mal seizures. It is of interest to note that the seizure began shortly after the PTZ injection, and lasted for about 15 minutes without interruption, until death occurred. All of these rats were dead 15 minutes after the PTZ injection. In contrast, rats pretreated with SR-3 and later challenged with 100 mg./kg. PTZ were very much protected. Only 3 out of 20 of this group showed grand mal seizures, and even in these 3 cases the attacks lasted only about 25 seconds; only 2 of the rats exhibiting seizures were dead 15 minutes after the PTZ injection. Another 12 rats from this group were found dead 4 hours after the PTZ injection.
Similar SR-3 protection was found in the group receiving the smaller dose. In the rats receiving saline solution, 8 out of the 20 had grand mal seizures with a latency of about 0.5 minute. The duration of the attacks was shorter than in the group receiving the larger PTZ dose. Only one rat from the SR-3 group showed a brief grand mal attack. In the group receiving the placebo, 9 rats were dead 15 minutes after the PTZ injection. No immediate deaths occurred in the SR-3 group and only 3 rats in this group were found dead 4 hours later; all others recovered.
EXAMPLE IVb
The use of ferrous or ferric salts injected into the cortex or amygdala-hippocampus complex for the induction of epileptogenic discharge (epileptic foci) is well-known. A group of 50 SPD male rats were prepared for this experiment by implantation of canules in their brain. The tip of the canules was in the amygdala according to Czennansky et al (Life Sciences, 1988, 32: 385-390). Then, 25 rats received SR-3 in daily injections as in Example IVa. The other 25 rats received a daily injection of saline (0.9% NaCl). After 3 weeks of treatment, all rats received 100 [f /M FeCl 3 intraventicularly through the canules into the amygdala. Only 2 out of 25 rats which were treated with saline did not exhibit epileptic seizure, while 21 of the 25 rats pretreated with SR-3 were protected from FeCl 3 -induced epileptic seizures.
EXAMPLE IVc
In this method of investigation according to Craig, C. R. and Colasanti, B. J., 1989 Pharmacol. Biochem Behav. 31: 667-70, PTZ was used to induce seizure and the ability of SR-3 to protect from seizure was examined. 15.0 mg./kg. was injected every 15 minutes until seizure occurred. For this purpose, seizure is defined as a single episode of colonic spasms of both forelimbs and hindlimbs lasting at least 5 secs., followed by loss of righting reflex. The results in the following table are expressed as the number of periods to seizure (mean + S.D.; ANOVA =P<0.001).
______________________________________ First Week Second Week Third Week______________________________________Control (n = 25) 4.32 ± 1.30 3.72 ± 1.41 3.50 ± 1.80SR-3 (n = 25) 18.64 ± 3.09 19.94 ± 3.21 22.75 ± 3.45p< 0.001 0.001 0.001______________________________________
The above results show that SR-3 afforded clear protection, insofar as many more injections of PTZ were needed to evoke seizure in the SR-3 group than in the control group, with no apparent habituation from one week to another.
EXAMPLE IVd
In this model, 55 non-audiogenic rate were made audiogenic (i.e. they are responsive with seizure to auditory stimuli) by chronic administration of p-cresol over a 7-week period; the effect of p-cresol lasts 4-5 weeks (see Yehuda, S. et al, 1577 Internat. J. Neurcsci. 7: 223-6). Then, 27 rats were treated with saline and 28 rats received SR-3, after which all rats were subjected to the same auditory stimuli and observed for 4 hours. Seizure were observed in 23 out of the 27 control rats, but in only 6 out of the 28 SR-3 treated rats.
The overall results in Example IV indicates that the composition of the invention affords protection from seizures even when the animals under investigation were already seizure-prone.
While the present invention has been particularly described with reference to certain embodiments, it will be apparent to those skilled in the art that many modifications and variations may be made. The invention is accordingly not to be construed as limited in any way by such embodiments, rather its concept is to be understood according to the spirit and scope of the claims which follow.
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A composition of matter which consists of (a) from about 13.0 to about 27.5% by weight of at least one compound selected from the group consisting of linolenic acid and derivatives thereof, calculated as the free acid, said derivatives of linolenic acid being both physiologically hydrolyzable and pharmacologically acceptable, and (b) about 87.0 to about 72.5% by weight of at least one compound selected from the group consisting of linoleic acid and derivatives thereof, calculated as the free acid, said derivatives of linoleic acid being both physiologically hydrolyzable and pharmacologically acceptable is utilized in a nutritional composition, in absence of an oily carrier or diluent which comprises at least one member of the group consisting of C- 8-18 saturated fatty acids, oleic acid and derivatives of these acids, in order to induce in a mammal (including humans) at least one physiological effect selected from memory enhancement, analgesia, sleep regulation and inhibition of the symptoms of senility. The invention also relates to a method for treating a mammal with the composition for the purpose of inducing such a physiological effect therein.
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This is a continuation, of application Ser. No. 08/227,786, filed Apr. 14, 1994, and now abandoned which, in turn, is a continuation of application Ser. No. 08/007,489, filed Jan. 25, 1993, which is a continuation-in-part of U.S. patent application Ser. No. 08/002,599, filed Jan. 11, 1993, each of which is abandoned.
TECHNICAL FIELD
The invention relates to a garment designed to protect the wearer from injury during athletic activity such as in-line roller skating, skiing, volleyball, mountain biking, basketball, hockey, field hockey, ice skating or gymnastics.
BACKGROUND ART
This invention concerns a solution to a problem encountered by many sports enthusiasts. The problem is injury to an athlete's body, including but not limited to the athlete's hips, coccyx, or buttocks, resulting from athletic activity such as volleyball, mountain biking, basketball, hockey, field hockey, ice skating, gymnastics or in-line roller skating. In performing these sports, athletes often fall backward or on their sides, many times causing serious injury and/or a cessation to the sport. The cause of the problem is a lack of adequate safety equipment available to the public. Existing protective padding tends to be restrictive, insufficient, or gives the illusion of obesity. Prior patents have addressed related problems, however, this invention presents a unique approach to solving each of these problems.
U.S. Pat. No. 2,266,886 describes a pair of thigh pads of the type used in the sport of football. The pads are not permanently affixed to any garment and are designed to protect the thigh. The pads contain a stiff board-like element. The present invention is distinguished by the affixed nature of its pads, the location of its pads, and the flexibility of the wearer due to the pliable nature of its pads.
U.S. Pat. No. 2,247,961 discloses a pair of shoulder pads and thigh pads of the type used in the sport of football. The pads are stiff and utilize inflatable sections. The shoulder pads are attached to the wearer using straps. The thigh pads are inserted into a pocket inside the wearer's pant leg over the thigh. The present invention is distinguished by the affixed nature of its pads to the garment, the location of its pads in relation to the garment and the wearer, and the flexibility of the wearer due to the pliable nature of its pads. Additionally, the present invention helps eliminate the illusion of obesity by locating its pads completely on the outside of the garment.
U.S. Pat. No. 4,810,559 describes the use of a plurality of platelets attached in a web like pattern to a garment. The object of the invention is to protect the wearer from scratches. The instant invention is different because it protects the wearer from the impact and abrasion of a fall. In addition, the instant invention uses foam, gel, air or elastic pads to absorb impact.
U.S. Pat. No. 4,961,233 discloses a design for cycling pants. The patent relates to a reinforced liner which is not a safety feature. It is dissimilar to the Protective Athletic Pants disclosed herein because the present invention utilizes padding to protect its wearer from the impact and abrasion of a fall.
U.S. Pat. No. 5,014,354 describes a device designed primarily to prevent abrasions. The device involves the general use of parallel strips of cushioning material, which yield in the direction of relative motion between the user's body and an abrading surface, to absorb friction related energy. It does not claim to protect against impact. In contrast, the present invention protects against impact and abrasion to fixed strategic locations throughout the lower torso and other body regions. Its protective pads are composed of foam, gel, air or elastic foam to absorb impact. In contrast to the 5,104,354 patent, the instant invention uses gel, air or a coated, hard, outer surface or abrasion resistant fabric to reduce friction by deflecting abrasive materials. The 5,104,354 patent claims to protect the wearer by absorbing friction related energy.
U.S. Pat. No. 5,038,408 describes the use of patches to reinforce conventional work pants. The primary object of that design is to increase the life of the pants and reduce abrasion. The 5,038,408 patent uses patches of leather and foam sewn to the pants to reduce abrasion. The present invention uses thicker gel, air or elastic foam pads to protect against impact and abrasion to fixed strategic locations on the wearer's lower torso. The coated, hard, outer surfaces of the pads deflect friction. The present invention protects against athletic falls, not prolonged abrasion, which is the object of the 5,038,408 patent.
U.S. Pat. No. 5,105,473 describes an athletic garment that uses removable pads to protect against impact. The garment, however, fails to provide a means to prevent abrasion. In contrast to the 5,105,473 patent, the pads of the present invention are fixed in shape and location to maximize protection and mobility. The present invention provides protection against abrasion, while the loose fit of the 5,105,473 garment would tend to promote it. Moreover, the present invention helps eliminate the illusion of obesity by locating the pads completely on the outside of the garment.
SUMMARY OF THE INVENTION
The present invention relates to a garment designed to protect the wearer from injury during athletic activity such as in-line roller skating, skiing, volleyball, mountain biking, basketball, hockey, field hockey, ice skating or gymnastics. The padding can be accomplished by means of foam, gel or air. The gel or air padding is accomplished by containing said gel or air a non-porous, stretchable covering such as a balloon or bladder apparatus affixed to the garment. The balloon or bladder can be affixed by means of a suitable adhesive or tacking. Although the invention can be applied to garments protecting the wearer's upper torso and limbs, the embodiment described herein applies the invention to shorts that protect the wearer's hips, coccyx, buttocks, and lower torso generally. The invention comprises a generally tubular garment such as shorts or above-the-knee pants made of elasticized or other stretch material (such as LYCRA spandex®), with strategically placed padding to protect the wearer from impact or abrasion resulting from a fall during the performance of a sport such as in-line roller skating, skiing, volleyball, mountain biking, basketball, hockey, field hockey, ice skating or gymnastics. An object of the invention is to provide comfortable and aesthetically pleasing protection to the wearer during sporting activities.
There currently exists for sports clothing padded and armored knee pads and protective wrist guards. Existing equipment is designed to protect the wearer's extremities during a forward fall. The present invention, however, is designed to protect the wearer from injury during a forward, side or backward fall. The invention, as applied to protect the lower torso, and as illustrated herein, provides protection to the wearer's hips, coccyx, and buttocks. This protection can be provided by strategically placed foam, gel, air, thermoformed or die cut, closed cell, high density foam pads that are affixed to the garment with at least one layer of fabric intervening between the pad and the wearer's body.
As applied to protect the lower torso, the invention uses a plurality of rib-like pads that are affixed to elasticized or LYCRA spandex® fabric shorts in parallel with each other and positioned substantially in line with the contour of each of the buttocks. These pads protect the wearer's hips and buttocks. A triangular pad is positioned over the coccyx to protect the wearer's coccyx and lower spine. The pads are positioned and shaped to minimize restriction of movement. The pads are thick enough to protect the wearer, but thickness is limited by a concern for aesthetics and agility.
The foam, gel or air composing the pads can be affixed to the garment with or without an encompassing outer elasticized or LYCRA spandex® fabric shell. The gel or air is contained in a non-porous elasticized envelope such as a balloon or bladder which comprises the outer surface of the pad. The wearer can control the thickness of the gel or air pads by determining the desired amount of gel or air to insert into the balloon or bladder. The outer front surface of each pad, in either event, can be coated with a hardened, but flexible substance such as polyethylene, vinyl laminate, or an epoxy based paint or abrasion resistant nylon fabric. This coated surface improves the padding in several regards. First, it provides a hardened surface to distribute the impact of concentrated objects such as rocks or a curb. Second, it allows the wearer to slide upon impact rather than absorbing the initial shock of the fall. Third, the coating protects the pad and/or garment from contact with abrasive surfaces such as pavement. The coating substantially prolongs the life of the garment by preventing wear and tear. And finally, the coating provides an aesthetically pleasing, finished look and may include contrasting designs and color, including high visibility colors for safety.
The protective pads do not impede the aesthetic value of the garment since they are interrupted and are separated by a fabric surface which conforms snugly to the wearer's body, presenting the appearance of being on the outside surface of the garment. Normally, a padded garment would tend to make the wearer appear overweight due to the shear bulk of the padding. The present invention, however, avoids such an appearance by affixing the pads so they appear to be on the outer surface of the garment. The garment forms and clings to the shape of the wearer. The pads are distinctly attached and displayed as outside padding. A person viewing the wearer can easily distinguish the wearer's shape versus the outside padding. This design eliminates the illusion of obesity inherent in other designs.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a rotated front view of the invention showing the form fitting characteristics of its elasticized fabric construction. This view illustrates the padded garment according to the invention on the front and side of the person wearing it.
FIG. 2 is a front view of a padded garment according to the invention showing the strategic placement and shape of the shock absorbent padding. This view illustrates the garment on the front of the person wearing it.
FIG. 3 is a rear view of a padded garment according to the invention showing the form fitting characteristics of a Lycra® fabric construction and the strategic placement and shape of the shock absorbent padding. This view illustrates a padded garment according to the invention on the back of the person wearing it.
FIG. 4 is a side view of a padded garment according to the invention showing the form fitting characteristics of a LYCRA spandex® fabric construction and the strategic placement and shape of the shock absorbent padding. This view illustrates a padded garment according to the invention on the side of the person wearing it.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring to FIGS. 1, 2, 3 and 4, the preferred embodiment of the invention is comprised of an athletic garment shown as a pair of protective fabric shorts 12 (preferably made of stretchable material such as LYCRA spandex® fiber), which can be manufactured and sized to fit a wide spectrum of users. The shorts 12 are equipped with a number of strategically placed rib-shaped pads 11, 13.
A plurality of pads 11a-11h and 11a'-11h', shaped and positioned as illustrated, are affixed to the shorts 12. The pads 11 are parallel to each other and are positioned in line with the contour of each of the buttocks of a user as shown in FIGS. 3 and 4. A single triangular pad 13 is affixed to the shorts 12 in a position covering the wearer's coccyx as shown in FIG. 3. The pads 11, 13 are positioned and shaped to minimize restriction of movement of the wearer's lower torso and legs. The pads 11, 13 have a thickness 14 sufficient to protect the wearer, but thickness 14 is limited by a concern for aesthetics and agility. The thickness of the pads which use gel or air may be controlled by the wearer.
Referring more specifically to FIGS. 3 and 4, it can be seen that the individual pads 11 are arranged in two mirror-image sets 11a-11h and 11a'-11h', the sets being disposed, respectively on the right and left halves of the wearer's anatomy in a pattern which is symmetrical with respect to a central vertical axis lying along the spine of the wearer. The pads 11a-11h, 11a'-11h' are spaced apart by a distance "d" (typically less than the width of an individual pad) and extend diagonally from an upper, rearwardly disposed end 18a-18h, 18a'-18h' to a lower, forwardly disposed end 19a-19h, 19a'-19h'. The pads 11a-11h, 11a'-11h' overlay the buttocks, hip and upper leg portions of the wearer. At least some of the pads (e.g., 11b-11e, 11b'-11e') are relatively longer and extend from the rear of the wearer's leg 20, 20' to the lower end 21, 21' of the leg portion of the garment 12 at the side or front of the leg 20, 20'. Others of the pads (such as 11a, 11a') are of relatively shorter length and are disposed substantially entirely in the rear portion of the garment 12 in the vicinity of the buttocks. The remainder of the pads (such as 11f-11h, 11f'-11h') are relatively shorter in length and are disposed in the vicinity of the forward portion of the hip of the wearer.
The upper ends 18f-18h, 18f'-18h40 generally are disposed just below the waist of the wearer. The upper ends 18a-18e, 18a'-18e' of the remaining pads diverge away from the central vertical axis along the wearer's spine and an additional triangular pad 13, arranged to provide significant protection to the coccyx and spinal area, is disposed between those upper ends.
The protective pads 11 and 13 preferably are composed of gel, air or thermoformed, closed cell, high density foam and are illustrated encased by a layer of, for example, LYCRA spandex® material. In the case of foam pads, the inner foam is highly elastic to promote the absorption of collision related energy. The pads 11, 13 provide the wearer with protection from the impact and abrasion of a fall. The pads 11, 13 are affixed onto the outer surface of the shorts 12 either mechanically or with adhesive. For example, the pads 11, 13 may be placed in the appropriate position on the shorts 12, fastened thereto with a suitable adhesive or by "tacking" with thread and thereafter, an additional layer or layers of similar or contrasting color Lycra® fabric and a wear surface (see below) are sewn to the pants 12 immediately around the edges of the pads 11, 13 and along the edges of the similarly shaped inner piece of fabric which forms pants 12.
The outer surfaces 15, 16 of each of the pads 11, 13 (or of the overlying fabric where used) preferably are coated with a hardened, relatively smooth substance such as polyethylene, vinyl laminate, or an epoxy based paint, or abrasion resistant nylon. The coated surfaces 15, 16 improve the pads 11, 13 in several regards. First they provide hardened surfaces to distribute the impact of concentrated objects such as rocks or a curb. Second they allow the wearer to slide upon impact rather than absorbing the initial shock of the fall. Third, the coatings protect the pad material from contact with abrasive surfaces such as pavement. The coating substantially prolongs the life of the garment 12 by preventing wear and tear. And finally, the coating provides an aesthetically pleasing, finished look and, for example, includes a high visibility color for safety and aesthetic reasons.
The protective pads 11, 13 do not adversely affect the aesthetic value of the garment 12 since they are located completely on an outside surface 17 of garment 12. Normally, a padded garment would tend to make the wearer appear overweight due to the shear bulk of the padding. The present invention, however, avoids such an appearance by affixing the pads on the outer surface 17 of the shorts 12. The shorts 12 when fabricated of elasticized or stretch material such as LYCRA spandex® material form and cling to the shape of the wearer. The pads 11, 13 are distinctly attached and displayed as outside padding. A person viewing the wearer can easily distinguish the wearer's shape from outside padding. This design eliminates the illusion of obesity inherent in other designs.
While the invention has been described in terms of a preferred embodiment, other configurations may occur to persons skilled in this art in the light of the foregoing teachings, which configurations may also fall within the scope of the invention as set forth in the following claims.
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An athletic garment designed to protect the wearer from injury during athletic activity such as in-line roller skating, skiing, volleyball, mountain biking, basketball, hockey, field hockey, ice skating or gymnastics. The invention uses strategically placed and rib-shaped gel, air or elastic foam padding to protect the wearer from the impact and abrasion of a fall caused by such activity.
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FIELD OF THE INVENTION
[0001] The present invention relates to a baking sheet and, more particularly, to a silicone baking sheet with removable thermo-resistant handles for easy handling and convenient storage.
BACKGROUND OF THE INVENTION
[0002] Baking sheets have long been widely and commonly used for supporting food products such as pre-baked food during the baking process. Patented designs include those disclosed in U.S. Pat. No. 6,279,771; and those in foreign Patents GB 2,154,860; EP 278055; DE 3832524.
[0003] Traditional baking sheets, however, pose handling problems after being exposed to high temperatures since they do not permit direct contact. In addition, with traditional baking sheets it is very difficult to place the sheet in an oven when it has cookie dough on it. In such case, the user generally must use a griddle or a metal tray. Many of the above references disclose enhanced baking sheets, for instance baking sheets adaptable to different widths, having an air pocket between the food product and the baking sheet, or eliminating the need for greasing the surface prior to baking. Nevertheless, there still is a need for a baking sheet enhanced to allow easy handling immediately following exposure to high temperatures as well as proper storage. There also is the need for a baking sheet that can be easily partially or completely removed from an oven for various purposes, such as to monitor baking status.
OBJECTS AND SUMMARY OF THE INVENTION
[0004] It is therefore an object of the present invention to provide a baking sheet enhanced for easy handling before, during and after exposure to high temperatures.
[0005] It is a further object of the present invention to provide a self-supported baking sheet allowing for quick disassembly and easy storage.
[0006] In accordance with the present invention, a baking sheet includes a cooking surface, first and second rods coupled to the cooking surface, and first and second thermo-resistant handles coupled to the first and second rods.
[0007] As a feature of the invention, the first and second rods are coupled to opposite ends of the cooking surface, and each of the first and second thermo-resistant handles is coupled to respective ends of the first and second rods.
[0008] As another feature of the invention, the cooking surface is substantially flat and made of silicone. The cooking surface may also be a sheet of metal.
[0009] As a further feature of the invention, each rod is encircled by the silicone cooking surface at respective opposite ends of the silicone cooking surface.
[0010] As yet a further feature, the ends of the two rods are exposed and the handles are coupled to the exposed ends of the rods.
[0011] As yet another feature, the two rods are made of thermo-resistant material.
[0012] As yet an additional feature, connectors attach the handles to the rods. The connectors may be rotatable to allow the handles to be moved adjacent to the rods to allow easy storage. The connectors may be thermo-resistant. The connectors may also be detachable to allow complete disassembly of the components.
[0013] Various other objects, advantages and features of the present invention will become readily apparent to those of ordinary skill in the art, and the novel features will be particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The following detailed description, given by way of example and not intended to limit the present invention solely thereto, will best be appreciated in conjunction with the accompanying drawings, wherein like reference numerals denote like elements and parts, in which:
[0015] FIG. 1 is a schematic illustration of the baking sheet of the present invention;
[0016] FIGS. 2 a and 2 b show different views of the baking sheet's cooking surface with attached rods;
[0017] FIGS. 3 a, 3 b and 3 c are schematic illustrations of the connector used to connect the handles to the baking sheet in accordance with the present invention;
[0018] FIG. 4 a shows part of the baking sheet of the present invention and FIG. 4 b is an enlarged view showing one connector used to connect a handle in accordance with the present invention;
[0019] FIG. 5 is a schematic illustration of a portion of another baking sheet in accordance with the present invention; and
[0020] FIGS. 6 a, 6 b and 6 c are schematic illustrations of yet a further baking sheet in accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention pertains to a novel baking sheet. As would be appreciated, the inventive baking sheet is intended for use by a layperson or a professional chef for the purpose of assisting in the baking of food products. Of course, the baking sheet may be utilized by other individuals for similar or different purposes.
[0022] Referring now to FIG. 1 of the drawings, the baking sheet 10 of the present invention includes a cooking surface 12 and a pair of handles 14 a and 14 b. Cooking surface 12 preferably is flat and made of silicone. However, cooking surface 12 may be made of other material, such as from a suitable metal sheet, coated or uncoated, appropriate for cooking functions. Handles 14 a and 14 b may be made of metal, but preferably are made of thermo-resistant material, so as to allow direct handling immediately after exposure to extreme temperatures, for example, after baking. Handles 14 a and 14 b may be completely straight or be slightly curved to provide tensile strength to the silicone sheet. Handles 14 a and 14 b are attached to the shorter sides of cooking surface 12 utilizing four sets of connectors 16 a, 16 b, 16 c and 16 d (an individual connector is sometimes referred to herein, for convenience, as connector 16 ).
[0023] Cooking surface 12 includes a pair of rods 18 a and 18 b, disposed along the longer sides, as shown in the respective top and side view drawings of FIGS. 2 a and 2 b. Rods 18 a and 18 b may be made of stainless steel or other appropriate material, but preferably are made of thermo-resistant material. In the preferred embodiment, cooking surface 12 is made of silicone and rods 18 a and 18 b are encircled within the silicone (see FIG. 2 b ) along the two longer surfaces of cooking surface 12 . Rods 18 a and 18 b extend slightly beyond the encircled silicone and, as discussed further below, each connector 16 attaches a handle to an exposed end of a rod.
[0024] The structure of each connector 16 will be described with reference to FIGS. 3 a, 3 b and 3 c of the drawings. As shown in FIG. 3 a, each connector ( 16 a, 16 b, 16 c and 16 d shown in FIG. 1 ) is identified herein, for convenience, as a coupled element 20 (also called connector 20 ). Coupled element 20 is comprised of two components coupled to one another: connector component 22 and connector component 24 (or simply components 22 and 24 ). FIG. 3 a shows components 22 and 24 coupled together. Components 22 and 24 may be disassembled from one another as further discussed below.
[0025] FIG. 3 b shows component 22 alone and FIG. 3 c shows component 24 alone. As shown in FIG. 3 b, component 22 includes connection end 22 a and connection end 22 b. Connection end 22 a (also called rod/handle connection end 22 a ) includes a round elongated inner core 22 a ′ for receiving an end of one of the rods or an end of one of the handles. Connection end 22 b (also called socket end 22 b ) includes a keyed, mostly round elongated inner core 22 b ′ (with notch 23 ) and is designed to receive the counterpart connection end of the other connector component 24 , discussed below. As shown, the apertures 22 a ′ and 22 b ′ within rod/handle connection end 22 a and socket end 22 b extend along longitudinal axes that are offset 90 degrees from one another.
[0026] Connector component 24 shown alone in FIG. 3 c includes connection ends 24 a and 24 b. Connection end 24 a (also called rod/handle connection end 24 a ) is similar to rod/handle connection end 22 a and includes a round elongated inner core 24 a ′ for receiving an end of one of the rods or an end of one of the handles. Connection end 24 b (also called plug end 24 b ) is in the form of a keyed plug (with projection 25 ) that is designed to be insertable into socket end 22 b. Plug end 24 b extends outwardly from rod connection end 24 a along an axis perpendicular the longitudinal axis of inner core 24 a′.
[0027] Connector components 22 and 24 are coupled together by inserting plug end 24 b of component 24 into socket end 22 b of component 22 . Since socket end 22 b and plug end 24 b are “keyed” by means of notch 23 and projection 25 , as shown, the components cannot be coupled together in an incorrect manner. That is, the components can only be coupled together to produce the coupled connector configuration shown in FIG. 3 a.
[0028] Baking sheet 10 of the present invention utilizes four of the connectors 20 shown in FIG. 3 a. As shown in FIG. 1 , two connectors ( 16 b and 16 c ) attach handle 14 a to rods 18 a and 18 b, and two connectors ( 16 a and 16 d ) attach handle 14 b to the other ends of rods 18 a and 18 b. Referring to FIG. 4 a, connector 16 b (shown as connector components 22 and 24 in FIG. 4 b ) connects handle 14 a to one end of rod 18 a. As described above, connector 16 b contains two rod/handle connection ends ( 22 a and 24 a ) and, in the preferred embodiment, rod/handle connection ends 22 a and 24 a are reversible, that is, each end can be coupled to an end of a handle and each end can be coupled to a disposed end of a rod. In this preferred embodiment, the dimensions of the ends of both handles 14 a and 14 b and the ends of both rods 18 a and 18 b are the same to provide for the versatility of allowing either end of each connector to be coupled to either a handle or a rod. Of course, in a non-preferred embodiment, it is contemplated that the ends of the handles and the rods are of different thicknesses (or other dimensions, as appropriate) and thus connection ends 22 a and 24 a are designed accordingly and thus may not be interchangeable.
[0029] The other three connectors 20 couple respective ends of handles to rods in a like manner as that described above. As would be appreciated, connectors generally are first connected to the handles with the orientations of the longitudinal axes of the exposed inner cores aligned in the same direction before coupling the connectors to the rods. In a preferred embodiment, the dimensions of the handles, rods and inners cores are appropriately sized to enable a user to be able to connect and disconnect the various connections with only a small amount of force. However, the connections should be relatively secure so as to prevent unintended disconnection. For example, gravity alone (i.e., holding the baking sheet in a slanted or vertical orientation) should not cause disconnection of the handles from the rods.
[0030] In the preferred embodiment, as further discussed below, a user of the baking sheet 10 of the present invention is able to disassemble the various components for further benefit discussed below. In a non-preferred embodiment, the baking sheet of the present invention is manufactured to produce connections between the handles, connectors and rods that cannot be disassembled by users of the baking sheet.
[0031] In accordance with the present invention, the handles can be partially disconnected or fully disconnected from the rods by users of the inventive baking sheet for various beneficial purposes. In particular, after use of the baking sheet 10 , one end of each handle 14 a, 14 b may be disconnected from the respective rods to which they are attached and then the handles may be rotated 90 degrees to place them alongside the longer sides of cooking surface 12 (partial disconnection) (i.e., the axes along which the handles extend are parallel to the axes along which the rods extend). To facilitate such rotation, core 22 b ′ of socket end 22 b of each connector (see FIG. 3 b ) is internally keyed to allow such 90 degree rotation. Since the particular internal design of the connector components to facilitate rotation is well within the capability of one of ordinary skill in the art, further description thereof is not provided. Thereafter, baking sheet 10 may be easily rolled or pushed into storage, as needed. It is appreciated that either end of the two handles may be removed and rotated by users.
[0032] To fully disconnect the handles from the cooking surface 12 , all of the connectors 16 a, 16 b, 16 c and 16 d are removed from the rods to which they are connected. The connectors may then be removed from the attached handles. The connectors themselves may further be dissembled (see FIGS. 3 a, 3 b, 3 c ). After disassembly, all of the components may be properly cleaned and stored for later use. Moreover, based upon the intended usage, benefits and preferences of users, multiple variations are possible. For example, various interconnections between the handles and the connectors, or the connectors themselves, may be made permanent during manufacture. That is, the baking sheet of the present invention may be manufactured to allow users to not be able to fully disassemble all of the components, as discussed above. For example, the connectors may be made to prevent their disassembly. For example, the rods and/or handles can be permanently secured to the connectors, e.g., via welding, permanent adhesive or other known technique. In yet another variation, connectors are not utilized, wherein the handles and rods are permanently secured together in any known manner.
[0033] As shown in the side view drawing of cooking surface 12 shown in FIG. 2 b, rods 18 a and 18 b are positioned and embedded within the silicone forming cooking surface 12 to provide a cooking surface that has a completely flat lower surface to maximize the area of contact between the cooking surface and an oven grid or plate. In a non-preferred variation, the rods may be maintained below the cooking surface so that only the silicone surrounding the rods contact a flat surface such as a table top after cooking. Moreover, the rods may be coupled to the cooking surface in other manners, such as by co-injection, adhesively secured, welded or other appropriate method. In yet another variation, the baking sheet may be made solely of silicone with a silicone frame provided around the sheet.
[0034] FIG. 5 of the drawings shows a variation of the baking sheet in accordance with the present invention. Like the embodiment shown in FIGS. 1-4 , the baking sheet in FIG. 5 includes a cooking surface 30 , a pair of handles 32 (only one handle shown in FIG. 1 ), four connectors 34 (only two shown in FIG. 5 ) and a pair of rods 36 . Each of these components has the same characteristics and features of the various components previously discussed, and may vary also as previously discussed. The embodiment shown in FIG. 5 differs, however, from the previously described embodiment in that each connector 34 is a single, unitary component with ends 34 a and 34 b for engagement with a respective end of a rod and a respective end of a handle. Each connector 34 provides for a fixed, that is, non-rotatable, engagement between a handle and rod. Each handle (with or without connectors 34 ) is removable from the cooking surface during use or storage of the baking sheet. Each handle 32 is curved as shown in FIG. 5 to maximize functionality and aesthetics. However, the curvature or shape of the handle may be different than that shown.
[0035] FIGS. 6 a, 6 b and 6 c of the drawings show another variation of the baking sheet of the present invention. The baking sheet shown is similar to the previously discussed variations. However, in this exemplary version, rods 42 disposed within cooking surface 40 include a bent at end 42 a (bent at about a 90 degree angle; other angles are possible) utilized for connection with handles 44 . Each handle 44 includes an end 44 a made of rubber, plastic or other suitable material that receives bent end 42 a. As shown, bent ends 42 a are directed slightly upwards to enable easy connection to a handle. The handle and rods can be permanently secured to one another, for example, by welding. They also can be detachable. In the version shown, a stable handle construction is provided. In a variation, each handle can include a bent portion for connection to a non-bent end of a rod.
[0036] While the present invention has been particularly shown and described in conjunction with preferred embodiments thereof, it will be readily appreciated by those of ordinary skill in the art that various changes may be made without departing from the spirit and scope of the invention. For example, the handles may be attached along the longer surfaces of the baking sheet's cooking surface. As another example, the rods may be positioned along the sheet in a different manner, such as crossed, and additional rods can be utilized, for example, to provide further enhanced support to the baking sheet.
[0037] The various dimensions shown may be modified. For example, the baking sheet may have a square cooking surface or other shape. The baking sheet may also have a non-flat structure, and may include waves or other structural design, and/or other textures, on one side or both sides of the baking sheet. In particular, with a non-flat design, different patterns of air circulation are provided. Still further, connectors used to attach the handles to the rods may have designs quite distinct from that shown and described. Moreover, the shapes/cross-sections of the various components may be modified. For example, the handles may have square, rectangular, oval or other appropriate cross-section, with connectors adapted to receive such shaped handles/rods. The rods can be flat, rectangular, square, round, oval, triangular, hexagonal or other appropriate shape. In a further variation, the inventiveness of the baking sheet as discussed is applicable to a serving tray. In such case, the serving tray is similar in appearance to the cooking surface shown in the figures.
[0038] As mentioned above, the handles preferably are manufactured from thermo-resistant material. Each of the connectors may be made from any appropriately strong material suitable for cooking applications. The connectors likewise preferably are made from thermo-resistant material. Each of the rods may be made from stainless steel or appropriately strong thermo-resistant material. The handles and rods can be made from the same material, thus providing tension strength to both the frame and sheet. Given the particular design of the baking sheet of the present invention with the preferred thermo-resistant components mentioned, the baking sheet advantageously allows users to make contact with the baking sheet's handles shortly, if not immediately, after exposure of the baking sheet to various cooking temperatures. This advantageous feature in turn allows the removal from an oven or other heat source of the baking sheet using the handles and without the need for the use of oven mitts or other protective device.
[0039] The baking sheet of the present invention further provides for a design that enables a user to partially or fully remove the handles. Partially removing the handles, as discussed above, allows the baking sheet to be conveniently slid, moved or rolled into storage when needed. The handles and connectors may also be fully removed to allow for easy cleaning of the various components as well as easy storage. In particular, the baking tray can be stored in close proximity to other baking trays (even those not embodying the present invention) without wasting space needed for the handles. The handles can also be removed prior to baking and then be re-attached to the sheet while it is still in the oven to allow easy “cold-handle” removal of the sheet. The baking sheet therefore may be utilized without the handles and, if desired, handles can be attached to provide easy handling.
[0040] In addition, the flexibility of having removable connectors and handles allows for the easy replacement of such components as needed. Handles of various shapes, styles and sizes can be utilized based upon cooking styles, personal preferences and other desires of users. Multiple pairs of handles can be utilized with a single baking sheet, with the user selecting the particular pair of handles to be used based upon whatever factors the user considers. It is further possible to provide different sets of connection ends for the connectors for use with different shaped/sized handles.
[0041] Therefore, it is intended that the appended claims be interpreted as including the embodiments described herein, the alternatives mentioned above, and all equivalents thereto.
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A baking sheet having a substantially flat silicone cooking surface, two rods extending along and coupled to opposite sides of the cooking surface, and two thermo-resistant handles coupled to and extending between the two rods. The thermo-resistant handles may be connected to the rods using thermo-resistant connectors. The connectors can disconnect from the rods and possibly be rotated to rotate the handles in positions adjacent to the rods to enable the baking sheet to be rolled into storage. The thermo-resistant characteristic of the handles enables users to lift the heated baking sheet without the use of oven mitts or other protective device.
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CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Priority is hereby claimed to provisional application Ser. No. 61/106,205, filed Oct. 17, 2008, and provisional application Ser. No. 61/229,325, filed Jul. 29, 2009, both of which are incorporated herein by reference.
FEDERAL FUNDING STATEMENT
[0002] This invention was made with United States government support awarded by the following agencies: National Institutes of Health (NIH) GM56414 and CA119875. The United States government has certain rights in this invention.
FIELD OF THE INVENTION
[0003] The invention is directed to a method of making polypeptide compounds comprising alpha- and beta-amino acid residues, the compounds produced thereby, and use of the compounds as pharmaceutically active agents to treat diseases in animals, including humans.
BACKGROUND
[0004] Many naturally occurring, biologically active compounds are proteins or peptides based upon α-amino acids (i.e., sequences of α-amino acids in which the α-carboxyl group of one amino acid is joined by an amide bond to the α-amino group of the adjacent amino acid). In recent years an approach to the discovery of new pharmaceutically active drugs has been to synthesize libraries of peptides and then to assay for compounds within the library which have a desired activity, such as a desired binding activity. However, α-amino acid peptides are not altogether satisfactory for pharmaceutical uses, in particular because they are often poorly absorbed and subject to proteolytic degradation in vivo.
[0005] Much work on β-amino acids and peptides synthesized from β-amino acids has been reported in the scientific and patent literature. See, for example, the work performed by a group led by current co-inventor Samuel H. Gellman, including: “Application of Microwave Irradiation to the Synthesis of 14-helical Beta-Peptides,” Murray & Gellman,” Organic Letters (2005) 7(8), 1517-1520; “Synthesis of 2,2-Disubstituted Pyrrolidine-4-carboxylic Acid Derivatives and Their Incorporation into Beta-Peptide Oligomers,” Huck & Gellman, J. Org. Chem . (2005) 70(9), 3353-62; “Effects of Conformational Stability and Geometry of Guanidinium Display on Cell Entry by Beta-Peptides,” Potocky, Menon, & Gellman, Journal of the American Chemical Society (2005) 127(11):3686-7; “Residue requirements for helical folding in short alpha/beta-peptides: crystallographic characterization of the 11-helix in an optimized sequence,” Schmitt, Choi, Guzei, & Gellman, Journal of the American Chemical Society (2005), 127(38), 13130-1 and “Efficient synthesis of a beta-peptide combinatorial library with microwave irradiation,” Murray, Farooqi, Sadowsky, Scalf, Freund, Smith, Chen, & Gellman, Journal of the American Chemical Society (2005), 127(38), 13271-80. Another group, led by Dieter Seebach in Zurich, Switzerland, has also published extensively in the beta-polypeptide field. See, for example, Seebach et al. (1996) Helv. Chim. Acta. 79:913-941; and Seebach et al. (1996) Helv. Chim. Acta. 79:2043-2066. In the first of these two papers Seebach et al. describe the synthesis and characterization of a β-hexapeptide, namely (H-β-HVal-β-HAla-β-HLeu) 2-OH. Interestingly, this paper specifically notes that prior art reports on the structure of β-peptides have been contradictory and “partially controversial.” In the second paper, Seebach et al. explore the secondary structure of the above-noted β-hexapeptide and the effects of residue variation on the secondary structure.
[0006] Dado and Gellman (1994) J. Am. Chem. Soc. 116:1054-1062 describe intramolecular hydrogen bonding in derivatives of β-alanine and γ-amino butyric acid. This paper postulates that β-peptides will fold in manners similar to α-amino acid polymers if intramolecular hydrogen bonding between nearest neighbor amide groups on the polymer backbone is not favored.
[0007] Suhara et al. (1996) Tetrahedron Lett. 37(10):1575-1578 report a polysaccharide analog of a β-peptide in which D-glycocylamine derivatives are linked to each other via a C-1 β-carboxylate and a C-2 α-amino group. This class of compounds has been given the trivial name “carbopeptoids.”
[0008] Regarding methods to generate combinatorial libraries, several reviews are available. See, for instance, Ellman (1996) Acc. Chem. Res. 29:132-143 and Lam et al. (1997) Chem. Rev. 97:411-448.
[0009] In the recent patent literature relating to β-polypeptides, see, for example, U.S. published patent applications 2008/0166388, titled “Beta-Peptides with Antifungal Activity”; 2008/0058548, titled Concise Beta2-Amino Acid Synthesis via Organocatalytic Aminomethylation”; 2007/0154882, titled “Beta-polypeptides that inhibit cytomegalovirus infection”; 2007/0123709, titled “Beta-amino acids”; and 2007/0087404, titled “Poly-beta-peptides from functionalized beta-lactam monomers and antibacterial compositions containing same.” See also U.S. published patent application 2003/0212250, titled “Peptides.”
SUMMARY OF THE INVENTION
[0010] The invention is directed to a method of fabricating biologically active, proteoloytic-resistant, unnatural polypeptides. The method comprises selecting a biologically or pharmacologically active polypeptide or biologically active fragment thereof (the “target”) having an amino acid sequence consisting essentially of α-amino acid residues. Then, a synthetic polypeptide is fabricated that has an amino acid sequence that corresponds to the α-amino acid sequence of the target. However, in the synthetic polypeptide, between about 14% and about 50% of the α-amino acid residues found in the target are replaced with β-amino acid residues. More preferably between about 20% and about 50% of the α-amino acid residues found in the target are replaced with β-amino acid residues. The β-amino acid residues are disposed in the synthetic polypeptide such that the β-amino acid residues and the α-amino acid residues are distributed in a repeating pattern throughout the amino acid sequence of the synthetic polypeptide. The resulting unnatural polypeptides preferably have a length of from about 10 to about 100 residues, and more preferably of from about 20 to about 50 residues. Preferably, at least two residues are β-amino acid residues.
[0011] In one version of the invention, at least one of the α-amino acid residues in the target is replaced with at least one β-amino acid residue that is cyclically constrained via a ring encompassing its β 2 and β 3 carbon atoms. In another version of the invention, most or all of the inserted β-amino acid residues are cyclically constrained via a ring encompassing its β 2 and β 3 carbon atoms. In another version of the invention, at least one of the β-amino acid residues is unsubstituted at its β 2 and β 3 carbon atoms. Alternatively all of the β-amino acid residues may substituted at their β 2 and β 3 carbon atoms (with linear, branched or cyclic substituents).
[0012] In another version of the invention between about 14% and about 50% of the α-amino acid residues found in the target are replaced with β-amino acid residues wherein each β-amino acid residue has at least one side chain identical to the α-amino acid residue it replaces. Thus, in this version, the method comprises selecting the target to be mimicked and then fabricating a synthetic polypeptide that has an amino acid sequence that corresponds to the sequence of the target, but wherein between about 20% and about 50% of the α-amino acid residues found in the target are replaced with analogous β-amino acid residues. In this version of the invention, each analogous β-amino acid residue has at least one side chain identical to the α-amino acid residue it replaces. Again, the β-amino acid residues and the α-amino acid residues are distributed in a repeating pattern in the amino acid sequence of the synthetic polypeptide.
[0013] Also included within the invention are isolated, unnatural polypeptides comprising a primary amino acid sequence as shown in SEQ. ID. NOS: 4-11, 16-22, and 25-30. These unnatural polypeptides can be used in a method of inhibiting fusion of human immunodeficiency virus to human cells. The method comprises contacting human cells with an isolated, unnatural polypeptide comprising a primary amino acid sequence as shown in SEQ. ID. NOS: 4-11, 16-22, and 25-30, whereby the cells are then resistant to entry of HIV through their cell membrane.
[0014] Another version of the invention is directed to a method of inhibiting fusion of human immunodeficiency virus (HIV) to human cells. The method comprises first selecting a natural, biologically active polypeptide or biologically active fragment thereof having an amino acid sequence comprising α-amino acid residues, and necessary for HIV fusion in vivo. A synthetic polypeptide is then fabricated that has an amino acid sequence that corresponds to the sequence of the biologically active polypeptide or fragment thereof. In the synthetic polypeptide, between about 14% and about 50% of the α-amino acid residues found in the biologically active polypeptide or fragment are replaced with β-amino acid residues. Further still, in the synthetic polypeptide the β-amino acid residues and the α-amino acid residues are distributed in a repeating pattern. Human cells are then contacted with the synthetic polypeptide.
[0015] In all embodiments of the invention, it is generally preferred (although not required) that the repeating pattern places the β-amino acid residues in alignment on one side of a helix in the unnatural polypeptides that adopt a helical conformation. That is, in the folded structure adopted by the polypeptides, the repeating pattern of α- and β-residues disposes the β-amino acid residues in alignment along one side of the folded molecular structure when the unnatural polypeptides adopt a helical conformation. The repeating pattern of β-amino acid residues and α-amino acid residues may be a pattern of from two to seven residues in length, such as (ααααααβ), (αααααβ), (ααααβ), (αααβ), (ααβ), (ααβαααβ), (ααβαβαβ), and (αβ). All unique patterns of α- and β-amino acid residues of from two to seven residues in length are explicitly within the scope of the invention.
[0016] The method can be used to fabricate polypeptide compounds via any means of polypeptide synthesis now known or developed in the future. Using current methods of peptide synthesis, polypeptides fabricated according to the present method are generally less than about 100 residues long, and more preferably from between about ten total residues and about 50 total residues, more preferably still between about 20 and about 50 total residues. Ranges above and below these stated ranges are within the scope of the invention. Many commercial services, such as Abgent (San Diego, Calif., USA) offer peptide synthesis services up to about 100 residues.
[0017] The sequence of side chains along the oligomer is preferably based on a prototype α-peptide (the target) having desirable biological activity against a disease state. The sequence of side chains may also be modified after translation onto the α/β-peptide backbone to optimize the desired properties of the compounds.
[0018] Each β-residue introduced into the unnatural α/β-peptide backbone can bear side chains at one of the two backbone carbons (β 3 or β 2 ) or both of the backbone carbons. The side chains may also be cyclically constrained via a ring connecting the two backbone carbons.
[0019] Of particular note in the present invention is that substitution of α-residues in the prototype target sequence with β-residues bearing side chains allows modification to the backbone without disrupting the sequence of side chains along the oligomer. Cyclic β-residues rigidify the backbone and promote helical structure.
[0020] It is preferred that β-residues be evenly spaced along the entire length of the sequence in order to maximize the protease resistance imparted to the oligomer by the backbone modifications. Examples of regularly repeating backbone patterns include, but are not limited to, (ααααααβ), (αααααβ), (ααααβ), (αααβ), (ααβ), (ααβαααβ), (ααβαβαβ), and (αβ).
[0021] Desirable properties in the final compounds include the ability to modulate a protein-protein interaction involved in the genesis or progression of a disease state in general and HIV entry into human cells in particular, and improved pharmacokinetic and pharmacodynamic properties relative to the target α-peptide sequence (e.g., better in vivo half-life, biodistribution, etc.). Many of the final compounds adopt a helical structure in solution, although a helical structure is not required.
[0022] Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, 5, 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
[0023] All references to singular characteristics or limitations of the present invention shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.
[0024] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
[0025] The methods, compounds, and compositions of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention as described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in synthetic organic chemistry.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A depicts sequences of α-peptides 1-3 and α/β-peptides 4-11. Bold residues indicate β 3 -residues corresponding to their α-amino acid counterparts; bold, underline residues are the cyclically constrained β-amino acid residues ACPC (X) and APC (Y). FIG. 1B depicts structures of an α-amino acid, the corresponding β 3 -amino acid analog, and cyclic β-residues ACPC (X) and APC (Z).
[0027] FIG. 2A depicts the gp41-5 protein, composed of three NHR segments and two CHR segments. FIG. 2B depicts the fluorescent CHR peptide used as a tracer in competition FP assays (Flu=5-carboxyfluorescein). FIG. 2C is a schematic of the interaction between the Flu-CHR peptide and the 5-helix bundle formed by gp41-5.
[0028] FIGS. 3A , 3 B, 3 C, and 3 D depict circular dichroism (CD) spectra of complexes formed between NHR peptide 1 and CHR analogs 3 ( FIG. 3A ), 4 ( FIG. 3B ), 5 ( FIG. 3C ), and 9 ( FIG. 3D ). Solid lines are spectra observed for a 1:1 mixture of the indicated oligomers at a total concentration of 20 μM in PBS at 25° C. Dashed lines are the spectra calculated for 1:1 non-interacting mixtures from CD of the individual components.
[0029] FIGS. 4A and 4B are a comparison of the six-helix bundles observed in the crystal structures of the newly characterized complex between α-peptides 1 and 3 ( FIG. 4A ) and the previously characterized complex between α-peptides 1 and 2 ( FIG. 4B ) (Chan, Fass, Berger, and Kim, Cell 1997, 89, 263-273). The RMSD of C α atoms between the two structures is 0.7 Å. FIG. 4C depicts the crystal structure of the 1+3 complex. FIG. 4D depicts the crystal structure of the 1+10 complex solved to 2.8 Å resolution. FIG. 4E depicts the crystal structure of the 1+8 complex solved to 2.8 Å resolution. FIGS. 4F and 4 G depict overlays of the all-α-peptide helix bundle-formed 1+3 with that formed by 1+10 ( FIG. 4F ) and 1+8 ( FIG. 4G ).
[0030] FIGS. 5A , 5 B, and 5 C depict circular dichroism (CD) spectra. FIG. 5A depicts superimposed CD data for NHR peptide 1 and CHR peptides 3, 4, 5, 8 and 10 at 20 μM concentration in PBS at 25° C. FIG. 5B depicts CD spectra of the indicated 1:1 mixtures at a total concentration of 20 μM in PBS at 25° C. (solid lines) along with the spectra calculated for 1:1 non-interacting mixtures from CD measurements on the individual components (dashed lines). FIG. 5C depicts temperature-dependent molar ellipticity at 222 nm for the indicated complexes at 20 μM concentration in PBS.
[0031] FIG. 6 is a graph depicting temperature dependent molar ellipticity at 222 nm for 1:1 mixtures of 1+3, 1+4, 1+5 and 1+9 at 20 μM total peptide in PBS.
[0032] FIG. 7A depicts the primary sequence of the Puma BH3 peptide (1) and α/β-peptide analogs 2-8 (gray circles and bold letters indicate β 3 residues). FIG. 7B depicts a helical wheel diagram of 1. Boxed residues in FIGS. 7A and 7B indicate hydrophobic positions most important for binding based on sequence homology. FIG. 7C presents schematic representations of 1-8, drawn in the same orientation as in FIG. 7B ; white and gray circles indicate heptad positions occupied by α-residues and β 3 -residues, respectively. FIG. 7D presents the structures of a generic α-amino acid and a generic β 3 -amino acid; the “R” substituent is conventionally referred to as the “side-chain.”
[0033] FIG. 8 is a histogram depicting inhibition constants for displacement of a fluorescently labeled Bak BH3 peptide bound to Bcl-x L or Mcl-1 by compounds 1-8. Broken bars indicate compounds binding tighter than discernable in the assay. The values for 8 were weaker than 100 μM for both proteins.
[0034] FIGS. 9A , 9 B, and 9 C depict proteolytic stability of 3, 4 and 10, respectively, whose structures are shown in FIG. 9D . Solutions of 20 μM peptide in TBS were incubated at room temperature with 10 μg/mL proteinase K. FIGS. 9A , 9 B, and 9 C depict time-dependent degradation data with curves resulting from fits to a simple exponential decay. FIG. 9D shows the structure of compounds 3, 4, and 10 and also depicts proteolysis products observed by mass spectrometry at the indicated time point. Vertical lines indicate observation by MALDI-MS of one or both products consistent with hydrolysis of the backbone amide bond between the indicated residues.
[0035] FIGS. 10A , 10 B, 10 C, and 10 D are graphs depicting inhibition of infection of TZM-bl cells by the indicated virus strains as a function of the concentration of gp41-derived fusion-blocking peptides. Each data point is the mean±S.E.M. from three independent experiments. FIG. 10A depicts inhibition of NL4-3 infection. FIG. 10B depicts inhibition of CC1/85 infection. FIG. 10C depicts inhibition of HC4 infection. FIG. 10D depicts inhibition of DJ258 infection.
DETAILED DESCRIPTION
[0036] The following abbreviations are used throughout the specification:
[0000] Ac 2 O=acetic anhydride, acetic oxide, acetylacetate.
ACPC=trans-2-aminocyclopentanecarboxylic acid.
APC=trans-3-aminopyrrolidine-4-carboxylic acid.
Boc=tert-butoxycarbonyl.
BOP=benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate.
β-Gal=β-galactosidase.
CD=circular dichroism.
CHR=C-terminal heptad repeat.
DIEA=N,N-diisopropylethylamine.
[0037] DMF=dimethylformamide.
DMSO=dimethylsulfoxide.
EDTA=ethylenediaminetetraacetic acid.
FKBP=FK506-binding protein.
Fmoc=9-fluorenylmethyl formyl.
FP=fluorescence poloarization.
Halogen=F, Cl, Br and I.
[0038] HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluoro-phosphate.
HIV=human immunodeficiency virus.
HOBT=N-hydroxybenzotriazole.
[0039] HPLC=high-performance liquid chromatography.
iPr 2 EtN=N,N-diisopropylethylamine.
IPTG=isopropyl β-D-1-thiogalactopyranoside.
MALDI-TOF-MS=matrix-assisted, laser-desorption, time-of-flight mass spectrometry.
MeOH=methanol.
NHR=N-terminal heptad repeat.
NMP=1-Methyl-2-pyrollidinone.
PTH1R and PTH2R=parathyroid hormone receptors 1 and 2.
RMSD=root mean square deviation.
RTKs=receptor tyrosine kinases.
TNF=tumor necrosis factor.
PBS=phosphate-buffered saline.
TBS=tris-buffered saline.
Tris=tris(hydroxymethyl)aminomethane.
TFA=trifluoroacetic acid.
TNBS=2,4,6-trinitrobenzene-sulfonic acid.
[0040] In the present description unless otherwise indicated terms such as “compounds of the invention” embrace the compounds in salt form as well as in free base form and also when the compounds are attached to a solid phase. Where a basic substituent such as an amine substituent is present, the salt form may be an acid addition salt, for example a dihydrochloride. Salts include, without limitation, those derived from mineral acids and organic acids, explicitly including hydrohalides, e.g., hydrochlorides and hydrobromides, sulfates, phosphates, nitrates, sulfamates, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane sulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates, and the like. Base addition salts include those derived from alkali or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N methylmorpholine, and the like. Other suitable salts are found in, for example, Handbook of Pharmaceutical Salts, P. H. Stahl and C. G. Wermuch, Eds., ©2002, Verlag Helvitica Chemica Acta (Zurich, Switzerland) and S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 66: p. 1-19 (January 1977), both of which are incorporated herein by reference.
[0041] The β-amino acid residues of the β-peptides of the invention are characteristically β-amino-n-propionic acid derivatives, typically further substituted at the 2-position carbon atom (the β 2 carbon) and/or the 3-position carbon atom (the β 3 carbon) in the backbone and may be further substituted, e.g., at the N-terminal amino nitrogen atom. The β 2 , β 3 , and amino substituents may include substituents containing from 1 to 43 carbon atoms optionally interrupted by up to 4 hetero atoms, selected from O, N or S, optionally containing a carbonyl (i.e., —C(O)—) group, and optionally further substituted by up to 6 substituents selected from halo, NO 2 , —OH, C 1-4 alkyl, —SH, —SO 3 , —NH 2 , C 1-4 -acyl, C 1-4 -acyloxy, C 1-4 -alkylamino, C 1-4 -dialkylamino, trihalomethyl, —CN, C 1-4 -alkylthio, C 1-4 -alkylsulfinyl, or C 1-4 -alkylsulfonyl.
[0042] Substituents on the β 2 and/or β 3 carbon atoms of β-amino acid residues may be selected from the group comprising the substituents which are present on the α-carbon atoms of natural α-amino acids, e.g., —H, —CH 3 , —CH(CH 3 ) 2 , —CH 2 —CH(CH 3 ) 2 , —CH(CH 3 )CH 2 CH 3 , —CH 2 -phenyl, CH 2 -pOH-phenyl, —CH 2 -indole, —CH 2 —SH, CH 2 —CH 2 —S—CH 3 , —CH 2 OH, —CHOH—CH 3 , —CH 2 —CH 2 —CH 2 —CH 2 —NH 2 , —CH 2 —CH 2 —CH 2 —NH—C(NH)NH 2 , —CH 2 -imidazole, —CH—COOH, —CH 2 —CH 2 —COOH, —CH 2 —CONH 2 , —CH 2 —CH 2 —CONH 2 or together with an adjacent NH group defines a pyrrolidine ring, as is found in the proteinogenic α-amino acid proline.
[0043] In accordance with the present invention it has been found that the compounds of the invention have desirable properties. For example, compounds described herein having approximately seven or more residues, three or more of which are cyclically constrained, are able to form stable helix structures in solution. Also the compounds described herein have much greater stability to the action of peptidases, such as pepsin, than do their corresponding α-peptides. As such the compounds described herein are expected exhibit correspondingly longer half lives, e.g., serum half lives, in vivo than corresponding α-peptides.
[0044] The invention includes the compounds of the invention in pure isomeric form, e.g., consisting of at least 90%, preferably at least 95% of a single isomeric form, as well as mixtures of these forms. The compounds of the invention may also be in the form of individual enantiomers or may be in the form of racemates or diastereoisomeric mixtures or any other mixture of the possible isomers.
[0045] The compounds of the invention may be prepared by the synthetic chemical procedures described herein, as well as other procedures similar to those which may be used for making α-amino acid peptides. Such procedures include both solution and solid phase procedures, e.g., using both Boc and Fmoc methodologies. Thus the compounds described herein may be prepared by successive amide bond-forming procedures in which amide bonds are formed between the β-amino group of a first β-amino acid residue or a precursor thereof and the α-carboxyl group of a second β-amino acid residue or α-amino acid residue or a precursor thereof. The amide bond-forming step may be repeated as many times, and with specific α-amino acid residues and/or β-amino acid residues and/or precursors thereof, as required to give the desired α/β-polypeptide. Also peptides comprising two, three, or more amino acid residues (α or β) may be joined together to yield larger α/β-peptides. Cyclic compounds may be prepared by forming peptide bonds between the N-terminal and C-terminal ends of a previously synthesized linear polypeptide.
[0046] β 3 -amino acids may be produced enantioselectively from corresponding α-amino acids; for instance, by Arndt-Eisert homologation of N-protected α-amino acids. Conveniently such homologation may be followed by coupling of the reactive diazo ketone intermediate of the Wolff rearrangement with a β-amino acid residue.
[0047] The method described herein can be used to establish discrete compound collections or libraries of compounds for use in screening for compounds having desirable activities, in particular biological activities indicative of particular pharmaceutical uses.
[0048] Thus the invention also includes discrete compound collections (typically comprising from 2 to about 1000 compounds) and libraries of compounds (typically comprising from 20 to 100 compounds up to many thousands of compounds, e.g., 100,000 compounds or more) comprising pluralities of the compounds described herein.
[0049] Compounds having desired biological activities may be identified using appropriate screening assays as described below.
[0050] The HIV protein gp41 is a canonical example of a class of proteins involved in the fusion of enveloped viruses to mammalian cells. During virus-cell fusion, the gp41 N-terminus inserts into the host cell membrane, and the trimeric protein undergoes a drastic structural rearrangement involving the formation a six-helix bundle composed of three copies of a N-terminal heptad repeat (NHR) domain and three copies of a C-terminal heptad repeat (CHR) domain. Formation of the gp41 six-helix bundle is an essential step for virus-cell fusion, and is therefore an attractive process to target for interruption using a rationally designed antiviral agent. To demonstrate the utility and functionality of the present invention, unnatural polypeptides analogous to gp41, but comprised of mixtures of α- and β-residues (α/β-peptides) were fabricated and shown to act as inhibitors of HIV-cell fusion.
[0051] A number of α-peptides based on either gp41 NHR or CHR sequences, e.g., compounds 1 and 2, (SEQ. ID. NOS: 1 and 2 respectively, see FIG. 1A ) have been investigated as fusion inhibitors. The most prominent example is the 36-residue α-peptide drug enfuvirtide (sold by Hoffmann-La Roche, Inc. under the registered trademark “FUZEON”), which is derived from the CHR domain. Several groups have tried to inhibit gp41 six-helix bundle formation with short α-helix mimics, including small molecules, cyclic peptides, terphenyls and β-peptides, that are intended to display three key CHR hydrophobic side chains in an α-helix-like fashion; however, these molecules display only modest anti-HIV activity in cell-based assays (IC 50 >1 μM vs.˜1 nM for enfuvirtide). Similar results have been seen with relatively short α-peptides that have been chemically predisposed toward α-helicity by internal cross-links.
[0052] The present inventors have discovered that systematically developing α/β-peptide foldamers that mimic key structural and functional properties of prototype α-peptide sequences, yields biologically active, unnatural polypeptides that are more stable to proteolytic degradation than analogous α-polypeptides. The method, referred to herein as “sequence-based design,” involves the systematic substitution of α-residues throughout a target sequence with β-amino acid residues in general, and preferably β 3 -amino residues bearing the side chain of the replaced α-residue. See FIG. 1B . The α→β modification alters the peptide backbone chemical composition while retaining the side chain sequence from the parent α-peptide. The systematic use of sequence-based design generates α/β-peptides that exhibit complex behaviors such as formation of protein-like quaternary assemblies and mimicry of protein helices involved in apoptosis. gp41-Mediated HIV-cell fusion was chosen as a model system to demonstrate the utility and functionality of sequence-based backbone modification because the target is of great biomedical importance. In short, a pharmacologically active agent that inhibits gp41-mediated HIV-cell fusion, designed using a rational and systematic method that can be repeated for other therapeutically important targets, is incredibly useful. The method provides an avenue to design pharmacologically active agents in less time, with less trial and error, and in a rational, directed fashion.
[0053] α-Peptides based on the native gp41 CHR sequence, such as compound 2, have been widely studied, and several groups have published efforts to improve the binding affinity and biological stability of CHR α-peptides by rational mutagenesis. To demonstrate the utility and functionality of the present invention, a recently reported gp41 CHR analog, α-peptide 3 (SEQ. ID. NO: 3, see FIG. 1A ) was chosen as the starting point for α→β modification. α-Peptide 3 includes numerous side chain mutations intended to enhance helical propensity by engineered intrahelical salt bridges and Xxx→Ala substitutions. In previous studies, 3 showed enhanced antiviral efficacy in cell culture and increased half-life relative to peptides based on the wild-type CHR sequence. Although the mutations in 3 were not intended to modify the structural nature of its binding interactions with the gp41 NHR domain, additional experimental evidence was sought that the six-helix bundle structure was unchanged. A co-crystal of α-peptides 1 and 3 was obtained by hanging drop vapor diffusion and the structure was solved to 2.0 Å resolution (see Table 1 and FIG. 4A ). The resulting six-helix bundle is essentially identical to that formed by native NHR+CHR peptide complex. Compare FIG. 4A (the 1+3 co-crystal and FIG. 4B (the 1+2 co-crystal).
[0000]
TABLE 1
Crystal Data Collection and Refinement Statistics.*
1+3 complex
Data Collection
Resolution (Å)
44.8-2.0 (2.1-2.0)
Total observations
137,233
Unique observations
15,938
Redundancy
8.6 (3.6)
Completeness (%)
99.9 (100)
I/σ
28.0 (4.7)
R sym † (%)
5.0 (26.2)
Refinement
Resolution (Å)
25.0-2.0
R (%)
21.1
R free ‡ (%)
26.0
Avg. B factor (Å 2 )
18.6
RMSD
Bonds (Å)
0.013
Angles (°)
1.1
*Values in parentheses are for data from the highest resolution shell;
† R sym = Σ n | I n − <I>|/Σ n I n where I n is the intensity of an independent observation of reflection n and <I> is the average of multiply recorded and symmetry related observations of reflection n;
‡ Free R reflections (~5% of total reflections) were held aside throughout refinement.
[0054] Among a variety of different α/β backbone patterns examined by the present inventors for use in sequence-based design, the most widely studied has been the ααβαααβ repeat. This pattern, which is tuned to the seven-residue repeat of the α-helix, places one β-residue per helical turn and results in a “stripe” of β-residues along one face of the helix. Crystal structures have shown that the helices formed by the ααβαααβ backbone are highly homologous to the α-helix. In the initial attempt at α→β modification of 3, eleven β-residues were incorporated in an ααβαααβ pattern (α/β-peptide 4, SEQ. ID. NO: 4, see FIG. 1A ). This resulted in the non-natural residues occupying positions opposite the hydrophobic face involved in binding to the gp41 NHR core.
[0055] To determine the affinity of α-peptide 3 and α/β analogues for gp41, a recently reported in vitro competition fluorescence polarization (FP) assay was employed. The assay uses a protein model of the gp41 fusion intermediate. See FIGS. 2A , 2 B, and 2 C. The model protein, gp41-5, is composed of three NHR segments (SEQ. ID. NO: 12) and two CHR segments (SEQ. ID. NO: 13) linked by short flexible loops. See FIG. 2A . The gp41-5 construct folds to form a five-helix bundle with a single binding site for a fluorescein-labeled CHR α-peptide. The fluorescein-labeled CHR α-peptide (SEQ. ID. NO: 14) is shown in FIG. 2B . Displacement of this fluorescent ligand provides the readout in competition FP. The reaction is depicted schematically in FIG. 2C and had a displacement constant (K d ) of 0.4 nM.
[0000]
TABLE 2
Summary of Data Obtained for gp41 CHR Analogs 3-9
gp41-5
NHR + CHR
Proteinase K
Cell-Cell Fusion
Oligomer
Ki (nM) a
Tm (° C.) b
t 1/2 (min) c
IC50 (nM) d
3
0.2
77
0.7
9
4
3,800
e
14
390
5
0.2
69
7
6
15
7
0.4
8
83
9
9
55
200
5
a Dissociation constant (K i ) for gp41-5 determined from competition FP experiments.
b Thermal unfolding transition observed by CD at 222 nm for a 1:1 mixture of NHR α-peptide 1 and the indicated CHR analog in PBS at 20 μM total peptide concentration.
c Half-life of a 20 μM solution of peptide in TBS in the presence of 10 μg/mL proteinase K.
d IC 50 in a cell-based fusion assay.
e No cooperative thermal transition was observed.
[0056] α-Peptide 3 showed binding affinity for gp41-5 in competition FP experiments (see Table 2) that was below the limit of detection of the assay (K i <0.2 nM). α/β-Peptide analog 4 (SEQ. ID. NO: 4) showed measurable affinity, but it bound the model protein more than 10,000-fold weaker than the prototype α sequence. Unpublished studies of the present inventors suggested that the W-W-I motif found near the N-terminus of 3 is critical for NHR binding. (See also Chan et al. (1998) Proc. Natl. Acad. Sci. USA 95:15613-7.) It was thus hypothesized that chimeric derivatives of α/β-peptide 4 which displayed these key hydrophobic side chains on a pure α backbone (i.e., oligomers 5 and 6, SEQ. ID. NOS: 5 and 6, respectively) would show tighter binding to gp41-5. Indeed, (α+α/β)-peptide 5 bound to gp41-5 with sub-nM affinity in the FP assay, indistinguishable from parent α-peptide 3. Extending the α/β segment in 5 toward the N-terminus (α/β-peptide 6) led to a diminution in binding affinity.
[0057] One of the fundamental motivations in the sequence-based α→β modification of a biomedically relevant sequence such as the gp41 CHR domain is to create oligomers that mimic the function of the parent α-peptide while displaying enhanced resistance to proteolytic degradation. As shown herein, the ααβαααβ backbone confers useful levels of resistance to protease; however, long α-peptide segments in chimeric oligomers are effectively cleaved by proteases.
[0058] To generate α/β-peptide analogs of the gp41 CHR domain with β-residues incorporated throughout the sequence, flexible substituted or unsubstituted β 2 - and/or β 3 -residues were replaced with cyclically constrained β-residues such as trans-2-aminocyclopentanecarboxylic acid (ACPC) and trans-2-aminopyrrolidinecarboxylic acid APC, see FIG. 1B , and those described in U.S. Pat. Nos. 6,060,585 and 6,613,876, incorporated herein by reference. Ring constraint of the C α -C β bond in a β-amino acid residue restricts torsional freedom, and promotes folding in diverse β-peptides and α/β-peptides. Substitution of three β 3 -hAla residues in chimeric (α+α/β)-peptide 6 (SEQ. ID. NO: 6) with ACPC generated α/β-peptide 7 (SEQ. ID. NO: 7), which showed a 40-fold improvement in binding to gp41-5 relative to 6. The same β 3 -hAla→ACPC substitutions were applied to oligomer 4 (SEQ. ID. NO: 4) yielding 8 (SEQ. ID. NO: 8). α/β-Peptide 8 showed a 50-fold higher affinity for gp41-5 than 4. To improve binding further, three β 3 -harg residues in 8 were mutated to APC, a cationic analog of ACPC, to produce 9 (SEQ. ID. NO: 9). Gratifyingly, α/β-peptide 9 showed gp41-5 binding affinity (K d =9 nM) that was impressive given its high degree of β-residue content.
[0059] α-Peptide 3 has been shown to be a potent inhibitor of HIV-cell fusion in cell culture. In the work described herein, the in vitro results obtained for the best α/β-peptide analog in the competition FP experiments compared favorably to 3. The gp41 CHR mimics were then tested for their ability to block gp41-mediated membrane fusion in a biological milieu. In order to compare the efficacy of α-peptide 3 to select foldamers in a more biologically relevant context, a previously described cell-cell fusion assay was employed. In this experiment, two cell lines are co-cultured. One cell line expresses HIV-1 Env (processed by cellular proteases to generate gp120+gp41) and Tat (an HIV transcriptional activator). The other cell line expresses CD4 (the primary cell surface receptor of HIV) and bears a gene for the enzyme β-galactosidase (β-Gal) preceded by an HIV long terminal repeat sequence (sensitive to activation by Tat). Env-mediated cell-cell fusion leads to expression of β-Gal, which can be quantified by a chemiluminescent enzymatic assay. Compounds 3, 4, 5 and 9 were tested for the ability to disrupt gp41-mediated membrane fusion in the above described assay. The results (Table 2) showed that the best foldamers, compounds 5 and 9 (SEQ. ID. NOS: 5 and 9 respectively), have IC 50 values that are indistinguishable from α-peptide 3.
[0060] The interactions of select α/β-peptides with the gp41 NHR domain were further investigated by circular dichroism (CD) spectroscopy. The CD spectra of 3, 4, 5, 8, 9 and were measured, both alone (see FIG. 5A ) and in a 1:1 mixture with NHR α-peptide 1 (see FIGS. 3A , 3 B, 3 C, and 3 D, respectively). See also FIGS. 5B and 5C for the superimposed spectra. The CHR analogs in isolation showed varying degrees of helicity. α-Peptide 3 ( FIG. 3A ) showed significant helical content at 20 μM in PBS, consistent with earlier published data. α/β-Peptide 9 ( FIG. 3D ), with seven β 3 →cyclic-β substitutions, revealed an intense CD minimum, consistent with a well-folded α/β-peptide helix. The observed CD spectrum for each 1:1 mixture of NHR+CHR peptide ( FIGS. 3A-3D , solid lines) was compared to that calculated by averaging spectra observed for the corresponding individual oligomers before mixing ( FIGS. 3A-3D , dashed lines). α/β-peptides 5 and 9, (SEQ. ID. NOS: 5 and 9, respectively) which showed nM or better affinity for gp41-5 in the competition FP assay, both showed a significant degree of induced helicity when mixed with NHR α-peptide 1. In contrast, α-peptide 3, which had only modest affinity for gp41-5 by FP, showed essentially no cooperative interaction with 1. The magnitude of the CD signatures among the well-folded mixtures (1+3, 1+5 and 1+9, FIGS. 3A , 3 C, and 3 D, respectively) are similar, but the ratio of intensities at 208 and 222 nm changes as a function of β-residue content (more β-residues tracks with a less intense peak at 222 nm). The well-folded NHR/CHR complexes (1+3, 1+5 and 1+9) each showed cooperative thermal transitions (see FIG. 6 ) with T m values that correlate with relative differences in affinity for gp41-5 by competition FP.
[0061] It has been shown (data omitted) that mixed α/β backbones (including the ααβαααβ pattern employed in the gp41 model) can impart resistance to degradation by proteases, a serious drawback of peptide-based HIV fusion inhibitors. The stability of α-peptide 3 and α/β-peptides 4 and 9 to degradation by the promiscuous serine protease proteinase K was tested. Under the conditions of the proteolysis assay, α-peptide 3 was completely degraded within minutes to yield products resulting from hydrolysis of at least ten different amide bonds in the sequence. α/β-Peptide 4, with simple α→β 3 substitution, showed 20-fold improvement in stability relative to prototype α-peptide 3. α/β CHR analog 9 showed an even greater improvement in stability over α-peptide 3 (280-fold). The relative improvement in proteolytic stability of α/β-peptide 9 over 4 likely results from a difference in their inherent helicity, as observed by CD.
[0062] The present invention is thus a method employing systematic α→β modifications to yield unnatural α/β polypeptides that retain the biological activity of an α-amino acid prototype, yet resist proteolytic degradation in cell culture and in vivo. As shown in the gp41 model, systematic α→β modifications in the HIV gp41 CHR domain, made in accordance with the present invention leads to α/β-peptide analogs with potent efficacy and enhanced proteolytic stability relative to the original α-peptide. The findings establish the scope of sequence-based backbone modification as a general method to create oligomers that mimic the structure and function of parent α-peptide sequences.
[0063] The gp41 model is presented herein as an illustration of how the present invention works in a specific environment. The method can be repeated, using any α-polypeptide as the target or prototype to be mimicked by a corresponding α/β-polypeptide fabricated according to the present method.
[0064] Thus, for example, the presently claimed method can be used to fabricate α/β-polypeptides, on a rational basis, to treat rheumatoid arthritis by targeting the interaction between tumor necrosis factor (TNF) and its receptor. See, for example, Williams, Ghrayeb, Feldmann, & Maini (1995) Immunology 84:433-439, for a discussion of this protein-protein interaction. Similarly, the presently claimed method can be used to fabricate α/β-polypeptides, on a rational basis, to treat central and peripheral nervous system disorders by targeting the interaction between gallanin and its receptor. See, for example, Mitsukawa, Lu, & Bartfai (2008) Cell. Mol. Life. Sci. (June 2008) 65(12):1796-17805 for a discussion regarding gallanin and its receptor and the suitability of using this interaction to design drug targets.
[0065] The presently claimed method can also be used to fabricate α/β-polypeptides, on a rational basis, to treat disorders relating to bone and calcium metabolism by targeting the interactions between parathyroid hormone and its receptors (PTH1R and PTH2R). See, for example, Usdin, Bonner, & Hoare (2002), “The parathyroid hormone 2 (PTH2) receptor,” Recept. Channels 8(3-4):211-218; and Mannstadt, Juppner, & Gardella (1999), “Receptors for PTH and PTHrP: their biological importance and functional properties,” Am. J. Physiol. 277(5 Pt. 2):F665-675.
[0066] The presently claimed method can also be used to fabricate α/β-polypeptides, on a rational basis, to treat disorders relating to serine protease reactions, such as the thrombin reaction. See, for example, EP1141022, which describes a series of α-polypeptide thrombin inhibitors. The present invention can be used to fabricate α/β-polypeptides that adopt similar conformations, have very similar anti-thrombin activity (as demonstrated in the case of the gp41 system), yet have much less susceptibility to proteolytic degradation in cell culture and in vivo.
[0067] The presently claimed method can also be used to fabricate α/β-polypeptides, on a rational basis, to inhibit the onset or progression of neoplasms by targeting, for example, the EPH receptors and their ephrin ligands. EPH receptors and their ephrin ligands constitute the largest sub-family of receptor tyrosine kinases (RTKs) and are components of cell signaling pathways involved in animal development. EPH signaling also plays an important role in oncogenic processes observed in several organs. These receptors are involved in a wide range of processes directly related to tumorigenesis and metastasis, including cell attachment and shape, migration, and angiogenesis. Accordingly, EPH expression and signaling activity is a critical system in the tumorigenic process. See, for example, Castano, Davalos, Schwartz & Arango (August 2008) Histol. Histopathol. 23(8):1011-1023. Thus, the present method can be used to fabricate α/β-polypeptides, on a rational basis, that mimic ephrin ligands.
[0068] Once suitable drug candidates are identified, their biological and/or pharmacological activities may be assayed using any number of well-known and industry-accepted assays.
[0069] The anti-inflammatory and immunosuppressive activities of the compounds described herein are determined by means of the following and similar assays: the IL-1β secretion inhibition, LPS fever, cytokine release from THP-1 cells, and functional IL-1 antagonist assays and the assay of carrageenan-induced paw edema in the rat (as described in EP0606044 and EP0618223); the macrophilin binding, Mixed Lymphocyte Reaction (MLR), IL-6 mediated proliferation, localized graft-versus-host (GvH) reaction, kidney allograft reaction in the rat, experimentally induced allergic encephalomyelitis (EAE) in the rat, Freund's adjuvant arthritis, FKBP binding, steroid potentiation and Mip and Mip-like factor inhibition assays (as described in WO94/09010, EP0296123 and EP0296122).
[0070] The central nervous system (CNS) activity of the compounds described herein is determined by means of the following and similar assays: serotonin ID (5HT 10) receptor agonist assays including the method of Weber et al., Schmiedeberg's Arch. Pharmacol. 337, 595-601 (1988), and as described in EP0641787; 5HT 3 receptor agonist assays (as described in GB2240476 and EP0189002); assays for activity in treatment of psychotic disorders and Parkinson's disease, such as the apomorphine-induced gnawing in the rat assay and dopamine receptor (D1 and D2) binding assays (as described in GB20206115 B); assays for dopamine receptor antagonist activity (in relation to schizophrenia and related diseases, as described in EP0483063 and EP0544240); assays for activity in relation to senile dementia and Alzheimer's disease (as described in EP0534904); assays for activity in relation to cerebral ischemia (as described in EP0433239), and assays in relation to gastrointestinal motility such as the peristaltic reflex in isolated guinea pig ileum and assays of anti-serotoninergic effects (specifically at the 5-HT 4 receptors) (as described in EP0505322).
[0071] Activity of the compounds described herein in relation to bone and calcium metabolism is determined by assays as (or similar to) those described in WO94/02510, GB2218102B and WO89/09786.
[0072] Activity of the compounds described herein in relation to asthma and other allergic and inflammatory conditions is determined by the following assay procedures: the PDE isoenzyme inhibition, inhibition of eosinophil activation by formyl-Met-Leu-Phe (fMLP), inhibition of TNFα secretion, inhibition of SRS-A production, bacterial endotoxin (LPS)-induced lethality in the guinea pig, arachidonic acid-induced irritant dermatitis in the mouse, relaxation of the human bronchus, suppression of SRS-A-induced bronchoconstriction, suppression of bombesin-induced bronchoconstriction, suppression of methacholine (MeCH)-induced bronchoconstriction in the rhesus monkey and suppression of airways hyperactivity in the guinea pig assays (as described in EP 0664289, WO94/12493 and GB2213482).
[0073] The serine protease (e.g., thrombin) inhibition activity of the compounds described herein is determined using assays such as those described in WO94/20526. The glycoprotein IIb/IIIa antagonist activity of the compounds described herein is determined using the assay procedures described by Cook et al., Thrombosis and Haemostasis, 70(3), 531-539 (1993) and Thrombosis and Haemostasis, 70(5), 838-847 (1993), and Müller et al. J. Biol. Chem., 268(9), 6800-6808 (1993).
[0074] Anticancer activity of the compounds described herein is determined by the anti-tumor activity assay as described in EP0296122 or by trial procedures, for instance as described in GB2239178. Multi-drug resistance (MDR)-reversing activity of the subject compounds is determined by the assays described in EP0296122.
[0075] The relevant teachings of the patent documents and other publications referred to above is incorporated herein by reference. Compounds fabricated according to the present invention which have appropriate levels of activity in these assays are useful as pharmaceuticals in relation to the corresponding therapies or disease states.
[0076] Thus the invention includes compounds as described herein for use as pharmaceuticals and the use of the compounds for the manufacture of a medicament for the treatment of any disease associated with any of the assays described herein, including infection by the HIV virus. The invention also includes the use of a compound fabricated according to the claimed method as a pharmaceutical, and pharmaceutical compositions comprising an effective amount of such a compound together with a pharmaceutically acceptable diluent or carrier.
[0077] The compounds of the invention may be synthesized using solid phase synthesis techniques.
[0078] Thus Fmoc-N-Protected β-amino acids can be used to synthesize poly-α/β-peptides by conventional manual solid-phase synthesis procedures under standard conditions on ortho-chloro-trityl chloride resin.
[0079] Esterification of Fmoc-β-amino acids with the ortho-chloro-trityl resin can be performed according to the method of Barlos et al., Tetrahedron Lett . (1989), 30, 3943. The resin (150 mg, 1.05 mmol Cl) is swelled in 2 ml CH 2 Cl 2 for 10 min. A solution of the Fmoc-protected β-amino acid in CH 2 Cl 2 and iPr 2 EtN are then added successively and the suspension is mixed under argon for 4 h. Subsequently, the resin is filtered and washed with CH 2 Cl 2 /MeOH/iPr 2 EtN (17:2:1, 3×3 min), CH 2 Cl 2 (3×3 min), DMF (2×3 min), CH 2 Cl 2 (3×3 min), and MeOH (2×3 min). The substitution of the resin is determined on a 3 mg sample by measuring the absorbance of the dibenzofulvene adduct at 300 nm. The Fmoc group is removed using 20% piperidine in DMF (4 ml, 2×20 min) under Ar bubbling. The resin is then filtered and washed with DMF (6×3 min). For each coupling step, a solution of the β-amino acid (3 equiv.), BOP (3 equiv.) and HOBT (3 equiv.) in DMF (2 ml) and iPr 2 EtN (9 eq) are added successively to the resin and the suspension is mixed for 1 h under Ar. Monitoring of the coupling reaction is performed with 2,4,6-trinitrobenzene-sulfonic acid (TNBS) (W. S. Hancock and J. E. Battersby, Anal. Biochem . (1976), 71, 260). In the case of a positive TNBS test (indicating incomplete coupling), the suspension is allowed to react for a further 1 h. The resin is then filtered and washed with DMF (3×3 min) prior to the following Fmoc deprotection step. After the removal of the last Fmoc protecting group, the resin is washed with DMF (6×3 min), CH 2 Cl 2 (3×3 min), Et 2 O (3×3 min) and dried under vacuum for 3 h. Finally the peptides are cleaved from the resin using 2% TFA in CH 2 Cl 2 (2 ml, 5×15 min) under Ar. The solvent is removed and the oily residues are triturated in ether to give the crude α/β-polypeptides. The compounds are further purified by HPLC.
[0080] The oral bioavailability of the compounds described herein is determined in the rat using standard procedures. The absolute oral bioavailabilty is expected to be about 1%.
[0081] In view of the stable structures which α/β-peptides exhibit in solution, their stability to enzymatic degradation and their encouraging pharmacokinetic properties, the compounds of the invention have the potential to provide useful pharmaceutical products.
[0082] As noted above, the gp41 CHR-derived α-peptide, 3 was used as the starting point for α→β modification ( FIG. 1A ). α-Peptide 3, also known as T-2635, is 50% mutated as compared to the wild type gp41 CHR domain and contains a combination of Xxx→Ala substitutions and engineered i→i+4 salt bridges that were intended to enhance α-helical propensity. α-Peptide 3 represents one of the most successful examples reported to date of improving the antiviral efficacy of gp41 CHR α-peptides via modification of the α-amino acid sequence. The initial studies began with the side chain sequence optimized in 3. Also explored were changes in backbone composition in the form of α→β residue substitution. In α/β-peptide 4, a subset of the α-residues in 3 has been replaced by β 3 -residues that bear the side chain of the replaced α-residue (see FIG. 1B ). Thus, α/β-peptide 4 has the sequence of side chains found in 3 displayed on an unnatural backbone. The β 3 -residues of 4 are incorporated in an ααβαααβ pattern, which, upon folding, generates a stripe of β-residues that runs along one side of the helix. This design places the β-stripe in 4 distal along the helix circumference to the molecular surface that packs against the gp41 NHR domain trimer in the six-helix bundle.
[0083] A competition fluorescence polarization (FP) assay based on a protein model of the gp41 six-helix bundle was used to compare 3 and 4. (See the Examples for details.) The assay measures displacement of a fluorescently-labeled CHR α-peptide from an engineered five-helix bundle protein, gp41-5, which contains three NHR segments and two CHR segments. Affinity for the gp41-5 protein construct correlates with the ability of CHR-mimetic agents to bind to the gp41 pre-hairpin intermediate formed just prior to HIV-cell fusion. As expected, α-peptide 3 binds very tightly to gp41-5 (K i <0.2 nM; Table 1). The analogous α/β-peptide 4, however, displays only weak affinity for gp41-5, >10,000-fold lower than that of 3. The modest potency of α/β-peptide 4 in this protein-based assay is comparable to that displayed by a number of small molecules and peptidomimetics in comparable experiments.
[0084] In an effort to understand the dramatic differences in binding between 3 and 4 and to improve the affinity of the α/β-peptide for gp41, chimeric α/β-peptides 5 and 6 were prepared and characterized. Both 5 and 6 contain a pure α segment at the N-terminus and an α/β segment at the C-terminus; these oligomers are chimeras of α-peptide 3 and α/β-peptide 4. α/β-Peptide 5 displays very high affinity for gp41-5, indistinguishable from that of α-peptide 3; however, extending the α/β segment toward the N-terminus (as in 6) causes a significant loss of affinity. The sensitivity of the N-terminal segment to α→β 3 modification is consistent with data showing that side chains in this region, especially those corresponding to Trp 3 , Trp 6 and Ile 10 in 3, play a crucial role in CHR binding to the NHR trimer.(29)
[0085] α/β-Peptides 5 and 6 represent an improvement in gp41 mimicry relative to 4, but it would be desirable to place β-residues throughout an α/β-peptide sequence in order to maximize resistance to proteolysis. Each α→β 3 replacement, however, adds a flexible bond to the backbone, which should increase the conformational entropy penalty associated with helix formation. The greater conformational entropy of the unfolded state of 4 relative to 3, arising from eleven α→β 3 replacements, may account for the large difference in binding affinity for gp41-5 between these two oligomers. Although β-residues are the source of this loss of stability, these residues provide an avenue for conformational pre-organization that is made uniquely possible by their chemical structure. Incorporation of cyclic β-residues (e.g., ACPC and APC, FIG. 1B ) can constrain the C α -C β backbone torsion and thereby enhance folding propensity without disrupting backbone amide hydrogen bonding.
[0086] The impact of conformational preorganization in the context of gp41 mimicry was probed by replacing a subset of β 3 -residues with cyclic analogues. The first comparison involved α/β-peptide 7, the analogue of 6 in which the three β 3 -hAla residues are replaced by ACPC ( FIGS. 1A and 1B ). Both β 3 -hAla and ACPC are non-polar, and this similarity was expected to maintain the physical properties that emerge from side chain sequence. The >30-fold higher affinity for gp41-5 displayed by 7 relative to 6 supports the hypothesis that residue-based rigidification is a useful complement to sequence-based design for developing peptide-mimetic foldamers. Replacement of two β 3 -hArg residues in oligomer 7 with APC, a heterocyclic analogue of ACPC, leads to α/β-peptide 8, which showed a very high affinity for gp41-5. APC 36 in α/β-peptide 8 is in a region of the CHR sequence that does not engage the NHR region contained in gp41-5; this observation may explain the similar K i values of 7 and 8. Additional evidence of the favorable contribution of cyclic β-residues comes from comparison of oligomers 4, 9 and 10, each of which has β-residues throughout the sequence. α/β-Peptide 9 was generated from 4 by four β 3 -hAla→ACPC replacements, which leads to a >45-fold improvement in K i . Replacement of the three β 3 -hArg residues of 9 with APC, to generate 10, improves K i by a further ˜10-fold. Relative to completely flexible α/β-peptide 4, rigidified analogue 10 (K i =9 nM) shows ˜380-fold enhanced binding to gp41-5.
[0087] The interactions of CHR α-peptide 3 and α/β-peptide analogues 4, 5, 8 and 10 with a peptide derived from the gp41 NHR domain (1) were investigated by circular dichroism (CD) spectroscopy. NHR α-peptide 1 forms a six-helix bundle when mixed with gp41 CHR α-peptides; this six-helix bundle is thought to represent the post-fusion state adopted by gp41 in the course of viral entry. α-Peptide 3 showed significant helical content at 20 μM in PBS, consistent with previously published data ( FIG. 5A ). α/β-Peptide 4 showed no significant helicity under similar conditions; however, analogue 10, with seven β 3 →cyclic-β substitutions, showed an intense CD minimum, consistent with a well-folded α/β-peptide helix ( FIG. 5A ). The observed CD spectrum for each 1:1 mixture of NHR+CHR peptide was compared ( FIGS. 3A through 3D , and 5 B, solid lines) to that calculated by averaging spectra for the corresponding individual oligomers ( FIGS. 3A through 3D and 5 B, dashed lines). α/β-Peptides 5, 8 and 10, which displayed high affinity for gp41-5 in the competition FP assay, each showed a significant degree of induced helicity when mixed with NHR α-peptide 1, which is consistent with six-helix bundle formation. By contrast, α/β-peptide 4, which has only modest affinity for gp41-5, showed essentially no interaction with 1. The magnitude of the CD signatures among the well-folded mixtures (1+3, 1+5, 1+8 and 1+10) are similar, but the ratio of intensities at 208 and 222 nm changes as a function of β-residue content (higher β-residue content is correlated with a less intense peak at 222 nm). This trend is consistent with previous studies on helical oligomers containing mixed α/β backbones. The complexes formed by 1+3, 1+5, 1+8 and 1+10 each showed highly cooperative thermal transitions ( FIG. 5C ). The trend in T m,app values (i.e., apparent T m ) correlates with differences in affinity among 3, 5, 8 and 10 for gp41-5 in the competition FP assay; that is, stronger binding to gp41-5 correlates with more stable assembly with NHR peptide 1.
[0088] Crystal Structures.
[0089] X-ray crystallography was employed to compare the heteromeric six-helix bundles formed by NHR α-peptide 1 with CHR α-peptide 3, chimeric CHR α/β-peptide 8 or CHR α/β-peptide 10 (see Table 3).
[0000] TABLE 3 X-ray data collection and refinement statistics 1+3 1+10 1+8 complex 10 complex complex Data collection Space group P2 1 2 1 2 C2 P4 1 32 H32 Cell dimensions a, b, c (Å) 37.6, 179.0, 71.3, 44.0, 84.9, 84.9, 57.0, 57.0, 33.1 58.1 84.9 186.3 α, β, γ (°) 90, 90, 90 90, 105.4, 90 90, 90, 90 90, 90, 120 Resolution (Å) 44.8-2.0 50.0-2.1 50.0-2.8 50.0-2.8 (2.1-2.0)* (2.18-2.10)* (2.9-2.8)* (2.9-2.8)* R sym (%) 5.0 (26.2) 6.7 (35.6) 6.1 (51.2) 5.8 (38.8) I/σI 28.0 (4.7) 16.1 (3.5) 31.6 (2.8) 16.8 (3.7) Completeness 99.9 (100) 99.8 (99.8) 99.8 (98.2) 99.5 (100) (%) Redundancy 8.6 (3.6) 3.5 (3.4) 7.8 (6.2) 5.9 (6.2) Refinement Resolution (Å) 25.0-2.0 25.0-2.1 25.0-2.8 25.0-2.8 No. reflections 15,123 9,769 2,730 2,947 R work /R free (%) 20.9/26.0 20.4/24.9 26.6/30.7 25.2/31.1 Avg. B factor (Å 2 ) RMSD Bond lengths 0.013 0.015 0.013 0.018 (Å) Bond angles (°) 1.1 2.0 1.7 1.8 *Highest resolution shell is shown in parenthesis.
Although the mutations to the native CHR sequence that lead to α-peptide 3 were not intended to modify the nature of its binding interactions with the gp41 NHR domain, direct evidence was sought that the six-helix bundle structure was unchanged relative to that formed by 1 and the native CHR sequence. A co-crystal of α-peptides 1 and 3 was obtained and its structure solved to 2.0 Å resolution. See FIG. 4A . The resulting six-helix bundle is essentially identical to that for 1+2 (see FIG. 4B ) which contains the native CHR sequence; the root mean square deviation (rmsd) is 0.73 Å for C α atoms.
[0090] A crystal of the 1+10 complex was also obtained and its structure solved to 2.8 Å resolution. See FIG. 4D . α-Peptide 1 and α/β-peptide 10 combine to form a six-helix bundle that is similar to the assembly formed by 1+3 (duplicated in FIG. 4C to allow a side-by-side comparison). A crystal containing only α/β-peptide 10 (not shown) was obtained as well. The structure of 10 alone, solved to 2.1 Å resolution, revealed a parallel trimeric helix bundle with a hydrophobic core comprising the residues that engage the gp41 NHR trimer in the six-helix bundle formed by 1+10. The self-assembly of α/β-peptide 10 in the crystalline state parallels the behavior previously observed for prototype α-peptide 3, which was shown to self-assemble in solution.
[0091] The core NHR trimers in the structures of 1+10 and 1+3 are highly homologous (0.65 Å C α rmsd for NHR residues 3-30). When the two bundles are aligned via the NHR trimer, the CHR helices track very closely in the C-terminal segment (0.84 Å C α rmsd for residues 16-33) but diverge near the N-terminus (4.2 Å C α rmsd for residues 2-15). This divergence reflects a greater superhelical twist in α-peptide 3 relative to α/β-peptide 10. The divergent portion of the helix formed by 10 contains the two Trp residues that, in CHR α-peptides, are essential for stable six-helix bundle formation. In the structure of 1+10, the side chains of Trp 3 and Trp 5 were not resolved in electron density, suggesting a high degree of disorder. In addition, significant disorder was observed in the side chains of NHR residues Lys 29 and Trp 26 , which pack around CHR Trp 5 in the 1+3 complex. FIGS. 4F and 4G depict overlays of the all-α-peptide helix bundle-formed 1+3 with that formed by 1+10 ( FIG. 4F ) and 1+8 ( FIG. 4G ).
[0092] Given the well-established role of the gp41 CHR domain Trp-Trp-Ile motif in six-helix bundle formation, the observation that the N-terminal segment of α/β-peptide 10 does not engage the NHR binding pocket in the crystal structure of the 1+10 complex is intriguing. Removal of the first ten residues of α/β-peptide 10 leads to oligomer 11, in which the Trp-Trp-Ile motif is not present (see FIG. 1A ). If the N-terminal region of 10 were not involved in binding to the NHR trimer in solution, as might be suspected based on the crystal structure of 1+10, then 11 should show affinity for gp41-5 that is comparable to that of 10. However, α/β-peptide 11 showed no measurable affinity for gp41-5 (K i >10 M), indicating that the N-terminal segment of 10 is essential for high-affinity binding to gp41-5 in solution.
[0093] Motivated by the differences between the CHR domain N-terminal segments in the 1+3 complex and the 1+10 complex, the structure of NHR peptide 1 in complex with CHR α/β-peptide 8, a chimera of α-peptide 3 and α/β-peptide 10, was investigated. The 1+8 complex was crystallized and its structure solved to 2.8 Å resolution. See FIG. 4E . Relative to α/β-peptide 10, chimeric α/β-peptide 8 tracks much more closely with the CHR helix (3) in the all-α-peptide, six-helix bundle formed by 1+3 ( FIG. 4C , 1.4 Å C α rmsd for residues 2-33). The side chains of the Trp-Trp-Ile motif in the N-terminal segment of 8 show the expected packing into the binding pocket on the NHR core trimer (data not shown). Based on this result and the behavior of truncated α/β-peptide 11, it is suspected that the lack of direct contact between the N-terminal portion of 10 and the NHR trimer in the 1+10 complex is an artifact of crystal packing.
[0094] Antiviral Activity.
[0095] Two sets of experiments were performed to evaluate the activities of α-peptide 3 and α/β-peptides 4, 5 and 10 in a biological context. The first experiment compared the oligomers in a cell-cell fusion assay based on expression of the env gene of the HIV-1 clone HxB2, an assay that is commonly used to model gp41-mediated HIV-cell fusion. (Deng Y Q, Zheng Q, Ketas T J, Moore J P, & Lu M (2007) Protein design of a bacterially expressed HIV-1 gp41 fusion inhibitor. Biochemistry 46(14):4360-4369.) The cell-cell fusion assay results (Table 4) showed that α/β-peptides and 10 have IC 50 values indistinguishable from that of α-peptide 3, while α/β-peptide 4 is much less effective. Compounds 3, 4, 5 and 10 were then evaluated for the ability to prevent HIV infection of the cell line TZM-bl. (Wei X P, et al. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46(6):1896-1905.) These studies employed one T-cell line adapted strain and three primary isolates; two of the strains are X4-tropic, and the other two are R5-tropic.
[0000] TABLE 4 Summary of physical and functional data obtained for gp41 CHR analogues 3-11. gp41-5 binding NHR + CHR Stability to Cell-cell affinity stability by Proteinase fusion Inhibition of HIV-1 infectivity, IC 50 (nM) e by FP a CD b K c inhibition d X4 strains R5 strains Oligomer K i (nM) T m,app (° C.) t 1/2 (min.) IC 50 (nM) NL4-3 HC4 CC 1/85 DJ258 3 <0.2 77 0.7 9 ± 3 5 ± 0.6 27 ± 4 140 ± 20 58 ± 6 4 3,800 — f 14 390 ± 40 700 ± 60 590 ± 100 1300 ± 100 960 ± 200 5 <0.2 67 7 ± 2 10 ± 2 55 ± 8 270 ± 20 280 ± 90 6 15 7 0.4 8 0.3 65 9 83 10 9 55 200 5 ± 2 28 ± 3 59 ± 10 180 ± 30 110 ± 40 11 >10,000 T-20 700 ± 100 250 ± 20 1400 ± 400 330 ± 60 a Dissociation constant (K i ) for binding to the protein gp41-5 as determined by competition FP experiments. b Melting temperature (T m,app ) for the thermal unfolding transition observed by CD at 222 nm for a 1:1 mixture of NHR α-peptide 1 and the indicated CHR analogue at 20 μM total peptide concentration in PBS. c Half-life (t 1/2 ) of a 20 μM solution of peptide in TBS in the presence of 10 μg/mL proteinase K. d Values are the means ± S.E.M. of IC 50 values obtained in three independent experiments. The envelope protein expressed was of the HxB2 clone, derived from the T-cell-line-adapted isolate IIIB of clade B. e Values are the means ± S.E.M. of IC 50 values obtained in three independent experiments. f The temperature dependent CD for the 1 + 4 mixture was not significantly different than that calculated from the average of the temperature dependent CD spectra of 1 alone and 4 alone.
The results of the infectivity assays (Table 4, FIGS. 10A , 10 B, 10 C, and 100 D) show similar biological potencies among 3, 5 and 10 for HIV-1 strains that use different co-receptors. This finding indicates the blocking of a necessary, shared step in entry through peptide interactions with conserved regions of gp41. It may be noted that there is imperfect correlation between K i for binding to gp41-5 and IC 50 values in cell-based assays among the compounds reported here. For example, the affinity of 10 for gp41-5 was >45-fold higher than that of 5, yet IC 50 values for 10 were sometimes lower than for 5. There are several possible reasons for this discrepancy. Sequence differences between the CHR and NHR domains found in gp41-5 and those found in the viruses tested may lead to better correlation between gp41-5 binding affinity and antiviral activity against some strains relative to others. In addition, it has previously been suggested that the association rates for CHR peptides binding to gp41 are a better predictor of relative antiviral potencies than are equilibrium binding affinities. (Steger H K & Root M J (2006) Kinetic dependence to HIV-1 entry inhibition. J Biol Chem 281(35):25813-25821.) The rigidified backbone in 10 may alter its association rate with gp41 relative to that of 5. Sensitivity to gp41-derived fusion inhibitors may be affected by many factors that differ among strains of virus, including the amount of Env incorporated into the virion, the strength of Env interactions with CD4 and with co-receptors, the kinetics and energetics of the fusion process, as well as amino acid variation in the binding site for inhibitory peptides. Overall, the antiviral assays results strongly support the hypothesis that CHR-derived α/β-peptides effectively mimic gp41 in a complex biological milieu.
[0096] Proteolytic Susceptibility.
[0097] An important motivation for developing foldamer antagonists of protein-protein interactions is the prospect of diminishing sensitivity to proteolytic degradation. Rapid destruction by proteolytic enzymes represents a significant drawback to the clinical use of α-peptide drugs. The susceptibilities of α-peptide 3 and α/β-peptides 4 and 10 to degradation by proteinase K, a promiscuous serine protease, were compared. Under the assay conditions, α-peptide 3 was completely degraded within minutes ( FIG. 9A ); mass spectrometry revealed hydrolysis of at least ten different amide bonds in the sequence ( FIG. 9D , top sequence). α/β-Peptide 4, with exclusively α→β 3 substitution, showed 20-fold improvement in stability relative to prototype α-peptide 3. See FIG. 9B and FIG. 9D , middle sequence. Rigidified α/β-peptide 10 showed an even greater improvement in stability over α-peptide 3 (280-fold). See FIG. 9C and FIG. 9D , bottom sequence. The greater stability of α/β-peptide 10 relative to α/β-peptide 4 likely results from the greater helical propensity of 10, as detected by CD. The small number of proteolysis products observed for α/β-peptide 10 by mass spectrometry ( FIG. 9D ) supports previous observations that β-residues in mixed α/β backbones tend to protect neighboring amides from proteolytic cleavage.
[0098] Many proteins display surfaces that participate in highly selective interactions. Information flow mediated by protein-protein interactions is essential for normal function of individual cells and entire organisms; such interactions can play key roles in disease as well. There is considerable motivation to identify strategies for inhibiting the formation of specific inter-protein complexes. At the clinical level, the most successful approach to this goal involves the use of engineered proteins or protein fragments, i.e., molecules constructed from the same building blocks as the protein targets themselves. The motivating hypothesis of the presently claimed method is that recognition surfaces displayed by proteins can be mimicked with unnatural oligomers that adopt protein-like conformations and display protein-like side chains, and that such oligomers will function as inhibitors of natural protein-protein associations. Natural protein sequences are logical starting points for designing folded oligomers with normatural backbones that have sophisticated functions. The data presented here provide strong support for these hypotheses in the context of a widely studied viral infection process.
[0099] The results presented herein indicate that a long α-helical segment, the CHR region of HIV protein gp41, can be structurally and functionally mimicked by oligomers composed of α- and β-amino acid residues. A two-stage process was required to generate an α/β-peptide that manifests a favorable profile of properties, including strong association with the intended binding partner, potent inhibition of HIV infection in a cell-based assay and resistance to proteolytic cleavage. The first design stage involves replacement of selected α-residues in a parent peptide sequence with homologous β-residues that retain the original side chains. The second design stage involves selective replacement of flexible β 3 -residues with cyclically preorganized β-residues. These modifications are intended to remove deleterious backbone flexibility that is unavoidably introduced with the initial α→β 3 modifications.
[0100] Using a two-stage approach for creation of an effective α/β-peptide mimic of the gp41 CHR segment is noteworthy in light of our previous findings in a different and inherently simpler protein recognition system. Mimicry of BH3 domains, short α-helical segments that mediate protein-protein interactions in the Bcl-2 protein family, required only the first stage of this design approach, simple α→β 3 substitution throughout the prototype sequence. (Horne W S, Boersma M D, Windsor M A, & Gellman S H (2008) Sequence-based design of α/β-peptide foldamers that mimic BH3 domains. Angew Chem Int Ed 47(15):2853-2856.) In contrast, α/β-peptide 4, which showed only modest affinity for gp41-5, was the most potent gp41 mimic identified among a series of α/β-peptides designed by exploring alternative α/β 3 backbone patterns in the native gp41 CHR domain and related sequences.
[0101] The results reported here represent a substantial advance relative to earlier efforts to develop unnatural oligomers that mimic α-helices involved in protein-protein recognition events. Previous work has been limited to relatively short α-helical targets, typically only two to four helical turns. Efficacies of oligomers developed in these prior studies have generally been modest (IC 50 values greater than 1 μM). Moreover, in most previously studied systems, effective inhibition has been possible with small molecule antagonists. The present results are distinctive because the data show that a long α-helix (˜10 turns) can be structurally and functionally mimicked with a rationally designed oligomer. To date, efforts to disrupt gp41 six-helix bundle assembly with small molecules have been relatively unsuccessful.
[0102] The present work demonstrates the value of designing unnatural oligomers that can “read” the sophisticated recognition signals that have been evolutionarily encoded in natural proteins. Potent inhibition of HIV infectivity by α/β-peptides is an important advance in the development of functional foldamers.
EXAMPLES
[0103] Reagents:
[0104] Protected α-amino acids and resins used in peptide synthesis were purchased from Novabiochem (a wholly owned subsidiary of EMD Chemicals Inc. and Merck KGaA, Darmstadt, Germany). Protected β 3 -amino acids were purchased from PepTech (Burlington, Mass., USA). Cyclically constrained β-residues, Fmoc-ACPC and Fmoc-APC(Boc), were prepared as previously described. Lee, LePlae, Porter, and Gellman, J. Org. Chem. 2001, 66, 3597-3599; LePlae, Umezawa, Lee, and Gellman, J. Org. Chem. 2001, 66, 5629-5632. 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluoro-phosphate (HBTU) was purchased from AnaSpec (San Jose, Calif., USA). 5-Carboxyfluorescein was purchased from Invitrogen (Carlsbad, Calif., USA). 1-Methyl-2-pyrollidinone (NMP) was purchased from Advanced Chemtech (Louisville, Ky., USA). All other reagents were purchased from Sigma-Aldrich Corp. (St. Louis, Mo., USA) or Fisher Scientific (Pittsburgh, Pa., USA) and used as received.
[0105] Synthesis:
[0106] All peptides were prepared on “NovaSyn TGR”-brand resin (Novabiochem). α-Peptides were prepared by standard Fmoc solid phase peptide synthesis methods on a Symphony Multiple Peptide Synthesizer (Protein Technologies, Inc., Tucson, Ariz., USA). α/β-Peptides were prepared by automated Fmoc solid phase peptide synthesis on a Synergy 432A automated synthesizer (Applied Biosystems, Foster City, Calif., USA). α/β-Peptides were also prepared manually by microwave-assisted Fmoc solid phase peptide synthesis. Erdelyi and Gogoll (2002) Synthesis 11:1592-1596. The N-terminus of each peptide was capped by treatment with 8:2:1 DMF/DIEA/Ac 2 O. The resin was washed thoroughly (3×DMF, 3×CH 2 Cl 2 , 3×MeOH) and then dried under vacuum. All peptides were cleaved from resin by treatment with 94:2.5:2.5:1 TFA/H 2 O/ethanedithiol/triisopropylsilane. The resin was filtered, washed with additional TFA, and the combined filtrates concentrated to ˜2 mL under a stream of dry nitrogen. Crude peptide was precipitated from the cleavage mixture by addition of cold ether (45 mL). The mixture was centrifuged, decanted, and the remaining solid dried under a stream of nitrogen. Peptides were purified by reverse phase HPLC on a prep-C 18 column using gradients between 0.1% TFA in water and 0.1% TFA in acetonitrile. The identity and purity of the final products were confirmed by MALDI-TOF-MS and analytical HPLC, respectively. Stock solution concentrations were determined by UV absorbance. Gill, S. C.; Vonhippel, P. H. Anal. Biochem. 1989, 182, 319-326. MALDI-TOF-MS (monoisotopic [M+H] + , m/z): 1: obsd.=4162.6, calc.=4162.4; 2: obsd.=4288.7, calc.=4288.0; 3: obsd.=4455.0, calc.=4455.3; 4: obsd.=4609.9, calc.=4609.5; 5: obsd.=4526.1, calc.=4525.4; 6: obsd.=4552.7, calc.=4553.4; 7: obsd.=4631.6, calc.=4631.4; 8: obsd.=4516.5, calc.=4515.3; 9: obsd.=4713.0, calc.=4713.5; 10: obsd.=4539.9, calc.=4539.4; 11: obsd.=3299.4, calc.=3299.8.
[0107] Synthesis of Flu-C38:
[0108] “NovaSyn TGR”-brand resin bearing the full-length C38 peptide with free N-terminus (WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDK; SEQ. ID. NO: 23) was prepared on a 25 μmol scale by standard Fmoc solid phase peptide synthesis methods on a Symphony Multiple Peptide Synthesizer (Protein Technologies, Inc.). Following synthesis, the resin was transferred to a fritted syringe. 5-Carboxyfluorescein (28 mg, 0.075 mmol) and HOBT.H 2 O (11 mg, 0.075 mmol) were dissolved in N-methyl-2-pyrrolidinone (0.75 mL). Diisopropylcarbodiimide (12 μL, 0.075 mmol) was added. The resulting solution was transferred to the peptide-bearing resin. The reaction vessel was covered in foil and placed on a shaker overnight. The resin was washed with DMF (3×), and the coupling reaction was repeated with fresh reagents. The resin was then washed with DMF (3×), 20% piperidine (2×), DMF (3×), CH 2 Cl 2 (3×), and MeOH (3×). Fischer, R.; Mader, O.; Jung, G.; Brock, R. Bioconjugate Chem. 2003, 14, 653-660. The crude peptide was cleaved and purified as described above. Stock solutions, prepared in water, were quantified by visible absorbance (ε 494 =68,000 M −1 cm −1 at pH 8). MALDI-TOF-MS (monoisotopic [M+H] + , m/z): obsd.=5089.3, calc.=5089.3.
[0109] Crystallization.
[0110] Hanging drops were prepared by mixing 1 μL of crystallization stock and 1 μL of reservoir buffer followed by room temperature equilibration over 0.7 mL buffer. Stock solutions of the 1+3 and 1+8 complexes were prepared by mixing concentrated stocks of the individual peptides in a 1:1 ratio to a final concentration of 2.2 mM total peptide in water. Crystals of 1+3 were obtained from a reservoir buffer comprising 0.1 M Tris pH 8.5, 1 M (NH 4 )H 2 PO 4 . Crystals of the 1+8 complex were grown a reservoir buffer comprising 0.4 M Li 2 SO 4 .H 2 O, 12% v/v PEG 8000, 20% v/v glycerol. In initial attempts to crystallize the 1+10 complex, a stock solution was prepared by mixing concentrated stocks of the individual peptides in a 1:1 ratio to a final concentration of 0.76 mM total peptide in water. Stocks of 1+10 prepared in this way were not fully soluble. However, the resulting viscous suspension yielded crystals of α/β-peptide 10 alone from a well buffer comprised of 0.5 M ammonium sulfate, 0.1 M HEPES-Na, pH 7.5, 30% v/v 2-methyl-2,4-pentanediol. For subsequent crystallization trials of 1+10, the stock solution of the complex was prepared by refolding the 1:1 peptide mixture at 130 μM total peptide in water followed by concentration to −1.1 mM by centrifugation at 4° C. through a 10 kDa molecular weight cutoff membrane. Crystals of 1+10 were obtained from a stock prepared in this way and a reservoir buffer comprised of 0.2 M NaCl, 0.1 M Tris pH 8.5, 25% w/v PEG 3350.
[0111] X-Ray Data Collection, and Structure Determination.
[0112] All crystals were flash frozen in liquid nitrogen. Crystals of the 1+3 complex were briefly soaked in 0.08 M Tris pH 8.5, 1.6 M (NH 4 )H 2 PO 4 , 20% v/v glycerol prior to freezing. Crystals of 10 and 1+8 were frozen directly from the crystallization drop. Crystals of the 1+10 complex were soaked briefly in 0.2 M NaCl, 0.1 M Tris pH 8.5, 25% w/v PEG 3350, 20% v/v glycerol prior to freezing. Diffraction data for the 1+3 and 1+8 complexes were collected on a Bruker X8 Proteum Diffractometer (Bruker AXS, Inc. Madison, Wis. USA) using Cu K α radiation and were processed with the Bruker Proteum2 software package. Diffraction data for the crystals of 10 and the 1+10 complex were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G at the Advanced Photon Source, Argonne National Laboratory, and were processed with HKL-2000-brand software (HKL Research, Inc., Charlottesville, Va., USA). Structure determination was carried out using the CCP4 software suite. Collaborative Computational Project Number 4 (1994) The CCP4 Suite—Programs for Protein Crystallography. Acta Crystallogr, Sect D 50:760-763. Molecular replacement was carried out with Phaser software (McCoy A J, Grosse-Kunstleve R W, Storoni L C, & Read R J (2005) Likelihood-enhanced fast translation functions. Acta Crystallogr, Sect D 61:458-464) or Molrep software (Vagin A & Teplyakov A (1997) MOLREP: An automated program for molecular replacement. J Appl Crystallogr 30(6):1022-1025). Refinement was accomplished by a combination of Refmac (Murshudov G N, Vagin A A, & Dodson E J (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr, Sect D 53:240-255) for automated refinement, Coot (Emsley P & Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta Crystallogr, Sect D 60:2126-2132) for manual model building, and ARP/wARP for automated water building and free atom density modification. (Lamzin V S & Wilson K S (1993) Automated refinement of protein models. Acta Crystallogr, Sect D 49:129-147.) The structure of the 1+3 complex was solved using a search model derived from a published gp41 hexamer structure (PDB ID: 1AIK). Chan D C, Fass D, Berger J M, & Kim P S (1997) Core structure of gp41 from the HIV envelope glycoprotein. Cell 89(2):263-273. The structure of α/β-peptide 10 was solved using a CHR helix from the 1+3 complex as a search model. The structure of the 1+10 complex was solved using two search models, an NHR helix from the 1+3 complex and a CHR helix from the structure of α/β-peptide 10 alone. The structure of the 1+8 complex was solved using two search models, an NHR helix from the 1+3 complex and a chimeric CHR helix prepared from the structures of 1+3 and 1+10. Molecular graphics were prepared using PyMOL (DeLano Scientific, Palo Alto, Calif., USA).
[0113] Protease Stability.
[0114] Stock solutions of peptides were prepared at a concentration of 25 μM (based on UV absorbance) in TBS. A solution of proteinase K was prepared at a concentration of 50 μg/mL (based on weight to volume) in TBS. For each proteolysis reaction, 40 μL of peptide stock was mixed with 10 μL of proteinase K stock. The reaction was allowed to proceed at room temperature and quenched at the desired time point by addition of 100 μL of 1% TFA in water. 125 μL of the resulting quenched reaction was injected onto an analytical reverse phase HPLC and run on a gradient between 0.1% TFA in water and 0.1% TFA in acetonitrile. The amount of starting peptide present quantified by integration of the peak at 220 nm. Duplicate reactions were run for each time point. Half-lives were determined by fitting time dependent peptide concentration to an exponential decay using GraphPad Prism-brand software (GraphPad Software, Inc., La Jolla, Calif., USA). Crude samples for some time points were analyzed by MALDI-MS, and the products observed were used to identify amide bonds cleaved in the course of the reaction.
[0115] Expression, Purification, and Refolding of gp41-5.
[0116] The sequence of the gp41-5 construct used herein is below.
[0000]
(SEQ. ID. NO: 24)
MSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILSGGSGGWMEWDREINNYTSLIH
SLIEESQNQQEKNEQELLGGSGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL
SGGSGGWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLGGSGGSGIVQQQNNLLRAI
EAQQHLLQLTVWGIKQLQARIL
[0117] Expression, purification, and refolding of gp41-5 were carried out as previously described. Frey, G.; Rits-Volloch, S.; Zhang, X. Q.; Schooley, R. T.; Chen, B.; Harrison, S. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13938-13943. A sample of the gp41-5 plasmid, provided by Prof. Stephen Harrison (Harvard University), was transfected into E. coli cells (Rosetta™ strain, Novagen) by electroporation. A single colony was grown overnight in 20 mL LB supplemented with 50 μg/mL ampicillin (resistance provided by the gp41-5 plasmid) and 30 μg/mL chloramphenicol (resistance provided by the plasmid for rare tRNAs included in the Rosetta™ strain). 500 mL of antibiotic supplemented LB was inoculated with 5 mL of the overnight starter culture. Cells were grown at 37° C. to an OD 600 of 0.75 and subsequently induced by addition of IPTG to a final concentration of 1 mM. The cells were grown for an additional 3 hr at 37° C., and then centrifuged at 12,000 g for 15 min at 4° C. The cell pellet was dissolved in ice cold glacial acetic acid and left on ice for 45 min with periodic agitation. The suspension was centrifuged at 39,000 g for min at 4° C. The supernatant was decanted and lyophilized. The crude protein was purified by preparative HPLC on a C 18 column eluted by a gradient between 0.1% TFA in water and 0.1% TFA in acetonitrile. Purified protein was lyophilized and stored at −40° C. until refolding. For refolding, purified gp41-5 (˜2 mg) was dissolved in 10 mL of 6 M guanidinium chloride. The resulting solution was dialyzed at room temperature against 0.1 M glyicne pH 3.6 (1×) followed by PBS (2×). Precipitate was removed by centrifugation and the resulting protein used without further purification.
[0118] Fluorescence Polarization.
[0119] Fluorescence polarization assays were conducted at room temperature in black polystyrene plates. All measurements were performed in duplicate wells. The assay buffer was composed of 20 mM phosphate, pH 7.4, 1 mM EDTA, 50 mM NaCl, 0.2 mM NaN 3 , 0.5 mg/mL “Pluronic F-68”-brand polyoxyalkylene ether surfactant. The binding affinity of Flu-C38 for gp41-5 was measured by titrating a fixed concentration of the labeled peptide (0.2 nM) with increasing concentrations of protein in 384-well plates with a final volume of 50 μL per well in assay buffer with 1% v/v DMSO (added to mimic the conditions of the competition FP experiments). All wells were run in duplicate. The plate was allowed to equilibrate for 30 min and analyzed on an Envision 2100 plate reader. The data were fit using Graphpad Prism software (Graphpad Software Inc., La Jolla, Calif.) to a FP direct-binding model. Roehrl, M. H. A.; Wang, J. Y.; Wagner, G. Biochemistry 2004, 43, 16056-16066. The K d of the tracer was determined to be 0.4±0.1 nM. The binding affinity measured is somewhat tighter than that previously reported for the gp41-5/Flu-CHR interaction (K d =3 nM), but the previous study utilized a much higher concentration of tracer in the direct binding experiment (5 nM). The lower limit of a K d value that can be accurately determined in a direct binding FP experiment is roughly equal to the concentration of tracer employed. Roehrl, Wang, & Wagner, supra.
[0120] Competition fluorescence polarization assays were conducted in black 96-well plates. A solution of 2 nM gp41-5, 1 nM Flu-C38 was prepared in FP assay buffer and arrayed into a 96-well plate (100 μL/well). A second stock plate was prepared containing serial dilutions of peptide inhibitors in DMSO. The peptide stock solutions were transferred to the assay plate (1 μL per well). Each assay plate also included 4 wells each of the following three controls: (1) 100 μL assay buffer+1 μL DMSO; (2) 100 μL of 1 nM Flu-C38+1 μL DMSO as an unbound tracer control; (3) 100 μL of the 2 nM protein/1 nM tracer solution+1 μL DMSO as a bound tracer control. All experimental conditions were carried out in duplicate, and each peptide was assayed in 2-3 independent experiments. Data analysis was carried out in GraphPad Prism. Raw mP data from each experiment were fit to a sigmoidal dose response and normalized to the resulting parameters for the top and bottom of the curve. All experiments included at least one compound showing complete inhibition at the highest concentrations tested. Normalized data from multiple independent runs of the each oligomer were combined and globally fit to an exact analytical expressions for FP competitive binding with K i as the only floating parameter. The lower bound for K i measurable in the competition FP experiment was considered to be 0.2 nM, based on the K d of the tracer. See Roehrl, Wang, & Wagner, supra.
[0121] Circular Dichroism Spectroscopy.
[0122] Circular dichroism measurements were carried out on an Aviv 202SF Circular Dichroism Spectrophotometer. Samples of each peptide were prepared at 20 μM concentration in PBS. Solutions of 1:1 peptide mixtures were prepared by mixing equal volumes from the same 20 μM stock solutions used for individual peptide measurements. Spectra were recorded in a 1 mm cell with a step size of 1 nm and an averaging time of 5 sec. All spectra are background corrected against buffer measured in the same cell. Thermal melts were carried out in 5-degree increments with an equilibration time of 10 min between each temperature change. Thermal unfolding data were fit to a simple two state folding model Shortle, D.; Meeker, A. K.; Freire, E. Biochemistry 1988, 27, 4761-4768) using GraphPad Prism.
[0123] Protease Stability.
[0124] Stock solutions of the peptides were prepared at a concentration of 25 uM (based on UV absorbance) in TBS. A solution of proteinase K was prepared at a concentration of 50 μg/mL (based on weight to volume) in TBS. For each proteolysis reaction, 40 μL of peptide stock was mixed with 10 μL of proteinase K stock. The reaction was allowed to proceed at room temperature and quenched at the desired time point by addition of 100 μL of 1% TFA in water. 125 μL of the resulting quenched reaction was injected onto an analytical reverse phase HPLC, and the amount of starting peptide present quantified by integration of the peak at 220 nm. Duplicate reactions were run for each time point. Half-lives were determined by fitting time dependent peptide concentration to an exponential decay using GraphPad Prism. Crude samples for some time points were analyzed by MALDI-MS, and the products observed were used to identify amide bonds cleaved in the course of the reaction.
[0125] Antiviral Assays.
[0126] A cell-to-cell-fusion assay based on the envelope glycoprotein of the HIV-1 clone HXB2 expressed in CHO cells and with U373-MAGI cells as targets was carried out as previously described. (Deng Y Q, Zheng Q, Ketas T J, Moore J P, & Lu M (2007) Protein design of a bacterially expressed HIV-1 gp41 fusion inhibitor. Biochemistry 46(14):4360-4369.) All the α/β peptides showed no cytotoxicity at 5 μM, as judged by measuring the basal level of β-galactosidase expression in the U373-MAGI target cells. Inhibition of HIV-1 infectivity was measured on TZM-bl (JC53BL) cells, which express CD4, CXCR4, CCR5 and the luciferase gene under the control of HIV-1 LTR (long terminal repeat). (Wei X P, et al. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46(6):1896-1905.) Viral stocks produced in PBMC of four HIV-1 strains were used: NL4-3, a clone derived from the X4-tropic T-cell line-adapted isolate IIIB of clade B; HC4, an X4 primary isolate of clade B (Trkola A, et al. (1998) Neutralization sensitivity of human immunodeficiency virus type 1 primary isolates to antibodies and CD4-based reagents is independent of coreceptor usage. J Virol 72(3):1876-1885); an R5 primary isolate, CC 1/85 (clade B) (Connor R I, Sheridan K E, Ceradini D, Choe S, & Landau N R (1997) Change in coreceptor use correlates with disease progression in HIV-1-infected individuals. J Exp Med 185(4):621-628); and another R5 primary isolate, DJ258 (clade A) (Louwagie J, et al. (1995) Genetic diversity of the envelope glycoprotein from human immunodeficiency virus type-1 isolates of African origin. J Virol 69(1):263-271).
[0127] Briefly, TZM-bl cells were seeded the day before inoculation at a density of 10 5 cells/ml, 100 μl/well. Serially diluted peptide in 50 μl (or medium alone as a control) was added to each well. Then the virus, 40 TCID 50 in 50 μl, or medium only as a background control, was added to each well. On the third day, the wells were inspected by light microscopy. Wells with and without peptide were compared for cell confluency and morphology. No signs of toxicity were discerned at the highest concentrations of peptide used. The infectivity was then quantified in relative light units with the Bright-Glo Luciferase Assay System (Promega Corporation, Madison, Wis., USA), according to the manufacturer's instructions. The experiment was performed three times. The signal of test wells was normalized to that of control wells without inhibitor after background subtraction from both. The % inhibition of infectivity was expressed as a function of the log 10 concentration of inhibitor in nM. A four-parameter sigmoid function was fitted to the data in Prism (Graphpad). The R 2 values for the fits were 0.95-1.0 for NL4-3; 0.98-1.0 for HC4; 0.95-0.98 for CC 1/85; and 0.92-0.98 for DJ258. Finally, the means±S.E.M. of the IC 50 values from the individual fits of the three repeat experiments were calculated. The results are depicted graphically in FIGS. 10A (NL4-3), 10 B (CC1/85), 10 C (HC4), and 10 D (DJ258).
[0128] Sequence-Based Design of α/β-Peptides that Mimic BH3 Domains:
[0129] As noted above, designing molecules that bind tightly and selectively to a specific site on a protein constitutes a fundamental challenge in molecular recognition. Thus, a systematic approach for identifying suitable molecules would be a distinct advantage. This Example is presented to show that systematic backbone modification throughout a natural protein-binding domain (i.e., sequence-based design) can be used to expeditiously generate α/β-peptide foldamers that bind tightly and selectively to target protein surfaces. In this Example, the sequence-based design approach was used to develop α/β-peptide foldamer ligands for the BH3-recognition cleft of the protein Bcl-x L . Bcl-x L . is a member of the Bcl-2 family, which controls programmed cell death pathways and includes both anti-apoptotic members (e.g., Bcl-2, Bcl-x L , Mcl-1) and pro-apoptotic members (e.g., Bak, Bad, Puma). See Adams & Cory (2007) Oncogene 26:1324-1337.
[0130] This Example describes a sequence-based design of α/β-peptide ligands for BH3-recognition clefts that differs fundamentally from the structure-based design approaches to foldamer ligands previously pursued by the present inventors and others. The approach involves replacing subsets of regularly spaced α-residues with β-residues bearing the original side chains. Each α to β replacement introduces an extra methylene unit into the backbone. This sequence-based approach does not directly aim to recapitulate the folded structure of an α-peptide prototype, although conformational mimicry is achieved as a byproduct of the replacement strategy employed. As shown in the earlier Example, it has been demonstrated that sequence-based design can be used to generate helix-bundle foldamer quaternary structure from an α-peptide prototype. In this Example, the method is used to mimic the protein-binding behavior of an α-helical BH3 domain. The results demonstrate that sequence-based design is more efficient than structure-based design for generating foldamers that bind tightly to the anti-apoptotic Bcl-2 family proteins, and that sequence-based design can deliver α/β-peptides that display significant resistance to proteolytic degradation.
[0131] Puma is a Bcl-2 homolog that binds promiscuously to anti-apoptotic family members. See Chen et al. (2005) Mol. Cell. 17:393-403. A 26-residue α-peptide corresponding to the Puma BH3 domain (1′) was prepared, along with seven α/β-peptide analogues (2′-8′) with the same primary sequence of side chains displayed on different α/β-peptide backbones. See FIG. 7A . Each α/β-peptide contained an ααβαααβ backbone repeat which was derived from the heptad pattern common among α-peptide sequences that form α-helices with a well-developed “stripe” of hydrophobic side chains running along one side. See FIG. 7B . Recent crystal structures demonstrate that the ααβαααβ backbone allows formation of an α-helix-like conformation. See Home, Price, Keck, & Gellman (2007) J. Am. Chem. Soc. 129:4178-4180. α/β-Peptides 2′-8′ represent all possible isomers of the Puma BH3 sequence with the ααβαααβ backbone pattern. These oligomers can be viewed as a series of analogs of Puma in which a band of β-residues moves around the helical periphery. See FIG. 7C .
[0132] Compounds 1′-8′ were tested for their ability to bind to two distinct Bcl-2 family targets, Bcl-xL and Mcl-1. Inhibition constants (K i for each compound were determined by competition fluorescence polarization (FP) assays (see FIG. 8 ) with a fluorescently labeled Bak-BH3 peptide as the tracer. The Puma-BH3 peptide (1′) showed affinities for Bcl-xL and Mcl-1 that are tighter than can be measured with these FP assays, which is consistent with previous work. K i values for α/β-peptides 2′-8′ vary from less than 1 nM to greater than 100 μM. Variation in the position of β-residue incorporation causes considerable changes in affinity for each protein: greater than 100,000-fold for Bcl-x L and greater than 700-fold for Mcl-1.
[0133] For both protein targets, 4′ is the tightest-binding foldamer, with K i <1 nM for Bcl-x L and K i =150 nM for Mcl-1. It is noteworthy that α/β-peptide 5′, which contains β-modifications at critical hydrophobic residues in the Puma BH3 sequence, shows nanomolar affinity for Bcl-x L . These data demonstrate that the location of β-residue incorporation strongly influences Bcl-x L versus Mcl-1 selectivity among the Puma-derived α/β-peptide isomers, in addition to affinity for these protein targets. For example, 3′ shows equal affinity for the two proteins, but 5′ displays greater than 4000-fold selectivity for Bcl-x L over Mcl-1. The validity of the conclusions regarding affinity and selectivity derived from the FP competition assays were tested for α-peptide 1′ and α/β-peptides 4′ and 5′ by performing direct-binding FP measurements with analogs in which the N-terminal acetyl group is replaced with a BODIPY-TMR fluorophore. The K d values determined by direct binding were consistent with the K i , values obtained from competition data (see Table 5). The differences in absolute values of K d versus K i may reflect modest contributions of the appended fluorophore to affinity as measured in the direct binding mode.
[0000]
TABLE 5
Binding affinity and protease stability data for
α-peptide 1′ and α/β-peptides 4′, 5′.
K 1 [nM] [a]
K d [nM] [b]
t 1/2 [min] [c]
Bcl-x L
Mcl-1
Bcl-x L
Mcl-1
Prot. K
Pronase
1′
<1
<10
<1
<2
0.7
1
2′
<1
150
2.2
110
>3000
100
3′
2.4
11000
1
1100
170
3.5
[a] Inhibition constants determined by competition FP.
[b] Dissociation constants of BODIPY-labeled analogues determined by direct binding FP.
[c] Measured half-life of a 50 μm solution of α-peptide or α/β-peptide in the presence of 10 μg mL −1 proteinase K or 5 μg mL −1 pronase.
[0134] Having established that certain α/β-analogs of the Puma BH3 domain can bind with high affinity to the natural protein partners, an experiment was performed to determine whether the α/β-peptides would be recognized and processed by proteolytic enzymes. α-Peptide 1′ and α/β-peptides 4′ and 5′ were tested for their susceptibility to two proteases with broad substrate profiles: (1) proteinase K, a non-specific serine protease that tends to cleave C-terminal to hydrophobic residues, and (2) “PRONASE”-brand proteinase, a mixture of aggressive endopeptidases and exopeptidases that digests proteins into individual amino acids. (“PRONASE” is a registered trademark of EMD Chemicals, Inc., Gibbstown, N.J.) The results, presented in Table 3, show that the ααβαααβ backbone can confer substantial resistance to proteolytic degradation. α/β-Peptide 4′, which binds tightly to both Bcl-x L and Mcl-1, showed a greater than 4000-fold improvement in stability to proteinase K and a 100-fold improvement in stability to “PRONASE”-brand proteinase relative to α-peptide 1′. Analysis of the cleavage products by mass spectrometry indicated that the β-residues tend to protect nearby amide groups from proteolysis, which is consistent with previous reports for isolated α to β 3 insertions. α/β-Peptide 5′ is more susceptible than is isomer 4′ to proteolytic degradation, but 5′ nevertheless shows significant improvement relative to α-peptide 1′.
[0135] Previous work has suggested that the α-helical propensity of BH3-derived α-peptides may be an important determinant of affinity for anti-apoptotic Bcl-2 family proteins. Circular dichroism (CD) spectroscopy was therefore employed to probe for conformational differences among two of the tight-binding α/β-peptides (4′ and 5′) and one of the weakest binding analogs (7′) described in this Example. Qualitative comparison of CD spectra for 4′, 5′, and 7′ indicates that the large differences in binding affinity among these three isomers cannot be explained by differences in helical propensity. Each of these three α/β-peptides shows a CD minimum at approximately 202 nm with per-residue ellipticity between −13,000 and −15,000 deg cm 2 dmol −1 in aqueous solution. Helix formation in the ααβαααβ backbone is reflected by a strong CD minimum at 206 nm with a maximum magnitude of approximately −40,000 deg cm 2 dmol −1 . Thus, the CD data for 4′, 5′, and 7′ alone in aqueous solution suggest relatively low population of the helical state. Similarly, the CD signature for Puma α-peptide 1 in aqueous solution ([θ] 222 =−10,000 deg cm 2 dmol −1 res −1 ) suggests little α-helical content. Without being limited to any specific mechanism, on the basis of the established precedent for induction of α-helix formation upon binding of BH3 domain α-peptides to Bcl-x L and Mcl-1, the co-inventors hypothesize that α/β peptides such as 4′ and 5′ are induced to adopt helical conformations upon binding to protein partners.
[0136] The work reported herein demonstrates that a straightforward principle of sequence-based design can be used to convert a helical α-peptide ligand into an α/β-peptide with comparable binding affinity for protein targets and substantially improved proteolytic stability. The strategy disclosed and claimed herein is a fundamental departure from previous work on the development of foldamer-based inhibitors of protein-protein interactions. The sequence-based approach disclosed herein has been shown by these Examples to be more efficient than the structure-based approach for generating foldamer mimics of α-helices.
[0137] In short, evaluating a series of just seven α/β-peptides designed purely on the basis of primary sequence information led to a compound that rivals the best of the previously described chimeric α/β+α ligands in binding affinity for Bcl-x L . See Sadowsky, Schmitt, Lee, Umezawa, Wang, Tomita, and Gellman (2005) J. Am. Chem. Soc 127:11966-11968; Sadowsky, Fairlie, Hadley, Lee, Umezawa, Nikolovska-Coleska, Wang, Huang, Tomita, and Gellman (2007) J. Am. Chem. Soc. 129:139-154; and Sadowsky, Murray, Tomita, and Gellman (2007) Chem Bio Chem 8:903-916. Moreover, the best α/β-peptide binds moderately well to Mcl-1, a biomedically important Bcl-2 family protein that is not targeted by oligomers identified through structure-based design. The implementation of multiple and systematic α-residue to β-residue replacements throughout a peptide sequence (7 of 26 positions substituted in the Puma BH3 domain) constitutes a significant advance beyond earlier precedents in the design of bioactive, proteolytically stable oligomers. The finding that one version of this substitution pattern is well-tolerated in terms of binding to anti-apoptotic proteins is surprising and noteworthy.
[0138] The sequence-based design illustrated herein can be implemented with commercially available α- and β-amino acid monomers and standard automated peptide synthesis methods. Thus, it is straightforward for others to undertake analogous efforts.
[0139] Comparisons of Chimeric α+α/β Foldamers:
[0140] Peptides 12, 13, and 8, below are chimeric α+α/β foldamers of a lead α/β foldamer 10. These peptides were created to determine the effect of beta substitution in the region near the N terminus. The beta residues were sequentially subtracted in the “f” and “c” positions along the heptad. The effect of α to β substitutions was monitored with a previously reported Fluorescence Polarization (FP) competition assay. (Frey, G.; Rits-Volloch, S.; Zhang, X. Q.; Schooley, R. T.; Chen, B.; Harrison, S. C. Small molecules that bind the inner core of gp41 and inhibit HIV envelope-mediated fusion. Proc. Natl. Acad. Sci., 2006, 103, 13938-43.) The results suggest that β substitution has a slow, cumulative effect of decreasing the binding.
[0000] Chimeric α+α/β Foldamers, Subtracting β Residues from the “f” and “c” Positions Near the N-Terminus:
[0000]
fgabcdefgabcdefg . . .
(SEQ. ID. NO: 10)
10: Ac- T TWE X WD Z AIA E YA X RIE X LI Z AAQ E QQ E KNE X AL Z EL-NH 2
(SEQ. ID. NO: 25)
12: Ac-TTWEAWD Z AIA E YA X RIE X LI Z AAQ E QQ E KNE X AL Z EL-NH 2
(SEQ. ID. NO: 26)
13: Ac-TTWEAWDRAIA E YA X RIE X LI Z AAQ E QQ E KNE X AL Z EL-NH 2
(SEQ. ID. NO: 8)
8: Ac-TTWEAWDRAIAEYA X RIE X LI Z AAQ E QQ E KNE X AL Z EL-NH 2
[0000]
[0141] Bold, underline residues,
[0000]
K i (nM):
compound 10=9
compound 12=8
compound 13=0.8
compound 8=0.3
[0147] To determine if β substitution disrupted binding in one region of the peptide, α+α/β chimeric peptides were synthesized with different alpha segments substituted in the beta stripe. The regions of focus were near the N terminus 8, middle 14, and C terminus of the peptide 15. The FP data showed that introducing an alpha segment did increase binding of the foldamer; however, the K i 's were all very similar, which suggested that β substitution slowly disrupted the binding across the entire length of the helix and not in a particular region.
Chimeric α+α/β Foldamers, Substitution of a Segments in the N-Terminal, Middle, and C-Terminal Regions:
[0148]
[0000]
(SEQ. ID. NO: 8)
8: Ac-TTWEAWDRAIAEYA X RIE X LI Z AAQ E QQ E KNE X AL Z EL-NH 2
(SEQ. ID. NO: 27)
14: Ac- T TWE X WD Z AIA E YAARIEALIRAAQ E QQ E KNE X AL Z EL-NH 2
(SEQ. ID. NO: 28)
15: Ac- T TWE X WD Z AIA E YA X RIE X LI Z AAQEQQEKNEAALREL-NH 2
K i (nM):
compound 8=0.3
compound 14=1.4
compound 15=0.2
[0153] Foldamer 10 showed that cyclic residues effectively constrained the Cα-Cβ torsional angles to aid in folding, but other tactics could be used to constrain a helix. Salt bridges of α residues were effective at pre-forming α helices. See Nishikawa, H.; Nakamura, S.; Kodama, E.; Ito, S.; Kajiwara, K.; Izumi, K.; Sakagami, Y.; Oishi, S.; Ohkubo, T.; Kobayashi, Y.; Otaka, A.; Fujii, N.; Matsuoka, M. Electrostatically constrained alpha-helical peptide inhibits replication of HIV-1 resistant to enfuvirtide. Int. J. Biochem. Cell Biol. 2009, 41, 891-9. Another design strategy positioned a stripe of arginines in the i position which interacted with a stripe of glutamates in the i+4 position, favoring an a helical structure. See Burkhard, P.; Meier, M.; Lustig, A. Design of a minimal protein oligomerization domain by a structural approach. Prot. Sci., 2000, 9, 2294-2301.
[0154] The following peptide 17 examined the ability of beta residues to form salt bridges that pre-organize a helix. Because it was previously found that the “f” and “c” positions were the most compliant with beta substitution, β-hArg was placed in the “f” position and β-hGlu was placed in the “c” position to maximize i and i+4 interactions. Peptide 16 was created to test if both cyclic beta residues and salt bridging beta residues worked synergistically in the beta stripe. The FP data suggested that α/β foldamers 16 and 17 were approximately equal inhibitors to foldamer 1.
[0000]
fgabcdefgabcdefg . . .
(SEQ. ID. NO: 10)
10: Ac- T TWE X WD Z AIA E YA X RIE X LI Z AAQ E QQ E KNE X AL Z EL-NH 2
(SEQ. ID. NO: 29)
16: Ac- R TWE E WD R AIA E YA X RIE X LI Z AAQ X QQ Z KNE X AL Z EL-NH 2
(SEQ. ID. NO: 30)
17: Ac- R TWE E WD R AIA E YA R RIE E LI R AAQ E QQ R KNE E AL R EL-NH 2
K i (nM):
compound 10=9
compound 16=3
compound 17=11
These results are significant in that compound 17 does not contain any cyclically constrained residues. While not being limited to any underlying mechanism or phenomenon, it appears that conformational stability is achieved by incorporating ion pairs along one side of the helical conformation.
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Described is a method of fabricating biologically active, unnatural polypeptides. The method includes the steps of selecting a biologically active polypeptide or biologically active fragment thereof having an amino acid sequence comprising α-amino acid residues, and fabricating a synthetic polypeptide that has an amino acid sequence that corresponds to the sequence of the biologically active polypeptide, but wherein about 14% to about 50% of the α-amino acid residues found in the biologically active polypeptide or fragment of step (a) are replaced with β-amino acid residues, and the α-amino acid residues are distributed in a repeating pattern.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a packaging article and corresponding method of manufacture.
2. Description of the Prior Art
U.S. Pat. No. 5,009,939 issued Apr. 23, 1991 to B. A. Goldberg describes a fluid tight packaging tray that is formed from machine laid paperboard. A pair of die-cut paperboard blanks are prefolded to a tray configuration and placed into a cooperative pair of blow molding cavities. A vacuum system within the respective cavities holds the blanks in place as a hot, extruded parison of polymer is positioned between the cavities. Sequentially, the paperboard blank lined cavities are closed upon the parison which is expanded by pressurized gas against the internal surface faces of the blanks. Here, the polymer is chilled to structurally unitize all the tray shaping panels of paperboard and to seal the tray walls with a continuous, fluid-tight barrier of polymer film.
The immediate product of a mold cavity pair is a singular, vessel enclosure having a circumferential band of unlaminated polymer. When the circumferential band is die-cut, the vessel enclosure opens into two completed trays, each having a stiff, tough, exterior paperboard cladding printed with sharp, bright, press-applied graphics.
Although the aforedescribed Goldberg tray and method of manufacture represents a hallmark in consumer packaging development, the further refinement of partitioning the tray volume within the perimeter walls has, until now, proven elusive. It has been the object of the present invention, therefore, to provide a planar, paperboard blank configuration, that, when folded and placed into a blow mold cavity set will produce a partitioned tray i.e., a tray in which the bottom area is divided by upstanding walls into separate, fluid-tight pool areas.
SUMMARY OF THE INVENTION
These and other objects of the invention are accomplished by a paperboard blank configuration wherein the finished product bottom area is divided into the desired number of pool areas. At least two adjacent pool areas are separated by a scored and folded wall that is of less height than the full tray depth.
When folded into a blow molding cavity and internally lined with a continuous film of hot, pressure formed polymer, a structurally rigid, partitioned tray results.
DESCRIPTION OF THE DRAWINGS
Relative to the drawings wherein like reference characters designate like or similar elements throughout the several drawing figures:
FIG. 1 is a pictorial view of the present invention tray product divided into two pools with a single, internal wall.
FIG. 2 is a sectional detail from FIG. 1 showing the folded internal wall intersection with an external wall.
FIG. 3 is a cut and score plan for the paperboard blank used to form the tray product of FIG. 1.
FIG. 4 is a plan view of the present invention tray product divided by three separate walls into three distinct pool areas.
FIG. 5 is a cut and score plan for the paperboard blank used to form the tray product of FIG. 4;
FIGS. 6 through 10 each represent respective stages of the blow molding operation relevant to the present invention, and;
FIG. 11 illustrates a trimming operation performed on the blow mold raw product.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The basic substrate material for the present invention is machine laid paperboard which may range in thickness from 0.007 to 0.035 inches. In some cases it may be desirable to coat the paperboard web stock on that web side to be formed to the tray interior with a film of extruded or press applied polymer selected on the basis of chemical and adhesive receptivity to the subsequently applied parison polymer. However, due to the fact that extruded parison polymers of the type and nature anticipated by the present invention are applied to the paperboard blank mold liner at such temperature, viscosity and pressure, many tie films on paperboard substrates become unnecessary. The parison bonds deeply and securely to the untreated paper surface.
Those of ordinary skill in the art understand the economic value to consumer packaging arising from the quality of graphics that may be press applied to a clay coated paperboard web. Moreover, the web may be printed and die cut into individual tray blanks in one continuous machine fed operation. Resultantly, display shelf graphics may be directly applied to the exterior side of a package wall that is in direct contact with the package contents on the opposite wall side. No outer packaging or post-fill labeling is required. These properties and consequences of paperboard as a packaging medium are distinct from and uniquely valuable over molded packaging, whether of solid plastic or pressed cellulosic pulp.
Proceeding from the foregoing understandings, FIG. 1 pictorially illustrates a finished form of the most simply executed embodiment of the present invention. Tray 10 is of nominally rectangular finished platform within the outer perimeter walls 11, 12, 13, 14, 15 and 16. An internal wall 17 divides the rectangular perimeter bottom into two smaller pool bottom areas 19 and 20.
Flanges 21, 22, 23, 24, 25 and 26 are integral with and turn outwardly from the plane of respective outer perimeter walls. The perimeter wall plane is set at a small angle, 10° for example, of departure from normal to the bottom panels 19 and 20 for the purpose of facilitating erected tray nesting.
The flanges 21 through 26 serve to structurally rigidify the upper edges of the outer walls 11 through 16 and to secure by thermal fusion a polymer coated tray cover panel 29 shown in phantom line.
Referring now to the sectional elevation of FIG. 2 which relates to the Detail II area of FIG. 1, and to the blank plan of FIG. 3, it is seen that the internal wall 17 comprises two panel sections 17a and 17b which are integral material continuations of bottom panels 19 and 20. Although the outer side walls 11 and 16 are also integral material continuations of bottom panels 19 and 20, the juncture 18 above the wall 17 pleat ridge is a discontinuous butt joint of the paperboard walls il and 16. Such butt joint is lapped by the blow parison polymer film 28. This detail is repeated on the opposite side of the tray between outer walls 13 and 14.
Functionally, the joint 18 is critical to the tray rigidity. It will be noted that if interior wall 17 is made the same height as the exterior walls 11 or 16, the wall 17 ridge would function as a hinge between pool areas 19 and 20. Such structural configuration may be useful to form a clamshell package having pool area 19 identical to pool areas 20 and one serving as the hinged lid for the other.
However, in the present invention the tensile strength of the polymer film 28 lapping the butt joint 18 and the planar compression strength of the paperboard combine to rigidify the joint. When combined with a top 29 that is heat fused around the entire flange perimeter, the configuration offers outstanding market place stress resistance.
Turning now to the more complex embodiment of the invention represented by FIGS. 4 and 5, the finished tray product 50 is a nominal rectangle having truncated corners and a bottom that is divided into three areas 51, 52 and 53 by interior walls 54, 55 and 56. Exterior perimeter walls 60, 61, 62, 63 and 70 are integral continuations of the bottom panel 51 delineated by respective score/fold lines. Similarly, exterior perimeter walls 64, 65 and 66 are integral continuations of the bottom panel 52. Exterior perimeter walls 67, 68, and 69 are integral continuations of bottom panel 53.
Interior wall panels 54a and 54b (FIG. 5) integrate bottom panels 51 and 52 and interior wall panels 56a and 56b integrate bottom panels 52 and 53. However, interior wall panels 55a and 55b are discontinuous between bottom panels 51 and 53.
Seven top flange areas 71 through 77 are integral extensions of outer perimeter wall panels 61, 63, 64, 66, 67, 69, and 70, respectively.
Included among the several characteristics common to all embodiments of the invention is the absence of lapped panels. Respective edges of adjacent panels fold to contiguous alignment with no overlap. Selective prefolding prior to placement of a cut blank into a mold cavity and vacuum held positionment within the mold cavity maintains such precise edge alignment until a blown parison film is chilled against the interior blank surface.
Another characteristic common to two of the embodiments includes an interior partition wall height that is less than the tray depth at the outer perimeter walls and a polymer film lapped butt joint of outer wall edges extending upwardly to the outer flange edge from the interior wall ridge apex.
Continuing with the invention description and the blow molded application of a polymer film to the paperboard blank of the FIG. 3 configuration. FIG. 6 illustrates a blank positioned in each of a pair of blow mold cavity halves 81 and 82. These cavity halves are mechanically linked to reciprocate from an open position represented by FIGS. 6, 7 and 10 to a closed position represented by FIGS. 8 and 9. At one end of the mold halves, the product cavities open into a plenum section 84 configured to confine an inflation bulb. Mold half 81 is also provided with a hollow inflating needle 85.
Both mold halves are provided with vacuum conduits 86 having orifices 87 opening into the mold cavities. This vacuum system secures the position of an erected tray blank in each mold cavity prior to film application; these tray blanks being placed within the respective cavities while the mold unit is open as represented by FIG. 6.
Also while the mold unit is open, a tubular length of melted polymer material, known to the art as a parison 90, is extruded between the open mold halves as shown by FIG. 7 at a temperature within the range of 250° F. to 600° F. More descriptively, the parison 90 is a continuous, vertically hanging extrusion around which the wheel mounted open mold pairs are positioned tangentially.
With the tray banks and parison 90 in place, the mold halves 81 and 82 are closed upon the parison 90 as represented by FIG. 8 thereby sealing the upper end of the parison along a fused seam 92. The lower or distal end of the parison 90 is sealed along seam 93 by the same mold closure movement.
Closure of the mold halves 81 and 82 also pushes the inflation needle 85 through the parison wall film of inflation bulb 91. In this condition, a charge of compressed air or other gas, in the order of 5 to 90 psi, is released through the inflation needle 85 into the inflation bulb 91 and, consequently, into the closed interior of parison 90. Such pressure within the parison 90 expands the hot, malleable polymer tube tightly against the mold cavity walls and inner surfaces of the tray blank as shown by FIG. 9.
Following a brief chilling interval, the two mold halves 81 and 82 are separated as represented by FIG. 10 leaving the two tray blanks securely bonded to the inflated parison 90 as a singular unit 100. This unit 100 is then separated from the extruded parison continuity by a cut 94 across the fused seam 92.
At this point in the process, unit 100 represents two semifinished trays joined by a continuous, unlaminated band 95 of polymer which includes the inflation bulb 91.
Following severance of the parison, the segregated unit 100 is placed upon the anvil element 31 of a cutting die 30 (FIG. 11). As shown by FIG. 11, striker element 32 engages the underside of the first tray flange area and presses it against the upper face of the second tray flange area. Held at this position by die 30, the excess polymer material represented by band 95 may be trimmed by a shear 34.
Although the extruded parison 90 has been generally described as a homogenous polymer material, which it may be, it should be understood that the invention is not so limited. The melted polymer extrusion art is capable of extruding multiple layers of diverse polymers in a single parison flow stream. Consequently, film 28 composites may be designed to include several different compound layers, each selected on the basis of maximum barrier properties and functions for a specific gas or combination of gases.
Having fully described the preferred embodiments of my invention,
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A composite food packaging article having internal partitioning walls and corresponding method of manufacture is constructed from flat, machine formed paperboard. A pair of die cut and folded paperboard blanks are inserted within the opposing cavities of a blow mold. A hot-flow parison between the cavities expands to structurally unitize the folded paper blank into a fluid impermeable tray vessel.
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BACKGROUND OF THE INVENTION
[0001] The subject invention relates to organizing structures for the truck beds of pick-up trucks. More particularly this invention is directed to a collapsible box that usually can be fastened to an upstanding wall of a pick-up truck bed. Most truck bed organizing structures, called truck boxes, tend to be large monolithic structures made of welded aluminum or injection molded structural foam. One problem with such boxes is that they tend to block much of the storage space in the truck bed by occupying usually the front two or three feet of the truck bed volume. Some truck boxes mitigate this blocking phenomenon by providing a so-called “cross bed” construction. A cross bed box is shallow so that it bridges across the truck bed since it is supported on either end by a protruding flange that rests on the upwardly facing surface of the sides of the truck bed. In this way the space beneath the cross bed box is freed for plywood sheets or other long, flat items. Such cross bed boxes are consequently quite shallow, even when they are built to project a substantial distance above the sides of the truck. Truck bed boxes tend to be quite heavy since they must structurally span the full width of the truck bed to keep the space below the box unrestricted.
[0002] Accordingly, it is an object of the subject invention to provide a truck box that permits a full depth box to collapse into a narrow compact stack against one wall of the truck bed, preferably the wall between the truck bed and the cab. It is another object of the invention to provide a truck box with bottom side walls and a lid that provides reasonable security and protection for the goods within the box, yet the box includes walls with hinged edges and surfaces that permit the truck box to selectively collapse into a narrow space within the truck bed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] [0003]FIG. 1 is a perspective view of the collapsible storage box in the collapsed position and positioned against the front end of a pick-up bed.
[0004] [0004]FIG. 2 is a perspective view of the lid of the collapsible storage box being lifted (pivoted) up to allow erection of the box from the collapsed condition.
[0005] [0005]FIG. 3 is another view of the lid being pivoted.
[0006] [0006]FIG. 4 shows the main compartment of the collapsible storage box being opened during erection.
[0007] [0007]FIG. 5 shows the floor of the collapsible storage box being moved into place for assembly forming a space for storage.
[0008] [0008]FIG. 6 shows the collapsible storage box fully erected with the lid open.
[0009] [0009]FIG. 7 shows a golf bag being placed in the storage formed by the fully erected storage box.
[0010] [0010]FIG. 8 shows the fully erected storage box with the lid in the closed position.
[0011] [0011]FIG. 9 is a section taken along line 9 - 9 of FIG. 8.
[0012] [0012]FIG. 10 is a section taken along line 10 - 10 of FIG. 1.
[0013] [0013]FIG. 11 is an alternative embodiment showing the box executed in patterned aluminum sheet.
[0014] [0014]FIG. 12 is another view of the second embodiment.
[0015] [0015]FIG. 13 shows the way the side walls fold in the second embodiment.
[0016] [0016]FIG. 14 shows the side walls and bottom wall in the erected second embodiment.
[0017] [0017]FIG. 15 shows the side walls, lid, front and back walls in the erected second embodiment.
[0018] [0018]FIG. 16 shows the hinged lip on the lid of the second embodiment.
[0019] [0019]FIG. 17 shows positioning the bracket on the truck bed wall.
[0020] [0020]FIG. 18 shows a pair of brackets positioned on the back wall of the truck bed ready to receive the collapsible truck bed box.
[0021] [0021]FIG. 19 shows the bracket positioned on the back wall of the truck box.
[0022] [0022]FIG. 20 shows the truck box and brackets, as they would appear installed in the truck bed.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0023] The instant invention pertains to a collapsible storage box in general, and particularly to a collapsible storage box suitable for use in a pick-up truck bed. While the description below focuses on the specific use of the box in a pick-up truck bed, it is contemplated that it could be used also in other settings, such as in a garage, car trunk, basement, and for general storage in virtually any location.
[0024] The first embodiment of the erected box, as shown in FIGS. 5, 6, 7 and 8 includes a front wall, a rear wall, opposing end walls, a bottom wall and a lid. Each of the walls is made of a lightweight, rigid material, such as plastic, metal or wood. It is important for the material to be resistant to the weather elements, and maintain operability (not be detrimentally affected by UV, salt, etc.) in extreme outdoor use conditions. For the purposes of this description, the uncollapsed or erected box is oriented so that the bottom wall preferably rests directly or indirectly horizontally on the truck bed support surface, the front, rear, and end walls extend substantially vertically from the bottom wall, and the lid rests horizontally on the top edges of the front, rear and end walls.
[0025] The front wall is pivotally attached along a pivot line at either end to a front edge of a respective end wall. Each of the ends of the front wall defines a rearwardly-extending flange (see FIGS. 4 and 5) to space the pivot line rearwardly from the back surface of the front wall. The flange allows enough space between the pivot line and the back surface of the front wall to allow the bottom wall to lay against the front wall without interfering with the folding of the end walls when the box is collapsed into its compact condition, as described below. The bottom edge of the front wall is pivotally attached to the front edge of the bottom wall. Alternatively the bottom wall could be pivotally attached to the bottom edge of the rear wall. The lock mechanism can alternatively be located on the lid so as to interact with a mating lock mechanism on the end wall(s). The front wall includes part of a lock mechanism which, when engaged with the mating lock mechanism on the front hinged lip of the lid (as shown in FIGS. 1 and 7), allows the lid to be locked closed if desired.
[0026] The rear wall is also pivotally attached at or near either end to the rear edge of a respective end wall. The lid is pivotally attached along a rear edge to the top edge of the rear wall. The top edge of the rear wall can define a horizontal plane, flange or rim, with the hinge being located at the front of the horizontal plane, flange or rim (See FIGS. 2, 6 and 7 ). The rear wall in the instant invention is positioned adjacent to and in possible contact with the front wall of the pick-up bed. The rear wall can be permanently fixed to the front wall of the pick-up bed by adhesives, through-bolts, or other such types of fasteners such as shown in FIG. 8. The rear wall can also be removably fixed to the front wall of the pick-up bed, such as by a hook and loop type fastener (such as with hook and loop type fastener systems) or other removable fasteners. This would allow the box to be securely positioned in the truck bed, or removed, as desired by the user.
[0027] The end walls each define a vertically extending pivot line, defined in the preferred embodiment by a hinge such as a piano hinge, located halfway along their length from front to back. The pivot line splits each end wall into a front section and a rear section, and allows the front and rear sections to pivot to a position where they are side-by-side (See FIG. 3). The hinge is oriented on the pivot line to cause the end walls to pivot inwardly into the interior of the box when collapsing the box from the erect condition.
[0028] The lid includes a main body and a front lip pivotally attached along a pivot line to the main body. The front lip bends from right angles to the main body (see FIGS. 4, 5, 6 , and 7 ) to extending in-line (substantially in a common plane with) with the lid main body (see FIGS. 1 and 2). A locking structure is positioned midway along the length of the lip. The locking structure works in conjunction with a first mating locking structure on the top edge of the front wall to selectively lock the lid when the box is in the erected condition. As mentioned before, the lock could be positioned elsewhere, and could also be made to work when the box is in the collapsed position. This is accomplished by providing a second mating lock structure, for example near the bottom edge of the front wall as shown in FIG. 3. The locking structure on the pivoting lip aligns with this second mating lock structure when the box is in its collapsed or stored position as shown in FIG. 1, for example.
[0029] In operation, the box is easily converted from the collapsed condition to the assembled condition. In the collapsed position and the assembled position the back wall remains in relatively the same position. The other walls pivot and move with respect to the back wall between the collapsed and assembled position.
[0030] In the collapsed position (see FIG. 1), the bottom wall is folded upwardly along its pivot line with the front wall to lay against the rear side of the front wall (see FIG. 4). The combination of the front and bottom walls lay adjacent to but not in contact with the front surface of the rear wall. The combination of the front wall and bottom wall is able to be in such a position because the end walls each bend inwardly along their respective center pivot lines (see FIG. 3). As mentioned above, the flange on either end of the front wall spaces the pivot line between the front wall and each end wall rearwardly to allow for the folded position of the bottom wall against the front wall without interfering with the end walls. Because each end wall is folded, the front portion and rear portion of each lay against each other.
[0031] As referenced earlier, the bottom wall could also pivotably attach to the bottom edge of the rear wall. However, pivotally attaching the bottom wall to the bottom edge of the front wall adds L-beam structure to the front wall, and also allows the user to more easily use one hand to collapse the front panel while keeping the bottom wall from undesirably falling down.
[0032] In the collapsed position, the front wall, bottom wall, and folded end walls are all stacked against the front surface of the rear wall. In this position these parts are under the rim formed along the top edge of the rear wall. The rim is dimensioned to receive these folded parts underneath it. The lid then folds downwardly over the other parts to encase them between the rear wall, rim and lid. The lid defines flanges extending downwardly from the side edges to somewhat envelope the folded parts when in the collapsed position. When folded down in the collapsed position, the lip on the lid extends in a common plane with the lid and helps cover the bottom edge of the front wall. The lip also adds L-beam structure to stiffen the lid in the uncollapsed or horizontal position. A second mating lock structure, like that used to engage the lock or locks on the lid when the box is erected, could be provided at or near the bottom edge of the front wall, so that the lip on the lid could be secured to hold the box in the collapsed position.
[0033] To convert the collapsed truck box to the assembled truck box, the lid, after unlocking the lock from the second mating lock structure, if provided, is pivoted upwardly out of the way, as shown in FIGS. 2 and 3. The front wall and bottom wall, still in their stacked configuration, are moved away from the rear wall, as shown in FIG. 4. This causes the end walls each to unfold along their pivot lines into substantially straight walls. See FIGS. 5 and 6. The bottom wall is then pivoted away from the front wall and into its horizontal position. In its horizontal position, the bottom wall mechanically interferes with and blocks the inward bending of the end walls about their pivot lines, thus keeping the box from accidentally collapsing. See FIG. 5. If the bottom wall is pivotally attached to the rear wall then these steps would be slightly altered accordingly.
[0034] The truck box is now in its assembled position and ready to receive any articles that fit into the recess formed by the walls. The lid closes over the top of the open box, and contacts the top edges of the front wall and both end walls to help keep out dirt and weather. The pivotal lip can now be turned down to embrace the top, front surface of the front wall, and the lock can be actuated to keep the lid closed and secure the articles placed in the box.
[0035] As shown, the instant invention can be utilized as a carrier inside of a pick-up bed. The truck box can be positioned against, and preferably affixed to, the front wall of the pick-up bed, and when in the collapsed position it takes up approximately 3-4 inches of space. When in the open position, the box extends rearwardly to about the front end of the wheel wells inside the truck bed (depending on the size of the truck bed and the truck box). A wheel, (not shown) could be provided near the juncture of the front wall and each of the side walls to help support the front wall when it is moved between its collapsed and expanded positions. Such wheels could also help the box ride over the initial sloping surfaces of the protruding wheel wells at the corners of the erected truck box.
[0036] Each of the walls is approximately ¼ to ½ inch in thickness if made of injection molded polymers or the like. The overall depth and width of the truck box is generally dictated by the height of the back wall of the truck box. The truck box is generally as tall as the front wall of the truck bed. Depth is defined as the distance from the closed lid to the bottom wall. Width is defined as the distance from the front wall to the back wall. Length is defined as the distance between the end walls. Assuming that the lid needs to cover the width of the truck box in the assembled condition, and the lid needs to extend generally vertically when in the collapsed position, the width and height of the truck box is dictated by the height of the back wall. If these two assumptions are not required, then the truck box can be virtually any size when in the assembled condition and still collapse to a significantly smaller size when not in use.
[0037] [0037]FIG. 9 is a section view through the truck box when in the assembled condition. FIG. 10 is a section view through the truck box when in the collapsed condition.
[0038] The truck box can include other features and still function in the intended manner. For instance, the bottom wall could be attached along its rear edge to the bottom edge of the rear wall. Further, the rear wall could be a little taller than the front wall, with the top edges of the end walls tapered to allow the lid to slant downwardly and drain any liquid toward the front wall. To further enhance the weatherproof capabilities, the edges that mate when in the assembled condition, as well as the hinges, can be sealed by some manner such as by weather stripping or other such suitable treatment.
[0039] Since the truck box can be attached to the front or side walls of the truck bed, it can be elevated a couple of inches above the truck bed to allow for storing things, such as long 2×4s, under the truck box. Further, the instant invention is believed to be the only such truck box with at least a floor, front wall and a lid that does not require fastening to the floor of the truck bed.
[0040] All pivot lines can be defined by piano hinges (continuous), or can be discrete hinges, living hinges, or any type of connection that allows the relative pivoting motion of two planar members with respect to one another. The piano hinge structure is preferred because it provides some structural rigidity to the storage box when in the assembled condition.
[0041] The instant invention has many advantages. There are no obtrusions on the floor of the truck box. The truck box folds to a collapsed position when desired by the user, and is in a vertical orientation when collapsed to avoid collecting water when not in use.
[0042] Alternatively, the end walls of the instant invention could be eliminated so the side walls of the truck box could be used to keep objects in the truck box when in the erected position. In this embodiment, at least one hinged link (brace) would need to be positioned to connect the front and rear walls. Since it is hinged it would allow the front and rear walls to collapse together. Likewise, the floor panel could be removed to use the bottom of the truck bed if desired.
[0043] FIGS. 11 , etc. show an alternative embodiment of the invention. Here, rather than using injection molded plastic or the like to construct the various walls, panels and hinges, these are made from metal, preferably from the common aluminum sheet used for similar truck boxes having fixed and thus non-expandable shapes. The structures and functions are essentially identical with that shown in the first embodiment. FIG. 13 shows a side wall of the second embodiment in a partially expanded or collapsed position. The front wall includes a flange that embraces the upper edge of the bottom wall, which is contained within the flange and the side flange in this partially collapsed position. These and other flanges are formed using a conventional metal brake, although die stamping could be used to form the flanges along the edges and other structurally enhancing ribs and the like in the major faces of the panels thus shaped. The side wall has a vertical flange carried by one of the mutually hinged portions of the side wall. This vertical flange helps stabilize the side wall in its fully erect position. Thus, the user's goods stored within the box will not tend to bow the side wall out thanks to this vertical flange which back stops the piano hinge positioned on the outside thereof. FIG. 14 shows the bottom wall now in its deployed position within the fully erected box. Note that this bottom wall has flanges around each side edge and along the edge furthest away from the side edge opposite its edge hinged to the front wall. FIG. 15 shows the lid of this embodiment with the pivotal lip in its opened position. Ideally the hinge connecting the pivotal lip with the rest of the lid is integrally formed with the sheet metal. In FIG. 16 the pivotal lip is in its closed position where the lock structure can engage the first mating lock mechanism (not shown) in a manner similar to that shown with regard to the first embodiment. This pivotal lip is preferably executed in a distinctive color or pattern so that the box can be customized or carry unique branding logos or trademarks.
[0044] As mentioned before, attaching the collapsible box structure to the side (in this case the front wall) of the truck box is an important step. Here a U-shaped bracket engages the front and back surface of that truck box wall and spans the lip connecting therebetween. FIG. 18 shows these brackets positioned. Each bracket preferably has a large setscrew, which can be tightened towards the track box wall to engage below its lip, thus holding the bracket in place on the bed wall and thus holding the box in the truck bed. The bracket also has protruding bolts, which engage corresponding holes in the back wall of the truck box. These brackets can be first positioned at an appropriate location along the truck box wall. Then the truck box itself can be partially expanded so that nuts matching the protruding bolts can be fastened once the bolts are passed through appropriately provided holes through the box's back wall. Alternatively the brackets can be pre-positioned on the back wall of the truck box as shown in FIG. 19. The resulting collapsible truck box is fastened to the truck bed by the front wall as shown in FIG. 20.
[0045] As mentioned previously, this bracket mounting system permits a properly rugged box to be suspended above the bottom wall of the truck box. In this way construction materials or lumber as represented by the element shown can pass below the bottom wall of even the fully expanded and erected truck box, giving extra versatility so that the truck box can expand over and can remain expanded or erected even though long elements being carried in the truck bed extend into thus occupied space. As mentioned before wheels may be provided near the juncture between the front wall and the side walls to further support and aid in moving the truck box from its collapsed to its fully erect position.
[0046] While the invention has been particularly shown and described with reference to a preferred embodiment thereof, it will be understood by those skilled in the art that various other changes in the form and details may be made without departing from the spirit and scope of the invention.
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Pick-up trucks are very popular vehicles. The versatility provided by the truck bed in the pick-up truck is aided by providing a truck box usually a monolithic elongated container fastened to the truck bed that usually spans the width of the truck bed. Each end of this type of truck box is support by laterally flanking walls of the truck bed. However, once installed, these boxes occupy a considerable space and usually must be removed so that large items can be carried in the truck bed. The disclosed collapsible truck box provides the functionality of a fixed sized truck box but can be easily collapsed by the user to occupy a very small portion of the truck bed space. Hinged connections between the lid wall, back wall, front wall and bottom wall provide this collapsing and erecting function. Preferably side walls at each end of the truck box, with each wall including its own vertically oriented hinge, further enhance the functionality of the disclosed box.
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BACKGROUND OF THE INVENTION
1. Technological Field
The present invention relates to a wheel slip control system for a vehicle, operable so as to control the rotation of the driven wheels to control the power of internal combustion engine, as well as control the braking system for driven wheels during an acceleration slip.
2. Prior Art
There have been known various kinds of anti-skid control systems (for example, Published unexamined patent application sho No. 59-2962 "Hydraulic Anti-skid Control System") for vehicles which control the slip of the wheels during braking and acceleration operation. On the other hand, another slip control system for vehicles which conducts traction control for improving running stability, acceleration, etc. has been considered. In such a device, idle running of driven wheels is prevented and rotation of driven wheels is controlled so as to maximize the frictional force between the tire and road surface during acceleration operation.
As for the latter traction control system, it is considered that the control of internal combustion engine power and that of rotation of driven wheels are carried out by adjusting, e.g., the ignition timing and the fuel injection amount when the degree of driven wheel slip is found to exceed a predetermined value.
However, the wheel slip control systems mentioned above are still inert, and the following problems remained unsolved.
The conventional wheel slip control systems aim at the prevention of slip during acceleration by reducing the fuel injection amount or setting a time lag against a change in the ignition timing so as to control the output of the internal combustion engine. In this case, there is a problem that the control capacity is limited, due to the necessity of taking preventive measures against abnormal vibration or fire due to abrupt changes in the operating conditions of the engine.
On the other hand, when the intake air amount is controlled, the ignition timing and the fuel injection amount, etc. are determined in accordance with it. Therefore, the output of internal combustion engine can be controlled smoothly to improve the drivability. But if the opening position of the throttle valve interlocked to the accelerator pedal, is controlled against the commanded position of the pedal commanded by being stepped on, some problems may occur. For example, discomfort like kickback to the driver, or incapability of keeping stability in case of failure with control parts of the throttle valve may exist.
In addition, another problem, that is, the rotation of driven wheels cannot be controlled immediately because of the bad responsiveness, should be pointed out when the output of internal combustion engine is used for controlling the rotation of driven wheels in the case above. Accordingly, it is possible to control the rotation of driven wheels directly by using the brake system mounted on the vehicle, when the slip of driven wheels occurs during acceleration operation and the rotation of them must be controlled instantly. But a special brake system will be required to control the rotation of driven wheels by using the brake system only. Namely, if the driving force is great, for example, when the gear position of transmission is set at the first speed, the special brake system which produces greater brake force will be necessary to give the corresponding brake force against the driving force. The conventional brake system may not work well. Also, the pressure source will be required for the purpose of increasing braking pressure supplied to the braking cylinder equipped with the driven wheels of the control system for vehicles. Therefore, an oil pump must be added to the conventional vehicles in order to activate the brake for throttle control (A small-type pump is acceptable).
SUMMARY OF THE INVENTION
The objective of the present invention is to solve the above-mentioned problems and offer a wheel slip control system capable of carrying out the fine control without any decrease in drivability and safety of vehicles as well as any delay in control when the slip of driven wheels occur during acceleration operation.
Another objective of the present invention is to offer a wheel slip control system wherein the braking force of wheels, including driven wheels, is controlled by braking means, so-called anti-skid control system in braking and traction control in accelerating and other pressure sources mounted on vehicles and the output of internal combustion engine is controlled by the second throttle valve which controls the intake air amount in longer period, thus the driven wheels can be operated with the most suitable driving force by controlling the driving force of driven wheels to prevent the occurrence of excessive slip like locking, accompanying skidding, etc.
It is another further objective of the present invention to offer a wheel slip control system wherein the necessary braking force for braking means is minimized by controlling the output of internal combustion engine as well as braking force, therefore, the system can be reduced in size and weight so as to save the fuel consumption.
The other objective of the present invention is to offer a wheel slip control system wherein the traction control system is easily added to vehicles equipped with the anti-skid control system and other pressure sources, as the device and constitution of the anti-skid control system are simplified by utilizing the hydraulic system, which leads to the capability of conducting the fine control of braking force and minimizing the necessary devices used for traction control only.
In order to achieve the above objectives thereby to solve the afforementioned prior art problems, the inventive wheel slip control system features comprise as shown in FIG. 1.
A wheel slip control system used for vehicle comprising:
(a) a pressure source selection means M3 monitoring a first pressure of a first pressure source M1 and a second pressure of a second pressure source M2 to measure the difference between two pressures, and adapted to select one of said first and second pressure sources depending on the measured differential pressure;
(b) a slip control means M5 receiving a first adjustment signal for anti-skid control to adjust the first pressure provided by said pressure source selection means M3 or receiving a second adjustment signal for traction control to adjust the second pressure provided by said pressure source selection means M3 and after adjustment operating to suppress slipping of wheels including driven wheels M4;
(c) a brake slip detection means M6 detecting the state of slipping of a driven wheel M4 during a braking operation of the vehicle and producing a brake slip signal representing the state of slipping;
(d) an acceleration slip detection means M7 detecting the state of slipping of a driven wheel M4 during an accelerating operation of the vehicle;
(e) a second throttle valve M10 provided to intake air path 3 where a first throttle valve M9 is interlocked to a vehicle speed increase means M8;
(f) an electronic control means M11 receiving said brake slip signal and providing the first adjustment signal based on said received signal for said slip control means M5 so that the driven wheel speed is within a first predetermined range, receiving said acceleration slip signal and providing the second adjustment signal based on said received signal for said slip control means M5 so that the driven wheel speed is within a second predetermined range, and outputting a third adjustment signal for traction control to a operating means for said second throttle valve M10 so that the driven wheel speed is within a third predetermined range.
The first pressure source M1 means indicates the pressure as obtained from a brake pedal directly or indirectly by driver. The artificial pressure amplified by machinery, like powerbrake, may be acceptable.
Also, the second pressure source M2 indicates any pressure sources mounted on vehicles, for example, the pressure sources for anti-skid or power steering, etc.
The pressure source selection means M3 may have a constitution which gives priority to the source of higher pressure. Thus, the combination of check valves and the use of a shuttle valve may be adopted. As a matter of course, the constitution which selects the suitable source compulsorily in accordance with the state of slip control of vehicle may be allowed.
The brake slip detection means M6 detects the slip condition of driven wheels M4 during braking operation, e.g. one judges the slip when the difference between vehicle speed and driven wheel speed exceeds a predetermined value, or the other detects the slip when the detected acceleration of driven wheels lowers a predetermined negative value.
On the other hand, an acceleration slip detection means M7 detects the slip (lock) of driven wheels during accelerating operation, e.g., one decides the slip when the difference between vehicle speed and driven wheel speed exceeds a predetermined value or the other detects the slip in accordance with the difference of speed (number of rotations) between driven wheels and non-driven wheels, further, the other judges the slip when acceleration of rotations of driven wheels is above a predetermined value.
It is possible for said brake slip detection means M6 and said acceleration slip detection means M7 to have the same constitution if only the parameter is changed. As the brake slip and the acceleration slip do not occur concurrently, there are no problems for using the system in common. Thus, that has such a remarkable merit as being capable of simplifying the system and the constitution.
An electronic control means M11 inputs signals from said acceleration slip detection means M7 and said brake slip detection means M6 and controls the hydraulic pressure of said slip control means M5 and the position of said second throttle valve M10. It is constituted so as to control the position of said second throttle valve M10 by actuator, etc. as well as to decrease, hold or increase the hydraulic pressure by 3-position 3-port valve, etc. Also, such a constitution as controlling the position of said 3-position 3-port valve and the operation amount of said actuator by microcomputer in addition to said brake slip detection means M6 and said acceleration slip detection means M7 or having discrete circuit construction for every means.
In the wheel slip control system as mentioned above, a part of so-called anti-skid brake control system, which controls the brake of wheels by changing the hydraulic pressure of hydraulic pressure system in accordance with the first pressure source operated by driver when the slip condition of vehicle is detected during braking operation of a vehicle, is used in common. In addition, when the slip condition is detected during acceleration of the vehicle, using the second pressure source mounted on the vehicle separately from the first one as the pressure source of the hydraulic pressure system for said brake control means, the position of said second throttle valve as well as the pressure of the hydraulic pressure system are controlled.
Namely, when the slip occurs during acceleration operation in the system, the suitable brake force is given to the wheels by pressure of the second pressure source and the output of internal combustion engine which operates the driven wheel is controlled by adjusting the intake air amount with said second throttle valve. Therefore, the quick response of traction control can be secured by the former and the unnecessary power from the internal combustion engine can be reduced by the latter when the traction control is conducted. In other words, the economic efficiency and the durability are satisfied.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a general block diagram according to the invention slip control system;
FIG. 2 is a schematic block diagram mainly showing the hydraulic system of the wheel slip control system embodying the first embodiment of the present invention;
FIG. 3 is a schematic block diagram showing the traction control section 30' of the electronic control circuit 30 in FIG. 2 according to the first embodiment;
FIG. 4 is a timing chart showing, as an example, the control operation of the first embodiment;
FIG. 5 is a block diagram showing the arrangement of the electronic control circuit 30 in FIG. 1 according to the second embodiment;
FIG. 6(A) and (B) are flowcharts showing, as an example, the control operation of the second embodiment;
FIG. 7 is a timing chart showing, as an example, the control operation of the second embodiment;
DESCRIPTION OF THE PREFERRED EMBODIMENT
Embodiments of the present invention will now be described with reference to the drawings.
In FIG. 2 showing generally a first embodiment of the inventive wheel slip control system, numeral 1 shows an internal combustion engine, 2 shows a fuel injection valve, 3 shows an intake air path, the intake air path 3 is provided with a first throttle valve 5 which interlocks to an accelerator pedal 4 and a second throttle valve 7 which is driven by a DC motor 6 so as to adjust the intake air amount. Normally, said second throttle valve 7 is fully opened. It is controlled from the full open to the full closed positions so as to control the power of the internal combustion engine for the traction control only. The slip control is executed when the valve is half-opened.
The arrangement also includes a brake pedal 11, a brake master cylinder 12 serving as the first pressure source for providing a brake hydraulic pressure in response to a displacement of the brake pedal 11, a sub-master cylinder 13 serving as the second pressure source for providing a brake hydraulic pressure derived from the power steering hydraulic system upon detection of an acceleration slip as described later, non-driven right and left wheels 14 and 15 of the vehicle, driven right and left wheels 16 and 17, wheel cylinders 18˜21 provided for the wheels 14˜17, a hydraulic system 23 used for anti-skid control, a power steering hydraulic system 24 for both anti-skid and traction controls, a non-driven wheel speed sensor 25 generating a pulse signal at frequencies in proportion to the rotational speed of the non-driven wheels 14 and 15, a driven-wheel speed sensor 26 generating a pulse signal at frequencies in proportion to the rotational speed of the driven wheels 16 and 17, and an electronic control circuit 30 implementing anti-skid control and traction control as well by controlling the hydraulic system 23 and 24.
The brake master cylinder 12 is of a tandem master cylinder assembly, supplying a brake oil pressure to the wheel cylinders 18 and 19 on the non-driven wheels 14 and 15 in one hydraulic system, and to the wheel cylinders 20 and 21 of the driven wheels 16 and 17 in another. The brake oil pressure produced by the sub-master cylinder 13 is used for the braking of driven wheels 16 and 17, and as to which of this brake hydraulic pressure or that produced by the brake master cylinder 2 is supplied by way of the anti-skid hydraulic system 23 to the wheel cylinders 20 and 21 is determined by a change valve 32 serving as the pressure source selection means. The change valve 32 has the structure of a shuttle valve, supplying the higher of the above two hydraulic pressures to the anti-skid hydraulic system 23.
The anti-skid hydraulic system 23 has major hydraulic paths for supplying the pressure from the change valve 32 to the wheel cylinders 20 and 21 via a 3-position valve 34, and operates to increase the pressure by means of an oil pump 36 and hold or decrease (release the pressure to a reservoir 38) depending on the position of the 3-position valve 34. The system 23 further includes check valves 39, 40 and 41, the hydraulic path via the valve 41 being used to decrease the pressure (decrease the braking force) by the operation of the brake pedal when the 3-position valve 34 is set at "hold" position. The 3-position valve 34 is operated by the electronic circuit 30, and positions a, b and c in FIG. 2 correspond to "increase pressure", "hold pressure" and "decrease pressure", respectively.
Next, the power steering hydraulic system 24 will be described. The power steering hydraulic system 24 consists of an oil pump 52 for pumping up the hydraulic oil used in the system from a reservoir tank 51, check valves 53 and 54 for conducting the oil flow in one way, a steering gear box 55, an oil pressure switch 56 which turns on (low level) when the oil pressure in the steering gear box 55 increases due to steering operation, a 2-position valve 57 (will be termed M/C up-pressure valve hereinafter) having e position for supplying the oil pressure increased by the oil pump 52 (will be termed steering oil pressure hereinafter) only to the power steering hydraulic system 24 and position f for supplying the oil to both of the system 24 and the traction control sub-master cylinder 13, and 2-position valve 58 (will be termed PS up-pressure valve hereinafter) having position h for supplying the hydraulic oil pumped by the oil pump 52 directly to the steering gear box 55 and position i for supplying the hydraulic oil with increased pressure to the gear box 55. The position of the oil pressure switch 56 is informed to the electronic control circuit 30, and the M/C up-pressure valve 57 and PS up-pressure valve 58 are controlled by the electronic control circuit 30.
Next, a section 30' of the electronic control 30 for implementing traction control will be described with reference to FIG. 3. Besides the traction control circuit 30', the electronic control circuit 30 includes the anti-skid control circuit (ESC), but this section unrelated directly to the present invention will be disregarded in the following description.
Briefly, the traction control circuit 30' operates to receive the pulse signal from the non-driven wheel speed sensor 25 on an F/V converter 70, differentiate its voltage output Vfr by a differentiator 71 to obtain the acceleration of the vehicle, and compare the differentiated output with a zero level by a comparator 72 to obtain a signal Vfc indicative of as to whether the vehicle is in acceleration or deceleration. The signal Vfc is sent to the anti-skid control circuit ESC, which is activated when Vfc is at a low level.
Based on the signal Vfr representing the nondriven wheel speed, a threshold-1 generator 73a and an adder 74a in unison produce the first slip threshold level Vfbl, and a threshold-2 generator 73b and an adder 74b in unison produce the second slip threshold level Vfbh, a threshold-3 generator 73c and an adder 74c in unison produce the third slip threshold level Vfsh, a threshold-4 generator 73d and an adder 74d in unison produce the fourth slip threshold level Vfsl.
These voltage levels are compared by comparators 80a, 80b, 80c and 80d respectively, with the voltage signal Vrr which is derived from the pulse signal generated by the driven wheel sensor 26 and converted into voltage by an F/V converter 79, and four slip threshold signals Vcb1, Vcbh, Vcsh and Vcsl are obtained. The relationship among those signals is as follows.
Vfbh>Vfsh>Vfsl>Vfbl
As explained later in FIG. 4, the first and second slip threshold signals, Vfbh and Vfbh, are employed as threshold levels for controlling the braking force by means of said anti-skid hydraulic system 23 and the third and fourth slip threshold signals, Vfsh and Vfsl, as threshold level for controlling intake air amount of said internal combustion engine 1 by means of said second throttle valve 7. In addition, the first two signals indicate comparisons of the driven wheel speed (Vrr) with the first threshold level Vcb1 and second threshold level Vcbh provided based on the non-driven wheel speed (Vfr) as shown in FIG. 4. The fourth slip threshold signal Vfbl gives a timing for the preparation of traction control, while the third slip threshold signal Vfbh gives a timing for carrying out traction control by increasing the brake oil pressure.
Also, the third slip threshold signal Vcsh generated by comparing the third slip threshold level Vfsh with the driving wheel speed indicates the timing when said throttle valve 7 is driven toward closing position by operating said motor 6 in normal direction. On the other hand, the fourth slip threshold signal Vcsl generated by comparing the fourth slip threshold level Vfsl with the driven wheel speed indicates the timing when said throttle valve 7 is driven toward opening position by operating said motor 6 in the opposite direction.
The traction control circuit 30' further operates to differentiate the signal Vrr representing the driven wheel speed to obtain a signal Vrg representing the acceleration of the driven wheels 16 and 17. The signal Vrg is compared with an output signal G1 from a threshold-5 generator 84 and an output signal G2 from a threshold-6 generator 85 by comparators 87 and 88, respectively, and the first slip control signal Vrgh and second slip control signal Vrgl are obtained.
The above three signal processing systems perform traction control as shown in FIG. 4.
In operation, when the vehicle accelerates to have a high Vfc signal, a lower slip threshold signal Vfbl goes high, causing a delay circuit 89 formed of a monostable multivibrator to produce a high output, and a 2-input AND gate 90a produces a high output Vrt as shown in FIG. 4. This signal goes through a 2-input OR gate 91 and an amplifier 92 to activate the oil pump 36 in the anti-skid hydraulic system 23 and, at the same time, goes through an amplifier 90b to switch the M/C up-pressure valve 57 to position f so that an oil path for conducting the power steering oil pressure to the sub-master cylinder 13 is formed. In another condition in which the oil pressure switch 56 is at a high level added to the above condition, a 3-input AND gate 90c provides an output Vrs, and when the Vrs becomes high, it goes through an amplifier 93 to switch the PS up-pressure valve 58 to position i and the steering gear box 55 is throttled and the power steering hydraulic pressure is conducted to the sub-master cylinder 13. The purpose of entering the output of the oil pressure switch 56 to the 3-input AND gate 90c is to switch the PS up-pressure valve 58 so as to prevent an unnecessary increase in the oil pressure which is raised automatically when the steering wheel is turned during traction control operation. The arrangement also works to supply the oil pressure to the power steering system in precedence over the traction control system.
The 3-position valve 34 in the anti-skid hydraulic system 23 has its three positions a, b and c controlled in response to the state of a transistor Tr1 driven by the output of an amplifier 95 and the state of a transistor Tr2 provided with a current limiting resistor R and driven by the output of another amplifier 96. The valve position is determined b the combination of the transistor outputs as follows.
______________________________________Tr1 Tr2 Valve Position Brake Oil Pressure______________________________________OFF OFF a IncreaseOFF ON b HoldON ON c Decrease______________________________________
The amplifier 96 for driving the transistor Tr2 receives the output Vru of a 3-point AND gate 97, which signal Vru is determined by the logical sum of the output of a 2-point NAND gate 98 receiving the upper slip threshold signal Vcbh and first slip control signal Vrgh, the signal Vrt, and the control signal from the anti-skid control circuit (ESC), as shown in FIG. 4. During which, the amplifier 95 for driving the transistor Tr1 receives the output signal Vrd of a 2-input OR gate 99, which signal Vrd is determined by the logical sum of the output of a 2-input AND gate 101 receiving the lower slip threshold signal Vcb1 inverted by an inverter 100 and the second slip control signal Vrgl, and the input signal from the ESC.
On the other hand, said second throttle valve 7 is controlled as follows by the third and fourth slip threshold signals Vcsh and Vcsl respectively. When the third and fourth slip threshold signals are in low level, the output of a 2-input NOR gate 105 Vcsc becomes high level and a normal-reverse amplifier 107 feeds the current so as to operate said motor 6 in the opposite direction until said throttle valve 7 is fully opened. Consequently, said second throttle valve 7 is fully opened and the intake air amount is controlled by said first throttle valve 5 interlocked to said accelerator pedal 4. When the fourth slip threshold signal Vcsl is high in level, the output of said 2-input NOR gate 105 Vcsc becomes low level, thus, said normal-reverse amplifier 107 does not supply the power to said motor 6. Next, when the third slip threshold signal Vcsh is in high level, the output of said 2-input NOR gate 105 Vcsc becomes low level, then said normal-reverse amplifier 107 supplies the power in the opposite direction compared with the former case so as to operate said motor 6 in normal direction until said second throttle valve 7 is fully closed.
As a result of the foregoing arrangement of this embodiment,
(A) When the vehicle accelerates as detected on the non-driven wheels 14 and 15, the power steering oil pressure is high enough, the control operation proceeds as follows:
(1) When the driven wheel speed Vrr has exceeded the lower threshold level Vfb1 based on the non-driven wheel speed, the oil pump 36 in the anti-skid hydraulic system 23 is activated, and the M/C up-pressure valve 57 and PS up-pressure valve 58 in the power steering hydraulic system 24 are switched to positions f and i, respectively;
(2) When the driven wheel speed Vrr has exceeded the third slip threshold level Vfsh based on the non-driven wheel speed, the power of said internal combustion engine 1 is judged as unnecessarily high, and is controlled by operating said motor 6 in normal direction and adjusting the position of said second throttle valve 7 toward the full closed position.
(3) If the driven wheel speed Vrr exceeds the upper threshold level Vfbh based on the non-driven wheel speed and at the same time the driven wheel acceleration Vrg exceeds a certain reference level G1, the 3-position valve 34 in the anti-skid hydraulic system 23 is set to position a (increase pressure) so as to increase the braking force of the driven wheels 16 and 17;
(4) If the driven wheel speed Vrr is below the lower threshold level vfb1 based on the non-driven wheel speed and at the same time the driven wheel acceleration Vrg is below a certain reference level G2 (a negative value), the 3-position valve 34 is set to position c (decrease pressure) so as to reduce the braking force;
(5) If the driven wheel speed Vrr is below the fourth slip threshold level Vfsl based on the non-driven wheel speed, the control of power of said internal combustion engine 1 is regarded as unnecessary and the power is increased by operating said motor 6 in reverse direction and controlling the position of said second throttle valve 7 toward the full open; and
(6) The control is conducted so as to hold the braking force with the position of said 3-position valve 34 set at position b (hold) under the conditions other than (3) and (4) mentioned above. On the other hand, the power of said internal combustion engine 1 is held without controlling the position of said second throttle valve 7 under the conditions except (2) and (5) mentioned above.
(B) When a steering operation takes place, causing the oil pressure in the steering gear box 55 to increase with the oil pressure switch 56 operating ON, the PS up-pressure valve 58 is restored to position h and the 3-position valve 34 is set to position a (increase pressure). Accordingly, if the driven wheel 16 or 17 slips during the acceleration of the vehicle, the existing anti-skid hydraulic system sourced by the power steering hydraulic pressure is used to brake the driven wheels 16 and 17 so as to prevent a slip running etc., whereby the driven wheels 16 and 17 are exerted to produce an optimal traction force.
Also, in view of the power of said internal combustion engine 1 being decreased by the second throttle valve 7, it is possible to prevent the lock of the driven wheels 16 and 17, etc. and give them the optimum driving force by traction control. Therefore, as a whole, the traction control with the good efficiency can be achieved, employing the braking system in accordance with the hydraulic pressure of the anti-skid hydraulic system 23 for the quick response, and the second throttle valve 7 for controlling the rotation in comparatively longer period, respectively.
Additionally, as the power of the internal combustion engine 1 is controlled by the second throttle valve 7, the necessary braking force for traction control is minimized and a reduction of the hydraulic system in size and weight becomes possible. Also, the fuel consumption is improved because no extra fuel is supplied to the internal combustion engine 1 when the braking force is generated, and at an same time, the exothermic condition of the brake system which supplies the braking force can be prevented.
This traction control is easily implemented without the need of a specialized hydraulic system and devices, but by utilization of the hydraulic system used for the anti-skid operation, the 3-position valve 34 used therein, and the power steering hydraulic system for obtaining the braking oil pressure independently of the driver's action. On this account, traction control can be established merely through a little modification for the piping and replacement of the electronic control circuit 30 for a vehicle which already installs the anti-skid control and power steering systems. It should be noted in FIG. 3 that the remaining one input to the 2-input OR gates 91 and 99 and the 3-point AND gate 97 respectively and to allow the anti-skid control circuit (ESC) to control the 3-position valve 34 and oil pump 36. Since anti-skid control and traction control do not take place concurrently, these systems can be shared by separate purposes through a simple logical sum process.
Next, the second embodiment of this invention will be described. The second embodiment is intended to control the hydraulic systems and associated devices similar to those of the first embodiment shown in FIG. 2 through an electronic control circuit 30 which is in this case constituted mainly by a microcomputer as shown in FIG. 5. The control is executed according to the flowchart shown in FIGS. 6 (A) and (B). The arrangement of FIG. 5 includes a central processing unit (CPU) 110 which is programmed to receive the sensor signals from the driven wheel speed sensor 26, non-driven wheel speed sensor 25 and oil pressure switch 56, and perform various calculations in response to these input signals. Other circuit components included are a read-only memory (ROM) 112 for storing the above-mentioned CPU control program, maps, and fixed data, a random access memory (RAM) 113 for performing reading and writing sensor input data temporarily, and calculation data, an input interface circuit 114 including waveform shaping circuits and a multiplexer for supplying sensor input signals selectively to the CPU 110, an output circuit 116 for driving the oil pump 36, M/C up-pressure valve 57 and PS up-pressure valve 58 and also driving the 3-position valve 34 through transistors Tr1 and Tr2, and a bus line for providing data communication between the CPU 110, ROM 112, RAM 113, and input/output interface circuits 114 and 116, and a power supply circuit 120 for all of the above circuit components.
In addition, the 3-position valve 34 and the motor 6 are constituted so as to be driven by the output circuit interface circuit 116 via transistors Tr1 and Tr2, a power restricting resistor R and PWM drive circuit 125 which conducts pulse width modulation respectively.
The traction control operation which is carried out together with anti-skid control on a time division basis by the electronic control circuit 30 arranged as described above will be described in connection with the flowchart shown in FIGS. 6 (A) and (B).
Though the flowcharts are drawn separately in the form of FIGS. 6 (A) and (B) due to the size of the drawing, a series of process is performed consistently. The routine of this traction control is performed every predetermined period together with the anti-skid control, etc. The process of every step will be described hereafter.
Step 200: Read input data on the input interface circuit 114 of the driven wheel speed vr from the driven wheel sensor 26, the non-driven wheel speed vf from the non-driven wheel sensor 25, and the switch state from the oil pressure switch 56.
Step 220: Perform differentiation process for the driven wheel speed vr to obtain acceleration α of the driven wheels.
Step 230: Test whether the driven wheel acceleration is above zero (acceleration).
Step 240: Calculate the slip threshold levels vs1, vs2, vs3 and vs4 from the non-driven wheel speed vf using predetermined constants K1, K2, K3, K4 (K2>K3>K4>K1), g1, g2, g3 and g4 as follows.
vs1=K1×vf+g1
vs2=K2×vf+g2
vs3=K3×vf+g3
vs4=K4×vf+g4
Step 242: Determine whether the driven wheel speed is above the third slip threshold level vs3 or not.
Step 244: Determine whether the driven wheel speed is equal or below the fourth slip thereshold level vs4 or not.
Slip 246: Output the normal signal via the output interface circuit 116 so as to control the position of the second throttle valve 7 toward the full close position by operating the motor 6 with the PWM drive circuit 125 in normal rotation.
Step 247: Output the reverse signal via the output interface circuit 116 so as to control the position of the throttle valve 7 toward the full open position by operating the motor 6 with the PWM drive circuit 125 in opposite rotation.
Step 248: Reset the output of the normal and the reverse signals mentioned above.
Each step of the flowchart shown in FIG. 6 (A) has been explained above and that in FIG. 6 (B) will be described below.
Step 250: Test whether the driven wheel speed vr is higher than the first threshold level vs1.
Step 260: Set a predetermined value to the counter C in the CPU.
Step 270: Reset the counter C to zero.
Step 280: Decrement the counter C by one.
Step 290: Test whether the content of counter C is above zero. The counter C used in the steps 260-290 is actually a programmed operation substituting the delay circuit 89 in the first embodiment, and its purpose is to continue traction control for a preset time length when the speed vr of driven wheels 16 and 17 has fallen below the first threshold level vs1 after traction control had been commenced.
Step 293: Test whether the oil pressure switch 56 is OFF. If the switch is OFF, indicating the steering operation being inert and the power steering hydraulic pressure not being increased, the sequence proceeds to step 350.
Step 296: If the oil pressure switch 56 is found ON in step 293, indicating the steering operation, the PS up-pressure valve 58 is reset to position h.
Step 300: Set or hold the M/C up-pressure valve 57 in the power steering hydraulic system 24 to position f, and activate the oil pump 36.
Step 303: Test whether the oil switch 56 is OFF.
Step 306: If the oil pressure switch 56 is OFF, switch the PS up-pressure valve 58 to position i so as to increase the oil pressure conducted to the sub-master cylinder 13.
Step 309: If the oil pressure switch 56 is ON, switch the PS up-pressure valve 58 to position h.
Step 310: Test whether the driven wheel speed vr is higher than the second threshold level vs2
Step 320: Reset or hold the M/C up-pressure valve 57 and PS up-pressure valve 58 to positions e and h, respectively, and deactivate the oil pump 36.
Step 330: Set or hold the 3-position valve 34 in the anti-skid hydraulic system 23 to position a (increase pressure).
Step 340: Set the 3-position valve 34 to position b (hold pressure).
Step 350: Set the 3-position valve 34 to position c (decrease pressure).
According to this embodiment, as shown in FIG. 7,
(A) When the driven wheels 16 and 17 are found accelerating (α=0), i.e., an affirmative decision made by step 230, the control operation takes place as follows.
(1) When the driven wheel speed vr has exceeded the third slip threshold level vs3 based on the non-driven wheel speed vf, i.e., an affirmative decision made by step 242, the motor 6 is operated in normal rotation so as to control the position of the second throttle valve 7 toward the full close position and the intake air amount of the internal combustion engine 1 is decreased so as to control the output (See period CL in FIG. 7).
(2) When the driving wheel speed vr has fallen below the fourth slip threshold level vs4 by the control mentioned in (1) or (5) hereafter, i.e., an affirmative decision made by step 244, the motor 6 is operated in opposite rotation so as to control the second throttle valve 7 toward the full open position and the intake air amount of the internal combustion engine 1 is increased so as to increase the output (See period OP in FIG. 7).
(3) When the relation of the driven wheel speed vr and the fourth slip thereshold level is vs4<vr<vs3, i.e., a negative decision made by both steps 242 and 244, both the normal and the reverse signals are reset so as to hold the position of the second throttle valve 7.
(4) When the driven wheel speed vr has exceeded the first thereshold level vs1 based on the non-driven wheel speed vf, i.e., an affirmative decision made by step 250, the oil pump 34 in the anti-skid hydraulic system 23 is activated, the M/C up-pressure valve 57 in the power steering hydraulic system 24 is set to position f, and the 3-position valve 34 in the anti-skid hydraulic system 23 is set to position b (hold pressure). (See period I in FIG. 7.)
In the above operation, if the oil pressure switch 56 is OFF, the PS up-pressure valve 58 is set to position i (steps 303 and 306), or if the oil pressure switch 56 is ON, the PS up-pressure valve 58 is set to position h (steps 303 and 309).
(5) When the driven wheel speed vr has exceeded the second threshold level vs2, i.e., an affirmative decision made by step 310, the 3-position valve 34 is set to position a (increase pressure) so as to increase the braking force of the driven wheels. (See period II in FIG. 7.)
(6) Following the above state (4) or (5), when the driven wheel speed vr has fallen below the first threshold level vs1, i.e., a negative decision made by step 250 and an affirmative decision made by step 290, the 3-position valve 34 is set to position c (decrease pressure) so as to reduce the braking force. (See period III in FIG. 7.) At this time, if the oil pressure switch 56 is found ON, the PS up-pressure valve 58 is reset to position h (steps 293 and 296).
(7) When the time length set in the counter C has expired (a negative decision made by step 290), while the driven wheel speed vr staying below the first threshold level vs1, the M/C up-pressure valve 57 and PS up-pressure valve 58 are set to positions e and h, respectively, the oil pump 34 is deactivated, and the 3-position valve 34 is switched to position a (increase pressure) to complete the traction control operation (the period not shown in FIG. 7.)
(B) If the vehicle acceleration α falls below zero, i.e., deceleration begins, (a negative decision made by step 230), the counter C is reset to zero, and the same control as above (4) takes place. (See period IV in FIG. 7.)
Accordingly, this embodiment performs identically to the first embodiment and has the same effects as those of the first embodiment, for example, the quick response achieved by the anti-skid hydraulic system 23, the reduction of fuel consumption by the second throttle valve 7, etc. and moreover, the use of the CPU 110 allows various schemes of traction control without changing the hardware structure of the electronic control circuit 30.
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A wheel slip control system used for a vehicle comprising: a pressure source selector being informed of a first pressure of a first pressure source and a second pressure of a second pressure source and adapted to select one of the first and second pressure source, a slip controller receiving a first adjustment signal for anti-skid control to adjust the first pressure provided by the pressure source selector or receiving a second adjustment signal for traction control to adjust the second pressure provided by the pressure source, selector a brake slip detector detecting the state of slipping of wheels during a braking operation of the vehicle and producing a brake slip signal, an acceleration slip detector detecting the state of slipping of wheels during an accelerating operation of the vehicle, a second throttle valve being placed at an intake air path provided with a first throttle valve interlocked to an acceleration increase member; and an electronic controller receiving the brake slip signal and providing the first adjustment signal for the slip controller so that the driven wheel speed is within a first predetermined range, receiving the acceleration slip signal and providing the second adjustment signal for the slip controller so that the driven wheel speed is within a first predetermined range, and providing the third adjustment signal to a driving member for driving the second throttle valve for the slip controller so that the driven wheel speed is within a second predetermined range.
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COPYRIGHT NOTICE
[0001] A portion of the disclosure of this patent contains material that is subject to copyright protection. The copyright owner has no objection to the reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a dispensing jar. In particular, the present invention relates to a food dispensing jar especially suited for thick or viscous food products.
[0004] 2. Description of Related Art
[0005] The dispensing of food products that have a viscous nature such as peanut butter, mayonnaise, thick sauces, jams, jellies and the like is difficult from the current standard straight sided containers. The typical peanut butter jar for example is a cylindrical straight sided container or plastic or glass which has a twist off lid. The jar is exceedingly difficult to get product out of as the volume of product decreases. Use of a spoon or knife is often difficult if not dangerous as removal tools as the last ounces of food are retrieved.
[0006] The concept of using a push up system for food has seen limited application. One such application is seen in U.S. Pat. No. 8,079,499 to Juteau issued Dec. 20, 2011. In this invention, a threaded center post is utilized and the bottom is pushed up the rotating around the center post. The user then can get product out of the top portion of the jar. This has several use problems in that the center post gets in the way of using a tool since the bottom is pushed up not to dispense but rather to make it easier to remove with a tool such as a knife or spoon.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention relates to the discovery that a rotatable base fit inside a threaded container can be utilized to overcome the problems and limitations of the prior art.
[0008] Accordingly, in one embodiment the present invention relates to a container for a food product that is viscous comprising:
[0009] a) a container having a cylindrical inside wall having a bottom of the inside wall defining an open bottom and a top of the inside wall defining an open top, the inside wall having a threaded surface extending continuously from the bottom of the wall to the top of the wall;
[0010] b) a rotatable circular base having a diameter which fits and is retained inside the container by a peripheral edge of the base adapted to engage the threaded surface, the base adapted and positioned to rotate by hand from the open bottom to the open top.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a top perspective view of the container of the invention.
[0012] FIG. 2 is a bottom perspective view of the container of the invention.
[0013] FIG. 3 is a perspective view of the cylinder of the invention.
[0014] FIG. 4 is a perspective view of the base of the invention.
[0015] FIG. 5 is a side view of a top of the present invention with screw threads.
[0016] FIG. 6 is a bottom perspective view showing a different design of hand grip and a lower sealing lip.
[0017] FIG. 7 is a side view of a clean container with peanut butter inside.
DETAILED DESCRIPTION OF THE INVENTION
[0018] While this invention is susceptible to embodiment in many different forms, there is shown in the drawings and will herein be described in detail specific embodiments, with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the specific embodiments shown and described. In the description below, like reference numerals are used to describe the same, similar or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments in order for those skilled in the art to practice the invention.
DEFINITIONS
[0019] The terms “about” and “essentially” mean ±10 percent.
[0020] The terms “a” or “an”, as used herein, are defined as one or as more than one.
[0021] The term “plurality”, as used herein, is defined as two or as more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language). The term “coupled”, as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.
[0022] The term “comprising” is not intended to limit inventions to only claiming the present invention with such comprising language. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of” claim language and is so intended.
[0023] Reference throughout this document to “one embodiment”, “certain embodiments”, and “an embodiment” or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases or in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments without limitation.
[0024] The term “or” as used herein is to be interpreted as an inclusive or meaning any one or any combination. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.
[0025] The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the present invention, and are not to be considered as limitation thereto. Term “means” preceding a present participle of an operation indicates a desired function for which there is one or more embodiments, i.e., one or more methods, devices, or apparatuses for achieving the desired function and that one skilled in the art could select from these or their equivalent in view of the disclosure herein and use of the term “means” is not intended to be limiting.
[0026] As used herein the term food product that is “viscous” refers to food products that are very thick liquids or gels such as peanut butter, jams, jellies, mayonnaise and similarly viscous thickened liquids.
[0027] As used herein the term “container” refers to a food compatible container adapted for containing the food product that is viscous. Accordingly, glass or food acceptable/compatible plastics would be among the materials however one skilled in the art would understand what materials to use. A container can have walls and the like sufficiently think to give rigidity to the container and be of a size sufficient to contain a desired amount of the food product. Making a container with the threads would be within the skill in the art in view of the disclosure herein and could be by molding techniques that are well known or the like.
[0028] As used herein the container is “cylindrical” and has an outside wall and an inside wall. The cylinder has a bottom of the inside wall defining the open bottom of the cylinder and a top inside wall defining an open top. See the figures for further explanation. In one embodiment, there is a stop or a lip at the bottom of the inside wall.
[0029] As used herein the inside wall “threaded surface” extends continuously from about the bottom of the wall to about the top of the wall. The threading is molded into the surface of the inside wall of the cylinder and can be either female threading (indented) or male threading (raised). The threading can be tightly space or openly spaced as desired with the closer together threading requiring more turns or rotations than further spreading of the threads.
[0030] As used herein the “rotatable circular base” refers to a flat disk like bottom to the cylinder which is essentially the same diameter as the inside diameter of the cylinder. The base on its side will have threading matching the threading in the cylinder wall. That is the base will have male threading if the cylinder has female and female if the cylinder has male. The side and threading is such that it fits into to the cylinder and is retained by the peripheral edge that is threaded to be adapted to engage the threaded container inside wall surface. The base is adapted and position to rotate by hand use in the threading to move up inside the cylinder from the bottom when full of food product to the top. In one embodiment, there is a grip means on the bottom of the base such that one can grab the bottom of the base and rotate it thus moving the base up the cylinder as well as the food product inside. The gripping surface can be anything that allows a firm grip such as a plurality of ridges as an X or Y type configuration shown in the figures or the like. In one embodiment where there is a lower lip as described above the base can be rotated down to the lip to seal the container.
[0031] In one embodiment, there is a top for the container. The top can be snap on or a screw type and can if a screw top matches the inside wall threading or separate threading intended just for the lid. One skilled in the art can make an appropriate lid for a food product jar in view of the present disclosure.
[0032] Now referring to the drawings. FIG. 1 a top perspective view of the container of the invention with the optional top shown. In this view, cylindrical container 1 is shown with female (indented) threads 2 on inside wall 5 which show as male threads on the outside of the container 1 but in other embodiments do not need to appear on the exterior. The inside wall define an open top 8 and an open bottom not seen in this view. The threads 2 extend from the bottom 3 of the container 1 to the top 4 of the container 1 in a spiral fashion. Seen within container 1 is rotatable circular base 6 which is attached to the threads 2 by the peripheral edge male threads not show in this view wherein the edge is adapted to engage the threads 2 and rotate by hand from the open bottom to the open top 8 . Also seen, is top cover 9 which can be used to close the cylindrical container 1 by matching the male threads 11 on the top cover 9 and screwing them into the female threads 2 on the container 1 .
[0033] FIG. 2 is a bottom perspective view of the container of the invention. In this view the bottom 3 of the container 1 can clearly be seen and the open bottom 22 can clearly be seen. Also seen in this view is a bottom view of the rotatable circular base 6 . The base 6 has ridged hand grips 23 which can be grasped by the fingers and twist the base 6 to push up the contents of container 1 .
[0034] FIG. 3 is a perspective view of the cylinder of the invention with the rotatable circular base 6 removed.
[0035] FIG. 4 is a top perspective view of the base 6 of the invention showing the male threads 41 on peripheral edge 42 . While this embodiment shows male threads on the base 6 to be mated with the female threads 2 of the container 1 in other embodiments female and male threads are reversed on each part of the device.
[0036] FIG. 5 is a side view of a top of the present invention with screw threads 11 seen.
[0037] Note that the product to be used within the device such as peanut butter would be placed in the open top and by twisting base 6 clockwise (in this embodiment) the base moves up the sides of the container thus pushing the food up the container towards the open top for easy removal. FIG. 6 is the bottom perspective view of the container 61 with a wing hand grip 62 . Also shown is a lower sealing lip 63 in both FIGS. 6 and 7 .
[0038] Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applicant.
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The present invention relates to a container for dispensing a viscous food product such as peanut butter. The container has a spiral groove in which a bottom fits into and can spiral upwards to dispense the product from the top of the container.
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This is a continuation in part application of U.S. Ser. No. 08/059,022, filed May 6, 1993, now abandoned.
BACKGROUND OF THE INVENTION
The invention relates to prophylactic or affirmative treatment of diseases and disorders of the musculature by administering polypeptides found in vertebrate species, which polypeptides are growth, differentiation and survival factors for muscle cells.
Muscle tissue in adult vertebrates will regenerate from reserve myoblasts called satellite cells. Satellite cells are distributed throughout muscle tissue and are mitotically quiescent in the absence of injury or disease. Following muscle injury or during recovery from disease, satellite cells will reenter the cell cycle, proliferate and 1) enter existing muscle fibers or 2) undergo differentiation into multinucleate myotubes which form new muscle fiber. The myoblasts ultimately yield replacement muscle fibers or fuse into existing muscle fibers, thereby increasing fiber girth by the synthesis of contractile apparatus components. This process is illustrated, for example, by the nearly complete regeneration which occurs in mammals following induced muscle fiber degeneration; the muscle progenitor cells proliferate and fuse together regenerating muscle fibers.
Several growth factors which regulate the proliferation and differentiation of adult (and embryonic) myoblasts in vitro have been identified. Fibroblast growth factor (FGF) is mitogenic for muscle cells and is an inhibitor of muscle differentiation. Transforming growth factor β (TGFβ) has no effect on myoblast proliferation, but is an inhibitor of muscle differentiation. Insulin-like growth factors (IGFs) have been shown to stimulate both myoblast proliferation and differentiation in rodents. Platelet derived growth factor (PDGF) is also mitogenic for myoblasts and is a potent inhibitor of muscle cell differentiation. (For a review of myoblast division and differentiation see: Florini and Magri, 1989:256:C701-C711).
In vertebrate species both muscle tissue and neurons are potential sources of factors which stimulate myoblast proliferation and differentiation. In diseases affecting the neuromuscular system which are neural in origin (i.e., neurogenic), the muscle tissue innervated by the affected nerve becomes paralyzed and wastes progressively. During peripheral nerve regeneration and recovery from neurologic and myopathic disease, neurons may provide a source of growth factors which elicit the muscle regeneration described above and provide a mechanism for muscle recovery from wasting and atrophy.
A recently described family of growth factors, the neuregulins, are synthesized by motor neurons (Marchioni et al. Nature 362:313, 1993) and inflammatory cells (Tarakhovsky et al., Oncogene 6:2187-2196 (1991)). The neuregulins and related p185 erbB2 binding factors have been purified, cloned and expressed (Benveniste et al., PNAS 82:3930-3934, 1985; Kimura et al., Nature 348:257-260, 1990; Davis and Stroobant, J. Cell. Biol . 110:1353-1360, 1990; Wen et al., Cell 69:559, 1992; Yarden and Ullrich, Ann. Rev. Biochem . 57:443, 1988; Holmes et al., Science 256:1205, 1992; Dobashi et al., Proc. Natl. Acad. Sci . 88:8582, 1991; Lupu et al., Proc. Natl. Acad. Sci . 89:2287, 1992). Recombinant neuregulins have been shown to be mitogenic for peripheral glia (Marchionni et al., Nature 362:313, 1993) and have been shown to influence the formation of the neuromuscular junction (Falls et al., Cell 72:801, 1993). Thus the regenerating neuron and the inflammatory cells associated with the recovery from neurogenic disease and nerve injury provide a source of factors which coordinate the remyelination of motor neurons and their ability to form the appropriate connection with their target. After muscle has been reinnervated the motor neuron may provide factors to muscle, stimulating muscle growth and survival.
Currently, there is no useful therapy for the promotion of muscle differentiation and survival. Such a therapy would be useful for treatment of a variety of neural and muscular diseases and disorders.
SUMMARY OF THE INVENTION
We have discovered that increased mitogenesis differentiation and survival of muscle cells may be achieved using proteins heretofore described as glial growth factors, acetylcholine receptor inducing activity (ARIA), heregulins, neu differentiation factor, and, more generally, neuregulins. We have discovered that these compounds are capable of inducing both the proliferation of muscle cells and the differentiation and survival of myotubes. These phenomena may occur in cardiac and smooth muscle tissues in addition to skeletal muscle tissues. Thus, the above compounds, regulatory compounds which induce synthesis of these compounds, and small molecules which mimic these compounds by binding to the receptors on muscle or by stimulating through other means the second messenger systems activated by the ligand-receptor complex are all extremely useful as prophylactic and affirmative therapies for muscle diseases.
A novel aspect of the invention involves the use of the above named proteins as growth factors to induce the mitogenesis, survival, growth and differentiation of muscle cells. Treating of the muscle cells to achieve these effects may be achieved by contacting muscle cells with a polypeptide described herein. The treatments may be provided to slow or halt net muscle loss or to increase the amount or quality of muscle present in the vertebrate.
These factors may be used to produce muscle cell mitogenesis, differentiation, and survival in a vertebrate (preferably a mammal, more preferably a human) by administering to the vertebrate an effective amount of a polypeptide or a related compound. Neuregulin effects on muscle may occur, for example, by causing an increase in muscle performance by inducing the synthesis of particular isoforms of the contractile apparatus such as the myosin heavy chain slow and fast isoforms; by promoting muscle fiber survival via the induction of synthesis of protective molecules such as, but not limited to, dystrophin; and/or by increasing muscle innervation by, for example, increasing acetylcholine receptor molecules at the neuromuscular junction.
The term muscle cell as used herein refers to any cell which contributes to muscle tissue. Myoblasts, satellite cells, myotubes, and myofibril tissues are all included in the term “muscle cells” and may all be treated using the methods of the invention. Muscle cell effects may be induced within skeletal, cardiac and smooth muscles.
Mitogenesis may be induced in muscle cells, including myoblasts or satellite cells, of skeletal muscle, smooth muscle or cardiac muscle. Mitogenesis as used herein refers to any cell division which results in the production of new muscle cells in the patient. More specifically, mitogenesis in vitro is defined as an increase in mitotic index relative to untreated cells of 50%, more preferably 100%, and most preferably 300%, when the cells are exposed to labelling agent for a time equivalent to two doubling times. The mitotic index is the fraction of cells in the culture which have labelled nuclei when grown in the presence of a tracer which only incorporates during S phase (i.e., BrdU) and the doubling time is defined as the average time required for the number of cells in the culture to C increase by a factor of two.
An effect on mitogenesis in vivo is defined as an increase in satellite cell activation as measured by the appearance of labelled satellite cells in the muscle tissue of a mammal exposed to a tracer which only incorporates during S phase (i.e., BrdU). The useful therapeutic is defined in vivo as a compound which increases satellite cell activation relative to a control mammal by at least 10%, more preferably by at least 50%, and most preferably by more than 200% when the mammal is exposed to labelling agent for a period of greater than 15 minutes and tissues are assayed between 10 hours and 24 hours after administration of the mitogen at the therapeutic dose. Alternatively, satellite cell activation in vivo may be detected by monitoring the appearance of the intermediate filament vimentin by immunological or RNA analysis methods. When vimentin is assayed, the useful mitogen is defined as one which causes expression of detectable levels of vimentin in the muscle tissue when the therapeutically useful dosage is provided.
Myogenesis as used herein refers to any fusion of myoblasts to yield myotubes. Most preferably, an effect on myogenesis is defined as an increase in the fusion of myoblasts and the enablement of the muscle differentiation program. The useful myogenic therapeutic is defined as a compound which confers any increase in the fusion index in vitro. More preferably, the compound confers at least a 2.0-fold increase and, most preferably, the compound confers a 3-fold or greater increase in the fusion index relative to the control. The fusion index is defined as the fraction of nuclei present in multinucleated cells in the culture relative to the total number of nuclei present in the culture. The percentages provided above are for cells assayed after 6 days of exposure to the myogenic compound and are relative to an untreated control. Myogenesis may also be determined by assaying the number of nuclei per area in myotubes or by measurement of the levels of muscle specific protein by Western analysis. Preferably, the compound confers at least a 2.0-fold increase in the density of myotubes using the assay provided, for example, herein, and, most preferably, the compound confers a 3-fold or greater increase.
The growth of muscle may occur by the increase in the fiber size and/or by increasing the number of fibers. The growth of muscle as used herein may be measured by A) an increase in wet weight, B) an increase in protein content, C) an increase in the number of muscle fibers, or D) an increase in muscle fiber diameter. An increase in growth of a muscle fiber can be defined as an increase in the diameter where the diameter is defined as the minor axis of ellipsis of the cross section. The useful therapeutic is one which increases the wet weight, protein content and/or diameter by 10% or more, more preferably by more than 50% and most preferably by more than 100% in an animal whose muscles have been previously degenerated by at least 10% and relative to a similarly treated control animal (i.e., an animal with degenerated muscle tissue which is not treated with the muscle growth compound). A compound which increases growth by increasing the number of muscle fibers is useful as a therapeutic when it increases the number of fibers in the diseased tissue by at least 1%, more preferably at least 20%, and most preferably, by at least 50%. These percentages are determined relative to the basal level in a comparable untreated undiseased mammal or in the contralateral undiseased muscle when the compound is administered and acts locally.
The survival of muscle fibers as used herein refers to the prevention of loss of muscle fibers as evidenced by necrosis or apoptosis or the prevention of other mechanisms of muscle fiber loss. Survival as used herein indicates an decrease in the rate of cell death of at least 10%, more preferably by at least 50%, and most preferably by at least 300% relative to an untreated control. The rate of survival may be measured by counting cells stainable with a dye specific for dead cells (such as propidium iodide) in culture when the cells are 8 days post-differentiation (i.e., 8 days after the media is changed from 20% to 0.5% serum).
Muscle regeneration as used herein refers to the process by which new muscle fibers form from muscle progenitor cells. The useful therapeutic for regeneration confers an increase in the number of new fibers by at least 1%, more preferably by at least 20%, and most preferably by at least 50%, as defined above.
The differentiation of muscle cells as used herein refers to the induction of a muscle developmental program which specifies the components of the muscle fiber such as the contractile apparatus (the myofibril). The therapeutic useful for differentiation increases the quantity of any component of the muscle fiber in the diseased tissue by at least 10% or more, more preferably by 50% or more, and most preferably by more than 100% relative to the equivalent tissue in a similarly treated control animal.
Atrophy of muscle as used herein refers to a significant loss in muscle fiber girth. By significant atrophy is meant a reduction of muscle fiber diameter in diseased, injured or unused muscle tissue of at least 10% relative to undiseased, uninjured, or normally utilized tissue.
Methods for treatment of diseases or disorders using the polypeptides or other compounds described herein are also part of the invention. Examples of muscular disorders which may be treated include skeletal muscle diseases and disorders such as myopathies, dystrophies, myoneural conductive diseases, traumatic muscle injury, and nerve injury. Cardiac muscle pathologies such as cardiomyopathies, ischemic damage, congenital disease, and traumatic injury may also be treated using the methods of the invention, as may smooth muscle diseases and disorders such as arterial sclerosis, vascular lesions, and congenital vascular diseases. For example, Duchenne's muscular dystrophy, Becker's dystrophy, and Myasthenia gravis are but three of the diseases which may be treated using the methods of the invention.
The invention also includes methods for the prophylaxis or treatment of a tumor of muscle cell origin such as rhabdomyosarcoma. These methods include administration of an effective amount of a substance which inhibits the binding of one or more of the polypeptides described herein and inhibiting the proliferation of the cells which contribute to the tumor.
The methods of the invention may also be used to treat a patient suffering from a disease caused by a lack of a neurotrophic factor. By lacking a neurotrophic factor is meant a decreased amount of neurotrophic factor relative to an unaffected individual sufficient to cause detectable decrease in neuromuscular connections and/or muscular strength. The neurotrophic factor may be present at levels 10% below those observed in unaffected individuals. More preferably, the factor is present at levels 20% lower than are observed in unaffected individuals, and most preferably the levels are lowered by 80% relative to unaffected individuals under similar circumstances.
The methods of the invention make use of the fact that the neuregulin proteins are encoded by the same gene. A variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show binding to P185 erbB2 and activation of the same. Products of this gene have been used to show muscle cell mitogenic activity (see Examples 1 and 2, below), differentiation (Examples 3 and 6), and survival (Examples 4 and 5). This invention provides a use for all of the known products of the neuregulin gene (described herein and in the references listed above) which have the stated activities as muscle cell mitogens, differentiation factors, and survival factors. Most preferably, recombinant human GGF2 (rhGGF2)is used in these methods.
The invention also relates to the use of other, not yet naturally isolated, splicing variants of the neuregulin gene. FIG. 29 shows the known patterns of splicing. These patterns are derived from polymerase chain reaction experiments (on reverse transcribed RNA), analysis of cDNA clones (as presented within), and analysis of published sequences encoding neuregulins (Peles et al., Cell 69:205 (1992) and Wen et al., Cell 69:559 (1992)). These patterns, as well as additional patterns disclosed herein, represent probable splicing variants which exist. The splicing variants are fully described in Goodearl et al., U.S. Ser. No. 08/036,555, filed Mar. 24, 1993, incorporated herein by reference.
More specifically, cell division, survival, differentiation and growth of muscle cells may be achieved by contacting muscle cells with a polypeptide defined by the formula WYBAZCX (SEQ ID NOS: 212-379)
wherein WYBAZCX is composed of the polypeptide segments shown in FIG. 30 (SEQ ID NOS: 185-211) wherein W comprises the polypeptide segment F (SEQ ID NO: 206 ), or is absent wherein Y comprises the polypeptide segment E (SEQ ID NO: 207), or is absent; wherein Z comprises the polypeptide segment G (SEQ ID NO: 210) or is absent; wherein X comprises the polypeptide segment C/D HKL (SEQ ID NO: 185), C/D H (SEQ ID NO: 186), C/D HL (SEQ ID NO: 187), C/F D (SEQ ID NO: 188), C/D′HL (SEQ ID NO: 189), C/D′HKL (SEQ ID NO: 190), C/D′H (SEQ ID NO: 191), C/D′D (SEQ ID NO: 192), C/D C/D′HKL (SEQ ID NO: 193), C/D C/D′H (SEQ ID NO: 194), C/D C/D′HL (SEQ ID NO: 195), C/D C/D′D (SEQ ID NO: 196), C/D D′H (SEQ ID NO: 197), C/D D′HL (SEQ ID NO: 198), C/D D′HKL (SEQ ID NO: 199), C/D′D′H (SEQ ID NO: 200), C/D′D′HL (SEQ ID NO: 201), C/D′D′HKL (SEQ ID NO: 202), C/D C/D′D′H (SEQ ID NO: 203), C/D C/D′D′HL (SEQ ID NO: 204), or C/D C/D′D′HKL (SEQ ID NO: 205).
Furthermore, the invention includes a method of treating muscle cells by the application to the muscle cell of a
30 kD polypeptide factor isolated from the MDA-MB 231 human breast cell line; or 35 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line to the glial cell or 75 kD polypeptide factor isolated from the SKBR-3 human breast cell line; or 44 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line; or 25 kD polypeptide factor isolated from activated mouse peritoneal macrophages; or 45 kD polypeptide factor isolated from the MDA-MB 231 human breast cell; or 7 to 14 kD polypeptide factor isolated from the ATL-2 human T-cell line to the glial cell; or 25 kD polypeptide factor isolated from the bovine kidney cells; or 42 kD ARIA polypeptide factor isolated from brain; 46-47 kD polypeptide factor which stimulates 0-2A glial progenitor cells; or 43-45 kD polypeptide factor, GGFIII,175 U.S. patent application Ser. No. 07/931,041, filed Aug. 17, 1992, incorporated herein by reference.
The invention further includes methods for the use of the EGFL1, EGFL2, EGFL3, EGFL4, EGFL5, and EGFL6 polypeptides, FIG. 37 to 42 and SEQ ID Nos. 150 to 155, respectively, for the treatment of muscle cells in vivo and in vitro.
Also included in the invention is the administration of the GGF2 polypeptide whose sequence is shown in FIG. 44 for the treatment of muscle cells.
An additional important aspect of the invention are methods for treating muscle cells using:
(a) a basic polypeptide factor also known to have glial cell mitogenic activity, in the presence of fetal calf plasma, a molecular weight of from about 30 kD to about 36 kD, and including within its amino acid sequence any one or more of the following peptide sequences:
F K G D A H T E (SEQ ID NO: 1) A S L A D E Y E Y M X K (SEQ ID NO: 2) T E T S S S G L X L K (SEQ ID NO: 3) A S L A D E Y E Y M R K (SEQ ID NO: 7) A G Y F A E X A R (SEQ ID NO: 11) T T E M A S E Q G A (SEQ ID NO: 13) A K E A L A A L K (SEQ ID NO: 14) F V L Q A K K (SEQ ID NO: 15) E T Q P D P G Q I L K K V P M V I G A Y T (SEQ ID NO: 165) E Y K C L K F K W F K K A T V M (SEQ ID NO: 17) E X K F Y V P (SEQ ID NO: 19) K L E F L X A K (SEQ ID NO: 32); and
(b) a basic polypeptide factor for use in treating muscle cells which is also known to stimulate glial cell mitogenesis in the presence of fetal calf plasma, has a molecular weight of from about 55 kD to about 63 kD, and including within its amino acid sequence any one or more of the following peptide sequences:
V H Q V W A A K (SEQ ID NO: 33) Y I F F M E P E A X S S G (SEQ ID NO: 34) L G A W G P P A F P V X Y (SEQ ID NO: 35) W F V V I E G K (SEQ ID NO: 36) A S P V S V G S V Q E L Q R (SEQ ID NO: 37) V C L L T V A A L P P T (SEQ ID NO: 38) K V H Q V W A A K (SEQ ID NO: 48) K A S L A D S G E Y M X K (SEQ ID NO: 49) D L L L X V (SEQ ID NO: 39)
Methods for the use of the peptide sequences set out above, derived from the smaller molecular weight polypeptide factor, and from the larger molecular weight polypeptide factor, are also aspects of this invention. Monoclonal antibodies to the above peptides are themselves useful investigative tools and therapeutics.
Thus, the invention further embraces methods of using a polypeptide factor having activities useful for treating muscle cells and including an amino acid sequence encoded by:
(a) a DNA sequence shown in any one of FIGS. 27A , 27 B or 27 C, SEQ ID Nos. 129-131, respectively; (b) a DNA sequence shown in FIG. 21 , SEQ ID No. 85; (c) the DNA sequence represented by nucleotides 281-557 of the sequence shown in FIG. 27A , SEQ ID No. 129; or (d) a DNA sequence hybridizable to any one of the DNA sequences according to (a), (b) or (c).
Following factors as muscle cell mitogens:
(a) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, whether in reducing conditions or not, of from about 30 kD to about 36 kD on SDS-polyacrylamide gel electrophoresis which factor has muscle cell mitogenic activity including stimulating the division of myoblasts, and when isolated using reversed-phase HPLC retains at least 50% of said activity after 10 weeks incubation in 0.1% trifluoroacetic acid at 4° C.; and (b) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, under non-reducing conditions, of from about 55 kD to about 63 kD on SDS-polyacrylamide gel electrophoresis which factor the human equivalent of which is encoded by DNA clone GGF2HBS5 and which factor has muscle cell mitogenic activity and when isolated using reversed-phase HPLC retains at least 50% of the activity after 4 days incubation in 0.1% trifluoroacetic acid at 4° C.
Thus other important aspects of the invention are the use of:
(a) A series of human and bovine polypeptide factors having cell mitogenic activity including stimulating the division of muscle cells. These peptide sequences are shown in FIGS. 30 , 31 , 32 and 33 , SEQ ID Nos. 132-133, respectively. (b) A series of polypeptide factors having cell mitogenic activity including stimulating the division of muscle cells and purified and characterized according to the procedures outlined by Lupu et al. Science 249: 1552 (1990); Lupu et al. Proc. Natl. Acad. Sci USA 89: 2287 (1992); Holmes et al. Science 256: 1205 (1992); Peles et al. 69: 205 (1992); Yarden and Peles Biochemistry 30: 3543 (1991); Dobashi et al. Proc. Natl. Acad. Sci. 88: 8582 (1991); Davis et al. Biochem. Biophys. Res. Commun. 179: 1536 (1991); Beaumont et al., patent application PCT/US91/03443 (1990); Bottenstein, U.S. Pat. No. 5,276,145, issued Jan. 4, 1994; and Greene et al. patent application PCT/US91/02331 (1990). (c) A polypeptide factor (GGFBPP5) having glial cell mitogenic activity including stimulating the division of muscle cells. The amino acid sequence is shown in FIG. 31 , SEQ ID No. 144.
Methods for stimulating mitogenesis of a myoblast by contacting the myoblast cell with a polypeptide defined above as a muscle cell mitogen in vivo or in vitro are included as features of the invention.
Muscle cell treatments may also be achieved by administering DNA encoding the polypeptide compounds described above in an expressible genetic construction. DNA encoding the polypeptide may be administered to the patient using techniques known in the art for delivering DNA to the cells. For example, retroviral vectors, electroporation or liposomes may be used to deliver DNA.
The invention includes the use of the above named family of proteins as extracted from natural sources (tissues or cell lines) or as prepared by recombinant means.
Other compounds in particular, peptides, which bind specifically to the p185 erbB2 receptor can also be used according to the invention as muscle cell mitogens. A candidate compound can be routinely screened for p185 erbB2 binding, and, if it binds, can then be screened for glial cell mitogenic activity using the methods described herein.
The invention includes use of any modifications or equivalents of the above polypeptide factors which do not exhibit a significantly reduced activity. For example, modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included. The statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent factors being part of the invention.
The human peptide sequences described above and presented in FIGS. 30 , 31 , 32 and 33 , SEQ ID Nos. 132-146, respectively, represent a series of splicing variants which can be isolated as full length complementary DNAs (cDNAS) from natural sources (cDNA libraries prepared from the appropriate tissues) or can be assembled as DNA constructs with individual exons (e.g., derived as separate exons) by someone skilled in the art.
The invention also includes a method of making a medicament for treating muscle cells, i.e., for inducing muscular mitogenesis, myogenesis, differentiation, or survival, by administering an effective amount of a polypeptide as defined above. Such a medicament is made by administering the polypeptide with a pharmaceutically effective carrier.
Another aspect of the invention is the use of a pharmaceutical or veterinary formulation comprising any factor as defined above formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form. In using the factors of the invention, conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions.
Thus, the formulations to be used as a part of the invention can be applied to parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal or vaginal administration.
The formulations of this invention may also be administered by the transplantation into the patient of host cells expressing the DNA encoding polypeptides which are effective for the methods of the invention or by the use of surgical implants which release the formulations of the invention.
Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are to be found in, for example, “Remington's Pharmaceutical Sciences.” Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.
The present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors.
The concentration of the present factors in the formulations of the invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.
In general terms, the factors of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. General dose ranges are from about 1 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage to be administered is likely to depend upon the type and extent of progression of the pathophysiological condition being addressed, the overall health of the patient, the make up of the formulation, and the route of administration.
The polypeptide factors utilized in the methods of the invention can also be used as immunogens for making antibodies, such as monoclonal antibodies, following standard techniques. These antibodies can, in turn, be used for therapeutic or diagnostic purposes. Thus, conditions perhaps associated with muscle-diseases resulting from abnormal levels of the factor may be tracked by using such antibodies. In vitro techniques can be used, employing assays on isolated samples using standard methods. Imaging methods in which the antibodies are, for example, tagged with radioactive isotopes which can be imaged outside the body using techniques for the art of tumor imaging may also be employed.
A further general aspect of the invention is the use of a factor of the invention in the manufacture of a medicament, preferably for the treatment of a muscular disease or disorder. The “GGF2” designation is used for all clones which were previously isolated with peptide sequence data derived from GGF-II protein (i.e., GGF2HBS5, GGF2BPP3) and, when present alone (i.e., GGF2 or rhGGF2), to indicate recombinant human protein encoded by plasmids isolated with peptide sequence data derived from the GGF-II protein (i.e., as produced in insect cells from the plasmid HBS5). Recombinant human GGF from the GGFHBS5 clone is called GGF2, rhGGF2 and GGF2HBS5 polypeptide.
Treating as used herein means any administration of the compounds described herein for the purpose of increasing muscle cell mitogenesis, survival, and/or differentiation, and/or decreasing muscle atrophy and degeneration. Most preferably, the treating is for the purpose of reducing or diminishing the symptoms or progression of a disease or disorder of the muscle cells. Treating as used herein also means the administration of the compounds for increasing or altering the muscle cells in healthy individuals. The treating may be brought about by the contacing of the muscle cells which are sensitive or responsive to the compounds described herein with an effective amount of the compound, as described above. Inhibitors of the compounds described herein may also be used to halt or slow diseases of muscle cell proliferation.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings will first be described.
Drawings
FIG. 1 is a graph showing the results of rhGGF2 in a myoblast mitogenesis assay.
FIG. 2 is a graph showing the effect of rhGGF2 on the number of nuclei in myotubes.
FIG. 3 is a graph of a survival assay showing the effect of rhGGF2 on survival of differentiated myotubes.
FIG. 4 is a graph of survival assays showing the effect of rhGGF2 on differentiated myotubes relative to human platelet derived growth factor, human fibroblast growth factor, human epidermal growth factor, human leucocyte inhibitory factor, and human insulin-like growth factors I and II.
FIG. 5 is a graph showing the increased survival on Duchenne muscular dystrophy cells in the presence of rhGGF2.
FIG. 6 is a graph of increasing human growth hormone (hGH) expression in C2 cells from an hGH reporter gene under control of the AchR delta subunit transcriptional control elements. This increase is tied to the addition of GGF2 to the media.
FIG. 7 is a graph of increasing hGH reporter synthesis and bungarotoxin (BTX) binding to AchRs following the addition of increasing amounts of GGF2 to C2 cells.
FIGS. 8 , 9 , 10 and 11 are the peptide sequences derived from GGF-I and GGF-II, SEQ ID Nos. 1-20, 22-29, 32-50 and 165, (see Examples 11-13 hereinafter).
FIG. 9 , Panel A, is the sequences of GGF-I peptides used to design degenerate oligonucleotide probes and degenerate PCR primers are listed (SEQ ID Nos. 1, 17 and 22-29). Some of the sequences in Panel A were also used to design synthetic peptides. Panel B is a listing of the sequences of novel peptides that were too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID Nos. 17 and 32);
FIG. 11 , Panel A, is a listing of the sequences of GGF-II peptides used to design degenerate oligonucleotide probes and degenerate PCR primers (SEQ ID Nos. 45-52). Some of the sequences in Panel A were used to design synthetic peptides. Panel B is a listing of the novel peptide that was too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID No. 53);
FIGS. 12 , 13 A, 13 B, 14 , 15 , 16 , 17 , 18 , and 19 relate to Example 8, below, and depict the mitogenic activity of factors of the invention;
FIGS. 20 , 21 , 22 , 23 , 24 , 25 , 26 , and 27 relate to Example 10, below and are briefly described below:
FIG. 20 is a listing of the degenerate oligonucleotide probes (SEQ ID Nos. 51-84) designed from the novel peptide sequences in FIG. 7 , Panel A and FIG. 9 , Panel A;
FIG. 21 (SEQ ID No. 85) depicts a stretch of the putative bovine GGF-II gene sequence from the recombinant bovine genomic phage GGF2BG1, containing the binding site of degenerate oligonucleotide probes 609 and 650 (see FIG. 20 , SEQ ID Nos. 66 and 69, respectively). The figure is the coding strand of the DNA sequence and the deduced amino acid sequence in the third reading frame. The sequence of peptide 12 from factor 2 (bold) is part of a 66 amino acid open reading frame (nucleotides 75272);
FIG. 22 is the degenerate PCR primers (Panel A, SEQ ID Nos. 86-104) and unique PCR primers (Panel B, SEQ ID Nos. 105-115) used in experiments to isolate segments of the bovine GGF-II coding sequences present in RNA from posterior pituitary;
FIG. 23 depicts of the nine distinct contiguous bovine GGF-II cDNA structures and sequences that were obtained in PCR amplification experiments. The top line of the Figure is a schematic of the coding sequences which contribute to the cDNA structures that were characterized;
FIG. 24 is a physical map of bovine recombinant phage of GGF2BG1. The bovine fragment is roughly 20 kb in length and contains two exons (bold) of the bovine GGF-II gene. Restriction sites for the enzymes Xbal, SpeI, Ndel, EcoRI, Kpnl, and SstI have been placed on this physical map. Shaded portions correspond to fragments which were subcloned for sequencing;
FIG. 25 is a schematic of the structure of three alternative gene products of the putative bovine GGF-II gene. Exons are listed A through E in the order of their discovery. The alternative splicing patterns 1, 2 and 3 generate three overlapping deduced protein structures (GGF2BPP1, 2, and 3), which are displayed in the various FIGS. 27A , 27 B, 27 C (described below);
FIG. 26 (SEQ ID Nos. 116-128 and 380-382) is a comparison of the GGF-I and GGF-II sequences identified in the deduced protein sequences shown in FIGS. 27A , 27 B, 27 C (described below) with the novel peptide sequences listed in FIGS. 9 and 11 . The figure shows that six of the nine novel GGF-II peptide sequences are accounted for in these deduced protein sequences. Two peptide sequences similar to GGF-I sequences are also found;
FIG. 27 (SEQ ID Nos. 129 and 409) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 1 in FIG. 25 . This partial cDNA of the putative bovine GGF-II gene encodes a protein of 206 amino acids in length. Peptides in bold were those identified from the lists presented in FIGS. 9 and 11 . Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA);
FIG. 27 (SEQ ID Nos. 130 and 410) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 2 in FIG. 25 . This partial cDNA of the putative bovine GGF-II gene encodes a protein of 281 amino acids in length. Peptides in bold were those identified from the lists presented in FIGS. 7 and 9 . Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA);
FIG. 27 (SEQ ID Nos. 131 and 411) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 3 in FIG. 25 . This partial cDNA of the putative bovine GGF-II gene encodes a protein of 257 amino acids in length. Peptides in bold were those identified from the lists presented in FIGS. 9 and 11 . Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA);
FIG. 28 which relates to Example 15 hereinafter, is an autoradiogram of a cross hybridization analysis of putative bovine GGF-II gene sequences to a variety of mammalian DNAs on a southern blot. The filter contains lanes of EcoRI-digested DNA (5 μg per lane) from the species listed in the Figure. The probe detects a single strong band in each DNA sample, including a four kilobase fragment in the bovine DNA as anticipated by the physical map in FIG. 24 . Bands of relatively minor intensity are observed as well, which could represent related DNA sequences. The strong hybridizing band from each of the other mammalian DNA samples presumably represents the GGF-II homologue of those species.
FIG. 29 is a diagram of representative splicing variants. The coding segments are represented by F, E, B, A, G, C, C/D, C/D′, D, D′, H, K and L. The location of the peptide sequences derived from purified protein are indicated by “o”.
FIG. 30 (SEQ ID Nos. 132-143, 156, 157, 159, 169-178, and 383-405) is a listing of the DNA sequences and predicted peptide sequences of the coding segments of GGF. Line 1 is a listing of the predicted amino acid sequences of bovine GGF, line 2 is a listing of the nucleotide sequences of bovine GGF, line 3 is a listing of the nucleotide sequences of human GGF (heregulin) (nucleotide base matches are indicated with a vertical line) and line 4 is a listing of the predicted amino acid sequences of human GGF/heregulin where it differs from the predicted bovine sequences, Coding segment E, A′ and K represent only the bovine sequences. Coding segment D′ represents only the human (heregulin) sequence.
FIG. 31 (SEQ ID Nos. 144 and 406) is the predicted GGF2 amino acid sequence and nucleotide sequence of BPP5. The upper line is the nucleotide sequence and the lower line in the predicted amino acid sequence.
FIG. 32 (SEQ ID Nos. 145 and 407) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP2. The upper line is the nucleotide sequence and the lower line in the predicted amino acid sequence.
FIG. 33 (SEQ ID Nos. 146 and 408) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP4. The upper line is the nucleotide sequence and the lower line in the predicted amino acid sequence.
FIG. 34 (SEQ ID Nos. 147-149) depicts the alignment of two GGF peptide sequences (GGF2BPP4 and GGF2BPP5) with the human EGF (hEGF). Asterisks indicate positions of conserved cysteines.
FIG. 35 depicts the level of GGF activity (Schwann cell mitogenic assay) and tyrosine phosphorylation of a ca. 200 kD protein (intensity of a 200 kD band on an autoradiogram of a Western blot developed with an antiphosphotyrosine polyclonal antibody) in response to increasing amounts of GGF.
FIG. 36 is a list of splicing variants derived from the sequences shown in FIG. 30 .
FIG. 37 is the predicted amino acid sequence, by bottom, and nucleic sequence, top, of EGFL1 (SEQ ID Nos. 150 and 412).
FIG. 38 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL2 (SEQ ID Nos. 151 and 413).
FIG. 39 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL3 (SEQ ID Nos. 152 and 414).
FIG. 40 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL4 (SEQ ID Nos. 153 and 415).
FIG. 41 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL5 (SEQ ID Nos. 154 and 416).
FIG. 42 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL6 (SEQ ID Nos. 155 and 417).
FIG. 43 is a scale coding segment map of the clone. T3 refers to the bacteriophage promoter used to produce mRNA from the clone. R=flanking EcoRI restriction enzyme sites. 5′ UT refers to the 5′ untranslated region. E, B, A, C, C/D′, and D refer to the coding segments. O=the translation start site. Λ=the 5′ limit of the region homologous to the bovine E segment (see Example 16) and 3′ UT refers to the 3′ untranslated region.
FIG. 44 is the predicted amino acid sequence (middle) and nucleic sequence (top) of GGF2HBS5 (SEQ ID No. 21). The bottom (intermittent) sequence represents peptide sequences derived from GGF-II preparations (see FIGS. 8 , 9 ).
FIG. 45 (A) is a graph showing the purification of rGGF on cation exchange column by fraction; FIG. 45 (B) is a photograph of a Western blot using fractions as depicted in (A) and a GGFII specific antibody.
FIG. 46 is the sequence of the GGFHBS5, GGFHFB1 and GGFBPP5 polypeptides (SEQ ID NOS: 166, 167, and 168).
FIG. 47 is a map of the plasmid pcDHRFpolyA.
DETAILED DESCRIPTION
The invention pertains to the use of isolated and purified neuregulin factors and DNA sequences encoding these factors, regulatory compounds which increase the extramuscular concentrations of these factors, and compounds which are mimetics of these factors for the induction of muscle cell mitogenesis, differentiation, and survival of the muscle cells in vivo and in vitro.
It is evident that the gene encoding GGF/p 185 erbB2 binding neuregulin proteins produces a number of variably-sized, differentially-spliced RNA transcripts that give rise to a series of proteins. These proteins are of different lengths and contain some common peptide sequences and some unique peptide sequences. The conclusion that these factors are encoded by a single gene is supported by the differentially-spliced RNA sequences which are recoverable from bovine posterior pituitary and human breast cancer cells (MDA-MB-231)). Further support for this conclusion derives from the size range of proteins which act as both mitogens for muscle tissue (as disclosed herein) and as ligands for the p185 erbB2 receptor (see below).
Further evidence to support the fact that the genes encoding GGF/p185 erbB2 binding proteins are homologous comes from nucleotide sequence comparison. Holmes et al., (Science 256:1205-1210, 1992) demonstrate the purification of a 45-kilodalton human protein (Heregulin-α) which specifically interacts with the receptor protein p185 erbB2 . Peles et al. (Cell 69:205 (1992)) and Wen et al. (Cell 69:559 (1992)) describe a complementary DNA isolated from rat cells encoding a protein called “neu differentiation factor” (NDF). The translation product of the NDF cDNA has p185 erbB2 binding activity. Several other groups have reported the purification of proteins of various molecular weights with p185 erbB2 binding activity. These groups include Lupu et al. ((1992) Proc. Natl. Acad. Sci. USA 89:2287); Yarden and Peles ((1991) Biochemistry 30:3543); Lupu et al. ((1990) Science 249:1552)); Dobashi et al. ((1991) Biochem. Biophys. Res. Comm. 179:1536); and Huang et al. ((1992) J. Biol. Chem. 257:11508-11512).
We have found that p185 erbB2 receptor binding proteins stimulate muscle cell mitogenesis and hence, stimulates myotube formation (myogenesis). This stimulation results in increased formation of myoblasts and increased formation of myotubes (myogenesis). The compounds described herein also stimulate increased muscle growth, differentiation, and survival of muscle cells. These ligands include, but are not limited to the GGF's, the neuregulins, the heregulins, NDF, and ARIA. As a result of this mitogenic activity, these proteins, DNA encoding these proteins, and related compounds may be administered to patients suffering from traumatic damage or diseases of the muscle tissue. It is understood that all methods provided for the purpose of mitogenesis are useful for the purpose of myogenesis. Inhibitors of these ligands (such as antibodies or peptide fragments) may be administered for the treatment of muscle derived tumors.
These compounds may be obtained using the protocols described herein (Examples 9-17) and in Holmes et al., Science 256: 1205 (1992); Peles et al., Cell 69:205 (1992); Wen et al., Cell 69:559 (1992); Lupu et al., Proc. Natl. Acad. Sci. USA 89:2287 (1992); Yarden and Peles, Biochemistry 30:3543 (1991); Lupu et al., Science 249:1552 (1990); Dobashi et al., Biochem. Biophys. Res. Comm . 179:1536 (1991); Huang et al., J. Biol. Chem . 257:11508-11512 (1992); Marchionni et al., Nature 362:313, (1993); and in the GGF-III U.S. application Ser. No. 07/931,041 all of which are incorporated herein by reference. The sequences are provided and the characteristics described for many of these compounds. For sequences see FIGS. 8-11 , 20 - 27 C, 29 - 34 , 36 - 44 , and 46 . For protein characteristics see FIGS. 12-19 , 28 35 , 45 A and 45 B.
Compounds may be assayed for their usefulness in vitro using the methods provided in the examples below. In vivo testing may be performed as described in Example 1 and in Sklar et al., In Vitro Cellular and Developmental Biology 27A:433-434, 1991.
OTHER EMBODIMENTS
The invention includes methods for the use of any protein which is substantially homologous to the coding segments in FIG. 30 (SEQ ID Nos.: 132-143, 156, and 157) as well as other naturally occurring GGF polypeptides for the purpose of inducing muscle mitogenesis. Also included are the use of: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid naturally occurring (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1-6.3.6, hereby incorporated by reference); and the use of polypeptides or proteins specifically bound by antisera to GGF polypeptides. The term also includes the use of chimeric polypeptides that include the GGF polypeptides comprising sequences from FIG. 28 for the induction of muscle mitogenesis.
As will be seen from Example 8, below, the present factors exhibit mitogenic activity on a range of cell types. The general statements of invention above in relation to formulations and/or medicaments and their manufacture should clearly be construed to include appropriate products and uses.
A series of experiments follow which provide additional basis for the claims described herein. The following examples relating to the present invention should not be construed as specifically limiting the invention, or such variations of the invention, now known or later developed.
The examples illustrate our discovery that recombinant human GGF2 (rhGGF2) confers several effects on primary human muscle culture. rhGGF2 has significant effects in three independent biological activity assays on muscle cultures. The polypeptide increased mitogenesis as measured by proliferation of subconfluent quiescent myoblasts, increased differentiation by confluent myoblasts in the presence of growth factor, and increased survival of differentiated myotubes as measured by loss of dye exclusion and increased acetylcholine receptor synthesis. These activities indicate efficacy of GGF2 and other neuregulins in inducing muscle repair, regeneration, and prophylactic effects on muscle degeneration.
EXAMPLE 1
Mitogenic Activity of rhGGF on Myoblasts Clone GGF2HBS5 was expressed in recombinant Baculovirus infected insect cells as described in Example 13, infra, and the resultant recombinant human GGF2 was added to myoblasts in culture (conditioned medium added at 40 μl/ml). Myoblasts (057A cells) were grown to preconfluence in a 24 well dish. Medium was removed and replaced with DMEM containing 0.5% fetal calf serum with or without GGF2 conditioned medium at a concentration of 40 μl/ml. Medium was changed after 2 days and cells were fixed and stained after 5 days. Total nuclei were counted as were the number of nuclei in myoblasts (Table 1).
TABLE 1 Total Number Nuclei in Fusion Treatment of Nuclei/mm 2 Myotubes Index Control 395 ± 28.3 204 ± 9.19 0.515 ± 0.01 GGF 40 μl/ml 636 ± 8.5 381 ± 82.7 0.591 ± 0.15
GGF treated myoblasts showed an increased number of total nuclei (636 nuclei) over untreated controls (395 nuclei) indicating mitogenic activity. rhGGF2 treated myotubes had a greater number of nuclei (381 nuclei) than untreated controls (204 nuclei). Thus, rhGGF2 enhances the total number of nuclei through proliferation and increased cell survival. rhGGF2 is also likely to enhance the formation of myotubes.
The mitogenic activity of rhGGF2 may be measured in vivo by giving a continuous supply of GGF2 and [ 3 H]thymidine to rat muscle via an osmotic mini pump. The muscle bulk is determined by wet weight after one and two weeks of treatment. DNA replication is measured by counting labeled nuclei in sections after coating for autoradiography (Sklar et al., In Vitro Cellular and Developmental Biology 27A:433-434, 1991) in sham and rhGGF2-treated muscle. Denervated muscle is also examined in this rat animal model via these methods and this method allows the assessment of the role of rhGGF2 in muscle atrophy and repair. Mean fiber diameter can also be used for assessing effects of FGF on prevention of atrophy.
EXAMPLE 2
Effect of rhGGF2 on Muscle Cell Mitogenesis
Quiescent primary clonal human myoblasts were prepared as previously described (Sklar, R., Hudson, A., Brown, R., In vitro Cellular and Developmental Biology 1991; 27A:433-434). The quiescent cells were treated with the indicated agents (rhGGF2 conditioned media, PDGF with and without methylprednisolone, and control media) in the presence of 10 μM BrdU, 0.5% FCS in DMEM. After two days the cells were fixed in 4% paraformaldehyde in PBS for 30 minutes, and washed with 70% ethanol. The cells were then incubated with an anti-BrdU antibody, washed, and antibody binding was visualized with a peroxidase reaction. The number of staining nuclei were then quantified per area. The results show that GGF2 induces an increase in the number of labelled nuclei per area over controls (see Table 2).
TABLE 2 Mitogenic Effects of GGF on Human Myoblasts Labelled T-Test Treatment Nuclei/cm 2 p value Control 120 ± 22.4 Infected Control 103 ± 11.9 GGF 5 μl/ml 223 ± 33.8 0.019 PDGF 20 ng/ml 418 ± 45.8 0.0005 IGFI 30 ng/ml 280 ± 109.6 0.068 Methylprednisolone 1.0 μM 142 ± 20.7 0.293
Platelet derived growth factor (PDGF) was used as a positive control. Methylprednisolone (a corticosteroid) was also used in addition to rhGGF2 and showed no significant increase in labelling of DNA.
rhGGF2 purified to homogeneity (<95% pure) is also mitogenic for human myoblasts (FIG. 1 ).
Recombinant human GGF2 also causes mitogenesis of primary human myoblasts (see Table 2 and FIG. 1 ). The mitogenesis assay is performed as described above. The mitotic index is then calculated by dividing the number of BrdU positive cells by the total number of cells.
EXAMPLE 3
Effect of rhGGF2 on Muscle Cell Differentiation The effects of purified rhGGF2 (95% pure) on muscle culture differentiation were examined (FIG. 2 ). Confluent myoblast cultures were induced to differentiate by lowering the serum content of the culture medium from 20% to 0.5%. The test cultures were treated with the indicated concentration of rhGGF2 for six days, refreshing the culture medium every 2 days. The cultures were then fixed, stained, and the number of nuclei counted per millimeter. The data in FIG. 2 demonstrate a large increase in the number of nuclei in myotubes when rhGGF2 is present, relative to controls.
EXAMPLE 4
Effect of rhGGF2 on the Survival of Differentiated Myotubes The survival of differentiated myotubes was significantly increased by rhGGF2 treatment. Muscle cultures were differentiated in the presence of rhGGF2 and at various times the number of dead myotubes were counted by propidium iodide staining. As can be seen in FIG. 3 , the number of dead myotubes is lower in the rhGGF2 treated culture at 4, 5, 6, and 8 days of differentiation. The number of nuclei in myotubes was significantly increased by GGF2 treatment compared to untreated cultures after 8 days of differentiation. Specifically, the control showed 8.6 myonuclei/mm 2 , while rhGGF2 treated cultures showed 57.2 myonuclei/mm 2 (p=0.035) when counted on the same plates after geimsa staining.
The survival assay was also performed with other growth factors which have known effects on muscle culture. The rhGGF2 effect was unique among the growth factors tested (FIG. 4 ). In this experiment cultures were treated in parallel with the rhGGF2 treated plates with the indicated concentrations of the various growth factors. Survival of myotubes was measured as above at 8 days of differentiation of 057A myoblast cells. Concentrations of factors were as follows: rhGGF2: 100 ng/ml; human platelet derived growth factor: 20 ng/ml; human basic fibroblast growth factor: 25 ng/ml; human epidermal growth factor: 30 ng/ml; human leucocyte inhibitory factor: 10 ng/ml; human insulin like growth factor I: 30 ng/ml; human insulin like growth factor II: 25 ng/ml.
The observed protection of differentiated myotubes from death indicates that rhGGF2 has promise as a therapy for intervention of muscle degeneration characterized by numerous muscle diseases. Thus, agents which increase the extramuscular concentration of neuregulins may have a prophylactic effect or slow the progress of muscle-wasting disorders and increase rates of muscle differentiation, repair, conditioning, and regeneration.
EXAMPLE 5
rhGGF2 Promotes Survival of Differentiated Myotubes With a Genetic Defect at the Duchenne Muscular Dystrophy Locus
The positive effects of rhGGF2 on myotube survival could reflect potential efficacy in degenerative disorders. These effects on myotube survival were tested on a clonally-derived primary Duchenne myoblast to determine if the response observed in normal muscle culture could also be demonstrated in cultures derived from diseased individuals. The data presented in FIG. 5 was obtained using the same muscle culture conditions (Example 4, above) used for normal individual. rhGGF2 significantly decreased the number of dead myotubes in the differentiated Duchenne muscle culture, compared to controls (p=0.032). Concentrations were as follows: GGF2: 100 ng/ml; human platelet derived growth factor: 20 ng/ml; human insulin like growth factor I: 30 ng/ml.
This example demonstrates that rhGGF2 can also promote survival of differentiated Duchenne myotubes and provides strong evidence that rhGGF2 may slow or prevent the course of muscle degeneration and wasting in mammals.
EXAMPLE 6
rhGGF2 Effect on the Differentiation Program: Induction of MHC Slow and Dystrophin Proteins
The effects of purified rhGGF2 on muscle culture differentiation was also examined by Western analysis of culture lysates. The levels of muscle specific proteins were determined in triplicate treated and untreated cultures. These cultures were prepared and treated as above except that the plate size was increased to 150 mm and the muscle culture layer was scraped off for Western analysis as described in Sklar, R., and Brown, R. ( J. Neurol. Sci . 101:73-81, 1991). The results presented in Table A indicate that rhGGF2 treatment increases the levels of several muscle specific proteins, including dystrophin, myosin heavy chain (MHC, adult slow and fast isoforms), but does not increase the levels of HSP72 or MHC neonate isoform to a similar level per amount of protein loaded on the Western. The levels of muscle specific proteins induced by rhGGF2 were similar to the quantitative increases in the number of myonuclei/mm 2 (Table 3).
TABLE 3
rhGGF2
Control ± SD
Treatment ± SD
p value
Total Protein (μg)
554 ± 38.4
798 ± 73.6
0.007
Myonuclei/mm 2
29.0 ± 12.2
106 ± 24.1
0.008
MHC fast/μg protein
1.22 ± 0.47
4.00 ± 0.40
0.001
MHC slow/μg protein
0.17 ± 0.13
1.66 ± 0.27
0.001
MHC neonate/μg protein
0.30 ± 0.27
0.55 ± 0.04
0.199
dystrophin/μg protein
6.67 ± 0.37
25.5 ± 11.0
0.042
HSP 72/μg protein
3.30 ± 0.42
4.54 ± 0.08
0.008
The rhGGF2 dependent increase in the adult myosin heavy chain isoforms (slow is found in type I human muscle fibers; fast is found in type 2A and 2B human muscle fibers) may represent a maturation of the myotubes, as the neonatal isoform was not significantly increased by rhGGF2 treatment.
During rat muscle development MHC isoforms switch from fetal to neonatal forms followed by a switch to mature adult slow and fast MHC isoforms (Periasamy et al. J. Biol. Chem . 259:13573-13578, 1984; Periasamy et al. J. Biol. Chem . 260:15856-15862, 1985; Wieczorek et al. J. Cell Biol . 101:618-629, 1985). While muscle can autonomously undergo some of these isoform transitions in the absence of neural cells or tissue, mouse muscle explants express the adult fast MHC isoform only when cultured in the presence of mouse spinal cord (Ecob-Prince et al. J. Cell Biol . 103:995-1005, 1986). Additional evidence that MHC isoform transitions are influenced by nerve was established by Whalen et al. ( Deve. Biol . 141:24-40, 1990); after regeneration of notexin treated rat soleus muscles only the adult fast MHC isoform was produced in the new denervated muscle, but innervated regenerated muscle made both fast and slow adult MHC isoforms. Thus the demonstration in Table 3 that rhGGF2 increases the synthesis of adult MHC isoforms indicates that rhGGF2 may induce a developmental maturation of muscle which may mimic neuronal innervation.
EXAMPLE 7
Neuregulins, Including rhGGF2, Induce the Synthesis of Acetylcholine Receptors in Muscle
The expression of acetylcholine receptor (AchR) subunit proteins can be induced by exposing muscle cells to neuregulins. More specifically, we have shown that contacting muscle cells with rhGGF2 can induce the synthesis of AchR subunit proteins. This induction following rhGGF2 exposure was observed in two ways: first, we detected increased expression of human growth hormone via the product of a reporter gene construct and second we detected increased binding of alpha-bungarotoxin to cells.
In the following example a mouse myoblast cell line C2 was used. C2 cells were transfected with a transgene that contained the 5′ regulatory sequences of the AChR delta subunit gene of mouse linked to a human growth hormone full-length cDNA (Baldwin and Burden, 1988. J. Cell Biol. 107:2271-2279). This reporter construct allows the measurement of the induction of AChR delta gene expression by assaying the quantity of growth hormone secreted into the media. The line can be induced to form myotubes by lowering serum concentration in the media from 20% to 0.5%.
Specifically, mouse C2 myoblasts transfected with an AChR-human growth hormone reporter construct and were assayed for expression of hGH following treatment with rhGGF2. The results of two separate experiments are summarized in Table 4 and in FIGS. 6 (hGH expression) and 7 (hGH expression and alpha-bungarotoxin binding). Shown are the dose response curves for secreted human growth hormone and for bungarotoxin binding from muscle cultures treated with rhGGF2.
TABLE 4
Effects of rhGGF2 on the expression of AChR delta
subunit/hGH transgene and the synthesis of AChR
Exp 1
Exp 2
GGF
hGH
hGH
AChR
(ul)
(ng/ml)
(ng/ml)
(cpm/mg protein)
0
9.3 + 2.1
5.7 + 2.1
822 + 170
0.1
—
6.8 + 1.5
891 + 134
0.5
—
12.0 + 0.9
993 + 35
1.0
—
9.7 + 2.3
818 + 67
5.0
17.5 + 2.8
14.7 + 3.5
1300 + 177
10.0
14.3 + 3.2
14.1 + 3.3
1388 + 137
15.0
22.0 + 1.4
—
—
C2 myotubes were treated with cold α-BTX (20 nM) for 1 hr. at 37° C., washed with culture medium twice and then treated with GGF2. Culture medium was adjusted with bovine serum albumin at the concentration of 1 mg/ml. 24 hours later, culture medium was removed and saved for hGH assay. Muscle cultures were treated with 125 I-α-BTX (20 nM) for 1 hour at 37° C., washed and scraped in PBS containing 1% SDS. Non-specific binding was determined in the presence of cold α-BTX (40 nM). The cell homogenate was counted for radioactivity and assayed for total protein amount.
The presence of rhGGF2 led to a greater than 2-fold increase in hGH gene expression, thereby indicating that rhGGF2 induced the synthesis of the delta subunit of the acetylcholine receptor. Furthermore, increased bungarotoxin binding is consistant with assembly of these subunit proteins into functional acetylcholine receptors. To strenthen the interpretation of these data the analysis was repeated on cultures that had the hGH reporter linked to a metallothiene promotor, which should not be responsive to rhGGF2. The results of that control experiment showed that the hGH response was mediated through transcriptional activation of the AchR delta subunit gene control elements.
These results indicate that rhGGF2 could be useful in replenishing AchRs as part of the therapy for the autoimmune disease Myasthenia gravis. This activity may also be beneficial in treatment of peripheral nerve regeneration and neuropathy by stimulating a key step in re-innervation of muscle.
EXAMPLE 8
Additional Mitogenic Activities of Purified GGF-I and GGF-II
The mitogenic activity of a highly purified sample containing both GGFs I and II was studied using a quantitative method, which allows a single microculture to be examined for DNA synthesis, cell morphology, cell number and expression of cell antigens. This technique has been modified from a method previously reported by Muir et al., Analytical Biochemistry 185, 377-382, 1990. The main modifications are: 1) the use of uncoated microtiter plates, 2) the cell number per well, 3) the use of 5% Foetal Bovine Plasma (FBP) instead of 10% Foetal Calf Serum (FCS), and 4) the time of incubation in presence of mitogens and bromodeoxyuridine (BrdU), added simultaneously to the cultures. In addition the cell monolayer was not washed before fixation to avoid loss of cells, and the incubation time of monoclonal mouse anti-BrdU antibody and peroxidase conjugated goat anti-mouse immunoglobulin (IgG) antibody were doubled to increase the sensitivity of the assay. The assay, optimized for rat sciatic nerve Schwann cells, has also been used for several cell lines, after appropriate modifications to the cell culture conditions.
I. Methods of Mitogenesis Testing
On day 1, purified Schwann cells were plated onto uncoated 96 well plates in 5% FBP/Dulbecco's Modified Eagle Medium (DMEM) (5,000 cells/well). On day 2, GGFs or other test factors were added to the cultures, as well as BrdU at a final concentration of 10 μm. After 48 hours (day 4) BrdU incorporation was terminated by aspirating the medium and cells were fixed with 200 μl/well of 70% ethanol for 20 min at room temperature. Next, the cells were washed with water and the DNA denatured by incubation with 100 μl 2N HCl for 10 min at 37° C. Following aspiration, residual acid was neutralized by filling the wells with 0.1 M borate buffer, pH 9.0, and the cells were washed with phosphate buffered saline (PBS). Cells were then treated with 50 μl of blocking buffer (PBS containing 0.1% Triton X 100 and 2% normal goat serum) for 15 min at 37° C. After aspiration, monoclonal mouse anti-BrdU antibody (Dako Corp., Santa Barbara, Calif.) (50 μl/well, 1.4 μg/ml diluted in blocking buffer) was added and incubated for two hours at 37° C. Unbound antibodies were removed by three washes in PBS containing 0.1% Triton X-100 and peroxidase-conjugated goat anti-mouse IgG antibody (Dako Corp., Santa Barbara, Calif.) (50 μl/well, 2 μg/ml diluted in blocking buffer) was added and incubated for one hour at 37° C. After three washes in PBS/Triton and a final rinse in PBS, wells received 100 μl/well of 50 mM phosphate/citrate buffer, pH 5.0, containing 0.05% of the soluble chromogen o-phenylenediamine (OPD) and 0.02% H 2 0 2 . The reaction was terminated after 5-20 min at room temperature, by pipetting 80 μl from each well to a clean plate containing 40 μl/well of 2N sulfuric acid. The absorbance was recorded at 490nm using a plate reader (Dynatech Labs). The assay plates containing the cell monolayers were washed twice with PBS and immunocytochemically stained for BrdU-DNA by adding 100 μl/well of the substrate diaminobenzidine (DAB) and 0.02% H 2 O 2 to generate an insoluble product. After 10-20 min the staining reaction was stopped by washing with water, and BrdU-positive nuclei observed and counted using an inverted microscope. occasionally, negative nuclei were counterstained with 0.001% Toluidine blue and counted as before.
II. Cell lines used for Mitogenesis Assays
Swiss 3T3 Fibroblasts: Cells, from Flow Labs, were maintained in DMEM supplemented with 10% FCS, penicillin and streptomycin, at 37° C. in a humidified atmosphere of 10% CO 2 in air. Cells were fed or subcultured every two days. For mitogenic assay, cells were plated at a density of 5,000 cells/well in complete medium and incubated for a week until cells were confluent and quiescent. The serum containing medium was removed and the cell monolayer washed twice with serum free-medium. 100 μl of serum free medium containing mitogens and 10 μM of BrdU were added to each well and incubated for 48 hours. Dose responses to GGFs and serum or PDGF (as a positive control) were performed.
BHK (Baby Hamster Kidney) 21 C13 Fibroblasts: Cells from European Collection of Animal Cell Cultures (ECACC), were maintained in Glasgow Modified Eagle Medium (GMEM) supplemented with 5% tryptose phosphate broth, 5% FCS, penicillin and streptomycin, at 37° C. in a humidified atmosphere of 5% CO 2 in air. Cells were fed or subcultured every two to three days. For mitogenic assay, cells were plated at a density of 2,000 cell/well in complete medium for 24 hours. The serum containing medium was then removed and after washing with serum free medium, replaced with 100 Al of 0.1% FCS containing GMEM or GMEM alone. GGFs and FCS or bFGF as positive controls were added, coincident with 10 μM BrdU, and incubated for 48 hours. Cell cultures were then processed as described for Schwann cells.
C6 Rat Glioma Cell Line: Cells, obtained at passage 39, were maintained in DMEM containing 5% FCS, 5% Horse serum (HS), penicillin and streptomycin, at 37° C. in a humidified atmosphere of 10% CO 2 in air. Cells were fed or subcultured every three days. For mitogenic assay, cells were plated at a density of 2,000 cells/well in complete medium and incubated for 24 hours. Then medium was replaced with a mixture of 1:1 DMEM and F12 medium containing 0.1% FCS, after washing in serum free medium. Dose responses to GGFs, FCS and αFGF were then performed and cells were processed through the ELISA as previously described for the other cell types.
PC12 (Rat Adrenal Pheochromocytoma Cells): Cells from ECACC, were maintained in RPMI 1640 supplemented with 10% HS, 5% FCS, penicillin and streptomycin, in collagen coated flasks, at 37° C. in a humidified atmosphere of 5% CO 2 in air. Cells were fed every three days by replacing 80% of the medium. For mitogenic assay, cells were plated at a density of 3,000 cells/well in complete medium, on collagen coated plates (50 μl/well collagen, Vitrogen Collagen Corp., diluted 1:50, 30 min at 37° C.) and incubated for 24 hours. The medium was then placed with fresh RPMI either alone or containing 1 mM insulin or 1% FCS. Dose responses to FCS/HS (1:2) as positive control and to GGFs were performed as before. After 48 hours cells were fixed and the ELISA performed as previously described.
III. Results of Mitogenesis Assays: All the experiments presented in this Example were performed using a highly purified sample from a Sepharose 12 chromatography purification step containing a mixture of GGF-I and GGF-II (GGFs).
First, the results obtained with the BrdU incorporation assay were compared with the classical mitogenic assay for Schwann cells based on [125]I-UdR incorporation into DNA of dividing cells, described by J. P. Brockes ( Methods Enzymol . 147:217, 1987).
FIG. 12 shows the comparison of data obtained with the two assays, performed in the same cell culture conditions (5,000 cells/well, in 5% FBP/DMEM, incubated in presence of GGFs for 48 hrs). As clearly shown, the results are comparable, but BrdU incorporation assay appears to be slightly more sensitive, as suggested by the shift of the curve to the left of the graph, i.e. to lower concentrations of GGFS.
As described under the section “Methods of Mitogenesis Testing”, after the immunoreactive BrdU-DNA has been quantitated by reading the intensity of the soluble product of the OPD peroxidase reaction, the original assay plates containing cell monolayers can undergo the second reaction resulting in the insoluble DAB product, which stains the BrdU positive nuclei. The microcultures can then be examined under an inverted microscope, and cell morphology and the numbers of BrdU-positive and negative nuclei can be observed.
In FIG. 13 A and FIG. 13B the BrdU-DNA immunoreactivity, evaluated by reading absorbance at 490 nm, is compared to the number of BrdU-positive nuclei and to the percentage of BrdU-positive nuclei on the total number of cells per well, counted in the same cultures. Standard deviations were less than 10%. The two evaluation methods show a very good correlation and the discrepancy between the values at the highest dose of GGFs can be explained by the different extent of DNA synthesis in cells detected as BrdU-positive.
The BrdU incorporation assay can therefore provide additional useful information about the biological activity of polypeptides on Schwann cells when compared to the (125) I-UdR incorporation assay. For example, the data reported in FIG. 15 show that GGFs can act on Schwann cells to induce DNA synthesis, but at lower doses to increase the number of negative cells present in the microculture after 48 hours.
The assay has then been used on several cell lines of different origin. In FIG. 15 the mitogenic responses of Schwann cells and Swiss 3T3 fibroblasts to GGFs are compared; despite the weak response obtained in 3T3 fibroblasts, some clearly BrdU-positive nuclei were detected in these cultures. Control cultures were run in parallel in presence of several doses of FCS or human recombinant PDGF, showing that the cells could respond to appropriate stimuli (not shown).
The ability of fibroblasts to respond to GGFs was further investigated using the BHK 21 C13 cell line. These fibroblasts, derived from kidney, do not exhibit contact inhibition or reach a quiescent state when confluent. Therefore the experimental conditions were designed to have a very low background proliferation without compromising the cell viability. GGFs have a significant mitogenic activity on BHK21 C13 cells as shown by FIG. 16 and FIG. 17 . FIG. 16 shows the BrdU incorporation into DNA by BHK 21 C13 cells stimulated by GGFS in the presence of 0.1% FCS. The good mitogenic response to FCS indicates that cell culture conditions were not limiting. In FIG. 17 the mitogenic effect of GGFs is expressed as the number of BrdU-positive and BrdU-negative cells and as the total number of cells counted per well. Data are representative of two experiments run in duplicates; at least three fields per well were counted. As observed for Schwann cells in addition to a proliferative effect at low doses, GGFs also increase the numbers of nonresponding cells surviving. The percentage of BrdU positive cells is proportional to the increasing amounts of GGFs added to the cultures. The total number of cells after 48 hours in presence of higher doses of GGFs is at least doubled, confirming that GGFs induce DNA synthesis and proliferation in BHK21 C13 cells. Under the same conditions, cells maintained for 48 hours in the presence of 2% FCS showed an increase of about six fold (not shown).
C6 glioma cells have provided a useful model to study glial cell properties. The phenotype expressed seems to be dependent on the cell passage, the cells more closely resembling an astrocyte phenotype at an early stage, and an oligodendrocyte phenotype at later stages (beyond passage 70). C6 cells used in these experiments were from passage 39 to passage 52. C6 cells are a highly proliferating population, therefore the experimental conditions were optimized to have a very low background of BrdU incorporation. The presence of 0.1% serum was necessary to maintain cell viability without significantly affecting the mitogenic responses, as shown by the dose response to FCS (FIG. 18 ).
In FIG. 19 the mitogenic responses to aFGF (acidic Fibroblast growth factor) and GGFs are expressed as the percentages of maximal BrdU incorporation obtained in the presence of FCS (8%). Values are averages of two experiments, run in duplicates. The effect of GGFs was comparable to that of a pure preparation of aFGF. aFGF has been described as a specific growth factor for C6 cells (Lim R. et al., Cell Regulation 1:741-746, 1990) and for that reason it was used as a positive control. The direct counting of BrdU positive and negative cells was not possible because of the high cell density in the microcultures. In contrast to the cell lines so far reported, PC12 cells did not show any evident responsiveness to GGFS, when treated under culture conditions in which PC12 could respond to sera (mixture of FCS and HS as used routinely for cell maintenance). Nevertheless the number of cells plated per well seems to affect the behavior of PC12 cells, and therefore further experiments are required.
EXAMPLE 9
Amino acid sequences of purified GGF-I and GGF-II Amino acid sequence analysis studies were performed using highly purified bovine pituitary GGF-I and GGF-II. The conventional single letter code was used to describe the sequences. Peptides were obtained by lysyl endopeptidase and protease V8 digests, carried out on reduced and carboxymethylated samples, with the lysyl endopeptidase digest of GGF-II carried out on material eluted from the 55-65 RD region of a 11% SDS-PAGE (MW relative to the above-quoted markers).
A total of 21 peptide sequences (see FIG. 8 , SEQ ID Nos. 1-20, 169) were obtained for GGF-I, of which 12 peptides (see FIG. 9 , SEQ ID Nos. 1, 22-29, 17, 19, and 32) are not present in current protein databases and therefore represent unique sequences. A total of 12 peptide sequences Q. (see FIG. 10 , SEQ ID Nos: 42-50 and 160-162) were obtained it for GGF-II, of which 10 peptides (see FIG. 11 , SEQ ID Nos. 42-50) are not present in current protein databases and therefore represent unique sequences (an exception is peptide GGF-II 06 which shows identical sequences in many proteins which are probably of no significance given the small number of residues). These novel sequences are extremely likely to correspond to portions of the true amino acid sequences of GGFs I and II.
Particular attention can be drawn to the sequences of GGF-I 07 and GGF-II 12, which are clearly highly related. The similarities indicate that the sequences of these peptides are almost certainly those of the assigned GGF species, and are most unlikely to be derived from contaminant proteins.
In addition, in peptide GGF-II 02, the sequence X S S is consistent with the presence of an N linked carbohydrate moiety on an asparagine at the position denoted by X.
In general, in FIGS. 8 and 10 , X represents an unknown residue denoting a sequencing cycle where a single position could not be called with certainty either because there was more than one signal of equal size in the cycle or because no signal was present. As asterisk denotes those peptides where the last amino acid called corresponds to the last amino acid present in that peptide. In the remaining peptides, the signal strength after the last amino acid called was insufficient to continue sequence calling to the end of that peptide. The right hand column indicates the results of a computer database search using the GCG package FASTA and TFASTA programs to analyze the NBRF and EMBL sequence databases. The name of a protein in this column denotes identity of a portion of its sequence with the peptide amino acid sequence called allowing a maximum of two mismatches. A question mark denotes three mismatches allowed. The abbreviations used are as follows:
HMG-1
High Mobility Group protein-1
HMG-2
High Mobility Group protein-2
LH-alpha
Luteinizing hormone alpha subunit
LH-beta
Luteinizing hormone beta subunit
EXAMPLE 10
Isolating and Cloning of Nucleotide Sequences Encoding Proteins Containing GGF-I and GGF-II Peptides
Isolation and cloning of the GGF-II nucleotide sequences was performed as outlined herein, using peptide sequence information and library screening, and was performed as set out below. It will be appreciated that the peptides of FIGS. 10 and 11 can be used as the starting point for isolation and cloning of GGF-I sequences by following the techniques described herein. Indeed, FIG. 20 , SEQ ID Nos. 50-84) shows possible degenerate oligonucleotide probes for this purpose, and FIG. 22 , SEQ ID Nos. 86-115, lists possible PCR primers. DNA sequence and polypeptide sequence should be obtainable by this means as with GGF-II, and also DNA constructs and expression vectors incorporating such DNA sequence, host cells genetically altered by incorporating such constructs/vectors, and protein obtainable by cultivating such host cells. The invention envisages such subject matter.
I. Design and Synthesis of Oligonucleotide Probes and Primers
Degenerate DNA oligomer probes were designed by backtranslating the amino acid sequences (derived from the peptides generated from purified GGF protein) into nucleotide sequences. Oligomers represented either the coding strand or the non-coding strand of the DNA sequence. When serine, arginine or leucine were included in the oligomer design, then two separate syntheses were prepared to avoid ambiguities. For example, serine was encoded by either TCN or AGY as in 537 and 538 or 609 and 610. Similar codon splitting was done for arginine or leucine (e.g. 544, 545). DNA oligomers were synthesized on a Biosearch 8750 4-column DNA synthesizer using β-cyanoethyl chemistry operated at 0.2 micromole scale synthesis. Oligomers were cleaved off the column (500 angstrom CpG resins) and deprotected in concentrated ammonium hydroxide for 6-24 hours at 55-60° C. Deprotected oligomers were dried under vacuum (Speedvac) and purified by electrophoresis in gels of 15% acrylamide (20 mono:1 bis), 50 mM Tris-borate-EDTA buffer containing 7 M urea. Full length oligomers were detected in the gels by UV shadowing, then the bands were excised and DNA oligomers eluted into 1.5 mls H20 for 4-16 hours with shaking. The eluate was dried, redissolved in 0.1 ml H 2 0 and absorbance measurements were taken at 260 nm.
Concentrations were determined according to the following formula:
(A 260×units/ml)(60.6/length=×μM)
All oligomers were adjusted to 50 μM concentration by addition of H 2 O.
Degenerate probes designed as above are shown in by FIG. 20 , SEQ ID Nos: 50-84.
PCR primers were prepared by essentially the same procedures that were used for probes with the following modifications. Linkers of thirteen nucleotides containing restriction sites were included at the 5′ ends of the degenerate oligomers for use in cloning into vectors. DNA synthesis was performed at 1 micromole scale using 1,000 angstrom CpG resins and inosine was used at positions where all four nucleotides were incorporated normally into degenerate probes. Purifications of PCR primers included an ethanol precipitation following the gel electrophoresis purification.
II. Library Construction and Screening
A bovine genomic DNA library was purchased from Stratagene (Catalogue Number: 945701). The library contained 2×10 6 15-20 kb Sau3Al partial bovine DNA fragments cloned into the vector lambda DashII. A bovine total brain cDNA library was purchased from Clonetech (Catalogue Number: BL 10139). Complementary DNA libraries were constructed (In Vitrogen; Stratagene) from mRNA prepared from bovine total brain, from bovine pituitary and from bovine posterior pituitary. In Vitrogen prepared two cDNA libraries: one library was in the vector lambda g10, the other in vector pcDNAI (a plasmid library). The Stratagene libraries were prepared in the vector lambda unizap. Collectively, the cDNA libraries contained 14 million primary recombinant phage.
The bovine genomic library was plated on E. coli K12 host strain LE392 on 23×23 cm plates (Nunc) at 150,000 to 200,000 phage plaques per plate. Each plate represented approximately one bovine genome equivalent. Following an overnight incubation at 37° C., the plates were chilled and replicate filters were prepared according to procedures of Maniatis et al. (2:60-81). Four plaque lifts were prepared from each plate onto uncharged nylon membranes (Pall Biodyne A or MSI Nitropure). The DNA was immobilized onto the membranes by cross-linking under UV light for 5 minutes or, by baking at 80° C. under vacuum for two hours. DNA probes were labelled using T4 polynucleotide kinase (New England Biolabs) with gamma 32P ATP (New England Nuclear; 6500 Ci/mmol) according to the specifications of the suppliers. Briefly, 50 pmols of degenerate DNA oligomer were incubated in the presence of 600 μCi gamma 32 P-ATP and 5 units T4 polynucleotide kinase for 30 minutes at 37° C. Reactions were terminated, gel electrophoresis loading buffer was added and then radiolabelled probes were purified by electrophoresis. 32P labelled probes were excised from gel slices and eluted into water. Alternatively, DNA probes were labelled via PCR amplification by incorporation of α-32P-dATP or α-32P dCTP according to the protocol of Schowalter and Sommer, Anal. Biochem 177:90-94 (1989). Probes labelled in PCR reactions were purified by desalting on Sephadex G-150 columns.
Prehybridization and hybridization were performed in GMC buffer (0.52 M NaPi, 7% SDS, 1% BSA, 1.5 mM EDTA, 0.1 M NaCl 10 mg/ml tRNA). Washing was performed in oligowash (160 ml 1 M Na 2 HP0 4 , 200 ml 20% SDS, 8.0 ml 0.5 M EDTA, 100 ml 5M NaCl, 3632 ml H20). Typically, 20 filters (400 sq. centimeters each) representing replicate copies of ten bovine genome equivalents were incubated in 200 ml hybridization solution with 100 pmols of degenerate oligonucleotide probe (128-512 fold degenerate). Hybridization was allowed to occur overnight at 5° C. below the minimum melting temperature calculated for the degenerate probe. The calculation of minimum melting temperature assumes 2° C. for an AT pair and 4° C. for a GC pair.
Filters were washed in repeated changes of oligowash at the hybridization temperatures four to five hours and finally, in 3.2 M tetramethylammonium chloride, 1% SDS twice for 30 min at a temperature dependent on the DNA probe length. For 20 mers, the final wash temperature was 60° C. Filters were mounted, then exposed to X-ray film (Kodak XAR5) using intensifying screens (Dupont Cronex Lightening Plus). Usually, a three to five day film exposure at minus 80° C. was sufficient to detect duplicate signals in these library screens. Following analysis of the results, filters could be stripped and reprobed. Filters were stripped by incubating through two successive cycles of fifteen minutes in a microwave oven at full power in a solution of 1% SDS containing 10 mM EDTA pH8. Filters were taken through at least three to four cycles of stripping and reprobing with various probes.
III. Recombinant Phage Isolation, Growth and DNA Preparation
These procedures followed standard protocol as described in Recombinant DNA (Maniatis et al 2:60-2:81).
IV. Analysis of Isolated Clones Using DNA Digestion and Southern Blots
Recombinant Phage DNA samples (2 micrograms) were digested according to conditions recommended by the restriction endonuclease supplier (New England Biolabs). Following a four hour incubation at 37° C., the reactions products were precipitated in the presence of 0.1M sodium acetate and three volumes of ethanol. Precipitated DNA was collected by centrifugation, rinsed in 75% ethanol and dried. All resuspended samples were loaded onto agarose gels (typically 1% in TAE buffer; 0.04M Tris acetate, 0.002M EDTA). Gel runs were at 1 volt per centimeter from 4 to 20 hours. Markers included lambda Hind III DNA fragments and/or ØX174HaeIII DNA fragments (New England Biolabs). The gels were stained with 0.5 micrograms/ml of ethidium bromide and photographed. For southern blotting, DNA was first depurinated in the gel by treatment with 0.125 N HCl, denatured in 0.5 N NaOH and transferred in 20×SSC (3M sodium chloride, 0.03 M sodium citrate) to uncharged nylon membranes. Blotting was done for 6 hours up to 24 hours, then the filters were neutralized in 0.5 Tris HCl pH 7.5, 0.15 M sodium chloride, then rinsed briefly in 50 mM Tris-borate EDTA.
For cross-linking, the filters were wrapped first in transparent plastic wrap, then the DNA side exposed for five minutes to an ultraviolet light. Hybridization and washing was performed as described for library screening (see section 2 of this Example). For hybridization analysis to determine whether similar genes exist in other species slight modifications were made. The DNA filter was purchased from Clonetech (Catalogue Number 7753-1) and contains 5 micrograms of EcoRI digested DNA from various species per lane. The probe was labelled by PCR amplification reactions as described in section 2 above, and hybridizations were done in 80% buffer B(2 g polyvinylpyrrolidine, 2 g Ficoll-400, 2 g bovine serum albumin, 50 ml 1M Tris-HCl (pH 7.5) 58 g NaCl, 1 g sodium pyrophosphate, 10 g sodium dodecyl sulfate, 950ml H 2 0) containing 10% dextran sulfate. The probes were denatured by boiling for ten minutes then rapidly cooling in ice water. The probe was added to the hybridization buffer at 10 6 dpm 32 P per ml and incubated overnight at 600C. The filters were washed at 60° C. first in buffer B followed by 2×SSC, 0.1% SDS then in 1×SSC, 0.1% SDS. For high stringency, experiments, final washes were done in 0.1×SSC, 1% SDS and the temperature raised to 65° C.
Southern blot data were used to prepare a restriction map of the genomic clone and to indicate which subfragments hybridized to the GGF probes (candidates for subcloning).
V. Subcloning of Segments of DNA Homologous to Hybridization Probes
DNA digests (e.g. 5 micrograms) were loaded onto 1% agarose gels then appropriate fragments excised from the gels following staining. The DNA was purified by adsorption onto glass beads followed by elution using the protocol described by the supplier (Bio 101). Recovered DNA fragments (100-200 ng) were ligated into linearized dephosphorylated vectors, e.g. pT3T7 (Ambion), which is a derivative of pUC18, using T4 ligase (New England Biolabs). This vector carries the E. coli β lactamase gene, hence, transformants can be selected on plates containing ampicillin. The vector also supplies β-galactosidase complementation to the host cell, therefore non-recombinants (blue) can be detected using isopropylthiogalactoside and Bluogal (Bethesda Research Labs). A portion of the ligation reactions was used to transform E. coli K12 XLl blue competent cells (Stratagene Catalogue Number: 200236) and then the transformants were selected on LB plates containing 50 micrograms per ml ampicillin. White colonies were selected and plasmid mini preps were prepared for DNA digestion and for DNA sequence analysis. Selected clones were retested to determine if their insert DNA hybridized with the GGF probes.
VI. DNA Sequencing
Double stranded plasmid DNA templates were prepared from 5 ml cultures according to standard protocols. Sequencing was by the dideoxy chain termination method using Sequenase 2.0 and a dideoxynucleotide sequencing kit (US Biochemical) according to the manufacturers protocol (a modification of Sanger et al. PNAS; USA 74:5463 (1977)]. Alternatively, sequencing was done in a DNA thermal cycler (Perkin Elmer, model 4800) using a cycle sequencing kit (New England Biolabs; Bethesda Research Laboratories) and was performed according to manufacturers instructions using a 5′-end labelled primer. Sequence primers were either those supplied with the sequencing kits or were synthesized according to sequence determined from the clones. Sequencing reactions were loaded on and resolved on 0.4 mm thick sequencing gels of 6% polyacrylamide. Gels were dried and exposed to X-Ray film. Typically, 35S was incorporated when standard sequencing kits were used and a 32P end labelled primer was used for cycle sequencing reactions. Sequences were read into a DNA sequence editor from the bottom of the gel to the top (5′ direction to 3′) and data were analyzed using programs supplied by Genetics Computer Group (GCG, University of Wisconsin).
VII. RNA Preparation and PCR Amplification
Open reading frames detected in the genomic DNA and which contained sequence encoding GGF peptides were extended via PCR amplification of pituitary RNA. RNA was prepared from frozen bovine tissue (Pelfreeze) according to the guanidine neutral-CsCl procedure (Chirgwin et. al. Biochemistry 18:5294(1979).) Polyadenylated RNA was selected by oligo-dT cellulose column chromatography (Aviv and Leder PNAS (USA) 69:1408 (1972)).
Specific DNA target sequences were amplified beginning with either total RNA or polyadenylated RNA samples that had been converted to cDNA using the Perkin Elmer PCR/RNA Kit Number: N808-0017. First strand reverse transcription reactions used 1 μg template RNA and either primers of oligo dT with restriction enzyme recognition site linkers attached or specific antisense primers determined from cloned sequences with restriction sites attached. To produce the second strand, the primers either were plus strand unique sequences as used in 3′ RACE reactions (Frohman et. al., PNAS (USA) 85:8998 (1988)) or were oligo dT primers with restriction sites attached if the second target site had been added by terminal transferase tailing first strand reaction products with dATP (e.g. 5′ race reactions, Frohman et. al., ibid). Alternatively, as in anchored PCR reactions the second strand primers were degenerate, hence, representing particular peptide sequences.
The amplification profiles followed the following general scheme: 1) five minutes soak file at 95° C.; 2) thermal cycle file of 1 minute, 95° C.; 1 minute ramped down to an annealing temperature of 45° C., 50° C. or 55° C.; maintain the annealing temperature for one minute; ramp up to 72° C. over one minute; extend at 72° C. for one minute or for one minute plus a 10 second auto extension; 3) extension cycle at 72° C., five minutes, and; 4) soak file 4° C. for infinite time. Thermal cycle files (#2) usually were run for 30 cycles. A sixteen μl sample of each 100 μl amplification reaction was analyzed by electrophoresis in 2% Nusieve 1% agarose gels run in TAE buffer at 4 volts per centimeter for three hours. The gels were stained, then blotted to uncharged nylon membranes which were probed with labelled DNA probes that were internal to the primers.
Specific sets of DNA amplification products could be identified in the blotting experiments and their positions used as a guide to purification and reamplification. When appropriate, the remaining portions of selected samples were loaded onto preparative gels, then following electrophoresis four to five slices of 0.5 mm thickness (bracketing the expected position of the specific product) were taken from the gel. The agarose was crushed, then soaked in 0.5 ml of electrophoresis buffer from 2-16 hours at 40° C. The crushed agarose was centrifuged for two minutes and the aqueous phase was transferred to fresh tubes.
Reamplification was done on five microliters (roughly 1% of the product) of the eluted material using the same sets of primers and the reaction profiles as in the original reactions. When the reamplification reactions were completed, samples were extracted with chloroform and transferred to fresh tubes. Concentrated restriction enzyme buffers and enzymes were added to the reactions in order to cleave at the restriction sites present in the linkers. The digested PCR products were purified by gel electrophoresis, then subcloned into vectors as described in the subcloning section above. DNA sequencing was done described as above.
VIII. DNA Sequence Analysis
DNA sequences were assembled using a fragment assembly program and the amino acid sequences deduced by the GCG programs GelAssemble, Map and Translate. The deduced protein sequences were used as a query sequence to search protein sequence databases using WordSearch. Analysis was done on a VAX Station 3100 workstation operating under VMS 5.1. The database search was done on SwissProt release number 21 using GCG Version 7.0.
IX. Results of Cloning and Sequencing of Genes Encoding GGF-I and GGF-II
As indicated above, to identify the DNA sequence encoding bovine GGF-II degenerate oligonucleotide probes were designed from GGF-II peptide sequences. GGF-II 12 (SEQ ID No. 49), a peptide generated via lysyl endopeptidase digestion of a purified GGF-II preparation (see FIGS. 16 and 12 ) showed strong amino acid sequence homology with GGF-I 07 (SEQ ID No. 39), a tryptic peptide generated from a purified GGF-I preparation. GGF-II 12 was thus used to create ten degenerate oligonucleotide probes (see oligos 609, 610 and 649 to 656 in FIG. 20 , SEQ ID Nos: 66, 67, 68 and75, respectively). A duplicate set of filters were probed with two sets (set 1=609, 610; set 2=649-5656) of probes encoding two overlapping portions of GGF-II 12. Hybridization signals were observed, but, only one clone hybridized to both probe sets. The clone (designated GGF2BG1) was purified.
Southern blot analysis of DNA from the phage clone GGF2BG1 confirmed that both sets of probes hybridized with that bovine DNA sequence, and showed further that both probes reacted with the same set of DNA fragments within the clone. Based on those experiments a 4 kb Eco RI sub-fragment of the original clone was identified, subcloned and partially sequenced. FIG. 21 shows the nucleotide sequence, SEQ ID No. 85) and the deduced amino acid sequence of the initial DNA sequence readings that included the hybridization sites of probes 609 and 650, and confirmed that a portion of this bovine genomic DNA encoded peptide 12 (KASLADSGEYM).
Further sequence analysis demonstrated that GGF-II 12 resided on a 66 amino acid open reading frame (see below) which has become the starting point for the isolation of overlapping sequences representing a putative bovine GGF-II gene and a cDNA.
Several PCR procedures were used to obtain additional coding sequences for the putative bovine GGF-II gene. Total RNA and oligo dT-selected (poly A containing) RNA samples were prepared from bovine total pituitary, anterior pituitary, posterior pituitary, and hypothalamus. Using primers from the list shown in FIG. 22 , SEQ ID Nos. 105-115, one-sided PCR reactions (RACE) were used to amplify cDNA ends in both the 3′ and 5′ directions, and anchored PCR reactions were performed with degenerate oligonucleotide primers representing additional GGF-II peptides. FIG. 29 summarizes the contiguous DNA structures and sequences obtained in those experiments. From the 3′ RACE reactions, three alternatively spliced cDNA sequences were produced, which have been cloned and sequenced. A 5′ RACE reaction led to the discovery of an additional exon containing coding sequence for at least 52 amino acids. Analysis of that deduced amino acid sequence revealed peptides GGF-II-6 and a sequence similar to GGF-I-18 (see below). The anchored PCR reactions led to the identification of (cDNA) coding sequences of peptides GGF-II-1, 2, 3 and 10 contained within an additional cDNA segment of 300 bp. The 5′ limit of this segment (i.e., segment E, see FIG. 30 ) is defined by the oligonucleotide which encodes peptide GGF-II-1 and which was used in the PCR reaction (additional 5′ sequence data exists as described for the human clone in Example 11). Thus this clone contains nucleotide sequences encoding six out of the existing total of nine novel GGF-II peptide sequences.
The cloned gene was characterized first by constructing a physical map of GGF2BG1 that allowed us to position the coding sequences as they were found (see below, FIG. 30 ). DNA probes from the coding sequences described above have been used to identify further DNA fragments containing the exons on this phage clone and to identify clones that overlap in both directions. The putative bovine GGF-II gene is divided into at least 5 coding segments. Coding segments are defined as discrete lengths of DNA sequence which can be translated into polypeptide sequences using the universal genetic code. The coding segments described in FIG. 36 and referred to in the present application are: 1) particular exons present within the GGF gene (e.g. coding segment a), or 2) derived from sets of two or more exons that appear in specific sub-groups of mRNAs, where each set can be translated into the specific polypeptide segments as in the gene products shown. The polypeptide segments referred to in the claims are the translation products of the analogous DNA coding segments. Only coding segments A and B have been defined as exons and sequenced and mapped thus far. The summary of the contiguous coding sequences identified is shown in FIG. 31 . The exons are listed (alphabetically) in the order of their discovery. It is apparent from the intron/exon boundaries that exon B may be included in cDNAs that connect coding segment E and coding segment A. That is, exon B cannot be spliced out without compromising the reading frame. Therefore, we suggest that three alternative splicing patterns can produce putative bovine GGF-II cDNA sequences 1, 2 and 3. The coding sequences of these, designated GGF2BPP1.CDS, GGF2BPP2. CDS and GGF2BPP3. CDS, respectively, are given in FIGS. 27A (SEQ ID NO: 129), 27 B (SEQ ID NO. 130), and 27 C (SEQ ID NO: 131), respectively. The deduced amino acid sequence of the three cDNAs is also given in FIGS. 27A , (SEQ ID NO: 129), 27 B (SEQ ID NO: 130), and 27 C (SEQ ID NO: 131).
The three deduced structures encode proteins of lengths 206, 281 and 257 amino acids. The first 183 residues of the deduced protein sequence are identical in all three gene products. At position 184 the clones differ significantly. A codon for glycine GGT in GGF2BPP1 also serves as a splice donor for GGF2BPP2 and GGF2BPP3, which alternatively add on exons C, C/D, C/D′ and D or C, C/D and D, respectively, and shown in FIG. 32 , SEQ ID NO: 145). GGFIIBPP1 is a truncated gene product which is generated by reading past the coding segment A splice junction into the following intervening sequence (intron). This represents coding segment A′ in FIG. 30 (SEQ ID NO: 136). The transcript ends adjacent to a canonical AATAAA polyadenylation sequence, and we suggest that this truncated gene product represents a bona fide mature transcript. The other two longer gene products share the same 3′ untranslated sequence and polyadenylation site.
All three of these molecules contain six of the nine novel GGF-II peptide sequences (see FIG. 11 ) and another peptide is highly homologous to GGF-I-18 (see FIG. 26 ). This finding gives a high probability that this recombinant molecule encodes at least a portion of bovine GGF-II. Furthermore, the calculated isoelectric points for the three peptides are consistent with the physical properties of GGF-I and II. Since the molecular size of GGF-II is roughly 60 kD, the longest of the three cDNAs should encode a protein with nearly one-half of the predicted number of amino acids.
A probe encompassing the B and A exons was labelled via PCR amplification and used to screen a cDNA library made from RNA isolated from bovine posterior pituitary. One clone (GGF2BPP5) showed the pattern indicated in FIG. 29 and contained an additional DNA coding segment (G) between coding segments A and C. The entire nucleic acid sequence is shown in FIG. 31 (SEQ ID NO: 144). The predicted translation product from the longest open reading frame is 241 amino acids. A portion of a second cDNA (GGF2BPP4) was also isolated from the bovine posterior pituitary library using the probe described above. This clone showed the pattern indicated in FIG. 29 . This clone is incomplete at the 5′ end, but is a splicing variant in the sense that it lacks coding segments G and D. BPP4 also displays a novel 3′ end with regions H, K and L beyond region C/D. The sequence of BPP4 is shown in FIG. 33 (SEQ ID NO: 146).
EXAMPLE 11
GGF Sequences in Various Species
The GGF proteins are the members of a new superfamily of proteins. In high stringency cross hybridization studies (DNA blotting experiments) with other mammalian DNAs we have shown, clearly, that DNA probes from this bovine recombinant molecule can readily detect specific sequences in a variety of samples tested. A highly homologous sequence is also detected in human genomic DNA. The autoradiogram is shown in FIG. 28 . The signals in the lanes containing rat and human DNA represent the rat and human equivalents of the GGF gene, the sequences of several cDNA's encoded by this gene have been recently reported by Holmes et al. (Science 256: 1205 (1992)) and Wen et al. (Cell 69: 559 (1992)).
EXAMPLE 12
Isolation of a Human Sequence Encoding Human GGF2 Several human clones containing sequences from the bovine GGFII coding segment E were isolated by screening a human cDNA library prepared from brain stem (Stratagene catalog #935206). This strategy was pursued based on the strong link between most of the GGF2 peptides (unique to GGF2) and the predicted peptide sequence from clones containing the bovine E segment. This library was screened as described in Example 8, Section II using the oligonucleotide probes 914-919 listed below.
914TCGGGCTCCATGAAGAAGATGTA (SEQ ID NO: 179) 915TCCATGAAGAAGATGTACCTGCT (SEQ ID NO: 180) 916ATGTACCTGCTGTCCTCCTTGA (SEQ ID NO: 181) 917TTGAAGAAGGACTCGCTGCTCA (SEQ ID NO: 182) 918AAAGCCGGGGGCTTGAAGAA (SEQ ID NO: 183) 919ATGARGTGTGGGCGGCGAAA (SEQ ID NO: 184)
Clones detected with these probes were further analyzed by hybridization. A probe derived from coding segment A (see FIG. 30 ), which was produced by labeling a polymerase chain reaction (PCR) product from segment A, was also used to screen the primary library. Several clones that hybridized with both A and E derived probes were selected and one particular clone, GGF2HBS5, was selected for further analysis. This clone is represented by the pattern of coding segments (EBACC/D′D as shown in FIG. 30 ). The E segment in this clone is the human equivalent of the truncated bovine version of E shown in FIG. 30 . GGF2HBS5 is the most likely candidate to encode GGF-II of all the “putative” GGF-II candidates described. The length of coding sequence segment E is 786 nucleotides plus 264 bases of untranslated sequence. The predicted size of the protein encoded by GGF2HBS5 is approximately 423 amino acids (approximately 45 kilodaltons, see FIG. 44 , SEQ ID NO: 21), which is similar to the size of the deglycosylated form of GGF-II (see Example 19). Additionally, seven of the GGF-II peptides listed in FIG. 26 have equivalent sequences which fall within the protein sequence predicted from region E. Peptides II-6 and II-12 are exceptions, which fall in coding segment B and coding segment A, respectively. RNA encoding the GGF2HBS5 protein was produced in an in vitro transcription system driven by the bacteriophage T7 promoter resident in the vector (Bluescript SK [Stratagene Inc.] see FIG. 47 ) containing the GGF2HBS5 insert. This RNA was translated in a cell free (rabbit reticulocyte) translation system and the size of the protein product was 45 Kd. Additionally, the cell free product has been assayed in a Schwann cell mitogenic assay to confirm biological activity. Schwann cells treated with conditioned medium show both increased proliferation as measured by incorporation of 125 I-Uridine and phosphorylation on tyrosine of a protein in the 185 kilodalton range.
Thus the size of the product encoded by GGF2HBS5 and the presence of DNA sequences which encode human peptides highly homologous to the bovine peptides shown in FIG. 11 confirm that GGF2HBS5 encodes the human equivalent of bovine GGF2. The fact that conditioned media prepared from cells transformed with this clone elicits Schwann cell mitogenic activity confirms that the GGFIIHBS5 gene produce (unlike the BPP5 gene product) is secreted. Additionally the GGFIIBPP5 gene product seems to mediate the Schwann cell proliferation response via a receptor tyrosine kinase such as p185 erbB2 or a closely related receptor (see Example 18).
EXAMPLE 13
Expression of Human Recombinant GGF2 in Mammalian and Insect Cells
The GGF2HBS5 cDNA clone encoding human GGF2 (as described in Example 12 and also referred to herein as HBS5) was cloned into vector pcDL-SRα296 and COS-7 cells were transfected in 100 mm dishes by the DEAE-dextran method. Cell lysates or conditioned media from transiently expressing COS cells were harvested at 3 or 4 days post-transfection. To prepare lysates, cell monolayers were washed with PBS, scraped from the dishes lysed by three freeze/thaw cycles in 150 μm of 0.25 M Tris-HCl, pH8. Cell debris was pelleted and the supernatant recovered. Conditioned media samples (7 mls.) were collected, then concentrated and buffer exchanged with 10 mm Tris, pH 7.4 using Centiprep-10 and Centricon-10 units as described by the manufactures (Amicon, Beverly, Mass.). Rat nerve Schwann cells were assayed for incorporation of DNA synthesis precursors, as described. Conditioned media or cell lysate samples were tested in the Schwann cell proliferation assay as described in Marchionni et al., Nature 362:313 (1993). The cDNA, GGF2HBS5, encoding GGF2 directed the secretion of the protein product to the medium. Minimal activity was detectable inside the cells as determined by assays using cell lysates. GGF2HFB1 and GGFBPP5 cDNA's failed to direct the secretion of the product to the extracellular medium. GGF activity from these clones was detectable only in cell lysates.
Recombinant GGF2 was also expressed in CHO cells. The GGF2HBS5 cDNA encoding GGF2 was cloned into the EcoRI site of vector pcdhfrpolyA and transfected into the DHFR negative CHO cell line (GG44) by the calcium phosphate coprecipitation method. Clones were selected in nucleotide and nucleoside free α medium (Gibco) in 96-well plates. After 3 weeks, conditioned media samples from individual clones were screened for expression of GGF by the Schwann cell proliferation assay as described in Marchionni et al., Nature 362:313 (1993). Stable clones which secreted significant levels of GGF activity into the medium were identified. Schwann cell proliferation activity data from different volume aliquots of, CHO cell conditioned medium were used to produce the dose response curve shown in FIG. 46 (Graham and Van Der Eb, Virology 52:456, 1973). This material was analyzed on a Western blot probed with polyclonal antisera raised against a GGF2 specific peptide. A band of approximately 65 Kd (the expected size of GGF2 extracted from pituitary) is specifically labeled ( FIG. 48 , lane 12).
Recombinant GGF2 was also expressed in insect cells using the Baculovirus expression. Sf9 insect cells were infected with baculovirus containing the GGF2HBS5 cDNA clone at a multiplicity of 3-5 (10 6 cells/ml) and cultured in Sf900-II medium. Schwann cell mitogenic activity was secreted into the extracellular medium. Different volumes of insect cell conditioned medium were tested in the Schwann cell proliferation assay in the absence of forskolin and the data used to produce a dose response curve.
This material was also analyzed on a Western blot ( FIG. 45B ) probed with the GGF II specific antibody described above.
The methods used in this example were as follows:
Schwann cell mitogenic activity of recombinant human and bovine glial growth factors was determined as follows: Mitogenic responses of cultured Schwann cells were measured in the presence of 5 μM forskolin using crude recombinant GGF preparations obtained from transient mammalian expression experiments. Incorporation of [ 125 I]-Urd was determined following an 18-24 hour exposure to materials obtained from transfected or mock transfected cos cells as described in the Methods. The mean and standard deviation of four sets of data are shown. The mitogenic response to partially purified native bovine pituitary GGF (carboxymethyl cellulose fraction; Goodearl et al., submitted) is shown (GGF) as a standard of one hundred percent activity.
cDNAs ( FIG. 46 , SEQ ID NOS: 166-168) were cloned into pcDL-SRα296 (Takebe et al., Mol. Cell Biol. 8:466-472 (1988)), and COS-7 cells were transfected in 100 mm dishes by the DEAE-dextran method (Sambrook et al., In Molecular Cloning. A Laboratory Manual , 2 nd. ed . (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)). Cell lysates or conditioned media were harvested at 3 or 4 days post-transfection. To prepare lysates, cell monolayers were washed with PBS, scraped from the dishes, and lysed by three freeze/than cycles in 150 μl of 0.25 M Tris-HCl, pH 8. Cell debris was pelleted and the supernate recovered. Conditioned media samples (7 mls) were collected, then concentrated and buffer exchanged with 10 mM Tris, pH 7.4 using Centriprep-10 and Centricon-10 units are described by the manufacturers (Amicon, Beverly, Mass.). Rat sciatic nerve Schwann cells were assayed for incorporation of DNA synthesis precursors, as described (Davis and Stroobant, J. Cell Biol. 110:1353-1360 (1990); Brockes et al., Brain Res. 165:105-118 (1979)).
Western blot of recombinant CHO cell conditioned medium were performed as follows: A recombinant CHO clone was cultured in MCDB302 protein-free for 3 days. 2 ml of conditioned medium was harvested, concentrated, buffered exchanged against 10 mM Tris-HCl, pH 7.4 and lyophilized to dryness. The pellet was resuspended in SDS-PAGE sample buffer, subjected to reducing SDS gel electrophoresis and analyzed by Western blotting with a GGF peptide antibody. A CHO control was done by using conditioned medium from untransfected CHO-DG44 host and the CHO HBS5 levels were assayed using conditioned medium from a recombinant clone.
EXAMPLE 14
Identification of Functional Elements of GGF
The deduced structures of the family of GGF sequences indicate that the longest forms (as represented by GGF2BPP4) encode transmembrane proteins where the extracellular part contains a domain which resembles epidermal growth factor (see Carpenter and Wahl in Peptide Growth Factors and Their Receptors I pp. 69-133, Springer-Verlag, NY 1991). The positions of the cysteine residues in coding segments C and C/D or C/D′ peptide sequence are conserved with respect to the analogous residues in the epidermal growth factor (EGF) peptide sequence (see FIG. 34 , SEQ ID NOs. 147-149. This suggests that the extracellular domain functions as receptor recognition and biological activation sites. Several of the variant forms lack the H, K, and L coding segments and thus may be expressed as secreted, diffusible biologically active proteins. GGF DNA sequences encoding polypeptides which encompass the EGF-like domain (EGFL) can have full biological activity for stimulating glial cell mitogenic activity.
Membrane bound versions of this protein may induce Schwann cell proliferation if expressed on the surface of neurons during embryogenesis or during nerve regeneration (where the surfaces of neurons are intimately associated with the surfaces of proliferating Schwann cells).
Secreted (non membrane bound) GGFs may act as classically diffusible factors which can interact with Schwann cells at some distance from their point of secretion. Other forms may be released from intracells by sources via tissue injury and cell disruption. An example of a secreted GGF is the protein encoded by GGF2HBS5; this is the only GGF known which has been found to be directed to the exterior of the cell. Secretion is probably mediated via an N-terminal hydrophobic sequence found only in region E, which is the N-terminal domain contained within recombinant GGF2 encoded by GGF2HBS5.
Other GGF's appear to be non-secreted. These GGFs may be injury response forms which are released as a consequence of tissue damage.
Other regions of the predicted protein structure of GGF2 (encoded by GGF2HBS5) and other proteins containing regions B and A exhibit similarities to the human basement membrane heparan sulfate proteoglycan core protein. The peptide ADSGEY, which is located next to the second cysteine of the C2 immunoglobulin fold in these GGF's, occurs in nine of twenty-two C-2 repeats found in that basal lamina protein. This evidence strongly suggests that these proteins may associate with matrix proteins such as those associated with neurons and glia, and may suggest a method for sequestration of glial growth factors at target sites.
EXAMPLE 15
Purification of GGFs From Recombinant Cells
In order to obtain full length or portions of GGFs to assay for biological activity, the proteins can be overproduced using cloned DNA. Several approaches can be used. A recombinant E. coli cell containing the sequences described above can be constructed. Expression systems such as pNH8a or pHH16a (Stratagene, Inc.) can be used for this purpose by following manufacturers procedures. Alternatively, these sequences can be inserted in a mammalian expression vector and an overproducing cell line can be constructed. As an example, for this purpose DNA encoding a GGF, clone GGF2BPP5 has been expressed in COS cells and can be expressed in Chinese hamster ovary cells using the pMSXND expression vector (Lee and Nathans, J. Biol. Chem. 263, 3521-3527, (1981)). This vector containing GGF DNA sequences can be transfected into host cells using established procedures.
Transient expression can be examined or G418-resistant clones can be grown in the presence of methotrexate to select for cells that amplify the dhfr gene (contained on the pMSXND vector) and, in the process, co-amplify the adjacent GGF protein encoding sequence. Because CHO cells can be maintained in a totally protein-free medium (Hamilton and Ham, In Vitro 13, 537-547 (1977)), the desired protein can be purified from the medium. Western analysis using the antisera produced in Example 16 can be used to detect the presence of the desired protein in the conditioned medium of the overproducing cells.
The desired protein (rGGF2) was purified from the medium conditioned by transiently expressing cos cells as follows. rGGF II was harvested from the conditioned medium and partially purified using Cation Exchange Chromatography (POROS-HS). The column was equilibrated with 33.3 mM MES pH 6.0. Conditioned media was loaded at flow rate of 10 ml/min. The peak containing Schwann cell proliferation activity and immunoreactive (using the polyclonal antisera was against a GGF2 peptide described above) was eluted with 50 mM Tris, 1M NaCl pH 8.0.
rhGGF2 is also expressed using a stable Chinese Ovary Hamster cell line. rGGF2 from the harvested conditioned media was partially purified using Cation Exchange Chromatograph (POROS-HS). The column was equilibrated with PBS pH 7.4. Conditioned media was loaded at 10 ml/min. The peak containing the Schwann Cell Proliferative activity and immunoreactivity (using GGF2 polyclonal antisera) was eluted with 50 mM Hepes, 500 mM NaCl pH 8.0. An additional peak was observed at 50 mM Hepes, 1M NaCl pH 8.0 with both proliferation as well as immunoreactivity (FIG. 45 ).
rhGGF2 can be further purified using Hydrophobic Interaction Chromatography as a high resolution step; Cation exchange/Reserve phase Chromatography (if needed as second high resolution step); A viral inactivation step and a DNA removal step such as Anion exchange chromatography.
Schwann Cell Proliferation Activity of recombinant GGF2 peak eluted from the Cation Exchange column was determined as follows: Mitogenic responses of the cultured Schwann cells were measured in the presence of 5 M Forskolin using the peak eluted by 50 mM Tris 1 M NaCl pH 8.0. The peak was added at 20 1, 10 1 (1:10) 10 1 and (1:100) 10 1. Incorporation of 125 I-Uridine was determined and expressed as (CPM) following an 18-24 hour exposure.
An immunoblot using polyclonal antibody raised against a peptide of GGF2 was carried out as follows: 10 1 of different fractions were ran on 4-12% gradient gels. The gels were transferred on to Nitrocellulose paper, and the nitrocellulose blots were blocked with 5% BSA and probed with GGF2-specific antibody (1:250 dilution). 125I protein A (1:500 dilution, Specific Activity=9.0/Ci/g) was used as the secondary antibody. The immunoblots were exposed to Kodax X-Ray films for 6 hours. The peak fractions eluted with 1 M NaCl showed an immunoreactive band at 69 K.
GGF2 purification on cation exchange columns was performed as follows: CHO cell conditioned media expressing rGGFII was loaded on the cation exchange column at 10 ml/min. The column was equilibrated with PBS pH 7.4. The elution was achieved with 50 mM Hepes 500 mM NaCl pH 8.0 and 50 mM Hepes 1M NaCl pH 8.0 respectively. All fractions were analyzed using the Schwann cell proliferation assay (CPM) described herein. The protein concentration (mg/ml) was determined by the Bradford assay using BSA as the standard.
A Western blot using 10 1 of each fraction was performed and immunoreactivity and the Schwann cell activity were observed to co-migrate.
The protein may be assayed at various points in the procedure using a Western blot assay. Alternatively, the Schwann cell mitogenic assay described herein may be used to assay the expressed product of the full length clone or any biologically active portions thereof. The full length clone GGF2BPP5 has been expressed transiently in COS cells.
Intracellular extracts of transfected COS cells show biological activity when assayed in the Schwann cell proliferation assay described in Example 8. In addition, the full length close encoding GGF2HBS5 has been expressed transiently in COS cells. In this case both cell extract and conditioned media show biological activity in the Schwann cell proliferation assay described in Example 8. Any member of the family of splicing variant complementary DNA's derived from the GGF gene (including the Heregulins) can be expressed in this manner and assayed in the Schwann cell proliferation assay by one skilled in the art.
Alternatively, recombinant material may be isolated from other variants according to Wen et al. (Cell 69:559 (1992)) who expressed the splicing variant Neu differentiation factor (NDF) in COS-7 cells. cDNA clones inserted in the pJT-2 eukaryotic plasmid vector are under the control of the SV40 early promoter, and are 3′-flanked with the SV40 termination and polyadenylation signals. COS-7 cells were transfected with the pJT-2 plasmid DNA by electroporation as follows: 6×106 cells (in 0.8 ml of DMEM and 10% FEBS) were transferred to a 0.4 cm cuvette and mixed with 20 μg of plasmid DNA in 10 μl of TE solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Electroporation was performed at room temperature at 1600 V and 25 μF using a Bio-Rad Gene Pulser apparatus with the pulse controller unit set at 200 ohms. The cells were then diluted into 20 ml of DMEM, 10% FBS and transferred into a T75 flask (Falcon). After 14 hr. of incubation at 37° C., the medium was replaced with DMEM, 1% FBS, and the incubation continued for an additional 48 hr. Conditioned medium containing recombinant protein which was harvested from the cells demonstrated biological activity in a cell line expressing the receptor for this protein. This cell line (cultured human breast carcinoma cell line AU 565) was treated with recombinant material. The treated cells exhibited a morphology change which is characteristic of the activation of the erbB2 receptor. Conditioned medium of this type also can be tested in the Schwann cell proliferation assay.
EXAMPLE 16
Isolation of a Further Splicing Variant
Methods for updating other neuregulins descsribed in U.S. patent application Ser. No. 07/965,173, filed Oct. 23, 1992, incorporated herein by reference, produced four closely related sequences (heregulin α, β1, β2, β3) which arise as a result of splicing variation. Peles et al. (Cell 69:205 (1992)), and Wen et al. (Cell 69:559 (1992)) have isolated another splicing variant (from rat) using a similar purification and cloning approach to that described in Examples 1-9 and 11 involving a protein which binds to p 185 erbB2 . The cDNA clone was obtained as follows (via the purification and sequencing of a p185 erbB2 binding protein from a transformed rat fibroblast cell line). A p185 erbB2 binding protein was purified from conditioned medium as follows. Pooled conditioned medium from three harvests of 500 roller bottles (120 liters total) was cleared by filtration through 0.2 4 filters and concentrated 31-fold with a Pelicon ultrafiltration system using membranes with a 20 kD molecular size cutoff. All the purification steps were performed by using a Pharmacia fast protein liquid chromatography system. The concentrated material was directly loaded on a column of heparin-Sepharose (150 ml, preequilibrated with phosphate-buffered saline (PBS)). The column was washed with PBS containing 0.2 M NaCl until no absorbance at 280 nm wavelength could be detected. Bound proteins were then eluted with a continuous gradient (250 ml) of NaCl (from 0.2 M to 1.0 M), and 5 ml fractions were collected. Samples (0.01 ml of the collected fractions were used for the quantitative assay of the kinase stimulatory activity. Active fractions from three column runs (total volume=360 ml) were pooled, concentrated to 25 ml by using a YM10 ultrafiltration membrane (Amicon, Danvers, Mass.), and ammonium sulfate was added to reach a concentration of 1.7 M. After clearance by centrifugation (10,000×g, 15 min.), the pooled material was loaded on a phenyl-Superose column (HR10/10, Pharmacia). The column was developed with a 45 ml gradient of (NH 4 ) 2 SO 4 (from 1.7 M to no salt) in 0.1 M Na 2 PO 4 (pH 7.4), and 2 ml fractions were collected and assayed (0.002 ml per sample) for kinase stimulation (as described in Example 18). The major peak of activity was pooled and dialyzed against 50 mM sodium phosphate buffer (pH 7.3). A Mono-S cation-exchange column (HR5/5, Pharmacia) was preequilibrated with 50 mM sodium phosphate. After loading the active material (0.884 mg of protein; 35 ml), the column was washed with the starting buffer and then developed at a rate of 1 ml/min. with a gradient of NaCl. The kinase stimulatory activity was recovered at 0.45-0.55 M salt and was spread over four fractions of 2 ml each. These were pooled and loaded directly on a Cu +2 chelating columns (1.6 ml, HR2/5 chelating Superose, Pharmacia). Most of the proteins adsorbed to the resin, but they gradually eluted with a 30 ml linear gradient of ammonium chloride (0-1 M). The activity eluted in a single peak of protein at the range of 0.05 to 0.2 M NH 4 Cl. Samples from various steps of purification were analyzed by gel electrophoresis followed by silver staining using a kit from ICN (Costa Mesa, Calif.), and their protein contents were determined with a Coomassie blue dye binding assay using a kit from Bio-Rad (Richmond, Calif.).
The p44 protein (10 ug) was reconstituted in 200 μl of 0.1 M ammonium bicarbonate buffer (pH 7.8). Digestion was conducted with L-1-tosyl-amide 2-phenylethyl chloromethyl ketone-treated trypsin (Serva) at 37° C. for 18 hr. at an enzyme-to-substrate ratio of 1:10. The resulting peptide mixture was separated by reverse-phase HPLC and monitored at 215 nm using a Vydac C4 micro column (2.1 mm i.d.×15 cm, 300 Å) and an HP 1090 liquid chromatographic system equipped with a diode-array detector and a workstation. The column was equilibrated with 0.1% trifluoroacetic acid (mobile phase A), and elution was effected with a linear gradient from 0%-55% mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) over 70 min. The flow rate was 0.2 ml/min. and the column temperature was controlled at 25° C. One-third aliquots of the peptide peaks collected manually from the HPLC system were characterized by N-terminal sequence analysis by Edman degradation. The fraction eluted after 27.7 min. (T27.7) contained mixed amino acid sequences and was further rechromatographed after reduction as follows: A 70% aliquot of the peptide fraction was dried in vacuo and reconstituted in 100 μl of 0.2 M ammonium bicarbonate buffer (pH 7.8). DTT (final concentration 2 mM) was added to the solution, which was then incubated at 37° C. for 30 min. The reduced peptide mixture was then separated by reverse-phase HPLC using a Vydac column (2.1 mm i.d.×15 cm). Elution conditions and flow rat were identical to those described above. Amino acid sequence analysis of the peptide was performed with a Model 477 protein sequencer (Applied Biosystems, Inc., Foster City, Calif.) equipped with an on-line phenylthiohydantoin (PTH) amino acid analyzer and a Model 900 data analysis system (Hunkapiller et al. (1986) In Methods of Protein Microcharacterization , J. E. Shively, ed. (Clifton, N.J.: Humana Press p. 223-247). The protein as loaded onto a trifluoroacetic acid-treated glass fiber disc precycled with polybrene and NaCl. The PTH-amino acid analysis was performed with a micro liquid chromatography system (Model 120) using dual syringe pumps and reverse-phase (C-18) narrow bore columns (Applied Biosystems, 2.1 mm×250 mm). RNA was isolated from Rat1-EJ cells by standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y. (1982) and poly (A)+was selected using an mRNA Separator kit (Clontech Lab, Inc., Palo Alto, Calif.). cDNA was synthesized with the Superscript kit (from BRL Life Technologies, Inc., Bethesda, Md.). Column-fractionated double-strand cDNA was ligated into an Sal1- and Not1-digested pJT-2 plasmid vector, a derivative of the pCD-X vector (Okayama and Berg, Mol. Cell Biol. 3: 280 (1983)) and transformed into DH10B E. coli cells by electroporation (Dower et al., Nucl. Acids Res. 16: 6127 (1988)). Approximately 5×105 primary transformants were screened with two oligonucleotide probes that were derived from the protein sequences of the N-terminus of NDF (residues 5-24) and the T40.4 tryptic peptide (residues 7-12). Their respective sequences were as follows (N indicates all 4 nt):
(1)
5′-ATA GGG AAG GGC GGG GGA AGG GTC NCC CTC NGC
A T
AGG GCC GGG CTT GCC TCT GGA GCC TCT-3′
(2)
5′-TTT ACA CAT ATA TTC NCC-3′
C G G C
(1: SEQ ID NO: 163; 2: SEQ ID NO: 164)
The synthetic oligonucleotides were end-labeled with [γ- 32 P]ATP with T4 polynucleotide kinase and used to screen replicate sets of nitrocellulose filters. The hybridization solution contained 6×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 2×Denhardt's solution, 50 μg/ml salmon sperm DNA, and 20% formamide (for probe 1) or no formamide (for probe 2). The filters were washed at either 50° C. with 0.5×SSC, 0.2% SDS, 2 mM EDTA (for probe 1) or at 37° C. with 2×SSC, 0.2% SDS, 2 mM EDTA (for probe 2). Autoradiography of the filters gave ten clones that hybridized with both probes. These clones were purified by replating and probe hybridization as described above. The cDNA clones were sequenced using an Applied Biosystems 373A automated DNA sequencer and Applied Biosystems Taq DyeDeoxy™ Terminator cycle sequencing kits following the =manufacture's instructions. In some instances, sequences were obtained using [ 35 S]dATP (Amersham) and Sequenase™ kits from U.S. Biochemicals following the manufacturer's instructions. Both strands of the cDNA clone 44 were sequenced by using synthetic oligonucleotides as primers. The sequence of the most 5′ 350 nt was determined in seven independent cDNA clones. The resultant clone demonstrated the pattern shown in FIG. 27 (NDF).
EXAMPLE 17
Purification and Assay of Other Proteins Which Bind p185 erbB2 Receptor
I. Purification of gp30 and p70
Lupu et al. (Science 249, 1552 (1990)) and Lippman and Lupu (patent application number PCT/US91/03443 (1990)), hereby incorporated by reference, have purified a protein from conditioned media of a human breast cancer cell line MDA-MB-231.
Lupu et al. (Proc. Natl. Acad. Sci. 89, 2287 (1992)) purified another protein which binds to the p185 erbB2 receptor. This particular protein, p75, was purified from conditioned medium used for the growth of SKBr-3 (a human breast cancer cell line) propagated in improved Eagle's medium (IMEM: GIBCO) supplemented with 10% fetal bovine serum (GIBCO).
II. Other p185 erbB2 Ligands
Peles et al. (Cell 69, 205 (1992)) have also purified a 185 erbB2 stimulating ligand from rat cells. Holmes et al. (Science 256, 1205 (1992)) have purified Heregulin α from human cells which binds and stimulates p185 erbB2 (see Example 5). Tarakovsky et al. Oncogene 6:218 (1991) have demonstrated bending of a 25 kD polypeptide isolated from activated macrophages to the Neu receptor, a p185 erbB2 homology, herein incorporated by reference.
III. NDF Isolation
Yarden and Peles (Biochemistry 30, 3543 (1991)) have identified a 35 kilodalton glycoprotein which will stimulate the 185 erbB2 receptor.
In other publications, Davis et al. (Biochem. Biophys. Res. Commun. 179, 1536 (1991), Proc. Natl. Acad. Sci. 88, 8582 (1991) and Greene et al., PCT patent application PCT/US91/02331 (1990)) describe the purification of a protein from conditioned medium of a human T-cell (ATL-2) cell line.
Huang et al. (1992, J. Biol. Chem. 257:11508-11512), hereby incorporated by reference, have isolated an additional neu/erb B2 ligand growth factor from bovine kidney. The 25 kD polypeptide factor was isolated by a procedure of column fractionation, followed by sequential column chromatography on DEAE/cellulose (DE52), Sulfadex (sulfated Sephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquid chromatography). The factor, NEL-GF, stimulates tyrosine-specific autophosphorylation of the neu/erb B2 gene product.
IV. Purification of Acetylcholine Receptor Inducing Activity (ARIA)
ARIA, a 42 kD protein which stimulates acetylcholine receptor synthesis, has been isolated in the laboratory of Gerald Fischbach (Falls et al., (1993) Cell 72:801-815). ARIA induces tyrosine phosphorylation of a 185 Kda muscle transmembrane protein which resembles p185 erbB2 , and stimulates acetylcholine receptor synthesis in cultured embryonic myotubes. ARIA is most likely a member of the GGF/erbB2 ligand group of proteins, and this is potentially useful in the glial cell mitogenesis stimulation and other applications of, e.g., GGF2 described herein.
EXAMPLE 18
Protein tyrosine phosphorylation mediated by GGF
Rat Schwann cells, following treatment with sufficient levels of Glial Growth Factor to induce proliferation, show stimulation of protein tyrosine phosphorylation. Varying amounts of partially purified GGF were applied to a primary culture of rat Schwann cells according to the procedure outlined in Example 9. Schwann cells were grown in DMEM/10% fetal calf serum/5 μM forskolin/0.5 μg per mL GGF-CM (0.5 mL per well) in poly D-lysine coated 24 well plates. When confluent, the cells were fed with DMEM/10% fetal calf serum at 0.5 mL per well and left in the incubator overnight to quiesce. The following day, the cells were fed with 0.2 mL of DMEM/10% fetal calf serum and left in the incubator for 1 hour. Test samples were then added directly to the medium at different concentrations and for different lengths of time as required. The cells were then lysed in boiling lysis buffer (sodium phosphate, 5 mM, pH 6.8; SDS, 2%, B-mercapteothanol, 5%; dithiothreitol, 0.1 M; glycerol, 10%; Bromophenol Blue, 0.4%; sodium vanadate, 10 mM), incubated in a boiling water bath for 10 minutes and then either analyzed directly or frozen at −70° C. Samples were analyzed by running on 7.5% SDS-PAGE gels and then electroblotting onto nitrocellulose using standard procedures as described by Towbin et al. (1979) Proc. Natl. Acad. Sci. USA 76:4350-4354. The blotted nitrocellulose was probed with antiphosphotyrosine antibodies using standard methods as described in Kamps and Selton (1988) Oncogene 2:305-315. The probed blots were exposed to autoradiography film overnight and developed using a standard laboratory processor. Densitometric measurements were carried out using an Ultrascan XL enhanced laser densitometer (LKB). Molecular weight assignments were made relative to prestained high molecular weight standards (Sigma). The dose responses of protein phosphorylation and Schwann cell proliferation are very similar (FIG. 33 ). The molecular weight of the phosphorylated band is very close to the molecular weight of p185 erbB2 . Similar results were obtained when Schwann cells were treated with conditioned media prepared from COS cells translates with the GGF2HBS5 clone. These results correlate well with the expected interaction of the GGFs with and activation of p185 erbB2 This experiment has been repeated with recombinant GGF2. Conditioned medium derived from a CHO cell line stably transformed with the GGF2 clone (GGF2HBS5) stimulates protein tyrosine phosphorylation using the assay described above. Mock transfected CHO cells fail to stimulate this activity.
EXAMPLE 19
N-glycosylation of GGF
The protein sequence predicted from the cDNA sequence of GGF-II candidate clones GGF2BPP1,2 and 3 contains a number of consensus N-glycosylation motifs. A gap in the GGFII02 peptide sequence coincides with the asparagine residue in one of these motifs, indicating that carbohydrate is probably bound at this site.
N-glycosylation of the GGFs was studied by observing mobility changes on SDS-PAGE after incubation with N-glycanase, an enzyme that cleaves the covalent linkages between carbohydrate and aspargine residues in proteins.
N-Glycanase treatment of GGF-II yielded a major band of MW 40-42 kDa and a minor band at 45-48 kDa. Activity single active deglycosylated species at ca 45-50 kDa.
Activity elution experiments with GGF-I also demonstrate an increase in electrophoretic mobility when treated with N-Glycanase, giving an active species of MW 26-28 kDa. Silver staining confirmed that there is a mobility shift, although no N-deglycosylated band could be assigned because of background staining in the sample used.
Further embodiments are within the following claims.
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The invention relates to methods of treating diseases and disorders of the muscle tissues in a vertebrate by the administration of compounds which bind the p185 erbB2 receptor. These compounds are found to cause increased differentiation and survival of cardiac, skeletal and smooth muscle.
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CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of provisional patent application Ser. No. 60/132,036, filed Apr. 30, 1999.
FIELD OF THE INVENTION
The present invention relates to highly selective phosphodiesterase (PDE) enzyme inhibitors and to their use in pharmaceutical articles of manufacture. In particular, the present invention relates to potent inhibitors of cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase type 5 (PDE5) that when incorporated into a pharmaceutical product are useful for the treatment of sexual dysfunction. The articles of manufacture described herein are characterized by selective PDE5 inhibition, and accordingly, provide a benefit in therapeutic areas where inhibition of PDE5 is desired, with minimization or elimination of adverse side effects resulting from inhibition of other phosphodiesterase enzymes.
BACKGROUND OF THE INVENTION
The biochemical, physiological, and clinical effects of cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase (cGMP-specific PDE) inhibitors suggest their utility in a variety of disease states in which modulation of smooth muscle, renal, hemostatic, inflammatory, and/or endocrine function is desired. Type 5 cGMP-specific phosphodiesterase (PDE5) is the major cGMP hydrolyzing enzyme in vascular smooth muscle, and its expression in penile corpus cavernosum has been reported (Taher et al., J. Urol., 149:285A (1993)). Thus, PDE5 is an attractive target in the treatment of sexual dysfunction (Murray, DN & P 6(3):150-56 (1993)).
A pharmaceutical product, which provides a PDE5 inhibitor, is currently available and marketed under the trademark VIAGRA®. The active ingredient in VIAGRA® is sildenafil. The product is sold as an article of manufacture including 25, 50, and 100 mg tablets of sildenafil and a package insert. The package insert provides that sildenafil is a more potent inhibitor of PDE5 than other known phosphodiesterases (greater than 80 fold for PDE1 inhibition, greater than 1,000 fold for PDE2, PDE3, and PDE4 inhibition). The IC 50 for sildenafil against PDE5 has been reported as 3 nM ( Drugs of the Future, 22(2), pp. 128-143 (1997)), and as 3.9 nM (Boolell et al., Int. J. of Impotence Res., 8 p. 47-52 (1996)). N.C. Sildenafil is described as having a 4,000-fold selectivity for PDE5 versus PDE3, and only a 10-fold selectivity for PDE5 versus PDE6. Its relative lack of selectivity for PDE6 is theorized to be the basis for abnormalities related to color vision.
While sildenafil has obtained significant commercial success, it has fallen short due to its significant adverse side effects, including facial flushing (10% incidence rate). Adverse side effects limit the use of sildenafil in patients suffering from visual abnormalities, hypertension, and, most significantly, by individuals who use organic nitrates (Welds et al., Amer. J. of Cardiology, 83 (5A), pp. 21(C)-28(C) (1999)).
The use of sildenafil in patients taking organic nitrates is believed to cause a clinically significant drop in blood pressure which could place the patient in danger. Accordingly, the package label for sildenafil provides strict contraindications against its use in combination with organic nitrates (e.g., nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, erythrityl tetranitrate) and other nitric oxide donors in any form, either regularly or intermittently, because sildenafil potentiates the hypotensive effects of nitrates. See C. R. Conti et al., Amer. J. of Cardiology, 83(5A), pp. 29C-34C (1999). Thus, even with the availability of sildenafil, there remains a need to identify improved pharmaceutical products that are useful in treating sexual dysfunction.
The present invention provides an article of manufacture for human pharmaceutical use, comprising a package insert, a container, and an oral dosage form comprising a selective PDE5 inhibitor at unit dosages between about 1 and about 20 mg/dosage form. The beneficial effects of the present invention were observed in clinical studies and through the discovery that a selective PDE5 inhibitor meeting the following criteria allows for the effective oral administration of about 1 to about 20 mg/dosage form without contraindications generally required for PDE5 inhibitor products, such as warnings directed to vision abnormalities. A selective PDE5 inhibitor of the present invention exhibits:
1) at least a 100 fold differential in the IC 50 values for the inhibition of PDE5 versus PDE6 for a particular PDE5 inhibitor (i.e., the IC 50 value versus PDE5 is at least 100 times less than the IC 50 value versus PDE6);
2) at least a 1000 fold differential in the IC 50 values for the inhibition of PDE5 versus PDE1c; and
3) an IC 50 value less than 10 nM.
Significantly, clinical studies also revealed that an effective product having a reduced tendency to cause flushing in susceptible individuals can be provided. Most unexpectedly, the product also can be administered with clinically insignificant side effects associated with the combined effects of a PDE5 inhibitor and an organic nitrate. Thus, the contraindication once believed necessary for a product containing a PDE5 inhibitor is unnecessary when a selective PDE5 inhibitor, as defined above, is used as disclosed herein. Thus, the present invention provides an effective therapy for sexual dysfunction in individuals who previously were untreatable or suffered from unacceptable side effects, including individuals having cardiovascular disease, such as in individuals requiring nitrate therapy, having suffered a myocardial infarction more than three months before the onset of sexual dysfunction therapy, and suffering from class 1 congestive heart failure as defined by the New York Heart Association (NYHA), or individuals suffering from vision abnormalities.
SUMMARY OF THE INVENTION
The present invention provides an article of manufacture for human pharmaceutical use, comprising a package insert, a container, and an oral dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor per dosage form.
The present invention further provides a method of treating conditions where inhibition of PDE5 is desired, which comprises administering to a patient in need thereof an oral dosage form containing about 1 to about 20 mg of a selective PDE5 inhibitor, as needed, up to a total dose of 20 mg/-day. The invention further provides the use of an oral dosage form comprising a selective PDE5 inhibitor at a dosage of about 1 to about 20 mg for the treatment of sexual dysfunction.
Specific conditions that can be treated by the method and article of the present invention, include, but are not limited to, male erectile dysfunction and female sexual dysfunction, particularly female arousal disorder, also known as female sexual arousal disorder.
In particular, the present invention provides an article of manufacture for human pharmaceutical use comprising:
(a) an oral dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor having
(i) at least a 100 fold differential in IC 50 values for the inhibition of PDE5 versus PDE6,
(ii) at least a 1000 fold differential in IC 50 values for the inhibition of PDE5 versus PDE1c,
(iii) an IC 50 less than 10 nM, and
(iv) sufficient bioavailability to be effective in about 1 to about 20 mg unit oral dosages;
(b) a package insert providing that the PDE5 inhibitor is useful to treat sexual dysfunction in a patient in need thereof, and that is free of contradictions associated with administration of organic nitrates; and
(c) a container.
The present invention further provides an article of manufacture for human pharmaceutical use comprising:
(a) an oral dosage form comprising about 1 to about 20 mg of selective PDE5 inhibitor having
(i) at least a 100 fold differential in IC 50 values for the inhibition of PDE5 versus PDE6,
(ii) at least a 1000 fold differential in IC 50 values for the inhibition of PDE5 versus PDE1c,
(iii) an IC 50 less than 10 nM, and
(iv) a sufficient bioavailability to be effective in about 1 to about 20 mg unit oral dosages;
(b) a package insert providing that the PDE5 inhibitor is useful to treat sexual dysfunction in a patient in need thereof and that is using an organic nitrate; and
(c) a container.
The present invention also provides an article of manufacture for human pharmaceutical use comprising:
(a) an oral dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor having
(i) at least a 100 fold differential in IC 50 values for the inhibition of PDE5 versus PDE6,
(ii) at least 1000 fold differential in IC 50 values for the inhibition of PDE5 versus PDE1c,
(iii) an IC 50 less than 10 nM, and
(iv) a sufficient bioavailability to be effective in about 1 to about 20 mg unit oral dosages;
(b) a package insert providing that the PDE5 inhibitor is useful to treat sexual dysfunction in a patient in need thereof and that is suffering from a condition selected from the group consisting of a retinal disease, proneness to flushing, proneness to vision abnormalities, class 1 congestive heart failure, a myocardial infarction 90 days or more before onset of the sexual dysfunction treatment, and combinations thereof; and
(c) a container.
DETAILED DESCRIPTION
For purposes of the present invention as disclosed and described herein, the following terms and abbreviations are defined as follows.
The term “container” means any receptacle and closure therefor suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.
The term “IC 50 ” is the measure of potency of a compound to inhibit a particular PDE enzyme (e.g., PDE1c, PDE5, or PDE6). The IC 50 is the concentration of a compound that results in 50% enzyme inhibition in a single dose-response experiment. Determining the IC 50 value for a compound is readily carried out by a known in vitro methodology generally described in Y. Cheng et al., Biochem. Pharmacol., 22, pp. 3099-3108 (1973).
The term “package insert” means information accompanying the product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product. The package insert generally is regarded as the “label” for a pharmaceutical product.
The term “oral dosage form” is used in a general sense to reference pharmaceutical products administered orally. Oral dosage forms are recognized by those skilled in the art to include such forms as liquid formulations, tablets, capsules, and gelcaps.
The term “selective PDE5 inhibitor” is defined as a PDE5 inhibitor having:
1) an IC 50 value for the inhibition of PDE5 at least 100 times less than the IC 50 value for the inhibition of PDE6;
2) an IC 50 value for the inhibition of PDE5 at least 1,000 times less than the IC 50 value for the inhibition of PDE1c; and
3) an IC 50 value for the inhibition of PDE5 less than 10 nM. Selective PDE5 inhibitors vary significantly in chemical structure, and their use in the present invention is not dependent on chemical structure, but rather on the selectivity and potency parameters disclosed herein.
The term “vision abnormalities” means abnormal vision characterized by blue-green vision believed to be caused by PDE6 inhibition.
The term “flushing” means an episodic redness of the face and neck attributed to vasodilation caused by the ingestion of a drug, usually accompanied by a feeling of warmth over the face and neck and sometimes accompanied by perspiration.
The term “free drug” means solid particles of drug not intimately embedded in a polymeric co-precipitate.
As previously stated, the present invention is directed to an article of manufacture for human pharmaceutical use, comprising a package insert, a container, and a dosage form comprising about 1 to about 20 mg of a selective PDE5 inhibitor per unit dosage form. A selective PDE5 inhibitor useful in the present invention is a PDE5 inhibitor having:
1) at least a 100 fold differential in IC 50 values for the inhibition of PDE5 versus PDE6;
2) at least a 1000 fold differential in IC 50 values for the inhibition of PDE5 versus PDE1c; and
3) an IC 50 value less than 10 nM; and is sufficiently bioavailable to be effective in about 1 to about 20 mg unit dosages.
The differential is expressed as a PDE6/PDE5 ratio of IC 50 values, i.e., the ratio of the IC 50 value versus PDE6 to the IC 50 value versus PDE5 (PDE6/PDE5) is greater than 100, more preferably greater than 300, and most preferably greater than 500.
Similarly, the ratio of IC 50 value versus PDE1c to IC 50 value versus PDE5 (PDE1c/PDE5) is greater than 1000. Preferred PDE5 inhibitors have a greater than 3,000 fold differential between the inhibition of PDE5 and PDE1c, more preferably greater than a 5,000 fold differential between IC 50 value versus PDE5 and PDE1c. The potency of the inhibitor, as represented by the IC 50 value versus PDE5, is less than 10 nM, preferably less than 5 nM, more preferably less than 2 nM, and most preferably less than 1 nM.
The package insert provides a description of how to administer a pharmaceutical product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding the use of the product. The package insert generally is regarded as the label of the pharmaceutical product. The package insert incorporated into the present article of manufacture indicates that the selective PDE5 inhibitor is useful in the treatment of conditions wherein inhibition of PDE5 is desired. The package insert also provides instructions to administer one or more about 1 to about 20 mg unit dosage forms as needed, up to a maximum total dose of 20 mg per day. Preferably, the dose administered is about 5 to about 20 mg/day, more preferably about 5 to about 15 mg, and most preferably an about 5 mg or about 10 mg dosage form administered once per day, as needed.
Preferred conditions to be treated include sexual dysfunction (including male erectile dysfunction; and female sexual dysfunction, and more preferably female arousal disorder (FAD)). The preferred condition to be treated is male erectile dysfunction.
Significantly, the package insert supports use of the product to treat sexual dysfunction in patients suffering from a retinal disease, for example diabetic retinopathy or retinitis pigmentosa, or in patients who are using organic nitrates. Thus, the package insert preferably is free of contraindications associated with these conditions, and particularly the administration of the dosage form with an organic nitrate. More preferably, the package insert also is free of any cautions or warnings both associated with retinal diseases, particularly retinitis pigmentosa, and associated with individuals prone to vision abnormalities. Preferably, the package insert also reports incidences of flushing below 2%, preferably below 1%, and most preferably below 0.5%, of the patients administered the dosage form. The incidence rate of flushing demonstrates marked improvement over prior pharmaceutical products containing a PDE5 inhibitor.
The container used in the present article of manufacture is conventional in the pharmaceutical arts. Generally, the container is a blister pack, foil packet, glass or plastic bottle and accompanying cap or closure, or other such article suitable for use by the patient or pharmacist. Preferably, the container is sized to accommodate 1-1000 solid dosage forms, preferably 1 to 500 solid dosage forms, and most preferably, 5 to 30 solid dosage forms.
Oral dosage forms are recognized by those skilled in the art to include, for example, such forms as liquid formulations, tablets, capsules, and gelcaps. Preferably the dosage forms are solid dosage forms, particularly, tablets comprising about 1 to about 20 mg of a selective PDE5 inhibitor. Any pharmaceutically acceptable excipients for oral use are suitable for preparation of such dosage forms. Suitable pharmaceutical dosage forms include coprecipitate forms described, for example, in Butler U.S. Pat. No. 5,985,326, incorporated herein by reference. In preferred embodiments, the unit dosage form of the present invention is a solid free of a coprecipitate form of the PDE5 inhibitor, but rather contains a solid PDE5 inhibitor as a free drug.
Preferably, the tablets comprise pharmaceutical excipients generally recognized as safe such as lactose, microcrystalline cellulose, starch, calcium carbonate, magnesium stearate, stearic acid, talc, and colloidal silicon dioxide, and are prepared by standard pharmaceutical manufacturing techniques as described in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). Such techniques include, for example, wet granulation followed by drying, milling, and compression into tablets with or without film coating; dry granulation followed by milling, compression into tablets with or without film coating; dry blending followed by compression into tablets, with or without film coating; molded tablets; wet granulation, dried and filled into gelatin capsules; dry blend filled into gelatin capsules; or suspension and solution filled into gelatin capsules. Generally, the solid dosage forms have identifying marks which are debossed or imprinted on the surface.
The present invention is based on detailed experiments and clinical trials, and the unexpected observations that side effects previously believed to be indicative of PDE5 inhibition can be reduced to clinically insignificant levels by the selection of a selective PDE5 inhibitor having the specific characteristics outlined herein, namely:
1) at least a 100 fold differential in the IC 50 values for the inhibition of PDE5 versus PDE6;
2) at least a 1000 fold differential in the IC 50 values for the inhibition of PDE5 versus PDE1c; and
3) an IC 50 value for the inhibition of PDE5 less than 10 nM.
This unexpected observation enabled the development of articles of manufacture that incorporate a selective PDE5 inhibitor in about 1 to about 20 mg unit dosage forms that, when orally administered, minimize undesired side effects previously believed unavoidable. These side effects include facial flushing, vision abnormalities, and a significant decrease in blood pressure when the PDE5 inhibitor is administered alone or in combination with an organic nitrate. The minimal effect of a present PDE5 inhibitor, administered in about 1 to about 20 mg unit dosage forms, on PDE6 also allows the administration of a selective PDE5 inhibitor to patients suffering from a retinal disease, like diabetic retinopathy or retinitis pigmentosa.
One such selective PDE5 inhibitor, i.e., (6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methylpyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione, alternatively named (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylene-dioxyphenyl)pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione, is disclosed in Daugan U.S. Pat. No. 5,859,006, and represented by structural formula (I):
The compound of formula (I) was demonstrated in human clinical studies to exert a minimal impact on systolic blood pressure when administered in conjunction with organic nitrates. By contrast, sildenafil, when administered with nitrates, demonstrates as much as a four-fold greater decrease in systolic blood pressure over a placebo, which leads to the contraindications in the VIAGRA® insert, and in warnings to certain patients.
Selective PDE5 inhibitors vary significantly in chemical structure, and the use of a selective PDE5 inhibitor as defined in the present invention is not dependent on a particular chemical structure, but rather on the critical parameters outlined herein. However, preferred compounds having the required potency and selectivity can be readily identified by tests described herein from those described in Daugan U.S. Pat. No. 5,859,006, Daugan et al. U.S. Pat. No. 5,981,527, and Daugan et al. U.S. Pat. No. 6,001,847, each of which is incorporated herein by reference.
Preferred compounds of Daugan U.S. Pat. No. 5,859,006 and Daugan et al. U.S. Pat. No. 5,981,527 are represented by structural formula (II):
wherein R 0 is selected from the group consisting of hydrogen, halogen, and C 1-6 alkyl;
R 1 is selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkynyl, halo-C 1-6 alkyl, C 3-8 cycloalkyl, C 3-8 cycloalkylC 1-3 alkyl, arylC 1-3 alkyl, wherein aryl is phenyl or phenyl substituted with one to three substituents selected from the group consisting of halogen, C 1-6 alkyl, C 1-6 alkoxy, methylenedioxy, and mixtures thereof, and heteroarylC 1-3 alkyl, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents selected from the group consisting of halogen, C 1-6 alkyl, C 1-6 alkoxy, and mixtures thereof;
R 2 represents an optionally substituted monocyclic aromatic ring selected from benzene, thiophene, furan, and pyridine, or an optionally substituted bicyclic ring
attached to the rest of the molecule via one of the benzene ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from the group consisting of oxygen, sulphur and nitrogen;
R 3 represents hydrogen or C 1-3 alkyl, or R 1 and R 3 together represent a 3- or 4-membered alkyl or alkenyl chain; and salts and solvates thereof.
Other preferred compounds are those of formula (II) wherein:
R 0 is hydrogen, halogen, or C 1-6 alkyl;
R 1 is hydrogen or C 1-6 alkyl;
R 2 is the bicyclic ring
which can be optionally substituted by one or more groups selected from halogen and C 1-3 alkyl; and
R 3 is hydrogen or C 1-3 alkyl.
The following Table 1 illustrates PDE5 and PDE6 IC 50 values for representative selective PDE5 inhibitors disclosed in U.S. Pat. No. 5,859,006, as determined by the procedures described herein.
TABLE 1
Compound
PDE5 IC 50 (nM)
PDE6 IC 50 (nM)
PDE6/PDE5
1
5
663
133
2
2
937
469
3
2
420
210
4
5
729
146
5
2.5
3400
1360
Compound 5 in Table 1 has the structural formula (I) and additionally demonstrates an IC 50 against PDE1c of 10,000 and a ratio of PDE1c/PDE5 of 4,000.
The structures of Compound Nos. 1-5 in Table 1 are as in structural formula (II) wherein R 0 , R 1 , R 2 , and R 3 are as follows:
Compound
R 0
R 1
R 2
R 3
1
H
H
2
H
CH 3
H
3
H
H
4
H
H
CH 3
5
H
CH 3
H
The data in Table 1 indicate that a compound of structural formula (I), wherein R 1 is hydrogen or C 1-6 alkyl, R 2 is
and R 3 is hydrogen is especially preferred. Preferably, A is
Preferred compounds are:
(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione; and
(3S,6R,12aR)-2,3,6,7,12,12a-hexahydro-2,3-dimethyl-6-(3,4-methylenedioxyphenyl)pyrazino[2′,1′:6,1]-pyrido[3,4-b]indole-1,4-dione; and physiologically acceptable salts and solvates (e.g., hydrates) thereof.
Other exemplary compounds useful in the present invention are those disclosed in Daugan et al. U.S. Pat. No. 6,001,847 and WO 97/43287, incorporated herein by reference.
Further exemplary compounds for use in the present invention are disclosed PCT application PCT/EP98/06050, which designates the U.S., entitled “Chemical Compounds,” inventors A. Bombrun and F. Gellibert, the disclosure of which is specifically incorporated herein by reference. This class of compounds has the structural formula (III):
and salts and solvates (e.g., hydrates) wherein
C represents a 5- or 6-membered heteroaryl group containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;
R 12 represents hydrogen or halogen;
R 13 is selected from the group consisting of
hydrogen,
nitro (NO 2 ),
trifluoromethyl,
trifluoromethoxy,
halogen,
cyano (CN),
a 5- or 6-membered heterocyclic group containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulphur, optionally substituted with C(═O)OR a or C 1-4 alkyl,
C 1-6 alkyl optionally substituted with OR h ,
C 1-3 alkoxy,
C(═O)R h ,
OC(═O)OR h ,
C(═O)OR h ,
C 1-4 alkyleneHet,
C 1-4 alkyleneC(═O)OR h ,
OC 1-4 alkyleneC(═O)OR h ,
C 1-4 alkyleneOC 1-4 alkyleneC(═O)OR h ,
C(═O)NR i SO 2 R j ,
C(═O)C 1-4 alkyleneHet,
C 1-4 alkyleneNR h R i ,
C 2-6 alkenyleneNR h R i ,
C(═O)NR h R i ,
C(═O)NR h R i ,
C(═O)NR h C 1-4 alkyleneOR i ,
C(═O)NR h C 1-4 alkyleneHet,
OR i ,
OC 2-4 alkyleneNR h R i ,
OC 2-4 alkyleneCH(OR h )CH 2 NR h R i ,
OC 1-4 alkyleneHet,
OC 2-4 alkyleneOR h ,
OC 2-4 alkyleneNR h C(═O)OR h ,
NR h R i ,
NR h C 1-4 alkyleneNR h R i ,
NR h C(═O)R i ,
NR h C(═O)NR h R i ,
N(SO 2 C 1-4 alkyl) 2 ,
NR h (SO 2 C 1-4 alkyl),
SO 2 NR h R i ,
and OSO 2 trifluoromethyl;
R 14 is selected from the group consisting of hydrogen, halogen, OR h , C 1-6 alkyl, NO 2 , and NR h R i ;
or R 13 and R 14 are taken together to form a 3- or 4-membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;
R 15 is selected from the group consisting of hydrogen, halogen, NO 2 , trifluoromethoxy, C 1-6 alkyl, OC 1-6 alkyl, and C(═O)OR h ;
R 16 is hydrogen,
or R 15 and R 16 are taken together to form a 3- or 4-membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;
Het represents a 5- or 6-membered heterocyclic group containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with C 1-4 alkyl;
R h and R i can be the same or different and are independently selected from hydrogen and C 1-6 alkyl;
R j represents phenyl or C 4-6 cycloalkyl, wherein the phenyl or C 4-6 cycloalkyl can be optionally substituted with one or more halogen atoms, one or more C(═O)OR h , or one or more OR h ;
n is an integer 1, 2, or 3; and
m is an integer 1 or 2.
Preparations
Human PDE5 Preparation
Recombinant production of human PDE5 was carried out essentially as described in Example 7 of U.S. Pat. No. 5,702,936, incorporated herein by reference, except that the yeast transformation vector employed, which is derived from the basic ADH2 plasmid described in V. Price et al., Methods in Enzymology, 1985, pages 308-318 (1990), incorporated yeast ADH2 promoter and terminator sequences rather than ADH1 promoter and terminator sequences and the Saccharomyces cerevisiase host was the protease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with the American Type Culture Collection, Manassas, Va., under accession number ATCC 74465. Transformed host cells were grown in 2×SC-leu medium, pH 6.2, with trace metals, and vitamins. After 24 hours, YEP medium containing glycerol was added to a final concentration of 2×YEP/3% glycerol. Approximately 24 hours later, cells were harvested, washed, and stored at −70° C.
Cell pellets (29 g) were thawed on ice with an equal volume of lysis buffer (25 mM Tris-Cl, pH 8, 5 mM MgCl 2 , 0.25 mM dithiothreitol, 1 mM benzamidine, and 10 μM ZnSO 4 ). Cells were lysed in a microfluidizer with N 2 at 20,000 psi. The lysate was centrifuged and filtered through 0.45 μm disposable filters. The filtrate was applied to a 150 mL column of Q Sepharose Fast Flow (Pharmacia). The column was washed with 1.5 volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1 mM MgCl 2 , 0.25 mM dithiothreitol, 10 μM ZnSO 4 ) and eluted with a step gradient of 125 mM NaCl in Buffer A followed by a linear gradient of 125-1000 mM NaCl in Buffer A.
Active fractions from the linear gradient were applied to a 180 mL hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane (pH 6.8), 1 mM MgCl 2 , 0.25 mM dithiothreitol, 10 μM ZnSO 4 , and 250 mM KCl). After loading, the column was washed with 2 volumes of Buffer B and eluted with a linear gradient of 0-125 mM potassium phosphate in Buffer B. Active fractions were pooled, precipitated with 60% ammonium sulfate, and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mM NaCl, 0.5 mM dithiothreitol, and 10 μM ZnSO 4 ). The pool was applied to a 140 mL column of Sephacryl S-300 HR and eluted with Buffer C. Active fractions were diluted to 50% glycerol and stored at −20° C. The resultant preparations were about 85% pure by SDS-PAGE.
Assay for PDE Activity
Activity of PDE5 can be measured by standard assays in the art. For example, specific activity of any PDE can be determined as follows. PDE assays utilizing a charcoal separation technique were performed essentially as described in Loughney et al., (1996), The Journal of Biological Chemistry, 271:796-806. In this assay, PDE5 activity converts [ 32 P]cGMP to [ 32 P]5′GMP in proportion to the amount of PDE5 activity present. The [ 32 P]5′GMP then is quantitatively converted to free [ 32 P] phosphate and unlabeled adenosine by the action of snake venom 5′-nucleotidase. Hence, the amount of [ 32 P] phosphate liberated is proportional to enzyme activity. The assay is performed at 30° C. in a 100 μL reaction mixture containing (final concentrations) 40 mM Tris-Cl (pH 8.0), 1 μM ZnSO 4 , 5 mM MgCl 2 , and 0.1 mg/mL bovine serium albumin. PDE5 is present in quantities that yield <30% total hydrolysis of substrate (linear assay conditions). The assay is initiated by addition of substrate (1 mM [ 32 P]cGMP), and the mixture is incubated for 12 minutes. Seventy-five (75) μg of Crotalus atrox venom then is added, and the incubation is continued for 3 more minutes (15 minutes total). The reaction is stopped by addition of 200 mL of activated charcoal (25 mg/mL suspension in 0.1 M NaH 2 PO 4 , pH 4). After centrifugation (750×g for 3 minutes) to sediment the charcoal, a sample of the supernatant is taken for radioactivity determination in a scintillation counter and the PDE5 activity is calculated. The preparations had specific activities of about 3 μmoles cGMP hydrolyzed per minute per milligram protein.
Bovine PDE6 Preparation
Bovine PDE6 was supplied by Dr. N. Virmaux, INSERM U338, Strasbourg. Bovine retinas were prepared as described by Virmaux et al., FEBS Letters, 12(6), pp. 325-328 (1971) and see also, A. Sitaramayya et al., Exp. Eye Res., 25, pp. 163-169 (1977). Briefly, unless stated otherwise, all operations were done in the cold and in dim red light. Eyes were kept in the cold and in the dark for up to four hours after slaughtering.
Preparation of bovine retinal outer segment (ROS) basically followed procedures described by Schichi et al., J. Biol. Chem., 224:529 (1969). In a typical experiment, 35 bovine retinas were ground in a mortar with 35 mL 0.066 M phosphate buffer, pH 7.0, made up to 40% with sucrose, followed by homogenization in a Potter homogenizer (20 up and down strokes). The suspension was centrifuged at 25,000×g for 20 minutes. The pellet was homogenized in 7.5 mL 0.006 M phosphate buffer (40% in sucrose), and carefully layered under 7.5 mL of phosphate buffer (containing no sucrose). Centrifugation was conducted in a swing-out rotor at 45,000×g for 20 minutes, and produced a pellet which is black at the bottom, and also a red band at the interface 0.066 M. phosphate—40% sucrose/0.066 M phosphate (crude ROS). The red material at the interface was removed, diluted with phosphate buffer, spun down to a pellet, and redistributed in buffered 40% sucrose as described above. This procedure was repeated 2 or 3 times until no pellet was formed. The purified ROS was washed in phosphate buffer and finally spun down to a pellet at 25,000×g for 20 minutes. All materials were then kept frozen until used.
Hypotonic extracts were prepared by suspending isolated ROS in 10 mM Tris-Cl pH 7.5, 1 mM EDTA, and 1 mM dithioerythritol, followed by centrifugation at 100,000×g for 30 minutes.
The preparation was reported to have a specific activity of about 35 nmoles cGMP hydrolyzed per minute per milligram protein.
PDE1c Preparation from Spodoptera fugiperda Cells (Sf9)
Cell pellets (5g) were thawed on ice with 20 ml of Lysis Buffer (50 mM MOPS pH 7.4, 10 μM ZnSO 4 , 0.1 mM CaCl 2 , 1 mM DTT, 2 mM benzamidine HCl, 5μg/ml each of pepstatin, leupeptin, and aprotenin). Cells were lysed by passage through a French pressure cell (SLM-Aminco) while temperatures were maintained below 10° C. The resultant cell homogenate was centrifuged at 36,000 rpm at 4° C. for 45 minutes in a Beckman ultracentrifuge using a Type TI45 rotor. The supernatant was discarded and the resultant pellet was resuspended with 40 ml of Solubilization Buffer (Lysis Buffer containing 1M NaCl, 0.1M MgCl 2 , 1 mM CaCl 2 , 20 μg/ml calmodulin, and 1% Sulfobetaine SB12 (Z3-12) by sonicating using a VibraCell tuner with a microtip for 3×30 seconds. This was performed in a crushed ice/salt mix for cooling. Following sonication, the mixture was slowly mixed for 30 minutes at 4° C. to finish solubilizing membrane bound proteins. This mixture was centrifuged in a Beckman ultracentrifuge using a type TI45 rotor at 36,000 rpm for 45 minutes. The supernatant was diluted with Lysis Buffer containing 10 μg/ml calpain inhibitor I and II. The precipitated protein was centrifuged for 20 minutes at 9,000 rpm in a Beckman JA-10 rotor. The recovered supernatant then was subjected to Mimetic Blue AP Agarose Chromatography.
In order to run the Mimetic Blue AP Agarose Column, the resin initially was shielded by the application of 10 bed volumes of 1% polyvinyl-pyrrolidine (i.e., MW of 40,000) to block nonspecific binding sites. The loosely bound PVP-40 was removed by washing with 10 bed volumes of 2M NaCl, and 10 mM sodium citrate pH 3.4. Just prior to addition of the solubilized PDE1c sample, the column was equilibrated with 5 bed volumes of Column Buffer A (50 mM MOPS pH 7.4, 10 μM ZnSO 4 , 5 mM MgCl 2 , 0.1 mM CaCl 2 , 1 mM DTT, 2 mM benzamidine HCl).
The solubilized sample was applied to the column at a flow rate of 2 ml/min with recycling such that the total sample was applied 4 to 5 times in 12 hours. After loading was completed, the column was washed with 10 column volumes of Column Buffer A, followed by 5 column volumes of Column Buffer B (Column Buffer A containing 20 mM 5′-AMP), and followed by 5 column volumes of Column Buffer C (50 mM MOPS pH 7.4, 10 μM ZnSO 4 , 0.1 mM CaCl 2 , 1 mM dithiothreitol, and 2 mM benzamidine HCl). The enzyme was eluted into three successive pools. The first pool consisted of enzyme from a 5 bed volume wash with Column Buffer C containing 1 mM cAMP. The second pool consisted of enzyme from a 10 bed volume wash with Column Buffer C containing 1 M NaCl. The final pool of enzyme consisted of a 5 bed volume wash with Column Buffer C containing 1 M NaCl and 20 mM cAMP.
The active pools of enzyme were collected and the cyclic nucleotide removed via conventional gel filtration chromatography or chromatography on hydroxy-apatite resins. Following removal of cyclic nucleotides, the enzyme pools were dialyzed against Dialysis Buffer containing 25 mM MOPS pH 7.4, 10 μM ZnSO 4 , 500 mM NaCl, 1 mM CaCl 2 , 1 mM dithiothreitol, 1 mM benzamidine HCl, followed by dialysis against Dialysis buffer containing 50% glycerol. The enzyme was quick frozen with the aid of dry ice and stored at −70° C.
The resultant preparations were about >90% pure by SDS-PAGE. These preparations had specific activities of about 0.1 to 1.0 μmol cAMP hydrolyzed per minute per milligram protein.
IC 50 Value Determinations
The parameter of interest in evaluating the potency of a competitive enzyme inhibitor of PDE5 and/or PDE1c and PDE6 is the inhibition constant, i.e., K i . This parameter can be approximated by determining the IC 50 , which is the inhibitor concentration that results in 50% enzyme inhibition, in a single dose-response experiment under the following conditions.
The concentration of inhibitor is always much greater than the concentration of enzyme, so that free inhibitor concentration (which is unknown) is approximated by total inhibitor concentration (which is known).
A suitable range of inhibitor concentrations is chosen (i.e., inhibitor concentrations at least several fold greater and several fold less than the K i are present in the experiment). Typically, inhibitor concentrations ranged from 10 nM to 10 μM.
The concentrations of enzyme and substrate are chosen such that less than 20% of the substrate is consumed in the absence of inhibitor (providing, e.g., maximum substrate hydrolysis of from 10 to 15%), so that enzyme activity is approximately constant throughout the assay.
The concentration of substrate is less than one-tenth the Michaelis constant (K m ). Under these conditions, the IC 50 will closely approximate the K i . This is because of the Cheng-Prusoff equation relating these two parameters: IC 50 =K i (1+S/K m ), with (1+S/K m ) approximately 1 at low values of S/K m .
The IC 50 value is estimated from the data points by fitting the data to a suitable model of the enzyme inhibitor interaction. When this interaction is known to involve simple competition of the inhibitor with the substrate, a two-parameter model can be used:
Y=A /(1 +x/B )
where the y is the enzyme activity measured at an inhibitor concentration of x, A is the activity in the absence of inhibitor and B is the IC 50 . See Y. Cheng et al., Biochem. Pharmacol., 22:3099-3108 (1973).
Effects of inhibitors of the present invention on enzymatic activity of PDE5 and PDE6 preparations as described above were assessed in either of two assays which differed from each other principally on the basis of scale and provided essentially the same results in terms of IC 50 values. Both assays involved modification of the procedure of Wells et al., Biochim. Biophys. Acta, 384:430 (1975). The first of the assays was performed in a total volume of 200 μl containing 50 mM Tris pH 7.5, 3 mM Mg acetate, 1 mM EDTA, 50 μg/mL snake venom nucleotidase and 50 nM [ 3 H]-cGMP (Amersham). Compounds of the invention were dissolved in DMSO finally present at 2% in the assay. The assays were incubated for 30 minutes at 30° C. and stopped by addition of 800 μl of 10 mM Tris pH 7.5, 10 mM EDTA, 10 mM theophylline, 0.1 mM adenosine, and 0.1 mM guanosine. The mixtures were loaded on to 0.5 mL QAE Sephadex columns, and eluted with 2 mL of 0.1 M formate (pH 7.4). The eluted radioactivity was measured by scintillation counting in Optiphase Hisafe 3.
A second, microplate, PDE assay was developed using Multiscreen plates and a vacuum manifold. The assay (100 μl) contained 50 mM Tris pH 7.5, 5 mM Mg acetate, 1 mM EDTA and 250 μg/mL snake venom nucleotidase. The other components of the reaction mixture were as described above. At the end of the incubation, the total volume of the assays were loaded on a QAE Sephadex microcolumn plate by filtration. Free radioactivity was eluted with 200 μl of water from which 50 μl aliquots were analyzed by scintillation counting as described above.
The following examples are presented to further illustrate the preparation of the claimed invention. The scope of the present invention is not to be construed as merely consisting of the following examples.
EXAMPLE 1
The compound of structural formula (I) was prepared as described in U.S. Pat. No. 5,859,006 and formulated in tablets using wet granulation. Povidone was dissolved in water to make a 10% solution. The active compound, microcrystalline cellulose, croscarmellose sodium, and sodium lauryl sulfate were added to a high shear mixer and mixed for 2 minutes. The powders were wet granulated with the povidone solution and extra water as required to complete the granulation. The resultant mixture was dried in a fluid bed drier with inlet air at 70° C.±5° C. until the loss on drying was below 2.5%. The granules were passed through a Comil with a suitable screen (or a sieve) and added to a suitable mixer. The extragranular croscarmellose sodium and sodium lauryl sulfate, and the colloidal anhydrous silica were passed through a suitable sieve (e.g., 500 micron) and added to the mixer and blended 5 minutes. Magnesium stearate was added and blended for 2 minutes. The blend was compressed to a target compression/weight of 250 mg using 9 mm round normal concave tooling.
The core tablets were coated with an aqueous suspension of Opadry OY-S-7322 using an Accelacota (or similar coating pan) using inlet air at 50° C. to 70° C. until the tablet weight was increased by approximately 8 mg. Opadry OY-S-7322 contains methylhydroxypropylcellulose Ph.Eur., titanium dioxide Ph. Eur., Triacetin USP. Opadry increases the weight of each tablet to about 258 mg. The amount of film coat applied per tablet may be less than that stated depending on the process efficiency.
The tablets are filled into blister packs and accompanied by package insert describing the safety and efficacy of the compound.
Formulations
Component
(mg per tablet)
Selective PDE5 Inhibitor 1)
1
5
Hydroxypropylmethylcellulose
1
5
phthalate
Microcrystalline Cellulose
221.87
213.87
Croscarmellose Sodium
5.00
5.00
Sodium Lauryl Sulfate
2.50
2.50
Sulfate Povidone K30
9.38
9.38
Purified Water, USP (water
q.s.
q.s.
for irrigation)
Croscarmellose Sodium
5.00
5.00
Sodium Lauryl Sulfate
2.50
2.50
Colloidal Anhydrous Silica
0.50
0.50
Magnesium Stearate
1.25
1.25
Total core subtotal
250.00
250.00
(Film coat Opadry OY-S-7322)
about 8 mg
about 8 mg
1) Compound of structural formula (I).
EXAMPLE 2
The following formula is used in preparing a finished dosage form containing 10 mg of the compound of structural formula (I).
Ingredient
Quantity (mg)
Granulation
Selective PDE5 Inhibitor 1)
10.00
Lactose Monohydrate
153.80
Lactose Monohydrate (spray dried)
25.00
Hydroxypropylcellulose
4.00
Croscarmellose Sodium
9.00
Hydroxypropylcellulose (EF)
1.75
Sodium Lauryl Sulfate
0.70
35.00
Outside Powders
Microcrystalline Cellulose (granular-102)
37.50
Croscarmellose Sodium
7.00
Magnesium Stearate (vegetable)
1.25
Total
250 mg
Film coat (approximately) 11.25
Purified Water, USP is used in the manufacture of the tablets. The water is removed during processing and minimal levels remain in the finished product.
Tablets are manufactured using a wet granulation process. A step-by-step description of the process is as follows. The drug and excipients to be granulated are security sieved. The selective PDE5 inhibitor is dry blended with lactose monohydrate (spray dried), hydroxypropylcellulose, croscarmellulose sodium, and lactose monohydrate. The resulting powder blend is granulated with an aqueous solution of hydroxypropylcellulose and sodium lauryl sulfate using a Powrex or other suitable high shear granulator. Additional water can be added to reach the desired endpoint. A mill can be used to delump the wet granulation and facilitate drying. The wet granulation is dried using either a fluid bed dryer or a drying oven. Once the material is dried, it can be sized to eliminate any large agglomerates. Microcrystalline cellulose, croscarmellose sodium, and magnesium stearate are security sieved and added to the dry sized granules. These excipients and the dry granulation are mixed until uniform using a tumble bin, ribbon mixer, or other suitable mixing equipment. The mixing process can be separated into two phases. The microcrystalline cellulose, croscarmellose sodium, and the dried granulation are added to the mixer and blended during the first phase, followed by the addition of the magnesium stearate to this granulation and a second mixing phase.
The mixed granulation then is compressed into tablets using a rotary compression machine. The core tablets are film coated with an aqueous suspension of the appropriate color mixture in a coating pan (e.g., Accela Cota). The coated tablets can be lightly dusted with talc to improve tablet handling characteristics.
The tablets are filled into plastic containers (30 tablets/container) and accompanied by package insert describing the safety and efficacy of the compound.
EXAMPLE 3
The following formula is used in preparing a finished dosage form of 5 mg of the compound of structural formula (I).
Ingredient
Quantity (mg)
Granulation
Selective PDE5 Inhibitor 1)
2.50
Lactose Monohydrate
79.395
Lactose Monohydrate (spray dried)
12.50
Hydroxypropylcellulose
2.00
Croscarmellose Sodium
4.50
Hydroxypropylcellulose (EF)
0.875
Sodium Lauryl Sulfate
0.35
Outside Powders
Microcrystalline Cellulose
18.75
(granular-102)
Croscarmellose Sodium
3.50
Magnesium Stearate (vegetable)
0.63
Total
125 mg
Film coat (approximately) 6.875
The dosage form of Example 3 was prepared in an identical manner to the dosage form of Example 2.
EXAMPLE 4
Solution Capsule
Ingredient
mg/Capsule
Percent (%)
Selective PDE5 Inhibitor 1)
10
2
PEG400 NF
490
98
Fill Weight
500
100
The gelatin capsules are precisely filled by pumping an accurate fill volume of pre-dissolved drug formulation into the partially sealed cavity of a capsule. Immediately following injection fill of the drug solution formulation, the capsule is completely heat sealed.
The capsules are filled into plastic containers and accompanied by a package insert.
EXAMPLE 5
This study was a randomized, double-blind, placebo-controlled, two-way crossover design clinical pharmacology drug interaction study that evaluated the hemodynamic effects of concomitant administration of a selective PDE5 inhibitor Study Drug (i.e., the compound of structural formula (I)) and short-acting nitrates on healthy male volunteers. In this study, the subjects received either the Study Drug at a dose of 10 mg or a placebo, daily for seven days. On the sixth or seventh day, the subjects received sublingual nitroglycerin (0.4 mg) while supine on a tilt table. The nitroglycerin was administered 3 hours after Study Drug dosing, and all subjects kept the nitroglycerin tablet under their tongue until it completely dissolved. The subjects were tilted to 70° head-up every 5 minutes for a total of 30 minutes with measurement of blood pressure and heart rate. There were no discontinuations among the twenty-two healthy male subjects (ages 19 to 60 years old) that entered this study.
In a preliminary analysis of this study, the Study Drug was well tolerated and there were no serious adverse events. There were no Study Drug-associated changes in laboratory safety assessments or 12-lead ECGs. The most common adverse events were headache, dyspepsia, and back pain. The study demonstrated minimal effects on mean systolic blood pressure and on mean maximal nitroglycerin-induced decrease in systolic blood pressure and the maximal nitroglycerin-induced decrease in systolic blood pressure among all patients.
EXAMPLE 6
In two randomized, double-blinded placebo controlled studies, the compound of structural formula (I), at a range of doses in both daily dosing and for on demand therapy for sexual encounters and intercourse in the home setting, was administered to patients in need thereof. Doses from 5 to 20 mg of the compound of structural formula (I) were efficacious and demonstrated no flushing and no reports of vision abnormalities. It was found that a 10 mg dose of the compound of structural formula (I) was fully efficacious and demonstrated minimal side effects (no flushing and no reports of blue vision).
Erectile function was assessed by the International Index of Erectile Function (IIEF) (Rosen et al., Urology, 49, pp. 822-830 (1997)), diaries of sexual attempts, and a global satisfaction question. The compound of structural formula (I) significantly improved erectile function as assessed by all endpoints. In both “on demand” and daily dose regimens, the compound of structural formula (I) significantly improved erectile function in doses between 1 and 20 mg.
EXAMPLE 7
A third clinical study was a randomized, double-blind, placebo-controlled study using a compound of structural formula (I) (Study Drug) administered “on demand” to patients with male erectile dysfunction. The Study Drug was administered over a period of eight weeks in the treatment of male erectile dysfunction (ED). Erectile dysfunction (ED) is defined as the persistent inability to attain and/or maintain an erection adequate to permit satisfactory sexual performance. “On demand” dosing is defined as intermittent administration of Study Drug prior to expected sexual activity.
The study population consisted of 212 men, at least 18 years of age, with mild to severe erectile dysfunction. The Study Drug was orally administered as tablets of coprecipitate made in accordance with Butler U.S. Pat. No. 5,985,326. The Study Drug was administered in 2 mg, 5 mg, 10 mg, and 25 mg doses, “on demand” and not more than once every 24 hours. Treatment with all nitrates, azole antifungals (e.g., ketoconazole or itraconazole), warfarin, erythromycin, or antiandrogens was not allowed at any time during the study. No other approved or experimental medications, treatments, or devices used to treat ED were allowed. Forty-one subjects were administered a placebo.
The two primary efficacy variables were the ability of a subject to penetrate his partner and his ability to maintain an erection during intercourse, as measured by the International Index of Erectile Function (IIEF). The IIEF Questionnaire contains fifteen questions, and is a brief, reliable measure of erectile function. See R. C. Rosen et al., Urology, 49, pp. 822-830 (1997).
Secondary efficacy variables were IIEF domain scores for erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction; the patient's ability to achieve an erection, ability to insert his penis into his partner's vagina, completion of intercourse with ejaculation, satisfaction with the hardness of his erection, and overall satisfaction, all as measured by the Sexual Encounter Profile (SEP) diary; and a global assessment question asked at the end of the treatment period. The SEP is a patient diary instrument documenting each sexual encounter during the course of the study.
The safety analysis of the study included all enrolled subjects, and was assessed by evaluating all reported adverse events, and changes in clinical laboratory values, vital signs, physical examination results, and electrocardiogram results.
At endpoint, patients who rated their penetration ability (IIEF Question 3) as “almost always or always” were as follows: 17.5% in the placebo group, 38.1% in the 2 mg group, 48.8% in the 5 mg group, 51.2% in the 10 mg group, and 83.7% in the 25 mg group. Comparisons revealed statistically significant differences in change in penetration ability between placebo and all dose levels of the Study Drug.
At endpoint, patients who rated their ability to maintain an erection (IIEF Question 4) during intercourse as “almost always or always” are as follows: 10.0% in the placebo group, 19.5% in the 2 mg group, 32.6% in the 5 mg group, 39.0% in the 10 mg group, and 69.0% in the 25 mg group. Comparison revealed statistically significant differences in change in penetration ability between placebo and the three higher dose levels of Study Drug.
Overall, this study demonstrated that all four doses of Study Drug, namely 2 mg, 5 mg, 10 mg, and 25 mg, taken “on demand” produced significant improvement, relative to placebo, in the sexual performance of men with erectile dysfunction as assessed by the IIEF, by patient diaries assessing frequency of successful intercourse and intercourse satisfaction, and by a global assessment. This improvement was demonstrated in a broad study population that included patients who exhibited all severities of erectile dysfunction. Most adverse events were mild or moderate in severity. Significantly, no adverse events related to color vision disturbances were reported by any patient.
The combined results from clinical studies showed that administration of a compound of structural formula (I) effectively treats male erectile dysfunction, as illustrated in the following table.
IIEF ERECTILE FUNCTION DOMAIN
(Change from Baseline)
Unit Dose
n
Mean ± SD
p
placebo
131
0.8 ± 5.3
2
mg
75
3.9 ± 6.1
<.001
5
mg
79
6.6 ± 7.1
<.001
10
mg
135
7.9 ± 6.7
<.001
25
mg
132
9.4 ± 7.0
<.001
50
mg
52
9.8 ± 5.5
<.001
100
mg
49
8.4 ± 6.1
<.001
n is number of subjects, SD is standard deviation.
However, it also was observed from the combined clinical studies that the percent of treatment-emergent adverse events increased with an increasing unit dose of the compound of structural formula (I), as illustrated in the following table.
Treatment-Emergent Adverse Events (%)
Unit Dose (mg)
Event
Placebo
2
5
10
25
50
100
Headache
10
12
10
23
29
34
46
Dyspepsia
6
3
14
13
19
20
25
Back Pain
5
3
3
15
18
24
22
Myalgia
3
0
3
9
16
20
29
Rhinitis
3
7
3
4
4
0
2
Conjunctivitis
1
0
1
1
0
2
5
Eyelid Edema
0
0
0
1
1
2
3
Flushing
0
0
0
<1
0
3
7
Vision Abnormalities
0
0
0
0
0
0
0
The above table shows an increase in adverse events at 25 mg through 100 mg unit doses. Accordingly, even though efficacy in the treatment of ED was observed at 25 mg to 100 mg doses, the adverse events observed from 25 mg to 100 mg doses must be considered.
In accordance with the present invention, a unit dose of about 1 to about 20 mg, preferably about 2 to about 20 mg, more preferably about 5 to about 20 mg, and most preferably about 5 to about 15 mg, administered up to a maximum of 20 mg per 24-hour period, both effectively treats ED and minimizes or eliminates the occurrence of adverse side effects. Importantly, no vision abnormalities were reported and flushing was essentially eliminated. Surprisingly, in addition to treating ED in individuals, with about 1 to about 20 mg unit dose of the compound of structural formula (I), with a minimum of adverse side effects, individuals undergoing nitrate therapy also can be treated for ED by the method and composition of the present invention.
The principles, preferred embodiments, and modes of operation of the present invention have been described in the foregoing specification. The invention intended to be protected herein, however, is not construed to be limited to the particular forms disclosed, because they are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.
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The present invention relates to highly selective phosphodiesterase (PDE) enzyme inhibitors and to their use in methods of treating sexual dysfunction in individuals suffering from a retinal disease, class 1 congestive heart failure, or myocardial infarction.
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[0001] This is national stage application under 35 U.S.C. section 371 of international application WO 03/061683 filed on Jan. 23 rd , 2003 and published on Jul. 31st, 2003, said international application claiming priority of the Finnish national patent application FI20020000121 filed on Jan. 23 rd , 2002.
FIELD OF INVENTION
[0002] The present invention is related to human and animal medicine, and more specifically, to improvement of the resistance of humans and animals to microbial infections and/or enhancement of the therapeutic effect of antibiotics. According to this invention, certain natural proteins contain peptide segments that can augment activity of macrophages and and other cells of immune system. These peptides can be used for the creation of novel drugs.
BACKGROUND OF INVENTION
[0003] A specific enzyme, leucokinase, that is located in the outer membrane of the neutrophils, spilits leucocinin, a leucophilic fraction of immunoglobulin G (IgG) and produces a phagocytosis-stimulating tetrapeptide (Najjar V. A. and Nishioka K., 1970, Nature 228, pp. 672-673). That tetrapeptide was subsequently named “tuftsin” (Najjar V. A. and Nishioka K., 1970, Nature 228, pp. 672-673; Sieminon I. Z. and Kluczyk A., 1999, Peptides 20, pp. 645-674). Tuftsin (Thr-Lys-Pro-Arg) is a 289-292 sequence in the C H2 domain of the Fc subunit of human IgG1 heavy (H) chain. It was originally found that tuftsin stimulates phagocytosis after binding to polymorphonuclear cells (Constantopoulos A. and Najjar V. A., 1972, Cyobios. 6, pp. 97-100; Najjar V. A. and Constantopoulos A. A., 1972, J. Reticulendothel. Soc. 12, pp. 197-215; Najjar V. A., 1979, Clin. Wochenschr. 57, pp. 751-756; Najjar V. A., 1980, Adv. Exp. Med. Biol. 121 A, 131-147; Najjar V. A., 1983, Ann. NY Acad. Sci. 419, pp. 1-11). Subsequently, tuftsin was also shown to stimulate the phagocytosis activity of monocytes-macrophages (Coleman D. L., 1986, Eur. J. Clin. Microbiol. 5, pp. 1-5). Potentially, tuftsin could be used as a drug component to increase phagocytosis activities. However, the drawback is that tuftsin activity is very low demanding its high concentrations in blood circulation. Another drawback is that half-lives of linear peptides in blood are short.
[0004] In 1980 the existence of a β-endorphin-like sequence in CH3 domain of the Fc subunit of human IgG1-4H-chain was reported (Julliard J. H. et al., 1980, Science 208, pp. 183-185). To isolate ACTH and β-endorphin from human placenta, the authors used immobilized antibodies to these hormones as affinity absorbents. A 50 kDa protein was thereby isolated and found to be an H-chain of IgG. Elucidation of the origin of such an effect led to the discovery of ACTH- and β-endorphin-like sequences in the H-chain. It was found that the human IgG1 H-chain fragment 364-377 (SLTCLVKGFYPSDI; see FIG. 1 ) was 40% homologous to β-endorphin fragment 10-23 (SQTPLVTLFKNAII). An artificial peptide (14 amino acid residues) corresponding to the β-endorphin-like human IgG1 sequence was synthesized and found to interact with rat brain receptors for β-endorphin (Houck J. C. et al., 1980, Science 207, 78-80). Our group synthesized a decapeptide SLTCLVLGFY (termed immunorphin) corresponding to the human IgG1 H-chain sequence 364-37. It was demonstrated to compete with [ 125 I]-endorphin for high-affinity receptors on murine peritoneal macrophages (Ki=2.5 nM; Zav'yalov V. P. et al., 1996, Immun. Lett. 49, 21-26). Later on it was also demonstrated to compete with [ 125 I]β-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K i =0.6 nM). Tests of the specificity of the receptors revealed that they are insensitive to an antagonist of opioid receptors naloxone and [Met 5 ]enkephalin, i.e. they are non-opioid receptors. The displacement assays demonstrated that pentapeptide VKGFY, termed hereinafter as pentarphin, was the shortest immunorphin fragment, capable of inhibiting [ 125 I]β-endorphin binding to non-opioid receptors on murine macrophages (Ki=12 nM) and human T lymphocytes (Ki=15 nM). According to the present invention, the primary effect of pentarphin, following binding to specific cell surface receptors, consists of the stimulation of the functions of macrophages and T lymphocytes.
BRIEF DESCRIPTION OF DRAWINGS
[0005] FIG. 1 . Comparison of amino acid sequences of [Met 5 ]enkephalin, β-endorphin, β-endorphin-like fragments of human immunoglobulin GI (HuIgG1) heavy (H) chain, immunorphin, and pentarphin.
[0006] FIG. 2 . Structure of cyclopentarphin.
DETAILED DESCRIPTION OF THE INVENTION
[0007] The present invention is related to novel bioactive compounds—pentapeptide Val-Lys-Gly-Phe-Tyr and its cyclic analog—cyclo(Val-Lys-Gly-Phe-Tyr), capable of enhancing phagocytic activity of mouse peritoneal macrophages against virulent bacterial strains and of binding to a variety of cells of immune system. The term pentarphin is used hereafter for the linear sequence described above. In a larger meaning the term pentarphins is used for the linear or cyclic sequences or for sequences containing mentioned sequences within their molecule and having related biological activities as the linear or cyclic pentarphin.
[0008] According to this invention, during phagocytosis pentarphins bind to the specific high-affinity receptors on macrophage surface and enhance the capacity of phagocytic cells to digest captured microbes. However, for example, a virulent bacterium Salmonella typhimurium has adapted, as many other virulent microorganisms, in the process of natural selection, to protect themselves against the bactericidal action of phagocytes. Such adapted microbes release in the surrounding medium so-called “virulence factors” that interfere with the process of formation of a junction between phagosomes (bubbles formed by cell membrane that contain captured microbes) and lysosomes. As a result, phagolysosomes are not formed and lysosomal enzymes have no access to microbes and therefore cannot digest them. Data presented in Table 1 show that in the absence of pentarphins (control) captured microorganisms were not digested but, in contrast, propagated themselves inside phagocytes (PN increases from 10.17±0.18 to 15.50±0.34 between 2 and 7 h of phagocytosis). Pentarphin and cyclopentarphin do not influence viability and growth of S. typhimurium , that is, they are not peptides-antibiotics. Similar to tuftsin, both peptides stimulate the capacity of phagocytes to digest captured microorganisms.
[0009] Pentarphins have following advantages as compared to classical antibiotics:
1. Unlike antibiotics that act directly on microorganism, pentarphins affect phagocytes stimulating their digestive function. Therefore, the activator peptides can be effective in microbial infections, i.e. to be universal antimicrobial agents. 2. Antibiotics are toxic and cause a number of undesirable side effects (allergy, disbacteriosis, changes in blood cellular content, impairments of liver, kidney and the central nervous system functions etc.). Pentarphins are non-toxic, since the products of their hydrolysis are natural amino acids, while the only effect is the stimulation of the immune system.
[0012] Antibiotics are widely used, commercially important drugs for treatment of a multitude of infectious diseases of human and higher animals. However, antibiotics have severe drawbacks for their toxicity, narrow effective dose range, and various side effects. Therefore, it is propitious to try to increase their activities by other means. High efficacy and safety of pentarphins form the basis for elaboration of novel effective therapeutic agents that enhance the resistance of human and animal organisms to pathogenic microbes or other microparticles eliminated by macrophages. In addition, it appeared promising to work out combined preparations of pentarphins and antibiotics. The combinatory drugs influence microbe and macrophage simultaneously. Such an approach will allow therapeutic doses of antibiotics and consequently their toxicity to be significantly reduced, whereas antimicrobial activity of preparation as a whole to be significantly increased.
[0013] In this invention, it has been shown that certain favourable physiological effects of antibiotics can be amplified by biologically active peptides, preferably cyclopeptides, corresponding to the β-endorphin-like sequences of human IgG1-4 subclasses. The mentioned cyclopeptides can be commercially produced by synthetic techniques. The present invention provides definite improvement of drugs based on antibiotics designed for treatment of humans and animals. The peptide parts of such drugs include the biologically active cyclopeptides corresponding to the β-endorphin-like sequence of IgG subclasses of different animal species. Although it was impossible to test experimentally all the potentially bioactive peptide structures, such structures were revealed by extensive comparisons of available amino acid sequences of IgG subclasses from different species by computer and molecular modeling techniques taking into account the data on the localization of the β-endorphin-like sequence of human IgG1-4 subclasses and the experimental data on the competition of pentarphin with [125I]β-endorphin for the common receptors. Therefore the present invention is not limited to the linear or cyclic structure of pentapeptide VLGFY, but also includes related structures from other animals with the same biological effects, i.e. the capability to enhance the activity of macrophages and/or bind to immunocompetent cells. It is reasonable to assume that in the process of natural selection the changes in the amino acid sequence of the pentarphin-like site of IgG were selected not to abolish the biological activities of the site. Consequently, all peptides corresponding to this site in all IgG subclasses of different species might reproduce the activity of pentarphin.
[0014] To test a synergy between antibiotics and pentarphins, we employed the classic macrophage assays with mouse peritoneal macrophages in a cell culture system. The cell culture conditions are strongly reminiscent of that of blood circulation. In fact, in cell cultures, which are commonly used for testing of potential drugs, the conditions are strictly maintained similar to the blood circulation as to the temperature, pH, buffer, minerals, CO 2 - and O 2 -partial pressures, and so on. Thus, it is highly predictable that the biologically active compositions of the present invention can be used as medical drugs for humans and animals. Such effective drugs are very desirable for treatment persons with a lowered resistance to microbial infections like those having different kinds of immunodeficiency, for example, AIDS.
[0015] It was demonstrated that pentarphins (see Example 4) have very low or no toxicity, because of their amino acid nature. On the other hand, they are not immunogenic. Therefore they are extremely suitable components for any drug formulations. For certain purposes derivatives of pentarphins may be useful. Larger pentarphin polymers (repeated polypeptide sequences) will possibly be immunogenic. However, such polymers or derivatives may possess relatively higher activity, which can make their usage favorable.
[0016] The role of macrophages is essentially to remove extremely small foreign particles, for example microbial cells, from the organism. The present invention indicates that this process can be accelerated by pentarphins. One would expect that macrophages activated so would also remove other particles from human and animal blood circulation, thus having a beneficial effect on, for instance, allergic reactions.
[0017] It is theoretically predictable that the peptides and their fragments, according to the present invention, will be useful for amelioration of allergic reactions, because the mentioned peptides activate T cells. Allergic reactions are mediated by activated B cells as well as interleukin-4-secreting T (T H2 ) cells; this cytokine is necessary for IgE production by B cells. There are literature data concerning role of β-endorphin in the regulation of cytokine production by T cells: β-endorphin stimulates IL-2, IL-4 and β-interferon production by murine CD4 + T cells (van den Bergh P. et al., 1994, Cell. Immunol. 154 109-122; van den Bergh P. et al., 1994, Lymphokine Cytokine Res., 13, 63-69). It was shown that β-endorphin could modulate the magnitude of an IL-4 induced IgE response (Aebisher I. 1996, Exp. Dermatol., 5, 38-44).
[0018] The present invention shows that pentarphins and certain other peptides have sequence homologies (see FIG. 1 ). All these peptides have strong physiological effects on animal and human organisms. According to this invention, at least macrophages and T-lymphocytes have receptors for pentarphins. Both of these cells are crucial for organism's resistance against microbial attacks. T-cell activation takes place at the first stage of the development of specific immune response to infection. Antigen-primed naive T cells differentiate into either helper cells (T H2 ) or inflammatory cells (T H1 ). T H2 cells participate in the development of humoral immunity (production of specific antibodies) and T H1 cells take place in macrophage activation. Immunorphin and its fragments are not species-specific, since our results show that these peptides bind with high affinity to specific receptors on both mouse and human cells. Immunorphin is a fragment of the constant part of the heavy chain of IgG. This fragment has the same sequence at least in human, mouse, and rat IgG.
[0019] Numerous methods of preparing drug formulations in the context of peptide drugs, including tuftsin, have been described in literature (see, for example, U.S. Pat. No. 4,816,560, EP0448811, EP0253190). High tolerance of cyclopentarphin to the action of proteolytic enzymes enables its administration not only intramuscularly but also per os and in nosal. According to the present invention, it is possible to find out appropriate formulations using pentarphins alone as effective substances, or combinations of pentaprphins with antibiotics, such as penicillins, cefalosporins, tetracyclins, streptomycins, laevomycetins, polymyxins, rifamycins, peptide antibiotics (exemplified by gramicidins, bacitracins, and polymycsins).
[0020] It is evident that pentarphins' structure (5-residue peptide, minimal) forms the very basis of the present invention. With the knowledge described in this invention, it is possible to design many other active structures. For example, it is possible to create polymeric forms of pentarphins with or without using synthetic or biological linker molecules between the active units. Further, it is possible to add flanking sequences outside of N- and C-terminal ends, or to add such ones to the amino acid side chains (to the Lys residue) to change solubility etc. It is also possible to prepare macromolecular carriers with one or more active peptides linked to them.
[0021] In the following the invention is further illustrated by non-limiting examples.
EXAMPLE 1
Pentarphins and Immunorphins Activate Cells of Immune System
[0022] (3-[ 125 I]iodotyrosyl 27 )β-endorphin (˜2000 Ci/mmol specific activity) was purchased from Amersham (UK). Na 125 I (2×10 6 Ci/M specific activity) was from Russian Scientific Center “Applied Chemistry” (St. Petersburg, Russia). All media, sera for culturing cells, 1,3,4,6-tetra-chloro-3α, 6α diphenyl glycoluril (Iodogen) and other chemicals were obtained from Sigma (St. Louis, Mo.). The decapeptide encompassing the sequence 364-373 of HuIgG (immunorphin) and its fragments were synthesized by using pentafluorophenyl ethers of N-protected amino acids. The peptides were purified by HPLC followed by fast atom bombardment mass spectrometric analysis. Only peptides that were >95% pure were used.
[0023] Mononuclear cells were separated from healthy donors blood according to the Boyum method. T lymphocytes were purified by a nylon wool column filtration method. The non-adherent fraction was eluted with RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum. This fraction contained T lymphocytes and small amounts of monocytes and B lymphocytes.
[0024] The binding of [ 125 I]β-endorphin to T lymphocytes was measured as follows: 10 6 cells per tube were incubated with labeled peptide at a concentration of 10 −7 -10 −11 M for 1 h at 4° C. in 1 ml RPMI-1640 medium containing 20 mM NaN 3 and 10 mM Hepes, pH 7.5. The incubation was terminated by rapid filtration through GF/A glass fiber filters (Whatman, UK) under vacuum pressure. Filters were rinsed twice with 5 ml volumes of ice-cold 0.15 M NaCl. The cell-bound radioactivity was measured using 121 I Minigamma counter (LKB, Sweden). Non-specific binding of [ 125 I]-endorphin was measured in the presence of 10 μM unlabeled β-endorphin. The equilibrium dissociation constant (K d ) was estimated by Scatchard analysis.
[0025] Immunorphin (10 μg) and the peptide H-VKGFY-OH (10 μg) were labeled by solid phase oxidation method using Iodogen and Na 125 , (1 mCi). The labeled peptides were purified by gel filtration on Sephadex G-10 (0.9×10 cm column, 0.05 M phosphate buffer, pH 7.5). The purity of the labeled peptides was tested by thin-layer chromatography on aluminium oxide glass with n-butano/acetic acid/water (4:1:1) solvent system, followed by autoradiography. The specific activity of [ 125 I]-immunorphin and [ 125 I]-H-VKGFY-OH were 232 Ci/mmol and 179 Ci/mmol respectively. Assay of [ 125 1]-immunorphin (10 −10 -10 −7 M) and [ 125 I]-H-VKGFY—OH (10 −10 -10 −7 M) binding to T lymphocytes (10 6 cells per tube) was carried out in 1 ml RPMI-1640 medium containing 10 mM Hepes, 20 mM NaN 3 , and 0.6 mg/L PMSF, pH 7.4 at 4° C. for 40 min. The reaction mixture was then filtered through GF/A filters (Whatman, UK). Filters were rinsed twice with 5 ml volumes of ice-cold 0.15 M NaCl, pH 7.4. Radioactivity was counted using 121 I Minigamma counter (LKB). Non-specific binding of the labeled peptide was measured in the presence of 10 μM of the unlabeled peptide.
[0026] To test the inhibitory effect of unlabeled naloxone, Met-enkephalin, and immunorphin fragments on the binding of [ 125 I]-endorphin, T lymphocytes (10 6 cells per tube) were incubated with 1 nM [ 125 I]β-endorphin and unlabeled ligands at various concentrations (10 −10 -10 −6 M) as described in Section 2.4. The results were plotted as percentage of specific binding vs. log of competitor concentration, and IC 50 values were determined graphically. The inhibition constant (Ks) was calculated according to the equation:
K i =IC 50 /(1 +[L]/K d ),
where [L] is a molar concentration of [ 125 I]β-endorphin, K d is the dissociation constant of [125I]α-endorphin/receptor complex, and IC 50 is the concentration of the competing ligand causing half-maximum displacement of [ 125 I]α-endorphin. β-Endorphin was found to interact specifically with T lymphocytes separated from normal human blood. The data propose the presence of one class binding sites with the K d value of 0.25±0.03 nM. Non-specific [ 125 I]β-endorphin binding that occurs in the presence of 10 μM unlabeled β-endorphin constitutes about 11% of the total binding value.
[0027] To examine the specificity of the β-endorphin binding sites, competition experiments were performed using the constant concentration of [ 125 I]β-endorphin and increasing concentrations of unlabeled ligands (naloxone, Met-enkephalin, immunorphin and eight synthetic immunophin fragments with various chain lengths; see section 2.5 of the previous part). Displacement curves indicated that only six unlabelled peptides (H-VKGFY-OH, H-LVKGFY-OH, -CLVKGFY-OH, H-TCLVKGFY-OH, H-LTCLVKGFY-OH and immunorphin) were able to compete with [ 125 I]-endorphin for the same binding sites. The K i values correlate with ligand-receptor affinity and inhibiting potential of ligands. The results of the displacement assay demonstrated that β-endorphin binding to this type of receptors is not inhibited by naloxone and Met-enkephalin. A minimum fragment of immunorphin retaining its inhibitory activity in the competition test was found to be the pentapeptide H-VKGFY—OH. The pentapeptide was characterized by lesser inhibiting capacity (Ki=15 nM) as compared to immunorphin (0.6 nM) and its longer fragments (H-LVKGFY-OH, Ki=8.0 nM; H-CLVKGFY-OH, Ki=3.4 nM; H-TCLVKGFY-OH, Ki=2.2 nM; H-LTCLVKGFY-OH, K i =1.0 nM). Thus, the β-endorphin receptors expressed by T lymphocytes are highly specific and naloxone-insensitive ones.
[0028] The Scatchard plots demonstrate the specific binding of [ 125 1]-immunorphin to T lymphocytes in the absence (plot 1; K d =7.0±0.3 nM) and in the presence (plot 2; K d =7.4±0.2 nM) of naloxone. The results of this experiment confirmed that naloxone does not influence the kinetic of [ 125 I]-immunorphin binding to the receptors on T lymphocytes.
[0029] The displacement assays demonstrated that pentapeptide H-VKGFY-OH was the shortest active immunorphin fragment. We prepared [ 125 I]-H-VKGFY-OH and studied its interaction with T lymphocytes. Scatchard analysis of the binding showed that the data best fit a one-site model. The K d value for [ 125 I]-H-VKGFY-OH/receptor complex was 36.3±0.5 nM. Non-specific binding of the labeled peptide to T lymphocytes was about 8% of its total binding to these cells.
[0030] The results of the binding assays confirmed that lymphocytes separated from normal human blood express high affinity binding sites for β-endorphin, K d =(0.25±0.03) nM. The binding of β-endorphin to these sites was naloxone- and Met-enkephalin-insensitive, but sensitive to immunorphin and its fragments H-VKGFY-OH, H-LVKGFY-OH, H-CLVKGFY-OH, H- TCLVKGFY-OH, H-LTCLVKGFY-OH ( FIG. 3 , Tab. 1). Thus, T lymphocytes from normal human blood express non-opioid receptors for β-endorphin.
[0031] Immunorphin (H-SLTCLVKGFY-OH) is homologous (50%) to the β-endorphin fragment 10 −19 (SQTPLVTLFK). The high affinity binding of immunorphin (K d =7.0±0.3 nM) and its fragment H-VKGFY-OH (K d =36.3±0.5 nM) to non-opioid receptors for β-endorphin on T lymphocytes shows that α-endorphin is a peptide with a dualistic nature: its C-terminal moiety binds to non-opiod receptors, whereas its N-terminal enkephalin sequence is responsible for β-endorphin binding to opioid receptors. Therefore, pentarphins can have a special function in organisms which is related to certain functions of β-endorphin. We have found that β-endorphin, immunorphin and pentarphin stimulate Con A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met 5 ]enkephalin and an antagonist of opioid receptors naloxone, tested in parallell, were not active. The stimulating effect of β-endorphin, immunorphin and pentarphin on T lymphocyte proliferation was not inhibited by naloxone. Thus, these peptides bind to common naloxone-insensitive binding sites on T lymphocytes and enhance Con A-induced proliferation of these cells.
EXAMPLE 2
Phagocytosis of S. typhimurium by Macrophages in the Absence or in the Presence of Pentarphin, Cyclopentarphin, and Tuftsin
[0032] Pentarphin was obtained by a solid-phase synthesis. The crude product was purified by HPLC on a Zorbax ODS column (4×150 mm, 5 μm particle size) using linear gradient of water acetonitrile (95%) in 0.2% TCA (10-25%, 20 min) at a flow rate of 1 ml/min. According to the absorbance at 220 nm, the main substance (pentarphin) content was 98%. The peptide structure was confirmed by an amino acid analysis under standard conditions with a D500 amino acid analyzer (Durrum, USA). Molecular mass of pentarphin was estimated by mass spectrometric analysis using Vision 2000 spectrometer (“Thermo Bioanalysis”, Great Britain).
[0033] Cyclopentarphin was obtained by the cyclization reaction of linear pentarphin (having the side chain groups protected) through amino group of Val to an activated carboxyl group of Tyr. The reaction product was unmasked from protective groups and purified by HPLC as described above. The molar yield was 15%. Mass spectrometric analysis showed the molecular peak of cyclopentarphin (594 Da) in the spectrum.
[0034] Peritoneal macrophages were isolated from CBA mice (16-18 g). The virulent strain Salmonella typhimurium 415 with typical morphological and functional properties was used. LD 50 was approximately 100 microbial cells injected intraperitoneally into white mice. S. typhimurium was grown in Hottinger's broth for 4-6 h at 37° C., then transferred to beef-extract agar and incubated at 37° C. for 18 h. Macrophage monolayers on cover glasses were cultivated in sterile test tubes in 199 medium supplemented with streptomycin and penicillin (100 μg/ml each) and inactivated fetal calf serum (5%) at 37° C. 24 h later macrophages were infected with 199 medium supplemented with serum and S. typhimurium 415 (108 microbial cells/ml final concentration). Microorganisms and peptides (tuftsin, pentarphin, or cyclopentarphin) at particular concentrations were added to the cultivation medium simultaneously. In 2 h the contact between microbes and macrophages was interrupted by replacing the infection medium with a fresh one supplemented with antibiotics. To prevent the recapture of bacteria released from destroyed phagocytes by other cells, cultivation medium was replaced with a fresh one every two hours. Macrophages on cover glasses (in triplicate for every time point, namely 1,2,4,7 and 12 h) were fixed in methanol for 7 min. After that, the preparations were stained with 0.1% azur II-eosin water solution for 5 min. Cells (300 per cover glass) were examined using light microscope and analyzed for following parameters: phagocytic activity (PA)—a percentage of macrophages, participating in phagocytosis; bacterial cytocidal activity (BCA)—a percentage of phagocytes, destroyed by intracellular bacteria; and phagocytic number (PN)- an average number of microbes per macrophage.
[0035] The values of the main characteristics (PA, PN, BCA) of S. typhimurium phagocytosis in the absence (control) or in the presence of pentarphin (1 nM), cyclopentarphin (1 nM) and tuftsin (100 nM) are shown in Table 1. BCA in control was more than 65% within 7h, and within 12 h all macrophage monolayer was destroyed by intracellular microorganisms. The presence of 1 nM pentarphin or cyclopentarphin in cultivation medium resulted in a significant increase in bactericidal activity of macrophages. In the presence of pentarphin, phagocytes completely digested the captured microbes within 12 h, and in the presence of cyclopentarphin—within 7 h of phagocytosis. Tuftsin acted the same way at a concentration of 100 nM, that is, its activity was 100 times lower. Thus, pentarphin enhances the bactericidal activity of peritoneal macrophages in relation to S. typhimurium 415, in its presence at a concentration of 1 nM, phagocytosis of this bacteria in vitro completes with total digestion of captured microbes. Cyclopentarphin is preferable in a drug composition, because its half-life in biological fluids is considerably longer than its linear analogs, as known from prior art.
EXAMPLE 3
Combined Action of Pentarphin and Streptomycin
[0036] Macrophage monolayers on cover glasses were cultivated and infected as described above in Example 1. Microorganisms, streptomycine and peptides (pentarphin, or cyclopentarphin) at particular concentrations were added to the cultivation medium simultaneously. In 2 h, the contact between microbes and macrophages was interrupted by replacing the infection medium with a fresh one supplemented with antibiotics. Macrophages on cover glasses (in triplicate for every time point, namely 1, 2, and 4 h) were fixed in methanol for 7 min. After that, the preparations were stained with 0.1% azur II-eosin water solution for 5 min. Cells (300 per cover glass) were examined under a light microscope and analyzed for following parameters: phagocytic activity (PA)—a percentage of macrophages, participating in phagocytosis; bacterial cytocidal activity (BCA)—a percentage of phagocytes, destroyed by intracellular bacteria; and phagocytic number (PN)—an average number of microbes per macrophage. The data given in Table 2 show that the combined action of streptomycine (10 μg/ml) pentarphin (1 nM) allows the antibiotic dose to be lowered 5 times.
EXAMPLE 4
Toxicity and Immunogenicity of Pentarphin and Immunorphin
[0037] The toxicity of pentarphin and immunorphin was estimated by intraperitoneal injection of peptides (10, 100, 250, 1000, 2500, and 3000 mg/kg of body weight) to BCA mice (16-18g). The LD 50 value was 2500 mg/kg for each peptide, i.e. this dose is not physiological. Active concentrations of both peptides are 10-100 μg/kg. Very low toxicity of the peptides can be attributed to the fact that aminoacids are the only degradation products of these compounds.
[0038] No immunogenicity was observed during several experiments with pentarphin and immunorphin utilizing mice as model animals. This was an expectable result, because the related peptides, Met-enkephalin and Leu-enkephalin, are known to be nonimmunogenic. In general, small peptides do not induce antibody production.
TABLE 1 Effect of cyclopentarphin, pentarphin and tuftsin on digestion of the virulent bacterial strain S. typhimurium 415 by mouse peritoneal macrophages in vitro. Phago- cytosis *PA, **BCA, ***PN, Peptide time, h % ± SEM % ± SEM n ± SEM Control 1 65.33 ± 1.11 1.33 ± 0.67 3.90 ± 0.13 2 72.67 ± 1.07 13.67 ± 0.69 10.17 ± 0.18 4 62.33 ± 1.29 35.67 ± 1.41 11.17 ± 1.03 7 29.00 ± 0.89 66.67 ± 0.62 15.50 ± 0.34 12 0 100 — Pentarphin 1 84.08 ± 2.12 1.08 ± 0.71 7.11 ± 0.15 (1 nM) 2 89.15 ± 1.78 1.55 ± 0.65 9.29 ± 0.74 4 66.82 ± 1.14 9.32 ± 1.32 6.02 ± 0.66 7 23.31 ± 0.84 10.24. ± 1.17 1.03 ± 0.24 12 3.73 ± 0.64 2.35 ± 1.21 0.66 ± 0.12 Cyclo- 1 87.81 ± 3.14 1.25 ± 0.64 7.78 ± 0.39 pentarphin 2 91.33 ± 2..19 1.06 ± 0.49 8.81 ± 0.27 (1 nM) 4 57.43 ± 2.01 2.13 ± 0.60 5.32 ± 0.51 7 1.74 ± 0.44 2.22 ± 1.31 0.27 ± 0.20 Tuftsin 1 66.07 ± 1.82 1.41 ± 0.27 5.84 ± 0.27 (100 nM) 2 74.11 ± 1.42 8.12 ± 0.99 7.31 ± 0.25 4 61.97 ± 1.18 29.34 ± 0.75 6.29 ± 0.48 7 38.48 ± 0.23 31.46 ± 1.23 3.32 ± 0.56 12 9.31 ± 0.29 7.64 ± 1.38 1.36 ± 0.86 *PA—phagocytic activity, a percentage of macrophages, participating in phagocytosis; **BCA—bacterial cytocidal activity, a percentage of phagocytes, destroyed by intracellular bacteria; ***PN—phagocytic number, an average number of microbes per macrophage.
[0039]
TABLE 2
Effect of streptomycine + pentarphin on the digestion of
the virulent bacterial strain S. typhimurium 415 by mouse
peritoneal macrophages in vitro.
Phagocytosis
PA,
BCA,
PN,
Peptide
time, h
% ± SEM
% ± SEM
n ± SEM
Streptomycine
1
86.45 ± 2.13
1.04 ± 0.32
8.56 ± 0.76
(50 μg/ml)
2
96.17 ± 1.76
1.12 ± 0.74
2.03 ± 0.46
4
0
0
0
Streptomycine
1
87.65 ± 2.19
0.94 ± 0.49
8.60 ± 0.58
(10 μg/ml) +
2
98.22 ± 1.94
1.05 ± 0.54
2.11 ± 0.44
pentarphin
4
0
0
0
(1 nM)
Streptomycine
1
91.66 ± 2.27
0.99 ± 0.56
8.92 ± 0.83
(10 μg/ml) +
2
99.31 ± 2.44
1.17 ± 0.61
2.01 ± 0.39
cyclo-
4
0
0
0
pentarphin
(1 nM)
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The present invention is related to novel bioactive pentapeptides, pentarphins, the main indication of which is enhancing phagocytic activity of macrophages against microbes. In particular, the cyclopentapeptide, cyclo(Val-Lys-Gly-Phe-Tyr), termed cyclopeptarphin, was 100 times more active than tuftsin. Cyclopentarphin was non-toxic even at concentrations 1000 times higher than the minimum active dose, while being non-immunogenic. Furthermore, cyclopentarphin is more stable to enzymatic cleavage in vitro as compared to linear pentarphin and tuftsin and, hence, its life span in vivo is also larger than that of linear peptides. High efficacy and safety of cyclopentarphin enable elaboration of novel drugs that enhance the resistance of human and animal organisms to microbes and micro particles.
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[0001] This application is a continuation of U.S. Non-Provisional Utility application Ser. No. 11/278,552, filed Apr. 4, 2006, and claims the benefit of U.S. Provisional Patent Application Ser. No. 60/669,356 filed Apr. 8, 2005, both of which applications are incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention generally relates to the field of medical devices. Some embodiments of the invention relate to spinal implants inserted in the spine of a patient during surgical procedures and to instruments used to insert the implants. Other embodiments of the invention relate to methods for positioning, rotating and advancing an implant during a surgical procedure.
[0003] A spinal implant may be used to stabilize a portion of a spine. The implant may promote bone growth between adjacent vertebra that fuses the vertebra together. The implant may include a spherical protrusion, a threaded pin and an angled surface to facilitate remote adjustment of the implant position using an insertion instrument.
[0004] The insertion instrument may include, but is not limited to, a threaded rod, an actuator and a lock knob. The insertion instrument can be attached and detached to the implant, rotate the implant by transferring torque from the actuator to the implant. The actuator can be used to lock the implant in relation to the instrument. The rod can be used to apply force to the implant and advance it. The implant and instruments may be supplied in an instrument kit.
[0005] An intervertebral disc may degenerate. Degeneration may be caused by trauma, disease, and/or aging. An intervertebral disc that becomes degenerated may have to be partially or fully removed from a spinal column. Partial or full removal of an intervertebral disc may destabilize the spinal column. Destabilization of a spinal column may result in alteration of a natural separation distance between adjacent vertebra. Maintaining the natural separation between vertebra may prevent pressure from being applied to nerves that pass between vertebral bodies. Excessive pressure applied to the nerves may cause pain and nerve damage.
[0006] During a spinal fixation procedure, a spinal implant may be inserted in a space created by the removal or partial removal of an intervertebral disc between adjacent vertebra. The spinal implant may maintain the height of the spine and restore stability to the spine. Bone growth may fuse the implant to adjacent vertebra.
[0007] A spinal implant may be inserted during a spinal fixation procedure using an anterior, lateral, posterior, or transverse spinal approach. A discectomy may be performed to remove or partially remove a defective or damaged intervertebral disc. The discectomy may create a space for a spinal implant. The amount of removed disc material may correspond to the size and type of spinal implant to be inserted.
[0008] Spinal implants are described in U.S. Pat. No. 5,653,763 to Errico et al.; U.S. Pat. No. 5,713,899 to Marney et al.; U.S. Pat. No. 6,143,033 to Paul et al.; U.S. Pat. No. 6,245,108 to Biscup; and U.S. Pat. No. 5,609,635 to Michelson, United States Patent Application 20050027360 to Webb.
BRIEF DESCRIPTION OF THE INVENTION
[0009] A spinal implant is disclosed comprising: a top, wherein at least a portion of the top is configured to contact a first vertebra; a bottom, wherein at least a portion of the bottom is configured to contact a second vertebra and a side having a releasable attachment to receive an insertion device and a cam surface to engage a cam on the insertion device. The spinal implant may include a hemispherical mount and a pin mounted within the spinal implant, wherein the insertion device attaches to the pin that serves as an axis of rotation and pivots around the pin with respect to the hemispherical housing.
[0010] A method is disclose comprising: inserting an implant between portions of bone, wherein the implant locked at a first angle relative to a shaft of the instrument; loosening the implant relative to the shaft; turning the shaft to pivot the implant relative to the shaft, and releasing the implant from the instrument so that the implant is in position between the bone. Turning the shaft rotates a cam fixed to the shaft across a cam surface on the implant, wherein the cam surface is slanted and the movement of the cam across the cam surface pivots the implant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a top-side perspective view of a spinal implant attached to an insertion instrument.
[0012] FIG. 2 is an exploded view showing the spinal implant separate from the insertion instrument.
[0013] FIG. 3 is a perspective view of the FIG. 3 illustrates the interaction between the Actuator 202 of the instrument and the implant 100 .
[0014] FIG. 4 is a perspective view of the implant releasably attached to the insertion instrument and positioned over a vertebra.
DETAILED DESCRIPTION OF THE INVENTION
[0015] FIG. 1 shows the spinal implant 100 releasably attached to an insertion instrument 200 . The implant 100 may be made by made of PEEK plastic commonly used in spinal implants. The implant includes a hemispherical mount 105 and slanted cam surface 106 from which the mount protrudes. The tip of rod 201 pivotably attaches to the mount such that the implant may pivot with respect to the axis of the instrument. The pivoting of the implant is controlled by the a knob on the instrument that rotates the cam wings 205 about the hemispherical surface. The rotation of the cam, slides the front edges of the cam wings across the and cam surface 106 and thereby forces the implant to pivot with respect to the axis of the instrument.
[0016] A knob (e.g. actuator wings) 206 on the on the proximal end of the instrument enables a surgeon to rotate the cam and thereby adjust the angle between the implant and the axis of the instrument. Pivoting of the implant is caused as the actuator pushers 205 (e.g., cam) act on the slanted surface 106 of the implant 100 . As the cammed actuator 202 rotate and slide across the slanted surface 106 , the implant makes a yaw movement with respect to the axis of the instrument. Actuator 202 is equipped with the actuator wings 206 used to rotate pushers 205 (cam) from outside of the patient's body.
[0017] Locking knob 207 may be tightened to bind the actuator against the implant effectively locking the implant with respect to the instrument. When locked, axial force and torque can be applied to the handle 204 to advance the implant into the spinal space and position the implant in the space. Turning the locking knob 207 that is threaded inside and engages threads on the proximal end of the rod causes the actuator 202 that is hollow to slide axially forward over the threaded rod 201 and thereby loosen or tighten the actuator against the implant.
[0018] FIG. 2 shows the details of the attachment of the implant 100 to the instrument 200 . Threaded pin 102 is inserted into the channel 107 in the spherical protrusion (mount) 105 and retained there by a snap ring 103 . A threaded hollow shaft 108 in the threaded pin 102 is aligned with the slot opening 109 of the implant so that the treaded rod 201 can be threaded into the shaft 108 of the pin 102 . Slot opening allows pivoting of the implant by accommodating the pendulum motion of the rod 201 . Pin 104 is made of a material that enhances X-ray imaging. Making the pin visible assists the physician in the positioning of the implant while viewing a real-time x-ray image of the implant and vertebra.
[0019] The actuator 202 may be a hollow tube that is coaxial with the rod 201 . The pushers are fixed to the distal end of the actuator. The pushers 205 include cams that engage a cam surface 106 on the implant. The proximal end of the tube has a knob (e.g. actuator wings) 206 to turn the tube and thereby move the cams against the cam surface. The angle of the implant with respect to the implant is adjusted by moving the cam against the cam surface. Adjusting the angle may allow the surgeon to properly place the implant in the spine area.
[0020] FIG. 3 illustrates the interaction between the Actuator 202 of the instrument and the implant 100 . The actuator 202 is rotated around the axis of the threaded rod 201 that is engaged in the threaded pin 102 . As the cammed pushers 205 rotate, they push against the surface 106 . As a result the implant 100 turns around the axis of the pin 102 . It can be envisioned as if the implant is performing a “dog wagging its tail” motion with respect to the insert instrument 200 .
[0021] If the locking knob 207 ( FIG. 1 ) is rotated, the actuator 202 is pushed against the implant 100 . Both pushers are advanced towards the surface 106 to bind the actuator against the implant so as to lock the implant with respect to the instrument. When locked, the assembly of the implant and instrument can be advanced while retaining the desired angle of the implant 100 in relation to the insertion instrument 200 .
[0022] FIG. 4 shows the implant 100 with the insertion instrument 200 attached and in position on a patient vertebra 401 . Rotation of the actuator 202 in relation to the axis of the threaded rod 201 results in the rotation of the implant 100 around the axis of the pin 102 . Rotation of the knob 207 pushes the actuator 202 into the implant locking the assembly. When the assembly is locked hammer tapping can be applied to the handle 204 to advance the assembly forward.
[0023] A spinal implant may be used to stabilize a portion of a spine. The implant may promote bone growth between adjacent vertebra that fuses the vertebra together. An implant may include an opening through a height of a body of the implant. The body of the implant may include curved sides. A top and/or a bottom of the implant may include protrusions that contact and/or engage vertebral surfaces to prevent backout of the implant from the disc space.
[0024] A spinal implant may be used to provide stability and promote fusion of adjacent vertebra. The implant may be used in conjunction with a spinal stabilization device such as a bone plate or rod-and-fastener stabilization system. The implant may establish a desired separation distance between vertebra. The implant may promote bone growth between adjacent vertebra that fuses the vertebra together. Instrument at is necessary for insertion of an implant in a patient and alignment of the implant in the space.
[0025] A discectomy may be performed to establish a disc space between vertebra. The disc space may be prepared for implant insertion by distraction of adjacent vertebra, rasping and filing of the bone to achieve the desired spacing.
[0026] It is desired to perform insertion of the implant and positioning of the implant using minimum number of inserted instruments and thought the smallest possible insertion channel in the body.
[0027] Implants may be constructed of biocompatible materials sufficiently strong to maintain spinal distraction. Implants may include, but are not limited to, allograft bone, xenograft bone, autograft bone, metals, ceramics, inorganic compositions, polymers such as PEEK, or combinations thereof. If the implant is not made of bone, surfaces of the implant that contact bone may be treated to promote fusion of the implant to the bone. Treatment may include, but is not limited to, applying a hydroxyapatite coating on contact surfaces, spraying a titanium plasma on contact surfaces, and/or texturing the contact surfaces by scoring, peening, implanting particles in the surfaces, or otherwise roughening the surfaces.
[0028] In some embodiments, an implant may include an opening that extends through a body of the implant. The opening may have a regular shape or an irregular shape. Bone graft may be placed in the opening. The bone graft may be autogenic bone graft, allogenic bone graft, xenogenic bone graft, and/or synthetic bone graft. Some implant embodiments may be constructed from allogenic bone, such as cortical bone from a femur, tibia, or other large bone. In some embodiments, an implant may be formed from one or more pieces of allograft bone cut to a desired shape.
[0029] In certain embodiments, sides of an implant may be shaped to increase contact between an implant and adjacent vertebra with notches, ribs and other similar features. Increasing contact of an implant with adjacent vertebra may inhibit movement of the implant after insertion. An increased contact area between an implant and adjacent vertebra may promote bone growth between adjacent vertebra.
[0030] In some embodiments, one or more sides of an implant may be curved. One or more curved sides of an implant may allow the implant to be maneuvered in a disc space during insertion of the implant. The curvature of a side may approximate a curvature of an anterior side of a vertebra adjacent to which the implant is inserted.
[0031] Instruments may be used to prepare a space for an implant between adjacent vertebra. An instrument may be used to insert an implant in a prepared space. Instruments may be supplied to a surgeon or surgical team in an instrument set. An instrument set may include one or more implants for use during an insertion procedure. An instrument set may include implants of various sizes and/or lordotic angles to allow selection of an implant to suit a patient during surgery. Instrument is attached to the implant before the insertion into the body. When the desired position of the implant is achieved, instrument is disengaged from the implant and can be extracted from the body.
[0032] An instrument acts as an implant inserter. The implant inserter may be used to push the implant and to rotate the implant. After insertion of the implant, the implant may be released from the inserter without the application of significant repositioning forces to the implant. It can be imagined that the insertion instrument can be screwed into the implant using threads or use other techniques such as a tightening collet, jamming or grabbing. In the disclosed embodiment the implant turns around the axis of the implant pin as a result of the rotation of cam pushers. It can be imagined that other mechanisms can be used to rotate the implant such as ratchets or threaded push rods. The implant inserter may have a low profile that allows for visualization of the implant and surrounding area during insertion of the implant. Implant is equipped to couple and uncouple from the instrument.
[0033] While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
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A spinal implant include a top, wherein at least a portion of the top is configured to contact a first vertebra, a bottom, wherein at least a portion of the bottom is configured to contact a second vertebra, a side having a releasable attachment to receive an insertion device and a cam surface to engage a cam on the insertion device
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CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of Korean Application No. 10-2011-0049482, filed on May 25, 2011, with the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a carrier for use with both younger and older infants. The carrier can be used by younger infants having a smaller build and older infants having a bigger build.
2. Description of Related Art
A baby, a younger infant, and even older infants were typically moved around in a baby wrapper carried on a parent's back. In recent years, a baby carrier to allow a parent to carry his baby on his back or shoulder has come into wide use.
However, such baby carrier has the disadvantage of exposure to sunlight. The baby carriers that use a sunlight screen present additional problems of screens that cannot be optionally mounted on the baby carrier. These disadvantages may be avoided by a baby carrier disclosed in Korean Patent No. 10-0763412 owned by the present applicant. However, the techniques disclosed in Korean Patent No. 10-0763412 do not overcome the following problems.
An M-shaped leg posture is the recommended leg posture of a baby in a baby carrier. As used herein, the phrase “M-shaped leg posture” refers to a posture where a baby's knee is located above its hip. This leg posture allows a baby's hip to be tilted toward the front slightly arching its back, to minimize pressure from above, thereby reducing the risk of backbone damage.
For the purpose of achieving this posture, a user (for example, a baby's parent) has to purchase and use different size baby carriers based on age of the baby in days.
For example, a small-built younger infant, up to 100 days old (particularly as old as from 30 days to 100 days) should be placed in the M-shaped leg posture in a baby carrier. As shown in FIG. 1 , a younger infant in an M-shaped leg position leaves spaces 31 on both sides of the lower part of a back support web body 30 . Shoulder supports 10 are lockstitched to both sides of the upper part of the back support with body 30 . A waist band 20 for fastening to a user's waist is lockstitched to the lower part of the back support web body 30 .
With this configuration, the legs of the younger infant in the carrier are placed in the M-shaped leg posture only when the lower part of the back support web body 30 supports the hip of the small-built younger infant.
Since the legs of the younger infant placed in the M-shaped leg posture are placed in spaces 31 formed in both sides of the lower part of the back support web body 30 , it is possible for the user to move with the younger infant in the baby carrier.
A bigger older infant, older than 100 days, should also be in an M-shaped leg posture in the baby carrier. However, when such a big-built older infant is put into the baby carrier having the same back support web body 30 sized for a younger infant, injury to the baby may occur. Since the lower part of the back support web body 30 is narrower than the hip of the older infant and the legs of the older infant stand up, there is a risk of backbone damage, or dislocation of a hip joint, and so on.
In order to avoid this problem, as shown in FIG. 2 , the M-shaped leg leading spaces are removed from the back support web body 40 . The shoulder supports 10 are lockstitched to both sides of the upper part of the back support web body 40 . The waist band 20 for fastening to the user's waist is lockstitched to the lower part of the back support web body 40 . The hips and some of the thigh of the big-built older infant can then be supported in the M-shaped leg posture.
The problem remains that the user has to buy at least two different baby carriers, one for use with a younger infant, and one for use with the older infant, as the small-built younger infant grows.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a carrier that can be used with both younger and older infants, maintaining an M-shaped leg posture for the different sized infants.
According to an aspect of the invention, there is provided a carrier for use with both younger and older infants, including a back support web body which supports the back of a baby and has an M-shaped leg leading space formed in each side of the lower part thereof. A waist band is attached to the bottom of the back support web body for fastening to a user's waist. A space opening/closing member which opens or closes the M-shaped leg leading space, is formed in each side of the lower part of the back support web body.
The space opening/closing member includes a hip supporting part, made of flexible material, with its upper and lower centers fixed to the outer side of the back support web body and the waist band. A pair of movable rails are respectively fixed to the back support web body and the waist band to which both sides of the hip supporting part are respectively fixed. A pair of horizontal moving connectors which are respectively fixed to the upper and lower parts of the hip supporting part, are slidably movable to allow the hip supporting part to be folded. The connectors have connecting projections forming a horizontal moving space, with a lead-in groove.
Preferably, the carrier further includes a binding member which fixes the hip supporting part in a position where the hip supporting part is unfolded or doubly folded to open or close the M-shaped leg leading space formed in both sides of the lower part of the back supporting body.
Preferably, the carrier further includes a space maintenance binding member which fixes the hip supporting part at a position where the edges of both sides of the hip supporting part are doubly folded to open the M-shaped leg leading space formed in both sides of the lower part of the back supporting body.
BRIEF DESCRIPTION OF THE DRAWINGS
The exact nature of this invention, as well as the objects and advantages thereof, will become readily apparent from consideration of the following specification in conjunction with the accompanying drawings in which like reference numerals designate like parts throughout the figures thereof and wherein:
FIG. 1 is a view showing a conventional younger infant carrier used for younger infants from birth till 100 days or so;
FIG. 2 is a view showing a conventional older infant carrier used for older infants above 100 days;
FIG. 3 is a view showing a carrier used for both of younger and older infants according to an embodiment of the present invention;
FIG. 4 is a longitudinal sectional view of a back support web body of the carrier according to the embodiment;
FIG. 5 is a perspective view showing an open position of an M-shaped leg space formed in the back support web body of FIG. 4 ;
FIG. 6 is a perspective view showing a closing position of an M-shaped leg space formed in the back support web body of FIG. 4 ;
FIG. 7 is a perspective view showing a closed position of an M-shaped leg space formed in the back support web of FIG. 4 ;
FIG. 8 is a view showing the younger infant M-shaped leg space formed in the back support web body opened to allow a younger infant having a small build to take an M-shaped leg posture;
FIG. 9 is a view showing the younger infant M-shaped leg space formed in the back support web body closed to allow an older infant having a big build to take an M-shaped leg posture;
FIG. 10 is a view showing main parts of a binding member provided in the back support web body;
FIG. 11 is a view showing main parts of a binding member provided in the back support web body;
FIG. 12 is a view showing main parts of a binding member provided in the back support web body;
FIG. 13 is a view showing main parts of a binding member provided in the back support web body;
FIG. 14 is a view showing main parts of a binding member provided in the back support web body;
FIG. 15 is a view showing main parts of a space maintenance binding member provided in the back support web body; and
FIG. 16 is a view showing main parts of a space maintenance binding member provided in the back support web body.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the following description, a baby placed in a carrier usable with both younger and older infants, according to the present invention, are generally divided into a small-built younger infant being about 30 days to 100 days old, and a big-built older infant over 100 days.
FIG. 3 shows a carrier usable for both younger and older infants, according to an embodiment of the present invention. FIG. 4 is a cross-section of a back support web body 310 of a carrier according to the embodiment. FIGS. 5 to 7 show phased closing of an M-shaped leg leading space formed in the back support web body of FIGS. 3 and 4 . FIG. 8 shows the carrier in use, with a younger infant. The M-shaped leg space 110 formed in the back support web body 310 opened to allow a younger infant having a small build to take an M-shaped leg posture. FIG. 9 shows the carrier in use with the younger infant M-shaped leg space 110 formed in the back support web body 310 closed to allow an older infant having a big build to take an M-shaped leg posture. FIG. 10 shows main parts of a binding member provided in the back support web body according to a first example. FIG. 11 shows main parts of a binding member provided in the back support web body according to another example. FIG. 12 shows main parts of a binding member provided in the back support web body according to another example. FIG. 13 shows main parts of a binding member provided in the back support web body according to yet another example. FIG. 14 shows main parts of a binding member provided in the back support web body according to another example. FIG. 15 shows main parts of a space maintenance binding member provided in the back support web body according to another example. FIG. 16 shows main parts of a space maintenance binding member provided in the back support web body according to another example.
As shown in FIG. 3 , a younger/older infant carrier according to the present invention generally includes a back support web body 100 , a waist band 200 , a space opening/closing member 300 and a pair of shoulder supports 700 .
The back support web body 100 serves to support the back of a baby. For a younger infant, M-shaped leg space 110 is formed in each side of the lower part thereof in such a manner that a small-built younger infant can take an M-shaped leg posture when the infant is put in the carrier.
The waist band 200 for fastening around a user's waist, is attached to the bottom of the back support web body 100 so that the baby can be supported by the back support web body 100 . A female buckle 212 and a male buckle 214 are connected to the ends of the waist band 200 , respectively, so that the waist band 200 can be fastened around a user's waist.
The pair of shoulder supports 700 each have one end connected to one end of the back supporting body 100 and the other end connected to the other end of the back support web body 100 , so that the user can move with the baby put in the carrier.
The space opening/closing member 300 serves to open/close the M-shaped leg space 110 , which is formed in each side of the lower part of the back support web body 100 , in phases. Since the M-shaped leg space 110 can be optionally opened or closed in phases with growth of the small-built younger infant into a big-built older infant, this space opening/closing member 300 allows both the younger and older infant to be placed into an M-shaped leg posture in the same carrier.
Such a space opening/closing member 300 includes a hip supporting part 310 which is made of flexible material and has its upper and lower centers fixed to the outer side of the back support web body 100 and the waist band 200 , respectively. A pair of movable rails 311 are respectively fixed to the back support web body 100 and the waist band 200 to which both sides of the hip supporting part 310 are respectively fixed. A pair of horizontal moving connectors 319 which are respectively fixed to the upper and lower parts of the hip support web part 310 , are slidably movable to allow the hip support web part 310 to be folded. The moving connectors have connecting projections 315 ( FIG. 4 ) to enclose a space 313 . A partial opening is formed in projections 315 by a lead-in groove 317 .
The hip support web part 310 is preferably made of cotton material. A shape retention pad 321 may be overlaid and lockstitched near both edges of the hip supporting part 310 so that the hip supporting part 310 made of the cotton material will not lose its shape when supporting a baby.
Such a shape retention pad 321 may be a combination of memory foam having high elasticity, such as a sponge, and an inflexible foamed plastic hardboard.
In order to fix the movable rails 311 to the back support web body 100 and the waist band 200 , respectively, the movable rails 311 are wrapped by a connection fabric 312 ( FIG. 4 ). An end portion of the connection fabric 312 is lockstitched to the back support web body 100 and the waist band 200 . In this case, although not shown in the figures, a connecting rod is integrated with the movable rails 311 and is lockstitched with the connection fabric 312 so that the movable rails 311 wrapped by the connection fabric 312 cannot be moved within the connection fabric 312 .
The horizontal moving connectors 319 are lockstitched and fixed to the rear side of the hip supporting part 310 by a connection band 318 which is locked in a locking groove of connection pieces 316 integrally formed in the connecting projections 315 of the horizontal moving connectors 319 .
A process of opening the younger infant M-shaped leg space 110 formed in both sides of the lower part of the back support web body 100 in phases using the above-configured space opening/closing member 300 for both a younger and older infant will be described with reference to FIGS. 5 to 7 .
FIG. 5 shows the carrier support with the younger infant M-shaped leg leading space 110 formed in both sides of the lower part of the back support web body 100 opened by sliding the horizontal moving connectors 319 on moving rails 311 to the center of the moving rails 311 . Non-lockstitched edges of both sides of the hip supporting part 310 are doubly folded, so that a small-built younger infant can take an M-shaped leg posture in the carrier.
In this case, the doubly-folded edges of both sides of the hip supporting part 310 are as wide as the shape retention pad 321 , and the doubly-folded edges of both sides of the hip supporting part 310 are folded such that inner sides of the shape retention pad 321 near the doubly-folded edges of both sides of the hip supporting part 310 can make contact with each other.
FIG. 8 shows the carrier adjusted for a small-built younger infant, with the infant positioned in the carrier.
The horizontal moving connectors 319 , fixed near an edge of the back side of the hip supporting part 310 , by virtue of the weight of the small-built younger infant, pull the upper and lower moving rails 311 in a direction perpendicular to the horizontally-arranged moving rails 311 and lock the moving connectors 319 in place.
FIG. 6 shows a carrier support web where the younger infant M-shaped leg space 110 formed in both sides of the lower part of the back support web body 100 is about half opened. The horizontal moving connectors 319 , fixed to the doubly-folded edges of both sides of the hip supporting part 310 , are moved towards the outside of the moving rails 311 . A middle-built baby between a small-built younger infant and a big-built older infant can take an M-shaped leg posture in a carrier so adjusted.
FIG. 7 shows a carrier support web where the M-shaped leg leading space 110 formed in both sides of the lower part of the back support web body 100 is closed by further sliding the horizontal moving connectors 319 , to the outside of the moving rails 311 . A big-built older infant can take an M-shaped leg posture in the carrier so adjusted.
FIG. 9 shows a carrier adjusted for a big-built younger infant with a baby located in the carrier.
In this embodiment, a binding member 400 (see FIGS. 10 to 14 ) is further provided to fix the hip supporting part 310 at a position where the edges of both sides of the hip supporting part 310 are doubly folded so that a younger or older infant can take an M-shaped leg posture in the carrier, thereby providing complete binding of the folded hip supporting part 310 .
This binding member 400 may be implemented with different configurations.
The binding member 400 may have a button structure of different configurations. First, as shown in FIG. 10 , the button structure of the binding member 400 includes a connection strap 401 having its one end fixed to an edge of the outer side of the hip supporting part 310 . A male button 403 is fixed to the other end of the connection strap 401 . A female button 405 is fixed to the back support web body 100 .
The female button 405 may be fixed to a finishing fabric 404 whose one side is lockstitched along the back support web body 100 and the waist band 200 . The reason for fixing the female button 405 to the finishing fabric 404 is to conceal the moving rails 311 , and the horizontal moving connectors 319 , and so on, thereby providing an aesthetic appearance. The female and male buttons may be positioned in consideration of a user's taste and convenience.
As shown in FIG. 11 , the button structure of the binding member 400 may be a simplified structure where the male button 403 is directly fixed to the hip supporting part 310 without being fixed to the connection strap 401 .
As shown in FIG. 12 , the binding member 400 may include a connection strap 401 having its one end fixed to an edge of the outer side of the hip supporting part 310 . A stud 407 is fixed to the other end of the connection strap 401 , and a stud hole 409 is formed in the back support web body 100 . The waist band 200 (not shown) fixed with both sides of the hip supporting part 310 is disposed to correspond to a position where the hip supporting part 310 is unfolded or doubly folded.
The stud hole 409 is preferably formed in a finishing fabric 404 whose one side is lockstitched along the back support web body 100 and the waist band 200 .
As shown in FIG. 13 , the binding member 400 may include a fixing loop 411 fixed to an edge of the outer side of the hip supporting part 310 , and a hook 413 which is fixed to the back support web body 100 .
The waist band 200 is fixed to both sides of the hip supporting part 310 and is disposed to correspond to a position where the hip supporting part 310 is unfolded or doubly folded.
Here, the fixing loop 411 is preferably made of elastic material and tightly fixed to the hook 413 by virtue of its elasticity.
As shown in FIG. 13 , the hook 413 may be an L shape or a stud shape, and is preferably formed in a finishing fabric 404 whose one side is lockstitched along the back support web body 100 and the waist band 200 .
As shown in FIG. 14 , the binding member 400 may include a male Velcro tape 415 provided on an edge of the outer side of the hip support part 310 , and a female Velcro tape 417 fixed to the back support web body 100 . The waist band 200 is fixed to both sides of the hip supporting part 310 and is disposed to correspond to a position where the hip supporting part 310 is unfolded or doubly folded.
The female Velcro tape 417 is preferably attached to a finishing fabric 404 whose one side is lockstitched along the back support web body 100 and the waist band 200 .
In a different embodiment, a space maintenance binding member 500 (see FIGS. 15 and 16 ) may be further provided to fix the hip supporting part 310 at a position where the edges of both sides of the hip supporting part 310 are doubly folded to contact each other so that a small-built younger infant can take an M-shaped leg posture in the carrier.
This space maintenance binding member 500 may be implemented in different configurations.
As shown in FIG. 15 , the space maintenance binding member 500 may include a zipper 510 lockstitched along both sides of a contact portion when the hip supporting part 310 is folded.
As shown in FIG. 16 , the space maintenance binding member 500 may include a pair of binding strings 520 which are respectively provided in both sides of a contact portion when the hip supporting part 310 is folded. Here, a pair of upper binding strings 520 and a pair of lower binding strings may be provided to further increase binding efficiency.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present invention. The exemplary embodiments are provided for the purpose of illustrating the invention, not in a limitative sense. Thus, it is intended that the present invention covers the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
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A carrier for use with both younger and older infants, is capable of placing the legs of younger infants having a smaller build and older infants having a bigger build into a desired M-shaped leg posture. A means for opening and closing the leg space formed in a back support web body of the carrier, adapts the carrier for continued use as the younger infants grow into older infants.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a windshield assembly for motorcycles and more particularly to a windshield assembly having a wiper mechanism for motorcycles, three-wheeled motor vehicles and the like, and a method of assembling the windshield assembly and mounting same on a vehicle.
2. Description of Relevant Art
A windshield assembly for motorcycles comprising an upwardly extending windshield member mounted in front of a handlebar, a wiper motor attached to an upper part of the windshield member and a wiper arm for supporting a wiper blade supported pivotably from above, is known.
In such a conventional windshield assembly for motorcycles, because the wiper motor is heavy relative to the windshield member which is supported at its lower portion, it is necessary to additionally provide a motor supporting stay, and consequently it has been difficult to attain a favorable external appearance. In view of this consideration, if the wiper motor is supported directly by the windshield without the additional provision of a supporting stay, it becomes necessary to increase the rigidity of the windshield and hence to thicken the shield, thus resulting in an increase in weight, which is contrary to the demand for reduction in weight of motorcycles.
Moreover, due to the size of the wiper motor, it is desirable to suitably dispose it while taking into account mounting space and its shielding thereof from an external view. Additionally, it is preferable that a wiper switch for manually turning on and off the wiper motor be disposed in a position where switching operation thereof can be performed easily as necessary.
Furthermore, if the windshield member is mounted on a fairing member, it is undesirable with respect to working efficiency to attach the fairing member and the wiper mechanism separately to the vehicle body through separate stays, and in order to maintain the contact angle of the blade of the wiper mechanism with the surface of the shield member and the mounting angle of the driving shaft of the wiper motor at a predetermined accuracy, it is desirable to avoid disassembly and assembly of the wiper mechanism itself, and if possible, to assemble the fairing member and the windshield member together in advance. Further, if an inner panel (ornamental panel) member mounted between the fairing member and the handlebar is also assembled together with the aforesaid members in advance, it becomes possible to improve the required man-hour time for mounting such assembled members to the vehicle body.
The present invention effectively overcomes the foregoing drawbacks of the prior art while meeting the demand mentioned above.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a windshield assembly in a motorcycle having a vehicle body and a handlebar attached to a front part of the vehicle body, which windshield assembly comprises a fairing member supported by the vehicle body in front of the handlebar, an upwardly extending windshield member mounted on the fairing member and permitting substantially a front view therethrough, an ornamental panel member extending rearwardly from the lower portion of the windshield member, a manual switch mounted on the upper surface of the ornamental panel member, a motor disposed below the ornamental panel member and capable of being turned on and off by the manual switch, a wiper coupled to the motor and attached to the windshield member and capable of wiping the front surface of the windshield member, and a stay member fixed to the vehicle body to support the motor.
It is an object of the present invention to provide a windshield assembly for motorcycles having characteristics such that the reduction in weight of a windshield member and a high operating accuracy of a wiper can be attained and the windshield member is assembled together with a fairing member and an inner panel member to constitute an integrally assembled member having a favorable external appearance while attaining a desirable arrangement of a wiper motor and a wiper switch, thereby permitting the integrally assembled member to be mounted to a vehicle body as necessary in a simple manner, which greatly contributes to a remarkable improvement in the mounting time.
Preferred embodiments of the present invention will be described in detail hereinunder with reference to the accompanying drawings, from which further features, objects and advantages of the present invention will become apparent.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic side view of a motorcycle having a windshield assembly according to a first embodiment of the present invention.
FIG. 2 is an enlarged side view of the windshield assembly shown in FIG. 1, partly broken away to show principal portions.
FIG. 3 is an enlarged transverse sectional view of the windshield assembly shown in FIG. 1.
FIG. 4 is a transverse sectional view of a windshield assembly according to a second embodiment of the present invention.
FIG. 5 is an enlarged sectional view taken along line 5--5 of FIG. 4.
FIG. 6 is an enlarged sectional view taken along line 6--6 of FIG. 4.
FIG. 7 is an enlarged sectional view taken along line 7--7 of FIG. 6.
FIG. 8 is a sectional view of a windshield device according to a third embodiment of the present invention.
FIG. 9 is an enlarged view of principal portions of the windshield assembly of FIG. 8.
FIG. 10 is a partially cut-away enlarged side view of principal portions of the windshield assembly of FIG. 8.
FIG. 11 is a side view of a windshield assembly according to a fourth embodiment of the present invention, partly broken away to show principal portions.
FIG. 12 is an enlarged longitudinal sectional view of principal portions of the windshield assembly of FIG. 11.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Referring first to FIGS. 1 through 3, reference numeral 1 represents the entirety of a motorcycle, and a front wheel 3 is attached to a lower part of the front portion of a vehicle body 2 of the motorcycle 1, while from the rear portion of the vehicle body 2 there extends upwardly a seat post 4 which supports a seat 5, with the front portion of a power unit 6 which supports a rear wheel 7 as a driving wheel being secured for swinging motion to a lower part of the rear portion of the vehicle body 2. At the front portion of the vehicle body 2 there extends upwardly a front cover 8, which also serves as a leg shield, in a rearwardly inclined manner. A transversely extending handlebar 10 is mounted on a bracket 9 secured to a steering stem (not shown) which passes through a head tube 2a. The handlebar 10 is provided at both ends thereof with grips 10a, and as shown in FIG. 3, a handlebar pipe 10b positioned between the grips 10a is covered with a cover 10c which incorporates a meter unit, etc. Furthermore, a fairing 11 is disposed in front of the handlebar 10, and is provided with a body 11a positioned intermediately in the transverse direction and grip cover portions 11b which, as shown in FIG. 2, extend to the right and left with respect to the body 11a, the body 11a and the grip cover portions 11b being integrally molded from a synthetic resin. On the fairing 11 there extends upwardly a transparent windshield 12 made of a synthetic resin. A lower portion 12a of the windshield 12 is overlapped with an upper portion 11c of the fairing body 11a, and both are secured together with a plurality of machine screws 13 as shown in FIG. 3 and further secured, also by means of the machine screws 13, to a support frame 14 which comprises a steel plate or the like and which is disposed on the back of the lower portion 12a of the windshield. The frame 14 is supported by tip ends of right and left side stays 15 whose base portons are fixed to the handlebar pipe 10b in positions more inwardly than the grips.
As is apparent from FIGS. 2 and 3, a wiper motor 16 is mounted at an intermediate or upper part and centrally in the transverse direction of the rear side of the fairing body 11a. The motor 16 is fixed with screws 17 to a central part in the transverse direction of the frame 14 (i.e., the motor 16 is fixed to the windshield 12, through the frame 14) and is also supported by a tip portion of a center stay 18 whose base portion is fixed to the steering stem bracket 9, whereby the motor 16 is held in place firmly enough to resist a reaction force induced by its operation. Thus the motor 16 is disposed below the windshield 12, and this arrangement is preferable from the standpoint of weight balance. The central location of motor 16 in the transverse direction is further preferable with respect to balance. From the front side of the motor 16 there forwardly projects a driving shaft 16a and a guide shaft 16b through the upper portion 11c of the fairing 11 and the lower portion 12a of the windshield 12, and base portions of arms 19a and 19b of a wiper 19 are connected to the driving shaft 16a and guide shaft 16b, respectively. To the tip ends of the arms 19a and 19b is connected a blade 19c which contacts the front surface of the windshield 12.
As shown in FIG. 3, the wiper motor 16 is thus disposed within a space S formed between the handlebar 10 and the fairing 11, and mounted thereover is an ornamental panel 20 which extends transversely along the upper portion 11c of the body 11a of the fairing 11. The panel 20 covers the motor 16 and the stays 15 and 18, and a switch 21 for driving the wiper motor 16 is fixed to one side end portion of the panel 20 in a position near the handlebar grip 10a. The switch 21 has a knob 22 which projects onto the outer surface of the panel 20, and its mounting base 23, as shown in FIG. 3, faces the lower surface of the panel 20 and is connected to the motor 16 through a harness 24. Thus, because the switch 21 is disposed adjacent the handlebar grip 10a, it can be easily operated together with other switches (not shown) disposed concentratedly in inside positions with respect to the grip 10a.
In the foregoing manner, the wiper assembly, including wiper motor 16, wiper switch 21 and wiper 19, can be operably attached to the windshield assembly in advance. In assembling the windshield assembly, the wiper motor 16 is mounted in a lower position on the inner side of the windshield assembly, and the panel 20 is secured in an inner and lower portion of the windshield so as to cover the wiper motor. The wiper switch 21 is mounted on the panel 20, and the wiper 19 is mounted adjacent the front surface of the windshield and operatively coupled to the wiper motor 16 as described above. Thereafter, the windshield assembly, having the wiper assembly operatively connected therewith, is mounted to the front porton of the vehicle body such that the fairing is positioned in front of the handlebar 10 of the vehicle and the wiper motor 16 is disposed within the space S defined between the handlebar 10 and the fairing 11, with the panel 20 extending substantially proximal to the handlebar 10. As part of such mounting operation, the wiper motor 16 is secured to the vehicle body through the stay 18 which is fixed to the steering stem bracket 9.
Referring now to FIGS. 4 through 7, reference numeral 112 shown in FIG. 4 designates a center stay fixed at its lower end to a steering stem bracket (not shown) mounted on the upper end of a front fork (not shown), and behind the center stay 112 are disposed right and left handlebar pipes 113 and 114 which are fixed to the steering stem bracket, with meters 116 being disposed on a cover 115 for the pipes 113 and 114. To the handlebar pipes 113 and 114 are connected rear ends of forwardly extending right and left side stays 117 and 118, respectively, and a support frame 119 is attached to the front ends of the center stay 112 and side stays 117, 118. The support frame 119, which is forwardly bulged and curved in horizontal section, has its length dimension extending in the transverse direction of the vehicle body, and a wiper motor 120 is attached thereto centrally in the transverse direction of the vehicle body.
The front of the handlebar cover 115 is covered with a windshielding fairing 121, and from the fairing 121 there extends upwardly a windshield 122 which is spread in front of the driver's face. Behind the fairing 121 is disposed an ornamental panel 123 in which is formed a downwardly projecting recess 123a which serves favorably as a container. The ornamental panel 123 covers the upper surface of the wiper motor 120. The fairing 121, windshield 122 and ornamental panel 123 are fixed to the support frame 119 and thereby supported, as shown in FIGS. 5 through 7.
The mounting operation for the above-mentioned members is performed in the following manner. First, a lower portion 122a of the windshield 122 is placed on a back 121b of an upper portion 121a of the fairing 121 through a rubber member 124, then the upper portion 121a, the rubber member 124 and the lower portion 122a are fixed to a front 119a of the support frame 119, as shown in FIG. 6, with bolts 126-129 and nuts 126a-129a which are disposed centrally in the transverse direction of the vehicle body among bolts 125-130 and nuts 125a-130a provided at a total of six places as shown in FIG. 4. Head portions 125b-130b of all the bolts 125-130 do not have a groove for insertion and engagement of a tool such as a screw-driver, i.e., the head portions 125b-130b exposed to the front of the fairing 121 are smooth and hence provide a favorable appearance. The tightening operation for the bolts 126-129 and nuts 126a-129a is effected by turning the nuts 126a-129a on the back side of the fairing 121. At this time, the ornamental panel 123 is not yet mounted.
An upper portion 119b of the support frame 119 is rearwardly bent stepwise as shown in FIG. 5, and consequently there is formed a gap S 1 between upper portion 119b and the back 121b of the fairing, or the back of the windshield 122 because the rubber member 124 and the lower portion 122a of the windshield 122 are disposed on the back 121b in this embodiment. The gap S 1 is formed throughout the overall length of the support frame 119 except the portion where the wiper motor 120 is mounted, and the fairing 121, etc. are fixed to the support frame 119 with bolts 126-129 and nuts 126a-129a in the above-described manner while maintaining the gap S 1 .
On the other hand, at a transversely extending front end 123b of the ornamental panel 123 there is formed a downwardly projecting rib 123c, which is formed on the peripheral edge of the front end 123b in correlation with the position of the gap S 1 , and on both end portions of the peripheral edge of the front end 123b of the ornamental panel 123 are formed projections 123d and 123e as shown in FIG. 4. The projections 123d and 123e, as shown in FIGS. 6 and 7, extend downwardly an amount larger than the extending amount of the rib 123c. On the other hand, in both end portions of the support frame 119 there is formed a hole 119c which extends vertically therethrough, in positions corresponding to the positions of the projections 123d and 123e.
For mounting the ornamental panel 123 to the support frame 119, first the rib 123c is inserted in the gap S 1 whereby it is held between the back 121b of the fairing 121 and the upper portion 119b of the support frame 119, and more concretely in this embodiment, between the lower portion 122a of the windshield 122 and the upper portion 119b of the support frame 119. As a result, in the portions of the bolts 126-129 and nuts 126a-129a, the ornamental panel 123 is supported by the support frame 119 without performing the nut tightening operation. When inserting the rib 123c into the gap S 1 , the projections 123d and 123e are also inserted into the hole 119c and allowed to project onto the back of the support frame 119. Thereafter, at the portions of the projections 123d and 123e, the ornamental panel 123 is fixed to the support frame 119 together with the fairing 121, rubber member 124 and windshield 122 by means of bolts 125, 130 and nuts 125a, 130a. This fixing operation with bolts 125, 130 and nuts 125a, 130a is attained by turning the nuts 125a and 130a because the head portions 125b and 130b are smooth as previously noted. In this case, the support frame 119 is positioned at both end portions of the front end peripheral edge of the ornamental panel 123 as shown in FIG. 4, and these end portions permit easy insertion of a nut tightening tool in the lower surface of the ornamental panel 123 without being impeded by the wiper motor 120 and the goods accommodating recess 123a, and therefore the tightening operation for the nuts 125a and 130a can be performed easily.
Thus, in this embodiment, when fixing the ornamental panel 123 with bolts and nuts to the support frame 119, the nut tightening operation may be performed only at both end portions of the front end peripheral edge of the ornamental panel 123, and the other portions of the panel 123 are supported by insertion of the rib 123c into the gap S 1 .
Moreover, because the rib 123c is formed substantially throughout the peripheral edge of the front end 123b of the ornamental panel 123, the circumferential portion of the panel 123 and hence the fairing 121 can be made stronger. Furthermore, if the windshield 122 is interposed between the fairing 121 and the ornamental panel 123 as in this embodiment, the insertion of the rib 123c into the gap S 1 can afford a junction surface having a favorable external appearance between the windshield 122 and the ornamental panel 123.
Also, it will be understood that in this embodiment the wiper motor 120 is fixed to the windshield 122, through the support frame 119.
Referring now to FIGS. 8 through 10, reference numeral 201 shown in FIG. 8 designates a handlebar pipe of a motorcycle. A windshield 216 and a grip cover 217 which covers right and left handlebar grips 201a are fixed firmly to the handlebar pipe 201 while being interconnected up and down by a contact panel 212.
More specifically, the windshield 216 is formed at its lower portion to have a width approximately equal to the handlebar width, and in a side position of the connection 216a with the contact panel 212 there is mounted a wiper arm 209 which is supported by a driving shaft 208, and a wiper blade 209a is connected to the wiper arm 209. The grip cover 217 is dependent at its right and left portions 217a so as to permit the placement of a headlight L therebetween, and the right and left dependent portions 217a are positioned ahead of the handlebar grips 201a and behind blinkers W.
Furthermore, in a predetermined position of each dependent portion 217a there is formed a ventilation slit mechanism 218 which has a plurality of openings capable of being opened and closed.
The windshield 216 and the grip cover 217, as shown in FIG. 10, are fixed to the handlebar pipe 201 through a wiper driving unit 206. The details of such mounting arrangement will be described hereunder.
A bracket 202 is fixed by welding to a predetermined position of the handlebar pipe 201, and to the bracket 202 is firmly fixed a stay 203 having a substantially L-shaped section by means of a bolt and nut 204, the stay 203 being fixed at the other end thereof to a support portion 207 of the wiper driving unit 206 by means of a bolt and nut 205.
On the other hand, the lower end portion of the windshield 216 and the upper end portion of the grip cover 217 are overlapped with each other and in this state firmly connected, by means of bolts and nuts (not shown), to the contact panel 212 which comprises front and rear rubber protection members 215, an outer panel 213 and a mounting panel 214. The thus-connected windshield 216 and grip cover 217 are fixed to the wiper driving unit 206 by means of a pair of bolts and nuts 211 as shown in FIG. 9 in a side position of the contact panel 212.
In the above described fixed portion, as shown in FIG. 10, there is formed an opening 212a which extends through the contact panel 212, windshield 216 and grip cover 217, and a bearing 210 is fitted in the opening 212a, and the driving shaft 208 is carried on the bearing 210 so that it can be pivoted by the wiper driving unit 206.
As will also be apparent from the above description, the windshield 216 and the grip cover 217 interconnected by the contact panel 212 are attached to the handlebar pipe 201 through the wiper driving unit 206.
For mounting the windshield assembly of the above-mentioned construction including the wiper driving unit 206 with respect to the handlebar pipe 201, the windshield 216 and the grip cover 217 are interconnected by the contact panel 212 and the driving shaft 208 is inserted into the bearing 210 of the thus-connected body, then the wiper driving unit 206 is fixed to the connected body with the paired bolts and nuts 211, and the wiper arm 209 is attached to the tip end portion of the wiper shaft 208.
Then, because the stay 203 is fixed with bolt and nut 205 to the support portion 207 of the wiper driving unit 206, the other end of the stay 203 may be fixed with bolt and nut 204 to the bracket 202 which is welded to the handlebar pipe 201.
Although in this embodiment the grip cover 217 is connected to the lower portion of the windshield 216, it is not always necessary to provide the grip cover 217.
Referring now to FIGS. 11 and 12, reference numeral 311 shown in FIG. 11 designates a fairing of a motorcycle, and a windshield 312 which comprises a transparent synthetic resin plate is mounted on the fairing 311 in an upwardly inclined manner. A lower portion 312a of the windshield 312 is overlapped with the back of an upper portion 311c of a transversely central portion 311a of the fairing 311 and is fixed thereto with machine screws (not shown) or the like, with a rubber seal 314 being interposed therebetween. The fairing 311 is supported by a plate-like fairing support frame 316 which is supported by the tip end of each of right and left side stays 315 whose base end portions are fixed to a handlebar pipe 310 in inside positions with respect to right and left grips 310a. Behind the lower portion of the windshield 312 is disposed a wiper motor 317 having a driving shaft 317a and a guide shaft (not shown). The driving shaft 317a and the guide shaft are projected to the front through the overlap of the lower portion 312a of the windshield 312 and the upper portion 311c of the fairing 311, and connected thereto is an arm 318a of a wiper blade 318 and the base end portion of a wiper link 318b, respectively. The arm 318a and the link 318b are adapted to pivot with the lower portion of the windshield 312 as a fulcrum, thereby allowing the blade 318 to wipe the front of the windshield 312. The wiper motor 317, as shown in FIG. 12, is supported at two right and left points on its front by a support portion 316a of the frame 316 which portion is somewhat concaved rearwardly from the central part of the frame, and at the same time, as shown in FIG. 11, a tip end portion 320a of a center stay 320 whose base end portion is fixed to a steering stem bracket 309 is secured to the central part of the front of the motor 317, and thus the motor 317 is supported at a total of three points, whereby it is made possible to suitably bear the reaction force induced by operation of the motor 317 and fix the motor relatively stationarily. The space behind the fairing 311 including the motor 317 is closed by a transversely disposed ornamental panel 321, thereby covering the stays 315, 320 and the motor 317. The panel 321 is disposed above a handlebar cover 322.
As shown in detail in FIG. 12, a mounting bolt 319a projects from a front surface 317b of the body 317a of the wiper motor 317, while in the plate-like support portion 316a of the frame 316 there is formed a mounting hole 316b having a diameter larger than that of the bolt 319a. Between the back of the support portion 316a and the front 317b of the motor 317 is interposed an elastic vibration insulator 323 such as a rubber member, and a mounting hole 323a which matches the mounting hole 316b is formed in the vibration insulator 323. Furthermore, the bolt 319a is loosely fitted in the mounting holes 323a and 316b, and a flanged collar 324 is fitted over the bolt 319a. The collar 324 is provided on its outer periphery with a flanged rubber bushing 325. A tubular portion 325a of the bushing 325 is fitted in the mounting holes 323a and 316b from the outside of the support portion 316a of the frame 316, and a flange portion 325b of the bushing 325 is brought into contact with a surrounding portion of the mounting hole 316b of the support portion 316a. The tubular portion 324a of the collar 324 surrounds the outer periphery of the bolt 319a, and its flange portion 324b faces the outer end face of the flange portion 325b of the bushing 325. A nut 319b is threadedly fitted over the tip portion of the bolt 319a projecting from the collar 324, and is tightened to clamp the collar 324, thereby clamping the wiper motor 317 to the support portion 316a of the frame 316 through the rubber bushing 325 and the vibration insulator 323. Furthermore, a sound absorbing material 326 is adhesively secured to the lower surface of the ornamental panel 321 which covers the wiper motor 317.
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A windshield assembly for motorcycles. A wiper motor is disposed in a lower position inside a windshield member which extends upwardly from a fairing member, and a wiper switch is mounted on an ornamental panel member which is disposed over the motor, and further a wiper arm is coupled to the motor. These windshield components are integrally assembled together in advance, and the assembled body is mounted to a vehicle body through a mounting stay.
Reduction in weight, ensuring of the operating accuracy, improvement of the external appearance and decrease of the mounting time is attained.
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This application is a Division of application Ser. No. 09/345,498 Filed on Jul. 1, 1999, now U.S. Pat. No. 6,209,856,which is a divisional application of Application Ser. No. 08/924,000 filed on Sep. 5, 1997, now U.S. Pat. No. 5,945,039.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a gas-liquid processing apparatus using a static fluid mixer (motionless fluid mixer) applicable for the substance transfer from a gas to a liquid, such as the gas absorption and the gas dissolution, the substance transfer from a liquid to a gas, such as the diffusion, the gas phase reaction with a substance to be processed in a liquid, such as the aeration and the incubation, the chemical reaction with a gas and a liquid, such as halogenation, hydrogenation, oxidation and sulfidization, and the three phase reaction of gas-liquid-solid, such as a bioreactor and a bioreacting apparatus.
2. Description of the Related Art
In the food industry, carbon dioxide is absorbed in water in the production process of refreshing beverages such as a carbonated water. In the petrochemical industry, a liquid and a gas are contacted in an oxidization reaction apparatus, a hydrogenation reaction apparatus, or a gas diluted water manufacturing apparatus. In the paper and pulp industry, a liquid and a sulfidized water are contacted for the absorption reaction of a sulfidized water. A contact process of a gas and a liquid is necessary also for environmental apparatus such as a deep aeration apparatus, a chlorine pasteurization apparatus of water, an exhaust gas processing apparatus, a purification apparatus for industrial waste water, water supply or sewage, a processing apparatus of industrial waste water with ozone gas, water supply or sewage, and an aerator. Furthermore, in the fishery industry, air is mixed in water by contacting air with water for charging oxygen in a pisciculture pond.
The gas-liquid processing apparatus is used particularly in a purifying apparatus for eliminating an organo-chloric compound such as 1-1-1-trichloroethane, trichloroethylene, and tetrachloroethylene from a waste liquid, a hazardous substance eliminating apparatus for eliminating a substance such as chlorine, trihalomethane and fumic acid from tap water or well water, a pasteurization apparatus for sterilization or pasteurization of dissolution and enrichment of oxygen gas, ozone, chlorine dioxide or chlorine gas in raw water, and a bioreactor where aerobic bacteria are used.
A conventional gas-liquid processing apparatus (a gas-liquid contacting apparatus) utilizing a static type fluid mixer, comprising a spiral blade body in a passage pipe and a plurality of fluid passage for passing a fluid in the pipe axis direction, arranged perpendicularly, for supplying liquid from a position higher than the fluid mixer by the hydrostatic pressure difference, and further, a gas can pass in the fluid mixer (Japanese Patent Application Laid Open No. 5-96144) is known.
However, since the liquid is supplied from the upper direction with respect to the liquid mixer by the hydrostatic pressure difference into the fluid mixer in the conventional gas-liquid processing apparatus, although the production cost and the running cost can be low for not requiring a motive power, it has the disadvantage of having a low gas-liquid contacting efficiency.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a gas-liquid processing apparatus with a high contacting efficiency between a gas and a liquid and a high reaction rate and a high mixing efficiency at a low production cost.
A first aspect of a gas-liquid processing apparatus according to the present invention comprises one or a plurality of static type fluid mixer arranged substantially perpendicularly in the longitudinal direction, a liquid supplying means for supplying a liquid in the passage pipe, and a gas supplying means for supplying a gas in the passage pipe, wherein a fluid consisted of said liquid and said gas is in the pressured state higher than the atmospheric pressure in the static type fluid mixer.
A second aspect of a gas-liquid processing apparatus of the present invention comprises a plurality of static type fluid mixers arranged parallel to each other and substantially perpendicularly in the longitudinal direction, a container for keeping a fluid and arranging the static type fluid mixers so as to be soaked at least partially in the fluid, partition members for partitioning the inside of the container for each of the static type fluid mixers so as to allow passage of a liquid but not allow passage of a gas, and pipes for connecting the gas space of each room of the container partitioned by the partition members and a gas introducing portion of an adjacent static type fluid mixer, wherein the fluid is in the pressured state higher than the atmospheric pressure in the static type fluid mixers.
Furthermore, it is preferable that a circulating means for returning the fluid from the bottom portion of the static type fluid mixer to the upper portion thereof for circulating the fluid is further provided in the first and second aspects of the present invention.
It is also preferable that the static type fluid mixer comprises a passage pipe for the passage of a fluid and a spiral blade body arranged in the passage pipe. An opening is formed in the spiral blade body at the center portion of the passage pipe.
In the first aspect of the present invention, a liquid and a gas are mixed and contacted for generating a certain reaction, a gas absorption or a dissolution while passing through the fluid mixer. In this case, the fluid is maintained in a pressured state in the static type fluid mixer. Preferably, the liquid and the gas are circulated and supplied in the static type fluid mixer. Accordingly, in the present invention, a fluid is maintained in a pressurized state higher than the atmospheric pressure, and preferably circulated and supplied into the static type fluid mixer. Therefore, a gas and a liquid are contacted and mixed with a high efficiency. Besides, since a motive power is not used for stirring the gas and the liquid, it has an advantage of a low production cost.
In the second aspect of the present invention, a plurality of the static type fluid mixers are arranged parallel to each other and the container is partitioned by the partition members so that a liquid can move freely among the rooms but a gas cannot move freely. Then the gas is introduced to the gas introducing portion of an adjacent static type fluid mixer by the pipe. Then, the gas passes through the static type fluid mixer of each room successively and contacts with the liquid. Accordingly, the gas and the liquid are introduced into the static type fluid mixer arranged at an end of the container so that a mixed fluid in the container is discharged from the room on the other end of the container. Similarly, a fluid is mixed in a pressured state in the static type fluid mixer as well as since the contacting length of a gas and a liquid is long in the second aspect, the contacting efficiency of the liquid and the gas is extremely high. By circulating and supplying a fluid in the static type fluid mixer in each room, the contacting efficiency of a liquid and a gas can be further higher.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of a first embodiment of the present invention;
FIG. 2 is a perspective view of a 90° rotation type mixing element;
FIG. 3 is a perspective view of a 90° rotation type mixing element;
FIG. 4 is a side view of a static type fluid mixer utilizing the mixing element;
FIG. 5 is a schematic diagram of a second embodiment of the present invention;
FIG. 6 is a schematic diagram of a third embodiment of the present invention;
FIG. 7 is a schematic diagram of a fourth embodiment of the present invention;
FIG. 8 is a schematic diagram of a fifth embodiment of the present invention;
FIG. 9 is a schematic diagram of a sixth embodiment of the present invention; and
FIG. 10 is a schematic diagram of a seventh embodiment of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter embodiments of the present invention will be explained concretely with reference to the accompanied drawings. FIG. 1 is a schematic diagram of the first embodiment of the present invention. FIGS. 2 and 3 are perspective views of a mixing element. FIG. 4 is a side view of a static type fluid mixer utilizing the mixing element. As shown in FIG. 2 to 4 , each of mixing elements 1 and 8 of a static type fluid mixer 30 used in this embodiment comprises a cylindrical passage pipe 2 or 9 , and spiral blade bodies 3 , 4 or 10 , 11 arranged in the passage pipe 2 or 9 . The blade bodies 3 , 4 and 10 , 11 are tw'sted clockwise (right-handed rotation) or counterclockwise (left-handed rotation) by 90°, respectively so as to form fluid passages 5 , 6 or 12 , 13 . The blade bodies 3 , 4 , or 10 , 11 do not exist on the axis of the passage pipe 2 or 9 . Accordingly, an opening portion 7 or 14 is formed on the axis of the passage pipe 2 , 9 when viewed two-dimensionally. Therefore, the fluid passages 5 , 6 and the fluid passages 12 , 13 communicate with each other via the opening portions 7 and 14 through the entire length of the passage pipes 2 and 9 .
The static type fluid mixer 30 can be assembled by fitting the mixing elements 1 and 8 in a cylindrical casing 15 alternately so as to have the edges of the blade bodies 3 , 4 and 10 , 11 of the mixing elements 1 and 8 orthogonal to each other.
While two kinds of fluids FA, FB pass through the fluid passages of the static type fluid mixer 30 of the above-mentioned configuration, part of the fluid rotates spirally by 90° to be partially sheared at the opening portion, joins the fluid passed through the other fluid passage and is further divided, and rotates spirally by 90° to the other direction. While repeating the rotation, shearing, confluence, and division as mentioned above, the fluids can be mixed. In the static type fluid mixer 30 , 180° rotation type blade bodies can be used in place of the 90° rotation type blade bodies of this embodiment.
In this embodiment, the static type fluid mixer 30 of the above-mentioned configuration is placed vertically with respect to its longitudinal direction in a sealed processing container 31 , as shown in FIG. 1 . In this case, an introducing portion 31 a is provided at the upper part of the container 31 as a space for introducing a gas and a liquid, and a storage portion 31 b is provided at the lower part of the container 31 for storing a liquid.
A pipe 32 , connected with a liquid supply, is connected with the introducing portion 31 a at the upper part of the container 31 . A flow regulating valve 34 is provided in the pipe 32 . A pipe 33 connected with a gas supply is connected with the introducing portion 31 a . A flow regulating valve 35 is provided in the pipe 33 . A liquid and a gas is supplied into the container 31 with pressure from the liquid supply and the gas supply. A spray nozzle 37 is provided in the introducing portion 31 a at the upper part of the container 31 for jetting the liquid.
On the other hand, a pipe 36 is connected with the storage portion 31 b at the lower part of the container 31 for discharging the liquid stored at the lower part of the container to outside the container via the pipe 36 . The pipe 36 is connected with the spray nozzle 37 at the upper part of the container so that the liquid discharged from the bottom portion of the container is supplied to the spray nozzle 37 at the upper part of the container via the pipe 36 to be jetted toward the inside of the container 31 via the nozzle 37 . Accordingly, the liquid in the container 31 is returned into the container 31 via the pipe 36 to be circulated and supplied to the static type fluid mixer 30 in the container 31 . A pump 38 is provided in the pipe 36 , and furthermore, a flow regulating valve 39 is provided therein. A pipe 40 branches out from the pipe 36 at the upstream side with respect to the flow regulating valve 39 . A switching valve 41 is provided in the pipe 40 .
The operation of the gas-liquid processing apparatus of the above-mentioned configuration will be explained. With the valve 41 closed, and the valve 39 opened, the valves 34 and 35 are opened at a predetermined angle for supplying the liquid and the gas into the container 31 via the pipes 32 and 33 at a predetermined rate with pressure. Then the liquid and the gas are stirred and mixed in the static type fluid mixer 30 so that the gas is dissolved in the liquid to be aerated or reacted by sufficiently contacting the gas and the liquid.
The mixture fluid stored in the container 31 is supplied to the spray nozzle 37 at the upper part of the container 31 by the pump 38 to be jetted into the container 31 by the spray nozzle 37 . Then a liquid and a gas supplied from the pipes 32 and 33 , and the mixture fluid from the spray nozzle 37 are mixed while passing through the static type fluid mixer 30 . After applying pressure to the gas and the liquid in the container 31 until the pressure becomes higher than the atmospheric pressure, the valves 34 and 35 are closed to seal the mixture fluid of the liquid and the gas in the container 31 . The fluid passing through the static type fluid mixer 30 in the container 31 circulates in the static type fluid mixer 30 in a pressured state. Accordingly, the liquid and the gas sufficiently contacts so that the gas is dissolved in the liquid, aerated or reacted.
Afterwards, the fluid after the mixing and contacting processing is discharged from the container 31 via the pipe 40 by closing the valve 39 and opening the valve 41 .
FIG. 5 is a schematic diagram of the second embodiment of the present invention. In a container 42 , a plurality of the static type fluid mixers 30 are interlocked via cylindrical spacers 43 having the same diameter size as the casing of the static type fluid mixers. A gap is formed between the container and the static type fluid mixers 30 or the spacers 43 for the passage of a fluid. That is, the container 42 , the static type fluid mixers 30 and the spacers 43 have a double-pipe structure. The spacers 43 are provided with holes 43 a for the passage of a fluid so that a fluid can flow into the spacers 43 from the gap via the holes 43 a . A mixture fluid of the gas and the liquid is stored at the bottom portion of the container 42 and the mixture fluid is returned to the spray nozzle 37 at the upper part of the container via the pump 38 .
In the gas-liquid processing apparatus of the above-mentioned configuration, a liquid is jetted from the spray nozzle 37 into the uppermost static type fluid mixer 30 to be mixed with a gas introduced from the top of the container to the inside of the container, contacted, and processed. The mixture fluid is also mixed with a gas introduced via the holes 43 a at the spacers 43 to be introduced further into the lower static type fluid mixer 30 .
In this embodiment, the contacting processing of a gas and a liquid is conducted in a pressured state higher than the atmospheric pressure, and thus the contacting efficiency is extremely high. Further, since a fluid is circulated and supplied in the static type fluid mixers 30 , the dissolution of the gas or the reaction between the gas and the liquid can sufficiently proceed.
FIG. 6 is a schematic diagram of the third embodiment of the present invention. A liquid is supplied in a container 50 to be stored. The static type fluid mixer 30 is arranged with the lower half thereof soaked in the liquid in the container 50 with the fluid passing direction vertically. An introducing portion 51 is provided at the upper part of the static type fluid mixer 30 for introducing a gas into the static type fluid mixer 30 , and the spray nozzle 37 is provided in the introducing portion 51 for circulating and supplying a liquid. The outer periphery of the static type fluid mixer 50 of the portion soaked in the fluid in the container 50 is fitted with spiral blade bodies so that the static type fluid mixer 53 is formed by the spiral blade bodies.
In the gas-liquid processing apparatus of the above-mentioned configuration, a liquid is supplied in the container 50 , and the liquid is pumped up into the introducing portion 51 by the pump 38 . The liquid is jetted inside the introducing portion 51 via the spray nozzle 37 so as to be supplied with pressure into the static type fluid mixer 30 with the air supplied to the introducing portion 51 . The liquid and the gas are mixed while passing through the static type fluid mixer 30 downward. The mixture fluid enters the container 50 from the lower end of the static type fluid mixer 30 and is further mixed while passing through the static type fluid mixer 53 in the rising process. In this embodiment, since the pressure supplied to the introducing portion 51 of the gas is adjusted so that the surface of the fluid in the container 50 is always above the lower end of the static type fluid mixer 30 , the fluid is applied with a pressure higher than the atmospheric pressure at a position lower than the fluid surface in the static type fluid mixer 30 . Accordingly, a fluid is mixed with a high efficiency in this embodiment.
FIG. 7 is a schematic diagram of the fourth embodiment of the present invention. The static type fluid mixer 30 is arranged in a container 60 . An introducing portion 62 is provided at the upper part of the static type fluid mixer 30 . The lower part of the introducing portion 62 and the static type fluid mixer 30 are surrounded by a container 61 . The container 61 is arranged in the container 60 between the container 60 and the static type fluid mixer 30 . A plurality of the holes 63 are provided in the container 61 so that the fluid in the container 61 is discharged to the outside via the holes 63 . On the other hand, the supply pressure of a liquid and a gas is selected so that the fluid surface in the container 60 is always above the static type fluid mixer 30 .
In the gas-liquid processing apparatus of the above-mentioned configuration, a liquid and a fluid are always supplied to the introducing portion 62 , mixed by the static type fluid mixer 30 and discharged into the container 61 . Furthermore, the fluid is discharged into the container 60 via the holes 63 , and again supplied into the introducing portion 62 from the spray nozzle 37 at the upper part of the static type fluid mixer 30 by the pump 38 . Accordingly, the fluid is circulated and supplied to the static type fluid mixer 30 . Since the static type fluid mixer 30 is below the fluid surface in the containers 60 , 61 , the fluid in the static type fluid mixer 30 is applied with the pressure based on the hydrostatic pressure difference. Therefore, the gas and liquid contacting efficiency of a fluid is high.
FIG. 8 is a schematic diagram of the fifth embodiment of the present invention. The fifth embodiment is another embodiment of the second embodiment, wherein a plurality of the gas-liquid processing apparatus of the first embodiment shown in FIG. 1 are provided parallel. Three static type fluid mixers 30 are arranged in the sealed container 70 with the lower part inserted. A fluid is stored in the container 70 . Partitioning members 71 comprising partition plates, standing from the bottom plate of the container to a position lower than the fluid surface, and partition plates, hanged from the upper plate of the container to deeper than the fluid surface, are arranged between the static type fluid mixers. An introducing portion 31 of a gas is provided at the upper part of the static type fluid mixer 30 . A pipe 32 for introducing a liquid into the introducing portion 31 and a pipe 33 for introducing a gas into the introducing portion 31 from the outside are connected with the introducing portion 31 of the static type fluid mixer 30 arranged at one end of the container 70 . A discharging pipe 73 for discharging a gas and a discharging pipe 74 for discharging a liquid are connected to the room having the static type fluid mixer 30 on the opposite end of the container 70 . In each room partitioned by the partitioning members 71 , a fluid is supplied to the spray nozzle 37 provided at the introducing portion 31 of the static type fluid mixer 30 of each room by the pump 38 via the pipe 36 so as to be circulated and supplied into the static type fluid mixer 30 .
In the gas-liquid processing apparatus of the above-mentioned configuration, a liquid and a gas are introduced into the introducing portion 31 of the static type fluid mixer 30 on the left end of the drawing via the pipes 32 , 33 . The liquid and the gas are mixed in the static type fluid mixer 30 and discharged into the container 70 . The pressure of the liquid and the gas introduced via the pipes 32 , 33 is determined so that the fluid surface in the container 70 is positioned at a comparatively high position in the container 70 as shown in the drawing for the static type fluid mixer 30 sufficiently soaked in the fluid.
In the container 70 , a liquid flows to an adjacent room beyond the partitioning members 71 , but a gas in each room is supplied from the upper space of the room to the introducing portion 31 of the static type fluid mixer 30 of an adjacent room via a pipe 75 . In each room, a liquid is circulated and supplied to the static type fluid mixer 30 via a pipe 36 by the pump 38 .
The gas processed by sufficiently contacting with the liquid is discharged from the container 70 via the pipe 73 , and the liquid is discharged via the pipe 74 . In this embodiment, a liquid and a gas are mixed and contacted in each static type fluid mixer, and a gas moves into the static type fluid mixers successively so as to contact with the liquid in each room. Accordingly, the contacting efficiency between a gas and a liquid is extremely high.
FIG. 9 is a schematic diagram of the sixth embodiment of the present invention. In the sixth embodiment, a pipe 81 for discharging the gas in the storage portion 31 b is connected to the storage portion 31 b of the container 31 . Also, a valve 82 is provided in the pipe 81 . The remaining construction is same as the first embodiment shown in FIG. 1 .
The operation of the gas-liquid processing apparatus of the above-mentioned configuration will be explained. With the valve 41 closed, and the valves 39 and 82 opened, the valves 34 and 35 are opened at a predetermined angle for supplying the liquid and the gas into the container 31 via the pipes 32 and 33 at a predetermined rate with pressure. Then the liquid and the gas are stirred and mixed in the static type fluid mixer 30 so that the gas is dissolved in the liquid to be aerated or reacted by sufficiently contacting the gas and the liquid.
The mixture fluid stored in the lower part of the container 31 is supplied to the spray nozzle 37 at the upper part of the container 31 by the pump 38 to be jetted into the container 31 by the spray nozzle 37 . Then a liquid and a gas supplied from the pipes 32 and 33 , and the mixture fluid from the spray nozzle 37 are mixed while passing through the static type fluid mixer 30 . After closing the valves 34 and 82 and applying pressure to the gas and the liquid in the container 31 until the pressure becomes higher than the atmospheric pressure thorough the pipe 33 , the valve 35 is closed to seal the mixture fluid of the liquid and the gas in the container 31 . The fluid passing through the static type fluid mixer 30 in the container 31 circulates in the static type fluid mixer 30 in a pressurized state. Accordingly, the liquid and the gas sufficiently contacts so that the gas is dissolved in the liquid, aerated or reacted.
Afterwards, the fluid after the mixing and contacting processing is discharged from the container 31 via the pipe 40 by closing the valve 39 and opening the valve 41 .
FIG. 10 is a schematic diagram of the seventh embodiment of the present invention. In the seventh embodiment, a mixture fluid of the gas and the liquid is extracted from the container 42 at the bottom 42 a thereof. Other construction of the seventh embodiment is same as that of the second embodiment shown in FIG. 5 .
The first embodiment shown in FIGS. 1 to 4 , the second embodiment shown in FIG. 5, the sixth embodiment shown in FIG. 9 and the seventh embodiment shown in FIG. 10 are effective as an apparatus for eliminating a nitrogen compound in an aqueous solution. They are effective also as an apparatus for eliminating an ammonium type nitrogen in the water supply or sewage, and an apparatus for diffusing a volatile matter in waste water. Furthermore, they are advantageous also as an apparatus for injecting and adding a chlorine gas or an apparatus for dissolving an oxygen gas.
The third embodiment shown in FIG. 6 and the fourth embodiment shown in FIG. 7 can be applied as an apparatus for processing water supply, sewage, or industrial waste water with ozone. Furthermore, the fifth embodiment shown in FIG. 8 is advantageous as a continuous type aeration apparatus or a waste water processing apparatus by the activated sludge method. Furthermore, it is advantageous also as a water processing apparatus with an ozone gas or a waste water processing apparatus by the activated sludge method.
As heretofore mentioned, according to the present invention, since a liquid and a gas are mixed in a pressured state in a static type fluid mixer, it is advantageous in that the contacting efficiency is high as well as the production cost is low.
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The present invention is to provide a gas-liquid processing apparatus having a high contact efficiency of a gas and a liquid and a high reaction efficiency at a low production cost. A static type fluid mixer includes a passage pipe for the passage of a fluid and a spiral blade body arranged in the passage pipe with the longitudinal direction substantially perpendicularly but being absent in the center portion of the passage pipe. A liquid and a gas are supplied into the static type fluid mixer and a fluid is returned from the bottom portion of the static type fluid mixer to the upper portion via the pipe for the fluid circulation. The fluid is maintained in the static type fluid mixer at a pressured state higher than the atmospheric pressure.
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BACKGROUND OF THE INVENTION
1. Field of Invention
This invention pertains to a burial casket having a capsule for identifying the remains contained therein, which capsule is accessible from the outside.
2. Description of the Prior Art
It is frequently desirable to provide burial caskets with some form of identifying the remains contained therein either just prior to burial, or in some cases relatively long periods of time thereafter. Heretofore, such identification could only be provided either on the outside or the inside of the casket. If the identification was on the outside, it had to be firmly secured to the casket so that it would not be separated. Because of various psychological, emotional and religious factors, the external identification had to be aesthetically pleasing, solemn, and discrete and at the same time must be in a form that could withstand rather extreme physical conditions for a long period of time. Obviously, external identification means are usually expensive and require a long time to make.
Internal identification need not be so rugged as the external one, however it still must be able to withstand strong chemical action due to the decaying body, as well as normal oxidation for a long period of time. Furthermore, internal identification is sometimes objectionable on an emotional and psychological basis because it can be checked only by opening the casket.
OBJECTIVES AND SUMMARY OF THE INVENTION
It is a principal objective of the present invention to provide a casket with an identification member which is externally accessible yet is easy and inexpensive to make.
A further objective is to provide a casket with an identification means which is easy to inspect and yet normally unobtrusive.
Another objective is to provide a casket with an identification means which is unaffected by normal chemical action, over a long period of time.
Other objectives and advantages of the invention shall become apparent from the following description. In accordance with this invention, a casket is provided with a a plurality of walls defining an enclosure and a capsule for holding information, substantially disposed in said enclosure, said capsule being attached attached to one of said walls and being removable without opening said enclosure.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a casket with an identification capsule constructed in accordance with this invention;
FIG. 2 shows an exploded view of the capsule incorporated in the casket of FIG. 1;
FIG. 3 shows the manner in which the capsule is affixed to the casket;
FIG. 4 shows the capsule inner tube being inserted into the casket; and
FIG. 5 shows the inner tube of FIG. 4 in its final position.
DETAILED DESCRIPTION OF THE INVENTION
Referring now to the Figures, and more particularly to FIG. 1, a casket 10 includes a bottom wall 12, side walls 14 and hinged top walls 16,18. One of these walls, such as for example one of the side walls 14 is provided with a capsule 20. As shown in FIG. 1, it is preferable to place the capsule at or toward the foot of the casket adjacent to an upper casket corner 22 so that it is inconspicuous but easily accessible. Of course, this location should be standardized for all caskets.
The individual elements of the capsule 20 are shown in FIG. 2. The capsule includes a sleeve 24, an outer tube 26, an inner tube 28 and a cap 30. Sleeve 24 is preferably made of galvanized metal or other relatively strong material which withstands corrosion. The sleeve has an axial length which conforms substantially to the thickness of side wall 14 and at one axial end, has an enlarged radial rim 32. As best shown in FIG. 3, the sleeve 24, has a first inner wall portion 34, adjacent rim 32, which is relatively smooth. Axially spaced from inner wall 34 there is a threaded portion 36, and a second inner wall portion 38 which is also relatively smooth but has a diameter which is larger than the diameter of first inner wall portion 34. A shoulder 40 is formed between inner wall portion 38 and threaded portion 36 as shown. As shown in FIG. 3, sleeve 24 passes through and is secured to casket side wall 14 to resist rotation, with rim 32 disposed inside the casket. Outer tube 26 has a cylindrical outside surface 42 which terminates at one end by an open threaded portion 44 which is adapted to engage the threads of sleeve portion 36. The tube 26 also has a substantially cylindrical inner surface 46 which terminates at a closed end 47 opposite threaded portion 44 with several reinforcing members 48 and open end 49.
Inner tube 28 included a cylindrical outer surface 50, and a rim 52 having two parallel, axially spaced shoulders 54 and 56. A threaded portion 58 is disposed axially adjacent to rim 52. Tube 26 is closed at end 60 and open at end 62. Inner tube 26 is adapted to hold a scroll or parchment 64 containing identification information. Preferably, one or both tubes 26, 28 are made of a transparent or translucent material such as a biologically inert plastic, i.e. a material which is not chemically affected by biological decomposition.
Finally, cap 30 comprises two axially spaced portions 66, 68. Preferably, cap 30, like sleeve 24, is made of a anti-corrosive material. Portion 66 has a threaded outer surface 70 which may be screwed into threaded sleeve portion 36, and a threaded inner surface 72 which accepts inner tube threaded portion 58. Cap portion 66 is preferably slightly longer than corresponding inner tube threaded portion 58 and ends in a rim 75. On outer surface 70 there is a provided a sealing O-ring 74 made of a resilient material. Cap portion 68 is provided with a knurled outer surface 76 so that cap 30 can be easily screwed or unscrewed.
The casket is assembled as follows. Sleeve 24 is first installed into side wall 14 so that it is securely attached thereto. Alternatively, a threaded hole corresponding to the inner wall portions 34, 36 and 38 shown in FIG. 3 is made in the side wall. Next, outer tube is screwed into the sleeve with the tube disposed substantially within the casket as shown in FIG. 4. The threaded portion 44 is advanced within the sleeve until it abuts inner wall portion 34. Scroll 64 is placed in inner tube 28, and cap 30 is then secured to the tube by screwing cap portion 72 into tube portion 58. Cap 30 is advanced until cap rim 75 contacts shoulder 56 on rib 52 thereby sealing tube 28. The inner tube 28 is then inserted into outer tube 26 (FIG. 4) and outer cap portion 70 is screwed into sleeve portion 36 until shoulder 54 contacts the center tube end 49 thereby sealing outer tube 26. Preferably, cap 30 is dimensioned so that just before shoulder 54 engages outer tube end 49, the resilient ring 74 disposed with annular space between the cap and sleeve wall portion 40 contacts sleeve shoulder 40. As the cap is tightened further the ring 74 further resiliently seals the capsule. Thus, in the final configuration of FIG. 5, the capsule is doubly sealed within the casket.
Scroll 64 may contain, biographical, medical or other type of information. Obviously, numerous improvements may be made to this invention without departing from its scope as defined in the appended claims.
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A burial casket is provided with a capsule which extends from a casket side wall inwardly and contains information regarding the remains contained in the casket. The capsule is removable from the casket to give access to the information without opening the casket.
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/EP2012/054507 filed Mar. 15, 2012, claiming priority based on European Patent Application No. 11 158 692.1 filed Mar. 17, 2011, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to methods for identifying and purifying multi-specific polypeptides, in particular bispecific antibodies.
PRIOR ART
Antibodies are widely used for therapeutic purposes such as treatment of cancer and rheumatoid arthritis. Typically the antibody molecules comprise two sites for antigen recognition, both recognizing the same antigen.
Bispecific antibodies have been developed for therapeutic purposes that are capable of simultaneously recognizing two different antigens. The first bispecific antibody approved by regulatory authorities was Catumaxomab, a rat-mouse hybrid IgG. Its antigen recognition sites target a molecule on tumor cells (EpCAM) and a killer T cell, respectively, leading to effective tumor destruction. Due to their additional Fc receptor function, which is common to IgG antibodies, the bispecific antibody is considered to be tri-functional or tri-specific.
The chromatographic purification of such multispecific antibodies remains a major challenge since the different antibody types typically exhibit very similar adsorptive properties during chromatography. Furthermore, the complexity of antibody isoform combinations that may arise after expression of the different antibody chains makes purification of a single isoform species amongst other similar isoforms challenging.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide an improved purification method in particular for the purification of multifunctional multimeric antibody structures. It is a further object of the present invention to provide a method to identify and isolate particularly suitable multifunctional multimeric antibody structures. The term “antibody” includes in general any polypeptide allowing multispecificity binding functions and in particular fully human monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, single domain antibodies, minibodies and multispecific antibodies.
The present invention achieves this object by proposing a method of purification as claimed in claim 1 as well as by providing a method for the identification according to claim 7 , i.e. by creating sufficiently diverse pools of heteromeric multi-specific antibodies or antibody-like scaffolds and then selecting, isolating and producing heteromultimeric polypetide species for pharmaceutical and therapeutical purposes.
Particularly, the present invention relates to a method for the purification of a ternary mixture of multimeric antibodies of the type AA, AB, BB (for the case of dimers), characterised in that for the separation of the three (or more) components and in particular for the isolation of the multi-specific fraction AB multicolumn counter current solvent gradient purification chromatography preferably with a stationary phase load of more than 1 mg antibody mixture per milliliter packed bed volume (stationary phase, firmly packed and including liquid) is used.
According to a first preferred embodiment, the antibodies are dimers formed from polypeptides A and B, the heterodimer being a bispecific antibody, each polypeptide A and B comprising a heavy polypeptide chain with at least one heavy chain variable region.
According to a further preferred embodiment, the antibody dimers may comprise a heavy polypeptide chain with at least one heavy chain variable region; and in addition a light polypeptide chain with a light chain variable region or a corresponding scaffold peptide.
According to a further preferred embodiment, the heavy polypeptide chain may also comprise at least one heavy chain constant region or a corresponding scaffold peptide. Such heavy chain constant regions can preferably be of the IgG, IgA, IgM, IgD or IgE class, wherein preferably the heavy chain constant regions are selected from the group of IgG1 and IgG4, or IgG1 and IgG2 subclasses.
According to another preferred embodiment of the method, a chromatographic stationary phase with a mean particle diameter of less than 70 micrometers is used, preferably of less than 50 micrometers, most preferably of in the range of 2-35 micrometers, wherein the width of the distribution (2σ) is preferably in the range of +/−50% of the mean value.
In single column chromatography, the process performance of the isolation of bispecific antibodies is often limited with respect to yield and productivity under a given purity constraint due to mass transfer and isotherm effects. In a chromatogram, this translates to peak overlap of the monoclonal and the bispecific antibody species which increases with increasing load. In order to improve yield and productivity and to better exploit the capacity of the stationary phase, a continuous process providing internal recycling of the overlapping fractions, such as the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process, can be used beneficially. As mentioned above, the present invention also pertains to an identification or selection method for finding particularly suitable systems, for example to be purified in a process as outlined above. FIG. 3 in a schematic representation shows is the essential elements of the invented selection method, wherein two libraries A and B of monoclonal antibodies recognizing two different antigens are produced (top left boxes). For the specific situation of a ternary mixture as illustrated in FIG. 3 it is normally important that the monoclonal antibodies A and B have the same light chain and only distinguish in the heavy chain. If the antibodies comprise different light chains, the corresponding recombinations can e.g. yield 10 antibody types. The antibodies from each library are analyzed separately using a suitable method such as cation exchange chromatography (bottom left boxes). One of the ideas is that the antibodies from each library are essentially equal (pareto-optimal) with respect to the criteria affinity, specificity and stability, so that one is sure that those which are selected by analytical chromatography are also optimal. By comparing characteristic data obtained from the chromatograms, one monoclonal antibody candidate is chosen from each library and the DNA encoding for its heavy chains is transfected into host cells by means of a suitable vector together with the DNA encoding for the common light chain, and the host cells express the DNA and produce heavy and light chain polypeptides that associate randomly and form the heterodimeric desired bispecific antibody (A-B) and two homodimeric monoclonal antibody species (A-A, B-B), wherein the bispecific antibody may be isolated by single column or multicolumn continuous countercurrent chromatography, the latter being beneficial if the peaks of the homodimers and the heterodimer overlap.
The method described by the present invention enables the selection and preparative production of bispecific antibodies by preparative chromatography without requiring targeted mutagenesis to modify the amino acid sequence of the antibodies, allowing to preserve the natural sequence and reducing potential antigenicity of mutated sequences.
According to this element of the present invention, for the identification of a purifyable multi-specific polypeptide species (AiBj) which is a multimer consisting of at least two different specificity polypeptide chains (Ai, Bj), in particular a heterodimeric bispecific antibody, a method is used comprising the following steps:
(A) generation of a library of expression systems for each of the different specificity polypeptide chains (A, B) forming the multimer, preferably using array technologies, phage display, yeast display, ribosome display and variations thereof, and narrowing the library size through antigen binding and/or activity assays; (B) individually expressing a sufficient amount of polypeptide chains (A1, A2, A3, . . . An; B1, B2, B3, . . . Bm) with each individual library member, and forming homomultimers therefrom, preferably homodimers (A1A1, A2A2, A3A3, . . . AnAn; B1B1, B2B2, B3B3, . . . BmBm) therefrom; (C) using analytical methods, preferably chromatography, particularly preferably chromatography in single-column mode or isoelectric focusing (inclusive of HPLC and/or gradient methods), for the determination of an analytically discriminative parameter, preferably the chromatographic retention time, for each of the homomultimers; (D) selecting at least one pair (AiAi, BjBj) of homomultimers of different specificity polypeptide chains which are analytically sufficiently discriminated; (E) expressing the nuclear acids corresponding to the homomultimers identified in step (D) in host cells such that a mixture of homomultimers (AiAi; BjBj) and heteromultimers (AiBj) is produced by culture of the host cells; (F) purifying the resulting ternary mixture by using chromatography, preferably using a chromatographic purification method as outlined above.
One of the important and preferred aspects here is that the antibodies from each library are essentially equal (pareto-optimal) with respect to selection criteria such as affinity, specificity, expressibility, avidity and stability (including aggregation properties, process stability, antibody stability etc.) and/or other criteria depending on the requirements of the specific case, so that one is sure that those which are selected by analytical chromatography are also optimal.
According to a first preferred embodiment of this method, the steps (A)-(D) are repeated until a pair can be identified, which is analytically sufficiently discriminated. For the discrimination in step (D) preferably a relative retention time in a chromatographic separation of smaller than 0.9 and larger than 1.1 is used. As a criterion for the selection in step (D) at least one of the following relative parameters can be used: resolution (Rs), relative retention time (RRT), retention time, retention time difference, retention volume purity.
According to yet another preferred embodiment, steps (A)-(F) are carried through repeatedly for the generation of a library of purifyable multi-specific polypeptide species, and out of these one or several are selected which show the best activity. Indeed sometimes it is not possible to discriminate between finally particularly active systems and less active systems using the discrimination in step (D). In this case the above mentioned steps are used for the generation of a library of systems and then these are scanned for active pairs which at the same time active and purifyable.
According to yet another preferred embodiment, the purifyable multi-specific polypeptide species (AiBj) is a bispecific antibody formed from polypeptides A and B, the heterodimer being a bispecific antibody, each polypeptide A and B comprising a heavy polypeptide chain with at least one heavy chain variable region.
According to yet another preferred embodiment, the antibodies are dimers formed from polypeptides A and B, the heterodimer being a bispecific antibody, each polypeptide A and B comprising: a heavy polypeptide chain with at least one heavy chain variable region; and a light polypeptide chain with a light chain variable region or a corresponding scaffold peptide.
Generally, the term “scaffold peptide” refers to any polymer of amino acids that exhibits properties desired to support the function of the antibody. This includes the addition of specificity, the enhancement of antibody function or the support of antibody structure and stability.
According to a further preferred embodiment, the heavy polypeptide chain comprises at least one heavy chain constant region or a corresponding scaffold peptide.
The heavy chain constant regions can be of the IgG, IgA, IgM, IgD or IgE class, wherein preferably the heavy chain constant regions are selected from the group of IgG1 and IgG4, or IgG1 and IgG2 subclasses. Each of the polypeptides (A,B) may comprise a heavy chain variable region and/or a corresponding scaffold peptide.
The multispecific antibody or scaffold may comprise a third polypeptide (C) comprising a light chain variable region and/or a scaffold, wherein a first polypeptide (A) and a second polypeptide (B) each form a multimer with said third polypeptide (C).
The first polypeptide (A) and the second polypeptide (B) may comprise a heavy chain constant region.
As mentioned above, the heavy chain constant regions of the polypeptides can be of different subclasses such as IgG1, IgG2 and IgG4.
Furthermore the present invention relates to a heterodimeric bispecific antibody (AiBj) determined using an identification method as outlined above and/or using a purification method as outlined above.
A preferred embodiment of the invention is the production of heterodimeric bispecific antibodies from two libraries of homodimeric monoclonal antibodies. The antibodies of each library are directed against different antigens as to allow for production of bispecific antibodies using the DNA of one antibody of each library. Each library should preferably contain more than 10 antibodies, preferably more than 30 antibodies and even more preferably more than 50 antibodies. All antibodies preferably contain the same L chain polypeptide and differ among each other in their H chains only.
All antibodies of each library are analyzed, preferably individually, with a suitable technique in order to obtain information allowing for a ranking of the antibodies for the production of bispecific antibodies. Preferably, the analysis of the monoclonal antibodies from each library is carried out by HPLC using a single column and linear gradient elution conditions. Preferably, in view of prospective semi-preparative or preparative scale manufacturing, the analysis is carried out using semi-preparative or preparative stationary phases. The mobile phase conditions for the analysis are chosen in such manner that the analysis for each of the selected antibodies is possible using the same mobile phase conditions. For this purpose the use of linear gradients from adsorbing to non-adsorbing conditions is preferred.
The detection of the eluting antibodies is done by spectroscopic methods including but not limited to UV absorption. The amount of antibody injected has to be above the detection limit of the instrument, preferably above 1 μg, more preferably above 10 μg.
Since the sequence and the structure of the antibodies are different, they exhibit different adsorptive properties with respect to the stationary phase and, consequently, different retention times. For each antibody the chromatogram is recorded and the retention time of the main peak maximum tRP, the retention time of the peak front start tRF, the retention time of the peak tail end tRT (for the definition of these parameters reference is made to the explanations given below, in particular to example 4 detailed below), the area of the main peak and the total area is recorded.
A preferred embodiment of the invented method includes a calculation of the purity of the antibody by dividing the area of the main peak which is given by the area in between the valleys between the main peak and the early and the late eluting neighboring peaks by the total area of all peaks together, as also indicated in FIG. 5 . Preferably the purity is determined using an analytical stationary phase.
Subsequently, the retention time data of pairs of antibodies with one antibody from a library of antibodies against a first antigen (A-A) and the second antibody from a library of antibodies against a second antigen (B-B) is compared. Thereby each antibody of the first library is compared with each antibody of the second library, as to evaluate each possible combination. For the sake of clarity, in the following the antibodies of the first library are designated A1-A1, A2-A2, A3-A3, etc, while the antibodies of the second library are designated B1-B1, B2-B2, B3-B3, etc, according to their order in the analytics, or in general Ai-Ai and Bj-Bj, where i and j are integer numbers running from 1 to the number of antibodies in the respective library.
In the following, with regard to each pair of antibodies, the antibody with the smaller value of tRT is referred to as the “first” antibody while the antibody with the larger value of tRT is referred to as the “second” antibody. For the methods described in the following it is irrelevant which one of the antibodies Ai-Ai and Bj-Bj is eluting before the other. The retention times of the first antibody shall be referred to as tRF,1; tRP,1 and tRT,1 in the following, the values of the second antibody shall be referred to as tRF,2; tRP,2 and tRT,2.
In the following, it is shown how pairs of antibodies (Ai-Ai, Bj-Bj) are evaluated according to a resolution criterion.
“Resolution” is defined by the following formula for symmetrical, Gaussian peak shapes: Rs=2*(tRP,2−tRP,1)/(W1+W2), where W1 and W2 are the widths of the first and the second peak, respectively. For asymmetric peaks, this formula is no longer valid, and the retention times of the second and the first main peak have to be replaced by the mid-point of the baseline. Thus tRP,2 has to be replaced by (tRF2+tRT2)/2 and tRP,1 has to be replaced by (tRF1+tRT1)/2. Furthermore, the widths are given by W1=tRT1−tRF1 and W2=tRT2−tRF2.
With this, the resolution equation can be rewritten as Rs=(tRF2+tRT2−tRF1−tRT1)/(tRT1+tRT2−tRF1−tRF2). The resolution can now be now calculated for each pair (Ai-Ai, Bj-Bj) and sorted in descending order. A value of Rs>1 indicates that the peaks are baseline-separated, a value of Rs=1 indicates a touching of the peaks at their baselines and a value of Rs<1 indicates a peak overlap.
The purity of each of the two antibodies of each pair, determined according to the method shown in Example 2 should be >70%, more preferably >80% and even more preferably >90%.
A simplified criterion, termed Δ(i,j) criterion, which is based on a single retention time difference can be used when all peaks Ai-Ai and Bj-Bj have similar widths, preferably differing by 25% at the most.
The calculations are carried out as follows: For each pair (Ai-Ai, Bj-Bj), the value of tRT,1 of the first antibody is subtracted from the value tRF,2 of the second antibody, the corresponding difference is called Δ(i,j). A value Δ(i,j)>0 indicates that the peaks are separated at the baseline. A value Δ(i,j)<0 indicates that the peaks of the two antibodies overlap. Subsequently the antibody pairs are ranked by their Δ(i,j) values in descending order starting with the largest positive values.
The purity of each of the two antibodies of each pair, determined according to the method shown in Example 2 should be >70%, more preferably >80% and even more preferably >90%. Antibodies from the pairs with the highest Rs or Δ(i,j) values, respectively, are preferred. In a preferred embodiment of the method, the purity criterion shall be prevailing over the Rs or Δ(i,j) criterion, respectively.
The difference between the Δ(i,j) and Rs criteria is that the Rs criterion directly takes into account the peak widths, that may strongly vary from antibody to antibody due to the presence of isoforms. Since, in most cases, the antibody purity and the peak width correlate, the Rs criterion is more powerful than the Δ(i,j) criterion and preferred since it contains information on the purity. Nevertheless, it is not recommended to abandon the independent evaluation of the purity and to apply the Rs criterion without the purity criterion.
The genes encoding for the H chains of the selected candidates are now transfected together with the genes encoding for the common L chain into host cells using a suitable vector. The cells are grown using suitable media and culture conditions. The host cells produce and secrete into the surrounding media a mixture of antibodies containing the original monoclonal antibodies from each library (A-A, B-B) and the bispecific antibodies combining the antigen specificities of both libraries (A-B). When the antibody concentration has reached the desired level the cell culture supernatant is harvested and clarified. Subsequently the bispecific antibody is purified by chromatography.
The bispecific antibody is purified from the mixture of the monoclonal antibodies and from other typical impurities such as host cell proteins, host cell DNA and media components by means of single column chromatography or continuous countercurrent chromatography, after a suitable pre-treatment or without such treatment.
In a preferred embodiment, the purification is carried out using the MCSGP process with cation-exchange stationary phases, preferably with the stationary phase that was used also in the cloning candidate selection process of the antibodies and using one or more additional chromatographic steps for polishing.
In another preferred embodiment, the antibody is purified by protein A affinity chromatography which removes a large part of the typical impurities. However, due to its specific mode of action of binding only certain IgG subclasses, in most cases the protein A step does not significantly contribute to the removal of the homodimeric monoclonal antibodies (A-A, B-B). After purification by protein A chromatography, the bispecific antibody A-B is isolated in a second chromatographic purification step, preferably using the stationary phase applied in the cloning candidate selection process. The second chromatographic step may be carried out either in single column batch mode or in continuous counter-current mode which is beneficial if an overlap between the antibodies A-A, A-B and B-B is observed in single column mode. The second chromatography step may be followed by a third purification step to produce bispecific antibody with the desired purity.
The purity of a bispecific antibody is defined as the mass percentage of the heteromeric antibody with respect to all antibodies of the mixture, heteromeric and homomeric.
Once the bispecific antibody has been purified it may be transferred into a suitable formulation for application as a pharmaceutical.
The term “stationary phase” refers to functionalized or non-functionalized particles of polymeric or inorganic composition, e.g. silica, that are typically sphere-shaped in the case of polymeric particles and amorphous in the case of inorganic particles and represent the backbone of the chromatographic material. In the case of functionalized particles, the adsorption of the product to be purified takes place through interaction with a ligand that is typically connected to the stationary phase backbone by linkers. The modes of interaction of the ligands include but are not limited to cation-exchange, anion-exchange, hydrophobic interaction, reversed phase, normal phase, multi-modality, affinity, hydrophobic charge interaction chromatography, and chromatofocusing.
These stationary phases are characterized by their average particle size and their relatively broad particle size distribution
The term “analytical stationary phase” refers to stationary phases with average particle sizes below 10 μm. The term “semi-preparative stationary phase” refers to stationary phases with average particle diameters in the range of 10 μm to 30 μm, while the term “preparative stationary phase” refers to stationary phases with particle diameters of 30 μm or larger.
Typically analytical stationary phases are commercially available only in the form of pre-packed columns. Suitable analytical pre-packed columns include but are not limited to Propac wCX-10, 4×250 mm, Dionex, Sunnyvale, Calif., USA; Tosoh SP Stat, 5×100 mm, Tosoh, Tokyo, Japan; YMC BioPro SP-10, Kyoto, Japan; Mono S 5/50 GL, 5×50 mm, GE Healthcare, Uppsala, Sweden.
Semi-preparative and preparative stationary phases are also available as bulk materials. Suitable stationary phases include but are not limited to materials of the Fractogel series (Merck, Darmstadt, Germany) and the Source series (GE Healthcare, Uppsala, Sweden).
The bulk materials can be packed in standard columns according to the manufacturer's instructions. The standard columns include but are not limited to columns of the Tricorn series (GE Healthcare, Uppsala, Sweden).
The term “load” refers to the mass of polypeptide that is injected onto the stationary phase for the purpose of purification. Typically, the load is refers to the column volume packed with stationary phase which is also referred to as packed bed volume and is given in mg polypeptide per mL of packed bed volume. Generally, the loading phase is followed by a washing step and before the purified product is recovered in a subsequent elution step and the stationary phase is cleaned and re-equilibrated for the next loading phase.
The analytical methods for ranking the antibodies of each library include HPLC methods and the evaluation such as described above but also isoelectric focusing (IEF) methods. HPLC may be carried out using a suitable instrument, preferably with optimized dead volumes, such as the Agilent 1100 or 1200 series (Agilent, Santa Clara, Calif., USA).
In the case of gel IEF, the method may be carried out using a Phast system (GE Healthcare, Uppsala, Sweden) or a comparable device using the operating parameters and conditions recommended by the manufacturer. The method may also be carried out using a capillary IEF (cIEF) device such as the iCE280 device (Convergent Bioscience, Toronto, Canada). Following the isoelectric focusing, a suitable method for detecting the antibody is carried such as UV absorption measurement, Coomassie or silver staining according to standard protocols. In IEF, each monoclonal antibody appears as series of bands as it comprises multiple charged isoforms. Nevertheless, the Δ(i,j) and the Rs ranking method as reported above can be carried out in a similar fashion:
Instead of the retention times tR, the distance of the bands on the IEF gel with respect to the edges of the working section can be used. The method is independent of the orientation of the working section.
For each antibody the distance of the band of the first isoform LFB and the distance of the band of the last isoform LLB are recorded.
In the following, with regard to each pair of antibodies, the antibody with the smaller value of LLB is referred to as the “first” antibody while the antibody with the larger value of LLB is referred to as the “second” antibody. In analogy to the calculation of Rs and Δ(i,j) based on the retention time values, the corresponding values RsIEF or ΔIEF(i,j) can be calculated by replacing tRF,1 by LFB,1; tRT,1 by LLB,1; tRF,2 by LFB,2 and tRT,2 by LLB,2.
Subsequently the antibody pairs are ranked in descending order by their RsIEF or ΔIEF values, respectively, starting with the largest positive values.
In IEF, the purity of the antibody is measured by comparing the intensity of the band of the main isoform with the band intensity of the remaining isoforms.
The advantage of isoelectric focusing over HPLC methods is that multiple samples can be analyzed in parallel.
The disadvantage of gel IEF is that predictions of the chromatographic behavior during the final purification are possible only to a limited extent and in the case of gel IEF that the estimation of the peak width and the purity is less accurate.
The term “mobile phase” refers to suitable running buffers for the operation of the chromatographic process, taking into account the functionality of the stationary phases. The mobile phases include but are not limited to phosphate buffers, acetate buffers and TRIS buffers. The buffer initiating the product elution from the column preferably contains a low modifier concentration while the buffer at the end of the product elution preferably contains a high modifier concentration, thus representing a change in modifier concentration over time, also termed “modifier gradient”. Typically the modifier gradient is generated by mixing a first buffer with low modifier concentration with a percentage of a second buffer with high modifier concentration that increases over time. The respective modifier includes but is not limited to inorganic salts such as sodium chloride in the case of ion exchange chromatography; acetonitrile, ethanol, iso-propanol in the case of reversed phase chromatography; water in the case of hydrophobic interaction chromatography; sodium citrate, sodium acetate and glycin in the case of affinity chromatography; sodium phosphate, potassium phosphate and sodium chloride in the case of multimodal chromatography.
The term “Multicolumn Countercurrent Solvent Gradient Purification”, abbreviated “MCSGP” refers to the class of chromatographic purification processes described in WO/2006/116886 as well as in WO 2010/079060, the disclosure of which is expressly included into this specification as concerns these purification processes using, chromatographic columns.
Generally, the term “polypeptides” refers to peptides and proteins or fragments thereof with more than ten amino acids. The polypeptides may be obtained through expression by genetically modified or unmodified living cells or by chemical synthesis. Moreover, the polypeptides may be produced by subjecting the expressed or synthesized amino acid chains to further processing such as enzymatic treatment or chemical modification.
The genes encoding for the H chain or L chain of antibodies can be obtained from known sequences or from antibody libraries. Many antibody libraries with antibodies directed against a single antigen are either known or can be generated by those skilled in the art using selection/amplification techniques such as array technologies, phage display, ribosome display, or lymphocyte immunization. Methods to produce antibody libraries by phage display are described in Clackson et al., Nature 1991, 352: 624-628; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Griffiths et al., EMBO J. 1994, 13: 3245-3260; Vaughan et al., Nature Biotechnology 1996, 14: 309-314. These methods include the use of single chain antibodies obtained from human antibody libraries. The libraries preferably contain antibodies comprising heavy chains with difference isoelectric points. Further difference in isoelectric points may be introduced by the use of different wild-type antibody subclasses for each library (e.g. IgG1, IgG2, IgG3, and IgG4 in the case of IgG) that naturally have different pIs.
In addition, known methods, such as methods that use eukaryotic cells as libraries (WO95/15393) can be used.
Methods based on lymphocyte immunization rely on the use of the antigen for performing the immunization of B-cells. The immunization may be carried out in vitro using human B-lymphocytes or in vivo using mammals by suitable methods that comprise injection of the antigen. The immunized monoclonal antibody-producing B-cells are isolated and fused to myeloma cells to obtain hybridoma cells (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46). DNA encoding for antibody variable regions can be then obtained from the mRNA of the monoclonal antibody expressing hybridoma cells using reverse transcriptase and PCR techniques. Genes encoding for complete antibody chains can be obtained by linking the DNA encoding for the variable regions with DNA encoding for antibody constant regions.
In addition, human antibodies with the desired specificity can be obtained by immunizing transgenic animals that carry a complete repertoire of human antibody genes (see e.g. WO93/12227, WO94/02602, WO96/34096, and WO96/33735). Furthermore, B-lymphocytes carrying human antibodies can be isolated from transgenic animals carrying human antibody gene repertoires (Mendez et al., Nat. Genet. 1997, 15: 146-156, US20100323401).
The term “antigen” refers to molecules that can be recognized by the variable region of antibodies. These molecules include but are not limited to proteins, peptides, fragments and aggregates of the latter two and lipopolysaccharides. The antigen may be linked to other molecules such as BSA or cells and administered with adjuvants for enhancement of the immunogenicity.
The terms “H chain” and “L chain” relate preferably to the immunoglobulin class IgG although they are not limited to the different immunoglobulin classes (IgA, IgD, IgE, IgG, and IgM) or subclasses such as IgG1, IgG2, IgG3, and IgG4.
The genes for H and L chains are integrated into suitable vectors, preferably expression vectors that are subsequently introduced into host cells by known methods such as electroporation, lipofection and calcium phosphate precipitation.
The genes encoding for the antibody H and L chains may be supplemented with DNA encoding for a signaling sequence that enhances the secretion of the antibody by the host cells. Furthermore, the antibody may be treated enzymatically within the cells by intrinsic enzymes or enzymes integrated by genetic recombination in order to change the amino acid sequence after the translation or to change the glycosylation of the antibodies. The host cells include mammalian cells (such as CHO, BHK, HeLa, HEK293), bacterial cells (such as E. coli ), yeast (such as Saccharomyces and Pichia ), fungal cells (such as Aspergillus ), insect cells and plant cells. The cells are grown in suitable growth media (such as DMEM) and suitable culture conditions such as suitable temperatures and pH values. Typically suitable temperatures are in the range of 35 to 40° C. and suitable pH values in between pH 6.0 and pH 8.0. Containers for cell culture include but are not limited to T-flasks, roller bottles, wave bioreactors, disposable bioreactors, and glass and steel bioreactors. The typical cultivation time is in between 1 day and 15 days. During the cultivation, nutrient solution may be added, media may be exchanged and the bioreactor contents may be agitated or aerated or otherwise adjusted in order to maintain suitable living conditions for the cells.
The antibodies may be produced also in transgenic animals, preferably mammals such as transgenic sheep, goats, cattle, pigs, rabbits or mice or in plants such as transgenic tobacco. The produced polypeptides of interest are either present within the cells or secreted into the cell culture media. In the former case the cells have to be destroyed in order to harvest the polypeptides.
The cell destruction can be carried out by various methods applying mechanical, physical, and chemical stress or combinations thereof in order to disrupt the cells.
If the polypeptide of interest is present in the cells in form of inclusion bodies the inclusion bodies are harvested from the cell lysate, solubilized and refolded using refolding agents such as guanidinium hydrochloride or urea before further purification.
In general, prior to chromatographic purification the lysate or cell culture supernatant is clarified by centrifugation, filtration or ultrafiltration or combinations thereof to remove insoluble components and cells. Numerous methods for purifying the polypeptide of interest are available.
If expanded bed chromatography is applied, the clarification step may be omitted. Furthermore, precipitation steps for the desired polypeptide or for impurities may be included in the purification process including but not limited such as ammonium sulfate precipitation or other salting out methods, ethanol precipitation and precipitation using caprylic acid. In addition solvent extraction or crystallization may be applied.
The chromatographic purification of the desired polypeptides is carried out using either functionality described above and may include one or multiple steps. The chromatography may be carried out in single column mode, or in continuous mode such as continuous annular chromatography, SMCC (sequential multicolumn chromatography), or in continuous countercurrent mode such as SMB or MCSGP.
Typically the antibody purification process includes at least one virus filtration step and one diafiltration step prior to formulation of the polypeptide product.
The formulation includes transfer of the polypeptide into a stable form. This may be achieved by freeze-drying or by transfer into sterile solutions such as oligosaccharide or sodium chloride solutions that can be administered to patients as pharmaceuticals.
Excipients, stabilizers and adjuvants may be included in the formulation.
The formulated polypeptides are administered to the patients for preventing or treating diseases or for diagnostic purposes. The administration of the formulated peptides takes place preferably parenteraly with a suitable dosage.
The term “multispecific antibody” refers to an antibody that can specifically bind to at least two different types of antigens. The multispecific antibody preferably comprises two polypeptides with heavy chain variable regions recognizing different antigens and a third polypeptide comprising a light chain variable region. Preferably, an oligomer is formed by the first polypeptide and the third polypeptide and another oligomer by the second polypeptide and the third polypeptide. This includes bispecific antibodies such as bispecific IgGs.
The term “different antigens” is not limited to different antigen molecules but refers also to different epitopes on a single antigen molecule. Thus, according to this definition, antibodies recognizing different antigens include antibodies recognizing different epitopes on the same antigen molecule.
The method described in the invention can be applied beneficially for the production of bispecific antibodies of various types. Thus, the term “antibody” includes in general any peptide scaffold allowing multispecificity binding functions and in particular fully human monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, minibodies and multispecific antibodies. The antibodies or scaffolds of the present invention may be derived from humans or animals, plants or microorganisms. The term “Chimeric antibodies” refers to antibodies that comprise amino acid sequences derived from different species. An example is an antibody containing mouse variable regions and human constant regions of the heavy and light chains, respectively. The constant regions of the antibodies including the CH1, CH2, CH3 and CL domains preferably are of human origin.
The term “humanized antibodies” refers to antibodies that essentially have the same amino acid sequence as fully human antibodies except for the CDR regions that are derived from animals. The humanization is carried out by extracting the DNA encoding for the CDR regions from an animal source and integrating it into DNA encoding for a human antibody. In order to aid this integration DNA sequences may be synthesized that provide overlapping portions between animal and human antibody DNA. The antibody genes containing the ligated DNA can be expressed in host cells after introduction via a suitable vector carrying the genes.
Further modifications of the DNA of the antibodies may be carried out, in order to improve their properties, such as the antigen binding capability. Among these modifications are site-directed PCR and cassette mutagenesis (see for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492). The sequence homology between the mutated variable region and the original variable region should be as high as possible, preferably 80% or higher, even more preferably 90% and higher.
The antibody binding site contains six CDRs, three in the H chain variable region and three in the L chain variable region.
Antigen binding can occur also if the number of CDRs is reduced, however with a lower affinity. Thus, in the present invention, the term “antibodies” also covers oligomers with a reduced number of CDRs in the H chain variable region and/or in the L chain variable region.
The term “minibodies” relates to polypeptides carrying antigen recognizing parts. The minibodies preferably contain the H and the L chain variable regions. They include also antibody fragments. Examples for antibody fragments are Fab and Fab2. Antibody fragments can be obtained from complete antibodies by enzymatic digestion, for example with papain or pepsin (see Brennan et al., Science (1985) 229:81) or by genetic recombination methods using DNA encoding for the H and the L chain variable regions. Minibodies can be produced from antibodies or antibody fragments by enzymatic treatment or by introducing vectors encoding for the complete amino acid sequence of the minibody into suitable host cells such as E. coli or yeast.
Minibodies include also single chain Fv (scFv) polypeptides that are obtained by linking the variable parts of the H and the L chain that form together an antigen recognizing site (Hu et al., Cancer Research (1996) 56: 3055-3061, Pluckthun and Pack, Immunology (1997) 3: 83-105). A minibody may also contain two variable parts of the H chain and two variable parts of the L chain that are linked together (tandem scFv). Suitable linkers typically are peptides themselves and do not negatively impact the function of the antigen recognizing site.
The advantages of minibodies and antibody fragments relate mainly to their small size compared to Immunoglobulin antibodies, which affects pharmacokinetics and to their low production costs, as they can be also produced using bacterial cells (Harrison and Keshavarz-Moore, 2006, Annals of the New York Academy of SciencesVolume 782, Issue 1; Arne Skerra Current Opinion in Immunology Volume 5, Issue 2, 1993, Pages 256-262), or yeast cells (Ridder et al. (1995) Nature Biotechnology 13, 255-260).
The minibody formats include Fab, Fab′, F(ab′)2, Fv, and scFv (single-chain Fv) (Holliger P and Hudson P J (2005) Nature Biotechnology 23 (9) 1126-1136, Pluckthun and Pack (1997) Immunotechnology Volume (3) 2: 83-105), diabodies, sc(Fv) 2 , triabodies (Hudson and Kortt (1999) Journal of Immunological Methods (1999) 231: 177-189), and tandem diabodies (Björn Cochlovius et al. 2000, Cancer Research (2000) 60:4336-4341).
The term “diabody” refers to an antibody that comprises two light chain variable regions and two heavy chain variable regions. The L chain variable regions are each linked to a H chain variable region by a peptide linker of a length that is too short to allow the formation of an antigen recognizing site. However, if the peptide linker length is in the range of five amino acids, two constructs of this type may associate to form a dimeric antibody with two antigen recognizing sites, which is called “diabody”. When the H chain variable regions or the L chain variable regions are different, the two antigen recognizing sites are different, and a bispecific diabody has been generated. (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); WO93/11161). Moreover, if the peptide linker length is in the range of one or two amino acids, three constructs of this type can associate to form a trimeric antibody with three antigen recognizing sites, which is called “triabody”. If the peptide linker length is zero, i.e. no linker is present, four constructs of this type can associate to form a tetrameric antibody with four antigen recognizing sites, which is called “tetrabody”. With increasing number of constructs, the size of the oligomer increases, improving the pharmacokinetics of the antibody (Le Gall, et al 1999. FEBS Letters 453 (1): 164-168.). Thus by using constructs with different H or L chain variable regions, tri- or tetraspecific antibodies may be generated.
In the present invention, the term “multispecific antibody” preferably relates to “bispecific antibodies” that have two different antigen recognizing sites. However, the antibodies may have additional functionality, such as effector functions of the Fc constant region in the case of bispecific IgGs,
The term “bispecific antibodies” includes antibody constructs that comprise two different scFv or tandem scFv parts linked to an Fc constant region. These constructs resemble an IgG molecule but lack the CH1 and/or the CL domain.
Furthermore, the term “bispecific antibodies” includes bispecific single domain antibodies. Single domain antibodies lack the L chain and thus comprise only two H chain variable regions and a constant part. Some single domain antibodies, such as the ones obtained from camelid animals also lack the CH1 domain.
Single domain antibodies can be obtained by immunization of camelid animals such as camels, dromedaries, llamas, alpacas, guanacos or cartilaginous fish such as sharks with the desired antigen. By isolation of the mRNA encoding for the antibodies, reverse transcription and PCR, an antibody library can be generated. Using screening techniques like phage display or ribosome display, antibodies that bind to the antigens can be found. Alternatively single domain antibodies can be produced from libraries containing the complete ensemble of antibodies of non-immunized animals and applying in vitro affinity maturation. The antibody may then be humanized by to decrease the risk of immunological reaction upon administration to humans. Due to the large homology between camelid VHH and human VH parts, the extent of required humanization can be minimized. (Muyldermans (2001) Reviews in Molecular Biotechnology 74: 277-302).
Single domain antibodies may also be generated by recombinant methods from genes encoding for full antibodies of humans or animals, comprising also light chain parts.
However, the derivation of single domain antibodies from camelid animals or cartilaginous fish is preferred.
Bispecific single domain antibodies are single domain antibodies comprising two different H chain variable regions. They can be produced using host cells that carry the DNA encoding for the two different heavy chain variable regions as described above.
Single domain antibodies share beneficial properties that may prove useful in therapeutic applications, such as their extended CDR3 loop that allows for targeting hidden antigens. (Current Opinion in Pharmacology. 2008, 8:600-608)
Further embodiments of the invention are laid down in the dependent claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
FIG. 1 shows a schematic illustration of a monoclonal antibody including two antigens;
FIG. 2 shows a schematic illustration of the effect of amino acid modification of the H chains on the chromatographic purification of a bispecific antibody, wherein the upper part of the figure shows the chromatogram for a separation of heterodimeric bispecific antibodies (A-B) and homodimeric monoclonal antibodies (A-A, B-B) expressed from non-modified H chains (A, B), and the lower part of the figure shows the chromatogram for a separation of heterodimeric bispecific antibodies (A′-B′) and homodimeric antibodies monoclonal (A′-A′, B′-B′) expressed from modified H chains (A′,B′) as described in US 20090263392;
FIG. 3 shows a schematic illustration of the invented method, wherein two libraries of monoclonal antibodies recognizing two different antigens are produced, the antibodies from each library are analyzed separately using a suitable method such as cation exchange chromatography, by comparing characteristic data obtained from the chromatograms, one monoclonal antibody candidate is chosen from each library and the DNA encoding for its heavy chains is transfected into host cells by means of a suitable vector together with the DNA encoding for the common light chain, and the host cells express the DNA and produce heavy and light chain polypeptides that associate randomly and form the heterodimeric desired bispecific antibody (A-B) and two homodimeric monoclonal antibody species (A-A, B-B), wherein the bispecific antibody may be isolated by single column or multicolumn continuous countercurrent chromatography, the latter being beneficial if the peaks of the homodimers and the heterodimer overlap;
FIG. 4 shows an overlay of five different simulated chromatograms obtained by applying the method described in Example 3 to five monoclonal antibodies from one library, wherein the chromatograms for the antibodies in all libraries are obtained using the same method, and wherein the chromatograms are processed and evaluated according to the method described in Example 4 and Example 5;
FIG. 5 shows an analytical chromatogram of a monoclonal antibody obtained using a Propac wCX-10 column, wherein the retention times are indicated as follows: peak front tRF, peak maximum of the tallest peak tRP, peak tail tRT. The area of the main peak in the chromatogram, AP, is confined by the valleys in the chromatogram that are closest to the main peak, indicated by the borders of the rectangles that face each other, the baseline and the UV signal, and wherein the total area AT is given by the area between the UV signal and the baseline;
FIG. 6 shows a schematic overview with the evaluation of pairs Ai-Ai and Bj-Bj of monoclonal antibodies, wherein the retention times are indicated as follows: peak front of the first antibody tRF,1; peak tail of the first antibody tRT,1; peak front of the second antibody tRF,2; peak tail of the second antibody tRT,2, and wherein the difference Δ(i,j) is given by (tRF,2−tRT,1). The peak widths are indicated by W1 and W2, respectively;
FIG. 7 shows a simulated analytical chromatogram produced with the method described in Example 3 for an injection of 0.02 mg antibody per mL of packed bed volume of the antibody mixture produced by the host cells (thick, solid line), wherein the mixture contains bispecific heterodimeric antibodies (A-B) and homodimeric monoclonal antibodies (A-A, B-B), wherein the simulated chromatograms of injections of pure homodimers A-A and B-B are overlaid and indicated by the dashed lines, and wherein the peak in between the homodimer peaks corresponds to the bispecific antibody A-B;
FIG. 8 shows a preparative single column chromatogram obtained applying the method described in Example 7 for an injection of 20 mg antibody per mL of packed bed volume of the antibody mixture of bispecific heterodimers (A-B) and homodimers (A-A, B-B) produced by the host cells; and
FIG. 9 shows a simulated internal MCSGP profile obtained applying the method described in Example 8 for preparative purification of the antibody mixture of bispecific heterodimers (A-B) and homodimers (A-A, B-B) produced by the host cells, wherein it is to be noted that the time axis is inverted; the profile is to be seen in analogy to a batch chromatogram and is cyclically repeated; the parts of the chromatogram indicated with the shaded rectangles correspond to sections of the chromatogram that are internally recycled; the part of the chromatogram in between the shaded rectangle corresponds to the product elution window.
DESCRIPTION OF PREFERRED EMBODIMENTS
Current antibody formats comprise heavy (H) and light (L) chains or at least their variable regions. IgG type antibodies are dimers consisting of two H and two L chains. The antigen recognition site is formed at the N terminus by each H and L chain pair ( FIG. 1 ). In the case of monoclonal IgG antibodies, identical H and identical L chains are present, thus giving rise to the two identical antigen binding sites. In the case of bispecific IgG antibodies, the H chains or the L chains or both are not identical, forming two different antigen recognition sites.
A number of techniques are used in order to identify DNA encoding for H and L chains that lead to the formation of antigen recognition sites with the desired specificity. Among these screening techniques are phage display techniques, ribosome display techniques or techniques for isolating the DNA from antibody-producing B-lymphocytes. As state of the art, antibodies are produced in genetically modified host cells.
Since the contribution of the H chain to antigen recognition is the predominant one, the screening is frequently limited to the H chain. Criteria for selecting H chain candidates include the activity and the affinity and/or avidity for the antigen, the antigen specificity, low off-rates, good solubility, little aggregation, and good expressibility in the host cell system. Typically, each screening delivers several H chain candidates that are satisfactory with regard to these criteria. However, these candidates differ with respect to their amino acid composition which leads to differences in their physico-chemical properties such as the isoelectric point.
For each different antigen recognition site of the desired antibody, one H chain library is generated. Thus, for bispecific antibodies, two libraries of H chains are generated.
For production of IgG bispecific antibodies, one H chain is selected from each library and the corresponding DNA is integrated into suitable vectors and transfected into the host cells together with DNA corresponding to L chains. The cells produce the H and the L chains which randomly associate within the cells, giving rise to full IgG antibodies consisting of two H and two L chains which finally are secreted. Thus, due to the random association, apart from the desired bispecific antibody a number of unwanted antibody types is produced. The presence substantial quantities of unwanted antibody types with close similarity increases the complexity of the production process and decreases the yield of the actual desired antibody type, leading to increased production costs and cost of goods.
In order to minimize the number of unwanted antibody types in bispecific antibody production, a number of approaches can be used.
Firstly a common L chain, that maintains the antigen specificity of both antigen recognizing sites, is used for pairing with the H chains. This reduces the number of antibody types from ten to three and the proportion of the bispecific H chain heterodimer increases up to 50%.
Secondly the knobs-into-holes technique is applied to promote heterologous association of the H chains. Essentially, this technique comprises genetic modification of the H chain genes in such manner that at the association site in the CH3 region the H chain with the first specificity (“chain A”) contains at least one amino acid with a bulky side chain (knob) while the H chain with the second specificity (“chain B”) contains at least one amino acid with a small side chain (hole). For bispecific IgG production by combining the former two techniques, the proportion of the desired A-B heterodimer can be increased up to 95% while the remaining 5% consist of A-A and B-B homodimers. However, for use as biopharmaceuticals the purity of the A-B heterodimer has to be increased even further, which is achieved by removal of the homodimers from the antibody mixture in one or more chromatographic steps. However, the chromatographic purification still remains a major challenge since the different antibody types typically exhibit very similar adsorptive properties during chromatography.
To overcome this challenge, a further method as disclosed in US 20090263392 to improve the chromatographic purification of bispecific antibodies comprises firstly the modification of the amino acids that constitute the two H chains of the antibody through genetic recombination techniques in order to increase their difference in their isoelectric points, secondly the expression of the antibodies in host cells and thirdly the purification of the bispecific antibodies by chromatography. An increased difference of the isoelectric points of the two H chains leads to increased charge differences of the A-A homodimer, the A-B heterodimer and the B-B homodimer, which can be exploited for instance by ion exchange chromatography. The effect of the amino acid modification on the chromatographic purification performance is illustrated in the schematic in FIG. 2 .
Use of different chromatographic functionalities is possible including affinity chromatography, hydrophobic interaction chromatography, thiophilic affinity chromatography, antigen affinity chromatography and ion-exchange chromatography.
The purification of the A-B heterodimer by protein A affinity chromatography is e.g. possible for the case of a heterodimer comprising a mouse IgG2a chain on the one hand and a rat IgG2b chain on the other. Since protein A has a high affinity for mouse IgG2a and a low affinity for rat IgG2b, the heterodimer has an intermediate affinity and can be eluted from the protein A stationary phase at intermediate elution strength.
The disadvantages of this method are its limitation to different IgG subclasses and to IgG subclasses from different non-human sources (mouse, rat), which increases the risk for antigenicity.
The purification of the A-B heterodimer by hydrophobic interaction chromatography has been demonstrated for the case of a heterodimer comprising a mouse IgG2a chain on the one hand and mouse IgG1 chain on the other. However, experimental results show that the separation between the desired heterodimer and the homodimers is not sufficient. A purification of an A-B heterodimer comprising two monomers of the same subclass, e.g. two IgG1 monomers, by hydrophobic interaction chromatography has so far not been described.
The purification of the A-B heterodimer by thiophilic affinity chromatography has been demonstrated for the case of a heterodimer comprising a mouse IgG1 chain on the one hand and rat IgG2a chain on the other. Since the technique requires the presence of free cysteines in the antibodies is not a general method and cannot be used for the heterodimers with H chains of the same subclass. Furthermore, proteins with free cysteines are prone to aggregation during the biopharmaceutical production process.
The purification of the A-B heterodimer by antigen affinity chromatography has been demonstrated nd is based on the use of immobilized antigens as ligands and requires two chromatographic steps. In the first step, the A-B heterodimer and one of the homodimers, for instance A-A, binds to the ligands, while the B-B homodimer is not bound to the ligand and removed by washing. In the second step, only the heterodimer is bound and the remaining homodimer is removed through washing. Since this method uses two chromatographic steps and requires customized stationary phases it is very costly at a manufacturing scale. Moreover it cannot be used for antibodies with high activity but low affinity.
The satisfactory isolation of A-B heterodimers with H chains belonging to the same subclass in combination with the targeted modification of the amino acid composition has been demonstrated wherein the isolation of the amino-acid-modified A-B heterodimer by ion exchange chromatography is used, using an analytical stationary phase and a preparative stationary phase. More precisely, in analytical cation exchange chromatography, the retention time for both the unmodified humanized A69 antibody homodimer and the humanized B26 antibody homodimer is the same, and thus separation of these homodimers and the desired bispecific antibody is impossible. On the other hand the chromatograms of antibodies with modified amino acids in the variable region of the H chains display peak shifts of the homodimers. This difference increases with the number of charge modifications made. Thus, the charge modification of the amino acid sequence of the H chains of the A69 and the B26 antibody, both belonging to the same IgG subclass, enables the separation of the homodimers and the isolation of the bispecific heterodimer. The modification may be carried out in the framework region (FR) or the complementary determining region (CDR) of the antigen recognizing part of the H chain requiring.
Chromatography as described in all cases above relates to single column batch mode and so far in this field no use of continuous counter-current chromatography has been reported. The two continuous counter-current chromatography processes reported include Simulated Moving Bed (SMB) chromatography and Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), (see e.g. 05405327.7, EP 05405421.8). The isolation of bispecific antibodies requires the purification of a product component from a mixture containing impurity components with very similar adsorptive properties. As the bispecific heteromeric antibody in terms of adsorptive properties represents an average between the homodimeric antibodies, it will elute between the homodimers which makes the isolation more challenging since it requires a three fraction (ternary) separation.
Although pseudo-ternary separations have been reported for SMB with one of the impurities having very different adsorptive properties as the product, SMB cannot be used to isolate a product overlapping with early and late eluting impurities. In contrast, this capability has been demonstrated by MCSGP. Due to the counter-current movement between the mobile and stationary phases and the internal recycling of impure side fractions, with MCSGP the product can be isolated from the accompanying early and late side components achieving high purity and high yield simultaneously, even if the corresponding single column chromatogram shows a strong overlapping of the product and the impurity peaks. Application examples for MCSGP include the separation of monoclonal antibody variants or the purification of peptides produced by chemical synthesis.
In summary, chromatographic approaches to isolate a bispecific heterodimeric antibody from a mixture containing also the homodimeric forms have been successful only in limited cases, for instance if different H chain subclasses were used in order to introduce diversity of the monomeric and the heteromeric antibodies with respect to adsorptive properties in chromatography. A method for production and chromatographic isolation of a heterodimeric bispecific antibody comprising a single H chain subclass, suitable for production scale manufacturing and satisfying pharmaceutical product standards has not been reported, apart from the method described in US 2009/0263392. The isolation of bispecific antibodies from a mixture of antibodies, all carrying the same H chain subclass has proven challenging, since the antibodies can be separated only by their differences in their variable regions. Since the sequence homology of the variable regions is large, US 20090263392 suggests the modification of the amino acid sequence of antibodies with the desired specificity in order to alter the charge differences between the variable regions of the H chains involved on the antibody formation. This decreases the homology and may potentially decrease the activity of the antibody while the risk of immunogenicity increases making subsequent activity testing imperative. Thus, beneficial properties of the antibody are sacrificed in favor of a better separability in the chromatographic purification process.
As mentioned above, the present invention takes a completely different approach to identify or select particularly suitable systems, for example to be purified in a process as outlined above, and this is summarized in FIG. 3 in a schematic representation: two libraries A and B of monoclonal antibodies recognizing two different antigens are produced (top left boxes). The antibodies from each library are then analyzed separately using a suitable method such as cation exchange chromatography (bottom left boxes). One of the key ideas here is that the antibodies from each library are essentially equal (pareto-optimal) with respect to selection criteria such as affinity, specificity, expressibility, avidity and stability (including aggregation properties, process stability, antibody stability etc.) and/or other criteria depending on the requirements of the specific case, so that one is sure that those which are selected by analytical chromatography are also optimal. By comparing characteristic data obtained from the chromatograms, one monoclonal antibody candidate is chosen from each library and the DNA encoding for its heavy chains is transfected into host cells by means of a suitable vector together with the DNA encoding for the common light chain, and the host cells express the DNA and produce heavy and light chain polypeptides that associate randomly and form the heterodimeric desired bispecific antibody (A-B) and two homodimeric monoclonal antibody species (A-A, B-B), wherein the bispecific antibody may be isolated by single column or multicolumn continuous countercurrent chromatography, the latter being beneficial if the peaks of the homodimers and the heterodimer overlap. This method enables the selection and preparative production of bispecific antibodies by preparative chromatography without requiring targeted mutagenesis to modify the amino acid sequence of the antibodies, allowing to preserve to a large extent the natural sequence and reducing potential antigenicity of mutated sequences. This shall be illustrated in more detail in the examples given below.
EXAMPLES
Example 1
Establishing Libraries of Monoclonal Antibodies by Phage Display
In the following the isolation of high affinity antibodies from a phage display library of the heavy chain variable and the light chain variable segments (VH and VL, respectively) is described.
In a first step, B-cells are obtained from human donors or from immunized animals such as mice or camelids as described in by Marks et al (1991) in J. Mol. Bio. 222, 581-597. Also synthetic libraries from cloned human variable chain segments may be used (Winter G. et al Annu. Rev. Immunol. 1994. 12:433-55).
Subsequently, VH and VL mRNA is obtained from the cells and transcribed separately into cDNA using PCR with suitable primers as described by Marks et al (1991 loc. cit.). Tagged primers to incorporate restriction sites in order to facilitate future ligations may be used advantageously as described by Vaughan et al. (1996), Nat. Biotech. 14, 309-314.
In order to obtain single chain Fv (scFv) fragments consisting of one VH and one VL chain, linker DNA such as DNA encoding for (Gly4Ser) 3 (Huston et al. (1988) Proc. Natl. Acad. Sci. 85, 5879-5883) is amplified by PCR either separately Marks et al (1991 loc. cit.), or together with the DNA encoding for one of the variable segments (Vaughan et al. (1996 loc. cit.)) to facilitate the construction of the library.
The VH, VL and linker DNA fragments are then assembled using PCR to form scFv genes. Afterwards, the scFV DNA is ligated with phage vector DNA such as pCantab 6 (Mc Cafferty et al. (1994) Appl Biochem Biotechnol. 47 (2-3): 157-171) or pHEN1 (Hoogenboom et al. (1991) Nucleic Acids Research, Vol. 19, No. 15 4133-4137) using restriction enzymes
Using this technique, libraries of more than 10 10 individual recombinants have been reported (Vaughan et al. (1996, loc cit)). By using combinatorial infection, even larger libraries of 10 12 individual recombinants have been reported (Winter G. et al 1994 loc cit) The DNA constructs are introduced into of E. coli bacterial cells by electroporation or other suitable means and the cells are grown using a suitable media. Depending on the locus of the fusion of the scFv DNA and the phage DNA, phage rescue is required (Winter G. et al 1994 loc cit). Phage rescue may be performed using a helper phage such as M13 KO7 (Marks et al (1991 loc cit)).
The obtained library phages are then applied to a surface containing immobilized antigen (Vaughan et al. (1996 loc cit)). By repeated washing, only the phage expressing the antibodies with the largest affinity for the antigen remain bound and are recovered in a separate elution step. Binding strength may be evaluated by methods such as ELISA and “equilibrium capture” (Clackson T et al. (1991) Nature 352, 624-628, Winter G. et al 1994 loc cit). The eluted phage are then used to infect E. coli and the cycle of rescue and selection is repeated. In order to increase mutations leading to antibodies with better binding properties, bacterial mutator strains may be used or mutations may be introduced in vitro using PCR (Winter G. et al 1994 loc cit). Once high-affinity scFv fragments are isolated, the encoding genes are combined with genes encoding for the desired antibody format, such as monoclonal IgG for the expression in suitable host cells. For the production of libraries of antibodies to be used for expression of bispecific antibodies, it is convenient to use the same VL library to construct binders against the two different target antigens, and to limit its size (Merchant A M et al (1998) Nat. Biotechnol., July 1998, Vol. 16(7), p. 677-681). For single domain antibodies it is sufficient to generate two VH libraries with different antigen specificity.
Example 2
Establishing Libraries of Monoclonal Antibodies by Ribosome Display
Ribosome display offers the opportunity to obtain libraries even more diverse than the ones obtained using phage display and libraries of the size of up to 10 13 individual recombinants have been reported (Hanes and Pluckthun, (1997) Proc. Natl. Acad. Sci. 94, 4937-4942).
For ribosome display, as a first step, B cells are obtained as described in example 1 and mRNA encoding for VH and VL segments is extracted. The mRNA is transcribed to cDNA and the DNA encoding for the VH and the VL chains is amplified by PCR, with primers providing restriction sites and purified. Subsequently the PCR products are ligated with DNA that comprise sequences required for ribosome display including a ribosome binding site, a transcription terminator such as T3Te and the T7 promoter (Krebber et al. (1997), J Immunol Methods. (1997) 201(1): 35-55., Hanes and Pluckthun, (1997) Proc. Natl. Acad. Sci. 94, 4937-4942), Hanes et al. (1998) Proc. Natl. Acad. Sci. 95 (24) 14130-14135). As in example 1 a linker sequence may be added in order to obtain scFv fragments. After amplification by PCR, the ligated PCR products are transcribed in vitro and the resulting mRNA is purified. The mRNA is now translated in vitro using E. coli extract containing ribosomes (Hanes and Pluckthun, (1997 loc cit), Hanes et al. (1998 loc cit)). The translation is stopped using a suitable buffer and the ribosomes carrying the mRNA and the scFv protein are isolated.
The selection of binding proteins is carried out by applying the ribosome mixture to a surface with immobilized antigen and by removing the unbound ribosomes through washing.
The retained ribosome complexes are then dissociated and the mRNA is recovered. The mRNA is then amplified by reverse transcription PCR, purified and used for the next round of ribosome display (Hanes and Pluckthun, (1997 loc cit), Hanes et al. (1998 loc cit)). In order to complete the screening and to obtain scFv protein, PCR products from the last round of ribosome display are cloned into a suitable vector, transcribed and translated in vitro. The scFv fragments are then detected by ELISA. During the various PCR steps mutations leading to diversity of the library are introduced mimicking the natural process of affinity maturation. Other methods to increase diversity are described in by Hanes et al. (1998 loc cit).
As in phage display, once high-affinity scFv fragments are isolated, the encoding genes are combined with genes encoding for the desired antibody format, such as monoclonal IgG for the expression in suitable host cells. For expression of bispecific antibodies two libraries with two different VH chains but the same VL chains are produced. For single domain antibodies, the presence of VL is not required.
Example 3
Chromatographic Analytics for Identification of Monoclonal Antibodies Suitable for Cloning of Bispecific Antibodies
Antibodies from two libraries containing monoclonal antibodies with different antigen recognizing sites were subjected to chromatographic analytics using an Agilent HP 1100 series instrument in order to find suitable candidates for the later expression of bispecific antibodies. The stationary phase was Fractogel SO3(S) (Merck, Darmstadt, Germany), that had been packed into a Tricorn column (GE Healthcare, Uppsala, Sweden) of 5 mm diameter and 100 mm length according to the manufacturer's instructions. The analytical method comprised a linear gradient elution using the following buffers: buffer A: 25 mM phosphate; buffer B: 25 mM phosphate, 1.0 M NaCl. The pH of both buffers had been adjusted to pH 6.0 using 8 M NaOH solution. The method was run at a flow rate of 0.5 mL/min at a temperature of 25° C.
Prior to the injection of the antibody, the column was equilibrated by running 0% B for 8 min. The gradient was run from 0% B (0 M NaCl) to 30% B (0.3 M NaCl) in 30 min, followed by a step to 100% B, a hold for 4 min at 100% B, followed by a further step to 0% B and a hold for 8 min. The linear gradient phase serves for eluting the antibody, while potential strongly adsorbed impurities are eluted during the high salt wash after which the column is re-equilibrated.
The injection volume was 40 μL which corresponded to amounts of 20-80 μg of monoclonal antibody, depending on the antibody concentration in the sample. Since these amounts are still far in the analytical range, the experimental method is insensitive to variations of the injection amounts in this magnitude.
The antibody was detected using the diode array detector of the Agilent system at wavelengths of 220 nm and 280 nm and the chromatogram was recorded.
The same analytical method was used to analyze all antibodies of the two libraries. An overlay of five simulated typical chromatograms obtained by analyzing five different mAbs using the method described above is show in FIG. 4 . Due to the presence of charged isoforms, the antibody may appear very heterogeneous, thus the chromatogram of a monoclonal antibody may display multiple peaks as shown in FIG. 5 .
Example 4
Processing of Chromatograms and Data Extraction
Prior to evaluation of the chromatograms recorded using the method described in example 1 a baseline is drawn under the antibody peak. The baseline is drawn such that a maximum of an estimated 0.5% of the total peak area is below baseline. It is important that the baseline be drawn in a consistent manner for the different analyzed chromatograms. For this purpose, also a computer software tool may be used, which is typically included in the software package provided with the HPLC instrument. Consequently, the baseline touches the chromatogram at the peak front and at the peak tail.
The information extraction from the chromatogram is demonstrated in FIG. 5 for an analytical chromatogram of a monoclonal antibody, obtained using a Propac wCX-10 column. The following parameters are obtained:
The retention time of the peak front which corresponds to touching point of the baseline and the chromatogram in the peak front and is termed tRF The retention time of the peak tail which corresponds to touching point of the baseline and the chromatogram in the peak tail and is termed tRT The retention time the peak maximum of the highest peak (main isoform peak), termed tRP The area of the main peak in the chromatogram, confined by the valleys that are closest to the main peak, indicated by inner borders of the rectangles in FIG. 5 , the baseline and the chromatogram, termed AP The total peak area, confined by the chromatogram and the baseline, termed AT
Example 5
Evaluation of Chromatographic Analytical Data and Antibody Ranking
After having extracted the data from the chromatograms, the purity P of each monoclonal antibody is calculated. In Table 1 the data is summarized for each antibody Ai-Ai from a first example library against a first antigen and Bj-Bj from a second example library second antigen. The antibodies were sorted by their main peak retention time and named A1-A1, A2-A2, A3-A3 etc.
TABLE 1
Evaluation of chromatographic analytical data and antibody
ranking according to the Rs and the Δ(i,j) criteria. The highlighted
fields indicate the top ranking antibody pairs according to the
respective criteria. In the two lower tables the index of A is increasing
from top to bottom (i designates a row), and the index of B is
increasing from left to right (j designates a column)
antibody library 1
Ai-Ai
i
tR
tRF
tRT
Purity
1
10.0
9.0
11.5
100%
2
12.0
10.5
14.0
100%
3
13.0
11.0
13.5
93%
4
14.0
13.0
15.5
83%
5
16.0
14.5
17.5
100%
antibody library 2
Bj-Bj
j
tR
tRF
tRT
Purity
1
9.0
8.0
11.0
100%
2
12.5
11.0
14.5
70%
3
14.0
13.0
14.5
100%
4
15.0
13.5
16.0
90%
5
17.0
16.0
19.5
97%
Subsequently, each antibody of the first library Ai-Ai is compared with each antibody of the second library Bj-Bj, as to evaluate each possible combination. The Rs and the Δ(i,j) values were calculated and for each pair (Ai-Ai, Bj-Bj). A purity criterion of 95% was imposed which excludes certain antibodies from the ranking. Both antibodies in the pair (Ai-Ai, Bj-Bj) are required to satisfy the purity criterion.
Finally the antibody pairs are ranked by their Rs values in descending order starting with the largest positive values. The top scoring antibody pairs according to the Rs criterion are (A1-A1, B5-B5), (A5-A5, B1-B1), (A1-A1, B3-B3). Note that some antibody pairs such as (A3-A3, B5-B5) would be ranked higher than (A1-A1, B3-B3) according to the Rs criterion (Rs=1.83 vs. Rs=1.75), but are excluded from the ranking because at least one of the antibodies of the pair does not fulfill the purity criterion.
The top scoring antibody pairs according to the Δ(i,j) criterion are (A1-A1, B5-B5), (A5-A5, B1-B1), (A2-A2, B5-B5). The deviation between the results of the Rs and the Δ(i,j) criterion are due to the different peak widths that are not taken into account by the Δ(i,j) criterion. Nevertheless for the two top scoring antibody pairs the two criteria deliver the same results. The top scoring antibody pairs with the largest Rs or Δ(i,j) values that are fulfilling the purity constraint are selected for cloning of bispecific antibodies.
Example 6
Expression of Bispecific Antibodies in Host Cells and Chromatographic Analytics of Bispecific Antibodies
Bispecific antibodies were expressed in host cells. The antibody mixture produced by the host cells contains the heterodimeric bispecific antibody A-B and the homodimeric monoclonal antibodies A-A and B-B which correspond to the original antibodies from the pair (Ai-Ai, Bj-Bj) that was selected for cloning as described in Example 5. The chromatographic method developed for analyzing all antibodies from the libraries is suited also to analyze the antibody mixture produced by the host cells as the bispecific antibody combines the properties of the A-A antibody and the B-B antibody and will therefore display an elution behavior that is in between that of A-A and B-B. This context is illustrated in FIG. 7 where the simulated chromatograms of the antibody mixture produced by the host cells containing the bispecific antibody and the chromatograms of the A-A and the B-B homodimers from the libraries are superimposed. For the sake of clarity, the heights of the simulated homodimer chromatograms were scaled to match the chromatogram of the mixture.
Example 7
Isolation of Bispecific Antibodies on a Preparative Scale Using Single Column Chromatography
After harvest, the cell culture supernatant was passed through two clarification steps comprising a centrifuge and a depth filtration step.
The bispecific antibody contained in the clarified cell culture supernatant was purified using a two-step chromatographic process.
The first chromatographic step was carried out based on protein A affinity chromatography. It served the purpose of removing the largest part of the impurities such as host cell proteins, DNA and media components. However, since protein A cannot distinguish among the different antibody species, it does not contribute to the purification of the bispecific antibody from the antibody mixture.
The second chromatographic step was carried out using the same stationary phase, i.e. Fractogel SO3(S), the same buffers and the same method that is described in Example 3 except for the column cleaning which was extended and included a cleaning-in-place step using 1 M NaOH and the protein load was increased by a factor of 1000 from 0.02 to 20 mg protein per mL of packed bed volume. The protein A eluate was loaded directly without buffer exchange. The linear flow rate was 150 cm/h throughout the run.
A simulated chromatogram of the purification is provided in FIG. 8 . The results of the simulations further show that the yield of the bispecific antibody with an ideal peak fractionation is 78% for a purity of 99.8% at a productivity of 12 mg pure product per mL of packed bed volume per hour. In practice the yield for the desired purity will be much lower since the product peak fractionation needs to be carried out leaving safety margins towards the product peak front and tail.
In FIG. 8 the portions of the chromatogram indicated by the grey rectangles contain parts of the bispecific antibody peak that are overlapping with the peaks of the homodimers and that have to be therefore discarded, which explains the low yield.
Example 8
Isolation of Bispecific Antibodies on a Preparative Scale Using Continuous Countercurrent Chromatography (MCSGP)
The clarification of the cell culture supernatant and the first chromatography step using protein A affinity chromatography was carried out as described in Example 7: Isolation of bispecific antibodies on a preparative scale using single column chromatography.
Subsequently, multicolumn countercurrent solvent gradient purification (MCSGP) was applied as a second purification step instead of single column chromatography.
MCSGP was carried out using the same stationary phase, i.e. Fractogel SO3(S) and the same buffers as described in Examples 3 and 7, respectively. The MCSGP process was operated in a three-column configuration as described in FIG. 3 in Biotechnology and Bioengineering 100(6): 1166-1177 with the operating parameters listed in Table 2.
TABLE 2
MCSGP operating parameters. Nomenclature as reported in
Biotechnology and Bioengineering 100(6): 1166-1177, FIG. 3
Q1
[cm/h]
150
Q2
[cm/h]
45
Q3
[cm/h]
75
Q4
[cm/h]
105
Qfeed
[cm/h]
138
Q6
[cm/h]
0
c1
[g/L]
60.0-60.0
c2
[g/L]
8.0-8.0
c3
[g/L]
8.0-8.0
c4
[g/L]
0.0-8.0
cFeed
[g/L]
Feed
c6
[g/L]
1.0-1.0
tCC
[min]
14.0
tBL
[min]
8.0
A simulated internal chromatogram of the purification using MCSGP is provided in FIG. 9 . The results of the simulations show that the yield of the bispecific antibody is 99.9% for a purity of 99.8% at a productivity of 25 mg pure product per mL of packed bed volume per hour. Thus by using MCSGP, the yield could be increased by more than 20% and maximized and the productivity could be more than doubled.
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The document pertains to a method for the purification of a ternary mixture of dimeric antibodies of the type AA, AB, BB, characterized in that for the separation of the three components and in particular for the isolation of the multi-specific fraction AB multicolumn counter current solvent gradient purification chromatography with a stationary phase load of more than 1 mg antibody mixture per milliliter stationary phase is used. It furthermore relates to a method for the identification of in particular bispecific antibody systems, which are particularly suitable for the application of such a purification method.
| 2 |
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a process of producing Maddrell salt, i.e. a long-chain, high-molecular weight sodium polyphosphate and more particularly to a simple and highly advantageous process of producing said salt in a form which is substantially free of water-soluble by-products and to such a substantially water-insoluble Maddrell salt.
(2) Description of the Prior Art
Heretofore, Maddrell salt has been produced by heating sodium orthophosphate or disodium pyrophosphate at a temperature between 250° C. and 350° C. However, only little attention has been paid to its preparation since due to its insolubility in water no utility had been found therefor. This is also the reason why no quantitative investigations regarding the amount and the type of its water-soluble components-- which always are obtained when producing the salt according to the methods known and used heretofore-- were carried out. The water-soluble components of said salt were simply eluted with water to produce a salt useful for scientific investigations.
However, a few years ago Maddrell salt has been found to be useful on a large scale as polishing and cleaning agent in toothpastes. For such a use the salt should have as low a water-soluble component as possible. When dehydrating mono-sodium orthophosphate in the known manner to Maddrell salt, there are always formed thereby the water-soluble compounds sodium trimetaphosphate and di-sodium pyrophosphate in an amount depending almost exclusively on the manner in which the temperature is adjusted during dehydration. Usually the amount of water-soluble components exceeds 5 %. Said water-soluble compounds have an extremely negative effect upon the consistency, effectiveness, and stability on storage of the toothpaste containing Maddrell salt.
In the meantime, several new processes of producing Maddrell salt have become known. Thus a process for producing Maddrell salt with up to 4 % of water-soluble components is described in U.S. Pat. No. 2,356,799. According to this process, monosodium orthophosphate must be shaped to pellets in a first reaction step. Said pellets are then converted into Maddrell salt by heating to a temperature between 300° C. and 460° C. This mode of operation, however, is quite complicated and expensive because an additional process step of producing pellets is required.
Furthermore, it is stated in "Journal American Chemical Society" vol. 81, p. 79 (1959) that neither pelletizing nor very fine comminution of monosodium orthophosphate or considerably prolonged thermal treatment are able to reduce the water-soluble portion in the final product below 5 % calculated for total substance.
J. R. Van Wazer in his book on "Phosphorus and its compounds" vol. 1, p. 668, has disclosed that, when converting monosodium orthophosphate to Maddrell salt at a temperature of 400° C., water vapor is formed by splitting off the water of constitution. Such formation of water vapor may even have a negative effect upon the formation of Maddrell salt.
German Published Application No. 1,667,569 describes a process of producing Maddrell salt from monosodium orthophosphate by heating the latter to 450° C. and removing by suction the water vapor formed due to the condensation reaction. The water vapor partial pressure is maintained during this reaction between 50 Torr. and 450 Torr.
As described in "Zeitschrift Anorg. Allg. Chemie", vol. 258, p. 52 (1949) and in "Analytical Chemistry", vol. 30, p. 1101 to 1110 (1958), Maddrell salt is produced by heating monosodium orthophosphate to a temperature of 350° C. or, respectively, 380° C. within a period of time between about 50 hours and about 168 hours. Thereby, a product is obtained which is contaminated to a considerable extent. It must be purified by washing out the by-products.
Another process of producing Maddrell salt is described in German Pat. No. 2,161,600. Said process consists in dehydrating monosodium orthophosphate at a temperature between about 300° C. and about 380° C. in the presence of water vapor. According to this process dehydration is carried out in a saturated water vapor atmosphere. This process is also rather complicated since it is necessary that a predetermined water vapor partial pressure is maintained during dehydration.
SUMMARY OF THE INVENTION
It is one object of the present invention to provide a simple, highly effective, and advantageous process of producing Maddrell salt by dehydration of monosodium orthophosphate and/or di-sodium pyrophosphate which process can be carried out in a simple manner and without having to maintain a predetermined water vapor partial pressure.
Another object of the present invention is to provide a substantially pure Maddrell salt containing water-soluble compounds in an amount not exceeding about 2% and more particularly to provide a Maddrell salt which is substantially free of water-soluble compounds.
Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.
In principle, and in contrast to the heretofore known processes, the Maddrell salt according to the present invention is produced in a simple manner and without having to maintain a predetermined water vapor partial pressure by heating monosodium orthophosphate or, respectively, di-sodium pyrophosphate at a temperature between about 250° C. and about 450° C. with the addition of a salt of phosphoric acid with a nitrogen-containing base. Said phosphoric acid salts with nitrogen-containing bases are added to the dehydration charge in catalytic amounts between about 0.25% and about 5.0%, by weight, of the starting material. The mixture is then heated and calcined. When proceeding in this manner, a Maddrell salt is obtained, the water-soluble components of which do not exceed about 2%, by weight. This low content of water-soluble components does not increase even on comminuting and grinding the salt, so that it is not necessary to subsequently temper the same or condition it by heating.
As stated above, phosphoric acid salts of nitrogen-containing bases are added when carrying out the process according to the present invention. Especially useful salts of this type are, for instance, ammonium dihydrogen orthophosphate, di-ammonium hydrogen orthophosphate, urea phosphate, guanidine phosphate, melaminephosphate, hydroxylammonium phosphate, hydrazine phosphate, or ammonium polyphosphate. These compounds can be added to the charge of monosodium orthophosphate and/or di-sodium pyrophosphate either as such or in mixture with each other, preferably in amounts between about 0.5% and about 2.0%.
It is an especially noteworthy advantage of the process according to the present invention that the nature and quality of the starting material is substantially of no importance. Thus, the same satisfactory results are achieved regardless whether a starting material is used which has been obtained by the spray-drying process or by crystallization. For instance, when starting with a spray-dried monosodium orthophosphate, the amount of catalyst can be added thereto already before spray-drying, provided the phosphoric acid salts of the nitrogen-containing bases as they are used in this process, are not sensitive to the spray-drying temperature. If a starting material is used which has been obtained by crystallization, the phosphoric acid salts of the nitrogen-containing bases can either be admixed to the starting material by means of a simple mixing device or they can be sprayed subsequently upon the starting material in the form of an aqueous solution or of an alcoholic solution or of a solution in a mixture of water and alcohol.
This process can be carried out, for instance, in a kneading device which can be heated, in a rotating cylindrical kiln which is directly heated, or in a rotating drum. When using a rotating kiln or furnace, the process can be carried out especially advantageously by making use of the so-called recirculation process, whereby part of the finished product is returned to the rotating kiln or oven. The advantage of this mode of operation is to be seen in the feature that it is possible to keep the amount of recirculated finished material and of the charge of freshly introduced starting mixture at such a proportion that adhering on and/or sticking of the reaction product to the rotating kiln or furnace and agglomeration during calcination can be prevented. When using a kneading device or a rotating kiln, calcination can be effected continuously. Most preferably, the temperature during calcination is kept between about 300° C. and about 420° C.
It is, of course, also possible to carry out the process in batch procedure by calcining the starting mixture in a muffle or retort furnace.
In conclusion, the advantage of the process according to the present invention over the known processes consists in the feature that a Maddrell salt is produced the water-soluble portion of which is below 2.0%, even after comminution and grinding and that neither subsequent tempering nor maintaining a pre-determined water vapor partial pressure is required. Furthermore, the process can be carried out continuously, of course, depending upon the apparatus used.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following examples serve to illustrate the present invention without, however, limiting the same thereto.
EXAMPLE 1
200 g. of spray-dried monosodium orthophosphate of a pH-value of 4.5 are well mixed with 2 g. (1%) of finely pulverized ammonium dihydrogen orthophsophate. The resulting mixture is heated in an electrically heated oven at an oven temperature of 400° C. for a period of time of 11/2 hours. After cooling and grinding, a Maddrell salt of a purity of 99.2% is obtained.
EXAMPLE 2
200 g. of spray-dried monosodium orthophosphate of a pH-value of 4.5 are intimately mixed with 2 g. (1%) of finely pulverized hydroxylammonium phosphate. The mixture is calcined at 400° C. within a period of time of about 2 hours as described in Example 1. The water-soluble component of the resulting cooled and comminuted Maddrell salt amounts to 1.1%.
EXAMPLE 3
4 g. (2%) of finely pulverized urea phosphate are admixed to 200 g. of spray-dried monosodium orthophosphate. The mixture is heated in an electrically heated oven at an oven temperature of 400° C. for a period of two hours. After cooling and grinding, the product has a Maddrell salt content of 98.1%.
EXAMPLE 4
A mixture of 500 kg. of spray-dried monosodium orthophosphate of a pH-value of 4.5 and of 5 kg. of finely pulverized ammonium dihydrogen orthophosphate is calcined in continuous operation by passing through a kneading device which is indirectly heated by means of oil. The temperature of the heat carrier is 330° C. The reaction product remains in the kneading device for about 41/2 hours. The resulting comminuted product has a content of 98.8% of Maddrell salt.
EXAMPLE 5
A rotating kiln is charged by means of a dosing groove or chute and a feed screw with 70 kg. to 100 kg./hour of a mixture of 500 kg. of spray-dried sodium dihydrogen phosphate of a pH-value of 4.5 and 10 kg. of finely pulverized ammonium dihydrogen phosphate. The temperature of the heating gas introduced into the rotating kiln or furnace is between about 430° C. and about 480° C. The temperature at the gas outlet is between about 100° C. and about 130° C. The charge remains in the rotating kiln or oven for about 45 minutes. The resulting Maddrell salt, after cooling and comminution, contains water-soluble components only in an amount of 1.8%.
Of course, many changes and variations in the calcination temperature and duration, in the apparatus used for calcination, in the kind of phosphoric acid salt of nitrogen containing bases admixed to the sodium dihydrogen phosphate and/or di-sodium pyrophosphate, in the amounts of said salts added to the charge, and the like may be made by those skilled in the art in accordance with the principles set forth herein and in the claims annexed hereto.
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An advantageous process of producing Maddrell salt which is substantially free of water-soluble by-products is described. The resulting substantially water-insoluble Maddrell salt is especially useful as polishing and cleaning agent in toothpaste.
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FIELD OF THE INVENTION
The present invention relates to an ornamental element to be applied to a lock of the hair of the user.
BACKGROUND OF THE PRIOR ART
It is known that since time immemorial people, particularly of female sex, have worn ornamental elements such as bracelets, necklaces, pins, earrings, pendants and similar articles.
Frequently ornamental articles to be arranged on the hair of the user have been used. By way of example there is mentioned the application of flowers, small beads and other decorative elements to the hair, a typical application among primitive people.
Actually the decoration of the hair of the user is not carried out in general with special devices but by utilizing hairpins, small combs and similar articles which, in addition to collecting in some manner the hair of the user, are shaped in a particular manner so as to guarantee a certain aesthetic effect.
In addition, particularly in the more recent years, the utilization of dyeing with coloring materials so as to give particular color to the hair has spread among women and to a lesser extent among men.
However, actual jewelry articles and articles of non-precious metals such as chains, pins and similar articles, are not applied to the hair of the user because of the difficulty of making sure that these objects remain in the hair in a stable manner.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an ornamental element which remains continuously on or in the hair of the user and which is simple from the constructive point of view and easy to apply to the hair of the user.
This object is achieved according to the invention by providing an element which has elastic means so that it may grasp a lock of hair of the user and at least a chain for decoration purpose hangs from the elastic means. In this manner the user has available a decorative element which may potentially acquire a great variety of aesthetic appearances in relation to the particular conformation of both the chain or chains present and the elastic means which grasp the hair.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention are described in detail by reference to two particular embodiments provided herein as non-limiting examples, by reference to the drawings of which:
FIG. 1 shows a schematic view of the first embodiment of the invention;
FIG. 2 shows a more detailed view of the first embodiment of the invention;
FIG. 3 is a schematic view of the second embodiment of the invention;
FIG. 4 is an enlarged view of the second embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 shows that according to the first embodiment of the invention, there are provided means capable of engaging a lock of hair of the user. The main feature of the device is constituted by the fact that at least a chain ( 1 ) hangs from the means which engage with the lock of hair and the chain gives a particular aesthetic effect to the hair of the user.
FIG. 2 shows that advantageously the means capable of engaging with a lock of hair of the user are constituted by a spiral spring ( 2 ), which may be shaped in a variety of ways, since the coils could be circular, square, oval, etc. Also the diameter of the coils may vary along the spring, thus giving rise to particular aesthetic effects. At the end of the spring terminal elements having a decorative effect may also be provided.
The chain may be of any type and may have, at the intermediate portion or at its extremities, one or more decorative elements, such as precious, semi-precious, or non-precious stones or other decorative elements, such as small hearts, small stars, etc.
Both the spring and the chain may be of any material, precious or non-precious. In addition both the spring and the chain may have particular colors, for aesthetic purpose.
It should be kept in mind that the spring may be replaced by any other element capable of grasping in a sufficiently stable manner a lock of hair of the user. By way of a non-limiting example, the function of the spring may be carried out by a hairpin, a clamp or similar articles.
FIG. 3 shows that the second embodiment of the invention comprises a spiral spring ( 2 ), capable of engaging with a lock of hair, and chain ( 1 ) inserted in the spring, with the extremities of the chain hanging down from the extremities of the spring.
Two terminals ( 3 ) are present corresponding to each of the two extremities of the chain. The terminals in the figure have the shape of small spheres but they could have different shapes, as long as they are capable of occupying totally the transversal section of the spring in a manner that they cannot be inserted within the spring.
The presence of the chain confers a particular aesthetic effect to the hair of the user.
Also in this case the observation already made with respect to the particular conformation of the spiral spring, the diameter of the coils, and the particular conformation of the chains are applicable.
With respect to the utilization of this particular form of accomplishment of the device, the user must position the chain in the desired manner with respect to the spiral spring, in such a way that the extremities of the chain are let out from the spiral spring in the quantity desired, after which the user will hook the spiral spring to a lock of hair.
It is emphasized that all the objects which conceptually contain the features discussed hereinabove are included in the present invention, with the exception of their particular conformation and configuration.
On the basis of what has been shown hereinabove, it is clear that by utilizing the device according to the present invention the user may obtain a valid decorative element which is simple from both the constructive and functional point of view and which in addition is intrinsically light so that the embodiments are intrinsically economical even if they are made of a precious metal and the decorative element in addition may be used easily.
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An ornamental coil-spring element is applied to a lock of hair of the user which engages with the lock of hair and one or more chains ( 1 ) hang from the spring element to confer a pleasant aesthetic effect to the hair of the user.
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FIELD OF THE INVENTION
[0001] The present invention relates to an apparatus, method and system for protecting hips from fracture, and for providing immediate response to hip fracture events.
BACKGROUND INFORMATION
[0002] Hip fractures may cause morbidity and mortality in persons, including, for example, elderly persons. With the progressive increase in the number of elderly persons in the United States, a concurrent surge in hip fractures is occurring. Hip fracture in elderly persons may result from a fall on the hip area. For example, Cummings et al. in “A Hypothesis: The Causes of Hip Fractures”, Journal of Gerontology: Medical Sciences, Vol. 44, No. 4 (1989), state that about 80 to 90% of hip fractures in elderly persons are due to falls, and that fewer than 10% occur before the fall. Consequently, hip protecting devices have been advocated to reduce the risk of sustaining a hip fracture.
[0003] Hip protective devices should provide both an effective and cost-saving strategy for reducing the risk of hip fractures. To be effective, however, a hip protecting device must be worn. A major issue is patient non-compliance and/or non-adherence with wearing of such hip protecting devices. It is has been found that the non-compliance in community and institutional settings ranges from 37% to 72%.
[0004] There may be several reasons why persons do not always wear a hip protecting device when instructed to do so. Reasons for non-compliance may include, for example, discomfort (e.g., too tight and/or a poor fit), and the extra effort and time required to put on and adjust the hip protecting device. It is believed that patient compliance would be substantially increased if hip protecting devices were more comfortable and provided a better fit. Patient compliance may be increased if hip protecting devices are presented in a form that those in need of such devices would be less inclined to resist the wearing of the device, due to, for example, vanity concerns or not wanting to admit the time has come to wear such a device.
[0005] There are hip protecting devices, for example, in which the device is worn underneath clothing because the wearer may not consider the protecting device aesthetically pleasing. However, in the past 10 years or so, it is understood that a very large proportion of the wearers need to be able to take the device off, sometimes urgently, such as in a need to visit the toilet, and this cannot be done with the type of the hip protector that is worn in or as underwear. This even applies if the caregiver has to take off the garment.
[0006] Wearers of hip protectors may have different capabilities with respect to possible movements of their limbs and agility. Certain existing hip protectors may not accommodate such individual needs and/or capabilities of the wearer, particularly if the wearer cannot move certain body parts in a particular direction, in agile fashion. For example, the wearer may have arthritis or muscular weakness in the hands. It has also been understood that certain existing hip protecting devices are not easily removed or put on by the wearer or the care giver.
[0007] The material of certain existing hip protecting devices using pads can stretch and therefore allow for undesired movement of the pads with respect to a particular desired area to be protected (e.g., pads that are included in a sweatpants arrangement). For example, if a person falls off a couch, the friction from contact with the couch may cause the padding of the hip protecting device to slide away from the point to be protected.
[0008] U.S. Pat. No. 5,545,128 purports to relate to a garment worn underneath clothing for bone fracture prevention during impact from a fall, in which the undergarment has an horseshoe-shaped pad arrangement for shunting a substantial portion of impact energy from the vulnerable region to the soft tissue region. However, such a design rests on the faulty notion that only falls with an impact at right angles to the greater trochanter cause hip fractures, which is not accurate. There are many other angles at which persons may fall. At some angles, contact will not be made near the greater trochanter. For example, one may fall flat backwards or half sideways on the buttocks. Accordingly, unless the fall occurs directly on the entire horseshoe-shaped pad, the thixotropy (hardening due to impact on the protective fluid/solid) will not occur fast enough, and the device is likely to do more harm than good. Moreover, the pads, which are about one inch thick, increase the perceived width of the wearer and thus may be esthetically unacceptable to the wearer. Moreover, the horseshoe-shaped design and the direct adherence to the skin is considered an impractical solution. Hence, the device discussed in U.S. Pat. No. 5,545,128 is understood to be functionally deficient, uncomfortable, or impractical for certain wearers, such as, for example, older persons.
[0009] Furthermore, another pad arrangement, in which the pads are fixed in a tight undergarment with straps around the legs and waist so that the pads can be held precisely over the greater trochanter, is not likely to be usable by an older person with arthritic fingers.
[0010] Certain hip protecting devices, which are designed to be worn underneath clothing, may include plastic shields or foam pads that may be held in place at the hips with specially designed underwear. However, such pads may provide only limited protection. For example, such pads do not protect from a rearward fall. In the human pelvis there are two large hip bones, each consisting of three fused bones, the illium, ischium, and pubis. The hip bones form a ring around a central cavity. The fused terminal segments of the spine, known as the sacrum and coccyx, connect the hip bones at the back of the central cavity; a fibrous band connects them at the front. A backward fall may cause injury or fracture of sacrum and/or coccyx. Moreover, with the internal force transmission occurring from bone to bone, falling backwards can not only hurt the sacrum and coccyx but hard impact on them can be passed onto other bones.
SUMMARY OF THE INVENTION
[0011] The present invention provides a device for protecting a hip bone or limiting a severity of a hip bone injury. In this regard, the device may include, for example, at least one pad arranged to protect the hip bone, and a wearable garment to hold the at least one pad with respect to the hip bone, in which the garment is configured to be worn over clothing and the pad configured to wrap around an area of the hip bone in a circumferential manner.
[0012] According to an exemplary embodiment and/or exemplary method of the present invention, the hip protecting device is configured to be more easily removed as compared to other existing hip protecting devices. This may be particularly important when the wearer encounters an urgent need to remove the device, such as a toilet visit. It is essential for many older people who require incontinence pads, because the device may be used in conjunction with them. In particular, the wrap-around aspect of the hip protecting device of the present invention provides a unique solution to the problem of wearing both a hip protecting device and incontinence pads.
[0013] According to an exemplary embodiment and/or exemplary method of the present invention, the hip protecting device is put on by laying the device flat on a bedside, and the wearer sits or slides on to the device, which may then be wrapped around and fastened. If the wearer has the capability, the device may also be donned in a standing position.
[0014] According to an exemplary embodiment and/or exemplary method of the present invention, a hip protecting device includes a wrap-around pad on each side so that protection may be provided for a rearward or partially rearward fall, in addition to a sideways fall. In this regard, a hip protecting device according to the present invention may provide protection against injury or fracture to certain hip bones, or other bones in the vicinity of the hip, including, for example, the sacrum and coccyx bones.
[0015] According to an exemplary embodiment and/or exemplary method of the present invention, a hip protecting device is easily adapted to the specific protections of a variety of wearers. In particular, the hip protecting device provides a fastening device that can accommodate multiple sized and shaped wearers. Accordingly, the hip protecting device may be provided in a one-size-fits all, or essentially a one-size-fits all, configuration.
[0016] According to an exemplary embodiment and/or exemplary method of the present invention, a hip protecting device may be worn over clothing, including, for example, all types of clothing, to provide improved comfort and/or convenience. The hip protecting device of the present invention may also be provided in a one-size fits all mode, which allows the user to personally adjust the device to improve the fit that may be obtained therewith. The hip protecting device may also include a pocket to hold a variety of fall/injury avoidance electronic devices, and help summoning devices, such as, for example, a postural sensor, personal emergency response system, etc.
[0017] According to an exemplary embodiment and/or exemplary method of the present invention, the hip protecting device includes at least one pad of about 7/16′ in thickness, which includes a closed cell material, such as, for example, polyvinyl chloride (PVC)-nitrile. The hip protecting device may also include two extended panels, which are configured to be self-fastening to each other so as to provide an one size fits essentially all wearers of the device. In this regard, the two panels may be fastened in various positions so as to accommodate a range of circumference of about 32 to 49 inches. Here, it is noted that to manufacture extremely large sizes, adjustments may also be made in the back of the garment in the area between the pads. The hip protecting device may further include an impact detector configured to signal for help upon detecting a fall or sudden impact.
[0018] According to an exemplary embodiment and/or exemplary method of the present invention, the hip-protecting device may be fastened by rotating two panels of the hip-protecting device around of an axis perpendicular to a fastening plane, overlapping the two panels, and pressing the two panels together. Here, the rotation is important because is makes the fastener adaptable to slight variations in an individual's anatomy. Moreover, the facing panels may be brought together at various angles with respect to the fastening plane
[0019] According to an exemplary embodiment and/or exemplary method of the present invention, the hip protecting device includes an impact detector to detect a fall. In this regard, the impact detector may include, for example, an accelerometer to detect a deceleration change, and a first processing arrangement to determine if the deceleration change is within a range in which a fracture may occur.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A A shows an exemplary method to protect a hip bone from fracture.
[0021] FIG. 1A shows a front view of an exemplary device for protecting the hip area in a human.
[0022] FIG. 1B shows an exemplary fastening device for securing the exemplary device of FIG. 1A to the hip area of the human.
[0023] FIG. 1C shows exemplary double seams of the exemplary fastening device of FIG. 1B , at which a length of the fastening device may be removed using scissors to better accommodate a particular sized individual wearer.
[0024] FIG. 1D shows a rear view of the exemplary device of FIG. 1A .
[0025] FIG. 2 shows an exemplary electronic device that may be provided with the exemplary device of FIG. 1A .
DETAILED DESCRIPTION
[0026] FIG. 1 AA shows an exemplary method to protect a hip bone from fracture or to limit a severity of a hip bone injury. In step S 101 , at least one pad is provided to protect the hip bone. For example, two pads may be provided, each pad approximately rectangular in shape with rounded edges and constructed to absorb a shock.
[0027] In step S 102 , the at least one pad is configured to wrap around an area of the hip bone. In particular, the at least one pad is configured to wrap around the area of the hip bone in a circumferential manner so as to conform to the particular wearer's shape.
[0028] In step S 103 , a one size fits all device is provided, which includes at least one pad to protect the hip bone, and which is configured to be worn over clothing. Here, the one size fits all aspect of the device may accommodate, for example, 95% of adult wearers, which may be important, for example, in terms of the application of the device in health institutions, such as nursing homes, because without such a one size fits all feature the device may be unnecessarily expensive with respect to storage and providing different sizes of the device to persons having differing sizes. Additionally, the configuration of the device so as to be worn over clothing provides certain benefits with respect to convenience and ease of use of the device. For instance, if a wearer decides to sit in once place for a particular time period, the device may be easily unfastened during this time and fastened again before rising.
[0029] In step S 102 , a pocket is provided on the device to hold an arrangement to notify a non-wearer of a fall occurrence. Here, the non-wearer may be, for example, a caregiver, who may assist the wearer of the device should he or she need assistance after a fall. The pocket may be configured to accommodate a wide variety of devices so that a customized solution may be provided to each wearer, if required.
[0030] In step S 103 , the arrangement to notify a non-wearer is provided so that both the device and the notification arrangement can be provided in one commercially available package so as to reap further economies of scale with respect to mass production of the device. Indeed, having a device that both protects hips from fracture and notifies a caregiver when the wearer falls or experiences a sudden impact to the hip area should be desirable in the context of elderly and/or injury-prone individuals who would otherwise not have access to such features with other existing hip protecting devices.
[0031] FIGS. 1A and 1D show a front and rear view of a human from slightly above the waistline to slightly below the buttocks, and a device 100 for protecting the hip area of a human. The protection may include, for example, protection against fracture, contusions, injuries to the skin and tissues lying below, which may occur, for example, from a fall. The device 100 includes a garment piece 106 , an electronic device 101 , a fastening device 103 , at least one pad 105 , and a slot/pocket 105 a to hold the pad 105 .
[0032] The garment 106 surrounds the human torso over the hip area to hold the device 100 in a suitable position. The garment 106 may be worn, for example, over clothing. In this regard, the garment 106 may be easily secured and/or removed. The garment 106 may be provided in a “one-size-fits-all” configuration, and may be made of an elastic spandex material, which may include a woven material from section to section as well, or any other suitably appropriate material.
[0033] The garment 106 may be provided in a color or pattern that is suitable for wearing over clothing. In particular, the garment 106 may be provided in a dark gray and/or brown color pattern. It is believed that the dark gray and/or brown color may be a desirable color since those who may wear the garment 106 , including elderly persons, may tend to dress in darker shades and thus the dark gray and/or brown color may not contrast so greatly. Moreover, a dark gray and/or brown color may not show dirt or stains or other discolorations as noticeably. Moreover still, a dark gray and/or brown color may improve compliance—that is, the tendency of the wearer to accept the recommendation by a physician and a caregiver, to wear the device. Any color may, however, be used.
[0034] The device 100 includes a fastening device 103 , which may be easily manipulated to adjust the device 100 to suit the particular needs of the wearer. In this regard, the device is configured for a wearer in a sitting position to be able to unfasten and refasten the device without rearrangement of the device with respect to the hips. Of course, the device may be unfastened and refastened in other positions as well, including the standing or lying down position.
[0035] The fastening device 103 may be provided in the form of two facing velcro panels 103 a and 103 b . In this regard, the overlap of the velcro panels 103 a / 103 b is such that the garment 106 may be opened widely or less widely to accommodate wearers of multiple sizes. Instead of being fastened with both panels in an aligning axial position they may also be fastened in a manner where the longitudinal axis of one pad is at an angle to the longitudinal axis of the other panel, which may provide a more snug fit to accommodate the wearer's unique shape. In this regard, the width of the two panels may be configured to increase an overlapping area of the panels when the panels are fastened in a non-parallel manner.
[0036] In one exemplary embodiment, the velcro panels 103 a / 103 b may be configured to accommodate nearly all potential wearers of the device. For example, certain potential wearers of the device may have a circumference at the hip area that is relatively large or, alternatively, relatively small as compared with the general population. Accordingly, providing a device that accommodates a wide variety of circumferences is believed to be desirable. In this regard, it is found that the potential wearers of the device include individuals whose circumference is as small as 32 inches or possibly even less, or as a large as 49 inches. Here, it is noted that the number of individuals whose circumference is less than 32 inches is expected to be quite small. It is also noted that most elderly people tend to lose weight as they age so that it is expected that most elderly wearers tend not to have a circumference that is nearly as large as 49 inches. Indeed, it is believed that most of the potential wearers who are elderly required a device that accommodates a much lesser circumference than 49 inches. Therefore, according to one exemplary embodiment, the velcro panels 103 a / 103 b may be configured to include a section 103 d that is easily removed or detached such that the potential wearer or caretaker may eliminate that portion of the panels that is not required to accommodate his circumference. For example, the velcro panels 103 a / 103 b may include a double seam 103 c to subdivide the velcro panel in such a way that a section 103 d of the velcro panels may be cut off using a pair of scissors, for example. (Note the double seam 103 c allows the end the velcro panels that remains after cutting to have an appearance of being quite reasonably neatly finished). Hence, potential wearers of the device can more easily customize the fit. This is what enables the “one-size-fits-all” feature.
[0037] The “one-size-fits-all” configuration may also provide certain benefits with respect to cost, distribution and/or stocking of the device. For example, nursing homes or other similar care facilities may more easily maintain an adequate stock of the devices for expected and unexpected needs of the residents since a supply of only one type of device is needed to accommodate all or nearly all its residents.
[0038] The body of the device 100 includes materials that are expandable and non-expandable. The expanding materials in conjunction with the velcro closure serve to adjust the device 100 to the form of the body of the wearer, both for comfort and retension of the protective pads in their operative location. Expanding material is provided particularly between the pads 105 and around the front areas where the pads 105 are held fastened.
[0039] The device 100 includes at least one pad 105 , which is constructed of a protective material within certain industry standards. In sports safety, automotive and other safety fields, a measure of protection that has become widely established is “G-max”, which describes the maximum number of multiples of the force of gravity that result from a reversal of momentum caused by an object hitting the protective material. For protection against human bone fracture, a G-max value of 200 or less is believed to be desirable so as to prevent the most fractures and yet not be too thick. In this regard, a 7/16″ thickness of, for example, the AMC material made by Armacell, is a closed cell polyvinyl chloride (PVC)-nitrile material that tests at this range.(Materials that absorb more force of impact are still being developed). In particular, the AMC material performs adequately when subjected to the ASTM F-355 prop Test, in which a steel cylinder is dropped on a sample of the material, which is situated on a steel surface connected to equipment which measures the impact over a period of time.
[0040] The at least one pad 105 may be constructed of a closed cell material that does not absorb body fluids such as urine and cleaning water when the pad is sponged or immersed in water.
[0041] Accordingly, the at least one pad 105 may provide certain benefits with respect to maintaining its cleanliness.
[0042] The at least one pad 105 may be shaped and/or adapted to the human torso in a wrap-around manner so as to protect certain bones of the human pelvis, including, for example, the greater trochanter or the two large hip-related bones, each consisting of three fused bones, the illium and ischium, which partially form a ring around a central cavity. The wrap-around construction additionally protects the fused terminal segments of the pelvis, known as the sacrum and coccyx. Accordingly, the at least one pad 105 may protect the wearer in the event of a backward or partially backward fall, which might otherwise cause injury or fracture of sacrum and coccyx, for example.
[0043] The at least one pad 105 may have an additional section that folds underneath the buttocks when the wearer assumes a sitting position. Accordingly, the at least one pad 105 is constructed to provide flexibility and protection at the same time.
[0044] The body device 100 includes an electronic device 101 to warn of risky movement and immediately alert the wearer, and also to detect a possible impending fall. In this regard, an alert may also be directed to a caregiver, such as, for example, a nurse at a nursing station, etc. If a person falls and fractures a hip, help cannot arrive too soon. Even if there is no hip fracture, help may nonetheless be needed and/or desired to provide prompt attention and aid to the patient, who may be distressed by the fall and/or may not be able to get up or even press a pendant button, such as has been provided in certain devices for various medial alerts, including those not connected with hip protecting features.
[0045] The electronic device 101 may include an inertial component 101 a to detect a change in position. In this regard, the detected change in position may indicate, for example, that the wearer has experienced a fall. The electronic device 101 may also include a transmitter 101 b to transmit a signal to a receiver (e.g., monitoring station, a nursing station, a home care giver, etc.), a receiver 101 c to receive a signal from a transmitter (e.g., a broadcast station, etc.) and a speaker 101 d to provide an audible alarm to the wearer of the device 100 .
[0046] The electronic device 101 may also include a haptic component 101 e to provide a vibration sensation to the wearer of the device 100 to indicate, for example, an impending fall or dangerous condition. In this regard, the haptic component 101 e may provide a desired feature for those individuals whose hearing is impaired, or where an audible signal may be disturbing to the wearer or others in the vicinity of the wearer.
[0047] The electronic device 101 may be fixedly arranged in the device 100 , or may be inserted or held in a pocket 107 of the garment 106 . In this regard, the electronic device 101 may be easily removed when desired; for example, when the garment 106 is to be washed. The pocket 107 may be located, for example, where it is least exposed to outside impact. For example, the pocket 107 may be located above the hip and slightly anterior with respect to the torso.
[0048] The electronic device 101 may also be arranged in a slot 105 a of the pad 105 . In particular, the electronic device 101 may be arranged, for example, in an upper outermost corner of the pad 105 . For this purpose, the garment need not require additional sewing operations. Moreover, the pad 105 may better protect the electronic device 101 .
[0049] The electronic device 101 may also include a power source to provide power. Here, the power source may be, for example, a battery.
[0050] The electronic device 101 may also include a manual alert mechanism 101 f so the wearer may manually activate an alarm condition to a nursing-station or a telephone line. In this regard, the telephone line may be, for example, a wireless connection, such as, for example, a cellular or mobile phone connection. The manual alert mechanism 101 f may be, for example, in the form of a button, a dial, switch, microphone, or any other suitable form for enabling manual activation by the wearer or any other nearby person. In this regard, the microphone may be used, for example, to provide a voice-activated manual alert.
[0051] The manual alert mechanism 101 f may be used to alert the nursing station to a fall, a fear of a fall, or any other condition that may require attention. In this regard, the wearer may alert, for example, that he or she may be experiencing a discomforting and/or life-threatening condition, such as, for example, a heat attack.
[0052] The manual alert mechanism 101 f may include an element to deactivate it so that wearers or others may prevent unintended alerts and alerts from patients or wearers that occur with excessive frequency because the wearer is regarded to have not the capacity to properly judge when an activation is required and/or necessary.
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A method and device for protecting a hip bone or limiting a severity of a hip bone injury, which includes at least one pad arranged to protect the hip bone, and a wearable garment to hold the at least one pad with respect to the hip bone, the garment configured to be worn over clothing, the pad configured to wrap around an area of the hip bone in a circumferential manner, the garment including two fastening panels, each configured to face and overlap with the other fastening panel, and fastenable together in a rotatable manner around an axis perpendicular to a fastening plane, a length of the two fastening panels configured to provide a one size fits essentially all wearers.
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TECHNICAL FIELD
[0001] The present invention is related generally to barium magnesium aluminate (BAM) phosphors and methods for enhancing their performance in lighting and display applications. More particularly, the present invention is related to increasing the thermal stability and radiance maintenance of europium-activated BAM phosphors in highly loaded fluorescent lamps and plasma display panels.
BACKGROUND OF THE INVENTION
[0002] Europium-activated barium magnesium aluminate (BAM) phosphors are widely used as the blue-emitting component of the phosphor blends in most fluorescent lamps intended for white light generation. BAM phosphors also serve as the blue-emitting pixels in plasma display panels (PDPs). Despite its wide use, BAM is notorious for its shortcomings in brightness and maintenance, particularly in those applications involving exposure to high ultraviolet (UV) and vacuum ultraviolet (VUV) fluxes. Because of these shortcomings, the blue BAM emission is reduced at a significantly faster rate over time than the emissions of the other color components in the blends or pixels. This results in a loss of lumens and a color shift in the overall light output.
[0003] Theoretical and experimental investigations of various BAM compositions over the past few years have yielded clues about the degradation mechanisms involved in the phosphor's maintenance. A prolonged exposure to radiation with photons having energies above 5 eV (wavelengths less than 254 nm) causes a reduction in the phosphor's brightness and changes in the spectral power distribution of the phosphor's emission. These effects can be observed by spectroscopic methods after hundreds of hours of lamp operation or by a short period of high-intensity laser irradiation (e.g., a 193 nm excimer laser). In addition to an approximate 25% decrease in brightness after 500 hours of operation, there is an increase in the long wavelength side of the emission band of the phosphor. Very likely, these effects are linked to electron and hole centers formed during the phosphor synthesis and/or later generated as a result of ion bombardment and UV/VUV irradiation during lamp operation. In particular, electron centers (oxygen vacancies that have captured zero, one or two electrons) are believed to compete with the europium activator ions for UV/VUV photons and may also absorb a portion of the visible light emissions from the phosphor. It is also possible that oxygen vacancies with zero or one electron may capture electrons produced, for example, from the photoionization of Eu 2+ to Eu 3+ upon 185 nm UV irradiation. If the number of defects capable of capturing electrons from the ionization of the europium activator ions is comparable to the number of europium ions in the lattice, or becomes so during the operating life of the phosphor, a serious reduction of the emission intensity will follow over time.
SUMMARY OF THE INVENTION
[0004] We have discovered that by replacing some of the cations (Ba 2+ , Eu 2+ , Mg 2+ and Al 3+ ) in europium-activated barium magnesium aluminate phosphors with tetravalent cations of silicon, hafnium, and zirconium (Si 4+ , Hf 4+ and Zr 4+ ) the performance of the BAM phosphors in certain UV/VUV applications is improved. It is believed that the introduction of the tetravalent cations into the BAM lattice reduces the probability of forming the oxygen vacancies which lead to the degradation of the phosphor. Silicon, hafnium and zirconium were chosen because of their stable 4+-valence state in varying conditions. The tetravalent dopants may be used individually or in combination.
[0005] The BAM phosphor of this invention preferably contains from about 1 to about 5 weight percent of the europium activator, and more preferably about 2 weight percent europium. The dopant amounts preferably range from greater than 0 to about 2000 parts-per-million (ppm) silicon by weight, from greater than 0 to about 12500 ppm hafnium by weight, and from greater than 0 to about 6500 ppm zirconium by weight. More preferably, the dopant amounts range from about 100 to about 400 ppm silicon by weight, from about 600 to about 2500 ppm hafnium by weight, and from about 300 to about 1300 ppm by weight zirconium. Even more preferred, the dopant amounts range from about 100 to about 200 ppm silicon by weight, from about 600 to about 1300 ppm hafnium by weight, and from about 300 to about 650 ppm zirconium by weight.
[0006] In an alternative embodiment, the phosphors of this invention may be represented by (Ba 1-x Eu x )MgAl 10 O 17 : (Hf, Zr, Si) y where 0.05≦x≦0.25 and 0<y≦0.05; preferably, 0.0025≦y≦0.01; and, more preferably, 0.0025≦y≦0.005.
DETAILED DESCRIPTION OF THE INVENTION
[0007] For a better understanding of the present invention, together with other and further objects, advantages and capabilities thereof, reference is made to the following disclosure and appended claims.
[0008] A series of europium-activated barium magnesium aluminate phosphors were prepared with Hf, Zr and Si dopants. The phosphors had a composition which may be represented by the formula (Ba 0.90 Eu 0.10 )MgAl 10-y O 17 : (Hf, Zr, Si) y . The performance of these phosphors was compared with a control phosphor, (Ba 0.90 Eu 0.10 )MgAl 10 O 17 , made under the same conditions. Three different dopant levels were used: 0.02, 0.1, and 0.5 mole %. In each case, the molar amount of the tetravalent dopant ion was substituted for an equal molar portion of aluminum.
[0009] In a preferred method, the phosphors were made by combining stoichiometric amounts of the phosphor precursor compounds: BaCO 3 (0.800 moles), BaF 2 (0.100 moles) Eu 2 O 3 (0.050 moles), MgO (1.000 mole), Al(OH) 3 (10.0000-x moles), HfOCl 2 .8H 2 O (x=0.0002, 0.0010, and 0.0050 moles), ZrO(NO 3 ) 2 (x=0.0002, 0.0010, and 0.0050 moles) and SiO 2 (x=0.002, 0.0010, and 0.0050 moles). Barium fluoride, BaF 2 , was added as a flux substituting for 10 mole percent of the BaCO 3 . The precursor compounds were mixed together and wet milled for 4 hours using YTZ beads. The pH of the milled slurry was adjusted with ammonia to have a pH of above 8.0 in order to cause the soluble additives, primarily Hf or Zr precursors, to precipitate. The milled mixture was then filtered, oven dried at 120° C., crushed and fired at 1625° C. for about 1 to about 4 hours in a 75% H 2 /25% N 2 atmosphere. The presence of barium magnesium aluminate was confirmed by x-ray diffraction; no minor phases were detected. The phosphors exhibited the characteristic blue emission peak at about 450 nm under UV and VUV excitation.
[0010] The initial brightness of each phosphor sample was measured to be within a few percent of the brightness of a standard BAM phosphor. The samples containing 0.5 mole % Hf, Zr and Si were hard pressed into a recess in a copper holder in order to dissipate excess heat during testing. The samples were then irradiated in a vacuum with 193 nm VUV radiation from a Lambda-Physik Compex 110 excimer laser. Incident power density was maintained at about 1.75 W/cm 2 . A ten-minute irradiation time was selected to avoid the nearly complete saturation in degradation of the top surface of the plaque observed with prolonged exposures (e.g., an hour or more). Compared to the control phosphor, the samples doped with Si, Hf and Zr exhibited about 10% greater brightness under the same conditions. These results are summarized in Table 1. Brightness was measured as the integrated visible radiance in the range 350-600 nm under 250 nm excitation and is reported in relative units.
TABLE 1 Initial Brightness after Brightness vs. Sample Brightness 193 nm irradiation control after 193 (mole %) (Rel. Units) (Rel. Units) nm irradiation Control 1.0 0.648 100.0% 0.5 Hf 1.0 0.707 109.1% 0.5 Zr 1.0 0.717 110.7% 0.5 Si 1.0 0.698 107.8%
[0011] The 0.5 mole % samples were also examined for their performance following exposure to a high-VUV-flux Xe discharge and an oxidizing heat treatment. In the first instance, the samples were exposed to 147/172 nm radiation from a Xe discharge for a 2-hour period in a vacuum. The power density of the incident VUV radiation was about 90 mW/cm 2 . As can be seen from the data in Table 2, the samples which were doped with Zr 4+ and Si 4+ cations exhibited a higher stability under the 2-hour exposure than the control sample. The sample doped with 0.5 mole % Hf exhibited a slightly inferior behavior under these conditions relative to the control. In the second test, the phosphor samples were heated in air for 1 hour at 450° C. to simulate conditions used in the manufacture of plasma display panels. The phosphors which were doped with Zr and Si exhibited better brightness than the control after the 1-hour heat treatment at 450° C. The sample doped with 0.5 mole % Hf did not enhance the stability of the phosphor under these conditions.
TABLE 2 Brightness under 147/172 nm Xe discharge VUV- Change Heat- Change untreated treated relative treated relative Sample (Rel. (Rel. to (Rel. to (mole %) Units) Units)) control Units) control control 99.5 71.9 100.0% 90.5 100.0% 0.5 Hf 100.7 69.7 96.9% 91.5 99.9% 0.5 Zr 99.7 74.2 103.2% 93.2 107.1% 0.5 Si 101.5 78.6 109.3% 97.6 105.7%
[0012] Fluorescent lamps were constructed in order to determine the performance of the phosphors under high wall loadings. The lamps were an electrodeless compact fluorescent type having a wall loading of about 0.1 to 0.2 W/cm 2 . The phosphors were applied to the interior surface of the lamp envelope using a conventional organic-based coating technique. Two test lamps were made for each phosphor. Radiance values (integrated from 350-700 nm) were measured for each lamp before and after ˜500 hours of operation. The average radiance values are given in Table 3 in arbitrary units (a.u.). The average radiance maintenance of the lamps (500 h radiance/0 h radiance) is given relative to the average radiance maintenance of control lamps.
TABLE 3 Sample Ave. 0 h Ave. 500 h Ave. Rel. (mole %) radiance (a.u.) radiance (a.u.) Maintenance control 8.977 5.815 100.0% 0.02 Hf 8.487 5.216 94.9% 0.5 Hf 9.105 5.545 94.1% 0.02 Si 10.08 6.398 98.1% 0.5 Si 9.512 6.079 98.6% 0.02 Zr 9.352 6.252 103.2 0.5 Zr 9.884 6.987 109.2
[0013] In almost all cases, the average 0 h radiance for the lamps containing the phosphors doped with the tetravalent ions was greater than the average for the lamps containing the control phosphor. Under these tests conditions, the lamps containing the Zr and Si-doped phosphors continued to exhibit a higher radiance than the control lamps after ˜500 hours of operation. In terms of relative radiance maintenance, the lamps coated with the Zr-doped phosphor outperformed the control lamps.
[0014] The data from the various test environments demonstrate that the tetravalent dopants can improve the stability of BAM phosphors under harsh conditions. The performance of the Hf, Si and Zr dopants varied depending on the test environment with the Zr-doped phosphors exhibiting an improved stability under all test conditions.
[0015] While there has been shown and described what are at the present considered the preferred embodiments of the invention, it will be obvious to those skilled in the art that various changes and modifications may be made therein without departing from the scope of the invention as defined by the appended claims.
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A europium-activated barium aluminate phosphor is described wherein the phosphor is doped with tetravalent ions of Hf, Zr, or Si. Preferably, the phosphor is represented by (Ba 1-x Eu x )MgAl 10 O 17 : (Hf, Zr, Si) y where 0.05≦x≦0.25 and 0<y≦0.05. The tetravalent dopant ions are shown to enhance the stability of the phosphor in UV/VUV applications.
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The present application is a Continuation In Part of U.S. patent application Ser. No. 12/610,181 filed Oct. 30, 2009, which application is incorporated in its entirety herein by reference.
BACKGROUND OF THE INVENTION
The present invention relates to coffee makers and in particular to a coffee maker utilizing a stream of hot water through tamped ground coffee.
Coffee is generally prepared in a coffee maker by measuring an amount of ground coffee into a coffee filter, closing a lid over the ground coffee, and providing a stream of hot water through the loosely packed ground coffee. Unfortunately, water passes freely through the loosely packed ground coffee and does not obtain the full flavor which might otherwise be obtained.
U.S. patent application Ser. No. 11/777,831 filed Jul. 13, 2007 for “Pod Adapter System for Single Service Beverage Brewers” by the present applicant overcomes this problem by packaging the coffee in closed filter paper commonly called a pod, and inserting the closed pod into a pod holder including a tamping spring and bottom tamper for tamping the pod between the bottom tamper and a coffee holder lid. While the pod adapter of the '831 application works well for coffee pods, it does not allow the simple use of bulk ground coffee not packaged in the closed pod.
BRIEF SUMMARY OF THE INVENTION
The present invention addresses the above and other needs by providing a self-tamping coffee holder which tamps loose ground coffee obtaining richer flavor. The coffee holder includes a holder base and a holder cap. Coffee is loosely deposited in the coffee holder and the holder cap is attached to the holder base. An internal filter chamber holds the coffee and allows tamping of the coffee into a compacted state. The filter chamber may be formed by a fixed filter or by a removeable filter constructed of filter paper, nylon mesh, metal mesh, or any material capable of holding the coffee while allowing a flow of heated water through the coffee. The tamping may be by a spring or by a resilient solid material attached to the coffee holder and may push the coffee down inside the filter or push the filter and the coffee up against the holder lid
In accordance with one aspect of the invention, there is provided a coffee making system for tamping coffee. The coffee holder receives a portion of untamped coffee and a holder lid closes the coffee holder after receiving the untamped coffee. A tamper resides inside the coffee holder and tamps the coffee after the holder lid is closed. After tamping, the coffee holder is places into a suitable coffee maker. A hot water nozzle is attached to the coffee maker for providing a flow of hot water to the tamped coffee to make a coffee drink.
In accordance with another aspect of the invention, there is provided
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
The above and other aspects, features and advantages of the present invention will be more apparent from the following more particular description thereof, presented in conjunction with the following drawings wherein:
FIG. 1A is a front view of a coffee maker according to the present invention.
FIG. 1B is a side view of the coffee maker according to the present invention.
FIG. 1C is a top view of the coffee maker according to the present invention.
FIG. 2 is a side view of the coffee maker with an open lid allowing placement of a coffee holder according to the present invention inside the coffee maker.
FIG. 2A is a functional diagram of the coffee maker.
FIG. 3 is a side view of a first coffee holder according to the present invention.
FIG. 4 is a cross-sectional side view of the first coffee holder according to the present invention taken along line 4 - 4 of FIG. 3 .
FIG. 5A is a cross-sectional side view of the first coffee holder according to the present invention taken along line 4 - 4 of FIG. 3 showing an empty coffee holder with the tamping spring and the bottom tamper according to the present invention.
FIG. 5B is a cross-sectional side view of the first coffee holder according to the present invention taken along line 4 - 4 of FIG. 3 showing the coffee holder with the tamping spring and bottom tamper, a portion of coffee, and the holder lid ready to attach to a holder body according to the present invention.
FIG. 5C is a cross-sectional side view of the first coffee holder according to the present invention taken along line 4 - 4 of FIG. 3 showing the coffee holder with the tamping spring and bottom tamper, the portion of coffee in the coffee holder, and the holder lid ready to attach to the holder body according to the present invention.
FIG. 5D is a cross-sectional side view of the first coffee holder according to the present invention taken along line 4 - 4 of FIG. 3 showing the coffee holder with the tamping spring and bottom tamper, the portion of coffee in the coffee holder, and the holder lid attached to the coffee holder body, according to the present invention.
FIG. 6 is a top view of the first holder lid.
FIG. 7A is a side view of a filter paper cup according to the present invention.
FIG. 7B is a top view of the filter paper cup according to the present invention.
FIG. 7C is a second embodiment of the filter paper cup with a lid.
FIG. 8A shows the first coffee holder ready for insertion into the coffee maker.
FIG. 8B shows the first coffee holder inserted into the coffee maker before tamping the coffee.
FIG. 8C shows the first coffee holder inserted into the coffee maker after tamping the coffee.
FIG. 9 is a side view of a second coffee holder according to the present invention.
FIG. 10 is a cross-sectional side view of the second coffee holder according to the present invention taken along line 10 - 10 of FIG. 9 .
FIG. 11A is a cross-sectional side view of the second coffee holder according to the present invention taken along line 10 - 10 of FIG. 9 showing an empty coffee holder with the tamping spring and the top tamper according to the present invention.
FIG. 11B is a cross-sectional side view of the second coffee holder according to the present invention taken along line 10 - 10 of FIG. 9 showing the coffee holder with the holder lid, tamping spring and top tamper, and a portion of coffee, ready to attach according to the present invention.
FIG. 11C is a cross-sectional side view of the second coffee holder according to the present invention taken along line 10 - 10 of FIG. 9 showing the portion of untamped coffee in the coffee holder, and the holder lid, tamping spring and top tamper, ready to attach to the holder base, according to the present invention.
FIG. 11D is a cross-sectional side view of the second coffee holder according to the present invention taken along line 10 - 10 of FIG. 9 showing the portion of coffee in the coffee holder and the tamping spring, top tamper, and the holder lid attached to the coffee holder, according to the present invention.
FIG. 12 is a top view of the second holder lid.
FIG. 13 is a top view of the bottom tamper.
FIG. 14A shows the second coffee holder ready for insertion into the coffee maker.
FIG. 14B shows the second coffee holder inserted into the coffee maker before tamping the coffee.
FIG. 14C shows the second coffee holder inserted into the coffee maker after tamping the coffee.
FIG. 15 is a side view of a third coffee holder according to the present invention.
FIG. 16 is a cross-sectional side view of the third coffee holder according to the present invention taken along line 16 - 16 of FIG. 15 .
FIG. 17A is a cross-sectional side view of the third coffee holder according to the present invention taken along line 16 - 16 of FIG. 15 showing the portion of coffee above the coffee holder and the top tamper and the holder lid ready to attach to the coffee holder, according to the present invention.
FIG. 17B is a cross-sectional side view of the third coffee holder according to the present invention taken along line 16 - 16 of FIG. 15 showing the portion of coffee in the coffee holder, and the top tamper and the holder lid ready to attach to the coffee holder, according to the present invention.
FIG. 17C is a cross-sectional side view of the third coffee holder according to the present invention taken along line 16 - 16 of FIG. 15 showing the portion of coffee in the coffee holder, and the bottom tamper, the top tamper, and the holder lid attached to the coffee holder, according to the present invention.
FIG. 18A shows the third coffee holder ready for insertion into a second coffee maker according to the present invention.
FIG. 18B shows the third coffee holder inserted into the coffee maker before tamping the coffee.
FIG. 18C shows the third coffee holder inserted into the coffee maker after tamping the coffee.
FIG. 19 is a side view of a fourth coffee holder according to the present invention.
FIG. 20 is a cross-sectional side view of the fourth coffee holder according to the present invention taken along line 20 - 20 of FIG. 19 .
FIG. 21A is a cross-sectional side view of the fourth coffee holder according to the present invention taken along line 20 - 20 of FIG. 19 showing the coffee holder with the bottom tamper, a portion of coffee, and the holder lid ready to attach, according to the present invention.
FIG. 21B is a cross-sectional side view of the fourth coffee holder according to the present invention taken along line 20 - 20 of FIG. 19 showing the coffee holder with the bottom tamper, a portion of coffee in the coffee holder, and the holder lid ready to attach, according to the present invention.
FIG. 21C is a cross-sectional side view of the fourth coffee holder according to the present invention taken along line 20 - 20 of FIG. 19 showing the coffee holder with the bottom tamper, a portion of coffee in the coffee holder, and the holder lid attached, according to the present invention.
FIG. 22A shows the fourth coffee holder ready for insertion into the coffee maker.
FIG. 22B shows the fourth coffee holder inserted into the coffee maker before tamping the coffee.
FIG. 22C shows the fourth coffee holder inserted into the coffee maker after tamping the coffee.
FIG. 23A shows the fourth coffee holder ready for insertion into the coffee maker having a tamping block according to the present invention.
FIG. 23B shows the fourth coffee holder inserted into the coffee maker having the tamping block before tamping the coffee.
FIG. 23C shows the fourth coffee holder inserted into the coffee maker having the tamping block after tamping the coffee.
FIG. 24 is a side view of a fifth coffee holder according to the present invention.
FIG. 25 is a cross-sectional side view of the fifth coffee holder according to the present invention taken along line 25 - 25 of FIG. 24 .
FIG. 26A is a cross-sectional side view of the fifth coffee holder according to the present invention taken along line 25 - 25 of FIG. 24 showing the portion of coffee above the coffee holder body, and the holder lid with the top tamper and tamping spring, ready to attach to the coffee holder body, according to the present invention.
FIG. 26B is a cross-sectional side view of the fifth coffee holder according to the present invention taken along line 25 - 25 of FIG. 24 showing the coffee holder with the portion of coffee in the coffee holder, and the holder lid with the top tamper and tamping spring ready to attach to the coffee holder body, according to the present invention.
FIG. 26C is a cross-sectional side view of the fifth coffee holder according to the present invention taken along line 25 - 25 of FIG. 24 showing the portion of coffee in the coffee holder, and the holder lid with the top tamper and tamping spring attached to the holder body, according to the present invention.
FIG. 27 is a side view of a sixth coffee holder according to the present invention.
FIG. 28 is a cross-sectional side view of the sixth coffee holder according to the present invention taken along line 28 - 28 of FIG. 27 .
FIG. 29A is a cross-sectional side view of the sixth coffee holder according to the present invention taken along line 28 - 28 of FIG. 27 showing the portion of coffee above the coffee holder, and the holder lid ready to attach to the holder body, according to the present invention.
FIG. 29B is a cross-sectional side view of the sixth coffee holder according to the present invention taken along line 28 - 28 of FIG. 27 showing the portion of coffee in the coffee holder, and the holder lid ready to attach to the holder body, according to the present invention.
FIG. 29C is a cross-sectional side view of the sixth coffee holder according to the present invention taken along line 28 - 28 of FIG. 27 showing the portion of coffee in the coffee holder, and the holder lid attached and tamping the coffee, according to the present invention.
FIG. 30 is a side view of a seventh coffee holder according to the present invention.
FIG. 31 is a cross-sectional side view of the seventh coffee holder according to the present invention taken along line 31 - 31 of FIG. 30 .
FIG. 32A is a cross-sectional side view of the seventh coffee holder according to the present invention taken along line 31 - 31 of FIG. 30 showing the portion of coffee above the coffee holder, and the holder lid ready to attach to the holder body, according to the present invention.
FIG. 32B is a cross-sectional side view of the seventh coffee holder according to the present invention taken along line 31 - 31 of FIG. 30 showing the portion of coffee in the coffee holder, and the holder lid ready to attach to the holder body, according to the present invention.
FIG. 32C is a cross-sectional side view of the seventh coffee holder according to the present invention taken along line 31 - 31 of FIG. 30 showing the portion of coffee in the coffee holder, and the holder lid attached to the holder body and the coffee tamped between the bottom tamper and spring and the holder lid, according to the present invention.
FIG. 33 is a side view of an eighth coffee holder according to the present invention.
FIG. 34A is a cross-sectional side view of the eighth coffee holder taken along line 34 - 34 of FIG. 33 showing a portion of coffee for placing inside the coffee holder and the holder lid with an insertable portion and an O-Ring inside the coffee holder for sealing according to the present invention.
FIG. 34B is a cross-sectional side view of the eighth coffee holder taken along line 34 - 34 of FIG. 33 showing the portion of coffee inside the coffee holder and the holder lid with the insertable portion inserted into the coffee holder and cooperating with the O-Ring inside the coffee holder for sealing.
FIG. 35 is a side view of a ninth coffee holder according to the present invention.
FIG. 36A is a cross-sectional side view of the ninth coffee holder taken along line 36 - 36 of FIG. 35 showing a portion of coffee for placing inside the coffee holder and a holder lid with a threaded portion for screwing inside the holder body for sealing according to the present invention.
FIG. 36B is a cross-sectional side view of the ninth coffee holder taken along line 36 - 36 of FIG. 35 showing the portion of coffee inside the coffee holder and a holder lid with the threaded portion screwed into the holder body and tamping the coffee according to the present invention.
FIG. 37A shows a third coffee maker having a coffee holder for receiving a portion of coffee and tamping spring according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 37B shows the third coffee maker with the coffee holder holding the portion of coffee and the tamping spring under the coffee holder according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 37C shows the third coffee maker with the coffee holder holding the portion of tamped coffee with the coffee maker lid closed for tamping the coffee according to the present invention.
FIG. 38A shows a third coffee maker having a coffee holder for receiving a portion of coffee and tamping spring attached to the coffee maker lid according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 38B shows the third coffee maker with the coffee holder holding the portion of untamped coffee according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 38C shows the third coffee maker with the coffee holder holding the portion of tamped coffee with the coffee maker lid closed to push the tamping spring into the coffee holder for tamping the coffee according to the present invention.
FIG. 39A shows a fourth coffee maker having a coffee holder for receiving a packet containing untamped coffee, a knife for cutting the packet open, and tamping spring attached to the coffee maker lid according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 39B shows the fourth coffee maker with the coffee holder holding the packet of untamped coffee according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 39C shows the fourth coffee maker with the coffee holder holding the packet of tamped coffee with the coffee maker lid closed to push the tamping spring into the coffee holder for tamping the coffee according to the present invention.
FIG. 40A shows a fifth coffee maker accepting a horizontal coffee holder and tamping spring residing horizontally in a coffee holder cavity according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 40B shows the fifth coffee maker with the coffee holder residing horizontally in the coffee holder cavity according to the present invention for tamping the coffee when the coffee maker lid is closed.
FIG. 40C shows the fifth coffee maker with the coffee holder residing horizontally in the coffee holder cavity with the coffee maker lid closed and the coffee holder pushed against the tamping spring for tamping the coffee, according to the present invention.
FIG. 41 is a side view of a tenth coffee holder with straight walls according to the present invention.
FIG. 42 is a cross-sectional view of the tenth coffee holder taken along line 42 - 42 of FIG. 41 showing an empty coffee holder.
FIG. 43 is a cross-sectional view of the tenth coffee holder taken along line 42 - 42 of FIG. 41 showing a full and tamped coffee holder.
FIG. 44 is a side view of an eleventh coffee holder with straight walls according to the present invention.
FIG. 45 is a cross-sectional view of the eleventh coffee holder taken along line 45 - 45 of FIG. 44 showing an empty coffee holder.
FIG. 46 is a cross-sectional view of the eleventh coffee holder taken along line 45 - 45 of FIG. 41 showing a full and tamped coffee holder.
FIG. 47A is a side view of a top tamper.
FIG. 47B is a top view of the top tamper.
FIG. 47C is a side view of a top tamper with a seal according to the present invention.
FIG. 47D is a top view of the top tamper with a seal.
FIG. 48 is a perspective view of a filter paper cup with a folding cup lid.
FIG. 49 is a side view of an twelfth coffee holder with straight walls according to the present invention.
FIG. 50 is a cross-sectional view of the twelfth coffee holder taken along line 50 - 50 of FIG. 49 showing an empty coffee holder.
FIG. 51A is a cross-sectional view of the twelfth coffee holder taken along line 50 - 50 of FIG. 49 showing a lid, coffee, a filter paper cup, above the base, and the coffee holder base.
FIG. 51B is a cross-sectional view of the twelfth coffee holder taken along line 50 - 50 of FIG. 49 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base.
FIG. 51C is a cross-sectional view of the twelfth coffee holder taken along line 50 - 50 of FIG. 49 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base with a filter paper cover folded over the coffee in the filter paper cup.
FIG. 51D is a cross-sectional view of the twelfth coffee holder taken along line 50 - 50 of FIG. 49 showing the lid attached to the base with the coffee and the filter paper cup residing in the coffee holder base with the coffee tamped.
FIG. 52 is a side view of a thirteenth coffee holder with a releaseable tamping latch according to the present invention.
FIG. 53 is a cross-sectional view of the thirteenth coffee holder taken along line 53 - 53 of FIG. 52 showing an empty coffee holder.
FIG. 54A is a cross-sectional view of the thirteenth coffee holder taken along line 53 - 53 of FIG. 52 showing a lid, coffee, a filter paper cup, above the base, and the coffee holder base, with the tamping latch retaining the bottom tamper.
FIG. 54B is a cross-sectional view of the thirteenth coffee holder taken along line 53 - 53 of FIG. 52 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base, with the tamping latch retaining the bottom tamper.
FIG. 54C is a cross-sectional view of the thirteenth coffee holder taken along line 53 - 53 of FIG. 52 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base with the tamping latch retaining the bottom tamper.
FIG. 54D is a cross-sectional view of the fourteenth coffee holder taken along line 53 - 53 of FIG. 52 showing the lid attached to the base with the coffee and the filter paper cup residing in the coffee holder base with tamping latch released and the coffee tamped.
FIG. 55 is a side view of a fourteenth coffee holder with a releaseable tamping latch according to the present invention.
FIG. 56 is a cross-sectional view of the fourteenth coffee holder taken along line 56 - 56 of FIG. 55 showing an empty coffee holder.
FIG. 57A is a cross-sectional view of the fourteenth coffee holder taken along line 56 - 56 of FIG. 55 showing a lid, coffee, a filter paper cup, above the base, and the coffee holder base, with the tamping latch retaining the bottom tamper.
FIG. 57B is a cross-sectional view of the fourteenth coffee holder taken along line 56 - 56 of FIG. 55 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base, with the tamping latch retaining the bottom tamper.
FIG. 57C is a cross-sectional view of the fourteenth coffee holder taken along line 56 - 56 of FIG. 55 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base, with the tamping latch released but just prior to tamping.
FIG. 57D is a cross-sectional view of the fourteenth coffee holder taken along line 56 - 56 of FIG. 55 showing the lid attached to the base with the coffee and the filter paper cup residing in the coffee holder base with tamping latch released and the coffee tamped.
FIG. 58 is a side view of a fourteenth coffee holder with a releaseable tamping lock according to the present invention.
FIG. 59 is a cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing an empty coffee holder.
FIG. 60A is a cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing a lid, coffee, a filter paper cup, above the base, and the coffee holder base, with the tamping lock retaining the bottom tamper.
FIG. 60B is a cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base, with the tamping lock retaining the bottom tamper.
FIG. 60C is a cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base prior to tamping.
FIG. 60D is a cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing the lid attached to the base with the coffee and the filter paper cup residing in the coffee holder base with tamping lock released and the coffee tamped.
FIG. 61 is a top view of a lock according to the present invention.
FIG. 62 is a bottom view of a second bottom tamper with cooperates with the tamping lock according to the present invention.
FIG. 63A is a side view of a filter cup according to the present invention.
FIG. 63B is a top view of the filter cup according to the present invention.
Corresponding reference characters indicate corresponding components throughout the several views of the drawings.
DETAILED DESCRIPTION OF THE INVENTION
The following description is of the best mode presently contemplated for carrying out the invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of describing one or more preferred embodiments of the invention. The scope of the invention should be determined with reference to the claims.
A front view of a coffee 10 maker according to the present invention is shown in FIG. 1A a side view of the coffee maker 10 is shown in FIG. 1B , and a top view of the coffee maker 10 is shown in FIG. 1C . The coffee maker 10 includes a body 12 , an opening lid 14 , a lid handle 16 , a water container 18 , a display 20 , controls 22 , and platform 24 . A coffee pitcher 26 rests on the platform 24 and has a pitcher lid 28 . The coffee maker 10 provides a flow of hot water through coffee grounds to produce a coffee drink. The flow of water may be heated by one of any known means, for example, an electrical heating coil or a conductive coating on tubing carrying the water.
A side view of the coffee maker 10 with an open lid 14 allowing placement of a coffee holder 30 according to the present invention inside the coffee maker 10 is shown in FIG. 2 . The lid 14 includes a lid hinge 14 a and a water tube 15 carries heated water into the lid 14 . A pad 17 resides on a bottom surface of the lid 14 and presses against the coffee holder 30 when the lid 14 is closed, and in cooperation with other means discloses hereafter, tamps coffee contained in the coffee holder 30 . A nozzle 19 extending down from the closed lid 14 directs the flow of hot water into the coffee holder 30 .
A functional diagram of the coffee maker 10 is shown in FIG. 2A . The preferred coffee maker 10 includes the water tank 18 , water pump 21 , a heater 13 , check valve 23 and the nozzle 18 . The pump 21 preferably provides at least one PSI water pressure. The water heater 13 may include a heating coil or a resistive coating or any other means for heating water. The check valve 23 limits the water pressure at the nozzle 19 by returning some of the water flow to the water tank 18 . While a the water pump 21 is a preferred method for providing a flow of water to the nozzle 19 , other methods include pressuring the water in the water tank 18 , and a coffee maker using any means to provide a forced flow of water is intending to within the scope of the present invention.
A side view of a first coffee holder 30 a according to the present invention is shown in FIG. 3 and a cross-sectional side view of the first coffee holder 30 a including a holder body 31 , a first holder lid 32 a , a bottom tamper 34 , and a tamping spring 36 according to the present invention taken along line 4 - 4 of FIG. 3 is shown in FIG. 4 . A volume (or coffee holder interior) 38 is provided inside the coffee holder 30 a to receive loose coffee 41 . A passage 33 in the lid 32 a is provided for the nozzle 19 (see FIG, 2 ).
A cross-sectional side view of the first coffee holder 30 a taken along line 4 - 4 of FIG. 3 showing an empty coffee holder 30 a with the tamping spring 36 and the bottom tamper 34 ready for filling are shown in FIG. 5A . A cross-sectional side view of the first coffee holder 30 a taken along line 4 - 4 of FIG. 3 showing the coffee holder 30 a with the tamping spring 36 and bottom tamper 34 , a portion of loose coffee 41 , and the holder lid 32 a ready to attach is shown in FIG. 5B . A cross-sectional side view of the first coffee holder 30 a taken along line 4 - 4 of FIG. 3 showing the coffee holder 30 a with the tamping spring 36 and bottom tamper 34 , a portion of coffee in the volume 38 , and the holder lid 32 a ready to attach is shown in FIG. 5C . A cross-sectional side view of the first coffee holder 30 a taken along line 4 - 4 of FIG. 3 showing the coffee holder 30 a with the tamping spring 36 and bottom tamper 34 , a portion of loose coffee 41 in the volume 38 , and the holder lid 32 a attached to the coffee holder 30 a , is shown in FIG. 5D .
A top view of the first holder lid 32 a showing the passage 33 provided for the nozzle 19 (see FIG. 2 ) is shown in FIG. 6 .
A side view of a filter paper cup 40 according to the present invention is shown in FIG. 7A and a top view of the filter paper cup 40 is shown in FIG. 7B . The filter paper cup 40 includes a bottom 40 b , sides 40 a , and a rim 40 c . The rim 40 c rests on a top edge of the holder body 31 and is held between the holder cap and body when the cap is placed on the body, thereby preventing or restricting the escape of coffee 41 from the cup 40 when hot water flows into the coffee holder 30 a.
A second embodiment of the filter paper cup 40 ′ with a folding paper lid 40 d is shown in FIG. 7C . The lid 40 d of the filter paper cup 40 ′ may be folded over the cup 40 ′ after loose coffee is poured into the cup. The lid 40 d preferably includes a perforation 40 e centered on the lid 40 d allowing the nozzle 19 to enter and/or inject the hot flow of water into the tamped coffee 41 .
The filter cups may be made from several materials including filter paper, nylon mesh, steel mesh, or any material suitable for filtration.
The first coffee holder 30 a is shown ready for insertion into a first coffee maker 10 a in FIG. 8A , the first coffee holder 30 a is shown inserted into the coffee maker 10 before tamping the coffee 41 in FIG. 8B , and the first coffee holder 30 a is shown in the coffee maker 10 after tamping the coffee 41 in FIG. 8C . The coffee maker includes a cavity 11 for accepting the coffee holder and has walls 11 a for aligning the coffee holder in the coffee maker. When the lid 14 is closed, the pad 17 on the bottom of the lid 14 and/or arms 25 attached to the bottom of the lid 25 , push the coffee holder 30 a down over the tamping spring 36 and the coffee 41 is tamped between the lid 32 a and the bottom tamper 34 . The arms 25 push the coffee holder 30 a down ahead of the nozzle 19 thereby seating the coffee holder 30 a in the cavity 11 for alignment of the nozzle 10 with the passage 33 in the lid 32 a.
A side view of a second coffee holder 30 b according to the present invention is shown in FIG. 9 and a cross-sectional side view of the second coffee holder 30 b taken along line 10 - 10 of FIG. 9 is shown in FIG. 10 . The coffee holder 30 b includes the holder body 31 , a second holder lid 32 b , a tamping spring 36 , a spring washer 35 a , and a top tamper 35 b.
A cross-sectional side view of the second coffee holder 30 b taken along line 10 - 10 of FIG. 9 showing an empty coffee holder 30 b is shown in FIG. 11A . A cross-sectional side view of the second coffee holder 30 b taken along line 10 - 10 of FIG. 9 showing the holder lid 32 b and a loose portion of coffee 41 above the empty coffee holder 30 b is shown in FIG. 11B . A cross-sectional side view of the second coffee holder 30 b taken along line 10 - 10 of FIG. 9 showing the holder lid 32 b above the coffee holder 30 b with the portion of loose coffee 41 in the coffee holder 32 b is shown in FIG. 11C . A cross-sectional side view of the second coffee holder 32 b taken along line 10 - 10 of FIG. 9 showing the coffee holder with the holder lid 32 b attached to the coffee holder 30 b and a portion of loose coffee 41 in the coffee holder 30 b is shown in FIG. 11D . The tamping spring 36 extends upward out of the coffee holder 30 b for tamping the loose coffee as disclosed hereafter.
A top view of the second holder lid 32 b is shown in FIG. 12 . The holder lid 32 b includes a larger passage 33 a allowing passage of the tamping spring 36 through the holder lid 32 b.
A top view of the bottom tamper 34 is shown in FIG. 13 . The bottom tamper 34 includes perforations 34 a to allow coffee drink to pass through the bottom tamper 34 .
The second coffee holder 30 b is shown ready for insertion into the coffee maker 10 in FIG. 14A , the second coffee holder 30 b is shown inserted into the coffee maker 10 before tamping the coffee 41 in FIG. 14B , and the second coffee holder 30 b is shown in the coffee maker 10 after tamping the coffee 41 in FIG. 14C . The coffee maker 10 may include a long nozzle 19 a to reach the top tamper 35 b for “injection” of the heated water into the tamped coffee, but may also include the nozzle 19 and the heated water may pass through the coffee 41 under the pull of gravity.
A side view of a third coffee holder 30 c according to the present invention is shown in FIG. 16 and a cross-sectional side view of the third coffee holder 30 c taken along line 16 - 16 of FIG. 15 is shown in FIG. 16 . The coffee holder 30 c includes the holder body 31 , the second holder lid 32 b , the bottom tamper 34 , and the top tamper 35 b.
A cross-sectional side view of the third coffee holder 30 c taken along line 16 - 16 of FIG. 15 showing the coffee holder 30 c with the holder lid 32 b , the top tamper 35 b , and a portion of coffee, ready to attach to the holder 31 , is shown in FIG. 17A . A cross-sectional side view of the third coffee holder taken along line 16 - 16 of FIG. 15 showing the coffee holder 30 c with the holder lid 32 b and the top tamper ready to attach, and a portion of coffee 41 in the coffee holder, is shown in FIG. 17B . A cross-sectional side view of the third coffee holder 30 c taken along line 16 - 16 of FIG. 15 showing the coffee holder with the holder lid and the top tamper attached and a loose portion of coffee 41 in the coffee holder is shown in FIG. 17C . The coffee holder 30 c is configured to use with a coffee make 10 b (see FIGS. 18A-18C ) including apparatus for entering the coffee holder for tamping the loose coffee 41 .
The third coffee holder 30 c ready for insertion into a second coffee maker 10 b in FIG. 18A , the third coffee holder 30 c is shown residing in the coffee maker 10 b before tamping the coffee 41 in FIG. 18B , and the third coffee holder 30 c is shown residing in the coffee maker 10 b after tamping the coffee 41 in FIG. 18C . The coffee maker 10 b includes the tamping spring 36 attached to the pad 17 on the bottom of the lid 14 . When the lid 14 is closed the tamping spring 36 enters the coffee holder 30 c through the lid passage 33 a (see FIG. 12 ) and pushes the top tamper 35 b against the coffee 41 to tamp the coffee 41 .
A side view of a fourth coffee holder 30 d according to the present invention is shown in FIG. 19 and a cross-sectional side view of the fourth coffee holder 30 d taken along line 20 - 20 of FIG. 19 is shown in FIG. 20 . The coffee holder 30 d includes the holder body 31 , the first holder lid 32 a , and the bottom tamper 34 .
A cross-sectional side view of the fourth coffee holder 30 d taken along line 20 - 20 of FIG. 19 showing the coffee holder with the bottom tamper 34 , and a portion of coffee 41 and the holder lid ready to attach is shown in FIG. 21A . A cross-sectional side view of the fourth coffee holder 30 d taken along line 20 - 20 of FIG. 19 showing the coffee holder 30 d with the bottom tamper 34 , the portion of coffee 41 in the coffee holder 30 d , and the holder lid 32 a ready to attach is shown in FIG. 21B . A cross-sectional side view of the fourth coffee holder 30 d taken along line 20 - 20 of FIG. 19 showing the coffee holder 30 d with the bottom tamper 34 , a portion of coffee in the coffee holder 41 , and the holder lid 32 a is shown in FIG. 21C .
The fourth coffee holder 30 d ready for insertion into another embodiment of the second coffee maker 10 b in FIG. 22A , the fourth coffee holder 30 d is shown residing in the coffee maker 10 b before tamping the coffee 41 in FIG. 22B , and the fourth coffee holder 30 d is shown residing in the coffee maker 10 b after tamping the coffee 41 in FIG. 22C . The coffee maker 10 b may include the tamping spring 36 residing in the bottom of the coffee holder cavity 11 . When the lid 14 is closed, the pad 17 pushed the coffee holder 30 d down over the tamping spring 36 and the tamping spring 36 enters the coffee holder 30 c through the bottom of the holder body 31 and pushes the bottom tamper 34 against the coffee 41 to tamp the coffee 41 .
The fourth coffee holder 30 d ready for insertion into another embodiment of the second coffee maker 10 b in FIG. 23A , the fourth coffee holder 30 d is shown residing in the coffee maker 10 b before tamping the coffee 41 in FIG. 23B , and the fourth coffee holder 30 d is shown residing in the coffee maker 10 b after tamping the coffee 41 in FIG. 23C . The coffee maker 10 b may include a resilient solid block 42 residing in the bottom of the coffee holder cavity 11 . When the lid 14 is closed, the pad 17 pushed the coffee holder 30 d down over the resilient solid block 42 and the resilient solid block 42 enters the coffee holder 30 c through the bottom of the holder body 31 and pushes the bottom tamper 34 against the coffee 41 to tamp the coffee 41 .
A side view of a fifth coffee holder 30 e according to the present invention is shown in FIG. 24 and a cross-sectional side view of the fifth coffee holder 30 e taken along line 25 - 25 of FIG. 24 is shown in FIG. 25 . The fifth coffee holder 30 e includes the holder body 31 , the holder lid 32 b , the tamping spring 36 and the top tamper 35 b attached to the holder lid 32 a.
A cross-sectional side view of the fifth coffee holder 30 e taken along line 25 - 25 of FIG. 24 showing the coffee holder 30 e with a portion of coffee 41 , and the holder lid 32 b with the top tamper 35 b and tamping spring 36 attached, above the holder body 31 , is shown in FIG. 26A . A cross-sectional side view of the fifth coffee holder 30 e taken along line 25 - 25 of FIG. 24 showing the coffee holder with the portion of coffee 41 in the coffee holder, and the holder lid 32 b with the top tamper 35 b and tamping spring 36 attached, above the holder body 31 , is shown in FIG. 26B . A cross-sectional side view of the fifth coffee holder 30 e taken along line 25 - 25 of FIG. 24 showing the coffee holder 30 e with the portion of coffee 41 in the coffee holder 30 e , and the holder lid 32 b with the top tamper 35 b and tamping spring 36 attached to the holder base 31 is shown in FIG. 26D . The tamping spring 36 and top tamper 35 b tamp the coffee 41 to provide a tamped coffee when the holder lid 32 b is attached to the holder base 31 .
A side view of a sixth coffee holder 30 f according to the present invention is shown in FIG. 27 and a cross-sectional side view of the sixth coffee holder 30 f taken along line 28 - 28 of FIG. 27 is shown in FIG. 28 . The sixth coffee holder 30 f includes the holder body 31 and a third holder lid 32 c . The third holder lid 32 c includes a recessed portion 32 ′ which reaches into the interior of the sixth coffee holder 30 f . The recessed portion 32 ′ is preferably a solid resilient material.
A cross-sectional side view of the sixth coffee holder 30 f taken along line 28 - 28 of FIG. 27 showing the sixth coffee holder 30 f with a portion of coffee 41 , and the holder lid 32 c , above the holder body 31 , is shown in FIG. 29A . A cross-sectional side view of the sixth coffee holder 30 f taken along line 28 - 28 of FIG. 27 showing the coffee holder with the portion of coffee 41 in the coffee holder, and the holder lid 32 c above the holder body 31 , is shown in FIG. 29B . A cross-sectional side view of the sixth coffee holder 30 f along line 28 - 28 of FIG. 27 showing the sixth coffee holder 30 f with the portion of coffee 41 in the coffee holder 30 e , and the holder lid 32 e attached to the holder base 31 is shown in FIG. 26D . A cushion 32 ′ tamps the coffee 41 to provide a tamped coffee when the holder lid 32 e is attached to the holder base 31 . The cushion 32 ′ is preferably made from a resilient material to cushion the tamping of the loose coffee.
A side view of a seventh coffee holder 30 g according to the present invention is shown in FIG. 30 and a cross-sectional side view of the seventh coffee holder 30 g taken along line 31 - 31 of FIG. 30 is shown in FIG. 31 . The seventh coffee holder 30 g includes the holder body 31 , the holder lid 32 b , the tamping spring 36 , and the bottom tamper 34 inside the holder base 31 .
A cross-sectional side view of the seventh coffee holder 30 g taken along line 31 - 31 of FIG. 30 showing the seventh coffee holder 30 g with a portion of coffee 41 and the holder lid 32 a above the holder body 31 , and with the bottom tamper 34 and tamping spring 36 inside the holder base 31 , is shown in FIG. 26A . A cross-sectional side view of the seventh coffee holder 30 g taken along line 31 - 31 of FIG. 30 showing the coffee holder with the portion of coffee 41 in the filter paper 40 in the holder base 31 resting on the bottom tamper 34 supported by the tamping spring 36 , and the holder lid 32 a above the holder body 31 , is shown in FIG. 26B . A cross-sectional side view of the seventh coffee holder 30 g taken along line 31 - 31 of FIG. 30 showing the seventh coffee holder 30 g with the portion of coffee 41 in the coffee holder 30 e , and the holder lid 32 a attached to the holder base 31 , is shown in FIG. 26D . The tamping spring 36 and bottom tamper 34 tamp the coffee 41 upward against the tamper lid 32 a to provide a tamped coffee when the holder lid 32 a is attached to the holder base 31 .
A side view of an eighth coffee holder 30 h according to the present invention is shown in FIG. 33 , a cross-sectional side view of the eighth coffee holder 30 h taken along line 34 - 34 of FIG. 33 showing a portion of coffee 41 for placing inside the coffee holder and a fourth holder lid 32 d with an insertable portion and an O-Ring 50 inside the coffee holder for sealing is shown in FIG. 34A , and a cross-sectional side view of the eighth coffee holder taken along line 34 - 34 of FIG. 33 showing the portion of coffee 41 inside the coffee holder 30 h and the holder lid 32 d with the insertable portion inserted into the coffee holder base 31 a is shown in FIG. 34B . The filter paper 40 extends up above the O-ring 50 and the O-Ring 50 cooperates with the holder lid 32 d to sandwich the top edge of the filter paper 40 for sealing the filter paper 40 to reduce or prevent the coffee 41 from escaping when the flow of hot water is provided to the coffee holder 30 h . The holder base 31 a is preferably cylindrical but may also be conical in shape.
A side view of a ninth coffee holder 30 i according to the present invention is shown in FIG. 35 , a cross-sectional side view of the ninth coffee holder 30 i taken along line 36 - 36 of FIG. 35 showing a portion of coffee 41 for placing inside the coffee holder and a fifth holder lid 32 e with a threaded portion for screwing inside the holder base 31 b for sealing is shown in FIG. 36A , and a cross-sectional side view of the ninth coffee holder 30 i taken along line 36 - 36 of FIG. 35 showing the portion of coffee 41 inside the coffee holder and the holder lid 32 e with the threaded portion screwed into the coffee holder and tamping the coffee 41 is shown in FIG. 36B . The threads both provide tamping and sealing the coffee to reduce or prevent the coffee 41 from escaping when the flow of hot water is provided to the coffee holder 30 h . The holder base 31 b is preferably cylindrical to facilitate having internal threads, and at least the threaded portion is preferably cylindrical.
A third coffee maker 10 c having a coffee holder 30 according to the present invention for receiving a portion of coffee and a tamping spring 36 for tamping the coffee is shown in FIG. 37A , the third coffee maker 10 c with the coffee holder 30 holding the portion of coffee 41 is shown in FIG. 37B , and the third coffee maker 10 c with the coffee holder 30 holding the portion of coffee 41 with the coffee maker lid 14 closed for tamping the coffee 41 is shown in FIG. 37C . When the lid 14 is closed, the pad 17 pushes the coffee holder 30 down and the tamping spring 36 enters the bottom of the coffee holder 30 to tamp the coffee 41 . While attaching the lid 32 a to the holder 30 is preferred in order to prevent coffee grounds from escaping the holder 30 , the coffee maker 10 c may also be used without the lid 32 a and the pad 17 may serve to seal the coffee 41 in the holder 30 . In this instance, the coffee maker lid 14 serves as a coffee holder lid.
A third coffee maker 10 c having a coffee holder for receiving a portion of coffee and tamping spring 36 attached to the coffee maker lid 14 according to the present invention for tamping the coffee 41 when the coffee maker lid 14 is closed is shown in FIG. 38A , the third coffee maker with the coffee holder 30 holding the portion of coffee 41 is shown in FIG. 38B , and the third coffee maker 10 c with the coffee holder 30 holding the portion of coffee 41 with the coffee maker lid 14 closed to push the tamping spring 36 into the coffee holder 30 for tamping the coffee 41 is shown in FIG. 38C .
A fourth coffee maker 10 d having a third holder base 31 c for receiving a packet 41 a containing untamped coffee, a knife 50 for cutting the packet 41 a open, and tamping spring 36 under the holder base 31 c according to the present invention for tamping the coffee when the coffee maker lid is closed is shown in FIG. 39A , the fourth coffee maker 10 d with the holder base 31 c holding the packet 41 a of untamped coffee is shown in FIG. 39B , and fourth coffee maker with the holder base 31 c holding the packet of tamped coffee 41 c with the coffee maker lid 14 closed to push the holder base down over the tamping spring 36 for tamping the coffee is shown in FIG. 39C . The coffee maker 10 d includes a somewhat pointed nozzle 19 a to puncture the packet 41 a to provide the flow of hot water to the tamped coffee in the packet 41 a . Known coffee packets include internal filters to allow a flow of hot water through the packet to make the coffee drink while preventing coffee grounds from escaping. The cut in the packet 41 a made by the knife 50 allows the coffee drink to escape from the packet while filter material in the packet 41 a prevent coffee grounds from escaping. The tamping spring 36 may also be attached to the lid 14 as in FIGS. 38A-38C .
The packet 41 a may be an air tight pod containing coffee in filter paper and positioning the knife on the side of the holder base 31 c results in less likelihood of the knife 50 cutting the filter paper. The packet 41 a is preferably air tight to maintain coffee freshness and may be plastic, metal foil, or other air tight material which is sufficiently flexible to allow the coffee contained in the packet 41 a to be tamped. Alternatively, the knife 50 may be eliminated when the packet 41 a is configured to burst under pressure to expose the coffee, for example, when the coffee maker tamps the coffee, the packet 41 a also bursts. In one embodiment, filter paper 41 is inserted into the holder base 31 c without the knife 50 , and the packet 41 a bursts during compacting to release the coffee into the filter paper.
Known coffee makers use a sealed cup or capsule having a somewhat ridged cup with a foil cover. Such cups might be compressible and used in the coffee maker 10 d , however, a similar cup or capsule having a less ridged cup which may be compressed in the coffee maker 10 d are more suitable for use in the coffee maker 10 d to allow tamping of the coffee contained in the cup or capsule.
A fifth coffee maker 10 e for horizontally receiving the coffee holder 30 is shown in FIG. 40A , the fifth coffee maker with the coffee holder 30 residing in the coffee maker is shown in FIG. 40B , and the fifth coffee maker with the coffee maker lid 14 closed and the tamping spring 36 entering the coffee holder 30 for tamping the coffee 41 is shown in FIG. 40C . The fifth coffee maker 10 d may alternatively include a tamping spring entering the coffee holder top, or a resilient solid block pushed into the coffee holder 30 to tamp the coffee. Preferably, a horizontal ram 42 a is actuated when the lid 14 is closed and pushed the coffee holder 30 against the spring 36 to tamp the coffee. The horizontal ram 42 a may actuated by an electrical solenoid, by pressure, or by mechanical levers connected to the lid 14 . The fifth coffee maker 10 e may further include any of the features described above for other embodiments of the coffee maker according to the present invention and may be configured to use any of the coffee holders described above according to the present invention.
A side view of a tenth coffee holder 30 j with straight walls according to the present invention is shown in FIG. 41 , and a cross-sectional view of the tenth coffee holder 30 j taken along line 42 - 42 of FIG. 41 showing an empty coffee holder is shown in FIG. 42 . The coffee holder 30 j provides straight cylindrical inside walls allowing a better fit between the top tamper 35 b and the inside walls to reduce or eliminate coffee 41 escaping past the top tamper 35 b during tamping.
A cross-sectional view of the tenth coffee holder 30 j taken along line 42 - 42 of FIG. 41 showing a full and tamped coffee holder is shown in FIG. 43 . The tamping spring 36 has been pushed down by the lid 32 b to tamp the coffee 41 .
A side view of an eleventh coffee holder 30 k with straight walls according to the present invention is shown in FIG. 44 , a cross-sectional view of the eleventh coffee holder 30 k taken along line 45 - 45 of FIG. 44 showing an empty coffee holder is shown in FIG. 45 , and a cross-sectional view of the eleventh coffee holder 30 k taken along line 45 - 45 of FIG. 41 showing a full and tamped coffee holder. As with the coffee holder 30 j , the coffee holder 30 k provides straight cylindrical inside walls allowing a better fit between the lid 32 f and the inside walls to reduce or eliminate coffee 41 escaping past the lid 32 f during tamping. The lid 32 f may be used with or without the top tamper 35 b.
A side view of a top tamper 35 b is shown in FIG. 47A and a top view of the top tamper 35 B is shown in FIG. 47B . A side view of a top tamper 35 b′ with a seal 60 according to the present invention is shown in FIG. 47C and a top view of the top tamper 35 b′ with the seal 60 is shown in FIG. 47D . In some instances, for example with a very fine ground coffee, an amount of coffee may escape past the top tamper 35 b . In such instances, a user may prefer to use the top tamper 35 b′ with the seal 60 to reduce or eliminate the escape of the coffee.
A perspective view of a filter paper cup 40 ′ with a folding cup lid 40 d is shown in FIG. 48 (also see FIG. 7C ). The cup lid 40 d may be folded over the rim 40 c to reduce or prevent coffee from escaping during tamping of subsequent processing. The lid 40 d may also include a perforation 40 e centered on the lid 40 d allowing the nozzle 19 to enter and/or inject the hot flow of water into the tamped coffee 41 , but in some embodiments, the lid 40 d does not include the perforation 40 e . The filter paper cup 40 ′ may be used in the coffee containers described herein, and may able be used in a coffee machine having a cavity for receiving the filter paper cup 40 ′. While the cup 40 ′ is preferably made from filter paper, the cup may also be made from a reusable mesh.
A side view of an twelfth coffee holder 30 l with straight walls according to the present invention is shown in FIG. 49 , and a cross-sectional view of the twelfth coffee holder 30 l taken along line 50 - 50 of FIG. 49 showing an empty coffee holder is shown in FIG. 50 . The twelfth coffee holder 30 l includes a straight walled base and the tamping spring below the coffee, and additionally uses a filter paper cup 40 ′ with the folding lid 40 d.
A cross-sectional view of the twelfth coffee holder 30 l taken along line 50 - 50 of FIG. 49 showing the lid 32 a , coffee 41 , the filter paper cup 40 ′ with lid 40 d , above the coffee holder base 31 a is shown in FIG. 51A , a cross-sectional view of the twelfth coffee holder 30 l taken along line 50 - 50 of FIG. 49 showing the lid 32 a , above the coffee 41 and the filter paper cup 40 ′ resting in the coffee holder base 31 a is shown in FIG. 51B , a cross-sectional view of the twelfth coffee holder 30 l taken along line 50 - 50 of FIG. 49 showing the lid 32 a , above the coffee 41 and the filter paper cup 40 ′ resting in the coffee holder base 31 a with the filter paper cover 40 d folded over the coffee 41 in the filter paper cup 40 ′ is shown in FIG. 51C , and a cross-sectional view of the twelfth coffee holder 30 l taken along line 50 - 50 of FIG. 49 showing the lid 32 a attached to the base 31 a with the coffee 41 and the filter paper cup 40 ′ residing in the coffee holder base 31 a with the coffee 41 tamped is shown in FIG. 51D . In embodiments with the coffee 41 partially exposed above the base 31 a , some coffee 41 may escape during tamping. Using the filter paper cup 40 ′ having the fold over paper lid 40 d reduces or eliminates such escape of coffee 41 .
A side view of a thirteenth coffee holder 30 m with a releaseable tamping latch 64 according to the present invention is shown in FIG. 52 and a cross-sectional view of the thirteenth coffee holder 30 m taken along line 53 - 53 of FIG. 52 showing an empty coffee holder is shown in FIG. 53 . The latch 64 is held in a latched position by a spring loaded lever 62 on the exterior of the base 31 a.
A cross-sectional view of the thirteenth coffee holder 30 m taken along line 53 - 53 of FIG. 52 showing the lid 32 a , coffee 41 , the filter paper cup 40 , above the base 31 a , and the coffee holder base 31 a , with the tamping latch 64 retaining the bottom tamper 34 is shown in FIG. 54A , a cross-sectional view of the thirteenth coffee holder 30 m taken along line 53 - 53 of FIG. 52 showing the lid 32 a above the coffee 41 and the filter paper cup 40 resting in the coffee holder base 31 a , with the tamping latch 64 retaining the bottom tamper 34 is shown in FIG. 54B , a cross-sectional view of the thirteenth coffee holder 30 m taken along line 53 - 53 of FIG. 52 showing the lid 32 a , above the coffee 41 and the filter paper cup 40 resting in the coffee holder base 31 a with the tamping latch 64 retaining the bottom tamper 34 is shown in FIG. 54C , and a cross-sectional view of the fourteenth coffee holder 30 m taken along line 53 - 53 of FIG. 52 showing the lid 32 a attached to the base 31 a with the coffee 41 and the filter paper cup 40 residing in the coffee holder base 31 a with tamping latch 64 released and the coffee 41 tamped is shown in FIG. 54D . The lever 62 thus holds the latch 64 until the lever 62 is pushed to release the latch 62 to release the bottom tamper 34 to tamp the coffee 41 .
A side view of a fourteenth coffee holder 30 n with a releaseable tamping latch 64 according to the present invention is shown in FIG. 55 and a cross-sectional view of the fourteenth coffee holder taken along line 56 - 56 of FIG. 55 showing an empty coffee holder is shown in FIG. 56 . The lever 62 holds the latch 64 until the arm 66 attached to the lid 32 g pushes the lever 62 to release the latch 64 .
A cross-sectional view of the fourteenth coffee holder 30 n taken along line 56 - 56 of FIG. 55 showing the lid 32 g , coffee 41 ,and the filter paper cup 40 , above the coffee holder base 31 a , with the tamping latch 64 retaining the bottom tamper 34 is shown in FIG. 57A , a cross-sectional view of the fourteenth coffee holder 30 n taken along line 56 - 56 of FIG. 55 showing the lid 32 g above the coffee 41 and the filter paper cup 40 resting in the coffee holder base 31 a , with the tamping latch 64 retaining the bottom tamper 34 is shown in FIG. 57B , a cross-sectional view of the fourteenth coffee holder 30 n taken along line 56 - 56 of FIG. 55 showing the lid 32 g , above the coffee 41 and the filter paper cup 40 resting in the coffee holder base 31 a with the tamping latch 64 released but just prior to tamping (the bottom tamper has been released but has not moved upward against the coffee 41 ) is shown in FIG. 57C , and a cross-sectional view of the fourteenth coffee holder 30 n taken along line 56 - 56 of FIG. 55 showing the lid 32 g attached to the base with the coffee 41 and the filter paper cup 40 residing in the coffee holder base 31 a with tamping latch 64 released and the coffee 41 tamped is shown in FIG. 57D . The lever 62 thus holds the latch 64 until the lever 62 is pushed by the arm 66 to release the latch 62 to release the bottom tamper 34 to tamp the coffee 41 .
A side view of a fourteenth coffee holder 30 o with a releaseable tamping lock according to the present invention is shown in FIG. 58 and a cross-sectional view of the fourteenth coffee holder 30 o taken along line 59 - 59 of FIG. 58 showing an empty coffee holder is shown in FIG. 59 . The coffee holder 30 o includes a tamping lock 70 which engages a second bottom tamper 34 ′ to hold the second bottom tamper in a down position for filling the coffee holder with coffee and releases the bottom tamper 34 ′ to be pushed upwards by the tamping spring 36 to tamp the coffee after the holder lid 32 b is attached to the base 31 a.
A cross-sectional view of the fourteenth coffee holder 30 o taken along line 59 - 59 of FIG. 58 showing a lid 32 b , coffee 41 , a filter paper cup 40 , above the coffee holder base 31 , with the tamping lock 70 retaining the bottom tamper 34 ′ is shown in FIG. FIG. 60A , cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base, with the tamping latch retaining the bottom tamper 34 ′ is shown in FIG. FIG. 60B , a cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing the lid, above the coffee and the filter paper cup resting in the coffee holder base prior to tamping is shown in FIG. 60A , and a cross-sectional view of the fourteenth coffee holder taken along line 59 - 59 of FIG. 58 showing the lid 32 b attached to the base 31 a with the coffee 41 and the filter paper cup 41 residing in the coffee holder base 31 a with tamping lock released and the coffee tamped is shown in FIG. 60D .
A top view of a tamping lock 70 according to the present invention is shown in FIG. 61 and a bottom view of a second bottom tamper 34 ′ which cooperates with the tamping lock 70 according to the present invention is shown ion FIG. 62 . The tamping lock 70 includes teeth 72 which are inserted between and turned to engage lips 74 on the bottom of the bottom tamper 34 ′ to hold the bottom tamper in the down position for filling the coffee holder 30 o with coffee 41 . After the coffee holder 30 o is filled with coffee and the holder lid 32 b attached, the tamping lock is twisted to release the bottom tamper 32 b to tamp the coffee.
A side view of a filter cup 80 according to the present invention is shown in FIG. 63A and a top view of the filter cup 80 is shown in FIG. 63B . The filter cup 80 includes a ring 84 made a of a material sufficiently strong to hold shape in the proposed use. Filter material 82 is attached to the ring 84 . The filter cup 80 is insertable into the coffee holder and in many embodiments is a replacement for the filter paper cup 40 .
While the present invention is described above as placing loose coffee in a coffee holder, the invention may also be practiced by placing prepackaged coffee, for example coffee pods, into the coffee holder. Further, while the coffee holder is generally described as having a snap on lid, a screw on lid may also be used, and in general the various elements of different embodiments described above may be mixed to provide new embodiments and such new embodiments are intended to come within the scope of the present invention.
Further, many embodiments are described as including a coffee chamber comprising a filter paper cup. In many cases, a filter cup made of nylon mesh or metal mesh is equally suitable, and any coffee holder or combination of coffee maker and coffee holder including a filter chamber which holds coffee and allows the coffee to be tamped as described above is intended to come within the scope of the present invention regardless of the specific filter material.
While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.
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A self-tamping coffee holder tamps loose ground coffee obtaining richer flavor. The coffee holder includes a holder base and a holder cap. Coffee is loosely deposited in the coffee holder and the holder cap is attached to the holder base. An internal filter chamber holds the coffee and allows tamping of the coffee into a compacted state. The filter chamber may be formed by a fixed filter or by a removeable filter constructed of filter paper, nylon mesh, metal mesh, or any material capable of holding the coffee while allowing a flow of heated water through the coffee. The tamping may be by a spring or by a resilient solid material attached to the coffee holder and may push the coffee down inside the filter or push the filter and the coffee up against the holder lid.
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BACKGROUND OF THE INVENTION
2. Field of the Invention
This invention relates to a device for monitoring the dispensing of medication to patients. More particularly it relates to a system for accurately detecting drug dispensing events.
2. Description of the Prior Art
A variety of devices and methods have been described for controlling, noting, and keeping track of dispensation of medicines to patients. These devices range from simple mechanical checklist systems, through pill containers equipped with alarm clocks and the like and pill containers having timer-controlled latching devices which regulate the patient's access to medication. Some typical examples of these devices include the timed medication dispenser described by Roy J. Machamer in U.S. Pat. No. 4,382,688 which shows a medical dispenser having an electronic reminder to take the medication it contains. In this device the electronic reminder is disabled when the user takes the medication. In U.S. Pat. No. 4,448,541, Jonathan D. Wirtschafter describes a magnetically responsive switch device which is activated when a medication dispenser is opened so as to give an indication of the drug dispensing event. U.S. Pat. No. 4,367,955 of Donald H. Ballew shows a combined timer and container for dispensing medications wherein the container and its lid coact to initiate the timer cycle upon interengagement of the cap and container. U.S. Pat. No. 4,034,757 of Glover shows a device having two switches, each of which must be activated simultaneously to register a drug delivery event.
The foregoing patents are merely representative. Other background patents relating to medication dispensers include for example U.S. Pat. Nos. 3,369,697 of Glucksman et al.; 3,395,829 of Cogdell et al.; 3,651,984 of Redenback; 3,722,739 of Blumberg; 3,762,601 of McLaughlin; 3,815,780 of Bauer; 3,911,856 of Ewing; 3,917,045 of Williams; 3,968,900 of Stambuk; 3,998,356 of Christensen; 4,207,992 of Brown; 4,223,801 of Carlson; 4,258,354 of Carmon et al.; 4,275,384 of Hicks et al.; 4,360,125 of Martindale et al.; 4,361,408 of Wirtschafter; 4,382,688 of Machamer; 4,419,016 of Zoltan; 4,448,541 Wirtschafter; 4,473,884 of Behl; 4,483,626 pof Nobel; 4,490,711 of Johnston; 4,504,153 of Schollmeyer et al. and 4,526,474 of Simon.
In the case of devices with which it is desired to monitor access to a multidose drug container it is of importance to be able to identify true access events and distinguish them from false events. A true event would include opening the container, removing a pill or other medicament and then closing the container. A false event could include leaving the container open and repeatedly removing pills or, in the case of the not-sure-handed, repeated attempts at reinstalling the cap after a single removal of a drug or dropping the closed container, thereby actuating the open-close switch by means of the force of impact.
It is an object of this invention to provide a detection system which will be capable of identifying true drug removal events and discriminating them from these false events.
It is important that a device capable of electronically identifying and recording drug dosing information be constructed in a manner which is sturdy and reliable. It is also important that the construction be such as to minimize even inadvertent contact between the medication contained in the device and the various electronic elements which note and record the dosing information. This avoids contamination of the drug by contact with the electronic component, on the one hand, and interference with the proper functioning of the electronics by contact with the drug, on the other. The construction should also minimize cost and advantageously permit reuse of expensive electronic components. To these ends, it is a further object of this invention to provide a device for measuring and recording drug dosing information which physically separates the majority of the electronic components from the drug storage chamber. It is also an object of this invention to provide a device in which major electronic components can be recycled.
STATEMENT OF THE INVENTION
In accord with the present invention, a device is provided which is capable of discriminating between true and false drug dispensing events. This device includes a drug container having an openable and reclosable cap, lid or other similar dispensing aperture. The container is equipped with a detector which generates a first electrical signal in response to the opening of the dispensing aperture and a second electrical signal in response to the reclosing of the aperture. The device additionally includes a timing mechanism which measures the time elapsed between the first electrical signal and the second electrical signal. The elapsed time is then compared to a predetermined accept/reject standard. Times shorter than the accepted range, and thus indicative of fumbling with the cap or an impact event, are rejected. In preferred embodiments times longer than the accepted range and thus indicative of an open container can also be rejected. In other embodiments the device can measure the time between a closing and the next opening and compare that period to a standard to validate a drug dispensing event. A time meeting the preset criteria, such as falling within the desired range, is considered to be a good indication of a true drug-dispensing event. The device further includes a system for using these indications of true drug-dispensing events. This system of use can include a memory for storing the number of such events. It can also include a timekeeping mechanism which can provide and record the time and date each time an elapsed time within the accept range is determined. The information so determined and stored can be accessed by the pharmacist, physician or other health care professional as needed to verify compliance with dosing regimens, to give an indication of the patient's condition, or the like. In alternative embodiments, the determination that an elapsed time has fallen outside the accept range can be used to activate an alarm, to deliver a message to the patient or to the patient's health care professional or to alter the delivery pattern of drugs from the device such as by disabling the ability of the device to deliver drug or the like.
In other aspects, this invention provides an improved construction for an electronic medication monitor. In this preferred construction, the electronics are present in a removable cap for a medication container. In this construction all the electronics, except for a switch, are isolated from the drug container so that contamination between the electronics and the drug is avoided. In other aspects, the electronics are positioned so that expensive components may be removed and recycled. In yet a further aspect, the device of this invention can include an electronic access port through which data and program information is loaded and off-loaded wherein this access port is in the form of a plurality of electrically conductive pads which are accessed by spring-loaded pins in a suitable probe.
DETAILED DESCRIPTION OF THE INVENTION
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be further described with reference being made to the accompanying drawings in which:
FIG. 1 is a perspective elevational view of a pill container incorporating the present invention.
FIG. 2 is a cutaway of the device shown in FIG. 1.
FIG. 3 is a simple circuit diagram of one form of electronics usable as part of the present invention.
FIG. 4 is an exploded cross-sectional side view of a cap for a drug container, which cap contains the electronics necessary for noting and recording drug delivery in accord with this invention.
FIG. 5 is a cross-sectional side view of the cap of FIG. 4 in unexploded format.
FIG. 6 is a top view of the cap of FIG. 4.
FIG. 7 is a cross-sectional view of a probe pin useful for making electrical contact with the electronic circuitry of the cap of FIG. 4 for data output or program input.
Description of Preferred Embodiments
Turning first to the drawings, In FIGS. 1 and 2, a drug container 10 is illustrated as including a pill vial 11 and removable/reclosable cap 12. Cap 12 serves as a drug access port and in the embodiment shown additionally includes an optional optical readout 13 which can be used to display messages, signals or the like. Container 10 can take on a variety of configurations. It can be a dry pill container, as shown, a fluid drug container with a removable or openable cap, an aerosol with its dispensing nozzle carried under a removable/replaceable cover, or the like. In any embodiment, device 10 includes means for noting opening and closing of the drug access port. This can take the form of switch 21 which is physically engaged when the top 12 is placed on vial 11 and which is disengaged when it is removed. Of course, other functionally equivalent magnet switches or the like could be used so long as they give an accurate indication of the opening and the closing of the drug container. The output of switch 21 is fed to circuit board 22. Latching tabs 23 are used to fasten the top to the vial.
The signal so generated by switch 21 is fed into an electronic circuit such as shown in FIG. 3. In FIG. 3, 3-volt power is supplied by lithium battery 30 to a variety of locations in the circuit, as noted in legend VCC. The circuit employs a general purpose microprocessor 32. A 32 kHz clock crystal frequency is fed to pins X1 and X2 of microprocessor 32.
An active analog filter, constructed to set the pair of times which validate an opening, is coupled to pin P60 of microprocessor 32. This filter functions as follows--when the cap is removed, switch 21 is closed. This sends current through resistor 34 to capacitor 36. This resistor and capacitor are matched so that it takes about 0.5 seconds for the capacitor to charge to a threshold voltage which can be read by the microprocessor. If the switch was not closed for at least this period, as would be the case with an instantaneous closing, such as if the device were dropped, an adequate charge to indicate cap removal would not be generated, and the microprocessor would not be signaled that the cap had been removed. As will be appreciated, resistor 34 and capacitor 36 can be altered in value to give other time constants, if desired.
After a "cap off" signal has been sent to the microprocessor, pin P60 remains above the threshold voltage. When the cap is replaced, eliminating the voltage source through resistor 34, capacitor 36 is drained at a set rate through resistor 38 to ground 40. The value of resistor 38 is selected in this particular case so that the voltage drains past the threshold voltage. In the circuit shown, this takes about 2 seconds. At that time, pin P60 notes that the cap has been replaced. Thus, the device provides that a valid cap closing occurs after 2 seconds. If the cap were to be jiggled back open, this would cause current to flow through switch 21 and resistor 34 to maintain pin P60 at a "cap open" voltage.
Returning to microprocessor 32, it is a general purpose which contains an internal clock function. It also contains a small amount of RAM and about 2K of 8-bit ROM. This contains custom code which is used to communicate with RAM memory 42 drug delivery information generated by the actuation of switch 21 and filtered with the validation circuit is stored in RAM 42 together with time information supplied by microprocessor 32. This information is accessible through data point 44. It may be used by the health care professional to determine dosage times so as to validate correct dosing or to determine incorrect dosing.
The time interval between opening and reclosing the top of drug container 10 has been shown to be measured and compared to a predetermined standard. In the case shown, if the time between the two events is shorter than about 0.5 seconds, the system logic determines that in fact the top was not removed and a drug dose was not dispensed simply because that time was too short. This event would be classed as an inadvertent or error signal. No indication of drug dosing would be noted. Similarly, if the time interval between the closing and the subsequent opening is too short, for example, less than about 2 seconds, the device will not register the event as a true closing of the device and instead record the event as a mere fumbling with the cap or the like. The device can additionally be equipped to compare the interval between a valid opening and valid closing an provide an indication as to whether or not this interval is consistent with a single dosing or not. Too long an interval would suggest that the device was left open for an extended period and that possibly multiple doses were taken. In a variation, the device may contain information indicating the usual time between successive doses. If the time period between a valid opening and a valid closing far exceeded the normal period of a few seconds, but rather corresponded to the period between successive doses, the device could be equipped to indicate the logical conclusion that the device was opened, a dose taken, and the device not reclosed until a subsequent time when a second dose was taken.
Correct drug dispensing events, that is a proper opening and a proper closing separated in time by a proper interval can be stored into a readable memory for use by the health care professional to verify proper dosing or to identify dosing errors. Incorrect events may in some cases be disregarded or may be noted in the memory as well, preferably with a suitable notation regarding their incorrectness, also for use by the health care professional The correct and incorrect opening and closing information can also be used on an interactive basis such as to modify the dosing regimen, to send signals to the patient or the health care professional alerting them of changes or deviations from the desired or expected regimen or the like.
Although not intended as a limitation on the structure of the device in which the present time filtering is employed, the device of FIGS. 1 and 2 can have several other useful features. These features, which find application in other drug compliance monitors, as well, are shown in FIGS. 4 through 7.
One such advantageous feature is to have a construction which separates the drug from the electronics of the medication event monitor. If the drug and electronics are allowed to come into contact with one another the drug may interfere with the electronics or the electronics may contaminate the drug such as by releasing noxious or toxic materials into the drugs. In the embodiment shown in FIGS. 4-6 the electronics are isolated in the cap of the drug dispenser. In this embodiment the cap 12 includes a cap body 41 having a continuous barrier 42. Barrier 42 has holes 43 and 43a through which electronic wires can be passed. The electronics employed in the device, save and except for a single switch 46 which is physically activated when the cap is removed or replaced on the drug container, are carried on a printed circuit board 45 which fits into body 41. Cap liner 48 is present shielding the switch 46 from the drug storage region. When the cap is placed on the drug container, the top lip of the container presses against the liner 48 and forces it upwards against the switch 46 causing it to open or close. The two leads on switch 46 pass through holes 43 and 43a and seal these holes, preferably so that there is no possible contact between the drug contained in the device with the electronics. A cap lid 49 is present covering the electronics. It is overlaid with a label 50 which can carry information about the drug, the device or the like.
Another useful feature of the device of this invention when configured as shown in FIGS. 4-5 is the ability to recycle electronics. The electronic circuitry employed in the present invention is relatively costly as it contains at least one general purpose microprocessor chip. While it is generally not preferred to reuse drug containers for a sequence of drugs, for fear of some risk or cross contamination, no matter how remote, it would be desirable to recycle the electronics. In the configuration shown, the single switch 46 can be uncoupled by removing two connections and then the entire electronics board, which has not been in contact with drug, can be removed and recycled.
Yet an additional feature of this preferred embodiment is shown with special reference to FIGS. 6 and 7. This feature relates to the way data is extracted from the memory of the device and programs are fed into the memory of the device. One typical way to do this is to use a telephone jack or the like. A preferred method is shown in the figures where a simpler less space consumptive coupling is shown. In this embodiment the coupling is effected through a plurality of electrically conductive pads 51, 51a, 51b, etc. these are aligned with a corresponding plurality of holes 52, 52a, 52b, etc in the cap lid 49. They also correspond in position to a plurality of spring-loaded pins 54, 54a, 54b, etc in a data probe 55. In use, the pins are thrust through the label 50, through the holes 52 until the sharp ends of the pins 54 contact the conductive pads 51. The pin 54 is loaded with spring 56 and held in place by stop 57 so that a firm engagement between the pin and the pad is possible. Conductor 58 carries data from the devices memory or feed program to the device, as appropriate. FIG. 7 shows a top view of one form of hole arrangement. In the arrangement shown, there are 5 holes, arranged i a configuration which allows only a single orientation of coupling of the connector. These five holes are located in a particular position relative to registration mark 59. In actual use, the cap could be placed in an automated reader of some sort with registration mark 59 properly aligned with a corresponding position in the reader. Then the test pins 54 could automatically align with and access the conductive pads through holes 52. This configuration has the advantages of small size, and low cost.
While the invention has been described with reference being made to certain preferred embodiments, these are not to be construed as limitations on the scope of the invention which is instead as defined by the following claims.
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A device for detecting the dispensing of drugs from a container in a way which eliminates false detection events due to mishandling of the container is disclosed. The device includes a container which may be opened and closed. It also contains a means for detecting the opening and separately detecting the closing of the container as well as means for measuring the time between these events and comparing this elapsed time to a predetermined standard indicative of drug dispensing event. The times of proper drug dispensing events are stored in the device for use by the health care professional following the patient's drug dosing compliance. Other opening and closing intervals which fall outside this time range give rise to an alternative response. They may be recorded with a notation of their probable error or they may be disregarded. In other aspects, the invention provides a preferred physical configuration for the electronic components of the device and a preferred means for accessing the data stored in the device.
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BACKGROUND
1. Field of the Invention
The present invention relates to a device and method for releasably connecting together two elements, such as two orthotic devices, or two elements of an orthotic device. More particularly, the invention relates to a quick connect apparatus that quickly and easily enables a user to attach and detach two devices or two elements of a device, such as the elements of a knee-ankle-foot orthosis.
2. Background of the Invention
Orthotic devices traditionally have been utilized to aid in supporting, guiding and limiting the range of motion of different joints in the human body, or in the case of prosthetic devices, to replace missing body joints and limbs or portions thereof. For example, if natural joints such as knees or ankles are congenitally impaired or become impaired due to disease or injury, an orthosis may be used to support the joints, and guide and limit their range of motion. Orthotic devices typically comprise one or more orthotic supporting members that support a limb or part of a limb, such as for example a foot, a leg, or a lower or upper portion of a leg. When more than one orthotic supporting member is used the members are typically connected with orthotic metal bars that may form a pivoting mechanical orthotic joint across, for example, the knee, elbow, wrist, hip, ankle, spine, torso or neck of a patient. Such orthotic metal bars are typically mounted on opposite sides of the orthotic supporting members and cannot usually be detached except by an orthotist using fabrication techniques. Orthotic devices are often custom fit specifically for an individual patient, such as by contouring orthotic supporting members and orthotic metal bars to a plaster mold of a patient's anatomy. Examples of such procedures are described in U.S. Pat. No. 6,171,535, the teachings of which are incorporated herein by reference.
In some cases a patient no longer needs part of their orthotic device, for instance where a patient with a knee-ankle-foot orthosis improves and requires only an ankle-foot orthosis. Alternatively, a patient with an ankle-foot orthosis may occasionally or temporarily need a knee-ankle-foot orthosis, for instance for certain physical activities. However, when orthotic metal bars are permanently or semi-permanently attached to the orthotic supporting members to create, for instance, a knee-ankle-foot orthosis, it is difficult and time consuming for the orthotist to later remove the various orthotic supporting members from the orthotic device. Because in the past they were not easily modified, patients have often required separate knee-ankle-foot and ankle-foot orthoses.
U.S. Pat. Nos. 6,129,689 and 6,736,567 to Dibello, both entitled Quick Connect Apparatus And Method For Orthotic And Prosthetics Devices, the teachings of which are incorporated herein by reference, are generally directed to a device for releasably connecting orthotic metal bars to orthotic supporting members. Each Dibello device includes two plate members attachable to different orthotic elements, and a slider plate that is movable between a first released position and a second locked position. The Dibello device also may include a stop to secure the slider plate in the locked position. While the Dibello device should theoretically be usable to allow a patient to releasably connect orthotic supporting members to his or her orthotic device, in practice the Dibello device has proven very difficult and tedious to implement and use, because it requires precise, simultaneous, three-dimensional alignment of complicated plate members. Specifically, two Dibello plate members must each be separately attached to orthosis metal bars, and then precisely aligned and connected with corresponding plate members attached to opposite sides of an orthotic supporting member. Such alignment and connection of the Dibello plate members is in practice very difficult even for a professional orthotist, due to the misalignment and dimensional variation inherent in custom fabrication, as well as normal flexing and deformation of the metal bars and polymer supporting members.
A need remains for a device and method of quickly and reliably attaching and detaching orthotic members to and from an orthotic device that can be performed easily, for instance by a patient. A need also exists for such a device and method allowing easy connection of orthotic members that is sturdy and reliable for the user of an orthotic device and will not inadvertently disconnect when the orthotic device is in use.
SUMMARY
For purposes of the present invention, the terms “orthotic elements” and “appliance elements” refer to orthotic bars, orthotic straps, cuffs or bands, prosthetic limbs or any other members which are desired to be connected in an orthotic or prosthetic device. Also as used herein, the term “appliance” shall mean an orthosis or orthotic device or a prosthesis or prosthetic device.
One aspect of the present invention relates to a quick connect device adapted to be affixed to a first element of an appliance for releasably connecting the first element of the appliance to a second element of the appliance, wherein the quick connect device comprises one or more engagement members that are drivable back and forth between a retracted position and an extended position by actuation of an actuatable member from a first position and a second position, the one or more engagement members being adapted to engage the second element of the appliance and connect the second element of the appliance to the first element of the appliance when the quick connect device is affixed to the first element of the appliance and the second element of the appliance is positioned adjacent the first element of the appliance and the actuatable member is actuated from the first position to the second position, where the one or more engagement members are further adapted to disengage the second element of the appliance and disconnect the second element of the appliance from the first element of the appliance when the actuatable member is then actuated from the second position to the first position. Alternatively, the quick connect device may comprise no fewer than two engagement members.
In accordance with one aspect of the invention the quick connect device may comprise a cam member adapted to drive the one or more engagement members back and forth between their retracted and extended positions when the actuatable member is actuated back and forth between its first position and second positions. The actuatable member may be adapted to drive the cam member back and forth between a lesser lift position and a greater lift position when the actuatable member is actuated back and forth between its first position and second positions.
Additionally, the quick connect device may comprise means to releasably lock the engagement members in an extended position.
One aspect of the present invention relates to an appliance with releasably connectable first and second elements, the appliance comprising a quick connect device affixed to the first element of the appliance for releasably connecting the first element of the appliance to the second element of the appliance, the quick connect device including one or more engagement members that are drivable back and forth between a retracted position and an extended position by actuation of an actuatable member from a first position and a second position, the one or more engagement members being releasably engagable with the second element of the appliance to connect the second element of the appliance to the first element of the appliance when the second element of the appliance is positioned adjacent the first element of the appliance and the actuatable member is actuated from the first position to the second position, the one or more engagement members being disengagable from the second element of the appliance to disconnect the second element of the appliance from the first element of the appliance when the actuatable member is actuated from the second position to the first position.
In accordance with one aspect of the invention the appliance may comprise a knee-ankle-foot orthosis.
In accordance with one aspect of the invention the first element of the appliance may be part of an ankle-foot orthosis.
In accordance with one aspect of the invention the appliance may comprise a prosthetic device.
One aspect of the present invention relates to a method of releasably connecting first and second elements of an appliance, the method comprising affixing a quick connect device to the first element of the appliance for releasably connecting the first element of the appliance to the second element of the appliance, the quick connect device including one or more engagement members that are drivable back and forth between a retracted position and an extended position by actuation of an actuatable member from a first position and a second position, the one or more engagement members being releasably engagable with the second element of the appliance to connect the second element of the appliance to the first element of the appliance when the second element of the appliance is positioned adjacent the first element of the appliance and the actuatable member is actuated from the first position to the second position, the one or more engagement members being disengagable from the second element of the appliance to disconnect the second element of the appliance from the first element of the appliance when the actuatable member is actuated from the second position to the first position; positioning the actuatable member such that the one or more engagement members are in the retracted position; and positioning the second element of the appliance adjacent the first element of the appliance and releasably connecting the first and second elements of the appliance by positioning the actuatable member such that the one or more engagement members are in the extended position and engaging the second element of the appliance.
In accordance with one aspect of the invention the method of releasably connecting first and second elements of an appliance further may further comprise the step of disconnecting the second element of the appliance from the first element of the appliance by positioning the actuatable member such that the one or more engagement members are in the retracted position and disengaged from the second element of the appliance.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of an example prior art ankle-foot orthosis (“AFO”) with an ankle joint;
FIG. 2 is a perspective view of an example prior art ankle-foot orthosis (“AFO”) without an ankle joint;
FIG. 3 is a perspective view of an example prior art knee-ankle-foot orthosis (“KAFO”) without an ankle joint;
FIG. 4 is a perspective view of an example appliance according to the present invention showing the quick connect device in the unlocked position;
FIG. 5 is a perspective view of the example appliance of FIG. 4 showing the quick connect device in the locked position;
FIG. 6 is a perspective exploded view of an example quick connect device mechanism according to the present invention;
FIG. 7 is a series of views of an example cam member of the example quick connect device mechanism of FIG. 6 , with FIG. 7-1 being a top perspective view, FIG. 7-2 being a bottom perspective view, FIG. 7-3 being a top view, FIG. 7-4 being a side view, and FIG. 7-5 being a bottom view thereof;
FIG. 8 is a series of views of an example actuatable member of the example quick connect device mechanism of FIG. 6 , with FIG. 8-1 being a bottom perspective view, FIG. 8-2 being a top perspective view, FIG. 8-3 being a bottom view, FIG. 8-4 being a side view, and FIG. 8-5 being a top view thereof;
FIG. 9 is a series of views of example engagement members of the example quick connect device mechanism of FIG. 6 , with FIG. 9-1 being a bottom perspective view of a left-side engagement member, FIG. 9-2 being a bottom perspective view of a right-side engagement member, FIG. 9-3 being a side view of the member of FIG. 9-2 , FIG. 9-4 being a bottom view of the member of FIG. 9-2 , and FIG. 9-5 being an end view of the member of FIG. 9-2 ;
FIG. 10 is a series of views of an example housing member of the example quick connect device mechanism of FIG. 6 , with FIG. 10-1 being a top perspective view, FIG. 10-2 being a bottom perspective view, FIG. 10-3 being a top view, FIG. 10-4 being a side view, FIG. 10-5 being a bottom view, and FIG. 10-6 being a side view thereof;
FIG. 11 is a series of views of an example housing cover member of the example quick connect device mechanism of FIG. 6 , with FIG. 11-1 being a top perspective view, FIG. 11-2 being a bottom perspective view, FIG. 11-3 being a top view, FIG. 11-4 being a side view, FIG. 11-5 being a bottom view, and FIG. 11-6 being a side view thereof;
FIG. 12 is a series of views of the example quick connect device mechanism of FIG. 6 , with FIG. 12-1 being a top perspective view showing the engagement members in a retracted position, and FIG. 12-2 being a top perspective view thereof showing the engagement members in an extended position.
DETAILED DESCRIPTION
While the quick-connect apparatus and method described below will be described with respect to ankle-foot and knee-ankle-foot orthoses, the principles will apply to other appliances as well.
Referring to FIG. 1 , an example prior art ankle-foot orthosis 10 (“AFO”) is shown including a foot support element 12 , a calf support element 14 , and an ankle joint 16 . Alternatively, an ankle-foot orthosis 20 can be one-piece or otherwise omit an ankle joint, as shown in FIG. 2 . Orthoses such as an ankle-foot orthosis typically include one or more straps 22 . Other example straps are shown in more detail in U.S. Pat. No. 7,018,352, all the teachings of which are incorporated herein by reference.
FIG. 3 depicts an example prior art knee-ankle-foot orthosis 30 (“KAFO”), comprising a one-piece ankle-foot orthosis 20 connected to a thigh support member 32 by metal bars 34 that are permanently or semi-permanently affixed to first and second sides of orthosis 20 and support member 32 by fasteners 36 and over-lamination areas 38 , which at least partially encapsulate the metal bars 34 . Straps 22 for holding a patient's leg (not shown) into the orthosis 30 are also depicted. The metal bars 34 may include joints 39 on each side to allow the ankle-foot orthosis 20 to pivot in a controlled manner relative to the thigh support member 32 , as is known in the art.
An example embodiment 40 will now be discussed with reference to FIGS. 4 , 5 , 6 , 10 and 12 . As an example first element of an appliance, FIG. 4 depicts a one-piece ankle-foot orthosis member 20 with an example quick connect device mechanism 60 (shown in FIGS. 6 and 12 ) attached to and at least partially encapsulated in the body of orthosis member 20 at location 42 , for instance by over-lamination. In one embodiment the example quick connect device mechanism 60 is completely encapsulated in the body of orthosis member 20 at location 42 , except openings are provided to allow a user to access actuatable member 80 and to allow engagement members 90 to be extended from retracted positions FIG. 12-1 to extended positions FIG. 12-2 . Alternatively, an example quick connect device mechanism 60 can be attached to orthosis member 20 with glue, fasteners, or any other suitable fastening means at or near location 42 , instead of or in addition to encapsulation in the body of the orthosis member 20 . In one embodiment the example quick connect device mechanism 60 is positioned on or in the orthosis member 20 at or near location 42 with the bottom 110 of the housing 100 positioned toward the leg (not shown) of the wearer of the orthosis member 20 , with the actuatable member 80 positioned away from the leg of the wearer, and with the tapered portion 102 of the housing 100 positioned upward to provide an outer profile decreasing in size as it extends upward toward the back of the wearer's knee. When so oriented the tapered portion 102 may provide additional clearance with respect to seating in which the orthosis wearer may sit. Alternatively the various elements of a quick connect device can be positioned in any suitable location and orientation.
Also shown in FIG. 4 is a second element of an appliance, in this example a calf support element 46 . Calf support element 46 may be connected to thigh support element 32 by orthotic metal bars 34 that may include joints 39 , as described above with respect to prior art connections of orthotic metal bars to other orthotic elements. In the example shown, calf support element 46 is contoured to be placed against and fit closely around the upper exterior of ankle-foot orthosis member 20 . In this example calf support element 46 includes a receiver portion 44 adapted to fit around the example quick connect device mechanism 60 . The receiver portion 44 may include an opening to allow a user to access actuatable member 80 when calf support element 46 is placed against ankle-foot orthosis member 20 as shown in FIG. 5 . The receiver portion 44 may further include recesses, detents, or openings adapted to receive and engage engagement members 90 when calf support element 46 is placed against ankle-foot orthosis member 20 as shown in FIG. 5 and engagement members 90 are extended from retracted positions FIG. 12-1 to extended positions FIG. 12-2 as explained herein. While the above embodiment is described with an example quick connect device mechanism 60 attached to orthosis member 20 and engaging calf support element 46 at receiver portion 44 , in an alternative embodiment a quick connect device could be attached to calf support element 46 and adapted to engage orthosis member 20 .
FIG. 5 depicts the first appliance element 20 and second appliance element 46 of FIG. 4 attached to form a knee-ankle-foot orthosis 40 ′. This attachment can easily be accomplished by a user, such as a patient wearing the appliance or any other person, by placing the calf support element 46 against ankle-foot orthosis member 20 as shown in FIG. 5 , and causing engagement members 90 to be extended from retracted positions FIG. 12-1 to extended positions FIG. 12-2 , thereby engaging engagement members 90 with corresponding and adjacent receiver portion 44 of calf support element 46 . Engagement members 90 can be caused to be extended from retracted positions FIG. 12-1 to extended positions FIG. 12-2 by actuating actuatable member 80 of quick connect device 60 as discussed below. Knee-ankle-foot orthosis 40 ′ can then easily be changed back to a stand-alone ankle-foot orthosis 20 by actuating actuatable member 80 of quick connect device 60 to cause engagement members 90 to be retracted from extended positions FIG. 12-2 to retracted positions FIG. 12-1 , and removing first appliance element 20 from second appliance element 46 .
The components of an example quick connect device 60 will now be described. FIG. 6 depicts an exploded view of an example quick connect device 60 , including housing 100 and housing bottom 110 encompassing and supporting cam member 70 , actuatable member 80 , and engagement members 90 . As shown in FIG. 7 , example cam member 70 includes a rack of gear teeth 72 , an interface surface 74 that interfaces with actuatable member 80 , a pilot slot 75 that interfaces with actuatable member 80 , and cam grooves 76 and cam ridges 78 that interface with engagement members 90 , 92 . When assembled as shown in quick connect device 60 , cam member 70 interfaces with and is driven by example actuatable member 80 shown in FIG. 8 . Example actuatable member 80 includes a pinion of gear teeth 82 that are adapted to mesh with cam member gear teeth 72 , a shoulder 84 that interfaces with and is axially restrained in one direction by inner surface 104 of housing 100 (shown in FIG. 10 ), a pilot protrusion 85 that interfaces with and is located by pilot slot 75 in cam member 70 , and a slot 86 adapted to interface with a flat screw driver, or a coin, such as a penny, nickel, dime, or quarter. Slot 86 could alternatively be replaced by any suitable structure, such as, for example, an external hex shape to be driven by a wrench, or a phillips-style interface, or a torx-style interface, or an allen-style interface. Example actuatable member 80 further includes an outer-diameter bearing surface 88 which interfaces with and is located by inner-diameter bearing surface 108 in housing 100 . Example quick connect device 60 further includes left and right, or first side and second side, engagement members 90 and 92 as shown in FIG. 9 . Left and right, or first side and second side, engagement members 90 and 92 are in this example mirror images of each other; accordingly, their features will be described with respect to right, or second side engagement member 92 . Engagement members 90 , 92 include engagement surfaces 94 that slidably interface with and are located by surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 . Engagement surfaces 94 are further adapted to engage receiver portion 44 of calf support element 46 , for instance by having holes (not shown) in receiver portion 44 dimensionally corresponding to engagement members 90 , 92 as located by surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 when first appliance element 20 and second appliance element 46 of FIG. 4 are attached to form a knee-ankle-foot orthosis 40 ′ as shown in FIG. 5 . Engagement members 90 , 92 may further include grooves 95 that interface with and catch on surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 when engagement members 90 , 92 are in extended positions FIG. 12-2 . This interface of grooves 95 with housing 100 and housing bottom 110 creates a locking effect, effectively locking the engagement members 90 , 92 in extended positions FIG. 12-2 such that first appliance element 20 does not inadvertently disconnect from appliance element 46 of FIG. 4 . This locking effect may be overcome, and the mechanism un-locked, by applying extra force to actuatable member 80 , to pop surfaces 106 and 114 out of grooves 95 . Engagement members 90 , 92 include engagement slots 96 and engagement ridges 98 that slidably interface with corresponding cam ridges 78 and cam grooves 76 , respectively, of cam member 70 . As shown in FIG. 10 , example housing 100 includes a top surface 101 , a tapered surface 102 that tapers away from top surface 101 , a raised boss 103 extending upward from the top surface 101 and designed to extend through overlamination material at least partially encapsulating connecting device 60 and connecting it to first appliance element 20 , an inner surface 104 , surfaces 106 to guide and engage members 90 , 92 , bottom edges 107 , and an inner-diameter bearing surface 108 for locating and interfacing with actuatable member 80 . Example housing bottom 110 is shown in FIG. 11 , and snap-fits between bottom edges 107 of housing 100 , and includes a bottom surface 112 , a top surface 113 , surfaces 114 on ears 116 to guide and engage members 90 , 92 , and a pilot hole or recess 115 to engage and locate pilot protrusion 85 of actuatable member 80 . The various components of quick connect device 60 discussed above may each be made from any suitable material, such as nylon 6/6 or any other suitable polymer or metal, and may be formed by any combination of machining, plastic injection molding, compression molding, vacuum molding, or any other suitable manufacturing process.
Operation of example quick connect device mechanism 60 will now be described. A user actuates actuatable member 80 by rotating it, for instance by engaging a screw driver or a coin in slot 86 . In other example mechanisms actuatable member 80 may be replaced by one or more switches, latches, buttons, levers, or any suitable mechanism adapted to drive one or more engagement members 90 , 92 . While two engagement members 90 , 92 are shown in this example, in other embodiments one engagement member may be used, or three or more engagement members may be used. When actuatable member 80 is rotated in a first or second rotational direction, gear teeth 82 drive cam member gear teeth 72 , slidably translating cam member 70 in a first or second axial direction within housing 100 . When cam member 70 is translated in a first or second axial direction, engagement slots 96 and engagement ridges 98 of engagement members 90 , 92 slidably interface with corresponding axially angled cam ridges 78 and cam grooves 76 , respectively, of cam member 70 , which then vary between lesser and greater lifts as cam member 70 is translated axially, and which cause engagement surfaces 94 of engagement members 90 , 92 to slide past surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 a distance corresponding to the change in lift of the cam member 70 . When actuatable member 80 is rotated in a first rotational direction, engagement surfaces 94 of engagement members 90 , 92 slide past surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 and extend to an extended position as shown in FIG. 12-2 . When fully extended or sufficiently extended, grooves 95 in engagement members 90 , 92 engage with surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 , locking the mechanism in the extended position as shown in FIG. 12-2 . Engagement members 90 , 92 may then be retracted by rotating actuatable member 80 in a second rotational direction, first applying sufficient force to disengage surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 from grooves 95 , and then causing engagement surfaces 94 of engagement members 90 , 92 to slide past surfaces 106 of housing 100 and surfaces 114 of housing bottom 110 to a retracted position as shown in FIG. 12-1 .
An example fabrication technique will now be discussed. A KAFO (Knee Ankle Foot Orthosis) cast or mold is created based on the shape of a patient's limb and may be fully modified and screened to the practitioner's expectations. Once a cast or mold is made, locate an area on the mold corresponding to where the quick connect device mechanism 60 will be located and trace with an indelible pencil, approximately three inches distal from the fibular head. An orthotist may use judgment on pediatric applications as they may vary. Using a flat sure form make a flat surface (using indelible marks as guide) on which to place the quick connect device mechanism 60 (screen smooth after satisfied with the way the box sits on the mold). Once the mold is fully shaped, seal it to reduce moisture, then place a nylon covering, such as nylon leg hosiery, over the mold. Later when a vacuum is applied, the vacuum will be communicated between the fibers and through the openings in the nylon material. Next, use plastic such as a trash bag to wrap and seal the nylon-covered upper thigh area of the KAFO, from the top just passed the knee center. Seal the ends of plastic material to the nylon with black electrical tape, covering several inches of nylon with the tape, and seal the other end of the plastic material to a vacuum source. Apply a small amount of spray adhesive to the nylon material where the quick connect device mechanism 60 will be placed, and place the quick connect device mechanism 60 there. Heat up 1/16 inch thick polyethylene, at least 7 inch by 7 inch in size, in an oven until it becomes substantially transparent, for instance about 18 minutes at 350 degrees Fahrenheit. Then remove the heated polyethylene from the oven and stretch it over the area on the mold where the quick connect device mechanism 60 is placed. The heated polyethylene may contact the quick connect device mechanism 60 , the nylon, the mold, and the black electrical tape, but may not touch the plastic bag material. The polyethylene may be stretched around the mold until it thins down to approximately 1/32 inch thick, and it should be compressed together to form an air-tight enclosure around the mold. Apply a strong vacuum to the interior of the plastic bag, which vacuum is then communicated through the nylon hosiery, under the electrical tape, and under the heated polyethylene that encloses the mold. The vacuum will then suck the heated polyethylene tightly to the nylon hosiery-covered mold, over and encapsulating the quick connect device mechanism 60 . After the material cools somewhat but while it is still hot to the touch, use a razor blade to cut the polyethylene away from the locations of engagement members 90 , 92 so that they may extend as shown in FIG. 12-2 , and grind away the polyethylene material covering the actuatable member 80 so that it may be actuated. After the material cools to substantially room temperature, the foregoing steps are repeated by heating, stretching, and vacuum-forming a second layer of polyethylene over the first layer of polyethylene at the location of the quick connect device mechanism 60 , while the engagement members 90 , 92 are extended as shown in FIG. 12-2 . This will form the corresponding engagement areas for engagement members 90 , 92 in the second layer of polyethylene. The second layer of polyethylene material covering the actuatable member 80 must also be ground away so that it may be actuated. In the present example discussed herein, the first layer of polyethylene will form the ankle-foot orthosis 20 , including protrusion 42 encapsulating quick connect device mechanism 60 , and the second layer of polyethylene will form calf support element 46 including receiver portion 44 form-fitted to fit around protrusion 42 ankle-foot orthosis 20 . After the material cools, excess material is cut away and typical orthosis hardware is added as is known in the art to produce an appliance as shown in FIGS. 4 and 5 .
While the foregoing invention has been described with reference to its preferred embodiments, various alterations and modifications will occur to those skilled in the art. All such alterations and modifications are intended to fall within the scopes of the appended claims.
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A device and method for releasably connecting together two elements, such as two orthotic devices, or two elements of an orthotic device is provided. A quick connect apparatus and method of use is disclosed that quickly and easily enables a user to attach and detach two devices or two elements of a device, such as the elements of an orthosis or a prosthetic. An example device that may easily be converted back and forth between an ankle-foot orthosis and a knee-ankle-foot orthosis is disclosed, along with steps for making and using same.
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BACKGROUND OF THE INVENTION
The invention relates to a packaging container for holding a plurality of articles such as chocolate bars which comprises a base part and a stack top.
Packaging containers of this kind are known in the art as shop display containers. They serve as transport and display means for articles, particularly for packaged confectionery such as chocolate bars, which are arranged inside the container in the form of several stacks. These goods are delivered inside the packaging containers to retailers and are sold to customers directly out of these containers.
In general, prior art packaging containers of this kind only permit a single use. Consequently, the packaging containers have to be disposed after the goods have been sold.
The pallet-like packaging containers are quite large which means that their disposal creates a considerable amount of waste.
SUMMARY OF THE INVENTION
The invention is therefore based on the object to provide a packaging container of the afore mentioned kind which is easily reusable.
To attain this object, a base part and a stack top of the packaging container are releasably connected to one another and the stack top is collapsible or foldable. Since the stack top can be taken off the base part, the base part itself, which only requires relatively little space, can be easily returned in order to be used again. Since the stack top can be collapsed and erected, it can also be returned without the loss of space and can then be reconnected to the base part. Thus, the whole packaging container is reusable.
According to a preferred embodiment of the invention, the stack top is collapsible or foldable in such a way that the surface area of the collapsed or folded stack top approximately corresponds to the surface area of the base part, and preferably, it is slightly smaller. Thus, the collapsed or folded stack top can be placed on or into the base part and the complete packaging container can be returned without the loss of space.
According to the invention, the stack top comprises several vertical partition walls. Some of these partition walls are pivotable about at least one preferably central (hollow) column. The column stabilizes the stack top and facilitates a pivoting of the partition walls in order to collapse and/or fold the stack top. Erecting the stack top is also facilitated by the column.
The column is preferably formed from two side-by-side part columns which are joined to one another in such a way that, starting from their confronting sides, the two part columns can be pivoted away from one another in order to fold together the stack top. In this process, the two part columns are collapsed, i.e. they loose their three-dimensional shape and assume a flat shape which only requires minimum space.
The part columns are joined to one another via two sides, which are located in juxtaposition in a common plane, by means of a portion of the stack top which forms two partition walls. This portion of the stack top also comprises a center folding line which forms a center hinge axis. This axis is located between the juxtaposed side faces of the interconnected part columns and allows to centrally fold together the collapsed stack top (approximately in a V-shaped manner). As a result, the surface area of the collapsed or folded stack top can be halved.
Expediently, the unfolded or erected stack top is connected to the base part via at least one plug connection. This connection is preferably formed from depressions which correspond to the outlines of the stack top. Thus, lower edge portions of the stack top can be easily and reliably inserted into the base part in a positive manner.
Within the base part, there are supports for the articles which are to be stacked up against the partition walls of the stack top. These supports downwardly incline towards the center column so that the articles are pressed against the partition walls of the stack top or the column. Consequently, the incline of the supports ensures that the articles which are stacked on top of one another are securely held in the packaging container. Even relatively high stacks of superposed articles can not collapse and fall out of the container.
The larger supports are divided into several support surface parts whose surface area preferably corresponds to that of a stack of articles which are to be arranged on top of one another. On the one hand, this results in an imbricated arrangement of the articles of individual side-by-side stacks, which increases the stability of the stacks, while, on the other hand, the inclined arrangement of the support surface parts only creates a small wasted space in the form of a cavity underneath the supports.
According to a preferred embodiment of the invention, the base part comprises an outer portion and at least one inner portion. Such a base part is particularly easy to manufacture, because the separate inner portion can be easily provided with the inclined supports or support surface parts for the articles which are to be stacked in the packaging container. Moreover, the depressions in which the unfolded stack top is to be inserted can be formed in the inner portion by simple longitudinally and transversely directed slots. The inner portion can have a single-piece or a multi-piece structure. In the case of a multi-piece inner portion, the individual parts extend in those regions of the base part which are either located between two parallel partition walls or between two partition walls which are located at right angles to one another (in corner regions of the packaging container).
It is another essential feature of the invention that the whole packaging container is made of a durable material which allows a repeated use of the container. Preferably, the base part as well as the stack top are made of plastic, preferably of a thermoplastic material. This choice of material makes the packaging container according to the invention particularly durable and thus near enough infinitely reusable. Moreover, the packaging container according to the invention can be manufactured particularly easy by means of deep-drawing, injection molding and/or by means of cutting its portions out of semi-finished sheets.
BRIEF DESCRIPTION OF THE DRAWINGS
A preferred embodiment of the packaging container according to the invention will be described below in detail with reference to the drawings, in which:
FIG. 1 is a perspective view of a packaging container arranged on a pallet, shown in an erected but empty condition,
FIG. 2 shows the packaging container of FIG. 1 in a disassembled condition in which the stack top is folded together,
FIG. 3 is a top plan view of a base part of the packaging container,
FIG. 4 is a section of the base part of FIG. 3, taken along the line IV--IV,
FIG. 5 is a section of the base part of FIG. 3, taken along the line V--V,
FIG. 6 is a perspective view of a stack top of the packaging container, shown in a partially folded condition, and
FIG. 7 is a top plan view of the partially folded stack top, on an enlarged scale.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The invention relates to a packaging container in the form of a reusable shop display. Articles such as chocolate bars 10 are packed in the container and delivered to retailers where they are directly sold to customers from the container. The packaging container is designed such that it can hold several stacks of a plurality of superposed chocolate bars 10. The outer sides of the packaging container are open and offer easy access to the chocolate bars 10 packed therein (FIG. 1).
The packaging container comprises a base part 11 and a collapsible stack top 12 which is releasably connected thereto.
In the packaging container illustrated in the drawings, the base part 11 is made from two portions specifically an outer portion 13 and an inner portion 14.
The outer portion 13 of the base part 11 has the shape of a box with low rims and an open top. Accordingly, the outer portion 13 has a closed bottom 15 which in this case has a rectangular surface area and (low) side panels 16 and transverse panels 17 adjoining the edges of the bottom 15. These panels are at right angles to the bottom 15 and completely surround this bottom 15. Corner portions of the side panels 16 and transverse panels 17 are upwardly extended in order to form stacking corners 18 (FIG. 1). Projections 19 which correspond to the stacking corners 18 are arranged underneath the bottom 15. In the present embodiment, the projections 19 are in the form of L-shaped ribs which are set back relative to the edge of the bottom 15 by the panel-thickness of the stacking corners 18 (FIGS. 4 and 5). As a result, several base parts 11 can be stacked on top of one another, such that, on the one hand, the projections 19 and, on the other hand, the stacking corners 18, form a lock which secures the stacked base parts 11 against transversely directed relative displacements. Moreover, the projections 19 underneath the bottom 15 of each outer portion 13 secure the packaging container with its base part 11 on a standardized pallet 20 (FIG. 1) against displacements. For this purpose, the projections 19 enter depressions in the top side of the pallet 20.
The inner portion 14 is formed like an insert which is placed inside the outer portion 13 and is positively held therein between the side panels 16 and transverse panels 17. The inner portion 14, which in the present embodiment is in the form of a single piece, is provided with continuous grooves extending from one side to the other, particularly with two parallel longitudinal grooves 21 and two parallel transverse grooves 22 (FIG. 3). These grooves divide the surface area of the inner portion 14 into altogether nine fields 23 to 26. The bottom of the longitudinal grooves 21 and transverse grooves 22 is defined by webs 54. These webs 54 connect the fields 23 to 26 of the inner portion 14 so that they form a single piece. The fields 23 to 25 form support surfaces for one or more stacks of chocolate bars 10. In the illustrated embodiment, each of the four identical corner fields 23 serves for holding four stacks, such that the longitudinal sides of the chocolate bars 10 abut one another. Each of the two fields 24 serve for holding six stacks which are located side-by-side and behind one another. Two small fields 25 hold one stack each. A center field 26 is surrounded by the outer fields 23, 24, 25 and remains free, i.e. it is not packed with articles, since it offers no access to the chocolate bars 10 (FIG. 3).
The supports of the fields 23, 24, 25 of the inner portion 14, on which the stacks of superposed chocolate bars 10 rest, incline towards the bottom 15 of the base part 11. This incline has been selected such that it is directed towards the flat bottom of the center field 26. As a result, the chocolate bars 10 in the packaging container are slightly inclined towards the center field 26, which prevents the stacks of superposed chocolate bars 10 from shifting towards the open outer sides of the packaging container and toppling over. The supports of the fields 24 have a continuous surface which is downwardly inclined towards the center field 26. The supports of the corner fields 23, on the other hand, are arranged in an imbricated manner. In particular, the supports of the fields 23 are formed from four consecutive support parts 27, each supporting one stack of superposed chocolate bars 10. Each support part 27 downwardly inclines towards the field 25, which gives the fields 23 a saw-toothed profile section (FIGS. 1 and 4). Moreover, the surface parts continuously incline towards the fields 24. As a result, the support parts 27 are on the whole directed towards the respective corner of the center field 26, so that the stacks of superposed chocolate bars 10 are held in a stable manner even in the corner fields 23.
The stack top 12 can not only be taken off the base part 11 but can also be collapsed or folded together thereafter. For this purpose, the stack top 12 comprises a center column 28 whose surface area corresponds to the surface area of the field 26. A partition wall 29, 30, 31 and 32 extends each side of the center column 28 at opposite ends. Altogether, the center column 28 is connected to four pairs of partition walls 29, 30, 31 and 32. According to the invention, the partition walls 29, 31, 32 are pivotable. In particular, they are hinged to the center column 28 by means of vertical hinge axes 33. The two partition walls 30, on the other hand, are rigidly joined to the center column 28. The partition walls 30 and the side of the center column 28 located between these two walls 30 form a folding surface 34. The pivotable partition walls 29, 31 and 32 which are hinged to the center column 28 can be moved against this folding surface 34 in order to collapse or fold together the stack top 12 (FIGS. 1 and 6).
The longitudinal grooves 21 and transverse grooves 22 in the inner portion 14 are defined such that their width and length corresponds to the outlines of the partition walls 29 to 32, so that the unfolded stack top 12 can be inserted into the longitudinal grooves 21 and transverse grooves 22 of the inner portion 14 from above.
It is an essential aspect of the invention that the center column 28 is centrally divided. For this purpose, the center column 28 comprises two hollow part columns 35 and 36 of equal size. The part columns 35 and 36 are joined to one another via two confronting vertical corners 37, i.e. via a corner 37 of the first part column 35 and an adjacent second corner 37 of the second part column 36 (FIGS. 1 and 6).
The illustrated stack top 12 is formed from altogether nine blanks which are durably connected to one another. Eight blanks are formed from four pairs of different types of blanks 38, 39, 40, 41, while there is only a single fifth blank 42. The structure of the blanks 38 to 42 will be described in the following in detail with reference to the partially unfolded stack top (FIGS. 6 and 7):
Each part column 35 and 36 is formed from the blank 40 and a portion of the blank 39. The blank 40 is bent in an L-shaped manner, while the blank 39 extends in the form of a double-L or a Z. The L-shaped blank 40 forms two panels 43, 44 of the respective part column 35, 36, while two perpendicular legs of each blank 39 form the other two panels 45 and 46 of each part column 35, 36. The free ends of the legs of blank 40 which form the panels 43 and 44 are provided with connecting flaps 47 and 48. These flaps 47, 48 connect the panels 43 and 44 of the blank 40 with the panels 45 and 46 of the blank 39. A third leg of the blank 39 forms one half of the partition wall 29. A second half of this partition wall 29 is formed from a connecting flap 49 of the L-shaped blank 38. This connecting flap 49 completely overlaps the first half of the partition wall 29 and is connected thereto. A second leg of the blank 38 extends perpendicular to the connecting flap 49 of each blank 38 and forms a partition wall 32. The panels 45 of the part columns 35 and 36 are located next to one another in one plane and are each connected to a flat blank 41 in such a way that each blank 41 completely overlaps the panel 45 of the respective part column 35 or 36. Moreover, each blank 41 projects from the respective part column 35, 36 at both ends in order to form the partition walls 30. The blank 42 is formed in a U-shaped manner. As a result, two parallel legs of the blank 42 form parallel partition walls 31 of equal size. A web 50 of the blank 42 connects the partition walls 31 to one another. This web 50 corresponds in width to the side-by-side part columns 35 and 36 and overlaps the sections of the blank 41 which abut the part columns 35, 36. A vertical center hinge axis 51 centrally extends across the web 50 of the blank 41. This axis allows to fold the part columns 35 and 36 away from one another in the region of their corners 37 and to halve the overal surface of the collapsed or folded stack top 12.
The stack top 12 is collapsed by means of pivoting individual sections of the blanks 38, 39, 40 and 42 about their hinge axes 33 or the center hinge axis 51. The hinge axes 33 and the center hinge axis 51 are formed in the blanks 38, 39, 40 and 42 by embossing or the like, so that the blanks can be easily pivoted in a well-defined manner.
The stack top 12 is collapsed or folded together in the following way (FIGS. 6 and 7):
After the stack top 12 has been released from the base part 11, the Z-shaped blanks 39 are spread out flat by means of pulling their free ends. The blanks 39 are thus placed against the blanks 41 which are located in the folding plane 34 and which remain flat even in the erected state of the container. In this process, the blanks 40 are deformed such that the part columns 35 and 36 are spread out flat. Subsequently, the oppositely located partition walls 32 of the blanks 38 are folded in opposed directions so that their free edges 52 are directed away from one another. In like manner, the partition walls 31 of the blank 42 are moved in opposed directions so that their free edges 53 are located on opposite outer sides of the collapsed stack top 12. Thereafter, the collapsed stack top 12 is again folded in a V-shaped manner about the center hinge axis 51 in order to halve its surface area. The dimensions of the blanks 38 to 42 of the stack top 12 are defined such that in the folded or collapsed state, the stack top 12 has a rectangular surface area which corresponds to the inside dimension of the outer portion 13 of the base part 11. Thus, the folded stack top 12 can be placed on the inner portion 14 of the base part 11 such that it is positively held therein. If required, other collapsed packaging containers can then be placed with their base parts 11 on the stacking corners 18 of the bottom base part 11, so that a plurality of empty collapsed packaging containers can be returned in a space-saving manner to the place where they are filled.
Alternatively it would be possible to dispense with the connecting flaps 47 to 49 and the web 50 and to directly hinge the partition walls 29 to 32 to the respective corners of the part columns 35 and 36, for example by means of hinge straps which are glued to corner portions and form hinge axes 33. In this case, the center hinge axis 51 is also formed from a hinge strap which directly joins the confronting edges 37 of the part columns 35 and 36.
It is another essential aspect of the invention that the whole packaging container is made of plastic, preferably of a thermoplastic material. For this purpose, the outer portion 13 of the base part 11 is preferably made by injection molding, whereas the inner portion 14 of the base part 11 is preferably made by deep-drawing. Alternatively, the outer portion 13 could also be made by deep-drawing. The blanks 39 to 42, on the other hand, are preferably cut from a sheet-like pastic material. The hinge axes 33 and the center hinge axis 51 are embossed so that they form hinges. The blanks 39 to 42 are interconnected via their connecting flaps 47 to 49 by means of glueing, welding or the like.
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A packaging container for holding a plurality of articles. Packaging containers of this kind serve as transport and display means for a plurality of packed articles. These articles are sold directly from the packaging container. The empty packaging containers are usually destroyed, because their material and their structure do not permit repeated usage. The invention is concerned with providing a reusable packaging container of this kind. The described packaging container comprises a base part (11) and a stack top (12) which is releasably connected to the base part (11) and which can be collapsed and/or folded together, preferably in such a way that the collapsed or folded stack top (12) can be placed on the base part (11). As a result, empty packaging containers can be returned in a space-saving manner and, if required, they can be refilled, which means they are reusable. The packaging container according to the invention is particularly suitable for holding several stacks of superposed packs of confectionery, particularly chocolate bars (10).
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FIELD OF THE INVENTION
[0001] The present invention relates to the field of cell culture. This invention provides a new culture system, for growing cells in general and plant cells in particular. Since this apparatus can be disposable and efficient at large scale, its use allows a great reduction of production costs in different kinds of applications.
BACKGROUND OF THE INVENTION
[0002] Conventional culture systems are generally composed of a rigid container (glass or stainless steel) having a means for aerating and mixing the culture content (air sparger, impeller). These systems are complex, and usual equipment and support facilities associated with aseptic bioprocess are extremely expensive because the large-scale production is based on stainless steel vessels, sterilized in situ. More than 60% of the production costs is due to the fixed costs: high capital costs of fermentation equipment, depreciation, interest and capital expenditure. The running costs are also high, due to low yields and the needs to clean and sterilize the bioreactor after each culturing cycle. In the particular industrial application of plant cell cultures, different well-known culture systems have been used such as stirred tank or airlift reactors. Despite many efforts to commercialise plant metabolites, few achieved commercial success. One reason is the low productivity in spite of the possibility to obtain higher content of desired compound than in whole plant (rosmarinic acid, shikonin, etc.), up to 20% of dry weight. The main constraint leading to a low productivity remains the low growth rate (below 0.7 day −1 , min 20 h doubled-time) compared to bacteria. Using batch culture in industrial fermentor means to operate no more than 10-20 runs per year with plant cell cultures in very high cost facilities. It means that the bottleneck for an industrial production is more an economical one than a biological one.
[0003] To overcome these problems and decrease production costs, new technologies recently appeared, based on the use of various disposable plastic bags instead of stainless steel fermentor. These new systems using pre-sterile disposable plastic bags are promising because they decrease capital investment since plastic is a low cost material and moreover they eliminate cleaning, sterilization, validation and maintenance of equipment, which is time and cost consuming. It also allows more flexibility in the process, which can be operated by people not skilled in the art since bags are provided pre-sterile.
[0004] Different aeration/mixing systems have been proposed in such disposable apparatus. Wave Biotech (Singh V, U.S. Pat. No. 6,190,913) has developed a system using an inflated bag placed on a rocking mechanism that moves the bag inducing a wave-like motion to the liquid contained therein. The rocking mechanism limits the size of the tank because such a mechanical agitation needs complex equipment to reach high volumes of culture.
[0005] Another suggestion is to use gas permeable plastic bags agitated with a mechanical system or not agitated at all. In U.S. Pat. No. 5,057,429 a gas permeable bag is rotated or shaked to diffuse oxygen and nutrients to the animal cells. A static gas-permeable bag is also described in U.S. Pat. No. 5,225,346. Up to now there is no industrial development of such culture systems mostly because on one hand there is a difficulty to scale-up an external agitation apparatus and on the other hand there are problems due to insufficient oxygen supply to the cells in a static bag containing several liters of culture medium.
[0006] A reactor can consist of a gas-sparged plastic bag in a tank with a head plate that has capabilities for inoculation and media sample removal. Disposable conical plastic bags produced by Osmotec are for small-scale use (few liters), using air bubbles for aeration through an inlet. U.S. Pat. No. 6,432,698 also describes a disposable bioreactor for culturing microorganisms or cells, comprising a gas bubbler, generating gas bubbles for mixing and providing gases, close to airlift bioreactor except it is herein made in plastic material.
[0007] In these inventions, there are two main constraints: at high density or high volumes of culture, there is a need to create smaller gas bubbles or fluid circulation in the whole reactor to achieve convenient mixing and aeration. This results in complex bubbling systems (gas diffusers, partitioned tanks . . . .), which are not in agreement with a simple disposable technology. Moreover, small bubbles are detrimental to sensitive cells, increase cell to wall adhesion and/or strip off some useful gases from the culture medium (ethylene for plant cell for example).
[0008] The use of gas bubbles for the aeration of bioreactor or fermenter is well known. Currently a diffuser injecting microbubbles is used to improved the gas transfer into the culture medium. Bioreactor where the aeration and also agitation is done through gas stream without mechanical agitation is also well known and currently named airlift bioreactor by the specialists. For example, U.S. Pat. No. 4,649,117 describes the culture system of airlift bioreactor, useful for carrying out cell culture and fermentation. Suitable gas flow rate are in the range of 10 to 300 cc/min, and the gas is gently continuously bubbled, without any reference to the size of bubbles or the periodic generation of single large bubble as in our present invention. Two chambers are used, a growth chamber and a mixing chamber.
[0009] The use of single bubble, noted as “large” but inferior to 3 cm 3 , is known for mixing and blending various materials such as chemicals, beverages or oils. WO-A-8503458 describes a method and apparatus for gas induced mixing and blending, not concerning the growth and cultivation of living cells. The method is based on gas bubbles of predetermined variable size and frequency injected into a tank through one or several air inlets. The goals are to reduce overall blending and mixing time, which is not the one of our present invention. The injection is done to obtain a single bubble or several single bubbles, the size of the bubble and the quantity of air being an empirical determination, and the bubble should not being too large (1 cubic inch (2.54 cm 3 ) cited), not being specifically a bubble with a diameter close to the one of the tank. This is quite different from our present invention where the size of the bubble and quantity of air is critical for the growth of the living cells. In WO-A-8503458, in case of several air inlets, several single bubbles are generated to have circular, vertical toroidal flow patterns. WO-A-8503458 invention is used for open or vented tanks, which is not compatible with the cultivation of living cells under sterile conditions.
[0010] U.S. Pat. No. 4,136,970 describes also a method and apparatus for regulating the size and frequency of bubbles employed for mixing liquids. It does not concern itself with the oxygenation and cultivation of living cells, not concern with maximising the size of the bubbles, and does not concern with large bubble higher than 1.5 cm 3 . The method described in U.S. Pat. No. 4,136,970 can be used for the counting of blood platelets but can in no case adapted, used or claimed for the cultivation and growing of living cells.
[0011] The aim of the present invention is to provide a low cost cell culture system via a disposable apparatus, which is efficient at large scale and easy to use.
SUMMARY OF THE INVENTION
[0012] The present invention consists in a pre-sterilized flexible or non flexible plastic bag in which cells are cultivated, being agitated/aerated by single large gas-bubble.
[0013] In the present invention, a single large gas-bubble is generated intermittently at the bottom of the column, partially filled with liquid medium and cells. As the large bubble almost fills the cross-section of the column, it creates a thin space between the bubble and the sidewalls of the cylindrical tank where the liquid can flow as the bubble rises. This trickling liquid film, in contact with gas-bubble, allows convenient mixing and aeration of the bulk in the apparatus during operation without damaging the cells. Such a mixing/aeration system allows an efficient scale-up since oxygen and mass transfer reactions occur at the thin liquid film level. Moreover, as the system is simply designed, capital and maintenance costs are greatly reduced.
[0014] This disposable apparatus is made of sterilizable and flexible plastic sheets sealed along their edges to form a column. Such a disposable system allows process flexibility and decreases dead time since no cleaning, sterilization, maintenance or validation are required like in traditional stainless steel devices.
[0015] As the present invention is disposable and efficient at large scale, it is a good alternative system to decrease production costs in industrial applications.
[0016] This culture system can be applied for plant, animal, insect or micro-organism cultures, in suspension or immobilized on different carrier systems. The process allows to produce a large variety of molecules like metabolites (de novo or via biotransformation) or recombinant proteins, or to multiply embryogenic plant cell line through batch, fed-batch or continuous culture, as well as any other use that could be obvious for the skilled person.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a side view of the apparatus, showing the bag and phenomena created by the rising bubble.
[0018] FIG. 2 is a side view of plastic bag to tubing connection.
[0019] FIG. 3 is a schematic of the pneumatic and electric circuits useful for generation and control of the frequency and size of bubbles.
[0020] FIG. 4 shows top of the upper part of the tank in the form of an inversed cone.
[0021] FIG. 5 shows growth kinetics of Soya cells in flasks, stirred tank reactor and Cell culture system, expressed in fresh weight per liter of liquid culture.
[0022] FIG. 6 shows growth kinetics of Soya cells in flasks, stirred tank reactor and Cell culture system, expressed in dry weight per liter of liquid culture.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention consists in the use of very large single bubbles, periodically produced (whatever the process to obtain them), having a diameter as close as possible from the one of the bioreactor itself for the aeration/agitation (providing a efficient oxygenation) of cell cultures. The consequence is that the culture medium flows out as a very thin film between the large bubble and the inner wall of the bioreactor.
[0024] In a basic design, as shown in FIG. 1 , the bioreactor (or reactor) is composed of different parts, comprising at least one tank ( 1 ) made of material, such as plastic sheets sealed along their edges ( 2 ), for example, to create an interior. The tank is stationary. In a preferred embodiment of the present invention, the tank(s) are made of flexible polypropylene for its sealable and autoclavable properties, so it can be sterilized in a small laboratory autoclave or by any other means well known in the art. However other kinds of materials are also suitable such as Pyrex®, stainless-steel, semi-flexible, rigid or molded plastics, among others and can be sterilized by any method known by people skilled in the art such as gamma radiation.
[0025] In a preferred embodiment of the invention, flexible biocompatible water proof material are heat-sealed along their edges ( 2 ), for example, with a thermic impulse sealer. However other sealing techniques can also be used, in accordance with methods well known in the art including, but not limited to, ultrasound or radio wave welding. Other kinds of plastics can be manufactured in a different manner such as mold injection for example.
[0026] In the present invention, as shown in FIG. 1 , the reactor can be cylindrical or can have an oval cross section, it can have 2 m height and its diameter can be 12 cm for a working volume of 20 liters.
[0027] Smaller or higher volumes can be used according to the present invention. For example, the diameter of the reactor can be as small as 5 cm and can go up to 40 cm or more. The height of the reactor can vary according to the needs of the user and the diameter chosen.
[0028] The reactor can also have different shapes but preferably the height of the shape is at least 5 times its width. It can be, for example a parallelepiped. The dimensions and shape of the tank ( 1 ) can be varied to suit the needs of the users; however, the cylindrical column shape is preferred. It is important to avoid dead space, where mixing does not occur, when culturing cells in suspension. Dead spaces appear preferentially at the corners, that's why it is preferred to manufacture rounded-bottoms mostly with cells, which tend to form dense aggregates (such as plant cells), which settle more rapidly than individual cells.
[0029] If the tank is made in a flexible matter, such as plastic, it is recommended to put the said tank in a rigid outer container to support shape and weight of the tank. This rigid container can be made of any material such as polycarbonate but this material will be chosen mostly for its rigidity and strength properties (assumed by thickness and/or formulation). This outer container can be translucent to facilitate observation of the culture ( 3 ) if the plastic bag is also translucent or to improve light transmission when growing photoautotrophic cells for example. Dimensions and shapes of outer containers are preferably designed according to dimensions and shapes of tank discussed above.
[0030] In the basic design shown in FIG. 1 , at least four tubes are connected to the tank. The first one, at the top, is used to remove excess of gases ( 4 ). The second one, at the bottom of the tank ( 5 ), is used to provide air to the liquid culture through gas-bubble ( 6 ). These tubes are equipped, in the most preferred embodiment, with filters ( 7 ), such as for example 0.22 μm filters, to prevent airborne contamination. Air inlet tubing can be equipped with a valve to prevent back flush of the liquid in the tube. Moreover, one inlet tube ( 8 ) located at the top of the tank allows to fill the bioreactor with sterile medium and inoculum and one outlet tube ( 9 ) located nearby the bottom may be needed to harvest and/or sample the culture bulk.
[0031] In a preferred embodiment, tubing is semi-flexible, made of autoclavable silicone but other types of tubing like C-flex or PVC can also be used. In the preferred embodiment of the present invention, inner diameters of tubing are 8 mm, except for air inlet tubing which is larger: 11 mm diameter. Lengths of tubing are about one to two meter in this invention but users, to meet requirements, can adjust these dimensions.
[0032] Tubing can be connected to the tank via an incorporation port welded on the plastic sheet according to standard techniques such as heat-sealing. In the preferred embodiment of the present invention, as shown in FIG. 2 , tubing is connected to the tank through a hole in the plastic sheet to autoclavable panel mount union ( 10 ) equipped with bolts ( 11 ) and seams ( 12 ). Imperviousness can be obtained by screwing bolts to clench seams on the plastic sheet. Inner diameters of panel mount union are equal to inner diameters of corresponding tubings in this invention but it is possible to adjust dimensions as needed.
[0033] However, it has to be understood that any means allowing air or gas to circulate can be adapted to the present invention. It is important, for the purpose of the present invention, that aeration and mixing of the medium is achieved by large gas/air bubbles, and preferably by a single large bubble created every few seconds, having its diameter dictated by the diameter of the tank. Consequently the preferred mixing and aeration means of the invention consists in a bubble that is more long than wide. However, the system also works when bubbles are as long as wide.
[0034] Preferably, the large bubble shape is dictated by the shape of the tank; in other words, the space between the bubble and the tank is restricted to a minimum: to a film of medium comprising cells. Preferably, the culture medium flows out as a very thin film between the large bubble and the inner wall of the bioreactor. However, the system also works when the film is less thin and the bubble represents from 50 to 99% of the width of the tank preferably from 60 to 99%, more preferably 98.5%.
[0035] By large bubbles, it has to be understood that the volume of each single and large bubble is at least of 65 cm 3 , more preferably of at least 500 cm 3 . For example, in reactors having a diameter of around 20 cm, preferred volumes for the large bubbles can vary between 2600 and 4100 cm 3 , or more preferably between 3000 and 4100 cm 3 , or even more preferably between 3500 or 3700 and 4100 cm 3 .
[0036] To create large bubbles, a bubble generator ( 13 ) is linked to the air inlet tube. The bubble generator, as shown in FIG. 3 , is for example, an electro-gate ( 17 ), controlled by a timer ( 18 ) and linked to a gas pump ( 19 ). In such a configuration, the electro-gate, controlled electrically by the timer, is directly linked to air inlet and gas pump. Regularly, the timer (programmed by users) sends an electrical signal to the electro-gate for a very short period of time. During this time, the electro-gate is open and allows gas supplied from the pump to enter the bioreactor. When a high flow of gas is supplied for a very short period of time in the column, it creates a single large bubble, which fills almost the cross section of the column. In the present invention, section of the electro-gate is 15 mm, air pressure at the gas pump is 0.5 bar and the electrical signal, during 0.1 second, is sent every 5 seconds, thus creating a large bubble every 5 seconds. Users, depending on their needs, can adjust these parameters.
[0037] This kind of bubble generator is preferred but other devices allowing creation of a large gas bubble in the column can also be used. In the present invention, the gas used is air but other gases alone or mixed or recycled from the bioreactor can be used to meet the requirements of the cells, for example CO 2 for photoautotrophic plant cells. When the bubble arrives at the top of the column, is somehow explodes, and some medium/cells can be lost on the walls of the tank ( 1 ). To avoid this disadvantage, in an embodiment of the present invention, the upper part of the tank is flared, for example in the preferred embodiment it is in the form of an inversed cone, so that the medium/cells can fall back into the tank again (symbolized on FIG. 4 by arrows 20 ).
[0038] During operation, evaporation occurs, reducing the culture volume and concentrating different compounds in the medium, which could be detrimental to the cells. To avoid these problems, it is possible to add devices such as condensers for exhaust gas or humidifiers for gas supply. Moreover, it is possible to connect more inlet and/or outlet tubing to the column, it can be useful, for instance, for acids, bases, anti-foam or elicitation solutions adding. Optional devices can be added to this culture system for control and/or regulation of culture conditions such as (but not limited to) thermometer, pH meter, gas evaluation systems, cell density, pressure control, and mass control . . . It is also possible to place a light generator apparatus around the bioreactor for photoautotrophic plant cells for example. Regulation of temperature in the bioreactor can be achieved by different systems such as (but not limited to) placing the bioreactor in a room where temperature is controlled via suitable air conditioning, using jacketed outer containers where a circulation of temperature regulated water or air is provided, or any other means known by the skilled person.
[0039] The present invention is based on the fact that liquid culture trickles between the rising gas-bubble ( 6 ) and the sidewalls of the bioreactor (as shown by arrows ( 14 ) in FIG. 1 ). This results in vortices ( 15 ) to mix the bulk, avoiding cells to settle and in a thin liquid film ( 16 ) in contact with gas bubbles ( 6 ) where mass transfer is easily achieved for aeration.
[0040] This culture system is easy to operate since user can choose the volume and the frequency of bubbles by programming the bubble generator as previously described.
[0041] The system of the invention can be used to grow living cells, such as for example plant cells, animal cells, or micro-organisms such as yeast cells, for example. Said cells can produce, for example, biomass cells, embryogenic plant cells, metabolites, secondary plant metabolites, and/or recombinant molecules.
EXAMPLE
[0042] The following example is illustrative of some of the products and methods of making the same falling within the scope of the present invention. It is not to be considered in any way limitative of the invention. Changes and modifications can be made with respect to the invention. That is, the skilled person will recognise many variations in this example to cover a wide range of formulas, ingredients, processing, and mixtures to rationally adjust the naturally occurring levels of the compounds of the invention for a variety of applications.
EXAMPLE
Comparison of Growth with Soya Cell Cultures
[0043] The ability of the invention to grow Soya cells has been demonstrated using batch cultures. This is comparable or better than in Erlenmeyer flask or stirred tank bioreactor, even at larger scale.
[0044] Tissue culture strains of Glycine max (L.) Merr. were initiated from different cultivars on Gamborg et al. medium (1968) supplemented with 20 g.L −1 sucrose, 7 g.L −1 agar (bacto-agar Difco) and 1 mg.L −1 2,4-Dichlorophenoxyacetic acid. The pH is adjusted to 5.8 prior autoclaving (30 min at 115° C.). One strain (13406, cv. Maple arrow) was transferred in liquid medium (same medium as for tissue cultures without agar and 30 g.L −1 sucrose) and subcultured in 250 mL Erlenmeyer flask (3 g.L −1 fresh weight with 100 mL medium) every two weeks, in the same conditions than tissue culture collection. The Erlenmeyer flasks were placed on an orbital shaker at 100 rpm (shaking diameter 20 mm).
[0045] A 14 L stirred tank bioreactor (New Brunswick Scientific) with two six flat blade impellers, was used with the same medium and conditions of temperature and pH as mentioned above. The bioreactor containing 9 L of fresh medium was autoclaved 40 min at 115° C. Fourteen day old Soya cells were filtered from two 1 L Erlenmeyer flasks (500 ml medium). 300 g fresh weight was put into 1 L of fresh medium in a sterile tank with a specific output to be connected aseptically to the bioreactor for inoculation. The stirrer speed was adjusted at 100 rpm. Dissolved oxygen was maintained at 30% by increasing or decreasing air flow rate, using a biocontroller equipped with a sterilizable oxygen probe (Ingold), and a mass flowmeter
[0046] A 25 L Cell culture system called large bubble column (as previously described), putted into a rigid outer container, was filled with 20 L of Soya cells in fresh culture medium (30 g/L fresh weight). Temperature of the room was regulated at 25° C. and a 12 cm diameter bubble (about 10 cm height) was generated every 5 seconds (by programming the bubble generator as mentioned above).
[0047] Growth measurements: Samples of cultivation bulk were taken at certain periods of growth from flasks, stirred tank bioreactor and large bubble column and sample volume was measured. Cells were then removed from liquid culture via filtration. Biomass was weighed (fresh weight). An aliquot of this biomass (about 1 g) was weighed precisely and putted into a drying room at 100° C. during 24 hours and then weighed precisely again (dry weight).
[0048] This example shows that the 20 L scale column provides a gentle environment to the cells, comparable with flasks and better than the stirred tank reactor. Cell damages are limited and mass and gas transfers are efficient in the operated conditions.
[0049] As already mentioned above, the present invention provides numerous advantages, which in turn are keys to economic benefits:
[0050] It provides a gentle environment to grow plant cells
[0051] Scale-up is easy
[0052] It is disposable
[0053] It is easy to operate
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The present invention provides a novel apparatus to grow cells where the cultivation chamber ( 1 ) is partially filled with liquid cultivation medium and cells. Mixing and aeration is achieved by generating intermittently one single large gas bubble ( 6 ) at the bottom of the column bioreactor, the single large bubble width representing from 50 99% of the tank width, preferably from 60 to 99%, more preferably 98.5%. The culture medium flows out as a film between the large bubble and the inner wall of the bioreactor. This rising bubble allows mixing and aeration of the bulk. As the design of the invention is very simple, it is possible to manufacture it with flexible plastic material and use the apparatus as a disposable system. Moreover, such a mixing/aeration principle minimizes cell damages usually due to shear stress and small bubbles and allows easy and efficient scale-up from small scale to a larger one. Such a large-scale, efficient and disposable culture system can largely reduce production costs.
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CROSS-REFERENCE TO RELATED APPLICATION
This application claims the priority, under 35 U.S.C. §119, of U.S. Provisional Patent Application No. 60/612,684 filed Sep. 24, 2004, the entire disclosure of which is hereby incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
n/a
FIELD OF THE INVENTION
The invention lies in the field of medical devices, namely, catheters. In particular, the invention relates to catheters that can change stiffness characteristics in use.
BACKGROUND OF THE INVENTION
To gain access to treatment sites in the body, catheters must be flexible enough to conform to and follow natural anatomical pathways as they are advanced. These pathways can be quite tortuous, made of soft and delicate tissues with many twists and turns. In the vasculature, this is especially the case, and even more so in certain areas of the vasculature such as the vessels of the brain and the coronary arteries.
When treating a site in the vasculature, the state-of-the-art practice is to first gain access to the treatment site with a flexible, steerable guidewire. Such a guidewire can be precisely controlled by the physician and steered into place using radiographic guidance. Once the guidewire is in-place, the catheter is advanced over the guidewire. The catheter must be flexible enough to smoothly follow the pathway of guidewire. The catheter can, then, be used to deliver the treatment.
In the case of arterial blockage, the catheter may be a balloon dilatation catheter that is used to open the blockage. The guidewire is, first, passed beyond the lesion, and the catheter is advanced over the guidewire and through the lesion. In the case of complete or nearly complete blockage, the force required to advance the guidewire through the lesion can be difficult for the physician to generate by pushing on the flexible guidewire from the arterial access site. Further, this access site may be far from the treatment site, such as in the case of coronary arterial treatment where access to the coronary arteries is gained though the femoral artery. In such a situation, the physician is trying to advance the flexible guidewire through an obstruction over 100 cm away from where he/she is pushing. The same flexibility that helped gain access to the treatment site now inhibits the advancement of the guidewire. The guidewire bends and buckles under the strain and very little thrust is delivered to the tip of the guidewire.
Current practice advances the balloon catheter up to the treatment site to provide support to the guidewire as it is advanced through the lesion. This is an improvement, but the catheter is also very flexible and provides little if any additional support. Specialty support catheters, which offer more support than balloon catheters, are also used. These provide an improvement over balloon catheters but are also limited by how flexible they must be to reach the treatment site.
The above-mentioned problems are compounded in the case of a total arterial blockage or Chronic Total Occlusion (CTO). Accordingly, most CTOs go untreated. And, there is no catheter-based standard accepted practice for CTO treatment. Currently, treatment of CTOs by catheter interventionalists is performed by attempting to pass a guidewire across the CTO. Once the guidewire is across, a low profile balloon catheter can be advanced over the guidewire to dilate the lesion. Such a procedure is almost always followed by placement of a stent. Specialty guidewires are available to aid the physician in this effort but they, too, are limited in their utility by the constraints of flexibility and compliance. It is noted that attempting to cross CTOs is a tedious practice with current equipment and is met with limited success.
Therefore, it would be beneficial to provide a catheter that can advance up to the treatment site with sufficient flexibility through a tortuous path and that can provide sufficient support to advance through a CTO lesion.
SUMMARY OF THE INVENTION
It is accordingly an object of the invention to provide a catheter with controllable stiffness and method for operating a selective stiffening catheter that overcome the hereinafore-mentioned disadvantages of the heretofore-known devices and methods of this general type and that can traverse a natural passage of the body in a first flexible state and can be made to change to a second stiffer state and cycle back and forth repeatedly between these states at will and that can traverse tortuous anatomy by conforming to it and, once in-place, can be made to stiffen and maintain its tortuous shape in the anatomy.
The catheter of the present invention provides a platform on which physicians can deliver tools to treatment sites to aid in the crossing of arterial blockages, especially, CTOs. In the stiff state, these tools can be used and the force applied by the tools on the treatment site can be enhanced and increased based upon the stiffness properties of the catheter.
The catheter according to the invention has a stiffness that can be controlled during use. The stiffness of the catheter can be changed during use from soft and flexible to firm and stiff and back again, if desired. The entire length of the catheter can be made to change its stiffness characteristics. Alternatively, and/or additionally, any portion or portions of the device can be configured to change its stiffness characteristics as well.
The catheter is delivered to the treatment site in the flexible state, in which, it will track over the guidewire and conform to the anatomical pathway, e.g., the vasculature. Once in-place, the catheter can be made to become stiff (either in whole or in part) without straightening and, thereby, maintain its conformance to the vasculature. In such a state, the catheter provides a stiff conduit to deliver tools to the treatment site without compromising the natural anatomy. This stiffness provides the support necessary to efficiently advance the guide or crossing wires without loss of motion and efficiently transmit thrust loads to the tools.
In the case of a guidewire as described above, the guidewire will, with use of the catheter according to the invention, not flex away from the treatment site when pushed and provides great increases in feel, control, and thrust. Such characteristics aid in the successful crossing of difficult-to-cross lesions and provide an opportunity to cross CTOs.
The vasculature example above has been used to describe the problem and embodiments of the present invention, but it can be appreciated that this same concept can be used in any part of the body.
With the foregoing and other objects in view, there is provided, in accordance with the invention, a controllable stiffness catheter, including a shaft having a stiffness sheath and an access lumen, a stiffness device having a heater and a melting binder in thermal contact with the heater, the stiffness device being disposed in the stiffness sheath, the binder having a transition temperature, being substantially solid at body temperature and softening as the heater warms the binder above the transition temperature, a first electrical conductor electrically contacting the stiffness device, a second electrical conductor electrically contacting the stiffness device and being electrically isolated from the first electrical conductor, a power supply for supplying power, a controller electrically connected to the first electrical conductor, to the second electrical conductor, and to the power supply to form an electrically resistive circuit having the stiffness device as a resistor of the circuit, the controller controlling power supplied to the stiffness device through the circuit and, thereby, controlling a temperature of the heater and the binder, and the stiffness device changes a stiffness of the stiffness sheath between a relatively stiff state when the heater is not energized by the controller and a relatively soft state when the heater is energized by the controller.
With the objects of the invention in view, there is also provided a controllable stiffness catheter, including a shaft having a stiffness sheath and an access lumen, a stiffness device having a heater and a melting binder in thermal contact with the heater, the stiffness device being disposed in the stiffness sheath, the binder having a transition temperature and being substantially solid at body temperature and softening as the heater warms the binder above the transition temperature, and a power controller electrically connected to the heater and selectively supplying power to the heater to change a stiffness of the stiffness sheath between a relatively stiff state when the heater is not powered by the power controller and a relatively soft state when the heater is powered by the power controller.
With the objects of the invention in view, there is also provided a controllable stiffness catheter, including a shaft having a stiffness sheath and an access lumen, means for controlling stiffness disposed in the stiffness sheath, the stiffness controlling means being substantially solid at body temperature and softening above body temperature, and means for supplying heat to the stiffness controlling means to change a stiffness of the stiffness sheath between a relatively stiff state and a relatively soft state. In one embodiment, the stiffness-controlling means is in the stiff state when the heat-supplying means is not activated and the heat-supplying means changes a stiffness of the stiffness sheath to the soft state when the stiffness controlling means is activated.
With the objects of the invention in view, there is also provided a controllable stiffness catheter, including a shaft having a stiffness sheath and an access lumen, a stiffness device disposed at the stiffness sheath and being in a relatively stiff state at or below a first temperature and being in a relatively soft state at or above a second temperature, a temperature-changing device in thermal contact with the stiffness device, the temperature-changing device changing a temperature of the stiffness device at least below the first temperature and above the second temperature, and a power controller electrically connected to the stiffness device and selectively supplying power to the temperature-changing device to change a stiffness of the stiffness device between the stiff state and the soft state.
With the objects of the invention in view, there is also provided a method for manufacturing a controllable stiffness catheter, includes the steps of providing a shaft with a stiffness sheath and an access lumen, disposing a heater inside the stiffness sheath, filling the stiffness sheath containing the heater with a binder to at least partially surround and thermally contact the heater, the binder being substantially solid at body temperature and softening above body temperature, and selectively supplying power to the heater to change a stiffness of the stiffness sheath between a relatively stiff state when the heater is not powered and a relatively flexible state when the heater is powered.
With the objects of the invention in view, there is also provided a method for treating a Chronic Total Occlusion, including the steps of extending a guidewire to a CTO treatment site in a body, providing a controllable stiffness catheter according to claim 39 and, with supplying power to the heater, threading the catheter along the guidewire up to the CTO, removing power from the heater to change stiffness of the stiffness sheath to the stiff state without straightening the catheter, and projecting the guidewire through the CTO. Preferably, the guidewire is a flexible, steerable guidewire.
With the objects of the invention in view, there is also provided a method for operating a selective stiffening catheter, including the steps of placing a selective stiffening catheter in a relatively flexible state, traversing a natural passage of the body with the catheter in the flexible state, and changing the catheter to a relatively stiffer state to substantially maintain a current shape of the catheter in the body.
With the objects of the invention in view, there is also provided a method for treating a Chronic Total Occlusion, including the steps of extending a guidewire to a CTO treatment site in a body, providing a controllable stiffness catheter with a shaft having a stiffness sheath and an access lumen, a stiffness device disposed at the stiffness sheath and being in a relatively stiff state at or below a first temperature and being in a relatively soft state at or above a second temperature, a temperature-changing device in thermal contact with the stiffness device, the temperature-changing device changing a temperature of the stiffness device at least below the first temperature and above the second temperature, and a power controller electrically connected to the stiffness device and selectively supplying power to the temperature-changing device to change a stiffness of the stiffness device between the stiff state and the soft state, threading the catheter along the guidewire up to the CTO in the soft state, changing a stiffness of the stiffness sheath to the stiff state without straightening the catheter, and projecting the guidewire through the CTO.
In accordance with another feature of the invention, the stiffness sheath is of a polymer, in particular, polyurethane, and the access lumen is of a polymer, in particular, PTFE.
In accordance with a further feature of the invention, the access lumen is disposed inside the stiffness sheath and is substantially concentric therewith to define an annulus therebetween. Accordingly, the stiffness device is disposed in the annulus. Preferably, the access lumen has an outer diameter of approximately 0.4 mm and the stiffness sheath has an outer diameter of between approximately 0.4 and 1.73 mm.
In accordance with an added feature of the invention, the stiffness device is disposed between the access lumen and the stiffness sheath. Preferably, the stiffness device is a resistive heating element, in particular, Nickel/Chromium wire.
In accordance with an additional feature of the invention, the binder is a mixture of discontinuous fibers, in particular, the discontinuous fibers are of chopped carbon fiber or glass. The binder is substantially solid up to approximately 105° F. and is soft at or above approximately 115° F.
In accordance with yet another feature of the invention, the stiffness device is one or more carbon fiber tows impregnated with the binder.
In accordance with yet a further feature of the invention, the shaft has a shaft distal end and a shaft proximal end, the power supply and the controller are disposed at the shaft proximal end, the at least one carbon fiber tow has a tow proximal end and a tow distal end, the first electrical conductor has a first distal end electrically connected to the tow distal end and a first proximal end electrically connected to the controller, and the second electrical conductor has a second distal end electrically connected to the tow proximal end and a second proximal end electrically connected to the controller.
In accordance with yet an added feature of the invention, the controller selectively applies a voltage to the first and second electrical conductors to cause current to flow through the circuit and resistively heat the at least one carbon fiber tow and to remove current flowing through the circuit and cool the at least one carbon fiber tow. The heated carbon fiber tow melts and softens the binder to allow individual carbon fibers of the tow to move with respect to each other and, thereby, increase flexibility of the shaft. A third electrical conductor having a distal end electrically connected to an intermediate point of the carbon fiber tow and a proximal end electrically connected to the controller. The controller selectively heats at least one portion of the carbon fiber tow dependent upon current supplied to at least two of the first, second, and third conductors.
In accordance with yet an additional feature of the invention, the heater is located only at a portion of the stiffness sheath. The portion can be a distal portion of the stiffness sheath. Alternatively, the stiffness device can be present throughout an entire length of the stiffness sheath.
In accordance with again another feature of the invention, the stiffness device is disposed between the access lumen and the stiffness sheath and the carbon fiber tow substantially surrounds the access lumen. Preferably, there are more than one carbon fiber tows. The tows can helically surround the access lumen and/or be braided around the access lumen.
In accordance with again a further feature of the invention, the binder is a low-melt-point wax, for example, paraffin, microcrystalline, or blended wax.
In accordance with again an added feature of the invention, the electrical conductors can be of copper wire.
In accordance with again an additional feature of the invention, one of the first and second electrical conductors electrically contact a distal end of heater and runs from the distal end of the catheter to the proximal end of the catheter and another of the first and second electrical conductors electrically contact a proximal end of heater.
In accordance with still another feature of the invention, the power supply is one of a battery and an electric mains.
In accordance with still a further feature of the invention, the controller limits heating of the binder by limiting current through the circuit. The controller includes a Proportional-Integral-Derivative current-sensing controller to limit at current supplied in the circuit. The controller includes a thermocouple for monitoring and regulating a temperature of the binder.
In accordance with still an added mode of the invention, power is supplied to the heater to change stiffness of the stiffness sheath to the soft state, the catheter is withdrawn from the guidewire while leaving the guidewire in the CTO, a balloon catheter is advanced over the guidewire and through the CTO, the balloon catheter having a stent surrounding a balloon, and the balloon is expanded to dilate the CTO and place the stent within the dilated CTO.
In accordance with still an additional mode of the invention, the projecting step is carried out by entirely withdrawing the guidewire from the catheter and replacing the guidewire with a CTO-opening tool having a relatively sharp distal end and the projecting step is carried out by opening the CTO with the CTO-opening tool.
In accordance with another mode of the invention, access to the treatment site is gained with the guidewire by steering the guidewire into place using radiographic guidance.
In accordance with a further mode of the invention, power is applying to the heater to place the catheter in the soft state, a natural passage of a body is traversed with the catheter in the soft state to deliver the catheter to a treatment site, and power is removed from the heater to harden the catheter into the stiff state and to substantially maintain a current shape of the catheter in the body.
In accordance with an added mode of the invention, a guidewire is first placed in the passage of the body to the treatment site and then the applying, traversing, and removing steps are carried out by traversing the passage with the catheter threaded on the guidewire.
In accordance with an additional mode of the invention, the removing step is carried out without straightening the catheter.
In accordance with a concomitant mode of the invention, in the stiff state, the catheter is provided as a relatively stiff conduit to deliver tools to the treatment site without compromising the natural anatomy, to support the tools and efficiently advance the tools therethrough without loss of motion, and to transmit thrust loads to the tools.
Other features that are considered as characteristic for the invention are set forth in the appended claims.
Although the invention is illustrated and described herein as embodied in a catheter with controllable stiffness and method for operating a selective stiffening catheter, it is, nevertheless, not intended to be limited to the details shown because various modifications and structural changes may be made therein without departing from the spirit of the invention and within the scope and range of equivalents of the claims.
The construction and method of operation of the invention, however, together with additional objects and advantages thereof, will be best understood from the following description of specific embodiments when read in connection with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Advantages of embodiments the present invention will be apparent from the following detailed description of the preferred embodiments thereof, which description should be considered in conjunction with the accompanying drawings in which:
FIG. 1 is a fragmentary, enlarged perspective view of a distal end of a shaft of a catheter according to the invention;
FIG. 2 is a block and schematic circuit diagram and a diagrammatic side elevational view of a proximal end of the catheter according to the invention;
FIG. 3 is a fragmentary, enlarged, cut-away, perspective view of a distal end an alternative embodiment of the catheter according to the invention; and
FIG. 4 is a fragmentary, enlarged, cut-away, perspective view of a distal end a further alternative embodiment of the catheter according to the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Aspects of the invention are disclosed in the following description and related drawings directed to specific embodiments of the invention. Alternate embodiments may be devised without departing from the spirit or the scope of the invention. Additionally, well-known elements of exemplary embodiments of the invention will not be described in detail or will be omitted so as not to obscure the relevant details of the invention.
While the specification concludes with claims defining the features of the invention that are regarded as novel, it is believed that the invention will be better understood from a consideration of the following description in conjunction with the drawing figures, in which like reference numerals are carried forward.
Before the present invention is disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
Referring now to the figures of the drawings in detail and first, particularly to FIG. 1 thereof, there is shown a distal portion of a shaft 10 of an exemplary embodiment of a catheter 1 according to the invention. The shaft 10 is configured with an outer sheath 11 made of a polymer tube such as polyurethane and an inner sheath 12 made of a polymer tube such as PTFE. The inner sheath 12 is assembled substantially concentrically with the outer sheath 11 . The annulus between the inner and outer sheaths 12 , 11 is filled with a stiffness device, in particular, at least one carbon fiber tow 13 (preferably, 2 to 4 tows 13 extending longitudinally in a helix or braided) impregnated with a binder such as a low-melt-point paraffin or microcrystalline wax or other temperature dependent phase change material. At body temperature, the binder is a solid and, therefore, the carbon fiber tow 13 behaves substantially as a solid carbon fiber rod. (As used herein, “body temperature” is defined to be approximately 40.5° C. (105° F.) or below). In such a condition, the catheter is stiff due the high modulus of the carbon fibers.
It is noted that concentricity is not a requirement. In another exemplary embodiment of the catheter 1 of the present invention, the inner sheath 12 can merely be off-center or the inner sheath 12 can be disposed at the inner wall of the outer sheath 11 . In the latter orientation, the space in which the stiffness device resides is somewhat crescent-shaped.
In one exemplary embodiment, an electrical conductor 14 , such as insulated copper wire, makes electrical contact with the distal end(s) of the carbon fiber tow(s) 13 and runs from the distal end of the catheter 1 to the proximal end of the catheter 1 . The proximal end(s) of the carbon fiber tow(s) 13 makes contact with a second electrical conductor 15 , such as copper wire, which extends to the proximal end of the catheter 1 at which resides a power supply 16 (e.g., a battery or an electric mains) and a controller 17 as shown in FIG. 2 . The proximal ends of the two conductors 14 , 15 are electrically connected to the power supply 16 through the controller 17 . These features make up a simple electrical circuit with the carbon fiber tow(s) acting like a resistor in the circuit. When voltage is applied to the two electrical conductors 14 , 15 , current flows through the circuit 13 , 14 , 15 and resistively heats the carbon fiber tow(s) 13 . When the tow 13 is heated to raise the temperature of the binder above a binder transition temperature, the binder softens (which can include a partial or a full melt) and allows the individual carbon fibers to move with respect to each other, thereby making the catheter shaft more flexible than before. (As used herein, the binder transition temperature is at or above approximately 46° C. (115° F.).) When the voltage is removed from the circuit 13 , 14 , 15 , the binder cools and solidifies. Thus, the catheter shaft 10 stiffens to its then constrained shape. This heating and cooling can be done repeatedly—making the catheter 1 flexible when navigating through a tortuous path and stiff when placed in a position for use, for example.
Another exemplary embodiment of the catheter 1 according to the invention is similar to that illustrated in FIG. 1 but, instead of a concentric configuration, the shaft 10 is constructed of a first hollow sheath 11 made of a polymer tube such as polyurethane and a second hollow sheath 12 made of a polymer tube such as PTFE. The first sheath 11 is assembled next to and outside of the interior of the second sheath 12 such that a cross-section of the two conduits is shaped like the number eight. The entire core of the second sheath 12 , therefore, can be used to house the stiffness device 13 .
Electrical power for supplying a voltage or current can be provided, for example, by at least one battery 16 . This battery 16 can be connected to the conductors 14 , 15 through the controller 17 , which is configured to limit heating of the binder by limiting current through the circuit 13 , 14 , 15 . Such current limiting can be achieved by using a Proportional-Integral-Derivative (PID) controller whereby a standard feedback loop measures the “output” of the process and controls the “input”, with a goal of maintaining the output at a target value, which is called the “setpoint”. Such a current-sensing controller, for example, could make the initial current through the circuit 13 , 14 , 15 high enough to achieve a rapid melt and, thus, a rapid softening, with a subsequent decrease and leveling in the current to just maintain the melt. A thermocouple 18 can be added to actively monitor temperature of the melt. A control switch 19 and indicator LEDs 20 are added to the handle of the catheter 1 to give control and feedback to the user.
The entire length of the catheter 1 can be controllable in stiffness or just a portion of it can be controlled. In the case of a coronary catheter, the distal 20 cm or so can be controllable. The remainder of the catheter 1 can be constructed to have a stiffness sufficient to deliver the controllable portion to the coronary arteries. In such an embodiment, the stiffness device 13 is only present in the distal quarter of the shaft, for example, and one conductor 14 is electrically connected to the distal end of the stiffness device 13 (located at approximately the distal end of the shaft) and the other conductor 15 is electrically connected to a point on the shaft approximately three-quarters of the way to the distal end of the stiffness device 13 .
The connection of conductors 14 , 15 need not only be at the two ends of the stiffness device 13 . Additional non-illustrated conductors can be electrically connected to different places along a single stiffness device 13 that extends the entire length of the catheter 1 to, thereby, subdivide the stiffness device 13 into different stiffening segments. The proximal ends of each of these additionally conductors are electrically connected to the controller 17 . Accordingly, only a portion or a set of portions of the stiffness device 13 can be softened depending upon which conductors are energized. Alternatively, the stiffness device 13 can be a set of tows 13 having different lengths with two conductors connected respectively to each tow.
In any embodiment of the conductors 14 , 15 and the stiffness device 13 , the conductors should be electrically isolated from one another. Even if one conductor contacts a first end of all of a plurality of stiffness devices 13 , the other conductors connected to the second end of each stiffness device must be electrically isolated from one another and the one conductor contacting the first end.
FIG. 1 shows a plurality of carbon tows 13 and distal conductors 14 , 15 wound around the inner sheath 12 . The pitch and the quantity of the carbon fiber tows 13 , and the properties of the binder, can be adjusted to affect the final stiffness of the catheter 1 . A stiffer binder or the addition of more carbon fiber would lead to a stiffer catheter and a less stiff binder or the subtraction of carbon fiber would lead to a less stiff catheter. A change in the pitch of the wind along the length of the catheter 1 would also vary the stiffness along its length.
The carbon fiber tows 13 can also be oriented longitudinally as rods without wrapping them around the inner sheath 12 . Or, a hollow braid of the carbon fiber tows 13 can be made to surround the inner sheath 12 . The distal conductor(s) 14 , 15 could be included anywhere along the in the rods or braid if desired.
A luer fitting 21 is located at the proximal end of the catheter 1 . This fitting 21 provides access to the central lumen of the catheter, for example, for a CTO-piercing tool. A hemostasis valve can be connected to the fitting 21 , for example, while the catheter is in use.
In another embodiment of the stiffness device, a mixture of shorter discontinuous fibers such as chopped carbon fiber or fiberglass and binder can be used instead of impregnated continuous carbon fiber tows 13 . In such a case, the fibers would no longer be used as the resistive heating measure. Heating of the binder can be achieved by wrapping the inner sheath 12 with a resistive heating element, such as Nickel/Chromium (e.g., NICHROME®) wire. In such a configuration, the wire passes the current and becomes warm, thus heating the surrounding fiber-loaded binder.
In the case of a coronary version of the catheter, the lumen diameter of the inner sheath is, at a minimum, 0.4064 mm (0.016″) to ensure free passage of 0.3556 mm (0.014″) diameter steerable coronary guidewires. It is preferred for the outer diameter of the catheter to be no greater than 1.651 mm (0.065″) to be compatible with an inner diameter of a standard 6 French coronary guide catheter (minimum inner diameter 1.7272 mm (0.068″)). Larger or smaller versions can be constructed to suit specific needs.
The catheter can also be stiffened using mechanical measures. The annulus between the inner and outer sheaths can be filled with a fine granular substance, such as aluminum oxide or silica, as shown in FIG. 3 . In its flexible state, the fine grains are loose and slide past each other as the catheter is flexed. When vacuum is applied to the annulus, however, the pressure is lowered inside the annulus and the flexible outer sheath begins to compress the grains of the filler together from the urging of the higher pressure outside the catheter. Under such compression, the grains are forced against each other and interlock, no longer sliding past each other and, thus, stiffening the catheter without straightening it. The magnitude of the pressure change between the outside of the catheter and the interior of the annulus affects the catheter stiffness: a small pressure difference (lower vacuum) for a more flexible catheter, a large pressure difference (higher vacuum) for a stiffer catheter. It is clear to see that the catheter could have multiple independent zones that could each be controlled by a different level of vacuum, thus, illustration of this feature is not necessary to understand the present invention. This allows the catheter to be stiff in some zones, and more flexible in others. Such stiffening could also be accomplished without using the granular substance by substituting a rough surface (such as ridges, grooves, bonded grit and combinations thereof) on the outside of the inner sheath and on the inside of the outer sheath as shown in FIG. 4 . In its flexible state, the two rough surfaces do not engage each other substantially. When vacuum is applied, the outer sheath is compressed, and the rough surface on the inside of the outer sheath begins to engage the rough surface on the outside of the inner sheath thereby stiffening the catheter.
The following text outlines exemplary procedures for using the catheter 1 of the present invention to pass a CTO.
First, a flexible, steerable guidewire is precisely controlled by the physician and steered into place at a treatment site in a body using, for example, radiographic guidance. Once the guidewire is in place, the catheter 1 of the present invention can be advanced over the guidewire. It is understood that body pathways can be quite tortuous and are made of soft and delicate tissues. This is especially true in the vasculature, in particular, in vessels of the brain and the coronary arteries. Therefore, using the catheter 1 to gain access to the treatment site in the body most likely requires that the catheter 1 start as being flexible to conform to and follow the natural anatomical pathways as it is advanced to the site.
In the case of a CTO, the guidewire is advanced only up to the blockage. Then, the access lumen 12 (whether inside the stiffness lumen 11 or outer sheath 11 ) is threaded on the guidewire. The catheter 1 is in its softened state or is caused to enter its softened state so that the catheter 1 can be threaded along the guidewire up to the CTO. At the point where the catheter 1 is near the CTO, the catheter 1 is caused to become stiff (without straightening).
In the stiff state, a CTO-opening tool will be used to open the CTO. For example, the CTO-opening tool can be the guidewire itself. Alternatively, a CTO-opening tool can be inserted through the access lumen 12 and into the CTO. If the tool is a device entirely separate from the catheter 1 , the guidewire can be removed from the catheter 1 and the CTO-opening tool can be threaded through the access lumen 12 . Preferably, the CTO-opening tool is hard (but flexible to traverse the catheter 1 ) and has a sharp distal end.
The guidewire or tool is pressed through the CTO with the stiffened catheter 1 efficiently transmitting the thrust loads to the tool as the CTO is providing resistance to puncture. Once the guidewire/tool is across the CTO, the guidewire/tool can be used to guide another device that will open and fix the blockage. To remove the catheter 1 , first, the catheter 1 is caused to soften. After softening, the catheter 1 is removed and the guidewire/tool is left in the position passed through the CTO. A low profile balloon catheter, for example, is advanced over the guidewire/tool and through the lesion. The balloon is expanded to dilate the lesion. A stent can, then, be placed in the lesion to fix the CTO.
With use of the catheter according to the invention, the guidewire/tool will not flex away from the treatment site when pushed and provides great increases in feel, control, and thrust. Such characteristics aid in the successful crossing of difficult-to-cross lesions and provide an opportunity to cross CTOs.
The foregoing description and accompanying drawings illustrate the principles, preferred embodiments and modes of operation of the invention. However, the invention should not be construed as being limited to the particular embodiments discussed above. Additional variations of the embodiments discussed above will be appreciated by those skilled in the art.
Therefore, the above-described embodiments should be regarded as illustrative rather than restrictive. Accordingly, it should be appreciated that variations to those embodiments can be made by those skilled in the art without departing from the scope of the invention as defined by the following claims.
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A method and device for treating a Chronic Total Occlusion provides a controllable stiffness catheter with a shaft having a stiffness sheath and an access lumen. The device has a stiffness device in a stiff state below a given temperature and in a soft state above the temperature, a temperature-changing device in thermal contact with the stiffness device, the temperature-changing device changing a temperature of the stiffness device below and above the temperature, and a power controller electrically connected to the stiffness device and selectively supplying power to the temperature-changing device to change a stiffness of the stiffness device between the stiff and soft states. A guidewire is moved to a treatment site and the catheter moves along the guidewire up to CTO in its soft state. The catheter stiffens without straightening and, in the stiff state, the catheter or the guidewire is projecting through the CTO.
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FIELD OF THE INVENTION
[0001] The present invention is referred to a method and an apparatus for producing direct reduced iron utilizing a source of reducing gas comprising hydrogen and carbon monoxide.
BACKGROUND OF THE INVENTION
[0002] In the recent years, the necessity of increasing the steelmaking process efficiency and productivity has become more urgent, due to rising production costs and also due to the restrictions imposed upon steel plants by environmental regulations.
[0003] One of the successful routes for steelmaking, which is being increasingly promoted, is the gas based Direct Reduction of Iron Ore to produce Direct Reduced Iron (DRI), also known in the steel industry as sponge iron, by circulating a reducing gas through a moving bed of particulate iron ore at temperature of the order of 700° C. to 1100° C. Oxygen is removed from the iron ore by chemical reduction for the production of highly metallized DRI.
[0004] Some of the advantages of direct reduction plants are the wide range of production capacity, that the metallic iron is produced in solid form with low sulfur and silicon content, and that the resulting DRI may be used as raw material for the electric furnace and may constitute the whole charge thereof.
[0005] Additionally, and as a peculiar advantage of the technology proposed, given that a part of the CO 2 produced as by product of the reduction reactions is selectively removed from the process, total CO 2 emitted in the atmosphere may be considerably reduced if compared with others routes for steel production.
[0006] The reducing agents utilized in the direct reduction plants are hydrogen and carbon monoxide, obtained by reformation of natural gas in an external catalytic reformer or “in situ” within the iron reduction system. Nevertheless, a direct reduction plant can be also designed for utilizing other sources of energy available in the form of gases from coke ovens, blast furnaces, coal or oil gasification, natural gas, exhaust gases containing hydrogen and carbon monoxide arriving from other chemical/metallurgical processes, etc.
[0007] A possible source of reducing gas is the excess gas produced in the combination of a plant for the production of pig iron based on the use of coal (for example a blast furnace or a plant known in the industry with the tradename Corex) and a direct reduction reactor. Corex plants or blast furnaces produce pig iron using gasified coal by partial combustion with an oxygen-containing gas. The exhausted reducing gas withdrawn from this process, still containing H 2 and CO, can be utilized for reduction, after removal of at least a portion of H 2 O and CO 2 .
[0008] U.S. Pat. No. 5,238,487 to Hauk et al. discloses a process comprising a melter-gasifier, a first reduction reactor and a second reduction reactor wherein DRI is produced using directly reducing gas effluent from said first reactor. As indicated in this patent, the effluent reducing gas, after being only cleaned, is mixed with dewatered spent reducing gas and treated in a CO 2 removal unit. The gas leaving the decarbonation station is then heated in a heat exchanger and finally subjected to a partial combustion to reach the right temperature required for the reduction reaction. Additionally, this patent teaches to use sulfur oxides and chlorine to inhibit carbon monoxide decomposition. All embodiments of this patent however utilize heat exchangers that consume a fuel for heating the reducing gas prior to the partial combustion heating stage.
[0009] U.S. Pat. No. 5,676,732 to Viramontes-Brown et al. discloses an improved method and an apparatus for utilizing in a direct reduction plant the excess exhausted gas from a first reduction reactor, which receives reducing gas from a melter-gasifier. Said method suggests to use a catalytic reactor, or shifter, for adjusting the composition of the gas stream effluent from said first reactor in order to avoid carbon deposition and corrosion in the gas heater required to heat fresh gas before feeding it into the reduction reactor. In order to get the maximum yield of H 2 product from the CO shift conversion, a special catalyst in a fixed bed reactor is used. For this reason, Syngas has to be further treated in order to remove substances that are poisonous for the catalyst.
[0010] Referring now to Syngas from a gasifier as alternative source of reducing gas, U.S. Pat. No. 6,149,859; and U.S. Pat. No. 6,033,456 to Jahnke et al. describe an integrated process for supplying high-pressure Syngas from a gasifier to a direct reduction plant. As in the prior art, this patent suggests to treat the Syngas in a shifter with the purpose of changing its composition in order to avoid carbon deposition when said gas is heated at a temperature higher than 400° C. (condition commonly achieved in a typical process gas heater of a Direct Reduction Plant). In this way, the conditioned gas stream, after being treated in a dedicated unit to remove CO 2 and being expanded to the pressure of the direct reduction circuit, is ready for being used as make up in the DRI process.
[0011] WO-A-2008/146112 discloses the additional possibility of having, in a process as described in U.S. Pat. No. 6,149,859 and U.S. Pat. No. 6,033,456, a single absorption unit wherein the acid-gas content is removed from a combined stream of both the Syngas produced in the gasifier and the recycle reducing gas from the reduction reactor.
[0012] U.S. Pat. No. 5,846,268 to Diehl et al. discloses a process for producing liquid pig iron or liquid steel pre-products and DRI from iron ore. The process shown in this patent is much similar to the process described by U.S. Pat. No. 5,238,487 to Hauk et al. where a reducing gas, derived from the gasification of coal is used for reducing iron ore in a first reduction shaft furnace and the exhausted reducing gas effluent from said first shaft furnace is utilized for producing more DRI in a second shaft furnace. This patent teaches several ways of using heat of the gas stream effluent from the second shaft furnace for preheating a portion of the same gas stream which is then utilized as fuel in a fired gas heater, but does not teach or suggests using said heat to preheat the stream of reducing gas fed to the reduction reactor.
[0013] None of the above patents teach or suggest the distinctive features of the present invention which overcome a number of disadvantages of the prior art and provide a more efficient method and apparatus for producing DRI utilizing gas derived from coal gasification in a gasifier or derived from a melter-gasifier, for example, using heat from the top gas effluent from the reduction reactor for heating the reducing gas to be fed to said reactor without consuming any additional fuel and within the practical limits of the degree of oxidation of the reducing gas for an efficient reduction of iron ore.
[0014] An additional advantage of the present invention is that the carbon dioxide emissions to the atmosphere can be decreased because there is no combustion in the heat exchanger for raising the temperature of the reducing gas prior to second heating stage of partial oxidation with oxygen.
OBJECTS OF THE INVENTION
[0015] It is therefore an object of the present invention to provide an improved process and apparatus for producing direct reduced iron (DRI) using a gas with high content of carbon monoxide possibly after being cleaned in order to remove dusts and TAR, and feeding this cleaned gas with high carbon monoxide content directly to the reduction circuit without additional treatment in a water gas shifter for changing its composition. This simplified process configuration has the further advantage that removal of compounds that are poisonous for the shifter catalyst is not required.
[0016] It is another object of the present invention to provide an improved method and apparatus for producing hot or cold DRI in which upgraded reducing gas, obtained treating a stream of Syngas previously mixed with dewatered and spent reducing gas in a CO 2 removal unit, is heated exclusively in a heat exchanger, without any additional fuel combustion and exploiting only sensible heat recovered from spent gas removed from the reactor; in this way the energy per ton of produced iron is decreased. Finally, said gas stream (heated CO 2 lean gas stream), available after heating at a temperature of less than 450° C., before being finally fed to the reactor, is subjected to a partial combustion in a combustion chamber with a stream of a molecular-oxygen-containing gas. Alternatively a portion of this gas stream is subjected to a total combustion and the combustion products are combined with the rest of said heated CO 2 lean gas stream. No additional gas heating means between said heat exchanger and said combustion chamber are included.
[0017] It is a further object of the invention to provide a method and apparatus for producing cold DRI utilizing a gas with a high content of carbon monoxide, and cooling said DRI by flowing through the conical discharge part of the reduction reactor a cooling gas with carburizing potential, that can be Coke Oven Gas.
SUMMARY OF THE INVENTION
[0018] The objects of the invention are generally achieved by providing a method and apparatus for producing DRI in a direct reduction system comprising a reduction reactor using a reducing gas with a high carbon monoxide content (for example a Syngas from any source which is cleaned of dust), wherein at least a part of the spent reducing gas removed from the reduction reactor is cleaned and cooled before being mixed with said cleaned Syngas to produce a combined gas stream which is subsequently fed to a CO 2 separation unit. Preferably, said CO 2 separation unit is of the adsorption type whereby a CO 2 laden gas stream and a CO 2 lean gas stream flow out of said CO 2 separation unit. The upgraded CO 2 lean reducing gas stream passes through a heat exchanger where only exchanging sensible heat recovered from said spent reducing gas removed from the reduction reactor and without any combustion, is heated at a temperature lower than 450° C. This heated CO 2 lean reducing gas stream is then partially combusted with a molecular-oxygen-containing gas in order to raise its temperature above 700° C. measured at the reactor inlet, thus dispensing the need of an additional heating in a conventional fired gas heater.
[0019] Another object of the invention is achieved by providing a method and apparatus for producing DRI as described above and having a desired amount of carbon, by cooling said DRI with a carburizing gas that is circulated in the lower part of said reduction reactor as for example Coke Oven Gas.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The detailed description of some preferred embodiments of the invention will be better understood with reference to the accompanying drawings wherein like numerals designate like elements for convenience of the reader.
[0021] The Figures disclose:
[0022] FIG. 1 shows a schematic process diagram of a direct reduction process incorporating one embodiment of the invention;
[0023] FIG. 2 shows a schematic process diagram of a direct reduction process incorporating a second embodiment of the invention;
[0024] FIG. 3 shows a schematic process diagram of a direct reduction process incorporating a third embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] FIG. 1 shows a Direct Reduction System where numeral 10 generally designates a vertical shaft, moving bed, iron ore gaseous reduction reactor, having a reduction zone 12 , to which iron ore 15 is fed through at least one inlet 16 in the form of lumps, pellets, or any blend thereof. This iron ore 15 descends by gravity through the reactor 10 in counter-current contact with a reducing gas at high temperature.
[0026] This reducing gas is introduced to the reactor through pipe 46 located in the lower part of the reduction zone 12 , and is mainly comprised of hydrogen and carbon monoxide which react with the iron ores to produce direct reduction iron (DRI) 18 , which is discharged from reactor 10 through its conical lower part 14 .
[0027] Spent reducing gas 44 , removed from the top of the reactor at a temperature ranging from 300° C. to 600° C., is treated to be upgraded in a recycle circuit and finally returned back to the reduction zone 12 through pipe 46 . In detail, this spent reducing gas stream 44 , with a partially depleted reducing capacity, passes through a heat exchanger 42 , where sensible heat removed from said gas stream 44 is recovered to preheat the upgraded portion of reducing gas 50 prior to being recycled back to the reactor 10 .
[0028] After passing through heat exchanger 42 , the partially-cooled spent gas 43 is conducted to a cleaning station 38 , where dust is removed by contact with a water stream 40 withdrawn as stream 36 , and the effluent clean gas 39 is then passed to a cooling station 30 , usually of the direct contact type, where the water by-product of the reduction reaction is condensed by contact with water 32 and then removed from the reducing gas as water stream 54 .
[0029] For maintaining a low N 2 concentration in the recycle circuit, a minor portion of the cleaned and dewatered spent gas is purged from the system through pipe 26 having a pressure control valve 28 (for pressure control). The purged gas stream 26 contains carbon monoxide, carbon dioxide, hydrogen and methane in quantities such that the gas can be used as fuel in standard combustion systems. The remaining main portion of this cleaned and dewatered reducing effluent gas is subsequently transferred, flowing through pipe 27 , to a compressor 24 wherein its pressure is raised to a level suitable for further treatment and use.
[0030] The compressed reducing gas stream 29 undergoes an additional cooling step in a heat exchanger or a quench tower packed vessel 22 , required to lower the gas temperature after compression; the stream of gas obtained 35 is mixed with make-up gas stream 23 containing carbon monoxide and hydrogen, for example Syngas derived from the gasification of coal or other hydrocarbon feedstock or export gas from a melter-gasifier system effluent from its associated reduction furnace.
[0031] This Syngas 23 , supplied from a suitable source 1 , is fed through pipe 2 to a gas cleaning system 6 where dust, tar and water are removed. The obtained stream 7 of clean Syngas, mainly composed of H 2 , CO, CO 2 and CH 4 , is first compressed in a Syngas compressor 20 and cooled in a dedicated equipment 21 , that can be a heat exchanger or a quench tower, before being added as make-up to the reduction circuit of reactor 10 as stream 23 .
[0032] After mixing the dewatered reducing gas stream 35 with the clean Syngas (make-up gas stream 23 ), the CO 2 contained in this resulting gas stream 31 is at least partially removed in the CO 2 removal unit 70 . Said unit is preferably of the type of PSA (Pressure Swing Adsorption) units or VPSA (Vacuum Pressure Swing Adsorption) whereby CO 2 is concentrated in a gas stream 33 , which is subsequently removed from the system as purge and eventually used as fuel; the gas stream 33 is adjusted by a pressure control valve 60 . The PSA unit, that utilizes adsorbent surfaces to block polar and less volatile molecules, removes from said stream 31 water and H 2 S molecules too.
[0033] According to a principle of the invention, the upgraded portion of the reduction gas 50 , with a low CO 2 concentration and an improved high reducing potential, leaves the CO 2 removal unit and is fed to the previously described heat exchanger 42 where it is heated at a temperature lower than 450° C. in order to prevent the onset of chemical corrosion reactions of the metal materials of the exchanger 42 (for example using the mechanism known as “metal dusting”). Since there is no combustion in exchanger 42 there is no additional emissions of carbon dioxide to the atmosphere.
[0034] The temperature of the resultant gas stream 45 , at a value below 450° C., is then increased up to the desired final value in a second stage by means of combustion of a portion of the preheated CO 2 lean gas stream. To this end, the preheated CO 2 lean gas stream 45 is divided in a first portion 132 which is directly sent to a combustion chamber 47 , where it is combusted with a stream of a molecular-oxygen-containing gas stream 48 , preferably oxygen of industrial purity, supplied from a suitable source 49 . The amount of oxygen is regulated by valve 52 in response to the level of temperature desired for the reducing gas flowing through pipe 46 . The amount of oxygen is also regulated so that the value of the ratio of reducing agents to oxidant agents (H 2 +CO)/(H 2 O+CO 2 ) in the heated gas stream is at least 7; or that the reducing index calculated as: (H 2 +CO)/(H 2 +CO+H 2 O+CO 2 ) of gas stream 46 fed to the reduction reactor is at least 0.87.
[0035] The combustion may be carried out by means of a dedicated burner or by injection of oxygen through injection lances located inside the combustion chamber 47 . The remaining portion of reducing gas 45 , or gas stream 130 , is then fed to the same combustion chamber 47 so that the partially or totally combusted gas 53 , mixed with the remaining reducing gas 130 , reaches a temperature higher than 700° C. at the reactor inlet. The gas stream 130 may also be fed to the same combustion chamber in order to protect the materials of the combustion chamber from the high temperatures that may be reached due to the stream 53 . Regulation of the amount of gas stream 132 is controlled by control valve 134 in response to the desired temperature for the reducing gas 46 to be introduced into the reduction zone 12 of the reactor 10 and in accordance with the maximum temperature allowed by the design and materials of the combustion chamber. In one example, the flow rate of gas stream 132 , which is partially or totally combusted in combustion chamber 47 , is in the range of 50% to 70% of the flow rate of gas stream 45 . The flow rate of reducing gas 132 and the quantity of oxygen 48 are controlled in accordance with the temperature desired for the reducing gas stream flowing through the pipe 46 by means of the valves, respectively 134 and 52 .
[0036] The gas stream 130 may also be combined with the partially or totally combusted gas stream 53 outside of the combustion chamber 47 to adjust the temperature of the combined reducing gas stream until it reaches the suitable value for being introduced into the reduction zone of the reactor 10 for reducing the iron ore contained therein.
[0037] The combustion chamber 47 is preferably preheated to temperatures above 600° C. for assuring that the mixture of reducing gas and oxygen is maintained under ignition in order to prevent the formation of any potential explosive mixtures.
[0038] Particulate solid iron ores 15 are contacted within the reduction zone 12 with said high-temperature upgraded reducing gas fed through pipe 46 into the reactor 10 . In this way the solid material, flowing counter-currently with this gas, reacts with hydrogen and carbon monoxide producing direct reduced iron (DRI). The DRI, flowing through the lower discharge zone 14 , is then discharged from said reactor 10 through the lower discharge zone 14 , hot or cold, depending on the type of subsequent utilization of the DRI.
[0039] When DRI is discharged at high temperature (as shown in FIG. 1 ), on the order of 400° C. to 750° C., it can be subsequently briquetted for further storage and handling or pneumatically transported, or alternatively by means of tanks or inertized belts, directly to a steelmaking furnace in a manner known in the art.
[0040] If DRI has to be cold produced (as shown in FIG. 2 , where identical components to those in FIG. 1 have the same reference numbers and are therefore not described again here), the DRI is cooled down by passing counter-currently a cooling gas stream 122 at a relatively low temperature through the conical part 14 of the reactor 10 , whereby the cooling gas temperature is increased and the temperature of the DRI is lowered to a temperature usually below 100° C. Cooling gas make-up 80 is fed to the cooling gas circuit from a suitable source 81 that can be for example Coke Oven Gas if available, natural gas or other hydrocarbon-containing gas so that said hydrocarbons are cracked in contact with the hot DRI and in this way DRI with the desired amount of combined carbon or graphite is produced.
[0041] In another embodiment of the invention shown in FIG. 3 (where identical components to those in FIG. 1 have the same reference numbers and are therefore not described again here), Coke Oven Gas from a source 81 is used as carburizing and cooling gas stream 80 and fed directly to the reactor cone at a desired location where the temperature of DRI is high. In this way, a further advantage is obtained because the hydrocarbons typically contained in Coke Oven Gas are destroyed by cracking.
[0042] The hot and spent cooling gas stream 90 may be cooled down and recycled in a manner well known in the art. Briefly, the warmed up gas withdrawn from the top of the cooling zone, is further treated in a cleaning station 92 to remove dust by washing with water 93 which is withdrawn through pipe 95 ; the clean gas 94 is treated in a cooling station 96 , where it is completely de-watered and cooled by contact with water 97 which is withdrawn through pipe 99 . The gas obtained 98 is compressed by means of compressor 100 before being fed to the reactor through pipe 120 .
[0043] In a further embodiment of the invention, the DRI may be hot discharged from the reduction reactor at a temperature in the order of 400° C. to 750° C., and it may be cooled down to a temperature lower than 100° C., to avoid its re-oxidation by atmospheric oxygen and water, in a separate DRI cooling vessel (not shown) external to the reduction reactor 10 , with a cooling gas system similar to the cooling gas system previously described. With this configuration, the iron reduction plant, designed to produce hot DRI for its immediate melting, can provide also for an emergency discharge of DRI in safe conditions, with the material available at an adequate temperature for storage and later utilization.
[0044] An alternative design for a direct reduction plant with the capacity of producing both hot or cold DRI provides the reduction reactor with a cooling gas system designed to optionally enable or not the operation of the cooling system, whereby the same reactor may cool the DRI inside the discharge cone or discharge it at high temperature.
[0045] According to an exemplary embodiment of the invention, in which the second heating stage of the CO 2 lean gas stream is combusted with 76 Nm3/t of DRI with 95% pure oxygen, the relative amounts of some of the components of the reducing gas which is heated to 838° C. suitable for reduction of iron ores, are presented in Table 1 below:
[0000]
TABLE 1
HYDROGEN
GAS
REDUCING
EFFLUENT
AND CO
EFFLUENT
GAS FED
GAS FROM
CONTAINING
FROM PSA
TO
REDUCTION
GAS
UNIT
REACTOR
ZONE
GAS STREAM
23
50
46
44
NO.
TEMPERATURE
° C.
45
48
838
519
FLOW
NCM/
1622
1997
1998
2060
TON
DRI
Composition
H2
VOL
18.070
33.926
26.354
23.196
%
CO
VOL
43.168
48.418
48.408
29.503
%
CO2
VOL
32.125
2.000
2.000
19.669
%
H2O
VOL
1.116
0.001
7.566
12.627
%
CH4
VOL
2.309
3.326
3.325
2.930
%
N2 and other
VOL
3.212
12.333
12.347
11.975
gases
%
REDUCING
0.65
0.98
0.89
0.62
INDEX
[0046] The reducing index is calculated as: (H 2 +CO)/(H 2 +CO+H 2 O+CO 2 ) and indicates the reducing power of each gas stream.
[0047] From table 1, it can be seen that the present invention provides an effective method and apparatus for producing DRI utilizing a gas containing H2 and CO with a low Reducing Index and an effective two-stages gas heating to the desired reduction temperature.
[0048] The present invention brings a number of advantages over the prior art, namely, a simpler iron ore reduction plant and process are possible because the fired heater, for preheating the reducing gas before raising its temperature to the reduction levels, is not needed. Therefore a direct reduction plant incorporating the invention has lower capital and operation costs because an important piece of equipment (the heater) requiring operation and maintenance materials and manpower is avoided.
[0049] It is of course to be understood that the above description of some embodiments of the invention has been made for purposes of illustration and not of limitation of the scope of the invention and that a number of changes may be made to the embodiments herein described as the application of the invention best fits a particular practical case without departing from the spirit and scope of the invention which is determined by the appended claims.
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The present invention concerns a method and an apparatus for producing DRI (Direct Reduced Iron) utilizing a high-oxidation reducing gas containing carbon monoxide and hydrogen, derived directly or indirectly from the gasification of hydrocarbons or coal, with a high content of oxidants (H 2 O and CO 2 ). The invention provides a more efficient method and plant comprising a reactor in which particulate material of iron ore comes into contact with a high temperature reducing gas to produce DRI, with lower investment and operating costs, avoiding the need for a fired heater for the reducing gas fed into the reduction reactor. The reducing gas is heated to a temperature above 700° C. in two steps, a first step at a temperature below about 400° C. to prevent the phenomenon of metal dusting, by exchange of sensible heat supplied by the stream of hot spent gas removed from the reduction reactor; and a second step by means of partial or total combustion with oxygen, maintaining the temperature of the combustion gas below the limits established by the construction materials of the combustion chamber.
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BACKGROUND OF THE INVENTION
The present invention relates to an ink jet writing apparatus, and in particular to an ink jet writing apparatus having means for preventing the clogging of ink when the writing head is not in operation.
Ink jet printing has been known in the art as exemplified by the system shown and described in U.S. Pat. No. 4,106,032. In the system described in this patent, a jet of writing fluid or ink is caused to issue from a nozzle in the form of a succession of tiny individual droplets of a varying size depending on the instantaneous value of an input signal which is to be recorded. The nozzle is moved across the surface of a recording medium so that the ink droplets are ejected to desired print positions.
In a writing system of the type described above, nozzle clogging has presented a well known problem. One of the reasons of clogging is due to the ink drying in the nozzle over a standstill period. The nozzle has an inside diameter in the order of 40 micrometers and ink allowed to stay therein tends to clog the opening when the ink has dried. Prior attempts have been made to solve this problem. For example, United States patent application No. 20,977 filed Mar. 16, 1979, now U.S. Pat. No. 4,223,324, discloses a system in which a solenoid-operated rubber-coated pad is brought up to close the nozzle opening to prevent the ink from drying when the system is not in use. In the disclosed system, moisture laden air is forced through a space between the pad and the nozzle opening when the system is at standstill to moisten the nozzle opening.
However, this system is still not satisfactory for a number of reasons. Firstly, since the contacting surface of the rubber-coated pad and the front face of the writing head on which the nozzle opening is provided are not strictly parallel to each other due to manufacturing errors, it is difficult to provide a fluid-tight chamber that encloses the nozzle opening, so that moisture laden air tends to escape through a space between the misaligned surfaces. Secondly, since the moisture laden air is supplied from a pressurized air source, it is necessary to keep the compressor operating even if the system is left unattended for a long period of time, which is undesirable from the power savings standpoint.
One approach to these problems would be to submerge the nozzle into water when the system is not in operation. However, the water tends to introduce into the nozzle and the ink in the nozzle would be excessively diluted with the result that upon resumption of the writing operation the initial printing is unsatisfactory.
SUMMARY OF THE INVENTION
According to the present invention, the ink jet writing apparatus comprises an ink jet writing head having a nozzle from which ink issues, an elastic enclosure engageable with a front face of the writing head to define a fluid-tight chamber enclosing the opening of the nozzle, a source of ink diluting fluid, a tubular member connected between the fluid source and the elastic enclosure including a capillary member therein for transmitting the fluid from the source to the enclosure by capillary action to permit evaporation of the fluid in the chamber, and means for producing a sealing contact between the writing head and the enclosure to create said fluid-tight chamber when the writing head is not in operation.
The use of the capillary tube provides advantages in that it allows a design flexibility whereby the source of ink diluting fluid or water can be located at any desired position and a plurality of such capillary tubes can be provided for a multiple-head writing system using a single source of water.
Preferably, a heating device is provided for heating a portion of the capillary tube to increase the rate of evaporation of water at the end of the tube.
Maintenance effort can be minimized by the use of a water-containing cartridge which is detachably mounted on the water source for refilling it when the water therein has been consumed. The cartridge includes a spring-loaded valve member which normally engages a valve seat when the cartridge is separated from the water source. Upon mounting on the water source, the valve member is automatically disengaged from contact with the valve seat allowing the water in the cartridge to be admitted into the lower chamber until the head of the water therein becomes flush with the opening of the nozzle. When the water in the lower chamber has been consumed so that its head becomes lower than the nozzle opening air is introduced into the upper chamber to allow the water therein to be admitted into the lower chamber until it is filled to the level of the nozzle opening of the upper chamber. Since the head of the water in the lower chamber or water supply source is maintained at a constant level, the rate of evaporation at the end of the capillary tube can also be maintained constant at all times. Since the vapor is confined within the fluid-tight chamber, the amount of water consumption is minimized so that the water supply source can be left unattended for a long period of time.
When the writing head is operated in response to an input signal having an amplitude close to the operating threshold level of the head which is also a function of the physical properties of the ink being used, the ink in the nozzle tends to spray around the nozzle opening as it emerges therefrom and accumulate therearound. This accumulated ink will then be dried and during this drying process it might collect dust and fine particles floating in the air and eventually becomes a thick layer of mixture of residual ink and such substances, or sludge. When this layer is exposed to the moisture produced by the capillary tube, it absorbs it and returns to the original state and is likely to narrow the nozzle opening, or produce stains on a writing surface, or could lead to an electrical circuit failure because of the conductive nature of the sludge.
This problem can be solved by the provision of a cleaning device mounted stationary with respect to the writing head for making a wiping contact with the front face of the writing head as the latter is moved between non-printing and printing positions to scrape off the sludge.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be further described by way of example with reference to the accompanying drawings, in which:
FIG. 1 is an illustration of a top plan view of the ink jet writing apparatus embodying the invention when the writing head is in non-printing position;
FIG. 2 is an illustration of the apparatus similar to FIG. 1 with the exception that the writing head is in a printing position;
FIG. 3 is an illustration of a cross-sectional view taken along the line 3--3 of FIG. 1;
FIG. 4 is an illustration of an alternative embodiment of the elastic enclosing member of FIG. 3;
FIG. 5 is an illustation of the apparatus embodying a cleaning device; and
FIG. 6 is an illustration of an example of the cleaning device of FIG. 5.
DETAILED DESCRIPTION
Referring now to FIG. 1, there is partially shown in a top plan view the ink jet writing apparatus in which the present invention is adapted for use. A support board 10 is provided to which a pair of guide rods 11, a pulse driven motor 12, a drum shaft 13, and a guide block 14 are mounted. On the guide rods 11 are slidably mounted a support member 15 which in turn carries an ink jet writing head 16 of the type described in the aforesaid U.S. Pat. No. 4,106,032. The writing head 16 is shown in non-printing position with its nozzle opening being enclosed by a cup-shaped enclosing member or lid 17 of an elastic material such as rubber or plastic. This enclosing member is attached to an end of a tube 18 which slidably supported by the guide block 14, the other end of the tube 18 coactively engaging the surface of a cam 19 mounted on the rotor shaft of the pulse driven motor 12. A compression spring 20 is provided on the tube 18 to urge it toward the cam 19. Adjacent to the closed end of the tube 18 is connected to a second tube 21 leading from a water supply source 22 from which water is fed to the enclosing member 17 in a manner as will be described. On the shaft 13 is mounted a drum 23 on the surface of which is rolled a sheet of recording paper. As is well known in the art, this drum is rotated by the width of line path along which ink jet is printed when the writing head 16 scans across the paper.
When the apparatus is in operation, the motor 12 is energized briefly to rotate the cam 19 so that the tube 18 is moved to a retracted position by the action of the spring 20. At the same time the writing head 16 is caused to move to a printing position as illustrated in FIG. 2.
As illustrated in more detail in FIG. 3 which is a cross-sectional view of FIG. 1, the water supply source 22 is located in a position lower than the writing head 16. According to the invention, the tubes 18 and 21 are filled with a porous capillary member 24 such as glass fibers or a material having an open-cell cellular structure. The capillary member 24 extends partly into the water container 22 to absorb water and transmit it by capillary action to the opposite end which partly extends into the enclosure 17 and terminates into a fan-shaped configuration to enhance evaporation. To ensure a sealing contact between the front face of the writing head 16 and the front edge of the enclosure 17, the latter has a forwardly increasing diameter portion with forwardly decreasing thickness. Due to the flexibility of the material that forms the member 17, the front edge of the latter expands as it makes a pressure contact with the head 16 by the action of cam 19 creating a completely sealed chamber between these contacting members, whereby the evaporated water rapidly fills the chamber and the wet condition is maintained for a substantial period of time without the need for supply from the source 22.
To further assist evaporation of water the guide block 14 includes a heating element 25 in the shape of a ring surrounding the front end portion of the tube 18. This heating element is supplied with a current from a voltage source 26 when the system is in the standby position to raise the temperature of the water inside the tube 18.
The enclosure 17 may also be in the form of a bellows as illustrated in FIG. 4 which obviously provides an intimate contact with the front face of the head 16 by a slight pressure acted upon the tube 18 by the cam 19.
Since the nozzle opening is completely shut off from the outside by the enclosure 17 when the apparatus is not in operation, the nozzle is also protection from dust or fine particles. When a dew point is reached in the confined moisture chamber, the rate of evaporation automatically decreases so that there is no possibility that the ink standing in the nozzle would be diluted excessively by the condensed water droplets.
For ease of maintenance the water supply source 22 is arranged to carry thereon a water cartridge 27 which comprises a container 28, a nozzle 30, a spring-biased valve member 29a and a valve seat 29b. The valve member 29a is normally seated on the valve seat 29b when the cartridge is detached from the container 22. When the cartridge is mounted on the container 22 as illustrated with its nozzle 30 projecting down into the container 22 through an opening thereof, the valve member 29a engages the bottom of the container 22, whereby the valve is disengaged from contact with the seat 29b to allow water to be admitted from the container 28 into the lower container 22 until the head of water therein becomes flush with the opening of the nozzle 30. When the water in the container 22 has been consumed reducing the water level from the nozzle opening, air is admitted through an opening 31 into the container 22 and thence into the upper container 28 in the form of bubbles, so that the water in the upper container 28 is admitted into the lower container to compensate for the amount of consumption. Therefore, the head of the water in the container 22 is maintained at a constant level at all times which is balanced against the atmospheric pressure, the rate of water feed to the enclosure 17 and hence the rate of evaporation is rendered constant regardless of the amount of water contained in the cartridge 27.
FIG. 5 is an illustration of another embodiment of the invention. In this embodiment, a cleaning device 40 is mounted on a lateral side of the enclosing member 17 on a spring board 41 which in turn is detachably mounted on the guide block 14 by means of a screw 42. The cleaning device 40 comprises a serrated elastic wiping member 43 formed of rubber or high-polymer compound, and a collector 44 mounted below the wiping member 43 to collect scraped-off sludges. The front edges of the serrated flexible member 43 are so positioned that they are brought into a wiping contact with the front face of the writing head 16 as the latter is moved between non-printing and printing positions. This wiping action scrapes off a sludge produced by the absorption of water vapor by the dried mixture of ink and dust which has accumulated around the nozzle opening of the head 16 as a result of the spraying action of ink when expelled from the nozzle in response to an input signal of a near threshold level (which is a function of the physical properties of the ink) or as a result of the splashing action of the expelled ink as it strikes the surface of the recording paper.
The ink jet writing head of the above-mentioned United States Patents provides a means for ejecting a stream of air along the path of the issued ink droplets in order for the latter to be assisted in arriving the writing surface in a small, sharply defined area by the confining action of the air flow. This air flow is advantageously employed for purposes of preventing the sludge from introducing into the nozzle as it is scraped off by the cleaning member 43.
The cleaning member 43 may take any one of various forms. One example is shown in FIG. 6. The exemplified cleaning member is formed with a plurality of serrated segments having increased lengths toward the center segment 43a to form a smooth wiping contact face against the front face of the head 16 as the latter is moved in opposite directions. For routine maintenance purposes, the screw 42 permits the maintenance personnel to detach the cleaning device 40 from the apparatus for flushing it with water, or replace it with a new one.
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In order to prevent clogging of the nozzle of an ink jet writing head, a nozzle moistening device is provided which includes an elastic enclosure fluid-tightly engageable with the front face of the writing head when not in use, a source of water, and a capillary tube for transmitting water from the source to the enclosure by capillary action to permit evaporation of water in the enclosure to moisten the nozzle.
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Provisional Patent Application No. 61/214,616 dated Apr. 27, 2009
BACKGROUND OF THE INVENTION
The scope of the present invention is the method and apparatus for breaking apart pistachio nut shells into two disjoined halves by an individual at the time of consumption. The pistachio nuts must have at least partially opened sutures.
Pistachios are the seed of a small tree of the cashew family. They are referred to as nuts and they come in a hard smooth shell. This shell tends to split naturally along a longitudinal seam, called a suture, when the nut matures. When a suture is open, the opening is a majority of the length longitudinally and distinguishes two halves attached around one end of the pistachio nut.
Usually, 80 to 85% of a normal crop of pistachio nuts will have open sutures when they are picked from the tree. A closed nut is one that is unopened or has a partially opened suture.
Consumers of pistachios demand the shells be open along the majority of the suture. Open nuts are easier to break apart into two disjoined halves by the consumer in order to access the edible portion and eat it. However, easier does not necessarily mean easy.
Many pistachios available in the US market have gone through a process after being harvested in order to separate the split nuts from those that are closed. The methods for commercially splitting closed nuts have varied, to include hand splitting them to moisturizing the shells and squeezing them through a splitting apparatus in large quantities to provide an open split in the shell for packaging and distribution to retail outlets and the consumer.
Approximately 25 to 75% of packaged pistachios reaching the consumer are still not easy to break apart at the time of consumption. As a result, an effort is required to break apart the pistachio into two disjoined halves in order to access and eat the edible portion.
This effort often entails a method whereby the consumer places either the fingertips or the fingernails against the edges of the shell of an open or partially open pistachio nut and applies outward pressure to force the halves to break apart. As pistachios are seldom eaten singly, the fingertips or the fingernails can become torn or broken after a short period. Another method is for the consumer to bite the pistachio nut in order to split it or break it apart. This approach has led to broken teeth on many occasions. Still other methods by the consumer for breaking apart pistachio nuts have been to use various types of nut crackers, hammers or pliers, which can crush the edible portion. None of these methods is optimal.
Additionally, as much as 15% of packaged pistachios may be fully closed.
There are several patents whereby methods and apparatuses have attempted to provide a better solution. U.S. Pat. No. 4,462,156—Himelhoch, 2002/0104219—Olsen, U.S. Pat. Nos. 5,339,525—Sawyer and 6,609,303—Rogel all provide for handheld apparatuses that insert protrusions into an opening in a pistachio nut and by utilizing a pivoting or rotational motion, the protrusions force the shell halves apart until they break into two disjoined halves and the edible portion is accessible. Utilizing a pivoting or rotational motion is less than optimal in that the protrusions actually move in a circular direction away from the pistachio nut the moment compression of the apparatus begins. Some pistachio nuts spread open far relative to their size before they break apart. The relationship between the aforementioned devices and a pistachio nut is further complicated if the open part of the suture is at the opposite end of the shell and not on the side, as the shell halves open rationally as well. U.S. Pat. No. 4,462,156—Himelhoch applies a raised portion to the protrusions to protect them from being inserted too far into the pistachio nut, but uses two flat protrusions facing one another, as does U.S. Pat. No. 6,609,303—Rogel. U.S. Pat. No. 5,339,524—Sawyer, aligns the edges of the protrusions in one horizontal plane, and by compressing the apparatus, forces the protrusions to move in a direction perpendicular to the horizontal plane of the protrusion tip blades. In this regard, U.S. Pat. No. 5,339,524—Sawyer improves over U.S. Pat. No. 6,609,303—Rogel, but does not resolve the rotational aspect of the nut openers, and does not prevent the protrusion tips from being inserted too far into the pistachio nut, possibly causing damage to the edible portion.
Moreover, the design of U.S. Pat. No. 5,339,524—Sawyer may not be comfortable for an individual to use as a wire is narrow and will cause pressure to be focused on a concentrated portion of the thumb and fingers when used repeatedly in a short period of time. This design also allows for the apparatus to twist in the hand during compression.
Of consequence is the habit of consumers to eat many pistachios in one sitting further exacerbating any frustration of breaking apart pistachio nuts through less than optimal means, and possibly intensifying any damage to fingertips or fingernails.
SUMMARY OF THE INVENTION
For the consumer, it is the method of applying outward pressure from within the suture of the pistachio nut shell to force apart and separate the two halves, until disjoined, of an open or partially open pistachio nut, and the hand held apparatus with protrusions that are inserted into the opening of a pistachio nut shell, without causing damage to the edible portion, using a translational vertical motion upon manual compression between the thumb and forefinger, to perform such method, that is the purpose of the present invention.
Furthermore, it is the purpose of the present invention to provide a device that is more portable in size, is easier to hold and operate, and is more comfortable to hold and operate.
Additionally, it is the purpose of the present invention to be more effective at splitting apart and disjoining the pistachio shell halves; and to provide a more satisfying experience for an individual while consuming pistachio nuts.
Moreover, it is the purpose of the present invention to be sufficiently durable in design, material and assembly to allow an individual to use the apparatus repeatedly and on multiple occasions.
It is intended that the apparatus, once assembled, not be disassembled.
Accordingly, the present invention is provided for an open or partially open pistachio nut to be held in one hand with the split towards the opposing hand, while the apparatus is held in the opposing hand positioned between the thumb and forefinger in their natural resting positions, and with the protrusions under the tip of the thumb. The pistachio nut is placed and held against the apparatus so that the protrusions are inserted into the opening of the split pistachio nut shell. The apparatus is compressed by the thumb and forefinger which moves the protrusions along the line of compression by translational vertical motion in opposite directions, the protrusions remaining engaged with the edges of the pistachio shell halves, until the pistachio breaks apart into two disjoined halves and the edible portion is freed from the shell, the arcs of the protrusions protecting the edible portion from being damaged.
DESCRIPTION OF THE DRAWINGS
FRONT PAGE VIEW shows the assembled apparatus in the extended position.
FIG. 1 shows the assembled apparatus in the extended position with an exploded view of the apparatus;
FIG. 2 shows the top piece from the front 2 A, top 2 B, side 2 C and bottom 2 D perspectives.
FIG. 3 shows the bottom piece from the front 3 A, top 3 B, side 3 C and bottom 3 D perspectives.
FIG. 4 shows the assembled apparatus in the compressed position.
FIG. 5 shows the apparatus in the extended position being held by the individual in one hand and oriented to the pistachio nut held by the other hand; and
FIG. 6 shows the apparatus in the compressed position being held by the individual in one hand and, in the other hand, two disjoined halves revealing the edible portion.
DESCRIPTION OF THE INVENTION
Referring now in detail to the drawings, the type apparatus being depicted in FIGS. 1 to 6 is illustrated for the pistachio nut opener 10 in accordance with the present invention and which is specifically for the handheld use between the thumb 34 and forefinger 35 of one hand by an individual while consuming pistachios to better and more reliably separate the pistachio nut 38 into two disjoined halves 40 and 41 to access the edible portion 42 within ( FIGS. 5 and 6 ). The pistachio nut opener 10 is comprised of three parts: the top piece 11 ; a compression spring 12 , and; the bottom piece 13 ( FIG. 1 ).
Referring to FIG. 2 , the top piece 11 having an over mold 19 in the channel 14 for comfort and grip, and having a protrusion 16 adapted vertically from the open end 23 of the top piece 11 to the channel 14 of the top piece 11 , and having a vertical cutout 17 adjacent to the protrusion 16 to accommodate the travel of the protrusion 25 of the bottom piece 13 when the apparatus is compressed ( FIG. 4 ).
Referring to FIG. 3 , the bottom piece 13 , also having an over mold 29 in channel 30 for comfort and grip, and having a protrusion 25 that aligns beside, and in contact with, protrusion 16 of the top piece 11 as in the assembled and extended position, the protrusion 25 is partially within the cutout 17 of the top piece 11 . The alignment of the tips 18 and 26 of protrusions 16 and 25 respectively, form a horizontal line perpendicular to the direction of travel during compression of the pistachio nut opener 10 when in the extended position ( FIG. 1 ).
Referring to FIGS. 2 and 3 , the internal open area 23 within the top piece 11 is adapted with a snap-fit cylinder 24 with two vertical grooves 22 cut into the internal wall of the snap fit cylinder 24 opposite of one another, while the internal open area 32 of the bottom piece 13 is adapted with a snap-fit cylinder 33 of a smaller diameter, so as to fit inside the snap-fit cylinder 24 , and having two tabs 31 adapted to the external wall of the snap-fit cylinder 33 , opposite of one another, which, when the top piece 11 and bottom piece 13 are assembled, the tabs 31 engaging the grooves 22 , causing an interlocking of the snap-fit cylinders 24 and 33 . The axis of the grooves 22 , being aligned with the axis of the tabs 31 and with the concave channel 30 of the bottom piece 13 , being perpendicular to the axis of the protrusions 16 and 25 , the protrusions 16 and 25 being aligned with the concave channel 14 of the top piece 11 . The assembled pistachio nut opener 10 having a compression spring 12 inserted into the snap-fit cylinder 33 of the bottom piece 13 . As the bottom piece 13 is inserted into the top piece 11 , the snap-fit cylinder 33 of the bottom piece 13 slides into the snap-fit cylinder 24 of the top piece 11 , becoming interlocked, enclosing the compression spring 12 . The assembled pistachio nut opener 10 is intended to be persistent, whereas disassembly will cause damage to the pistachio nut opener 10 and may render it inoperable.
Referring to FIGS. 2 and 3 , the protrusions 16 and 25 are adapted vertically in a right angle triangle, the bases 21 and 28 of the triangle forming a right angle with the side of the top piece 11 and the side of the bottom piece 13 respectively, having a concave arc 20 and 27 along the hypotenuse of the triangle, transitioning from the side of the top piece 11 and the bottom piece 13 respectively, to the open ends 23 and 32 so that the junction of the hypotenuse 20 and 27 of the triangle and the base 21 and 28 of the triangle form the tip 18 and 26 of the protrusion 16 and 25 respectively. The line formed by joining the protrusion tips 18 and 26 is horizontal ( FIG. 1 ), being perpendicular to the direction of travel of the protrusions 16 and 25 when the top piece 11 and the bottom piece 13 are compressed by an individual ( FIG. 4 ).
Referring to FIGS. 2 to 4 , when an individual manually compresses the assembled pistachio nut opener 10 , the top piece 11 and the bottom piece 13 move simultaneously in a translational vertical motion until the travel is stopped by either the individual relenting the force of compression or the base 28 of the protrusion 25 of the bottom piece 13 comes in contact with the top 15 of the cutout 17 of the top piece 11 , physically preventing further travel. The concave arcs 20 and 27 of the protrusions 16 and 25 protecting the edible portion 42 of the pistachio nut 38 . Upon release of pressure from the thumb 34 and the forefinger 35 the pistachio nut opener 10 is resilient, having a compression spring 12 , and returns to the extended position.
Referring to FIGS. 5 and 6 , an individual holds the pistachio nut opener 10 between the thumb 34 and forefinger 35 of one hand, being that the apparatus can be used by either the left or right hand. The thumb 34 rests in the channel 14 of the top piece 11 with the thumb 34 in a natural position extended from the wrist along the axis of the forearm; the forefinger 35 rests in the channel 30 of the bottom piece 13 perpendicular to the channel 14 of the top piece 11 and the thumb 34 , so the forefinger 35 has a natural curve inwards to the palm of the hand. The thumb 34 and forefinger 35 being in a position, and in relation to one another, natural as they would be if the arm was by one's side in the military position of attention.
Referring to FIGS. 5 and 6 , when the pistachio nut opener 10 is held by an individual in one hand, the other hand grasps an open or partially open pistachio nut 38 so that the suture 39 is facing the protrusions 16 and 25 of the pistachio nut opener 10 . The individual brings the two hands toward one another positioning the horizontally aligned protrusion tips 18 and 26 along the horizontally aligned suture 39 of the open or partially open pistachio nut 38 and coaxes the protrusion tips 18 and 26 within the opening so that the protrusion tips 18 and 26 engage the edges of the pistachio nut 38 shell halves 40 and 41 from inside the suture 39 . Where the pistachio nut 38 is fully open, the protrusion tips 18 and 26 are positioned against the place where the shell halves 40 and 41 are still joined in order to provide the greatest leverage to the shell halves 40 and 41 when the pistachio nut opener 10 is compressed and the protrusion tips 18 and 26 travel in opposite directions perpendicular to the horizontal nature of the pistachio nut suture 39 . The protrusion tips 18 and 26 are prevented from entering the pistachio nut 38 so far as to damage the edible portion 42 within by the concave arcs 20 and 27 of the triangle hypotenuse of the protrusions 18 and 26 ( FIGS. 2 and 3 ). During or upon compression of the pistachio nut opener 10 , the shell halves 40 and 41 will be forced to break apart and become two disjoined halves allowing the edible portion 42 to be accessed.
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A handheld pistachio nut opener that is box-shaped comprising an upper and lower piece, each with one protrusion that are oriented vertically but aligned horizontally and inserted into an open or partially open pistachio nut, and through manual compression of the apparatus between the thumb and forefinger, the protrusions travelling in opposite directions through translational vertical motion while remaining engaged with the pistachio nut shell halves until the shell halves break apart into two disjoined pieces revealing the edible portion inside, the arcs of the protrusions protecting the edible portion from being damaged.
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FIELD OF THE DISCLOSURE
The present invention relates to automotive refinish compositions, to methods for preparing and using such compositions, to refinish coatings on a substrate, and to articles such as automotive vehicles with refinish coatings on them.
INTRODUCTION TO THE DISCLOSURE
Automotive topcoat finishes include basecoat/clearcoat topcoats, in which the topcoat is applied in two layers, a first layer of a pigmented basecoat composition and a second layer of a clearcoat composition, as well as single-stage or monocoat topcoats, which are one-layer, pigmented, glossy topcoats. Basecoat/clearcoat coatings are desirable for their high level of gloss and depth of color. In addition, basecoats having special effect pigments, e.g., flake pigments such as metallic and pearlescent pigment, can achieve excellent gonioapparent effect in basecoat/clearcoat composite coatings.
Polyurethane clearcoat and single stage topcoat (or monocoat) systems have been widely used for many years for refinish coatings. These systems contain hydroxyl-functional resins that cure by reaction with polyisocyanates to form polyurethanes with generally excellent film properties including durability, toughness, and solvent resistance. In automotive refinish coating compositions, the polyisocyanates are not blocked so that the reaction with the hydroxyl groups will take place within a reasonable amount of time without heating or with heating at low temperatures of perhaps up to 150° F. Given the reactivity between the unblocked polyisocyanate and the hydroxyl-functional polyol under typical storage conditions, these materials are segregated into separately stored components until mixing just shortly before application of the coating composition to the substrate to be coated. This type of coating composition, in which the materials that react to cure the coating (resin and crosslinker) are segregated in separately stored components designed to be combined just before application, is referred to in the art as a “two-component” or “multi-component,” “two-package” or “multi-package,” or “2K” coating composition. Automotive refinish clearcoats may include other separately stored components, such as reducers used to provide desirable application characteristics for the particular application conditions (e.g., a fast reducer for cold weather, a slower reducer for hot weather). For a single stage topcoat or monocoat systems, a multi-component or multi-package coating composition includes multiple, differently colored bases containing pigment and hydroxyl-functional resin, one or more of which is combined with a polyisocyanates crosslinker component and, optionally, a reducer or other component just before application.
The polyisocyanate crosslinker has been used with a hydroxyl-functional acrylic resin or polymer. Once applied and cured, the outer coating layer, whether it is a clearcoat layer or a pigmented monocoat layer, should be resistant to weathering degradation (e.g., retain its gloss on exposure to sunlight) and resistant to scratching and marring that can detract from the appearance of the coated vehicle. Proposals for improving scratch and mar resistance have included using silicone or fluorinated polymers or additives, which are relatively expensive and can cause other problems (e.g., cratering in the coating and difficulty for recoat).
SUMMARY OF THE DISCLOSURE
We have invented a composition for a scratch-resistant coating, which may be a clearcoat or single-stage topcoat, a coating prepared from the composition, and an article coated with the coating. We also disclose methods of making and using the composition.
Disclosed is a method of increasing scratch resistance and/or reflow of a refinish topcoat, comprising preparing the topcoat from a refinish topcoat coating composition comprising an unsaturated fatty acid ester polyol having at least two hydroxyl groups that does not undergo oxidative cure when the applied refinish topcoat coating composition is cured because the refinish topcoat coating composition is free of any drier. In various embodiments of this method, the unsaturated fatty acid ester polyol is included in an amount from about 10% to about 50% by weight of the total nonvolatile weight of hydroxyl-functional materials in the refinish topcoat coating composition. In various embodiments of this method, the unsaturated fatty acid ester polyol may be a soy oil- or castor oil-based polyol and/or the unsaturated fatty acid ester polyol may have three or four hydroxyl groups.
A refinish, multi-component topcoat coating composition includes at least one first package including (a) of an unsaturated fatty acid ester polyol having at least two hydroxyl groups and (b) an acrylic polymer having a hydroxyl number of about 37 to about 170, wherein the composition comprises from about 10% to about 50% by weight, based on the total nonvolatile weight of components (a) and (b). of component (a). The at least one first package may optionally include a pigment. In certain embodiments, components (a) and (b) together may be the only hydroxy-functional components in the first package; in other embodiments, components (a) and (b) together are at least about 90% by weight, preferably at least about 95% by weight, of all hydroxy-functional components in the first package. In this description, “polymer” with be used to include both oligomeric and polymeric materials. The composition also has a polyisocyanate crosslinker separated during storage from the hydroxyl-functional components (a) and (b). The coating composition is free or essentially free of any drier, by which is meant that no drier is intentionally added to the composition (although such a compound may be present through an impurity in another component of the coating composition); no drier is present in an amount effective to catalyze oxidative cure. The refinish, multi-component coating composition includes a second package containing a polyisocyanate crosslinker, and, optionally, one or more reducers. If pigment is present in the first package, the refinish, multi-component topcoat coating composition produces a single stage, pigmented topcoat.
A method of refinishing a substrate includes combining the at least one first package (whether an unpigmented first package or one or more pigmented first packages, known as “bases”), the second package, and optionally one or more reducers to form a refinish topcoat coating composition mixture including components (a) and (b) and the polyisocyanate crosslinker, applying the refinish coating composition mixture to all of a surface area of a substrate or to a part of the surface area of the substrate, and curing the applied composition mixture to form a cured refinish topcoat layer from the applied refinish topcoat coating composition mixture. The refinish topcoat coating composition mixture has unexpectedly improved scratch resistance and/or reflow (annealing of film deformation). Also provided is the cured refinish coating and the article (particularly, an automotive vehicle or vehicle trailer) having on it the cured refinish coating.
“A,” “an,” “the,” “at least one,” and “one or more” are used interchangeably to indicate that at least one of the item is present; a plurality of such items may be present. Other than in the working examples provides at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range.
DETAILED DESCRIPTION
The following description includes details of particular inventive embodiments.
The first component of the refinish, multi-component topcoat coating composition includes a component (a) of an unsaturated fatty acid ester polyol having at least two hydroxyl groups. In various embodiments, the unsaturated fatty acid ester polyol may have two, three, or four hydroxyl groups. In various embodiments, the unsaturated fatty acid polyols may have from one to twelve, preferably from 1 to 4, unsaturated carbon-carbon bonds. The unsaturated fatty acid ester polyol may be prepared by reacting one or more unsaturated fatty acids or reactive derivatives thereof such as methyl esters or triglycerides with a polyol. In various embodiments, the component (a) may be a soy oil- and/or castor oil-based polyol provided by transesterifcation of soy oil, castor oil, or a combination of these oils. Castor oil fatty acids include ricinoleic acid. Examples of such unsaturated fatty acid-based polyols include the polyol esters of unsaturated fatty acids with polyols having at least a plurality of hydroxyl groups. Nonlimiting examples suitable polyols having at least three hydroxyl groups include trimethylolpropane, di-trimethylolpropane, triethylolpropane, di-triethylolpropane, pentaerythritol, dipentaerythritol, tetrakis(2-hydroxyethyl)methane, diglycerol, xylitol, glucitol, dulcitol, sucrose, and combinations of these.
The polyol or polyols having a plurality of hydroxyl groups are reacted with the unsaturated fatty acid(s) to esterify the fatty acid(s) or with the esterifiable unsaturated fatty acid derivative (e.g., the glyceride oil) to transesterify the fatty acids, generating byproduct such as methanol from fatty acid methyl esters or glycerol from a triglyceride such as castor or soy oil. Techniques for esterification of fatty acids or transesterification of fatty acid triglycerides are well known and described, for example, in Treasurer, U.S. Pat. No. 5,504,145 and Xiao, U.S. Pat. No. 7,462,679, the disclosures of each of which are incorporated herein by reference.
Some castor-oil based polyols are commercially available. Mention may be made of the POLYCIN™ M-365 and −280 castor oil based products, sold by Vertelllus Performance Materials Inc., Greensboro N.C., USA, and RENUVA™ soy-based polyols, sold by the Dow Chemical Company.
The first component of the refinish, multi-component coating composition also includes (b) an acrylic polymer having a hydroxyl number of about 37 to about 170. In various embodiments, the hydroxyl number may be from about 120 to about 160 or any of the ranges contained within these limits. In various embodiments the acrylic polymer may have a number average molecular weight of about 1100 to about 8200.
Suitable hydroxyl-functional acrylic resins may be prepared by polymerizing one or more hydroxyl-functional, ethylenically unsaturated monomers with one or more other ethylenically unsaturated monomers. Suitable examples of hydroxy-functional ethylenically unsaturated monomers include hydroxy alkyl esters of acrylic or methacrylic acid. (In the context of this description, the term “(meth)acrylate” will be used to indicate that both the methacrylate and acrylate esters are included.) Nonlimiting examples of hydroxyl-functional monomers include hydroxyethyl(meth)acrylate, hydroxypropyl(meth)acrylates, hydroxybutyl(meth)acrylates, hydroxyhexyl(meth)acrylates, other hydroxyalkyl(meth)acrylates having branched or linear alkyl groups of up to about 10 carbons, and mixtures of these. ε-Caprolactone esters of these hydroxyl-functional monomers may also be used. The hydroxyl groups may also be esterified with ε-caprolactone following polymerization. Generally, at least about 5% by weight hydroxyl-functional monomer is included in the polymer. Example embodiments include up to about 15% by weight hydroxyl-functional monomer in the polymer. In certain embodiments, a hydroxyl-functional acrylic polymer polymerized from hydroxyethyl(meth)acrylate, hydroxypropyl(meth)acrylates, and mixtures of these may be used in the first component. The person skilled in the art will appreciate that hydroxyl groups can be generated by other means, such as, for example, the ring opening of a glycidyl group, for example from glycidyl methacrylate, by an organic acid or an amine. Hydroxyl functionality may also be introduced through thio-alcohol compounds, including, without limitation, 3-mercapto-1-propanol, 3-mercapto-2-butanol, 11-mercapto-1-undecanol, 1-mercapto-2-propanol, 2-mercaptoethanol, 6-mercapto-1-hexanol, 2-mercaptobenzyl alcohol, 3-mercapto-1,2-proanediol, 4-mercapto-1-butanol, and combinations of these. Any of these methods may be used to prepare a useful hydroxyl-functional acrylic polymer.
Examples of suitable comonomers that may be polymerized along with a hydroxyl-functional, ethylenically unsaturated monomer include, without limitation, acrylic acid, methacrylic acid, and crotonic acid; esters, nitriles, and amides of acrylic acid, methacrylic acid, and crotonic acid; vinyl esters, vinyl ethers, vinyl ketones, vinyl amides, and aromatic and cycloaliphatic vinyl compounds. Representative examples include, without limitation, acrylic and methacrylic acid amides and aminoalkyl amides; acrylonitrile and methacrylonitriles; esters of acrylic and methacrylic acid, particularly those with saturated aliphatic and cycloaliphatic alcohols containing 1 to 20 carbon atoms such as methyl, ethyl, propyl, butyl, 2-ethylhexyl, isobutyl, isopropyl, cyclohexyl, tetrahydrofurfuryl, isobornyl, 2-tert-butyl cyclohexyl, 4-tert-butyl cyclohexyl, acrylates and methacrylates; unsaturated dialkanoic acids and anhydrides such as fumaric, maleic, itaconic acids and anhydrides and their mono- and diesters with alcohols such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and tert-butanol, like maleic anhydride, maleic acid dimethyl ester and maleic acid monohexyl ester; vinyl acetate, vinyl propionate, vinyl ethyl ether, and vinyl ethyl ketone; styrene, α-methyl styrene, vinyl toluene, 2-vinyl pyrrolidone, and p-tert-butylstyrene. The co-monomers may be used in any desired combination to obtain desired coating properties.
The acrylic polymer may be prepared using conventional techniques, such as by heating the monomers in the presence of a polymerization initiating agent and optionally a chain transfer agent. The polymerization is preferably carried out in solution, although it is also possible to polymerize the acrylic polymer in bulk or as an emulsion.
Typical initiators are organic peroxides such as dialkyl peroxides such as di-t-butyl peroxide, peroxyesters such as t-butyl peroxy 2-ethylhexanoate, and t-butyl peracetate, peroxydicarbonates, diacyl peroxides, hydroperoxides such as t-butyl hydroperoxide, and peroxyketals; azo compounds such as 2,2′azobis(2-methylbutanenitrile) and 1,1′-azobis(cyclohexanecarbonitrile); and combinations of these. Typical chain transfer agents are mercaptans such as octyl mercaptan, n- or tert-dodecyl mercaptan; halogenated compounds, thiosalicylic acid, mercaptoacetic acid, mercaptoethanol and the other thiol alcohols already mentioned, and dimeric alpha-methyl styrene.
The reaction is usually carried out at temperatures from about 20° C. to about 200° C. The reaction may conveniently be done at the temperature at which the solvent or solvent mixture refluxes, although with proper control a temperature below the reflux may be maintained. The initiator should be chosen to match the temperature at which the reaction is carried out, so that the half-life of the initiator at that temperature should preferably be no more than about thirty minutes. Further details of addition polymerization generally and of polymerization of mixtures including (meth)acrylate monomers is readily available in the polymer art.
The first package including the components (a) and (b) may also include other materials, such as solvent and conventional coating additives. When the refinish, multi-component coating composition is for a single stage topcoat, a plurality of first packages will be included as color bases as part of a mixer system that may be combined in predetermined amounts to provide a refinish coating of a desired color. An unpigmented first package may also be combined with the one or more color base first packages in making a single stage topcoat. Each color base includes one or more pigments dispersed according to known methods in the art. The first package or packages comprise from about 10% to about 50% by weight of component (a), the unsaturated fatty acid polyol, based on the total nonvolatile weight of component (a) and component (b), the acrylic polymer with hydroxyl number 37 to 170. In various embodiments, the first package (or each first package, if there are a plurality of first packages) comprises at least about 15%, at least about 20%, or at least about 25% by weight of component (a), based on the total nonvolatile weight of components (a) and (b); and in various embodiments, the first package comprises up to about 45%, up to about 40%, or up to about 35% by weight of component (a), based on the total nonvolatile weight of components (a) and (b).
The refinish, multi-component coating composition includes a second package including a polyisocyanate. Examples of suitable polyisocyanate crosslinkers include, without limitation, alkylene polyisocyanates such as hexamethylene diisocyanate, 2,2,4- and/or 2,4,4-trimethylhexamethylene diisocyanate, dodecamethylene diisocyanate, 1,4-diisocyanatocyclohexane, 1-isocyanato-3,3,5-trimethyl-5-isocyanatomethylcyclohexane (isophorone diisocyanate), 2,4′- and/or 4,4′-diisocyanatodicyclohexylmethane, 3-isocyanato-methyl-3,5,5-trimethyl cyclohexyl isocyanate, aromatic polyisocyanates such as 2,4′- and/or 4,4′-diisocyanatodiphenylmethane, 2,4- and/or 2,6-diisocyanatotoluene, naphthylene diisocyanate, and mixtures of these polyisocyanates. Generally, polyisocyanates having three or more isocyanate groups are used; these may be derivatives or adducts of diisocyanates. Useful polyisocyanates may be obtained by reaction of an excess amount of an isocyanate with water, a polyol (for example, ethylene glycol, propylene glycol, 1,3-butylene glycol, neopentyl glycol, 2,2,4-trimethyl-1,3-pentane diol, hexamethylene glycol, cyclohexane dimethanol, hydrogenated bisphenol-A, trimethylolpropane, trimethylolethane, 1,2,6-hexanetriol, glycerine, sorbitol or pentaerythritol), or by the reaction of the isocyanate with itself to give an isocyanurate. Examples include biuret-group-containing polyisocyanates, such as those described, for example, in U.S. Pat. No. 3,124,605 and U.S. Pat. No. 3,201,372 or DE-OS 1,101,394; isocyanurate-group-containing polyisocyanates, such as those described, for example, in U.S. Pat. No. 3,001,973, DE-PS 1,022,789, 1,222,067 and 1,027,394 and in DE-OS 1,929,034 and 2,004,048; urethane-group-containing polyisocyanates, such as those described, for example, in DE-OS 953,012, BE-PS 752,261 or U.S. Pat. Nos. 3,394,164 and 3,644,457; carbodiimide group-containing polyisocyanates, such as those described in DE-PS 1,092,007, U.S. Pat. No. 3,152,162 and DE-OS 2,504,400, 2,537,685 and 2,552,350; allophanate group-containing polyisocyanates, such as those described, for example, in GB-PS 994,890, BE-PS 761,626 and NL-OS 7,102,524; and uretdione group-containing polyisocyanates, such as those described in EP-A 0,377,177, each reference being incorporated herein by reference.
Certain embodiments of the second component package include one of aliphatic biurets and isocyanurates, such as the isocyanurates of hexamethylene diisocyanate and isophorone diisocyanate.
A third, optional package includes a reducing solvent, optionally a further resin or polymer, and optionally a catalyst for the isocyanate-hydroxyl curing reaction. The multi-component refinish composition may include multiple reducer packages, which may each be designed to be used under different weather conditions. For example, the multi-component refinish composition may include one reducer package having a relatively fast solvent for use in cold weather to speed evaporation of solvent from the applied coating layer, while a second reducer package has a relatively slow solvent for use in hot weather to allow the coating layer to flow out properly before all the solvent evaporates. In general, the solvent can be any organic solvent or solvents suitable for the binder materials. The solvent or solvents may be selected from aliphatic solvents or aromatic solvents, for example ketones, esters, acetates, toluene, xylene, aromatic hydrocarbon blends, or a combination of any of these. Generally, each of the first and second packages will also include one or more organic solvents.
The refinish composition may contain other materials, including additives such as pigments (which, as already described, are included in the case of single stage topcoat color bases), rheology control agents, surfactants, stabilizers, UV absorbers, hindered amine light stabilizers, and so on. Curing catalysts for the urethane reaction such as tin catalysts can be used in the coating composition. Typical examples are without limitation, tin and bismuth compounds including dibutyltin dilaurate, dibutyltin oxide, and bismuth octoate. When used, catalysts are typically present in amounts of about 0.05 to 2 percent by weight tin based on weight of total nonvolatile vehicle.
The pigment or filler may be any organic or inorganic compound or colored material, metallic or other inorganic flake material such as pearlescent mica flake pigments or metallic flake pigments such as aluminum flake, and the like that the art normally includes in such coatings. Such pigments may be used singly or in combination to provide a desired color of color base. Pigments and other insoluble particulate compounds such as fillers may be used in the refinish monocoat composition mixture in an amount of 1% to 100%, based on the total nonvolatile vehicle (i.e., a pigment-to-binder ratio of 0.1 to 1). The fillers or pigments can be introduced by first forming a mill base (also called pigment grind) with the hydroxyl functional resin or with other compatible polymers or dispersing resins by conventional techniques, such as sandgrinding, ball-milling, attritor grinding, and two roll milling, to disperse the pigments.
The refinish clearcoat or single stage topcoat is applied in a layer to a desired area of the substrate to be refinished and cured. The clearcoat is applied over an applied basecoat layer. The basecoat layer is allowed to dry before the clearcoat composition is applied. The clearcoat or single stage topcoat composition is then cured, at ambient or low temperature bake conditions. Because the coating composition is free or essentially free of any drier the clearcoat or single stage topcoat does not undergo oxidative curing at all or at least not to any appreciable extent.
The refinished substrate may be an automotive vehicle or a component of an automotive vehicle. The refinish coating compositions may, however, be applied to other articles for which a protective and/or decorative coating is desirable. Such articles may be those having parts or substrates that cannot withstand high temperature curing conditions or that cannot easily be placed in a high-bake oven.
The coatings and methods are further described in the following example. The examples are merely illustrative and not limiting. All parts are by weight unless otherwise indicated.
Example 1
Comparative Example
A two-component clearcoat refinish coating composition was prepared by combining the following materials in a separate first component and a separate second component.
Component 1: A 56% non-volatile clearcoat formulation was prepared, comprised of a styrenated acrylic resin with a T g of 24° C., a hydroxyl number of 136 mg. KOH/gm., and a number average molecular weight of 1400 daltons, a styrenated acrylic resin with a T g of 78° C., a hydroxyl number of 73 mg. KOH/gm., and a number average molecular weight of 6500 daltons, in a ratio of 84:16 first: second acrylic. The remainder of the clearcoat formulation was comprised of solvents, such as methyl iso-amyl ketone, n-butyl acetate, aromatic 100, xylene, ethyl 3-ethoxypropionate and acetone, and additives well known in the art, such as UV absorbers, silicone based flow agents, plasticizers and tin based catalysts.
Component 2: A 72% non-volatile hardener formulation was prepared, comprised of a solution of a hexamethylene diisocyanate based trimer with a percent isocyanate content of 22.6%. The remainder of the hardener formulation was comprised of solvents, such as n-butyl acetate, xylene, and toluene, and additives, such as tin based catalysts.
Component #1 and Component #2 were combined in a 73.5:26.5 weight ratio, resulting on a hydroxyl:isocyanate ratio of 1:1.3, reduced further with the above mentioned solvents to a non-volatile content of 34-38%, and spray applied over a test panel precoated with a BASF 90-Line black basecoat. The panel was allowed to dry at ambient temperatures for 15 minutes, then placed in a 140° F. for 18 hours before testing.
Example 2
Example of the Invention
A two-component clearcoat refinish coating composition was prepared by combining the following materials in a separate first component and a separate second component.
Component #1: To Component #1 of Example 1, Polycin M-365® (Vertellus Specialties Incorporated) was added in a weight ratio of 83:17.
Component #2: Component #2 was the same as Component #2 in Example 1.
Component #1 and Component #2 were combined in a 63:37 weight ratio, resulting on a hydroxyl:isocyanate ratio of 1:1.2, reduced further with the above mentioned solvents to a non-volatile content of 34-38%, and spray applied over a test panel precoated with a BASF 90-Line black basecoat. The panel was allowed to dry at ambient temperatures for 15 minutes, then placed in a 140° F. for 18 hours before testing.
Example 3
Comparative Example
Component 1: A 49% non-volatile clearcoat formulation was prepared, comprised of a styrenated acrylic resin with a T g of 9° C., a hydroxyl number of 125 mg. KOH/gm., and a number average molecular weight of 2700 daltons. The remainder of the clearcoat formulation was comprised of solvents, such as ethylene glycol butyl ether, methyl acetate, parachlorobenzotrifluoride, ethyl 3-ethoxypropionate and acetone, and additives well known in the art, such as UV absorbers, silicone based flow agents, plasticizers and tin based catalysts.
Component 2: A 37% non-volative hardener formulation was prepared, comprised of a solution of a hexamethylene diisocyanate based trimer with a percent isocyanate content of 23.5%. The remainder of the hardener formulation was comprised of solvents, such as methyl acetate, parachlorobenzotrifluoride.
Component #1 and Component #2 were combined in a 63:37 weight ratio, resulting on a hydroxyl:isocyanate ratio of about 1:1.2, reduced further with the above mentioned solvents to a non-volatile content of 42-45%, and spray applied over a test panel precoated with a BASF 90-Line black basecoat. The panel was allowed to dry at ambient temperatures for 15 minutes, then placed in a 140° F. for 18 hours before testing.
Example 4
Example of the Invention
Component #1: To Component #1 of Example 3, Polycin M-365® (Vertellus Specialties Incorporated) was added in a weight ratio of 7:3.
Component #2: Component #2 was the same as Component #2 in Example 3.
Component #1 and Component #2 were combined in a 46:54 weight ratio, resulting on a hydroxyl:isocyanate ratio of 1:0.9, reduced further with the above mentioned solvents to a non-volatile content of 42-45%, and spray applied over a test panel precoated with a BASF 90-Line black basecoat. The panel was allowed to dry at ambient temperatures for 15 minutes, then placed in a 140° F. for 18 hours before testing.
Examples 1-4 were tested with the following results. The scratch test was carried out with an Atlas A.A.T.C.C. Crockmeter mounted with a ⅝″ dowel covered with felt and 9 μm 3M 281Q WETODRY polishing paper, which was used to abrade the coating surface with ten (10) double strokes. The gloss before and after testing was measured according to ASTM method D523 for the tested surface area. The gloss was measured again according to ASTM method D523 after a re-flow bake.
Crockmeter Test
Example 1
Example 2
Example 3
Example 4
Initial 20° gloss after
85
85.7
86.7
83.5
bake:
Final 20° gloss after
47
63.5
64.9
71.9
scratch test:
Re-flow: Panels were baked again at 140 F. for
1 hour. 20 degree gloss is re-measured.
20° gloss after re-flow
60
80
80
82
bake
The invention has been described in detail with reference to preferred embodiments thereof. It should be understood, however, that variations and modifications can be made within the spirit and scope of the invention.
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Scratch resistance and/or reflow of a refinish topcoat is improved by preparing the topcoat from a refinish topcoat coating composition comprising an unsaturated fatty acid ester polyol that does not undergo oxidative cure when the applied refinish topcoat coating composition is cured.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a laminated rubber support assembly utilized for earthquake-proofing buildings and other structures.
2. Description of the Prior Art
As for a foundation material for protecting buildings and other structures against earthquakes, a laminated rubber support assembly as shown in FIG. 4 is known (Japanese Patent Application Disclosure No. 209347/1982). This laminated rubber support assembly 1 comprises a plurality of steel plates 2 and thin rubber plates 3 alternating with and vulcanization-bonded to the steel plates 2. In this assembly, since the ratio of the vertical spring rigidity to the horizontal spring rigidity can be greatly increased, it supports a building 4, which is a heavy object, in a stable manner and if an earthquake should occur, it allows the building to swing horizontally at a low speed with a period which is longer than the period of the earthquake, thus decreasing the input acceleration of the earthquake. Therefore, the earthquake resisting strength required of buildings can be made much lower than in the case of conventional rigid structure foundations which fix a building directly to the ground. Particularly, it facilitates the construction of high-rise buildings.
When the behavior of the rubber plates 3 of the laminated rubber support assembly 1 is observed, the following is found.
In the no-load state before the vulcanization-bonded laminate is installed, the laminate has been finished such that each rubber plate 3 is inwardly recessed with respect to the steel plates 2, as shown in FIG. 6 (a). In the installed state in which it is interposed between the building and the foundation, each rubber plate 3 is compressed and its peripheral region is arcuately bulged, as shown in FIG. 6 (b). After installation, if an earthquake should occur to cause a horizontal displacement of each steel plate 2, then, as shown in FIG. 6 (c), since each rubber plate 3 has its upper and lower surfaces bound by the steel plates 2, the whole is deformed under shearing stress. At this time, the exposed portion of the peripheral region of each rubber plate is obliquely stretched, as shown in FIG. 6 (c); however, since this peripheral region is also subjected to lateral tension from the inner region, it is in the highly tensioned state as compared with the inner region. Particularly when it is deformed to a large extent, it becomes harder, thus increasing the horizontal spring constant, a fact which decreases the earthquake-proofing capacity while causing the peripheral region to break as at 3a in FIG. 6 (c).
SUMMARY OF THE INVENTION
Accordingly, the invention provides a construction which prevents the peripheral region of the rubber plate from being subjected to high tension during great deformation. With this construction, the horizontal spring constant is held substantially constant during great deformation, eliminating the danger of the rubber plates breaking during great deformation, thereby increasing the earthquake-proofing capacity and durability of the laminated rubber support assembly.
The invention discloses a laminated rubber support assembly comprising a plurality of rigid plates arranged to alternate with rubber-like elastic plates and adapted to support a heavy object in such a manner as to allow the object to swing horizontally, the laminated rubber support assembly being characterized in that at least the peripheral region of each rubber-like elastic plate interposed between rigid plates is not bonded to the rigid plates.
In the above arrangement, the peripheral region of each rubber-like elastic plate is simply in contact with and held between rigid plates and not fixed to them. Therefore, as compared with the conventional completely bonded type, it can move rather freely with respect to the upper and lower rigid plates.
Therefore, when the upper and lower rigid plates make horizontal relative movement, the peripheral region follows the movement and starts rolling, so that the lateral tensile force from the inner region is absorbed by the material of the peripheral region being deformed. Further, since the surface area of the peripheral region is large, the tensile force acting on this portion is dispersed and minimized.
As a result, the phenomenon of the peripheral region being hardened during great deformation no longer occur, preventing the peripheral region from breaking and the horizontal spring constant from increasing during great deformation.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 (a), (b) and (c) are fragmentary sectional views of a laminated rubber support assembly according to the present invention, showing the no-load state, the compressed state and the compressed sheared state thereof, respectively;
FIG. 2 is a sectional view showing the deformed state of the laminated rubber support assembly of the invention;
FIG. 3 is a graph showing the shearing characteristics m of the laminated rubber support assembly of the invention with respect to the horizontal deformation of the laminated rubber support assembly of the invention in comparison with the characteristics n of a conventional laminated rubber support assembly;
FIG. 4 is a front view showing the common construction of laminated rubber support assemblies;
FIG. 5 is a front view showing a construction for earthquake-proofing buildings by using laminated rubber support assemblies; and
FIGS. 6 (a), (b) and (c) are fragmentary sectional views of a conventional laminated rubber support assembly, showing the no-load state, the compressed state and the compressed sheared state thereof, respectively.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
A laminated rubber support assembly 5 according to the present invention, as shown in FIGS. 1 (a), (b) and (c) and FIG. 2, comprises a rigid plates 6, such as steel plates, and rubber-like elastic plates 7 of natural rubber, synthetic rubber or the like. The plates 6 and 7 are alternately laminated to each other in such a manner that the peripheral region of each rubber-like elastic plate 7 is not bonded to the rigid plates 6. That is, aside from the peripheral region, the central region alone is fixed to the rigid plates as by vulcanization, the peripheral region being simply in contact therewith. In this case, the lateral end of the rubber-like elastic plate 7 is bulged outward and connected to the upper and lower surfaces by an arc, as shown in FIG. 1 (a). This is preferable in that it facilitates rolling. When the laminated rubber support assembly 5 constructed in the manner described above is installed between a heavy object, such as a building, and a foundation therefor, the lateral end thereof projects outward owing to the compression load, as shown in FIG. 1 (b). When the laminated rubber support assembly 5 is horizontally deformed for earthquake-proofing operation, as shown in FIG. 2, the peripheral region of each rubber-like elastic plate is deformed as shown in FIG. 1 (c). This will now be described in more detail.
The non-bonded surface 7b of the peripheral region 7a follows the movement of the rigid plates 6 with predetermined friction force, so that the material of the peripheral region starts rolling. At this time, since the material of the peripheral region is not bound by the rigid plates 6, it can be elastically deformed more freely than the material of the bonded inner region. Therefore, the tensile force directed to the lateral end 7c due to the horizontal relative displacement of the upper and lower rigid plates 6 and the tensile force directed laterally from the inner region are dispersed, so that the peripheral region 7a will not be hardened even during great deformation. Therefore, the phenomenon of the outer surface of the peripheral region being broken during great deformation no longer occur, and the horizontal spring constant is maintained substantially constant even during great deformation, developing earthquake-proofing characteristics effective also in withstanding great earthquakes.
The non-bonded type construction described above has characteristics such that the greater the load, the greater the friction force of the non-bonded surface 7b acting on the rigid plate 6, so that the area which performs the function of a spring increases, thus increasing the spring constant. This means that a single type of laminated rubber support assembly can be used both for heavier buildings and for less heavy buildings. That is, to obtain the primary intrinsic period effective in withstanding earthquakes, a greater spring constant is required for heavier buildings while a smaller spring constant is required for less heavy buildings.
In addition, in the above embodiment, the rubber-like elastic plate 7, aside from its peripheral region, has been bonded to the rigid plates 6. However, in the case where the laminated rubber support assembly is utilized for earthquake-proofing buildings, since the friction force produced by surface pressure due to load is generally greater than the shearing force produced, all surface may be left non-bonded; the holding of the upper and lower rigid plates 6 and rubber-like elastic plate 7 is effected by this friction force only. In this case, the bonding process becomes unnecessary, lowering the manufacturing cost. In addition, in this complete-surface non-bonded construction, it is necessary that the positional relation between the upper and lower rigid plates 6 and the rubber-like elastic plate 7 be maintained constant during the time the assembly is installed under a building. As for means therefor, there may be contemplated the use of temporary fixing members for temporarily fixing the assembly over its upper and lower end rigid plates, the use of a vibration energy absorbing rubber member made of such material as elastoplastic body, viscoelastic body, lead or highly attenuating rubber, to surround the laminated rubber support assembly so as to provide a holding construction having a vibration attenuating effect, or the use of a soft heat-resistant element to surround the laminated rubber support assembly to provide a holding construction which is also fire-resistant. In the holding construction having a vibration attenuating effect and the holding construction which is fire-resistant, since the laminated rubber support assembly in the interior is protected from the open air, its life is prolonged. Particularly in the case of the holding construction having a vibration attenuating effect, the psychological anxious feeling, associated with the complete-surface non-bonded type laminated rubber support assembly, that the component layers would be displaced sideways (which has been proved to be nothing more than overanxiousness) can be wiped away; thus, the residents feel greater confidence in the building to withstand earthquakes.
In addition, besides serving for earthquake-proofing buildings, the laminated rubber support assembly of the invention can be used also as a damper for machines.
In the present invention, since the peripheral region of each rubber-like elastic plate is made independent of the rigid plates rather than bonded thereto, the peripheral region is prevented from being hardened during great deformation, avoiding damage and an increase in horizontal spring constant, thereby providing an increased capacity to withstand earthquakes.
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A laminated rubber support assembly which earthquake-proofs buildings. It comprises a plurality of rigid plates arranged to alternate with rubber-like elastic plates and adapted to support a heavy object in such a manner as to allow the object to swing horizontally. This laminated rubber support assembly is characterized in that at least the peripheral region of each rubber-like elastic plate interposed between rigid plates is not bonded to the rigid plates.
| 1 |
BACKGROUND OF THE INVENTION
[0001] This invention is directed to compounds of formula I described herein, to a pharmaceutical composition comprising such compounds, and to methods of treatment of disorders or conditions that may be treated by antagonizing histamine-3 (H3) receptors using such compounds.
[0002] Histamine is a well-known mediator in hypersensitive reactions (e.g. allergies, hay fever, and asthma) that are commonly treated with antagonists of histamine or “antihistamines.” It has also been established that histamine receptors exist in at least two distinct types, referred to as H1 and H2 receptors.
[0003] A third histamine receptor (H3 receptor) is believed to play a role in neurotransmission in the central nervous system, where the H3 receptor is thought to be disposed presynaptically on histaminergic nerve endings (Nature, 302, S32-837 (1983)). The existence of the H3 receptor has been confirmed by the development of selective H3 receptor agonists and antagonists (Nature, 327, 117-123 (1987)) and has subsequently been shown to regulate the release of the neurotransmitters in both the central nervous system and peripheral organs, particularly the lungs, cardiovascular system and gastrointestinal tract.
[0004] A number of diseases or conditions may be treated with histamine-3 receptor ligands wherein the H3 ligand may be an antagonist, agonist or partial agonist, see: (Imamura et al., Circ. Res., (1996) 78, 475-481); (Imamura et. al., Circ. Res., (1996) 78, 863-869); (Lin et al., Brain Res. (1990) 523, 325-330); (Monti et al., Neuropsychopharmacology (1996) 15, 31 35); (Sakai, et al., Life Sci. (1991) 48, 2397-2404); (Mazurkiewiez-Kwilecki and Nsonwah, Can. J. Physiol. Pharmacol, (1989) 67, 75-78); (Panula, P. et al., Neuroscience (1998) 44, 465-481); (Wada et al., Trends in Neuroscience (1991) 14, 415); (Monti et al., Eur. J. Pharmacol. (1991) 205, 283); (Mazurkiewicz-Kwilecki and Nsonwah, Can. J. Physiol. Pharmacol. (1989) 67, 75-78); (Haas et al., Behav. Brain Res. (1995) 66, 41-44); (De Almeida and Izquierdo, Arch. Int. Pharmacodyn. (1986) 283, 193-198); (Karnel et al., Psychopharmacology (1990) 102, 312-318); (Kamei and Sakata, Japan. J. Pharmacol. (1991) 57, 437-482); (Schwartz et al., Psychopharmacology; The fourth Generation of Progress, Bloom and Kupfer (eds.), Raven Press, New York, (1995) 397); (Shaywitz et al., Psychopharmacology (1984) 82, 73-77); (Dumery and Blozovski, Exp. Brain Res. (1987) 67, 61-69); (Tedford et al., J. Pharmacol. Exp. Ther. (1995) 275, 598-604); (Tedford et al., Soc. Neurosci. Abstr. (1996) 22, 22); (Yokoyama et al., Eur. J. Pharmacol. (1993) 234, 129); (Yokoyama and Iinuma, CNS Drugs (1996) 5, 321); (Onodera et al., Prog. Neurobiol. (1994) 42, 685); (Leurs and Timmerman, Prog. Drug Res. (1992) 39, 127); (The Histamine H3 Receptor, Leurs and Timmerman (ed.), Elsevier Science, Amsterdam, The Netherlands (1998); (Leurs et al., Trends in Pharm. Sci. (1998) 19, 177-183); (Phillips et al., Annual Reports in Medicinal Chemistry (1998) 33, 31-40); (Matsubara et al., Eur. J. Pharmacol. (1992) 224, 145); (Rouleau et al., J. Pharmacol. Exp. Ther. (1997) 281, 1085); (Adam Szelag, “Role of histamine H3-receptors in the proliferation of neoplastic cells in vitro”, Med. Sci. Monit., 4(5): 747-755, (1998)); (Fitzsimons, C., H. Duran, F. Labombarda, B. Molinari and E. Rivera, “Histamine receptors signalling in epidermal tumor cell lines with H-ras gene alterations”, Inflammation Res., 47 (Suppl. 1): S50-651, (1998)); (R. Leurs, R. C. Volling a and H. Timmerman, “The medicinal chemistry and therapeutic potentials of ligand of the histamine H3 receptor”, Progress in Drug Research 45: 170-165, (1995)); (R. Levi and N. C. E. Smith, “Histamine H3-receptors: A new frontier in myocardial ischemia”, J. Pharm. Exp. Ther., 292: 825-830, (2000)); (Hatta, E., K Yasuda and R. Levi, “Activation of histamine H3 receptors inhibits carrier-mediated norepinephrine release in a human model of protracted myocardial ischemia”, J. Pharm. Exp. Ther., 283: 494-500, (1997); (H. Yokoyama and K. Iinuma, “Histamine and Seizures: Implications for the treatment of epilepsy”, CNS Drugs, 5(5); 321-330, (1995)); (K. Hurukami, H. Yokoyama, K. Onodera, K. Iinuma and T. Watanabe, AQ-0 145, “A newly developed histamine H3 antagonist, decreased seizure susceptibility of electrically induced convulsions in mice”, Meth. Find. Exp. Clin. Pharmacol., 17(C): 70-73, (1995); (Delaunois A., Gustin P., Garbarg M., and Ansay M., “Modulation of acetylcholine, capsaicin and substance P effects by histamine H3 receptors in isolated perfused rabbit lungs”, European Journal of Pharmacology 277(2-3):243-50, (1995)); and (Dimitriadou, et al., “Functional relationship between mast cells and C— sensitive nerve fibres evidenced by histamine H3-receptor modulation in rat lung and spleen”, Clinical Science 87(2):15163, (1994). Such diseases or conditions include cardiovascular disorders such as acute myocardial infarction; memory processes, dementia and cognitive disorders such as Alzheimer's disease and attention-deficit hyperactivity disorder; neurological disorders such as Parkinson's disease, schizophrenia, depression, epilepsy, and seizures or convulsions; cancer such as cutaneous carcinoma, medullary thyroid carcinoma and, melanoma; respiratory disorders such as asthma; sleep disorders such as narcolepsy; vestibular dysfunction such as Meniere's disease; gastrointestinal disorders, inflammation, migraine, motion sickness, obesity, pain, and septic shock.
[0005] H3 receptor antagonists have also been previously described in, for example, WO 03/050099, WO 02/0769252, WO 02/12224, and U.S. Patent Publication No. 2005/0171181 A1. The histamine H3 receptor (H3R) regulates the release of histamine and other neurotransmitters, including serotonin and acetylcholine. H3R is relatively neuron specific and inhibits the release of certain monoamines such as histamine. Selective antagonism of H3R receptors raises brain histamine levels and inhibits such activities as food consumption while minimizing non-specific peripheral consequences. Antagonists of the receptor increase synthesis and release of cerebral histamine and other monoamines. By this mechanism, they induce a prolonged wakefulness, improved cognitive function, reduction in food intake and normalization of vestibular reflexes. Accordingly, the receptor is an important target for new therapeutics in Alzheimer disease, mood and attention adjustments, including attention deficit hyperactive disorder (ADHD), cognitive deficiencies, obesity, dizziness, schizophrenia, epilepsy, sleeping disorders, narcolepsy and motion sickness, and various forms of anxiety.
[0006] The majority of histamine H3 receptor antagonists to date resemble histamine in possessing an imidazole ring that may be substituted, as described, for example, in WO 96/38142 Non-imidazole neuroactive compounds such as beta histamines (Arrang, Eur. J. Pharm. 1985, 111:72-84) demonstrated some histamine H3 receptor activity but with poor potency. EP 978512 and EP 0982300A2 disclose non-imidazole alkyamines as histamine H3 receptor antagonists. WO 02/12224 (Ortho McNeil Pharmaceuticals) describes non-imidazole bicyclic derivatives as histamine H3 receptor ligands. Other receptor antagonists have been described in WO 02/32893 and WO 02/06233.
[0007] This invention is directed to histamine-3 (H3) receptor antagonists of the invention useful for treating the conditions listed in the preceding paragraphs. The compounds of this invention are highly selective for the H3 receptor (vs. other histamine receptors), and possess remarkable drug disposition properties (pharmacokinetics). In particular, the compounds of this invention selectively distinguish H3R from the other receptor subtypes H1R, H2R. In view of the increased level of interest in histamine H3 receptor agonists, inverse agonists and antagonists in the art, novel compounds that interact with the histamine H3 receptor would be a highly desirable contribution to the art. The present invention provides such a contribution to the art being based on the finding that a novel class of tetraline amines has a high and specific affinity to the histamine H3 receptor.
SUMMARY OF THE INVENTION
[0008] This invention is directed to a compound of formula I:
[0000]
[0000] or a pharmaceutically acceptable salt thereof, wherein
[0009] Z, Y, Q, X are independently nitrogen or carbon;
[0010] R 1 and R 2 are independently hydrogen, (C 1 -C 8 )alkyl optionally substituted with 1 to 4 halogens, or (C 3 -C 7 )cycloalkyl-(C 0 -C 4 )alkyl, wherein each (C 0 -C 4 ) is optionally substituted with one to four (C 1 -C 4 )alkyl;
[0011] or optionally R 1 and R 2 , together with the nitrogen to which they are attached, form a 4 to 7-membered heterocycloalkyl ring, wherein one of the carbons of said heterocycloalkyl ring that is separated by at least two atoms from said nitrogen in said heterocycloalkyl ring is optionally replaced by O, S, NR 6 , or C═O, wherein R 6 is hydrogen, (C 1 -C 3 )alkyl, or —C(═O)(C 1 -C 3 )alkyl; and wherein said heterocycloalkyl ring is optionally substituted with halo, one or two (C 1 -C 4 )alkyl, phenyl, (C 1 -C 8 )alkyl optionally substituted with 1 to 4 halogens, or (C 3 -C 7 )cycloalkyl-(C 0 -C 4 )alkyl, and wherein each (C 0 -C 4 )alkyl is optionally substituted with one to four (C 1 -C 4 )alkyl;
[0012] R 3 is hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, halo, 5 to 6-membered aryl, 5 to 6-membered heteroaryl, hydroxyl, methylene hydroxyl, —(C═O)NR 4 R 5 , and S(O) p (C 1 -C 4 )alkyl, where p is 1 or 2;
wherein R 4 and R 5 are independently selected from the group consisting of hydrogen, (C 1 -C 8 )alkyl optionally substituted with 1 to 4 halogens; (C 1 -C 4 )alkyl group optionally substituted with a substituent selected from the group consisting of OH, 1 to 4 (C 1 -C 4 )alkyl, (C 3 -C 7 )cycloalkyl, (C 1 -C 4 )dialkylamino, (C 6 -C 14 )aryl optionally substituted with a halogen and optionally substituted with (C 6 -C 10 )aryloxy optionally substituted with 1 to 2 halogens, and 5 to 10-membered heteroaryl optionally substituted with a (C 6 -C 10 )aryl group and optionally substituted with 1 to 3 (C 1 -C 4 )alkyl groups; (C 3 -C 7 )cycloalkyl; (C 6 -C 14 )aryl; -(C 2 -C 8 )alkyl-O—(C 1 -C 3 )alkyl optionally substituted with (C 1 -C 3 )alkyl; —(C 1 -C 3 )alkyl-C(═O)O—(C 1 -C 3 )alkyl; 3-8-membered heterocycloalkyl optionally substituted with one or more (C 1 -C 4 )alkyl-carbonyl groups; (C 6 -C 10 )arylsulfonyl optionally substituted with one or more (C 1 -C 2 )alkyl; 5-10-membered heteroaryl; and (C 6 -C 14 )aryl-(C 0 -C 4 )alkylene-O—(C 0 -C 4 )alkyl, wherein each (C 0 -C 4 )alkyl and each (C 0 -C 4 )alkylene is optionally substituted with 1 to 4 (C 1 -C 4 alkyl); or optionally R 4 and R 5 , together with the nitrogen to which they attached, form a 4 to 6-membered heterocyclic ring, wherein one of the carbons of said heterocyclic ring that is separated by at least two atoms from said nitrogen in said heterocyclic ring is optionally replaced by O or NR 6 , wherein R r is hydrogen, (C 1 -C 3 )alkyl, or —C(═O)(C 1 -C 3 )alkyl; and wherein said heterocyclic ring is optionally substituted with halo, (C 1 -C 3 )alkyl, or hydroxyl;
[0025] R 7 is hydrogen;
[0026] or optionally R 3 and R 7 together with two adjacent atoms in the ring comprising Z, Y, Q and X to which they are attached, form a 5 or 6-membered heterocyclic ring; wherein one of the carbons of said heterocyclic ring that is separated by at least two atoms from said nitrogen in said heterocyclic ring is optionally replaced by O or NR 8 ; wherein R 8 is hydrogen or (C 1 -C 3 )alkyl.
[0027] A preferred embodiment includes compounds of formula I, wherein, the invention is directed to a compound of formula I, wherein Y and X are carbon; Q and Z are carbon or nitrogen; R 7 is hydrogen; R 1 and R 2 together form a 5-membered heterocycloalkyl ring, optionally substituted with (C 1 -C 4 )alkyl; and R 3 is selected from the group consisting of methoxy, —(C═O)NR 4 R 6 , and S(O) p (C 1 -C 4 )alkyl; wherein R 4 and R 5 are independently hydrogen or (C 1 -C 4 )alkyl; and wherein p is 1 or 2.
[0028] Another preferred embodiment includes compounds of formula I, wherein Z, Y, X, and Q are carbon; R 1 and R 2 together with the nitrogen to which they are attached form a 5 membered heterocycloalkyl ring optionally substituted with methyl;
R 7 is hydrogen; R 3 is —(C═O)NR 4 R 5 ; wherein R 4 and R 5 are independently selected from the group consisting of hydrogen; (C 1 -C 8 )alkyl optionally substituted with 1 to 4 halogens; (C 1 -C 4 )alkyl group optionally substituted with a substituent selected from the group consisting of OH, 1 to 4 (C 1 -C 4 )alkyl, (C 3 -C 7 )cycloalkyl, (C 1 -C 4 )dialkylamino, (C 6 -C 14 )aryl optionally substituted with a halogen and optionally substituted with (C 6 -C 10 )aryloxy optionally substituted with 1 to 2 halogens, and 5 to 10-membered heteroaryl optionally substituted with a (C 8 -C 10 )aryl group and optionally substituted with 1 to 3 (C 1 -C 4 )alkyl groups; (C 3 -C 7 )cycloalkyl; (C 6 -C 14 )aryl; -(C 2 -C 3 )alkyl-O—(C 1 -C 3 )alkyl optionally substituted with (C 1 -C 3 )alkyl; -(C 1 -C 3 )alkyl-C(═O)O—(C 1 -C 3 )alkyl; 3-8-membered heterocycloalkyl optionally substituted with one or more (C 1 -C 4 )alkyl-carbonyl groups; (C 6 -C 10 )arylsulfonyl optionally substituted with one or more (C 1 -C 2 )alkyl; 5-10-membered heteroaryl; and (C 6 -C 14 )aryl-(C 0 -C 4 )alkylene-O—(C 0 -C 4 )alkyl, wherein each (C 0 -C 4 )alkyl and each (C 0 -C 4 )alkylene is optionally substituted with 1 to 4 (C 1 -C 4 alkyl); or optionally R 4 and R 5 , together with the nitrogen to which they attached, form a 4 to 6-membered heterocyclic ring, wherein one of the carbons of said heterocycloalkyl ring that is separated by at least two atoms from said nitrogen in said heterocycloalkyl ring is optionally replaced by C or NR 8 , wherein R 8 is hydrogen or (C 1 -C 3 )alkyl; and wherein said heterocycloalkyl ring is optionally substituted with halo, (C 1 -C 3 )alkyl, or hydroxyl.
[0043] Another preferred embodiment includes compounds of formula I, wherein Z, Y, X, and Q are carbon;
[0044] R 1 and R 2 together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl ring optionally substituted with methyl;
[0045] R 7 is hydrogen;
[0046] R 3 is —(C═O)NR 4 R 5 ; wherein R 4 and R 6 are independently selected from the group consisting of hydrogen, (C 1 -C 5 )alkyl, (C 3 -C 5 )cycloalkyl.
[0047] Another preferred embodiment includes compounds of formula I, wherein Z, Y, X, and Q are carbon;
[0048] R 1 and R 2 together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl ring optionally substituted with methyl;
[0049] R 7 is hydrogen;
[0050] R 3 is —(C═O)NR 4 R 5 ; wherein R 4 and R 5 , together with the nitrogen to which they are attached, form a 4 to 6-membered heterocycloalkyl ring, and wherein said heterocycloalkyl ring is optionally substituted with halo, hydroxy, or (C 1 -C 5 )alkyl.
[0051] Another preferred embodiment includes compounds of formula I, wherein X, Y, Z are carbon;
[0000] Q is nitrogen;
[0052] R 1 and R 2 together with the nitrogen to which they are attached form a 5-membered heterocyclic ring optionally substituted with methyl; R 3 is selected from the group, consisting of hydrogen, methyl, ethyl, methoxy, and ethoxy; and R 7 is hydrogen.
[0053] Another preferred embodiment includes compounds of formula I, wherein X is carbon; Z and Q are nitrogen; R 3 is selected from the group consisting of hydrogen, methyl, ethyl, methoxy, and ethoxy; and R 7 is hydrogen.
[0054] Another preferred embodiment includes compounds of formula I, wherein said compound has the following structure:
[0000]
[0055] Another preferred embodiment includes compounds of formula I, wherein said compound has the following structure:
[0000]
[0000] Another preferred embodiment includes compounds of formula i, selected from the group consisting of
3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; Azetidin-1-yl-[4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenyl]-methanone; N-Cyclobutyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; N-Isobutyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; 4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydronaphthalen-2-yl)-pyridine; N-(2-Methoxy-ethyl)-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide methanone; N-(2-Hydroxy-ethyl)-N-methyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; N-Cyclopropyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydronaphthalen-2-yl)-benzamide; 3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yloxy)-benzamide; 5-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydronaphthalen-2-yl)-oxazole; 1-[6-(4-Methanesulfonyl-phenoxy)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; N-(2-Hydroxy-ethyl)-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; 4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-N-(tetrahydro-pyran-4-yl)-benzamide; N-(2-Methoxy-ethyl)-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; N-isobutyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; 1-[6-(4-Methoxy-phenoxy)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; N,N-dimethyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; Azetidin-1-yl-[4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenyl]-methanone; N-Ethyl-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; (+)-N-Ethyl-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; (−)-N-Ethyl-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; 2-Methoxy-5-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine; 3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine; 2-Methoxy-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine; 6-Methoxy-2-methyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine; N-Isopropyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; N-Cyclobutyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; 4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenol; 1-[6-(4-Methoxy-2,6-dimethyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; 1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; (R)-1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; (S)-1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; 3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; (S)-3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; (R)-3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; 1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; (R)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; (S)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; 1-Isopropyl-4-[6-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-piperazine; (S)-(−)-1-[6-(4-Chloro-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; (R)-(+)-1-[6-(4-Chloro-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; 3-Fluoro-1-[6-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; 1-[4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenyl]-ethanone; 3,4-Difluoro-1-[6-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine; 1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine; (R,R)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine; (S,R)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine; (R)-1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine; (S)-1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine; (R. R)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine; (S,R)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine; (R,S)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine; (S,S)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine; (R)-N,N-Dimethyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide: (R)-N,N-Dimethyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; (S,R)-3-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-pyridine; (R,R)-3-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-pyridine; 1-[6-(3,4-Dimethoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine; 1-[6-(3-Fluoro-4-methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine; N-Methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide; 4-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide; 3-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide; (R,R)-3-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide; (S,R)—[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide; 1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine;
and pharmaceutically acceptable salts thereof.
[0121] This invention is also directed to pharmaceutical composition for treating a disorder or condition that may be treated by antagonizing histamine-3 receptors, the composition comprising a compound of formula I and optionally a pharmaceutically acceptable carrier.
[0122] This invention is also directed to a method of treatment of a disorder or condition that may be treated by antagonizing histamine-3 receptors, the method comprising administering to a mammal in need of such treatment a compound of formula I.
[0123] This invention is also directed to a method of treatment of a disorder or condition selected from the group consisting of depression, mood disorders, schizophrenia, anxiety disorders, cognitive disorders, Alzheimer's disease, attention-deficit disorder (ADD), attention-deficit hyperactivity disorder (ADHD), psychotic disorders, sleep disorders, obesity, dizziness, epilepsy, motion sickness, respiratory diseases, allergy, allergy-induced airway responses, allergic rhinitis, nasal congestion, allergic congestion, congestion, hypotension, cardiovascular disease, diseases of the GI tract, hyper and hypo motility and acidic secretion of the gastro-intestinal tract, the method comprising administering to a mammal in need of such treatment a compound of formula I.
[0124] This invention is also directed to a pharmaceutical composition for treating allergic rhinitis, nasal congestion or allergic congestion comprising: (a) an H3 receptor antagonist compound of formula I or a pharmaceutically acceptable salt thereof; (b) an HI receptor antagonist or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier; wherein the active ingredients (a) and (b) above are present in amounts that render the composition effective in treating allergy rhinitis, nasal congestion or allergic congestion.
[0125] This invention is also directed to a pharmaceutical composition for treating ADD, ADHD, depression, mood disorders, or cognitive disorders comprising: (a) an H3 receptor antagonist compound of Formula I or a pharmaceutically acceptable salt thereof; (b) a neurotransmitter re-uptake blocker or a pharmaceutically acceptable salt thereof; (c) a pharmaceutically acceptable carrier; wherein the active ingredients (a) and (b) above are present in amounts that render the composition effective in treating depression, mood disorders, and cognitive disorders.
[0126] In the general formula I according to the present invention, when a radical is mono- or poly-substituted, said substituent(s) can be located at any desired position(s), unless otherwise stated. Also, when a radical is polysubstituted, said substituents can be identical or different, unless otherwise stated.
[0127] The histamine-3 (H3) receptor antagonists of the invention are useful for treating, in particular, ADD, ADHD, obesity, anxiety disorders and respiratory diseases. Respiratory diseases that may be treated by the present invention include adult respiratory distress syndrome, acute respiratory distress syndrome, bronchitis, chronic bronchitis, chronic obstructive pulmonary disease, cystic fibrosis, asthma, emphysema, rhinitis and chronic sinusitis.
[0128] The pharmaceutical composition and method of this invention may also be used for preventing a relapse in a disorder or condition described in the previous paragraphs. Preventing such relapse is accomplished by administering to a mammal in need of such prevention a compound of formula I as described above.
[0129] The disclosed compounds may also be used as part of a combination therapy, including their administration as separate entities or combined in a single delivery system, which employs an effective dose of a histamine H3 antagonist compound of general formula I and an effective dose of a histamine H1 antagonist, such as cetirizine (Zyrtec™), chlorpheniramine (Chlortrimeton™), loratidine (Claritin™), fexofenadine (Allegra™), or desloratadine (Clarinex™) for the treatment of allergic rhinitis, nasal congestion, and allergic congestion.
[0130] The disclosed compounds may also be used as part of a combination therapy, including their administration as separate entities or combined in a single delivery system, which employs an effective dose of a histamine H3 antagonist compound of general formula I and an effective dose of a neurotransmitter reuptake blocker. Examples of neurotransmitter reuptake blockers will include the serotonin-selective reuptake inhibitors (SSRI's) like sertraline (Zoloft™), fluoxetine (Prozac™), and paroxetine (Paxil™), or non-selective serotonin, dopamine or norepinephrine reuptake inhibitors for treating ADD, ADHD, depression, mood disorders, or cognitive disorders.
[0131] The compounds of the present invention may have optical centers and therefore may occur in different enantiomeric configurations. Formula I, as depicted above, includes all enantiomers, diastereomers, and other stereoisomers of the compounds depicted in structural formula I, as well as racemic and other mixtures thereof. Individual isomers can be obtained by known methods, such as optical resolution, optically selective reaction, or chromatographic separation in the preparation of the final product or its intermediate.
[0132] The present invention also includes isotopically labeled compounds, which are identical to those recited in formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 15 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl, 123 I respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i., 2 H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances.
[0133] Substitution with positron emitting isotopes, such as 11 C, 18 F, 15 O and 13 N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
[0134] Anxiety disorders include, for example, generalized anxiety disorder, panic disorder, PTSD, and social anxiety disorder. Mood adjustment disorders include, for example, depressed mood, mixed anxiety and depressed mood, disturbance of conduct, and mixed disturbance of conduct and depressed mood. Attention adjustment disorders include, for example, in addition to ADHD, attention-deficit disorders or other cognitive disorders due to general medical conditions. Psychotic disorders include, for example, schizoaffective disorders and schizophrenia; sleep disorders include, for example, narcolepsy and enuresis.
[0135] Examples of the disorders or conditions which may be treated by the compound, composition and method of this invention are also as follows: depression, including, for example, depression in cancer patients, depression in Parkinson's patients, post-myocardial infarction depression, depression in patients with human immunodeficiency virus (HIV), Subsyndromal Symptomatic depression, depression in infertile women, pediatric depression, major depression, single episode depression, recurrent depression, child abuse induced depression, post partum depression, DSM-IV major depression, treatment-refractory major depression, severe depression, psychotic depression, post-stroke depression, neuropathic pain, manic depressive illness, including manic depressive illness with mixed episodes and manic depressive illness with depressive episodes, seasonal affective disorder, bipolar depression BP I, bipolar depression BP II, or major depression with dysthymia; dysthymia; phobias, including, for example, agoraphobia, social phobia or simple phobias; eating disorders, including, for example, anorexia nervosa or bulimia nervosa; chemical dependencies, including, for example, addictions to alcohol, cocaine, amphetamine and other psychostimulants, morphine, heroin and other opioid agonists, phenobarbital and other barbiturates, nicotine, diazepam, benzodiazepines and other psychoactive substances; Parkinson's diseases, including, for example, dementia in Parkinson's disease, neuroleptic-induced parkinsonism or tardive dyskinesias; headache, including, for example, headache associated with vascular disorders; withdrawal syndrome; age-associated learning and mental disorders; apathy; bipolar disorder; chronic fatigue syndrome; chronic or acute stress; conduct disorder; cyclothymic disorder; somatoform disorders such as somatization disorder, conversion disorder, pain disorder, hypochondriasis, body dysmorphic disorder, undifferentiated disorder, and somatoform NOS; incontinence; inhalation disorders; intoxication disorders; mania; oppositional defiant disorder; peripheral neuropathy; post-traumatic stress disorder; late luteal phase dysphoric disorder; specific developmental disorders; SSRI “poop out” syndrome, or a patient's failure to maintain a satisfactory response to SSRI therapy after an initial period of satisfactory response; and tic disorders including Tourette's disease.
[0136] As an example, the mammal in need of the treatment or prevention may be a human. As another example, the mammal in need of the treatment or prevention may be a mammal other than a human.
[0137] Pharmaceutically acceptable salts of the compounds of formula I include the acid addition and base salts thereof.
[0138] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include the acetate, aspantate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts.
[0139] Suitable base salts are formed from bases that form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
[0140] Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
[0141] For a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
[0142] The compounds of the invention may exist in both unsolvated and solvated forms. The term ‘solvate’ is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water.
[0143] Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D 2 O, d 6 -acetone, d 6 -DMSO.
[0144] Included within the scope of the invention are complexes such as clathrates, drug-host inclusion complexes wherein, in contrast to the aforementioned solvates, the drug and host are present in stoichiometric or non-stoichiometric amounts. Also included are complexes of the drug containing two or more organic and/or inorganic components, which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of such complexes, see J Pharm Sci, 64 (8), 1269-1288 by Haleblian (August 1975).
[0145] Hereinafter all references to compounds of formula I include references to salts, solvates and complexes thereof and to solvates and complexes of salts thereof.
[0146] The compounds of the invention include compounds of formula I as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) as hereinafter defined and isotopically-labeled compounds of formula I.
[0147] As indicated, so-called ‘pro-drugs’ of the compounds of formula I are also within the scope of the invention. Thus certain derivatives of compounds of formula I which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula I having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as ‘prodrugs’. Further information on the use of prodrugs may be found in ‘Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and ‘Bioreversible Carriers in Drug Design’, Pergamon Press, 1987 (ed. E. B Roche, American Pharmaceutical Association).
[0148] Compounds of formula I containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism (‘tautomerism’) can occur. This can take the form of proton tautomerism in compounds of formula I containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds that contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.
[0149] Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of formula I, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counterion is optically active, for example, d-lactate or l-lysine, or racemic, for example, dl-tartrate or dl-arginine.
[0150] Unless otherwise indicated, the term “halo”, as used herein includes fluoro, chloro, bromo and iodo.
[0151] Unless otherwise indicated, the term ‘alkyl’, as used herein includes includes saturated monovalent hydrocarbon radicals having straight or branched moieties. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, and t-butyl.
[0152] Unless otherwise indicated, the term “alkoxy”, as used herein, includes straight-chain and branched alkoxy groups and includes for example methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, secbutoxy and t-butoxy.
[0153] Unless otherwise indicated, the term “alkylene”, as used herein, includes a divalent radical derived from straight-chain or branched alkane. Examples of alkylene radicals are methylene, ethylene (1,2-ethylene or 1,1-ethylene), trimethylene (1,3-propylene), tetramethylene (1,4-butylene), pentamethylene and hexamethylene.
[0154] Unless otherwise indicated, the term “cycloalkyl”, as used herein, unless otherwise indicated, includes non-aromatic saturated cyclic alkyl moieties wherein alkyl is as defined above. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
[0155] Unless otherwise indicated, the term “heterocycloalkyl”, as used herein, refer to non-aromatic cyclic groups containing one or more heteroatoms, preferably from one to four heteroatoms, each preferably selected from oxygen, sulfur and nitrogen. The heterocycloalkyl groups of this invention can also include ring systems substituted with one or more oxo moieties. Examples of non-aromatic heterocycloalkyl groups are aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepinyl, piperazinyl, 1,2,3,6-tetrahydropyridinyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholino, thiomorpholino, thioxanyl, pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, quinolizinyl, quinuclidinyl, 1,4-dioxaspiro[4.5]decyl, 1,4-dioxaspiro[4.4]nonyl, 1,4-dioxaspiro[4.3]octyl, and 1,4-dioxaspiro[4.2]heptyl.
[0156] Unless otherwise indicated, the term “aryl”, as used herein, includes and organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl, napthyl, indenyl, and fluoroenyl. ‘Aryl’ encompasses fused ring groups wherein at least one ring is aromatic.
[0157] Unless otherwise indicated, the term “heteroaryl” as used herein, includes monocyclic or bicyclic heteroaryl groups having 5 to 9 and 9 to 14 ring members respectively, which contain 1, 2, 3 or 4 heteroatom(s) selected from nitrogen, oxygen and sulphur. The heteroaryl group can be unsubstituted, monosubstituted or disubstituted. Examples of heteroaryl groups include, but are not limited to thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyranyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiadiazinyl, isobenzofuranyl, benzoturanyl, chromenyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, quinolinyl, isoquinolyl, cinnolinyl, phthalazinyl, naphthyridinyl, quinazolinyl, quinoxalinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, pyrrolopyrazinyl, pyrrolopyridinyl, and imidazopyridinyl.
[0158] Unless otherwise indicated, the term “heterocyclic ring”, as used herein, refers to both heteroaryl and heterocycloalkyl groups, as defined above.
DETAILED DESCRIPTION OF THE INVENTION
[0159] The compounds of the Formula I may be prepared by the methods described below, together with synthetic methods known in the art of organic chemistry, or modifications and derivatisations that are familiar to those of ordinary skill in the art. Preferred methods include, but are not limited to, those described below.
[0160] During any of the following synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by means of conventional protecting groups, such as those described in T. W. Greene, Protective Groups in Organic Chemistry, John Wiley & Sons, 1981; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Chemistry, John Wiley & Sons, 1991, which are hereby incorporated by reference.
[0161] Compounds of formula I, or their pharmaceutically acceptable salts, can be prepared according to the following reaction Schemes I through II as discussed herein below. Unless otherwise indicated X, Q, Y, Z and R 1 through R 5 are defined as above. Isolation and purification of the products is accomplished by standard procedures, which are known to a chemist of ordinary skill.
[0162] The following schemes are exemplary of the processes for making compounds of formula I.
[0163] Scheme I illustrates a method for the preparation of compounds having the basic structure of formula I, where R 1 , R 2 , R 3 , Y, Q, Z and X are defined as above.
[0164] Referring to Scheme I, a compound of formula (II) can be prepared by treatment of a bromo-tetralone compound of formula (I) with an appropriately substituted amine reagent of formula (II) and a suitable reducing agent such as NaHB(OAc) 3 in a solvent such as CH 2 Cl 2 or DCE, at temperatures ranging from −5° C. to room temperature, preferably at about room temperature, to produce the desired amine of formula (III). Other suitable reducing agents for this reaction include NaCNBH3 or NaBH 4 , in solvents such as MeOH or EfOH. Other suitable conditions for this transformation include treatment of the corresponding tetralone of formula (I) with the amine reagent (II) in CH 2 Cl 2 or DCE in the presence of 4 A molecular sieves and a base such as TEA at room temperature, followed by treatment with NaBH 4 or NaHB(OAc) 3 . Compounds of formula (III) can then be treated with an appropriately substituted boronic acid of formula (IV), in the presence of a suitable palladium catalyst such as 1,1-bis(diphenylphosphino)ferrocene palladium (II) chloride and a suitable aqueous solution of an alkali base such as sodium carbonate and in solvents such as dimethoxy ethane, at temperatures ranging from room temperature to about 100° C., preferably at about 90° C., to produce the desired compound of formula (V). Other suitable conditions for this transformation include treatment of the compound of formula (III) and the appropriately substituted boronic acid of formula (IV) with tetrakis(triphenylphosphine)palladium(0) and sodium carbonate in ethanol/water mixture at temperatures ranging from 30° C. to 110° C., preferably at about the reflux temperature, to produce the corresponding compound of formula (V).
[0000]
[0165] Scheme II illustrates an alternative method for the preparation of compounds having the basic structure of formula i, where R 5 is CONR 4 R 5 and R 1 , R 2 , Y, Z, Q and X are defined as above. Coupling of the bromide (III) and a suitable boronic acid reagent of formula (VI) can be carried out as described above in scheme I to produce the desired compound of formula (VII). Treatment of the corresponding t-butyl ester derivative of formula (VII) with trifluoroacetic acid in methylene chloride at room temperature produces the corresponding carboxylic acid (not depicted). Treatment of the carboxylic acid with an amine of formula (VIII), in the presence of a suitable coupling reagent such as HOBT and EDCl, and a tertiary amine such as triethyl amine, can produce the desired compounds of formula (IX).
[0000]
[0166] Alternatively, compounds of formula (IX) can also be prepared by treatment of the carboxylic acid and suitable amine of formula (VIII) with 2-chloro-1,3-dimethyl imidazoline chloride and a suitable base such as diisopropylethyl amine, in solvents such as methylene chloride.
[0167] The following examples and preparations illustrate the present invention. It is to be understood, however, that the invention, as fully described herein and as recited in the claims, TO is not intended to be limited by the details of the following examples.
Preparation 1
1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine
[0168] To a solution of 10.0 g (43.84 mmol) 6-bromo-3,4-dihydronaphthalen-2-(1H)-one in 550 mL of methylene chloride in a round-bottomed flask was added dropwise 5.5 mL pyrrolidine (65.76 mmol) at room temperature. The solution became dark purple in color. After cooling the solution to 0° C., sodium triacetoxy borohydride (20.0 g, 87.68 mmol) was added in small portions. The reaction mixture was allowed to warm to room temperature and let stir overnight (15 hours). The reaction was then quenched with water (300 mL). Saturated sodium bicarbonate was added (200 mL) bringing the pH to 7. Solid sodium bicarbonate was added until the reaction became basic (pH 9). The organic layer was separated and washed with saturated sodium bicarbonate, water, then brine, and was dried (MgSO 4 ), filtered, and concentrated.
[0169] The crude product was then dissolved in ethyl acetate (200 mL). HCl in ethyl acetate was added and the reaction was stirred for a few minutes and then filtered to collect solid that crashed out. The solid product was washed with 50:50 hexanes/ethyl acetate and dried in vacuo to give 13.7 g, a 98.4% yield of 1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine. 400 MHz 1 H NMR (CD 3 OD) δ 7.3 (s, 1H), 7.3 (d, J=8.29 Hz, 1H), 7.1 (d, J=8.29 Hz, 1H), 3.7 (m, 2H), 3.6 (m, 1H), 3.3 (m, 3H), 2.9-3.0 (m, 3H), 2.4 (m, 1H), 2.2 (m, 2H), 2.1 (m, 2H), 1.9 (m, 1H). MS (M+1) 280.3, 282.3. TLC (Silica Gel GF); R f =0.50 in methylene chloride-methanol (4:1).
Preparation 2
Tert-butyl 3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzoate
[0170] To a solution of 1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine (9.0 g, 28.417 mmol) in dimethoxy ethane (165 mL) was added 3-t-butoxycarbonyl phenyl boronic acid (9.465 g, 42.6255 mmol). 2 M sodium carbonate solution (71 mL) and 1,1-bis(diphenylphosphino)ferrocene palladium (II) chloride (0.23 g, 0.248) were then added to the solution. The reaction was warmed to 90° C. and refluxed for 6 hours. LCMS and TLC analysis showed no starting material. The reaction was cooled to room temperature and concentrated. The reaction was diluted with ethyl acetate, washed with water ×3, brine, and was dried (MgSO 4 ), filtered, and concentrated in vacuo to give a crude yield of 13.45 g. The resulting solid was purified by flash column chromatography on 330 g silica gel, eluting with methylene chloride/methanol/NH 4 OH (10:1:0.1). The pure fractions were collected and concentrated to yield 12.23 g Tert-butyl 3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzoate. 400 MHz 1 H NMR (CDCl 3 ) δ 8.2 (m, 1H), 7.9 (m, 1H), 7.7 (m, 1H), 7.4-7.5 (m, 1H), 7.3-7.4 (m, 2H) 7.1 (m, 1H), 2.7-3.1 (m, 6H), 2.5 (brs, 1H), 2.2 (m, 1H), 1.8 (m, 3H), 1.7-1.8 (m, 1H), 1.6 (s, 9H), 1.5 (m, 3H). MS (M+1) 378.3, 379.2.
Preparation 3
3-(6-(Pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzoic Acid
[0171] Crude tert-butyl 3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzoate was diluted in methylene chloride Trifluoroacetic acid was added. The reaction stirred over night, LCMS showed no starting material, The reaction was concentrated to give a quantitative yield (14.0 g) of the desired TFA salt 3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzoic acid. 400 MHz 1 H NMR (CD 3 OD) δ 8.2 (s, 1H), 8.0 (d, J=7.88 Hz, 1H), 7.8 (m, 1H), 7.5 (m, 1H), 7.4-7.5 (m, 2H), 7.3 (d, J=7.8 Hz, 1H), 3.8 (m, 2H), 3.6 (m, 1H), 3.3-3.4 (m, 1H), 3.2-3.3 (m, 2H), 3.0-3.1 (m, 3H), 2.4 (m, 1H), 2.2 (m, 2H), 2.0-2.1 (m, 2H), 1.9-2.0 (m, 1H). MS (M+1) 322.2, 323.2.
[0172] Other examples prepared according to the described procedure in preparation 3.
4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzoic acid
[0173] 400 MHz 1 H NMR (CD 3 OD) δ 8.1 (d, J=8.3 Hz, 2H), 7.7 (d, J=8.7 Hz, 2H), 7.6 (m, 2H), 7.3 (d, J=8.7 Hz, 1H), 3.8 (m, 2H), 3.6 (m, 1H), 3.2-3.4 (m, 2H), 3.0-3.2 (m, 4H), 2.4 (m, 1H), 2.2 (m, 2H), 1.9-2.1 (m, 3H).
General Procedure for Boronic Acid Coupling Reactions
[0174] To a solution of the bromo-amino-tetraline intermediate of formula (III) (1 equiv) in dimethoxy ethane (0.18 M) was added the boronic acid (1.5 equiv). 2 M sodium carbonate solution (5 equiv) and 1,1-bis(diphenylphosphino)ferrocene palladium (II) chloride (0.01 equiv) were then added to the solution. The reaction was warmed to 90° C. and refluxed for S6 hours. Reaction monitored by LCMS and TLC analysis. The reaction was cooled to room temperature and concentrated. The reaction was diluted with ethyl acetate, washed with water (×3), brine, and was dried (MgSO 4 ), filtered, and concentrated in vacuo to give the crude product. The resulting solid was purified by flash column chromatography eluting with methylene chloride/methanol/NH 4 OH (10:1:0.1). The pure fractions were collected and concentrated to yield the desired product.
Example 1
N,N-dimethyl-3-(6-(pyrrolidin-1-Vi)-5,6,7,8-tetrahydronaphthalen-2-yl)benzamide
[0175] To a solution of 1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine (10.0 g, 31.575 mmol) in dimethoxy ethane (180 mL) was added the boronic acid (9.14, 47.36 mmol). 2 M sodium carbonate solution (78 mL, 157.88 mmol) and Pd(dppf)Cl 2 (0.316 g, 0.3157 mmol) were then added to the solution. The reaction was warmed to 100° C. and refluxed overnight (20 hours). LCMS and TLC analysis showed no starting material. The reaction was cooled to room temperature and concentrated. The reaction was diluted with ethyl acetate, washed with water (×3), brine, and was dried (MgSO 4 ), filtered, and concentrated in vacuo. The resulting solid was purified by flash column chromatography on 330 g silica gel, eluting with methylene chloride/methanol/NH 4 OH (10:1:0.1). Fractions 30-65 gave 7.72 g pure N,N-dimethyl-3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzamide, a 70.3% yield. 400 MHz 1 H NMR (CDCl 3 ) δ 7.5-7.6 (m, 2H), 7.4 (m, 1H), 7.3 (m, 3H), 7.1 (d, J=7.9 Hz, 1H), 3.1 (s, 3H), 2.7-3.0 (m, 11H), 2.6 (m, 1H), 2.2 (m, 1H), 1.8-1.9 (m, 3H), 1.7-1.8 (m, 2H). MS (M+1) 349.3.
Example 2
N-Ethyl-N-methyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0176] To a solution of 3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzoic acid (prepared above, 1.5 g, 3.44 mmol) in 27.5 mL methylene chloride was added N-ethylmethyl amine (0.355 mL, 4.13 mmol) followed by HOBT (0.512 g, 3.79 mmol), EDCl (0.86 g, 4.478 mmol), and triethyl amine (2.4 mL, 17.224 mmol). The reaction stirred overnight at room temperature. The reaction was quenched with water and extracted (×3) with methylene chloride, dried (MgSO 4 ), filtered, and concentrated. The resulting solid was purified by flash column chromatography on 220 g silica gel, eluting with methylene chloride/methanol/NH 4 OH (10:1:0.1). The pure fractions were collected and concentrated to give 775 mg N,N-dimethyl-3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzamide, a 62% yield. 400 MHz 1 H NMR (CDCl 3 ) δ 7.6 (m, 2H), 7.4 (m, 1H), 7.3 (m, 3H), 7.1 (d, J=7.8 Hz, 1H), 3.6 (m, 1H), 3.3 (m, 1H), 3.0-3.1 (s, 3H), 2.7-3.0 (m, 9H), 2.5 μm, 1H), 2.2 (m, 1H), 1.8-1.9 (m, 4H), 1.6-1.7 (m, 1H), 1.1-1.2 (m, 2H). MS (M+1) 363.3, 364.3. TLC (Silica Gel GF): R f =0.25 in methylene chloride/methanol/NH 4 OH (10:1:0.1).
Alternative Amide Coupling Conditions for Preparation of N,N-dimethyl-3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzamide
[0177] To a solution of 3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzoic acid
[0178] (prepared above, 11.35 g, 26.06 mmol) in 130 mL of methylene chloride at a C (due to large scale of reaction) was added diisopropyl ethyl amine (22 mL, 130.3 mmol), followed by dimethyl amine HCL (3.19 g, 39.1 mmol). 2-chloro-1,3-dimethyl imidazoline chloride (4.4 g) was added to the reaction in portions, using methylene chloride to transfer the hydroscopic reagent. The reaction was removed from the ice bath and stirred at room temperature over night. (18 h). LCMS showed no starting material. The reaction was concentrated, then diluted with ethyl acetate and water. The organic layer was washed (3×) H 2 O and brine, dried (MgSO 4 ), filtered, and concentrated to give 3.9 g crude N,N-dimethyl-3-(6-(pyrrolidin-1-yl)-5,6,7,8-tetrahydronaphthalen-2-yl)benzamide, 400 MHz 1 H NMR (CDCl 3 ) δ 7.5-7.6 (m, 2H), 7.4 (m, 1H), 7.3 (m, 3H), 7.1 (d, J=7.9 Hz, 1H), 3.1 (s, 3H), 2.7-3.0 (m, 11H), 2.6 (m, 1H), 2.2 (m, 1H), 1.8-1.9 (m, 3H), 1.7-1.8 (m, 2H); 349.3.
General Salt Formation:
[0179] The free base was dissolved in ethyl acetate. Saturated HCl in ethyl acetate was added to the solution and allowed to stir for five minutes, then concentrated in vacuo giving the resulting HCL salt.
[0180] The following examples were prepared utilizing the procedures and examples described above.
Example 3
3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0181] 400 MHz 1 H NMR (CD 3 OD) δ 8.1 (t, J=1.7 Hz, 1H), 7.8 μm, 2H), 7.5 (d, J=7.9 Hz, 1H), 7.4 (m, 2H), 7.2-7.3 (d, J=7.9 Hz, 1H), 3.7-3.8 (m, 2H), 3.6 (m, 1H), 3.3-3.4 (m, 1H), 3.2-3.3 (m, 2H), 3.0-3.1 (m, 3H), 2.4 (m, 1H), 2.2 (m, 2H), 1.9-2.1 (m, 3H).
Example 4
N-Isopropyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0182] 400 MHz 1 H NMR (CD 3 OD) δ 7.9 (t, J=1.7 Hz, 1H), 7.6-7.7 (m, 2H), 7.4-7.5 (t, J=7.6 Hz, 1H), 7.3-7.4 (m, 2H), 7.1 (d, J=7.9 Hz, 1H), 5.9-6.0 (m, 1H), 4.2-4.3 (m, 1H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 3H), 2.7-2.8 (m, 4H), 2.5 (brs, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.7 (m, 1H), 1.2-1.3 (d, J=6.6 Hz, 6H).
Example 5
Azetidin-1-yl-[4-(6-pyrrolidin-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenyl]-methanone
[0183] 400 MHz 1 H NMR (CD 3 OD) δ 7.8 (t, J=1.7 Hz, 1H), 7.6 (m, 1H), 7.5 (m, 1H), 7.4 (t, J=7.6 Hz, 1H), 7.3 (m, 2H), 7.1 (d, J=7.9 Hz, 1H), 4.3 (t, J=7.6 Hz, 2H), 4.2 (t, J=7.6 Hz, 2H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.4-2.5 (m, 1H), 2.3-2.4 (m, 2H), 2.2-2.3 (m, 1H), 1.8-1.9 (m, 4H), 1.7 (m, 1H).
Example 6
N-Cyclobutyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0184] 400 MHz 1 H NMR (CD 3 OD) δ 7.9 (t, J=1.7 Hz, 1H), 7.6-7.7 (m, 2H), 7.4-7.5 (t, J=7.4 Hz, 1H), 7.3 (m, 2H), 7.1-7.2 (d, J=7.5 Hz, 1H), 6.3 (d, J=7.5 Hz, 1H), 4.6 (m, 1H), 2.8-3.1 (m, 4H), 2.7 (m, 4H), 2.4-2.5 (m, 3H), 2.2 (m, 1H), 1.7-2.0 (m, 8H).
Example 7
N-Isobutyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0185] 400 MHz 1 H NMR (CD 3 OD) δ 7.9 (t, J=1.7 Hz, 1H), 7.6-7.7 (m, 2H), 7.4-7.5 (t, J=7.9 Hz, 1H), 7.3-7.4 (m, 2H), 7.1-7.2 (d, J=7.5 Hz, 1H), 6.1 (m, 1H), 3.3 (m, 2H), 3.0-3.1 (dd, J=16.2, 3.7 Hz, 1H), 2.8-3.0 (m, 7H), 2.6 (brs, 1H), 2.2-2.3 (m, 1H), 1.8-1.9 (m, 6H), 1.2-1.3 (d, J=6.6 Hz, 6H).
Example 8
4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine
[0186] 400 MHz H NMR (CDCl 3 ) δ 8.6 (m, 2H), 7.4-7.5 (m, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 2.8-3.1 (m, 4H), 2.7 (m, 4H), 2.4-2.5 (m, 1H), 2.2 (m, 1H), 1.7-1.9 (m, 5H).
Example 9
N-(2-Methoxy-ethyl)-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0187] 400 MHz 1 H NMR (CDCl 3 ) 8.0 (t, J=1.7 Hz, 1H), 7.6-7.7 (m, 2H), 7.4-7.5 (t, J=7.6 Hz, 1H), 7.3-7.4 (m, 2H), 7.1-7.2 (d, J=7.9 Hz, 1H), 6.6 (brs, 1H), 3.6-3.7 (m, 2H), 3.5-3.6 (m, 2H), 3.4 (s, 3H), 2.8-3.2 (m, 4H), 2.7-2.8 (m, 4H), 2.5 (m, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.6-1.8 (m, 1H).
Example 10
(3-Fluoro-azetidin-1-yl)-[3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenyl]-methanone
[0188] 400 MHz 1 H NMR (CDCl 3 ) δ 7.8 (t, J=1.4 Hz, 1H), 7.6-7.7 (m, 1H), 7.5 (m, 1H), 7.4-7.5 (t, J=7.7 Hz, 1H), 7.3 (m, 2H), 7.1-7.2 (d, J=7.9 Hz, 1H), 5.2-5.4 (m, 1H), 4.2-4.6 (m, 4H), 3.1 (m, 1H), 2.9-3.0 (m, 3H), 2.7-2.9 (m, 4H), 2.5 (m, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.7-1.8 (m, 1H).
Example 11
N-(2-Hydroxy-ethyl)-N-methyl-3-(6-pyrrolidin-1-VI-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0189] 400 MHz 1 H NMR (CDCl 3 ) δ 7.6 (m, 2H), 7.3-7.5 (m, 4H), 7.1-7.2 (d, J=7.9 Hz, 1H), 3.9 (brs, 1H), 3.7 (m, 2H), 3.3-3.5 (m, 1H), 2.7-3.1 (m, 10H), 2.5 (brs, 1H), 2.2 (m, 1H), 1.6-1.8 (m, 7H).
Example 12
N-Cyclopropyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0190] 400 MHz 1 H NMR (CDCl 3 ) δ 7.9 (t, J=1.7 Hz, 1H), 7.6-7.7 (m, 2H), 7.4-7.5 (t, J=7.7 Hz, 1H), 7.3 (m, 2H), 7.1-7.2 (d, J=7.9 Hz, 1H), 6.3 (brs, 1H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 4H), 2.7-2.8 (m, 4H), 2.5 μm, 1H), 2.2-2.3 (m, 1H), 1.8-1.9 (m, 4H), 1.7-1.8 (m, 1H), 0.8-1.9 (m, 2H), 0.6-0.7 (m, 2H).
Example 13
3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yloxy)-benzamide
[0191] 400 MHz 1 H NMR (CDCl 3 ) δ 7.4-7.5 (m, 1H), 7.3-7.4 (m, 2H), 7.1 (m, 1H), 7.0 (d, J=8.3 Hz, 1H), 6.7-6.8 (2H), 6.1 (brs, 1H), 5.8 (brs, 1H), 2.7-3.0 (m, 8H), 2.4-2.5 (m, 1H), 2.1-2.2 (m, 1H), 1.8 (m, 4H), 1.6-1.7 (m, 1H).
Example 14
5-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-oxazole
[0192] 400 MHz 1 H NMR (CDCl 3 ) δ 7.9 (s, 1H), 7.4 (d, J=7.9 Hz, 2H), 7.3 (s, 1H), 7.0-7.1 (m, 1H), 2.7-3.1 (m, 4H), 2.6-2.7 (m, 4H), 2.4 (m, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.6-1.7 (m, 1H).
Example 15
1-[6-(4-Methanesulfonyl-phenoxy)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0193] 400 MHz 1 H NMR (CDCl 3 ) δ 7.8-7.9 (m, 2H), 7.1 (d, J=8.2 Hz, 1H), 7.0-7.1 (m, 2H), 6.7-6.8 (m, 2H), 3.0 (s, 3H), 2.7-3.0 (m, 8H), 2.4-2.5 (m, 1H), 2.2 (m, 1H), 1.8-1:9 (m, 4H), 1.6-1.7 (m, 1H).
Example 16
N-(2-Hydroxy-ethyl)-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0194] 400 MHz 1 H NMR (CDCl 3 ) δ 7.6 (m, 2H), 7.5 (m, 2H), 7.3 (m, 2H), 7.1-7.2 (d, J=7.9 Hz, 1H), 3.9 (brs, 1H), 3.7 (m, 2H), 3.4-3.5 (m, 1H), 3.0-3.1 (m, 3H), 2.8-3.0 (m, 8H), 2.6 (brs, 1H), 2.2-2.3 (m, 1H), 1.8-1.9 (m, 6H).
Example 17
4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-N-(tetrahydro-pyran-4-yl)-benzamide
[0195] 400 MHz 1 H NMR (CDCl 3 ) 7.8 (m, 2H), 7.6 (m, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 6.0 (d, J=7.5 Hz, 1H), 4.2 (m, 1H), 4.0 (m, 2H), 3.5-3.6 (m, 2H), 3.03 (dd, J=16.6, 4.2 Hz, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.5 (m, 1H), 2.2 (m, 1H), 20 (dd, J=12.4, 2.5 Hz, 2H), 1.5-1.8 (m, 7H).
Example 18
N-(2-Methoxy-ethyl)-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0196] 400 MHz 1 H NMR (CDCl 3 ) δ 7.8 (d, J=8.3 Hz, 2H), 7.6 (d, J=8.3 Hz, 2H), 7.4 (d, J=7.9 Hz, 1H), 7.3 (s, 1H), 7.1-7.2 (d, J=7.9 Hz, 1H), 6.6 (m, 1H), 3.6-3.7 (m, 2H), 3.5-3.6 (m, 2H), 3.4 (s, 3H), 2.9-3.4 (m, 8H), 2.4 (m, 1H), 2.1-2.2 (m, 5H), 1.8-1.9 (m, 1H).
Example 19
N-Isobutyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0197] 400 MHz 1 H NMR (CDCl 3 ) δ 7.8 (m, 2H), 7.6 (m, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 6.6 (t, J=5.8 Hz, 1H), 3.3 (t, J=7.9 Hz, 2H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.4-2.5 (m, 1H), 2.1-2.3 (m, 1H), 1.6-1.9 (m, 6H), 1.0 (d, J=6.6 Hz, 6H).
Example 20
1-[6-(4-Methoxy-phenoxy)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0198] 400 MHz 1 H NMR (CDCl 3 ) S 7.0 (m, 3H), 6.8-6.9 (m, 2H), 6.7 (m, 1H), 6.6 (m, 1H), 3.8 (s, 3H), 3.0 (m, 1H), 2.7-2.8 (m, 7H), 2.4-2.5 (m, 1H), 2.1-2.2 (m, 1H), 1.8-1.9 (m, 4H), 1.6-1.7 (m, 1H).
Example 21
N,N-dimethyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0199] 400 MHz 1 H NMR (CDCl 3 ) δ 7.6 (m, 2H), 7.4-7.5 (m, 2H), 7.31 (m, 2H), 7.1-7.2 (d, J=7.5 Hz, 1H), 2.7-3.1 (m, 12H), 2.4 (m, 1H), 2.2 (m, 1H), 1.6-1.8 (m, 7H).
Example 22
Azetidin-1-yl-[4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenyl]-methanone
[0200] 400 MHz 1 H NMR (CDCl 3 ) δ 7.6-7.7 (m, 2H), 7.6 (m, 2H), 7.3 (m, 2H), 7.1-7.2 (d, J=7.9 Hz 1H), 4.3-4.4 (d, J=7.7 Hz, 2H), 4.2 (d, J=7.7 Hz, 2H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 4H), 2.7 (m, 4H), 2.4-2.5 (m, 1H), 2.2-2.4 (m, 2H), 2.2 (m, 1H), 1.7-1.8 (m, 5H).
Example 23
N-Ethyl-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0201] 400 MHz 1 H NMR (CDCl 3 ) δ 7.5-7.6 (m, 2H), 7.4 μm, 2H), 7.3 (m, 2H), 7.1-7.2 (d, J=7.9 Hz, 1H), 3.6 (m, 1H), 3.3 (m, 1H), 2.6-3.1 (m, 10H), 2.4 (m, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.6-1.7 (m, 2H), 1.1-1.3 (m, 3H).
Example 24
(+)-N-Ethyl-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0202] 400 MHz 1 H NMR (CD 3 OD) δ 7.7 (d, J=7.9 Hz, 1H), 7.6 (d, J=7.5 Hz, 1H), 7.5 (t, J=7.8 Hz, 1H), 7.4 (m, 2H), 7.3 (m, 1H), 7.2-7.3 (d, J=7.9 Hz, 1H), 3.7-3.8 (m, 2H), 3.6 (m, 2H), 3.2-3.3 (m, 3H), 3.0-3.1 (m, 6H), 2.4 (m, 1H), 2.2 (m, 2H), 1.9-2.0 (m, 3H), 1.2-1.3 (t, J=7.1 Hz, 1H), 1.1-1.2 (m, 3H). MS (M+1) 363.4. [_]=(+) 34.32. Chiralcel OJ, Mobile Phase 9515 Heptane/EtOH, T R =15.162 min.
Example 24
(−)-N-Ethyl-N-methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0203] 400 MHz 1 H NMR (CD 3 OD) δ 7.7 (d, J=7.9 Hz, 1H), 7.6 (d, J=7.5 Hz, 1H), 7.5 (t, J=7.8 Hz, 1H), 7.4 (m, 2H), 7.3 (m, 1H), 7.2-7.3 (d, J=7.9 Hz, 1H), 3.7-3.8 (m, 2H), 3.6 (m, 2H), 3.2-3.3 (m, 3H), 3.0-3.1 (m, 6H), 2.4 (m, 1H), 2.2 (m, 2H), 1.9-2.0 (m, 3H), 1.2-1.3 (m, 2H), 1.1-1.2 (m, 2H). MS (M+1) 363.4. [_]=(−) 31.56. Chiralcel OJ, Mobile Phase 95/5 Heptane/EtOH, T R =18.131 min.
Example 25
2-Methoxy-5-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine
[0204] 400 MHz 1 H NMR (CDCl 3 ) δ 8.3 (dd, J=2.5, 0.8 Hz, 1H), 7.7-7.8 (dd, J=8.3, 2.5 Hz, 1H), 7.2-7.3 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 6.8 (m, 1H), 4.0 (s, 3H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.5 (m, 1H), 2.2-2.3 (m, 1H), 1.8-1.9 (m, 4H), 1.7-1.8 (m, 1H).
Example 26
3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine
[0205] 400 MHz 1 H NMR (CDCl 3 ) δ 8.8 (d, J=1.7 Hz, 1H), 8.5 (d, J=4.5 Hz, 1H), 7.8 (m, 1H), 7.3 (m, 3H), 7.2 (d, J=7.9 Hz, 1H), 3.0-3.1 (m, 1H), 2.7-3.0 (m, 7H), 2.5 (m, 1H), 2.2-2.3 (m, 1H), 1.7-1.9 (m, 5H).
Example 27
2-Methoxy-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine
[0206] 400 MHz 1 H NMR (CDCl 3 ) δ 8.1 (m, 1H), 7.5-7.6 (m, 1H), 7.3 (m, 1H), 7.2 (d, J=1.7 Hz, 1H), 7.1 (d, J=7.9 Hz, 1H), 7.0 (m, 1H), 4.0 (s, 3H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.5 (m, 1H), 2.2 (m, 1H), 1.8-1.9 (m, 4H), 1.7-1.8 (m, 1H).
Example 28
6-Methoxy-2-methyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pyridine
[0207] 400 MHz 1 H NMR (CDCl 3 ) δ 7.4 (d, J=8.3 Hz, 1H), 7.1 (d, J=7.7 Hz, 1H), 7.0 (m, 2H), 6.6 (d, J=7.9 Hz, 1H), 3.9 (s, 3H), 3.1 (m, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.5 (m, 1H), 2.4 (s, 3H), 2.2 (m, 1H), 1.8-1.9 (m, 4H), 1.7 (m, 1H).
Example 29
N-Isopropyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0208] 400 MHz 1 H NMR (CDCl 3 ) δ 7.8 (d, J=8.3 Hz, 2H), 7.6 (d, J=8.3 Hz, 2H), 7.3 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 6.0 (d, J=7.5 Hz, 1H), 4.3 (m, 1H), 3.1 (dd, J=15.8, 4.1 Hz, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.5 (m, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.6-1.8 (m, 1H), 1.3 (d, J=6.6 Hz, 6H).
Example 30
N-Cyclobutyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0209] 400 MHz 1 H NMR (CDCl 3 ) δ 7.8 (m, 2H), 7.6 (m, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.5 Hz, 1H), 6.2 (d, J=7.9 Hz, 1H), 4.6 (m, 1H), 3.0-3.1 (m, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.4-2.5 (m, 2H), 2.2 (m, 1H), 1.6-2.0 (m, 10H).
Example 31
4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenol
[0210] 400 MHz 1 H NMR (DMSO) δ 7.4 (m, 2H), 7.2 (m, 2H), (7.0 (m, 1H), 6.8 (m, 2H), 2.5-2.9 (m, 8H), 2.3 brs, 1H), 2.0 (brs, 1H), 1.5-1.6 (m, 5H).
Example 32
1-[6-(4-Methoxy-2,6-dimethyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0211] 400 MHz 1 H NMR (CD 3 OD) δ 7.2 (d, J=7.5 Hz, 1H), 6.9 (m, 2H), 6.6 (s, 2H), 3.7-3.8 (m, 5H), 3.6 (m, 1H), 3.4 (m, 1H), 3.0-3.1 (m, 4H), 2.4 (m, 1H), 2.2 (m, 2H), 2.1 (m, 2H), 1.9-2.0 (m, 8H). MS (M+1) 336.4.
Example 33
1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0212] 400 MHz 1 H NMR (CDCl 3 ) δ 8.0 (d, J=8.3 Hz, 2H), 7.7 (d, J=8.3 Hz, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 3.1-3.2 (m, 1H), 3.1 (s, 3H), 2.8-3.0 (m, 3H), 2.8 (m, 4H), 2.5 (m, 1H), 2.2-2.3 (m, 1H), 1.7-1.9 (m, 5H). MS-(M+1) 356.3.
Example 34
(R)-1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0213] 400 MHz 1 H NMR (CDCl 3 ) δ 8.0 (m, 2H), 7.7 (m, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 3.1-3.2 (m, 1H), 3.1 (s, 3H), 2.8-3.0 (m, 3H), 2.8 (m, 4H), 2.6 (m, 1H), 2.2-2.3 (m, 1H), 1.7-1.9 (m, 5H); MS (M+1) 356.2. Chiralcel OJ, Mobile Phase 30170 Heptane/EtOH, T R =9.431 min.
Example 35
(S)-1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0214] 400 MHz 1 H NMR (CD 3 OD) δ 8.0 (d, J=8.3 Hz, 1H), 7.8-7.9 (d, J=8.3 Hz, 1H), 7.5 (t, J=7.1 Hz, 1H), 7.3 (m, 3H), 7.1 (d, J=8.3 Hz, 1H), 3.3-3.8 (m, 5H), 2.9-3.2 (m, 7H), 2.4 (m, 1H), 1.8-2.2 (m, 5H); MS (M+1) 356.4. Chiralcel OJ, Mobile Phase 30170 Heptane/EtOH, T R =13.911 min.
Example 36
3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0215] 400 MHz 1 H NMR (CDCl 3 ) δ 8.1 (s, 1H), 7.7-7.8 (m, 2H), 7.5 (m, 1H), 7.4 (m, 2H), 7.2 (d, J=8-3 Hz, 1H), 3.1 (m, 1H), 2.7-2.9 (m, 7H), 2.5 (m, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.6-1.7 (m, 1H); MS (M+1) 321.3, 322.4.
Example 37
(S)-3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0216] Chiralpak AS, Mobile Phase 80/20 Heptane/EtOH T R =14.006 min. MS (M+1) 321.4.
Example 38
(R)-3-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0217] Chiralpak AS, Mobile Phase 80/20 Heptane/EtOH T R =9.253 min, MS (M+1) 321.4.
Example 39
1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0218] 400 MHz 1 H NMR (CDCl 3 ) δ 7.5 (m, 2H), 7.2-7.3 (m, 2H), 7.1 (d, J=7.9 Hz, 1H), 6.9-7.0 (m, 2H), 3.8 (s, 3H), 3.0-3.1 (dd, J=16.2, 3.7 Hz, 1H), 2.8-3.0 (m, 3H), 2.7 (m, 4H), 2.4 (m, 1H), 2.2 (m, 1H), 1.8 (m, 4H), 1.6-1.7 (m, 1H). MS (M+1) 308.2.
Example 40
(R)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0219] Chiralpak AS, Mobile Phase 80/20 Heptane/EtOH T R =10.248 min. MS (M+1) 308.3.
Example 41
(S)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0220] Chiralpak AS, Mobile Phase 80/20 Heptane/EtOH T R =12.360 min. MS (M+1) 308.2.
Example 42
1-Isopropyl-4-[6-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-piperazine
[0221] 400 MHz 1 H NMR (CDCl 3 ) δ 7.5 (m, 2H), 7.3 (d, J=7.9 Hz, 2H), 7.1 (d, J=7.9 Hz, 1H), 7.0 (m, 2H), 3.8 (s, 3H), 2.6-3.0 (m, 13H), 2.2 (m, 1H); 1.6 (m, 2H), 1.0 (d, J=6.2 Hz 6H). MS (M+1) 365.3.
Example 43
(S)-(−)-1-[6-(4-Chloro-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0222] 400 MHz 1 H NMR (CD 3 OD) δ 7.6 (d, J=8.3 Hz, 2H), 7.4 (d, J=8.7 Hz, 4H), 7.2 (d, J=7.5 Hz, 1H), 3.8 (m, 2H), 3.6 (m, 1H), 3.2-3.3 (m, 3H), 3.0-3.1 (m, 4H), 2.4 (m, 1H), 2.2 (m, 2H), 2.0 (m, 2H); MS (M+1) 312.3, 313.4. [_]=(−) 41.39. Chiralpak OJ, Mobile Phase 90110 Heptane/IPO, T R =6.488 min.
Example 44
(R)-(+)-1-[6-(4-Chloro-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0223] 400 MHz 1 H NMR (CD 3 OD) δ 7.6 (m, 2H), 7.3-7.4 (m, 4H), 7.2 (d, J=7.9 Hz, 1H), 3.8 (m, 2H), 3.6 (m, 1H), 3.2-3.3 (m, 3H), 3.0-3.1 (m, 4H), 2.4 (m, 1H), 2.2 (m, 2H), 2.0 (m, 2H): MS (M+1) 312.3, 313.4, [_]=(−) 39.58. Chiralpak OJ, Mobile Phase 90110 Heptane/IPO, T R =5.573 min.
Example 45
3-Fluoro-1-[6-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0224] 400 MHz 1 H NMR (CD 3 OD) δ 7.5 (d, J=8.7 Hz, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 7.0 (d, J=8.7 Hz, 2H), 5.4-5.6 (m, 1H), 3.9-4.1 (m, 2H), 3.5-3.7 (m, 3H), 3.3 (m, 4H), 3.0-3.1 (m, 3H), 2.3-2.6 (m, 3H), 1.9-2.0 (m, 1H); MS (M+1) 326.3.
Example 46
1-[4-(6-Pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-phenyl]-ethanone
[0225] 400 MHz 1 H NMR (CD 3 OD) δ 8.0-8.1 (d, J=8.7 Hz, 2H), 7.7 (d, J=8.3 Hz, 2H), 7.5 (d, J=8.3 Hz, 2H), 7.3 (J=7.5 Hz, 1H), 3.6 (m, 2H), 3.4-3.5 (dd, J=14.1, 7.1 Hz, 1H), 3.0-3.3 (m, 6H), 2.4-2.6 (m, 4H), 1.9-2.1 (m, 4H), 2.0 (m, 1H). MS (M+1) 320.4.
Example 47
3,4-Difluoro-1-[6-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-pyrrolidine
[0226] 400 MHz 1 H NMR (CD 3 OD) δ 7.5 (m, 2H), 7.4 (d, J=7.9 Hz, 1H), 7.3 (s, 1H), 7.2 (d, J=7.9 Hz, 1H), 7.0 (m, 2H), 5.6 (m, 1H), 5.5 (m, 1H), 3.8 (s, 3H), 3.7 (m, 1H), 3.2-3.3 (m, 6H), 3.0-3.1 (m, 2H), 2.4 (m, 1H), 2.0 (m, 1H). MS (M+1) 344.3.
Example 48
1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine
[0227] 400 MHz 1 H NMR (CD 3 OD) δ 7.5 (d, J=8.7 Hz, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 7.0 (d, J=8.7 Hz, 2H), 3.8-3.9 (m, 1H), 3.7 (s, 3H), 3.6 (m, 1H), 3.3-3.4 (m, 3H), 3.0-3.1 (m, 3H), 2.3-2.4 (m, 2H), 2.0-2.1 (m, 2H), 1.8-2.0 (m, 2H), 1.5 (m, 3H). MS (M+1) 322.4.
Example 49
(R,R)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine
[0228] 400 MHz 1 H NMR (CD 3 OD) δ 7.5 (d, J=8.7 Hz, 2H), 7.3-7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 7.0 (d, J=8.7 Hz, 2H), 3.9 (m, 1H), 3.8 (s, 3H), 3.7 (m, 1H), 3.6 (m, 1H), 3.2-3.4 (m, 2H), 3.0-3.1 (m, 3H), 2.4 (m, 2H), 2.0-2.1 (m, 2H), 1.8-2.0 (m, 2H), 1.5 (dd, J=6.6, 2.1 Hz, 3H). MS (M+1) 322.4. Chiralcel OJ, Mobile Phase 85115, Heptane/EtOH, T R =13.213 min.
Example 50
(S,R)-1-[6-(4-Methoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine
[0229] Chiralcel OJ, Mobile Phase 85/15 Heptane/EtOH, T R =13.659 min, MS (M+1) 322.4
Example 51
(R)-1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine
[0230] Chiralcel OD, Mobile Phase 80120 Heptane/IPA, T R =9.378 min.
Example 52
(S)-1-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)pyrrolidine
[0231] Chiralcel OD, Mobile Phase 80120 Heptane/IPA, T R =14.325 min.
Example 53
(R,R)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine
[0232] Chiralcel OD, Mobile Phase 85/15 Heptane/IPO, T R =19.591 min.
Example 54
[0233] (S,R)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine
[0234] 400 MHz 1 H NMR (CDCl 3 ) δ 7.2 (m, 2H), 6.9 (d, J=7.9 Hz, 1H), 2.6-3.0 (m, 8H), 1.5-2.1 (m 5H), 1.4 (m, 1H), 1.0-1.1 (m, 3H). GCMS 293.0. Chiralcel OD, Mobile Phase 85/15 Heptane/IPO, T R =24.109 min.
Example 55
(R,S)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine
[0235] Chiralcel OD, Mobile Phase 85/15 Heptane/IPO, T R =18.050 min.
Example 56
(S,S)-1-(6-Bromo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-methyl-pyrrolidine
[0236] 400 MHz 1 H NMR (CDCl 3 ) δ 7.2 (m, 2H), 6.9 (d, J=7.9 Hz, 1H), 2.6-3.0 (m, 8H), 1.5-2.1 (m 5H), 1.4 (m, 1H), 1.0-1.1 (m, 3H). GCMS 293.0. Chiralcel OD, Mobile Phase 85/15 Heptane/IPO, T R =20.109 min.
Example 57
(R)-N,N-Dimethyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0237] Chiralcel OJ, Mobile Phase 90/10 Heptane/EtOH, T R =11.814 min. MS (M+1) 349.3.
Example 58
(R)-N,N-Dimethyl-3-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide
[0238] Chiralcel OJ, Mobile Phase 90/10 Heptane/EtOH, T R =13.677 min. MS (M+1) 349.3
Example 59
(S,R)-3-[6-(2-Methyl-Pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-pyridine
[0239] 400 MHz 1 H NMR (CD 3 OD) δ 9.0 (d, J=1.2 Hz, 1H), 8.6-8.7 (m, 2H), 7.9 (m, 1H), 7.6 (m, 2H), 7.4 (d, J=8.3 Hz, 1H), 3.9 (m, 1H), 3.8 (m, 1H), 3.6 (m, 1H), 3.4 (m, 1H), 3.0-3.2 (m, 4H), 2.3-2.4 (m, 2H), 1.8-2.1 (m, 4H), 1.5 (d, J=6.6 Hz, 3H). MS (M+1) 293.4. Chiralpak AD, Mobile Phase 85/15 Heptane/EtOH, T R =8.512 min.
Example 60
(R,R)-4-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-pyridine
[0240] 400 MHz 1 H NMR (CD 3 OD) δ 9.0 (d, J=1.2 Hz, 1H), 8.6-8.7 (m, 2H), 7.9 (m, 1H), 7.6 (m, 2H), 7.4 (d, J=8.3 Hz, 1H), 3.9 (m, 1H), 3.8 (m, 1H), 3.6 (m, 1H), 3.4 (m, 1H), 3.0-3.2 (m, 4H), 2.3-2.4 (m, 2H), 1.8-2.1 (m, 4H), 1.5 (d, J=6.6 Hz, 3H). MS (M+1) 293.4. Chiralpak AD, Mobile Phase 85/15 Heptane/EtOH, T R =6.445 min.
Example 61
1-[6-(3,4-Dimethoxy-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine
[0241] 400 MHz 1 H NMR (CD 3 OD) δ 7.4 (m, 2H), 7.2 (d, J=7.9 Hz, 1H), 7.1 (m, 2H), 7.0 (d, J=8.7 Hz, 1H), 3.9 (s, 3H), 3.8 (s, 3H), 3.3-3.8 (m, 4H), 3.0 (m, 4H), 2.4 (m, 2H), 2.1 (m, 2H), 1.8-2.0 (m, 2H), 1.5 (m, 3H). MS (M+1) 352.1.
Example 62
1-[6-(3-Fluoro-4-methoxy-phenyl)-1,2,3,4-tetrahydronaphthalen-2-yl]-2-methyl-pyrrolidine
[0242] 400 MHz 1 H NMR (CD 3 OD) δ 7.3-7.4 (m, 3H), 7.2 (d, 7.9 Hz, 1H), 7.1 (m, 2H), 3.9 (s, 3H), 3.8 (m, 1H), 3.6 (m, 1H), 3.2-3.4 (m, 2H), 3.0-3.1 (m, 4H), 2.3-2.4 (m, 2H), 1.8-2.2 (m, 4H), 1.5 (m, 3H). MS (M+1) 340.1.
Example 63
N-Methyl-4-(6-pyrrolidin-1-yl-5,6,7,8-tetrahydroaphthalen-2-yl)-benzamide
[0243] 400 MHz 1 H NMR (CD 3 OD) δ 7.8-7.9 (d, J=8.7 Hz, 2H), 7.7 id, J=8.7 Hz, 2H), 7.5 (m, 2H), 7.2-7.3 (d, J=7.9 Hz, 1H), 3.8 (m, 2H), 3.6 (m, 1H), 3.3-3.4 (dd, J=16.2, 4.2 Hz, 1H), 3.2-3.3 (m, 2H), 3.0-3.1 (m, 3H), 2.9 (s, 3H), 2.4 (m, 1H), 2.2 (m, 2H), 1.9-2.2 (m, 3H). MS (M+1) 335.4.
Example 64
4-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide
[0244] 400 MHz 7H NMR (CD 3 OD) δ 8.0 (d, J=8.3 Hz, 2H), 7.7 (d, J=8.3 Hz, 2H), 7.4 (m, 2H), 7.2 (d, J=7.5 Hz, 1H), 2.8-3.5 (m, 8H), 2.3 (m, 1H), 2.2 (m, 1H), 2.0 (m, 2H), 1.8 (m, 1H), 1.6 (m, 1H), 1.3 (brs, 3H). MS (M+1) 335.4.
Example 65
3-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide
[0245] 400 MHz 1 H NMR (CD 3 OD) δ 8.1-8.2 (m, 1H), 7.8-7.9 (m, 2H), 7.4-7.6 (m, 3H), 7.2-7.3 (m, 1H), 3.9 (m, 1H), 3.8 (m, 1H), 3.6 (m, 1H), 3.2-3.4 (m, 1H), 3.0-3.2 (m, 4H), 2.3-2.4 (m, 2H), 1.8-2.2 (m, 4H), 1.5 (dd, J=6.6, 2.9 Hz, 3H). MS (M+1) 335.4.
Example 66
(R,R)-3-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide
[0246] Chiralpak AD, Mobile Phase 85/15 Heptane/EtOH, T R =10.934 min. MS (M+1) 335.4.
Example 67
(S,R)-3-[6-(2-Methyl-pyrrolidin-1-yl)-5,6,7,8-tetrahydro-naphthalen-2-yl]-benzamide
[0247] Chiralpak AD, Mobile Phase 85/15 Heptane/EtOH, T R =14.219 min. MS (M+1) 335.4.
Example 68
1-[6-(4-Methanesulfonyl-phenyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-2-methyl-pyrrolidine
[0248] 400 MHz 1 H NMR (CD 3 OD) δ 8.0 (d, J=8.3 Hz, 2H), 7.8-7.9 (d, J=8.7 Hz, 2H), 7.5 (m, 2H), 7.3 (d, J=7.9 Hz, 1H), 3.7-3.9 (m, 2H), 3.6 (m, 1H), 3.4 (m, 1H), 3.0-3.2 (m, 7H), 2.3-2.4 (m, 2H), 1.8-2.1 (m, 4H), 1.5 (m, 3H). MS (M+1) 369.3. GCMS 369.0.
[0249] The following tables of examples were also prepared according to the procedures and examples described above.
[0000]
Experimental
MS
Ex-
Observed
MW
ample
IUPAC
(M + 1)
calc.
69
1-[6-(2,5-Dimethyl-phenyl)-1,2,3,4-
306.137
305.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
70
2-(6-Pyrrolidin-1-yl-5,6,7,8-
306.139
305.18
tetrahydro-naphthalen-2-yl)-
benzaldehyde
71
1-[6-(2-Phenoxy-phenyl)-1,2,3,4-
370.124
369.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
72
1-[6-(4-Fluoro-3-methyl-phenyl)-
310.115
309.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
73
1-[6-(4-Methoxy-3,5-dimethyl-
336.176
335.22
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
74
1-[6-(3-Fluoro-4-methoxy-phenyl)-
326.137
325.18
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
75
1-[6-(2,3,4-Trimethoxy-phenyl)-
368.131
367.21
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
76
1-(6-Phenoxathiin-4-yl-1,2,3,4-
400.12
399.17
tetrahydro-naphthalen-2-yl)-
pyrrolidine
77
[3-(6-Pyrrolidin-1-yl-5,6,7,8-
308.134
307.19
tetrahydro-naphthalen-2-yl)-phenyl]-
methanol
78
3-(6-Pyrrolidin-1-yl-5,6,7,8-
303.104
302.18
tetrahydro-naphthalen-2-yl)-
benzonitrile
79
1-(6-Phenyl-1,2,3,4-tetrahydro-
278.107
277.18
naphthalen-2-yl)-pyrrolidine
80
N,N-Diisopropyl-2-(6-pyrrolidin-1-yl-
405.177
404.28
5,6,7,8-tetrahydro-naphthalen-2-yl)-
benzamide
81
1-[6-(4-Methylsulfanyl-phenyl)-
324.1
323.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
82
1-(6-m-Tolyl-1,2,3,4-tetrahydro-
292.137
291.2
naphthalen-2-yl)-pyrrolidine
83
1-[6-(3-Chloro-4-fluoro-phenyl)-
330.069
329.13
1,2,3,4-tetrahydo-naphthalen-2-yl]-
pyrrolidine
84
1-[6-(3-Nitro-phenyl)-1,2,3,4-
323.087
322.17
tetrahydro-naphthalen-2-yl]-
pyrrolidine
85
1-[6-(3,5-Dimethyl-phenyl)-1,2,3,4-
306.141
305.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
86
1-[6-(3-Fluoro-phenyl)-1,2,3,4-
296.109
295.17
tetrahydro-naphthalen-2-yl]-
pyrrolidine
87
1-[6-(3,4-Dichloro-phenyl)-1,2,3,4-
346.063
345.11
tetrahydro-naphthalen-2-yl]-
pyrrolidine
88
1-[6-(2-Methoxy-phenyl)-1,2,3,4-
308.12
307.19
tetrahydro-naphthalen-2-yl]-
pyrrolidine
89
1-[6-(3-Ethoxy-phenyl)-1,2,3,4-
322.136
321.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
90
1-[6-(3,4-Difluoro-phenyl)-1,2,3,4-
314.106
313.16
tetrahydro-naphthalen-2-yl]-
pyrrolidine
91
1-[6-(4-Fluoro-phenyl)-1,2,3,4-
296.095
295.17
tetrahydro-naphthalen-2-yl]-
pyrrolidine
92
1-[6-(4-Trifluoromethoxy-phenyl)-
362.117
361.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
93
1-(6-Benzo[1,3]dioxol-5-yl-1,2,3,4-
322.121
321.17
tetrahydro-naphthalen-2-yl)-
pyrrolidine
94
1-(5,6,7,8-Tetrahydro-
328.128
327.2
[2,2′]binaphthalenyl-6-yl)-pyrrolidine
95
1-[6-(2-Chloro-phenyl)-1,2,3,4-
312.078
311.14
tetrahydro-naphthalen-2-yl]-
pyrrolidine
96
1-[6-(4-Ethyl-phenyl)-1,2,3,4-
306.162
305.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
97
1-[6-(2-Ethoxy-phenyl)-1,2,3,4-
322.148
321.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
98
1-[4-(6-Pyrrolidin-1-yl-5,6,7,8-
INPAT
319.19
tetrahydro-naphthalen-2-yl)-phenyl]-
ethanone
99
1-[4-(6-Pyrrolidin-1-yl-5,6,7,8-
320.143
319.19
tetrahydro-naphthalen-2-yl)-phenyl]-
ethanone
100
1-[6-(2,6-Dimethyl-phenyl)-1,2,3,4-
306.161
305.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
101
1-[6-(4-Ethoxy-phenyl)-1,2,3,4-
322.137
321.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
102
1-[3-(6-Pyrrolidin-1-yl-5,6,7,8-
320.148
319.19
tetrahydro-naphthalen-2-yl)-phenyl]-
ethanone
103
1-[6-(2-Methoxy-5-methyl-phenyl)-
322.151
321.21
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
104
Dimethyl-[4-(6-pyrrolidin-1-yl-5,6,7,8-
161.077
320.23
tetrahydro-naphthalen-2-yl)-phenyl]-
amine
105
[4-(6-Pyrrolidin-1-yl-5,6,7,8-
308.141
307.19
tetrahydro-naphthalen-2-yl)-phenyl]-
methanol
106
1-[6-(2-Fluoro-3-methoxy-phenyl)-
326.1
325.18
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
107
[2-(6-Pyrrolidin-1-yl-5,6,7,8-
308.135
307.19
tetrahydro-naphthalen-2-yl)-phenyl]-
methanol
108
1-(4-Methyl-5′,6′,7′,8′-tetrahydro-
342.129
341.21
[1,2′]binaphthalenyl-6′-yl)-pyrrolidine
109
N-[3-(6-Pyrrolidin-1-yl-5,6,7,8-
335.142
334.2
tetrahydro-naphthalen-2-yl)-phenyl]-
acetamide
110
3-(6-Pyrrolidin-1-yl-5,6,7,8-
350.162
349.2
tetrahydro-naphthalen-2-yl)-benzoic
acid ethyl ester
111
1-[6-(2-Benzyloxy-phenyl)-1,2,3,4-
384.129
383.22
tetrahydro-naphthalen-2-yl]-
pyrrolidine
112
1-[6-(3-Methylsulfanyl-phenyl)-
324.088
323.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
113
1-[6-(4-Methoxy-3-methyl-phenyl)-
322.122
321.21
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
114
4-(6-Pyrrolidin-1-yl-5,6,7,8-
303.11
302.18
tetrahydro-naphthalen-2-yl)-
benzonitrile
115
1-[6-(3,4-Dimethyl-phenyl)-1,2,3,4-
306.132
305.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
116
1-(6-Biphenyl-2-yl-1,2,3,4-tetrahydro-
354.123
353.21
naphthalen-2-yl)-pyrrolidine
117
1-[6-(4-Benzyloxy-phenyl)-1,2,3,4-
384.132
383.22
tetrahydro-naphthalen-2-yl]-
pyrrolidine
118
1-[6-(2-Fluoro-biphenyl-4-yl)-1,2,3,4-
372.114
371.2
tetrahydro-naphthalen-2-yl]-
pyrrolidine
119
1-[6-(3,4,5-Trimethoxy-phenyl)-
368.122
367.21
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
120
1-[6-(3-Benzyloxy-phenyl)-1,2,3,4-
384.134
383.22
tetrahydro-naphthalen-2-yl]-
pyrrolidine
121
1-[6-(4-Fluoro-2-methyl-phenyl)-
310.103
309.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
122
1-[6-(2-Ethyl-phenyl)-1,2,3,4-
306.167
305.21
tetrahydro-naphthalen-2-yl]-
pyrrolidine
123
4-(6-Pyrrolidin-1-yl-5,6,7,8-
294.101
293.18
tetrahydro-naphthalen-2-yl)-phenol
124
1-[6-(2-Methylsulfanyl-phenyl)-
324.101
323.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
125
1-[6-(4-Benzyloxy-2-fluoro-phenyl)-
402.13
401.22
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
126
1-[6-(4-Isopropoxy-phenyl)-1,2,3,4-
336.162
335.22
tetrahydro-naphthalen-2-yl]-
pyrrolidine
127
3,5-Dimethyl-1-[3-(6-pyrrolidin-1-yl-
186.585
371.24
5,6,7,8-tetrahydro-naphthalen-2-yl)-
phenyl]-1H-pyrazole
128
1-(5′,6′,7′,8′-Tetrahydro-
328.112
327.2
[1,2′]binaphthalenyl-6′-yl)-pyrrolidine
129
1-[6-(2,3-Dihydro-benzo[1,4]dioxin-6-
336.102
335.19
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-pyrrolidine
130
1-{6-[4-(1-Methoxy-ethyl)-phenyl]-
336.166
335.22
1,2,3,4-tetrahydro-naphthalen-2-yl}-
pyrrolidine
131
1-[6-(3-Chloro-phenyl)-1,2,3,4-
312.062
311.14
tetrahydro-naphthalen-2-yl]-
pyrrolidine
132
1-[6-(2,3-Dihydro-benzofuran-5-yl)-
320.137
319.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
133
1-[3-(6-Pyrrolidin-1-yl-5,6,7,8-
344.113
343.2
tetrahydro-naphthalen-2-yl)-phenyl]-
1H-pyrazole
134
1-[6-(3,4-Dihydro-2H-
350.111
349.2
benzo[b][1,4]dioxepin-7-yl)-1,2,3,4-
tetrahydro-naphthalen-2-yl]-
pyrrolidine
135
1-[6-(3-Methoxy-phenyl)-1,2,3,4-
308.146
307.19
tetrahydro-naphthalen-2-yl]-
pyrrolidine
136
2-Isopropyl-5-(6-pyrrolidin-1-yl-
181.08
360.26
5,6,7,8-tetrahydro-naphthalen-2-yl)-
2,3-dihydro-1H-isoindole
137
1-[6-(4-Chloro-phenyl)-1,2,3,4-
312.1
311.14
tetrahydro-naphthalen-2-yl]-
pyrrolidine
138
1-[6-(5-Chloro-2-fluoro-phenyl)-
330.074
329.13
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
139
1-[6-(5-Chloro-2-methoxy-phenyl)-
356.028
355.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
140
4-Methoxy-3-[6-(2-methyl-pyrrolidin-
350.056
349.2
1-yl)-5,6,7,8-tetrahydro-naphthalen-
2-yl]-benzaldehyde
141
3-[6-(2-Methyl-pyrrolidin-1-yl)-
317.064
316.19
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzonitrile
142
1-(6-Biphenyl-3-yl-1,2,3,4-tetrahydro-
368.078
367.23
naphthalen-2-yl)-2-methyl-pyrrolidine
143
N-tert-Butyl-2-[6-(2-methyl-pyrrolidin-
427.061
426.23
1-yl)-5,6,7,8-tetrahydro-naphthalen-
2-yl]-benzenesulfonamide
144
2-Methyl-1-(6-phenyl-1,2,3,4-
292.071
291.2
tetrahydro-naphthalen-2-yl)-
pyrrolidine
145
1-[6-(2,5-Dimethoxy-phenyl)-1,2,3,4-
352.073
351.22
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
146
2-Methyl-1-(6-thianthren-1-yl-1,2,3,4-
429.99
429.16
tetrahydro-naphthalen-2-yl)-
pyrrolidine
147
2-Methyl-1-(6-p-tolyl-1,2,3,4-
306.079
305.21
tetrahydro-naphthalen-2-yl)-
pyrrolidine
148
2-Methyl-1-[6-(3-nitro-phenyl)-
337.043
336.18
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
149
1-[6-(5-Fluoro-2-methoxy-phenyl)-
340.051
339.2
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
150
N,N-Diisopropyl-2-methoxy-6-[6-(2-
449.133
448.31
methyl-pyrrolidin-1-yl)-5,6,7,8-
tetrahydro-naphthalen-2-yl]-
benzamide
151
1-[6-(2,6-Dimethoxy-phenyl)-1,2,3,4-
352.073
351.22
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
152
N,N-Diisopropyl-2-[6-(2-methyl-
419.122
418.3
pyrrolidin-1-yl)-5,6,7,8-tetrahydro-
naphthalen-2-yl]-benzamide
153
1-[6-(4-Fluoro-phenyl)-1,2,3,4-
310.053
309.19
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
154
1-[6-(3,5-Bis-trifluoromethyl-phenyl)-
427.998
427.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
155
2-Methyl-1-(6-m-tolyl-1,2,3,4-
306.083
305.21
tetrahydro-naphthalen-2-yl)-
pyrrolidine
156
1-{6-[4-(4-Methoxy-phenoxy)-
414.069
413.24
phenyl]-1,2,3,4-tetrahydro-
naphthalen-2-yl}-2-methyl-pyrrolidine
157
1-[6-(3-Chloro-4-fluoro-phenyl)-
344.007
343.15
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
158
2-Methyl-1-[6-(4-trifluoromethyl-
360.041
359.19
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
159
2-Methyl-1-[6-(2,3,4-trimethoxy-
382.069
381.23
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
160
1-[6-(3,5-Dichloro-phenyl)-1,2,3,4-
359.98
359.12
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
161
2-Methyl-1-(6-o-tolyl-1,2,3,4-
306.084
305.21
tetrahydro-naphthalen-2-yl)-
pyrrolidine
162
1-[6-(2-Methoxy-phenyl)-1,2,3,4-
322.084
321.21
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
163
2-Methyl-1-(6-thiophen-3-yl-1,2,3,4-
298.034
297.16
tetrahydro-naphthalen-2-yl)-
pyrrolidine
164
2-Methyl-1-[6-(4-phenoxy-phenyl)-
384.074
383.22
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
165
1-[6-(3,5-Dimethyl-phenyl)-1,2,3,4-
320.099
319.23
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
166
2-Methyl-1-(5,6,7,8-tetrahydro-
342.085
341.21
[2,2′]binaphthalenyl-6-yl)-pyrrolidine
167
1-[6-(2-Ethoxy-phenyl)-1,2,3,4-
336.092
335.22
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
168
2-Methyl-1-[6-(3-trifluoromethyl-
360.042
359.19
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
169
1-[6-(3-Ethoxy-phenyl)-1,2,3,4-
336.096
335.22
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
170
1-[6-(3,4-Difluoro-phenyl)-1,2,3,4-
328.066
327.18
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
171
1-[6-(3-Fluoro-phenyl)-1,2,3,4-
310.066
309.19
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
172
2-Methyl-1-[6-(2-trifluoromethyl-
360.044
359.19
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
173
1-[6-(4-Ethyl-phenyl)-1,2,3,4-
320.103
319.23
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
174
1-[6-(2-Chloro-phenyl)-1,2,3,4-
326.033
325.16
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
175
1-[6-(3,4-Dichloro-phenyl)-1,2,3,4-
360.002
359.12
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
176
1-(6-Benzo[1,3]dioxol-5-yl-1,2,3,4-
336.073
335.19
tetrahydro-naphthalen-2-yl)-2-
methyl-pyrrolidine
177
1-[6-(4-Ethoxy-phenyl)-1,2,3,4-
336.103
335.22
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
178
1-[6-(2-Fluoro-phenyl)-1,2,3,4-
310.064
309.19
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
179
1-{3-[6-(2-Methyl-pyrrolidin-1-yl)-
334.097
333.21
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-ethanone
180
{4-[6-(2-Methyl-pyrrolidin-1-yl)-
322.091
321.21
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-methanol
181
1-[6-(4-tert-Butyl-phenyl)-1,2,3,4-
348.134
347.26
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
182
1-[6-(2,6-Dimethyl-phenyl)-1,2,3,4-
320.108
319.23
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
183
2-Methyl-1-[6-(4-trifluoromethoxy-
376.049
375.18
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
184
1-[6-(2,5-Difluoro-phenyl)-1,2,3,4-
328.065
327.18
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
185
1-[6-(4-Fluoro-3-methyl-phenyl)-
324.1
323.2
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
186
{3-[6-(2-Methyl-pyrrolidin-1-yl)-
322.103
321.21
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-methanol
187
1-[6-(2,4-Difluoro-phenyl)-1,2,3,4-
328.062
327.18
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
188
1-[6-(4-Methoxy-3,5-dimethyl-
350.128
349.24
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-2-methyl-pyrrolidine
189
1-[6-(2,3-Dimethyl-phenyl)-1,2,3,4-
320.12
319.23
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
190
2-Methyl-1-[6-(2-trifluoromethoxy-
376.057
375.18
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
191
2-Methyl-1-[6-(3-trifluoromethoxy-
376.053
375.18
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
192
1-[6-(4-Isobutyl-phenyl)-1,2,3,4-
348.143
347.26
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
193
2-Methyl-1-[6-(2-phenoxy-phenyl)-
384.1
383.22
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
194
1-[6-(3,5-Difluoro-phenyl)-1,2,3,4-
328.067
327.18
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
195
4-[6-(2-Methyl-pyrrolidin-1-yl)-
378.112
377.24
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzoic acid isopropyl ester
196
1-[6-(2,5-Dimethyl-phenyl)-1,2,3,4-
320.12
319.23
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
197
1-{2-[6-(2-Methyl-pyrrolidin-1-yl)-
334.102
333.21
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-ethanone
198
4-[6-(2-Methyl-pyrrolidin-1-yl)-
335.096
334.2
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzamide
199
3-[6-(2-Methyl-pyrrolidin-1-yl)-
308.094
307.19
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenol
200
2-Methyl-1-(4-methyl-5′,6′,7′,8′-
356.136
355.23
tetrahydro-[1,2′]binaphthalenyl-6′-yl)-
pyrrolidine
201
1-[6-(2-Benzyloxy-phenyl)-1,2,3,4-
398.135
397.24
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
202
3-[6-(2-Methyl-pyrrolidin-1-yl)-
293.115
292.19
5,6,7,8-tetrahydro-naphthalen-2-yl]-
pyridine
203
{2-[6-(2-Methyl-pyrrolidin-1-yl)-
322.121
321.21
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-methanol
204
1-[6-(2-Fluoro-3-methoxy-phenyl)-
340.106
339.2
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
205
3-[6-(2-Methyl-pyrrolidin-1-yl)-
364.121
363.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzoic acid ethyl ester
206
1-(6-Furan-3-yl-1,2,3,4-tetrahydro-
282.095
281.18
naphthalen-2-yl)-2-methyl-pyrrolidine
207
2-Methyl-1-[6-(3-methylsulfanyl-
338.086
337.19
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
208
1-[6-(4-Methoxy-3-methyl-phenyl)-
336.132
335.22
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
209
{4-[6-(2-Methyl-pyrrolidin-1-yl)-
441.14
440.25
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-carbamic acid benzyl ester
210
2-Methyl-1-[6-(5-methyl-furan-2-yl)-
293.998
295.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
211
4-[6-(2-Methyl-pyrrolidin-1-yl)-
364.116
363.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzoic acid ethyl ester
212
2-Methoxy-5-[6-(2-methyl-pyrrolidin-
323.118
322.2
1-yl)-5,6,7,8-tetrahydro-naphthalen-
2-yl]-pyridine
213
1-(6-Biphenyl-2-yl-1,2,3,4-tetrahydro-
368.123
367.23
naphthalen-2-yl)-2-methyl-pyrrolidine
214
N-{3-[6-(2-Methyl-pyrrolidin-1-yl)-
349.118
348.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-acetamide
215
1-[6-(2,6-Dichloro-phenyl)-1,2,3,4-
360.026
359.12
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
216
1-(6-Dibenzothiophen-1-yl-1,2,3,4-
398.08
397.19
tetrahydro-naphthalen-2-yl)-2-
methyl-pyrrolidine
217
1-[6-(2-Methoxy-5-methyl-phenyl)-
336.127
335.22
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
218
2-Methyl-1-[6-(3,4,5-trimethoxy-
382.127
381.23
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
219
1-[6-(4-Benzyloxy-3-fluoro-phenyl)-
416.117
415.23
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
220
1-[6-(3,4-Dimethyl-phenyl)-1,2,3,4-
320.135
319.23
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
221
4-[6-(2-Methyl-pyrrolidin-1-yl)-
426.128
425.24
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzoic acid benzyl ester
222
1-[6-(2-Fluoro-biphenyl-4-yl)-1,2,3,4-
386.118
385.22
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
223
4-[6-(2-Methyl-pyrrolidin-1-yl)-
317.105
316.19
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzonitrile
224
1-[6-(4-Isopropyl-phenyl)-1,2,3,4-
334.15
333.25
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
225
1-[6-(2,3-Difluoro-phenyl)-1,2,3,4-
328.089
327.18
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
226
1-[6-(5-Isopropyl-2-methoxy-phenyl)-
364.155
363.26
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
227
2-Methyl-1-[6-(4-pentyl-phenyl)-
362.177
361.28
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine
228
1-[6-(2,3-Dichloro-phenyl)-1,2,3,4-
360.028
359.12
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
229
1-[6-(4-Methanesulfonyl-phenyl)-
370.078
369.18
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
230
4-[6-(2-Methyl-pyrrolidin-1-yl)-
308.107
307.19
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenol
231
2-Methyl-1-[6-(2-methylsulfanyl-
338.091
337.19
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
232
1-[6-(2,4-Bis-trifluoromethyl-phenyl)-
428.059
427.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
233
1-[6-(3-Benzyloxy-phenyl)-1,2,3,4-
398.14
397.24
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
234
1-[6-(4-Fluoro-2-methyl-phenyl)-
324.113
323.2
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
235
3-[6-(2-Methyl-pyrrolidin-1-yl)-
336.1
335.19
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzoic acid
236
1-[6-(2-Ethyl-phenyl)-1,2,3,4-
320.142
319.23
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
237
1-[6-(4-Benzyloxy-phenyl)-1,2,3,4-
398.139
397.24
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
238
1-[6-(4-Benzyloxy-2-fluoro-phenyl)-
416.124
415.23
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
239
1-[6-(4-Butyl-phenyl)-1,2,3,4-
348.172
347.26
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
240
2-Methyl-1-(5′,6′,7′,8′-tetrahydro-
342.124
341.21
[1,2′]binaphthalenyl-6′-yl)-pyrrolidine
241
1-[6-(3-Chloro-phenyl)-1,2,3,4-
326.073
325.16
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
242
1-[6-(4-Ethanesulfonyl-phenyl)-
384.1
383.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
243
1-[6-(2,4-Dichloro-phenyl)-1,2,3,4-
360.034
359.12
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
244
1-{3-[6-(2-Methyl-pyrrolidin-1-yl)-
358.124
357.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
phenyl}-1H-pyrazole
245
3-[6-(2-Methyl-pyrrolidin-1-yl)-
335.112
334.2
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzamide
246
1-(6-Dibenzofuran-4-yl-1,2,3,4-
382.109
381.21
tetrahydro-naphthalen-2-yl)-2-
methyl-pyrrolidine
247
1-[6-(4-Chloro-phenyl)-1,2,3,4-
326.075
325.16
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
248
1-[6-(4-Isopropoxy-phenyl)-1,2,3,4-
350.144
349.24
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
249
3-Chloro-4-[6-(2-methyl-pyrrolidin-1-
327.068
326.15
yl)-5,6,7,8-tetrahydro-naphthalen-2-
yl]-pyridine
250
1-[6-(3-Methoxy-phenyl)-1,2,3,4-
322.115
321.21
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
251
2-Methyl-1-[6-(4-methyl-3-nitro-
351.108
350.2
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-pyrrolidine
252
2-Methyl-1-(6-methyl-1,2,3,4-
230.113
229.18
tetrahydro-naphthalen-2-yl)-
pyrrolidine
253
1-[6-(3,4-Dihydro-2H-
364.115
363.22
benzo[b][1,4]dioxepin-7-yl)-1,2,3,4-
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
254
1-[6-(2,3-Dihydro-benzo[1,4]dioxin-6-
350.105
349.2
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-2-methyl-pyrrolidine
255
3,5-Dimethyl-1-{3-[6-(2-methyl-
193.569
385.25
pyrrolidin-1-yl)-5,6,7,8-tetrahydro-
naphthalen-2-yl]-phenyl}-1H-
pyrazole
256
5-[6-(2-Methyl-pyrrolidin-1-yl)-
294.106
293.19
5,6,7,8-tetrahydro-naphthalen-2-yl]-
pyrimidine
257
1-[6-(4-Methoxy-phenyl)-1,2,3,4-
322.122
321.21
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
258
1-[6-(4-Methoxy-2,6-dimethyl-
350.134
349.24
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-2-methyl-pyrrolidine
259
1-[6-(2,3-Dihydro-benzofuran-5-yl)-
334.111
333.21
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
260
1-[6-(4-Ethylsulfanyl-phenyl)-1,2,3,4-
352.101
351.2
tetrahydro-naphthalen-2-yl]-2-
methyl-pyrrolidine
261
1-{6-[4-(1-Methoxy-ethyl)-phenyl]-
350.136
349.24
1,2,3,4-tetrahydro-naphthalen-2-yl}-
2-methyl-pyrrolidine
262
1-[6-(2-Chloro-5-fluoro-phenyl)-
344.043
343.15
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
263
1-Benzenesulfonyl-3-[6-(2-methyl-
471.077
470.2
pyrrolidin-1-yl)-5,6,7,8-tetrahydro-
naphthalen-2-yl]-1H-indole
264
1-[6-(5-Chloro-2-fluoro-phenyl)-
344.049
343.15
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
265
2-Isopropyl-5-[6-(2-methyl-pyrrolidin-
188.087
374.27
1-yl)-5,6,7,8-tetrahydro-naphthalen-
2-yl]-2,3-dihydro-1H-isoindole
266
2-[Ethyl-(6-pyridin-3-yl-1,2,3,4-
296.189
296.19
tetrahydro-naphthalen-2-yl)-amino]-
ethanol
267
[1-(6-Pyridin-3-yl-1,2,3,4-tetrahydro-
322.205
322.2
naphthalen-2-yl)-piperidin-3-yl]-
methanol
268
3-(6-Azetidin-1-yl-5,6,7,8-tetrahydro-
264.163
264.16
naphthalen-2-yl)-pyridine
269
4-(6-Azetidin-1-yl-5,6,7,8-tetrahydro-
307
306.17
naphthalen-2-yl)-benzamide
270
1-[6-(6-Methoxy-2-methyl-pyridin-3-
394.67
393.24
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-pyrrolidine-2-carboxylic acid
dimethylamide
271
1-[6-(4-Methanesulfonyl-phenyl)-
427.62
426.2
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine-2-carboxylic acid
dimethylamide
272
1-[6-(4-Methanesulfonyl-phenyl)-
342.59
341.14
1,2,3,4-tetrahydro-naphthalen-2-yl]-
azetidine
273
1-[6-(4-Ethanesulfonyl-phenyl)-
441.64
440.21
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine-2-carboxylic acid
dimethylamide
274
1-[6-(4-Methanesulfonyl-phenyl)-
400.59
399.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methoxymethyl-pyrrolidine
275
1-[6-(4-Ethanesulfonyl-phenyl)-
414.65
413.2
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methoxymethyl-pyrrolidine
276
1-[6-(4-Ethanesulfonyl-phenyl)-
356.58
355.16
1,2,3,4-tetrahydro-naphthalen-2-yl]-
azetidine
277
1-[6-(6-Methoxy-pyridin-3-yl)-1,2,3,4-
380.67
379.23
tetrahydro-naphthalen-2-yl]-
pyrrolidine-2-carboxylic acid
dimethylamide
278
1-[6-(3-Methanesulfonyl-phenyl)-
400.2
399.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methoxymethyl-pyrrolidine
279
2-Methoxy-5-[6-(2-methoxymethyl-
353.68
352.22
pyrrolidin-1-yl)-5,6,7,8-tetrahydro-
naphthalen-2-yl]-pyridine
280
3-[6-(2-Methoxymethyl-pyrrolidin-1-
393.67
392.25
yl)-5,6,7,8-tetrahydro-naphthalen-2-
yl]-N,N-dimethyl-benzamide
281
1-[6-(3-Methanesulfonyl-phenyl)-
342.2
341.14
1,2,3,4-tetrahydro-naphthalen-2-yl]-
azetidine
282
4-[6-(2-Methoxymethyl-pyrrolidin-1-
393.67
392.25
yl)-5,6,7,8-tetrahydro-naphthalen-2-
yl]-N,N-dimethyl-benzamide
283
3-[6-(2-Methoxymethyl-pyrrolidin-1-
365.62
364.22
yl)-5,6,7,8-tetrahydro-naphthalen-2-
yl]-benzamide
284
4-[6-(2-Methoxymethyl-pyrrolidin-1-
379.67
378.23
yl)-5,6,7,8-tetrahydro-naphthalen-2-
yl]-N-methyl-benzamide
285
1-[6-(4-Dimethylcarbamoyl-phenyl)-
420.3
419.26
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidine-2-carboxylic acid
dimethylamide
286
3-[6-(2-Phenyl-pyrrolidin-1-yl)-
397.62
396.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzamide
287
3-(6-Azetidin-1-yl-5,6,7,8-tetrahydro-
307.57
306.17
naphthalen-2-yl)-benzamide
288
1-Isopropyl-4-[6-(6-methoxy-2-
380.71
379.26
methyl-pyridin-3-yl)-1,2,3,4-
tetrahydro-naphthalen-2-yl]-
piperazine
289
1-[6-(4-Methanesulfonyl-phenyl)-
370.61
369.18
1,2,3,4-tetrahydro-naphthalen-2-yl]-
piperidine
290
1-Isopropyl-4-[6-(4-methanesulfonyl-
413.66
412.22
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-piperazine
291
1-[6-(6-Methoxy-2-methyl-pyridin-3-
353.52
352.22
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-piperidin-3-ol
292
1-[6-(4-Ethanesulfonyl-phenyl)-
427.68
426.23
1,2,3,4-tetrahydro-naphthalen-2-yl]-
4-isopropyl-piperazine
293
1-[6-(4-Methanesulfonyl-phenyl)-
386.65
385.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
piperidin-3-ol
294
1-[6-(4-Ethanesulfonyl-phenyl)-
384.63
383.19
1,2,34-tetrahydro-naphthalen-2-yl]-
piperidine
295
4-[6-(6-Methoxy-2-methyl-pyridin-3-
355.59
354.18
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-thiomorpholine
296
[6-(4-Ethanesulfonyl-phenyl)-1,2,3,4-
372.62
371.19
tetrahydro-naphthalen-2-yl]-diethyl-
amine
297
1-[6-(4-Ethanesulfonyl-phenyl)-
400.60
399.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
piperidin-3-ol
298
6-Methoxy-2-methyl-3-(6-piperidin-1-
337.64
336.22
yl-5,6,7,8-tetrahydro-naphthalen-2-
yl)-pyridine
299
Diethyl-[6-(4-methanesulfonyl-
358.62
357.18
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-amine
300
4-[6-(4-Methanesulfonyl-phenyl)-
386.61
385.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
3-methyl-morpholine
301
Diethyl-(6-pyrimidin-5-yl-1,2,3,4-
282.54
281.19
tetrahydro-naphthalen-2-yl)-amine
302
4-[6-(4-Ethanesulfonyl-phenyl)-
402.57
401.15
1,2,3,4-tetrahydro-naphthalen-2-yl]-
thiomorpholine
303
4-[6-(6-Methoxy-2-methyl-pyridin-3-
353.64
352.22
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-3-methyl-morpholine
304
4-[6-(4-Ethanesulfonyl-phenyl)-
400.65
399.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
3-methyl-morpholine
305
1-Isopropyl-4-[6-(6-methoxy-pyridin-
366.74
365.25
3-yl)-1,2,3,4-tetrahydro-naphthalen-
2-yl]-piperazine
306
4-[6-(6-Methoxy-pyridin-3-yl)-1,2,3,4-
341.60
340.16
tetrahydro-naphthalen-2-yl]-
thiomorpholine
307
5-[6-(4-Isopropyl-piperazin-1-yl)-
337.64
336.23
5,6,7,8-tetrahydro-naphthalen-2-yl]-
pyrimidine
308
4-[6-(4-Isopropyl-piperazin-1-yl)-
378.73
377.25
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzamide
309
1-(6-Pyrimidin-5-yl-1,2,3,4-
310.52
309.18
tetrahydro-naphthalen-2-yl)-
piperidin-3-ol
310
1-Isopropyl-4-[6-(3-methanesulfonyl-
413.66
412.22
phenyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl]-piperazine
311
1-[6-(6-Methoxy-pyridin-3-yl)-1,2,3,4-
339.62
338.2
tetrahydro-naphthalen-2-yl]-piperidin-
3-ol
312
5-(6-Piperidin-1-yl-5,6,7,8-
294.62
293.19
tetrahydro-naphthalen-2-yl)-
pyrimidine
313
4-[6-(3-Hydroxy-piperidin-1-yl)-
351.64
350.2
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzamide
314
2-Methoxy-5-(6-piperidin-1-yl-
323.66
322.2
5,6,7,8-tetrahydro-naphthalen-2-yl)-
pyridine
315
1-[6-(3-Methanesulfonyl-phenyl)-
386.61
385.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
piperidin-3-ol
316
4-(6-Thiomorpholin-4-yl-5,6,7,8-
3.53.57
352.16
tetrahydro-naphthalen-2-yl)-
benzamide
317
3-[6-(4-Isopropyl-piperazin-1-yl)-
406.66
405.28
5,6,7,8-tetrahydro-naphthalen-2-yl]-
N,N-dimethyl-benzamide
318
3-[6-(3-Hydroxy-piperidin-1-yl)-
379.68
378.23
5,6,7,8-tetrahydro-naphthalen-2-yl]-
N,N-dimethyl-benzamide
319
3-[6-(2-Isopropyl-pyrrolidin-1-yl)-
365.39
364.25
5,6,7,8-tetrahydro-naphthalen-2-yl]-
6-methoxy-2-methyl-pyridine
320
1-[6-(4-Ethanesulfonyl-phenyl)-
ND
383.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-methyl-pyrrolidine
321
2-[6-(4-Ethanesulfonyl-phenyl)-
418.26
417.18
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2,3-dihydro-1H-isoindole
322
2-[6-(6-Methoxy-2-methyl-pyridin-3-
371.32
370.2
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-2,3-dihydro-1H-isoindole
323
1-[6-(4-Ethanesulfonyl-phenyl)-
412.32
411.22
1,2,3,4-tetrahydro-naphthalen-2-yl]-
2-isopropyl-pyrrolidine
324
5-[6-(2-Isopropyl-pyrrolidin-1-yl)-
322.36
321.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
pyrimidine
325
4-[6-(4-Methanesulfonyl-phenyl)-
372.28
371.16
1,2,3,4-tetrahydro-naphthalen-2-yl]-
morpholine
326
4-[6-(4-Ethanesulfonyl-phenyl)-
386.28
385.17
1,2,3,4-tetrahydro-naphthalen-2-yl]-
morpholine
327
4-[6-(6-Methoxy-2-methyl-pyridin-3-
339.34
338.2
yl)-1,2,3,4-tetrahydro-naphthalen-2-
yl]-morpholine
328
4-(6-Pyrimidin-5-yl-1,2,3,4-
296.31
295.17
tetrahydro-naphthalen-2-yl)-
morpholine
329
3-[6-(2-Isopropyl-pyrrolidin-1-yl)-
391.37
390.27
5,6,7,8-tetrahydro-naphthalen-2-yl]-
N,N-dimethyl-benzamide
330
1-[6-(4-Methanesulfonyl-phenyl)-
372.27
371.16
1,2,3,4-tetrahydro-naphthalen-2-yl]-
pyrrolidin-3-ol
331
4-[6-(1,3-Dihydro-isoindol-2-yl)-
397.35
396.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
N,N-dimethyl-benzamide
332
2-Methoxy-5-[6-(3-phenyl-pyrrolidin-
385.33
384.22
1-yl)-5,6,7,8-tetrahydro-naphthalen-
2-yl]-pyridine
333
3-[6-(1,3-Dihydro-isoindol-2-yl)-
397.35
396.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
N,N-dimethyl-benzamide
334
4-[6-(6-Methoxy-pyridin-3-yl)-1,2,3,4-
325.32
324.18
tetrahydro-naphthalen-2-yl]-
morpholine
335
3-[6-(3-Phenyl-pyrrolidin-1-yl)-
397.34
396.22
5,6,7,8-tetrahydro-naphthalen-2-yl]-
benzamide
336
5-[6-(2-Isopropyl-pyrrolidin-1-yl)-
351.37
350.24
5,6,7,8-tetrahydro-naphthalen-2-yl]-
2-methoxy-pyridine
337
4-[6-(Benzyl-methyl-amino)-5,6,7,8-
399.35
398.24
tetrahydro-naphthalen-2-yl]-N,N-
dimethyl-benzamide
338
4-[6-(3-Methanesulfonyl-phenyl)-
372.27
371.16
1,2,3,4-tetrahydro-naphthalen-2-yl]-
morpholine
339
1-[6-(3-Methanesulfonyl-phenyl)-
432.28
431.19
1,2,3,4-tetrahydro-naphthalen-2-yl]-
3-phenyl-pyrrolidine
[0250] The composition of the present invention may be a composition comprising a compound of formula I and optionally a pharmaceutically acceptable carrier. The composition of the present invention may also be a composition comprising a compound of formula I, a histamine H 1 antagonist and optionally a pharmaceutically acceptable carrier. The composition of the present invention may also be a composition comprising a compound of formula I, a neurotransmitter re-uptake blocker and optionally a pharmaceutically acceptable carrier.
[0251] The composition of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers. The composition may be formulated for oral, buccal, intranasal, parenteral (e.g., intravenous, intramuscular, intraperitoneal, or subcutaneous or through an implant) nasal, vaginal, sublingual, rectal or topical administration or in a form suitable for administration by inhalation or insufflation.
[0252] Pharmaceutically acceptable salts of compounds of formula I may be prepared by one or more of three methods: (i) by reacting the compound of formula I with the desired acid or base; (ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of formula I or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound of formula I to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
[0253] All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
[0254] Also included within the scope of the invention are metabolites of compounds of formula I, that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites in accordance with the invention include: (i) where the compound of formula (I) contains a methyl group, an hydroxymethyl derivative thereof (—CH 3 —CH 2 OH); (ii) where the compound of formula (I) contains an alkoxy group, an hydroxy derivative thereof (—OR→—OH); (iii) where the compound of formula (I) contains a tertiary amino group, a secondary amino derivative thereof (—NR a R b →—NHR a or —NHR b ); (iv) where the compound of formula (I) contains a secondary amino group, a primary derivative thereof (—NHR a →—NH 2 ); (v) where the compound of formula (I) contains an amide group, a carboxylic acid derivative thereof (—CONR c R d →—COOH).
[0255] Isotopically labeled compounds of formula I of this invention can generally be prepared by carrying out the procedures disclosed in the preceeding Schemes and/or in the Examples and Preparations, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
[0256] For oral administration, the pharmaceutical composition may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents such as pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose; fillers such as lactose, microcrystalline cellulose or calcium phosphate; lubricants such as magnesium stearate, talc or silica; disintegrants such as potato starch or sodium starch glycolate; or wetting agents such as sodium lauryl sulphate. The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents such as sorbitol syrup, methyl cellulose or hydrogenated edible fats; emulsifying agents such as lecithin or acacia, non-aqueous vehicles such as almond oil, oily esters or ethyl alcohol; and preservatives such as methyl or propyl p-hydroxybenzoates or sorbic acid.
[0257] For buccal administration, the composition may take the form of tablets or lozenges formulated in conventional manner.
[0258] The composition of the invention may be formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient or ingredients in a composition may be in powder form for reconstitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. The term “active ingredient” as used herein refers to a compound of the formula I, a histamine H 1 antagonist, or a neurotransmitter re-uptake blocker.
[0259] The composition of the invention may also be formulated in a rectal composition such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. A composition for vaginal administration is preferably a suppository that may contain, in addition to the active ingredient or ingredients, excipients such as cocoa butter or a suppository wax. A composition for nasal or sublingual administration is also prepared with standard excipients well known in the art.
[0260] For intranasal administration or administration by inhalation, the composition may be conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of the active ingredient or ingredients. Capsules and cartridges, made, for example, from gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of an active ingredient or ingredients and a suitable powder base such as lactose or starch. The active ingredient or ingredients in the composition may range in size from nanoparticles to microparticles.
[0261] An exemplary dose of the composition of the invention comprising a compound of formula I for oral, parenteral or buccal administration to the average adult human for the treatment of the conditions referred to herein is about 0.01 to about 1000 mg of the compound of formula I per unit dose which could be administered, for example, 1 to 3 times per day.
[0262] An exemplary dose of the composition of the invention comprising a compound of formula I and a histamine H 1 antagonist or a neurotransmitter re-uptake blocker for oral, parenteral or buccal administration to the average adult human for the treatment of the conditions referred to herein is about 0.01 to about 500 mg of the compound of formula I and of about 0.01 mg to about 500 mg of the histamine H 1 antagonist or the neurotransmitter re-uptake blocker per unit dose which could be administered, for example, 1 to 3 times per day.
[0263] Aerosol formulations for treatment of the conditions referred to herein in the average adult human are preferably arranged so that each metered dose or “puff” of aerosol contains about 20 μg to about 1000 μg of the compound of formula I. The overall daily dose with an aerosol will be within the range about 100 μg to about 10 mg. Administration may be several times daily, for example 2, 3, 4 or 8 times, giving for example, 1, 2 or 3 doses each time. Aerosol formulations containing a compound of formula I and a histamine H 1 antagonist or a neurotransmitter re-uptake blocker are preferably arranged so that each metered dose or “puff” of aerosol contains about 100 μg to about 10,000 μg of the compound of formula I and about 100 μg to about 30,000 μg of the histamine H 1 antagonist or the neurotransmitter re-uptake blocker. Administration may be several times daily, for example 1, 3, 4 or 8 times, giving for example, 1, 2 or 3 doses each time. The composition of the invention comprising a compound of formula I and a histamine H 1 antagonist or a neurotransmitter re-uptake blocker may optionally contain a pharmaceutically acceptable carrier and may be administered in both single and multiple dosages as a variety of different dosage forms, such as tablets, capsules, lozenges, troches, hard candies, powders, sprays, aqueous suspension, injectable solutions, elixirs, syrups; and the like. The pharmaceutically acceptable carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Oral pharmaceutical formulations can be suitably sweetened and/or flavored by means of various agents of the type commonly employed for such purposes. In general, the compound of formula I is present in such dosage forms at concentration levels ranging from about 0.1% to about 99.9% by weight of the total composition, i.e., in amounts which are sufficient to provide the desired unit dosage, and the histamine H 1 antagonist or the neurotransmitter re-uptake blocker is present in such dosage forms at concentration levels ranging from about 0.1% to about 99.9% by weight of the total composition, i.e., in amounts which are sufficient to provide the desired unit dosage.
[0264] The compound of formula I and the histamine H 1 antagonist may be administered together or separately. When administered separately, the compound of formula I and the histamine H 1 antagonist may be administered in either order, provided that after administration of the first of the two active ingredients, the second active ingredient is administered within 24 hours or less, preferably 12 hours or less.
[0265] The compound of formula I and the neurotransmitter re-uptake blocker may be administered together or separately. When administered separately, the compound of formula I and the neurotransmitter re-uptake blocker may be administered in either order, provided that after administration of the first of the two active ingredients, the second active ingredient is administered within 24 hours or less, preferably 12 hours or less.
[0266] A preferred dose ratio of compound of formula I to the histamine H 1 antagonist or to the neurotransmitter re-uptake blocker for oral, parenteral or buccal administration to the average adult human for the treatment of the conditions referred to herein is from about 0.001 to about 1000, preferably from about 0.01 to about 100.
[0267] The composition may be homogeneous, wherein by homogeneous it is meant that the active ingredient or ingredients are dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid composition is then subdivided into unit dosage forms of the type described herein containing from about 0.1 to about 1000 mg of the active ingredient or ingredients. Typical unit dosage forms contain from about 1 to about 300 mg, for example about 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient or ingredients. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
[0268] The dosage of the active ingredient or ingredients in the composition and methods of this invention may be varied; however, it is necessary that the amount of the active ingredient or ingredients in such a composition be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, the particular compounds administered, the duration of the treatment, and other factors. All dosage ranges and dosage levels mentioned herein refer to each active ingredient present in the pharmaceutical composition of the present invention, as well as those used in the methods of the present invention. Generally, dosage levels of between about 0.01 and about 100 mg/kg of body weight daily are administered to humans and other mammals. A preferred dosage range in humans is about 0.1 to about 50 mg/kg of body weight daily which can be administered as a single dose or divided into multiple doses. A preferred dosage range in mammals other than humans is about 0.01 to about 10.0 mg/kg of body weight daily which can be administered as a single dose or divided into multiple doses. A more preferred dosage range in mammals other than humans is about 0.1 to about 5.0 mg/kg of body weight daily which can be administered as a single dose or divided into multiple doses.
[0269] The pharmaceutical composition comprising the compound of formula I and the histamine H 1 antagonist or the neurotransmitter re-uptake blocker may be administered at dosages of a therapeutically effective amount of the compound of formula I and of the second active ingredient in single or divided doses.
[0270] The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age. However, some variation in dosage will necessarily occur depending upon the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
[0271] The dosage amounts set forth in this description and in the appended claims may be used, for example, for an average human subject having a weight of about 65 kg to about 70 kg. The skilled practitioner will readily be able to determine any variation in the dosage amount that may be required for a subject whose weight falls outside the about 65 kg to about 70 kg range based upon the medical history of the subject. The pharmaceutical combinations may be administered on a regimen of up to 6 times per day, preferably 1 to 3 times per day, such as 2 times per day or once daily.
Determination of Biological Activity
[0272] The in vitro affinity of the compounds in the present invention at the rat or human histamine H3 receptors can be determined according to the following procedure. Frozen rat frontal brain or frozen human post-mortem frontal brain is homogenized in 20 volumes of cold 50 mM Tris HCl containing 2 mM MgCl 2 (pH to 7.4 at 4° C.). The homogenate is then centrifuged at 45,000 G for 10 minutes. The supernatant is decanted and the membrane pellet resuspended by Polytron in cold 50 mM Tris HCl containing 2 mM MgCl 2 (pH to 7.4 at 4° C.) and centrifuged again. The final pellet is resuspended in 50 mM Tris HCl containing 2 mM MgCl 2 (pH to 7.4 at 25° C.) at a concentration of 12 mg/mL. Dilutions of compounds are made in 10% DMSO/50 mM Tris buffer (pH 7.4) (at 10× final concentration, so that the final DMSO concentration is 1%). Incubations are initiated by the addition of membranes (200 microliters) to 96 well V-bottom polypropylene plates containing 25 microliters of drug dilutions and 25 microliters of radioligand (1 nM final concentration 3H-N-methyl-histamine). After a 1 hour incubation, assay samples are rapidly filtered through Whatman GF/B filters and rinsed with ice-cold 50 mM Tris buffer (pH 7.4) using a Skatron cell harvester. Radioactivity is quantified using a BetaPlate scintillation counter. The percent inhibition of specific binding can then be calculated,
[0273] A person of ordinary skill in the art could adapt the above procedure to other assays.
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This invention is directed to a compound of formula (I), as defined herein, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing a compound of formula (I) a process of en preparation of a compound of formula (I), a method of treatment of a disorder or condition that may be treated by antagonizing histamine H3 receptors, the method comprising administering to a mammal in need of such treatment a compound of formula (I) as described above, and a method of treatment of a disorder or condition selected from the group consisting of depression, mood disorders, schizophrenia, anxiety disorders, Alzheimer's disease, attention-deficit hyperactivity disorder (ADHD), psychotic disorders, cognitive disorders, sleep disorders, obesity, dizziness, epilepsy, motion sickness, respiratory diseases, allergy, allergy-induced airway responses, allergic rhinitis, nasal congestion, allergic congestion, congestion, hypotension, cardiovascular disease, diseases of the GI tract, hyper and hypo motility and acidic secretion of the gastro-intestinal tract, the method comprising administering to a mammal in need of such treatment a compound of formula (I) as described above.
| 2 |
This is a continuation of application Ser. No. 140,165, filed on Dec. 31, 1987, now abandoned.
FIELD OF THE INVENTION
The present invention relates generally to extrusion processes and apparatus, and more particularly to the type of extrusion apparatus generally known as conform machines designed to permit continuous extrusion of a feedstock material into various shapes and sizes.
DESCRIPTION OF THE PRIOR ART
In the typical conform extrusion machine, solid feedstock such as an aluminum rod or other solid or powdered material to be extruded is fed in an unheated state into the machine along a rotating wheel. The wheel has an endless groove at its periphery to receive the feedstock. A portion of the circumference of the wheel, typically about one-quarter of the length thereof, is maintained in close contact with a fixed heavy metal block known as an extrusion shoe. At the end of the contacting portion, a blocking abutment that enters the groove obstructs the path of the feedstock, preventing it from being carried further along the groove in the rotating wheel. As the extrusion material is pushed against the abutment by the frictional force exerted by the continuously rotating wheel, sufficient force is produced to extrude the material through a die retained at the end of a chamber in the shoe adjacent to the blocking abutment.
The advantages of the conform extrusion machine over heretofore conventional extrusion apparatus include the provision of a theoretically continuous extruding process, with attendant simplification of subsequent handling techniques and elimination of billet discards, and the use of cold solid or powdered feedstock with avoidance of any need to preheat the material prior to extrusion thereof. Examples of prior art conform extrusion apparatus of the aforementioned type are described in U.S. Pat. Nos. 3,765,216 to Green and 4,055,979 to Hunter et al.
Considerable heat is generated by the enormous frictional resistance and resulting axial stress encountered by the feedstock as it is fed along the groove by the rotating wheel, as a consequence of the close contact of the latter with the extrusion shoe. The frictional force and attendant heat cause the feedstock to yield and flow through the die. In a typical process, the extruded product may be fed into a water quench tank located some five to ten feet from the exit die. It has been found that such prior art conform machines produce extruded products having non-uniform grain size and large surface grains which cause "orange peel" of the product when it is subjected to mechanical bending or other similar high stress working operations. Accordingly, it is a principal object of the present invention to provide a conform extrusion machine for producing extruded products with uniform small grain structure and improved mechanical properties.
In a typical conform extrusion process, an expansion chamber may be provided in the extrusion shoe, located adjacent to the blocking abutment and upstream of the die, to allow extrusion of product of larger cross-section than the feed material. The shearing forces on the feed material are higher along the extrusion shoe, which is fixed relative to the moving material, than along the grooved rotating wheel with which the material is moved. As a result, the temperature of the feed material is higher in the region adjacent to the shoe than in the region adjacent to the rotating wheel. In the conformed product (i.e., the extruded product), the portion subjected to the higher temperature during the extrusion process has a larger grain size. As a result of the orientation of the movable and stationary components of the conform machine, the region of the feed material adjacent the stationary shoe experiences higher temperatures and, thus, the corresponding portion of the conform product has larger grains than other portions of the product as it leaves the die.
The surface of the conform product recrystallizes more rapidly than the product interior because of the hardening process. Additionally, because of the high exit temperature of the conform product as it leaves the die, it undergoes a spontaneous secondary recrystallization along the edges of its surface, with consequent further grain growth. The resulting product suffers seriously inconsistent grain size and attendant structural deficiencies.
Therefore, a further object of the present invention is to provide an improved conform machine employing special cooling systems to enhance the structural properties of the final product.
It is a more specific object of the invention to provide a cooling system for conform extrusion apparatus which allows the extrusion process to be carried out at a preselected desired temperature and which maintains the material in the extrusion chamber at a uniform temperature.
Another object of the present invention is to provide a conform machine with plural cooling systems for maintaining a preset extruding temperature and for inhibiting secondary recrystallization of the product.
SUMMARY OF THE INVENTION
The present invention resides, in one aspect, in a conform extrusion apparatus employing a first cooling system for maintaining a desired temperature in the extrusion chamber of the apparatus. This first cooling system provides means at both sides of the extrusion chamber for sensing the temperature thereat, a coolant supply system to both sides of the chamber, and control means responsive to changes in the temperature at either side relative to a predetermined extrusion chamber temperature for varying the flow of coolant to each side respectively. Thus, there are expected temperature values at a plurality of points within the extrusion chamber and a predetermined extrusion chamber temperature, resulting in several possible temperature differences. In this manner, the temperature of the material is maintained substantially uniform throughout the extrusion chamber, to produce a conform product having a substantially uniform small grain structure and consequent improved mechanical properties.
According to another aspect of the invention, a second cooling system is provided to cool the conform product as it exits the die and thereby inhibit secondary recrystallization and grain growth at the surface of the product.
In conventional extrusion processes which do not use the conform technique, it is customary to provide cooling within the die. However, the heating problem in the conform process is different from that encountered in the conventional extrusion process and requires a vastly different solution which takes into account the presence of localized hot spots contributing to the different grain sizes in the final product. It has been proposed in the past to use various types of cooling systems in the type of conform extrusion apparatus which uses entry feed material in molten rather than solid or powdered form, but in those instances the proposed cooling has been for purposes of solidifying the molten material. Examples of such techniques are found in U.S. Pat. No. 4,393,917 to Fuchs, Jr., and European patent application publication No. EP 0110653. In contrast to the prior art proposals, the present invention provides a cooling system for conform extrusion apparatus which maintains uniform temperature of the extrudable material at both sides of the extrusion chamber to prevent non-uniform grain sizes in the extruded product.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and still further objects, features, and attendant advantages of the present invention will become apparent from a consideration of the following detailed description of a preferred embodiment thereof, taken in conjunction with the accompanying drawings in which:
FIG. 1 is a partial view of the sectional side elevation of a conform extrusion apparatus including a first cooling system according to a preferred embodiment of the invention;
FIG. 2 is a partial view of a sectional end elevation of the apparatus of FIG. 1 taken along the line 2--2, and schematically illustrating the control system for the cooling system therein;
FIG. 3 is a partial sectional view of the apparatus of FIG. 1 taken along the line 3--3; and
FIG. 4 is a sectional side elevational view of a second cooling system for the apparatus of FIGS. 1 and 2.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring now to FIGS. 1 and 2, an apparatus for continuously extruding material into a desired conform product includes a wheel 10 mounted for rotation on a shaft 12. Wheel 10 has an endless channel or groove 15 suitably formed in its circumferential edge 17. The wheel 10 rotates counterclockwise in close proximity to an extrusion shoe 20 which remains stationary relative to the wheel. A channel blocking abutment 22 is affixed to shoe 20 and enters the channel 15 in close proximity to the walls thereof, so that the wheel is free to rotate but a barrier is formed by abutment 22 to anything that may be carried in that passageway. The extrusion shoe 20 includes an extrusion chamber 25 disposed adjacent to the blocking abutment 22. A die block 28 at the end of the extrusion chamber forms a wall of the chamber and retains a die 29 therein to permit material to be extruded therethrough into a desired shape.
Thus far, everything which has been described is completely conventional structure in conform extrusion machines of the prior art. Such conventional structure is shown and described in U.S. Pat. No. 3,765,216 to Green, the teaching of which is hereby incorporated by reference. In operation, a solid feedstock or feed material 30, which may be an aluminum rod, for example, of a size adequate to be received within channel 15 is fed into the channel. It will, of course, be appreciated by those skilled in the art that the orientation of the extrusion shoe 20 and abutment 22 may be inverted such that the feed material 30 is fed into the channel 15 from above the wheel 10 which would then be rotated clockwise.
The feed material 30 is upset into the channel 15 with the assistance of a coining roll 33 such that it frictionally engages the walls of the channel 15 as the wheel 10 rotates in a counterclockwise direction, as viewed in FIG. 1. The material eventually encounters the blocking abutment 22 in channel 15, typically located about one-quarter of the circumference of the wheel from the entry point for the material 30. The abutment 22 stops the movement of material and continued feed of the material causes it to fill the channel 15 until the material engages the stationary surface of the shoe 20 confronting the channel 15. Under the frictional forces and the pressure exerted on the feed material, and the accompanying axial stress set up in the material it will begin to yield at a point which depends on the heat generated by the process and the yielding strength of the particular material used.
The yielding material commences to flow and is thereby forced into the extrusion chamber 25 and ultimately extruded through the die 29, to produce the desired conform product 37. If desired, an expansion chamber may be used with an appropriately larger die, to permit extrusion by the conform apparatus. Viewing the orientation of the various components of the apparatus described thus far, which is a typical orientation for conform machines of the prior art, the feed material experiences greater frictional force along the stationary member side of the machine; that is, on the side toward the extrusion shoe 20. The portion of the material encountering this higher frictional force is heated to a higher temperature than that portion of the material subjected to lower frictional force. Accordingly, in the illustrated apparatus, the flowable material in the lower part of the extrusion chamber is typically substantially hotter than that in the upper part of the chamber. As a result, the grain size of the extruded product is irregular, with the larger grains at the lower part of the product relative to the size of the grains in the upper part, and with a concomitant deleterious effect on the mechanical properties of the final extruded product. Similarly, if the orientation of the shoe is the reverse of that shown in the drawings, i.e., with the stationary shoe above the abutment, the larger grains will be at the upper part of the product.
According to a preferred embodiment of the present invention, the conform extrusion apparatus is provided with a system for controlling the temperature of the flowable material in the extrusion chamber to remove the undesired hot spots, in the manner shown in FIGS. 1, 2 and 3. First, it should be noted that although the extrusion chamber 25 may be integrally formed in the extrusion shoe 20 by machining the latter, it may alternatively be provided in a separate component or components which are fitted into or on the shoe, and which maintain the above-described relationship of the chamber to the blocking abutment 22 in the channel 15.
As shown most clearly in FIG. 1, such a component is an expansion chamber member 40 having an arcuate surface 43 conforming to an arc at the circumference of rotatable wheel 10 and confronting the channeled edge of the wheel when member 40 is fastened to the shoe 20. The extrusion chamber (expansion chamber) 25 is formed in part in chamber member 40 and in further part in a pair of feeder blocks 45,46 which are also fitted and secured in the shoe. That portion of chamber 25 provided by the openings in the feeder blocks 45,46 may be larger than the chamber portion in member 40, and the portion in feeder block 46 may be tapered down toward the die block 28 forming the end wall of the extrusion chamber.
Thermocouples 49 and 50 are housed at or near the longitudinal surface of chamber 25 in feeder block 46, preferably close to the die block 28. Each of the thermocouples is formed in conventional manner from a pair of dissimilar thermoelectric materials, and each generates an electrical signal representative of the temperature at the junction of the dissimilar materials. The thermocouples are electrically insulated from the feeder block and from each other, and have their respective junctions positioned as close as practicable to the surface of chamber 25 to detect the temperature of feed material in the chamber or of that portion of the feeder block immediately adjacent to the chamber. The location of the thermocouples next to the die block assures that the temperature of the material in the extrusion chamber is sensed at a point or points reasonably close to the point from which the material is extruded from the chamber to form the desired conform product 37.
In the preferred embodiment, thermocouple 49 is positioned at the upper side of the extrusion chamber 25 and thermocouple 50 is positioned at the lower side of the chamber 25 to sense the temperature of regions of the material which are, in the orientation shown in the drawings, typically at the lowest and highest temperatures, respectively, in the selected portion of the chamber, for reasons discussed above. Although only two thermocouples are shown in the embodiment of FIGS. 1 and 2, it will be understood that additional thermocouples may be employed at spaced locations about portions of or the entire periphery of the chamber close to the die block 28.
Feeder blocks 45,46 are provided with ducts or passageways 52 and 53 therethrough, respectively running adjacent to the upper and lower sides of chamber 25 so as to be in heat exchange relationship principally with those portions of the chamber. Each of the ducts is adapted to carry a coolant fluid therethrough, such as water or liquid nitrogen. Obviously, these examples of suitable coolant fluids are virtually opposite ends of the range of coolants which could be employed; in the case of liquid nitrogen, the handling and distribution requirements are more rigorous, albeit entirely conventional. Ducts 52 are joined together at a single inlet having an electrically controlled valve 56, such as a solenoid valve, to regulate the flow of coolant fluid therethrough. A corresponding but completely separate cooling system arrangement is provided for lower ducts 53 which are joined at a single inlet having an electrically controlled flow regulating valve 57. At the opposite ends of the upper and lower ducts, suitable conventional means (not shown) are provided for recirculating the coolant fluid back to the source thereof.
Each of the thermocouples 49, 50 is electrically connected via leads 59,60, respectively, to a sampling circuit and/or an analog/digital (A/D) converter 63 (FIG. 2) to properly condition the electrical signal outputs of the thermocouples, which are representative of the temperature values at the respective thermocouple junctions, for application to a microprocessor 65. Preferably, the microprocessor is adapted to compare the sensed temperature signal value from thermocouple 50 to the signal value deriving from thermocouple 49 and to null the difference by generating an output which is converted by D/A converter 68 to an analog signal for that purpose. Since the temperature of the material in one region (in this case, the lower region) of the extrusion chamber attributable to the conform extrusion process is almost invariably higher than the temperature of the material in the opposite region (in this case, the upper region) of the chamber, the analog control signal deriving from the microprocessor may be used to control the valve 57 to allow flow of the coolant fluid through ducts 53 until the temperature sensed by thermocouple 50 is reduced to the temperature sensed by thermocouple 49. If this preferred process of nulling the difference between the two temperature readings is chosen, it is possible to perform the nulling using only the one cooling system including ducts 53 and valve 57 and associated electronic control, because temperature reduction will be necessary only in the one (lower) region of the extrusion chamber.
Alternatively, a preselected temperature at which the material is to be extruded may be set into a memory associated with the microprocessor 65, and constitute the temperature which is to be maintained at both thermocouple locations. Such predetermined temperature will be selected to be always less than or equal to the actual anticipated temperature of material in the upper portion of the chamber attributable to the extrusion process alone (i.e., before cooling). In this alternative arrangement, separate upper and lower cooling systems are required. The microprocessor compares the sensed temperature at each thermocouple location to the predetermined temperature, and generates separate control signals which are applied via the converter 68 to the two valves 56,57 to regulate the flow of coolant fluid therethrough. Thereby, the temperatures of the upper and lower regions of the chamber are adjusted as necessary to bring them to predetermined temperature.
The extruded product 37 resulting from the provision of a substantially uniform temperature of material at the point of extrusion, according to the invention, has a considerably more uniform grain size throughout and consequent improved mechanical properties. Nevertheless, it has been found that the extruded product undergoes secondary recrystallization at and near its surface, attributable to the high exit temperature of the product. While this secondary recrystallization is not as extensive as occurs in conventional conform extrusion processes without the cooling system of the present invention, it does cause some grain growth in the affected region near the product surface. According to a further feature of the invention, a second system is provided for cooling the product as it is extruded from the die, to inhibit the secondary recrystallization.
Referring to FIG. 4, the second cooling system includes a cylindrical member 80 positioned against the surface of the die block 28 or of the shoe 20 holding the die block. Member 80, which constitutes a backer plate, has a pair of cooling devices operatively associated therewith. One of these cooling devices is a ring conduit 83 supported internally in the member 80 to be in close proximity to the die block 28 when the member is assembled against the extrusion shoe (e.g., under hydraulic pressure). Conduit 83 is coupled to an inlet 84 to receive nitrogen gas or other suitable inert cooling gas, and to direct the inert gas onto the extruded product as it exits the die, from an array of aligned holes or nozzles 87 in the interior surface of the ring. This provides both cooling of the product surface and an inert atmosphere to inhibit oxidation of the product surface.
The second of the pair of cooling devices in the die exit cooling system includes an array of sprayheads 90 coupled to a reservoir 92 about the inner surface of member 80. The reservoir is connected to a pipe 95 for delivery of cooling water under pressure to the reservoir and the sprayheads, from which the water is sprayed directly onto the surface of the extruded product for rapid cooling thereof as the product exits the die 29. The latter cooling device is most effective for inhibiting the secondary recrystallization at the surface region of the product and the accompanying grain growth in that region. The gaseous streams produced by the ring conduit 83 also serve to prevent water spray from contacting the surface of the die, and thereby protect against spalling of the die. Although two alignments of sprayheads 90 are shown in FIG. 4, additional strings of sprayheads or different arrays thereof may be utilized depending on the size and shape of the extruded product. The exit cooling system comprising the inert gas and the water spray may be controlled in any conventional manner to be and remain operative throughout the extrusion of product through die 29.
Although certain preferred embodiments have been described herein, it will be apparent to those of ordinary skill in the field to which the invention pertains that variations and modifications of the described embodiments may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims.
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Apparatus for continuously extruding material includes a moving member and a stationary member forming a passageway therebetween for frictional feeding of the material to be extruded under pressure into the passageway. An abutment in the passageway forms a barrier to the material being fed therein, whereby the forces on the material heat it and cause it to yield. The heated material flows into an extrusion chamber adjacent to the abutment and is extruded from a die in a wall of the chamber. A cooling system in heat exchanging relationship with the chamber in proximity to the die maintains the material in that region of the chamber at a substantially uniform temperature to provide uniformity of grain size in the extruded product. A further cooling system at the point of egress cools the extruded product immediately as it exits from the die to inhibit secondary recrystallization.
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/725,184, filed Dec. 21, 2012, which claims the priority of U.S. Provisional Application No. 61/578,706 filed Dec. 21, 2011, U.S. Provisional Application No. 61/607,900 filed Mar. 7, 2012, and U.S. Provisional Application No. 61/661,688 filed Jun. 19, 2012, the entire disclosures of which are hereby incorporated by reference.
INCORPORATION OF SEQUENCE LISTING
The entire contents of a paper copy of the “Sequence Listing” and a computer readable form of the sequence listing on optical disk, containing a file named FINAL_APSE_SEQ_ST25.txt, which is 10 kilobytes in size and was created on May 15, 2014, are herein incorporated by reference.
TECHNICAL FIELD
The invention relates to virus-like particles, and in particular to processes for producing, isolating and purifying virus-like particles containing heterologous cargo molecules.
BACKGROUND OF THE INVENTION
Virus-like particles (VLPs) are particles derived in part from viruses through the expression of certain viral structural proteins which make up the viral envelope and/or capsid, but VLPs do not contain the viral genome and are non-infectious. VLPs have been derived for example from the Hepatitis B virus and certain other viruses, and have been used to study viral assembly and in vaccine development.
Viral capsids are composed of at least one protein, several copies of which assemble to form the capsid. In some viruses, the viral capsid is covered by the viral envelope. Such viral envelopes are comprised of viral glycoproteins and portions of the infected host's cell membranes, and shield the viral capsids from large molecules that would otherwise interact with them. The capsid is typically said to encapsidate the nucleic acids which encode the viral genome and sometimes also proteins necessary for the virus' persistence in the natural environment. For the viral genome of a virus to enter a new host, the capsid must be disassembled. Such disassembly happens under conditions normally used by the host to degrade its own as well as foreign components, and most often involves proteolysis. Viruses take advantage of normal host processes such as proteolytic degradation to enable that critical part of their cycle, i.e. capsid disassembly and genome release.
It is therefore unsurprising that the literature has not previously described capsids resistant to hydrolases that act on peptide bonds. A very limited number of certain specific peptide sequences which are part of larger proteins are known to be somewhat resistant to certain proteases, but the vast majority of peptide sequences are not. Viruses that resist proteolysis have been reported, but these are all enveloped viruses, in which the capsid is shielded by the viral envelope. In such viruses the capsids are not in contact with, i.e. they are shielded from the proteases described. Thus the role, if any, of the proteolytical stability of the virus capsid in such cases is unknown.
In large-scale manufacturing of recombinant molecules such as proteins, ultrafiltration is often used to remove molecules smaller than the target protein in the purification steps leading to its isolation. Purification methods also often involve precipitation, solvent extraction, and crystallization techniques. These separation techniques are inherently simple and low cost because, in contrast to chromatography, they are not based on surface but on bulk interactions. However, these techniques are typically limited to applications to simple systems, and by the need to specify a different set of conditions for each protein and expression system. Yet each target recombinant protein presents a unique set of interactions, thereby making its isolation process unique and complex. The separation efficiency for recombinant proteins using these simple isolation processes is therefore low.
Nucleic acids, including siRNA and miRNA, have for the most part been manufactured using chemical synthesis methods. These methods are generally complex and high cost because of the large number of steps needed and the complexity of the reactions which predispose technical difficulties, and the cost of the manufacturing systems. In addition, the synthetic reagents involved are costly and so economy of scale is not easily obtained by simply increasing batch size. In large-scale manufacturing of recombinant molecules such as proteins, ultrafiltration is often used to remove molecules smaller than the target protein in the purification steps leading to its isolation. Purification methods also often involve precipitation, solvent extraction, and crystallization techniques. These separation techniques are inherently simple and low cost because, in contrast to chromatography, they are not based on surface but on bulk interactions. However, these techniques are typically limited to applications to simple systems, and by the need to specify a different set of conditions for each protein and expression system. Yet each target recombinant protein presents a unique set of interactions, thereby making its isolation process unique and complex. The separation efficiency for recombinant proteins using these simple isolation processes is therefore low.
BRIEF SUMMARY OF THE INVENTION
In one aspect the present disclosure provides a method for producing a virus-like particle (VLP) comprising a capsid enclosing at least one heterologous cargo molecule.
VLPs according to the present disclosure may comprise a capsid which comprises a wild type viral capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4, or a capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). The capsid may comprise a wild type Enterobacteria phage MS2 capsid protein having the amino acid sequence of SEQ ID NO: 3.
VLPs according to the present disclosure may comprise a heterologous cargo molecule comprising a peptide or polypeptide. A VLP may further comprise an oligonucleotide linker coupling the heterologous cargo peptide or polypeptide molecule and the viral capsid.
In another aspect, the present disclosure provides a nucleic acid construct comprising a nucleotide sequence that encodes an RNA. The RNA may be for example an siRNA or an shRNA. The present disclosure also encompasses a vector comprising any such nucleic acid constructs, and host cells comprising such a vector, as well as host cell stably transformed with such a vector. Host cells may be a bacterial cell, such as but not limited to an Escherichia coli cell, a plant cell, a mammalian cell, an insect cell, a fungal cell or a yeast cell. A host cell may further be stably transfected with a second vector comprising a second nucleic acid sequence encoding a viral capsid. The second nucleic acid sequence may encode for example a viral protein encoding a viral capsid having at least 40% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). A nucleic acid construct as described herein may also encode a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). The present disclosure also encompasses a plant or plant tissue transformed to contain a nucleic acid construct described herein, and seed or progeny of such a plant or plant tissue, wherein the seed or progeny comprises the nucleic acid construct.
In another aspect, the present disclosure provides a composition comprising: a) a plurality of virus-like particles each comprising a viral capsid enclosing at least one heterologous cargo molecule; and b) one or more cell lysis products present in an amount of less than 4 grams for every 100 grams of capsid present in the composition, wherein the cell lysis products are selected from proteins, polypeptides, peptides and any combination thereof. In the composition, the capsid is for example resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. Such VLPs in a composition may further comprise an oligonucleotide linker coupling the heterologous cargo molecule and the viral capsid.
In another aspect, the present disclosure provides method for isolating and purifying a target cargo molecule, the method comprising: (a) obtaining a whole cell lysate comprising a plurality of virus-like particles (VLPs) each comprising a capsid enclosing at least one target cargo molecule, wherein the capsids are resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4; (b) subjecting the VLP's to hydrolysis using a peptide bond hydrolase category E.C. 3.4, for a time and under conditions sufficient for at least 60, at least 70, at least 80, or at least 90 of every 100 individual polypeptides present in the whole cell lysate but not enclosed by the capsids to be cleaved, while at least 60, at least 70, at least 80, or at least 90 of every 100 capsids present in the whole cell lysate before such hydrolysis remain intact following the hydrolysis. In the method, the capsids may each comprise a viral capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). The capsids may each comprise a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). In the method, the heterologous cargo molecule may comprise an oligonucleotide which may be an oligoribonucleotide, or a peptide or a polypeptide. An oligoribonucleotide may be selected for example from siRNA, shRNA, sshRNA, lshRNA and miRNA. The method may further comprise purification of the capsids following hydrolysis. Purification may include at least one of a liquid-liquid extraction step, a crystallization step, a fractional precipitation step, and an ultra filtration step. The present disclosure also encompasses a composition produced by such a method.
In another aspect, the present disclosure provides a method for protecting a target molecule from hydrolysis in a whole cell lysate following intracellular production of the target molecule in a host cell, the method comprising: (a) selecting a viral capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4; (b) stably transfecting the host cell with a first vector comprising a nucleic acid sequence encoding a viral protein forming the viral capsid, and a second vector comprising a nucleic acid sequence comprising a sequence encoding a cargo molecule; and (c) maintaining the cells for a time and under conditions sufficient for the transformed cells to express capsid protein and assemble capsids encapsidating the cargo molecule. In the process, the capsids may each comprise a viral capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3).
In another aspect, the present disclosure provides a process for purifying VLPs enclosing at least one heterologous cargo molecule, the process comprising: (a) obtaining a cell lysate comprising a plurality of the VLPs; (b) contacting the cell lysate with a protease for a time and under conditions sufficient to hydrolyze cell lysis products other than the VLPs to form a hydrolysate; and (c) isolating the VLPs from the hydrolsyate. Step (c) may comprise (i) performing a first precipitation with ammonium sulfate followed by a first centrifugation to obtain a first precipitate and a first supernatant; and (ii) performing a second precipitation on the first supernatant with ammonium sulfate followed by a second centrifugation to obtain a second precipitate, wherein the second precipitate comprises at least about 70%, 80% or 90% by weight of the VLPs. Step (c) may comprise (i) performing a first precipitation with ethanol followed by a first centrifugation to obtain a first precipitate and a first supernatant; and (ii) performing a second precipitation on the first supernatant with ammonium sulfate followed by a second centrifugation to obtain a second precipitate, wherein the second precipitate comprises at least about 70%, 80% or 90% by weight of the VLPs. Step (c) may comprise ultracentrifuging the hydrolysate to obtain a precipitate comprising at least about 70%, 80% or 90% by weight of the VLPs. In the process, the VLPs may each comprise a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4, which can comprise a capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). The VLPs may each comprise a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). In the process, step (b) can be performed for at least about 30 minutes at about 37° C. The process may further comprise, before step (b), contacting the cell lysate with at least one of a nuclease, an amylase and a lipase for at least about 30 minutes at about 37° C. In the process, the protease can be for example a peptide bond hydrolase category E.C. 3.4, which can be selected for example from Proteinase K, Protease from Streptomyces griseus , Protease from Bacillus licheniformis , pepsin and papain. In the process, the heterologous cargo molecule enclosed by the VLPs may comprise an oligonucleotide which may be an oligoribonucleotide, or a peptide or a polypeptide. An oligoribonucleotide may be selected for example from siRNA, shRNA, sshRNA, lshRNA and miRNA. The process may further comprise preparing the cell lyaste before step (a) by centrifuging cells following expression of the VLPs in the cells; resuspending the cells; lysing the cells and centrifuging the cell lysate to obtain a supernatant, wherein the supernatant is used as the cell lysate for step (a).
In another aspect, the present disclosure provides VLPs comprising a capsid enclosing at least one heterologous cargo molecule and a packing sequence wherein the capsid comprises a capsid protein which is a variant of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) except that the A residue at position 1 is deleted. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) except that the A residue at position 1 is deleted and the S residue at position 2 is deleted. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) except that the A residue at position 1 is deleted, the S residue at position 2 is deleted and the N residue at position 3 is deleted. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) except that the Y reside at position 129 is deleted. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) but having a single (1) amino acid deletion in the 112-117 segment. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) but having a single (1) amino acid deletion in the 112-117 segment. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) but having a 1-2 residue insertion in the 65-83 segment. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) but having a 1-2 residue insertion in the 44-55 segment. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) but having a single (1) residue insertion in the 33-43 segment. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) but having a 1-2 residue insertion in the 24-30 segment. The capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO:3) but having a single (1) residue insertion in the 10-18 segment. The capsid may comprise a capsid protein monomer sequence concatenated with a second capsid protein monomer sequence which assembles into a capsid which resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. The capsid may comprise a capsid protein monomer sequence whose C-terminus is extended with a 0-6 residue linker segment whose C-terminus is concatenated with a second capsid protein monomer sequence, all of which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. A linker segment may have a sequence such as, for example, -(Gly) x , wherein x=0-6. A linker segment may be a Gly-Ser linker selected from -Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly- The capsid may comprise the capsid protein concatenated with a third capsid protein monomer sequence which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. The capsid may comprise a capsid protein wherein the C-terminus is extended with a 0-6 residue linker segment whose C-terminus is concatenated with a third capsid protein monomer sequence, all of which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. The capsid may comprise a capsid protein wherein the capsid comprises a capsid protein in which one or both linker sequences is -(Gly) x , wherein x=0-6, including -Gly-; -Gly-Gly-; and -Gly-Gly-Gly-. A linker segment may be a Gly-Ser linker selected from -Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly-. Such a capsid protein assembles for example into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. For example, the capsid may comprise a capsid protein in which one or both linker sequences is -(Gly) x -, x=1, which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. The capsid may comprise a capsid protein in which one or both linker sequences is -(Gly) x -, x=2, which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. The capsid may comprise a capsid protein in which one or both linker sequences is -(Gly) x -, x=3, which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. The capsid may comprise one or more capsid protein sequences which is N-terminally truncated by 1-3 residues and a linker segment as described herein is lengthened by the number of residues deleted. The capsid may comprise one or more capsid protein sequences which is C-terminally truncated by 1 residue, and linker segments as described herein are lengthened by the one residue, wherein the capsid is resistant to hydrolysis catalyzed by a peptide bond hydrolase category E.C. 3.4. The capsid may comprise a first capsid protein sequence in a concatenated dimer which is C-terminally truncated by 1 residue and the linker segments lengthened by the one residue or wherein the first and/or second capsid protein sequence in a concatenated trimer is C-terminally truncated by 1 residues. The capsid may comprise a capsid protein having N- and C-terminal truncations.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plot of Optical Density (OD; filled diamonds) and pH (open squares) over time, showing propagation of wild type MS2 bacteriophage (ATCC No. 15597-B1, from American Type Culture Collection, Rockville, Md.) in its E. coli host (ATCC No. 15669).
FIG. 2 is a gel showing results of SDS-PAGE analysis of MS2 bacteriophage samples obtained following propagation in E. coli and purified using Proteinase K and ultrafiltration, showing that Proteinase K purification yields phage purified to higher than 99% (band at 14 kDa corresponds to MS2 bacteriophage coat protein).
FIG. 3 is a gel showing results of SDS-PAGE analysis of partially purified MS2, showing complete degradation of the phage and results obtained after 1× or 2× ultrafiltration of the lysate (Lanes 4 and 6).
FIG. 4 is a gel showing results of SDS-PAGE analysis of MS2 samples purified using ultrafiltration and Proteinase K treatment.
FIG. 5 is a gel showing results of SDS-PAGE analysis of MS2 samples purified using Proteinase K treatment, precipitation at acidic conditions, precipitation using ethanol at basic and acidic conditions, and ultrafiltration.
FIG. 6 is a graph showing the UV spectrum of MS2 samples purified using Proteinase K treatment, precipitation at acidic conditions, precipitation using ethanol at basic and acidic conditions, and ultrafiltration.
FIG. 7 is a plot of Optical Density (OD; filled diamonds) over time, obtained with a control sample (open diamonds) and an MS2 sample following purification described for FIGS. 5 and 6 (filled squares), showing that the purified sample contained phage that retained high infectivity.
FIG. 8 is a gel showing results of SDS-PAGE analysis of VLP samples following expression of MS2 capsids encapsidating RNA coding for the capsid protein attached to a coat-specific 19-mer RNA hairpin.
FIG. 9 is a chromatograph of PCR products obtained from an MS2 sample following purification described for FIGS. 5 and 6 , chromatographed in 1.5% agarose gel stained with Ethidium Bromide (1.2 kbp for primers F1201 — 1223-R1979 — 2001 in Lane 1, 800 bp for primers F1201 — 1223-R1979 — 2001 in Lane 2, and 304 bp for primers F1401 — 1426-R1680 — 1705 in Lane 3), showing consistency with an intact MS2 bacteriophage genome.
FIG. 10 is a chromatograph of PCR products from PCR interrogation of an VLP sample for presence or absence of a section of the MS2 capsid protein following purification, chromatographed in 2% agarose gel stained with Ethidium Bromide (304 bp in Lane 1; the leftmost Lane corresponds to 1 kb plus ladder from Life Technologies), showing consistency with an intact MS2 capsid protein gene.
FIG. 11 is a gel showing results of SDS-PAGE analysis of VLP samples following simple precipitation with ethanol.
FIG. 12 is a gel showing results of SDS-PAGE analysis of VLP samples following use of Proteinase K and simple precipitation with ethanol for purification of MS2 VLPs.
FIG. 13 is a gel showing results of SDS-PAGE analysis of MS2 samples following use of constitutive hydrolases, fractional precipitation with ethanol, and ultrafiltration for purification of VLPs.
FIG. 14 is a gel showing results of SDS-PAGE analysis of VLP samples following use of various hydrolases, and fractional precipitation with ammonium sulfate for purification of VLPs.
FIG. 15 is a gel showing results of PAGE analysis of RNA obtained from RNA encapsidated in VLPs.
FIG. 16 is a gel showing results of PAGE analysis of RNA products obtained from RNA encapsidated in VLPs, following purification of the VLP's and isolation of the RNA from the VLPs.
FIG. 17 is a series of gels showing results of SDS-PAGE analyses of VLP's comprising MS2 capsids, following purification and suspension of the VLPs, and exposure to various proteases for 1 hour and 4 hours of incubation.
DETAILED DESCRIPTION OF THE INVENTION
Section headings as used in this section and the entire disclosure herein are not intended to be limiting.
A. Definitions
A wide variety of conventional techniques and tools in chemistry, biochemistry, molecular biology, and immunology are employed and available for practicing the methods and compositions described herein, are within the capabilities of a person of ordinary skill in the art and well described in the literature. Such techniques and tools include those for generating and purifying VLPs including those with a wild type or a recombinant capsid together with the cargo molecule(s), and for transforming host organisms and expressing recombinant proteins and nucleic acids as described herein. See, e.g., MOLECULAR CLONING, A LABORATORY MANUAL 2nd ed. 1989 (Sambrook et al, Cold Spring Harbor Laboratory Press); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al, Greene Publ. Assoc., Wiley-Interscience, NY) 1995. The disclosures in each of these are herein incorporated by reference.
As used herein, the term “cargo molecule” refers to an oligonucleotide, polypeptide or peptide molecule, which is or may be enclosed by a capsid.
As used herein, the term “oligonucleotide” refers to a short polymer of at least two, and no more than about 70 nucleotides, preferably no more than about 55 nucleotides linked by phosphodiester bonds. An oligonucleotide may be an oligodeoxyribonucleotide (DNA) or a oligoribonucleotide (RNA), and encompasses short RNA molecules such as but not limited to siRNA, shRNA, sshRNA, lshRNA, and miRNA.
As used herein, the term “peptide” refers to a polymeric molecule which minimally includes at least two amino acid monomers linked by peptide bond, and preferably has at least about 10, and more preferably at least about 20 amino acid monomers, and no more than about 60 amino acid monomers, preferably no more than about 50 amino acid monomers linked by peptide bonds. For example, the term encompasses polymers having about 10, about 20, about 30, about 40, about 50, or about 60 amino acid residues.
As used herein, the term “polypeptide” refers to a polymeric molecule including at least one chain of amino acid monomers linked by peptide bonds, wherein the chain includes at least about 70 amino acid residues, preferably at least about 80, more preferably at least about 90, and still more preferably at least about 100 amino acid residues. As used herein the term encompasses proteins, which may include one or more linked polypeptide chains, which may or may not be further bound to cofactors or other proteins. The term “protein” as used herein is used interchangeably with the term “polypeptide.”
As used herein, the term “variant” with reference to a molecule is a sequence that is substantially similar to the sequence of a native or wild type molecule. With respect to nucleotide sequences, variants include those sequences that may vary as to one or more bases, but because of the degeneracy of the genetic code, still encode the identical amino acid sequence of the native protein. Variants include naturally occurring alleles, and nucleotide sequences which are engineered using well-known techniques in molecular biology, such as for example site-directed mutagenesis, and which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention have at least 40%, at least 50%, at least 60%, at least 70% or at least 80% sequence identity to the native (endogenous) nucleotide sequence. The present disclosure also encompasses nucleotide sequence variants having at least about 85% sequence identity, at least about 90% sequence identity, at least about 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
Sequence identity of amino acid sequences or nucleotide sequences, within defined regions of the molecule or across the full-length sequence, can be readily determined using conventional tools and methods known in the art and as described herein. For example, the degree of sequence identity of two amino acid sequences, or two nucleotide sequences, is readily determined using alignment tools such as the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990), which are readily available from multiple online sources. Algorithms for optimal sequence alignment are well known and described in the art, including for example in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988). Algorithms for sequence analysis are also readily available in programs such as blastp, blastn, blastx, tblastn and tblastx. For the purposes of the present disclosure, two nucleotide sequences may be also considered “substantially identical” when they hybridize to each other under stringent conditions. Stringent conditions include high hybridization temperatures and low salt hybridization buffers which permit hybridization only between nucleic acid sequences that are highly similar. Stringent conditions are sequence-dependent and will be different in different circumstance, but typically include a temperature at least about 60° C., which is about 10° C. to about 15° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Salt concentration is typically about 0.02 molar at pH 7.
As used herein with respect to a given nucleotide sequence, the term “conservative variant” refers to a nucleotide sequence that encodes an identical or essentially identical amino acid sequence as that of a reference sequence. Due to the degeneracy of the genetic code, whereby almost always more than one codon may code for each amino acid, nucleotide sequences encoding very closely related proteins may not share a high level of sequence identity. Moreover, different organisms have preferred codons for many amino acids, and different organisms or even different strains of the same organism, e.g., E coli strains, can have different preferred codons for the same amino acid. Thus, a first nucleotide acid sequence which encodes essentially the same polypeptide as a second nucleotide acid sequence is considered substantially identical to the second nucleotide sequence, even if they do not share a minimum percentage sequence identity, or would not hybridize to one another under stringent conditions. Additionally, it should be understood that with the limited exception of ATG, which is usually the sole codon for methionine, any sequence can be modified to yield a functionally identical molecule by standard techniques, and such modifications are encompassed by the present disclosure. As described herein below, the present disclosure specifically contemplates protein variants of a native protein, which have amino acid sequences having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity to a native nucleotide sequence.
The degree of sequence identity between two amino acid sequences may be determined using the BLASTp algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which an identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
One of skill will recognize that polypeptides may be “substantially similar”, in that an amino acid may be substituted with a similar amino acid residue without affecting the function of the mature protein. Polypeptide sequences which are “substantially similar” share sequences as noted above except that residue positions, which are not identical, may have conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
A nucleic acid encoding a peptide, polypeptide or protein may be obtained by screening selected cDNA or genomic libraries using a deduced amino acid sequence for a given protein. Conventional procedures using primer extension procedures, as described for example in Sambrook et al, can be used to detect precursors and processing intermediates.
B. Virus-Like Particles (VLPs) Composed of a Capsid Enclosing a Cargo Molecule
The methods and compositions described herein are the result in part of the appreciation that certain viral capsids can be prepared and/or used in novel manufacturing and purification methods to improve commercialization procedures for nucleic acids, peptides and proteins. The methods described herein use recombinant viral capsids which are resistant to readily available hydrolases, to enclose heterologous cargo molecules such as nucleic acids, peptides, or polypeptides including proteins.
The capsid may be a wild type capsid or a mutant capsid derived from a mutant capsid protein or a wild type capsid protein, provided that the capsid exhibits resistance to hydrolysis catalyzed by at least one hydrolase acting on peptide bonds when the capsids are contacted with the hydrolase. As used interchangeably herein, the phrases “resistance to hydrolysis” and “hydrolase resistant” refer to any capsid which, when present in a whole cell lysate also containing polypeptides which are cell lysis products and not enclosed in the capsids, and subjected to hydrolysis using a peptide bond hydrolase category E.C. 3.4 for a time and under conditions sufficient for at least 60, at least 70, at least 80, or at least 90 of every 100 individual polypeptides present in the lysate (which are cell lysis products and not enclosed in the capsids) to be cleaved (i.e. at least 60%, at least 70%, at least 80%), or at least 90% of all individual unenclosed polypeptides are cleaved), yet at least 60, at least 70, at least 80, or at least 90 of every 100 capsids present before such hydrolysis remain intact following the hydrolysis. Hydrolysis may be conducted for a period of time and under conditions sufficient for the average molecular weight of cell proteins remaining from the cell line following hydrolysis is less than about two thirds, less than about one half, less than about one third, less than about one fourth, or less than about one fifth, of the average molecular weight of the cell proteins before the hydrolysis is conducted. Methods may further comprise purifying the intact capsid remaining after hydrolysis, and measuring the weight of capsids and the weight of total dry cell matter before and after hydrolysis and purification, wherein the weight of capsids divided by the weight of total dry cell matter after hydrolysis and purification is at least twice the weight of capsids divided by the weight of total dry cell matter measured before the hydrolysis and purification. The weight of capsids divided by the weight of total dry cell matter after hydrolysis and purification may be at least 10 times more than, preferably 100 times more than, more preferably 1,000 times more than, and most preferably 10,000 times more than the weight of capsids divided by the weight of total dry cell matter measured before such hydrolysis and purification.
Hydrolases are enzymes that catalyze hydrolysis reactions classified under the identity number E.C. 3 by the Enzyme Commission. For example, enzymes that catalyze hydrolysis of ester bonds have identity numbers starting with E.C. 3.1. Enzymes that catalyze hydrolysis of glycosidic bonds have identity numbers starting with E.C. 3.2. Enzymes that catalyze hydrolysis of peptide bonds have identity numbers starting with E.C. 3.4. Proteases, which are enzymes that catalyze hydrolysis of proteins, are classified using identity numbers starting with E.C. 3.4, including but not limited to Proteinase K and subtilisin. For example, Proteinase K has identity number E.C. 3.4.21.64. The present disclosure encompasses VLPs with capsids which are resistant, in non-limiting, example, Proteinase K, Protease from Streptomyces griseus , Protease from Bacillus licheniformis , pepsin and papain, and methods and processes of using such VLPs.
The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology also recommends naming and classification of enzymes by the reactions they catalyze. Their complete recommendations are freely and widely available, and for example can be accessed online at http://enzyme.expasy.org and, www.chem.qmul.ac.uk/iubmb/enzyme/, among others. The IUBMB developed shorthand for describing what sites each enzyme is active against. Enzymes that indiscriminately cut are referred to as broadly specific. Some enzymes have more extensive binding requirements so the description can become more complicated. For an enzyme that catalyzes a very specific reaction, for example an enzyme that processes prothrombin to active thrombin, then that activity is the basis of the cleavage description. In certain instances the precise activity of an enzyme may not be clear, and in such cases, cleavage results against standard test proteins like B-chain insulin are reported.
The capsids can be further selected and/or prepared such that they can be isolated and purified using simple isolation and purification procedures, as described in further detail herein. For example, the capsids can be selected or genetically modified to have significantly higher hydrophobicity than a surrounding matrix as described herein, so as to selectively partition into a non-polar water-immiscible phase into which they are simply extracted. Alternatively, a capsid may be selected or genetically modified for improved ability to selectively crystallize from solution.
Use of simple and effective purification processes using the capsids is enabled by the choice of certain wild type capsids, or modifications to the amino acid sequence of capsid proteins comprising the wild type capsids, such that the capsid exhibits resistance to hydrolysis catalyzed by at least one hydrolase acting on peptide bonds as described herein above. Such wild type capsids, such as the wild type MS2 capsid, can be used in a purification process in which certain inexpensive enzymes such as Proteinase K or subtilisin are used for proteolysis. A non-limiting example is the Enterobacteria phage MS2 capsid protein, encoded by nucleic acid sequence SEQ ID NO: 2 producing amino acid sequence SEQ ID NO: 3. A non-limiting example is the Enterobacteria phage MS2 wild type genome (SEQ ID NO: 1) MS2 wild type coat protein DNA sequence (SEQ ID NO: 2); and MS2 wild type coat protein amino acid sequence (SEQ ID NO: 3).
Surprisingly, the unmodified, wild type MS2 capsid though lacking an envelope is resistant to a variety of category E.C. 3.4 hydrolases, including but not limited to Proteinase K and subtilisin, such that a highly purified VLP composition comprising the capsid, which may contain a cargo molecule, can be prepared from a whole cell lysate. Accordingly, the present disclosure provides VLPs comprising viral capsids comprising the wild type MS2 capsid protein, and/or capsid proteins sharing homology with wild type MS2 capsid proteins, which viral capsids encapsidate the cargo molecule. The cargo molecule may comprise one or more heterologous nucleic acids, peptides, polypeptides or proteins. These VLPs can then be isolated and purified from a whole cell lysate after a hydrolysis step using a category E.C. 3.4 hydrolase, to produce a composition of VLPs of high purity, for example at least 60%, at least 70%, a least 80%, or at least 85% by weight VLPs. Compositions having a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and 98% by weight of VLPs are expressly contemplated.
The present disclosure encompasses a composition comprising: a) a plurality of virus-like particles each comprising a wild type viral capsid and at least one target heterologous cargo molecule enclosed in the wild type viral capsid; and b) one or more cell lysis products present in an amount of less than 40 grams, less than 30 grams, less than 20 grams, less than 15 grams, less than 10 grams, and preferably less than 9, 8, 7, 6, 5, 4, 3, more preferably less than 2 grams, and still more preferably less than 1 gram, for every 100 grams of capsid present in the composition, wherein the cell lysis products are selected from proteins, polypeptides, peptides and any combination thereof. Subsequently the cargo molecules can be readily harvested from the capsids. Accordingly, such compositions are highly desirable for all applications where high purity and/or high production efficiency is required.
Hydrolase resistant capsids as described herein may be used to enclose different types of cargo molecules to form a virus-like particle. The cargo molecule can be but is not limited to any one or more oligonucleotide or oligoribonucleotide (DNA, RNA, LNA, PNA, siRNA, shRNA, sshRNA, lshRNA or miRNA, or any oligonucleotide comprising any type of non-naturally occurring nucleic acid), any peptide, polypeptide or protein. A cargo molecule which is an oligonucleotide or oligoribonucleotide may be enclosed in a capsid with or without the use of a linker. A capsid can be triggered for example to self-assemble from capsid protein in the presence of nucleotide cargo, such as an oligoribonucleotide. In non-limiting example, a capsid as described herein may enclose a target heterologous RNA strand, such as for example a target heterologous RNA strand containing a total of between 1,800 and 2,248 ribonucleotides, including the 19-mer pack site from Enterobacteria phage MS2, such RNA strand transcribed from a plasmid separate from a plasmid coding for the capsid proteins, as described by Wei, Y. et al. (2008) J. Clin. Microbiol. 46: 1734-1740.
RNA interference (RNAi) is a phenomenon mediated by short RNA molecules such as siRNA molecules, which can be used for selective suppression of a target gene of interest, and has multiple applications in biotechnology and medicine. For example, short RNA molecules can be employed to target a specific gene of interest in an organism to obtain a desirable phenotype. Short RNA molecules, including siRNA, are however easily degraded by ubiquitous enzymes called RNAses. Capsids, such as those described herein, protect encapsidated RNA from enzymatic degradation.
One or more RNA sequences can also be encapsidated into a viral capsid, either wild type or genetically modified, which has been modified to insert an external peptide tag, to deliver a protein or drug molecule to a specific class of cell. Wild type capsids may also be genetically modified to insert external peptide sequences acting as ligands for certain surface protein cell receptors can be advantageously used to encapsidate short RNA sequences aimed at inducing RNAi in specific target cells. Such compositions are much simpler, less expensive and more reliably manufactured than current alternatives for RNA delivery.
VLPs as described herein may alternatively enclose at least one target peptide, polypeptide or protein within a capsid. When the target heterologous cargo molecule is a peptide, polypeptide or protein, an oligonucleotide linker can be used to couple the target heterologous cargo molecule and the viral capsid. A cargo molecule which is a peptide, polypeptide or protein, preferably is packaged in a capsid using a linker. The packaging process is promoted by the linker, consisting of a short RNA aptamer sequence, which forms a link between the coat protein and a peptide tag fused to the target cargo molecule. (See Fiedler, J. et al, RNA-Directed Packaging of Enzymes within Virus-like Particles, Angew. Chem. Int. Ed. 49: 9648-9651 (2010)). The oligonucleotide linker may consist of DNA, RNA, LNA, PNA, and the like. The linker is for example a 50- to 100-mer having a short sequence, for example about 20 nt long, at a first end with binding specificity for the inside of the capsid coat, and another sequence, for example about 70 nt long, at the second, opposite end which has a binding specificity for the cargo peptide, polypeptide or protein. Alternatively, a capsid as described herein may enclose at least one target protein N-terminally tagged with a peptide able to non-covalently bind to an aptamer- and capsid pack sequence-containing RNA strand, for example an N-terminal tag and aptamer- and pack sequence-containing RNA strand as described by Fiedler, J. et al. (2010).
VLPs as described herein may be assembled by any available method(s) which produces a VLP with an assembled, hydrolase resistant capsid encapsidating one or more cargo molecule(s). For example, capsids and cargo molecules may be co-expressed in any expression system. Recombinant DNA encoding one or more capsid proteins, one or more cargo molecule(s) can be readily introduced into the host cells, e.g., bacterial cells, plant cells, yeast cells, fungal cells, and animal cells (including insect and mammalian) by transfection with one or more expression vectors by any procedure useful for introducing such a vector into a particular cell, and stably transfecting the cell to yield a cell which expresses the recombinant sequence(s).
The host cell is preferably of eukaryotic origin, e.g., plant, mammalian, insect, yeast or fungal sources, but non-eukaryotic host cells may also be used. Suitable expression systems include but are not limited to microorganisms such as bacteria {e.g., E. coli ) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequences for the VLP elements. In non-limiting example, for VLPs using the MS2 capsid protein, expression in E. coli is a suitable expression system.
The present disclosure expressly contemplates plant cells which have been transformed using a nucleic acid construct as described herein, and which expresses a capsid coat protein and cargo molecule. Means for transforming cells including plant cells and preparing transgenic cells are well known in the art. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments can be used to transform cells and will as generally recognized include promoters, enhancers, and/or polylinkers. Transgenic cells specifically contemplated include transgenic plant cells including but not limited to cells obtained from corn, soybean, wheat, vegetables, grains, legumes, fruit trees, and so on, or any plant which would benefit from introduction of a VLP as described herein. Also contemplated are plants, plant tissue obtained from cells transformed as described herein, and the seed or progeny of the plant or plant tissue.
Expression of assembled VLPs can be obtained for example by constructing at least one expression vector including sequences encoding all elements of the VLP. Sometimes two vectors are used, a first vector which includes a sequence encoding the cargo molecule(s); and a second vector which includes a sequence encoding the capsid protein. In an exemplary process for generating exemplary VLPs including siRNA, two vectors may be co-expressed in the host cell for generation of the VLP, as further detailed in the Examples. Methods and tools for constructing such expression vectors containing the coding sequences and transcriptional and translational control sequences are well known in the art. Vector(s) once constructed are transferred to the host cells also using techniques well known in the art, and the cells then maintained under culture conditions for a time sufficient for expression and assembling of the VLP's to occur, all using conventional techniques. The present disclosure thus encompasses host cells containing any such vectors, and cells which have been transformed by such vectors, as well as cells containing the VLP's.
When the VLP's have been expressed and assembled in the host cell they may be isolated and purified using any method known in the art for virus purification. For example, the cells can be lysed using conventional cell lysis techniques and agents, and the cell lysate subjected to hydrolysis using at least one peptide bond hydrolase category E.C. 3.4 such as but not limited to Proteinase K or subtilisin. Intact capsids remaining in the cell lysate following hydrolysis can be removed and purified using conventional protein isolation techniques.
Purification of capsids, VLPs or proteins may also include methods generally known in the art. For example, following capsid expression and cell lysis, the resulting lysate can be subjected to one or more isolation or purification steps. Such steps may include for example enzymatic lipolysis, DNA hydrolysis, and proteolysis steps. A proteolysis step may be performed for example using a blend of endo- and exo-proteases. For example, after cell lysis and hydrolytic disassembly of most cell components, such capsids with their cargo molecules can be separated from surrounding matrix by extraction, for example into a suitable non-polar water-immiscible solvent, or by crystallization from a suitable solvent. For example, hydrolysis and/or proteolysis steps transform contaminants from the capsid that are contained in the lysate matrix into small, water soluble molecules. Hydrophobic capsids may then be extracted into an organic phase such as 1,3-bis(trifluoromethyl)benzene. Purification of capsids, VLPs or proteins may include for example at least one liquid-liquid extraction step, at least one fractional precipitation step, at least one ultrafiltration step, or at least one crystallization step. A liquid-liquid extraction may comprise for example use of an immiscible non-aqueous non-polar solvent, such as but not limited to benzene, toluene, hexane, heptane, octane, chloroform, dichloromethane, or carbon tetrachloride. Purifying may include at least one crystallization step. Use of one or more hydrolytic steps, and especially of one or more proteolytic steps, eliminates certain problems observed with current separation processes used for cargo molecules, which are mainly result from the large number and varying degree of binding interactions which take place between cargo molecules and components derived from the cell culture in which they are produced. The capsids described herein resist hydrolytic steps such that the matrix which results after hydrolysis includes intact capsids which safely partition any cargo molecules from the surrounding matrix, thereby interrupting the troublesome binding interactions which interfere with current purification processes.
Following purification, the capsid can be opened to obtain the cargo molecule, which maybe a protein or polypeptide, a peptide, or a nucleic acid molecule as described herein. Capsids can be opened using any one of several possible procedures known in the art, including for example heating in an aqueous solution above 50° C.; repeated freeze-thawing; incubating with denaturing agents such as formamide; or by a combination of any of these procedures.
Capsids which are resistant to hydrolases and useful in the VLPs and methods according to the present disclosure can also be variants of, or derived from the wild type MS2 capsid. Capsid proteins may comprise, for example, at least one substitution, deletion or insertion of an amino acid residue relative to the wild type MS2 capsid protein amino acid sequence. Such capsid proteins may be naturally occurring variants or can be obtained by genetically modifying the MS2 capsid protein using conventional techniques, provided that the variant or modified capsid protein forms a non-enveloped capsid which is resistant to hydolysis catalyzed by a peptide bond hydrolase category E.C. 3.4 as described herein.
Genetically modified MS2 capsid proteins which can assemble into capsids which are resistant to hydrolysis as described herein can be engineered by making select modifications in the amino acid sequence according to conventional and well-known principles in physical chemistry and biochemistry as described herein and in the Examples herein below.
It is common knowledge for example that the shape or global fold of a functional protein is determined by the amino acid sequence of the protein, and that the fold defines the protein's function. The global fold is comprised of one or more folding domains. When more than one folding domain exists in the global fold, the domains generally bind together, loosely or tightly along a domain interface. The domain fold can be broken down into a folding core of tightly packed, well-defined secondary structure elements which is primarily responsible for the domain's shape and a more mobile outer layer typically comprised of turns and loops whose conformations are influenced by interactions with the folding core as well as interactions with nearby domains and other molecules, including solvent and other proteins. An extensive public domain database of protein folds, the Structural Classification of Proteins (SCOP) database (Alexey G Murzin, Curr Opin Struct Biol (1996) 6, 386-394) of solved protein structures in the public domain is maintained online at http://scop.berkeley.edu and regularly expanded as new solved structures enter the public domain (Protein Data Bank (F. C. Bernstein, T. F. Koetzle, G. J. Williams, E. E. Meyer Jr., M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi, M. Tasumi, “The Protein Data Bank: A Computer-based Archival File For Macromolecular Structures,” J. of. Mol. Biol, 112 (1977): 535), http://www.rcsb.org) database. Members of a family which are evolutionarily distant, yet have the same shape and very similar function, commonly retain as few as 30% identical residues at topologically and/or functionally equivalent positions. In some families, sequences of distant members have as few as 20% of their residues unchanged with respect to each other, e.g. levi- and alloleviviridae capsid proteins. Further, the fold and function of a protein is remarkably tolerant to change via directed or random mutation, even of core residues (Peter O. Olins, S. Christopher Bauer, Sarah Braford-Goldberg, Kris Sterbenz, Joseph O. Polazzi, Maire H. Caparon, Barbara K. Klein, Alan M. Easton, Kumnan Paik, Jon A. Klover, Barrett R. Thiele, and John P. McKearn (1995) J Biol Chem 270, 23754-23760; Yiqing Feng, Barbara K. Klein and Charles A. McWherter (1996), J Mol Biol 259, 524-541; Dale Rennell, Suzanne E. Bouvier, Larry W. Hardy and Anthony R. Poteetel (1991) J Mol Biol 222, 67-87), insertion/deletion of one or more residues (Yiqing Feng, Barbara K. Klein and Charles A. McWherter (1996), J Mol Biol 259, 524-541), permutation of the sequence (Multi-functional chimeric hematopoietic fusion proteins between sequence rearranged c-mpl receptor agonists and other hematopoietic factors, U.S. Pat. No. 6,066,318), concatenation via the N- or C-terminus or both (to copies of itself or other peptides or proteins) (Multi-functional chimeric hematopoietic fusion proteins between sequence rearranged g-csf receptor agonists and other hematopoietic factors, US20040171115; Plevka, P., Tars, K., Liljas, L. (2008) Protein Sci. 17: 173) or covalent modification, e.g., glycosylation, pegylation, SUMOylation or the addition of peptidyl or nonpeptidyl affinity tags as long as the residues critical to maintaining the fold and/or function are spared.
VLPs according to the present disclosure and as used in any of the methods and processes, thus encompass those comprising a capsid protein having at least 15%, 16%, 21%, 40%, 41%, 52%, 53%, 56%, 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). Such VLPs include for example a VLP comprising a capsid protein having at least 52% sequence identity with SEQ ID NO: 3 as described above. Also included is a VLP comprising a capsid protein having at least 53% sequence identity to SEQ ID NO:3, which can be obtained substantially as described above but not disregarding the FR capsid sequence, representing 53% sequence identity to wild-type enterobacteria phage MS2 capsid protein (SEQ ID NO:3). Also included is a VLP comprising a capsid protein having at least 56% sequence identity to SEQ ID NO:3, when it is considered that when the structures identified as 1AQ3 (van den Worm, S. H., Stonehouse, N. J., Valegard, K., Murray, J. B., Walton, C, Fridborg, K., Stockley, P. G., Liljas, L. (1998) Nucleic Acids Res. 26: 1345-1351), 1GAV (Tars, K., Bundule, M., Fridborg, K., Liljas, L. (1997) J. Mol. Biol. 271: 759-773), 1FRS (Liljas, L., Fridborg, K., Valegard, K., Bundule, M., Pumpens, P. (1994) J. Mol. Biol. 244: 279-290) and 2VTU (Plevka, P., Tars, K., Liljas, L. (2008) Protein Sci. 17: 1731) (Protein Data Bank identifiers described above), only 56% of the sequence positions have identical sequence and topologically equivalent positions with respect to the backbone overlays when all three sequences are considered together. Also included is a VLP comprising a capsid protein having at least 59% sequence identity to SEQ ID NO:3, when it is considered that the sequence of the MS2 viral capsid protein compared to that of the GA viral capsid protein is 59%. Also included is a VLP comprising a capsid protein having at least 86% sequence identity to SEQ ID NO:3, when it is considered that the sequence of the MS2 viral capsid protein compared to that of the FR capsid protein is 86%. VLPs according to the present disclosure thus encompass those comprising a capsid protein having at least 15%, 16%, or 21% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) based on a valid structure anchored alignment.
A VLP may thus comprise any of the MS2 capsid protein variants as described herein. Genetically modified capsid proteins consistent with those described herein can be produced for example by constructing at least one DNA plasmid encoding at least one capsid protein having at least one amino acid substitution, deletion or insertion relative to the amino acid sequence of the wild type MS2 capsid protein, making multiple copies of each plasmid, transforming a cell line with the plasmids; maintaining the cells for a time and under conditions sufficient for the transformed cells to express and assemble capsids encapsidating nucleic acids; lysing the cells to form a cell lysate; subjecting the cell lysate to hydrolysis using at least one peptide bond hydrolase, category E.C. 3.4; and removing intact capsids remaining in the cell lysate following hydrolysis to obtain capsids having increased resistance to at least one hydrolase relative to the wild type capsid protein. Following purification of the resulting, intact capsids, an amino acid sequence for each capsid protein may be determined according to methods known in the art.
The specialized capsids described herein can be used in research and development and in industrial manufacturing facilities to provide improved yields, since the purification processes used in both settings have the same matrix composition. Having such same composition mainly depends on using the same cell line in both research and development and manufacturing processes. However, differences in matrix composition due to using different cell lines are greatly reduced after proteolytic steps used in both research and development and manufacturing stages. This feature enables use of different cell lines in both stages with a minimal manufacturing yield penalty.
EXAMPLES
The following non-limiting examples are included to illustrate various aspects of the present disclosure. It will be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the Applicants to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the instant disclosure, appreciate that many changes can be made in the specific examples described, while still obtaining like or similar results, without departing from the scope of the invention. Thus, the examples are exemplary only and should not be construed to limit the invention in any way. To the extent necessary to enable and describe the instant invention, all references cited are herein incorporated by reference.
Example A
Propagation of MS2 Bacteriophage
MS2 bacteriophage (ATCC No. 15597-B 1, from American Type Culture Collection, Rockville, Md.) and its E. coli host (ATCC No. 15669) were obtained from ATCC and propagated using the procedure described by Strauss and Sinsheimer (1963) J. Mol. Biol 7:43-54 J. Mol. Biol 7:43-54. Results are plotted in FIG. 1 . Optical Density (OD) at 600 nm and pH were followed during the reaction. ODi represents OD immediately after inoculation with host. Infection was done at 2.3 hours. Ln(OD/ODi) was plotted on the left axis (full diamonds) and pH was plotted on the right axis (open squares). This experiment was ended 5.3 hours after inoculation with host. Lysate obtained was centrifuged at 2,000 g and filtered through a 0.2 μm membrane to eliminate remaining bacteria and bacterial debris.
Example B
Purification of MS2 Bacteriophage Using Proteinase K and Ultrafiltration
Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 2 . Eight milliliters of lysate obtained at end of Example A (sample in Lane 1, FIG. 2 ) was filtered through a 300 kDa membrane (Vivaspin 2, from Sartorius Stedim, Bohemia, N.Y.) and the filtrate was filtered through a 100 kDa membrane, from which 1 mL of retentate was obtained (sample in Lane 2, FIG. 2 ). This retentate was divided in two equal parts. To one half (control) 206 μL, 20 mM CaCl 2 aqueous solution at pH=7.5 were added. To the second half (Proteinase) 0.15 mg Proteinase K (Sigma Aldrich, St. Louis, Mo.) dissolved in 206 μL, 20 mM CaCl 2 aqueous solution at pH=7.5 was added. Both tubes were incubated at 37° C. and after 1 hour they were placed in an ice-water bath. Samples were then taken and analyzed: control sample in Lane 3, FIG. 2 , and Proteinase sample in Lane 5, FIG. 2 . Each product was then diluted to 2 mL with deionized (DI) water and filtered through a 100 kDa membrane. Each retentate (150 μL) was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration was repeated one more time for each product. Samples of each retentate were then taken and analyzed: control sample in Lane 4, FIG. 2 , and Proteinase sample in Lane 6, FIG. 2 . Band at 14 kDa corresponds to MS2 bacteriophage's coat protein. Band at 30 kDa corresponds to Proteinase K. Product from control experiment yields a highly impure phage. Product from the Proteinase experiment yields a product containing phage with purity higher than 99%.
Example C
Degradation of MS2 Bacteriophage
Treatment of MS2 bacteriophage was conducted as follows. Samples were taken during treatment and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 3 . Four milliliters of lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. A sample of the aqueous solution after extraction with Freon 11 was taken and analyzed (sample in Lane 1, FIG. 3 ). To the partially purified phage solution (130 μL) 370 μL, of 20 mM CaCl 2 aqueous solution was added. The mixture was incubated at 37° C. and after 1 hour it was placed in an ice-water bath. A sample was then taken and analyzed: sample in Lane 2, FIG. 3 . The incubation product was diluted to 2 mL with deionized (DI) water and filtered through a 100 kDa membrane. The retentate (150 μL) was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration of the retentate was repeated one more time. A sample of the retentate was then taken and analyzed: sample in Lane 3, FIG. 3 . Only weak bands at lower than 10 kDa were observed, indicating complete degradation of phage.
Example D
Purification of MS2 Bacteriophage Using Ultrafiltration
Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 3 . Four milliliters of lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. The aqueous solution containing partially purified phage was diluted to 2 mL with deionized water, filtered through a 300 kDa membrane and the filtrate was filtered through a 100 kDa membrane, from which 150 μl of retentate was obtained. The retentate was then diluted to 2 mL with deionized (DI) water and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (150 μL) was repeated one more time. A sample of the retentate was then taken and analyzed: sample in Lane 4, FIG. 3 . 370 μL of 20 mM CaCl 2 aqueous solution was added to the retentate (130 μL). The mixture was incubated at 37° C. and after 1 hour it was placed in an ice-water bath. A sample was then taken and analyzed: sample in Lane 5, FIG. 3 . The product was then diluted to 2 mL with deionized (DI) water and filtered through a 100 kDa membrane. The retentate (150 μL) was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration of the retentate was repeated one more time. A sample of the retentate was then taken and analyzed: sample in Lane 6, FIG. 3 . MS2's capsid protein, of 14 kDa, retained by a membrane through which permeate proteins with less than 100 kDa molecular weight is clearly visible, indicating the presence of intact MS2 capsids. The product obtained contained phage with purity higher than 99%.
Example E
Purification of MS2 Bacteriophage Using Proteinase K and Ultrafiltration
Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 4 . Four milliliters of lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. The aqueous solution containing partially purified phage was diluted to 2 mL with deionized water, filtered through a 100 kDa membrane, from which 150 μL of retentate was obtained. The retentate was then diluted to 2 mL with deionized (DI) water and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (150 μL) was repeated one more time. A sample of the retentate was then taken and analyzed: sample in Lane 1, FIG. 4 . 0.15 mg of Proteinase K dissolved in 370 μL of 20 mMCaCl 2 aqueous solution was added to the retentate (130 μL). The mixture was incubated at 37° C. and after 1 hour it was placed in an ice-water bath. A sample was then taken and analyzed: sample in Lane 2, FIG. 4 . The product was then diluted to 2 mL with deionized (DI) water and filtered through a 100 kDa membrane. The retentate (150 μL) was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration of the retentate was repeated one more time. A sample of the retentate was then taken and analyzed: sample in Lane 3, FIG. 4 . The product obtained contained phage with purity higher than 99%.
Example F
Purification of MS2 Bacteriophage Using Proteinase K, Precipitation at Acidic Conditions, Precipitation Using Ethanol at Basic and Acidic Conditions, and Ultrafiltration
Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 5 . Fifty milliliters of lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. A sample of the aqueous solution after extraction with Freon 11 was taken and analyzed (sample in Lane 1, FIG. 5 ). To the partially purified phage solution (1.2 mL) 0.9 mg of Proteinase K dissolved in 1.24 mL of 20 mM CaCl 2 aqueous solution was added. The mixture was incubated at 37° C. and after 1 hour 60 μL of 0.2M Phenylmethanesulfonyl fluoride (PMSF) solution in ethanol was added to inactivate Proteinase K. The mixture was then placed in an ice-water bath. A sample was taken and analyzed: sample in Lane 2, FIG. 5.0 . Six hundred and eighty microliters of 0.1% phosphoric acid aqueous solution was slowly added with vigorous agitation in an ice/water bath to bring the pH of the liquid to 4. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 30 min. The supernatant was allowed to reach room temperature and 130 μL of 1% NaOH was added to bring the pH of the liquid to 8. 0.81 mL of ethanol at room temperature was slowly added with vigorous agitation to bring the ethanol concentration in the liquid to 20%. The liquid was kept at room temperature for 30 min and centrifuged at 16,000 g at room temperature for 30 min. The supernatant was placed in an ice/water bath for 15 min and 1.3 mL of 1% acetic acid was slowly added at 0° C. with vigorous agitation to bring the pH of the liquid to 4. 1.5 mL of ethanol at 0° C. was slowly added with vigorous agitation to bring the ethanol concentration in the liquid to 34%. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 30 min. The pellet was resuspended in 200 μL of DI water and a 20 μL sample was taken and analyzed: Lane 3, FIG. 5 . The rest (180 μL) was diluted with DI water to 2 mL and filtered through 100 kDa membrane. The retentate (150 μL) was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration of the retentate was repeated one more time. A sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 5 . MS2's coat protein, of 14 kDa, retained by a membrane through which proteins with less than 100 kDa molecular weight are able to permeate, is clearly visible, consistent with the presence of intact MS2 capsids. A UV spectrum on the same retentate is shown in FIG. 6 , which is consistent with results published by G. F. Rohrmann and R. G. Krueger, (1970) J. Virol, 6(3):26 for pure MS2 phage. A Superdex 200 (GE Healthcare, Piscataway, N.J.) size exclusion chromatography was run on the same retentate using Tris-buffered saline at pH 7.4 and 150 mM NaCl. It showed 280 nm absorbance only at the void volume of the column. There was no absorbance in the elution volume for proteins of 600 kDa to 2 kDa. This test is consistent with intact phage particles. RNA was isolated from another sample of the same retentate using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, Calif.) and a DNA-free kit (Life Technologies, Grand Island, N.Y.), and reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Life Technologies). The presence or absence of three different sections of the MS2 genome was then interrogated in PCR experiments. The following pairs of primers were used, each primer named for the position of its first and last base in the MS2 genome, forward (F) and reverse (R) respectively:F1001 — 1021-R2180 — 2201, F1201 — 1223-R1979 — 2001, F1401 — 1426-R1680 — 1705. Platinum Taq DNA Polymerase High Fidelity (Life Technologies) was used for amplification. PCR products, analyzed in 1.5% agarose gel stained with Ethidium Bromide, as shown in FIG. 9 (1.2 kbp for primers F1201 — 1223-R1979 — 2001 in Lane 1, 800 bp for primers F1201 — 1223-R1979 — 2001 in Lane 2, and 304 bp for primers F1401 — 1426-R1680 — 1705 in Lane 3), were consistent with an intact MS2 bacteriophage genome. An infectivity test was also run on the same retentate as follows. Five microliters of retentate were used to infect 1 mL of bacterial culture as described in Example A at the point it reached OD (600 nm)=0.22. OD (600 nm) was 0.82 1 hour after infection and dropped to 0.21 after 2 additional hours, while during the same time a control sample attained OD (600 nm) of 0.82 1 hour after infection and 1.2 after 2 additional hours, as shown in FIG. 7 . This test showed a highly infectious phage in the retentate and therefore demonstrated that the purification processes used to isolate it did not compromise its integrity. In conclusion, the product obtained contained MS2 bacteriophage with purity higher than 99%.
Example G
Purification of MS2 Bacteriophage Using Different Exogenous Proteases, and Ultrafiltration
Purification of MS2 bacteriophage using different exogenous proteases was attempted substantially as described in Example E, with the exception that proteases other than Proteinase K were used. MS2 bacteriophage was successfully purified after proteolysis promoted by Protease from Bacillus licheniformis (P5380, Sigma Aldrich). However, a proteolysis reaction using Pepsin from porcine gastric mucosa (P6887, Sigma Aldrich) at pH of 6 was found to significantly degrade MS2 bacteriophage. On the other hand, proteolysis reactions using Papain from papaya latex (P3125, Sigma Aldrich) at pH 6 did not extensively degrade MS2 bacteriophage.
Example H
Production of VLP Capsids Encapsidating RNA Coding for MS2 Capsid Protein Attached to its Specific 19-Mer RNA Hairpin
Production of VLP capsids was conducted as follows. Samples were taken during the course of expression and SDS PAGE analysis was run on the samples to monitor capsid production. Results obtained are shown in FIG. 8 . A DNA sequence, SEQ ID NO: 4, encoding MS2's capsid protein and its specific RNA 19-mer PAC site was cloned into pDEST14 A252 plasmid (Life Technologies).
One Shot BL21(DE3) Chemically Competent E. coli (Life Technologies) cells were transformed using such plasmid. BL21(DE3) containing the plasmid were grown in 750 mL of LB medium containing ampicillin at 37° C., to OD (600 nm) equal to 0.8. A pre-induction sample was then taken and analyzed: sample in Lane 1, FIG. 8 . Isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich) was then added to a final concentration of 1 mM. Four hours post-induction cells were harvested by centrifugation at 3,000 g and 4° C. for 40 min. A sample was then taken and analyzed: sample in Lane 2, FIG. 8 .
Example I
Purification and Characterization of VLP Capsids Encapsidating RNA Coding for MS2 Capsid Protein Attached to its Specific 19-Mer RNA Hairpin
Purification of VLP capsids was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 8 . A fraction of the pellet from Example H equivalent to 115 mL of culture was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. To the partially purified VLP capsid solution (1.05 mL) 0.3 mg of Proteinase K dissolved in 1.05 mL of 20 mM CaCl 2 aqueous solution was added. The mixture was incubated at 37° C. and after 2.5 hours it was placed in an ice-water bath. A sample was then taken and analyzed: sample in Lane 3, FIG. 8 . Fifteen minutes afterwards, 0.14 mL of 1% phosphoric acid aqueous solution was slowly added with vigorous agitation in an ice/water bath to bring the pH of the liquid to 4.1. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. To the supernatant, kept at 0° C., 100 μL, of 1% NaOH was added to bring the pH of the liquid to 7.9. Five hundred microliters of ethanol at 0° C. was then slowly added with vigorous agitation to bring the ethanol concentration in the liquid to 20%. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. After adding 1% acetic acid to adjust the pH of the solution to 7, the supernatant was filtered through a Vivaspin 2 (Sartorius) 300 kDa membrane and the filtrate was filtered through a 100 kDa membrane, from which 150 μL of retentate was obtained. The retentate was then diluted to 2 mL with phosphate buffered saline and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (150 μL) was repeated four more times. A sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 8 . MS2's capsid protein, of 14 kDa, retained by a membrane through which proteins with less than 100 kDa molecular weight are able to permeate, is clearly visible, consistent with the presence of intact VLP capsids. RNA was isolated from another sample of the same retentate using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, Calif.) and a DNA-free kit (Life Technologies, Grand Island, N.Y.), and reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Life Technologies). The presence or absence of a section of the MS2 capsid protein was then interrogated in PCR experiments. The following pair of primers was used, each primer named for the position of its first and last base in the MS2 genome, forward (F) and reverse (R) respectively: F1401 — 1426-R1680 — 1705. Platinum Taq DNA Polymerase High Fidelity (Life Technologies) was used for amplification. The PCR product, analyzed in 2% agarose gel stained with Ethidium Bromide, as shown in FIG. 10 (304 bp in Lane 1; the leftmost Lane corresponds to 1 kb plus ladder from Life Technologies), was consistent with an intact MS2 capsid gene. In conclusion, the product obtained contained VLP capsids with purity higher than 99%.
Example J
Simple Precipitation with Ethanol for Purification of VLPs
Purification of VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 11 . One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. A sample was taken and analyzed: sample in Lane 1, FIG. 11 . A strong band at about 14 kDa was found, consistent with the capsid protein of MS2 phage. Other bands—impurities—mostly of higher molecular weight, represent about 27% of the sample weight. To the partially purified MS2 VLP solution (1.35 mL) 1.36 mL of 20 mM CaCl 2 aqueous solution was added and placed in an ice-water bath. Fifteen minutes afterwards, 50 μL of 10% acetic acid aqueous solution was added to bring the pH of the liquid to 4.1. Then, at the same temperature and with vigorous agitation, 1.44 mL of ethanol was slowly added. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. The pellet was suspended in 2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mMMgCl2 adjusted to pH 7.5. A sample was taken and analyzed by SDS PAGE: sample in Lane 2, FIG. 11 . Impurities in this sample represented about 24% of the sample weight. The diluted sample was filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 200 μL of retentate was obtained. The retentate was then diluted to 2 mL with the same buffer and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (200 μL) was repeated four more times. A sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 3, FIG. 11 . Impurities in this sample represented about 9.7% of the sample weight. In conclusion, the product obtained contained MS2 VLPs with purity higher than 90%.
Example K
Use of Proteinase K (PK) and Simple Precipitation with Ethanol for Purification of VLPs
Purification of VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 12 . One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. A sample was taken and analyzed: sample in Lane 1, FIG. 12 . A strong band at about 14 kDa was found, consistent with the capsid protein of MS2 phage. Other bands—impurities—mostly of higher molecular weight represent about 26% of the sample weight. To the partially purified MS2 VLP solution (1.35 mL) 0.6 mg of Proteinase K dissolved in 1.36 mL of 20 mM CaCl 2 aqueous solution was added. The mixture was incubated at 37° C. and after 2.5 hours placed in an ice-water bath. A sample was taken and analyzed by SDS PAGE: sample in Lane 2, FIG. 12 . Impurities in this sample represented about 14% of the sample weight. Fifteen minutes afterwards, about 50 μL of 10% acetic acid aqueous solution was added in an ice/water bath to bring the pH of the liquid to 4.1. Then, at the same temperature and with vigorous agitation, 1.54 mL of ethanol was slowly added. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. The pellet was suspended in 2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM MgCl 2 adjusted to pH 7.5. A sample was taken and analyzed by SDS PAGE: sample in Lane 3, FIG. 12 . Impurities in this sample represented about 10% of the sample weight. The diluted sample was filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 200 μL of retentate was obtained. The retentate was then diluted to 2 mL with the same buffer and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (200 μL) was repeated four more times. A sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 12 . Impurities in this sample represented about 5.1% of the sample weight. In conclusion, the product obtained contained VLPs with purity of about 95%.
Example L
Use of Constitutive Hydrolases, Fractional Precipitation with Ethanol, and Ultrafiltration for Purification of VLPs
Purification of VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 13 . One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. To the partially purified VLP solution (1.35 mL) 1.36 mL of 20 mM CaCl 2 aqueous solution was added. The mixture was incubated at 37° C. for 2.5 hours (to allow constitutive hydrolases to act) and afterwards was placed in an ice-water bath. A sample was taken and analyzed by SDS PAGE: sample in Lane 1, FIG. 13 . Impurities in this sample represented about 12% of the sample weight. Fifteen minutes afterwards, about 120 μL, of 1% sodium hydroxide aqueous solution was added in an ice/water bath to bring the pH of the liquid to 7.86. Then, at the same temperature and with vigorous agitation, 0.81 mL of ethanol was slowly added. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. About 100 of 10% acetic acid aqueous solution was slowly added to the supernatant with vigorous agitation in an ice/water bath to bring the pH of the liquid to 4.01. Then, at the same temperature and with vigorous agitation, 1.3 mL of ethanol was slowly added. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. The pellet was suspended in 2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM MgCl 2 adjusted to pH 7.5. The diluted sample was filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 200 μL of retentate was obtained. The retentate was then diluted to 2 mL with the same buffer and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (200 μL) was repeated four more times. A sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 3, FIG. 13 . Impurities in this sample represented about 4.7% of the sample weight. In conclusion, the product obtained contained VLPs with purity higher than about 95%.
Example M
Use of Proteinase K (PK), Fractional Precipitation with Ethanol, and Ultrafiltration for Purification of VLPs
Purification of VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 13 . One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol 7:43-54. To the partially purified VLP solution (1.35 mL) 0.3 mg of Proteinase K dissolved in 1.36 mL of 20 mM CaCl 2 aqueous solution was added. The mixture was incubated at 37° C. for 2.5 hours and afterwards was placed in an ice-water bath. A sample was taken and analyzed by SDS PAGE: sample in Lane 2, FIG. 13 . Impurities in this sample represented about 8.1% of the sample weight. Fifteen minutes afterwards, about 120 μL of 1% sodium hydroxide aqueous solution was added in an ice/water bath to bring the pH of the liquid to 7.86. Then, at the same temperature and with vigorous agitation, 0.81 mL of ethanol was slowly added. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. About 100 μL of 10% acetic acid aqueous solution was added to the supernatant in an ice/water bath to bring the pH of the liquid to 4.0. Then, at the same temperature and with vigorous agitation, 1.3 mL of ethanol was slowly added. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. The pellet was suspended in 2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM MgCl 2 adjusted to pH 7.5. The diluted sample was filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 200 μL of retentate was obtained. The retentate was then diluted to 2 mL with the same buffer and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (200 μL) was repeated four more times. A sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 13 . Impurities in this sample represented about 0.9% of the sample weight. In conclusion, the product obtained contained VLPs with purity higher than about 99%.
Example N
Use of Various Hydrolases, and Factional Precipitation with Ammonium Sulfate for Purification of VLPs
Purification of VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 14 . One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. A sample of the supernatant was taken and analyzed by SDS PAGE: sample in Lane 1, FIG. 14 . Impurities in this sample represented about 70% of the sample weight. Four other identical fractions of the pellet obtained from such experiment identical to Example H were processed in the same manner.
The five centrifuged cell lysates obtained, each 3.7 mL in volume, were further processed in five different manners, as follows. The first centrifuged cell lysate was placed in an ice-water bath for 15 minutes and 0.1 grams of ammonium sulfate was added. The mixture was vortexed until complete dissolution of ammonium sulfate was achieved. The liquid was kept at 0° C. for 2 hours and centrifuged at 16,000 g at 4° C. for 30 min. 0.4 grams of ammonium sulfate was added to the supernatant and vortexed until complete dissolution of ammonium sulfate was achieved. The liquid was kept at 0° C. for 2 hours and centrifuged at 16,000 g at 4° C. for 30 min. The purified VLPs pellet was suspended in 0.2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM MgCl 2 adjusted to pH 7.5. The second centrifuged cell lysate was incubated at 37° C. for five hours, placed in an ice-water bath for the same amount of time as the first centrifuged cell lysate and subsequently processed in identical manner as the first centrifuged cell lysate. One hundred and fifty micrograms of Proteinase K (Sigma Aldrich, St. Louis, Mo.) was added to the third centrifuged cell lysate which was then incubated at 37° C. for five hours, placed in an ice-water bath for the same amount of time as the first centrifuged cell lysate and subsequently processed in identical manner as the first centrifuged cell lysate. The fourth centrifuged cell lysate was incubated at 37° C. for two hours. 0.15 mg of Proteinase K was then added. It was incubated at 37° C. for an additional three hours, placed in an ice-water bath for the same amount of time as the first centrifuged cell lysate and subsequently processed in identical manner as the first centrifuged cell lysate.
Five hundred units of Benzonase® Nuclease (Sigma Aldrich, St. Louis, Mo.) and 35 units of Lipase from Candida rugosa (Sigma Aldrich, St. Louis, Mo.) was added to the fifth centrifuged cell lysate and incubated at 37° C. for one hour. 15 units of α-Amylase from Bacillus sp. (Sigma Aldrich, St. Louis, Mo.) was then added and incubated at 37° C. for one additional hour. 0.15 mg of Proteinase K was then added. The mixture was incubated at 37° C. for an additional three hours, placed in an ice-water bath for the same amount of time as the first centrifuged cell lysate and subsequently processed in identical manner as the first centrifuged cell lysate.
A sample was taken of the second centrifuged cell lysate after its 5 hours incubation and analyzed by SDS PAGE: sample in Lane 2, FIG. 14 . A sample was taken of the third centrifuged cell lysate after its 5 hours incubation and analyzed by SDS PAGE: sample in Lane 3, FIG. 14 . A sample was taken of the fourth centrifuged cell lysate after its 5 hours incubation and analyzed by SDS PAGE: sample in Lane 4, FIG. 14 . A sample was taken of the fifth centrifuged cell lysate after its 5 hours incubation and analyzed by SDS PAGE: sample in Lane 5, FIG. 14 .
A sample was taken of the purified VLPs suspension for the first centrifuged cell lysate and analyzed by SDS PAGE: sample in Lane 6, FIG. 14 . The product obtained contained VLPs with purity of about 88%. Protein concentration (Pierce® BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, Ill.) of this sample was 18.5 mg/mL Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.553 and OD-280 nm was 0.303. These measurements are consistent with RNA yield of about 9 mg per liter of culture.
A sample was taken of the purified VLPs suspension for the second centrifuged cell lysate and analyzed by SDS PAGE: sample in Lane 7, FIG. 14 . The product obtained contained VLPs with purity of about 75%. Protein concentration of this sample was 25.4 mg/mL. Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.784 and OD-280 nm was 0.453. These measurements are consistent with RNA yield of about 11 mg per liter of culture.
A sample was taken of the purified VLPs suspension for the third centrifuged cell lysate and analyzed by SDS PAGE: sample in Lane 8, FIG. 14 . The product obtained contained VLPs with purity of about 94.3%. Protein concentration of this sample was 21.0 mg/mL. Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.632 and OD-280 nm was 0.321. These measurements are consistent with RNA yield of about 10 mg per liter of culture.
A sample was taken of the purified VLPs suspension for the fourth centrifuged cell lysate and analyzed by SDS PAGE: sample in Lane 9, FIG. 14 . The product obtained contained VLPs with purity of about 95.6%. Protein concentration of this sample was 19.4 mg/mL. Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.666 and OD-280 nm was 0.353. These measurements are consistent with RNA yield of about 11 mg per liter of culture.
A sample was taken of the purified VLPs suspension for the fifth centrifuged cell lysate and analyzed by SDS PAGE: sample in Lane 10, FIG. 14 . The product obtained contained VLPs with purity of about 96%. Protein concentration of this sample was 19.8 mg/mL Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.661 and OD-280 nm was 0.354. These measurements are consistent with RNA yield of about 11 mg per liter of culture.
Example O
Isolation of RNA Encapsidated in VLPs Obtained in Example N
RNA encapsidated VLPs purified as described in Example N was extracted from each experiment using TRIzol® reagent according to the protocol supplied by the manufacturer (Life Technologies, Grand Island, N.Y.). RNA obtained was denatured by heating for 5 min at 95° C. in formamide and analyzed by electrophoresis in 17.6 cm×38 cm×0.04 cm (W, L, T) gels composed of 8% polyacrylamide, 8 M urea, 1.08% Tris base, 0.55% Boric acid, and 0.093% EDTA. The running buffer had the same concentrations of Tris base, Boric acid and EDTA as the gel. Power was delivered at about 40 W. Gels were stained using a 0.025% solution of Stains-All dye (Sigma-Aldrich, St. Louis, Mo.) in an aqueous mixture containing 25% formamide, 19% isopropanol and 15 mM Tris at pH 8. Results obtained are shown in FIG. 15 . Lane numbers for RNA electrophoresis in FIG. 15 refer to the same lane numbers for protein electrophoresis in FIG. 14 . A single RNA band can be observed in each lane, consistent with high purity RNA recovered in each case.
Example P
Production of VLPs Using a Transcript Coding for shRNA Against EGFP Flanked by a Long Hammerhead (HH) Ribozyme at its 5′ End and Another Long HH Ribozyme Attached to MS2 19-Mer RNA Hairpin at its 3′ End
Production of MS2 capsids was conducted as follows. The DNA sequence (SEQ ID NO: 5), encoding MS2 capsid protein was cloned into pDEST14 (Life Technologies) plasmid:
Construct T7-Rz4 (SEQ ID NO: 4) encodes aT7 promoter sequence upstream of an shRNA against EGFP flanked by a HH ribozyme designed to cut its 5′ end having 12 nucleotides hybridizing to the shRNA and a HH ribozyme designed to cut its 3′ end having 23 nucleotides hybridizing to the shRNA was cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of SEQ ID NO: 4 to form pT7-Rz4.
One Shot BL21(DE3) Chemically Competent E. coli (Life Technologies) cells were transformed with the two plasmids, one containing MS2 coat protein SEQ ID NO: 5 in pDEST14 and one containing the T7-Rz4 construct SEQ ID NO: 4 in pACYC184, and selecting for chloramphenicol and ampicillin resistant transformants. For capsid production these transformants were grown at 37° C. in 750 mL LB medium containing both ampicillin and chloramphenicol. When the culture density reached OD (600 nm)=0.8, isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich) was added to a final concentration of 1 mM. Cells were harvested 4 hours post-induction by centrifugation at 3,000 g and 4° C. for 40 min. A sample was taken prior to induction and at the time of harvest for analysis.
Example Q
Purification of VLPs Obtained in Example P
Purification of VLPs produced in Example P. was conducted as in Example N.
Example R
Isolation of RNA in VLPs Obtained in Example Q
RNA encapsidated in VLPs purified as described in Example Q were extracted using TRIzol® reagent according to the protocol supplied by the manufacturer (Life
Technologies, Grand Island, N.Y.). RNA obtained was denatured by heating for 5 min at 95° C. in formamide and analyzed by electrophoresis in Novex® denaturing 15% polyacrylamide TBE-Urea gels (Life Technologies) run at 70° C. RNA bands were visualized using 0.5 μs of Ethidium Bromide (Sigma-Aldrich, St. Louis, Mo.) per mL of aqueous solution. Results obtained are shown in lane 3, FIG. 16 . Lane 1 shows a set of molecular standards. Lane 2 shows a chemically synthesized shRNA 49 nucleotides long.
Example S
VLPs Obtained in Example Q are Resistant to Proteinase K from Engyodontium Album , Protease from Bacillus Licheniformis , Pepsin from Porcine Gastric Mucosa, and Papain from Papaya Latex
VLPs obtained from 250 mL of culture and purified as described in Example-Q were suspended in 400 μL 20 mM CaCl 2 aqueous solution at pH=7.5.
A 66 μL aliquote of this suspension was diluted to 0.25 mL with 20 mM CaCl 2 aqueous solution at pH=7.5 and incubated at 37° C. Samples were taken for protein concentration (Pierce® BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, Ill.) and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 3,086, and 4,656 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes IB, and 6 respectively. The same amount of protein was loaded in each lane (4 μg). This set of experiments was used as a negative control. Two μg Protease from Streptomyces griseus (Sigma Aldrich, St. Louis, Mo.) was diluted to 0.25 mL with 20 mM CaCl 2 aqueous solution at pH=7.5 and incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 361 and 324 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 1, and 7 respectively. The same amount of protein was loaded in each lane (4 μg). This set of experiments was used as another negative control.
Two μg of Protease from Streptomyces griseus was added to another 66 μL aliquote of the VLPs comprising MS2 capsids suspension, diluted to 0.25 mL with 20 mM CaCl 2 aqueous solution at pH=7.5 and incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 2,940, and 3,012 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 2, and 8 respectively. The same amount of protein was loaded in each lane (4 μg). This set of experiments was used to test the proteolytic stability towards Protease from Streptomyces griseus of MS2 capsids forming the VLPs. Less than 10% degradation was observed.
Another 66 aliquote of the VLPs comprising MS2 capsids suspension, diluted to 0.25 mL with 20 mM CaCl 2 aqueous solution at pH=7.5 was subjected to three cycles of heating to 95° C. for 10 minutes and cooling on wet ice for 10 min to achieve the disassembly of the VLPs. Two μg of Protease from Streptomyces griseus was then added to this suspension and was incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 2,601, and 3,033 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 3, and 9 respectively. The same amount of protein was loaded in each lane (4 μg). Disassembled particles were degraded to a significant extent by Protease from Streptomyces griseus . This set of experiments was used as a positive control.
Two μg of Protease from Streptomyces griseus dissolved in 0.002 mL of 20 mM CaCl 2 aqueous solution at pH=7.5 was added to 0.248 mL of bacterial cell lysate obtained from 41 mL of cell culture from example P and incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 3,192, and 4,837 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 4, and 10 respectively. The last lane of FIG. 17 , labeled L, shows untreated bacterial cell lysate. The same amount of protein was loaded in each lane (4 μg). More than 90% of proteins other than MS2 capsid protein were degraded by Protease from Streptomyces griseus . This set of experiments was used as another positive control.
This set of five experiments demonstrate that MS2 capsids form VLPs resistant to proteolysis by Protease from Streptomyces griseus.
Two μg Protease from Bacillus licheniformis (Sigma Aldrich, St. Louis, Mo.) was diluted to 0.25 mL with 10 mM Na acetate and 5 mM Ca acetate aqueous solution at pH=7.5 and incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 976, and 1,003 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 2B, and 7B respectively.
The same amount of protein was loaded in each lane (4 μg). This set of experiments was used as another negative control.
Two μg of Protease from Bacillus licheniformis was added to another 66 μL, aliquote of the VLPs comprising MS2 capsids suspension, diluted to 0.25 mL with 10 mM Na acetate and 5 mM Ca acetate aqueous solution at pH=7.5 and incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 3,144, and 3,727 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 3B, and 8B respectively. The same amount of protein was loaded in each lane (4 μg). This set of experiments was used to test the proteolytic stability towards Protease from Bacillus licheniformis of MS2 capsids forming the VLPs. Less than 10% degradation was observed.
Another 66 μL aliquote of the VLPs comprising MS2 capsids suspension, diluted to 0.25 mL with 10 mM Na acetate and 5 mM Ca acetate aqueous solution at pH=7.5 was subjected to three cycles of heating to 95° C. for 10 minutes and cooling on wet ice for 10 min to achieve the disassembly of the VLPs. Two μg of Protease from Bacillus licheniformis was then added to this suspension and was incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 1,769, and 1,785 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 4B, and 9B respectively. The same amount of protein was loaded in each lane (4 μg). Disassembled particles were degraded by Protease from Bacillus licheniformis . This set of experiments was used as a positive control.
Two μg of Protease from Bacillus licheniformis dissolved in 0.002 mL of 10 mM Na acetate and 5 mM Ca acetate aqueous solution at pH=7.5 was added to 0.248 mL of bacterial cell lysate obtained from 41 mL of cell culture from example P and incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 3,696, and 4,078 mg/L respectively. SDS PAGE analyses are shown in FIG. 17 , Lanes 6B, and 10B respectively. The last lane of FIG. 17 , labeled L, shows untreated bacterial cell lysate. The same amount of protein was loaded in each lane (4 μg). More than 90% of proteins other than MS2 capsid protein were degraded by Protease from Bacillus licheniformis . This set of experiments was used as another positive control.
This set of four experiments demonstrated that MS2 capsid proteins in VLPs are resistant to proteolysis by Protease from Bacillus licheniformis.
Three additional sets of equivalent experiments demonstrated that MS2 capsid proteins in VLPs are resistant to proteolysis by any of the following three proteases: Proteinase K from Engyodontium album , Pepsin from porcine gastric mucosa (CAS Number 9001-75-6), and Papain from papaya latex (CAS Number 9001-73-4) (Sigma-Aldrich, St. Louis, Mo.). Each protease was used according the manufacturer's instructions. Proteinase K was used at pH=7.5, Pepsin was used at pH=1.6, and Papain was used at pH=6.6.
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Processes and compositions to produce, package, and purify virus like particles containing heterologous cargo molecules utilizing self assembling proteins and protease treatment coupled with simple precipitation and filtration methods are described.
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TECHNICAL FIELD
[0001] The present invention relates to oily paclitaxel composition and formulation for transcatheter arterial chemoembolization (TACE) by solubilizing paclitaxel and the preparation method thereof. The present invention also relates to oily paclitaxel composition and formulation additionally comprising chemicals that prevent paclitaxel precipitation for prolonged preservation and the preparation method thereof.
BACKGROUND ART
[0002] TACE is a cancer treatment method that prevents the nutrition supplies to the cancer tissue by injecting embolizing materials and anticancer agents though the feeding artery of tumor while visualizing the operation process with contrast medium. Since the composition of the present invention solubilizes paclitaxel effectively, it can be used for TACE to treat hepatoma and other solid tumors.
[0003] The most widely used TACE is transcatheter arterial chemoembolization through hepatic artery for the treatment of hepatoma. The contrast medium serves as a visualization tool during and after the operation and also causes embolism in the tumor. The anticancer drugs such as doxorubicin (adriamycin), cisplatin and carboplatin are dissolved or suspended in oily contrast medium.
[0004] One of the most frequently used contrast media in TACE is iodized oils such as Lipiodol®. The suspension system comprising Lipiodol and above-mentioned anticancer drugs, however, is physically unstable and therefore has many limitations during the operation. The anticancer agents such as doxorubicin and epirubicin are used conventionally for the treatment of hepatoma in Radiology. Most of the anticancer agents, however, are water-soluble materials. Therefore, suspension type formulation, rather than oily solution, was used in TACE (Yoshihiro Katagiri et al., Cancer Chemother. Pharmacol 1989, 23, 238-242). The suspension type formulation, however, cannot be stored for a prolonged period of time since particles aggregate upon storage.
[0005] To overcome this stability problem, the anticancer drug is dissolved in the aqueous contrast medium before dispersing the aqueous phase in the oily contrast medium such as Lipiodol®. In other words, the anticancer drug is dissolved in the aqueous contrast medium and mixed with oily contrast medium by pumping method just before administering to a patient. To maximize the stability of the emulsion-, aqueous contrast media such as Urografin (specific gravity 1.328-1.332) or lopamiro (specific gravity 1.17-1.41) are used since they have similar specific gravities with Lipiodol (1.275-1.290) (Takashi Kanematsu et al., Journal of surgical oncology 1984, 25, 218-226, Takafumi Ichida et al., Cancer Chemother. Pharmacol 1994, 33, 74-78). However, only a transient emulsion that phase-separates in a few minutes after preparation is produced by the above method. Unstable emulsion system does not provide enough embolizing effect. In reality, phase separation can be observed inside the catheter during the operation. When this unstable emulsion is administered, adriamycin is absorbed immediately to the tissue and therefore does not provide an effect of sustained delivery of anticancer drug.
[0006] One of the ideal hepatoma treatments uses a synthetic polymeric anticancer agent, poly(styrene-co-maleic acid)-conjugated neocarzinostatin (SMANCS). SMANCS can be solubilized in Lipiodol since it has both hydrophilic and hydrophobic properties (Konno, T. and Maeda, H., Targetting chemotherapy of hepatocellular carcinoma. Neoplasms of the liver, Eds. Okuda, K., and Ishak, K. G., Springger-Verlag, Berlin, P 343-352). Even though SMANCS/Lipiodol formulation has solved the stability problems of adriamycin/Lipiodol formulation, SMANCS/Lipiodol formulation is not widely used due to the high price and severe toxic side effects.
[0007] On the other hand, paclitaxel, an anticancer agent, shows excellent cytotoxicity to ovarian cancer, breast cancer, esophagus cancer, melanoma and leukemia. Paclitaxel has been commerciallized as intravenous injection Taxol® by Bristol-Myers Squibb Company.
[0008] Paclitaxel is one of the water-insoluble drug and therefore the solubilization technique has been developed along with the drug itself. One of the examples in the solubilization technique is the use of solubilizing agent for systemic administration such as intravenous injection. The above-mentioned Taxol® uses Cremophor EL (polyoxyethylene 35 castor oil) and ethanol as solubilizing agents. Taxol® is a pre-concentrate type emulsion formulation that forms microemulsion spontaneously when dispersed in excess amount of water (U.S. Pat. No. 5,438,072). It is known, however, that solubilizing agent in Taxoli causes toxic side effects. Therefore, many studies are performed to develop new paclitaxel formulations with high anticancer activity and low toxic effects.
SUMMARY OF THE INVENTION
[0009] The object of the present invention is to use paclitaxel in transcatheter arterial chemoembolization by solubilizing paclitaxel.
[0010] Therefore, one of the objects of the present invention is to provide a new composition of paclitaxel that can solubilize paclitaxel.
[0011] More particularly, the object of the present invention is to provide an oily paclitaxel formulation that can be used for the treatment of solid tumors by transcatheter arterial chemoembolization
[0012] Also, another object of the present invention is to provide an oily paclitaxel formulation that can maintain the original composition stably during the transcatheter arterial chemoembolization process.
[0013] Another object of the present invention is to provide a preparation process of the above composition of paclitaxel.
[0014] Another object of the present invention is to provide a paclitaxel composition for transcatheter arterial chemoembolization comprising an additional component to prevent paclitaxel precipitation.
DETAILED DESCRIPTION OF THE INVENTION
[0015] While trying to find a paclitaxel formulation that can be used in transcatheter arterial chemoembolization to meet the above mentioned expectations, the present inventors have found unexpectedly that paclitaxel is soluble in the oily contrast medium to form a homogeneous single phase viscous oily liquid of viscosity ranging 40˜180 centipoises (cP).
[0016] Also the paclitaxel/oily contrast medium composition can be stored for a long period of time without changing the composition since it is chemically and physically stable. This paclitaxel/oily contrast medium composition has superior physical properties to the conventional Lipiodol formulations using water-soluble anticancer drugs such as doxorubicin. The paclitaxel/oily contrast medium composition of the present invention has similar physical characteristics to SMANCS/Lipiodol formulation. In contrast to the SMANCS/Lipiodol formulation that is too expensive and has toxic side effects, however, the paclitaxel/lipiodol composition uses two relatively inexpensive raw materials and is very easy to prepare reducing the production cost. Also the obtained formulation is stable upon storage.
[0017] The oily paclitaxel formulation of the present invention can maintain the original composition stably during the transcatheter arterial chemoembolization process while the conventional Lipiodol/lopamiro/doxorubicin formulation phase-separated immediately after mixing. Therefore, the paclitaxel/oily contrast medium formulation of the present invention can deliver the anticancer drug in a sustained release fashion to the tumor. Also, the formulation can be stored for a long period of time due to its excellent stability. Moreover, the result described hereinbelow shows that the formulation of the present invention has an excellent embolization effect and anticancer activity when TACE was performed through hepatic artery in an animal model. Therefore, it is expected that the formulation of the present invention can be used in TACE.
[0018] Even though the most typical TACE is TACE through hepatic artery, it can be applied to a variety of solid tumors. For instance, SMANCS/Lipiodol formulation has been used for the targeted therapy of renal cancer by performing TACE through renal artery (K. Tsuchiya, Tumor-targeted chemotherapy with SMANCS in Lipiodol for renal cell carcinoma: longer survival with larger size tumors. Urology. 2000 April; 55(4):495-500).
[0019] The object of the present invention is to use paclitaxel in transcatheter arterial chemoembolization by solubilizing paclitaxel.
[0020] An example of an oily contrast medium that can be used in preparing the paclitaxel/oily contrast medium composition is iodized oil. The iodized oils include iodized poppy seed oil such as Lipiodol (Laboratoire Guerbet, France), Ethiodol (Savage Laboratories, Melville, N.Y.) and iodized soybean oil. The iodized soybean oil is described by Ma Tai (The effect of oral iodized oil on prevention and treatment of endemic goiter. Chinese Med. J. 61 (9):533, 1981).
[0021] The iodine content of the iodized oil used as oily contrast medium in the present invention is preferably 30˜50% by weight. More preferably, the iodine content is 35˜45% by weight. It is the most preferable to use Lipiodol as the oily contrast medium.
[0022] The amount of paclitaxel in the paclitaxel/oily contrast medium of the present invention is 0.0001˜10 mg per 1 ml of oily contrast medium. When the amount of paclitaxel exceeds 10 mg per 1 ml of oily contrast medium, it is not preferable since the excess paclitaxel precipitates. On the other hand, anticancer activity is too low when the amount of paclitaxel is lower than 0.0001 mg per 1 ml of oily contrast medium.
[0023] Also, animal oils such as squalene or vegetable oils such as soybean oil can be included additionally in the paclitaxel/oily contrast medium composition of the present invention. By substituting parts of the oily contrast medium with animal oils, vegetable oils or their mixture, the cost of producing the formulation can be lowered without sacrificing the efficacy or stability. The ratio of oily contrast medium: animal oil and/or vegetable oil is 1:0.01˜1 by volume. More preferably, the above ratio is 1:0.01˜0.5.
[0024] The paclitaxel/oily contrast medium composition of the present invention can be easily prepared by adding paclitaxel to the oily contrast medium according to the above composition range and solubilizing paclitaxel by stirring the mixture at room temperature. To speed up the solubilization process, it is acceptable to raise the temperature to 35˜45° C. or to sonicate in a bath type sonicator. The prepared paclitaxel/oily contrast medium composition is stored after sterilization process. It is acceptable to use sterilized raw materials and to mix them under a sterile environment. Or the paclitaxel/oily contrast medium composition can be sterilized by injecting through a sterile syringe filter (pore size 200 μm, PVDF sterile filter). It is also acceptable to sterilize and to mix the oily contrast medium and paclitaxel or to sterilize the composition by using gamma ray or EO gas sterilization protocols.
[0025] The paclitaxel/oily contrast medium composition of the present invention prepared as above was stable for more than 60 days at room temperature.
[0026] In the above oily composition, paclitaxel is precipitated out of the oily solution eventually even though paclitaxel is stably solubilized for 2 months. The precipitation is formed by inter- and intra-molecular hydrogen bonding between paclitaxel molecules. The present inventors have found that the precipitation can be effectively prevented by adding chemicals that form hydrogen bonding with paclitaxel or that disturb inter- and intra-molecular hydrogen bonding between paclitaxel molecules. The oily paclitaxel composition does not form precipitation after 2 months if the oily contrast medium itself can form hydrogen bonding with paclitaxel.
[0027] When Lipiodol, one of the most popularly used oily contrast media, was used, Lipiodol cannot form hydrogen bonding with paclitaxel due to the chemical nature of Lipiodol molecules. In this case, the chemicals which can form hydrogen bonding with paclitaxel in Lipiodol solution can prevent paclitaxel precipitation. For example, paclitaxel precipitation was prevented when tricaprylin was added to the oily paclitaxel composition since the hydrogen bonding between paclitaxel and tricaprylin was formed instead of that between paclitaxel molecules.
[0028] The contents of paclitaxel and the oily contrast medium in the oily paclitaxel composition after prolonged storage depend on the preparation process. If the composition was prepared in the absence of moisture or oxygen and also without being heated, the composition is stable for longer period of time since oxidation and hydrolysis of the components can be minimized. The precipitation process, however, is a thermodynamically driven process unlike other destabilization processes. Therefore, precipitation formation is unavoidable for the present oily paclitaxel composition no matter what precaution is taken during and after preparation. The rate of precipitation formation depends on the concentration of paclitaxel in the oily composition. In case paclitaxel concentrations are 10 mg/ml and 5 mg/ml in the oily composition, the precipitation is formed in approximately 60 and 120 days, respectively, at ambient temperatures. Therefore, the oily paclitaxel formulation can be stable for more than 1 year only when additional component that inhibits paclitaxel precipitation is added to the composition.
[0029] Therefore, the oily paclitaxel composition of the present invention can additionally comprise a component that inhibits paclitaxel precipitation. The solubility of paclitaxel in the oily composition increases up to 13 mg/ml in this case.
[0030] In other words, the amount of paclitaxel in the paclitaxel/oily contrast medium of the present invention is 0.0001˜13 mg, and the amount of the chemical that prevents paclitaxel precipitation is 0.01˜1 ml per 1 ml of oily contrast medium.
[0031] An example of the oily contrast medium is the same as described above.
[0032] The chemicals that can prevent paclitaxel precipitation in preparing the paclitaxel/oily contrast medium composition include an agent that forms hydrogen bonding with paclitaxel or a chaotropic agent that disturbs hydrogen bonding between paclitaxel molecules.
[0033] Chemicals that can form hydrogen bonding with the above paclitaxel molecule include alcohols, polyols, oils, lipids, polymers or peptides. Alcohols include methanol, ethanol, propanol, isopropanol, butanol and fatty alcohols. Polyols include ethylene glycol, propylene glycol and polyethyleneglycol. Oils include triglycerides, diglyceride, monoglycerides, tocopherol and animal or plant oils which are the mixtures of triglycerides, diglyceride, monoglycerides and other minor components. Lipids include phospholipid, neutral lipid, cationic lipid, anionic lipid and fatty acid. Polymers include poly(lactic acid), poly(glycolic acid) and their copolymers, chitosan, alginate, hyaluronate, daxtran and poly(ε-caprolatone). Chaotropic agents include dimethylsulfoxide (DMSO) and amides.
[0034] The paclitaxel/oily contrast medium of the present invention was stable for more than 200 days at ambient temperatures when a chemical that prevents paclitaxel precipitation was added.
[0035] The paclitaxel/oily contrast medium of the present invention can be used for TACE to treat solid tumors and has a viscosity of 40˜180 cP.
[0036] Also the amount and the method of the administration of the paclitaxel/oily contrast medium composition of the present invention can be varied up to the decision of the doctor depending on the age, sex, weight, and severeness of the patient. Generally, TACE can be performed once in 1˜4 months and can be repeated. Two to 15 ml of the formulation is injected through the feeding artery of a solid tumor, for instance through hepatic artery in case of hepatoma.
[0037] The invention will be further illustrated by the following examples. It should be understood that these examples are intended to be illustrative only and the present invention is not limited-to the conditions, materials or devices recited therein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a computed tomography (CT) picture obtained 1 week after selectively administering 0.3 cc of paclitaxel/lipiodol formulation of the present invention to the rabbit hepatoma by transcatheter arterial chemoembolization. The amount of the administered paclitaxel corresponds to A) 1 mg, B) 3 mg and C) 0 mg.
[0039] FIG. 2 is a graph showing the concentration of paclitaxel in the hepatoma and neighboring normal liver tissues one week after selectively administering 0.3 cc of paclitaxel/lipiodol formulation of the present invention to the rabbit hepatoma by transcatheter arterial chemoembolization. The quantitative analysis of paclitaxel was performed by high performance liquid chromatography (HPLC). The amount of the administered paclitaxel corresponds to A) 1 mg and B) 3 mg.
[0040] FIG. 3 is a graph showing the percent ratio of the viable tumor in total hepatoma tissue one week after selectively administering 0.3 cc (the groups administered with 1 mg and 3 mg of paclitaxel) and 0.4 cc (the group administered with 4 mg of paclitaxel) of paclitaxel/lipiodol formulation of the present invention to the rabbit hepatoma by transcatheter arterial chemoembolization. In case of the negative control group, 0.3 cc of Lipiodol was administered.
[0041] FIG. 4 is a graph showing the concentration of paclitaxel in hepatoma, left lobe and right lobe one week after selectively administering 0.4 cc (the group administered with 4 mg of paclitaxel) of paclitaxel/lipiodol formulation of the present invention to the rabbit hepatoma by transcatheter arterial chemoembolization.
--; concentration of paclitaxel in hepatoma, -◯-; concentration of paclitaxel in left lobe, -▾-; concentration of paclitaxel in right lobe.
[0045] FIG. 5 is a photograph of paclitaxel/lipiodol and paclitaxel/lipiodol/tricaprylin formulations after 200 days of storage at ambient temperature.
A; photograph of paclitaxel/lipiodol formulation, B; photograph of paclitaxel/lipiodol formulation under polarized light microscope, C; photograph of paclitaxel/lipiodol/tricaprylin formulation, D; photograph of paclitaxel/lipiodol/tricaprylin formulation under polarized light microscope.
[0050] FIG. 6 is a graph showing the thickness of mice footpad after injecting 20 μl of paclitaxel/lipiodol/tricaprylin formulation (the group administered with 200 μg of paclitaxel) 5 days after inoculating melanoma cells. In case of the control group, 20 μl of lipiodol/tricaprylin was administered Untreated group was also used as a negative control.
--; group administered with 20 μl of paclitaxel/lipiodol/tricaprylin formulation (200 μg of paclitaxel),. -◯-; group administered with 20 μl of lipiodol/tricaprylin formulation, -▾-; untreated group.
[0054] FIG. 7 is a graph showing the number of surviving mice after injecting 20 μl of paclitaxel/lipiodol/tricaprylin formulation (the group administered with 200 μg of paclitaxel) 5 days after inoculating melanoma cells. Untreated group was used as a negative control.
--; group administered with 20 μl of paclitaxel/lipiodol/tricaprylin formulation (200 μg of paclitaxel), -◯-; untreated group.
EXAMPLES
Example 1
[0057] Preparation of Paclitaxel/Lipiodol Composition
[0058] One milliliter of Lipiodol (Lipiodol Ultra-fluid, Laboratoire Guerbet, France, Iodine content 38% by weight) was used as an oily contrast medium. Lipiodol and 2, 4, 6, 8, 10 or 11 mg of paclitaxel (Samyang Genex, Korea) were added in test tubes (micro test tubes with safety lock, polyethylene, 1.5 ml, Eppendorf AG, Germany) and solubilized by stirring at room temperature. To speed up the solubilization process, it is acceptable to raise the temperature to 35˜45° C. or to sonicate in a bath type sonicator. When 2˜10 mg of paclitaxel was added in 1 ml of Lipiodol, paclitaxel was completely solubilized in Lipiodol as evidenced by the formation of clear single liquid phase. When 11 mg of paclitaxel was added to 1 ml of Lipiodol, however, clear liquid was formed initially but the turbidity of the solution increased after overnight storage at room temperature. Paclitaxel precipitation was observed under a microscopy. Therefore, it was confirmed that the solubility of paclitaxel in Lipiodol is approximately 10 mg/ml at room temperature (24˜28° C.). Viscosity of the paclitaxel/lipiodol (10 mg/1 ml) formulation was measured using a Kinematic viscometer Cannon-Fenske Type, Calibrated, Cat. No. 13-617E, Size 200, Fisher Scientific, Pittsburgh, Pa.) by measuring the falling time of the liquid formulation and was 67 cP at 25° C. Since the viscosity was higher than 45 cP, embolization effect is maximized, it is expected that paclitaxel/Lipiodol composition has an excellent embolization effect.
Example 2
[0059] Physical Stability of Paclitaxel/Lipiodol Composition
[0060] One milliliter of Lipiodol (Lipiodol Ultra-fluid, Laboratoire Guerbet, France, Iodine content 38% by weight) and 10 mg of paclitaxel (Samyang Genex, Korea) were added in test tubes and solubilized by stirring at room temperature. To speed up the solubilization process, the temperature of the mixture was raised to 40° C. Paclitaxel was completely solubilized in Lipiodol as evidenced by the formation of clear single liquid phase. The prepared composition was sterilized by injecting through a syringe filter (200 μm pore size, PVDF filter) and stored at room temperature and at 4° C. for 60 days to observe the physical stability and the degradation of paclitaxel. There was no change in the color and odor of the formulation. Phase separation or precipitation did not occur. Degradation of paclitaxel was not observed as evidenced by the analysis performed by HPLC.
[0061] The HPLC conditions were as follows.
Pump: SP8810 precision isocratic pump (Spectra-Physics Inc., San Jose, Calif.) Column: Waters Bondpack C18 Column (3.9 mm×300 mm, Waters Corp., Milford, Mass.) Mobile phase: acetonitrile and water 50% (w/w) each Flow rate: 1 ml/min Detector: Spectra 100 variable wavelength (Spectra-Physics)
Example 3
[0067] Physical Stability of Paclitaxel/Ethiodol Composition
[0068] Except that Ethiodol (Savage Laboratories, Melville, N.Y.) was used instead of Lipiodol as an oily contrast medium, the oily paclitaxel composition was prepared as described in Example 2. Paclitaxel was completely solubilized in Ethiodol as evidenced by the formation of clear single liquid phase. The physical stability of the prepared composition was tested by the same methods as in Example 2. The prepared composition was sterilized and stored at room temperature and at 4° C. for 60 days to observe the physical stability and the degradation of paclitaxel. There was no change in the color and odor of the formulation. Phase separation or precipitation did not occur. Degradation of paclitaxel was not observed as evidenced by the analysis performed by HPLC.
Experimental Example 1
[0069] Preparation of Hepatoma Animal Model
[0070] VX2 tumor provided by Deutsches Krebsforschungszentrum Tumorbank (Germany) was transplanted into the thigh of rabbits (New Zealand White). After 2 weeks, the rabbits having 1˜2 cm tumors were sacrificed by intravenous injection of 10 ml of pentothal sodium solution (62.5 mg/kg). The tumors were excised along with the tissues around them after disinfection with Iodine solution and alcohol, removing the hair and cutting the skin over the tumor site. The tumor was cut to remove the central necrotic portion. The viable peripheral tumor tissue was mixed with calcium and magnesium-free Hank's balanced salt solution (Grand Island Biological Co., Grand Island, N.Y.) and cut into very small pieces with scissors and surgical mess. The tumor solution was mixed with 5 ml of RMPI-1640 (Rosewell Park Memorial Institute, Rosewell Park, N.Y.). The mixture was diluted to 1×10 6 tumor cells/mm 3 .
[0071] Injection of Tumors Cell Solution into Rabbit Liver
[0072] Five hundred milliliters of phosphate buffered saline was administered through the vein of the ear via 23 G needle as a first step. Through this rabbit vein, 40 ml of phosphate buffered saline mixed with 500 mg of pentothal sodium was injected at a flow rate of 1 ml/min to anesthetize a rabbit. The total dose of the solution was 1.5 ml/kg. The hair in the abdomen was removed, and the skin was disinfected with Iodine solution and alcohol. Under the ultrasound guide, 0.1 ml of the tumor tissue solution was injected to the liver parenchyma of the left lobe with a 1 ml syringe through a 22 G needle. The tumor tissue solution was injected to the left lobe among the 5 lobes in the rabbit liver since it is the easiest to observe with the ultrasound ( FIG. 1 ). To prevent secondary infection, antibiotic (PenbrexR, 250 mg) was injected intravenously. After the injection of the tumor tissue solution, the rabbits were grown in a rabbit cage with normal meals. In two weeks after the transplantation of tumor cells, tumor was identified by ultrasound observation and CT. The tumor growth could be roughly predicted by the growth curve. The ultrasound observation was performed every 3 days, and CT was performed every week starting 2 weeks after the transplantation to follow up the position and size of the tumor.
Example 4
[0073] Transcatheter Arterial Chemoembolization with Paclitaxel/Lipiodol Composition in Hepatoma Animal Model
[0074] One milliliter of Lipiodol and 3.33 mg or 10 mg each of paclitaxel (Samyang Genex, Korea) were added in test tubes and solubilized by stirring at room temperature. To speed up the solubilization process, the temperature of the mixture was raised to 40° C. Paclitaxel was completely solubilized in Lipiodol as evidenced by the formation of clear single liquid phase. The prepared composition was sterilized by injecting through a syringe filter (200 μm pore size, PVDF filter).
[0075] In the hepatoma animal model prepared in Experimental Example 1, TACE was performed through a catheter into the feeding artery of the tumor 0.3 ml of the paclitaxel/Lipiodol formulation of the present invention. Therefore, the dose of paclitaxel corresponds to 1 mg and 3 mg, respectively. As a negative control group, 0.3 cc of Lipiodol was injected to the hepatoma animal model. Lipiodol was taken up selectively into the tumor tissue in one week after the surgery as shown by the computed tomographic picture in FIG. 1 .
Example 5
[0076] Analysis of Paclitaxel Concentration in the Hepatoma Tissue After the Transcatheter Arterial Chemoembolization with Paclitaxel/Lipiodol Composition
[0077] The rabbits were sacrificed in one week after the transcatheter arterial chemoembolization in Example 4, and livers were taken out. The paclitaxel concentration was determined in the tumor tissue that Lipiodol was visually identified, the tumor tissue that Lipiodol is not visually identified and the normal liver tissue neighboring the tumor. Each liver tissue was mixed with a lysis buffer solution [62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 5% β-mercaptoethanol, 10% glycerol] and homogenized. After the homogenized mixture was centrifuged, the supernatant was obtained to analyze the paclitaxel concentration by HPLC. The conditions for HPLC were identical to those in Example 2. As explained in Example 4, the paclitaxel concentrations in the liver of the rabbits administered with the formulation corresponding to 1 mg or 3 mg of paclitaxel are shown in FIGS. 2A and 2B , respectively. The concentration of paclitaxel in the hepatoma tissue that Lipiodol was visually identified was the highest. The concentration was relatively high in the hepatoma tissue that Lipiodol was not visually identified. On the other hand, the paclitaxel concentration was negligible in the normal liver tissue neighboring the tumor. Therefore, it was confirmed that paclitaxel distributes selectively in the tumor one week after the operation with the paclitaxel/Lipiodol formulation of the present invention.
Example 6
[0078] Determination of Viable Tumor After the Transcatheter Arterial Chemoembolization with Paclitaxel/Lipiodol Composition
[0079] One milliliter of Lipiodol and 3.33 mg or 10 mg each of paclitaxel (Samyang Genex, Korea) were added in test tubes and solubilized by stirring at room temperature. To speed up the solubilization process, the temperature of the mixture was raised to 40° C. Paclitaxel was completely solubilized in Lipiodol as evidenced by the formation of clear single liquid phase. The prepared composition was sterilized by injecting through a syringe filter (200 μm pore size, PVDF filter).
[0080] In the hepatoma animal model prepared in Experimental Example 1, TACE was performed through a catheter into the feeding artery of the tumor 0.3 ml (3.33 or 10 mg/ml formulations) or 0.4 ml (10 mg/ml formulation) of the paclitaxel/Lipiodol formulation of the present invention. Therefore, the dose of paclitaxel corresponds to 1 mg, 3 mg or 4 mg, respectively. As a negative control group, 0.3 cc of Lipiodol was injected to the hepatoma animal model. Lipiodol was taken up selectively into the tumor tissue in one week after the surgery as shown by the computed tomographic picture in FIG. 1 . The rabbits were sacrificed in one week after the transcatheter arterial chemoembolization, and livers were taken out. The size of the tumors in the groups administered with the paclitaxel/Lipiodol formulations was similar to the negative control group administered with Lipiodol and was 32±5 mm. Pathological examination was performed to distinguish necrotic tumor and viable tumor in the tumor tissue. The viable tumor portion in the total tumor tissue is shown in FIG. 3 . In the negative control group, more than 30% of the tumor was viable whereas the viable tumor was 13.2%, 10.4% and 0.6% in the groups of rabbits administered with 1 mg, 3 mg and 4 mg, respectively, of paclitaxel. These result indicate that paclitaxel in the paclitaxel/Lipiodol formulation of the present invention effectively destroys tumor cells.
Example 7
[0081] Preparation of Lipiodol/Soybean Oil/Paclitaxel Composition
[0082] One milliliter of Lipiodol, 0.2 ml of soybean oil and 10 mg each of paclitaxel were added in test tubes and solubilized by stirring at room temperature. To speed up the solubilization process, the mixture was sonicated in a bath type sonicator. Paclitaxel was completely solubilized in the mixed oil system of Lipiodol/soybean oil as evidenced by the formation of clear single liquid phase.
Example 8
[0083] Preparation of Lipiodol/Squalene/Paclitaxel Composition
[0084] Except that squalene was used instead of soybean oil, and the mixture was heated to 40° C. to speed up the solubilization process, Lipiodol/squalene/paclitaxel composition was prepared by using the same preparation method in Example 6. Paclitaxel was completely solubilized in the mixed oil system of Lipiodol/soybean oil as evidenced by the formation of clear single liquid phase.
Example 9
[0085] Preparation of Paclitaxel/Lipiodol/Tricaprylin Composition and Determination of Its Physical Stability
[0086] An oily mixture of 1 ml of Lipiodol (Lipiodol Ultra-fluid, Laboratoire Guerbet, France, Iodine content 38% by weight) and 0.01 ml of tricaprylin (Sigma Chemical Co.) and 10 mg of paclitaxel (Samyang Genex, Korea) were added in a test tube and solubilized by stirring at room temperature. To speed up the solubilization process, the composition was sonicated in a bath type sonicator. Paclitaxel was completely solubilized in the oil mixture of Lipiodol/tricaprylin as evidenced by the formation of clear single liquid phase. The prepared composition was sterilized by injecting through a syringe filter (200 μm pore size, PVDF filter) and stored at room temperature and at 4° C. for 200 days to observe the physical stability and the degradation of paclitaxel. There was no change in the color and odor of the formulation. Phase separation or precipitation did not occur. Degradation of paclitaxel was not observed as evidenced by the analysis performed by HPLC. In case of paclitaxel/lipiodol formulation in Example 1, the composition became turbid due to the precipitation of paclitaxel ( FIG. 5A ) after 200 days of storage at ambient temperatures. Paclitaxel precipitation was observed under polarized light microscope for paclitaxel/lipiodol composition ( FIG. 5B ). In contrast, paclitaxel/lipiodol/tricaprylin composition stayed clear ( FIG. 5C ) without forming paclitaxel precipitation ( FIG. 5D ). Therefore, the paclitaxel/lipiodol composition can be stabilized for a long period of time by adding tricaprylin as a component to inhibit paclitaxel precipitation.
Example 10
[0087] Preparation of Paclitaxel/Lipiodol/Tricaprylin Composition and Determination of Its Physical Stability
[0088] A mixture of 1 ml of Lipiodol (Lipiodol Ultra-fluid, Laboratoire Guerbet, France, Iodine content 38% by weight) and 0.01 ml of tricaprylin (Sigma Chemical Co.) and 12 mg of paclitaxel (Samyang Genex, Korea) were added in a test tube and solubilized by stirring at room temperature. To speed up the solubilization process, the composition was sonicated in a bath type sonicator. Since paclitaxel was completely solubilized in the oil mixture of Lipiodol/tricaprylin as evidenced by the formation of clear single liquid phase, the solubility of paclitaxel is higher in a mixed oil system of lipiodol/tricaprylin than in lipiodol alone.
Experimental Example 2
[0089] Preparation of Melanoma Animal Model
[0090] Melanoma cell line, B16F10, spontaneously occurring in C57BL/6J mice was obtained from American Type Culture Collection (ATCC, USA). The cells were cultivated in Dulbeccos Modified Eagle Medium (DMEM, Gibco BRL/Life Technologies, New York, N.Y.), supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin/Streptomycin (Gibco). To prepare melanoma animal model, 1×10 6 cells were dispersed in 100 μl of DMEM and inoculated into rear left footpad of 8-week old C57BL/J mice (Samtaco, Korea).
Example 11
[0091] Determination of Melanoma Size After Injecting Paclitaxel/Lipiodol/Tricaprylin Composition
[0092] The paclitaxel/lipiodol/tricaprylin composition prepared in Example 9 was sterilized by injecting through a syringe filter (200 μm pore size, PVDF filter). Twenty microliters of the composition was injected into the inoculation site of rear left footpad 5 days after inoculation of melanoma as in Experimental Example 2. As negative controls, a group injected with 20 μl of lipiodol/tricaprylin (100:1 by volume) and untreated group were used. The size of the melanoma was quantified by measuring the thickness of the footpad and is shown in FIG. 6 . Melanoma began to grow 18 and 22 days after inoculation in case of the untreated group and the group treated with lipiodol/tricaprylin, respectively. In contrast, melanoma did not grow at all in the group treated with paclitaxel/lipiodol/tricaprylin proving the marked anticancer activity.
Example 12
[0093] Determination of Survival Time After Injecting Paclitaxel/Lipiodol/Tricaprylin Composition
[0094] The paclitaxel/lipiodol/tricaprylin composition prepared in Example 9 was sterilized by injecting through a syringe filter (200 μm pore size, PVDF filter). Twenty microliters of the composition was injected into the inoculation site of rear left footpad 5 days after inoculation as in Experimental Example 2.
[0095] Untreated group was used as a negative control. The number of surviving mice is shown in FIG. 7 as a function of time. In-the untreated group, mice began to die 20 days after inoculation. All of the mice died in 48 days after inoculation (n=6). All of the mice treated with paclitaxel/lipiodol/tricaprylin composition stayed healthy and alive showing the marked anticancer activity of the present composition.
INDUSTRIAL APPLICABILITY
[0096] The paclitaxel/oily contrast medium composition of the present invention is a single phase viscous liquid. The composition of the present invention opens up a new administration route for paclitaxel, which has been conventionally administered mainly through intravenous injection. The composition of the present invention can be used for the treatment of hepatoma by transcatheter arterial chemoembolization. The paclitaxel/Lipiodol formulation of the present invention is easy to prepare and to sterilize and is physically and chemically more stable than conventional doxorubicin/Lipiodol formulation. Therefore, the composition is stable during and after the TACE for the treatment of solid tumors, and is stable for at least 60 days at room temperature. Also, the solubility of paclitaxel can be increased in the paclitaxel/lipiodol composition, which became stable for more than at least 200 days by adding a component that can inhibit paclitaxel precipitation.
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Oily paclitaxel composition and formulation for chemoembolization and preparation method thereof solubilizing paclitaxel in an oily contrast medium. The composition of the present invention solubilizes paclitaxel and has an advantage of delivering anticancer drug to the target cells by chemoembolization since it is possible to visualize the blood vessel during the chemoembolization process. The present invention also relates to oily paclitaxel composition and formulation additionally comprising chemicals that prevent paclitaxel precipitation for prolonged preservation and the preparation method thereof. Since the composition of the present invention solubilize paclitaxel effectively and can be visualized during chemoembolization, it can be used for TACE to treat hepatoma and other solid tumors.
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REFERENCE TO RELATED APPLICATIONS
[0001] This application is copending with application Ser. No. ______ filed substantially concurrently herewith entitled Dry Ice Draw Through Galley Cooling having attorney docket no. 13-1283 and application Ser. No. ______ filed substantially concurrently herewith entitled CO 2 Shut Off Method for Dry Ice Sublimation Inside a Galley Cart having attorney docket no. 13-1515 both having a common assignee with the present invention, the disclosures of which are incorporated herein by reference.
BACKGROUND INFORMATION
[0002] 1. Field
[0003] Embodiments of the disclosure relate generally to the galley cart systems for transportation vehicles and more particularly to a collapsible bulb seal with one or more orifices for collection of CO 2 when expanding from a collapsed to expanded position and expulsion of CO 2 when collapsing from the expanded position.
[0004] 2. Background
[0005] Galley carts employed for food service in transportation vehicles such as aircraft and trains often require cooling contain food and beverages at a temperature that is cooler than a cabin of the vehicle. At least some known carts include or connect to a refrigeration system (a chiller) that provides cool air to an interior volume of the cart to cool the food/beverages. However, the chiller is powered by the vehicle systems, reducing the amount of power available to the vehicle for propulsion, thrust, etc. As such, the chiller is an inefficient draw on the power supply system of the vehicle. Further, such a chiller system adds weight and complexity to the vehicle. Accordingly, some galley carts are configured to contain dry ice that cools the food/beverages as it sublimates. One drawback with the use of dry ice is the carbon dioxide gas (CO 2 ) sublimate that is released. At least in aircraft, the Federal Aviation Administration has set forth requirements for the maximum CO 2 concentration in a cabin of the aircraft. The sublimation of the dry ice may cause the CO 2 concentration in the cabin to exceed the maximum parts-per-million (ppm). For example, the CO 2 gas may escape from the cart into the cabin when the door of the cart is opened in the galley area or in the aisle as food/beverages are served (a transient condition). Further, the CO 2 gas may escape from the cart through provided leak paths to ensure that the pressure within the cart does not exceed a maximum threshold as the dry ice sublimates (a steady-state condition). Dry ice, providing CO 2 sublimation as a coolant, is a commonly available, cost effective and volumetrically efficient refrigerant for such use. However, limiting venting of CO 2 gas from the galley carts to avoid undesirable buildup of CO 2 in passenger compartments is preferred.
[0006] It is therefore desirable to provide structurally simple and cost effective structure for control of CO 2 sublimation in galley carts.
SUMMARY
[0007] Exemplary embodiments provide a galley cart having a housing defining a cavity. A door is coupled to the housing, the door configured to be moved between a closed position and an open position. A seal assembly is coupled between the housing and the door with at least one bulb seal configured to draw gas in when the door is in the open position and to exhaust the gas when the door is in the closed position.
[0008] The embodiments provide a method for collection of CO 2 sublimate in a galley cart by compressing a bulb seal having at least one aperture between the galley cart door and housing. Upon opening the door, the bulb seal expands drawing in CO 2 sublimate into the bulb seal through the aperture. Upon closing the door, the bulb seal is compressed exhausting CO 2 .
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The features, functions, and advantages that have been discussed can be achieved independently in various embodiments of the present invention or may be combined in yet other embodiments further details of which can be seen with reference to the following description and drawings.
[0010] FIG. 1A is a perspective view of an example a galley cart in which the described embodiments may be employed;
[0011] FIG. 1B is a second perspective view of the galley cart of FIG. 1A with a first door in a partially open position;
[0012] FIG. 2A is a partial bottom section view of the galley cart showing the door in a closed position with a compressed bulb seal;
[0013] FIG. 2B is a detailed partial bottom section view of the galley cart showing the door in a closed position with a compressed bulb seal
[0014] FIG. 3A is a detailed partial bottom section view of the galley cart showing the door in a partially open position for a first embodiment with the bulb seal attached to the door and with the compressed bulb seal expanded;
[0015] FIG. 3B is a detailed partial bottom section view of the galley cart showing the door in a partially open position for a second embodiment with the bulb seal attached to the door seal relief and with the compressed bulb seal expanded;
[0016] FIG. 4 is a perspective view of an example bulb seal;
[0017] FIG. 5A is a partial bottom perspective view of the bulb seal showing the compressed seal and orifice;
[0018] FIG. 5B is a section view of the bulb seal of FIG. 5A demonstrating the interior volume of the compressed seal;
[0019] FIG. 6A is a partial bottom perspective view of the bulb seal showing the partially compressed seal and orifice;
[0020] FIG. 6B is a section view of the bulb seal of FIG. 6A demonstrating the interior volume of the partially compressed seal;
[0021] FIG. 7A is a partial bottom perspective view of the bulb seal showing the fully expanded seal and orifice;
[0022] FIG. 7B is a section view of the bulb seal of FIG. 7A demonstrating the interior volume of the fully expanded seal;
[0023] FIG. 8 is an interior perspective view of the door with a bottom bulb seal;
[0024] FIG. 9 is an interior perspective view of the door with a peripheral bulb seal;
[0025] FIG. 10 is a cross section of a bulb seal with a first exemplary attachment tab;
[0026] FIG. 11 is a cross section of a bulb seal with a second exemplary attachment tab;
[0027] FIG. 12 a cross section of a bulb seal with an alternative shape and attachment tab; and,
[0028] FIG. 13 is a flow chart of a CO 2 capture method enabled by the disclosed embodiments.
DETAILED DESCRIPTION
[0029] The embodiments described herein provide a galley cart for use in a transportation vehicle that includes a seal assembly having a hollow bulb seal positioned between the cart housing and the door of the cart. The bulb seal is coupled to the housing or the door at any suitable location. In one embodiment, the bulb seal is positioned vertically along an edge of the door opposite of the door hinges. The bulb seal defines a hollow cavity, and two end caps coupled to the seal further enclose the cavity. At least one of the end caps includes at least one aperture; however, the end cap(s) can include any suitable number and/or arrangement of apertures.
[0030] When the cart door opens, the bulb seal expands and draws gas through the aperture into the seal cavity. As such, when the cart door opens, the expanding bulb seal helps prevent escape of CO 2 gas into the cabin by drawing the CO 2 gas, and possibly other gas, into the cavity (the CO 2 gas will be near the bottom of the cart because of its relative weight). When the cart door closes, the bulb seal is compressed and exhausts the gas from the seal cavity. In the example embodiment, the bottom end cap includes the aperture(s) such that gas is exhausted out of the bottom of the cart because the gas (CO 2 ) is heavier than the air in the cabin and the gas will be lower than a breathing height of the passengers. In alternative embodiments, the orifice in the bulb seal may be positioned to exhaust the gas back into the galley cart interior. Accordingly, this invention reduces CO 2 in the cabin during transient conditions and exhausts CO 2 gas in a manner to avoid inhalation by the passengers. The terms “CO2 gas”, “CO2 sublimate”, and “sublimate” are used interchangeably herein. [Please be consistent when referring to CO2 as CO2 (compound), CO2 (solid), and CO2 (gas)].
[0031] Referring to the drawings, FIG. 1A is an isometric view of a galley cart 100 which may be employed in the embodiments disclosed herein. In one aspect of this embodiment, the galley cart 100 includes a housing 102 . In the illustrated embodiment, the housing 102 has a first side 104 , a second side 106 , a top 108 , and a bottom 110 . The galley cart 100 further includes a first door 112 positioned on one end of the housing 102 , and, for certain embodiments, a second door (not shown) is positioned on an opposite end of the case 102 . Each of the doors 112 can further include one or more hinges 114 and a latch 116 . The hinges 114 pivotally attach the doors 112 to the housing 102 . The latch 116 can be configured to releasably engage corresponding receivers 118 attached to the housing 102 when the doors 112 are in closed positions as illustrated in FIG. 1A .
[0032] By disengaging the latch 116 from the corresponding receiver 118 , the doors 112 can be opened outwardly providing access to an interior cavity 120 of the housing 102 as shown in FIG. 1B . Positioning the doors 112 at respective ends of the housing 102 allows flight attendants to conveniently access food stored within the housing 102 from either end of the galley cart 100 . In other embodiments, the second door can be omitted if desired. Additionally, the doors 112 are received in a recess 122 in the housing 102 to be described in greater detail subsequently. Wheels or casters 124 allow the galley cart to be easily maneuvered within the service areas and aisles of the aircraft.
[0033] Dry ice may be stored in the galley cart to provide CO 2 sublimate in the interior cavity as a coolant for food or beverages stored in the cart. The CO 2 sublimate will tend to pool near the bottom of the interior cavity 120 in the cart. A hollow bulb seal 126 is mounted in the recess 122 into which the doors 112 are received as shown in FIGS. 2A , 2 B and 3 A or 3 B. In the embodiment shown, the bulb seal 126 is mounted in the recess 122 opposite the hinge attachment. With the door 112 in a closed position, the bulb seal is compressed as shown in FIGS. 2A and 2B . With the door open as shown in FIGS. 3A or 3 B, the bulb seal is expanded. The bulb seal 126 may be attached to the recess 122 as in FIG. 3A or to the door 112 as in FIG. 3B . An aperture 128 placed in a bottom cap 130 of the bulb seal 126 , shown in detail in FIG. 4 , provides a port into which gas, such as CO 2 gas, in the cart is drawn during opening of the door 112 .
[0034] Opening of the door 112 results in the bulb seal 126 transitioning from a compressed condition to an expanded condition thereby increasing interior volume and creating a reduced pressure within the bulb seal. This transition is shown in FIGS. 5A , 5 B, 6 A, 6 B, 7 A and 7 C. In FIG. 5A the bulb seal 126 is in a compressed condition with a resulting cross sectional area 130 as shown in FIG. 5B . As the door 112 is opened, the bulb seal begins to expand as shown in FIG. 6A , resulting in an increased cross sectional area 132 as shown in FIG. 6B . The increased cross sectional area results in a greater volume within the bulb. When fully expanded as shown in FIGS. 7A and 7B , the maximum (?) cross sectional area 134 results. The perimeter of the seal remains the same but internal volume (and vacuum) increases when removing compression in the system.
[0035] Upon closing the door 112 , the bulb seal 126 is recompressed expelling the accumulated CO 2 gas. A relief channel 136 (seen in FIG. 1B ) may be provided to vent the CO 2 gas from the compressing of bulb seal downward from the bottom 110 of the cart. This release of CO 2 gas at essentially floor level precludes undesirable distribution of CO 2 to be breathed by passengers in the vehicle.
[0036] As shown in FIG. 4 , additional apertures 138 may be provided along the length of the bulb seal to draw CO 2 gas into the expanding seal from a greater portion of the interior cavity of the cart.
[0037] In an alternative embodiment, the bulb seal 126 may be placed along the bottom edge of the door 112 and cart housing 102 as shown in FIG. 8 with apertures 138 as shown in FIG. 4 oriented along the bottom of the door. In another alternative embodiment, the bulb seal 126 may extend around the door 112 as a complete peripheral seal around the door 112 as shown in FIG. 9 . Multiple apertures 138 may be directed downward for collecting CO 2 gas from the lower portion of the internal cavity as the door 112 is opened expanding the seal and exhausting the CO 2 gas downward as the door is closed compressing the seal. Additional apertures along the periphery for collecting CO 2 gas from within the internal cavity may also be provided.
[0038] In other alternative embodiments, the bulb seal may be positioned with the apertures in communication with the interior cavity 120 to exhaust the collected CO 2 gas back into the interior volume upon closing of the door 112 .
[0039] As shown in FIG. 4 and FIG. 10 , the seal assembly may incorporate a T-rib 140 for attachment of the bulb seal 126 to the galley cart door or recess. As seen in FIGS. 3A and 3B , the T-rib 140 is received within a T-slot 142 in either the recess 122 or door 112 to constrain the bulb seal 126 . An alternative attachment of the bulb seal may be accomplished with a flat flange 144 as shown in FIG. 11 . Alternative cross sectional shapes may also be employed for the bulb seal such as a rectangular seal 146 as shown in FIG. 12 .
[0040] As shown in FIG. 13 , the embodiments herein provide for capture of CO 2 gas during opening of a galley cart door and directed exhausting of the collected CO 2 gas upon closing the galley cart door. A bulb seal is compressed between the galley cart door and housing, step 1302 . The door is opened, step 1304 , expanding the bulb seal and drawing in CO 2 gas through one or more apertures, step 1306 . Upon closing the door, step 1308 , the bulb seal is compressed exhausting CO 2 gas, step 1310 . In certain embodiments, the exhausted CO 2 gas is directed downward out of the galley cart, step 1312 . In alternative embodiments, the CO 2 gas is exhausted into the internal cavity of the cart, step 1314 .
[0041] Having now described various embodiments of the invention in detail as required by the patent statutes, those skilled in the art will recognize modifications and substitutions to the specific embodiments disclosed herein. Such modifications are within the scope and intent of the present invention as defined in the following claims.
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A galley cart system employs a dry ice compartment and a refrigeration compartment in a galley cart in flow communication with the dry ice compartment. A ventilation system is in interruptible flow communication with at least the refrigeration compartment and configured to receive gas discharged from at least the refrigeration compartment.
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PRIORITY CLAIM
[0001] This application claims priority to U.S. Provisional Application Ser. No. 62/356,357, filed Jun. 29, 2016. The above referenced application is incorporated herein by reference as if restated in full.
BACKGROUND
[0002] Approximately 10-20 percent of the United States population suffers from inverted nipples. The condition can adversely affect self-esteem, sexuality, and, in severe cases, the ability to breastfeed. Inverted nipples, which retract into the breast, can occur in both men and women. An inverted nipple is characterized in the medical industry as Grades 1, 2, and 3. Less severe Grade 1 and Grade 2 inverted nipples may be protracted through various methods of pressure to the areola to evert the nipple, however, most return to the inverted state. The treatment method depends on the grade of the inversion, the cause of the inversion, and whether breastfeeding is planned. There are several ways to reverse the condition, ranging from manual manipulation to plastic surgery.
[0003] As of this writing, many of the solutions which appear in prior art generally involve suction, vacuum, a pump, piercing, a syringe, or multiple parts, some of which rely on a combination thereof. Problems known to be associated with the aforementioned devices and methods include skin irritation, ulceration, discomfort, unreliable attachment, poor concealability, multiple procedures, and sterilization or other maintenance. While the prior art may have fulfilled their respective particular objectives and requirements, they have not addressed the needs of today's consumer in search of an effective “user-friendly” alternative for the treatment of inverted nipples. Therefore, it is an important object of the present invention to provide a solution which minimizes the discomfort, adverse effects, number of parts, number of procedures, and amount of physical manipulation to the breast or nipple associated with prior art.
[0004] To this end, the present invention provides a single-unit apparatus of ultralight construction primarily developed for the purpose of everting inverted nipples and maintaining the protracted state with a more “user-friendly” solution which is easy to apply, unrestrictive, more comfortable to wear, more concealable beneath clothing, and better capable of remaining securely in place when active or asleep. Further adding to its “user-friendly” attributes, the present invention is constructed of ultralight and cost-effective materials to allow for disposable use.
[0005] As such, the device of the present invention for an eversion assist of inverted nipples and maintaining the protracted state substantially departs from the conventional concepts and designs of the prior art. Therefore, it can be appreciated that there exists a continuing need for a new and improved device for eversion assist and maintaining the nipples in the everted orientation. In this regard, the present invention substantially fulfills this need.
SUMMARY
[0006] In view of the disadvantages inherent in the known types of nipple everting devices now present in the prior art, the general purpose of the present invention is to provide a new and improved device and method for an eversion assist of inverted nipples which overcomes the many disadvantages associated with the prior art. The present invention is a “user-friendly” solution comprised of a generally annular single-unit device with adhesive back, which relies on the interplay between the adhesive, the areola tissue and the flat base of the device to create a gentle, yet focused nudge for effectively manipulating nipple orientation and maintaining a protracted state. As such, the present invention substantially departs from the conventional use of vacuum, suction, pumps, syringes, strong manipulation of the breast or nipple, encapsulation, or multiple parts to treat inverted nipples.
[0007] In this respect, the ultralight apparatus of the present invention offers a solution with many advantages afforded by its unique design and approach to eversion assist, including a more comfortable and securely attached device with improved concealability, which is particularly advantageous when only one nipple is inverted and requires treatment.
[0008] Adding to the aforementioned “user-friendly” attributes, the present invention provides for a new and improved eversion assist device fabricated of ultralight and cost-effective materials which lend themselves well to being configured and manufactured as a disposable utility, thus eliminating the need for sterilization or other maintenance known to prior art.
[0009] There has thus been outlined, rather broadly, the features of the invention in order that the present contribution to the art may be better appreciated. These, together with other objects of the invention, are pointed out in the accompanying drawings and descriptive matter hereinafter. There are, of course, additional features of the invention that will be described and which will form the subject matter of the claims attached. Further, the device is capable of other embodiments and being carried out in various ways, so it is to be understood that the phraseology and terminology employed herein are for the purpose of descriptions and should not be regarded as limiting. As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a side elevational view of a device for an eversion assist of inverted nipples, the device constructed in accordance with the principles of the present invention, the device being in use.
[0011] FIG. 2 is an exploded side elevational view of the device shown in FIG. 1 .
[0012] FIG. 3 is a front elevational view taken along line 3 - 3 of FIG. 2 .
[0013] FIG. 4 is a cross sectional view taken along line 4 - 4 of FIG. 3 .
[0014] FIG. 5 is a front perspective illustration of the device shown in the prior Figures.
[0015] FIG. 6 is a rear perspective illustration of the device shown in the prior Figures.
DETAILED DESCRIPTION
[0016] With reference now to the drawings, and in particular to FIGS. 1 and 4 thereof, the preferred embodiment of the new and improved device for an eversion assist of inverted nipples embodying the principles and concepts of the present invention and generally designated by the reference numeral 10 will be described.
[0017] The present invention, the device for an eversion assist of inverted nipples 10 , is comprised of a plurality of components. Such components in their broadest context include upper and lower surfaces, a central aperture, and an exterior periphery. Such components are individually configured and correlated with respect to each other so as to attain the desired objective.
[0018] More specifically, the present invention is a uniquely “user friendly” apparatus of ultralight materials comprising a multi-layered single-unit construction with adhesive back. The device of the present invention includes a substantially annular raised composite with aperture which fits over the areola and relies upon the interplay between the device's flat base, the adhesive and the areola tissue to manipulate the nipple orientation and maintain a protracted state. Unlike products comprising the prior art, many of which utilize suction, vacuum and are often cumbersome methods/devices, the present “user friendly” invention effectively treats inverted nipples by means of a continuous gentle, yet focused nudge effect achieved when the adhesive-back flat base of the device meets with the areola tissue.
[0019] Further benefits of this new and improved solution for treating inverted nipples relate to the device's ultralight construction and adhering properties, which make this device easy to apply, comfortable to wear for extended periods, capable of remaining securely in place while active or asleep, as well as affording improved concealability over prior art, which can be particularly advantageous when only one nipple is inverted. Adding to its “user-friendly” attributes, this self-adhering ultralight device can be configured to be efficiently manufactured for disposable use, including peel-and-stick properties, thus eliminating the need for maintenance.
[0020] The present invention comprises a 3-dimensional, substantially annular, multi-tiered composite areolar device measuring from 0.25″-1″, (6.35 mm-25.4 mm) thickness. The topcoat is comprised of a thin membrane of latex, felt, moleskin, silicone or other lightweight polymer. The topcoat is bonded to an inner layer of ultralight material to provide firmness and cushioning. This ultralight material substance is firm enough to allow the device to be firmly pressed against the surface of the areola. The flat bottom layer is a self-sticking medical grade adhesive that is bonded to the bottom external layer of the device.
[0021] This self-sticking adhesive is characterized as having: a tackiness of 10-450 grams as determined by a ChemInstruments polyken probe tack tester and a tensile strength of 0.14-5.52 mega Pascals (20-800 pounds/square inch), a minimum elongation of 250-1100 percent and a tear strength of 0.88-35.2 kN/m, 5-200 pounds/square inch.
[0022] The overall diameter of the device measures between 1″-4″, (25.4 mm-101.1 mm) diameter with a center aperture between 0.25″-1″, (6.35 mm-25.4 mm) that are manufactured into rings to conform to various areola and nipple sizes.
[0023] Specifically, the device is designed to address the condition presented by Grade 1 and Grade 2 inverted nipples, i.e. those protractible with light inward pressure to the areola.
[0024] This “user-friendly” single-unit device can be applied with little time or effort involved. First, by using a method that is most comfortable to the user, the nipple is protracted. If the nipple cannot be everted easily, a doctor should be consulted. Second, remove the protective backing to expose the adhesive and apply the device so the nipple protrudes through the center opening, then gently press onto the areola. The affected result is that the device applies a slight pressure inward and upward to the areola tissue around the nipple base creating a continuous gentle, yet focused nudge-effect, while also keeping the areola tissue from spreading and thereby allowing for possible tightening around the nipple base which may prove beneficial for lasting results. Once applied, the device may be worn comfortably and securely, with or without clothing, for extended periods of time.
[0025] With continued use, results can vary, to include long lasting protraction in some users. Because of the small size and ultralight design of this device, one can expect the least amount of discomfort or intrusion upon the natural freedom of the breast and nipple.
[0026] Several attributes include its easy of application and use. The device may be worn comfortable for extended periods of time. The device is light weight and nearly invisible under clothing. The device need not rely on suction, creams, multiple parts, or piercing, but instead may rely on an ultra-light adhesive backing. The device does not require obtrusive hardware. Nor does the device require on-going cleaning or sterilization.
[0027] The present invention is a device 10 for an eversion assist of inverted nipples. The device is fabricated of ultralight material and has a generally three-dimensional annular configuration with an upper layer or upper planar surface 14 and a parallel lower planar surface 16 .
[0028] A cylindrical aperture 28 extends centrally through the device. The central aperture has an interior periphery or interior surface of the aperature 30 in a cylindrical configuration extending between the lower planar surface and the upper planar surface. The cylindrical aperture has a second central axis co-extensive with the first central axis. The cylindrical aperture generally has a diameter of 25 percent (25%) of the lower diameter.
[0029] The lower planar surface 16 has a radially interior edge or aperture opening 18 and a radially exterior edge 20 . The upper planar surface 14 has a radially interior edge 22 and a radially exterior edge 24 . The device has a first central axis. The lower peripheral surface has a lower diameter of from 1 to 4 inches. The upper planar surface has a diameter of from 70 to 100 percent (70%-100%) of the lower diameter. The device generally has a height of from 12.5 to 25 percent (12.5%-25%) of the lower diameter.
[0030] The device has a lower exterior periphery 36 in a cylindrical configuration extending upwardly from the radial exterior edge of the lower planar surface and terminating at an intermediate circle 44 . The lower exterior periphery has a peripheral height of from 65 to 100 percent (65%-100%) of the height of the device.
[0031] The device may have an upper exterior periphery 40 in a frusto-conical configuration extending upwardly from the intermediate circle and terminating at the radially exterior edge of the upper planar surface.
[0032] An upper layer 14 of a cover material is adhered to the upper surface and the upper exterior periphery. The upper layer is comprised of a thin membrane chosen from the class comprised of latex, elastomer, felt, moleskin, silicone, or other lightweight polymer.
[0033] A lower layer 48 is a self-sticking tacky adhesive or adhesive layer adhered to the lower surface. The lower layer is comprised of a medical grade adhesive.
[0034] As to the manner of usage and operation of the present invention, the same should be apparent from the above description. Accordingly, no further discussion relating to the manner of usage and operation will be provided.
[0035] With respect to the above description then, it is to be realized that the optimum dimensional relationships for the parts of the invention, to include variations in size, materials, shape, form, function and manner of operation, assembly and use, are deemed readily apparent and obvious to one skilled in the art, and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention.
[0036] Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.
[0037] The device can be described as a combination of other features as well. The device 10 may be a single-unit mold or a composite of multiple plies adhered together. The device may be made of a latex layer or be entirely latex-free. The device may be made of a synthetic or natural polymer. The device may be made of Poron. The diameter of the device may be from 1.5 to 4 inches. The thickness of the device may be from 0.125-1 inches.
[0038] The device may comprise an upper layer 14 , an adhesive layer 48 , and an aperture 28 , as shown in FIGS. 3 and 4 . The device may also feature a substantially flat portion which may be conterminous with the lower planar surface 16 . The exterior, which may be conterminous with lower exerior periphery 36 , the upper exerior periphery 40 , and the upper layer 14 , may have a straight, tapered, spherical, conical, frustoconical, angular edge, polygonal, or ovoid shape.
[0039] The upper layer may feature a substantially flat upper surface and a flat lower surface, which may be the same as the lower planar surface 16 , and may be made of felt, moleskin, foam, latex, elastomer, silicone, another flexible polymer, or a combination of these materials. The upper layer may serve as an exterior of the device. The lower surface of the upper layer may be disposed flush against the adhesive layer.
[0040] The adhesive layer may be configured to adhere to an areola tissue to manipulate nipple orientation. Specifically, it may be configured to adhere at least a portion of the areola tissue into a position parallel to the substantially flat portion. It may be formed from woven fabric, and/or feature a plastic portion or latex portion. The adhesive layer may be protected by a protective back material (not shown). This protective back material may be removably attached to the adhesive layer. The protective back material may feature a tag enabling easy removal.
[0041] The aperture may be disposed centrally in the device. It may be contoured to receive a nipple when at least a portion of the areola tissue is oriented in a position parallel to the substantially flat portion. The configuration of the aperture may be such that the nipple may enter though a gap in the adhesive layer. The aperture may be cylindrical, conical, frustoconical, or spherical in shape. The inner surface of the aperture 30 may feature a continuation of the adhesive layer or the adhesive layer may terminate at or near the opening of the aperture 18 . The diameter of the aperture may range from 0.25 to 1 inch.
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The invention is a single-unit areolar device of ultralight construction with an adhesive back that relies on no additional parts or components to effectively manipulate nipple orientation for eversion assist. The device builds upon the interplay between the adhesive-back flat base with the areola tissue to create a gentle, focused nudge to evert assist and maintain protraction of inverted nipples. The ultralight single-unit device is comfortable to wear for extended periods of time, easily concealed beneath clothing, and remains securely in place while active or asleep. The disposable attributes of the cost effective materials utilized lend further advantage to the device.
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This application is a Rule 371 continuation of PCT/US91/00857, filed Feb. 13, 1991, which is a continuation of U.S. Ser. No. 07/487,491, filed Mar. 1, 1990, now abandoned.
FIELD OF INVENTION
This invention relates to novel microorganisms. More particularly, it relates to a lactic acid consuming ruminal bacterium which can prevent acute lactic acidosis. particularly in cattle abruptly switched from forage to concentrate (high grain) diets.
BACKGROUND OF THE INVENTION
Intensive beef production involves feeding energy dense, high concentrate diets to cattle. These concentrate diets contain a high percentage of corn, wheat, milo or other starchy components. When starter cattle are switched from forage to concentrate diets, acute indigestion can result, Elam, C. J., J. Anim. Sci., 43, pp. 898-901 (1976); Huber, T. L., J. Anim. Sci., 43, pp. 902-909 (1976); Uhart, B. A. and F. D. Carroll, J. Anim. Sci., 26, pp. 1195-1198 (1967). This indigestion is due to the rapid and extensive fermentation of the starch grain by the rumen microbial community which results in production of large amounts of organic acids, including lactic acid. The production of organic acids can be so great that the balances between ruminal acid production and utilization and ruminal buffering capacity are disrupted. This condition is termed acidosis. Acute acidosis is characterized by a rapid drop in pH and a sharp increase in the level of lactic acid in the rumen and in the blood, Elam, C. J., (supra); Slyter, L. L., J. Anim. Sci., 43, pp. 910-929 (1976); Uhart, B. A. and F. D. Carroll, (supra). If sufficiently severe, the over-production of lactic acid and other acids can contribute to a decrease in ruminal pH such that the normal microbial flora are upset. Often the result is that only a few bacterial species, which are tolerant of the acidic conditions, survive, Krogh, N., Acta Vet. Scand 2, pp. 102-119 (1961); Mackie, R. I. and F. M. C. Gilchrist, Appl. Environ. Microbiol., 38, pp. 422-430 (1979); Mann, S. O., J. Appl. Bacteriol., 33, pp. 403-409 (1970).
INFORMATION DISCLOSURE STATEMENT
To control the problem of acute lactic acidosis, several researchers have investigated adding viable lactate consuming bacteria or rumen bacteria from animals adapted to high grain diets, to the rumens of cattle that were abruptly changed from low to high concentrate diets, Allison, M. J., et al., J. Anim. Sci., 23, pp. 1164-1171 (1964); Chandler, P. T., et al., J. Dairy Sci., 38, pp. 1660-1665 (1975); Cook, M. K., et al., Am. J. Vet. Res., 38, pp. 1015-1017 (1977); Huber, T. L., Am. J. Vet. Res., 35, pp. 639-641 (1974). They predicted that the added bacteria would consume the higher levels of lactate produced, maintaining the balance between production and consumption and thereby lessening or eliminating the problem of acidosis. Allison et al. and Huber, supra, found that if the rumen of a roughage fed animal was inoculated with rumen fluid from a high concentrate adapted animal the problem of acute acidosis was alleviated when the abrupt shift of ration was made. U.S. Pat. No. 4,138,498, refers to feeding rumen bacterial cultures from an animal adapted to a concentrate diet to a roughage adapted animal then fed a concentrate diet, and claims a reduction or elimination of the symptoms of lactic acidosis. Increases in weight gains and feed conversions also were alleged in cattle receiving these cultures as compared to control cattle. U.S. Pat No. 3,857,791, refers to rumen inoculation with "adapted rumen microorganisms," or a mixture of Megasphaera elsdenii and Selenomonas ruminantium to reduce or eliminate the symptoms of lactic acidosis during the adaptation of ruminants to high grain rations. Additionally, it has been reported that milk production was increased by the intraruminal inoculation of certain live adapted rumen microorganisms to dairy cows, Chandler et al., supra; U.S. Pat. No. 3,956,482.
Despite the fact that these documents describe these methods of preventing acidosis or aiding the adaptation of cattle to high concentrate diets, no related product has been marketed to date. To overcome the problems referred to above, we have isolated a specific lactic acid consuming rumen bacterium from concentrate fed cattle for use as ruminal inocula to prevent acidosis in cattle abruptly switched from a high forage to a high concentrate ration.
SUMMARY OF THE INVENTION
The present invention relates to a bacterial culture, NRRL B-18624. The bacterium is characterized in that it consumes lactic acid, is resistant to monensin, lasalocid 2-deoxy-glucose (2-DG) and low pH (5.3), it uses lactate in the presence of other sugars and it produces butyrate. Culture NRRL B-18624 is also referred to herein as isolate 407A and as culture UC-12497.
The invention also relates to a composition for facilitating the adaptation of ruminants from roughage or normal pasture rations to a high energy starch ration, consisting essentially of the bacterial culture NRRL B-18624.
Also disclosed is a method of facilitating the adaptation of ruminants from a roughage or normal pasture ration to a high energy ration comprising administering to said ruminant an amount of a bacterial culture, NRRL B-18624, during said adaptation.
Also disclosed is a method of preventing acute lactic acidosis in ruminant animals comprising administering to said ruminant an amount of a bacterial culture NRRL B18624 sufficient to prevent such acidosis.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE 1
Isolation and Enrichment of Ruminal Lactic Acid Consuming Bacteria
Animals and diets: Hereford X Angus crossbred steers were used. Four animals were fed a ration of 60% cracked corn, 30% silage and 10% B282 supplement (Table 1) at maintenance level once daily. Three rumenfistulated crossbred cattle were fed a ration of 90% B-376 and 10% chopped hay (Table 2) at maintenance once daily. Animals had free access to water at all times.
Media: Anaerobic dilution solution (ADS) was prepared according to the methods of Bryant, M. P. and L. A. Burkey, J. Dairy Sci., 36, pp. 205-212 (1953). Inoculations and transfers of bacteria were performed in an anaerobic glove box (Coy Lab. Products, Ann Arbor, Mich. atmosphere: 85% N 2 , 10% H 2 and 5% CO 2 , ambient temperature). All media were prepared under 100% CO 2 by methods described previously, Bryant, M. P., Am. J. Clin. Nutr., 25, pp. 1324-1328 (1972); Hungate, R. E., Academic Press, New York (1966). Media for the study are listed in Table 3. Differential carbohydrate medium plates were prepared as the L medium but without lactate and with 0.2 to 0.5% of the following compounds: glucose, maltose, mannitol or soluble starch (Difco). 2-deoxy-D-glucose (2-DG, grade II, Sigma) was added to the L medium at 0.5%, and monensin or lasalocid was added to L medium with a final concentration of 6 ppm. A series of media (designated B) was prepared without rumen fluid and with varying pH values and reducing solutions (media B 1, B2, B3 and B 14). For other tests, ultra-clarified rumen fluid was added back to the B media and designated B11-R, B12-R, and B13-R (Table 3).
Collection of rumen fluid: For in vitro enrichments, rumen fluid was collected 2 to 5 hours after feeding from the four animals on B282 supplement. For direct plating experiments, rumen fluid was collected 5 hours after feeding from the three animals on B-376 supplement. All rumen contents samples were collected under a flow of CO 2 into flasks and held on ice until delivered to the laboratory. Each animal was represented a separate source for potential bacterial isolations.
Enrichments and plating: In the in vitro enrichments, equal weights of rumen fluid and 2% (w/w) amylopectin (Sigma A-7780) in ADS were mixed under CO 2 and 10 ml aliquots of each of the 4 prepared rumen fiuid:1% amylopectin mixtures was pipeted into 50 ml serum bottles and sealed with slotted rubber stoppers. Bunsen valves were inserted through the stoppers to relieve excess pressure. After 4 hours of incubation at 38° C., 0.55 ml of the bottle contents was added to 5 ml B2 medium and incubated for 18 hours. Ten-fold dilutions were made of each of the four enrichments which were transferred to fresh B2 daily for 3 consecutive days. Afterwards, serial tenfold dilutions from each source were made in B3 medium to 10 -9 . The 10 -5 to 10 -9 dilutions were plated (0.1 ml/plate) onto B2 agar medium. Plates were incubated at 38° C. for 5 days under 5 p.s.i. CO 2 .
For the direct plating experiments, ten-fold dilutions (to 10 -9 ) of the rumen fluid collected from each of the 3 animals on B-376 supplement were made in B14 medium. One tenth ml aliquots of each dilution series (10 -5 to 10 -9 ) were plated onto B14 agar and incubated as above.
Isolation sequence: Well isolated, representative colonies were picked and spotted onto L agar plates in an array that corresponded to grids on a stainless steel replicator. Plates were incubated for 48 hours as above. Growth on the L agar plates served as the "master" plates and was used to inoculate the differential plates with the replicator.
Characterization of isolates: The scheme for selection of the lactic acid consuming isolates is presented in Chart 1. Initially, the isolates were tested for carbohydrate utilization and resistance to 6 ppm monensin or lasalocid. Those isolates that did not grow on L medium in the presence of either of the two antibiotics were eliminated. Those that grew in the presence of monensin or lasalocid were grown on lactate at pH 5.3 in B2 broth to test for low pH tolerance. Those isolates that did not grow were eliminated. Those that grew in B2 medium at pH 5.3 were assessed for their fermentation acid profiles in B2 broth. Growth rates of the isolates were determined in L broth. Finally, media with low level lactate (0.55 mm B11-R, B12-R and B13-R) in the presence and absence of 0.2% glucose or maltose were inoculated to determine if lactate was utilized in the presence of these two sugars.
Analytical: Volatile fatty acids (VFA) analysis was by standard methods. Lactic acid concentrations were determined by the method of Barker, S. B. and W. H. Summerson, J. Biol. Chem., 138, pp. 535-554 (1941).
The enrichment and direct plating techniques yielded lactic acid consuming rumen bacterial isolates. From the enrichment step, the B2 medium selected for those isolates that could grow rapidly on lactate at pH 5.3. The pH of the liquid in the in vitro enrichment bottles after 4 hours of incubation, (at which time aliquots from the bottle cultures were transferred into B2) was 5.1 to 5.4.
For the direct plating experiments, isolates were grown on the low pH medium (B14) in the presence of monensin and 2-DG. This medium selected for those lactate utilizers that were resistant to 6 ppm monensin and could grow in the presence of 2-DG at pH 5.3. 2-DG was chosen as a selective agent because it can inhibit those organisms that transport glucose or maltose as well as lactic acid (Romano, A. H. et al., J. Bacteriol., 139, pp. 93-97 (1979); Thompson, J. Biochimie, 70, pp. 325-336 (1988)).
A total of 142 colonies from both the enrichment and direct plating isolation techniques was picked and evaluated on the differential carbohydrate plates. From these, 14 representative isolates from the enrichment series and 8 from the direct plating series were tested as outlined in Chart 1. Ten of the 22 isolates were retained for further testing. Substrate utilization profiles of these 10 are shown in Table 4. Isolates #252 through 320 grew poorly on soluble starch. Mannitol was included because relatively few rumen bacteria can metabolize it. Mannitol differentiates two important rumen lactate utilizing species, Megasphaera elsdenii and Selenomonas ruminantium, from lactate utilizing bacteria that do not use it.
Of the 6 isolates tested, all except #298 grew well on mannitol. All 10 grew well on lactate medium in the presence of compounds that inhibit other bacteria (monensin, lasalocid, and 2-DG). Monensin and lasalocid were included as selection criteria because these ionophorc antibiotics are currently fed to feedlot cattle. Most rumen lactate utilizing bacteria arc resistant to at least 48 ppm of these antibiotics (Dennis, S. M. et at., J. Animi. Sci., 52, pp. 418-426 (1981)).
Growth curves of typical isolates in L medium were measured. Specific growth rates and generation times, respectively, ranged from 0.187 and 3.71 h for isolate #394 to 0.644 and 1.08 h for isolate #382 (Table 5).
When the isolates were grown on the B2 lactate medium the major VFA produced were butyric and propionic acids with minor amounts of valeric and isovaleric acids (Table 6). The negative values for acetate and isobutyrate indicate their utilization. Large amounts of butyrate were produced by all of the isolates except #394. Small amounts of caproic acid were produced by most of the isolates.
Lactate consumption in low lactate media (0.55 mM initial concentration) in the presence and absence of maltose or glucose was measured. Lactate was utilized by the isolates even in the presence of either of the two sugars. The apparent differences in initial concentration of DL-lactic acid in the cultures containing carbohydrate were due to an interference of the sugars with the lactate assay. In all 5 isolates tested, the initial concentration of lactic acid (35 μg/ml) was reduced 60% to approximately 15 μg/ml after 24 hours. The isolates also fermented the added glucose or maltose since the background absorbance value decreased with time.
Morphologically, all the isolates were either large cocci, (1.2 μm) occurring as singles or pairs, or coccobacilli (1.0×3.0 μm). This information along with resistance to lasalocid and monensin at 6 ppm, suggests that the isolates resemble M. elsdenii (Holdeman, L. V. et at., Anaerobe Laboratory Manual, 4th ed., Virginia Polytechnic Institute and State University, Blacksburg, (1977); Krieg, N. R. and I. G. Holt, Bergey's manual of systematic bacteriology, Vol. 1, Williams and Wilkins, Baltimore, (1984)).
In summary, using enrichment and direct plating techniques, we isolated 10 lactic acid consuming rumen bacteria. These isolates are resistant to monensin, lasalocid, 2-deoxy-glucose and low pH (5.3). Most grow as well on glucose, maltose and mannitol as they do on lactate, but they continue to utilize lactate even in the presence of these sugars. All the isolates produce butyrate, an unexpected result since morphologically they resemble M. elsdenii which produces propionate.
TABLE 1______________________________________Animal DietIngredients % in diet.sup.1______________________________________Cracked corn 60Corn silage (Harvestore) 30B-282 protein supplement.sup.2 10______________________________________.sup.1 On a dry matter basis.sup.2 Contains the following: % by wt.Soybean oil meal, 49% 57.145Alfalfa meal, dehydrated, 17% 17.000Molasses, dried 17.000Yellow tallow, stabilized 2.000Defluorinated phosphate (CDP), 18% P 1.500Limestone, ground 1.900Salt 2.000Trace Mineral Mix (CCC, 10% Zn) .250Vitamin A, 30,000 IU/gram .100Vitamin D, 15,000 IU/gram .005Vitamin E, 20,000 IU/lb .050Sodium selenite, .2 mg/g .050
TABLE 2______________________________________Animal DietIngredients % as fed______________________________________B-376.sup.1 90Hay, chopped to 3" lengths 10______________________________________.sup.1 Contains the following: % by Wt.Corn rolled (8.5 as is) 89.639Soybean meal (48.5% as is) 6.758Fat (animal) 0.973Dical (18.5%) 0.200Limestone 1.298Salt 0.291Dyna K (Potassium Chloride) 0.600Selenium premix (0.2%) 0.048Trace mineral premix 0.096Vitamin A (30,000 IU/gram) 0.007Vitamin D (15,000 IU/gram) 0.002Vitamin E (20,000 IU/lb) 0.088
TABLE 3__________________________________________________________________________Media Composition Medium.sup.1 L B B1 B2 B14 B3 B11-R B12-R B13-R g or ml per l__________________________________________________________________________Mineral I, ml 37.5 37.5 37.5 37.5 37.5 37.5 -- -- --Mineral II, ml 37.5 37.5 37.5 37.5 37.5 37.5 -- -- --Resazurin, 0.1% solution, ml 1.0 1.0 1.0 1.0 1.0 1.0 -- -- --Trypticase (BBL), g 2.0 2.0 2.0 2.0 2.0 2.0 -- -- --Yeast Extract (Difco), g 1.0 0.5 0.5 0.5 0.5 0.5 -- -- --VFA Solution, ml 10.0 10.0 10.0 10.0 10.0 10.0 -- -- --Hemin Solution, ml 10.0 10.0 10.0 10.0 10.0 10.0 -- -- --Trace minerals, ml 10.0 10.0 10.0 10.0 10.0 10.0 -- -- --Vitamin Solution, ml -- 10.0 10.0 10.0 10.0 10.0 -- -- --DL-Lactate Solution A.sup.2, ml 50 -- 50 50 50 -- -- -- --Acetic Acid, ml -- -- -- 1.2 1.2 1.2 -- -- --Sodium acetate, anhydrous, g -- -- -- 6.5 6.5 6.5 -- -- --Reducing agent.sup.3, ml 10 10 10 -- -- -- -- -- --Sodium carbonate, 8% solution, ml 50 50 50 -- -- -- -- -- --DL-Lactate solution B.sup.4, ml -- -- -- -- -- -- 10 10 10Ultra clarified rumen fluid.sup.5, ml 100 -- -- -- -- -- 1000 1000 1000Distilled water, ml 780 830 780 840 830 890 -- -- --Cysteine HCl.H.sub.2 O, g -- -- -- 0.5 0.5 0.5 0.25 0.25 0.25Glucose, g -- -- -- -- -- -- -- 2.0 --Maltose, g -- -- -- -- -- -- -- -- 2.02-deoxy-D-glucose, g -- -- -- -- 5 -- -- -- --Monensin solution.sup.6, ml -- -- -- -- 10.0 -- -- -- --Final pH 6.8 6.8 6.8 5.3 5.3 5.3 5.8 5.8 5.8__________________________________________________________________________ .sup.1 All media were prepared under 100% CO.sub.2. Agar was added at 1.75-2.0% w/v for solid media. .sup.2 DLSodium lactate 60% syrup (Baker):83.3 g, qs to 500 ml with distilled water to give a 10% w/v solution. Final concentration in the media was 0.5% w/v. .sup.3 Reducing agent: 2.5% each of cysteine HCL.H.sub.2 O and Na.sub.2 S.9H.sub.2 O added after boiling the medium and before autoclaving. .sup.4 DLSodium lactate 60% syrup (Baker): 1.0 ml mixed in 332.3 ml distilled water to give 1.8 mg/ml. Final concentration in media B11R, B12 and B13R was 200 μM (0.0018%). .sup.5 Rumen fluid was collected from concentrate fed steers, 5 hours after feeding, autoclaved, centrifuged and passed through a 0.2 μM filter. .sup.6 Monensin solution: Final concentration in medium was 6 ppm.
TABLE 4__________________________________________________________________________Growth of lactate utilizing isolates on various substrates.sup.1Substrate Lactate plusIsolate None.sup.2 Lactate Glucose Maltose Mannitol Sol. Starch Monensin Lasalocid 2-DG.sup.3__________________________________________________________________________252 + ++ -- +++ +++ + ++ ++ ++280 + ++ -- +++ ++++ + ++ ++ ++298 + ++ -- ++ + + ++ ++ ++310 + ++ -- +++ +++ + ++ ++ ++314 + ++ -- +++ +++ + ++ ++ ++320 + ++ -- +++ +++ + ++ ++ ++382 + ++ +++ +++ -- -- +++ +++ +++394 + ++ ++ + -- -- ++ ++ +++407 + ++ ++ +++ -- -- ++ ++ ++414 + ++ +++ + -- -- ++ ++ +++__________________________________________________________________________ .sup.1 Degree of growth on agar plates: -- = Not determined, + = very slight growth, ++ = good growth, +++ = heav growth, ++++ = luxuriant growth .sup.2 L medium without lactate .sup.3 Monensin and lasalocid at 6 ppm; 2DG at 0.5%, adjusted to pH 5.8
TABLE 5______________________________________Growth rate determinations ofseveral lactate utilizing bacteria.sup.1 Specific GenerationIsolate Growth Rate Time (h)______________________________________382 0.644 1.08394 0.187 3.71407 0.580 1.19414 0.543 1.27______________________________________ .sup.1 Isolates grown in L medium
TABLE 6______________________________________Volatile fatty acid profiles oflactate utilizing bacteria grown in B2 medium.sup.1Fermentation acids (mM) Iso- Iso-Isolate Acetic Propionic butyric Butyric valeric Valeric______________________________________252 -47.86 5.56 -0.30 21.51 0.73 2.82280 -46.08 8.83 -0.36 17.61 0.32 2.37298 -48.21 6.34 -0.46 19.23 0.47 2.57310 -43.94 5.70 -0.52 22.83 0.47 2.78314 -46.17 5.28 -0.52 21.90 0.43 2.67320 -62.38 0.44 -0.28 28.75 0.85 3.17382 -40.63 3.54 -0.38 32.08 0.84 3.09394 -13.18 -4.51 -0.58 4.29 -0.28 1.75407 -50.38 -4.34 -0.38 43.92 0.80 2.93414 -40.21 2.47 -0.30 34.61 0.94 3.16______________________________________ .sup.1 Net production of acids, compared with uninoculated B2 medium.
______________________________________Chart 1. Scheme for Selection of Lactate Utilizing Bacteria______________________________________Representative colonies from enrichment or direct plating↓Test for carbohydrate use and growth on lactate in presenceof 6 ppm monensin or lasalocid↓Re-streak for purity on L medium↓Test for low pH tolerance↓Assess fermentation acidsafter growth on lactate↓Utilization of 50 μg/ml (0.55 mM) lactatein presence of 0.2% glucose or maltose(B11-R, B12-R and B13-R)↓Store isolates in B medium with 20% w/v glycerol, at -153°______________________________________C.
EXAMPLE 2
In vitro Acidosis Test System to Assess Lactic Acid Consumption by Lactate Consuming Ruminal Bacteria
This example shows the development of an in vitro test system to mimic the acidotic process in rumens and to use it to select strains of lactic acid consuming rumen bacteria for use as ruminal inocula to prevent acidosis in vivo.
The in vitro test system is summarized as follows: Ruminal fluid, collected from forage fed animals, is mixed (50:50) with 2% (wt/vol) amylopectin in anaerobic dilution solution and incubated with shaking at 38° C. for 12 h. The response variables, pH and lactic acid concentrations, are measured at hourly intervals. During the incubation period, the pH of the rumen fluid mixture decreases to <6.0 and lactic acid accumulates to >35 mM within 6 h. These responses are similar to those observed in the rumens of cattle switched from forage- to grain-based diets. We used this in vitro system to test selected strains of lactic acid consuming rumen bacteria (Example 1) for their ability to prevent the decrease in pH and (or) the increase in lactic acid. Criteria for success were maintenance of pH above 6.0 and at least an 80% reduction in the amount of lactic acid produced relative to uninoculated controls. Six anaerobic, lactic acid consuming bacteria previously isolated from the rumens of grain fed cattle from Example 1 were tested. Four strains met the criteria for success. These 4 strains were further compared with respect to growth rate on lactic acid, lactic acid consumption in the presence of sugars, and sensitivity to antibiotics commonly used in the feedyard. Through this test scheme, 2 strains were selected: #320 and #407.
Animals and diets: Hereford X Angus crossbred steers fed high forage diets (>70% alfalfa hay or corn silage, dry matter basis) fed ad libitum, were used. Ruminal contents were collected by stomach tube (non-fistulated animals) or by rumen fistula. Animals had free access to water at all times. For each experiment, ruminal fluid was collected from 3 animals.
Media: Amylopectin (Sigma) was prepared as a 2% (wt/vol) suspension in anaerobic dilution solution (ADS, 5). Strains of lactic acid consuming bacteria were maintained on B1 medium containing 0.5% (wt/vol) sodium (DL-)lactate. Inoculations and transfers of bacteria were performed in an anaerobic glove box (Coy Lab. Products, Ann Arbor, Mich.; atmosphere: 85% N 2 , 10% H 2 and 5% CO 2 , ambient temperature). All media were prepared under 100% CO 2 by methods described previously (Example 1).
In vitro acidosis test procedure: Ruminal contents samples were collected 1.5 h postfeeding under a flow of CO 2 into flasks and held on ice until delivered to the laboratory. Ruminal samples were strained through 2 mm nylon mesh, pooled in equal weights, and chilled on ice for 20 min. When added, 400 ml of B1 broth cultures of the test strains of lactic acid consuming bacteria were grown to mid-log phase, harvested by anaerobic centrifugation, and resuspended in 14 ml ADS for use as inoculants. Under a flow of CO 2 , 10 ml pooled rumen fluid and 0.5 ml bacterial inoculant (or 0.5 ml ADS) were placed in 60 ml serum bottles, sealed with gray stoppers, and chilled on ice. Twenty two bottles were filled for each strain (control or treatment) tested. When the last bottle was filled, all bottles were fitted with bunsen valves and placed in a rotary shaker-incubator (New Brunswick, 160 rpm) at 38° C. The bottles were allowed to equilibrate 20 min, then 2 bottles of each treatment were collected at 0, 2, and at hourly intervals thereafter through 12 h.
Growth measurements: Growth rates of selected strains were compared on B15 broth medium. B15 was a modification of the basal (B) medium previously described (Example 1), and contained additionally (percentage in final medium): sodium lactate (99.5+% L-lactate, Baker, 0.18%), glucose (0.2%), and maltose (0.2%). Optical densities were measured at 650 nm in a Perkin-Elmer spectrophotometer, Model 55, with a path length of 18 min.
Analytical: The pH was recorded after transferring the bottle contents to a plastic centrifuge tube and immersing a standard combination pH electrode. Then the samples were frozen at -20° C. for later analysis of fermentation acids. Lactic acid concentrations were determined by the methods of Barker, S. B. and W. H. Summerson, supra; or a modification thereof. Both the D(-) and L(+) forms of lactic acid were measured unless otherwise stated. References to lactic acid in this report are to the total of D(-) and L(+) forms. Volatile fatty acids were analyzed as described previously in Example 1.
Antibiotic susceptibility: Minimal inhibitory concentrations (MICs) of selected antibiotics were determined following NCCLS guidelines (National Committee for Clinical Laboratory Standards, Approved standard M11-A: reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria, National Committee for Clincial Laboratory Standards, Villanova, Pa. (1985)) with Wilkins-Chalgren agar medium (Difco) containing 1% (vol/vol) reducing agent (2.5% L-cysteine hydrochloride/2.5% sodium sulfide, wt/vol). The following antibiotics were tested: NAXCEL Sterile Powder (The Upjohn Co.), oxytetracycline (P-L Biochemicals), erythromycin (Sigma), penicillin G (Sigma), lincomycin (hydrochloride, The Upjohn Co.), tylosin (tartrate, Sigma), rifampin (Sigma), thiopeptin (Fugisawa), thiostrepton (Sigma), and chlortetracycline (ICN Nutritional Biochemicals). NAXCEL Sterile Powder, penicillin G, lincomycin, and tylosin were dissolved in water. All others were dissolved in DMSO. Antibiotic concentrations were prepared from 0.25 to 128 μg/ml (or Units/ml) correcting for the specified purity of each agent, or, if no information was available, the purity was assumed to be 100%. Three strains of anaerobic bacteria were included in the MIC determination as positive controls (known MICs for the antibiotics used). These bacteria were: Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Clostridium perfringens ATCC 13124.
The decrease in pH to below 6.0 and increase in lactic acid concentration to above 35 mM was reproducible among replicate bottles and between experiments. Preliminary evaluations of candidate strains of ruminal lactic acid consuming bacteria were conducted on the "best" 6 strains of lactic acid consuming bacteria identified through our selection scheme in Example 1.
Rumen fluid for Experiment #8 was collected from all pasture fed animals. Test strain numbers #310, #320, and #382 were compared against ADS-inoculated control bottles. The pH of the control bottles of the incubating rumen fluid-amylopectin mixture declined after 3 h from its initial value of 6.7 to 5.8 at 6 h. Thereafter the pH remained below 6.0. Meanwhile, the concentration of lactic acid in the control bottles increased from the level of detection (ca. 0.5 mM) to 39 mM at 6 h. The concentration of lactic acid decreased after 6 h to 24 mM at 12 h. In bottles inoculated with any of the three test strains, the pH of the incubating mixture remained above 6.0, and the concentration of lactic acid never rose above 7 mM (4-5 h). Any lactic acid produced was consumed by the isolates within a,3 h period.
Fermentation acids produced during the incubation period also were measured. Fermentation acid accumulation plateaued after 6 h but comparisons were made at 12 h. Total acid concentrations at 12 h were 468 mM in the control bottles, and 542, 511, and 526 mM for strains #310, #320, and #382, respectively. In the uninoculated control bottles, acetate, propionate, butyrate and valerate were present at 59, 24, 15, and 2 molar percent, respectively. With the test strains added, these molar percentages changed to (averages of) 50, 17, 26, and 7%, respectively. All test strains yielded similar fermentation acid profiles. On average, bottles with added test strains produced 58 mM more total acids compared to control bottles. Lactic acid in the control bottles comprised a portion of this difference (at 12 h) although it is likely that the fermentation in the control bottles was limited in extent by the low pH after 6 h.
Rumen fluid for the next Experiment was collected from all pasture fed animals. Test strains #394, #407 and #414 were compared against ADS-inoculated control bottles. The pH of the control bottles declined after 4 h from its initial value of 6.8 to 5.7 at 6 h. As in the previous Experiment, the pH remained below 6.0 thereafter. In this experiment, test strains #407 and #414 kept the pH above 6.0, whereas strain #394 did not. Lactic acid began to accumulate in the bottles after 4 h of incubation. The concentration of lactic acid in the control bottles increased from the level of detection to 45 mM at 6 h. After 6 h, the control bottle concentration of lactic acid remained around 45 mM until 12 h when the concentration increased to 66 mM. With strains #407 and #414 added, lactic acid accumulated to 15 mM by 6 h but within an hour was consumed to the level of detection. With strain #394 however, lactic acid accumulated to 36 mM at 6 h and only gradually decreased to 12 mM at 12 h.
In this second Experiment the fermentation acids were more variable; individual acid concentrations did not plateau after 6 h as they had in the first Experiment. Total acid concentrations at 12 h were 476 mM in the control bottles, and 513, 561, and 579 mM for strains #394, #407, and #414, respectively. In the control bottles, acetate, propionate, butyrate and valerate were present at 63, 26, 9, and 1 molar percent, respectively. With test strain #394 added, these molar percentages were 62, 17, 11, and 10%, respectively. With strains #407 and #414 added, these molar percentages were 45, 15, 30 and 10%, respectively. On average, bottles with added test strains produced 75 mM more total acids compared to control bottles. If strain #394 is omitted, strains #407 and #414 each produced an average of 94 mM more total acids than did controI bottle fermentations. As before, the concentration of lactic acid in the control bottles comprised a potion of this difference although it is likely that the control bottle fermentations were limited by low pH.
None of the 6 strains tested in this in vitro incubation system produced propionate, the preferred fermentation product (based on positive correlation with improved animal performance), as the predominant fermentation acid. All appeared to produce large amounts of butyrate which decreased the molar percentages of acetate and propionate by 30%. Likewise, the presence of added test strains increased the molar percentages of butyrate and valerate by 2- to 3-fold and 3- to 10-fold, respectively. These data agree with the fermentation acids produced by these test strains in pure culture (Example 1 ).
Five of the 6 test strains maintained the pH of the bottle contents above 6.0 which is advantageous for the continuance of a balanced ruminal fermentation. Continuance of the fermentation under these conditions was supported by the higher total acid concentrations in the bottles containing test strains compared to control bottles (including lactic acid).
More importantly however, lactic acid produced by the indigenous rumen bacteria on the added amylopectin (starch substrate) was consumed by 5 of the 6 added test strains of lactic acid consuming bacteria. We chose the best strains from the two Experiments for further evaluation. These were strains #320, #382, #407, and #414.
These 4 were assessed for their ability to consume lactic acid in the presence of glucose and matlose (medium B15). Comparisons of their growth curves and consumption rate of lactate and glucose showed that all strains grew as fast or at a faster rate than other rumen bacteria. Generation (doubling) times in B15 medium were 0.93, 1.21, 0.87, and 1.56 h for strain #320, #382, #407, and #414, respectively. All 4 strains were able to cometabolize lactate and glucose (maltose concentrations in the medium were too low to allow interpretation), although #320 and #407 used both substrates at faster rates. Thus, strains #320 and #407 with doubling times of 1 h or less were "better" than #382 and #414. Their faster growth rates might enable them to better compete in the rumen.
The minimal inhibitory concentrations for the 4 selected strains were determined with an array of antibiotics (Table 7). The MIC results can be interpreted as the same within plus or minus two 2-fold dilutions. Data for the control microorganisms agreed with their known MICs. We found strain differences for NAXCEL Sterile Powder, erythromycin, penicillin G, lincomycin and tylosin. All 4 test strains were equally sensitive to rifampin and were tolerant of oxytetracycline, thiopeptin, and thiostrepton (Table 7). Strain #414 was the most tolerant to tylosin, a common cattle feed additive, while strain #320 was most sensitive to lincomycin, a common swine feed additive.
We selected strains #320 and #407 for further evaluation as potential acute acidosis preventatives based on the results of the in vitro lactic acidosis tests, strain growth rates, and the MIC data.
Methods for administering microbial cultures to animals are well known to those in the art (see e.g., U.S. Pat. No. 4,138,498 which is incorporated herein by reference.)
TABLE 7__________________________________________________________________________Minimal inhibitory concentrations (μg/ml) of selectedantibotics.sup.1 Reference Strains ATCC ATCC ATCC 25285 29741 13124 Lactic acid utilizing test strainsAntibiotic B. Fragilis B. theta. Cl. perf. #407 #320 #382 #414__________________________________________________________________________Naxcel ® 128 >128 16 16 >128 8 32Oxytetracycline 1 64 ≦0.25 32 128 64 64Erythromycin 64 64 8 32 8 32 128Penicillin G 64 64 1 8 128 2 64Lincomycin 32 64 1 32 ≦0.25 32 32Tylosin 8 16 2 16 16 16 128Rifampin 0.5 2 ≦0.25 4 2 4 4Thiopeptin 128 >128 ≦0.25 >128 >128 >128 >128Thiostrepton 128 >128 ≦0.25 >128 >128 >128 >128Chlortetracycline 1 16 ≦0.25 16 64 32 32__________________________________________________________________________ .sup.1 Method as in (17). Penicillin in units/ml.
EXAMPLE 3
Characterization and Presumptive Identification of Ruminal Lactate Utilizing Bacterium 407A.
Ruminal lactate utilizing bacterial isolate 407A (NRRL B-18624; UC-12497) and two strains of Megasphaera elsdenii (strains B 159 and ATCC 25940) were characterized according to published guidelines. Isolate 407A was similar to M. elsdenii strain B 159 and the type strain ATCC 25940 in that it was a large, non-motile, gram-negative coccus that produced isobutyric, butyric, isovaleric, valerie and caproic acids. Hydrogen was produced by all three bacteria from the metabolism of lactate. All grew at pH 5.4 on lactate but only isolate 407A grew on lactate at pH 5.0 in the presence of 6 ppm monensin. All converted threonine to propionate, were indole negative, did not reduce nitrate or hydrolyze starch. Isolate 407A was inhibited by bile whereas 25940 and B159 were not. Strain B159 produced acid in, media containing glucose, maltose or mannitol but 407A did not, whereas type strain 25940 produced only weak acid from maltose and mannitol. The results obtained for isolate 407A show that it is similar to the descriptions of M. elsdenii. However, growth of 407A in lactate-containing media at pH 5.0 in the presence of monensin may place this bacterium in a taxonomic group other than M. elsdenii.
Bacteria and their cultivation: Strain 407A (UC-12497) was isolated according to Examples 1 and 2 and was stored under liquid nitrogen. The type strain of Megasphaera elsdenii, strain number 25940, was received from the American Type Culture Collection, Rockville, Md. and was originally isolated from the rumen of a sheep. Strain B159 was obtained from the culture collection of Dr. M. P. Bryant, Univ. of Illinois and also was originally isolated from a sheep rumen. All strains were routinely cultivated on B18 medium (Table 8) at 38° C. under a CO 2 headspace.
Test media: Commercial preparations of anaerobic peptone yeast extract fermentation media conforming to the formulations and methods of the Virginia Polytechnic Institute (V.P.I.) Anaerobe Laboratory manual, Holdeman, L. V., E. P. Cato and W. E. C. Moore (ed.). Anaerobe laboratory manual, 4th ed., Virginia Polytechnic Institute and State University, Blacksburg, were purchased from Carr-Scarborough Microbiologicals, Inc. (Stone Mountain, Ga). Low pH lactate containing media were prepared without and with 0.5% 2-deoxy-D-glucose or 6 ppm monensin and designated B20, B21 and B22 respectively (Table 8). The final pH values of the B20, B21 and B22 media were 5.43, 5.65 and 5.04, respectively.
Procedures: Young (7 h) cultures grown in B18c were used to determine the Gram stain and to inoculate chopped meat carbohydrate (CMC) medium (Carr-Scarborough Microbiologicals, Inc.). The CMC cultures were incubated overnight (17 h) and then used to inoculate the test media tubes (4 drops/tube) and B18c agar plates. The tubes were inoculated under CO 2 to comply with the V.P.I. methodology, Holdeman, L. V., et al., supra. The stoppers were removed under a flow of anaerobic grade CO 2 , the tubes were inoculated and then resealed. After the tubes were incubated statically and upright at 38° C. for 46 to 47 h they were opened, the pH of the contents was measured and the appropriate biochemical tests were performed (Table 9).
Analytical: Measurement of pH was done with a Corning Model 12 pH meter equipped with a Corning semi-micro combination electrode, model 476541. PY lactate cultures were assayed for D(-) and L(+) lactic acid. PY basal, lactate, glucose and threonine cultures were analyzed for volatile fatty acids via flame ionization gas chromatography. Hydrogen was measured with a gas chromatograph equipped with a thermal conductivity detector using conditions similar to those described by Nelson, D. R. and J. G. Zeikus, Appl. Environ. Micro., 28, pp. 258-261 (1974).
The biochemical tests chosen for the identification of 407A were based on the key for anaerobic genera of bacteria as described in the V.P.I. manual, Holdeman, L. V., et al., supra. The key in the manual indicates that strain 407A is likely from the Megasphaera genus because it is a large gram-negative coccus producing isobutyric, butyric, isovalerie, valerie and caproic acids. Consequently, those tests were done which would sufficiently differentiate Megasphaera from all the other characterized anaerobic cocci. Additionally, we knew that isolate 407A would grow in the low pH, lactate-containing media (B20, B21 and B22). We tested these media to see if they were inhibitory to M. elsdenii strains 25940 and B159.
Results of the biochemical tests and VFA analyses used to characterize the bacteria are presented in Table 9. Examination of the three strains via phase contrast microscopy revealed large (ca. 2.3 μm diameter) cells that occurred singly, in pairs or short chains. In older cultures, chains were more commonly seen. All three bacteria stained gram-negatively. Surface colonial morphologies after 2 days of incubation for the three strains were similar. They were 2 to 3 mm in diameter, circular and entire, buff colored, and had a butter-like consistency.
All bacteria failed to produce acid when cellobiose, esculin, lactose, starch, sucrose and xylose were the carbon sources in the test media. Catalase was not detected, esculin was not hydrolyzed and all were obligately anaerobic. None of the bacteria digested meat, reduced nitrate, hydrolyzed starch, produced indole or were motile. B159 produced more acid in glucose, maltose and mannitol than did 407A and 25940. Bile inhibited growth of 407A, but not of the other two strains. All grew well on lactate at pH 5.3-5.7 in the presence or absence of 2-deoxy-D-glucose, but only 407A grew at pH 5.0 in the presence of 6 ppm monensin. Threonine was converted to propionate by all three bacteria. Hydrogen was produced by all three at levels of 6.2, 12.9 and 26.7% for 407A, 25940 and B159, respectively. VFA profiles were similar for each microorganism grown on the basal, lactate, glucose and threonine media. Butyrate was the main fermentation end product in the three bacteria tested. This is in agreement with data reported by others for M. elsdenii.
The tests unique to strain 407A were 1) its lack of acid production from glucose, maltose and mannitol, 2) its poorer growth in the presence of bile and 3) growth in lactate containing medium at pH 5.0 in the presence of 6 ppm monensin (Table 9). Acid production (or the lack of it) from carbohydrates by a bacterium is one of the key traits for its identification. Only the tests done for B159 fully conformed to the results in the V.P.I. manual. The type strain 25940 did not fully conform because glucose was negative, and maltose and mannitol produced only intermediate amounts of acidity ("weak acidity", Table 9).
Only two other genera of the anaerobic cocci, Veillonella and Acidaminococcus are gram negative. Isolate 407A differs from Veillonella because it is much larger, produces isobutyric, burytic, isovaleric and caproie acids and converts threonine to propionate. It differs from Acidaminococcus because the latter is smaller, does not produce caproute and does not utilize lactate. Thus, the characteristics of isolate 407A are more comparable with the taxonomy of M. elsdenii, but because it can grow well at pH 5.0 in the presence of monensin, 407A may be a new species or subspecies of the genus Megasphaera.
The phylogenetic relationship of Megasphaera to other anaerobic cocci is poorly known, Hill, G. B., (1981) The anaerobic cocci, pp. 1631-1650, In M. P. Starr, H. Stolp, H. G. Truper, A. Balows and H. G. Schlegel (ed.), The prokaryotes: a handbook on habitats, isolation, and identification of bacteria. Springer-Verlag, New York; Rogosa, M., Anaerobic gram-negative cocci, pp. 680-685. In N. R. Kreig and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore, (1984). To date, only one species of Megasphaera is known, M. elsdenii. Although there are some discrepancies between the results of our tests and those of the V.P.I. manual, there may not be enough evidence at this point to support that 407A is other than a new strain of M. elsdenii. However, identification of isolate 407A on the basis of biochemical tests done in this study should be regarded as presumptive.
TABLE 8______________________________________Composition of B18c, B20, B21, and B22 media Amount per liter B18c B20 B21 B22______________________________________sodium lactate, 16.7 g 16.7 g 16.7 g 16.7 g60% syrup.sup.aMineral 1.sup.b 37.5 g 37.5 ml 37.5 ml 37.5 mlMineral 2.sup.c 37.5 g 37.5 ml 37.5 ml 37.5 mlresazurin 1.0 ml 1.0 ml 1.0 ml 1.0 ml(0.1% solution)Trypticase (BBL) 5.0 g 5.0 g 5.0 g 5.0 gPeptone 5.0 g 5.0 g 5.0 g 5.0 g(Bacto, Difco)Yeast extract (Difco) 5.0 g 5.0 g 5.0 g 5.0 gDistilled water 865.0 ml 910.0 ml 910.0 ml 910.0 mlboil under CO.sub.2, cool,then add:cysteine HCL.H.sub.2 O 0.5 g 0.5 g 0.5 g 0.5 gsodium carbonate 50.0 ml -- -- --(8% wt./vol,prepared under CO.sub.2)Acetic acid -- 1.2 ml 1.2 ml 1.2 ml -- (1.3 g) (1.3 g) (1.3 g)Sodium acetate -- 6.5 g.sup.d 6.5 g.sup.d 6.5 g.sup.d(anhydrous)autoclave at 121° C.for 20 min2-deoxy-D-glucose -- -- 5.0 g.sup.e --Monensin solution.sup.f -- -- -- 10 ml______________________________________ .sup.a Sodium lactate: Sigma No. L1375 contained approximately equal amounts of D(-) and L(+) isomers .sup.b Mineral 1:6 g K.sub.2 HPO.sub.4 per liter distilled water .sup.c Mineral 2:6 g KH.sub.2 PO.sub.4 ; 6 g (NH.sub.4).sub.2 SO.sub.4 ; 12 g NaCl; 2.45 g MgSO.sub.4.7H.sub.2 O and 1.59 g CaCl.sub.2.2H.sub.2 O per liter distilled water .sup.d pH of medium adjusted to 5.2 before autoclaving .sup.e Prepared in an anaerobic glovebox with deoxygenated water, and the added aseptically to sterile, but cooled medium by using filtration. .sup.f Added 32.4 mg monensin sodium (Sigma M2513) to 50 ml 200 proof ethanol. Mixed then added filter sterilized solution to sterile medium. Final concentrations of monensin was 6 ppm.
TABLE 9__________________________________________________________________________Tests and methods used for the characterization of bacterial isolate 407Aand Megasphaera elsdenii,strains 25940 and B159..sup.aTest Media.sup.b Procedures and interpretation__________________________________________________________________________pH Basal (PY) +: measure 48 h culture with pH electrode - if pH >6.0 then score as negative (-), if cellobiose pH 5.5 to 6.0 then score as weak acid (w), if pH <5.5 then score as strong acid (a). esculin glucose lactose maltose mannitol starch sucrose xylosebile glucose + bile compare growth to PY glucose - record as inhibited (no growth), growth (+ to ++++) or stimulated (growth better than in PY glucose).catalase chopped meat add 0.5 ml culture to small tube - add 0.5 ml 3% H.sub.2 O.sub.2 - continuous bubbles = +.esculin hydrolysis esculin add 3 drops 1% ferric ammonium citrate - black color = +.growth aerobically B18c agar streak plates with overnight culture, incubate aerobically, check for growth, 48 h.growth on lactate at pH 5.4 B20 record as inhibited (no growth) or growth (+ to ++++).growth on lactate at pH 5.6 in B21 record as inhibited (no growth) or growth (+ to ++++).presence of 2-DGgrowth on lactate at pH 5.0 in B22 record as inhibited (no growth) or growth (+ to ++++).presence of 6 ppm monensinhydrogen production from B18c measure % H.sub.2 in the headspace.lactateindole indole nitrate 2 ml culture +1 ml xylene, mix, let stand 2 min. Slowly add 0.5 ml Erlich's reagent. Pink color = positive, yellow = negative.lactate utilization lactate measure total lactate - compare with uninoculated PY lactate and in PY basal - decrease in lactate = utilization (+).meat digestion chopped meat disintegration of meat particles = positive reaction. Incubate 14-21 days.motility B18c observe 4-6 h culture via phase contrast microscopy.nitrate reduction indole nitrate add 1 ml nitrate Reagent A and 0.5 ml nitrate Reagent B to culture. Red = positive (nitrite present). If no red color, add Zn dust - red color = negative, no color = complete reduction.starch hydrolysis starch add 2 or 3 drops of Gram's iodine to culture. Observe immediately. Black = no hydrolysis.VFA profiles from: PY basal gas chromatography - compare results with uninoculated media - if acids produced glucose in amounts > 1 meg./100 ml then the abbreviation for that acid capitalized, if lactate acids produced in amounts < 1 meg./100 ml then the abbreviation for that acid is threonine presented in lower case. Abbreviations as in the V.P.I. manual (5).__________________________________________________________________________ .sup.a Most tests and procedures as described in the V.P.I. anaerobe laboratory manual (5). .sup.b Prereduced, anaerobically sterilized media see Appendix for formulation.
TABLE 10__________________________________________________________________________Biochemical reactions and gas chromatographic profiles for lactateutilizingisolate 407A and Megasphaera elsdenii strains 25940 and B159.sup.a Reactions/Profiles isolate strain strainTest 407A 25940 B159__________________________________________________________________________gram stain - - -colonial morphology 2-3 mm dia. 2-3 dia. 2-3 mm dia. circular, entire, circular, entire, circular, entire, biege, butyrous biege, butyrous biege, butyrouspH in:PY (basal) - - -cellobiose - - -esculin - - -glucose - - alactose - - -maltose - w amannitol - w astarch - - -sucrose - - -xylose - - -bile + ++++ ++++catalase - - -esculin hydrolysis - - -growth aerobically - - -growth at pH 5.4 ++++ ++++ ++++growth at pH 5.6 ++++ ++++ ++++in presence of 2-DGgrowth at pH 5.0 ++++ ± ±in presene of 6 ppmmonesinhydrogen production 6.2 12.9 26.7from lactate.sup.bindole - - -lactate utilization + + +meat digestion - - -motility - - -nitrate reduction - - -starch hydrolysis - - -threonine conversion + + +to propionateVFA from:.sup.cPY (basal) p, ib, b, iv, v, c p, ib, b, iv, v, c a, p, ib, b, iv, v, clactate A, P, ib, B, iv, V, c A, P, ib, B, iv, V, c A, P, ib, B, iv, v, cthreonine p, ib, b, iv, v, c p, ib, b, iv, v, c a, p, ib, b, iv, vglucose a, p, ib, b, iv, v, c a, p, b, ib, iv, v, c a, p, ib, B, iv, v, c__________________________________________________________________________ .sup.a Refer to Table 2 and text for interpretation and keys. Methods based on those of the V.P.I manual (5). .sup.b Percent H.sub.2 in the head space. .sup.c Compared to uninoculated media.
EXAMPLE 4
This example summarizes data from 3 pairs of animals ruminally inoculated with strain 407A, each pair inoculated at a different time during acidosis induction, compared to their contemporary (block) controls.
Experimental design: The parent study was designed based on an in vivo model of acute lactic acidosis. Four blocks of 4 animals each were used. Each block was separated in time by 2 to 4 weeks. In each of blocks 2, 3 and 4, two additional ruminally fistulated steers were added to make a group of 6. Steers were randomly assigned to either control or inoculated (407A-dosed) treatment groups. Block 1 was not included as 407A cells were unavailable. The data summarized below were derived from the steers in blocks 2, 3 and 4.
Animals and their management: Hereford x Angus crossbred steers were used. These cattle (about 300 to 380 kg body weight) were ruminally fistulated, housed in motels and fed ad libitum a low quality alfalfa hay-wheat straw diet (5:2; minimal gain of 0.1 to 0.2 kg per day) for a minimum of three weeks. Just prior to use, each animal was weighed (Table 11 ). Subsequent feedings of chopped hay or acidsis-inducing grain meals were based on this bodyweight (BW).
Feeding regimen and rumen sampling: Cattle were moved into the measurement room in the afternoon prior to the experimental period and not fed until the following day. Water was available thwughout the feeding and fasting period. On Day 1, the animals were fed 0.5 % BW meals consisting of chopped alfalfa at 7 AM and 3 PM and at 7 AM on Day 2. Any feed remaining was delivered intraruminally via the fistula. No feed was offered on Day 3. Acute acidosis was initiated on Day 4 starting at 6 AM. The fistula was opened and two 5 ml samples of ruminal contents were collected, frozen immediately in plastic tubes immersed in dry ice-ethanol, and stored at -20° C. for lactate and volatile fatty acid (VFA) analysis. Ruminal pH was measured with an immersible flat surface electrode (Sensorex 450C, Stranton, CA) and an MP-815 pH meter (Fisher Scientific) with the probe held approximately midpoint in the rumen. Immediately following, the first of four 0.5% BW meals consisting of 90% ground corn and 10% molasses, hand-mixed with an equal weight of water, was introduced as a bolus dose into each rumen via the fistula. The remaining meals were delivered similarly at 7, 8 and 9 AM. In addition to the rumen samples collected at 6 AM, subsequent rumen samples were obtained through the fistula at intervals after bolus dosing. In such cases, no more than 1250 ml of ruminal fluid was collected per animal after the pH had been measured.
Animal recovery after initiation of acidosis: After the last sample from each animal was taken, the steers were offered chopped hay ad libitum. The following day, the animals were returned to the motels where they had free access to long hay and minimal supplement.
Preparation of Strain 407A for ruminal inoculation: Lactic acid consuming ruminal bacterium strain 407A (UC-12497) was grown in static culture on medium B18c (Table 8) at 37° C. Routine transfers were done using (crimp seal) serum tubes (5 ml medium/tube). Using a 1% inoculum level, an overnight culture was used to inoculate 2, 60 ml broths in 100 ml serum bottles. These scale up cultures were incubated 6 to 7 h and then used to inoculate a 20 l glass carboy containing 12 l of B 18c medium. The inoculated carboy was incubated statically overnight (16 to 18 h) at 37° C. After that time a sample was withdrawn aseptically for OD measurement (650 nm, path length 18 mm) and for viable count determination (see below).
Anaerobic harvesting and preservation of viable cells: 407A cells were harvested by anaerobic centrifugation. Air-fight, o-ring sealed, centrifuge bottles (DuPont), deoxygenated by previous equilibration within an anaerobic glovebox, were used. The bottles were filled in the glovebox using house vacuum to dispense the carboy culture, balanced (weighed) and sealed. The bottles then were centrifuged at 6,250× g in a CS-3 rotor (Sorvall) for 20 rain at room temperature. In the glovebox, the supernatants were discarded and the pelleted cells were resuspended in a small amount of B18c medium with no lactate but with 20% (v/v) glycerol. After all the pellets were resuspended, the suspensions were pooled, the total volume recorded, and a viable count determination made as described below. The avenge total volume of concentrated cell suspension from each 12 l carboy was 123 ml representing about a 100-fold concentration factor. The B18c-glycerol cell suspensions were distributed into 100 ml serum bottles and frozen at -70° C. Maximum time frozen before use was 3 weeks. Viability tests showed that the frozen suspensions were stable for at least 3 months. Immediately after thawing and prior to inoculation of the suspension into cattle, another viable count was made.
Viable count determination: One ml of culture or concentrated cell suspension was serially diluted in 10-fold steps in ADS buffer (Bryant, M. P. and L. A. Burkey, J. Dairy Sci., 36, pp. 205-217 (1953)). B18c agar (1.5% w/v) plates were inoculated with 50 and 100 μl quantifies of selected dilutions. The plates were incubated at 38° C. under 5 lbs. CO 2 for 48 h in stainless steel incubation vessels and then the colonies were counted to the population of viable cells in the culture or fresh or thawed cell suspensions.
Selection of ruminal dose: Previously, we determined that an inoculation level of 2×10 8 viable cells per ml was an effective dose to reduce the accumulation of lactic acid and to prevent a decrease in pH in vitro. Thus, we targeted this concentration for our in vivo dose level. Since actual rumen volumes of the experimental steers were unknown, we assumed that each had a nominal liquid volume of 30 1. The target was to inoculate 6×10 12 viable 407A cells per rumen, or approximately 2×10 8 viable cells per ml ruminal fluid. Since each carboy yielded a different volume and viability of 407A cells, concentrated cell suspensions from 3 carboys were combined for each block. Thus, in addition to being inoculated at a different time after acidosis induction, each block received a slightly different dose (see Table 12).
Rumen inoculation procedure: On the day of acidosis initiation for each experimental block, concentrated cell suspensions from 3 separate carboy cultures were thawed at room temperature and combined in the anaerobic glovebox. A viable count of the combined suspension was determined Crable 12). The suspension subsequently was divided into 2 equal portions (one for each of the 2 experimental animals) and sealed in erlenmeyer flasks. These were removed from the glovebox and, at the designated time, opened within the ruen and thoroughly hand-mixed with the ruminal contents of each animal. The inoculum was ready for ruminal administration no earlier than 30 min ahead of time. The treated animals received their ruminal inoculum at 4, 3 and 2 h after the initiation (0600 h) of acidosis in blocks 2, 3 and 4, respectively.
Analytical procedures: Lactic acid concentrations were determined using the method previously described. Volatile fatty acid concentrations were determined via an HPLC method (Dionex Corporation, Ion chromatography cookbook: a practical guide to quantitative analysis by ion chromatography, pp. II15-16 (1987)).
In block 2 the treated cattle were inoculated at 4 h post-initiation of acidosis. Strain 407A had previously been shown to reduce the in vitro accumulation of lactic acid at this timepoint.
In block 2, ruminal pH decreased rapidly in the control animals, from a mean of about 8.0 to below 5.0 within 6 h after the initiation of acute acidosis. Ruminal pH in the 2 steers dosed with strain 407A also declined to 5.0 by 6 h but then stayed at pH 5.0 or above for the remaining time while the control animals' pH continued to decrease.
Ruminal lactic acid in the control animals accumulated to over 100 mM between 4 and 8 h post-acidosis induction, and lactic acid remained high throughout the remaining observation period. Ruminal lactate accumulated in the dosed steers rose to about 60 mM between 4 and 6 h post-acidosis induction. Thereafter, lactic acid concentrations continually decreased to less than 20 raM.
Ruminal concentrations of VFAs were monitored for the first 12 h. Before acidosis induction, very little mminal fermentation activity was present in any of the experimental animals. For example, acetate concentrations were about 10 mM. Ruminal fermentation activity, reflected in acetate accumulation, increased steadily with each meal. Between 5 and 12 h, the ruminal acetate concentration in the control animals steadily declined to a level of about 5 mM. This temporal decrease coincided with the large increase in lactic acid production. In contrast, ruminal acetate concentrations in 407A-dosed steers increased over the first 6 h then appeared to hold at 28 mM over the 12 h interval monitored.
Very low levels of propionate, butyrate and valerate (less than 5, 2 and 1 mM, respectively) were detected in control animals over the observation period. However, after ruminal inoculation at 4 h with strain 407A, propionate increased about 8-fold. An 8-fold increase also was observed for butyrate and valerate both of which increased steadily from the 4 h timepoint, reaching mean concentrations of 14 and 4.5 mM, respectively, by 12 h. Less than 1 mM concentrations of isobutyrate and isovalerate were observed in control or dosed animals.
In block 3, mininat inoculation time was moved up one hour; the animals were inoculated 3 h post-initiation of acidosis.
Ruminal pH in the control animals again decreased rapidly from a mean of 8.4 to less than 5.0 within 9 h post-acidosis induction. Ruminal pH in the dosed steers however, decreased less rapidly than in block 2 and remained around 5.5. A low mean pH value of 5.2 was reached at the 16 h timepoint for the dosed steers. Ruminal lactic acid concentrations in control steers again climbed above 100 mM between 4 and 8 h and remained high. In dosed animals, lactate concentrations rose to about 45 mM between 4 and 6 h but declined after 12 h to near zero by 24 h.
In control steers, ruminal acetate concentrations rose during the period of meal feeding but fell off thereafter as in block 2, while acetate concentrations remained around 20 mM in the dosed steers. Propionate, butyrate and valerate concentrations in control animals again were quite low (less than 5, 2 and 1 mM, respectively). In the dosed steers, propionate increased 5-fold after 5 h post-acidosis initiation while butyrate and valerate increased steadily about 10-fold each after 3 h, the time of ruminai inoculation. Less than 1 mM concentrations of isobutyrate and isovalerate were observed in control or dosed animals.
With the analysis of the lactic acid samples being completed from the earlier blocks, it appeared that 407A was able to metabolize lactate immediately upon inoculation and did not appear to suffer if the ruminal concentration of lactic acid was low. With the success of inoculating one hour earlier in block 3, the ruminai inoculation time was moved up one additional hour for block 4 to 2 h post-acidosis induction.
In block 4, mechanical failure caused the loss of ruminai pH data. Ruminal lactic acid concentrations showed that induction of acute acidosis was as successful as in previous blocks. Control animals again showed the rapid accumulation of ruminal lactic acid between 4 and 8 h post-acidosis induction. However, inoculation with 407A 2 h post-acidosis induction, appeared to arrest most of the lactic acid accumulation. The highest level detected in dosed steers was less than 20 mM at 5 to 6 h. The lactic acid concentration was less than 4 mM thereafter.
In block 4, the profiles of acetate concentration in both control and dosed steers were similar to those of previous blocks although the dosed steers' acetate concentration was higher. Propionate, butyrate and valerate concentrations in control animals were similar to those of previous blocks. In contrast to previous blocks, little propionate was produced in the dosed animals. However, butyrate and valerate increased steadily about 40-fold from the 2 h time of ruminal inoculation in dosed steers to 50 and 10 mM, respectively. Less than 1 mM concentrations of isobutyrate and isovalerate were observed in control or dosed animals.
The acidosis animal model used in this example was used to test the lactic acid consuming bacterium 407A. First, the animals were backgrounded on a very low quality diet, then fed hay at 1% BW, and then starved for 24 h prior to acidosis induction. This predisposed these steers to very low levels of endogenous ruminal fermentation activity typified by high ruminal pH (>8.0) possibly due to excess bicarbonate, lower amounts of CO 2 produced and low concentrations of VFAs. Second, the introduction of grain meals (2% BW total) into this system initiated metabolism of the surviving ruminal microbes evidenced by the steady increase in all major VFAs over the first 4 h. After 4 h however, it was obvious that the faster growing, lactic acid producing microbes had overwhelmed their VFA-preducing counterparts, and a lactic acid fermentation was established. Third, although there was some variability in the time required for the ruminal pH to drop below 5.0 and the lactic acid concentration to increase above 100 mM, the model was consistent. Introduction of lactic acid consuming bacterium 407A into this (lactic) acidosis model was a rigorous test of its integrity and metabolism.
In block 2, 407A was inoculated at 4 h post-acidosis induction. This timepoint coincided with the greatest rates of decrease in ruminal pH and increase in ruminal lactic acid. Except for the fact that the ruminal pH did not decline below 5.0, we concluded that 407A was inoculated too late to have a significant effect on pH. In block 3, with ruminal inoculation at 3 h after initiation of acidosis induction, the effect on ruminal pH was more obvious. Block 3 data indicated a mean ruminal pH of 5.5 or above in 407A-dosed animals. Due to technical difficulties, block 4 pH data could not be included. However, the differences in pH between control and 407A-dosed steers may have been even greater because the lactic acid levels in these dosed animals were much less than those in block 3.
The effect of 407A on lactic acid accumulation was large in all blocks tested. In block 2, not only did its concentration reach half that of control animals, but lactic acid was continually reduced with time in the 407A-dosed animals. These data indicated that frozen and thawed 407A cells were capable of immediate lactic acid metabolism upon ruminal inoculation. With the earlier inoculations of blocks 3 and 4, ruminal lactic acid accumulations were effectively blocked by 407A, thus acute lactic acidosis conditions did not develop.
As mentioned, low VFA concentrations were present in all animals prior to acidosis induction. In acidotic control animals, the increase in major VFAs was replaced by lactic acid after 4 h post-induction. Regardless of the time of ruminal inoculation of 407A-dosed animals, butyrate and valerate, the major fermentation products of 407A when grown on lactate, increased immediately after inoculation. The increase in propionate followed 1 to 2 h later in blocks 2 and 3.
Butyrate levels were highest when propionate levels were lowest (block 4). In contrast, butyrate levels were lowest when propionate levels were highest (block 2). When the rumens were dosed at the 4 h timepoint (block 2), the soluble sugar concentration (mostly glucose and maltose) was high, nearly 50 mM. Thus, sugars as well as lactic acid were available to 407A. In block 4 when the rumens were inoculated at the 2 h timepoint, correspondingly less sugar and more lactic acid was available for fermentation. Since 407A also can grow on some sugars these ruminal conditions may be responsible for the differences in VFA profiles observed. This phenomenon has been observed previously in vitro with Megasphaera elsdenii, Marounek, M., et al., Appl. Environ. Microbiol, 55, pp. 1570-1573 (1989). M. elsdenii can utilize several substrates simultaneously and make propionate, butyrate and valerate, Marounek, M., et al., supra, and Russell, J. B. and R. L. Baldwin, Appl. Environ. Microbiol., 36, pp. 319-329 (1978).
In summary, prevention of acute lactic acidosis was progressively more successful in terms of maintaining a more neutral ruminal pH and low lactic acid concentration, when the rumen was inoculated with 1-2×10 8 viable 407A cells per ml ruminal fluid, 4, 3 and 2 h after acidosis induction. From these data it appears that lactic acid consuming bacterium 407A UC-12497) is useful as an acidosis preventative or adaptation aid.
Isolate 407A has been deposited at The Upjohn Culture Collection, The Upjohn Company, Kalamazoo, Mich. 49001 and has been assigned deposit number UC-12497. Isolate 407A was also deposited at the ARS Patent Culture Collection, Agricultural Research Service Culture Collection, Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604 on Feb. 15, 1990 under accession number NRRL B- 18624.
TABLE 11__________________________________________________________________________Animal numbers, body weights, meal size and the time of ruminalinoculationBlock Experiment Trial Actual Body weight 0.5% BW meals Time of ruminalno. date animal no. animal no. (kg) (kg) inoculation.sup.a__________________________________________________________________________1 30 1 2046 355 1.6.sup.b ND.sup.c March 2 2063 325 1.4 1989 3 2102 338 1.4 4 2011 354 1.62 18 5 2099 317 1.6 April 6 2049 357 1.8 1989 7 2051 336 1.7 8 2030 329 1.6 101 2093 364 1.8 4 102 2028 337 1.7 4 9 2088 370 1.83 03 10 2040 388 1.9 May 11 1087 364 1.8 1989 12 2042 369 1.8 103 2039 361 1.8 3 104 2029 368 1.8 3 13 2021 356 1.84 07 14 2092 308 1.5 June 15 2062 296 1.5 1989 16 2001 304 1.5 105 2075 340 1.7 2 106 2004 350 1.75 2__________________________________________________________________________ .sup.a Hours after initiation of acidosis .sup.b Calculation of size of 0.5% BW meals were done incorrectly in this block .sup.c Not done
TABLE 12__________________________________________________________________________Viability data - 407A Carboy Combined cell susp. Rumen inoculant/BlockBlock V.C..sup.a Volume V.C. Volume.sup.b V.C. Est. doseNo. No. OD.sub.650 × 10.sup.8 (ml) × 10.sup.10 (ml) × 10.sup.10 × 10.sup.8__________________________________________________________________________2 A.sup.c 1.345 4.45 70 2.41 1 1.339 ND.sup.d --.sup.e -- 2 1.303 ND -- -- 3 1.287 ND 372 3.41 221 1.64 1.203 4 1.266 4.20 120 4.20 5 1.384 5.10 105 4.20 6 1.265 2.60 137 7.44 178 3.21 1.904 7 1.065 3.21 100 3.78 8 1.267 3.91 126 3.68 9 1.292 4.44 145 3.19 185 3.48 2.15__________________________________________________________________________ .sup.a Viable count .sup.b Per steer .sup.c 6 l carboy .sup.d Not determined .sup.e Carboys in this block were done collectively on same day
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This invention relates to a bacterial culture, NRRL B-18624, method for facilitating adaptation of ruminants from roughage or normal pasture diet to a higher energy diet, and a composition therefor comprising the bacterial culture.
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FIELD OF THE INVENTION
[0001] This application relates to particle handling and, in particular, to an apparatus for separating particles, such as cereal grains, oil seeds and pulse crops, from a mixture of particles and gas.
BACKGROUND OF THE INVENTION
[0002] When commodities such as cereal grains, oil seeds and pulse crops are harvested, they are often stored in large storage bins, such as silos, to await transportation to market. Typically, the transportation is by truck. In order to transfer the particles or grains of the commodity from a silo, in which it is stored, into a truck, which will transport the grain to market, a grain vacuum may be used.
[0003] An inlet end of the grain vacuum has a hose that is inserted into the silo. An outlet end of the grain vacuum is connected to a grain transport system, such as an auger. The vacuum is turned on to generate a vacuum to pull the grain from the silo into the hose. The operation of the vacuum causes both grain and air to be pulled into the hose and into the grain vacuum. It is desirable to send only the grain and not the air to the grain transport means. To do this, a separation of the air from the grain within the grain vacuum is required.
[0004] One means of effecting the separation of the grain and air is to use a separator assembly with an inner surface shape that draws the air off in a direction perpendicular to the direction of flow of the mixture of grain and air. Such a separator, however, has significant manufacturing cost.
SUMMARY OF THE INVENTION
[0005] An aspect of the invention relates to an apparatus for effecting separation of particles from a mixture of particles and gas, the apparatus comprising: a housing that defines therethrough a channel; an inlet in said housing to admit the mixture of particles and gas into the channel; a deflector in the channel; and a gas outlet port in the channel and downstream of the deflector; wherein the gas outlet port is open in an upstream direction and the deflector is shaped to direct the particles downstream in the channel past the gas outlet port.
[0006] In some embodiments the apparatus further comprises a guide in the channel downstream of the deflector and the guide is shaped to direct the gas to the gas outlet port.
[0007] In some embodiments the deflector is shaped to deflect the particles outwardly.
[0008] In some embodiments the deflector gradually increases in diameter in the downstream direction.
[0009] In some embodiments a longitudinal axis of the deflector extends substantially parallel to the longitudinal axis of the channel.
[0010] In some embodiments the guide gradually decreases in diameter in the downstream direction.
[0011] In some embodiments the deflector is suspended in the channel.
[0012] In some embodiments the sides of the deflector are conical and define an angle of approximately 12° to 30° from the longitudinal axis of the channel.
[0013] In some embodiments a longitudinal axis of the deflector and the gas outlet port are substantially coaxial with the longitudinal axis of the channel.
[0014] In some embodiments the gas outlet port comprises a tube projecting into the chamber.
[0015] In some embodiments the gas outlet port is spaced about 5 to 7 inches from a downstream end of the deflector.
[0016] In some embodiments the particles are cereal grain, oil seed or pulse crop particles.
[0017] In some embodiments the apparatus further comprising a vacuum generator for drawing the combination of particles and gas into the channel and for drawing the gas out through the gas outlet port.
[0018] Another aspect of the invention relates to a method for separating particles from a mixture of particles and gas comprising: drawing the mixture into a channel in a downstream direction; directing the flow of the particles downstream past a gas outlet in the channel, the gas outlet being open in an upstream direction; drawing the gas out through the gas outlet.
[0019] In some embodiments the gas outlet is centrally located in the channel and directing the flow of particles comprises directing the flow of particles radially outwards.
[0020] In some embodiments drawing the gas through the gas outlet comprises guiding the gas radially inwardly towards the gas outlet.
[0021] A further aspect of the invention relates to a separator for use in the separation of particles from a mixture of particles and gas, the separator comprising: a deflector at a first end shaped to deflect the particles radially outwardly; and a guide at a second end shaped to guide the gas inwardly.
[0022] In some embodiments the deflector increases in diameter in a direction from the first end to the second end.
[0023] In some embodiments the guide decreases in diameter in a direction from the first end to the second end.
[0024] In some embodiments the deflector and guide comprise a single component.
[0025] A further aspect of the invention relates to a grain vacuum comprising a vacuum generator and the separator assembly described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Embodiments of the invention will now be described with reference to the attached drawings in which:
[0027] FIG. 1 is an isometric view of a grain vacuum according to an embodiment of the invention;
[0028] FIG. 2 is a top view of the grain vacuum of FIG. 1 ;
[0029] FIG. 3 is a cross-sectional view of the grain vacuum of FIG. 2 taken along line AA;
[0030] FIG. 4 is an enlarged view of detail B from FIG. 3 ;
[0031] FIG. 5 is a perspective view of a structure for use with the embodiment of FIG. 1 ; and
[0032] FIG. 6 is a perspective view of detail C from FIG. 2 .
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0033] This invention relates to an assembly for separating particles, such as particles of grain, from a combination of particles and gas. The gas may be air. The assembly can form part of a grain vacuum for transporting grain from a storage bin, such as a silo, to a transportation container, such as the back of a truck.
[0034] The apparatus includes a channel into which the combination of particles and gas will be drawn from an inlet. A deflector is located in the channel. The apparatus includes a gas outlet port in the channel downstream from the deflector. The gas outlet port is open in an upstream direction. The deflector is shaped to direct the particles downstream past the gas outlet port. There may also be a guide located in the channel downstream of the deflector and upstream of the gas outlet port. If present, the guide is shaped to help to direct the gas to the gas outlet port.
[0035] Turning to the figures in detail, FIG. 1 shows an isometric view of an exemplary grain vacuum 10 . The grain vacuum 10 , of this example, has a body 12 , an inlet 14 at one end of the body and an outlet 16 at an opposite end. The grain vacuum 10 also includes a fan assembly 18 for generating a vacuum.
[0036] The grain vacuum 10 of this embodiment includes a separator assembly 20 , as shown in FIGS. 2 to 4 . The separator assembly 20 is positioned in this embodiment adjacent the inlet end 14 of the grain vacuum 10 . In other embodiments, the separator assembly may be further downstream. The body 12 of the grain vacuum 10 is a housing which defines a channel 22 . In other embodiments, the channel 22 may be formed by a separate element inside the body 12 .
[0037] In this embodiment, a separator 24 consists of a deflector portion 26 and a guide portion 28 . The diameter of the deflector portion 26 in this example gradually increases from the inlet end direction towards the outlet end direction along its longitudinal axis. The diameter of the guide portion 28 gradually decreases from the inlet end direction towards the outlet end or downstream direction along its longitudinal axis. In this embodiment the separator is symmetrical about its longitudinal axis. The deflector portion 26 is conical and the guide portion 28 has a truncated conical shape. The conical shapes that define the deflector portion 26 and the guide portion 28 are oriented in opposite directions and smoothly flow from one to the other.
[0038] In this embodiment the angle α of the deflector 26 to its longitudinal axis may be about 12° to 30° and may be 18° and the angle β of the guide portion 28 to its longitudinal axis may be about 34°. The relative angles and the shapes of the deflector portion 26 and the guide portion 28 may be varied as long as they function as a separator. For example, the sides of the deflector portion 26 may have a concave or a convex rather than a straight shape and the guide portion 28 may be semi-spherical.
[0039] Additionally, the separator 24 need not be completely symmetrical around its longitudinal axis. For example, the angle from the longitudinal axis on the top portion may be different from the angle of the bottom portion to compensate for the effects of gravity. Also, the deflector portion 26 and the guide portion 28 may be separate components, rather than forming a single component may be spaced apart. The guide portion may also be eliminated such that the separator 24 comprises only the deflector portion 26 .
[0040] The separator 24 is located in the channel 22 . In this embodiment, the inlet end 30 of the separator 24 is aligned with the inlet to the channel 22 . Again, this positioning may be varied. The inlet end 30 of the separator 24 may extend out of the channel 22 or may be set back further into the channel 22 . Although the figures depict the tip of the cone of the deflector portion 26 separately, this in merely a manufacturing option and not essential to the invention. The leading or inlet end 30 of the separator may also be other shapes including rounded or flattened.
[0041] As can be seen from FIGS. 3 and 4 , the sides of the channel 22 need not track the shape of the separator 24 . The shape of the channel need only allow the particles and gas to flow as directed by the deflector 26 and the guide 28 .
[0042] The fan assembly 18 is provided to generate the vacuum to pull grain and air into the grain vacuum. As best seen in FIGS. 2 and 6 , a conduit 32 which may, for example, be a pipe, connects from the fan assembly 18 which is external to the body 12 to the interior of body 12 and into the channel 22 . In this embodiment, the conduit 32 has a rounded elbow section so that the orientation of the portion of the conduit 32 which is external to the body 12 is at right angles to the portion of the conduit 32 which is internal to the body 12 .
[0043] An inlet end 34 of the conduit 32 of this example is located adjacent to the end of the guide portion 28 of the separator 24 . In this embodiment, the inlet end 34 is straight and the diameter of the inlet end is smaller than the maximum diameter of the separator 24 . In this embodiment, the separator 24 has a length of 20 inches and a maximum diameter of 10.5 inches and the inlet end 34 of the conduit has a diameter of 8 to 10 inches. However, the size of the conduit may be larger or smaller than the maximum diameter of the separator 24 and configuration of the conduit 32 between the inlet end 34 and the vacuum generator may vary. The inlet end 34 of the conduit 32 may, for example, be flared rather than straight.
[0044] In this embodiment, the separator 24 is suspended by a shaft 25 which extents out from the conduit 32 . The shaft 25 may be bolted to the separator 24 . The separator 24 and the opening of the conduit 32 in this example are centred in the channel 22 along a longitudinal axis of the channel. However, the separator 24 may be otherwise positioned in the channel, for example, it may be slightly offset from centre as long as the position of the separator 24 effects the separation as described further below.
[0045] The shaft 25 , by which the separator 24 is suspended, is connected to the conduit 32 . In this embodiment, three spokes 35 are equally distributed around the conduit and the shaft 25 is suspended from the point of intersection of the spokes along the central axis of the conduit 32 as best seen in FIG. 6 .
[0046] The opening of inlet end 34 of the conduit 32 is substantially co-axial with longitudinal axis of the channel 22 and the longitudinal axis of the separator 24 in this embodiment. These various components need not be co-axial. However, the efficiency of the separator may be higher if the inlet end 34 of the conduit 32 is substantially parallel to and co-axial with the longitudinal axis of the channel and the separator 24 and the conduit 32 are no more than ¼ to ½ inch off axis from each other.
[0047] In this embodiment, the inlet end 34 is spaced from the downstream end of the deflector 26 by a dimension X which is approximately 5 to 7 inches.
[0048] The fan assembly 18 also includes an air outlet 36 through which air that travels through the conduit 32 may exit the grain vacuum 10 . A conventional fan 37 may be utilized for this purpose.
[0049] An auger 38 or other grain transport means may be provided in the channel downstream of the inlet end 34 of the conduit 32 . As best seen in FIG. 3 , in this embodiment, the auger 38 is positioned at an upward angle along an upwardly angled outlet end 39 of the body 12 then through an auger housing 41 to the outlet end 16 of the grain vacuum 10 . In this embodiment, the auger 38 is positioned entirely downstream of the separator assembly 20 . The auger may include an air lock or other means at the outlet end 16 to prevent air from coming in from the outlet end 16 .
[0050] A bottom side 42 of the body 12 of the grain vacuum 10 in this example is downwardly angled such that any grain that falls against the bottom side 42 will be directed to the auger 38 as explained in further detail below. In other embodiments, the bottom may, for example, be flat and the auger positioned horizontally.
[0051] In this embodiment, the body portion of the separator assembly 20 is provided with a hinge 43 to allow the separator assembly 20 to be opened by rotating the upstream end of the separator assembly 20 about the hinge 43 . This allows easy access to the interior of the body 12 to, for example, replace the separator 24 with a differently sized or shaped separator 24 for use in different operating conditions or grain types.
[0052] The flow of the gas and particles through the separator assembly 20 is illustrated in FIG. 4 . The separator assembly 20 is divided here into an inlet section 20 a , a deflector section 20 b , and a guide section 20 c . In the inlet section 20 a , a combination of air 46 and grain 44 are drawn into the inlet 14 of the grain vacuum 10 by operation of the fan assembly 18 . This mixture is drawn axially in a downstream direction along the channel 22 until this mixture contacts the separator 24 . In section 20 b , the vacuum continues to pull the air and grain in a downstream direction of the channel 22 but because the separator 24 is in the way, the air and grain will accelerate up the sides of the separator 24 along the deflector 26 .
[0053] When the air and grain reaches the end of the deflector 26 and moves into section 20 c , the momentum of the heavier grain particles will cause them to continue along outwardly in the direction defined by the deflector 26 past the inlet end 34 . The air being lighter will be pulled by the fan suction on a path defined by the shape of the separator 24 . The air will therefore follow the inwardly shaped guide portion 28 , if present, and proceed out through the conduit 32 and out through the air outlet 36 . Even if the guide portion 28 is not present, the fan suction will draw the air out through the conduit 32 . The inlet end 34 is therefore the outlet port of the air from the channel 22 . The grain, once it passes the inlet end 34 of the conduit 32 will lose its momentum and fall to the bottom of the body 12 where it will land either on the bottom side 42 and slide into the auger 38 or land directly on the auger 38 . The auger 38 once powered will rotate to move the grain outwardly through the outlet 16 .
[0054] In some embodiments, internal to the conduit 32 , there may be spiralling channels defined to minimize dead air spots. This may be achieved, for example, by including a structure 70 , such as shown in FIGS. 5 and 6 , internal to the conduit 32 . The structure 70 has a number of webs 72 , eight are shown in this embodiment, which are connected along the axis 74 of the conduit 32 and extend to the wall of the conduit 32 . These webs spiral such that eight spiralling channels are defined within the conduit 32 .
[0055] It will be appreciated other means of handling the grain once separated from the air may be used.
[0056] Similarly, other means of generating a vacuum may be used and the separator may be used with the other machinery.
[0057] The separator assembly may be used to separate other granular particles and gases of differing mass.
[0058] Numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
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An apparatus for effecting separation of particles from a mixture of particles and gas, the apparatus comprises: a housing that defines therethrough a channel; an inlet in said housing to admit the mixture of particles and gas into the channel; a deflector in the channel; and a gas outlet port substantially parallel to a longitudinal axis of the channel and downstream of the deflector; wherein the deflector is shaped to direct the particles past the gas outlet port. A method of separation is also disclosed.
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BACKGROUND
[0001] 1. Technical Field
[0002] The present invention relates to packaging and, in various embodiments, to chewing gum packaging that may have an attached supplemental compartment, in order to permit proper disposal of wrappers and provide for a cleaner environment.
[0003] 2. Background
[0004] Chewing gum is popular throughout culture for a myriad of reasons. However, once a piece of chewing gum is unwrapped, consumers have the dilemma of what to do with the gum wrapper. To dispose of it the consumer turns to a variety of convenient options be it a pocket, waste basket, purse, or in most instances, littering.
[0005] Accordingly, there is a need for a solution to the disposal of chewing gum wrappers, a potential place for disposal of chewed gum itself if properly wrapped for disposal, and lastly prevention of littering and the promotion of a healthier, green environment.
[0006] The foregoing discussion is intended only to illustrate some of the shortcomings present in the field of the invention at the time, and should not be taken as a disavowal of claim scope.
SUMMARY
[0007] In accordance with general aspects of at least one form, there is provided a gum package device wherein the back side of the gum package has two side members extending from the back side, an additional face connecting the two side members creating an expanding secondary compartment that is open at the top. The secondary compartment is able to close and reopen as needed by the consumer in order to dispose of gum wrapper and gum waste.
[0008] In accordance with other general aspects of at least one form, there is provided a gum package wherein the back side of the gum package has two lateral side members extending from the back side, an additional face connecting the two lateral side members and having an extending flap in order to close both the primary compartment, wherein the edible products are contained, and the secondary compartment, by folding the extending flap over both compartments, and is sealed by placing the tab of the extending face in a slit on the front face of the primary compartment.
BRIEF DESCRIPTION OF DRAWINGS
[0009] The above-mentioned and other features and advantages of this invention, and the manner of attaining them, will become more apparent and the invention itself will be better understood by reference to the following description of embodiments of the invention taken in conjunction with the accompanying drawings, wherein:
[0010] FIG. 1 is a rear perspective view of a package for an edible product in an open position;
[0011] FIG. 2 is a side elevational view of the package of FIG. 1 ;
[0012] FIG. 3 is a front perspective view of the package of FIG. 1 ;
[0013] FIG. 4 is a rear perspective view of another form of package for an edible product;
[0014] FIG. 5 is a side elevational view of the package of FIG. 4 ;
[0015] FIG. 6 is a front perspective view of the package of FIG. 4 ;
[0016] FIG. 7 is a rear perspective view of yet another form of package device for an edible product;
[0017] FIG. 8 is a side elevational view of the package of FIG. 7 ;
[0018] FIG. 9 is a front perspective view of the package of FIG. 7 ;
[0019] FIG. 10 is a rear perspective view of yet another form of package device for an edible product;
[0020] FIG. 11 is a side elevational view of the package device of FIG. 10 ;
[0021] FIG. 12 is a front perspective view of the package device of FIG. 10 ;
[0022] FIG. 13 is a side elevational view of yet another form of package device;
[0023] FIG. 14 is a side elevational view of yet another form of package device;
[0024] FIG. 15 is a side elevational view of still another form of package device; and
[0025] FIG. 16 is a top elevational view of the form of package device in FIG. 15 .
DETAILED DESCRIPTION
[0026] Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the devices and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those of ordinary skill in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the various embodiments of the present invention is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present invention.
[0027] Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” or “an embodiment”, or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments”, “in one embodiment”, or the like in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, the particular features, structures, or characteristics illustrated or described in connection with one embodiment may be combined, in whole or in part, with the features, structures, or characteristics of one or more other embodiments without limitation. Such modifications and variations are intended to be included within the scope of the present invention.
[0028] Reference will now be made to the accompanying drawings, which assist in illustrating the various pertinent features of the package design. While the following descriptions will be referencing gum packaging, this invention is applicable to other food or other packaging devices. Also, the materials to form the package device may be paperboard blanks, cardboard, hard paper, light cardboard, foil paper, etc; however, other similar and known materials are also applicable.
[0029] Referring to FIGS. 1 , 2 , and 3 there is shown one embodiment of a package device, generally represented as 10 , for pieces of an edible product such as chewing gum. Package device 10 includes a primary compartment 12 for the edible product, and has front member 16 , side members 18 and 20 , and bottom member 25 as seen in FIG. 2 . A secondary compartment 14 for receiving waste materials is attached adjacent to primary compartment 12 , and has a separator member 19 between compartments 12 and 14 that may be the same material as the other members of the packaging device, and two lateral side members 26 and 28 , which may be triangular in shape, extending from side members 18 and 20 . Side members 26 and 28 may fold down their respective centerlines, which allows the secondary compartment 14 to expand and contract. The secondary compartment may include a back member 22 attached to lateral side members 26 and 28 . Back member 22 may have an extending flap 24 , having a tab 30 extending from its leading edge. The package device 10 may be closed by folding flap 24 over the top sides of both primary and secondary compartments, 12 and 14 respectively, utilizing the tab 30 that is inserted in slot 32 formed in front face 16 shown in FIG. 3 . However, other closure members such as temporary adhesive, Velcro®, Ziploc®, double-sided tape, etc. may be used. The perspective view of FIG. 1 and the side view of FIG. 2 of embodiment 10 are shown in the open position. FIG. 3 shows a frontal perspective view of embodiment 10 of the package device in the closed position.
[0030] The embodiment of the package device 100 depicted in FIGS. 4 , 5 , and 6 includes a primary compartment 112 for edible products and having a front member 116 , side members 118 and 120 , and bottom member 125 as seen in FIG. 5 . A secondary compartment 114 is attached adjacent to primary compartment 112 , and may include a separator member 119 between compartments 112 and 114 that may be the same material as the other members of the packaging device, and two lateral side members 140 and 142 , which may be rectangular in shape, extending from sides 118 and 120 . Secondary compartment 114 may have side members 140 and 142 and bottom member 143 , which may fold down their respective centerlines, to expand and contract. The secondary compartment may include a back member 122 connected to lateral side members 140 and 142 . Back member 122 may have an extending flap 124 having a tab 130 extending from its leading edge. The package device of embodiment 100 may be closed by folding flap 124 over the top sides of both primary and secondary compartments 112 and 114 , respectively, utilizing tab 130 that is inserted in slot 132 formed in front face 116 shown in FIG. 6 . However, other closure members such as temporary adhesive, Velcro®, Ziploc®, double-sided tape, etc. may be used. The perspective view of FIG. 4 and the side view in FIG. 5 of embodiment 100 are in the open position. FIG. 6 shows a frontal perspective view of embodiment 100 of the package device in the closed position.
[0031] The package device of embodiment 200 depicted in FIGS. 7 , 8 , and 9 includes a primary compartment 212 for the edible product, and having a front member 216 , side members 218 and 220 , a separator member 219 , that may be the same material as the other members of the packaging device, and having extending flap 250 , and bottom member 224 . Extending from flap 250 is tab 252 . Flap 250 may fold over primary compartment 212 in order to close the primary compartment 212 . FIG. 9 shows tab 252 placed in slot 254 on front member 216 in order to close the first compartment 212 . A secondary compartment 214 for receiving waste materials is attached adjacent to primary compartment 212 , and may have lateral side members 226 and 228 , which may be triangular in shape, a back member 222 , and shares the lower portion of separator member 219 with the primary compartment 212 . Side members 226 and 228 may fold down their respective centerlines, which may expand and contract secondary compartment 214 . The secondary compartment 214 may be closed at 230 using a temporary adhesive, Velcro®, Ziploc®, double-sided tape, or other suitable closure device. The side view of embodiment 200 in FIG. 8 shows the packaging device with compartment 212 open and compartment 214 open. FIG. 13 generally represented as 200 ′, depicts the package design as generally represented by 200 of FIGS. 7 , 8 , and 9 ; however, the package design 200 ′ may utilize a secondary flap 256 extending from or adjacent to separator member 219 that folds over the secondary compartment 214 on to face 222 in order to close the secondary compartment 214 by means of temporary adhesive, Velcro®, Ziploc®, double-sided tape, a tab, or other suitable closing device 258 on secondary extended flap 256 .
[0032] The embodiment of packaging device 300 depicted in FIGS. 10 , 11 , and 12 includes a primary compartment 312 having a front member 316 , side members 318 and 320 , separator member 319 having an extending flap 350 , and bottom member 324 as seen in FIG. 11 . Extending from flap 350 is tab 352 and flap 350 folds over the primary compartment 312 in order to close the primary compartment 312 . The secondary compartment 314 is attached adjacent to primary compartment 312 and may have lateral side members 340 and 342 that may be rectangular in shape, extending from side members 318 and 320 , a back member 322 , and a bottom member 343 . A separator member 319 , that may be the same material as the other members of the packaging device may separate primary compartment 312 from the secondary compartment 314 . Side members 340 and 342 may fold down their respective centerlines, which may expand and contract secondary compartment 314 . The secondary compartment 314 may be closed at 330 using a temporary adhesive, Velcro®, Ziploc®, double-sided tape, or other suitable closing device. The side view of embodiment 300 in FIG. 12 shows the packaging device with primary compartment 312 open and secondary compartment 314 open. FIG. 12 shows tab 352 placed in slot 354 on frontal face 316 in order to close the first compartment 312 . FIG. 14 shows another form of the packaging device 300 ′. In this form, the package design may have an extending flap 356 that is a secondary flap extending from or adjacent to separator member 319 that folds over the secondary compartment 314 on to face 322 in order to close compartment 314 by means of a temporary adhesive, Velcro®, Ziploc®, double-sided tape, a tab, or other suitable closing device 358 on secondary extending flap 356 .
[0033] The embodiment of packaging device 400 depicted in FIGS. 15 and 16 includes a primary compartment 412 having front member 416 , side members 418 and 420 , separator member 419 , that may be the same material as the other members of the packaging device, and has an extending flap 424 , and bottom member 425 as seen in FIG. 15 . Extending from flap 424 is tab 452 and flap 424 may fold over primary compartment 412 in order to close the primary compartment. The secondary compartment 414 is attached adjacent to primary compartment 412 and has a back member 422 that is attached to separator 419 along side members 418 and 420 and bottom member 425 . The top view of FIG. 16 shows the packaging device 400 in an expanded position where flap 424 is folded over primary compartment 412 and secondary compartment 414 is expanded yet sealed by means of closure device 430 which may include and is not limited to a temporary adhesive, Velcro®, Ziploc®, double-sided tape, or other suitable closure device.
[0034] Thus, various embodiments provide for gum packaging devices each having a separate waste disposal compartment. While the various embodiments have been described as having exemplary designs, these embodiments may be further modified within the spirit and scope of the disclosure. This application is, therefore, intended to cover any variations, uses, or adaptations of the various embodiments using their general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains.
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A package for chewing gum and other edible products having one compartment which may hold the pieces of chewing gum, and an attached rear second compartment, which may serve as a pocket for waste.
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[0001] This invention relates in general to glider type exercise equipment, such as the equipment described and claimed in the inventor's U.S. Pat. Nos. 5,795,268, 5,857,940, D390,628 and D403,033, and in particular to a novel suspension system for such equipment. The patented exercise gliders feature very low impact in a device that simulates a full range of natural striding motion, including aggressive striding, for achieving an upper and lower body workout.
BACKGROUND OF THE INVENTION AND PRIOR ART
[0002] With the patented exercise devices, a full range of striding motion is very closely simulated while impact on the user's body is practically eliminated. Significantly, the aerobic effect experienced is readily controllable by merely accelerating the striding action and lengthening the stride, precisely as can be done when aggressively striding over a stationary surface. However, unlike striding, with the inventive device a user can lean backward and forward to transfer significant weight to his arms without loss of balance or control. This not only increases the aerobic effort and enables an upper body workout, but also varies the muscle groups that are being exercised.
[0003] The spring suspension system of the present invention adds a slight cushion effect to the rear of each foot platform for enhancing the gliding action. Essentially, tension springs permit the heel ends of the foot platforms to move up and down (within defined limits) to resiliently modify the radial paths traversed by the foot platforms. The effect is to further reduce the stress on both the user's body and the exercise machine structure. The novel suspension system is achieved with a simple, low cost, spring structure that may be readily added to the fold-away versions of the patented gliders.
OBJECTS OF THE INVENTION
[0004] A principal object of the invention is to provide a novel suspension system for a low impact glider exercise apparatus.
[0005] Another object of the invention is to provide a novel glider exercise apparatus.
[0006] A further object of the invention is to provide an improved suspension system for a low impact glider exercise apparatus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] These and other objects and advantages of the invention will become apparent upon reading the following description in conjunction with the drawings, in which:
[0008] FIG. 1 is a perspective of a fold-away impact glider apparatus incorporating the suspension system of the invention;
[0009] FIG. 2 shows the spring cage of the invention;
[0010] FIG. 3 is a side elevation of the spring cage of FIG. 2 ;
[0011] FIG. 4 is a plan view of the spring retainer 55 of the spring cage;
[0012] FIG. 5 is a cross section of the spring retainer of FIG. 4 , taken along the line 5 - 5 ;
[0013] FIG. 6 is a plan view of the base 45 of the spring cage;
[0014] FIG. 7 is an end view of base 45 ; and
[0015] FIG. 8 is a partial cross section taken along line 8 - 8 of FIG. 1 illustrating the attachment of the spring cage to the rear leg of the glider apparatus.
SUMMARY OF THE INVENTION
[0016] The invention comprises a shock absorbing arrangement in the link that supports the swingable foot platform from the frame of a glider type exercise machine.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0017] Referring to FIG. 1 , a glider type exercise apparatus 10 as shown in the above-mentioned patents includes a fold-away frame consisting of front legs 12 , 13 and rear legs 14 , 15 that are arranged for swingably supporting a pair of foot platforms 20 , 21 . A pair of swing arms, 16 , 17 , pivotally supported on a crossmember 18 of the frame, is pivotally connected to the toe ends of foot platforms 20 , 21 , respectively. The heel ends of foot platforms 20 , 21 are coupled by heel end pivots 34 , 35 to a pair of links 24 , 26 , respectively. The links 24 , 26 are in turn coupled, via spring cages 36 , 38 to a pair of rear pivots 30 , 31 on legs 12 , 13 , respectively. While the links 24 , 26 may be rigid, in the preferred embodiments of the apparatus, the links comprise steel cables. The apparatus functions, in a well-known manner to enable a user standing on the foot platforms and grasping the swing arms to engage in an aerobic upper and lower body workout with minimal impact to his body.
[0018] FIGS. 2-7 illustrate various features of spring cage 36 of the invention, it being understood that spring cage 38 is a mirror image thereof. A top plate 40 , preferably made of steel, is welded to the upper ends of a pair of steel tie rods 43 , 44 that are welded at their lower ends to a steel base plate 45 ( FIGS. 6 and 7 ) to form a generally cylindrical structure. Top plate 40 includes a hole 41 for pivotal mounting to rear pivot 30 and a small hole 42 for attaching the upper end of a tension spring 50 . The lower end of tension spring 50 is connected to an intermediate member 52 , preferably made of steel, that serves as a coupler for the upper end of link 24 . In practice, link 24 is a cable having a threaded stud secured to its upper end for secure engagement with intermediate member (cable coupler) 52 . A compression spring 54 , which encircles link 24 , has its lower end seated in a spring retainer 55 ( FIGS. 4 and 5 ). A polyurethane washer 53 is positioned atop compression spring 54 and serves to cushion the impact between intermediate member 52 and compression spring 54 upon elongation of tension spring 50 . A plastic shield 56 covers the major portions of spring cage 36 .
[0019] As more clearly shown in FIGS. 4 and 5 , spring retainer 55 includes a pair of edge notches 59 , 60 that partially encircle the round circumferences of tie rods 43 , 44 . The spring retainer has a central orifice 57 through which link 24 freely passes and a circular recess 58 for receiving the bottom end of compression spring 54 . Spring retainer 55 is preferably made of a plastic material and is dimensioned such that it is a force fit between tie rods 43 , 44 .
[0020] FIGS. 6 and 7 show details of base plate 45 , in particular the end notches 47 , 48 which are welded to the ends of tie rods 43 , 44 , respectively and the central orifice 46 , through which link 24 freely passes.
[0021] In FIG. 8 , details of the pivotal attachment of the spring cage to the upper (rear) part of front leg 12 are shown. Pivot 30 comprises a cylindrical pin 62 which passes through leg 12 and is engaged by a threaded screw 63 which includes a collar 64 and a head 65 . A contoured support 67 , through which pin 62 passes, engages the circular periphery of leg 12 and presents a flat surface that engages a plastic spacing washer 68 . A plastic washer 66 , having a stepped diameter for engaging hole 41 in top plate 40 and an inner hole engaging screw collar 64 , centers the spring cage 36 on pin 62 . A plastic cover 70 has a stepped orifice 71 for accepting a washer 66 , with everything being secured together by the screw head 65 . The arrangement enables free pivotal movement of spring cage 36 about pin 62 , thus defining the rear pivot 30 .
[0022] It will be appreciated by those skilled in the art that the spring cage may be located anywhere in the link, although its placement as shown at rear pivot 30 is preferred. In the preferred embodiment of the invention tension spring 50 has an overall length of 3.375 in. and a spring rate of 76 lbs/in. and compression spring 54 has an overall length of 1.5 in. and a spring rate of 108 lbs/in.
[0023] What has been described is a novel suspension system for a glider type exercise device that further reduces the stress on the user's body and the exercise apparatus when performing provides a low impact simulation of walking and striding, including aggressive striding, aerobic upper and lower body exercises. It is recognized that numerous changes to the described embodiment of the invention will be apparent to those skilled in the art without departing from its true spirit and scope. The invention is to be limited only as defined in the claims.
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A glider device includes a pair of swingably mounted foot platforms pivotally supported by links at their heel ends. A spring cage, including a tension spring and a compression spring, is included in each link to resiliently support the heel end of the foot platform. The spring cage limits displacement of the tension spring and includes a compression spring that cushions the displacement of the tension spring.
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FIELD OF THE INVENTION
The present invention relates to methods and apparatus for vacuum coating plastic parts, and especially, for reactive sputter coating of plastic ophthalmic lens elements. As used herein, lens elements include, according to context, edged lenses, semi-finished lenses and lens blanks. Also included are wafers for forming laminate lenses or wafer blanks therefor. Ophthalmic uses of the lens elements include uses in eyeglasses, goggles and sunglasses.
BACKGROUND AND OBJECTS OF THE INVENTION
Ophthalmic lens elements are frequently coated to achieve special properties. Anti-reflection coatings improve the transmittance of visible light and the cosmetic appearance of the lenses. Reflective coatings may be employed in sun lenses to reduce light transmittance to the eye, to protect the eye from UV radiation and/or to impart cosmetic colorations to the lens. Coatings may also provide other beneficial properties such as increased hardness and scratch resistance and anti-static properties.
Desirable lens coatings may be created by applying single or multiple layers of metal or semi-metal oxides to surfaces of the lens element. Such materials include oxides of silicon, zirconium, titanium, neobium and tantalum. Examples of such multilayer coatings are given, for example, in U.S. Pat. No. 5,719,705 to Machol entitled “Anti-static Anti-reflection Coatings”, assigned to applicant. Interference filter coatings for sunglasses are disclosed, for example, in U.S. Pat. No. 2,758,510 to Auwarter.
Various methods are disclosed in the prior art for applying metal and semi-metal oxide coatings to ophthalmic lenses. Ritter et al. U.S. Pat. No. 4,172,156 discloses vacuum evaporation in an oxygen atmosphere of Cr and Si to form coating layers on a plastic lens. Reactive sputter deposition of various oxide layers on lens elements is disclosed in the above-mentioned '705 patent to Machol.
Reactive sputtering in general is a conventional technique often used, for example, in providing thin oxide coatings for such items as semi-conductor wafers or glass lamp reflectors. Examples of various conventional vacuum deposition systems for the formation of coatings by reactive sputtering are disclosed in the following patents: U.S. Pat. Nos. 5,616,224 to Boling; U.S. Pat. No. 4,851,095 to Scobey et al.; U.S. Pat. No. 4,591,418 to Snyder; U.S. Pat. No. 4,420,385 to Hartsough; British Patent Application GB 2,180,262 to Wort et al.; Japanese Kokai No. 62-284076 to Ito; and German Patent No. 123,714 to Heisig et al.
Most ophthalmic lenses produced today are made from a single plastic body or laminated plastic wafers. The plastic material may include thermoplastic material such as polycarbonate or thermoset material such as diallyl glycol carbonate types, e.g. CR-39 (PPG Industries). The material may also be a cross linkable polymeric casting composition such as described in U.S. Pat. No. 5,502,139 to Toh et al and assigned to applicant. The challenge is to adapt conventional vacuum deposition systems to high volume production of plastic lens elements, while ensuing a high degree of control over the thickness and composition of the coating.
Accordingly, it is an object of the present invention to improve the degree of control over the thickness and composition of thin metal and semi-metal oxide coatings deposited on plastic lenses.
Anti-reflection coating of plastic ophthalmic lenses by physical vapor deposition has traditionally been performed by means of thermal evaporation, or more recently, e-beam evaporation of metal and semi-metal oxides in a vacuum of typically significantly better than 10E-5 Torr. Anti-reflection coating of plastic lenses in spinning drum coaters by means of sputter technology is a relatively recent development. A conventional vacuum system used for this purpose is shown in FIG. 1 . The system includes a vacuum coating chamber 11 , which contains a hollow workpiece holder or drum 12 . Lens elements, such as lens 13 are arranged in columns on an external surface of the drum 12 . A coating applicator 14 is located in a wall of the vacuum chamber adjacent the drum 12 . In a preferred embodiment, the coating applicator may be a combination of magnetron sputtering targets, microwave plasma generator, reactive gas supply, and reversing power supply such as disclosed in U.S. Pat. No. 5,616,224 to Boling, which is hereby incorporated by reference.
A pumping plenum 15 is attached to vacuum pumps (not shown) which evacuate the vacuum chamber 12 . A cryopumping surface is provided in the form of cryocoils 16 in the plenum 15 . The cryopumping surface is also known as a “Meissner trap”. Conventionally the Meissner trap takes the form of a coiled or serpentine metal tube through which a coolant passes. Advantageously, the coolant is maintained at a temperature well below the freezing point of water. The Meissner trap is used to remove water vapor from the system.
In most such drum coaters the placement of the cryocoils in the pumping chamber plenum is favored. The prime reason for this particular placement is that it is done with a view to protecting the subsequent pumps, especially large roughing pumps, from excessive water contamination which can reduce the life and efficacy of such pumps. A secondary reason for such placement appears to be the desire to keep the cryocoils away from the rotating drum to avoid somewhat greater mechanical complexity, the danger of the parts held on the drum colliding with the cryocoils and to provide greater ease of maintenance of both the coils and the drum. However, Meissner traps have been located in the vacuum chamber rather than the plenum in systems for vacuum coating work pieces on holders other than plastic lenses on drums as disclosed in U.S. Pat. No. 4,647,361 to Bauer and U.S. Pat. No. 5,121,707 to Kanoo.
Plastic lenses are usually thoroughly baked at temperatures in excess of 90° C. for one to several hours prior to introduction to the vacuum system to reduce water vapor outgassing. Load sizes of plastic lenses have been limited to about 200. Pump-down times to base vacuums in the 10E-6 Torr regime are typically in the order of 30 minutes or more.
It is another object of the present invention to reduce the processing time required to deposit vacuum coatings on plastic parts.
It is another object of the present invention to provide an apparatus for depositing a high quality vacuum coating on large numbers of plastic lens elements in a system which is relatively inexpensive to construct and operate.
These and other objects and features of the present invention will be apparent from the written description and drawings presented herein.
SUMMARY OF THE INVENTION
One apparently unrecognized problem with the vacuum coating of plastics is the ongoing effect of large amounts of water vapor (or other gas or solvent) outgassing from the plastic in the presence of plasmas, even after a base pressure is reached which would be considered satisfactory for beginning to process low outgassing materials (e.g. glass). For instance, a particular problem has been found in the deposition of multi-layer anti-reflection (AR) coatings of metal and semi-metal oxides on plastic ophthalmic lenses by means of sputter deposition in a drum coater. The problem is that conventionally designed coaters do not provide sufficient process control in the presence of the outgassing from plastics whereas the same coater design is found to be perfectly acceptable for coating glass components. The problem arises from the breakdown of water molecules into their constituent atoms in the presence of sputter plasmas. The problem may well be exacerbated by the presence of specialized plasmas such as those in processes such as described in U.S. Pat. No. 5,616,224 to Boling, in which a microwave excited plasma is used to increase the rate of oxidation of freshly deposited metal surfaces and to overcome some problems which arise with sputter magnetrons utilizing polarity reversing power supplies.
Applicant has determined that the conventional placement of the cryocoil in the plenum ignores, to a large extent, the different requirement that drum coaters and plastic workpieces place on such systems compared with conventional evaporative box coaters. In the latter the vast majority of molecules in the vacuum chamber have unobstructed access to (i.e. can “see”) the cryocoil in the plenum. In a drum coater this is not true. Applicant has observed that, in the reactive sputtering drum coater design previously used by applicant to provide coated lenses in the prior art, the vast majority of molecules in the chambers were obstructed from direct access to the plenum—they could not “see” the cryocoil because the drum wall runs close (within a few inches) of the mouth of the plenum.
As noted above, the original use of the drum coating system was in a process to deposit multi-layer coatings on glass objects, especially lamps and reflectors. The amount of water vapor outgassed by glass components in a vacuum, especially if they have been preheated or outgassed in a heated oven, is believed to be considerably less than that outgassed by a large load of plastic lens elements (typically about 400 lenses, each 3″ diameter). These lens elements absorb water throughout the material of which they are composed in contrast to the situation with glass components where water is merely adsorbed on the surface. Some plastic lenses absorb up to several percent by weight of water.
Conventional wisdom has it that once a vacuum system has been pumped down to a satisfactory base pressure for a particular process then that is sufficient. However, applicant has determined that process instability results from the very substantially greater water out-gassing from plastics (as compared to glass components). Even after reaching a base pressure which had been shown to be perfectly satisfactory for glass coating, continued out-gassing and instability are believed to be present. The increased stability and improved pump down speed provided by the present invention were surprising.
A preferred embodiment of the present invention is a method and system for sputter coating plastic ophthalmic lens elements. The system includes a vacuum chamber containing a hollow, apertured drum with a substantially hollow interior. Large numbers of plastic ophthalmic lens elements (for example 200 to 400) are located in a two dimensional array on a radially outwardly facing surface of the drum so that radially inwardly facing surfaces of the lens elements are exposed through apertures in the drum, to the hollow interior of the drum. Conduits for circulating coolant are located in at least one end wall of the vacuum chamber adjacent the hollow interior of the drum. A majority (i.e. at least 50%) of the water vapor outgassed by the plastic lens elements when placed under vacuum condenses on the conduits, whereby it is removed from active areas of the vacuum system. The system is configured so that at least one face of substantially all of the plural lens elements lies on an unobstructed line of sight with the at least one coolant conduit. The drum and at least one sputtering station are moved relatively to one another to apply various sputter coatings to the radially outward surfaces of the plastic lens elements.
Advantageously, in such a system the sputter coating is performed by a reactive DC or mid frequency magnetron sputter process in which sputter material reacts with a reactant gas to form an insulating layer on the radially outward surfaces of the lens elements and on portions of a sputter target. Oxides may be formed on electrode surfaces of the sputtering apparatus and may require arc suppression. The sputter coating may be performed using a microwave plasma generator and at least one applicator or sputter target located adjacent one another and radially outwardly from the drum.
In preferred embodiments of the present invention at least 200 lens elements are loaded onto the work holder before drawing a vacuum in the system and pump down is achieved in less than 10 minutes.
The present invention also includes apparatus for reactive sputtering of a thin oxide coating onto surfaces of plural plastic lens elements. The apparatus may include a vacuum chamber and a lens element holder located in the vacuum chamber and rotatable about an axis intersecting at least one wall of the vacuum chamber. The holder rotates the plural plastic lens elements past an elongated sputtering electrode. A source of oxygen is provided to facilitate formation of oxide layer(s) on the lens element. An elongated microwave plasma generator may be located adjacent to the sputtering electrode. The holder rotates the plural plastic lens element past the elongated plasma generator which produces a plasma to facilitate the reaction of the oxygen with material sputtered from the sputtering electrode to thereby provide an oxide coating on surfaces of the plural plastic lens elements.
At least one cooled surface is located in at least one wall of the vacuum chamber intersected by the axis of rotation of the holder. The cooled surface condenses substantially all the water vapor released into the vacuum chamber by exposed surfaces of the plural plastic lens elements. In a more preferred embodiment, the axis of rotation of the holder intersects two end walls of the vacuum chamber. Cryocoils may extend through at least about half of the portions of the two end walls facing the open ends of the rotating holder.
The lens holder may be a hollow drum rotated about its central axis, for example the drum may be generally cylindrical in shape. The plural lens elements may be arranged in columns on an outside surface of the drum. Advantageously, the drum is formed with apertures through which water vapor passes from an uncoated back surface of each lens element. The cryocoils on the end walls of the vacuum chamber may extend adjacent to edges of the external surface of the drum to facilitate condensing water vapor which would otherwise pass into the sputtering and reaction zones adjacent the external surface of the drum.
The cooled conduits employed in the present invention are arranged in a coil in each of the end walls of the vacuum chamber. The coils may be in serpentine form or in the form of loops, spirals or helices. The apparatus may also employ a second sputtering electrode located outside the holder and adjacent to at least one of the microwave plasma generator or first sputtering electrode. The second sputtering electrode may sputter a different metal or semi-metal than the first sputtering electrode to produce alternating coating layers of different oxides. The magnetron and sputtering electrode(s) may be located on a door through which lens elements are loaded onto the holders.
The foregoing has been provided as a convenient summary of aspects of the invention. The invention intended to be protected is, however, defined by the claims and equivalents thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a pictorial view in partial phantom of a system known in the prior art for vacuum coating plural plastic lens elements.
FIG. 2 is a pictorial view in partial phantom of drum vacuum coating system in which the cryocoils do not lie on a direct line of sight with surfaces of the workpieces.
FIG. 3 is a cross-sectional view of a vacuum system in which the cryocoils do not lie on a direct line of sight with surfaces of the workpieces.
FIG. 4 is a pictorial view in partial phantom of a system for vacuum coating plastic lens elements employing cryocoils in the upper and lower walls of the vacuum chamber in accordance with the present invention.
FIGS. 5 ( a ) and ( b ) are top and bottom views of a vacuum system in accordance with the present invention for coating plural plastic lens elements.
FIG. 6 is a pictorial view of the cryocoils employed in the system of FIG. 5 .
DETAILED DESCRIPTION
The disclosed embodiments address the need for effective cryopumping to handle large and continuing outgassing for plastic substrates, particularly in systems containing drum workpiece holders.
The conventional placement of the cryocoils, either in the pumping chamber plenum or on the inside vertical walls of the chamber is ineffective because the cold surface cannot be seen by the majority of molecules inside the chamber, i.e. inside the hollow spinning drum which holds the plastic parts as discussed above in connection with FIG. 1 .
The effects of large water vapor outgassing loads throughout the process are especially deleterious when sputter deposition is employed. The plasma employed dissociates water vapor (and carbon dioxide) to create uncontrolled sources especially of oxygen but also of hydrogen. Both oxygen and hydrogen take part in the physical and/or chemical interactions of sputtering and oxidization in the growing metal or semi-metal oxide film. It should be noted that the uncontrolled source of hydrogen has deleterious effects on the process in that every hydrogen ion accelerated toward the sputter target contributes to the sputter ion current but not measurably to the sputter yield. It has also been conjectured that hydrogen may be incorporated in the growing film in a manner which may cause undesirable changes to its optical properties.
FIG. 2 represents a cryocoil configuration suggested to applicant by a third party supplier. In the system serpentine cryocoils 21 are located in side walls of the vacuum chamber 22 . Coolant for the coils enters and exits the coils through lines 23 which pass through the plenum 24 .
When used with a drum workpiece holder, the drum blocks most line-of-sight paths between the coils and the plastic parts mounted on the drums. Furthermore, the cryocoils cannot obstruct the sputter applicator 25 . It is difficult to locate a substantial amount of cooled surface in the vicinity of the sputter applicator 25 .
FIG. 3 represents a cryocoil configuration similar to that shown in U.S. Pat. No. 4,647,361 to Bauer, in which a coil of coolant conduit 31 is located adjacent to a bottom wall 32 of a vacuum chamber 33 . If a workpiece holder drum 34 were used in such a system (not shown in Bauer) and mounted for rotation on shaft 35 , it will be seen that the line-of-sight access from plastic parts mounted on the drum would be oblique and quite limited.
In preferred embodiments of the present invention cryocoils of appropriate size are placed in a drum coater in locations whereby the majority of vapor molecules have direct and unobstructed access to the cryocoil. Such locations and coils at the top and bottom of the hollow rotating drum are illustrated in FIG. 4 .
In FIG. 4, a first serpentine cryocoil 41 is located in an upper wall 42 of a drum coater vacuum chamber 43 and covers a substantial area of the upper wall including a central portion thereof. Similarly, a second serpentine cryocoil 44 is located in a lower wall 45 of the vacuum chamber and covers a substantial area of the lower wall including a central portion.
A flow of refrigerant is provided to the cryocoil through inlets 46 passing through the plenum 47 of the vacuum chamber. The refrigerant may be hydrofluorocarbon, liquid nitrogen, liquid air or other coolant having sufficient capacity to cool the surface of the conduits to facilitate vapor condensation.
A hollow, cylindrical workpiece holder or drum, such as shown in FIG. 1, is located in the vacuum chamber. A portion of the drum is indicated at 48 . The drum is mounted for rotation about an axis A—A which passes generally perpendicularly through the upper and lower walls 42 and 45 of the vacuum chamber.
Plural plastic lens elements such as elements 49 (for example 400 such lens elements) are mounted on the drum. Radially outwardly facing optical surfaces of the lens element may be coated by repeatedly rotating the elements past various sputtering applicators. The lens elements are mounted such that radially inwardly facing surface of the lens elements is exposed to the hollow interior of the drum through apertures in the drum wall. A direct line of sight path exists between these exposed surfaces and substantial portions of the cryocoils 41 and 44 . Several such lines of sight for lens elements 49 are indicated by the dotted lines 50 .
Approximately the same total length of cryocoil (as compared to that in the plenum in FIG. 1) is placed in the system illustrated in FIG. 4 . The effect on pumpdown time and, more particularly for process stability, for reactive AR sputter coating of plastic lenses is dramatic. Furthermore, the stabilization of the process, due to continued removal of water vapor and thus of the uncontrolled evolution of oxygen, allows clear, fully oxidized films to be produced with a full load of lenses. This had not been achieved with the cryocoil placement of the prior art. Plastic lenses of higher refractive index materials often have significantly more water uptake than CR39 and thus the advantages of the invention are even more significant in that case.
Vacuum Flow Regimes, Placement of Cryocoils
The following is from Leybold's Vacuum Notes:
Vacuum Flow Regimes
Mean Free Path (L)
L × P
= 5.07E−3 (Torr cm)
is given by
┘
= 50.7 (m Torr mm) (for air at 20° C.)
where p
= pressure.
Viscous Flow
Pd>4600 mTorr mm L<d/100
where d=the shortest distance across a conducting member.
Intermediate (Transitional, Knudsen) Flow
100<P d <4600 mTorr mm d/100<L<d/2
Molecular Flow
Pd<100 mTorr mm L>d/2
Sputter Drum Coater Flow Regimes
Argon and air have very similar mean free paths (=to within 5% at 1 Torr). The Mean Free Path L for water vapor is almost exactly ⅔ that of air at 1 Torr).
Table I sets out the various flow regimes during deposition for a typical sputter drum coater at various working pressures in the mTorr regime and for two characteristic distances is the radial spacing from drum to chamber wall and from drum to sputtering target.
TABLE I
Flow Regimes
Characteristic
Pressure (P)
MFP (L)
Distance (d)
Where is d
Pd (mTorr
d/100
d/2
(mTorr)
(mm)
(mm)
measured
mm)
Pd Regime
(mm)
L (mm)
(mm)
L Regime
3
17
25
drum to chamber
75
Molecular Flow
0.25
17
12.5
Molecular
wall
Flow
4
10
25
drum to chamber
125
Transitional
0.25
10
12.5
Transitional
wall
Flow
Flow
8
6
25
drum to chamber
200
Transitional
0.25
6
12.5
Transitional
wall
Flow
Flow
12
4
25
drum to chamber
300
Transitional
0.25
4
12.5
Transitional
wall
Flow
Flow
3
17
67
drum to target
201
Transitional
0.67
17
33.5
Transitional
Flow
Flow
5
10
67
drum to target
335
Transitional
0.67
10
33.5
Transitional
Flow
Flow
8
6
67
drum to target
536
Transitional
0.67
6
33.5
Transitional
Flow
Flow
12
4
67
drum to target
804
Transitional
0.67
4
33.5
Transitional
Flow
Flow
Viscous Flow
Pd
>4600 mTorr
d/100<
L
<d/100
mm
Transitional
4600>
Pd
>100 mTorr
L
<d/2
Flow
mm
Molecular
Pd
<100 mTorr
L
>d/2
Flow
mm
Note that the process does not operate very close to the Viscous Flow regime and in fact is usually bordering on the Molecular Flow regime or occasionally in it. Whether the drum to chamber wall distance is the typical 25 mm (1″) or 30 mm (1¼) (or somewhere near that figure) will make no difference to the conclusions regarding operating flow regime.
Placement of Cryocoils
Advantageously, the cryocoils are placed so that they are at the top and bottom of the drum. They should also be placed as near as possible to the portion of the chamber where the sputter applicators and plasmas are located.
As shown above, the process is usually operating in the transitional flow regime and closer to the molecular flow regime than the viscous flow regime. The gas conductance on both sides of the drum, from a vertical midpoint on the drum, to the top and bottom of the drum, is demonstrably greater on average than that to the first available position for cryocoil tubes on the inside walls of the chamber beyond the edges of the coating applicator. Conventionally, the coating applicators are located in the vacuum chamber door. Placing cryocoils in the door is fraught with mechanical difficulties of placement, design and manufacture. Their total effective area (near the operational plasma zones) will be very limited.
In contrast, standard ⅝″ OD cryocoils of some 30 feet in length (or more) at the top and bottom of the drum are feasible and relatively straightforward to install with copper tubing in positions which are at less risk to mechanical damage. In a 45 inch diameter vacuum system, four coils of ⅝″ tubing starting at 36″ diameter and spaced 2.125″ center to center [1.5″ from OD to OD] will provide about 30 ft of tubing at the top of the drum—a similar arrangement may be placed at the bottom of the drum.
In the first few seconds of sputtering silica on the lenses there is very good reason to believe that the outgassing from the coated (outwardly facing) lens surfaces will decrease markedly, probably to negligible proportions, due to the excellent moisture barrier properties of silica. This being the case, the remaining major source of outgassing during most of the deposition cycle will be the rear surfaces of the plastic lenses.
Cryocoils placed top and bottom of the drum will deal with this outgassing very effectively and can act as a trap to stop water vapor diffusing from the inside of the drum over the top and bottom of the drum to the outside where it is difficult to provide an effective Meissner trap in the operational region near the plasmas.
Another preferred embodiment of the present invention is illustrated in FIGS. 5 and 6. FIGS. 5 ( a ) and ( b ) are, respectively, top and bottom views of a vacuum system 100 for coating plastic lens elements. The system employs an arrangement of cryocoils 102 , which are shown in isolation in perspective view in FIG. 6 .
The vacuum chamber has an outer wall 104 in the shape of a twelve-sided prism. A cylindrical drum 105 is located inside the vacuum chamber. A chamber door 106 is hinged at 108 and provides access to the outer cylindrical surface of the drum 105 for loading lens elements onto the drum. The drum 105 is mounted for rotation about an axis passing through points B. Lens elements (not shown) may be mounted in registration with apertures on the drum so that one side is exposed to system coating applicators and the other side is exposed to direct lines of sight with the cryocoils in the top and bottom walls of the system.
The vacuum coating applicators may be located in the door 106 . In a preferred embodiment the coating applicators may include a first sputter magnetron 112 , a microwave plasma generator 114 and a second sputter magnetron 116 . Alternatively the positions of the first sputter magnetron 112 and the microwave plasma generator 114 may be reversed. Advantageously, the first and second sputter magnetrons may include targets of different metal and/or semi-metal materials to form sequential coatings of diverse oxides on the lens elements, the coatings having different indices of refractions. Layers are built up by repeatedly rotating the lens elements on the drum past the vacuum coating applicators. For example, the system may be used to apply a multi-layer oxide coating to a lens element whose radially outwardly facing optical surface has been treated with a hard coat. A five layer coating may comprise alternating layers of silicon oxide and zirconium oxide, silicon oxide layers being outermost and innermost.
The outer cylindrical face of the drum 105 is typically 1 to 2 inches from the inner wall of the vacuum chamber and typically 2 to 3 inches from the target surface of the coating applicators. The drum itself may be on the order of 40 inches in diameter and 40 inches high and carry hundreds of lens elements on its outer surface. Initially the lens elements may present on the order of 5600 square inches of exposed surface, approximately half of which (one side of each lens element) is coated during a coating run. Using the system depicted in FIGS. 5 and 6 pump down has been achieved in less than 10 minutes with a full load of 400 baked-out, uncoated 3″ lenses. This represents an approximately three fold reduction in pump down time in comparison to a system with cryocoils located in the plenum. Smaller loads would present smaller uncoated surface area or the order of 1000 square inches (about 1400 square inches for a load of 200 3″ lenses).
FIG. 6 is a perspective view of the cryocoils used in the system of FIG. 5 . The cryocoils on the upper wall and the cryocoils on the lower wall are indicated at 118 and 120 , respectively. Conduits running along the side walls are indicated at 122 . Cryocoils in the plenum are indicated at 124 .
The instant invention has been described with respect to particular preferred embodiments. The invention to be protected, however, is intended to be defined by the literal language of the claims and equivalents thereof.
|
A method and apparatus for vacuum coating plastic lens elements employs Meissner traps and a drum work holder configuration for effectively condensing water vapor in the system.
| 2 |
FIELD OF THE INVENTION
[0001] The present invention refers to the field of Genetic Engineering and Biotechnology, and particularly to the use of a polypeptide that incorporates the amino acid sequence of the gonadotrophin-releasing hormone (GnRH-I) for mammal immunocastration.
BRIEF SUMMARY OF THE INVENTION
[0002] The polypeptide of the present invention is a chimeric polypeptide or glycopeptide, formed by fusion of the amino acid sequence of the gonadotrophin-releasing hormone (GnRH-I) or variants thereof, and a non pathogen-derived theoretical sequence that enhances GnRH immunogenicity. The present fusion protein, its glycosilated version, as well as its tandem repetitions, may be used, together with different types of adjuvants, to immunoneutralize the gonadotrophin-releasing hormone (GnRH-I) producing a block in steroidogenesis, oogenesis and spermatogenesis in different animal species.
PRIOR ART COMMENTS
[0003] In most animal species, the reproductive capacities of both sexes suffer from temporary cyclic fluctuations as a result of the effects generated by sex hormones on gonads and on the reproductive system in general. Gonadotrophin-releasing hormone or GnRH plays a central part in this process.
[0004] The GnRH-I hormone is a decapeptide possessing an amino acid sequence evolutively well preserved and common for most mammals. GnRH-I is liberated from the mesiobasal portion of the hypothalamus and enters into the blood stream, where it induces in the hypophysis liberation of LH and FSH from gonadotroph cells. For several years efforts have been made to generate immunoneutralization of the GnRH-I hormone as a control of steroidogenesis, oogenesis and spermatogenesis. GnRH block with the accompanying reduction of gonadotrophin levels has various applications; thus, in human medicine the reduction of androgen production in patients with prostatic carcinoma has been a treatment target for several years. On the other hand, in veterinary medicine reproductive capability blocking in pets or wild species that may become plagues, with minimum side effects, has been a subject of research and development. In the field of animal breeding, surgical castration of males is a routine procedure to avoid an aggressive sexual behavior or to prevent their flesh from acquiring undesirable organoleptic characteristics through the effect of pheromones. In all these settings, the use of a vaccine capable of blocking GnRH-I hormone function represents an important tool.
[0005] The effect of different vaccines against GnRH hormone has been assessed in a great number of animal species, using various types of molecules associated to GnRH, together with different types of adjuvants. Most of these approaches are based on chemical synthesis of haptens linking GnRH to a highly immunogenic molecule such as bovine albumin (BSA), ovalbumin (OVA), tetanic toxoid (TT) or hemocyanin (KLH)(Sad, Chauhan et al. 1993; Beekman, Schaaper et al. 1999; Dunshea, Colantoni et al. 2001; Miller, Gionfriddo et al. 2008). However, a phenomenon of an antigenic dominance has been described, wherein these “carrier” proteins suppress the response to epitopes of the molecule of interest after successive immunizations, in a mechanism of tolerance to the GnRH antigen (Sad, Gupta et al. 1991; Sad, Gupta at al. 1991; Sad, Talwar at al. 1991). Epitope suppression may result from a defect in the hapten presentation by specific B-lymphocytes developing an immune response of the “helper” Type 2 (Th2) (Renjifo, Wolf et al. 1998). The exclusion of epitopes with high antigenicity, as stated in the present invention, reduces the risk of antigenic suppression and supports an immune response in favor of the GnRH antigen, allowing it to be used efficiently in repeated immunizations. Other obstacle to the use of the model of “carrier” proteins is the high cost in antigen synthesis and conjugation.
[0006] Recombinant ADN technology has been used to create tandem repeated GnRH molecules, linked to different protein sequences as immunogens for T helper lymphocytes (Hannesdottir, Han et al. 2004; Jinshu, Jingjing et al. 2004; Khan, Ferro et al. 2007; Zhang, Xu at al. 2007; Khan, Ogita et al. 2008). Recombinant proteins with multiple GnRH inserts have shown that immunogenicity is increased with the number of inserted GnRH sequences [15], and may use this advantage by incorporating in the formulation a greater number of repetitions of the fusion peptide. Multiple epitopes of B or T cells as lipopeptides (Pam3Cys) or different peptide sequences of pathogens such as Plasmodium falsiparum, Mycobacterium , syncytial respiratory virus or influenza virus, flanking GnRH sequences have been used in various “vaccinal” models and have proved to be effective (Khan, Ferro et al. 2007). In this connection, the present antigen does not incorporate pathogen sequences that may interfere with the liberation of an immune response against the GnRH sequence, since the intergenic sequence used between GnRH repetitions has been designed to improve antigenicity of the GnRH sequence.
[0007] In this connection, document US 2005/0239701 A1 is directed to the use as vaccine of GnRH multimers linked to “carrier” proteins or fragments thereof as bacterial toxins, and to the use of recombinant vectors that incorporate genetic sequences coding for GnRH multimers, by themselves or combined with genetic sequences coding for “carrier” proteins such as titanic toxin C fragment. Said recombinant vectors are directed to modify sexual behavior, fertility or both, in vertebrates through the induction of an immune response that changes the normal physiologic sexual function. The present invention does not incorporate gene or peptide sequences of “carrier” proteins, or corresponds to GnRH multimers by themselves, as it incorporates an intergenic sequence that is not associated with pathogens or “carrier” proteins which operates improving GnRH immunogenicity as an antigen; furthermore, this sequence possesses the potential of being glycosilated when the recombinant protein is expressed in eukaryotic systems capable of making post-translational modifications to proteins.
[0008] International publication WO 01/85763 discloses chimeric peptides with immunogenic effectiveness comprising the GnRH hormone sequence and epitope mixtures for T “helper” cells obtained from different pathogens or peptides of known immunogenicity known as the titanic toxin, Plasmodium falciparum, or the Measles virus F protein, for the production of anti-GnRH antibody titers.
[0009] Generally, in most of the publications that disclose the use of fusion proteins, the method is focused on the use of pathogen sequences that function as T “helper” lymphocyte epitopes, linked to a different number of GnRH repetitions or as in the case of a chemical synthesis, GnRH repetitions linked to an immunogenic molecule per se. An example of this is the document “Use of recombinant gonadotrophin-releasing hormone antigens for immunosterilization of beef heifers”, Journal of Animal Science, 2006; 84(2): 343-50, Geary T W, Grings E E, MacNeil M D, de Avila D M, Reeves J J.
[0010] A great number of studies have been conducted in pigs and cattle to do research on the use of immunization against GnRH as a method to improve growth rate and the meat product obtained from the animals. See, for example, Adams and Adams, J. Animal Sci. (1992) 70:1691-1698; Caray and Bonneau, C. R. Acad. Sc. Paris (1986) 303:673-676; Chaffaux at al, Recueil de Medicine Veterinaire (1985) 161:133-145; Finnerty et al., J. Repro. Fertil. (1994) 101:133-343. Castration eliminates the source of endogenous anabolic steroids and feed conversion becomes less efficient, animals need to eat more in order to generate dressed carcasses of the same weight and produce a greater fat cover. To this effect, it has been shown that growth of a non-castrated animal is more efficient than that of a castrated animal. The presence of sexual steroids in the animal acts as natural anabolics, allowing this animal to have a better growth and muscle development performance, thanks to a substantial improvement in feed conversion efficiency. This better efficiency in feed conversion has also positive environmental implications at world level, because it is expressed in a lesser amount of food intake with less pressure on farm lands and a reduction in waste production, and promotes a more sustainable industry using less feed and generating less waste per meat kilogram produced. The target of many of these studies has been to let the animals grow intact as males until they reach the end of the fattening stage, and to subject them after that to an immunologic castration. The use of anti-GnRH vaccines has been proposed as a viable alternative to maintain in production non castrated males that are vaccinated at the end of the productive stage, allowing metabolization of sexual hormones and their associated smell. Several patents that approach this problem have been granted (U.S. Pat. No 4,975,420 1990; U.S. Pat. No. 6,045,799 2000; U.S. Pat. No. 6,761,890 B1 2004, among others); however, the molecules used therein as antigens are chemical conjugations of the GnRH hormone amino acid sequence to a “carrier” molecule. In this sense, patent application US 2005/0239701 A1 protects the use of a vaccination comprising two doses 4 to 8 weeks before slaughter of the animal to ensure effectiveness of the vaccine, for a short period of time; this limits the application of vaccines against GnRH with an unsatisfactory effectiveness, needing revaccinations to obtain the neutralizing antibody titers to block the hormone effect.
[0011] In like manner, the following scientific articles also refer to conjugation of the GnRH sequence and a “carrier” molecule:
[0012] Beekman, N. J., W. M. Schaaper, et al. (1999). “Highly immunogenic and fully synthetic peptide-carrier constructs targeting GnRH.” Vaccine 17 (15-16): 2043-50. It specifies that in order to use peptides as synthetic vaccines, they have to be coupled to a “carrier” protein to make them more immunogenic. However, coupling efficiency between the “carrier” protein and a protein is hard to control with regard to the peptide charge density. As a result, these “carrier” proteins are not very suitable in practice. Attempts have been reported to find “carrier” molecules or delivery systems that allow an easy coupling or peptide incorporation, reproducible charge density and well-defined products. The authors have compared several promising constructs or delivery systems for male pigs immunization using a GnRH peptide in tandem as a branched polylysine construction, a lipo-thioester, a lipo-amide or a KLH conjugate in CFA, and the lipo-amide peptide in an immuno simulator complex (ISCOM). The authors found that lipo-thioester and branched polylysine constructs constituted the most effective “carrier” molecules to induce antibodies anti-GnRH and immunocastration in pigs.
[0013] Khan, M. A., K. Ogita, et al. (2008). “Immunization with a plasmid DNA vaccine encoding gonadotrophin-releasing hormone (GnRH-I) and T-helper epitopes in saline suppresses rodent fertility.” Vaccine 26 (10): 1365-74. It states that research on active immunization against the gonadotrophin-releasing hormone (GnRH-I) is gaining acceptance as a means to control reproduction and behavior in livestock, pets or wild animals. Many studies have described the use of multiple copies of the same peptide aligned and conjugated to a larger “carrier” protein, to enhance the immune response of said peptide. However, the problems that result from suppressing the “carrier” protein epitope have caused a decline of interest in the use of genetic materials that may initiate an optimal immune response. In the study conducted by the authors, a vaccine with 533 DNA base pairs was constructed in pcDNAVS-HisB coding for 18,871 kDa GnRH-I-T-helper-V5 epitopes of fusion proteins. Transfected COS1 cells were found with the vaccine construct, that liberate fusion protein into the culture supernatant. The vaccine construct (100 μg/mouse) in saline solution administered into the anterior quadriceps muscle of male and female ICR rats stimulated the response to the specific IgG antigen antibody. Testosterone levels in vaccinated males were significantly (p=0.021) reduced. A significant reduction was noticed in uterine implants after mating of immunized males and control females (p=0.028), as well as of immunized females and control males (p=0.004). Histological examination of gonads from both males and females in the study in week 13 showed atrophy of the seminiferous epithelium and foliclegenesis suppression.
[0014] Miller, L. A., J. P. Gionfriddo, et al. (2008). “The single-shot GnRH immunocontraceptive vaccine (GonaCon) in white-tailed deer: comparison of several GnRH preparations.” Am J Reprod Immunol 60 (3): 214-23. It specifies that the problem lies in the requirement of a single, effective and multi-annual injection of a GnRH contraceptive agent to control reproduction of the overabundant white-tailed deer population. The study method in this investigation refers to two GnRH conjugates, GonaCon (GnRH-KLH) and GonaCon-B (GnRH-Blue® protein), which were prepared in an emulsion as immunocontraceptive vaccine formulations for a single injection and for two injections. Besides, the GnRH-KLH protein conjugate was freeze-dried and suspended in AdjuVac adjuvant to produce a formulation of a fifth vaccine. Each formulation was administered to a group of captive adult female white-tailed deer. The reproductive performance of the treated females was monitored for 5 years to determine the comparative effectiveness of the different treatments. The results obtained in the study indicate that the long life of the contraceptive response (2 to 5 years) was strongly influenced by the design of the conjugated antigen, the adjuvant used, and the delivery form of the vaccine. The authors concluded that formulations in one and two injections of GonaCon and GonaCon-B produce multi-annual contraception in the adult female white-tailed deer. GonaCon-B produces a longer lasting contraceptive effect.
[0015] Sad, S., V. S. Chauhan, et al. (1993). “Synthetic gonadotrophin-releasing hormone (GnRH) vaccines incorporating GnRH and synthetic T-helper epitopes.” Vaccine 11 (11): 1145-50. It refers to the development of a vaccine against gonadotrophin-releasing hormone (GnRH) as an immunologic method for the treatment of prostatic hypertrophy, based on the observation that active immunization against GnRH leads to the production of anti-GnRH antibodies resulting in a reduction of the prostate gland. The authors have done research on the regulation of anti-GnRH antibody response by “carrier” molecules. In earlier studies, the authors have shown that the use of molecules of large proteins as “carriers” limits the use of said vaccines owing to the potential problems of anti-hapten of suppression induced by the carrier. In this study, the authors show that synthetic T-helper epitopes may be used as “carriers” for the generation of anti-GnRH antibody response.
[0016] However, according to the present invention, the use of immunopotentiators has allowed to obtain long term “vaccinal” effects using a single vaccine dose; therefore, the use of the antigen of the present invention in different formulations allows to modify the vaccination scheme.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 . Shows the immune response against the recombinant protein designated GnRXG/Q, of the present invention, measured by ELISA technique, as an increase of IgG immunoglobulins, in vaccinated animals versus control, observed as specific optic density against the recombinant peptide GnRXG/Q in immunized animals, using an aqueous adjuvant in the formulation. The serum dilution used was of 1:250 and the sample size of 10 subjects per group. The animals were immunized on days 0 and 15. In FIG. 1 -▪- corresponds to Control PBS and -▴- corresponds to GNRXG/Q+Adjuvant.
[0018] FIG. 2 . Shows the decrease of testosterone serum concentration, measured by ELISA technique, in immunized animals with the recombinant protein designated GnRXG/Q, of the present invention, versus control on days 0 and 15 in a number of 10 subjects per group. In FIG. 2 -▪- corresponds a Control PBS and -▴- corresponds to GNRXG/Q+Adjuvant.
[0019] FIG. 3 . Shows the immune response against the recombinant protein designated GnRXG/Q, of the present invention, as an increase of specific immunoglobulins, measured by ELISA technique, using different adjuvants in the formulation, in a 15-week assay. The animals (n=5) were immunized on days 0 and 30 and the increase in immunoglobulins was evaluated until day 110. In FIG. 3 -▪- corresponds to control PBS, -▴- corresponds to GNRXG/Q+Chi-H MW, -Δ- corresponds to GNRXG/Q+Chi-L MW, and -- corresponds to GNRXG/Q+CFA.
[0020] FIG. 4 . Shows testicular atrophy provoked by immunization with the recombinant protein designated GnRXG/Q, of the present invention and an adjuvant in its formulation. A exhibits testicles of a control mouse (1) and of a mouse immunized with the peptide GnRX G/Q (2) a scale in centimeters may be observed in the lower portion of the photograph; S exhibits testicle histological sections under two amplification levels.
[0021] FIG. 5 . Shows a decrease in testosterone serum concentration, measured by ELISA technique, in dogs immunized with the recombinant peptide designated GnRXG/Q of the present invention, in association with an adjuvant. The animals (n=7) were immunized on days 0 and 30 and the effect of the vaccine was evaluated for 3 months.
[0022] FIG. 6 . 10% SDS polyacrylamide gel, where the recombinant protein designated GnRX G/Q, of the present invention, is shown tandem repeated, purified from a total extract of bacterial proteins, with an approximate weight of 29 kiloDalton
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention comprises design, expression and purification of the following recombinant protein (Sequence SEQ ID No. 1 and Sequence SEQ ID No. 2) with a primary structure that incorporates the amino acid sequence of the gonadotrophin-releasing hormone (GnRH-I) fused to a glycosilable theoretical sequence and with immunogenic activity, that does not include pathogen or “carrier” protein sequences in its structure:
[0024] In sequences SEQ ID No. 1 and SEQ ID No. 2, the 10 amino acid peptide sequence of the GnRH-I hormone is observed in bold characters, fused to the 14 amino acid glycosilable theoretical sequence; this 24 amino acid chimeric peptide has been designated GnRX G/Q.
[0025] Another aspect of the invention comprises the vaccine that includes the peptide designated GnRX G/Q, to be used by itself or in a tandem repetition, the vaccine producing process, use thereof and method of mammal immunocastration.
[0026] The “theoretical” sequence may be flanking the GnRH-I sequence in any order (amino or carboxyl end of the peptide, Sequence SEQ ID No. 1 and SEQ ID No. 2).
[0027] Another aspect of the invention comprises the construct, see sequence SEQ ID No. 3 and SEQ ID No. 4 see sequence listing, formed by chimeric peptide GnRX G/Q tandem repeated 10 times, and observed as a recombinant protein migrating electrophoretically in a 10% SOS PAGE gel in FIG. 6 .
[0028] Another aspect of the invention comprises nucleotide sequences and corresponding vectors. The nucleotide sequences were designed by inverse genetics to be used as templates in the recombinant expression of the peptide GnRX G/Q; these were inserted in prokaryotic and eukaryotic expression vectors (sequences SEQ ID No. 5 and SEQ ID No. 6 see sequence listing), the result being the protein of sequence SEQ ID No. 7 sequence listing.
[0029] The present protein has been conceived as a recombinant or chimeric fusion protein where the GnRH amino acid sequence may be found as a percentage (40%) of the total molecule, the rest of the percentage (60%) corresponding to a sequence designed from the bioinformatic analysis of different peptides; a unique sequence has been designed that allows to improve immunogenicity of the segment that corresponds to the GnRH sequence, avoiding the incorporation of immunodominant segments such as pathogen, toxin or “carrier” protein- derived immunogens; this differentiates it from other molecules that have been patented in the prior art.
[0030] The designed sequence possesses a notable hydrofobicity and incorporates a consensus sequence that may be O-glycosilated in eukaryotic protein expression systems such as yeast or insect cells. This modification is oriented to improve peptide antigenicity in order to enhance the peptide capability of being recognized by the immune system. Moreover, the incorporation of this glycosilable segment differentiates the present protein from other GnRHs that incorporate fusion proteins, as the present glycopeptide is treated as a proteoglycan. Accordingly, the immune system will recognize the_entire molecule as a hapten and not just as an immunogenic segment thereof.
[0031] Another aspect of the invention comprises the preparation process of the fusion protein, wherein the nucleotide sequence coding for the recombinant protein (sequences SEQ ID No. 5 and 6) has been inserted in an expression vector with an inducible promoter for E. coli B121 bacteria (pQE 801, Qiagen) or a vector with an inducible promoter for S. cerevisiae yeast (pYES, invitrogen). The protein has been purified by affinity chromatography in Ni sepharose columns, which allows to eliminate possible contaminants of the expression system, mainly pyrogens, such as Lipopolysacharide (LPS).
[0032] This “theoretical” sequence has been designed using the following 10 bioinformatic algorithms that evaluate the hydrophobicity, hydrophylicity and antigenicity properties of a peptide sequence: 1) Fauchere-Pliska hydrofobicity algorithm, which generates a property profile using a hydrofobicity scale based on experimental octanol/water partitions of N-acetyl amino acid amides of each residue at a neuter pH; 2) Goldman/Engelman/Steitz hydrophylicity algorithm, which generates a property profile calculating the non-polar residues in α-helices; 3) Janin hydrofobicity algorithm, which generates a hydrofobicity property profile based on the molar fraction of hidden or exposed residue occurrence in known proteins; 4) Kyte Doolittle hydrofobicity algorithm, which generates a hydrophobicity and hydrophylicity property profile based on Kyte Doolittle values for individual residues in inner or outer regions of a globular protein; 5) Manavalan hydrofobicity algorithm, which generates a property profile based on the hydrophobicity of an individual residue modified by the presence of other residues in an 8 angstrom radius; 6) von Heijne hydrophylicity algorithm, which generates a property profile using a scale that reflects the free transference energy estimated when a α-helix moves from an aqueous phase to a non-polar one; 7) Hopp and Woods antigenicity algorithm: Hopp-Woods scale was designed to predict antigenic determinant sites in a protein, assuming that these are exposed on a protein surface and are confined to hydrophylic regions; 8) Parker antigenicity algorithm: this tool predicts the presence of antigenic determinants by the presence of areas with great local hydrophobicity, using a scale based on HPLC model peptide retention times; 9) Protrusion Index antigenicity algorithm: this tool generates a property profile using a protrusion index which is an antigenicity scale based on the study of known 3D structure proteins; and 10) Welling antigenicity algorithm: this tool calculates an antigenicity value as the log of the quotient between the percentage of a sample with known antigenic regions and the mean protein percentage.
[0033] Different amino acid sequences were evaluated for their potential capability of improving antigenicity and hydrophylicity of the sequence for GnRH-I when they are fused at the amino or carboxyl end of GnRH, as well as in tandem repetitions, comparing them to a tandem repeated GnRH-I sequence in the absence of intergenic sequences. Amino acid sequence NH 2 -GPPFSGGGGPPFSA-COOH was designed using the above mentioned parameters; it represents a hydrophobicity score in most algorithms, higher than 0 and greater than that of GnRH-I sequence. In the same way, owing to its hydrophobic condition, it exhibits limited antigenicity allowing, when analyzing the global molecule, to improve considerably GnRH-I sequence antigenicity in comparison to a tandem repeated GnRH-I sequence without intergenic sequences. Its design incorporated the consensus signal sequence SGGG, which corresponds to an O-glycosilation site, susceptible of receiving this post-translational modification when the protein is expressed in yeast or other eukaryotic cells. This tetrapeptide, which possesses the general sequence Ser-Gly-Xaa-Gly (wherein Xaa may be any amino acid), corresponds to a recognition site for the incorporation of a glycosaminoglycan (Burdon M., et al., 1987). Finally, in the design of this sequence it was considered to exclude similarities with pathogens or “carrier” proteins. To this end, a search and alignment analysis of this sequence was conducted with databases present in GenBank using BLAST (Basic Local Alignment Search Tool) tool. In none of these sequences 1a and 1b of the present invention were present.
[0034] To manufacture and express the recombinant protein, the double strand nucleotide sequence was linked to obtain tandem repetitions and was subsequently inserted in IPTG or glucose inducible prokaryotic and eukaryotic expression vectors. The recombinant protein obtained possesses a 6-hystidine tag repetition that allows it to be purified from endogenous proteins of another host by affinity chromatography with nickel or cobalt.
[0035] Another aspect of the invention comprises those nucleotide sequences where the codon to be used in the translation is varied; these may generate the same chimeric peptide, as it may be observed in Sequences SEQ ID Nos. 8-13 list of sequences.
TECHNICAL BACKGROUND OF THE INVENTION
[0036] To prove the effectiveness of the protein of the present invention, specifically that of Sequence 3c, in its capability to block steroidogenesis, oogenesis and spermatogenesis in laboratory animals via GnRH immunoneutralization, the above mentioned protein generated and purified was inoculated into laboratory animals in quantities from 50 to 500 μg in an oil adjuvant, particularly the complete or incomplete Freund's Adjuvant or an experimental adjuvant, specifically chitosan. Various parameters were analyzed such as the capability of animals to develop antibodies against the protein, their reproductive activity, spermatogenesis, oogenesis and androgen levels.
Experiment 1
[0037] The molecule as a vaccine was tested in a sample size of 18 laboratory animals, and significant differences (p<0,01) were obtained with regard to the expected physiologic effect and the adaptive immune response with the control group, using different adjuvants. (See FIGS. 1 to 4 ).
[0038] Ten 8-week-old male mice were subcutaneously immunized with 100 μg of recombinant protein GnRX G/Q (sequence 3c) in 100 μl of a commercial adjuvant, particularly complete Freund's adjuvant, on days 0 and 15. Blood was extracted from the animals every 15 days to evaluate effectiveness of the vaccine and its capability to increase immunoglobulin titers against the GnRH-I hormone. FIG. 1 shows the increase of immunoglobulin levels specific against the GnRH-I hormone, measured by ELISA technique, of immunized animals with regard to control. FIG. 2 shows the fall in serum testerone levels, measured by ELISA technique, of animals immunized with the above-mentioned GnRH GQ protein in comparison to a control group. At the end of the essay the animals were slaughtered and both testicles were compared macroscopically and microscopically to a group of control animals ( FIG. 4 .).
Experiment 2
[0039] Fifteen 8-week old male rats, were immunized with 100 μg of recombinant protein GnRX G/Q in 200 μl of a commercial adjuvant, specifically Freund's complete adjuvant, and 2 experimental adjuvants, particularly Chitosan of high and low molecular weight, 0,5% v/v, on days 0 and 30 of the experiment. Blood was extracted from the animals every 15 days, evaluating the effect of the vaccine in the increase of immunoglobulins specific against the GnRH-I hormone in time measured by ELISA technique ( FIG. 3 ).
Experiment 3
[0040] Seven adult male crossbred dogs were immunized with 200 μg of the recombinant protein GnRX G/Q (sequence 3c) in 1 ml of a commercial adjuvant, specifically Freund's incomplete adjuvant, on days 0 and 30. Blood was extracted from the animals every 30 days to evaluate the effect of the vaccine in testosterone plasma levels. FIG. 5 shows the fall in testosterone levels in time at values approaching surgical castration (0.1 ng/ml), measured by ELISA technique.
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Fusion protein for immunocastration (Sequences 1a and 1b) that comprises the primary amino acid sequence of the gonadotrophin-liberating protein fused to a theoretical sequence:
NH 2 -QHWSYGLRPGGPPFSGGGGPPFSA-COOH Sequence 1a
NH 2 -GPPFSGGGGPPFSAQHWSYGLRPG-COOH; Sequence 1b
DNA sequences coding for said fusion protein; vaccine comprising said fusion protein; use of the fusion protein for mammal immunocastration; process for producing the vaccine; process for preparing the fusion protein that comprises fusing the amino acid sequence of the gonadotrophin-liberating hormone (GnRH-I) to a theoretical glycosilable sequence having immunogenic activity that does not include pathogen or “carrier” protein sequences in its structure.
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TECHNICAL FIELD
This invention relates to the provision of contact center services to the players of networked games, and to the facilitation of financial transactions by such players when dealing with contact centers.
BACKGROUND ART
Networked multiplayer games are known by a variety of different acronyms from the earliest MUDs (multi-user domains or dungeons), to the current variety of MMOGs (massively multiplayer online games), the most popular genres of which include MMORPG (massively multiplayer online role playing game), MMORTS (massively multiplayer online real time strategy) and MMOFPS (massively multiplayer online first-person shooter).
These games, which are all networked multiplayer games, may be referred to herein for convenience as MMOGs, without any intended or implied limitation as to the number of players or the objectives, characteristics or nature of any such game, other than the fact that it is played over a network and involves multiple players.
A typical MMOG will employ the Internet as its network and will support several hundred or several thousand players. These players will frequently interact with one another and with the game environment through the medium of characters (also called avatars) who “live” within the game environment. Far from being a small niche market, the total number of current subscriptions to MMOGs as of July 2006 was thought to be approximately 15 million. Certain games have internal economies which generate substantial activity, and because the items and currency generated in-game can be sold for hard cash, the more popular games have sufficient economic activity to enable dedicated players to earn a living solely through the economic activity they generate in-game.
With the increasing value of virtual economies, issues arise with regard to taxation, legal rights, and the interaction between the virtual and real economies. It is understandably difficult for companies and interested individuals to participate in the economic activity occurring within virtual economies, although certain companies have begun to sponsor advertisements in social MMOGs such as “Second Life”, and indeed the Reuters news agency has a full time reporter in the Second Life game world.
As this trend increases, companies which have traditionally been confined to the real world will face increasing challenges in trying to successfully integrate virtual world activity with their day-to-day operations. The present invention is directed to providing methods and systems to assist in this process.
DISCLOSURE OF THE INVENTION
The invention provides, in a first aspect, a method of operating a networked multiplayer game. This method includes the steps of:
a) providing to a player a controllable game character which the player may control to interact with the game's environment; b) providing a mechanism as part of the game which can be operated by game characters to initiate a request for contact with a contact center agent; c) upon operation of the mechanism by a character, issuing a request to a contact center which is separate from the game; d) opening a communications channel between an agent or employee of the contact center and the game environment; and e) directing this communications channel to the locality of the player's character whereby the player and the contact center agent may communicate within the game environment.
According to this method, a game operator may provide enhanced experiences and opportunities to players of its games. By firstly enabling a request for contact with a contact center to be made within the context of a game, i.e. by employing an in-game mechanism, players are encouraged to stay within the game rather than being directed to visit an external website or to make a separate call or send an email to a contact center. Secondly, by enabling the agent to communicate with the player within the context of the game environment (at least from the player's point of view), the gaming experience is greatly enhanced and the player can remain “in character” throughout the interaction. In effect, the player perceives that the agent is “visiting” the game to respond to the player's request. A skilled agent may tailor the conversation to avoid this illusion being broken.
Preferably, there is also provided, in the game environment, a visual representation of an agent character and the communications channel is directed to the agent character.
In this way, the agent is “physically” embodied within the game in the sense that the player's character can now converse and interact with another character, that of the agent.
Preferably, the method also includes the step of animating the agent character in response to input from the agent.
Thus, the agent character can be enhanced to increase the realism by causing the character to apparently speak when a speech signal is detected from the agent. This can be automatically performed by equipment running the game or it can be performed by equipment at the contact center which augments the agent's communications with instructions for animating the agent character.
More preferably, the agent character is controlled by said agent.
The agent may control the character through a simple interface allowing the agent to enter simple commands to control the character, or the agent may be provided with a game client or game interface equivalent to that provided to players of the game, whereby the agent can fully control the agent character in the same way as the players control their own characters. Any such agent interface can of course be enhanced or simplified as appropriate to the situation.
Alternatively, the agent character can be automated.
The game engine can be provided with functionality to automate such agent characters. it is common within games to have non-player characters or NPCs which are controlled by the game and which can simulate the characteristics and actions of player-controlled characters. Such techniques can be used to augment the agent character with automated actions.
Another method of automation is where the agent's character is controlled/automated by the contact center server, by manipulating a game client interface or using other game control APIs (application programming interfaces) as may be available in the game. This mode is similar to when the agent controls the game character; the difference is that the agent's character is automated by the contact center. This may be used in conjunction with a live agent on the call, and for part of the time, it may be completely automated with no live agent participating, such as before the agent is allocated to the call.
A contact center providing a service or providing goods which are very much consistent with the game (e.g. a tailoring service for the clothes or a service for altering the appearance of characters e.g. a tattooing service) might benefit from employing well-trained agents having a full game client available to them. Other contact centers providing more limited services, e.g. in-game banking, might not need such functionality and might either automate their bank tellers and officials or might provide a “light” version of the client so that agents do not need the same degree of skill with the game mechanics to service contact requests.
Optionally, the method also includes the step of providing to the contact center information regarding the player making the request and/or regarding the player's character.
It will be appreciated that contact centers generally require information about the customer to properly service a call. In addition to the information which is strictly required to service a call, contact centers will often prompt the customer for additional information which helps to better service the contact. Often the information which might be most useful for an agent can be difficult to collect, such as a customer's spending habits, or the likelihood of a contact between a given customer and a contact center actually giving rise to a sale or transaction. Where a player has a history of interacting with businesses within a game, however, the game may log such interactions and can provide such information (subject to the customer's permission, if required, or the terms of service of the game) to a contact center to enhance the information available. The information can relate to the player of the game, and can include account information and history, time spent in game, history with other contact centers accessed in game, etc., or can relate to the character of the player such as the character's bank balance, details of the character's possessions, appearance and characteristics. Such additional information can greatly enhance the interaction between the agent and the player.
The method can also include the step, subsequent to the operation of the mechanism by the players s character, of placing the character in a simulation of a waiting environment.
This environment could be a simulation of a queue, for example, or of a waiting room, or of any other environment chosen to represent a waiting environment. To give a one example, if characters in the game are aircraft pilots (or indeed sentient aircraft), then the mechanism to request contact might be a radio frequency accessed to request a landing slot, and the waiting environment might be an aircraft holding pattern for a landing strip or airport.
Preferably the method further includes receiving from the contact center an indication of a wait time parameter at the contact center, and modifying said simulation of said waiting environment on the basis of said indication.
Thus, the length of the queue in which the contact sits, or the current average waiting time for contacts in that queue, or in the contact center generally, or any other wait time measurement, might be notified to the game and the game might modify the population of a waiting room, length of a queue or number of holding aircraft, based on this metric.
The invention also provides a system for use in a networked multiplayer game, the system including:
a) a game engine providing to a player a controllable representation of a game character in a game environment whereby the player may control the character to interact with the environment; b) within the game environment, a mechanism operable by game characters to initiate a request for contact with a contact center agent; c) a request generator which, upon operation of the mechanism by a character of the player, issues a request to a contact center external to the game; d) a communications interface for providing a communications channel between an agent of the contact center and the game environment; and e) communications direction means for directing the communications channel to the game environment in the locality of the player's character whereby the player and the contact center agent may communicate within the game environment.
The invention further provides, in this aspect, a computer program product comprising instructions which when executed in a networked multiplayer game system are effective to:
a) provide to a player a controllable representation of a game character in a game environment whereby the player may control the character to interact with the environment; b) provide, within the game environment, a mechanism operable by game characters to initiate a request for contact with a contact center agent; c) upon operation of the mechanism by a character of the player, issue a request to a contact center external to the game; d) open a communications channel between an agent of the contact center and the game environment; and e) direct the communications channel to the game environment in the locality of the player's character whereby the player and the contact center agent may communicate within the game environment.
It will be appreciated that such a computer program product can be distributed across different items of processing equipment associated with a game and need not be exclusively executed on a single game server. Typically, the program instructions will be distributed at least between the game engine and the communications server. The computer program product can be a physical carrier of a program on one or more media carriers such as hard drives, or optical or magnetic disks, or it can be embodied as an electrical signal or optical signal encoding the program instructions.
It should be noted that the agent may be unable to (and is not required to) differentiate between an MMOG-originating contact and a real world contact. One of the advantages provided by the invention is that it allows a contact center provider to offer existing contact center customers access to a new market without the need to retrain their agents. Agents may thus remain oblivious to the fact that a contact originated in a MMOG. However, there are advantages in some scenarios for having agents who are fully aware of the fact that a contact is a player of a networked online game, and this aspect of the invention, which has been alluded to previously, where the agent is aware of and may even participate in the game, will be described next in more detail.
In another aspect the invention provides a method of operating a contact center, including the steps of:
a) receiving at the contact center a request for contact from a networked multiplayer game which is separate from the contact center; b) allocating the request to an agent of the contact center; c) providing to the agent a representation of the game environment so that the agent may see a representation of the character who was responsible for the request for contact which issued from the game; and d) opening a communications channel between the agent and the game environment
In this aspect of the invention, the contact center provides an enhanced experience for the agent servicing requests received from players immersed in a game. While the agent need not be similarly immersed, it is extremely useful for such an agent to see a representation of the player's character so that the agent can provide a more realistic experience to the player who is immersed in the game. Thus an agent can note the character's appearance, demeanor, posture, actions and gestures, facial expressions, surroundings, and so on, and can employ such information usefully when interacting.
The method may also include the step of providing the agent with an interface enabling interaction with the representation of the environment of the game.
Preferably, the interface is a client program providing the agent with control of an agent character in the environment.
Thus, the agent can be provided with a full game client or a game client modified or enhanced for the agent, whereby the agent also controls a character within the game during the interaction between the agent and the player.
Further, preferably, the communications channel is directed to the agent character within the game environment.
In preferred embodiments, the method also includes the steps of providing to the agent a mechanism for interacting with a payment system of the game, and maintaining an account of payments involving the agent made using the payment system of the game, whereby financial transactions may be carried out between the player and the agent by employing the payment system of the game with such transactions recorded in the account.
The term “payment” as used herein is intended to denote transfers of currency having value in the real world, currency having value only within a game, transfers of bearer items such as cheques and credit notes written by a character in a game, and transfers of any other item of value such as artefacts created in game or forming part of a game (e.g. certain weapons and items of equipment within certain games will have defined or agreed values and can thus be used to effect barter-type payments).
This additional feature enables a contact center to capture some of the considerable wealth generated in game. While transactions can occur in either direction (e.g. a contact center might pay a player for completing a survey), the primary utility of this feature is that it provides a contact center agent with a way of obtaining payment in an alternative to the usual credit card type of transaction. Game systems will typically have payment mechanisms whereby one character can give money or other valuables to another character, and such mechanisms can be employed to allow the transfer of wealth or valuables between the player's character and the agent's character or a mechanism associated with the agent or the contact center (e.g. a deposit box). When such a transaction is completed an account will be updated and the contact center can thus give and receive valuable consideration for transactions agreed in game.
In some circumstances, as described above, the agent may not be aware of the contact's origins within an MMOG. Such agents may be provided with a payments interface which can map the in-game currency and other MMOG artefacts to real world values so that the transaction can be presented to the player in the terms of the game and can be presented to the agent in real world terms. In sufficiently sophisticated embodiments, each will be oblivious of the other person's view of the transaction, and automated “foreign exchanges2 can be used to convert sums between real currency and game currency. Obviously the applicability of such techniques would be dependent on the type of product/service being offered.
In this aspect there is also provided a contact center system, comprising;
a) an interface with a networked multiplayer game external to the contact center for receiving requests for contact from the game; b) a contact allocation system for allocating a received request from the game to an agent of the contact center; c) an agent interface for providing to the agent a representation of the environment of the game whereby the agent may see a representation of a character responsible for the request from the game; and d) a communications system for providing a communications channel between the agent and the game environment
There is further provided a computer program product comprising instructions which when executed in a contact center system are effective to cause the system to:
a) await receipt at the contact center of a request for contact from a networked multiplayer game external to the contact center; b) allocate the request to an agent of the contact center; c) provide to the agent a representation of the environment of the game whereby the agent may see a representation of a character responsible for the request from the game; and d) open a communications channel between the agent and the game environment
In another aspect the invention provides a method of enabling a transaction between a player of a networked multiplayer game and an agent of a contact center external to the game, comprising the steps of:
a) opening a communications channel between the agent and a character of the player within an environment of the game; b) providing a payment mechanism within the environment of the game whereby the player may receive a payment from or issue a payment to the contact center agent; and c) upon operation of the payment mechanism, causing an account held by the player within the game to be credited or debited by an agreed amount.
The method can also include the step of:
d) upon operation of the payment mechanism, causing an account held by the contact center within the game to be debited or credited by an agreed amount.
Alternatively, the method can include the further step of:
d) upon operation of the payment mechanism, causing an account held by the contact center outside the game to be debited or credited by an agreed amount.
Thus, the contact center may be prepared either to receive or pay in-game credit or may prefer to receive or pay real currency held outside the game and converted at a market rate to in-game currency.
Preferably, said payment mechanism further comprises automatically converting a payment amount between a currency valid within the game and a currency valid in the real world.
Automatic foreign exchanges may be employed for the conversion of game money. This is to enable the agent (if the contact center administrator so chooses, and if the product/service of relevance lends itself to such) to be unaware that he/she is dealing with a contact from an MMOG.
There is further provided a payment system for enabling a transaction between a player of a networked multiplayer game and an agent of a contact center external to the game, comprising:
a) a communications channel between the agent and a character of the player within an environment of the game; b) a payment mechanism provided within the environment of the game whereby the player may receive a payment from or issue a payment to the contact center agent; and c) an account recording system which, upon operation of the payment mechanism, causes an account held by the player within the game to be credited or debited by an agreed amount.
There is also provided in this aspect a computer program product comprising instructions which when executed in a networked multiplayer game system enable a transaction between a player of the networked multiplayer game and an agent of a contact center external to the game, by causing the game system to:
a) ensure an open communications channel between the agent and a character of the player within an environment of the game; b) provide a payment mechanism within the environment of the game whereby the player may receive a payment from or issue a payment to the contact center agent; and c) upon operation of the payment mechanism, cause an account held by the player within the game to be credited or debited by an agreed amount.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic architecture of a network environment for one implementation of the present invention;
FIG. 2 is a flowchart illustrating a method of operating a game and a contact center in accordance with the present invention;
FIG. 3 is a flowchart illustrating a variation on the method of FIG. 2 ;
FIG. 4 is a flowchart illustrating a first method of completing a financial transaction; and
FIG. 5 is a flowchart illustrating a second method of completing a financial transaction.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In FIG. 1 there is indicated, generally at 10 , a network architecture comprising a massive multiplayer online game (MMOC) environment 12 running on the Internet 14 , and a contact center environment 16 also connected to the Internet.
Within the MMOG environment 12 , individual players 18 connect via the Internet to a game server 20 following an authentication process carried out by an associated login server 22 (the functions of login server 22 may of course also be carried out by game server 20 ). The game server 20 , which is shown as a single server but will more often be implemented as a network of servers, executes a number of processes in order to host a game for the players 18 , such processes typically including a game engine 24 , a representation of the simulated world or game environment 26 , representations and data relating to player characters or avatars 28 (which inhabit the world representation 26 and interact with it and with each other according to the rules of the game engine 24 and the actions of players), and communication functions shown generically at 30 , such functions often including voice, video and instant messaging facilities whereby player characters may interact with one another, or players themselves may interact with one another in “out of game” communication channels which do not involve their characters.
The login server 22 is provided with player account information including the player's subscription and real world contact and billing details. In addition, if the game in question has an economy, the player's character(s) may also have separate accounts in the game currency, and in the illustrated embodiment, this character account information is stored with the character/avatar data 28 on game server 20 . It will be appreciated that the player's account and the character's account need not be separate from one another and a unified account could be employed.
The contact center environment 16 is separate from the game and comprises a contact center server 32 which is connected to the Internet and to which a plurality of trained human contact center agents 34 are connected. Agents 34 may make and receive voice calls to external parties or customers. Such calls may be carried over the public switched telephone network (PSTN), not shown, or may be carried over the Internet with which the contact center server connects via an Internet telephony gateway 36 . The contact center may additionally be enabled with technology such as the session initiation protocol (SIP), allowing communications to be made in a range of media types. In addition to voice calls, agents may communicate with customers using video, or using data media such as instant messaging or email.
Incoming communications (known as contacts) are managed by the contact center server 32 in order to route contacts to agents in the most efficient manner for the contact center's purposes. The management of contacts is controlled by workflows 38 which determine the routing of contacts through automated treatments such as an interactive voice response (IVR) application 40 or a music-on-hold server 42 . The IVR application provides information useful for classifying a contact in order to best route it to an available and competent agent. Other information gleaned from the source of the contact, the number called, etc. can also be used in this way.
Each contact can therefore be allocated to one or more skillsets which are serviced by the agents 34 according to skillset abilities noted for each agent in a set of agent resource records 44 . At busy times, the contacts are placed in queues according to skillset and the contact center server also therefore manages the set of queues 46 . In this way, all incoming calls are classified and directed to agents according to the treatment specified in the workflows. The system as described thus far is conventional and known in the art.
Referring additionally to FIG. 2 , a flowchart is shown detailing a sequence of steps carried out by a player 18 (left hand column of flowchart), the game server and login server 20 , 22 (center column of flowchart), and the contact center 16 (right hand side of flowchart).
The process begins when a player logs into the game, step 50 , and following a successful login on the login account server 22 , step 52 , the game engine 24 sends the player's avatar data and the world simulation data for the avatar's environment to a game client resident on the player's machine, step 54 .
The client program running on the player's machine renders the game world and the avatar within the game world to the player, step 56 , and the player can control this avatar in order to play the game as normal.
Within the game world provided according to the present invention, a mechanism has been provided for access to the services of contact center 16 . There may be any number of reasons why a player might want to access services which might be provided by a contact center, and the mechanism used to access the contact center can be any mechanism which a player avatar could normally access within the game. Thus it could be a doorway or an arch through which the avatar moves, or a lever or a button, or a textual, menu or clickable command available through the client program. The mechanism might be labelled with “real world” information relating to the contact center, or the mechanism can appear to be entirely within the terms of reference of the game world, i.e. without any real world references.
Assuming that there is a portal or doorway provided in this particular game, the player avatar accesses this portal to initiate communication with the contact center, step 58 . The game engine recognises this action as a command to send a contact request to a contact center associated with that portal mechanism, step 60 . A communications link is created or accessed over the Internet between the game and the contact center, i.e. the game server is programmed to formulate requests to a network address associated with the contact center. The request will typically include an identifier of the player, but may include additional details of the request, such as details entered by the player when accessing the mechanism in step 58 . Thus, a player might write a letter or note, or record a voice message when accessing the mechanism, and any such details would be passed along with the request to the contact center in step 60 .
In step 62 , the contact center, upon receiving a request for contact initiation, may request additional information to assist in queuing and directing the contact. Such additional information might already be available to the game server from, for example, the login/account server 22 . Other sources of data can also be used, such as the player history (how often the player has requested similar services, details of the player's in-game bank balance, details of previous services purchased or requested by the player, etc.), step 64 . When this information is returned to the contact center, a contact is placed in a queue, step 66 .
The contact center then returns details of the successful queuing of the contact and returns on-hold content to the game, step 68 . This on-hold content can be interactive voice response treatments, music on hold, or automated game content such as an automated character who appears to the player and asks questions analogous to those which would typically have been provided in an interactive voice response environment in a telephone call. The automated game content is supplied by a module 48 within the contact center environment 16 .
When these details are returned to the game in step 68 , the game engine optionally simulates a queue and passes on the on-hold content in step 70 . Thus, using the example of a series of question which are to be asked by the contact center, the game engine may in fact generate an automated character which is made to speak those question to the player's avatar. In step 72 the player receives any queue content or any on-hold content and optionally, an interactive session may follow in which the player's interaction with this content is processed by the game and/or the contact center to improve the queuing details or to add to the player's data stored by the game and/or the contact center. A representation of the queue might be generated for the player, so that the player sees a line of other characters ahead of his own character, which reduces as the player's contact approaches the top of the queue maintained by the contact center.
Another example is a representation of a waiting room, as at a doctor's office. This environment allows for non-sequential/out-of-sequence processing of contacts (such as when a nurse calls out the name of the next patient to be seen, which is not always the person waiting the longest).
As with traditional contacts in contact centers, the contact sooner or later reaches the top of a queue for which an agent is available, step 74 . The agent workstation is provided with a client program which performs a game login and launches the game client, step 76 . If the agent deals exclusively with contacts from one particular game, then the agent can be logged in permanently. Alternatively, the agent may be servicing contacts from other channels as well as from the game, in which case it is more likely that the agent's game client will log in to the game only when responding to such contacts.
In step 78 , the game engine, following the agent's login, sends avatar and world data to the agent's client program, step 78 . The agent avatar then enters the contact center portal where the player's avatar is situated, step 80 . In steps 82 and 84 , the agent avatar communicates with the player avatar and vice versa. The player is thus provided, in game, with an experience of dealing with an agent which is to all intents and purposes part of the game and does not require him or her to “break character”. From the point of view of the contact center, skilled agents can provide an enhanced experience to their customers which is not provided when a player has to log out of the game or take his or her attention away from the game in order to dial a contact center number or access a website.
FIG. 3 illustrates a variation on this process, beginning at step 74 , when the contact reaches the top of the queue and an agent becomes available. Whereas the process of FIG. 2 was immersive for the agent as well as for the player, the process of FIG. 3 is not immersive for the agent, i.e. the agent does not have a full game client on his or her workstation.
In step 86 of FIG. 3 , the agent workstation performs a game login without a game client being presented to the agent. The game engine acknowledges this login in step 88 , and then the game itself generates an automated avatar in step 90 which is associated with that agent login. In step 92 , the automated avatar generated by the game enters the contact center portal (i.e. it appears there to the player) and from this point on the player avatar can communicate with this automated avatar, i.e. the player will see and can speak to the automated avatar in the contact center portal.
Form the agent's point of view, the agent communicates with the game engine, in step 94 , such that any communications from the agent or to the agent using the normal communications equipment employed by that agent, are channelled to the game engine. The game engine intercepts such communications and uses them, step 96 , to automate the avatar with the agent's communication. Such automation can be as simple or as sophisticated as the game engine permits. Thus, the voice of the agent can be augmented by physical gestures, emotions, and so on. Alternatively, a video image of the agent can be converted to or merged with the avatar's appearance and actions.
In step 98 , it can be seen that the player avatar communicates with the automated avatar, and thus communication proceeds between the player and the agent with the game engine acting as an intermediary controlling the agent's avatar.
FIG. 4 shows an example of a transaction carried out once communications have been established according to FIG. 3 . The same principles apply, however, to the process of FIG. 2 . In steps 94 , 96 and 98 , the agent, game and player communicate with one another as described above. When it is agreed between the player and agent that the player will pay for a product or service, step 100 , the player provides credit card or other payment details and address details or other authentication details verbally or using secure instant messaging, step 102 . These details are communicated either through the game server or via a different communications channel set up specifically for the transaction, and the agent verifies the transaction details as an agent would in a communications session in a contact center which had been initiated using more conventional channels, step 104 . Once the agent is satisfied as to the financial details of the transaction and as to any agreed delivery of products or services, the transaction is completed, step 106 .
It will be appreciated that the process of FIG. 4 is essentially a conventional transaction piggybacked onto a contact center session carried out through the medium of a game according to the invention. However, FIG. 5 describes a further integration between the contact center and the game.
In the process of FIG. 5 , steps 94 , 96 , 98 and 100 are as described above, with the player agreeing to pay for a product or service. However, rather than employing real-world payment mechanisms, the player in this case uses an in-game payment mechanism to pay for a product or service (which may be a real world product or service) with game currency, step 108 . The game verifies that the player is in a position to make such a payment according to the game rules, and performs a financial transaction deducting the credit from the player's account, optionally taking a commission from the transaction, and crediting a contact center account held on the game server or held in the real world.
For games where there is an open exchange mechanism converting between in-game currency and real world currency (such as for the MMOG called “Second Life” where the in-game currency of “Linden dollars” are freely exchangeable on various websites to U.S. dollars), the contact center may choose to be paid in real world currency rather than in in-game currency. However, it may also suit the contact center to maintain an in-game account balance.
Once this transaction has been completed according to the game server's records, the agent is notified by the payment authorisation, step 112 , and the transaction then completes with the agreement of both player and agent, step 114 .
The invention is not limited to the embodiment(s) described herein but can be amended or modified without departing from the scope of the present invention.
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A mechanism is provided in a networked multiplayer game for use by playable characters. When operated by a player's character, this mechanism causes the game to send a request for contact to an external contact center. The contact request is queued and allocated to an agent, optionally with the assistance of information provided by the game. When a contact center agent answers the contact request, a communications channel is opened from the agent directly into the game where the game system directs the communication to the local environment of the player's character enabling the player to communicate with the agent of a contact center without leaving the game environment. The agent or contact center may additionally have a representation in the game so that the player can interact with the agent or contact center. The agent representation may be a character in the game controlled by an agent who is logged into the game, or by the contact center manipulating a game client or API, or by the game engine itself. A representation of the contact center may be a telephone graphic, portal, door, sign, lever, button or any other manipulable game object, and optionally a multimedia representation of the contact queuing system. Payments can be made to or from the player using in-game currency which is credited or debited to an account held by the contact center.
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[0001] This application claims priority from U.S. Ser. No. 60/182,476 filed Feb. 15, 2000, the contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention is in the field of vaccine preparation. New and improved techniques are illustrated for the preparation of a vaccine against influenza, which techniques are applicable to protein-based vaccines generally.
BACKGROUND OF THE INVENTION
Flue Incidence
[0003] Vaccination is the most effective way of reducing the high morbidity and mortality rates as well as diminishing the enormous social and economic impact associated with influenza infection. Although detergent-containing split influenza vaccines are available, the level of vaccination compliance especially in the high-risk groups such as infants and the elderly is low. For example, it is estimated that less than half of the eligible population over the age of 65 actually receives the vaccine. In addition, despite being 70-90% effective in inducing immunity in healthy adults, the current injectable influenza vaccines are poorly immunogenic as a single dose in infants and the geriatric population. Seroconversion rates as low as 20-50% have been reported amongst the elderly. This reduced response in the elderly is believed due to a decline in the Type 1 T cell response, including cytotoxic T lymphocyte activity in this age group. The combination of reduced compliance and poor immunogenicity ensures that large sectors of the general population remain at high risk of infection and complications caused by influenza. Numerous efforts to enhance the immunogenicity of injectable influenza subunit vaccines by co-administering them with adjuvants have proved unsuccessful due to unacceptable rates of local reactogenicity following immunization and the inability to reproduce the strong immunostimulatory effects seen in animal models in humans.
Advantages of Nasal Vaccines
[0004] Since influenza infections are restricted to the upper and lower respiratory tracts, nasally-delivered influenza vaccines offer a more benign approach to vaccination that should increase immunization compliance in all ages of the population. Furthermore, immunization by the nasal route may be more effective compared with intramuscular injection because the production of local secretory IgA in the upper respiratory tract can protect against influenza infection, while injectable influenza vaccines are inefficient at inducing mucosal IgA. Influenza specific secretory IgA shows a broader cross-reactivity for variant strains of virus and thus may offer a greater degree of protection against mutant influenza viruses. In particular, nasal flu vaccines may be more effective in the elderly since, unlike the systemic immune system, mucosal immune responses do not deteriorate with age. Nasal flu vaccines that also stimulate systemic immune responses may protect the lower respiratory tract (lungs) due to transudation of antibodies from the serum. In addition, influenza-specific cytotoxic T cells (CTL) in nasal associated lymphoid tissue can contribute to recovery from infection.
[0005] Live attenuated cold adapted (CAV) influenza vaccines conventionally have been used via the nasal route in humans. These influenza strains are genetic reassortants combining the HA and NA genes of the current strains of flu virus with the 6 genes encoding the other internal and structural proteins from an influenza donor virus adapted to grow at lower temperatures (25° C.) thereby allowing only minimal replication in the nasopharyngal respiratory tract. These vaccines have the advantage of inducing protective immune responses similar to those elicited by natural infection with influenza, including induction of secretory IgA in the nasal washes, interferon gamma production in restimulated PMNC's and activation of CTL specific for internal viral proteins that may broaden the cross-reactivity against viruses within the same sub-type. CAV influenza vaccines are close to commercialization and have been demonstrated to be well-tolerated and immunogenic in children and healthy adults. In recent studies in healthy children, one or two doses of CAV flu vaccine have been shown to induce equivalent systemic antibody as injectable split flu vaccines. The ability of a single dose of CAV to induce >80% protection in seronegative children is an advantage over injectable split vaccines that require two immunizations to achieve similar protection in this age group. While pre-existing circulating antibodies in healthy adults and the elderly prevent efficient seroconversion in these age groups (see below), CAV's have been demonstrated to significantly reduce the number of febrile illnesses, days lost at work and visits to healthcare providers compared with placebo. In the elderly, CAV's in combination with an injectable split subunit vaccine significantly reduced laboratory documented influenza compared to placebo.
[0006] Despite the benefits of described above CAV vaccines for influenza have a number of drawbacks: healthy adults and the elderly who have been previously exposed to influenza viruses respond poorly to CAV vaccines and often do not reach the levels of serum hemagglutination inhibition (HAI) activity that correlate with protection. This is particularly significant for the elderly who are amongst the highest risk group and currently the only group where global vaccination is advised. In addition, due to the potential problems with reversion to wild-type stains and/or recombination with circulating strains, CAV's are not recommended for use in immunosuppressed or pregnant women. Despite 20 years of clinical evaluation of CAV influenza vaccines licensing has been delayed due to production and quality control issues.
[0007] In order to circumvent the potential safety concerns with CAV influenza vaccines, there are currently attempts to develop nasal inactivated “split” influenza vaccines (ISIV). Inactivated split influenza vaccines contain purified influenza hemagglutinin (HA). Inactivated split influenza vaccines given alone or with various particulate delivery vehicles or enterotoxin-based adjuvants have induced influenza specific mucosal and systemic immune responses in animals and humans.
Nasal Formulation of ISIV
[0008] At doses equivalent to those given via the injectable route, nasal ISIV containing antigen alone reproducibly induce significantly higher levels of nasal IgA in animals and in limited studies in humans. However, two or more doses of nasal ISIV at higher amounts of HA are required to induce levels of serum HAI equivalent to injectable ISIV which make such vaccines less viable commercially.
Enterotoxin Addition
[0009] Increased influenza specific mucosal and serum immune responses can be achieved in mice by administering ISIV nasally with enterotoxins such as cholera toxin B subunit (CTB) Tamura, et al., J. Immunol . (1992) 149:981-988 (which contained a significant amount of active cholera toxin even if referred to as CTB, since a recombinant source of CTB was not used in these studies) and recombinant heat-labile toxin from E. coli (rLT), Barchfield, et al., Vaccine (1999) 17:695-704.
[0010] In mice these enterotoxins are powerful mucosal adjuvants that are capable of inducing both enhanced secretory IgA and serum immune responses against associated antigens including inactivated split influenza vaccine. Recombinant LT was also shown to enhance the local and systemic HA specific response against ISIV in humans (Hashigucci, et al., Vaccine (1996) 14:113-119). However, the evaluation of enterotoxin-based adjuvants nasally in humans has been halted by the US FDA due to the results from pre-clinical toxicity studies in mice, showing that the enterotoxins reach the olfactory bulb region of the CNS and induce strong inflammatory reactions in that tissue following nasal administration. This finding has significantly hampered development of flu vaccines with these adjuvants (McGhee, et al., J. Immunol . (2000) 165:4778-4782) and would likely preclude the use of this type of adjuvant in human vaccines for the foreseeable future.
Lipid Based Formulations
[0011] Particulate species such as the virosome (a liposome formulation with influenza antigens) have also been tested in animal studies and in humans as effective nasal delivery vehicles for inactivated influenza antigens. Particulate antigens may enhance uptake by antigen presenting cells in nasal associated lymphoid tissue. Virosomes are liposomes containing influenza virus antigens associated with spheres consisting of lipids. These vaccines have been licensed in Europe as injectables. In mice, nasal virosomes induce serum titers to the same levels as equivalent amounts of injectable split antigen together with significantly higher levels of mucosal secretory IgA. Virosomes have been also shown to be immunogenic in humans following nasal immunization, however in two clinical trials it was demonstrated that recombinant LT was necessary to achieve specific titers of serum antibody equivalent to injectable vaccine following nasal immunization with 30 μg total HA given in two doses (Gluck, et al., J. Infect. Dis . (2000) 181:1129-1132). Although currently licensed in Switzerland, the requirement for the potentially neurotoxic rLT to achieve immunogenic equivalency with injectable flu vaccines precludes the vaccine in many territories including North America.
[0012] Another particulate delivery vehicle under development is the Biovector system that comprises an inner core of carbohydrate surrounded by lipid envelope. In clinical studies, nasal ISIV together with Biovectors demonstrated higher serum HAI and mucosal IgA compared with placebo. However, two doses of the highest level tested of influenza antigen with Biovectors elicited an increase HAI titers that were not significant enough to warrant continued development of this product by a major vaccine manufacturing partner who discontinued cooperative involvement with this technology after examining the data, suggesting the need to supplement the Biovectors with an immunostimulant to achieve the levels of serum HAI that correlate with protection.
[0013] ISIV formulated with MF59, a lipid based emulsion, has not elicited responses significantly different enough from control influenza articles to warrant continued development. Another technology, monophosphoryl lipid A (MPLA), is a lipoplysachharide adjuvant consisting of oil-based or aqueous formulations of a lipid isolated from the lipopolysaccharide of Salmonella Minnesota R595. This technology has also been used in mice to make nasal influenza vaccines with moderate success in pre-clinical studies.
Proteosome Technology
[0014] “Proteosome” has been used to describe preparations of outer membrane proteins of Meningococci and similar preparations from other bacteria. Lowell, G. H., et al, J. Exp. Med . (1988) 167:658-663; Lowell, G. H., et al., Science (1988) 240:800-802; Lynch, E.C., et al., Biophys. J. (1984) 45:104-107; U.S. Pat. No. 5,726,292 issued Mar. 10, 1998; U.S. Pat. No. 4,707,543 issued 17 November 1987. The use of proteosomes for formulation of vaccines has been reviewed by Lowell, G. H., in “New Generation Vaccines” 2nd ed., Marcel Dekker, Inc., New York, Basil, Hong Kong (1997) pages 193-206, the contents of which are incorporated herein by reference. Proteosomes are described as comparable in size to certain viruses which are hydrophobic and safe for human use. Proteosomes are said to be useful in formulating vaccines with a variety of proteins and peptides. The review describes formulation of compositions comprising non-covalent complexes between various antigens and proteosomes which are formed when solubilizing detergent is selectively removed using exhaustive dialysis technology. With respect to the bacterial shigella vaccine, ultrafiltration was reported to be successful. Vaccines wherein the antigens are shigella lipopolysaccharide, Brucella lipopolysaccharide, Staphylococcal enterotoxin B toxoid, human immunodeficiency virus envelope protein, E. coli pilus adhesion proteins, and various peptides such as those derived from rice and influenza virus. These formulations are intended for mucosal application. Parenteral vaccines were also formulated. In particular, peptides derived from influenza (not the entire antigen) were used in vaccine preparation. Levi, R., et al., Vaccine (1995) 13:1353-1359. An additional description of outer membrane vesicles from Meningococcus acting as mucosal adjuvants for influenza virus antigens is described by Dalseg, R., et al., Vaccines (1998) 96:177-182.
[0015] Despite the multiplicity of efforts to formulate successful vaccines, there remains a need for efficient methods and effective compositions to immunize individuals, particularly against infection by influenza.
Disclosure of the Invention
[0016] The present invention describes proteosome-influenza vaccine compositions and processes for their production. These vaccines are straightforward to produce and are able to protect against influenza infection. A preferred embodiment is a nasal proteosome influenza vaccine that contains inactivated influenza antigens, preferably HA, non-covalently formulated with proteosomes formed from the purified outer membrane proteins of gram negative bacteria such as Neisseria meningitides . Although vaccines directed against influenza are exemplified herein, the processes employed are useful generally in preparing vaccines which contain viral protein antigens.
[0017] Thus, in one aspect, the invention is directed to a method to prepare a vaccine composition which method comprises providing a mixture of at least one viral protein antigen with a proteosome preparation in the presence of detergent and removing the detergent from the mixture by ultrafiltration. In preferred embodiments, the proteosome to viral antigen ratio in the mixture is greater than 1:1, preferably greater than 2:1, more preferably greater than 3:1 and more preferably greater than 4:1.
[0018] In other aspects, the invention is directed to vaccines prepared by the foregoing method, and in particular those vaccines where aggregates are formed between the viral antigen, preferably influenza hemagglutinin, and the proteosomes. Preferred size ranges are also described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1 A-C show serum immune responses induced by the invention vaccines.
[0020] [0020]FIG. 2 shows mucosal immune responses induced by these vaccines.
[0021] [0021]FIG. 3 is a graph showing protection of mice immunized with the recombinant form of the invention vaccine.
[0022] [0022]FIG. 4 is a graph showing the shift of immune response induced by split antigen vaccine from a Type 2 response to a balanced Type 1/Type 2 response in mice.
[0023] FIGS. 5 A- 5 F are graphic representations of responses in serum and nasal mucosa to trivalent split influenza vaccines.
[0024] [0024]FIGS. 6A and 6B are graphs showing serum HAI and IgA signal in nasal washes, respectively, from humans immunized with the invention vaccines.
[0025] [0025]FIG. 7 shows a particle size analysis of proteosome-HA vaccine complexes.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Peptide and lipopolysaccharide antigens from a diverse range of pathogenic organisms complexed to proteosomes have been demonstrated to induce enhanced mucosal and systemic immune responses following nasal or parenteral immunization in a variety of animal species. The invention herein describes improved compositions of, and improved processes for production of, proteosome-protein based vaccines as illustrated by vaccines designed to protect against influenza infection. The illustrated proteosome influenza vaccines, at equivalent doses of HA to those in injectable vaccines, induce comparable or enhanced serum virus specific immune responses, whereas the HA-influenza antigen without proteosomes induces significantly lower serum responses. Proteosome-influenza vaccines also generate high levels of specific mucosal nasal and lung IgA, whereas injected or nasal administration of influenza antigen alone induces trivial or very low levels of respiratory mucosal IgA. In addition, proteosome influenza vaccines convert immune responses to influenza antigens from a predominantly Type 2 response to a more balanced Type 1/Type 2 response or a predominant Type 1 response, whereas influenza antigens alone, given mucosally or by injection, elicit predominantly Type 2 responses. Type 1 responses promote the induction of cytotoxic T lymphocytes that are important for the resolution of influenza infections. In the past, Type 1 responses required live virulent or attenuated CAV nasal influenza vaccines. Prior reported ISIV administered either alone, or formulated with Biovector or virosome (with or without rLT), induce preferentially Type 2 immune responses.
[0027] In addition, proteosome nasal flu vaccines have been shown to be extremely well tolerated in mice and humans. No olfactory bulb or other central nervous system (CNS) involvement was seen in GLP mouse studies conducted with proteosome vaccines indicating that proteosome-flu vaccines are demonstrably inherently safer than the enterotoxin-based adjuvanted flu vaccines described above.
[0028] Finally, nasal proteosome influenza vaccine is immunogenic in humans and induces significant increases in serum HAI in healthy adults at a frequency and level not observed in subjects of this age group given CAV. At doses similar to those given by the injectable ISIV vaccines, the proteosome-influenza vaccine induces significant levels of secretory IgA in the nasal washes of humans. Thus, nasal proteosome-influenza vaccine has utility as an inactivated nasal influenza vaccine with immunogenic and safety properties superior to live CAV's and other nasally delivered or adjuvanted inactivated influenza vaccines.
[0029] The demonstration of the foregoing advantages of proteosome formulations with inactivated influenza antigens is typical of proteosomal compositions containing other antigen proteins and such compositions would be similarly effective in protecting against other respiratory or non-respiratory diseases using other viral or non-viral antigens.
[0030] The vaccines and compositions of the invention comprise two major components. The first component is a preparation of proteosomes. The second component is a protein antigen, preferably a viral antigen. Thus, bacterially derived antigens which are protein in nature can be used in the preferred formulations as well as viral antigens. The compositions are illustrated herein by use of a partially or fully purified preparation of influenza virus antigen. The antigen can be purified using detergent extractions and sucrose density gradient centrifugation to contain quantifiable amounts of influenza hemagglutinin (HA). Recombinant influenza proteins such as the hemagglutinin protein (HA) expressed in and purified from cell culture such as baculovirus or mammalian cell lines may also be used. The influenza component is generally referred to as influenza split-product or split-flu (for the antigen purified from natural sources) or recombinant HA (rHA).
[0031] By “proteosomal preparation” is meant an extract of outer membrane protein subjected to purification processes which result in the obtention of hydrophobic particles or vesicles as desired in, for example, U.S. Pat. No. 5,726,292, incorporated herein by reference, or in U.S. Pat. No. 4,707,543. Alternative and improved methods to prepare proteosomes are described in the examples below and illustrated with flowcharts. Any preparation method which results in the outer wall protein component in vesicular form is included within the definition of “proteosomal preparation.”
[0032] The two components are formulated at specific initial ratios by the processes described so as to optimize interaction between the components resulting in non-covalent association of a significant portion of the two components to each other. The processes generally involve the mixing of the components in a selected solution of detergent(s) and then removing the detergent(s) by diafiltration/ultrafiltration methodologies using flow and membrane parameters optimized for the vaccines of the invention.
[0033] One feature of the present invention is that the ratio of proteosomes to antigen contained in the composition is preferably greater than 1:1, more preferably greater than 3:1, more preferably greater than 4:1. The ratio can be as high as 8:1 or higher. The detergent-based solutions of the two components may contain the same detergent or different detergents and more than one detergent may be present in the mixture subjected to ultrafiltration/diafiltration. Suitable detergents include Triton, Empigen, and Mega- 10. Other detergents can also be used. The detergents serve to solubilize the components used to prepare the composition. The use of a mixture of detergents may be particularly advantageous. This mixture is, of course, removed by diafiltration/ultrafiltration prior to final formulation.
[0034] Another feature of the process for preparing the compositions of the invention which may then be formulated into vaccines is that the resultant composition is such that it can be filtered through a 0.8μ filter, a 0.45μ filter or a 0.2 μfilter. This permits sterilization to be performed by filtration, obviating the necessity of adding an antiseptic such as thimerasol. This is highly advantageous as it is desirable to eliminate any complications by virtue of the presence of such contaminants.
[0035] The compositions prepared by the method of the invention are ultimately formulated into vaccines by, if desired, filtration as described above, addition of diluents and carriers, buffers, and the like.
[0036] As will be illustrated below, vaccines wherein HA is the antigen, or indeed vaccines containing any protein antigen, can be made as multivalent vaccines. This can be accomplished in two ways. The initial mixture prior to diafiltration/ultrafiltration may contain a mixture of the desired antigens provided initially as separate components optionally in the presence of different detergents or in the presence of the same detergent; the mixture of antigens is then mixed with the detergent- 0 containing proteosome preparation and processed as described above. Alternatively, the composition obtained after diafiltration from a single (or multiple) antigens can be mixed with similarly prepared preparations from one or more additional antigens. Thus, illustrated below is a trivalent vaccine composed of three different HA antigens.
[0037] In addition to the features of the process for preparing the composition to be formulated into vaccines, the proteosomal composition itself may be prepared by an improved process. Thus, the multiplicity of steps set forth in the prior art may be short circuited, or additional steps or substituted steps may be employed. In one important embodiment, the preparation process involves one or more precipitations in the presence of ethanol as described in the examples below, followed by re-extracting of the proteosomes in 0.1 -1% detergent solutions, typically using Empigen, thus resulting in a more uniform product. In addition, the ammonium sulfate precipitation steps described on the prior art processes may be eliminated, whether or not the ethanol precipitation steps are employed. Thus, the compositions prepared by the method of the invention can be formulated into vaccines that can be delivered by a mucosal (such as nasal, oral, oropharyngeal, or rectal) or parenteral (such as intramuscular or subcutaneous) or transdermal route to induce both serum and mucosal antibodies and immune responses.
[0038] As shown below, nasal vaccine delivered by liquid or spray to mice induces specific anti-influenza immune responses including serum IgG antibodies and hemagglutination inhibition (HAI) antibodies. HAI responses are significant since their induction is known to correlate with protection against influenza in humans. The vaccines also result in mucosal antibodies including IgA in mucosal secretions collected from the nasal cavity or lungs and in switching of predominant Type 2 type responses to balanced or predominant Type 1 responses as measured by IgG1/IgG2a ratios and induction of Th1 cytokines such as interferon gamma without Th2 cytokines such as IL-5. Such responses are predictive of other cellular mediated responses such as development of cytotoxic T cells (CTLs). The ability of a nasal vaccine of the instant invention to elicit these three types of responses indicates that the vaccine can provide a more complete immunity since functional serum antibodies (including HAI antibodies), functional nasopharyngeal and lung IgA antibodies that can neutralize influenza virus and Th1 responses that help provide elimination of residual or intracellular virus are all important mediators of protection against influenza virus infection. This is consistent with the results showing that the vaccines described protect mice against weight loss and death associated with challenge of mice with virulent influenza virus.
[0039] In addition to administration by mucosal routes, such as nasal administration, the vaccines of the invention can also be administered parenterally, for example, by injection (e.g. intramuscularly or s.c.). Intramuscular injection is demonstrated below to provide higher levels of serum antibodies than provided by administering split-flu vaccine without proteosomes.
[0040] As shown below, administration of the vaccines of the invention by the nasal route to mice even using a greater number of immunizations (three) than typical for clinical applications (one or two immunizations) and using doses up to twenty fold, the highest expected human dose was well-tolerated. Importantly there was no evidence of inflammation in the olfactory bulb region of the CNS unlike other enteroxigenic mucosal adjuvants described above.
[0041] As further shown below, in humans, the invention vaccine prepared with split influenza antigen given by nasal spray was well tolerated without any serious adverse effects. At optimal doses the vaccine induced serum HAI responses in more than 50% of volunteers (even in volunteers profoundly seronegative to the influenza strain tested), the majority with titers equivalent or exceeding those that correlate with protection against disease caused by influenza virus. The serum HAI titers were significantly higher than those induced by split antigen alone given intranasally, which induced an HAI response in less than 13% of volunteers. The vaccine also induced nasal wash secretory IgA at levels in significantly more volunteers than, and significantly higher than, that produced following immunization with split vaccine alone given nasally or by injection. The doses of proteosome-flu vaccines that induced mucosal and systemic immune responses in humans (7.5-30 μg) were similar to those of the current injectable vaccines (15 μg) and would not have been predicted. In previous human studies, using proteosome shigella vaccines, to obtain optimal serum and mucosal immune responses following nasal immunization in humans, it was necessary to give the proteosome-shigella vaccines at doses of shigella antigen of 1,000 μg to 1,500 μg (fifty to 100 (50-100) fold higher than the average doses of influenza hemagglutinin antigen used for the proteosome-flu vaccines prepared by the methods of the present invention.
[0042] As set forth above, the invention includes monovalent and multi-valent (including, bi- or tri-valent) vaccines. The multivalent preparation can be obtained by combining individual monovalent proteosome-flu vaccines, or monovalent influenza antigens can be combined together to form a multivalent antigen mixture, then complexed with proteosomes to produce the composition to be formulated as a multi-component proteosome-flu vaccine.
[0043] For parenteral, nasal, oral or suppository use, the vaccine may contain the active ingredients plus potentially large amounts of a variety of excipients or adjuvants including oils, emulsions, nano-emulsions, fats, waxes, buffers, or sugars, as diluents or vehicles customary in the art to provide stable delivery of the product in the desired delivery format.
[0044] As is well-known in the art, a variety of protocols for administering the vaccines of the invention can be employed. The vaccines may be used in an individual protocol comprising several administrations of the vaccines of the invention, or the invention vaccines can be used in combination protocols with other formulations. The selection of antigens is governed by the nature of the infective agent; the design of a particular protocol for administration, including dosage levels and timing of dosing are determined by optimizing such procedures using routine methods well known to the skilled artisan.
[0045] While illustrated for influenza vaccination, vaccines similar to those exemplified but containing other antigens are successful in protecting humans or animals (as in veterinary applications) against viral or microbial diseases or against certain toxins or biologic threat agents or allergies acquired by mucosal routes, i.e., by inhalation, and also by ingestion or sexual transmission. The invention includes preventive or therapeutic vaccines delivered by mucosal or parenteral routes using cell surface or internal protein antigens for vaccines against microbial diseases, allergies or cancer.
[0046] The compositions resulting from the process of the invention are clearly different from the technologies known in the art. For instance, unlike live attenuated cold-adapted vaccines (CAV), the vaccines described herein contain non-living antigens which are purified or recombinant components. The compositions are clearly different from MF59 emulsions, liposome, virosome, monophosphoryl lipid A (MPLA) or Biovector technologies since proteosomes are essentially composed of bacterial outer membrane proteins and contain only trivial or minor amounts of native bacterial lipids, whereas MF59 lipid emulsions, liposomes or virosomes consist of many lipids while MPLA and Biovector technologies are lipid-saccharide entities with small (MPLA) or larger (Biovector) amounts of saccharides. None of these adjuvants contain substantial amounts of proteins (bacterial or otherwise).
[0047] A comparison of the nature and properties of the vaccines of the present invention with those described by Dalseg, R., et al., Vaccines (1998) 96:177-182, cited above, demonstrates the advantages of the present invention. The Dalseg compositions suffer from drawbacks set forth above with respect to attenuated virus; the antigenic component in the Dalseg vaccines is formalin-inactivated whole influenza virus, as opposed to the purified proteins used in the vaccines of the present invention. Vesicles obtained as an extracted outer membrane preparation from Neisseria meningitides by an unspecified method were mixed with formalin-inactivated influenza virus and either sonicated or simply mixed. As no diafiltration or ultrafiltration process is applied to the mixture, detergent present in the composition comprising the vesicles remains in the composition. The compositions thus prepared by Dalseg provide inferior results to those of the vaccines of the present invention; four doses of the Dalseg compositions were required in order to observe the results and the vaccine was not shown to be protective.
[0048] Prior reported compositions utilizing proteosomes as outlined in the review article by Lowell cited above, employed ratios of proteosomes to antigens of 1:1 or less; ratios as low as 1:20 were used. Prior art vaccines therein described showed optimal responses required that optimal responses required antigen doses of up to 1,000 μg or 15,000 μg whereas vaccines of the invention are effective in humans using antigen doses in the 7.5-30 μg range.
[0049] As to the process for preparation per se, it has been shown that it is possible to use a 100,000 molecular weight cutoff in the diafiltration/ultrafiltration procedure thus resulting in enhanced efficiency; similarly more efficient is the possibility to subject several antigens simultaneously in the presence of proteosomes to a one-step diafiltration/ultrafiltration procedure.
[0050] The following examples are intended to illustrate but not to limit the invention.
EXAMPLE 1
Production of Proteosomes
[0051] Outer membrane protein proteosome preparations were purified from Group B type 2 Neisseria meningitides by extraction of phenol-killed bacterial paste with a solution of 6% Empigen BB (EBB) (Albright and Wilson, Whithaven, UK) in 1 M calcium chloride followed by precipitation with ethanol, solubilization in 1% EBB-Tris/EDTA-saline and then precipitation with ammonium sulfate. The precipitates were re-solubilized in the 1% EBB buffer, dialyzed and stored in 0.1% EBB at −70° C. A flow chart of the process (Flowchart 1) is shown on the following pages. Proteosomes may also be prepared by omitting the ammonium sulfate precipitation step to shorten the process (Flowchart 1A). An alternative process that is also successful is shown in Flowchart 1B.
EXAMPLE 2
Preparation of Influenza Antigen (Influenza HA or Flu-HA) Containing Quantified Amounts of Influenza Hemagglutinin (HA)
Split Antigen
[0052] Preparation was performed as outlined in Flowchart 2. Briefly, preparation involved harvesting allantoic fluid from virus inoculated eggs followed by clarification, inactivation of the virus, concentration by diafiltration/ultrafiltration, banding the virus on sucrose gradient density centrifugation, pelleting, extracting the re-suspended pellet with Triton X-100, or NP-40 or other suitable detergent, and centrifuging and collecting the supernatant. This process was repeated as required, analyzed as described in Flowchart 2, pooled and stored at 2-8 degrees C.
Recombinant Baculovirus Expressed Influenza HA
[0053] Briefly, Influenza HA (A/Texas/36/91) was expressed and purified by conventional techniques as described in (Ref Gail Smith, et. al.). The resultant protein was >95% HA as determined by PAGE reducing gels. HA was quantified in the final complex using densitometry and comparing the intensity of the recombinant HA bands in the complex with the intensity of the bands of known concentrations of the recombinant protein.
EXAMPLE 3
Preparation of Proteosome-Influenza HA Vaccine
[0054] Portions of stock influenza split product antigens were complexed to and formulated with proteosomes using diafiltration/ultrafiltration methods described in Flowchart 3 or by using dialysis. For either method, the influenza split product was dissolved in saline buffered solution containing the desired detergent e.g. Empigen BB (EBB) at 1% or, at 0.1%-2% of EBB or other suitable detergent depending on the type of detergent used and was then mixed with proteosomes in the saline buffered 1% Empigen solution (or other appropriate detergent at appropriate concentrations as described above) at various proteosome:HA (wt/wt) ratios ranging from 4:1 to 8:1 including 1:4, 1:1, 2:1, 4:1 and 8:1. To remove Empigen, the mixture was then subjected to ultrafiltration/diafiltration technology as described in the Flowchart 3 or was exhaustively dialyzed across a dialysis membrane with a 10,000 Molecular Weight cut-off (MWCO) or functionally similar membranes with MWCO ranges of 1,000-30,000 against buffered saline for 1-2 weeks at 4° C. exchanging at least 500 parts buffer each day.
[0055] At various steps, single radial immunodiffusion (SRID) was used to measure potency. The halo immunodiffusion technique was used to accurately determine the content of formulate split-flu antigen with proteosomes at various ratios. This methodology is the classical potency assay for split-flu products based on hemagglutinin content for the final vialed materials. Reagents were obtained from National Institute for Biological Standards and Control (NIBSC), Hertfordshire, United Kingdom. Reference: Hudson, L. and Hay, F. C., Practical Immunology , ed. Blackwell Scientific Publication: Third Edition; pages 230-233.
[0056] Multivalent vaccines may be prepared by making individual monovalent proteosome vaccines and then combining them at the required proportions prior to final formulation and fill. Multivalent preparations may also be formulated by pooling individual antigens in the desired proportions and formulating the mixture with proteosomes as outlined in Flowchart 3. Vaccines were passed through membrane filters of 0.8 μm pore size and stored at 4° C. prior to and during the immunizations.
EXAMPLE 4
This Example Describes the Mouse Immunization Protocols Used
[0057] One day prior to the first immunization randomly selected mice were pre-bled. BALB/c mice were immunized intranasally or intramuscularly on days 1 and 21 with antigens in volumes of 25 or 100 μl respectively containing between 0.3 and 10 μg HA A/Taiwan/1/86 or A/Beijing/262/95 as split influenza antigen or A/Texas/36/91 as baculovirus recombinants, alone or formulated with proteosomes (proteosome-flu vaccine or proteosome-rHA) at proteosome:HA ratio's at complex initiation of 1:4, 1:1, 2:1, 4:1 and 8:1 wt/wt. In some examples control mice were given a single intranasal immunization with either phosphate buffered saline or 0.04 LD 50 mouse-adapted live influenza A/Taiwan/12/86 on day 1. Animals were bled on days 20 and 35 via the orbital sinus vein or by cardiac puncture. Nasal and lung lavage samples were taken on day 35. The lungs of each mouse were surgically exposed and a canula inserted in the trachea. Using a syringe containing phosphate buffered saline supplemented with 0.1% bovine serum albumin and protease inhibitors (0.2 mM AEBSF, 1 μg/ml Aprotinin, 3.25 μM Bestatin and 10 μM Leupeptin), 1 nasal lavage sample (approximately 1 ml) and 2 lung lavage samples (2×1 ml) were collected. The lung lavage fluids were combined and lavage fluids from individual animals vortexed and centrifuged to remove cell debris and supernatants stored at −70° C. until assayed by ELISA.
EXAMPLE 5
This Example Describes the Serum Hemagglutination Inhibition Assay (HAI)
[0058] Prior to determination of HAI activity, mouse or human sera were heated at 56° C. to inactivate complement. Elimination of non-specific agglutination was achieved by treating mouse sera with receptor destroying enzyme (RDE). To 0.1 ml of serum was added 0.4 ml of RDE (100 units/ml) for 12 to 18 hr at 37° C. Three hundred ml of sodium citrate (2.5%) was added for 30 min at 56° C. to inactivate the RDE. The sample volume was made up to 1 ml with PBS (to give final sample dilution of 1:10). Two-fold serial dilutions of each sample were tested for their ability to inhibit the agglutination of 0.5% chick red blood cells by A/Taiwan/1/86 virus in a standard HAI assay.
EXAMPLE 6
This Example Describes the Serum ELISA Assay to Measure Specific Anti Flu Antibodies in Sera. in Lung and Nasal Cavity Fluids
[0059] Sera were collected after each immunization; lung and nasal cavity lavage fluids were collected after the last immunization. Nasal wash and lung lavage starting dilutions were 1 in 4 and serum starting dilutions were 1/100. ELISA was performed using whole virus as the detecting antigen. Briefly, 96 well round bottom microtiter plates (Immulon 2, Dynatech, Chantilly, Va.) were coated with antigen and incubated overnight. After aspiration of the antigen using a plate washer, plates were washed once with PBS containing Tween (PBS-T) and incubated with blocking solution containing PBS-T plus plus 2% powdered milk. After aspirating the blocking solution and washing with PBS-T, samples of sera, lung or nasal cavity lavage fluids, serially diluted 2-fold in blocking solution, were added and the plates were incubated for two hours at 37° C. After washing with PBS-T, affinity purified horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or IgA was added and plates were incubated at 37° C. for 30 min. After aspirating and washing twice with PBS-T, developing solution was added and plates were incubated for 15 min at r.t. prior to determining the absorbance values using a microtiter ELISA plate reader (Molecular Devices, Menlo Park, California). Absorbances in the ELISA plate reader were determined at specified times. Antibody titers in the Figures are expressed as ng/ml of specific IgG or IgA determined from a standard curve produced using an ELISA capture assay using affinity purified mouse IgG and IgA standards (Sigma).
EXAMPLE 7
This Example Describes the in Vitro Neutralization Assay to Measure Influenza Virus Neutralizing Antibodies in Serum and Lung Lavage Fluids
[0060] Neutralization of virus infectivity was determined by direct observation of cell lysis and cytopathic effect (CPE) in MDCK cells. The assay was performed in 96-well plates. Each sample was run in octuplicate. Serial dilutions of test samples (sera or lung lavage fluids) were incubated with 100 TCID 50 of live influenza virus homologous to the vaccine strain, incubated for 90 minutes at room temperature and added to 2.4×10 5 MDCK cells/well. Plates were incubated at 32° C./5%CO 2 for the remainder of the assay. Viral neutralization was determined during the virus growth phase (5-7 days of incubation) by evaluation of CPE using an inverted microscope. Neutralizing titers were determined by the Kärber formula (TCID 50 =Δ−δ(S−0.5)) where “Δ” is the log 10 of the dilution with 100% positive cultures, “δ” is the log 10 of the dilution factor and “S” is the sum of positive cultures per dilution including those at dilution with 100% infected cultures.
EXAMPLE 8
Evidence of Enhanced Immunogenicity and Immunity as Measured by Enhanced Serum HAI and Virus Specific IgG Titers Elicited by Proteosome-HA Vaccines
[0061] This example shows the serum and mucosal antibody responses induced by proteosome-flu vaccines following nasal immunization with monovalent vaccines prepared with A/Taiwan/91 influenza split antigen (FIGS. 1 and 2) or purified baculovirus recombinant HA (A/Texas/36/91) (Table 1) by the dialysis method. Similar results were obtained using proteosome-flu vaccines prepared by the scalable diafiltration method (See Example 12 below).
[0062] Anti-influenza IgG antibodies in sera where analyzed by HAI; IgG in sera and IgA antibodies in lung and nasal cavity fluids were analyzed by ELISA; and IgG in serum and IgA and IgG in lung lavage fluids were tested for virus neutralizing activity. The responses were compared to the collections of samples from saline immunized animals and from animals immunized with influenza split product delivered alone without proteosomes or with animals immunized with control vaccines containing proteosomes and an irrelevant antigen (HBsAg). Results are shown and summarized in FIGS. 1 - 2 and Table 1. Briefly: nasal proteosome-flu and proteosome-rHA vaccines at the optimum ratio of proteosomes to HA. The optimal immune responses were obtained for proteosome:HA formulation ratio's between 4:1 and 8:1.
[0063] 1. elicited 6-32-fold higher serum HAI responses than Split Flu alone given nasally and titers that are equivalent to HAI titers elicited by giving the split product HA vaccine alone by injection (FIG. 1A and Table 1),
[0064] 2. elicited up to 250-fold higher Serum IgG responses than Split Flu alone given nasally and elicits responses comparable to nasal live virus or equivalent or up to 5-fold greater than split flu given by injection (i.m.) (FIG. 1B. and Table 1),
[0065] 3. induced serum neutralization titers equivalent to injectable split influenza vaccine and >100-fold greater than split flu antigen alone by the nasal route (FIG. 1C),
[0066] 4. elicited >1,000-fold higher IgA responses in the nasal cavity than Split Flu alone given nasally or by injection (i.m.) (FIG. 2A),
[0067] 5. elicited 20-1000-fold higher specific IgA responses in the lung than Split Flu alone given nasally or by injection (i.m.) (FIG. 2B and Table 1),
[0068] 6. elicited responses equal to or better than live virus (FIGS. 1 - 2 ),
[0069] 7. elicited neutralizing antibodies in the lung fluid secretions. Following nasal immunization only the 4:1 proteosome-flu vaccine induced functional antibodies in lung lavage fluids capable of completely inhibiting the cytopathic effect of the virus in 8/8 replicates at <1 in 2 dilution. No in vitro neutralization was observed for lung lavage fluids from mice immunized with the Flu antigen alone either after nasal or intramuscular immunization, and
[0070] 8. induced enhanced serum IgG and equivalent serum HAI titers compared to split antigen alone after parenteral immunization (Table 2).
TABLE 1 Serum IgG and Mucosal IgA induced by nasal proteosome-rHA vaccine (10 μg HA per dose @ 4:1 Pr:HA ratio) in mice Pr-rHA nasal rHA nasal rHA IM PBS Serum IgG (ng/mL)* 188,956 6,006 43,885 50 HAI (GMT)** 160 20 40 10 Lung IgA (ng/mL)*** 500 20 20 20
[0071] [0071] TABLE 2 Serum IgG and HAI responses Induced by intramuscular proteosome Split flu vaccine (3 μg HA per dose @ 4:1 Pr:HA ratio) in mice Pr-HA 4:1 Pr-HA 1:1 Pr-HA 1:4 HA Serum IgG (ng/mL)* 373,400*** 189,600 155,400 81,110 HAI (GMT)** 320 320 320 320
EXAMPLE 9
This Example Describes the Mouse Immunization Live Virus Challenge Protocols and Results
[0072] To demonstrate vaccine-induced protection against live virus challenge, groups of vaccine immunized and control animals (treated as described in example 4 above with nasal proteosome-flu (A/Taiwan/12/86) vaccine) were challenged on day 36 with specific 4 LD 50 of live mouse-adapted influenza. Mouse protection was assessed by monitoring weight changes in the animals over 14 days following challenge. Mice that lost 30% or more of their starting weight and that showed severe signs of clinical morbidity were sacrificed. Data showing protection elicited by the proteosome flu vaccine are shown and summarized in FIG. 3.
[0073] Briefly, complete protection against significant or lethal weight loss from challenge with virulent homologous virus is shown for the nasal proteo some-flu vaccines prepared at Pr:HA ratio's of between 4:1 and 8:1 whereas the HA without proteosomes showed a significant weight loss during the experiment. Furthermore, the protection induced is equal to that induced by the split flu vaccine alone given by injection. Protection that may be obtained for vaccines formulated at lower Pr:HA ratio's (such as 1:1) even though such formulations induce sub-optimal serum and mucosal immune responses may be due to the inability of the animal protection model to differentiate effectively between formulations prepared at sub-optimal initial formulation ratios.
EXAMPLE 10
[0074] This Example Describes the Shift of Immune Responses from Type 2 to Type 1 by Nasal Proteosome Influenza Vaccines
[0075] The IgG1/IgG2a ratio in mouse serum is a surrogate marker for the type of T cell response induced by a vaccine. Th1(IgG1/IgG2a ratio's<1) correlates with the induction of strong cell mediated immune responses (in addition to serum antibodies); while Th2 (IgG1/IgG2a ratio's >1) predict the induction of strong humoral responses. Levels of murine IgG sub-types, IgG1 and IgG2a were determined in the sera using ELISA assay kits (SBA Clonotyping System/HRP, Southern Biotech Assoc.) following nasal or intramuscular immunization with the proteosome-flu vaccines or flu antigen alone using either monovalent split influenza vaccine or recombinant baculovirus derived HA.
[0076] As shown in FIGS. 4 and Table 3, the IgG1/IgG2a ratio was shifted from 14-20 (for Flu antigen alone) down to the 1-2 range when the vaccine contained proteosomes for both nasal and injected vaccines for split flu antigens; and from 6-60 to 1.7 for the baculo HA antigen. This shift of immunity from a Th2 to Th1 response was confirmed for the recombinant HA antigen by measuring cytokines produced after re-stimulating spleen cells from immunized animals with inactivated purified influenza virus. Briefly, Balb/c mice were euthanized 14 days after the second immunization and the spleens from 5 mice from each group were harvested and cells teased into a single cell suspension using a 100-μm nylon cell strainer (Becton Dickinson, N.J.). Spleen cells were cultured at 2.0×10 6 cells/ml (200 μl/well) in RPMI 1640 medium (Gibco BRL, Life technologies, Burlington, ON) containing 8% fetal bovine serum (heat-inactivated for 1 hr at 56° C.; Gibco BRL), 2 mM glutamine (Gibco BRL), 50 μM 2-mercaptoethanol (Sigma Chemical Co., St-Louis, Mo.) and 50 μg/ml gentamycin (Gibco BRL) with or without UV-inactivated X-113 (A/Texas/36/94 (H1N1) and X-31 (H3N2) reassortant); influenza virus (NIBSC, Hertfordshire, UK) in 96-well cell culture cluster (Corning, N.Y.). Cells were incubated for 72 hrs at 37° C. and supernatants harvested and frozen at −80° C. Murine cytokines levels were measured using sandwich ELISAs (OptEIA set) purchased from Pharmingen (San Diego, Calif.). according to manufacturer's intructions. Recombinant cytokine were used as standards.
TABLE 3 Nasal proteosome baculo HA vaccine shifts the immune response induced by rHA alone from a Type 2 to a balanced Type 1/Type 2 immune response in mice Pr-rHA (IN) rHA (IN) rHA (IM) G1/G2a* INFγ** IL-5** G1/G2a INFγ IL-5 G1/G2a INFγ IL-5 1.7 4432 0 6.1 3769 390.5 60.1 6084 119.2
[0077] As shown in Table 3, the nasal proteosome HA vaccine induced the Th1 cytokine, interferon gamma without the Th2 cytokine IL-5; while the recombinant antigen administered by either the nasal or intramuscular route induced both IL-5 and interferon gamma. These data suggest that the nasal proteosome HA vaccine is creating a cytokine environment that favors the induction of other arms of immunity such as cytotoxic T cells. This may be advantageous since CTL are important for recovery from virus infection by eliminating virus from infected cells and for cross-protection against variant influenza strains.
EXAMPLE 11
Immunogenicity of Trivalent Formulations
[0078] Trivalent proteosome influenza vaccines were prepared using the procedure outlined in Example 3 using detergent split antigens from the A/Beijing/26/95 (H1N1), A/Sydney/05/97 (H3N2) and B/Yamanashi/166/98 sub-types of influenza virus. As shown in FIGS. 5 A-F for proteosome-flu vaccines made with each strain individually and combining them as a trivalent, strain specific serum IgG (FIG. 5A, C and E) and nasal IgA (FIGS. 5B, D and E) responses are enhanced compared to their non-proteosome complexed controls. The immunoglobulin titers induced by the monovalent and trivalent proteosome-flu vaccines are not significantly different. Thus vaccines comprising multivalent influenza antigens induce serum and mucosal immune responses against each component, equivalent to that induced by the individual univalent vaccines.
[0079] Vaccines can also be prepared by combining the desired amounts of each individual antigen into a trivalent antigen pool and subsequently complexing the combined antigen pool to proteosomes to produce a multivalent proteosome-flu vaccine. Evidence for the particle size uniformity and consistency suitable for such a vaccine is shown in example 14 below. Evidence for the potency of such vaccines was found using the standard potency test for influenza vaccines viz. the SRID test described in example 3. Using the SRID test, substantial retention of potent HA was found for each of the three strains used in the multivalent vaccines made at either 8:1, 4:1 or 2:1 proteosome:HA ratios in both unfiltered samples as well as in samples filtered using 0.8 urn or 0.2 um filters. For example, using the 0.8 um filter, at each of three different proteosome:HA ratios (8:1, 4:1 and 2:1), 80% to 86% average retention of HA was found from the three influenza strains, H1N1, H3N2 and B in three trivalent vaccines. These data show that a multivalent vaccine can be made using this methodology.
EXAMPLE 12
Induction of Serum HAI and Nasal Wash sIgA in Humans
[0080] A Phase I dose escalating safety and immunogenicity study was performed in healthy sero-negative adults. Groups of patients (8 to 13 per group) received either 2 nasal doses of 7.5, 15 or 30 μg HA as a GMP grade proteosome-A/Beijing/262/95 vaccine or A/Beijing/262/95 antigen alone at 14 day intervals. HAI GMT were determined as described in Example 5. Secretory IgA specific for the antigen of interest in human nasal wash specimens were measured as follows. Nasal wash specimens were mixed vigorously and then concentrated four to five-fold in centrifugal concentrators with 50 kD cutoff membranes. Total secretory antibody (overwhelmingly dimeric secretory IgA, sIgA) was measured by single radial immunodiffusion in agarose containing antibody to human secretory piece using purified human sIgA standards. Antigen-specific sIgA was detected in a kinetic enzyme-linked immunosorbent assay (KELISA). Microtiter plates were coated with a predetermined concentration of antigen. After washing of the plates, samples of each concentrated nasal wash were placed in triplicate wells at a single dilution (selected in preliminary experiments to yield signals in the dynamic range of the assay for >95% of typical specimens). After incubation, the plates were washed and bound sIgA detected by sequential incubations with biotinylated goat anti-human secretory piece and avidin conjugated with horseradish peroxidase. Following a final wash, TMB substrate was added and optical density at 650 nm measured every 9 seconds for five minutes. A rate of color development (mOD/min) was calculated which, in the presence of excess detection reagents, is directly proportional to the concentration of sIgA bound to antigen. Results for each specimen are normalized to a standard sIgA concentration of 150 μg/mL by the formula:
Normalized KELISA rate=(specimen KELISA rate×150)÷specimen total sIgA conc.
[0081] The resultant normalized rates provide a linear (not geometric as, for example, titers) readout proportional to the amount of antigen-specific sIgA contained in a standard concentration of total sIgA in nasal fluid.
[0082] The proteosome vaccine was well-tolerated at each antigen dose, allowing completion of the full dosing regime. Table 4 and FIG. 6A show the results for the GMT serum HAI titers and FIG. 6B shows nasal wash secretory IgA measurements at 42 days and 0 to 42 days respectively. Briefly, even in this profoundly seronegative population, approximately 50% of subjects had rises in the GMT serum HAI and most had post immunization titers of ≧40 that correlate with protection (Table 4). Furthermore, as shown by the time course of the serum HAI immune responses in FIG. 6A, strong responses were found in sera obtained from subjects immunized with each of the three dose levels (7.5, 15 or 30 μg) on day 14 before the second dose was administered indicating that one dose of vaccine may be sufficient in most individuals.
TABLE 4 Serum A/Beijing/262/95 HAI titers in humans following nasal immunization with proteosome-flu ≧4- fold rises ≧40 HAI titer on or before on or before HAI GMT Treatment Group N day 42 (%) day 42 (%) day 42 15 μg A/Beijing 8 1 (13) 1 (13) 7.7 7.5 μg proteo-flu 8 2 (25) 2 (25) 10.9 15 μg proteo-flu 13 6 (46) 5 (38) 14.5 30 μg proteo-flu 13 7 (54) 6 (46) 21.1
[0083] In addition to serum HAI, the proteosome influenza vaccines induced significant rises in mucosal sIgA (≧2.9 fold) in more than 85% of the total subjects (FIG. 6B) including 75% of those individuals that received the lowest (7.5 μg) dose of vaccine. These data demonstrate the ability of the said invention to induce protective immune responses in humans. These responses are superior to those observed for CAV influenza vaccines in this age group which induced mucosal, but poor serum responses following nasal immunization.
[0084] The doses of proteosome-flu vaccine that give significant immune responses in humans are low and would not have been predicted from previous results where a 67 to 100-fold higher dose of antigen was required for significant systemic and mucosal responses following nasal immunization with proteosome shigella LPS vaccines (Ref. Abstract or manuscript submitted for publication).
EXAMPLE 13
SDS-PAGE Analysis for Proteosome-HA Vaccine Complexes Demonstrate Complexing of Proteosomes to Influenza-HA Antigen
[0085] Uncomplexed proteosomes are insoluble in aqueous systems in the absence of surfactant; complexation with a soluble antigen solubilizes the proteosomes. By centrifuging the sample, the insoluble fraction is separated from the soluble fraction, and the identity of the contents of each is determined by SDS-PAGE. The presence of proteosome proteins in the supernatant with the soluble antigen is evidence of complexing with the antigen since in the absence of detergent or surfactant, the proteosome proteins are not soluble when not complexed by antigen. In order to determine the aggregation-state of an antigen-proteosome complex, a sample of the complex is spun in a centrifuge to pellet precipitated particles that may be present. The supernatant is transferred to another container and the pellet may be washed with TNS buffer. Both the supernatant and the pellet are then analyzed by SDS-PAGE with the non-complexed antigen run on the same gel as a reference. The gel is stained with Coomassie Blue stain, photographed, and re-stained with silver stain to enhanced sensitivity.
[0086] Non-complexed antigens are run as the reference standards. proteosome reference standard and molecular weight markers were: OMP001 reference standard: Mixture of GMP proteosome lots: 0175, 0566, 0621, 0621.
[0087] Molecular weight marker: Broad Range SDS-PAGE Standard
[0088] Proteosome-flu vaccines with complexes containing Pr:HA ratios ranging from 1:4 to 8:1 were made. Vaccines were tested for immunogenicity and for biochemical evidence of complexing as shown by the presence of proteosome proteins in the supernatants of samples centrifuged as described above. The data showed evidence of complexing of the proteosomes with the HA Flu antigen since characteristic bands of proteosome proteins were found in the SDS-PAGE gels in the supernatant with the HA influenza antigen. The presence of proteosomes in the supernatant is evidence of complex formation, since the proteosomes would otherwise be insoluble in the aqueous matrix. Surprisingly, proportionately more proteosomes were found in the supernatant when the preferred embodiment containing higher proteosome to HA ratios e.g. 4:1 (especially) or 8:1 were used whereas less proteosomes were found in the supernatant when lower ratios were used. Clearly, formulation at higher Pr:HA ratios (e.g. 4:1) allowed for more complexing and the lower ratios did not contain dose-limiting amounts of proteosomes that could be successfully complexed with the influenza antigen.
EXAMPLE 14
Particle Size Analysis of Proteosome-HA Vaccine Complexes Demonstrate Complexing of Proteosomes to Influenza-HA Antigen
[0089] Number-weighted log analyzed particle size distributions for various ratios of Pr-HA complex were measured with a Brookhaven Instruments model 90 plus particle size analyzer. As shown in FIG. 8, monovalent and trivalent proteosome-flu vaccines with Pr:HA ratios greater than 1:1 contained particle size distributions that were significantly larger than that of the split flu HA control vaccine without proteosomes. Note that the range of sizes within each vaccine formulation was narrow and characteristic of the parameters of the vaccine formulation. Effective mean sizes may range from ca. 150 to 1,000 nm (with typical bell curve particle size distributions around these means) depending on the proteosome:HA ratio and characteristics of the specific antigen(s), as well as formulation parameters such as the type(s) of detergent(s) or membrane filter size used.
EXAMPLE 15
Demonstration of Complexing by Electron Microscopy
[0090] EM images of labeled proteosome-flu (monovalent A/Beijing) complex were obtained. A transmission electron microscope (TEM) image of the 4:1 Pr-HA vaccine complex, which was then labeled with anti-HA monoclonal antibody and protein A-gold shows that most of the HA is associated with the vesicular structures of the particles or particle aggregates of the complex vaccine. Few labeled sites are not associated with the particles.
[0091] A scanning electron microscope (SEM) image of the 4:1 Pr-HA complex incubated with the anti-HA monoclonal antibody followed by protein A-gold shows evidence of the three-dimensional structure of the vesicles. The apparent brightness of the gold particles is dependent on their orientation in the vesicle—gold particles on the back of the vesicle appear blurred and more faint than those on the front of the vesicle.
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Improved forms of vaccines which comprise proteosomes and protein antigens are described. Vaccines which contain influenza HA as the antigen are used for illustration as to demonstrate efficacy. Improvements in the preparation of the vaccines themselves and the proteosome component are also included.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the technical area of recording physiological and physical data of a patient so that, in particular, monitoring, screening, or diagnosis can be performed. The invention relates in particular to recording the electrical activity of the heart muscle of a patient and the temperature of the patient.
2. Description of Related Art
In the above technical area, numerous instruments are known that take the patient's temperature and, by use of cells placed on the body of a patient, send an electrocardiogram signal, known as ECG, corresponding to the electrical currents produced by heart muscle contractions. A range of instruments have been developed, with the drawback of being fixed and with limited independent operation, requiring data to be acquired at times that do not necessarily respond to a characteristic phase of the heart rhythm. Moreover, data are recorded when the person is under conditions that do not exactly reproduce actual situations in the patient's daily life. It thus appeared that an ambulatory device was needed, adapted to a patient, and designed for picking up both physical and physiological data over a period of several days to several weeks.
One of the drawbacks of ambulatory devices is their inaccuracy, because they cannot follow the instantaneous heart rate. Moreover, these devices have a short power supply life, limiting their utilization time.
SUMMARY OF THE INVENTION
The object of the invention is to remedy the above drawbacks by offering a method for recording the instantaneous heart rate of a patient, as well as the patient's temperature.
To achieve this object, the method according to the invention includes the steps of detecting the peaks of the ECG signal with two integrators having different time constants and connected to a comparator delivering a signal representing the cardiac cycles; recording the times of the cardiac cycles; and recording the temperature as soon as the start of the cardiac cycle is detected.
Another object of the invention is to provide a portable module for recording the instantaneous heart rate and temperature of the patient while also providing high operating autonomy.
According to the invention, the module includes a main energy source connected to a second energy source supplying the temperature sensor by a controlled circuit; a unit that picks up and processes the ECG signal, having an ECG signal filter stage, connected to two integrators with different time constants, the integrator outputs being connected to a comparator delivering a signal representing cardiac cycles; a mechanism for placing the ECG signal pickup and processing unit in operation; and a processor. The processor includes an analog-digital converter designed to receive the analog signal delivered by the temperature sensor; a counter to which the comparator output is connected to determine the times of the cardiac cycles; and a control unit allowing the circuit controlling the operation of the secondary energy source to be controlled and the times of the cardiac cycles and the associated temperature values to be recorded.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other aspects and advantages of the invention will become apparent from the following detailed description of preferred embodiments when taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a block diagram illustrating the ambulatory module according to the invention.
FIG. 2 is a block diagram illustrating a component of the ambulatory module according to the invention.
FIG. 3 is a graph showing an example of curves illustrating the heart rate detection principle according to the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
While the invention will hereinafter be described in connection with preferred embodiments thereof, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents that may be included within the spirit and scope of the invention as defined by the appended claims.
For a general understanding of the features of the invention, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to designate identical elements.
As shown in FIG. 1, portable module 1 is designed to record the heart activity and temperature of a person. Module 1 is designed to be worn by a patient by any appropriate device, (not shown). Portable module 1 is designed to be connected to a known device (not shown) having electrodes placed on the patient so as to deliver an electrical signal produced by the heart muscle contractions. Such an electrocardiogram signal 2 (ECG), is received by a pickup and processing unit 3 which is part of module 1.
According to the invention, module 1 is designed to pick up at least one temperature from the patient simultaneously with the acquisition of the ECG signal. Module 1 is thus designed to be connected to sensors 4 placed such that, for example, one or more skin, esophageal, tympanic, or rectal temperatures can be taken.
Module 1 has a unit for processing and calculating the signals received and is designed around a processor P, for example of the PIC 16C74 type, made by Microchip Technology Inc. Module 1 also has a main power supply A that delivers a voltage of 3.6 V, for example, a memory unit M connected to outlet port D, C of processor P, a clock H connected to a serial port SC1 of the processor, and a transmitting circuit T connected to serial port SC2 of the processor and designed to be connected with a delocalized computer O by means of a link, for example RS232. Module 1 is also provided with a pushbutton PB connected to a waiting unit V of processor P.
Processor P also has a counter B connected to pickup and processing unit 3. As shown in FIG. 2, pickup and processing unit 3 has, at the input, an ECG signal filter stage 5. Filter stage 5 has a passband of preferably between 1 and 24 Hz, and preferably between 5 and 16 Hz. The output of filter stage 5 is connected to a circuit 6 that amplifies the alternating component of the signal. Thus, the output of filter stage 5 is connected by a resistor 6 1 and a capacitor 6 2 connected in series, at the inverting input of an operational amplifier 6 3 whose noninverting input is connected to ground. The inverting input of operational amplifier 6 3 is connected at its output through a circuit composed of a resistor 6 4 and a capacitor 6 5 connected in parallel. The output of operational amplifier 6 3 and hence of stage 6 is connected to two integrators 7 and 8 that have different time constants.
Each integrator 7 and 8 thus has an operational amplifier 7 1 , 8 1 whose noninverting inputs are connected to the output of filter stage 5 and circuit 6. The inverting inputs of amplifiers 7 1 , 8 1 are connected to the cathodes of diodes 7 2 , 8 2 whose anodes are connected to the outputs of the corresponding operational amplifiers. The cathodes of diodes 7 2 , 8 2 are connected to ground through capacitors 7 3 , 8 3 connected in parallel respectively with resistors 7 4 , 7 5 , having time constants τ 1 , τ 2 , respectively, of different values. For example, signal E 1 has a time constant of τ 1 =3 seconds while signal E 2 has a time constant of τ 2 =0.2 seconds. The outputs of integrators 7, 8 drive a comparator 11, that at its output, delivers a square-wave signal E 3 representing the cardiac cycle.
As shown in FIG. 3, pickup and processing unit 3 detects peaks "R" of the ECG signal with two signals E 1 and E 2 that detect the envelope of the signal with different fidelities. Thus, comparator 11 changes from a high level to a low level as soon as signal E 1 , which has the largest time constant, has reached its maximum value. Comparator 11 retains its low state as long as the value of signal E 2 is greater than the value of signal E 1 . As soon as the value of signal E 2 becomes less than the value of signal E 1 , comparator 11 switches from a low level to a high level. Pickup and processing unit 3 thus enables the peaks "R" of the ECG signal to be detected with great fidelity and hence the heart rate of a person to be detected.
Thus, measurement of each elementary period t separating two consecutive heart pulses, combined with counting the number of peaks or pulses, allows the instantaneous heart rate to be determined. The instantaneous heart rate is obtained by summing the elementary periods t, and dividing this sum by the number of cardiac peaks determined. It should be noted that it is possible to determine this instantaneous heart rate by considering a period T corresponding to a number n×t rather than the elementary period t.
As shown in FIG. 1, processor P has an analog-digital converter ADC connected to a multiplexer 15 whose analog inputs are connected to temperature sensors 4. Multiplexer 15 is controlled by a signal delivered for example by output port E of processor P. According to one aspect of the invention, port E controls a secondary energy source 18 designed to power temperature sensors 4 of any known type.
The portable module 1 described above is used as described below, considering that processor P incorporates an appropriate programming unit to operate it according to the invention.
When processor P is not operating, it is considered to be in a sleep mode when secondary energy source 18 does not supply temperature sensors 4. Processor P incorporates a programming unit for monitoring interruptions that might occur via clock H, pushbutton PB, or computer system O. Prior to running a measurement series, module 1 receives operating parameters from computer system O. This module 1 initialization phase defines recording parameters, namely the measurement recording start and end times as well as the definition of the number n of cardiac periods to be taken into account. Where the latter parameter is concerned, the value n can be selected between 1 and 16, with T=n×t and t being the elementary period between two consecutive pulses. Thus the module offers the advantage of choosing the cardiac cycle selection frequency between values 1 and 16. The higher the value of n, the greater the possible recording time for a given measurement storage capacity. However, by the same token, the measurement is coarser because each cardiac period measured corresponds to an average of n heart pulses. In other words, it may be considered that the module measures the instantaneous heart rate corresponding to the actual instantaneous heart rate, or measures the heart rate with a weighting factor n.
Once the parameters have been recorded, module 1 is ready to check the status of pushbutton PB and read the clock to see whether the data recording start time chosen in the initialization phase has been reached. As soon as the recording period start time has been reached or the pushbutton is pressed, processor P terminates the sleep mode and enters a standby mode waiting for an interrupt generated by the counter and corresponding to a low level of the ECG signal. As soon as the ECG signal low level has been detected, processor P reads a clock time H 1 and controls secondary energy source 18 supplying temperature sensors 4. Multiplexer 15 is switched such that it allows selected temperatures to be recorded. As soon as the temperatures have been acquired, secondary energy source 18 is switched so that it no longer supplies sensors 4 with energy. Processor P then enters the sleep mode until there is a further interrupt. As soon as another low level of signal E 3 corresponding to the nth pulse after the pulse previously detected appears, the temperature sensor values are acquired and a clock time H 2 is read. The difference between values H 2 and H 1 is then used to determine the time T of a selected cycle. As explained above, the instantaneous heart rate is obtained by dividing time T by the parameter n selected.
The portable module according to the invention allows the instantaneous heart rate to be detected with great accuracy while offering long autonomous operation. Thus, for example, for n=1, it may be possible to make measurements over a period of two months while selection of a higher value for n would multiply the measuring period by that amount.
While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations may be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth herein are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention as defined in the following claims.
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The invention relates to a portable module for recording the cardiac activity of a person with the aid of a device delivering an ECG signal. The device includes a main energy source connected to a second energy source supplying a temperature sensor by a controlled circuit, a unit that picks up and processes an ECG signal, having an ECG signal filter stage, connected to two integrators with different time constants. The integrator outputs are connected to a comparator delivering a signal (E 3 ) representing cardiac cycles. The device also includes a mechanism for placing the ECG signal pickup and processing unit in operation and a processor.
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FIELD OF THE INVENTION
[0001] The present invention relates to surgical stapling instruments, and in particular to instruments for performing a vascular anastomosis procedure.
BACKGROUND OF THE INVENTION
[0002] The term “anastomosis” covers a variety of procedures in which blood vessels such as veins and arteries, or other tubular members, such as part of the colon, intestines, stomach etc., are joined or reconnected. These vessels may be joined in a variety of relative orientations, including end-to-end and end-to-side and side-to side.
[0003] Recent advances made in the field of microsurgery and beating heart surgery have led to the development of alternatives to conventional suturing processes for joining vessels in order to accommodate the minute size of the vessels and in particular towards achieving a rapid anastomosis during beating heart (off-pump) coronary artery bypass surgery.
[0004] An alternative to suturing is the use of surgical clips which are applied along the junction between the vessels or tissue portions which are to be joined, and the clips perform a holding function similar to that of sutures. Two such non-penetrating clips are shown in U.S. Pat. Nos. 4,586,503 and 4,733,664.
[0005] The former patent discloses a surgical micro clip composed of plastically deformable metal or plastic material having minimal spring back when crimped. The clip has a pair of parallel curved legs joined by a bridge at one end and terminating in round tips at the other end. The clip grips the edges of the everted tissue and joins them by crimping the legs together.
[0006] The latter patent discloses a vascular surgical clip comprising a plastically deformable body portion, a tang for deforming the body, and the neck connecting the tang to the body. The body is designed to deform upon application to the tang of a predetermined tensile force, and the neck is designed to break upon application of a force in excess of the predetermined force to the tang.
[0007] As described in the above patents, the non-penetrating clips are applied over opposed edges of the vessels, the edges being first everted, or turned outward, to form flanges that are gripped between the jaws of the clips. A disadvantage of the above non-penetrating clip is the necessity to apply these clips to the outside of the everted tissues. The anastomosed vessels being repaired need to be returned to the intended function as quickly as possible, particularly where critical blood flow is involved.
[0008] The need therefore, exists for an instrument for rapidly applying surgical staples from either within the lumen or from outside the site of the anastomosis.
SUMMARY OF THE INVENTION
[0009] Accordingly, the present invention provides a surgical stapling instrument for stapling edges of tissue to be joined, the instrument comprising an elongated body and, carried by the body, a rigid member having a hooked end for penetrating the edges of tissue to be joined and stapling means for applying a staple to the edges held by the hooked end of the rigid member.
[0010] The invention also provides a device which accomplishes the same result with folds in tissue, i.e. rather than stapling two edges of tissue, an unbroken area of tissue may be folded and the folds stapled together in the same manner.
[0011] Preferably the stapling means comprises means for driving a staple longitudinally of the body against the inside of the hooked end of the needle for deformation of the staple into penetrating engagement with the everted edges.
[0012] The present invention may be used to perform a variety of vascular anastomosis including peripheral vascular surgical anastomosis, arterial venous fistula formation for dialysis, and coronary artery bypass anastomosis. More particularly, the present invention may be used to perform a coronary artery bypass anastomosis utilising a number of approaches including an open-chest approach (with and without cardiopulmonary bypass), a closed-chest approach under direct viewing and/or indirect thorascopic viewing (with and without cardiopulmonary bypass).
[0013] In an embodiment of the invention the instrument includes an elongated body with a handle at one end (herein referred to as the rear end) and which terminates at the other (front) end in a vascular staple delivery mechanism and a tissue grasping needle having a sharp hooked end. The elongated body portion includes two manually slidable members, the first to extend and retract the needle relative to the front end of the body and the second to deliver a staple which is deformed around an anvil on the inside of the hooked end of the needle. The staple is advanced by a spring biased pusher member coupled to the second slider.
[0014] Upon approximation of one of the tissue walls to be anastomosed by a suitable vascular forceps, the needle is extended so that the sharp hooked end of the needle is advanced free of the front end of the body so that, by manipulation by the user, it can penetrate and hook the tissue wall. When one tissue wall has been hooked, the forceps are used to approximate the other tissue wall which is then also hooked by the extended needle. The needle is configured so that when the tissue wall has been hooked it is inclined to slide back towards the narrow hooked end. The width of the hooked end is optimally equivalent to the combined wall thicknesses of the tissue walls being anastomosed. The needle is then retracted so that the hooked end grasping the tissue walls engages the front end of the body for stability during the subsequent staple delivery.
[0015] Once the tissue to be anastomosed has been grasped and approximated against the front end of the body the pusher member is advanced forwardly along a track in which a staple from a stack of 20 or more is positioned. The pusher member advances the staple along the track until the staple legs engage the inside edge of the hooked end of the needle. As the staple is further advanced the legs are deformed inward and toward each other by the anvil through the hole in the tissue walls created by the needle. Once the staple is deployed the pusher member returns so that its front end is positioned proximal to the staple stack.
[0016] The needle slider is then advanced so as to move the needle and stapled tissue away from the front end of the body to allow the needle to be unhooked from the stapled tissue.
[0017] In a further aspect the invention provides a method of stapling the edges of tissue to be joined, comprising the steps of:
[0018] a) penetrating the edges of tissue to be joined with a rigid member having a hooked end; and
[0019] b) applying a staple to the edges held by the hooked end of the rigid member.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The embodiment of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:
[0021] [0021]FIGS. 1A to 1 C are, respectively, a side view, a top plan view and an opposite side view of an instrument for applying a surgical staple to a blood vessel during a microsurgical anastomosis procedure;
[0022] [0022]FIGS. 2A to 2 C are longitudinal sectional views of the instrument, similar to those of FIGS. 1A to 1 C, with the needle extended in preparation for penetrating and grasping the edges of tissue to be anastomosed;
[0023] [0023]FIGS. 3A to 3 C are longitudinal sectional views of the instrument, similar to those of FIGS. 2A to 2 C, with the needle retracted after having penetrated and grasped the edges of the tissue;
[0024] [0024]FIGS. 4A to 4 C are longitudinal sectional views of the instrument, similar to those of FIGS. 2A to 2 C, showing a staple driven forwardly into the hook of the needle just prior to closing the staple onto the tissue;
[0025] [0025]FIG. 4D is an enlarged detailed view of the circled part of FIG. 4C;
[0026] [0026]FIGS. 5A to 5 C are longitudinal sectional views of the instrument, similar to those of FIGS. 2A to 2 C, just after closure of the staple;
[0027] [0027]FIG. 5D is an enlarged detailed view of the circled part of FIG. 5C;
[0028] [0028]FIG. 5E is an enlarged detailed view of the circled part of FIG. 5D;
[0029] [0029]FIG. 5F is an enlarged cross-section taken on the line A-A of FIG. 5D;
[0030] [0030]FIGS. 6A to 6 C are longitudinal sectional views of the instrument, similar to those of FIGS. 2A to 2 C, with the needle extended once again to release the stapled tissue;
[0031] [0031]FIG. 6D is an enlarged detailed view of the circled part of FIG. 6C;
[0032] [0032]FIG. 7 is a schematic side view of the tip of the instrument during the creation of a pleat in tissue; and
[0033] [0033]FIG. 8 is a sectional side view of the pleat when created by the instrument.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0034] Referring now to the drawings, an instrument for applying a surgical staple to a blood vessel during a microsurgical anastomosis procedure comprises an elongated hollow body 10 having a front “business” end 10 a and a rear handle end 10 b . A needle 12 is mounted within the body 10 and has a straight body portion 12 a and a hooked front end 12 b (hereinafter referred to simply as a hook), the hook 12 b terminating in a sharp outwardly inclined tip 12 c.
[0035] The body portion 12 a of the needle is parallel to the longitudinal axis of the body 10 and is slidable longitudinally of the body 10 between an extended position, FIGS. 2 and 6, wherein the hook 12 b is free of the front end 10 a of the body 10 and a retracted position, FIGS. 1, 3, 4 and 5 , wherein the opposite sides 12 b 1 and 12 b 2 of the hook are engaged with the front end 10 a of the body.
[0036] The needle 12 is actuated between its extended and retracted positions by a thumb-operated slider 14 mounted on the outside of the body 10 and fixed to the rear end of the needle portion 12 a through a longitudinal slot 16 (FIG. 1A) in the body. In its extended position the needle 12 is able to penetrate and evert tissue walls to be anastomosed, while in its retracted position the needle allows a staple to be delivered to the everted tissue walls, as will be described.
[0037] The slider 14 also operates a needle lock 18 via a push rod 20 which is slidable longitudinally of the body 10 in a bearing 22 . The needle lock comprises a U-shaped member 18 which embraces the front end 10 a of the body 10 and whose opposite arms 18 a , 18 b are pivoted to the front end of the push rod 20 for rotation about an axis 24 normal to the axis of the body 10 . The arms 18 a , 18 b also slide in respective bearings 26 which are mounted on opposite sides of the front end 10 a of the body 10 for rotation about an axis 28 parallel to the axis 24 .
[0038] When the needle 12 is fully retracted (FIGS. 1, 3, 4 and 5 ) the base 18 c of the U-shaped member 18 engages in a groove 30 in the front end 10 a of the body 10 just behind the needle tip 12 c.
[0039] This maintains the inside edge of the side 12 b 1 of the hook in alignment with one edge 32 a of a narrow staple guide slot 32 in the body 10 , the straight portion 12 a of the needle extending along the opposite edge 32 b of the guide slot. As will be described, this provides continuous guidance for a staple along the guide slot 32 , out of the front end 10 a of the body 10 and between the opposite sides 12 b 1 and 12 b 2 of the hook fully to the curved base 12 b 3 of the hook.
[0040] When the slider 14 is pushed forwardly to extend the needle 12 , the push rod 20 pushes the pivot axis 24 forwardly so that the U-shaped member 18 rotates and slides in the bearings 26 so that it is lifted out of the groove 30 to allow free forward movement of the tip 12 c of the needle, FIGS. 2 and 6.
[0041] A stack 34 of staples 36 are accommodated in the body 10 , the stack 34 being pressed laterally towards the guide slot 32 by a leaf spring 38 so that the lowermost staple in the stack (as seen in FIG. 2B) is aligned with the staple guide slot 32 with its legs pointing forward (FIG. 2C).
[0042] Staples are contained in a removable cartridge-like housing. When the contents of the cartridge have been exhausted, the empty cartridge is ejected from the device and replaced with a new cartridge pre-loaded with the desired quantity of staples.
[0043] A staple pusher 40 is slidable in the guide slot 32 behind the staple 36 , so that, when the needle 12 is fully retracted, by sliding the pusher 40 forwardly the staple 36 currently aligned with the slot 32 is pushed forwardly along the slot, toward the forward end 10 a of the body 10 , between the opposite sides 12 b 1 and 12 b 2 of the hook 12 b and finally up against the curved base 12 b 3 of the hook. The pusher 40 is actuated by a further thumb-operated slider 42 mounted on the outside of the body 10 and fixed to the rear end of the pusher 40 through a further longitudinal slot 44 (FIG. 1C) in the body.
[0044] The slider 42 is coupled to the rear end 10 b of the body 10 by a tension spring 46 which biases the pusher 40 towards the rear end 10 b . Therefore, the user has to push against the bias of the spring 46 when advancing the pusher 40 . However, a ratchet 48 engaged by a ratchet spring 50 fixed to the slider 42 ensures that the pusher 40 cannot inadvertently return towards the rear end 10 b of the body 10 until a full forward stroke of the pusher 40 has been completed, at which point the ratchet spring disengages from the front end 48 a of the ratchet 48 (FIG. 5B) to allow return of the pusher.
[0045] Except at the curved base 12 b 3 of the hook 12 b the needle 12 has a generally C-shaped cross-section along its full length. This defines a channel 52 along the inside edge of the needle 12 . When the needle 12 is fully retracted and a staple 36 is pushed forwardly by the pusher 40 as described, within the body 10 the staple is guided towards the hook 12 b by sliding along the slot 32 with one leg of the staple engaging in the channel 52 in the straight portion 12 a of the needle and the other leg of the staple engaging the edge 32 a of the slot. When the staple 36 leaves the front end 10 a of the body 10 the leg previously engaging the edge 32 a of the slot 32 now enters and slides along the channel 52 in the side 12 b 1 of the hook which is held in alignment with the edge 32 a by the needle lock 18 . At the same time the other leg of the staple 36 continues along the channel 52 in the side 12 b 2 of the hook (FIGS. 5E and 5F).
[0046] At the curved base 12 b 3 of the hook 12 b the inside edge of the needle has an anvil bump 54 , FIG. 5E. As a staple 36 is driven up against the base 12 b 3 of the hook by the pusher 40 , the legs of the staple are deformed so that they close to penetrate the everted tissue walls held by the hook 12 b (FIG. 5D).
[0047] In use of the instrument, one of the tissue walls 56 to be anastomosed is grasped by a suitable vascular forceps. Then the needle 12 is extended so that the needle lock 18 is rotated out of the groove 30 and the hook 12 b is advanced free of the front end 10 a of the body 10 (FIG. 2) so that, by manipulation by the user, it can penetrate and hook the tissue wall 56 . When one tissue wall has been hooked, the forceps are used to grasp the other tissue wall 58 which is then also hooked by the extended needle.
[0048] The needle is manipulated so that the hooked tissue flaps slide toward the curved base. The needle 12 is then retracted so that the hook 12 b engages the front end 10 a of the body 10 and the needle lock 18 rotates back into the groove 30 , FIG. 3. It will be noted that retraction of the needle automatically everts the tissue walls 56 , 58 . The front end 10 a of the body 10 has a V-shaped slot 60 which guides the side 12 b 1 of the hook to its final position in alignment with the edge 32 a of the slot 32 .
[0049] Now the pusher 40 is advanced forwardly to drive the lowermost staple 36 in the stack 34 along the track 32 until the staple legs engage the channel 52 in the inside edges of the opposite sides 12 b 1 and 12 b 2 of the hook 12 b , FIG. 4. As the staple is further advanced its legs are deformed inward and toward each other by the anvil bump 54 so that the legs of the staple pass through the holes in the tissue walls 56 , 58 created by the needle 12 , FIG. 5. Once the staple is deployed the pusher 40 returns so that its front end is once more positioned behind the staple stack 34 ready for a future deployment.
[0050] The needle slider 14 is then advanced so as to move the needle hook 12 b and stapled tissue away from the front end 10 of the body 10 to allow the needle 12 to be unhooked from the stapled tissue, FIG. 6.
[0051] The staple is made from a biocompatible material such as titanium or stainless steel. Specialist materials such as nitinol (memory metal) may also be used. Typically the material used will be ductile, easily formed, and will have minimum spring back. Preferably, the staple will be generally U-shaped with a curved base and straight sides, the sides being angled outward with respect to its centre-line. When loaded in the cartridge, the legs are compressed inwards until approximately parallel with the centre-line. This outward bias on the legs ensures they remain stacked tightly in position within the cartridge and prevents inadvertent forward movement of the staple when advancing along guide slot 32 .
[0052] While the staple legs are preferably pointed as shown, pointed ends are not necessarily required as the tissue grasping needle will already have punctured the tissue when the staple is deployed.
[0053] In another embodiment an adjustment feature is added to the device which allows the user to vary the forward movement of the staple pusher 40 . it can be seen that advancing the pusher beyond its normal stop will close the staple further. This has advantage where the user finds that the factory setting is insufficient to form a tight anastomosis. The device can then be adjusted to allow the staple pusher 40 advance further thereby closing the staple more tightly and providing a better quality anastomosis.
[0054] In another application the device may be used to create folds or pleats in tissue. An example of this is the creation of folds at the gastro-oesophagal junction as a possible cure of gastro-oesophagal reflux disease (GERD). In this instance, as illustrated in FIG. 7, the needle 12 is displaced forward from the front end 10 a of the stapler and is used to penetrate a pair of convex tissue folds 70 defining a concave fold 72 between them. A staple 36 is then applied onto the needle 12 in the manner described previously, and the staple deformed as shown in FIG. 8 to capture the concave fold 72 .
[0055] The invention is not limited to the embodiment described herein which may be modified or varied without departing from the scope of the invention.
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A surgical stapling instrument for performing a vascular anastomosis procedure comprises an elongated body 10 and a needle 12 having a hook 12 b for penetrating and everting the edges 56, 58 of tissue to be joined. The needle 12 is slidable in the body 10 between an extended position as shown and a retracted position wherein the hook is engaged with the end 10 a of the body. A stapling mechanism includes a slidable pusher 40 for driving a staple 36 longitudinally of the body 10 against the inside of the hook 12 b for deformation of the staple into penetrating engagement with the everted tissue edges 56, 58.
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PRIORITY TO RELATED APPLICATION(S)
This application claims the benefit of U.S. Provisional Application No. 61/557,455, filed Nov. 9, 2011, which is hereby incorporated by reference in its entirety.
RELATED APPLICATIONS
This application is related to U.S. application Ser. Nos. 12/614,485, filed Nov. 9, 2009; 12/614,478, filed Nov. 9, 2009; and 12/614,497, filed Nov. 9, 2009. The entire contents of these applications are expressly incorporated by reference herein.
BACKGROUND OF THE INVENTION
The present invention relates to novel substituted naphthalene-2-yl acetic acids, their manufacture, pharmaceutical compositions containing them and their use as CRTH2 antagonists, partial agonists, inverse agonists or partial inverse agonists.
Prostaglandin D 2 (PGD2) is the major prostanoid produced by activated mast cells and has been implicated in the pathogenesis of allergic diseases such as allergic asthma and atopic dermatitis. Chemoattractant Receptor-homologous molecule expressed on T-helper type cells (CRTH2) is one of the prostaglandin D 2 receptors and is expressed on the effector cells involved in allergic inflammation such as T helper type 2 (Th2) cells, eosinophils, and basophils (Nagata et al., FEBS Lett 459: 195-199, 1999). It has been shown to mediate PGD2-stimulated chemotaxis of Th2 cells, eosinophils, and basophils (Hirai et al., J Exp Med 193: 255-261, 2001). Moreover, CRTH2 mediates the respiratory burst and degranulation of eosinophils (Gervais et al., J Allergy Clin Immunol 108: 982-988, 2001), induces the production of proinflammatory cytokines in Th2 cells (Xue et al., J Immunol 175: 6531-6536), and enhances the release of histamine from basophils (Yoshimura-Uchiyama et al., Clin Exp Aller 34:1283-1290). Sequence variants of the gene encoding CRTH2, which differentially influence its mRNA stability, are shown to be associated with asthma (Huang et al., Hum Mol Genet. 13, 2691-2697, 2004). Increased numbers of circulating T cells expressing CRTH2 have also been correlated with severity of atopic dermatitis (Cosmi et al., Eur J Immunol 30, 2972-2979, 2000). These findings suggest that CRTH2 plays a proinflammatory role in allergic diseases. Therefore, antagonists of CRTH2 are believed to be useful for treating disorders such as asthma, allergic inflammation, COPD, allergic rhinitis, and atopic dermatitis.
SUMMARY OF THE INVENTION
The invention provides a compound of formula (I):
wherein:
R1 is halogen;
R2 is lower alkyl; and
R3 is cycloalkyl, unsubstituted phenyl or phenyl substituted with halogen,
or a pharmaceutically acceptable salt thereof.
The invention also provides for pharmaceutical compositions comprising the compounds, methods of using the compounds and methods of preparing the compounds.
All documents cited to or relied upon below are expressly incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise indicated, the following specific terms and phrases used in the description and claims are defined as follows:
The term “moiety” refers to an atom or group of chemically bonded atoms that is attached to another atom or molecule by one or more chemical bonds thereby forming part of a molecule. For example, the variables R 1 -R 7 of formula I refer to moieties that are attached to the core structure of formula I by a covalent bond.
In reference to a particular moiety with one or more hydrogen atoms, the term “substituted” refers to the fact that at least one of the hydrogen atoms of that moiety is replaced by another substituent or moiety. For example, the term “lower alkyl substituted by halogen” refers to the fact that one or more hydrogen atoms of a lower alkyl (as defined below) is replaced by one or more halogen atoms (e.g., trifluoromethyl, difluoromethyl, fluoromethyl, chloromethyl, etc.).
The term “optionally substituted” refers to the fact that one or more hydrogen atoms of a moiety (with one or more hydrogen atoms) can be, but does not necessarily have to be, substituted with another substituent.
The term “alkyl” refers to an aliphatic straight-chain or branched-chain saturated hydrocarbon moiety having 1 to 20 carbon atoms. In particular embodiments the alkyl has 1 to 10 carbon atoms.
The term “lower alkyl” refers to an alkyl moiety having 1 to 7 carbon atoms. In particular embodiments the lower alkyl has 1 to 4 carbon atoms and in other particular embodiments the lower alkyl has 1 to 3 carbon atoms. Examples of lower alkyls include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.
“Aryl” means a monovalent cyclic aromatic hydrocarbon moiety having a mono-, bi- or tricyclic aromatic ring. The aryl group can be optionally substituted as defined herein. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, phenanthryl, fluorenyl, indenyl, pentalenyl, azulenyl, oxydiphenyl, biphenyl, methylenediphenyl, aminodiphenyl, diphenylsulfidyl, diphenylsulfonyl, diphenylisopropylidenyl, benzodioxanyl, benzofuranyl, benzodioxylyl, benzopyranyl, benzoxazinyl, benzoxazinonyl, benzopiperadinyl, benzopiperazinyl, benzopyrrolidinyl, benzomorpholinyl, methylenedioxyphenyl, ethylenedioxyphenyl, and the like, including partially hydrogenated derivatives thereof, each being optionally substituted.
The terms “halo”, “halogen” and “halide”, which may be used interchangeably, refer to a substituent fluoro, chloro, bromo, or iodo.
“Cycloalkyl” means a monovalent saturated carbocyclic moiety having mono- or bicyclic rings. The cycloalkyl moiety can optionally be substituted with one or more substituents. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, including partially unsaturated (cycloalkenyl) derivatives thereof.
The term “halogen” refers to a moiety of fluoro, chloro, bromo or iodo.
Unless otherwise indicated, the term “hydrogen” or “hydro” refers to the moiety of a hydrogen atom (—H) and not H 2 .
Unless otherwise indicated, the term “a compound of the formula” or “a compound of formula” or “compounds of the formula” or “compounds of formula” refers to any compound selected from the genus of compounds as defined by the formula (Including any pharmaceutically acceptable salt or ester of any such compound If not otherwise noted).
The term “pharmaceutically acceptable salts” refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. Salts may be formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, preferably hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, salicylic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, N-acetylcystein and the like. In addition, salts may be prepared by the addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, and magnesium salts and the like. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins and the like.
The compounds of the present invention can be present in the form of pharmaceutically acceptable salts. The compounds of the present invention can also be present in the form of pharmaceutically acceptable esters (i.e., the methyl and ethyl esters of the acids of formula Ito be used as prodrugs). The compounds of the present invention can also be solvated, i.e. hydrated. The solvation can be effected in the course of the manufacturing process or can take place i.e. as a consequence of hygroscopic properties of an initially anhydrous compound of formula I (hydration).
Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers.” Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Diastereomers are stereoisomers with opposite configuration at one or more chiral centers which are not enantiomers. Stereoisomers bearing one or more asymmetric centers that are non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center or centers and is described by the R- and S-sequencing rules of Cahn, Ingold and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.
The term “a therapeutically effective amount” of a compound means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art. The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 0.1 mg to about 5,000 mg, 1 mg to about 1,000 mg, or 1 mg to 100 mg may be appropriate, although the lower and upper limits may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion.
The term “pharmaceutically acceptable carrier” is intended to include any and all material compatible with pharmaceutical administration including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other materials and compounds compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Useful pharmaceutical carriers for the preparation of the compositions hereof, can be solids, liquids or gases; thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g. binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, aerosols, and the like. The carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic with the blood) for injectable solutions. For example, formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers and the like. Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for proper administration to the recipient.
In the practice of the method of the present invention, an effective amount of any one of the compounds of this invention or a combination of any of the compounds of this invention or a pharmaceutically acceptable salt or ester thereof, is administered via any of the usual and acceptable methods known in the art, either singly or in combination. The compounds or compositions can thus be administered orally (e.g., buccal cavity), sublingually, parenterally (e.g., intramuscularly, intravenously, or subcutaneously), rectally (e.g., by suppositories or washings), transdermally (e.g., skin electroporation) or by inhalation (e.g., by aerosol), and in the form of solid, liquid or gaseous dosages, including tablets and suspensions. The administration can be conducted in a single unit dosage form with continuous therapy or in a single dose therapy ad libitum. The therapeutic composition can also be in the form of an oil emulsion or dispersion in conjunction with a lipophilic salt such as pamoic acid, or in the form of a biodegradable sustained-release composition for subcutaneous or intramuscular administration.
In detail, the present invention provides for compounds of formula (I):
wherein:
R1 is halogen;
R2 is lower alkyl; and
R3 is cycloalkyl, unsubstituted phenyl or phenyl substituted with halogen,
or a pharmaceutically acceptable salt thereof.
In another embodiment, the invention provides for a compound according to formula (I), wherein R1 is fluorine.
In another embodiment, the invention provides for a compound according to formula (I), wherein R2 is methyl or ethyl.
In another embodiment, the invention provides for a compound according to formula (I), wherein R2 is methyl.
In another embodiment, the invention provides for a compound according to formula (I), wherein R3 is cyclohexyl.
In another embodiment, the invention provides for a compound according to formula (I), wherein R3 is chloro-phenyl.
In another embodiment, the invention provides for a compound according to formula (I), wherein the compound is:
{4-[3-(2-Chloro-benzenesulfonylamino)-1-methylene-propyl]-6-fluoro-3-methyl-naphthalen-2-yl}-acetic acid, or [4-(3-Cyclohexanesulfonylamino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid.
In another embodiment, the invention provides for a pharmaceutical composition, comprising a therapeutically effective amount of a compound according to formula (I) and a pharmaceutically acceptable carrier.
In another embodiment, the invention provides for a compound according to formula (I) for use as a therapeutically active substance.
In another embodiment, the invention provides for the use of a compound according to formula (I) for the treatment or prophylaxis of a respiratory disorder.
In another embodiment, the invention provides for the use of a compound according to formula (I) for the preparation of a medicament for the treatment or prophylaxis of a respiratory disorder.
In another embodiment, the invention provides for a compound according to formula (I) for the treatment or prophylaxis of a respiratory disorder.
In another embodiment, the invention provides for a method for treating a respiratory disorder selected from chronic obstructive pulmonary disorder (COPD), asthma, and bronchospasm, comprising the step of administering a therapeutically effective amount of a compound according to formula (I) to a subject in need thereof.
In another embodiment, provided is an invention as hereinbefore described.
The starting materials and reagents used in preparing these compounds generally are either available from commercial suppliers, such as Aldrich Chemical Co., or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis ; Wiley & Sons: New York, 1991, Volumes 1-15 ; Rodd's Chemistry of Carbon Compounds , Elsevier Science Publishers, 1989, Volumes 1-5 and Supplementals; and Organic Reactions , Wiley & Sons: New York, 1991, Volumes 1-40.
The following synthetic reaction schemes are merely illustrative of some methods by which the compounds of the present invention can be synthesized, and various modifications to these synthetic reaction schemes can be made and will be suggested to one skilled in the art having referred to the disclosure contained in this Application.
The starting materials and the intermediates of the synthetic reaction schemes can be isolated and purified if desired using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including physical constants and spectral data.
Unless specified to the contrary, the reactions described herein preferably are conducted under an inert atmosphere at atmospheric pressure at a reaction temperature range of from about −78° C. to about 150° C., more preferably from about 0° C. to about 125° C., and most preferably and conveniently at about room (or ambient) temperature, e.g., about 20° C.
Starting with 3-iodo-pyrrolidine-1-carboxylic acid tert-butyl ester II, reaction with the naphthalene intermediate III affords compound IV. Deprotection of the tert-butyl carbamate generates the amine intermediate V. Reaction of V with sulfonyl chlorides of type VI furnishes compounds of type VII. Hydrolysis of the methyl ester affords the compounds of interest I. R1 can be, for example, halogen; R2 can be, for example, lower alkyl; and R3 can be, for example cycloalkyl, unsubstituted phenyl or phenyl substituted with halogen.
The reaction of 3-iodo-pyrrolidine-1-carboxylic acid tert-butyl ester II with the naphthalene intermediate III to form compound IV can be accomplished by first treating compound II with zinc dust and lithium chloride that was activated with 1,2-dibromoethane and chlorotrimethylsilane. This process can be carried out in an inert solvent such as tetrahydrofuran (THF) at temperatures between room temperature and 60° C. for several hours. The reagent thus formed can undergo a coupling reaction with intermediate III in the presence of a palladium catalyst such as palladium(II) acetate and a suitable phosphine ligand such as 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (S-Phos). The reaction can be carried out in an inert solvent such as tetrahydrofuran (THF) at temperatures between 55° C. and 65° C. for reaction times ranging from 5 hours to 65 hours.
Deprotection of the tert-butyl carbamate group in IV to afford amine V can be carried out in the presence of an acid such as trifluoroacetic acid in a suitable solvent such as methylene chloride at room temperature for reaction times ranging from one to several hours (Lundt, B. F.; Johansen, N. L.; Volund, A.; Markussen, J. International Journal of Peptide and Protein Research, 12 (1978) 258).
The reaction of amine V with sulphonyl chlorides of type VI to provide compounds of type VII can be accomplished using methods that are well known to someone skilled in the art. For example, the reaction can be carried out in the presence of an amine base such as N,N-diisopropylethylamine in a suitable solvent such as tetrahydrofuran at temperatures between 0° C. and room temperature for reaction times ranging from two hours to 24 hours.
Hydrolysis of the methyl ester in VII to give the compounds of interest I can be readily accomplished using methods that are well known to someone skilled in the art. For example, the reactions can be carried out in the presence of an aqueous solution of base such as lithium hydroxide, in an inert solvent such as tetrahydrofuran, water, or mixtures thereof, at a temperature between room temperature and reflux temperature for several hours.
The 3-iodo-pyrrolidine-1-carboxylic acid tert-butyl ester intermediate II can be prepared according to Scheme 2.
Starting with 3-hydroxy-pyrrolidine-1-carboxylic acid tert-butyl ester VIII, conversion of the hydroxyl group to an iodide affords 3-iodo-pyrrolidine-1-carboxylic acid tert-butyl ester II. This conversion can be carried out in the presence of iodine, triphenylphosphine, and imidazole in an inert solvent such as methylene chloride at room temperature for several hours (US 2010/0160280 A1).
The naphthalene intermediate III can be prepared according to Scheme 3 (see U.S. application Ser. Nos. 12/614,485; 12/614,478; and 12/614,497).
Starting with benzaldehydes 1× and succinate esters X, a Stobbe condensation reaction followed by basic hydrolysis of the Stobbe hemi-ester affords the dicarboxylic acids XI. Cyclization of compounds XI gives 4-hydroxy-naphthalene carboxylic acid intermediates XII. Esterification of the carboxylic acid in XII generates the 4-hydroxy-naphthalene carboxylic acid esters XIII. Protection of the hydroxyl group in XIII as a benzyl ether gives 4-benzyloxy-naphthalene carboxylic acid esters XIV. Reduction of the ester group in XIV affords naphthalen-2-yl methanol compounds XV, which can be transformed to chloromethyl substituted naphthalenes XVI. A carbonylation reaction of XVI in the presence of methanol gives 4-benzyloxy-naphthylacetic acid esters XVII. Deprotection of the benzyl ether in XVII affords 4-hydroxy-naphthylacetic acid esters XVIII which can undergo sulfonylation to afford the trifluoromethanesulfonate intermediates III.
Stobbe condensation reactions of IX with succinate esters X to give intermediates XI can be carried out using a base such as sodium methoxide, sodium ethoxide, potassium tert-butoxide, or sodium hydride in a suitable solvent such as methanol, ethanol, tert-butanol, toluene, benzene, or mixtures thereof, at temperatures between room temperature and 80° C. for one hour to several hours (Bloomer, J. L.; Stagliano, K. W.; Gazzillo, J. A. J. Org. Chem. 58 (1993) 7906). The resulting hemi-ester intermediates can readily undergo hydrolysis to afford the dicarboxylic acid intermediates XI. This reaction can be carried out in the presence of an aqueous solution of base such as sodium hydroxide or lithium hydroxide, in an inert solvent such as toluene, water, or mixtures thereof, at a temperature between room temperature and the reflux temperature for several hours.
Cyclization of the dicarboxylic acids XI to form 4-hydroxy-naphthalene carboxylic acids XII can be accomplished in neat trifluoromethanesulfonic acid at room temperature over several hours (Hong, W. P.; Lim, H. N.; Park, H. W.; Lee, K.-J. Bull. Korean Chem. Soc. 26 (2005) 655).
Intermediates XII can be readily converted to the 4-hydroxy-naphthalene carboxylic acid ester intermediates XIII in the presence of a catalytic amount of concentrated sulfuric acid and an alcohol solvent such as methanol or ethanol at temperatures between room temperature and 80° C. (or the reflux temperature) for several hours. Alternatively, the esterification reaction can be carried out in the presence of thionyl chloride and a suitable alcohol solvent such as methanol or ethanol at temperatures between 65° C. and 80° C. (or the reflux temperature) for several hours.
Preparation of intermediates XIV can be accomplished by treating XIII with benzyl chloride or benzyl bromide in the presence of a base such as potassium carbonate, sodium carbonate, or cesium carbonate. This reaction may occur in an inert organic solvent such as acetone, acetonitrile, or N,N-dimethylformamide at a temperature between room temperature and 80° C. for several hours.
Reduction of the ester group in XIV with lithium aluminum hydride gives the naphthalen-2-yl methanol compounds XV. This reaction can be carried out in an inert organic solvent such as tetrahydrofuran, diethyl ether, toluene, or mixtures thereof, at a temperature between 0° C. and 80° C. for several hours.
The chloromethyl naphthalene intermediates XVI can be prepared by the reaction of compounds XV with carbon tetrachloride and triphenylphosphine in an inert organic solvent such as toluene, acetonitrile, dichloromethane, N,N-dimethylformamide, or tetrahydrofuran at a temperature between 0° C. and 120° C. (or the reflux temperature) for several hours. Alternatively, the chlorination reaction may be accomplished using thionyl chloride either neat or in a suitable solvent such as dichloromethane, chloroform, N,N-dimethylformamide, benzene, or toluene at temperatures between 0° C. and 80° C. (or the reflux temperature) for several hours.
Conversion of chlorides XVI to the naphthylacetic acid esters XVII can be accomplished by a palladium catalyzed carbonylation reaction under one atmosphere of carbon monoxide in the presence of a base such as potassium carbonate in methanol and in the presence or absence of a co-solvent such as tetrahydrofuran. This transformation can be carried out using a palladium catalyst such as bis(triphenylphosphine)dichloropalladium(II) at a temperature between room temperature and 90° C. for 10 minutes to several hours (Schoenberg, A.; Bartoletti, I.; Heck, R. F. J. Org. Chem. 39 (1974) 3318).
Removal of the benzyl protecting group in XVII through catalytic hydrogenolysis affords the 4-hydroxy-naphthylacetic acid esters XVIII. This reaction can be carried out under one atmosphere of hydrogen in the presence of a catalyst such as 10% palladium on carbon or 20% palladium hydroxide on carbon in a solvent such as methanol or ethanol at room temperature for several hours. Alternatively, the benzyl ether can be removed in the presence of boron trifluoride diethyl etherate. This reaction can be performed in acetonitrile using sodium iodide as an additive at temperatures between 0° C. to room temperature for reaction times between one hour to several hours (Vankar, Y. D.; Rao, T. J. Chem. Research ( S ) (1985) 232).
Compounds XVIII can be converted to the trifluoromethanesulfonate esters III through a reaction with trifluoromethanesulfonic anhydride in the presence of an amine base such as pyridine, triethylamine, or diisopropylethylamine and in the presence or absence of an inert solvent such as dichloromethane for several hours at temperatures between 0° C. and room temperature.
EXAMPLES
Although certain exemplary embodiments are depicted and described herein, they should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof. The compounds of the present invention can be prepared using appropriate starting materials according to the methods described generally herein and/or by methods available to one of ordinary skill in the art.
Intermediates and final compounds were purified by either flash chromatography and/or by reverse-phase preparative HPLC (high performance liquid chromatography). Unless otherwise noted, flash chromatography was performed using (1) the Biotage SP1™ system and the Quad 12/25 Cartridge module (from Biotage AB), (2) the ISCO CombiFlash® chromatography instrument (from Teledyne Isco, Inc.), or (3) an Analogix® IntelliFlash280™ chromatography instrument (from Analogix Inc., a subsidiary of Varian Inc.). Unless otherwise noted, the silica gel brand and pore size utilized were: (1) KP-SIL™ 60 Å, particle size: 40-60 micron (from Biotage AB); (2) Silica Gel CAS registry No: 63231-67-4, particle size: 47-60 micron; or (3) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore size: 200-300 mesh or 300-400 mesh.
Mass spectrometry (MS) or high resolution mass spectrometry (HRMS) was performed using a Waters® ZQ™ 4000 (from Waters Corporation), a Waters® Quattro micro™ API (from Waters Corporation), a Micromass® Platform II (from Micromass, a division of Waters Corporation), a Bruker® Apex®II FTICR with a 4.7 Tesla magnet (from Bruker Corporation), a Waters® Alliance® 2795-ZQ™2000 (from Waters Corporation), or an MDS Sciex™ API-2000TMn API (from MDS Inc.). Mass spectra data generally only indicate the parent ions unless otherwise stated. MS or HRMS data is provided for a particular intermediate or compound where indicated.
Nuclear magnetic resonance spectroscopy (NMR) was performed using a Varian® Mercury300 NMR spectrometer (for the 1 H NMR spectra acquired at 300 MHz), a Varian® Inova400 NMR spectrometer, a Bruker® 300 MHz NMR spectrometer, or a Bruker® 400 MHz NMR spectrometer. 1 H NMR data is provided for a particular intermediate or compound where indicated.
All reactions involving air-sensitive reagents were performed under an inert atmosphere. Reagents were used as received from commercial suppliers unless otherwise noted.
Part I
Preparation of Starting Materials and Intermediates
Preparation of 3-iodo-pyrrolidine-1-carboxylic acid tert-butyl ester (Intermediate II)
A round bottom flask was charged with 3-hydroxy-pyrrolidine-1-carboxylic acid tert-butyl ester (5.0 g, 0.027 mol), triphenylphosphine (10.5 g, 0.0401 mol), iodine (10.16 g, 0.0401 mol), imidazole (2.72 g, 0.0401 mol), and methylene chloride (90 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the collected solids were washed with methylene chloride. The combined organic layers were concentrated. The resulting crude product was dissolved in ethyl acetate, and the organic phase was washed with water. The ethyl acetate layer was washed with a 3:1 mixture of water and methanol to remove triphenylphosphine oxide. The ethyl acetate layer was then washed with brine, dried over anhydrous MgSO 4 , filtered, and concentrated. The crude product was loaded onto a 330 gram silica gel column. Flash chromatography afforded 3-iodo-pyrrolidine-1-carboxylic acid tert-butyl ester (6.9 g, 87%). 1 H NMR (400 MHz, CDCl 3 ) δ ppm 4.31-4.42 (m, 1H), 3.84 (dd, J=12.60, 6.30 Hz, 1H), 3.67-3.79 (m, 1H), 3.54-3.64 (m, 1H), 3.40-3.49 (m, 1H), 2.20-2.32 (m, 2H), 1.48 (s, 8H). MS cald. for C 5 H 71 NO 2 [(M-C 4 H 9 ) + ] 241, obsd. 241.7, 282.7 [(M-C 4 H 9 +41) + ].
Preparation of (6-fluoro-3-methyl-4-trifluoromethanesulfonyloxy-naphthalen-2-yl)-acetic acid methyl ester
2-[1-(4-Fluoro-phenyl)-meth-(E)-ylidene]-3-methyl-succinic acid
To a suspension of sodium hydride (60% in paraffin oil, 31g, 686 mmol) in toluene (150 mL) was added a solution of 4-fluorobenzaldehyde (30 g, 214 mmol) and dimethyl methylsuccinate (58 g, 312 mmol) in toluene (150 mL) over 1 hour at 0° C. under nitrogen. The reaction was initiated by addition of a drop of methanol at room temperature and was stirred at room temperature for 2 hours. The reaction was quenched by slow addition of 2.0 N aqueous NaOH (300 mL) at 0° C. The resulting mixture was stirred at 110° C. for 4 hours. The mixture was then cooled to room temperature and the aqueous layer was diluted with water (300 mL) and washed with Et 2 O (2×300 mL). The aqueous phase was cooled in an ice-water bath. Addition of concentrated HCl was followed by extraction with ethyl acetate (2×100 mL). The combined organic extracts were washed with water (50 mL) followed by brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was crystallized from ethyl acetate-hexanes to give 2-[1-(4-fluoro-phenyl)-meth-(E)-ylidene]-3-methyl succinic acid. The procedure above was repeated using a separate amount of 4-fluorobenzaldehyde (30 g, 214 mmol). The products of the two reactions were combined to provide 2-[1-(4-fluoro-phenyl)-meth-(E)-ylidene]-3-methyl succinic acid as a pale yellow solid (28g, 27.5% overall). MS cald. for C 12 H 12 FO 4 [(M+H) + ]: 239, obsd. 239.2.
6-Fluoro-4-hydroxy-3-methyl-naphthalene-2-carboxylic acid
A solution of 2-[1-(4-fluoro-phenyl)-meth-(E)-ylidene]-3-methyl succinic acid (28 g, 119 mmol) in trifluoromethanesulfonic acid (140 mL) was stirred at room temperature for 16 h. The resulting mixture was carefully poured into ice cooled water with continuous stirring to obtain a solid precipitate, which was filtered, washed with water and dried in vacuo to yield 6-fluoro-4-hydroxy-3-methyl-naphthalene-2-carboxylic acid (28g, >100% crude) as yellow solid. This crude product was used in the next step without further purification. MS cald. for C 12 H 8 FO 3 [(M−H) + ]: 219, obsd. 218.9.
6-Fluoro-4-hydroxy-3-methyl-naphthalene-2-carboxylic acid methyl ester
To a 0° C. solution of 6-fluoro-4-hydroxy-3-methyl-naphthalene-2-carboxylic acid (28 g, 127 mmol) in MeOH (240 mL) was added concentrated sulfuric acid (18.9 mL, 382 mmol) dropwise. The reaction mixture was then warmed to room temperature and refluxed overnight. After this time, the methanol was distilled off under reduced pressure, and the crude mixture was diluted with ethyl acetate. This solution was washed with saturated aqueous NaHCO 3 . The organic phase was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product. Silica gel column chromatography (6% ethyl acetate-hexane) afforded 6-fluoro-4-hydroxy-3-methyl-naphthalene-2-carboxylic acid methyl ester (14.8 g, 50%) as light yellow solid. MS cald. for C 13 H 12 FO 3 [(M+H) + ]: 235, obsd. 235.2.
4-Benzyloxy-6-fluoro-3-methyl-naphthalene-2-carboxylic acid methyl ester
To a solution of 6-fluoro-4-hydroxy-3-methyl-naphthalene-2-carboxylic acid methyl ester (21.7 g, 92.7 mmol) in dry DMF (250 mL) was added K 2 CO 3 (17.9 g, 130 mmol), benzyl bromide (13 mL, 111 mmol) and Bu 4 NI (0.250 g) at room temperature under nitrogen. The reaction mixture was stirred for 3 hours at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic extracts were washed with water, brine, dried over anhydrous sodium sulfate and concentrated to give the crude product, which was purified using silica gel column chromatography (2-5% ethyl acetate-hexane) to yield 4-benzyloxy-6-fluoro-3-methyl-naphthalene-2-carboxylic acid methyl ester (25.4 g, 84%) as an off-white solid. MS cald. for C 20 H 18 FO 3 [(M+H) + ]: 325, obsd. 325.1.
(4-Benzyloxy-6-fluoro-3-methyl-naphthalen-2-yl)-methanol
To a suspension of LiAlH 4 (8.8 g, 235 mmol) in dry THF (120 mL) was added a solution of 4-benzyloxy-6-fluoro-3-methyl-naphthalene-2-carboxylic acid methyl ester (25.4 g, 78.4 mmol) in THF (180 mL) dropwise at 0° C. under nitrogen. The reaction mixture was stirred at room temperature for 3 hours. After this time, the reaction mixture was cooled to 0° C. and quenched carefully via addition of cold water (10 mL) followed by 15% NaOH solution (10 mL) and additional water. The resulting solution was stirred for one hour, then filtered through a sintered glass funnel. The filter pad was washed with THF (50 mL). The combined filtrates were dried over Na 2 SO 4 , filtered, and concentrated to afford (4-benzyloxy-6-fluoro-3-methyl-naphthalen-2-yl)-methanol (21.5 g, 92%, crude) as a white solid. The crude product was used in the next step without further purification. MS cald. for C 19 H 16 FO 2 [(M−H) + ]: 295, obsd. 294.9.
1-Benzyloxy-3-chloromethyl-7-fluoro-2-methyl-naphthalene
To a solution of triphenylphosphine (41.6 g, 159 mmol) in dry THF (190 mL) was added CCl 4 (59 mL). The reaction mixture was stirred for 10 minutes and (4-benzyloxy-6-fluoro-3-methyl-naphthalen-2-yl)-methanol (21.5 g, 79.4 mmol) was introduced as a solid at room temperature under nitrogen. The resulting solution was refluxed for 2 hours. The solvent was distilled off under reduced pressure, and the residue was diluted with water. The resulting mixture was extracted twice with ethyl acetate. The combined organic extracts were washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Silica gel column chromatography (100-200 mesh, 5% ethyl acetate in hexanes) provided 1-benzyloxy-3-chloromethyl-7-fluoro-2-methyl-naphthalene (18.5 g, 81%) as an off-white solid.
(4-Benzyloxy-6-fluoro-3-methyl-naphthalen-2-yl)-acetic acid methyl ester
To a stirred solution of 1-benzyloxy-3-chloromethyl-7-fluoro-2-methyl-naphthalene (18.5 g, 58.9 mmol) in a THF-methanol mixture (2:3; 500 mL) was added K 2 CO 3 (8.94 g, 64.8 mmol) and PdCl 2 (PPh 3 ) 2 (2.06 g, 2.96 mmol) at room temperature. The solution was degassed by purging with argon for 5 minutes. The reaction mixture was stirred under a balloon of carbon monoxide overnight at room temperature. After this time, the reaction progress was monitored by TLC (5% ethyl acetate in hexanes). The reaction mixture was concentrated, and the obtained crude residue was diluted with water and extracted with ethyl acetate. The combined organic extracts were washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. Silica gel chromatography (100-200 mesh, 5% ethyl acetate-hexanes) yielded (4-benzyloxy-6-fluoro-3-methyl-naphthalen-2-yl)-acetic acid methyl ester (5.5 g, 97.8%) as a pale yellow solid. MS cald. for C 21 H 20 FO 3 [(M+H) + ]: 339, obsd. 339.0.
(6-Fluoro-4-hydroxy-3-methyl-naphthalen-2-yl)-acetic acid methyl ester
To a stirred solution of (4-benzyloxy-6-fluoro-3-methyl-naphthalen-2-yl)-acetic acid methyl ester (15.8 g, 46.7 mmol) in MeOH (150 mL) was added 10% palladium on carbon (2.4 g). The resulting mixture was vigorously stirred under a balloon of hydrogen overnight. The reaction mixture was filtered through celite. The filtrate was concentrated to give the crude product, which was purified by silica gel chromatography (10% ethyl acetate in hexanes) to yield (6-fluoro-4-hydroxy-3-methyl-naphthalen-2-yl)-acetic acid methyl ester (9.5 g, 83%) as a white solid. MS cald. for C 14 H 13 FO 3 [(M+H) + ] 249, obsd. 249.1.
(6-Fluoro-3-methyl-4-trifluoromethanesulfonyloxy-naphthalen-2-yl)-acetic acid methyl ester
A light yellow solution of (6-fluoro-4-hydroxy-3-methyl-naphthalen-2-yl)-acetic acid methyl ester (12.2 g, 49.1 mmol) in methylene chloride (500 mL) was cooled to 0° C. using an ice-acetone bath. Pyridine (5.17 mL, 63.9 mmol) was added and then trifluoromethanesulfonic acid anhydride (20.8 g, 73.7 mmol) was added dropwise to the cold solution over 40 minutes. The resulting light yellow solution was stirred for two hours at 0° C. before being warmed to room temperature. The reaction mixture was stirred for another 30 minutes at room temperature. The mixture was quenched with water (300 mL) and the two layers were separated. The aqueous layer was extracted with dichloromethane (200 mL). The combined organic layers were washed with brine, dried over anhydrous MgSO 4 , filtered, and concentrated to give the crude product as a light yellow solid. The crude product was dissolved in dichloromethane (−50 mL) with heating and then the mixture was diluted with hexanes (˜100 mL). Some of the solvent was removed by heating with a heat gun. The resulting light brown solution was stored in the freezer for 15 hours. A white solid precipitated, which was collected by filtration and washed with hexanes. After air drying, (6-fluoro-3-methyl-4-trifluoromethanesulfonyloxy-naphthalen-2-yl)-acetic acid methyl ester (14.32 g, 77%) was isolated. 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 7.83 (dd, J=9.03, 5.52 Hz, 1H), 7.75 (s, 1H), 7.65 (dd, J=10.29, 2.51 Hz, 1H), 7.31 (td, J=8.60, 2.38 Hz, 1H), 3.85 (s, 2H), 3.74 (s, 3H).
Preparation of [4-(3-amino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester
Step 1: [4-(3-tert-butoxycarbonylamino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester
To an oven-dried three neck round-bottom flask equipped with an addition funnel and a magnetic stir bar was added zinc (3.03 g, 46.4 mmol) and lithium chloride (1.96 g, 46.4 mmol). The solids were mixed together, and the reaction flask was placed under high vacuum. The flask was heated to 171° C., and the solids were stirred under high vacuum at this temperature for 1.5 hours. The mixture was cooled to room temperature, and the flask was back-filled with nitrogen gas. Tetrahydrofuran (THF) (3 mL) was added followed by 1,2-dibromoethane (0.40 mL, 4.6 mmol). The suspension was stirred and gently heated with a heat gun until gas evolution and foaming occurred. This process was repeated three times to completely activate the zinc. Chlorotrimethylsilane (0.59 mL, 4.6 mmol) was added and the suspension was stirred for 15 minutes at room temperature. A solution of 3-iodo-pyrrolidine-1-carboxylic acid tert-butyl ester (6.9 g, 23 mmol) in 15 mL THF was added dropwise. After addition of only 5 mL, the reaction temperature increased to 63° C., then the remaining iodide was added dropwise at ˜50° C. for 10 minutes. The reaction mixture was heated to ˜55° C. with heat gun and then the very thick reaction mixture was stirred for 3 hours. Tetrahydrofuran (10 mL) was added. Stirring was halted, and the suspension was allowed to settle, giving a clear solution above the unreacted zinc dust.
In a separate 3 neck round-bottom flask, palladium (II) acetate (0.26 g, 1.2 mmol), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (S-Phos) (0.95 g, 2.3 mmol), and THF (10 mL) were combined under nitrogen gas. A solution of 6-fluoro-3-methyl-4-trifluoromethanesulfonyloxy-naphthalen-2-yl)-acetic acid methyl ester (Intermediate III) (4.86 g, 12.8 mmol) in THF (10 mL) was added. The zinc solution from above was added to the reaction flask to form a brown solution. The reaction mixture was heated to 55° C. and stirred at this temperature over the weekend. After cooling to room temperature, the reaction mixture was poured into a solution of saturated ammonium chloride and brine solution. The organic compound was extracted into ethyl acetate (3×150 mL). The combined extracts were washed with brine solution and dried over anhydrous MgSO 4 , filtered, and concentrated to provide 9.4 g of the crude product as an oil. The crude product was partially dissolved in acetonitrile (20 mL), producing some solids as a precipitate. The solids were collected by filtration and washed with acetonitrile. The filtrate was concentrated under vacuum to obtain a dark brown crude oil (8.9 g) which was purified using flash chromatography (330 g ISCO column, eluting with 100% hexanes ramped to 60% diethyl ether in hexanes). The third eluting product was [4-(3-tert-butoxycarbonylamino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester (0.89 g, 9.6%). 1 H NMR (400 MHz, CDCl 3 ) δ ppm 7.75 (dd, J=8.80, 5.80 Hz, 1H), 7.63 (s, 1H), 7.45 (dd, J=11.40, 2.50 Hz, 1H), 7.19 (td, J=8.70, 2.80 Hz, 1H), 5.60 (br. s, 1H), 5.09-5.12 (m, 1H), 3.82 (s, 2H), 3.73 (s, 3H), 3.26-3.34 (m, 2H), 2.53-2.59 (m, 2H), 2.37 (s, 3H), 1.43 (s, 8H).
Step 2: [4-(3-Amino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester
To a solution of [4-(3-tert-butoxycarbonylamino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester (0.79 g, 2.0 mmol) in methylene chloride (28.5 mL) was added trifluoroacetic acid (7.5 mL, 98 mmol). The reaction mixture was stirred at room temperature for 4 hours. The solvent was removed under vacuum and the resulting brown residue was dissolved in toluene. The solvent was again removed under vacuum, then dissolved in methylene chloride (−5 mL) with heating. The mixture was diluted with hexanes (˜10 mL). As a result, some oil precipitated. The mixture was diluted with dichloromethane and the dark brown solution was stored in the refrigerator for 15 hours; however, further precipitation of the product was not successful. The solvent was removed and the crude mixture (1.1 g) was purified using flash chromatography (80 g ISCO column, 0-100% methylene chloride in hexanes followed by 0-20% methanol in methylene chloride) to afford [4-(3-amino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester (0.502 g, 75%) as a white, amorphous, and hygroscopic solid. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 7.64-7.71 (m, 1H), 7.55-7.59 (m, 1H), 7.32-7.38 (m, 1H), 7.16 (td, J=8.5, 2.3 Hz, 1H), 5.53 (s, 1H), 5.09 (s, 1H), 3.77-3.81 (m, 2H), 3.67-3.71 (m, 4H), 2.99 (d, J=6.0 Hz, 2H), 2.65 (t, J=7.4 Hz, 2H), 2.28 (s, 4H).
Part II
Preparation of Certain Compounds of the Invention
Example 1
{4-[3-(2-Chloro-benzenesulfonylamino)-1-methylene-propyl]-6-fluoro-3-methyl-naphthalen-2-yl}-acetic acid
Step 1: {4-[3-(2-Chloro-benzenesulfonylamino)-1-methylene-propyl]-6-fluoro-3-methyl-naphthalen-2-yl}-acetic acid methyl ester
A solution of [4-(3-amino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester (120 mg, 0.398 mmol) in THF (4 mL) was cooled to 0° C. and the solid 2-chlorobenzene-1-sulfonyl chloride (168 mg, 0.796 mmol) was added followed by N,N-diisopropylethylamine (154 mg, 209 μL, 1.19 mmol). After stirring for 2 hours at 0° C., the cooling bath was removed and the reaction mixture was warmed to room temperature. The reaction mixture was stirred at room temperature for 15 hours, then diluted with water and brine. The resulting mixture was extracted with ethyl acetate (2×50 mL). The combined organic layers were dried over MgSO 4 , filtered, and concentrated to afford a viscous oil. Flash chromatography (40 g ISCO column, 100% hexanes ramped to 50% ethyl acetate in hexanes) provided {4-[3-(2-chloro-benzenesulfonylamino)-1-methylene-propyl]-6-fluoro-3-methyl-naphthalen-2-yl}-acetic acid methyl ester as a viscous oil. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 8.04-8.07 (m, 1H), 7.74 (dd, J=9.00, 5.80 Hz, 1H), 7.62 (s, 1H), 7.49-7.54 (m, 2H), 7.40 (dd, J=7.65, 2.89 Hz, 1H), 7.32 (dd, J=11.20, 2.60 Hz, 1H), 7.19 (td, J=8.60, 2.40 Hz, 1H), 5.52 (q, J=1.40 Hz, 1H), 5.08-5.10 (m, 1H), 5.03 (t, J=6.40 Hz, 1H), 3.80 (s, 2H), 3.73 (s, 3H), 3.09-3.16 (m, 2H), 2.51 (t, J=7.00 Hz, 2H), 2.31 (s, 3H). MS cald. for C 24 H 24 ClFNO 4 S [(M+H) + ] 476.1, obsd. 476.1.
Step 2: {4-[3-(2-Chloro-benzenesulfonylamino)-1-methylene-propyl]-6-fluoro-3-methyl-naphthalen-2-yl}-acetic acid
To a solution of {4-[3-(2-Chloro-benzenesulfonylamino)-1-methylene-propyl]-6-fluoro-3-methyl-naphthalen-2-yl}-acetic acid methyl ester (140 mg, 0.294 mmol) in THF (6 mL) was added a solution of LiOH (141 mg, 5.88 mmol) in water (1.5 mL). The resulting mixture was warmed with a heat gun to produce a clear solution, which was stirred for 15 hours at room temperature. At this time, LCMS analysis indicated the complete conversion of starting material. The THF was evaporated and the aqueous layer was diluted with water and slowly neutralized with 1.0 N HCl to obtain a white precipitate which was collected by filtration and washed with water and hexanes. After air drying, 125 mg of {4-[3-(2-chloro-benzenesulfonylamino)-1-methylene-propyl]-6-fluoro-3-methyl-naphthalen-2-yl}-acetic acid was isolated as an off-white solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 12.43 (bs, 1H), 7.96 (t, J=5.6 Hz, 1H), 7.87-7.93 (m, 2H), 7.73 (s, 1H), 7.59-7.62 (m, 2H), 7.45-7.51 (m, 1H), 7.29-7.38 (m, 2H), 5.50 (dd, J=3.3, 1.2 Hz, 1H), 4.93 (d, J=1.0 Hz, 1.2H), 3.72-3.80 (m, 2H), 3.00-3.12 (m, 2H), 2.30-2.39 (m, 2H), 2.17 (s, 3H). MS cald. for C 23 H 22 ClFNO 4 S [(M+H) + ] 462.1, obsd. 462.0.
Example 2
[4-(3-Cyclohexanesulfonylamino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid
[4-(3-Cyclohexanesulfonylamino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yf]-acetic acid was prepared according to the method described above for Example 1, starting from [4-(3-amino-1-methylene-propyl)-6-fluoro-3-methyl-naphthalen-2-yl]-acetic acid methyl ester and cyclohexanesulphonyl chloride. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 7.77 (dd, J=8.80, 6.00 Hz, 1H), 7.67 (s, 1H), 7.45 (dd, J=11.20, 2.60 Hz, 1H), 7.21 (td, J=8.60, 2.40 Hz, 1H), 5.61-5.63 (m, 1H), 5.12-5.15 (m, 1H), 4.04-4.10 (m, 1H), 3.87 (s, 2H), 3.28 (q, J=6.80 Hz, 2H), 2.65 (t, J=6.80 Hz, 2H), 2.40 (s, 3H), 1.79-1.88 (m, 4H), 1.64-1.72 (m, 4H), 1.36-1.47 (m, 4H). MS cald. for C 23 H 27 FNO 4 S [(M−H) − ] 432.2, obsd. 432.2.
Example 3
DK-PGD 2 -Induced IL-13 Production Assay in Th2 Cells
Inhibition of 13,14-dihydro-15-keto Prostaglandin D 2 (DK-PGD 2 )-induced IL-13 production in T helper type 2 (Th2) cells was applied to evaluate compound cellular potency.
Cultures of Th2 cells were established from blood of healthy human volunteers according to the following procedure. Peripheral blood mononuclear cells (PBMC) were first isolated from 50 mL of fresh blood by Ficoll-Hypaque density gradient centrifugation, followed by CD4′ cell purification using a CD4′ T Cell Isolation Kit II (from Miltenyi Biotec Inc.). The CD4′ T cells were then differentiated to Th2 cells by culturing the cells in X-VIVO 15® medium (from Cambrex BioScience Walkersville Inc.) containing 10% human AB serum (serum of blood type AB from Invitrogen Corporation), 50 U/mL of recombinant human interleukin-2 (rhIL-2) (from PeproTech Inc.) and 100 ng/mL of recombinant human interleukin-4 (rhIL-4) (from PeproTech Inc.) for 7 days. The Th2 cells were isolated using a CD294 (CRTH2) MicroBead Kit (from Miltenyi Biotec Inc.) and amplified in X-VIVO 15® medium containing 10% human AB serum and 50 U/mL of rhIL-2 for 2 to 5 weeks. In general, 70% to 80% of the Th2 cells used in the assay are CRTH2-positive when analyzed by fluorescence-activated cell sorting using the BM16 antibody (as previously described) conjugated to Alexa Fluor 647.
To determine cellular inhibitory potency, compounds at various concentrations were incubated with 2.5×10 4 Th2 cells and 500 nM DK-PGD 2 for 4 hrs at 37° C. in 200 L of X-VIVO 15® medium containing 10% human AB serum. IL-13 production to the medium was detected by ELISA (enzyme-linked immunosorbent assay) using an “Instant ELISA™” kit (from Bender MedSystems Inc.) according to the procedure suggested by the vendor. The spontaneous production of IL-13 by Th2 cells was determined in the absence of DK-PGD2 stimulation and the value was subtracted from that in the presence of each compound for percent inhibition and IC 50 calculations.
The percent inhibition of interleukin 13 (IL-13) production for a compound at various concentrations was calculated according to the following formula, [1-(IL-13 production in the presence of compound)/(IL-13 production in the presence of 0.15% DMSO)] x100 . The IC 50 value, defined as the concentration of a compound that is required for 50% inhibition of IL-13 production, was calculated by fitting the percent inhibition data for 7 concentrations to a sigmoidal dose-response (4 parameter logistic) model in the XLfit® software Excel add-in program [ID Business Solutions Ltd., model 205, where F(x)=(A+(B−A)/(1+((C/x)^D)))].
The compounds of interest were tested in the foregoing DK-PGD 2 -induced IL-13 production assay. The results of the DK-PGD 2 -induced IL-13 production are shown in the table below:
Example Number
IC 50 (mM)
1
0.0046
2
0.0144
It is to be understood that the invention is not limited to the particular embodiments of the invention described above, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.
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The invention is concerned with the compounds of formula (I):
and pharmaceutically acceptable salts thereof, wherein R1, R2 and R3 are defined in the detailed description and claims. In addition, the present invention relates to methods of manufacturing and using the compounds of formula I as well as pharmaceutical compositions containing such compounds. The compounds of formula I are antagonists or partial agonists at the CRTH2 receptor and may be useful in treating diseases and disorders associated with that receptor such as asthma.
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CROSS REFERENCE TO RELATED APPLICATION
The present application is a continuation of U.S. patent application Ser. No. 11/239,956, filed Sep. 29, 2005, now U.S. Pat. No. 7,452,208 and claims priority from pending European Patent Application No. 04023260.5, filed Sep. 30, 2004, which are hereby incorporated herein by reference.
BACKGROUND
1. Field
The present application concerns a dental handpiece for the treatment of root canals and the measurement of the root canal length.
2. Description of Prior Art
Such a handpiece is part of a device for the determination of the length (depth) of a root canal and of the position of the treatment instrument attached to the handpiece, respectively. The device comprises, together with the handpiece, two electrodes and a control and evaluation unit. The first electrode, usually in the form of a clip-on electrode, is attached to the oral mucosa of the patient. The instrument attached to the handpiece, for example a file moving back and forth (executing reciprocating movements), a reamer or a rotating dental drill, serves as the second electrode. Both electrodes are connected to the control and evaluation unit. The control and evaluation unit is connected to a current source, which generates a measuring signal, and furthermore includes a measuring device, preferably a measuring circuit, which determines the length of the root canal and the position of the dental instrument, respectively, on the basis of the change in the electrical resistance or the change in the impedance of the measuring signal between the two electrodes.
From EP 1 444 966 A1, a handpiece is known with which the signal line is implemented by a conducting wire outside of the handpiece, the wire being connected to the dental instrument, serving as the second electrode, with a clip-on connection and a clip holder. The disadvantage with this arrangement is the conducting wire is attached to the sleeve of the handpiece handle, which restricts the manageability of the handpiece.
In order to avoid this disadvantage, handpieces are manufactured with which conductors or components of the handpiece implement the signal line within the handpiece. DE 197 02 370 A1 (DE '370 A1) discloses an arrangement of conducting wires within the handpiece or in the sleeve of the handpiece which make contact with the instrument serving as an electrode by way of a contact device. In the DE 195 20 765 A1 (DE '765 A1) components serving for the transmission of the drive power, especially shafts and gears, parts of the outer sleeve and components in contact with these components are used for signal transmission.
With both designs, it is necessary that at least parts of the outer sleeves are insulated in order to prevent the impairment of the measuring circuit due to contact between the handpiece and the oral cavity of the patient or the hand of the user. Insulation may be achieved by manufacturing part of the handle from plastic, as seen in the DE '370 A1. Alternatively, an insulating film can be attached to the outer surface of the outer sleeve, as explained in connection with the DE '765 A1.
These known handpieces have the disadvantage on the one hand of the greater manufacturing costs due to the coating and on the other hand that plastics used for coating or as a material for the handle sleeve sections are not stable relative to the prevailing ambient conditions for sterilization, particularly steam-sterilization. Since however the handpieces must be sterilized following each use, the life of such coatings or handle sleeve sections is very limited.
The present application is therefore in response to the need for a dental handpiece for the treatment of root canals and the measurement of the root canal length while at the same time avoiding the disadvantages discussed above. In particular, the handpiece must be insensitive to the prevailing ambient conditions for sterilization.
SUMMARY
In the present application, the outer sleeve of the handpiece described below is comprised of non-insulating, preferably metallic, material and also has substantially no insulating coating. In relation to the prevailing ambient conditions for sterilization, it is therefore insensitive. Furthermore, to ensure faultless measurement of the root canal length and signal line integrity, all components of the handpiece which are part of the device for the transmission of the measuring signals or the contact device, and therefore “live,” are insulated from the outer sleeve by insulating material. The components which establish a conductive path between the device for the transmission of the measuring signals and the instrument serving as electrode are part of the contact portion.
Parts of the contact device may also be disposed outside of the handpiece. In particular, this is necessary when a dental instrument having a non-conductive shaft is used. Such an instrument, for example a file, is comprised of a metallic—and thus conductive—working area and a shaft attached to this, which is at least partly surrounded (especially in the area in which the spindle is connected to the instrument carrier of the handpiece) by a plastic sleeve. In order to establish a connection between the conductive section of the dental instrument and the device for the transmission of the measuring signals, a contact device is required which makes physical contact with the dental instrument outside of the handpiece.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a sectional view of a first embodiment for the handpiece.
FIG. 2 shows the head of the handpiece in FIG. 1 on an enlarged scale.
FIG. 3 shows a sectional view of a second embodiment for the handpiece.
The same components are numbered identically in all figures.
DETAILED DESCRIPTION
The contra-angle handpiece 1 illustrated in FIG. 1 is comprised of an outer sleeve 2 with a head section 3 and a handle section 4 . The entire outer sleeve 2 (including pushbutton 29 ) is not insulating, i.e. it is made substantially of a non-insulating, preferably metallic, material and also has substantially no insulating coating. At the proximal end of the outer sleeve 2 is a connecting device 5 for coupling the handpiece to a control and evaluation unit, a measuring circuit with a current source for the measurement of the root canal length and drive unit. The connecting device 5 is preferably designed in the form of the twist-on connection familiar to dental specialists, so that no further description is necessary here.
As used herein, the terms “non-conductive,” “insulating” and “insulated” mean that the component or material so described reduces or prevents the transmission of electricity. Conversely, the terms “conductive,” “conducting” and “non-insulating” mean that the component or material so described has the quality of conducting electricity. As would be understood by those of ordinary skill in the art, these terms are used to describe relative rather than absolute qualities.
The inside of the handle section 4 is interspersed with several elements for the transmission of the drive movement, including a first spindle designated the central drive spindle 6 , a second spindle designated the drive spindle 7 , and a hollow spindle 8 . The drive spindle 7 is received by the distal end of the hollow spindle 8 and fixed by a straight grooved pin 9 . A spring 10 preloads the hollow spindle 8 against the drive spindle 7 and allows an axial play of hollow spindle 8 relative to drive spindle 7 . At the proximal end, the hollow spindle 8 is detachably connected by a carrier 11 to the rotating spindle of the drive unit, preferably an electrical motor.
The first spindle 6 and second spindle 7 are connected through a gear unit 12 , consisting of two gearwheels 13 , 14 pressed onto the two spindles 6 , 7 and supported by several rolling bearings 15 , 16 , 17 , 18 in the coupling and bearing sleeves 19 , 20 , 21 . The rolling bearings 15 , 16 , 17 , 18 are pre-stressed by spring washers, as illustrated by way of example for the bearing 18 in FIG. 2 . Here, the spring washer 56 is mounted in an annular groove of the spacer disk 55 . Another gear wheel 22 is pressed onto the distal end of the first spindle 6 , which meshes with a gear wheel 23 , connected to a hollow spindle, designated the head spindle 24 . The head spindle 24 is simultaneously part of the instrument carrier 25 , which detachably receives a dental instrument in the known manner. The rotary motion of the drive device is thus transmitted via the elements for the transmission of the drive movement 6 , 7 , 8 to the head spindle 24 , the instrument carrier 25 and the dental instrument, preferably a rotating dental drill. Instead of the gear wheels 22 , 23 of course an eccentric gear can be placed between the first spindle 6 and the head spindle 24 , so that the dental instrument, preferably a file, executes stroke (reciprocating) movements.
A pushbutton 29 , the cover 30 of which is pre-stressed by a spring 31 and the inside of which is provided with wedges 36 ( FIG. 2 ), is provided for releasing the dental instrument from the instrument carrier 25 . Pressing the pushbutton cover 30 in the direction of the opening 31 A of the instrument carrier 25 up to the collar 32 of the supporting ring 33 moves the cylindrical slider 34 , pre-stressed by another spring 35 , radially through the wedges 36 (to the right in FIG. 2 ) until it is positioned coaxially with the instrument carrier 25 and the user can remove the dental instrument from the instrument carrier 25 .
The inside of the handpiece 1 also has a device 26 for the transmission of the measuring signal for the length measurement. For the handpiece 1 illustrated in FIG. 1 , the device 26 is formed by a conducting wire 27 , to which a voltage is applied while performing the length measurement and which is surrounded by a casing 28 of insulating material, in particular plastic. The proximal end of the conducting wire 27 in the region of the connecting device 5 is in the form of the sliding contact 57 , which is joined through the connecting device 5 to a slip ring of the connecting element, for example the drive unit or a coupling. If the connecting device 5 is a non-rotating plug-in connector, the proximal end of the conducting wire 27 is in the form of a contact pin to be seated in a socket connector of the connecting element coupled to the handpiece 1 .
The distal end of the connecting wire 27 is split into two ends, 27 A and 27 B. At each of the two ends 27 A, 27 B is a contact device 37 A, 37 B, respectively, which establishes a conductive path between the dental instrument serving as electrode and the connecting wire 27 . The contact device 37 B is intended for dental instruments having a non-conductive section and is in the familiar U-shaped form, comprising an elastic wire 38 with two legs 39 (in the sectional view only one leg can be seen). In the frontal end region both legs 39 are curved inward. This curvature region is essentially under the opening 31 of the instrument carrier 25 , so that a dental instrument placed in the instrument carrier 25 runs between the two legs and makes physical contact on two sides of the legs 39 .
The base 40 of the U-shaped wire 38 is connected to a sleeve 41 , preferably using a terminal connection. At one end, the sleeve 41 has a flange 42 attached, preferably by a cement bond, to the inside of the outer sleeve 2 . A stem 43 of the sleeve 41 projects outwards through a bore 44 in the outer sleeve 2 , with the diameter of the bore 44 smaller than that of the flange 42 . The sleeve 41 on the end opposite the flange 42 has a groove 45 with two ledges 46 and 47 , with the diameter of the groove 45 smaller than the diameter of the base 40 of the U-shaped wire 38 . Since the side walls of the groove 45 have a slight spring mounting, when the user applies pressure while connecting the base 40 to the ledges 46 , 47 they are pushed aside, so that the base 40 reaches the connective region 45 A of the groove 45 and is fixed by the ledges 46 , 47 returning as a result of the spring mounting to their original positions. To disconnect the base 40 , the user pulls on the U-shaped wire 38 , causing the ledges 46 , 47 to again move aside and release the base 40 .
The end 27 B of the connecting wire 27 is received inside the sleeve 41 and makes contact with the base 40 of the U-shaped wire 38 clamped in the section 45 A of the groove 45 , so that a conductive path is established between the dental instrument serving as electrode, the legs 39 and the base 40 of the wire 38 , the end 27 B and the connecting wire 27 to the sliding contact 57 .
In order to prevent disturbances in the measuring and signal wire, the sleeve 41 should be in the form of an insulating element, i.e. made of non-conductive material, preferably plastic, or coated with plastic. It is of course possible to use a separate component made of conductive material, for example a spring or a flexing element making contact with the connecting wire 27 , in place of the end 27 B of the conducting wire.
When using a dental instrument consisting entirely of a conductive material, the user may disconnect the wire 38 from the sleeve 41 . In this case, the contact device 37 A with the end 27 A of the connecting wire 27 serves to connect the dental instrument to the sliding contact 57 . The end 27 A makes contact with the outer ring 48 A of the spring washer 48 via a bore 54 in the socket 49 . The spring washer 48 in turn makes a sliding contact with the head spindle 24 , as part of the instrument carrier 25 , and the dental instrument attached. A voltage is applied to the spring washer 48 with outer ring 48 A and head spindle 24 while executing the measurement of the root canal length. The spring washer 48 is bearing-mounted in the socket 49 and serves for the pre-stressing of the rolling bearing 50 , which is pressed onto the head spindle 24 and supports the head spindle 24 rotatably. A second rolling bearing 51 is analogously arranged on the pushbutton side of the head spindle 24 and fixed by an O-ring 52 , which is bearing-mounted in a ring groove 53 of the supporting ring 33 . As the sliding contact between the head spindle 24 and the connecting wire 27 other elements, such as a brush or a contact pin, can also be used.
In order to ensure fault-free measurement of the root canal length and signal transmission through the contact device 37 A, at least the following components must serve as insulating means, i.e. must be made of non-conductive material: the socket 49 , the supporting ring 33 and the gearwheel 22 and/or 23 . To obtain better insulation of the outer sleeve 2 , in a preferred embodiment, in addition one or more of the rolling bearings 18 , 50 , 51 and/or the spacer disk 55 and/or the first spindle 6 should be made of insulating, i.e. non-conductive, material. In another embodiment, the first spindle 6 is comprised of several spindle sections, at least one of which is in the form of a non-conductive, insulating element.
The materials used for the components serving as the insulating portion are in particular plastic, preferably PEEK (polyetheretherketone) or silicone, or ceramic materials. Coatings with these materials applied to the components serving as the insulating means also ensure sufficient insulation. Preferably, the raceways and/or the rolling elements of the rolling bearings 18 , 50 , 51 are made of ceramic materials, such as silicon nitride, zirconium nitride or silicon carbide. The gearwheel 22 , the supporting ring 33 , the socket 49 and at least part of the first spindle 6 and spacer disk 55 are preferably made of plastic.
A handpiece can of course be equipped with only a contact device 37 A or 37 B as well, however it is advantageous to implement both contact devices 37 A and 37 B in the handpiece, since this handpiece 1 may then be used with dental instruments having an insulated instrument shaft as well as those not having an insulating instrument shaft.
The handpiece 100 shown in FIG. 3 in principle has the same design as handpiece 1 , so that it is not necessary to repeat the detailed description. The entire outer sleeve 2 with handle section 4 and head section 3 (including pushbutton 29 ) is once again made of completely non-insulating, preferably metallic, material and has no insulating coating.
With the handpiece 100 , the device for the transmission of the measuring signal 26 for the determination of the root canal length consists of the elements for the transmission of the drive movement (first spindle 6 , second spindle 7 , hollow spindle 8 ). A voltage is applied to these elements while executing the measurement of the root canal length, which must therefore be made of conductive material. For dental instruments with a conductive section, the head spindle 24 with gearwheel 23 and gearwheel 22 serve as the contact device 60 A with applied voltage. For dental instruments with a non-conductive section of the instrument shaft, the contact device 60 B must be used in order to establish a conductive path of the dental instrument with the elements 6 , 7 , 8 for the transmission of the drive movement via the head spindle 24 and the gearwheels 22 , 23 . The contact device 60 B consists of a U-shaped elastic wire 38 , connected through its base 40 to the sleeve 41 , preferably using a terminal connection, and a conducting wire 61 with a first end 61 A and a second end 61 B. The first end 61 A is taken up in the sleeve-shaped stem 43 of the sleeve 41 , which makes contact with the base 40 of the U-shaped wire 38 , clamped in section 45 A of the groove 45 . The second end 61 B of the conducting wire 61 is connected through the bore 54 of the socket 49 to the outer ring 48 A of the spring washer 48 ( FIG. 2 ). Via the sliding contact between the spring washer 48 and the head spindle 24 , as part of the instrument carrier 25 , a connection is established through the gearwheels 22 , 23 to the elements for the transmission of the drive movement 6 , 7 , 8 .
The retransmission of the measuring signals takes place from the hollow spindle 8 , via the driver 11 to the connecting piece coupled to the handpiece 100 through the connecting device 5 , for example the rotor shaft of the drive unit or the shaft of a coupling.
In order to ensure fault-free measurement of the root canal length and signal transmission through the contact device 60 A, at least the following components should be insulating, i.e., must be made of non-conductive material: the supporting ring 33 and the socket 49 . When using the contact device 60 B, in addition the sleeve 41 should be in the form of an insulating material. Preferably, the conducting wire 61 is also surrounded by an insulating sheath 62 . To obtain better insulation of the head section 3 of the outer sleeve 2 , in another preferred embodiment, in addition one or more of the rolling bearings 50 , 51 should be made of insulating, i.e. non-conductive, material.
To insulate the handle section 4 of the outer sleeve 2 , at least the rolling bearings 15 , 16 , 17 , 18 should be in the form of insulating means. Preferably, one or more of the coupling and bearing sleeves 19 , 20 , 21 are also made of non-conductive material. The materials for the components comprising the insulating portion are the same as described for handpiece 1 .
The invention is not limited to the embodiments discussed above, but encompasses all embodiments which do not change the fundamental functional principle of the invention. In particular, the handpiece according to the invention for the measurement of the root canal length does not depend on the type of drive unit and includes pneumatic, piezoelectric or magnetostrictive vibration drives as well. Depending on the type of drive unit, the elements for the transmission of the drive movement to the instrument carrier are different in form and can, for example, also include or vibrating rods or flexible spindles.
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A handpiece for measuring root canal length having an outer sleeve that is substantially non-insulating, a connecting device, an instrument carrier, at least one transmission element within the outer sleeve, a length measuring circuit portion, a contact portion and an insulating portion. The instrument carrier is configured to receive a dental instrument. The transmission element transmits force to move the instrument carrier so that a dental instrument received in the instrument carrier executes a rotating, reciprocating and/or vibrating working movement. The length measuring circuit portion is disposed inside the handpiece and operable to transmit measuring signals for measuring length. The contact portion is operable to establish a conductive path between the dental instrument serving as an electrode and the length measuring circuit portion. The insulating portion insulates the length measurement circuit portion and the contact device, through which a voltage is applied during measurement, from the outer sleeve.
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CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the U.S. provisional application 60/570,510 filed May 11, 2004, the entire content of which is expressly incorporated herein by reference thereto.
FIELD OF INVENTION
[0002] The invention describes an electrolyte for deposition of eutectic gold-tin alloy useful in many microelectronic applications including chip bonding and wafer bump plating. The use of 80-20 wt % (70-30 at atom %) eutectic gold-tin alloy is particularly desirable as a solder. At present time vacuum deposition or 80-20-wt % AuSn alloy eutectic gold-tin alloy pre-forms are the existing method for manufacture of electronic parts. However, electro-deposition, due to its low cost and versatility, is a preferred method of application.
BACKGROUND OF THE INVENTION
[0003] Electroplating baths for the deposition of gold-tin alloy have been found by the current inventors to be incapable of depositing the eutectic alloy over a usable current density range. This was clearly demonstrated in “Film growth characterization of pulse electro deposited Au/Sn tin films” by Djurfors and Ivey (GaAs MANTECH, 2001), where they show a step transition from 16 at % Sn to 50 at % at a current density of around 1.5 mA/cm 2 . According to the authors this is a result of the deposition of two distinct phases; Au 5 Sn (16 at % Sn) at low current density and AuSn (50 at % Sn) at high current density. This has been further confirmed by our work which has shown that prior art electrolytes will not typically yield the desired eutectic alloy.
[0004] The prior art electrolytes, using complexing agents such as citric acid, pyrophosphate, gluconic acid, ethylene diamine tetra acetic acid (“EDTA”), and the like, typically yield alloys which are either tin rich (<50% Au) or gold rich (95% Au), or have tin rich or gold rich regions at different current densities. An 80/20 wt % eutectic gold-tin alloy cannot be deposited over a usable current density range. Moreover, many prior art baths suffer from poor stability making them of little practical interest.
[0005] U.S. Pat. No. 4,634,505 by Kuhn, et al. describes an electrolyte using trivalent gold cyanide complex and a tin IV oxalate complex, which operates at pH below 3. The formulation also uses oxalic acid as a conducting salt. However, this bath gives deposits with less than 1% Sn, and therefore it is not useful for depositing a eutectic alloy.
[0006] U.S. Pat. No. 4,013,523 by Stevens et al. describes a bath using a trivalent gold complex and tin as a stannic halide complex. The pH is less than 3 and the bath is claimed to be capable of depositing an 80-20 wt % gold alloy.
[0007] U.S. patent application 2002063063-A1 by Uchida et al. describes a non-cyanide formulation where the gold complexes include gold chloride, gold sulfite and gold thiosulfate among others. The electrolyte includes stannic and stannous salts of sulfonic acids, sulfosuccinates, chlorides, sulfates, oxides and oxalates. The tin is complexed with EDTA, DTPA, NTA, IDA, IDP, HEDTA, citric acid, tartaric acid, gluconic acid, glucoheptonic acid among others. The deposit is brightened by a cationic macromolecular surfactant. Oxalate is listed among the possible buffer compounds.
[0008] Japanese patent application 56136994 describes a solution, which uses sulfite gold complex in combination with stannous tin pyrophosphate complex at a pH of 7 to 13.
[0009] German patent DE 4406434 uses trivalent gold cyanide complex in conjunction with stannic tin complexes. The pH is 3-14 and an 80-20 eutectic alloy is reported.
[0010] U.S. Pat. No. 6,245,208 by Ivey et al. discloses a non-cyanide formulation which uses gold chloride in combination with sodium sulfite, stannous tin, a complexing agent (ammonium citrate), and uses ascorbic acid to prevent oxidation of divalent tin. Eutectic alloy deposits are claimed and bath stability on the order of weeks is reported.
[0011] As noted, the prior art electrolytes are not always stable and have been found to be ineffective in providing eutectic gold tin alloy, particularly for electroplating of small parts for electronic components or composite substrates.
[0012] Accordingly, there is a need for a stable electroplating bath for the deposition of a eutectic gold-tin alloy on various substrates, and this is now provided by the present invention.
SUMMARY OF THE INVENTION
[0013] The invention relates to an aqueous electrolyte for use in connection with the deposition of a gold-tin alloy on an electroplatable substrate. This electrolyte generally comprises a solution that includes water, complexed gold ions, tin ions, a complexing compound to render the tin ions soluble in the solution, and an alloy stabilization agent present in an amount sufficient to stabilize the alloy composition that is deposited. Advantageously, the solution has a pH of between about 2 to 10 and the gold ions and tin ions are present in relative amounts sufficient to provide a deposit having a gold content of less than about 90% by weight and a tin content greater than about 10% by weight. Preferably, the gold ions and tin ions are present in relative amounts sufficient to provide a deposit having a gold content of between 75 and 85% by weight and a tin content of between 15 and 25% by weight.
[0014] The alloy stabilization agent is present in an amount sufficient to stabilize the deposited alloy and enables a eutectic gold-tin deposit to be provided over a usable current density range. The alloy stabilization agents of the current invention comprise anionic surfactants based on phosphate esters of the general formula:
wherein R is alkyl or alkyl aryl group, n is 7 to 10 moles of ethylene and/or propylene oxide, M is hydrogen, sodium, potassium, ammonium or other counter ion, and R′ is ethyl and/or propyl group.
[0015] The electrolyte of the current invention may also contain a brightening agent, which may act alone or in conjunction with the alloy stabilization agent to achieve a synergistic effect. Brightening agents include but are not limited to amphoteric imidazoline derivative having the general structural formula:
wherein R is fatty acid alkyl group and the derivative is soluble in the electrolyte. Alkali metal salts of hexacyano ferrate are not only powerful brightening agents but powerful tin antioxidants as well.
[0016] Lastly it has been found that ascorbic acid, or its alkali metal or ammonium salts, in combination with oxalic acid and its alkali metals or ammonium salts provide powerful synergistic brightening and Au—Sn eutectic alloy stabilizing effect.
[0017] The invention also relates to a method for electroplating of a eutectic gold-tin (80 wt % Au and 20 wt % Sn) alloy deposit which comprises contacting the substrate with one of the solutions disclosed herein and passing a current though the solution to provide a gold-tin alloy deposit thereon. This method is applicable for electroplating a eutectic alloy deposit on composite articles that include electroplatable and non-electroplatable portions. To do so, such articles are contacted with the solution and a current is passed though the solution to provide a gold-tin alloy electrodeposit on the electroplatable portions of the articles without deleteriously affecting the non-electroplatable portions of the articles.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] It has now been found that an alloy with a significant tin content, exemplified by the eutectic 80/20 gold-tin alloy, can be deposited over a usable range of current densities from the electrolytes disclosed herein. Thus, while alloys such as 70 at % gold—30 at % tin and 90 wt % gold—10 wt % tin are obtainable, the eutectic alloy, or as close to the eutectic alloy as possible, is preferred due to the well known advantages of such an alloy.
[0019] The source of gold ions can be mono- or tri-valent potassium or ammonium gold cyanide. In the presence of divalent tin compounds, the tri-valent gold is almost immediately reduced to mono-valent potassium, sodium or ammonium gold cyanide complex. The following equation elucidates the reduction process:
K[Au(CN) 4 ]+Sn(C 2 O 4 )+K 2 C 2 O 4 =K[Au(CN) 2 ]+Sn(C 2 O 4 ) 2 +2KCN
[0020] The complexing agents for gold ions are generally organic acids or a salt thereof, with oxalic, citric, gluconic, malonic, ascorbic, iminodiacetic acid or a solution soluble salt thereof being preferred. The complexed gold ions are advantageously gold cyanide or gold sulfite complexes. Preferably, the complexed gold ions are present in amount between about 0.1 and 100 g/l.
[0021] The tin ions can be added in any soluble form which provides stannous or stannic ions. The overall tin ion concentration in the solution is generally between about 0.1 and 50 g/l although this can vary depending upon other solution components. Any di or tetravalent tin salt, including; sulfate, chloride, methane sulfonate, oxalate, or any other suitable stannous or stannic tin salt, can be used to provide these stannous or stannic ions, and the specific tin salt is not critical. Stannic tin may also be added to the solution, however, some stannous tin must be present in the electrolyte for the invention to function properly. The stannous tin ion concentration in the inventive solution is between 1 and 30 g/l and more preferably between 2 and 10 g/l. The stannic tin may be present in the inventive electrolyte between 0 g/l to 40 g/l.
[0022] The concentration of stannous ions may be adjusted in relation to the gold ion concentration to provide the desired alloy. One of ordinary skill in the art can optimize the metal concentrations in any particular solution to obtain the desired gold-tin alloy.
[0023] The complexing agent for the tin ions is present in the electrolyte to assist in rendering and maintaining the stannous and/or stannic tin ions soluble at the operational pH. Any suitable organic acid can be used for this purpose. Examples of complexing agents useful in the present invention include but are not limited to oxalic acid, citric acid, ascorbic acid, gluconic acid, malonic acid, tartaric acid and iminodiacetic acid. Solution soluble salts of these acids can also be used. Generally, carboxylic acids are preferred, but ascorbic acid, which is not a carboxylic acid, is also a preferred complexor. Moreover, any other complexing agent which can complex the stannous and/or stannic tin in the solution, can be used. The most preferred complexing agents are oxalic, citric, gluconic, heptagluconic and malonic acids and their salts. Solution soluble oxalate, citrate, tartrate, glycerate, ascorbate, gluconate, heptagluconate, malonate, iminodiacetate, nitrilotriacetate, ethylene di-amino-tetra acetate or pyrophosphate salts are also useful.
[0024] The complexing agent is present in the solution in a sufficient concentration to maintain the stannous and/or stannic tin soluble at the electrolyte's pH. It is desirable to maintain an excess of complexing agent beyond the minimum concentration to improve solution conductivity and to provide pH buffering. The complexing agent for the tin ions is generally present in the solution from about 5 g/l to about saturation. The tin ion complexor concentration is typically between 10 and 300 g/l and is most preferably between 40 and 150 g/l.
[0025] The gold ions are preferably provided in the electrolyte as a gold cyanide complex, most preferably monovalent gold cyanide, although, trivalent gold cyanide may also be used. Non-cyanide sulfite gold complex can also be used in the present invention when short life span of the electrolyte is acceptable; otherwise this complex would not be preferred as the stability of this complex is inferior to the others. The most preferred is potassium, sodium, lithium and ammonium gold cyanide complex. The preferred concentration of gold ion complex in the present invention is between 2 and 40 g/l and most preferably between 3 and 10 g/l.
[0026] It has been found that the addition of an alloy stabilization agent comprising of anionic surfactants based on phosphate esters of the general formula:
wherein R is alkyl or alkyl aryl group, n is 7 to 10 moles of ethylene and/or propylene oxide, M is hydrogen, sodium, potassium or other counter ion, and R′ is ethyl and/or propyl group will produce an electrolyte which deposits the desired eutectic or similar gold-tin alloys over an acceptable range of current densities. In the absence of such an additive or additives the deposit may be either tin or gold rich or may have tin or gold rich regions in different areas caused by different current densities.
[0027] The concentration of the alloy stabilization agent in the electrolyte is in the range of 0.01 to 10 ml/l and most preferably in the range of 0.05 to 1 ml/l.
[0028] Other additives can be added to the solution to modify the grain structure of the deposit. These include metallic additives such as nickel, cobalt, arsenic, lead, thallium, or selenium. Organic additives such as those described in U.S. patent application 2002063063 may also be used, if desired. Brightening agents, generally comprising anionic or amphoteric surfactants, or a combination thereof, can be used if desired. In particular, it is preferred to use brightening agents of amphoteric imidazoline derivatives having the general structural formula:
wherein R is fatty acid alkyl group as these derivatives are soluble in the electrolyte. A most preferred brightener is the anionic surfactant is poly(oxy-1,2-ethanediyl).alpha.-tridecyl-. O.mega.-hydroxy-.phosphate at a concentration of 0.1 to 10 grams per liter.
[0029] It has been further found that alkali metals salts of hexacyanoferrate are also very effective brightening agents in the present invention. Thus, the use of brightening agents in conjunction with the alloy stabilizing agent is in accordance with the invention. For this combination, the alloy stabilizing agent is preferably present in a concentration of about 0.1 to 10 grams per liter and the brightening agent is preferably present in a concentration of about 0.05 to 5 grams per liter.
[0030] An antioxidant is by its nature a reducing agent. The invention preferably includes an antioxidant to assist in maintaining the tin ions as stannous tin. For the purpose of illustration and not limitation, the antioxidant can include catechol, hydroquinone, ascorbic acid, hexacyanoferrate, or phenolsulfonic acid, or other agents, such as potassium ferro-hexacyanide, hydrazine, hydroxylamine, pyrogallol, tiron, cresolsulphonic acid, pyrocatechol, resorcinol, phloroglucinol, 2-aminodiphenylmethane or p-hydroxyanisole, can be used to prevent tin oxidation. The preferred antioxidant is hydroquinone. The antioxidant is present in an amount of between about 0.1 and 5 g/l of the solution, and is preferably between about 0.5 and 2 g/l.
[0031] Other salts or buffers may be optionally added to the electrolyte to improve conductivity or pH stability. Examples, of such additives include simple salts such as potassium methane sulfonic acid, potassium sulfate, as well as others that are well known in the art.
[0032] The pH of the electrolyte is between about 2 and 10, most preferably between 3 and 5.5. The preferred pH of the solution depends upon the gold complex that is used. For instance, potassium gold cyanide is not stable below a pH of 3, but a trivalent gold cyanide complex is stable at lower pH values. Sulfite gold complexes are generally not stable below pH 6 and are most stable at pH 8 and higher. Since the solution of the present invention is useful in microelectronics applications, it is desirable to have a pH of less than 8 and preferably less than 7 to prevent solution attack on photoresist masks that are often applied to the electrodeposited substrates. Additionally, it has been found that deposit appearance of eutectic tin-gold alloy begins to degrade at pH values greater than 4.7. For these reasons, the electrolytes preferably have a pH value of about 4.
[0033] The solution temperature is typically between 20 and 70° C. and is most preferably between 38 and 60° C. Temperature has a direct effect on the composition of the deposited alloy, with higher temperature resulting in higher gold contents in the deposited eutectic alloy.
[0034] The electrolyte of the present invention may be operated using insoluble anodes including platinized titanium, platinized niobium, or iridium oxide electrode. It is also possible to use soluble anodes, however, this is not typically practiced in precious metals plating.
EXAMPLES
[0035] The following examples are merely illustrative of the present invention and they should not be considered as limiting the scope of the invention in any way, as these examples and other equivalents thereof will become apparent to those skilled in the art in light of the present disclosure and the accompanying claims.
Example 1
[0036] A eutectic gold-tin alloy electrodeposit is obtained from the following electrolyte;
Citric acid 52 g/l Potassium citrate 67 g/l Tin (as tin sulfate) 3 g/l Gold (as potassium gold cyanide) 6 g/l Ethoxylated phenol ester 0.15 ml/l Catechol 1 g/l pH adjusted with KOH 4.0
The citric acid electrolyte deposits 80-20 wt % gold-tin alloy of semi bright appearance. The current density was 10 ASF and temperature 140° F.
Example 2
[0037] Ascorbic acid 100 g/l Tin (as tin sulfate) 3 g/l Gold (as potassium gold cyanide) 13 g/l Ethoxylated phenol ester 0.15 ml/l pH adjusted with KOH 4
The ascorbic acid electrolyte deposits 80-20 wt % gold-tin alloy of semi bright appearance. The current density was 10 ASF and temperature 120° F.
Example 3
[0038] Potassium malonate 100 g/l Tin (as tin sulfate) 1 g/l Gold (as potassium gold cyanide) 6 g/l Ethoxylated phenol ester 0.35 ml/l Ascorbic acid 2 g/l pH adjusted with KOH 4
The potassium malonate electrolyte deposits 80-20 wt % gold-tin alloy of semi bright appearance. The current density was 10 ASF and temperature 130° F.
Example 4
[0039] Di-sodium-di-hydrogen pyrophosphate 100 g/l Tin (as tin sulfate) 5 g/l Gold (as potassium gold cyanide) 3 g/l Ethoxylated phenol ester (1% solution) 0.35 ml/l Ascorbic acid 2 g/l pH adjusted with KOH 3.7
The pyrophosphate electrolyte deposits 80-20 wt % gold-tin alloy of semi bright appearance. The current density was 7.5 ASF and temperature 130° F.
Example 5
[0040] Potassium oxalate 100 g/l Tin (as tin sulfate) 5 g/l Gold (as potassium gold cyanide) 5 g/l Ethoxylated phenol ester (1% solution) 0.30 ml/l Di-sodium Cocoampho-dipropionate 0.1 ml/l pH 4
The oxalate electrolyte deposits 80-20 wt % gold-tin alloy of bright appearance. The current density was 10 ASF and temperature 140° F.
Example 6
[0041] Potassium oxalate 100 g/l Ascorbic acid 24 g/l Tin (as tin sulfate) 5 g/l Gold (as potassium gold cyanide) 3.5 g/l Ethoxylated phenol ester (1% solution) 0.30 ml/l Di-sodium Cocoampho-dipropionate 0.1 ml/l pH 4
The oxalate/ascorbate electrolyte deposits 80-20 wt % gold-tin alloy of semi bright appearance. The current density was 5 ASF and temperature 130° F.
Example 7
[0042] Potassium oxalate 100 g/l Tin (as tin sulfate) 3.5 g/l Gold (as potassium gold cyanide) 5 g/l Ethoxylated phenol ester (1% solution) 0.3 ml/l Di-sodium cocoampho-dipropionate 0.1 g/l Potassium hexacyanoferrate 0.3 g/l Ascorbic acid 0.5 g/l pH 4
The current density was 5 ASF and temperature 130° F. The 80-20 gold-tin alloy deposit was of bright appearance.
[0043] While the invention has been described and pointed out in detail with reference to operative embodiments thereof, it will be understood by those skilled in the art that various changes, modifications, substitutions, and omissions can be made without departing from the spirit of the invention. It is intended therefore, that the invention embrace those equivalents within the scope of the claims that follow.
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The invention relates to an electrolyte used in connection with the deposition of a gold-tin alloy on an electroplatable substrate. This solution generally includes water; stannous and/or stannic tin ions, a complexing agent to render the stannous and/or stannic tin ions soluble, complexed gold ions, and an alloy stabilizing agent that includes ethoxylated compounds with phosphate ester functional group, brightening additives based on ethoxylated phosphate esters and alkali metal fatty acids dipropionates. The brighteners may be used alone or in conjunction with each other to achieve beneficial synergistic effect. The alloy stabilizing agent is present in an amount sufficient to stabilize the composition of the gold-tin deposit over a usable current density range. The solution has a pH of between 2 and 10 and the gold ions and tin ions are present in relative amounts sufficient to provide a deposit having a gold content less than 90% by weight and a tin content greater than 10% by weight.
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BACKGROUND OF THE INVENTION
[0001] The present invention relates to aircraft and more specifically to aircraft with a strut-braced foldable wing, and to methods of folding the wing on such an aircraft.
[0002] There is a trend towards increasingly fuel efficient passenger aircraft, for which it is desirable to have correspondingly large wing spans. However, the maximum aircraft span is often effectively limited by airport operating rules which govern various clearances required when manoeuvring around the airport (such as the span and/or ground clearance required for gate entry and safe taxiway usage).
[0003] In some suggested designs, aircraft are provided with wings which may be folded upwardly to reduce the span of the aircraft on the ground (compared to when the aircraft is configured for flight). However, a disadvantage with such arrangements is that they tend to be unduly heavy. For example, they may require high capacity (and therefore heavy) actuators to fold the wing. Alternatively or additionally, the wing structure near the folding joint may need to be significantly reinforced in order to withstand and transfer the wing loading across the joint.
[0004] Another problem encountered on large wing span aircraft, is that the magnitude of the bending moments generated at the wing root tend to be correspondingly large. The structure at the wing root must be sufficiently strong to withstand these large bending moments, which can lead to an undesirable weight increase in the aircraft.
[0005] To address this problem, it is known to provide aircraft with strut-braced wings in which a strut structure transfers wing loadings in the outer region of the wing, away from the inner region of the wing (and thus away from the wing root). Such an arrangement does not require the wing root structure to be as strong and can lead to a weight saving, which in some cases can be sufficient to offset the weight and/or drag penalties associated with the strut structure, especially for very large wingspan aircraft.
SUMMARY OF THE INVENTION
[0006] According to a first aspect of the invention, there is provided an aircraft comprising a wing, the wing comprising an inner region and an outer region, the inner and outer regions being connected by a hinge defining a hinge line about which the outer region is foldable to reduce the span of the wing, and the aircraft comprising an actuator arranged to actuate the folding of the outer region of the wing with an actuation force, wherein the wing is braced by an external strut structure for transferring some of the wing loadings in the outer region of the wing away from the inner region of the wing and characterised in that the actuator is arranged to exert the actuation force via the strut structure.
[0007] The invention recognises that the existence of a strut structure can be exploited when actuating a folding wing to reduce its span. More specifically, by exerting the actuation force (for folding the outer region of the wing) via the external strut structure, the nature of the actuation and the actuator, are no longer constrained by the geometry of the wing (for example its thickness) at the hinge, and therefore may be able to be of a lower capacity, and therefore lighter. This is especially beneficial where the wing is of relatively low thickness at the hinge. By virtue of the strut being external, it will be appreciated that the actuator is arranged to exert the actuation force to a location outside of the confines of the wing thickness.
[0008] The external strut structure is for transferring some of the wing loadings in the outer region of the wing away from the inner region of the wing. Thus, the strut structure is arranged to relieve the wing root bending moment.
[0009] The bending moment for folding the outer region of the wing is preferably effected by the actuation force acting about a moment arm that extends beyond the thickness of the wing. The invention recognises that when the aircraft comprises a strut structure external to the wing, this strut structure can be used to provide a load path that extends beyond the thickness of the wing; the moment arm need no longer be constrained by the thickness of the wing.
[0010] The actuator is preferably a linear actuator.
[0011] The actuator may be incorporated into the strut structure. For example, the actuator may be arranged to form part of the primary load path within the strut structure such that it transfers loads during flight (when the wing is unfolded). The actuator may be in the form of an extendable strut. The extendable strut may be connected at one end to the outer region of the wing, and connected at the other end to the strut structure. The extendable strut may be pivotably connected (for example via a pin joint) at one or both ends.
[0012] In some embodiments, the actuator may be ancillary to the strut structure, such that is lies off the primary load transfer path during flight. For example the actuator may be arranged to move the part of the strut structure through which the primary load path passes.
[0013] The strut structure may have an outer end. The outer end is preferably connected to the outer region of the wing. The outer end is preferably connected to the outer region of the wing at between 35% and 75% span, and more preferably between at between 40% and 70% span.
[0014] The strut structure may have an inner end. The inner end is preferably remote from the inner region of the wing. The inner end may bypass the fuselage and connect directly to the opposite wing, but more preferably it is connected to the aircraft fuselage. The inner end may be connected to the underside of the fuselage. The strut structure is preferably arranged such that some of the wing loadings, preferably the bending moments in the outer wing, are transferred to the fuselage.
[0015] The aircraft is preferably a passenger aircraft. The passenger aircraft preferably comprises a passenger cabin comprising a plurality of rows and columns of seat units for accommodating a multiplicity of passengers. The aircraft may have a capacity of at least 20, more preferably at least 50 passengers, and more preferably more than 50 passengers. The aircraft is preferably a powered aircraft. The aircraft preferably comprises an engine for propelling the aircraft. The aircraft may comprise wing-mounted, and preferably underwing, engines.
[0016] The aircraft may be in a mid-wing configuration, but is more preferably in a high-wing configuration.
[0017] The outer region of the wing may be part of the main wing. The outer region of the wing may be a wing tip device. In some embodiments, the outer region of the wing may be a part of the main wing to which a wing tip device is connected. The wing tip device may be a wing tip extension; for example the wing tip device may be a planar tip extension. In other embodiments, the wing tip device may comprise, or consist of, a non-planar device, such as a winglet.
[0018] The trailing edge of the inner region of the wing is preferably a continuation of the trailing edge of the outer region of the wing. The leading edge of the inner region of the wing is preferably a continuation of the leading edge of the outer region of the wing. The upper and the lower surfaces of the inner region of the wing are preferably continuations of the upper and lower surfaces of the outer region of the wing, such that there is a smooth transition from the inner region of the wing to the outer region of the wing.
[0019] The wing may be arranged such that the outer region is foldable downwards to reduce the span of the wing, but is more preferably arranged such that the outer region is foldable upwards to reduce the span of the wing.
[0020] The hinge line may lie substantially parallel to the plane of the wing. The hinge line preferably lies substantially within the plane of the wing.
[0021] According to another aspect of the invention, there is provided a method of folding a strut-braced wing on an aircraft, the wing comprising an inner region and an outer region, the inner and outer regions being connected by a hinge defining a hinge line about which the outer region is foldable to reduce the span of the wing, and wing being braced by an external strut structure for transferring some of the wing loadings in the outer region of the wing away from the inner region of the wing, characterised in that the method comprises the step of exerting an actuating force via the strut structure, such that the outer region of the wing folds about the hinge to reduce the span of the wing.
[0022] According to another aspect of the invention, there is provided a strut structure and actuator for use as the strut structure and actuator as described herein.
[0023] It will of course be appreciated that features described in relation to one aspect of the present invention may be incorporated into other aspects of the present invention. For example, the method of the invention may incorporate any of the features described with reference to the apparatus of the invention and vice versa.
DESCRIPTION OF THE DRAWINGS
[0024] Embodiments of the present invention will now be described by way of example only with reference to the accompanying schematic drawings of which:
[0025] FIGS. 1 a and 1 b are a schematic frontal views of an aircraft with a strut-braced wing according to a first embodiment of the invention, the Figures shown the wing in an unfolded and folded configuration respectively;
[0026] FIGS. 2 a and 2 b are perspective views showing part of the wing on the aircraft in FIGS. 1 a and 1 b;
[0027] FIG. 3 is a schematic frontal view of an aircraft with a strut-braced wing according to a second embodiment of the invention; and
[0028] FIG. 4 is a schematic frontal view of an aircraft with a strut-braced wing according to a third embodiment of the invention.
DETAILED DESCRIPTION
[0029] FIG. 1 a is a schematic front view of a passenger aircraft 1 according to a first embodiment of the invention. The aircraft 1 comprises a fuselage 3 , a wing 5 , an engine 7 and a strut structure 9 . For the sake of clarity only one side of the aircraft is shown; it will be appreciated that a corresponding wing, strut structure etc. also exist on the other side of the fuselage.
[0030] The aircraft is in a high-wing configuration, and accordingly the wing 5 root joins the top of the fuselage 3 . The aircraft 1 has a large wing span, and in order to relieve the loading at the wing root, and thus facilitate a lighter structure at said root, the wing 5 is braced against the underside of the fuselage 3 by the strut structure 9 . The strut structure 9 comprises a main strut 9 a extending from the fuselage 3 to an outer region 5 b of the wing 5 , and a jury strut 9 b extending from part-way along the main strut 9 a to the end of an inner region 5 a of the wing 5 .
[0031] The aircraft has a wing span that is too large to comply with many airport operating rules which govern various clearances required when manoeuvring around the airport (such as the span and/or ground clearance required for gate entry and safe taxiway usage). Accordingly, the wing 5 is foldable about a hinge 11 between the inner region 5 a and outer region 5 b of the wing 5 . FIG. 1 b shows the wing in a folded configuration in which the outer region 5 b is folded upwardly to reduce the span. The aircraft 1 is able to adopt this folded wing configuration after it has landed, in order to comply with, for example, airport gate limits.
[0032] A problem with folding wings in the prior art is that the actuator for folding the wing tends to be very heavy. The mechanism and the supporting structure may also be relatively inefficient. This is because the actuator must necessarily be contained within the wing thickness and, for example, it may have to act on a very small lever arm (less than the wing thickness) to fold the wing.
[0033] The first embodiment of the invention recognises that the existence of a strut structure can be exploited when actuating a folding wing. More specifically, the invention recognises that by exerting an actuation force (for folding the outer region 5 b of the wing 5 ) via the strut structure 9 , the nature of the actuation and the actuator, need no longer constrained by the geometry of the wing at the hinge, and therefore may be able to be of a lower capacity, and therefore lighter. This will now be demonstrated with reference to FIG. 1 b.
[0034] FIG. 1 b shows the wing in the folded configuration. Movement to this configuration is effected by a linear actuator 13 which has been incorporated into the strut structure 9 as an extendable strut (shown as two parallel lines). As the actuator 13 extends, it pushes the outer region 5 b of the wing upwards such that it rotates about the hinge 11 (it will be appreciated that the actuator is pin jointed at either end such that it is pivotably connected at either end to the respective structures, thus allowing it to rotate. Since the actuation force acts via the strut structure 9 (i.e. along the length of the extendable strut) it is acts about a moment arm that extends beyond the thickness of the wing 5 . The actuation force may therefore be relatively low, thereby increasing mechanical efficiency and enabling a lighter actuator to be used.
[0035] FIGS. 2 a and 2 b are close up perspective views of the hinge 11 , jury strut 9 b and actuator 13 . As shown in the Figures, the strut structure has an aerodynamic fairing to minimise its friction and form drag. The actuator is an extendable piston within the fairing at the end of the main strut 9 a . The end of the actuator is exposed (i.e. not covered by a fairing) when the wing is folded. However, this is not a problem because the wing is only folded when the aircraft is on the ground and stationary (or low speed taxiing).
[0036] FIG. 3 is a schematic front view of a passenger aircraft 101 according to a second embodiment of the invention. In FIG. 3 , the wing 105 is shown both folded and unfolded (dotted lines) in the same picture to illustrate the folding movement. Features in the second embodiment of the invention that correspond to similar features in the first embodiment of the invention, are shown with the same reference numerals as in the first embodiment, but with the addition of the prefix ‘1’ (or ‘10’ where appropriate).
[0037] In contrast to the first embodiment, the actuator 113 (shown as two parallel lines) is instead inboard of the jury strut 109 b and is arranged to push the distal end of the jury strut 109 b about the hinge 111 . The outer part of the strut structure 109 (including the jury strut 109 b ) is thus arranged to rotate as the wing is folded.
[0038] FIG. 4 is a schematic front view of a passenger aircraft 201 according to a third embodiment of the invention. As with FIG. 3 , the wing 205 is shown both folded and unfolded (dotted lines) in the same picture to illustrate the folding movement. Features in the third embodiment of the invention that correspond to similar features in the first embodiment of the invention, are shown with the same reference numerals as in the first embodiment, but with the addition of the prefix ‘2’ (or ‘20’ where appropriate).
[0039] In contrast to the first and second embodiments, engine nacelle 215 is used as a replacement of the jury strut. This avoids the drag penalty of using a separate jury strut. It also means that the outer region 205 b of the wing is relatively large, such that only a small rotation about the hinge is required to achieve a notable span reduction (or for the same rotation, a larger span reduction is achieved).
[0040] Whilst the present invention has been described and illustrated with reference to particular embodiments, it will be appreciated by those of ordinary skill in the art that the invention lends itself to many different variations not specifically illustrated herein. By way of example the outer region of the wing may incorporate a wing tip device. The aircraft need not necessarily be a high-wing aircraft. The strut structure need not necessarily be the configuration illustrated and may be any arrangement that braces the wing. Where in the foregoing description, integers or elements are mentioned which have known, obvious or foreseeable equivalents, then such equivalents are herein incorporated as if individually set forth. Reference should be made to the claims for determining the true scope of the present invention, which should be construed so as to encompass any such equivalents. It will also be appreciated by the reader that integers or features of the invention that are described as preferable, advantageous, convenient or the like are optional and do not limit the scope of the independent claims. Moreover, it is to be understood that such optional integers or features, whilst of possible benefit in some embodiments of the invention, may not be desirable, and may therefore be absent, in other embodiments.
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An aircraft ( 1 ) including a wing ( 5 ), the wing having an inner region ( 5 a ) and an outer region ( 5 b ), the inner and outer regions ( 5 a, 5 b ) being connected by a hinge ( 11 ) defining a hinge line about which the outer region ( 5 b ) is foldable to reduce the span of the wing. The aircraft ( 1 ) includes an actuator ( 13 ) arranged to actuate the folding of the outer region ( 5 b ) of the wing with an actuation force. The wing ( 5 ) is braced by an external strut structure ( 9 ) for transferring some of the wing loadings in the outer region ( 5 b ) of the wing away from the inner region of the wing ( 5 a ). The actuator ( 13 ) is arranged to exert the actuation force via the strut structure ( 9 ).
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims all benefits of Korean Patent Application No. 10-2007-0075211 filed on Jul. 26, 2007 in the Korean Intellectual Property Office, the disclosures of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a carbon nano-tube (CNT) thin film treated with chemical having an electron withdrawing functional group and a manufacturing method thereof, and more particularly, to a CNT material treated with chemical having an electron withdrawing functional group to reduce a contact resistance between the CNTs and capable of increasing conductivity of an electrode prepared using the process, and a treating method thereof.
2. Description of the Prior Art
A transparent electrode that is transparent and allows electric current to conduct is necessary for a display device. Indium tin oxide (ITO) is currently most used for the electrode. Although the ITO is currently most used, the cost thereof is increased as the consumption of indium is increased. In particular, since the resistance is increased due to cracks occurring when the ITO electrode is bent, it is difficult to apply the ITO to a flexible device in future.
Accordingly, it is needed to develop a transparent electrode that can be applied to a flexible device. Regarding this, the CNT is most spotlighted in recent years. Since the CNT has excellent electric conductivity and strength and an easily flexible property, a flexible transparent electrode using the CNT can be widely applied, as electrode material, to energy devices such as solar cell and secondary cell as well as display devices such as LCD, OLED and paper-like display.
The most important properties required for the CNT transparent electrode include conductivity, transparency and flexibility. In general, the CNT transparent electrode is prepared by dispersing CNT powders in a solution including a dispersing agent to manufacture CNT ink and then applying the CNT ink on a plastic substrate. In order to increase the conductivity of the CNT transparent electrode, it is important for carriers to move through the CNT itself and to freely move between the CNT and the CNT.
According to the recent research, when an amount of CNT is enough for the CNT to contact each other in a transparent electrode having a CNT network structure, i.e., in a state of percolation threshold or more, the resistance of the CNT itself little influences on the CNT network film. To the contrary, the contact resistance between the CNT and the CNT has a main influence on the resistance of the CNT network film. Therefore, the formation of the CNT network and the decrease in the contact resistance between the CNT and the CNT are important for increase of the conductivity of the CNT transparent electrode.
The CNT is classified into metallic and semiconducting types. The CNT of armchair having no chirality is a metallic CNT having a bandgap of 0 and the CNT is again classified into metallic and semiconducting types depending on a degree of the chirality.
The chirality can be expressed in accordance with a wrapping direction in a carbon plate structure of the CNT. In coordinates of (n, m) indicating the direction, when n and m are multiples of 3, it is referred to as metallic. When n and m are not multiples of 3, it is referred to as semiconducting. In growing the CNT, it is probabilistically said that ⅓ or less of the entire CNTs is metallic and ⅔ or more of the entire CNTs is semiconducting. Since the metallic CNT of armchair is formed very rarely, the amount thereof may be negligible.
When a transparent electrode is formed with CNTs in which the CNTs of the above two types are mixed, a barrier is formed between the CNTs having different bandgaps and the electrons flow. Thereby, the contact resistance between the CNTs is increased to lower the conductivity of the entire CNTs. Therefore, when a transparent electrode is formed with CNTs in which the CNTs of the two types are mixed, it is necessary to lower the contact resistance between the CNTs having different bandgaps.
SUMMARY OF THE INVENTION
Accordingly, the present invention has been made to solve the above problems occurring in the prior art.
An object of the invention is to treat the CNT with chemical having an electron withdrawing functional group, thereby improving the conductivity of a flexible transparent nano-electrode.
In other words, the main object of the invention is to treat CNT with chemical having an electron withdrawing functional group to reduce a contact resistance between the CNTs, thereby improving conductivity of an electrode comprising a CNT thin film prepared using the process.
In order to achieve the above object, the invention provides a CNT thin film treated with chemical having an electron withdrawing functional group and a manufacturing method thereof, and more particularly, a CNT material treated with chemical having an electron withdrawing functional group to reduce a contact resistance between the CNTs and capable of increasing conductivity of an electrode prepared using the process, and a treating method thereof.
Specifically, according to the invention, there is provided a CNT thin film comprising: a plastic substrate; and a CNT composition applied on the plastic substrate. The CNT composition includes a CNT; and chemical connected to the CNT and having an electron withdrawing functional group.
In addition, according to a first embodiment of the invention, there is provided a method of manufacturing a CNT thin film, comprising steps of: preparing a CNT; treating the CNT with chemical having an electron withdrawing functional group; mixing the CNT treated with the chemical with a dispersing agent or dispersing solvent to prepare a CNT dispersed solution; and forming a CNT thin film with the CNT dispersed solution.
Further, according to a second embodiment of the invention, there is provided a method of manufacturing a CNT thin film, comprising steps of mixing a CNT with a dispersing agent or dispersing solvent to prepare a CNT dispersed solution; forming a CNT thin film with the CNT dispersed solution; and treating a surface of the CNT thin film with chemical having an electron withdrawing functional group.
In the mean time, it is possible to implement a CNT electrode comprising the CNT thin film having the structure described above and to realize a thin film transistor (TFT) constituting an electrode and a channel material using the CNT thin film.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features and advantages of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows that a contact resistance is generated between CNTs when a CNT thin film is prepared with CNTs in which metallic CNTs and semiconducting CNTs are mixed;
FIG. 2A shows that an aromatic compound is connected to a CNT surface according to an embodiment of the invention;
FIG. 2B shows that an aliphatic compound is connected to a CNT surface according to an embodiment of the invention;
FIG. 3 shows that electron donating or withdrawing functional groups are arranged depending on functional strengths thereof;
FIG. 4A shows a charge transfer mechanism between chemical having an electron withdrawing functional group and a CNT according to an embodiment of the invention;
FIG. 4B is a schematic view showing barrier changes between CNTs depending on a charge transfer mechanism between chemical having an electron donating or withdrawing functional group and a CNT;
FIG. 5 is a graph showing a Raman result of RBM for a CNT thin film treated with chemical having an electron withdrawing functional group according to an embodiment of the invention;
FIG. 6 is a graph showing a Raman result of BWF for a CNT thin film treated with chemical having an electron withdrawing functional group according to an embodiment of the invention; and
FIG. 7 is a graph showing an XPS π plasmon for a CNT thin film treated with chemical having an electron withdrawing functional group according to an embodiment of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, a preferred embodiment of the present invention will be described with reference to the accompanying drawings. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.
FIG. 1 shows that a contact resistance is generated between CNTs when a CNT thin film is prepared with CNTs in which metallic CNTs and semiconducting CNTs are mixed. A Schottky barrier is generated between a metallic CNT and a semiconducting CNT having different bandgaps. As shown in FIG. 1 , electrons flow between the metallic CNT and the semiconducting CNT contacting each other and having different properties. Thus, the contact resistance is generated between the metallic CNT and the semiconducting CNT having different properties. This results in the decrease of conductivity of the CNTs in which metallic CNTs and semiconducting CNTs are mixed. Therefore, when a transparent electrode is formed using a CNT thin film in which metallic CNTs and semiconducting CNTs are mixed, it is necessary to decrease the contact resistance between the CNTs having different bandgaps.
Calculating the conductivity relating to the contact resistance between the metallic CNT and the semiconducitng CNT, the conductivity of the metallic CNT itself is about 4 e 2 /h and the conductivity of the semiconducting CNT is about 0.1 e 2 /h. The conductivity when the CNTs having different properties are contacted is calculated as shown in a table 1. An arrow in the table 1 indicates the flow direction of the electrons.
TABLE 1
metallic CNT → semiconducting CNT
0.26 e 2 /h
semiconducting CNT → semiconducting CNT
0.06 e 2 /h
semiconducting CNT → metallic CNT
0.008 e 2 /h
(Schottky barrier)
For the above three cases, the electric conductivity is lowest in the third case wherein the electrons flow from the semiconducting CNT to the metallic CNT. In this case, the Schottky barrier is formed, thereby lowering the electric conductivity of the entire CNTs.
Like this, for the CNTs in which the metallic and semiconducting CNTs are mixed, a barrier between the metallic CNT and the semiconducting CNT can be adjusted by treating the CNTs with chemical having an electron withdrawing functional group. This will be described in more detail.
FIG. 2A shows that aromatic compounds 21 , 22 , 23 are adsorbed to a CNT surface 20 according to an embodiment of the invention. Generally, in an aromatic compound, it is possible to introduce a variety of functional groups having benzene as backbone, instead of hydrogen. The aromatic compounds 21 , 22 , 23 shown in FIG. 2A have functional groups of NH 2 , CH 3 and NO 2 that are connected to each aromatic ring thereof, respectively.
From FIG. 2A , it can be seen that each of the aromatic compounds 21 , 22 , 23 is adsorbed on the CNT surface 20 in a direction that the aromatic ring thereof is horizontally contacted to a carbon plate-shaped structure of the CNT. Like this, in the course of the adsorption of the aromatic compounds 21 , 22 , 23 on the CNT surface 20 , electrons are transferred between the aromatic compounds 21 , 22 , 23 and the CNT surface 20 . Depending on the properties of the functional groups of the aromatic compounds 21 , 22 , 23 , the entire resistance of the CNT can be reduced by the electron transfer.
FIG. 2B shows that aliphatic compounds 24 , 25 , 36 are adsorbed to the CNT surface 20 according to an embodiment of the invention. Generally, in an aliphatic compound, it is possible to introduce a variety of functional groups, instead of hydrogen of an alkyl group. The aromatic compounds 24 , 25 , 26 shown in FIG. 2B have functional groups of NH 2 , CH 3 and NO 2 that are connected to each alkyl group thereof, respectively.
From FIG. 2B , it can be seen that each of the aliphatic compounds 24 , 25 , 26 is adsorbed on the CNT surface 20 in a form that it is connected in a carbon ring of a carbon plate-shaped structure of the CNT. As described above with regard to the aromatic compound, in the course of the adsorption of the aliphatic compounds 24 , 25 , 26 on the CNT surface 20 , the electrons are transferred between the aliphatic compounds 24 , 25 , 26 and the CNT surface 20 . Depending on the properties of the functional groups of the aliphatic compounds 24 , 25 , 26 , the entire resistance of the CNT can be reduced by the electron transfer.
FIG. 3 shows that electron donating or withdrawing functional groups are arranged depending on functional strengths thereof. As described above, for the aromatic compound, it is possible to introduce a variety of functional groups having benzene as backbone, instead of hydrogen. The functional groups can be classified into a functional group capable of donating an electron, which is arranged in the right side on the basis of hydrogen in FIG. 3 , and a functional group capable of withdrawing an electron, which is arranged in the left side.
In the arrangements of the respective functional groups shown in FIG. 3 , the functional group capable of donating an electron has the stronger tendency to be oxidized by donating an electron the farther the right side based on the hydrogen. This means that the reductive force for the CNT is higher. In addition, the functional group capable of withdrawing an electron has the stronger tendency to be reduced by taking an electron, the farther the left side based on the hydrogen. This means that the oxidative force for the CNT is higher.
FIG. 4A shows a charge transfer mechanism between chemical having an electron with drawing functional group and a CNT according to an embodiment of the invention. FIG. 4A shows a process in which —CN functional group obtains an electron from backbone of CH 3 CH 2 — to make an extra electron in the functional group and the aromatic compound, in which the backbone is deficient in the electron, is adsorbed on the CNT surface to take an electron from the CNT.
It is described a general mechanism how chemical acts on the CNT when the chemical is adsorbed on the CNT as shown in FIG. 4A .
When a functional group capable of withdrawing an electron is introduced to the backbone of the aromatic or aliphatic compound, the functional group attracts the electron from the backbone, so that the backbone is deficient in the electron and the functional group has the sufficient electrons. In this case, when the chemical is adsorbed to the CNT and the backbone thus acts, the CNT loses an electron. Since the CNT apt to relatively lose an electron is metallic CNT, it reduces a barrier when the barrier is generated, thereby increasing the conductivity.
FIG. 4B is a schematic view showing barrier changes between CNTs depending on a charge transfer mechanism between chemical having an electron donating or withdrawing functional group and a CNT. In FIG. 4B , the case where the CNT is treated with chemical having an electron donating functional group is shown in the left view (a) based on the central view (b) of FIG. 4B , and the case where the CNT is treated with chemical having an electron withdrawing functional group is shown in the right view (c).
In each view of FIG. 4B , the left side based on the central line shows the metallic CNT and the right side shows the semiconducting CNT. Among them, the view (c) shows that the barrier of the CNT is reduced in accordance with the mechanism of FIG. 4A : In other words, it can be seen from the view (c) that for the electron withdrawing functional group, the metallic CNT loses an electron, so that it is moved to the arrow direction and the barrier between the metallic CNT and the semiconducting CNT is thus decreased.
Regarding this, the acid treatment is generally performed for the refinement purpose when manufacturing the CNT. After the acid treatment, the CNT is lightly p-doped as shown in the view (b). When such CNT is treated with chemical having an electron withdrawing functional group, the CNT is more thickly p-doped as shown in the view (c). To the contrary, when the CNT is treated with chemical having an electron donating functional group, the CNT can be de-doped as shown in the view (a).
In followings, with respect to an experiment for manufacturing a CNT thin film related to the mechanism as described above, experimental procedures according to each embodiment is sequentially described.
In the method for manufacturing a CNT thin film according to a first embodiment of the invention, a high-purity single-wall CNT (Southwest) 1 mg is first put in a 20 mL glass bottle, into which 10 ml chemical having an electron withdrawing functional group is then put. Then, it is put in an ultrasonic bath that is then subject to a sonification process for 10 hours to prepare a CNT solution.
Then, a filtering method is used in which a vacuum filtering device is used to pass the CNT solution 10 ml., to an aluminum film (anodisc, 200 nm) to filter it, thereby preparing a CNT bucky paper.
The CNT prepared according to the above manner is treated with chemical having an electron withdrawing functional group, so that the chemical is adsorbed on the CNT surface. At this time, the chemical may be connected to the CNT surface by the electron transfer.
According to an embodiment of the invention, a liquid-phase compound is used as the chemical. For a solid compound, it should be dissolved and then used by using another solvent. Therefore, an effect thereof may be deteriorated by the functional group present in the solvent. In addition, the chemical may include a mono-functional group only, or may include plural functional groups (bi-functional group and tri-functional group etc) which are homogeneous or heterogeneous.
Furthermore, according to an embodiment of the invention, as the functional group, a group capable of withdrawing an electron may be used such as —CF 3 , —CN, —S═O, —SO 3 H, —NO 2 , —NR 4 + , —COR, —COOR, —CONR 2 , —F, —Cl and —Br where R is H or alkyl group or aryl group of C 1 -C 15 . In addition, the chemical including the functional group may include dichloroethane, dibromoethane, iodobenzene, formic acid, acetic acid, formanide, dimethyl sulfoxide, nitromethane, nitrobenzene, nitric acid, acetonitrile, benzonitrile, perfluoro alkane and the like.
The CNT bucky paper treated with the chemical is mixed with a dispersing agent or dispersing solvent to be re-dispersed, so that a CNT dispersed solution is prepared. Using the CNT dispersed solution, a CNT thin film is manufactured.
After the CNT thin film is manufactured through the respective steps of the experimental process, a surface resistance is measured with a surface resistance measurer. The resistance is measured for each of the various functional groups.
In addition, in a method for manufacturing a CNT thin film according to a second embodiment of the invention, a CNT dispersed solution is first prepared. Firstly, a high-purity CNT 1 mg is put in a 20 mL glass bottle, into which 10 ml N-methylpyrrolidone (NMP) is then put. Then, it is put in an ultrasonic bath that is then subject to a sonification process for 10 hours. The CNT-NMP solution is put in a 50 mL conical tube, which is then centrifugally separated at 10,000 rpm for 10 minutes. After the centrifugal separation, only the CNT dispersed solution that is not deposited is taken to prepare a CNT dispersed solution.
Then, a filtering method as the method for manufacturing a CNT thin film is used in which a vacuum filtering device is used to pass the CNT solution 2 ml, to an aluminum film to filter it, thereby preparing a CNT thin film.
The CNT prepared according to the above manner is treated with chemical having an electron withdrawing functional group, so that the chemical is adsorbed on the CNT surface. At this time, the chemical may be connected to the surface of the CNT by the electron transfer.
After the CNT thin film is manufactured through the respective steps of the experimental process, a surface resistance is measured with a surface resistance measurer. The resistance is measured for each of the various functional groups.
As the measure results of the surface resistance for the CNT thin films prepared according to the respective embodiments, the surface resistance of the CNT thin film treated with the chemical having an electron withdrawing functional group was decreased. In other words, the contact resistance between the CNTs can be reduced by treating the CNT with the chemical having an electron withdrawing functional group.
A table 2 shows the measure results of the resistance for the CNT bucky paper treated with the chemical according to the experimental processes described with reference to the first embodiment of the invention. At this time, the hexane, which is the aliphatic compound having no functional group, and the benzene, which is the aromatic compound having no functional group, were used as comparative examples. Also, it is shown the surface resistances resulting from the introduction of the various electron withdrawing functional groups (unit: Ω/sq).
TABLE 2
From the data of the table 2, when comparing the hexane and benzene having no functional group as comparative compounds with the compounds having the other functional groups, it can be seen that the surface resistance was much decreased in the compounds having an electron withdrawing functional group.
In other words, the functional group capable of withdrawing an electron attracts the electron from the backbone, so that the backbone deficient in the electron and the chemical including the backbone is adsorbed to the CNT to take an electron from the CNT. Like this, as the CNT loses an electron, the barrier between the CNTs is reduced and the contact resistance between the CNTs is decreased. As a result, the surface resistance is reduced.
The mechanism and the experimental result can be also confirmed in graphs described below.
FIG. 5 is a graph showing a Raman result of RBM (Radial Breathing Mode) for a CNT thin film treated with chemical having an electron withdrawing functional group according to an embodiment of the invention. Referring to points indicated with an arrow in FIG. 5 , peaks of strength depending on the wavelengths are rapidly decreased, which means the decrease of RBM. Like this, the decrease of RBM in the Raman result is a proof that there is something strongly adsorbed on the CNT surface. From this, it can be seen that the chemical having a functional group on the CNT surface is connected by the electron transfer.
FIG. 6 is a graph showing a Raman result of BWF for a CNT thin film treated with chemical having an electron withdrawing functional group according to an embodiment of the invention. Referring to points indicated with an arrow in FIG. 6 , it can be seen that the strengths are decreased depending on the wavelengths, i.e., the line widths of BWF are decreased. Like this, the decrease of the BWF line width in the Raman result is a proof that the CNT loses an electron. In other words, it means that the chemical adsorbed on the CNT surface obtains an electron (i.e., reduced).
FIG. 7 is a graph showing an XPS π plasmon for a CNT thin film treated with chemical having an electron withdrawing functional group according to an embodiment of the invention. Referring to FIG. 7 , the CNT thin film treated with chemical having an electron withdrawing functional group is changed into the low energy states as π plasmon is changed from 5.0 to 4.0, which means that the CNT loses an electron. Therefore, from the change in π plasmon of XPS, it can be seen that the CNT loses an electron (i.e., oxidized).
As described above, according to the invention, the CNT is treated with the chemical having an electron withdrawing functional group. Through the process, the contact resistance between the CNTs is reduced. In addition, the conductivity of an electrode prepared using the process can be increased.
Furthermore, the CNT treated with the chemical having an electron withdrawing functional group can be applied to a variety of fields such as sensor, memory and cell using the CNT.
While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made thereto without departing from the spirit and scope of the invention as defined by the appended claims.
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Disclosed are a carbon nano-tube (CNT) thin film treated with chemical having an electron withdrawing functional group and a manufacturing method thereof. Specifically, the CNT thin film comprises a CNT composition to be applied on a plastic substrate. The CNT composition comprises a CNT; and chemical connected to the CNT and having an electron withdrawing functional group. In addition, the method for manufacturing a CNT thin film comprises steps of preparing a CNT; treating the CNT with chemical having an electron withdrawing functional group; mixing the CNT treated with the chemical with a dispersing agent or dispersing solvent to prepare a CNT dispersed solution; and forming a CNT thin film with the CNT dispersed solution. According to the CNT thin film and the manufacturing method thereof, a resistance of an electrode is decreased to improve the electric conductivity of the electrode.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a light emitting diode package and a method of manufacturing the same.
2.Description of the Related Art
A light emitting diode (LED) is a semiconductor device that can realize a light source of various colors using a compound semiconductor material such as GaAs, AlGaAs, GaN, InGaN, and AlGaInP.
Generally, color, brightness, luminosity, etc. determine a characteristic of an LED. The characteristic of the LED is generally determined by a compound semiconductor material used in the LED, but is greatly influenced by a package structure for mounting chips.
In particular, as information telecommunication devices have become small-sized and slim, elements such as a resistor, a condenser, and a noise filter have become small-sized even more and been directly mounted in a printed circuit board (PCB) (a surface mount device (SMD) type).
FIG. 1 is a view illustrating an LED package structure according to a related art.
Referring to FIG. 1 , an LED package according to a related art includes a PCB 200 having a reflective hole 202 in which an LED 210 is mounted, a reflective coating layer 201 coated with Ag in the reflective hole 202 , a cathode electrode 220 and an anode electrode 230 connected with the reflective coating layer 201 , and wires 218 connecting the LED 210 with the reflective coating layer 201 .
A central region of the reflective coating layer 201 is electrically cut and the reflective coating layer 201 is electrically connected with the cathode electrode 220 and the anode electrode 230 . That is, a P electrode and an N electrode of the LED 210 are electrically connected with the reflective coating layer 201 such that the P and N electrodes are connected with the cathode electrode 220 and the anode electrode 230 .
The cathode electrode 220 and the anode electrode 230 may be formed by solder bonding. The cathode electrode 220 and the anode electrode 230 are formed, and then a mold lens 250 is formed on the reflective coating layer 201 of the PCB 200 in order to prevent an oxidation of the wires 218 , reduce a light loss due to an air resistance, improve thermal conductivity.
Meanwhile, light emitted from the LED 210 is reflected a lot inside the mold lens 250 , that is, internal reflection occurs a lot inside the mold lens, decreasing the transmittance and eventually decreasing the light efficiency.
SUMMARY OF THE INVENTION
Accordingly, the present invention is related to a light emitting diode and a method of manufacturing the same that substantially obviates one or more problems due to limitations and disadvantages of the related art.
The invention provides an LED package with an excellent light efficiency.
The invention provides an LED package with a simplified manufacturing method thereof.
The invention provides an LED package with an excellent physical stability.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
The embodiment of the invention provides an LED package, comprising: a PCB; an electrode pad formed on the PCB; an LED formed on the PCB; a first molding formed on the LED; and a second molding formed on the first molding.
The embodiment of the invention provides an LED package comprising: a PCB; an electrode pad formed on the PCB; an LED formed on the electrode pad; a first molding formed on the LED; and a second molding formed on the electrode pad and the first molding.
The embodiment of the invention provides a method of manufacturing an LED package, the method comprising: forming an electrode pad on a PCB; forming an LED on the PCB; forming a first molding on the LED by a dispensing method; and forming a second molding on the first molding by a dispensing method.
It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principle of the invention. In the drawings:
FIG. 1 is a view illustrating an LED package structure according to a related art;
FIG. 2 is a sectional view of an LED package according to an embodiment of the present invention;
FIG. 3 is a flowchart illustrating a method of forming an LED package according to an embodiment of the present invention; and
FIG. 4 is a view illustrating a first molding and a second molding of another shape in an LED package according to an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
It will be understood that when an element is referred to as being ‘on’ a layer, it can be directly on the layer, and one or more intervening layers may also be present.
Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings.
FIG. 2 is a sectional view of an LED package according to an embodiment of the present invention.
Referring to FIG. 2 , the present invention exemplarily illustrates a SMD type LED package manufactured using a metal core printed circuit board (MCPCB) as an LED package.
An LED package according to an embodiment of the present invention includes an MCPCB 1 , two electrode pads 2 formed on the MCPCB 1 , an LED 3 formed on the electrode pads 2 , two wires 4 connecting the LED 3 with the electrode pads 2 , and a first molding 6 formed outside the LED 3 and the wires 4 and a second molding 7 formed outside the first molding 6 .
The LED package may further include a guide unit 5 guiding the first and second moldings 6 and 7 to be easily formed.
The electrode pads 2 may be formed of a conductive metal material such as Ag, Au, and Cu on the MCPCB 1 and are connected with the LED 3 through the wires 4 . The two electrode pads 2 are not electrically connected, power is supplied to the LED 3 through the two wires 4 .
Also, the electrode pads 2 reflect light emitted from the LED 3 to increase the light efficiency.
Also, the LED 3 may be formed on the MCPCB 1 or may be formed on the electrode pads 2 , as illustrated in FIG. 2 .
In the present invention, the LED 3 is mounted on a planar PCB. That is, the LED 3 is formed on a planar portion of the PCB, and the portion in which the LED 3 is formed and the electrode pads 2 connected with the wires 4 are on the same plane. The MCPCB 1 may be used as the PCB.
In the present invention, the second molding 7 is formed in a hemispheric shape. That is, an outer circumference of a contact surface between the second molding 7 and the MCPCB 1 is formed in a circular shape.
In particular, a maximum distance of the contact surface between the second molding 7 and the MCPCB 1 , that is, a diameter of a circle formed of the outer circumference of the contact surface is 1.6-2.4 times larger than a maximum distance between the MCPCB 1 and the second molding 7 , that is, a height of the second molding 7 . In such a condition, the light efficiency may be maximized.
In other words, a contact surface between the first and second moldings 6 and 7 and the MCPCB 1 is formed in a circular shape, and a diameter of the circular contact surface is formed 1.6-2.4 times larger than the height of the second molding 7 .
FIG. 4 is a view illustrating a first molding and a second molding of another shape in an LED package according to an embodiment of the present invention.
Referring to FIG. 4 , the first molding 6 may be formed to contact the MCPCB 1 and the second molding 7 may be formed to contact only a surface of the first molding 6 without contacting the MCPCB 1 .
In this case, a maximum distance of a contact surface between the first molding 6 and the MCPCB 1 , that is, a diameter of a circle formed of an outer circumference of the contact surface is formed 1.6-2.4 times larger than a maximum distance between the MCPCB 1 and the second molding 7 , that is, a height of the second molding 7 .
In other words, the contact surface between the first molding 6 and the MCPCB 1 is formed in a circular shape, and a diameter of the circular contact surface is formed 1.6-2.4 times larger than the height of the second molding 7 .
Meanwhile, a jig may be used to form the second molding 7 in a hemispheric shape having a height and a diameter in the ratio of 1:1.6-2.4, but a manufacturing method using a jig is complicated.
Therefore, in the present invention, the first and second moldings 6 and 7 are formed by dispensing silicone of different kinds, respectively.
Silicone having a high strength and an excellent interfacial adhesion may be used for the first molding 6 in order to protect the LED 3 and the wires 4 , and silicone having an excellent adhesion with respect to the first molding 6 may be used as the second molding 7 in order to protect the first molding 6 against external impact.
The first molding 6 may be formed by dispensing a resin including siloxane and phenyl, and the second molding 7 may be formed by dispensing a resin including siloxane, platinum, and silica to the outside of the first molding 6 .
The guide unit 5 may be selectively formed to prevent the resin from flowing down during the forming of the second molding 7 using a dispensing method.
The guide unit 5 may be formed of a nonconductive material in a predetermined shape, for example, a shape of a circular ring or a rectangular ring with a predetermined height on the electrode pads 2 .
As described above, the LED package according to the present invention includes the LED 3 formed on the planar MCPCB 1 and the first and second moldings 6 and 7 formed in a hemispheric shape outside the LED 3 using a dispensing method.
In the present invention, when a material with a high refraction index (RI) is used in the first molding, compared to the second molding, for example, when a material with an RI of 1.51 is used in the first molding and a material with an RI of 1.46 is used in the second molding, the transmittance is improved and thus the light efficiency is improved. This is because the first molding serves as a buffer, improving the transmittance since an RI of air is 1. On the other hands, in a molding structure of the related art, although a material with an RI of 1.51 or 1.46 is used, internal reflection occurs a lot inside the molding, compared to the present invention, and thus the light efficiency decreases by approximately 10%.
Therefore, the light efficiency of the LED package may be maximized and a manufacturing method thereof is simple.
In particular, the present invention uses materials with an excellent adhesion with respect to each other for the first and second moldings 6 and 7 and thus may prevent a decrease in an adhesion between the first and second moldings 6 and 7 caused by heat generated from the LED 3 .
Therefore, a physical stability of the LED is excellent.
Hereinafter, a method of forming an LED package according to the present invention will be described.
FIG. 3 is a flowchart illustrating a method of forming an LED package according to an embodiment of the present invention.
Conductive electrode pads 2 of a predetermined thickness are formed on an MCPCB 1 (S 301 ). The electrode pads 2 are formed by plating a conductive metal material such as Ag, Au, and Cu to a predetermined thickness on the MCPCB 1 using electroplating and etching the metal material such that the electrode pads are spaced from each other by a predetermined distance as illustrated in FIG. 2 .
Next, an LED 3 is mounted on the electrode pads 2 . The LED 3 is connected with the electrode pads 2 through wires 4 (S 302 ).
Here, the LED 3 is mounted and connected with the wires 4 , and then a guide unit 5 of a shape of circular ring may be additionally formed around the LED 3 on the electrode pads 2 .
A first molding 6 is formed on the LED 3 using a dispensing method (S 303 ). Silicone having a high strength and a good interfacial adhesion may be used for the first molding 6 .
Next, a second molding 7 is formed on the first molding 6 (S 304 ). Silicone having an excellent adhesion with respect to the first molding 6 may be used for the second molding 7 .
The present invention exemplarily illustrates the SMD type LED package, but is not limited thereto and may be applied to an LED package where an LED is formed in a reflective hole formed in an MCPCB.
According to the LED package of the present invention, since the first and second moldings are easily formed using a dispensing method, additional processes are not necessary. Also, adherence between the first and second moldings is improved to increase the physical stability.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention. Thus, it is intended that the present invention covers the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
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A light emitting diode (LED) package is provided. The LED package includes a printed circuit board (PCB), an electrode pad, an LED, a wire, and first and second moldings. The electrode pad and the LED are formed on the PCB. The wire electrically connects the LED with the electrode pad. The first molding is formed on the LED and the second molding is formed on the first molding.
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RELATED APPLICATIONS
This application is a continuation of application Ser. No. 10/342,379, filed on Jan. 14, 2003, now abandoned, which is a continuation of application Ser. No. 10/180,399, filed on Jun. 26, 2002, now abandoned, which is a division of application Ser. No. 09/724,522, filed on Nov. 28, 2000, now U.S. Pat. No. 6,441,002, which claims priority of Provisional Application No. 60/167,922, filed Nov. 29, 1999.
BACKGROUND OF THE INVENTION
The present invention relates to polymorphic, amorphous and hydrated forms of the title compound which has the chemical structure shown below:
The compound is a potent and selective cyclooxygenase-2 (COX-2) inhibitor, useful primarily in the treatment of inflammation, pain and fever as well as other COX-2 mediated diseases, such as described in PCT Publication Nos. WO96/10012 and WO96/16934. Compound A is described in U.S. Pat. No. 5,861,419 granted on Jan. 19, 1999 (Example 23) incorporated by reference in its entirety.
Bipyridyl compounds generally are highly crystalline, poorly water soluble and hydrophobic, resulting in difficulties in the preparation of pharmaceutical formulations and problems associated with bioavailability. Accordingly, efforts were made to discover other forms of Compound A and to investigate the properties thereof. There were discovered three additional polymorphic forms, an amorphous form and two hydrates.
SUMMARY OF THE INVENTION
Polymorphic forms of Compound A, for purposes of this invention, are identified as Form I (onset of melting, m.p. 134-136° C., peak m.p. 138° C.), Form II (onset of melting, m.p˜131° C., peak m.p. 133° C.), Form III (onset of melting, m.p.˜133° C., peak m.p. 135° C.) and Form IV (onset of melting, m.p.˜134° C., peak m.p. 136° C.). Forms I through IV are anhydrous. An amorphous form and two hydrates have also been identified.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is described in connection with the appended drawings in which:
FIG. 1 is the X-ray powder diffraction (XRPD) pattern of Form I;
FIG. 2 is the XRPD pattern of Form II;
FIG. 3 is the XRPD pattern of Form III;
FIG. 4 is the XRPD pattern of Form IV;
FIG. 5 is the XRPD pattern of the hemihydrate;
FIG. 6 is the XRPD pattern of the sesquihydrate;
FIG. 7 is a thermogravimetric (TG) scan of the hemihydrate, and
FIG. 8 is a TG scan of the sesquihydrate.
DETAILED DESCRIPTION
Polymorphic forms of Compound A, for purposes of this invention, are identified as Form I (onset of melting, m.p. 134-136° C., peak m.p. 138° C.), Form II (onset of melting, m.p˜131° C., peak m.p.˜133° C.), Form III (onset of melting, m.p.˜133° C., peak m.p. 135° C.) and Form IV (onset of melting, m.p.˜134° C., peak m.p. 136° C.). Forms I through IV are anhydrous. An amorphous form and two hydrates have also been identified.
The polymorphs of the present invention are synthesized in accordance with the following examples which are illustrative.
PREPARATIVE EXAMPLE 1
The starting material compound A is made in accordance with Example 23 of U.S. Pat. No. 5,861,419 granted on Jan. 19, 1999.
EXAMPLE 1
Form II
Form II is obtained by crystallizing compound A obtained in accordance with Preparative Example 1 from ethyl acetate.
Differential Scanning Calorimetry showed an extrapolated onset of melting at 131±1° C., and a peak melting point of 132.5±0.1° C.
EXAMPLE 2
Form I
Form I was obtained by recrystallizing Form II obtained as described in Example 1 from a solvent mixture of isopropanol/hexane.
EXAMPLE 3
Form IV
Form IV appeared spontaneously in batches of compound A prepared as in Preparative Example 1.
Form IV is alternatively prepared by contacting Form I as described in Example 2 with an organic solvent, e.g., toluene and heptane, and then recrystallizing at a temperature less than 45° C., such as about 15° C.
Form IV is also alternatively prepared by dissolving Form II in an organic solvent, such as toluene and heptane, and then recrystallizing at a temperature less than 45° C., such as about 15° C.
EXAMPLE 4
Form III
Form III was prepared by stirring Form IV from Example 3 in water for 1 day, and then dehydrating at 90° C. in vacuo until Form III is present. The melting temperature onset was ca. 133° C. with an enthalpy of fusion of approximately 24 kJ/mol. The peak melting temperature was 135° C.
Alternatively, using the hemihydrate of Example 5, conducting a temperature XRPD of the hemihydrate at 130° C. resulted in the production of Form III.
EXAMPLE 5
Hemihydrate
The hemihydrate form of compound A is produced by stirring Form IV obtained in accordance with Example 3 in water for at least 1 day. XRPD analysis of the solid produced a diffractogramidentical to previous hemihydrate samples obtained for Form II. Thermogravimetry confirmed that Form IV had converted to the hemihydrate form, exhibiting a sharp weight loss of 2.45% on heating, which corresponds to a mole ratio of water to drug of 0.50%.
EXAMPLE 6
Sesquihydrate
The sesquihydrate of compound A is obtained by combining Form I in accordance with Example 2 and water (approximately 1.5 mol/mol compound).
EXAMPLE 7
Amorphous
The amorphous form of compound A is obtained by heating any polymorph to above its melting temperature (for example to 145° C.) under nitrogen, followed by quench cooling to room temperature under a dry atmosphere.
Characterization of Polymorphs
The polymorphic forms of compound A are characterized using the following procedures.
X-Ray Powder Diffraction Pattern Analysis
Polymorph 1 is crystalline by XRPD using a Scintag XDS-2000, Si(Li) Peltier-cooled solid state detector using a Cu K alpha source at 45 kV and 40 mA, and divergent beam (2 mm and 4 mm) and receiving beam slits (0.5 mm and 0.2 mm). Peak positions were calibrated using a standard silicon disk (97.5% pure).
Temperature XRPD studies were carried out under nitrogen, using a gold-plated copper stage with a Beryllium window on the cover. A Micristar temperature controller monitored and controlled the temperatures.
Temperature XRPD studies demonstrated that the compound did not undergo any transitions prior to melting, which was complete at 140° C., and that there was no conversion to a different polymorphic form. Similar results were obtained for Form II. The material remained amorphous and did not recrystallize.
Table 1 below lists the XRPD peak locations for Forms I, II, III and IV.
TABLE 1 X-ray Crystalline Reflections in °2 theta That Are Characteristic of Polymorphs I, II, III and IV using Cu K alpha Form I Form II Form III Form IV 7.1 5.6 5.7 9.7 9.4 10.5 11.8 10.7 16.1 15.5 17.6 15.2 20.1 17.1 19.5 22.7 22.4 21.7 24.1 23.5 23.6
XRPD patterns for Forms I-IV are shown in FIGS. 1-4 .
XRPD patterns for the two hydrate forms are shown in FIGS. 5 and 6 .
Differential Scanning Calorimetry (DSC)
The extrapolated melting temperature onset of Form I was 134.0±0.6° C. with an enthalpy of fusion of 27.2±0.9 kJ/mol at 10 deg/min under nitrogen in crimped aluminum pans (FIG. 1 ). The peak melting temperature was 138° C.
When measured using a TA Instruments DSC2910 instrument, at 10° C./min under a nitrogen atmosphere in an open aluminum pan, the onset of melting was 136° C. and the peak melting temperature was as described above. There were no significant changes with DSC scanning rate other than the expected shift in peak temperature. DSC thermal behaviour of Form I in crimped sample pans under nitrogen (60 mL/min) was measured using a Seiko robotic DSC (RDC-220) at 2, 10 and 20 deg/min. The DSC was calibrated for temperature and heat flow with gallium, indium and tin.
The melting temperature onset and enthalpy of fusion of Form I were slightly higher than those observed for Form II. These polymorphic forms do not recrystallize upon cooling from the melt nor do they recrystallize on reheating. The glass transition temperature of the amorphous form (mid-point, 10K/min, crimped aluminum pan) is 55° C.
Table 2 provides a comparison of the extrapolated melting temperature onset, T o , and the enthalpy of fusion, ΔH, for Forms I, II, III and IV.
TABLE 2
Extrapolated melting temperature onset, T o , and Enthalpy of
Fusion obtained by DSC at 10K/min in crimped pans under nitrogen
Polymorphic form
T o (° C.)
Enthalpy of fusion, kJ/mol
Form I
134.0 ± 0.6
27.2 ± 0.9
Form II
131 ± 1
25.8 ± 0.2
Form III
133
22.7
Form IV
134.0 ± 0.1
27.9 ± 0.2
The DSC thermogram for Form IV, obtained at a scanning rate of 10° C./min under nitrogen in crimped aluminum pans, consisted of a single symmetrical endotherm with a mean onset melting point of 134.0±0.1° C. and a heat of fusion of 27.9 kJ/mol. A scanning rate of 2° C./min confirmed that the observed endotherm was due to a single endothermic transition. The enthalpy of fusion of the different polymorphs are also similar.
Forms I and IV have similar solubilities. Form IV is slightly less soluble and slightly more stable at temperatures below 45° C. Forms I and IV are enantiotropic with Form IV converting to Form I at temperatures greater than 45° C. when in contact with organic solvents.
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Polymorphic, amorphous and hydrated forms of the title compound having the following structure:
are disclosed. The compound is a potent and selective cyclooxygenase-2 (COX-2)inhibitor.
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The Government has rights in this invention pursuant to contract Number F1962885-C-0002 awarded by the Department of the Air Force.
This is a division of application Ser. No. 07,124,346, filed Nov. 20, 1987, U.S. Pat. No. 4,889,583, which is a continuation of Ser. No. 06/805,117, filed Dec. 4, 1985, now abandoned.
BACKGROUND OF THE INVENTION
This invention relates generally to the conversion of amorphous or polycrystalline semiconductor materials to substantially single crystal semiconductor material by a process known as zone-melting recrystallization.
From transistors to very large scale integration of complex circuitry on a single chip, the field of solid state electronics has been built largely upon the abundant nonmetallic element silicon. Large diameter single crystal boules of silicon are sliced into wafers on which dopants, insulators and conductors are applied today using a variety of processes. Over the past few years, a major effort has been devoted to developing a new silicon-based technology involving the preparation of very thin films of pure single crystal silicon on the order of one-half micron thick, compared to the one-half millimeter thickness of typical silicon wafers. The new technology is called silicon-on-insulator (SOI) technology because the thin silicon film is supported by an insulating substrate. An efficient, reliable and economical process for producing thin film single crystal silicon has eluded researchers until now.
In comparison to device performance in bulk silicon, SOI promises significant advantages:
(1) improved speed performance in discrete devices and circuits resulting from reduced parasitic capacitance;
(2) simplified device isolation and design layout, yielding potentially higher packing densities; and
(3) radiation hard circuits for space and nuclear application.
In addition, new SOI technologies may also be utilized for three-dimensional integration of circuits.
At present, there is one mature SOI technology, silicon-on-sapphire (SOS). However, the commercial utilization of SOS has been severely limited by its high cost, relatively poor crystalline quality, and difficulty in handling and processing in comparison to bulk Si.
Recently, a new SOI technology called zone-melting recrystallization (ZMR) based on standard silicon wafers rather than sapphire crystals has exhibited the potential for displacing SOS and for utilization on a much larger scale by the semiconductor industry. The development of ZMR has been frustrated by processing problems related to the physical chemistry of the interface between the molten silicon and adjacent silicon dioxide layers which gives rise to the so-called silicon beading phenomenon during ZMR.
SOI by the ZMR technique is produced by recrystallizing a fine-grained Si film on an insulating substrate. A typical sample structure consists of a silicon wafer coated with a 1 micron thermally grown SiO 2 insulating layer, a half micron polycrystalline silicon (poly-Si) layer formed by low pressure chemical vapor deposition (LPCVD), topped by a 2 micron layer of CVD SiO 2 . The last layer forms a cover to encapsulate the polysilicon film constraining it while the film is being recrystallized.
SOI by the ZMR technique is described in a paper entitled "Zone Melting Recrystallization of Silicon Film With a Movable Strip Heater Oven" by Geis et al, J. Electrochem. Soc. Solid State Science and Technology, Vol. 129, p. 2813, 1982.
The sample is placed on a lower graphite strip and heated to a base temperature of 1100°-1300° C. in an argon gas ambient. Silicon has a melting point of about 1410° C.; SiO 2 has a higher melting point, about 1710° C. Additional radiant energy is typically provided by a movable upper graphite strip heater which produces localized heating of the sample along a strip to a temperature between the two melting points. Moving like a wand, the upper heater scans the molten zone across the sample leaving a recrystallized SOI film beneath the solid SiO 2 cap.
One of the major problems with this procedure arises out of an interaction between the surface tension of the molten silicon and the interface with the adjacent capping and insulating SiO 2 layers resulting in poor wetting by the molten silicon. The silicon breaks apart and agglomerates into small beads or stripes. The resulting delamination can fracture the cap and cause defects in the crystalline structure of the silicon.
The silicon beading phenomena during ZMR is described in Weinberg al, "Investigation of the Silicon Beading Phenomena During Zone Melting Recrystallization", Applied Physics Letters 43(12) 15 December 1983, page 1105. This article also refers to the apparently beneficial effect of a silicon nitride (Si 3 N 4 ) CVD overlay on top of the SiO 2 cap. This arrangement appeared to improve the wetting properties of the molten silicon on the silicon dioxide cap. A similar result is described in U.S. Pat. No. 4,371,421 to Fan et al entitled "Lateral Epitaxial Growth by Seeded Solidification", assigned to the assignee of the present application. The Weinberg article attributes the apparent wetting enhancement to the presence of nitrogen atoms not only in the encapsulation layers but in particular at the interface between the silicon layer and the overlying cap. Atomic nitrogen from the silicon nitride cladding probably diffuses through the 2 micron SiO 2 cap to the poly-Si/cap interface and promotes wetting of the molten silicon on the SiO 2 surface. However, problems with uniformity and reproducibility have arisen because of difficulty in controlling deposition of the nitride layer by CVD or by sputtering. Moreover, at best, this cladding technique does not readily lend itself to high volume simultaneous mass production or batch processing.
SUMMARY OF THE INVENTION
Accordingly, the general purpose of the present invention is to introduce the right amount of nitrogen to the poly-Si/SiO 2 cap interface in a uniform, controlled fashion to improve wetting for encapsulated SOI films undergoing ZMR. Instead of using a nitride cladding layer, the SiO 2 cap is exposed to a high temperature anneal in a reactive nitrogen-containing ambient. Annealing the capped SOI in ammonia (NH 3 ) with an intermediate oxidation step yields excellent wetting properties during ZMR. A wide range of process parameters is effective. The preferred anneal consists of:
(1) 1-3 hours in 100% NH 3 ;
(2) 20 minutes in O 2 ; and
(3) 1-3 hours in 100% NH 3 ,
all at 1100° C. The oxygen anneal appears to shorten the annealing time by enabling more nitrogen to reach the interface more quickly. A further variation on this process is to use an intermediate annealing step following deposition of a portion of the cap, i.e., to a partial depth (e.g., 0.2 micron) followed by deposition of the remainder of the cap to the full depth (e.g., 2 microns). The NH 3 anneal produces better ZMR samples and can be performed as a batch process.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a schematic isometric view of an encapsulated SOI wafer undergoing ZMR.
FIG. 2 is a schematic diagram of the cross-section of a typical SOI structure.
FIG. 3 is an overall process flow diagram.
FIG. 4 is a detailed process flow diagram for the capped SOI forming step of FIG. 3.
FIG. 5 is a detailed process flow diagram for the annealing step of FIG. 3.
FIG. 6 is a schematic diagram of the SOI structure in cross-section illustrating the diffusion of nitrogen to the Si/SiO 2 cap interface in the annealing atmosphere.
FIG. 7 is a process flow diagram of an alternate technique involving formation of the cap in steps with an intervening NH 3 anneal.
FIG. 8 is a process flow diagram of an alternate technique involving the NH 3 anneal before forming the cap.
DETAILED DESCRIPTION
The following description generally relates to silicon semiconductors. While silicon is by far the most important semiconductor material in use today, the invention is applicable by analogy in the epitaxial growth of other semiconductor materials such as gallium arsenide and germanium.
FIG. 1 illustrates the thermal components of a typical ZMR apparatus, namely, the stationary lower strip heater and the movable upper strip heater. The lower strip heater is formed by a thin rectangular plate of graphite. Opposite ends of the strip heater are connected in circuit to a source of electrical current in order to achieve controlled heating of a single wafer with formed SOI structure. The movable upper heater typically comprises an elongated graphite strip with a square cross-section of about 1 sq mm in area. The length of the upper heater more than spans the wafer and is oriented parallel to the reference face of the wafer spaced about 1 mm above the wafer surface. The ends of the upper heater are connected in an electrical circuit for resistive heating.
The layers of an encapsulated SOI wafer are diagramed in cross-section in FIG. 2. A typical sample consists of a silicon wafer coated with a 1 micron thermally grown SiO 2 layer, a 0.5 micron poly-Si layer formed by LPCVD, topped by a 2 micron layer of CVD SiO 2 . Prior to the present process development, an additional 30 nm cladding layer Si-rich Si 3 N 4 was deposited by RF sputtering or CVD on top of the SiO 2 cap to promote wetting of the molten Si film on SiO 2 during ZMR. The otherwise useless nitride layer is obviated by the present invention.
The wafer of FIG. 2 is placed cap-side-up on the lower strip heater (FIG. 1). The sample is heated to a base temperature of 1100°-1300° C., typically in an Argon gas ambient. The upper heater is heated to about 2200° C. The strip-like zone beneath the rod is heated to a temperature just above the poly-Si melting point, e.g., 1430° C., well below the melting point of SiO 2 , thus melting the polysilicon in a band beneath the solid SiO 2 cap. As the upper heater moves across the face of the wafer, the molten zone is scanned across the sample leaving behind a recrystallized SOI film. In this manner, the polycrystalline silicon layer is converted to a single crystal layer suitable for semiconductor devices.
The interaction between the surface of the molten silicon and the adjacent SiO 2 surfaces involves a property known as wetting. Water, for example, beads up on a hydrophobic surface like wax due to poor wetting. The angle formed between the outer skin of a liquid droplet and the solid surface is called the wetting angle or contact angle. For water, for example, the more hydrophobic the surface, the higher the contact angle. The contact angle of mercury on glass, for example, is so high (greater than 90°) that a convex miniscus is formed at the top of a mercury column in a glass tube. For silicon, like other materials, the contact angle is not solely a function of the molten material but is affected by the nature of the solid SiO 2 surface as well. Molten silicon on silicon dioxide exhibits a high contact angle, nominally, 87°, characteristic of high beading potential. In contrast, molten silicon on silicon nitride (Si 3 N 4 ) exhibits a low contact angle of about 25°. A description of the physical chemistry which accounts for this difference in contact angle is beyond the scope of this discussion although it appears that the lower surface energy of the nitride enables the molten silicon to wet the nitride surface better. That is, silicon beads up less on nitride than on silicon dioxide. Analysis has shown that wetting of molten Si in the SOI structure is best for small contact angles, i.e., much less than 90°. The molten Si is susceptible to beading or agglomeration as the contact angle approaches or exceeds 90°.
During ZMR of encapsulated SOI films, beading up of the molten silicon at the SiO 2 cap interferes with recrystallization and causes stress fractures in the cap itself. Either type of defect is unacceptable in that it destroys the uniformity of the single crystal silicon.
It is believed that nitrogen atoms in the nitride cladding layer used in the past diffused through the 2 micron CVD SiO 2 cap to the interface with poly-Si. The presence of nitrogen at the interface apparently promoted wetting of the molten silicon on the SiO 2 cap by lowering the contact angle.
As shown in FIG. 3, the present invention represents a better technique for introducing an appropriate amount of nitrogen to the poly-Si/CVD SiO 2 cap interface in a uniform, controlled fashion resulting in improved SOI films after ZMR. The capped SOI structure of FIG. 2 with no nitride cladding is annealed before ZMR at a temperature substantially below the melting point of silicon in an atmosphere rich in reactive nitrogen. A wide range of process parameters are effective, however, a 100% NH 3 atmosphere at 1100° appears to work best. At this temperature, the gas decomposes sufficiently at the SiO 2 surface allowing nitrogen atoms to diffuse through the SiO 2 cap. The cap/silicon interface appears to have an affinity for nitrogen atoms. After the high temperature anneal, a concentration of nitrogen exists at the interface with a concomitant improvement in wetting properties. After annealing, the treated wafer is inserted in the heating apparatus of FIG. 1 for ZMR of the silicon. Because of the lowered contact angle at the Si/SiO 2 cap boundary, beading or stripping of the molten silicon is suppressed and stress on the cap is thereby sufficiently diminished to leave the cap intact. If too much N is present at the Si/SiO 2 cap boundary, although wetting is excellent, ZMR results in poor crystal quality of the SOI film.
The process is described in more detail in FIGS. 4 and 5. Forming the encapsulated SOI structure begins with preparing a standard 3-inch silicon wafer typically 500 microns thick. The insulating layer is formed by growing SiO 2 thermally or by CVD on top of the wafer to a depth of 0.5 to 3.0 microns. If desired, before applying the next layer, a seeding pattern can be created as described in U.S. Pat. No. 4,371,421. Then, the polysilicon layer is formed via LPCVD on top of the insulating layer in a thin film from 0.1 to 100 microns (preferably, 0.5 micron). Over the poly-Si layer the silicon dioxide cap is formed preferably by CVD or grown thermally (like the insulating layer) to a thickness of from 2.0 to 3.0 microns. The layered wafer then proceeds to the annealing process as shown in FIG. 5.
Two different pre-ZMR annealing processes are shown in FIG. 5, the preferred one involving annealing the SOI structure in NH 3 with an intermediate oxidation step. In particular, the better method consists of exposing the cap side of the SOI structure for:
(1) 1-3 hours in 100% NH 3 ;
(2) 20 minutes in O 2 ; and
(3) 1-3 hours in 100% NH 3 ;
all at 1100° C.
Interruption of the NH 3 anneal with a short oxidation yields SOI structures with excellent wetting properties. During the initial NH 3 exposure, nitrogen is incorporated into the SiO 2 cap, with peak nitrogen accumulation at both surfaces, that is, the exterior and interior surface of the cap. It is believed that accumulation of nitrogen at the exterior gas interface of the cap progressively inhibits further rapid incorporation of nitrogen at the interior poly-Si interface. To eliminate this surface nitrogen-rich boundary layer (FIG. 6), the sample is briefly oxidized followed by an additional anneal in NH 3 . Multiple NH 3 anneals with intervening oxidation have been found to be more effective than a single uninterrupted eight-hour 100% NH 3 anneal as shown in the alternative process of FIG. 5 in promoting wetting of the SOI film.
Alternatively, an NH 3 anneal can be carried out before all of the thickness of the cap is deposited. That is, the cap can be deposited in two or more steps with intervening anneals.
After deposition of the poly-Si layer and a thinner CVD SiO 2 layer 500 A to 10,000 A thick, a single anneal in 100% NH 3 or NH 3 in N 2 , followed by additional CVD SiO 2 to a total thickness of 2.0 microns yields samples with excellent wetting characteristics during ZMR.
After deposition of the Si layer, we annealed in 4% NH 3 and then deposited the cap. Although wetting was excellent, crystal quality was poor presumably because of too much N at the interface. By reducing the amount of N introduced, either by reducing annealing temperature annealing time or NH 3 partial pressure, the amount of N can be adjusted to achieve good crystal quality as well as wetting. It is believed that this anneal introduced N to the native oxide on the polysilicon film.
With even higher annealing temperatures (1100°-1400° C.) it may be possible to introduce sufficient nitrogen into the two micron SiO 2 cap without the intermediate oxidation step. It has been found, however, that a high temperature anneal of the SOI structure in a relatively unreactive gas N 2 , does not produce samples which wet well during ZMR. It is believed that other nitrogen containing compounds, possibly alkylamines, which decompose at the SiO 2 surface at elevated temperatures may also be effective in introducing sufficient nitrogen into the SiO 2 capping layer.
Auger spectroscopy of good samples annealed according to the invention indicate that far less than a full monolayer of nitrogen is present at the interface between the SiO 2 cap and the poly-Si film. The sensitivity of the Auger spectroscopy instrument used was one-half monolayer at the Si/capping SiO 2 interface. The instrument gave no reading for a nitrogen layer thus indicating that less than one-half monolayer of nitrogen atoms was present at the interface.
EXAMPLE I
An encapsulated SOI was formed having a one-half micron polysilicon layer topped with a 2.0 micron SiO 2 cap. Prior to ZMR the sample was annealed for one hour in 100% NH 3 , oxidized in O 2 for twenty minutes and annealed for another hour in 100% NH 3 ambient all at 1100° C. The cap was observed to be intact after ZMR and the recrystallized silicon film was observed to have good crystal quality on gross inspection.
EXAMPLE II
In this experiment, all parameters were the same as Example I except that the annealing time in NH 3 is extended to three hours both before and after the intervening oxidation step. After ZMR, the sample was inspected and the cap found to be intact. Marginally better crystal quality than that in Example I was observed.
EXAMPLE III
Five samples were prepared in accordance with FIG. 2 and subjected to pure NH 3 anneals for one, two, three, four and eight hours, respectively, without the intermediate oxidation step. The results were moderately successful, however, not qualitatively as good as in Example I or II. In particular, there was a slight tendency of beading of the silicon film during ZMR.
EXAMPLE IV
To test the partial cap anneal technique of FIG. 7, samples with the following initial cap thicknesses were prepared, annealed and finished prior to ZMR in accordance with the following table:
______________________________________Initial Cap Thickness Anneal Time Final Cap Thickness(Micron) (Hours) (Micron)______________________________________0.2 4 2.00.2 8 2.00.5 4 1.50.5 8 1.51.0 4 2.01.0 8 2.0______________________________________
Identical sample pairs were annealed for four and eight hours respectively. The oxidation step was not used. Following ZMR, each sample was inspected and found to have a good cap and good crystal quality.
EXAMPLE V
A sample wafer was prepared in accordance with FIG. 2 except that the cap thickness was reduced to 500 Angstroms. The thinly capped SOI was subjected to a 4% NH 3 atmosphere for thirty minutes at 1100°. The remainder of the thickness of the SiO 2 cap was deposited to a full thickness of 2.0 microns before undergoing ZMR. Excellent results were obtained.
EXAMPLE VI
A sample was prepared in accordance with FIG. 2, except that before depositing the SiO 2 capping layer, the poly-Si layer was exposed to a 4% NH 3 anneal at 1100° C. The sample had excellent wetting characteristics but much nitrogen was incorporated into the silicon layer too, resulting in poor crystal quality upon ZMR.
EXAMPLE VII
A sample prepared in accordance with FIG. 2 was subjected to a high temperature anneal in a nitrogen gas (N 2 ) with no improvement in the wetting characteristics during ZMR. The resulting beading degraded the crystal quality and overstressed the cap. It was concluded that N 2 did not decompose at 1100° C. sufficiently to allow nitrogen atoms to diffuse to the interface. It was concluded that a gas is required which sufficiently decomposes well below the semiconductor melting point to release the reactive element, here N atoms.
The advantages of the foregoing annealing processes are important in terms of future commercial exploitation of SOI technology. The reactive nitrogen annealing process has relatively noncritical process parameters and can be performed as a batch process permitting a large throughput at low cost producing a much more uniform distribution of nitrogen than the existing technique of sputtered or CVD silicon nitride cladding with better properties and reproducibility.
The foregoing description of specific process parameters and materials is intended to be illustrative rather than restrictive. Many variations, additions, omissions or rearrangements with respect to the specific processes described herein are, of course, possible without departing from the spirit and principle of the invention. For example, while ammonia is preferred, other reactive nitrogen bearing gases may be used. While the foregoing examples were carried out at atmospheric pressure, different pressures will also work. In addition, while nitrogen appears to be beneficial with silicon, other elements which have a similar effect on wetting characteristics may be applied via the same process. Moreover, while silicon is by far the most important application known at present, other semiconductor material such as germanium and galium arsenide can be treated in a similar manner.
The temperature and duration of the anneal can be varied depending on the permeability (density) and thickness of the capping layer. The maximum temperature of 1250° C. attainable by conventional furnaces should be more than adequate.
The intermediate oxdiation step can be employed more than once and can be used in combination with the multistage cap formation system of FIG. 7. The process described herein, of course, is not restricted to any particular type of ZMR apparatus; other conventional means exist for generating a travelling molten zone besides those described and shown in FIG. 1 in this application. In any case, the scope of the invention is defined not by the specific processes disclosed herein but by the appended claims and equivalents thereto.
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Wetting of encapsulated silicon-on-insulator (SOI) films during a zone-melting recrystallization (ZMR) process is enhanced by a high temperature anneal of the SOI structure in a reactive nitrogen-containing ambient to introduce nitrogen atoms to the polysilicon/silicon dioxide cap interface. The technique is not only more effective in present in cap fracture and enhancing crystal quality but is also susceptible to batch processing with noncritical parameters in a highly efficient, uniform manner. Preferably, the cap is exposed to 100% ammonia at 1100° C. for one to three hours followed by a pure oxygen purge for twenty minutes. The ammonia atmosphere is reintroduced at the same temperature for another one to three hour period before ZMR. The process is believed to result in less than a half monolayer of nitrogen at the interior cap interface thereby significantly lowering the contact angle and improving the wetting character of the SOI structure.
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[0001] This invention relates to overhead conveyor systems in which transfer of individual conveyors from one line to an adjacent line is accomplished by a direct line transfer.
BACKGROUND OF THE INVENTION
[0002] Overhead conveyors are utilized in various production, transportation, assembly and treatment environments to transport parts or products through various operational stages. One type of overhead conveyor employs a rotating, generally horizontal drive tube or shaft that supports trolleys from which the load is suspended. Drive wheels on the trolleys ride on the upper surface of the rotating drive tube, and each is mounted for rotation about a driven wheel axis that is non-parallel and non-perpendicular to the drive tube axis, preferably at an acute angle to the drive axis. To support the load, the trolleys are also provided with wheels that ride on rails that define the load track. In layouts where the trolleys repeatedly traverse side-by-side, supply and return sides of the conveyor (or a loop), a powered curve cannot be used unless the supply and return sides of the line are spaced apart a sufficient distance to accommodate two 90-degree turns to form a 180-degree turn at each end of the line. This typically consumes six or seven feet of floor space at each 180-degree turn, resulting in excessive dead space between the lines and restricting the design of an efficient conveyor layout.
SUMMARY OF THE INVENTION
[0003] In an embodiment of the present invention the aforementioned problem is addressed by providing a lateral transfer apparatus for the trolleys of an overhead conveyor. Closely spaced, side-by-side load tracks, which may define the supply side (infeed) and the return side (outfeed) of the conveyor, receive load-bearing trolleys for movement along a first track in one direction, and along a second, typically parallel track in the same or another direction. A transfer shuttle unit is provided which has a normal position aligned with one of the tracks and a transfer position aligned with the other track, and is actuated to shift the unit between a normal position and a transfer position at an acute angle to the supply track and the receiving track, thereby transferring trolleys on the supply track to a position aligned with the receiving track for movement along the receiving track.
[0004] In another aspect of the invention the shuttle unit includes a track section aligned with the supply track when the unit is in a normal position, and aligned with the receiving track when the unit is in a transfer position. A transfer zone is defined by guide structure spanning the first and second tracks and supporting the shuttle unit for movement between the normal and transfer positions.
[0005] In a further aspect of the present invention, each of the supply and receiving tracks has a pair of load rails presenting staggered ends at the transfer zone defining an acute angle of approximately 45 degrees with the direction of movement of the trolleys. The shuttle unit has a pair of transfer rails presenting staggered ends at the transfer zone aligned with one another at the acute angle to define a path of travel of the shuttle unit along this acute angle between normal and transfer positions. Accordingly, the track section of the shuttle unit substantially abuts the ends of the first track when the shuttle unit is in its normal position, and substantially abuts the ends of the second track when the shuttle track is in its transfer position, whereby load-bearing trolleys are transferred by the shuttle unit from a first side of a line to a second side of the line for travel in a desired direction along the second track. Thereafter, the shuttle unit returns to its normal position for the next transfer operation.
[0006] Other advantages of this invention will become apparent from the following description taken in connection with the accompanying drawings, wherein is set forth by way of illustration and example, embodiments of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a fragmentary, plan view of an overhead conveyor showing the supply side and the return side of the line and a lateral transfer apparatus at one end of the conveyor, a transfer shuttle unit being shown in full lines aligned with the load rails on the supply side.
[0008] FIG. 2 is an end elevational view of the conveyor of FIG. 1 and additionally shows the overhead supports for the load rails.
[0009] FIG. 3 is a flow diagram showing the operation of a system controller.
[0010] FIG. 4 is a diagrammatic plan view of a particular track configuration showing the supply and return sides of a stretch of an overhead conveyor, and illustrates transfer units of the present invention at the respective ends thereof.
[0011] FIG. 5 is a plan view similar to FIG. 4 showing an alternative embodiment.
[0012] FIG. 6 is a diagrammatic plan view illustrating a transfer with a two-way outfeed.
[0013] FIG. 7 is a diagrammatic plan view illustrating a two-way infeed.
[0014] FIGS. 8 and 9 are diagrammatic plan views illustrating an infeed and an outfeed with multiple lane selection.
DETAILED DESCRIPTION
[0015] Referring initially to FIGS. 1 and 2 of the drawings, one of the terminal ends of a pair of spaced, parallel tracks 10 and 12 of an overhead conveyor is shown. It may be appreciated that the tracks 10 and 12 extend to the opposite end of the conveyor (not illustrated) and thus the tracks define an overhead conveyor line that may be employed, for example, to transport parts or products through various operational stages. The track 10 has a pair of spaced, parallel load rails 14 a and 14 b and the track 12 is presented by a pair of spaced, parallel load rails 16 a and 16 b . Each pair of load rails 14 a , 14 b and 16 a , 16 b is supported in a conventional manner by a series of horizontally spaced pairs of hanger rods 18 and 20 spaced along the conveyor line (partially shown in FIG. 2 ) and secured at their respective upper ends to overhead I-beams 22 a and 22 b.
[0016] In the present invention the track 10 presents a supply track or infeed for the trolleys of the conveyor line, and the track 12 presents a return track or outfeed for the trolleys of the conveyor line. As is conventional in an overhead conveyor, a rotating drive tube 24 of the line is shown in broken lines in FIG. 1 and terminates at the end of the track 10 at a transfer zone 26 where, as will be set forth in detail hereinbelow, trolleys are sequentially transferred to the return track 12 . Similarly, a rotating drive tube 28 for the return track 12 extends from the end of track 12 at the transfer zone 26 .
[0017] In FIG. 1 it may be seen that the load rails 14 a and 14 b present staggered ends 30 and 32 respectively at the transfer zone 26 defining an acute angle with the direction of movement of trolleys along supply track 10 , the staggered ends 30 and 32 defining a 45 degree angle with the line of the track 10 that defines the direction of movement of the trolleys (not shown) that are advanced by the rotating drive tube 24 in the direction of the infeed indicated by the arrow 34 . Preferably, the ends 30 and 32 define a 45 degree angle (as shown in FIG. 1 ) with the direction of movement of the trolleys. Similarly, the return track 12 terminates at the transfer zone 26 at ends 36 and 38 in linear alignment with ends 30 and 32 . The ends 36 and 38 define a 45 degree angle with respect to the return track 12 that provides the outfeed for the trolleys transferred by a shuttle 40 that, in its home position shown in full lines, receives individual trolleys delivered to the transfer zone 26 via supply track 10 , and then shifts the trolley at a 45 degree angle into alignment with the return track 12 . Arrow 42 illustrates the direction of movement of the shuttle 40 into alignment with return track 12 and return to the supply track 10 .
[0018] More particularly, as seen in FIGS. 1 and 2 , the shuttle 40 comprises a pair of spaced, inverted U-shaped hanger assemblies 44 and 46 supporting a pair of laterally spaced load rail sections 48 and 50 having forward end portions 48 ′ and 50 ′ terminating at a 45 degree angle and abutting supply track ends 30 and 32 in the receiving position thereof shown in FIG. 1 . The outer surface of a drive tube or shaft 52 is engaged by four driven wheels 54 carried by a yoke plate member 56 supported on a trolley having load wheels 57 that run on load rails 48 and 50 . As is conventional, driven wheels 54 have axes at an acute angle with respect to the axis of the drive tube 52 in order to propel a trolley thereon in an axial direction along drive tube 52 when the latter is driven by a motor 58 via a belt and pulley drive 60 . Two of the wheels 54 are seen in FIG. 2 in engagement with drive tube 52 . This drive arrangement is employed in the present invention to convey trolleys from the supply track 10 to the shuttle 40 for transfer to the return track 12 . The 45 degree angle established by the ends 30 and 32 of the supply track 10 and the aligned ends 36 and 38 of the return track 12 provides a continuous track for the trolley load wheels as individual trolleys are delivered to the transfer zone 26 from track 10 and then shifted into alignment with return track 12 and advanced onto track 12 in the direction of arrow 90 without traversing a discontinuity in either direction when advancing over ends 30 and 32 onto rail end portions 48 ′ and 50 ′, and subsequently propelled from the shuttle 40 over ends 32 and 38 of the return track 12 . A continuous load track is thus presented in both directions of transfer to and from the shuttle 40 . Although an acute angle to each of the tracks 10 and 12 in the range of approximately 15 to 75 degrees could be employed, the 45-degree angle is preferred as laterally aligned load wheels 48 and 50 do not simultaneously roll over ends 30 and 32 , or 36 and 38 . For example, load wheel 50 clears end 32 before load wheel 48 reaches end 30 .
[0019] Transfer is accomplished by a linear actuator or pneumatic cylinder 62 having a drive rod 64 shown retracted in FIG. 1 . Rod 64 is connected at its outer end to a shuttle push bar 66 shown in cross section in FIG. 1 . A pair of spaced, parallel, horizontally extending guide rods 68 and 70 are mounted on the top of respective hanger assemblies 44 and 46 and extend across the transfer zone 26 . The guide rod 68 receives a bushing 72 slidable thereon and, similarly, the guide rod 70 receives a bushing 74 slidable thereon, both of the bushings 72 and 74 being secured to the respective ends of the push bar 66 . When cylinder 40 is actuated, its piston rod 64 , connected to push bar 66 , shifts the shuttle to the right as indicated by arrow 42 to the position thereof shown in broken lines in FIG. 1 aligned with the return track 12 . At this time as will be discussed in more detail below, the motor 58 is energized to drive the transferred trolley on to return track 12 to the receiving drive tube 28 . After transfer, actuator 62 returns the transfer shuttle to its home position in alignment with the supply track or infeed 10 . Although not shown, it will be appreciated that a support is provided for actuator 62 to maintain it in a horizontal position at the transfer angle.
[0020] A programmable logic controller (PLC) may be employed as a system controller for the shuttle unit in response to sensors associated with the supply and return tracks and the rail sections of the shuttle. Referring to FIGS. 1 and 2 , five inductive proximity sensors are shown and comprise a shuttle present sensor 80 near the termination of supply track 10 , a carrier present sensor 82 spaced above rail section 48 , a shuttle present sensor 84 below rail section 48 of the shuttle 40 , a shuttle present sensor 86 for sensing the shuttle 40 in the transferred position thereof aligned with the return track 12 , and a carrier clear sensor 88 adjacent the end of the return track 12 .
[0021] Referring to the flow diagram of FIG. 3 showing the operation of the system controller, a carrier is approaching the transfer (block 90 ) and is detected by the sensor 80 ( FIG. 1 ). If sensor 82 indicates that the shuttle 40 is present at the home position, it produces a “Shuttle Home” output at 92 (YES) to initiate shuttle motor 58 to drive shaft 52 and propel trolleys on to load rail sections 48 and 50 of transfer zone 26 . Shuttle present sensor 84 stops motor 58 when the shuttle is in its unload position, and initiates actuator 62 to transfer load rail sections 48 and 50 to an unload position in alignment with load rails 16 a and 16 b of the return track 12 . Sensor 86 detects the shuttle in its unload position. If the return track 12 is clear (sensor 88 ) and the drive motor (not shown) for track 12 is in operation, motor 58 starts and drives shaft 52 to propel the trolleys onto return track 12 in the direction of arrow 90 . Motor 58 is de-energized when the carrier clears sensor 88 . Actuator 62 then returns the shuttle to its home position shown in full lines in FIGS. 1 and 2 where load rail sections 48 and 50 are in alignment with parallel rails 14 a and 14 b of the supply track 10 . The shuttle 40 is thus returned to its home position for sequentially receiving additional trolleys from the supply or infeed track 10 and sequentially transferring them to the return or outfeed track 12 .
[0022] Referring to FIGS. 4-9 , six track configurations are shown diagrammatically and comprise examples of conveyor configurations that may be employed with the angular lateral transfer apparatus of the present invention. FIG. 4 illustrates a supply track 100 having a drive tube 102 partially shown) driven by a motor 104 for advancement of trollies in the direction of arrow 106 . A transfer zone 108 at one end is provided with the shuttle 40 a of the present invention for transfer of trolleys at a 45 degree angle to a return track 112 for movement in the opposite direction as shown by arrow 114 . A drive tube 118 associated with return track 112 is diagrammatically illustrated and powered by a motor 116 . The opposite end of the conveyor configuration has a transfer zone 110 where a shuttle 40 b shifts the trolleys at a 45-degree angle in the direction of arrow 120 into alignment with supply track 100 for movement in the direction indicated by arrow 106 . Accordingly, utilizing the 45-degree shuttles 40 a and 40 b , a continuous loop is provided utilizing parallel, closely spaced tracks 100 and 112 .
[0023] FIG. 5 is an illustration similar to FIG. 4 except that a shuttle 40 c at the end of the supply track shifts the trolleys at a 45-degree angle in the direction of arrow 122 at a 90-degree angle with respect to the directional arrow 109 in FIG. 4 . Operation is otherwise the same as FIG. 4 with the return to the supply track being executed by shuttle 40 d.
[0024] Referring to FIG. 6 , in this illustration the transfer is effected at a mid-point in parallel tracks or at another location spaced from the ends thereof. Aligned tracks 122 and 122 a terminate at 126 at a 45-degree angle and define a transfer zone where a shuttle 40 e may deliver trolleys to either of the aligned tracks 128 and 130 for movement along outfeed track 128 in the direction illustrated by arrow 132 , or movement along outfeed track 130 in the direction indicated by arrow 134 . The direction of delivery is controlled by the shuttle drive motor 136 .
[0025] The track configuration shown in FIG. 7 is similar to FIG. 6 , but with track 130 omitted. FIG. 7 illustrates trolleys advancing along the supply track either from direction 136 or the opposite direction 138 , and then transferring via shuttle 40 f to an outfeed track 139 .
[0026] FIG. 8 illustrates a multiple track outfeed. Trolleys advance along the infeed track 140 in either of two opposing directions 146 and 148 to a shuttle 40 g for transfer to either outfeed track 142 or 144 . In FIG. 9 , an arrangement similar to FIG. 8 but reversed in flow is shown wherein infeed tracks 150 or 152 deliver trolleys to shuttle 40 h for transfer as indicated by arrow 154 to either of the outfeed tracks 156 or 158 for movement in either direction 160 or in the opposite direction 162 . From the foregoing it may be appreciated that various supply and return combinations can be employed with the lateral transfer apparatus of the present invention as dictated by the design of a conveyor layout.
[0027] It is to be understood that while certain forms of this invention have been illustrated and described, it is not limited thereto except insofar as such limitations are included in the following claims.
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A lateral transfer apparatus for an overhead conveyor comprises generally horizontally extending, side-by-side load tracks for receiving load-bearing trolleys for movement along a first track, and for movement along a second track. A transfer shuttle unit has a normal position aligned with the first track and provides a continuation thereof, and a transfer position aligned with the second track to provide a continuation of the second track. An actuator connected with the shuttle unit shifts it between the normal and transfer positions thereof at an acute angle to the tracks, thereby transferring trolleys to a position aligned with the receiving track for movement therealong.
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