Patent Application: US-2898898-A

Abstract:
the present invention provides activator compounds , including agonists , to the peroxisome proliferator - activated receptor gamma . particular pparγ activators are set forth , as are a pharmaceutical composition for treating diabetes , non - insulin - dependent diabetes mellitus , cardiovascular disorders , and methods for such treatment .

Description:
we have found that certain chemical compounds including thiazolidinediones are efficacious and selective activators of the pparγ . furthermore , while it has been shown that the ppars are activated by micromolar concentrations of fatty acids and hypolipidemic drugs such as clofibric acid and wy14 , 643 ( isseman et al , 1990 , dreyer et al , 1992 , schmidt et al , 1992 , kliewer et al . 1994 , gottlicher et al , 1992 ), none of these compounds has been shown previously to interact directly with the ppars . however , we have discovered that thiazolidinediones can bind directly with the pparγ . these data show that pparγ is the molecular target for the therapeutic effects of these compounds on insulin resistance , niddm and lipid metabolism with its implications for cardiovascular disease . the identification of novel pparγ - selective activators may lead to more effective drugs for the treatment of niddm and obesity as well as associated cardiovascular complications such as atherosclerosis , hypertension . preferably , the invention comprises an activator of pparγ selected from the group consisting of formula ( i )-( xiii ): ## str1 ## wherein : r 1 is selected from the group consisting of hydrogen , c 1 - 8 alkyl , aminoc 1 - 8 alkyl , c 1 - 8 alkylaminoc 1 - 8 alkyl , heteroarylaminoc 1 - 6 alkyl , ( heteroaryl )( c 1 - 8 alkyl ) aminoc 1 - 6 alkyl , ( c 1 - 8 cycloalkyl ) c 1 - 8 alkyl , c 1 - 8 alkylheteroaryllc 1 - 8 alkyl , 9 or 10 membered heterobicycle which is partially aromatic or substituted 9 or 10 membered heterobicycle which is partially aromatic ; x is selected from the group consisting of s , nh or o ; r 2 is selected from the group consisting of hydrogen , c 1 - 8 alkyl or c 1 - 8 alkenyl ; r 3 and r 4 are independently selected from the group consisting of hydrogen , hydroxy , oxo , c 1 - 8 alkyl , c 1 - 8 alkoxy or amino ; r 5 is selected from the group consisting of hydrogen , c 1 - 8 alkyl , c 1 - 8 alkenyl , ( carbonyl ) alkenyl , ( hydroxy ) alkenyl , phenyl , c 1 - 8 alkylr 6 , ( hydroxy ) c 1 - 8 alkylr 6 , c 1 - 8 alkylc 1 - 8 cycloalkylr 6 , ( hydroxy ) c 1 - 8 alkylc 1 - 8 cycloalkylr 6 or c 1 - 8 cycloalkylthior 6 ; r 6 is selected from the group consisting of phenyl or phenyl substituted with hydroxy , c 1 - 8 alkyl or c 1 - 8 alkoxy substituents ; r 7 is selected from the group consisting of hydrogen , hydroxy , carboxy or carboxyc 1 - 8 alkyl ; r 8 is selected from the group consisting of hydrogen , c 1 - 8 alkyl , phenyl , phenylc 1 - 8 alkyl , phenyl mono - or di - substituted with halo substituents or phenylc 1 - 8 alkyl wherein the phenyl is mono - or disubstituted with halo substituents ; r 9 is selected from the group consisting of hydrogen , c 1 - 8 alkyl , carboxyc 1 - 8 alkenyl mono - or disubstituted with hydroxy , phenyl or phenyl mono - or disubstituted with halo substituents ; r 11 is selected from the group consisting of hydrogen , c 1 - 8 alkyl or cycloc 1 - 8 alkylc 1 - 8 alkyl ; r 12 is selected from the group consisting of hydrogen , halo or fluorinatedc 1 - 8 alkyl ; r 13 is selected from the group consisting of hydrogen , c 1 - 8 alkoxycarbonyl or c 1 - 8 alkoxycarbonylc 1 - 8 alkylaminocarbonyl ; a dashed line (- - - - - -) is none or one double bond between two of the carbon atoms ; fluorinated alkyl in more detail is an alkyl wherein one or more of the hydrogen atoms is replaced by a fluorine atom ; heteroaryl in more detail is a 5 , 6 or 7 membered aromatic ring optionally interrupted by 1 , 2 , 3 or 4 n , s , or o heteroatoms , with the proviso that any two o or s atoms are not bonded to each other ; substituted heteroaryl in more detail is a 9 or 10 membered heterobicycle mono -, di -, or trisubstituted independently with hydroxy , oxo , c 1 - 6 alkyl , c 1 - 6 alkoxy or amino substituents ; 9 or 10 membered heterobicycle which is partially aromatic in more detail is a heterobicycle interrupted by 1 , 2 , 3 , or 4 n heteroatoms ; substituted 9 or 10 membered heterobicycle which is partially aromatic in more detail is a 9 or 10 membered heterobicycle mono -, di -, tri - or tetrasubstituted independently with hydroxy , oxo , c 1 - 8 alkyl , c 1 - 8 alkoxy , phenyl , phenylc 1 - 8 alkyl or amino substituents ; more preferably , the pparγ activator is selected from the group consisting of formula ( i ) or ( ii ): ## str2 ## wherein : r 1 is selected from the group consisting of hydrogen , c 1 - 8 alkyl , aminoc 1 - 8 alkyl , c 1 - 8 alkylaminoc 1 - 8 alkyl , heteroarylaminoc 1 - 6 alkyl , ( heteroaryl ) ( c 1 - 8 alkyl ) aminoc 1 - 6 alkyl , ( c 1 - 8 cycloalkyl ) c 1 - 8 alkyl , c 1 - 8 alkylheteroaryllc 1 - 8 alkyl , 9 or 10 membered heterobicycle which is partially aromatic or substituted 9 or 10 membered heterobicycle which is partially aromatic ; a dashed line (- - - - - -) is none or one double bond between the two carbon atoms . while it is possible that , for use in therapy , a compound of the invention may be administered as the raw chemical it is possible to present the active ingredient as a pharmaceutical formulation . the invention thus further provides a pharmaceutical formulation comprising a compound of formula ( i ) or a physiologically acceptable salt or solvate thereof together with one or more pharmaceutically acceptable carriers or excipients . the carrier ( s ) or excipient ( s ) must be acceptable in the sense of being compatable with the other ingredients of the formulation and not deleterious to the recipient thereof . according to another aspect of the invention , there is provided a process for the preparation of a pharmaceutical formulation comprising admixing a compound of formula ( i ) or a pharmaceutically acceptable salt or solvate thereof with one of more pharmaceutically acceptable carriers or excipients . compounds of formula ( i ) and physiologically acceptable salts and solvates thereof may be formulated for administration by any route , and the appropriate route will depend on the disease being treated . suitable pharmaceutical formulations include those for oral , rectal , nasal , topical ( including buccal and sublingual ), vaginal or parental ( including intramuscular , sub - cutaneous , intravenous , and directly into the affected tissue ) administration or in a form suitable for administration by inhalation or insufflation . the formulations may , where appropriate , be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy . all methods include the step of bringing into association the active compound with liquid carriers or finely divided solid carriers or both and then , if necessary , shaping the product into the desired formulation . pharmaceutical formulations suitable for oral administration may conveniently be presented as discrete units such as capsules , cachets , or tablets each containing a predetermined amount of the active ingredient ; as a powder or granules ; as a solution , a suspension or as an emulsion . the active ingredient may also be presented as a bolus , electuary , or paste . tablets and capsules for oral administration may contain conventional excipients such as binding agents , fillers , lubricants , disintegrants , or wetting agents . the tablets may be coated according to methods well known in the art . timed release formulations which are known in the art may also be suitable . oral liquid preparations may be in the form of , for example , aqueous or oily suspensions , solutions , emulsions , syrups , or elixirs , or may be presented as a dry product for constitution with water or other suitable vehicle before use . such liquid preparations may contain conventional additives such as suspending agents , non - aqueous vehicles ( which may include edible oils ), or preservatives . the compounds according to the invention may also be formulated for parental administration ( e . g . by injection , for example bolus injection or continuous infusion ) and may be presented in unit dose form in ampules , pre - filled syringes , small volume infusion or in multi - dose containers with an added preservative . the compositions may take such forms as suspensions , solutions , or emulsions in oily or aqueous vehicles , and may contain formulatory agents such as suspending , stabilizing , and / or dispersing agents . alternatively , the active ingredient may be in powder form , obtained by asceptic isolation of sterile solid or by lyophilization from solution , for constitution with a suitable vehicle , e . g . sterile , pyrogen free water , before use . for topical administration to the epidermis the compounds according to the invention may be formulated as ointments , creams or lotions , or as a transdermal patch . ointments and creams may , for example , be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents . lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents , stabilizing agents , suspending agents , thickening agents , or coloring agents . formulations suitable for topical administration in the mouth include lozenges comprising active ingredient in a flavored base , usually sucrose and acacia or tragacanth ; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia ; and mouth washes comprising the active ingredient in a suitable liquid carrier . for topical administration to the eye , the compounds according to the invention may be made up in a solution or suspension in a suitable sterile aqueous or non - aqueous vehicle . additives such as buffers ( e . g . sodium metabisulphite or disodium edeate ) and thickening agents such as hypromellose may also be included . pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are possibly presented as unit dose suppositories . suitable carriers include cocoa butter and other materials commonly used in the art , and the suppositories may be conveniently formed by admixture of the active compound with the softened or melted carrier ( s ) followed by chilling and shaping in moulds . formulations suitable for vaginal administration may be presented as pessaries , tampons , creams , gels , pastes , foams , or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate . for intra - nasal administration the compounds of the invention may be used as a liquid spray or dispersible powder or in the form of drops . drops may be formulated with an aqueous or non - aqueous base also comprising one or more dispersing agents , solubilizing agents , or suspending agents . liquid sprays are conveniently delivered from pressurized packs . for administration by inhalation the compounds according to the invention are conveniently delivered from an insufflator , nebulizer or a pressurized pack or other convenient means of delivering the aerosol spray . pressurized packs may comprise a suitable propellant such as 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 . alternatively , for administration by inhalation or insufflation , the compounds according to the invention may take the form of a dry powder composition , for example a powder mix of the compound and a suitable powder base such as lactose or starch . the powder composition may be presented in unit dosage form in , for example , capsules or cartridges or e . g . gelatin of blister packs from which the powder may be administered with the aid of an inhalator or insufflator . when desired the above described formulations adapted to give sustained release of the active ingredient may be employed . the compounds and pharmaceutical compositions of the invention may also be used in combination with other therapeutic agents . the combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof comprise a further aspect of the invention . the individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations . appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art . the amount of a compound of the invention required for use in treatment will of course vary not only with the particular compound selected but also with the route of administration , the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician . in general , however , a suitable dose will be in the range of from about 0 . 1 to 300 mg / kg of bodyweight per day , particularly from about 1 to 100 mg / kg of bodyweight per day . an appropriate dosage unit involved in oral administration may generally contain from about 1 to 250 mg , particularly from about 25 to 250 mg , of a compound of formula ( i ). the dosage employed for the topical administration will , of course , depend on the size of the area being treated . for the eyes each dose will be typically in the range of from 10 to 100 mg of the compound of formula ( i ). for use in the treatment of ppar related disorders the compounds of the invention can be administered by any of the aforementioned routes , particularly by the oral route or by injection . the daily dosage for a 70 kg mammal will be in the range of about 5 mg to 5 g of a compound of formula ( i ). as used herein the symbols and conventions used in these examples are consistent with those used in the contemporary scientific literature , for example , the journal of medicinal chemistry . a transient cotransfection assay was used to screen for activators of pparγ . as mammalian cell lines contain endogenous nuclear receptors that can complicate interpretation of the results , we used an established chimera system in which the ligand - binding domain of the murine pparγ was fused to the dna binding domain of the yeast transcription factor gal4 . the gal4 - pparγ chimera was cotransfected into cv - 1 cells with a reporter construct containing five copies of the gal4 binding site upstream of the thymidine kinase promoter driving secreted placental alkaline phosphatase ( spap ) as reporter . data is seen in the table below . gal4 - ppar chimeras and uas - tk - cat / spap reporters . the gal4 - ppar expression constructs contain the translation initiation sequence and amino acids 1 - 76 of the human glucocorticoid receptor fused to a nucleotide sequence encoding for amino acids 1 - 147 of gal4 in the psg5 expression vector ( stratagene ). a nucleotide sequence encoding for amino acids 167 - 468 , 138 - 440 , and 174 - 475 of pparα , nuc - 1 , and pparγ1 , respectively , were amplified by polymerase chain reaction ( pcr ) using vent polymerase ( new england biolabs ) and inserted c - terminal to the gal4 sequences . the uas - tk - cat / spap reporters contain 5 copies of the gal4 binding site inserted into pblcat2 or pg12 - tk - spap ( luckow , b . et al , 1987 and berger et al , 1988 ). nucleotide sequences encoding for murine pparα , nuc - 1 , and pparγ were inserted into the expression vector psg5 ( stratagene ). the 518 bp ecor1 / xba1 fragment containing the enhancer of the ap2 gene ( graves et al ., 1992 ) was inserted into pblcat2 ( luckow and schutz , 1987 ). cv - 1 cells were plated in dme medium supplemented with 10 % delipidated fetal calf serum at a density of 2 . 4 × 10 4 cells per well in a 96 - well plate ( costar ) 16 - 24 h before transfection . in general , 8 . 0 ng of reporter plasmid , 25 . 0 ng of β - galactosidase expression vector ( pch110 , pharamacia ), and 2 . 0 ng of gal4 - pparγ expression vector were mixed with carrier dna ( pbluescript , stratagene ) to a total of 80 ng of dna per well in a volume of 10 μl optime medium ( gibco brl ). to this , a second mix , containing 9 . 3 μl optime medium and 0 . 7 μl of lipofectamine ™ ( gibco brl ), was added . after 30 min . an additional 80 μl of optime medium were added and the combined mix was then applied to the cells . 16 h later the medium was exchanged to dme medium supplemented with 10 % delipidated fetal calf serum and the test compound at a concentration of 10 - 5 m . after incubation for an additional 24 h spap activity and β - galactosidase activity were measured as previously described ( pfahl et al ., 1990 ). cv - 1 cells were plated in dme medium supplemented with 10 % delipidated fetal calf serum at a density of 1 . 2 × 10 5 cells per well in a 24 - well plate ( costar ) 16 - 24 h before transfection . in general , 100 ng of reporter plasmid , 200 ng of β - galactosidase expression vector ( pch 110 , pharamacia ), and variable amounts amounts of receptor expression vector were mixed with carrier dna ( pbluescript , stratagene ) to 500 ng of total dna per well in a volume of 50 μl optime medium ( gibco brl ). to this mix , 50 μl of a 1 : 10 dilution of lipofectamine ™ ( gibco brl ) in optime medium was added . after 30 min . an additional 400 μl of optime medium was added and the total mix applied to the cells . 16 h later , the medium was exchanged to dme medium supplemented with 10 % delipidated fetal calf serum and the test compound at the indicated concentration . after incubation for an additional 24 h , chloramphenicol acetyl transferase ( cat ) activity and β - galactosidase activity were measured as previously described ( pfahl et al ., 1990 ). cat activity was normalized for transfection efficiency by using the cotransfected β - galactosidase expression plasmid ( pch110 , pharmacia ) as internal standard . c3h10t1 / 2 cells were seeded at a density of 2 . 5 × 10 - 6 per 225 cm 2 flasks ( costar ). 16 h later 0 . 1 % dmso ( vehicle ), pioglitazone at 10 - 5 m or the mhi differentiation cocktail ( 50 μm hydrocortisone , 0 . 5 mm 3 - isobutyl - 1 - methylxanthine , 60 μm indomethacin ) was added . medium was exchanged every three days and fresh compound added . cells were harvested on day nine and poly ( a )+ rna prepared using the polyatract system 1000 ( promega ). 41 μg of poly ( a )+ rna were loaded per lane for northern analysis . c3h10t / 1 / 2 cells were grown in dme medium ( gibco brl ) supplemented with 10 % fetal calf serum . cells were plated at a density of 1 . 0 × 10 5 cells per well in a 24 - well plate ( costar ). 16 h later , compound was added at the indicated concentration . medium and compound were exchanged every three days . cells were stained at day 7 with oil - red - o and photographed . ______________________________________results from transfection assay : spap reporter . compound fold activation______________________________________5 -{ 4 - 2 -( methyl - pyridine - 2 - yl - amino )- ethoxy !- 47 . 6benzyl }- thiazolidine - 2 , 4 - dione5 - 4 -( 1 - methyl - cyclohexylmethoxy )- benzyl )- 46 . 1thiazolidine - 2 , 4 - dione5 -{ 4 - 2 -( 5 - ethyl - pyridine - 2 - yl )- ethoxy !- 42 . 1benzyl }- thiazolidine - 2 , 4 - dione7 -{ 2 - 4 -( 1 - methyl - hexyl )- phenyl !- cyclopent - 27 . 02 - enyl }- heptanoic acid2 -( 6 , 6 , 9 , 9 - tetramethyl - 6 , 7 , 8 , 9 - tterahydro - 25 . 9benzo g ! quinolin - 3 - yl )- benzoic acid { 4 - chloro - 6 -( 2 , 3 - dimethyl - phenylamino )- pyrimidin - 25 . 72 - ylsulfanyl }- acetic acid7 - 43 - hydroxy - 5 - oxo - 2 -( 3 - oxo - oct - 1 - enyl )- cyclopentyl !- 25 . 6heptanoic acid7 -{ 2 - 4 -( cyclopentyl - hydroxy - methyl )- phenyl !- 24 . 65 - oxo - cyclopent - 1 - enyl }- heptanoic acid2 - 3 - bromo - 5 -( 2 - butyl - 4 - chloro - 5 - 23 . 9ethoxycarbonylmethylcarbamoyl - imidazol - 1 - ylmethyl )- benzofuran - 2 - yl !- benzoic acid7 - 5 - hydroxy - 2 -( 3 - hydroxy - oct - 1 - enyl )- 3 - oxo - 23 . 8cyclopentyl !- hept - 5 - enoic acid ( 2 - phenyl - 3 - propyl - 1h - indol - 5 - yl )- acetic acid 21 . 7 ( 3 - phenethyl - 2 - phenyl - 1h - indol - 5 - yl )- acetic acid 21 . 25 - 5 - hydroxy - 4 -( 3 - hydroxy - oct - 1 - enyl )- hexahydro - 19 . 6pentalen - 2 - ylidene !- pentanoic acid5 -( 2 - benzyl - chroman - 6 - ylmethyl )- thiazolidine - 2 , 4 - dione 18 . 2 5 - dimethylamino - 2 -( 4 - dimethylamino - benzyl )- phenyl !- 17 . 8acetic acid9 - 2 -( 4 - carboxy - butyl )- 4 , 6 - diisopropoxy - phenyl !- 7 - oxo - 11 . 3non - 8 - enoic acid______________________________________ to test for direct interactions between thiazolidinediones and pparγ , the ligand binding domain of pparγ was expressed in e . coli as a fusion protein with glutathionine - s - transferase ( gst - pparγ lbd ). radiolabeled 5 -{ 4 - 2 -( methyl - pyridine - 2 - yl - amino )- ethoxy !- benzyl }- thiazolidine - 2 , 4 - dione bound specifically and saturably to gst - pparγ lbd with a k d of 43 nm . no binding was detected in extracts from bacteria expressing glutathione - s - transferase . cdna encoding amino acids 174 to 475 of pparg1 was amplified via polymerase chain reaction and inserted into bacterial expression vector pgex - 2t ( pharmacia ). gst - pparγ lbd was expressed in bl21 ( de3 ) plyss cells and extracts prepared as previously described ( mangelsdorf , 1991 ). bacterial extracts containing the glutathione - s - transferase - pparγ ligand binding domain fusion protein ( gst - pparγ lbd ) were incubated with increasing concentrations of tritiated 5 -{ 4 - 2 -( methyl - pyridine - 2 - yl - amino )- ethoxy !- benzyl }- thiazolidine - 2 , 4 - dione ( specific activity , 40 ci / mmol ) in the absence or presence ( of a 500 - fold excess of nontritiated 5 -{ 4 - 2 -( methyl - pyridine - 2 - yl - amino )- ethoxy !- benzyl }- thiazolidine - 2 , 4 - dione . specific binding of tritiated 5 -{ 4 - 2 -( methyl - pyridine - 2 - yl - amino )- ethoxy !- benzyl }- thiazolidine - 2 , 4 - dione to gst - pparγ lbd was 14 , 500 dpm at 200 nm with a non - specific binding of 1 , 900dpm , and 13 , 500 dpm at 100 nm with a non - specific binding of 1 , 700 dpm . for saturation binding analysis , bacterial extracts ( 100 μg protein ) were incubated at 40 ° c . for 3 hr in buffer containing 10 mm tris , ph 8 . 0 , 50 mm kcl , 10 mm dtt with 3 h !- 5 -{ 4 - 2 -( methyl - pyridine - 2 - yl - amino )- ethoxy !- benzyl }- thiazolidine - 2 , 4 - dione ( specific activity , 40 ci / mmol ) in the presence or absence of unlabeled 5 -{ 4 - 2 -( methyl - pyridine - 2 - yl - amino )- ethoxy !- benzyl }- thiazolidine - 2 , 4 - dione . bound was separated from free radioactivity by elution through 1 ml sephadex g - 25 desalting columns ( boehringer mannheim ). bound radioactivity eluted in the column void volume and was quantitated by liquid scintillation counting . berger , j ., hauber , r ., geiger , r ., and cullen , b . r . 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