Patent Application: US-201414896269-A

Abstract:
presented herein are methods for treatment of diseases or conditions related to fibrosis . compounds , or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof provide for the treatment of diseases or conditions related to fibrosis . the methods utilize the compounds kynurenine , kynurenic acid and xanthurenic acid , and various analogues , related structures and pharmaceutical compositions thereof , wherein the compounds are represented by one or more compounds represented by formulas i , ii , or ii as set out below :

Description:
any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention . as employed throughout the specification , the following terms , unless otherwise indicated , shall be understood to have the following meanings . as used herein a ‘ subject ’ refers to an animal , such as a bird or a mammal . specific animals include rat , mouse , dog , cat , cow , sheep , horse , pig or primate . a subject may further be a human , alternatively referred to as a patient . a subject may further be a transgenic animal . a subject may further be a rodent , such as a beaver , mouse or a rat . as used herein , an ‘ inhibitor ’ refers to a drug , compound or an agent that restrains or retards a physiological , chemical or enzymatic action or function . an inhibitor may cause at least 5 % decrease in enzyme activity . an inhibitor may also refer to a drug , compound or agent that prevents or reduces the expression , transcription or translation of a gene or protein . ‘ indoleamine 2 , 3 - dioxygenase ’, or ‘ ido ’, is a heme - containing rate limiting enzyme that catalyzes tryptophan to n - formylkynurenine and then to kynurenine ( kyn ), and is found in non - hepatic cells mainly in macrophages and trophoblasts . recent findings have implicated catabolism of tryptophan , an essential amino acid , by ido as being involved in immune tolerance ( kahari and saarialho - kere 1997 ). as demonstrated herein , kynurenine , as well as its breakdown products kynurenic acid and xanthurenic acid , induce mmp - 1 and mmp - 3 , as well as showing a reduction of fibrosis in vitro and in vivo . the ‘ matrix metalloprotease ’, or ‘ mmp ’ family consist of 25 zinc - and calcium - dependent proteinases in the mammalian system . according to their substrate specificity , primary structure and cellular localization , 5 different subfamilies of closely related members known as collagenases , gelatinases , stromelysins , matrilysins , and membrane - type mmps have been identified ( murphy et al . 2002 ). from all of these mmps , mmp1 is the major enzyme involved in the collagenolytic process , breaking down the interstitial collagens such as types i , ii , and iii , while mmp - 3 ( stromelysin - 1 ) is a protease known to degrade mainly the noncollagenous portion of the ecm such as fibronectin , proteoglycans , and laminin ( kahari and saarialho - kere 1997 ). increases in both mmp1 and mmp - 3 expressions and released by fibroblasts can initiate degradation of almost all major components of the ecm ( saus et al . 1988 ). it is now accepted that mmps produced by keratinocytes facilitate epithelial migration , while mmps expressed by fibroblasts promote tissue remodeling ( salo et al . 1991 ). ‘ fibrosis ’ is a general terms that involves the formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process , as opposed to a formation of fibrous tissue as a normal constituent of an organ or tissue . scarring is confluent fibrosis that obliterates the architecture of the underlying organ or tissue . there are many diseases and / or conditions that are characterized by or associated with fibrosis , including , but not limited to : keloid , hypertrophic scar , pulmonary fibrosis , kidney fibrosis , liver cirrhosis , chronic inflammation of tunica albugenia ( cita ), endomyocardial fibrosis , mediastinal fibrosis , myelofibrosis , retroperitoneal fibrosis , progressive massive fibrosis , nephrogenic systemic fibrosis , crohn &# 39 ; s disease , old myocardial infarction , scleroderma , and systemic sclerosis . there are provided herein a number of compounds for use in the treatment of diseases or conditions characterized by or related to fibrosis . in the context of the current description , the term ‘ treatment ’ may refer to treatment of existing fibrosis or fibrotic disease , or alternately may refer to treatment which occurs before or during the fibrotic process in order to prevent the development or progression of fibrosis . the compounds described herein may be in isolation , or may be linked to or in combination with tracer compounds , liposomes , carbohydrate carriers , polymeric carriers or other agents or excipients as will be apparent to one of skill in the art . in an alternate embodiment , such compounds may comprise a medicament , wherein such compounds may be present in a pharmacologically effective amount . the compounds may be suitable for administration to a subject in need thereof , by virtue of the fact that the subject may benefit from prophylaxis or treatment of fibrosis or fibrotic disease . the compounds may also include tautomers or stereoisomers . as used herein “ fs ” refers to fibrostops ( for example , fs1 is used as an abbreviation for kynurenine ( or dl - kynurenine or dl - kyn ) and fs2 or ka may be used as an abbreviation for kynurenic acid ). l - kynurenine may be represented herein as l - kyn and d - kynurenine may be represented herein as d - kyn . similarly , xanthurenic acid may be represented herein as xa . the term ‘ medicament ’ as used herein refers to a composition that may be administered to a patient or test subject and is capable of producing an effect in the patient or test subject . the effect may be chemical , biological or physical , and the patient or test subject may be human , or a non - human animal , such as a rodent or transgenic mouse , or a dog , cat , cow , sheep , horse , hamster , guinea pig , rabbit or pig . the medicament may be comprised of the effective chemical entity alone or in combination with a pharmaceutically acceptable excipient . the term ‘ pharmaceutically acceptable excipient ’ may include any and all solvents , dispersion media , coatings , antibacterial , antimicrobial or antifungal agents , isotonic and absorption delaying agents , and the like that are physiologically compatible . an excipient may be suitable for intravenous , intraperitoneal , intramuscular , subcutaneous , intrathecal , topical or oral administration . an excipient may include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersion . use of such media for preparation of medicaments is known in the art . compositions or compounds according to some embodiments may be administered in any of a variety of known routes . examples of methods that may be suitable for the administration of a compound include orally , intravenous , inhalation , intramuscular , subcutaneous , topical , intraperitoneal , intra - rectal or intra - vaginal suppository , sublingual , and the like . the compounds described herein may be administered as a sterile aqueous solution , or may be administered in a fat - soluble excipient , or in another solution , suspension , patch , tablet or paste format as is appropriate . a composition comprising the compounds described herein may be formulated for administration by inhalation . for instance , a compound may be combined with an excipient to allow dispersion in an aerosol . examples of inhalation formulations will be known to those skilled in the art . other agents may be included in combination with the compounds described herein to aid uptake or metabolism , or delay dispersion within the host , such as in a controlled - release formulation . examples of controlled release formulations will be known to those of skill in the art , and may include microencapsulation , embolism within a carbohydrate or polymer matrix , and the like . other methods known in the art for making formulations are found in , for example , “ remington &# 39 ; s pharmaceutical sciences ”, ( 19th edition ), ed . a . gennaro , 1995 , mack publishing company , easton , pa . the dosage of the compositions or compounds of some embodiments described herein may vary depending on the route of administration ( oral , intravenous , inhalation , or the like ) and the form in which the composition or compound is administered ( solution , controlled release or the like ). determination of appropriate dosages is within the ability of one of skill in the art . as used herein , an ‘ effective amount ’, a ‘ therapeutically effective amount ’, or a ‘ pharmacologically effective amount ’ of a medicament refers to an amount of a medicament present in such a concentration to result in a therapeutic level of drug delivered over the term that the drug is used . this may be dependent on mode of delivery , time period of the dosage , age , weight , general health , sex and diet of the subject receiving the medicament . methods of determining effective amounts are known in the art . in one embodiment , there is provided a method for treatment of a subject having or suspected of having a fibrotic disease , the method comprising administering to the subject a therapeutically effective amount of a compound having a structure corresponding to formula i , ii , or iii . the fibrotic disease may be one of the following : keloid , hypertrophic scar , pulmonary fibrosis , kidney fibrosis , liver cirrhosis , chronic inflammation of tunica albugenia ( cita ), endomyocardial fibrosis , mediastinal fibrosis , myelofibrosis , retroperitoneal fibrosis , progressive massive fibrosis , nephrogenic systemic fibrosis , crohn &# 39 ; s disease , old myocardial infarction , scleroderma , systemic sclerosis , uterine fibroids , restenosis . neonatal foreskin and joints used as the sources of fibroblasts , keratinocytes and synoviocytes . the procedures were done based on the approval of human ethics committee of the university of british columbia . cultures of human foreskin fibroblasts were established as described previously ( li et al ., 2006 ). briefly , foreskin was collected and washed three times with dulbecco &# 39 ; s modified eagle medium ( dmem ; gibco ™, grand island , n . y .) supplemented with antibiotic - antimycotic preparation ( 100 u / ml penicillin , 100 μg / ml streptomycin , 0 . 25 μg / ml amphotericin b ) ( invitrogen life technologies ™, gaithersburg , md .). specimens were dissected free of fat and minced into small pieces less than 2 . 0 mm in diameter , washed six times with dmem , distributed into 60 × 15 - mm petri dishes and incubated at 37 ° c . in a water - jacked humidified incubator in an atmosphere of 5 % co 2 . the medium was replaced twice weekly . upon reaching confluence , the cells were released by trypsinization ( 0 . 1 % trypsin , invitrogen life technologies ™) and ( 0 . 02 % edta , sigma ™, st . louis , mo . ), split for subculture at a ratio of 1 : 6 , and reseeded onto 75 - cm 2 flasks . fibroblasts from passages 3 - 7 were used for this study . human foreskin keratinocytes were established as previously described ( ghahary et al ., 1998 ). cells were cultured in serum - free keratinocyte medium ( ksfm ; invitrogen life technologies ™) supplemented with bovine pituitary extract ( 50 μg / ml ) and egf ( 0 . 2 ng / ml ). these cells were used at passages 2 - 5 . synoviocytes were obtained by enzymatic digestion of synovial membrane from patients with rheumatoid arthritis during joint replacement with 1 mg / ml collagenase ( sigma ™) in rpmi1640 ( invitrogen life technologies ™) for 4 hours at 37 ° c . dissociated cells were plated in synoviocyte growth medium ( cell applications inc .™, san diego , calif .) supplemented with penicillin g sodium ( 100 u / ml ), streptomycin sulfate ( 100 μg / ml ), and amphotericin b ( 0 . 25 μg / ml ). synoviocytes were found to be morphologically homogenous fibroblast - like cells and were used at passages 2 - 5 . the squamous cell carcinoma ( umscc ) cell line derived from patients with head and neck cancer ( atcc ™, manassas , va .) were maintained in rpmi - 1640 medium with 10 % fbs . the human keratinocyte cell line hacat ( atcc ) and the carcinomic human alveolar basal epithelial cell line a549 ( atcc ™) were cultured in dmem with 10 % fbs . the diploid lung fibroblasts imr - 90 ( atcc ™) were maintained in minimum essential medium ( mem , invitrogen ™) with 10 % fbs . the construction of indoleamine 2 , 3 - dioxygenase ( ido ) expressing adenoviral vector has been previously described ( li et al ., 2004 ). recombinant adenoviruses were used to infect human skin fibroblasts at the multiplicity of infection ( moi ) of 100 . free viral particles were removed from culture medium 30 hours after infection . the success of infection was determined by fluorescent microscopy using a motic ™ inverted microscope equipped with a fluorescein isothiocyanate ( fitc ) filter ( motic instruments ™, richmond , bc , canada ) to view the reporter gene gfp . the expression of ido was assessed by western blot using anti - human ido antibody as described previously ( li et al ., 2004 ). the biologic activity of ido was evaluated by measuring the levels of tryptophan degrading product , kynurenine , present in conditioned medium . the levels of kynurenine were measured by a method previously described ( tokikawa et al ., 1988 ). in brief , about 2 ml of conditioned media was collected from the same cell number initiated culture 3 days post transfection . proteins from conditioned media were precipitated by trichloroacetic acid . after centrifugation to remove precipitated proteins , about 0 . 5 ml of supernatant was transferred into a new 1 . 5 ml tube and incubated with equal volume ehrich &# 39 ; s reagent ( sigma ™) for to minutes at room temperature . the absorption of resultant solution was measured at 490 nm by spectrophotometer within 2 hours . the values of kynurenine in conditioned media were calculated by a standard curve with defined kynurenine concentration ( 0 - 20 μg / ml ). for collection of conditioned media , fibroblasts were transduced by either none or control mock vector or ido adenovirus for 30 hours . viruses were removed by washing with pbs . fresh dmem containing 10 % fbs and antibiotics were added and cells were continued to be cultured for another 48 hours . conditioned media from either untreated , mock vector , or ido adenovirus transduced fibroblasts were then collected . fibroblasts at 80 % confluence were treated with media containing 90 % of conditioned media plus 10 % fresh media in the presence of 10 % fbs . cells were then harvested after 48 hours and western blot analysis was performed . in another set of experiments , fibroblasts at 80 % confluence were treated with either kynurenine or tryptophan at the indicated concentrations as mentioned in the result section in dmem containing 2 % fbs and antibiotics for 48 hours . cells were then harvested by trypsinization and western blot analysis was performed . similarly , other cells such as synoviocytes , imr - 90 , keratinocytes , umscc and a549 were treated with kynurenine at concentrations of 12 . 5 to 150 μg / ml in appropriate media for each cell type as described above for 48 hours . cells were then harvested for western blot analysis . cells were harvested by trypsin / edta and lysed with cell lysis buffer containing 50 mm tris - hcl ( ph7 . 40 ), 150 mm nacl , 10 mm edta , 5 mm egta , 1 % tritonx - 100 ™, 0 . 5 % igepal ca - 630 , 0 . 025 % nan 3 and protease inhibitor cocktail ( sigma ™). cell debris was removed by centrifugation at 20 , 000 × g for to minutes . the protein concentration in supernatant was determined using the microbca ™ method ( pierce ™, rockford , ill .). proteins in supernatant were mixed with protein sample loading buffer ( final concentration : 60 mm tris - hcl ( ph 6 . 80 ), 2 % sds , 10 % glycerol , 1 . 5 % 3 - mercaptoethanol , 0 . 002 % bromophenol blue ) and size fractioned by 10 % of sds - polyacrylamide gel . after proteins were transferred onto nitrocellulose membrane by iblot ™ ( invitrogen life technologies ™), non - specific binding were blocked with phosphate buffer saline twenty 20 ( pbs - t ) containing 5 % skim milk for 1 hour . the membrane was then incubated with primary antibody overnight . after incubation with a secondary antibody for 1 hour , protein bands were visualized by an enhanced chemiluminescence ( ecl ™) detection system ( santa cruz biotechnology ™, santa cruz , calif .). the primary antibodies used in this study were : mouse monoclonal anti - human mmp - 1 ( r & amp ; d systems ™, minneapolis , minn . ), mouse monoclonal anti - human mmp - 3 ( r & amp ; d system ™), rabbit monoclonal anti - human mmp - 2 ( epitomics ™, burlingame , calif . ), rabbit polyclonal anti - phospho - mek1 / 2 ( ser217 / 221 ™) ( cell signaling technology ™, danvers , mass . ), rabbit polyclonal anti - phospho - p44 / 42 mapk ( thr202 / tyr204 ) ( cell signaling technology ™), monoclonal anti - β - actin ( sigma ™), and mouse anti - type - 1 procollagen ( developmental studies hybridoma bank ™, iowa city , iowa ). the secondary antibodies were either goat anti - mouse igg ( h + l ) hpr conjugate or goat anti - rabbit igg ( h + l ) hpr conjugate ( bio - rad laboratory ™ ( mississauga , on , canada ). secondary antibodies were used at a concentration of 1 : 3000 . the activity of mmps was assessed using a f - fam / qxl ™ 520 fluorescence resonance energy transfer ( fret ) peptide as the mmp substrate ( sensolyte 520 ™ generic mmp assay kit , anaspec , inc .™, fremont , calif .) according to the manufacturer &# 39 ; s protocol . in brief , cells were treated with or without 50 μg / ml of kynurenine for 48 hours . conditioned media were collected and incubated with 1 mm of apma ( 4 - aminophenyl - mercuric acetate , in component c , anaspect ™) at 37 ° c . for 3 hrs . after activation mmps with apma , 50 μl / well in 96 - well plate of conditioned media was mixed with 50 μl of mmp substrate solution . after incubated at room temperature for 60 minutes , the fluorescence intensity at ex / em = 490 nm / 520 nm in each sample including the substrate control were measured using infinite f500 ™ fluorescence microplate reader ( tecan group ltd ™, morrisville , n . c .). human fibroblasts at 90 % confluence were starved in dmem without fbs overnight followed by the treatment with or without 100 μg / ml of kynurenine for 2 hours . protein phosphorylation was evaluated using the human phospho - kinase array ™ ( r & amp ; d system ™) according to the manufacturer &# 39 ; s instructions . briefly , capture and control antibodies were spotted in duplicate on nitrocellulose membranes ( total 46 kinase phosphorylation sites ). cell lysates ( 300 μg of total protein per array ) were incubated with array overnight . the array was washed to remove unbound proteins , followed by incubation with the cocktail of biotinylated detection antibodies . after incubation with streptavidin - hpr for 30 minutes , signals were visualized by ecl detection system ( santa cruz ™). blots were analyzed by densitometry , and protein phosphorylation was normalized to a positive control which was represented in each membrane . female rabbits ( new zealand white ) weighing 4 . 5 - 5 kg were used for this study . the protocol was reviewed and approved by the university of british columbia animal care committees . the rabbit ear model of hypertrophic scar was created as described previously ( rahmani - neishaboor , et al ., 2010 ). briefly , 2 rabbits were anesthetized by intramuscular injection of ketamine ( 22 . 5 mg / kg ) and xylazine ( 2 . 5 mg / kg ) followed by isoflurane gas through tracheal intubation . four wounds were created down to bare cartilage on the ventral side of each ear using an 8 - mm dermal biopsy punch to remove full - thickness sections of skin . antibiotics were applied on wounds daily until kynurenine treatment was started . kynureine in cmc gel ( rahmani - neishaboor et al ., 2010 ) with a concentration of 500 μg / ml was applied topically to the wounds of the experimental group ( 0 . 1 ml per wound ) daily for 3 weeks starting at 1 week post wounding . the wounds of the control group were received the treatment with an equal amount of cream alone daily . animals were sacrificed on weeks 3 after treatments . scars ( 10 mm punch biopsies ) were harvested . each scar was sectioned in two along its longitudinal axis and half of which was processed for routine histological analysis and another half was kept at − 80 ° c . for future use . scar elevation was quantified by measuring scar elevation index ( sei ) from the h & amp ; e stained tissue section . the sei is a ratio of total height in the wound tissue to the normal tissue below the hypertrophic scar . a sei of 1 indicates that the scar height is equal to the surrounding unwounded dermis ; an sei & gt ; 1 indicates a raised hypertrophic scar . the effect of kynurenine on human dermal fibroblast proliferation was detected by mtt [ 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ] assay . in brief , 10 , 000 cells were seeded on a 24 well - plate and incubated with different concentrations of kynurenine for 48 hours . media were removed and 0 . 2 ml of mtit ( 5 mg / ml in dmem containing 2 % fbs ) was added . cells were incubated with mit for 4 hours . after washing 3 times with pbs , 0 . 2 ml of dmso was added to dissolve the crystals . absorbance was measured at 570 nm . according to a method previously reported ( gawronskao - kozak b . et al . 2006 ), half of 8 mm diameter skin punches were weighed and frozen in − 80 ° c . skins were homogenized in 2 ml of pbs and stored at 4 ° c . overnight . the next day , 1 ml of 6n hcl was added and the mixture was heated at 120 ° c . for 5 hours . 20 μl of cooled samples and 50 μl of chloramine t solution were added to the 96 - well plate and incubated at room temperature for 20 minutes . 50 μl of erlich solution was then added and the mixture was incubated at 65 ° c . for 15 minutes . absorbance was read at 570 nm . rna extraction , cdna synthesis and quantitative rt - pcr rna was extracted by trizol ™ ( invitrogen life technologies ™). briefly , 1 ml of trizol ™ was added to the homogenized skin tissue . 250 μl of chloroform was added after the mixture was standed at room temperature for 5 minutes . top aqueous phase was transferred into a new eppendorf tube after centrifugation for to minute at 20 , 000 × g . equal volume isopropanol was added to the aqueous phase and mixed gently . the pellet was washed with 1 ml of 75 % ethanol after centrifugation for 20 minutes . rna was dissolved in depc treated h 2 o and its concentration was measured by nanodrop2000 ™. cdna was synthesized by cdna synthesis kit from roche according to manufacture &# 39 ; s introduction using1 g of total rna in each sample . quantitative real - time pcr for rabbit type - 1 α1 collagen , mmp - 1 and housekeeper gene β - actin were performed in viia 7 ( invitrogen ™). cdna samples were added to a pcr reaction master mix containing stbr green master mix ™ ( rox ) ( roche ™, indianapolis , ind .). all reactions were performed in duplicate using the following cycle conditions : 1 cycle of 95 ° c . for 10 minutes , 40 cycles of 95 ° c . for 15 seconds and 60 ° c . for 1 minute . the expression level of type - 1 α1 collagen and mmp - 1 in each sample was normalised to β - actin . rt - pcr primers : rabbit type - 1 α1 collagen : 5 ′- acaagggtgagacaggcgaac - 3 ′ ( forward ), 5 ′- gccgttgagtccatctttccc - 3 ′ ( reverse ); mmp - 1 , 5 ′- tctggccacatctgccaatgg - 3 ′ ( forward ), 5 ′- agggaagccaaaggagctgtg - 3 ′ ( reverse ); β - actin , 5 ′- aacgagcgcttccgttggccc - 3 ′ ( forward ), 5 ′- cttctgcatgcggtccgcga - 3 ′( reverse ). to assess the effect of ido on mmp - 1 expression , a human ido recombinant adenoviral vector was used for gene transduction in human dermal fibroblasts by a procedure previously reported ( li et al ., 2004 ). transfection efficiency was evaluated by detecting ido protein expression and its activity through western blot analysis and the kynurenine measurement in conditioned media , respectively . as shown in fig1 a left panel , the ido protein was expressed in ido adenovirus - transduced fibroblasts , but undetectable in control and mock adenovirus - transduced fibroblasts . the level of kynurenine , an index for ido activity , was significantly higher in ido adenovirus - transduced fibroblasts ( 14 . 3 ± 0 . 46 μg / ml , n = 3 ) compared to those in untransduced or mock - transduced controls ( figure a , right panel ). the expression of mmp - 1 in control , mock - transduced and ido - expressing fibroblasts was examined by using western blot analysis . as shown in fig1 b , there was a more than nine fold increase in mmp - 1 expression in ido - expressing fibroblasts ( 12 . 56 ± 2 . 37 , n = 3 ) as compared to those in mock - transduced ( 1 . 37 ± 0 . 59 , n = 3 ) and untreated control fibroblasts ( 1 ± 0 , n = 3 ). this finding suggests that up - regulation of mmp - 1 expression in ido - expressing fibroblasts is not due to adenovirus infection , since the mock - transduced fibroblasts showed no significant difference in mmp - 1 expression from the untreated fibroblasts . ido is an intracellular enzyme that converts tryptophan into kynurenine . therefore , it must be clarified whether the effect of mmp - 1 stimulation in ido - expressing fibroblasts is due to the ido protein itself or to tryptophan metabolites . to address this , conditioned media from both ido - expressing fibroblasts and controls were collected after 48 hours . a combination of 90 % collected conditioned media and 10 % fresh media was then used to treat dermal fibroblasts . cells were harvested 48 hours after treatment . as shown in fig1 c , a significant increase in mmp - 1 expression was observed in cells treated with conditioned media from ido - transduced fibroblasts ( 2 . 06 ± 0 . 62 , n = 3 ) as compared to those in either mock - transduced ( 1 . 16 ± 0 . 31 , n = 3 ) or untreated control fibroblasts ( 1 ± 0 , n = 3 ). this result suggests that a factor or factors in conditioned media from ido adenovirus infected fibroblast rather than intracellular ido protein is responsible for an increased level of mmp - 1 expression in fibroblasts . kynurenine but not depletion of tryptophan induces mmp - 1 expression in human dermal fibroblasts ido is an enzyme converting tryptophan into kynurenine . to examine what factor ( either depletion of tryptophan or increase of kynurenine ) is responsible for ido up - regulation of mmp - 1 expression . to examine what factor is responsible for ido up - regulation of mmp - 1 expression , fibroblasts were grown in either tryptophan - depleted cultured media or regular media with various concentrations of kynurenine . cells were then evaluated for mmp - 1 expression by western blotting . as shown in fig2 c , there was no significant difference in the expression of mmp - 1 between fibroblasts grown in the presence of 25 g / ml tryptophan or in the tryptophan - depleted cultured media . however , the mmp - 1 expression was significantly increased in response to different doses ( 25 - 150 μg / ml ) of kynurenine ( fig2 a and fig2 b ). these findings suggest that the presence of kynurenine , but not tryptophan depletion , contributes to the up - regulation of mmp - 1 in ido - expressing cells . furthermore , we found that as little as 12 . 5 μg / ml of kynurenine could stimulate mmp - 1 expression in dermal fibroblasts ( data not shown ). this concentration of kynurenine is similar to that detected in conditioned media from ido expressing fibroblasts ( fig1 a right panel ). the stimulation of mmp - 1 in fibroblasts is thus clearly specific to kynurenine as the addition of various concentration of tryptophan with a similar structure failed to increase the expression of mmp - 1 in dermal fibroblasts ( fig2 d ). effects of kynurenine on mmp - 2 and - 3 expression in dermal fibroblasts to investigate whether kynurenine also affects the expression of other mmps , we treated dermal fibroblasts with kynurenine at similar concentrations to those used in fig2 . western blotting was used to detect mmp - 2 and - 3 expression using untreated cells as controls . as shown in fig3 a , there was no significant difference in mmp - 2 expression between kynurenine - treated and untreated fibroblasts . however , under similar conditions , kynurenine treatment significantly increased mmp - 3 expression in dermal fibroblasts in a dose - dependent manner ( fig3 b / 3 c ). furthermore , to test whether the increased levels of mmps in kynurenine - treated fibroblasts were followed by increased mmp activity , conditioned media from fibroblasts in the presence or absence of 50 μg / ml of kynurenine were collected 48 hours after treatment . the mmp activity in the conditioned media was detected by a sensolyte 520 ™ generic mmp assay kit using a 5 - fam / qxl ™ 520 fluorescence resonance energy transfer ( fret ) peptide as a mmp substrate . as shown in fig4 , the mean activity of mmps in conditioned media from the kynurenine treated fibroblast was significantly higher than in the control media . this indicates that the increased mmps in fibroblasts treated by kynurenine have enzymatic activity . to determine what types of cells are sensitive to kynurenine - induced mmp - 1 expression , both mesenchymal cells ( such as an immobilized lung fibroblast cell line imr - 90 and fibroblast - like synoviocytes ) and epithelial cells ( such as lung epithelial carcinoma cell line a549 , primary dermal keratinocytes , human immobilized keratinocyte cell line hacat , and head and neck squamous cell carcinoma cell line umscc ) were used . as with the dermal fibroblasts , mmp - 1 expression in synoviocytes and imr - 90 were up - regulated by kynurenine treatments at concentrations of 12 . 5 μg / ml to 150 μg / ml , as shown in fig5 . however , the expression of mmp - 1 in all epithelial cells tested , including dermal keratinocytes , hacat , a549 and umscc , did not significantly differ from the untreated controls in response to the various concentration of kynurenine ( fig6 ). these results suggest that there is a difference between mesenchymal and epithelial cells in response to kynurenine - stimulating mmp - 1 expression . identification of the phosphorylated signal molecules by phospho - kinase array in cells treated with kynurenine to determine the possible mechanism of kynurenine up - regulated mmp - 1 expression in dermal fibroblasts , we analyzed the activation of multiple serine , threonine or tyrosine kinases , using a phosphor - kinase array . this array gives the possibility of simultaneously detecting the activation status of 46 different protein kinases and their downstream transcript factors . as shown in fig7 , after 1 hour of treatment in dermal fibroblasts with kynurenine , extracellular signal - regulated kinases 1 / 2 ( erk1 / 2 ) was activated . to confirm these results from the phospho - kinase array , dermal fibroblasts were treated with 100 μg / ml of kynurenine at different times . immunoblotting analysis , using a different antibody from those placed on the array , was then used to detect the phosphorylation of erk1 / 2 and its upstream molecule mitogen - activated protein / extracellular signal - regulated kinase kinase ( mek ). as shown in fig8 , erk1 / 2 was phosphorylated in cells treated with kynurenine . the result was further confirmed by detection of the erk1 / 2 upstream signal molecule mek phosphorylation in cells treated with kynurenine ( fig8 ). both erk1 / 2 and mek showed similar patterns of activation , with a peak at 8 hours following kynurenine treatments ( fig8 ). addition of inhibitors for mek - erk1 / 2 phosphorylation negates the effects of kynurenine stimulated mmp - 1 expression in dermal fibroblasts in another set of experiments , we tested whether the activation of the mek - erk1 / 2 mapk pathway by kynurenine is associated with kynurenine - stimulating mmp - 1 expression in dermal fibroblasts . to do this , we examined the effects of inhibitors of either mek or erk1 / 2 phosphorylation on kynurenine - stimulating mmp - 1 expression . as shown in fig9 a , the addition of pd98059 , a specific inhibitor for erk1 / 2 activation effectively prevented the stimulatory effect of kynurenine on mmp - 1 expression , in a dose - dependent manner . similarly , treatment of cells with to m and 30 μm of u0126 , a specific inhibitor for mek activation , also significantly reduced the up - regulation of mmp - 1 expression by kynurenine ( fig9 b ). these results demonstrate that the activation of the mek - erk1 / 2 signaling pathway contributes to the up - regulation of mmp - 1 expression induced by kynurenine in dermal fibroblasts . effects of kynurenine on collagen expression in dermal fibroblasts and fibroblast proliferation before studying its anti - fibrotic role in vivo , kynurenine was tested for its effect on collagen expression and cell proliferation . as shown in figure to ( top ), the addition of kynurenine 25 - 150 μg / ml remarkably decreases the expression of type 1 procollagen . however , it had no significant effect on fibroblast proliferation , even when the cells were cultured at concentrations up to 150 μg / ml of kynurenine ( fig1 ). also , testing of the kynurenine analogues / metabolites , kynurenic acid and xanthurenic acid , demonstrate that these compounds are also effective at inhibiting expression of type 1 procollagen ( figure to ( bottom )). since treatment of dermal fibroblasts with kynurenine showed an increase in both the mmp - 1 and - 3 expression as well as a decrease in type - 1 procollagen expression , we were interested to know whether kynurenine can be used as an anti - fibrotic agent for the treatment or prevention of hypertrophic scarring . to achieve this , as described previously ( rahmani - neishaboor et al ., 2010 ; kloeters et al ., 2007 ; xie et al ., 2008 ), a rabbit ear hypertrophic scar model was used . wounds were treated daily with 0 . 1 ml of carboxymethyl cellulose ( cmc ) gel containing 50 g of kynurenine for three weeks starting at day 8 post - wounding . the dose of 50 mg kynurenine per wound was matched with that used in an in vitro system with an optimum outcome . the result showed no significant difference to wound closure in kynurenine - treated wounds as compared to that of either untreated or cmc gel treated controls ( data not shown ). however , as shown in fig1 a , significantly less scarring was seen in wounds treated with kynurenine than either non - treated wounds or the vehicle - only control wounds after three weeks . the average scar elevation index ( sei ) was significantly reduced in the kynurenine - treated group ( 1 . 172 + 0 . 156 , n = 8 ) as compared to the vehicle - only control group ( 1 . 978 ± 0 . 442 , n = 4 , p & lt ; 0 . 01 ) and the untreated group ( 2 . 098 ± 0 . 324 , n = 4 , p & lt ; 0 . 001 ) ( fig1 b ). massons &# 39 ; trichrome staining for collagen revealed a significant reduction in collagen content in wounds treated with kynurenine , compared to those wounds receiving either no treatment or gel alone ( fig1 c ). consistent with this finding , the hydroxyproline content ( used as an index for tissue collagen content ) was significant lower in wounds treated with kynurenine compared to those wounds receiving either no treatment or gel alone ( fig1 d ). finally , we demonstrated that topical application of kynurenine in a rabbit ear fibrotic model decreased the expression of type - 1 α1 collagen and increased the expression of mmp - 1 , as compared to those in wounds received either no treatment or gel alone ( fig1 ). these results further support the supposition that kynurenine could potentially be used as an anti - fibrotic factor for treating hypertrophic scarring and even keloid , as frequently seen in patients with burn injuries or surgical incisions . effect of kynurenine isoforms on mmp - 1 expression in human dermal fibroblasts different isoforms of kynurenine were tested for their ability to affect mmp - 1 expression . isoforms tested were dl - kynurenine ( dl - kyn ) or d - kynurenine ( d - kyn ) and l - kynurenine ( l - kyn ). the result showed that all isoforms increase the mmp - 1 expression in dermal fibroblasts , however , l - kynurenine seems to have more activity compared to other two isoforms — see fig1 . effects of different kynurenine isoforms / analogues on collagen expression in human dermal fibroblasts dermal fibroblasts were treated with either fs - 1 ( dl - kynurenine ) or d - kynurenine or l - kynurenine or fs - 2 ( kynurenic acid ) as shown in fig1 . type - 1 , α1 - collagen expression was detected by real - time pcr . results indicate that these isoforms / analogues have similar efficacy in reducing collagen expression . dermal fibroblasts were treated with various concentration of either dl - kynurenine ( fs1 ), l - kynurenine , d - kynurenine or kynurenic acid ( fs2 ) as shown in fig1 . the expression of fibronectin was detected by real - time pcr . results demonstrate that kynurenine , dl - kynurenine , and l - kynurenine are all capable of down - regulating fibronectin expression , indicating that kynurenine metabolites may be also suitable for prevention or treatment of fibroproliferative disorders . the findings in fig1 showed that , there was almost 5 - fold reduction in cona - induced splenocyte proliferation following treatment with 100 and 150 g / ml d - kynurenine , l - kynurenine or dl - kynurenine after 96 hours ( p & lt ; 0 . 05 ), although splenocyte proliferation significantly reduced about 2 - fold by d - kynurenine , l - kynurenine and dl - kynurenine at 100 and 150 μg / ml after 48 hours . fs2 has less effect on proliferation than other metabolites . the findings in fig1 showed that fs1 has immune suppressive effect on some of the proinflammatory cytokine and chemokine production , like il - 1 , il - 2 , cxcl9 , and cxcl10 . besides it can significantly decrease il - 17 production which is thought to have an important role in inflammation . lasting effect of kynurenic acid and kynurenine on mmp1 expression in fibroblasts to determine the lasting effect of kynurenic acid ( kyna ) and kynurenine ( kyn ) on mmp1 expression in fibroblasts , cells were treated with 100 μg / ml of the drug . following 48 hours of treatment , the medium was changed with fresh medium and cells were then harvested at 0 , 12 , 24 or 48 hours post treatment removal . there was a marked increase in mmp1 expression in fibroblasts in response to either kyna or kyn treatment at 48 hours after treatment . following the removal of kyn and kyna , the mmp1 expression remained significantly higher than the untreated cells for another 24 hours ( fig1 a ). interestingly , while the mmp1 protein expression gradually reduced to normal levels within 48 hours after kyn removal , the mmp1 expression in response to kyna remained higher than controls ( fig1 a ). fig1 b represents the quantitative analysis of data in fig1 a (* p - value & lt ; 0 . 05 , ** p - value & lt ; 0 . 01 , n = 4 ). from these results it appears that kyna has a longer lasting effect on expression of mmp - 1 relative to kyn in treated fibroblasts . although various embodiments are disclosed herein , many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art . such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way . numeric ranges are inclusive of the numbers defining the range . the word “ comprising ” is used herein as an open ended term , substantially equivalent to the phrase “ including , but not limited to ”, and the word “ comprises ” has a corresponding meaning . as used herein , the singular forms “ a ”, “ an ” and “ the ” include plural referents unless the context clearly dictates otherwise . thus , for example , reference to “ a thing ” includes more than one such thing . citation of references herein is not an admission that such references are prior art to an embodiment of the present invention . the invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings . gawronskao - 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