Patent Application: US-48160504-A

Abstract:
in a preferred embodiemnt the present invention features a composition and method of delivery comprising glycosaminoglycans encapsulated in a liposomal delivery system for intraarticular administration for the treatment of osteoarthritis in a more preferred embodiemnt the present invention features a composition and method of delivery comprising hyaluronic acid encapsulated in a liposomal delivery system for intraarticular administration for the treatment of osteoarthritis .

Description:
it is believed that one skilled in the art can , based upon the description herein , utilize the present invention to its fullest extent . the following specific embodiments are to be construed as merely illustrative , and not limitative of the remainder of the disclosure in any way whatsoever . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs . also , all publications , patent applications , patents , and other references mentioned herein are incorporated by reference . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs . also , all publications , patent applications , patents , and other references mentioned thoroughout this application are herein expressly incorporated by reference . glycosaminoglycans ( gags ) are defined as biopolymers consisting of repeating polysaccharide units , and are present on the cell surface as well as in the extracellular matrix of animals . gags are long unbranched polysaccharides containing a repeating disaccharide unit . the disaccharide units contain either of two modified sugars , n - acetylgalactosamine or n - acetylglucosamine and a uronic acid such as glucuronate or iduronate . gags are highly negatively charged molecules , with extended conformation that imparts high viscosity to the solution . gags are located primarily on the surface of cells or in the extracellular matrix . along with the high viscosity of gags comes low compressibility , which makes these molecules ideal for a lubricating fluids in the joints . at the same time , their rigidity provides structural integrity to cells and provides passageways between cells allowing for cell migration . hyaluronic acid ( ha ) is defined as a high molecular weight polysaccharide of n - acetyl glucosamine and glucuronic acid molecules that is naturally occurring in all mammals in a variety of tissue and some bacterial species . for purposes of this applications ha includes any derivatives such as hyaluronan and hyaluronic acid itself with h + ion attached to the coo — group . and salts of hyaluronic acid whereby another positive ion replaces the h + ion , as for example with na + which forms sodium hyaluronate . also included in the definition is any physically or chemically cross - linked hyaluronic acid and deriviatives . ha is unique among the gags in that it does not contain any sulphate and is not found covalently attached to proteins as a proteoglycan . ha polymers are very large with molecular weights of between about 100 , 000 - 10 , 000 , 000 , and can displace a large volume of water . for purposes of this invention a most preferred embodiment includes a non cross - linked hyaluronic acid with a molecular weight of 0 . 5 - 10 mda . liposomes are defined as small spheres whose walls are layers of lipids with water . as they form , liposomes entrap water and any water soluble solutes that are present . because of this entrapping ability , they are useful as drug delivery systems . for purposes of the present invention a most preferred embodiment includes the use of a multilamellar vesicle . for purposes of this invention a preferred embodiment includes any naturally occurring phospholipid and a most preferred embodiment includes dipalmitoylphosphatidylcholine ( dppc ). intra - articular delivery is defined as a method whereby a treatment is delivered , directly or indirectly , into the synovial capsule of an articulating joint . what is meant by a liposome is a vesicle having at least one lipid bilayer surrounding an inner liquid phase ( e . g ., either a lipid bilayer surrounding either a liquid core or a liquid phase dispersed between it and another lipid bilayers ). the liposome may have various structures such as multilamellar ( mlvs ), unilamellar ( luvs or suvs ), and paucilamellar ( plvs ) as discussed above . the resulting structure of the liposome is dependent , in part , on the choice of materials forming the hydrophobic phase and the manufacturing parameters such temperature and incubation times . liposomes manufactured according to the present invention comprise at least one amphiphilic bilayer - forming substance and may comprise a benefit agent . the benefit agent may be contained either within the lipid bilayer or the hydrophilic or hydrophilic compartments of the liposome . what is meant by amphiphilic bilayer - forming substance is a lipid that is comprised of both a hydrophilic and lipophilic group and is capable of forming , either alone or in combination with other lipids , the bilayer of a liposome . the lipid can have single or multiple lipophilic side chains being either saturated or unsaturated in nature and branched or linear in structure . the amphiphilic bilayer - forming agent can be phospholipid or a ceramide . multiple lipophilic side chain amphiphilic bilayer - forming substances are amphiphilic bilayer - forming substances have two or more lipophilic side chains ( e . g ., that are attached to a polar head group ). such lipids may be nonionic , cationic , anionic , zwitterionic in nature . examples of suitable multiple lipophilic side chain amphiphilic bilayer - forming substances include , but are not limited to , those bilayer - forming cationic lipids that contain two saturated or unsaturated fatty acid chains ( e . g ., side chains having from about 10 to about 30 carbon atoms ) such as di ( soyoylethyl ) hydroxyethylmonium methosulfate ( dshm ), n -[ i -( 2 , 3 - dioleyloxy ) propyl ]- n , n , n - trimethylammonium bromide ( dotma ), 1 , 2dimyristyloxypropyl - n , n - dimethyl - hydroxyethyl ammonium bromide ( dmrie ), [ n —( n , n ′- dimethylaminoethane ) carbamoyl ] cholesterol ( dc - chol ), dioctadecylamidoglycyl spermidine ( dogs ), dimethyl dioctadecylammonium bromide ( ddab ), dioleoyl phosphatidylethanolamine ( dope ), 2 , 3 - dioleoyloxy ]- n [ 2 ( sperminecarbozamide - o - ethyl ]- n , n - dimethyl - propanaminium trifluoroacetate ( dospa ), i -[ 2 -( oleoyloxy )- ethyl ]- 2 - oleyl - 3 -( 2hydroxyethyl ) imidazolinium chloride ( dotim ), 1 , 2 - dioleoyloxy - 3 -( trimethylammonio ) propane ( dotap ), 1 , 2 - diacyl - 3 - trimethylammonium propane ( tap ), 1 , 2 - diacyl - 3 - dimethylammonium propane ( dap ), fatty acid salts of quaternary amines such as dicocodimonium chloride ( quaternium 34 ), and quaternary dimethyldiacyl amines wherein the acyl groups have from about 8 carbon atoms to about 30 carbon atoms ( e . g ., from about 10 carbon atoms to about 30 carbon atoms ), and derivatives thereof such as ammonium derivatives , i . e . dimethyl dihydrogenated tallow ammonium chloride ( quaternium 18 ), and decyl dimethyl octyl ammonium chloride ( quaternium 24 ), and derivatives thereof . other suitable cationic dual chain lipids are further described in the following references : fasbender et al ., 269 am j physiol l45 - l5 1 ( 1995 ); solodin et al ., 34 biochemistry 13537 - 13544 ( 1995 ); felgner et al ., 269 j biol chem 2550 - 2561 ( 1994 ); stamatatos et al ., 27 biochemistry 3917 - 3925 ( 1988 ); and leventis and silvius , 1023 biochim biophys acta 124 - 132 ( 1990 ), and jouani et al ., 9 j . liposome research 95 - 114 ( 1999 ), which are all incorporated by reference herein . examples of suitable nonionic multiple lipophilic side chain amphiphilic bilayer - forming substances include , but are not limited to , glyceryl diesters , and alkoxylated amides . examples of suitable glyceryl diesters include , but are not limited to , those glyceryl diesters having from about 10 carbon atoms to about 30 carbon atoms ( e . g ., from about 12 carbon atoms to about 20 carbon atoms ), glyceryl dilaurate (“ gdl ”), glyceryl dioleate , glyceryl dimyristate , glyceryl distearate (“ gds ”), glyceryl sesuioleate , glyceryl stearate lactate , and mixtures thereof , with glyceryl dilaurate , glyceryl distearate and glyceryl dimyristate , and derivatives thereof . examples of anionic multiple lipophilic side chain amphiphilic bilayer - forming substances include , but are not limited to , phosphatidic acids such as 1 , 2 dimyristoyl - sn - glycero - 3 - phosphate , sodium salt ( dmpa ), 1 , 2 dipalmitoyl - sn - glycero - 3 - phosphate , sodium salt ( dppa ), 1 , 2 distearoyl - sn - glycero - 3 - phosphate , sodium salt ( dspa ) and negatively charged phospholipids such as dipalmitoyl phosphatidylglycerol . the amount of multiple lipophilic side chain amphiphilic bilayer - forming substances in the vesicle bilayer may range from , based upon the total weight of the substances in the lipid bilayer ( s ), from about 0 . 001 percent to about 95 percent ( e . g ., from about 5 percent to about 65 percent ). the amount of multiple lipophilic side chain amphiphilic bilayer - forming substances based upon the total weight of the components in the liposome will depend upon the type of liposome ( e . g ., unilamellar or paucilamellar liposomes ), and may range from about 0 . 001 percent to about 95 percent ( e . g ., from about 1 to about 65 percent ). a single lipophilic chain amphiphilic bilayer - forming substance is a amphililic bilayer forming substance containing a single lipophilic side chain ( e . g ., attached to a polar head group ). the single chain lipids may be nonionic , cationic , anionic , or zwitterionic . examples of suitable nonionic single lipophilic chain amphiphilic bilayer - forming substances include , but are not limited to , glyceryl monoesters ; polyoxyethylene fatty ethers wherein the polyoxyethylene head group has from about 2 to about 100 groups and the fatty acid tail group has from about 10 to about 26 carbon atoms ; alkoxylated alcohols wherein the alkoxy group has from about 1 carbon atoms to about 200 carbon atoms and the fatty alkyl group has from about 8 carbon atom to about 30 carbon atoms ( e . g ., from about 10 carbon atoms to about 24 carbon atoms ); alkoxylated alkyl phenyls wherein the alkoxy group has from about 1 carbon atoms to about 200 carbon atoms and the fatty alkyl group has from about 8 carbon atom to about 30 carbon atoms ( e . g ., from about 10 carbon atoms to about 24 carbon atoms ); polyoxyethylene derivatives of polyol esters ; alkoxylated acids wherein the alkoxy group has from about 1 carbon atoms to about 200 carbon atoms and the fatty alkyl group has from about 8 carbon atom to about 30 carbon atoms ( e . g ., from about 10 carbon atoms to about 24 carbon atoms ); and alkoxylated acids . examples of suitable glyceryl monoester nonionic single lipophilic chain amphiphilic bilayer - forming substances include , but are not limited to , those glyceryl monoesters having from about 10 carbon atoms to about 30 carbon atoms ( e . g ., from about 12 carbon atoms to about 20 carbon atoms ), glyceryl caprate , glyceryl caprylate , glyceryl cocoate , glyceryl erucate , glyceryl hydroxystearate , glyceryl isostearate , glyceryl lanolate , glyceryl laurate , glyceryl linolate , glyceryl myristate , glyceryl oleate , glyceryl paba , glyceryl palmitate , glyceryl ricinoleate , glyceryl stearate , and glyceryl thiglycolate , and derivatives thereof . examples of suitable polyoxyethylene fatty ether nonionic single lipophilic chain amphiphilic bilayer - forming substance include , but are not limited to , polyoxyethylene cetyl ether , polyoxyethylene stearyl ether , polyoxyethylene cholesterol ether , polyoxyethylene laurate , polyoxyethylene dilaurate , polyoxyethylene stearate , polyoxyethylene distearate , polyoxyethylene lauryl ether , polyoxyethylene stearyl ether , polyoxyethylene myristyl ether , and polyoxyethylene lauryl ether , e . g ., with each ether having from about 3 to about 10 oxyethylene units , and derivatives thereof . suitable examples of an alkoxylated alcohol nonionic single lipophilic chain amphiphilic bilayer - forming substance include , but are not limited to , those having the structure shown in formula i below : wherein r 5 is an unbranched alkyl group having from about 10 to about 24 carbon atoms and y is an integer between about 4 and about 100 ( e . g ., from about 10 and about 100 ). an example of such an alkoxylated alcohol is the species wherein r 5 is a lauryl group and y has an average value of 23 , which is known by the ctfa name “ laureth 23 ” and is available from uniqema , inc . of wilmington , del . under the tradename brij 35 ®. suitable examples of an alkoxylated alkyl phenyls nonionic single lipophilic chain amphiphilic bilayer - forming substance include , but are not limited to , those having the structure shown in formula ii below : wherein r 6 is an unbranched alkyl group having from about 10 to about 24 carbon atoms and z is an integer of from about 7 and 120 ( e . g ., from about 10 to about 100 ). an example of this class of materials is the species wherein r 6 is a nonyl group and z has an average value of about 14 . this material is known by the ctfa name “ nonoxynol - 14 ” and is available under the tradename , makon 14 ® from the stepan company of northfield , ill . suitable polyoxyethylene derivatives of polyol ester nonionic single lipophilic chain amphiphilic bilayer - forming substance are those wherein the polyoxyethylene derivative of polyol ester that : ( 1 ) is derived from ( a ) a fatty acid containing from about 8 to about 22 ( e . g ., from about 10 to about 14 carbon atoms ) and ( b ) a polyol selected from sorbitol , sorbitan , glucose , α - methyl glucoside , polyglucose having an average of about 1 to about 3 glucose residues per molecule , glycerine , and pentaerythritol ; ( 2 ) contains an average of from about 10 to about 120 ( e . g ., from about 20 to about 80 ) oxyethylene units ; and ( 3 ) has an average of from about 1 to about 3 fatty acid residues per mole of polyoxyethylene derivative of polyol ester . examples of polyoxyethylene derivatives of polyol esters include , but are not limited to , peg - 80 sorbitan laurate and polysorbate 20 . peg - 80 sorbitan laurate , which is a sorbitan monoester of lauric acid ethoxylated with an average of about 80 moles of ethylene oxide , is available commercially from ici surfactants of wilmington , del . under the tradename atlas g4280 ®. polysorbate 20 , which is the laurate monoester of a mixture of sorbitol and sorbitol anhydrides condensed with approximately 20 moles of ethylene oxide , is available commercially from ici surfactants of wilmington , del . under the tradename tween 20 ®. another exemplary polyol ester is sorbitan stearate , which is available from uniqema , inc . under the tradename span 60 ®. suitable examples of alkoxylated acid nonionic single lipophilic chain amphiphilic bilayer - forming substance include , but are not limited to , the esters of an acid ( e . g ., a fatty acid ) with a polyalkylene glycol . an exemplary material of this class has the ctfa name peg - 8 laurate ®. examples of suitable cationic single lipophilic chain amphiphilic bilayer - forming substance include , but are not limited to , quaternary trimethylmonoacyl amines wherein the acyl groups have from about 8 carbon atoms to about 30 carbon atoms ( e . g ., from about 10 carbon atoms to about 24 carbon atoms ), and derivatives thereof such as ammonium derivatives , e . g ., stearamidopropyl dimethyl ( myristyl acetate ) ammonium chloride ( quaternium 70 ), triethyl hydrogenated tallow ammonium chloride ( quaternium 16 ), and benzalkonium chloride , and derivatives thereof . examples of suitable anionic single lipophilic chain amphiphilic bilayer - forming substances include , but are not limited to , fatty acids such as oleic acid and negatively charged single chained phospholipids such as phosphatidylserine and phosphatidylglycerol . the amount of single lipophilic chain amphiphilic bilayer - forming substance in the vesicle bilayer may range from , based upon the total weight of the substances in the lipid bilayer ( s ), from about 0 . 001 percent to about 70 percent ( e . g ., from about 1 percent to about 30 percent ). the amount of single lipophilic chain amphiphilic bilayer - forming substance based upon the total weight of the components in the liposome will depend upon the type of liposome ( e . g ., unilamellar or paucilamellar liposomes ), and may range from about 1 percent to about 95 percent ( e . g ., from about 1 percent to about 30 percent ). the above single and multiple lipophilic chain amphiphilic bilayer - forming substance may also be a phospholipid , which may be zwitterionic in nature . examples of phospholipids include , but are not limited to , natural and synthetic phospholipids . examples of natural phospholipids include , but are not limited to , egg phosphatidylcholine , hydrogenated egg phosphatidylcholine , soybean derived phospholipids such as soybean phosphatidylcholine , phospholipids from plant sources , sphingomyelin . examples of synthetic phospholipids include , but are not limited to , synthetic phosphatidylcholines such as 1 , 2 - dilauroyl - sn - glycero - 3 - phosphocholine ( dlpc ), 1 , 2 - dimyristoyl - sn - glycero - 3 - phosphocholine ( dmpc ), 1 , 2 - dipalmitoyl - sn - glycero - 3 - phosphocholine ( dppc ), 1 , 2 - distearoyl - sn - glycero - 3 - phoshpocholine ( dspc ), 1 , 2 - didioleoyl - sn - glycero - 3 - phoshpocholine ( dopc ), 1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phoshpocholine ( popc ), phosphatidylethanolamines include , but are not limited to , 1 , 2 - dimyristoyl - sn - glycero - 3 - phoshpethanolamine ( dmpe ), 1 , 2 - dipalmitoyl - sn - glycero - 3 - phoshpethanolamine ( dppe ), 1 , 2 - distearoyl - sn - glycero - 3 - phoshpethanolamine ( dspe ), 1 , 2 - dioleoyl - sn - glycero - 3 - phoshpethanolamine ( dope ), negatively charges phospholipids such as dipalmitoyl phosphatidylglycerol ( dppg ), dipalmitoyl phosphatidylcholine ( dppc ), dipalmitoyl phosphatidic acid ( dppa ), and phosphatidylserine ( ps ), and derivatives thereof . the above single and multiple lipophilic chain amphiphilic bilayer - forming substance may also be a cermide . examples include , but are not limited to , n - acetyl d - erythro - sphinogsine ( c2cer ), n - octanoyl d - erythro - sphinogsine ( c8cer ), n - myristoyl d - erythro - sphinogsine ( c14cer ), n - stearoyl d - erythro - sphinogsine ( c18cer ), n - arachidoyl d - erythro - sphinogsine ( c20cer ). other suitable lipids are further described in the following references : avanti polar lipids , inc ., alabaster , ala . interim catalog 13 - 92 and 105 - 127 ( 1999 ); polyglycerol such as those described in u . s . pat . no . 4 , 772 , 471 , french patent nos . 1 , 477 , 048 and 2 , 091 , 516 ;; amide - based oligomeric cationic lipids such as those described in u . s . pat . no . 5 , 877 , 220 ; cationic lipids such as those described in u . s . pat . nos . 5 , 980 , 935 , 5 , 851 , 548 , 5 , 830 , 430 , and 5 , 777 , 153 ; phosphonic acid - based cationic lipids such as those described in u . s . pat . no . 5 , 958 , 901 ; quaternary cytofectins such as those described in u . s . pat . no . 5 , 994 , 317 ; ether lipids such as those described in u . s . pat . no . 5 , 989 , 587 ; and polyethylene glycol modified cermide lipids such as those described in u . s . pat . no . 5 , 820 , 873 . sterols may be added to the lipid bilayer of the liposome . the presence of a rigid steroid alongside the fatty acid chains of the lipid in the bilayer may reduce the freedom of motion of these carbon chains , creating better packing of the lipid bilayers . examples of suitable sterols include , but are not limited to , cholesterol and salts and esters thereof , cholesterol 3 - sulfate , phytocholesterol , hydrocortisone , alpha - tocopherol , betasitosterol , bisabolol and derivatives thereof . the amount of sterol in the vesicle bilayer may range from , based upon the total weight of the substances in the vesicle bilayer , from about 0 . 001 percent to about 95 percent ( e . g ., from about 1 percent to about 65 percent ). the amount of sterol , based upon the total weight of the components in the liposome will depend upon the type of liposome ( e . g ., unilamellar or paucilamellar liposomes ), and may range from about 0 . 001 percent to about 95 percent ( e . g ., from about 1 percent to about 65 percent ). the benefit agent may be contained within the lipid bilayer ( e . g ., if it is a lipophilic agent ) or within a hydrophilic component of the liposome ( e . g ., within the hydrophilic regions within the lipid bilayers of within the core ). the hydrophilic component may contain water and / or other polar solvents . examples of polar solvents include , but are not limited to , glycols such as glycerin , alcohols ( e . g ., those alcohols having from about 2 carbon atoms to about 6 carbon atoms ), propylene glycol , sorbitol , oxyalkylene polymers such as peg 4 , and derivatives thereof . the liposomes of the present invention may be included within pharmaceutical ( e . g ., compounded with a pharmaceutically compatible carrier ). the resulting composition may be in the form of a cream , ointment , lotion , gel for therapeutic use . it is also envisaged that the liposomal formulation of the present invention may include additional benefit agents for the treatment of osteoarthritis , including analgesics , anti - inflammatory agents , or chondroprotective agents . typical examples of such benefit agents include , but are not limited to , non - steroidal anti - inflammatory drugs , p38 kinase inhibitors , tnf - α inhibitors , corticosteroids , inhibitors of enzymes that are involved in the destruction of articulating joints or synovial fluid components , such as hyaluronidase inhibitors , matrix metalloproteinase inhibitors , or aggrecanase inhibitors , apoptosis inhibiors such as epo , and cartilage enhancing factors such as tgf - β1 , and bone morphogenetic proteins . the extra benefit agent thus described may be either co - encapsulated with the gag , bound to the liposome but not encapsulated , or present as free drug outside the liposome bilayers . the following is a description of the manufacture and testing of liposomes of the present invention . other liposomes of the invention can be prepared in an analogous manner by a person of ordinary skill in the art . the desciption of formulation methodology outlined below is considered only as an example , and it is understood that other methods of producing formulations encapsulating gags in liposomes may also be effective . examples of such methods are described in detail by vemuri and rhodes ( 1995 ) and include , but are not limited to , mixing of suv with aqueous phase containing the benefit agent , subsequent lyophilisation and rehydration to yield mlv [ e . g . kirby and gregoriadis , 1984 ], reverse - phase evaporation [ szoka and papahadjopoulos , 1978 ], high pressure extrusion [ vemuri et . al , 1990 ], freeze - thaw of liposomes [ pick , 1981 ] and dehydration / rehydration [ shew and deamer , 1985 ]. ha may be prepared by any method . however , the preferred method would be to produce ha to a high purity through a bacterial fermentation route such as that described in wo86 / 04355 . hyaluronic acid was incorporated into 1 , 2 - dipalmitoyl - sn - glycero - 3 - phosphocholine ( dppc ) liposomes was carried out by a film hydration method . briefly , dppc ( 400 mg ) was dissolved in 40 ml of ethanol . the dppc / ethanol mixture was then placed in a round bottom flask and attached to a rotary evaporator apparatus and then the round bottom flask was lowered into a water bath ( buchi labortechnik ag , switerland ). at a rotation speed of 54 rpm , the ethanol in the mixture was slowly removed by vacuum and the resulting film was stored under vacuum for 1 hour . the thin film was then hydrated with 4 ml of a phosphate buffer saline ( pbs ) solution at 55 ° c . after the lipid film was hydrated , 4 ml of 20 mg / ml hyaluronic acid solution was added to the lipid / pbs mixture and was then vortexed in 15 second interval for 5 minutes at 55 ° c . the newly formed vesicles were allowed to equilibrium ambient condition before testing . the final liposomal concentration was 50 mg / ml dppc and 10 mg / ml hyaluronic acid . blank liposomes ( without hyaluronic acid ) were prepared using the same method above but without the addition of ha . the final liposomal concentration was 100 mg / ml dppc . to prepare liposomal mixture where the hyaluronic acid was not encapsulated , the 4 ml of the blank liposomes of example 2 were mixed with 4 ml of 20 mg / ml hyaluronic acid solution . the final liposomal concentration was 50 mg / ml dppc and 10 mg / ml hyaluronic acid . the compositions were subsequently examined using a freeze - fracture transmission electron microscope ( ff - tem ). ff - tem samples of each formulation were prepared in accordance with techniques described in chapter 5 of “ low temperature microscopy and analysis ” by patrick echlin ( 1992 ). the sample was mounted between thin metal sheets and rapidly cooled with liquid propane to − 196 ° c . the sample was then transferred under liquid nitrogen to a pre - cooled cold stage of a balzers baf - 301 high vacuum freeze - etch unit ( techno trade international , lichtenstein ). the sample was fractured at low temperature and etched at − 150 ° c . to remove a surface layer of water . the fracture faces were shadowed at an angle of 45 ° with platinum to create selective electron contrast . a thin layer of carbon was deposited over the entire fracture surface to create a continuous replica . the replicas were then examined using a jeol 100cx2 electron microscope ( japanese electronic optical laboratories , japan ). liposomes of example 2 showed the presence of intact vesicles with bilayers . there was no evident on hyaluronic acid in the external phase . all the hyaluronic acid appeared to be encapsulated inside the dppc liposomes . liposomal mixture showed liposomal structures and string like structures indicating the presence of hyaluronic acid not encapsulated inside the dppc liposomes . adams , m . e . an analysis of clinical studies of the use of crosslinked hyaluronan , hylan , in the treatment of osteoarthritis . j . rheumatol . 20 ( suppl . 39 ): 16 - 18 , 1993 . adams , m . e ., atkinson , m . h ., lussier , a ., schulz , j1 , siminovitch , k . a ., wade , j . p ., and zummer , m . 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