Patent Application: US-57421504-A

Abstract:
this invention relates to inhibition of αv integrins , especially αvβ3 and αvβ6 integrins , by specific antagonists , preferably non - peptidic antagonists , related compounds and compounds with comparable specificity , that downregulate fibrogenesis by inhibiting cell migration and production of pro - fibrogenic molecules and cytokines by activated hepatic stellate cells / myofibroblasts , activated epithelia and endothelia . these antagonists alone or in combination with other agents can effectively prevent , mitigate or even reverse development of advanced fibrosis , such as fibrosis / cirrhosis of the liver and fibrosis of other organs , such as lungs , kidneys , intestine , pancreas , skin and arteries .

Description:
briefly , the liver was perfused in situ through the portal vein using a 16 - 18 g cannula with calcium - free hbss ( gybco , uk ) for s min followed by 0 , 1 % pronase e ( sigma ) and subsequently 0 . 025 % type iv collagenase ( sigma ) in dulbecco &# 39 ; s modified eagle &# 39 ; s medium for 10 - 15 min each . the digested liver was excised , gently minced and incubated further with 0 . 04 % pronase , 0 , 025 % collagenase , 0 , 002 % dnase ( sigma ) in dulbecco &# 39 ; s modified eagle &# 39 ; s medium , supplemented with 25 mm hepes at 37 ° c . for 10 - 30 min with gentle agitation . after filtration through 100 μm nylon gauze , parenchymal cells were removed by low speed centrifugation . the hsc fraction was collected from the gradient interface after enrichment by a two - step centrifugation through a 11 and 13 % gradient of nycodenz ( sigma ) at 1500 g for 15 min without braking , and plated at a density 0 . 5 × 10 6 / cm 2 in dmem supplemented with 10 % fcs , penicillin and streptomycin . medium was changed after 24 h , and further every 48 hours . viability of the isolated cells was assessed by trypan blue exclusion and was routinely greater then 95 - 98 %. purity of hsc isolates was confirmed by their typical morphological appearance : lipid - droplets in the cytoplasm showing greenish autofluorescence at 390 nm excitation and star - like shape . contamination with kupffer cells was assessed by the ability to engulf 3 μm latex beads and was below 3 - 5 % after isolation , and almost undetectable after the 1 st passage . for experiments cells were used between the 1st and 3rd passage , if not stated otherwise . in addition the hsc cell lines cfsc - 2g ( moderately activated , kind gift of dr . m . rojkind , washington dc , usa ) were used . animal experimentation : male adult wistar rats , average weight 206 ± 19 g , underwent the following microsurgical procedure under an operating microscope ( opmi 6 - s , zeiss , germany ) ( 45 - 47 ): 1 . midline abdominal incision following anesthesia with 100 mg / kg ketamine - hydrochloride ( ketanest ®, parke - davis , germany ) and 10 mg / kg 5 , 6 - dihydro - 2 -( 2 , 6 - xylidino )- 4h - 1 , 3 - thiazine - hydrochloride ( rompun ®, bayer , germany ); 2 . dissection of the common bile duct , insertion of a teflon catheter ( abbocath ®- t 26 g , venisystems , usa ) and placement of a distal complete and a proximal incomplete ligature with 5 - 0 silk ( perma - hand ®, ethicon , germany ); 3 . retrograde injection of sodium - amidotrizoate ( ethibloc ®, ethicon germany ) at a dose of 0 . 02 ml / l 100 g body weight ; 4 . removal of the catheter , closure of the proximal ligature , scission of the bile duct between the ligatures and wound closure . after bile duct occlusion ( bdo ), animals received normal chow ( altromin ®, lage , germany ) and were allowed free access to water . early mortality ( within 1 h to 3 days ) in rats with bdo was due to bile leakage and amounted to 9 %. since in this model significant fibrosis is evident only after two weeks of bdo , animals which died before did not have to be considered for statistical analysis . after 6 weeks rats were sacrificed under ketanest / rompun - anesthesia by puncture of the right ventricle and exsanguination . liver and spleen were weighed , and 1 - 2 g pieces of the left and the right liver lobes fixed in 4 % formalin or snap frozen in liquid nitrogen for histology , mrna and hydroxyproline ( hyp ) determinations . scratch assay : cell migration was estimated by measuring the reduction of scratch area . cells were seeded on a 24 - well plate at a density of 60 , 000 cells / well . after reaching confluency cells were starved in serum - free medium for 24 h before making a scratch through the cell monolayer using a sterile pipet tip . after wounding cells were pre - treated by the αvβ3 - integrin inhibitor at increasing concentrations ( 10 − 10 m - 10 − 6 m ) for 30 min and then treated by pdgf - bb at a concentration of 10 ng / ml in the absence of serum . the reduction of scratch area was measured at three different points per well using a special ocular with net - micrometer after 15 - 20 h . brdu incorporation : to assess cell proliferation de novo dna synthesis was determined as brdu incorporation . cells were seeded at a density 20 . 000 cells / well in 96 - well plates in growth medium containing 10 % fetal calf serum ( fcs ). after 24 h medium was changed to 0 % fcs for the next 24 h , followed by incubation with medium containing pdgf - bb at a concentration of 10 ng / ml , αvβ3 - integrin inhibitor ( 10 − 9 - 10 − 6 ) or no inhibitor for 20 h . during last the urs of incubation cells were pulse - labeled with brdu and its incorporation measured using an elisa kit ( roche ) and a micro - plate reader . real - time pcr total rna was isolated from the cell lysates using the rnapure commercial kit ( peqlab , erlangen , germany ) according to the manufacturer &# 39 ; s recommendations . template cdna was obtained by reverse transcription of 0 . 5 mg of total rna . relative transcript levels were quantified by real time rt - pcr using the lightcycler system ( roche ), using 1 , 5 μl of template cdna dilution in a total reaction volume of 15 μl that included taq dna - polymerase , dntp - mix , reaction buffer , and 3 . 0 mm mgcl 2 provided by the “ lightcycler faststart dna master hybridization probes kit ” ( roche molecular biochemicals , mannheim , germany ) according to the manufactor &# 39 ; s instructions . for every measured transcript a 1 : 2 to 1 : 32 dilution series of one sample was used as standard . data were analyzed with the lightcycler software using the “ proportional second derivative maximum ” option . the housekeeping genes beta - 2 microglobulin or gapdh were amplified in a parallel reaction for normalization of the results . taqman probes and primer sets were designed using the primer express software ( perkin elmer ) based on published sequences . sense and antisense primers ( each at 0 , 5 μm ) and 0 , 125 μm 5 ′- phoshorylated probe , labeled at its 5 ′- end with the reporter dye ( fam ) and at the 3 ′ end with the quencher molecule ( tamra ), were synthesized at mwg biotech ag ( ebersberg , germany ). primers sets that were used to measure expression of specific target genes are summarised in the table : taqman probe labelled with 5 ′- fam + 3 ′- target tamra sybr molecule 5 ′- primer green ) 3 ′- primer procollagen tccggctcct ttcttggccatgcg gtatgcagctgac α1 ( i ) gctcctctta tcaggaggg ttcagggatgt mmp - 2 ccgaggacta tctgccccgagacc cttgttgcccagg tgaccgggat gctatgtcca aaagtgaag aa mmp - 3 ccgtttccat agatggtattcaat cagagagttagat ctctctcaag ccctctatggacct ttggtgggtacca atga cc mmp - 13 ggaagaccct tctggttagcatca tcatagacagcat cttcttctca tcataactccacac ctactttgtc gt timp - 1 tcctcttgtt ttctgcaactcgga cgctggtataagg gctatcattg cctggttataagg tggtctcgat atagctt tgfβ1 agaagtcacc accgcaacaacgca tcccgaatgtctg cgcgtgctaa atctatgacaaaac acgtattga ca ctgf atccctgcga ctcccccgccaacc caactgctttgga cccacacaag gcaagat aggactcgc β2 ccgatgtata aaccgtcacctggg cagatgattcaga mikroglob . tgcttgcaga accgagacatgta gctccataga gttaa β3 integrin tccaagtgcg sybr green cagactgtagcct gcaggtgg gcatgatgg β6 integrin catttggatt - - - gatattccaagac caagcacatt agttgacatgg ttgc for quantification of β3 and β36 integrin steady - state transcript levels , real - time pcr with sybr green as the fluorophore ( molecular probes , eugene , oreg .) was used . for distinguishing the specific pcr product from non - specific products and primer dimers , melting curve analyses were performed . because different dna product melt at different temperatures , it was possible to distinguish genuine products from primer dimers or nonspecific products . all experiments were performed in cell culture with the myofibroblasts - like cell line cfsc - 2g ( hsc cell line derived from cirrhotic rat liver ) and primary rat hsc / mf . to investigate the effect of the αvβ3 integrin inhibitor on cell migration cells were seeded on 24 well plates and after reaching confluence starved in the absence of fcs for 24 h . scratches were done and the cells treated with pdfg - bb at 10 ng / ml in the presence or absence of the αvβ3 inhibitor at 10 − 6 -− 10 9 m . the rate of migration was measured after 17 - 20 h as a reduction of initial scratch width . the αvβ3 inhibitor strongly inhibited pdgf - bb induced cell migration into the scratch area in a dose - dependent manner . complete abrogation of migration was observed at 10 − 6 m in all cell types used ( fig1 ). surprisingly , there was no effect of the αvβ33 integrin inhibitor on cell migration stimulated by fcs ( fig2 ) which could be due to stimulation of other , apparently more normal pathways involved in migration triggered by fcs . emd 409915 did not show any significant inhibition of migration even at reduced concentrations of fcs ( 0 , 25 %) ( data not shown ). these observations suggest that pdgf - induced hsc / mf migration is specifically and strongly αvβ3 - dependent . thus it is attractive to block hscmf migration in fibrotic liver and other fibrotic organs in a very specific manner , not interfering to a significant degree with “ normal ” migration by using β3 integrin inhibition . to check whether the αvβ33 inhibitor showed similar effects on cell proliferation , cells were starved in serum - free medium for 24 h and then treated with pdgf - bb at a concentration of 10 ng / ml and the αvβ33 inhibitor at 10 − 8 - 10 − 6 m for 24 h . under these conditions the αvβ3 inhibitor did not have any effect on pdgf - bb stimulated cell proliferation in all cell types ( fig3 ). the same absence of effect was observed for serum - stimulated cell proliferation ( fig4 ). to investigate whether the αvβ13 integrin inhibitor had any effect on ecm expression in hsc / mf mrna , expression of a spectrum of the major pro - and anti - fibrogenic molecules like procollagen α1 ( i ) i , tgfβ1 , tgfβ2 , mmp - 3 , mmp - 13 , mmp - 2 , connective tissue growth factor ( ctgf ) and timp - 1 was measured by real - time pcr . at confluency cells were starved for 24 h in serum - free medium , then pretreated with the αvβ3 inhibitor for 30 min and stimulated with pdfg - bb ( 10 ng / ml ) for the next 24 h . as shown in fig5 , the αvβ33 inhibitor downregulated ctgf expression in cfsc - 2g cells in a dose - dependent manner , with a maximum of inhibition at 10 − 6 m . there was no change in transcripts levels of other ecm molecules ( data not shown ). to investigate patterns of integrin expression during liver fibrosis , the rat model of complete biliary obstruction for six weeks , which results in cirrhosis with a 4 or 10 - 12 fold accumulation of relative ( per g of liver ) and absolute ( per total liver ) liver collagen , resp ., was used . after 6 weeks of bile duct occlusion a dramatic upregulation of mrna expression of procollagen α1 ( i ), tgfβ1 , tgfβ2 and ctgf ( 25 -, 10 -, 200 - and 190 - fold , resp .) was observed as compared to sham - operated animals ( fig6 ). at the same time αvβ3 mrna was upregulated moderately ( fig7 a ), while a dramatic ( 180 - fold ) overexpression of β6 integrin subunit was observed in cirrhotic livers ( fig7 b ). this strong upregulation of β6 integrin has never been shown in fibrotic diseases and in liver fibrosis in particular . the cells responsible for this upregulation are mainly proliferating bile duct epithelial cells ( but also activated hsc / myofibroblasts ), those cells that secrete large amounts of profibrogenic cytokines , such as tgf β , and ctgf and which therefore are prime targets for liver fibrosis therapy , apart from activated hsc / mf . moreover , our recent pilot experiments in the relentlessly progressive rat fibrosis model of bile duct occlusion ( 5 - 6 animals per group ) suggests that specific β6 integrin inhibition by emd 409849 can significantly ameliorate secondary biliary liver fibrosis by reducing total liver collagen by 50 - 70 % after 5 - 6weeks of bile duct occlusion . taken together these data show that inhibition of integrins αvβ3 and αvβ6 by specific low molecular weight peptides and in particular their non - peptide analogues is a powerful tool to ameliorate , block or even reverse fibrosis of the liver and of other organs the treatment of which remains largely elusive ( 11 - 19 , 33 - 44 ). the compounds for use according to the present invention and / or their physiologically acceptable salts and / or their physiologically acceptable derivatives can therefore be used for the production of pharmaceutical compositions or preparations by bringing them into the suitable dose form together with at least one excipient or auxiliary and , if desired , with one or more further active compounds . the compositions or preparations thus obtained can be employed as medicaments in human or veterinary medicine . suitable excipients are organic or inorganic substances which are suitable for enteral ( e . g . oral or rectal ) or parenteral administration and do not react with the compounds for use according to the present invention , for example water , vegetable oils , benzyl alcohols , polyethylene glycols , glycerol triacetate and other fatty acid glycerides , gelatin , soya lecithin , carbohydrates such as lactose or starch , magnesium stearate , talc or cellulose . for oral administration , in particular tablets , coated tablets , capsules , syrups , juices or drops are used . of interest are especially coated tablets and capsules having enteric coatings or capsule shells . for rectal administration , suppositories are used , and for parenteral administration , solutions , preferably oily or aqueous solutions , and also suspensions , emulsions or implants are used . the compounds for use according to the invention can also be lyophilized and the lyophilisates obtained used , for example , for the production of injection preparations . the compositions or preparations indicated can be sterilized and / or contain auxiliaries such as preservatives , stabilizers and / or wetting agents , emulsifiers , salts for affecting the osmotic pressure , buffer substances , colourants and / or flavourings . if desired , they can also contain one or more further active compounds , e . g . one or more vitamins , diuretics , anti - inflammatorial compounds , anti - diabetics , analgetics , anti - phlogistics or compounds other than the compounds for use according to the invention , such as compounds that are not subject of the present invention , that can be used in the diagnosis , prophylaxis and / or treatment of the disorders , clinical pictures and / or symptoms as described herein , for example to improve or enhance the therapeutic effect and / or tolerance of the compounds further . pharmaceutical compositions according to the invention can be obtained or produced according to methods known in the art or analogously to these methods . usually , the pharmaceutical compositions according to the invention are produced with non - chemical methods , for example by mixing the active ingredients with , e . g . physiologically acceptable excipients , auxiliaries , adjuvants and carriers , and converting the mixture into the desired dosage form , for example into tablets by molding methods or into solutions by solving the active ingredients in a solvent . in general , the active ingredients are converted into a pharmaceutical composition together with one or more excipient , for example a solid , liquid and / or semiliquid excipient , or one or more auxiliaries and , if desired , in combination with one or more further active ingredients . these preparations can be used as medicaments in human or veterinary medicine . suitable excipients are organic or inorganic substances which are suitable for enteral ( for example oral ), parenteral or topical administration and do not react with the novel compounds , for example water , vegetable oils , benzyl alcohols , alkylene glycols , polyethylene glycols , glycerol triacetate , gelatine , carbohydrates , such as lactose or starch , magnesium stearate , talc or vaseline . suitable for oral administration are , in particular , tablets , pills , coated tablets , capsules , powders , granules , syrups , juices or drops , suitable for rectal administration are suppositories , suitable for parenteral administration are solutions , preferably oily or aqueous solutions , furthermore suspensions , emulsions or implants , and suitable for topical application are ointments , creams or powders . the novel compounds can also be lyophilized and the resultant lyophilizates used , for example , for the preparation of injection preparations . the preparations indicated may be sterilized and / or comprise assistants , such as lubricants , preservatives , stabilizers and / or wetting agents , emulsifiers , salts for modifying the osmotic pressure , buffer substances , dyes , flavours and / or a plurality of further active ingredients , for example one or more vitamins . for administration as an inhalation spray , it is possible to use sprays in which the active ingredient is either dissolved or suspended in a propellant gas or propellant gas mixture ( for example co 2 or chlorofluorocarbons ). the active ingredient is advantageously used here in micronized form , in which case one or more additional physiologically acceptable solvents may be present , for example ethanol . inhalation solutions can be administered with the aid of conventional inhalers . accordingly , the antagonists are preferably administered in doses between about 0 . 001 mg and 200 mg , more preferably between about 0 . 01 mg and 100 mg , even more preferably between about 0 . 01 mg and 50 mg and in particular between 0 . 01 and 30 mg , per dose unit . the daily dose is preferably more than or equal to about 0 . 0001 mg / kg , more preferably more than or equal to about 0 . 001 mg / kg , even more preferably more than or equal to about 0 . 005 mg / kg , more than or equal to about 0 . 01 mg / kg or more than or equal to about 0 . 1 mg / kg . the daily dose is preferably less than or equal to about 30 mg / kg , more preferably less than or equal to about 20 mg / kg , even more preferably less than or equal to about 15 mg / kg , less than or equal to about 5 mg / kg or less than or equal to about 1 mg / kg of body weight . 1 . hood j d , cheresh d a . role of integrins in cell invasion and migration . nat . rev . cancer 2002 ; 2 : 91 - 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7 .