BR112015030569B1 - USE OF ANTI-FIBROGENIC COMPOUNDS - Google Patents

Abstract

USES OF ANTI-FIBROGENIC COMPOUNDS, AND PHARMACEUTICAL COMPOSITION COMPRISING ANTI-FIBROGENIC COMPOUNDS Use of kynurenine or related compounds, such as kynurenic acid or xanthurenic acid, in the treatment of conditions or diseases related to fibrosis. Diseases and conditions include keloid, hypertrophic scarring, pulmonary or renal fibrosis, Crohn's disease and scleroderma. The active compounds can be in various pharmaceutical compositions for delivery routes.

Description

CROSS-REFERENCE TO THE RELATED PATENT APPLICATION

[001] This application claims priority from US Provisional Application Series No. 61/831,404, entitled "ANTI-FIBROGENIC COMPOUNDS AND METHODS" filed on June 5, 2013.

FIELD OF THE INVENTION

[002] The present invention relates to new methods for treating fibrosis. More specifically, the description provided herein relates to the use of kynurenine, kynurenic acid, xanthurenic acid, and/or related compounds for the treatment of fibrotic disease, in particular skin diseases or conditions such as keloids and hypertrophic scars.

FUNDAMENTALS OF THE INVENTION

[003] Fibrosis, a disorder that belongs to a group of fibroproliferative conditions, is seen in different organs such as, skin, liver, lung, kidneys and arteries. It is estimated that approximately 40% of all deaths in the United States are caused, in part, by fibroproliferative disorders. Excessive accumulation of extracellular matrix due to overproduction of matrix such as fibronectin, type I and III collagens, low levels of matrix-degrading enzymes such as matrix metalloproteinases (MMP) or both are common features of all these fibrotic conditions.

[004] As in all other organs, skin wound healing is a dynamic process that involves tissue response to different types of injuries. This process involves a continuous sequence of signals and responses in which platelets, fibroblasts, epithelial cells, endothelial cells and immune cells come together outside of their usual domain in order to orchestrate the very complex process of tissue repair. These signals, which are primarily growth factors (GFs) and cytokines, orchestrate the initiation, continuation, and termination of wound healing (Scott et al., 1994). An imbalance in the synthesis and release of cytokines and GFs at the wound site can result in either delayed wound healing (e.g., in diabetic and elderly populations) or over-healing (e.g., fibroproliferative disorders, complication following surgical incision, traumatic wounds). , and severe thermal injuries). Thus, an important component of wound healing is its timely cessation, and without such timely cessation there can be an accumulation of excess matrix, a deleterious fibrotic condition seen in millions of patients worldwide.

[005] Matrix metalloproteinases (MMPs) represent a group of diverse proteolytic enzymes involved in ECM volume and connective tissue remodeling during physiological conditions such as embryonic growth and development, uterine involution, bone growth, bone resorption and wound healing. The expression level of MMP in normal cells is low and that allows healthy connective tissue remodeling. However, an imbalance in the expression of MMPs has been implicated in a number of pathological conditions such as dermal fibrosis, rheumatoid arthritis, atherosclerosis, and tumor invasion and metastasis.

[006] Current treatment modalities for any fibrotic condition including dermal fibroproliferative disorders such as hypertrophic scars (HSc) and keloid continue to be unsatisfactory. Therefore, it would be desirable to have therapeutic strategies for the treatment of various fibrotic diseases and conditions.

SUMMARY

[007] The present invention is based, in part, on the surprising discovery that certain compounds - kynurenine and its analogues/isoforms, kynurenic acid, and xanthurenic acid - are able to stimulate the expression of MMP1 and MMP3, while inhibiting the expression of collagen and fibronectin. Furthermore, as described herein these compounds, when applied in vivo, are capable of inhibiting, preventing or reducing keloid scar formation.

[008] In one embodiment, there is provided the use of a compound, the compound having the structure of formula I: wherein, E1 can be H, OH, NH2, R, OR, NHR, NR2, SH, SR, F, Cl, Br, or I; E2 can be H, OH, NH2, R, O, NHR, NR2, SH, SR, F, Cl, Br, or I; E3 can be H, OH, NH2, R, OR, NHR, NR2, SH, SR, F, Cl, Br, or I; E4 can be H, OH, NH2, R, OR, NHR, NR>, SH, SR, F, Cl, Br, or I; R can be a group of 1 to 20 carbons which can optionally be saturated, unsaturated, linear, branched linear, cyclic, cyclic branched, aromatic, partially aromatic or non-aromatic, where each carbon can optionally be replaced by O, S, SO, SO2, NH or NR', and each carbon can be optionally substituted with one or more of: OH, OR', R', F, Cl, Br, I, =0, SH, SR', NH2, NHR' , N(R')2, OSO3H, OPO3H3, CO2H, CON(R')2 and CO2R';R' may independently be selected from the group consisting of: a group of one to ten carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic; A can be H, or an NH2 group; d can be ; or the NH2 or °NΠ? A and D can form a 6-member ring selected from the following: G can be CH or N; J can be S or 0; Li can be OH, OQ, NH2, NHQ, NQ2, SH, or SQ; L2 can be 0, SQ' or NQ'; L3 can be OH, OQ, NH2, NHQ, NQ2, SH, or SQ; Q can be a group of 1 to 20 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic, where each carbon can optionally be replaced by 0, S, SO, SO2, NH, or NQ' , and each carbon may be optionally substituted with one or more of: OH, OQ', Q', F, Cl, Br, I, =0, SH, SQ', NH2, NHQ' , N(Q')2, OSO3H, OPO3H3, CO2H, CON(Q')2 and CO2Q';Q' may be independently selected from the group consisting of: a group of one to ten carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic; Mi can be H, OH, NH2, T, OT, NHT, NT2, SH, ST, F, Cl, Br, or I; M2 can be H, OH, NH2, T, OT, NHT, NT2, SH, ST, F, Cl, Br, or I; M3 can be H, OH, NH2, T, OT, NHT, NT2, SH, ST, F, Cl, Br, or I; M4 can be OH, NH2, T, OT, NHT, NT2, SH, ST, F, Cl, Br, or I; T can be H, or a group of 1 to 20 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic, where each carbon can optionally be replaced by 0, S , S0, S02, NH, or NT' , and each carbon may be optionally substituted with one or more of: OH, OT', T', F, Cl, Br, I, =0, SH, ST', NH2, NHT', N(T')2, OSO3H, OPO3H3, CO2H, CON(T')2 and CO2T';T' may be independently selected from the group consisting of: a group of one to ten carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic; Xi can be H, OH, NH2, Z, OZ, NHZ, NZ2, SH, SZ, F, Cl, Br, or I; X2 can be H, OH, NH2, Z, OZ, NHZ, NZ2, SH, SZ, F, Cl, Br, or I; X3 can be H, OH, NH2, Z, OZ, NHZ, NZ2, SH, SZ, F, Cl, Br, or I; and Z can be a group of 1 to 20 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, cyclic branched, aromatic, partially aromatic or non-aromatic, where each carbon can be optionally substituted by O, S, SO , SO2, NH, or R', and each carbon may be optionally substituted with one or more of: OH, OZ' , Z', F, Cl, Br, I, =0, SH, SZ', NH2, NHZ' , N(Z')2, OSO3H, OPO3H3, CO2H, CON(Z')2 and CO2Z'; and Z' may independently be selected from the group consisting of: a group of one to ten carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic; for either the treatment of fibrotic disease or for the manufacture of a medicament for treating fibrotic disease.

[009] In a further embodiment, there is provided a method of treating fibrotic disease, the method comprising administering to a mammalian cell a compound or pharmaceutically acceptable salt thereof, the compound having the structure of formula I.

[0010] In a further embodiment, there is provided a method of treating fibrotic disease, the method comprising administering to a mammalian cell a compound or pharmaceutically acceptable salt thereof, the compound having the structure of formula II.

[0011] In a further embodiment, there is provided a method of treating fibrotic disease, the method comprising administering to a mammalian cell a compound or pharmaceutically acceptable salt thereof, the compound having the structure of formula III.

[0012] In a further embodiment, there is provided a method of treating fibrotic disease, the method comprising administering to a subject in need thereof, a compound or pharmaceutically acceptable salt thereof, the compound having the structure of formula I.

[0013] In a further embodiment, there is provided a method of treating: fibrotic disease, the method comprising administering to a subject in need thereof, a compound or pharmaceutically acceptable salt thereof, the compound having the structure of formula II .

[0014] In a further embodiment, there is provided a method of treating fibrotic disease, the method comprising administering to a subject in need thereof, a compound or pharmaceutically acceptable salt thereof, the compound having the structure of formula III.

[0015] In a further embodiment, there is provided a pharmaceutical composition for the treatment of fibrotic disease, the pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the compound has the structure of formula I.

[0016] In a further embodiment, there is provided a pharmaceutical composition for the treatment of fibrotic disease, the pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the compound has the structure of formula II.

[0017] In a further embodiment, there is provided a pharmaceutical composition for the treatment of fibrotic disease, the pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the compound has the structure of formula III.

[0018] In a further embodiment, there is provided a pharmaceutical composition, pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the compound has the structure of formula I.

[0019] In a further embodiment, there is provided a pharmaceutical composition, pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the compound has the structure of formula II.

[0020] In a further embodiment, there is provided a pharmaceutical composition, pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the compound has the structure of formula III.

[0021] In a further embodiment, there is provided a commercial package comprising (a) a pharmaceutical composition described herein; and (b) instructions for its use for the treatment of fibrotic diseases.

[0022] In a further embodiment, there is provided a commercial package comprising (a) a compound of formula I; and (b) instructions for its use for the treatment of fibrotic diseases.

[0023] In a further embodiment, there is provided a commercial package comprising (a) a compound of formula II; and (b) instructions for its use for the treatment of fibrotic diseases.

[0024] In a further embodiment, there is provided a commercial package comprising (a) a compound of formula III; and (b) instructions for its use for the treatment of fibrotic diseases.

[0025] In a further embodiment, there is provided a compound of formula I for the treatment of fibrotic diseases.

[0026] In a further embodiment, there is provided a compound of formula II for the treatment of fibrotic diseases.

[0027] In a further embodiment, there is provided a compound of formula III for the treatment of fibrotic diseases.

[0028] Fibrotic disease can be selected from one or more of the following: keloids; hypertrophic scarring; pulmonary fibrosis; renal fibrosis; hepatical cirrhosis; chronic inflammation of tunica albuginea (CITA); endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis; progressive massive fibrosis; systemic nephrogenic fibrosis; Crohn's disease; old myocardial infarction; scleroderma; systemic sclerosis; uterine fibroids; and restenosis.

[0029] Q may be a group of 1 to 6 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic. R can be a group of 1 to 6 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic. T can be a 1 to 6 carbon group which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic. Z can be a group of 1 to 6 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic.

[0030] Q' can be a group of 1 to 6 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic. R' can be a group of 1 to 6 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic. T' can be a group of 1 to 6 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic. Z' can be a group of 1 to 6 carbons which is optionally saturated, unsaturated, linear, linear branched, cyclic, branched cyclic, aromatic, partially aromatic or non-aromatic.

[0031] Ei may be H, OH, NH2, OCH3, CH3, SH, F, Cl, Br, or I; E2 can be H, OH, NH2, OCH3, CH3, SH, F, Cl, Br, or I; E3 can be H, OH, NH2, OCH3, CH3, SH, F, Cl, Br, or I; E4 can be H, OH, NH2, OCH3, CH3, SH, F, Cl, Br, or I; A can be H or NH2; d can be

[0032] Alternatively, A and D may form a 6-membered ring selected from the following: G is CH or N. Li is OH, NH2, or SH. Mi can be H, OH, NH2, SH, F, Cl, Br, or I. M4 is OH, NH2, SH, F, Cl, Br, or I. Xi is H, OH, NH2, SH, F, Cl , Br, or I.

[0033] Ei can be H, OH, NH2, OCH3 or CH3. E2 can be H, OH, NH2, OCH3 OR CH3. E3 can be H, OH, NH2, OCH3 or CH3. E4 can be H, OH, NH2, OCH3 or CH3. A can be H or NH2. d can be . Alternatively, A and D can form a 6-membered ring selected from the following: G can be CH or N. Li can be OH or NH2. Mi is H, OH or NH2.

[0034] Ei can be H, OH, NH2, OCH3 or CH3; E2 can be H, OH, NH2, OCH3 OR CH3; E3 can be H, OH, NH2, OCH3 or CH3; E4 can be H, OH, NH2, OCH3 or CH3. A can be H, or NH2. d can be Alternatively, A and D form a 6-membered ring that has the following structure:

[0035] Ei can be H, OH, or NH2. E2 can be H, OH, or NH2. E3 can be H, OH, or NH2. E4 can be H, OH, or NH2. A is H, or an NH2 group. d can be . Alternatively, A and D form a 6-membered ring that has the following structure:

[0036] The compound may have the structure of formula II:

[0037] The compound may have the structure of formula III:

[0038] Li can be OH or NH2. Li can be OH. Hey can be H or OH. E2 can be H, OH, or NH2. E3 can be H, OH, or NH2. E4 can be H, OH, or NH2.

[0039] Ei can be H, OH, or NH2. E2 can be H or OH. E3 can be H, OH, or NH2. E4 can be H, OH, or NH2.

[0040] Ei can be H, OH, or NH2. E2 can be H, OH, or NH2. E3 can be H or OH. E4 can be H, OH, or NH2.

[0041] Ei can be H, OH, or NH2. E2 can be H, OH, or NH2. E3 can be H or OH. E4 can be H or NH2.

[0042] Hey can be H or OH. E2 can be H or OH. E3 can be H or OH. E4 can be H or NH2.

[0043] The compound can be selected from one or more of the following:

[0044] The compound can be selected from one or more It is .The compound can be .The compound can be .The compound can be The compound can be The compound can be The compound can be The compound can be .

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1: Indoleamine 2,3-dioxygenase (IDO) upregulation of MMP-1 expression in human dermal fibroblasts. Panel A shows fibroblasts that were transduced with either nothing (C), adenoviral vector (V) or a vector carrying the recombinant IDO gene (IDO) for 48 hours, where IDO and its activity was detected by Western blotting (left panel) and measurement. of kynurenine levels (right panel), respectively (N.D. indicates the level of kynurenine that was not detectable). Panel B shows both untreated adenoviral vector and IDO-transduced fibroblasts, which were lysed after being cultured for 48 hours, and MMP-1 expression was detected by Western blotting. Panel C shows that fibroblasts were incubated with conditioned medium taken from either control, empty vector or fibroblasts transduced by IDO adenoviral vector for 48 hours, where MMP-1 expression was analyzed by Western blotting, β-actin was used as a loading control in panels A, B and C. * indicates p < 0.001.

[0046] Figure 2: Effects of kynurenine and tryptophan on MMP-1 expression in human dermal fibroblasts. Panels A and B show dermal fibroblasts that were cultured in the presence of various concentrations of kynurenine for 48 hours, when cells were harvested and subjected to lysis, before Western blotting was performed, showing the relationship of MMP-1 to β-actin is shown in panel B. Panel C shows dermal fibroblasts, which were cultured in the presence or absence of tryptophan (25 mg/ml) for 48 hours, when cells were harvested and lysed and MMP-1 expression was assessed by Western blotting. Panel D shows fibroblasts that were cultured in the presence of different concentrations of tryptophan for 48 hours, when MMP-1 expression was evaluated by Western blotting, β-actin was used for a loading control in all panels.

[0047] Figure 3: Effects of kynurenine on MMP-2 and -3 expression in human dermal fibroblasts - shows dermal fibroblasts that were cultured in the presence of various concentrations of kynurenine for 48 hours, before which cells were harvested and subjected to lysis, and Western blotting was performed using either a rabbit anti-human monoclonal MMP-2 antibody (Panel A) or a mouse anti-MMP-3 monoclonal antibody (Panel B). Panel C shows the ratio of MMP-3 expression to β-actin from three independent experiments, β-actin was used as a loading control in all experiments.

[0048] Figure 4: Detects the activity of MMPs in conditioned medium of human dermal fibroblasts using SensoLyte 520 generic MMP from the fluorimetric assay kit - shows fibroblasts that were cultured in the presence (Kyn) or absence (CTL) of 50 μg/mL of kynurenine for 48 hours, when the conditioned medium of cells was collected, then, after centrifugation at 1000 x g for 10 minutes, the supernatant was used to detect the activity of MMPs according to the manufacturer's instructions (medium were incubated with 1 mM APMA, at 37 °C for 3 hours 50 µl/well of MMP containing sample was then mixed with 50 µL of MMP substrate solution and medium before cell culture was mixed with 50 µL of MMP substrate solution. MMP and used as a substrate control and after one hour of incubation, the fluorescence intensity at EX/EM = 490 nm/520 nm was measured). The activities of MMPs are represented as relative fluorescence units (RFU). Data were expressed as mean ± SD (n = 3). * indicates P<0.05.

[0049] Figure 5: Effects of kynurenine on MMP-1 expression in different types of mesenchymal cells - shows cells that were cultured and treated with kynurenine at concentrations from 12.5 to 150 μg/mL for 48 hours and the expression of MMP-1 was analyzed by Western blotting and β-actin was used as a loading control in all experiments. Panel A shows MMP-1 expression in synoviocytes. Panel B shows MMP-1 expression in IMR-90 lung fibroblast cell lines.

[0050] Figure 6: Effects of kynurenine on MMP-1 expression in different types of epithelial cells - shows cells were cultured and treated with kynurenine at concentrations of 12.5 to 150 μg/mL for 48 hours and the expression of MMP- 1 was analyzed by Western blotting and β-actin was used as a loading control in all in experiments, where the top panels and the bottom left panel, showing lysed fibroblasts, either untreated or treated kynurenine were used as negative controls and positive, respectively.

[0051] Figure 7: Kynurenine stimulates ERK1/2 phosphorylation in human dermal fibroblasts - shows dermal fibroblasts that were cultured in the absence or presence of 100 μg/mL kynurenine for 60 minutes, then the cells were harvested and lysed with cell lysis buffer, before an antibody array was performed using an array of human phosphokinase kit (R&D System™), with point 1, positive control; point 2, phospho-P38a; point 3, phosphorus-ERKl/2; point 4, phosphorus-GSK-3a; point 5, phosphorus-P53; point 6, positive control.

[0052] Figure 8: Kynurenine stimulation of MEK and ERK1/2 phosphorylation in human dermal fibroblasts - shows dermal fibroblasts that were cultured in the presence of 100 µg/mL kynurenine at indicated time points, when cells were harvested and lysed , before Western blotting was performed using either MEK-phosphorylated or ERK1/2-phosphorylated antibodies (β-actin was used as a loading control).

[0053] Figure 9 - Addition of MEK or ERK1/2 phosphorylation inhibitors negates the effect of kynurenine-stimulating MMP-1 expression in dermal fibroblasts. Panel A: shows dermal fibroblasts were cultured in the absence or presence of 100 µg/mL kynurenine with or without various concentrations of PD98059. Panel B: shows dermal fibroblasts were cultured in the absence or presence of 100 µg/ml kynurenine with or without 30 µM PD98059 (ERK1/2 inhibitor), 30 µM Uol26 (MEK inhibitor) or 10 µM U0126 . MMP-1 expression was detected by Western blot (β-actin was used as a loading control for all experiments).

[0054] Figure 10 - Effect of kynurenine, kynurenine and xanthurenic acid on type 1 procollagen expression in dermal fibroblasts - shows human dermal fibroblasts were treated with the indicated concentrations of kynurenine for 48 hours (top), at which time the cells were harvested and lysed with cell lysis buffer and a total of 50 μg of protein was fractionated by 8% SDS-PAGE, before Western blotting was performed using antibodies against pro-collagen. β-actin was used as a loading control, with kynurenic acid (KA) and xanthurenic acid (XA) also tested (background).

[0055] Figure 11 - Effect of kynurenine on fibroblast proliferation - shows human dermal fibroblasts were cultured in the presence of indicated concentrations of kynurenine for 48 hours. MTT cell proliferation assay was performed as described herein, with cell proliferation indicated as cell index (OD570nm) in the MTT assay.

[0056] Figure 12 - Clinical appearance and histology of the wound and scarring - shows wounds in the rabbit ear that were treated daily or with nothing (CTL), CMC gel alone (Gel), or 50 μg kynurenine (Kyn) in 0 .1 mL of CMC gel started from day 8, for a total of 3 weeks. Panel A: shows the microscopic histology of wounds that received either nothing (CTL), CMC gel (Gel) or kynurenine in CMC gel (Kyn) on day 28 at x25 magnification. Panel B: Shows Scar Lift Index (SEI) as measured (mean ± SD SEI for untreated, CMC gel, and kynurenine gel from wounds treated with CMC gel)* shows a significant difference between controls treated with kynurenine and untreated (P < 0.001); ** shows a significant difference between the kynurenine and CMC gel control groups (P < 0.01). Panel C: Shows Massons' trichrome stained full-thickness skin sections from either untreated skin with skin wound (left panels), cream-treated skin wound (middle panels), or kynurenine-treated wound ( right panels), both at x25 and x100 magnification. Panel D: Shows total hydroxyproline content of skins from any untreated wounds (total of 4 wounds), cream-treated wounds (total of 4 wounds) , or wounds treated with kynurenine (total of 8 wounds) - * indicates p<0.01.

[0057] Figure 13 - Topical application of kynurenine decreases type-1 collagen and increases MMP-1 expression in rabbit ear skin - shows wounds in rabbit ear which were treated either with nothing (CTL) or gel alone (Gel) or kynurenine (Kyn) gel as described above, where skin wounds were used to extract total RNA by Trizol™ and 1 μg of RNA was used to synthesize cDNA for quantitative RT-PCR for al-type collagen 1, MMP-1 and β-actin. Panel A: shows the relative expression level for type-1 collagen in rabbit ear skin tissue. Panel B: shows the relative expression level of MMP-1 in rabbit ear skin tissue - * indicates p < 0.05.

[0058] Figure 14 - Effect of the kynurenine isoform on the expression of MMP-1 in 15 human dermal fibroblasts - shows dermal fibroblasts that were cultured in the absence (CTL) or in the presence of 50 μg/mL of either DL-kynurenine (DL- Kyn) or D-kynurenine (D-Kyn) or L-kynurenine (L-Kyn) for 48 hours, whereby time cells were harvested and lysed in protein lysis buffer (50 µg of total protein was loaded on gel of 10% SDS acrylamide) before Western blotting was performed with anti-human MMP-1 antibodies, with β-actin as a loading control, which demonstrates that all tested kynurenine isoforms have increased expression in dermal fibroblasts MMP-1, however, L-kynureine appears to have more activity compared to two other isoforms.

[0059] Figure 15 - Effects of kynurenine (FS1) analogues on collagen expression in human dermal fibroblasts - shows dermal fibroblasts that were treated with various concentrations of either DL-kynurenine (FS1), L-kynurenine, D-kynurenine or kynurenic acid (FS2) and corresponding collagen expression at mRNA levels as detected by real-time PCR, with β-actin as a loading control.

[0060] Figure 16 - Effects of kynurenine analogues (FS1) on fibronectin expression in human dermal fibroblasts - shows dermal fibroblasts were treated with various concentrations of either DL-kynurenine (FSL), L-kynurenine, D-kynurenine or acid kynurenic (FS2) and corresponding fibronectin expression at mRNA levels as detected by real-time PCR, with β-actin as a loading control.

[0061] Figure 17 - Comparison of the suppressive effect of metabolites of 50, 100, 150 μg/mL of tryptophan (FS1, LK, FS2, DK) on the proliferation of splenocytes simulating ConA - shows that there was an almost 5-fold reduction in proliferation of splenocytes after treatment with 100 and 150 μg/mL of D-Kyn, L-Kyn, DL-Kyn (FS-1) and kynurenic acid (FS2) after 96 hours (P<0.05), although the proliferation of splenocytes Splenocytes significantly reduced about 2-fold by D-Kyn, L-Kyn and DL-Kyn at 100 and 150 μg/mL after 48 hours, but FS2 showed a lesser effect on proliferation.

[0062] Figure 18 - Immune factor protein microarray in splenocytes from treated and untreated FS1 (DL-kynurenine) mice - shows that FS1 has an immune suppressive effect on some pro-inflammatory cytokine and chemokine production, such as IL -1, IL-2, CXCL9, and CXCL10 and FS1 show a significant decrease in the production of IL-17, which is thought to play an important role in inflammation. Panel A: shows activated splenocytes that were either left untreated (ConA) or treated with 100 μg/mL Kyn (ConA + Kyn) for 48 hours, at which time conditioned medium (CM) was collected from non-treated cells. treated and treated cells and was then exposed to a Proteome Profiler Antibody Array™ membrane with percentages of the density value are shown for both untreated and treated cells for each reference point as shown in Panel B. Panel B: shows signals identified by Proteome Profiler Antibody Array membrane. Panel C: shows the local number shown in panel B represents reference protein.

[0063] Figure 19 - Long-lasting effect of FS1 and FS2 on MMP1 expression in human dermal fibroblasts. Panel A: shows the lasting effect of kynurenine (FS1) and kynurenic acid (FS2) on MMP1 expression, where fibroblasts were treated with FS1 or FS2 (100 μg/mL) for 48 hours and the medium was replaced and the cells were harvested immediately, and at 12, 24 and 48 hours after treatment removal, followed by evaluation of MMP1 expression in dermal fibroblasts using Western blotting. Panel B: shows the MMPl/β-actin expression ratio as calculated in treated fibroblasts. Data are the mean ± SEM of 4 independent experiments (*P-value<0.05 and **P-value<0.01, n = 4).

DETAILED DESCRIPTION

[0064] Any terms not defined herein directly are to be understood to have the meanings commonly associated with them as understood in the art of the invention. As used throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

[0065] As used herein, a "subject" refers to an animal, such as a mammal or a bird. 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 even be a transgenic animal. A subject may further be a rodent, such as a beaver, mouse or rat.

[0066] As used herein, an "inhibitor" refers to a drug, compound, or agent that prevents or delays a physiological, chemical, or enzymatic action or function. An inhibitor can cause at least a 5% decrease in enzyme activity. An inhibitor can also refer to a drug, compound or agent that prevents or reduces the expression, transcription or translation of a gene or protein.

[0067] "Indoleamine 2,3-dioxygenase", or "IDO", is a heme-containing ratio-limiting enzyme that catalyzes tryptophan to N-formylkynurenine and then to kynurenine (Kyn), and is found in non-liver cells primarily in macrophages and trophoblasts. Recent discoveries have implicated the catabolism of tryptophan, an essential amino acid, by IDO as being involved in immune tolerance (Kahari Saarialho-Kere 1997). As shown here, kynurenine, as well as its degradation products kynurenic acid and xanthurenic acid, induce MMP-1 and MMP-3, as well as shown to reduce fibrosis in vitro and in vivo.

[0068] The 'matrix metalloprotease', or MMP family consists of 25 zinc and calcium dependent proteinases in the mammalian system. According to their substrate specificity, primary structure, and cellular location, 5 different subfamilies of closely related members known as collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs have been identified (Murphy et al., 2002). Of all these MMPs, MMP1 is the main enzyme involved in the collagenolytic process, breaking down interstitial collagens such as type I, II, and III, while MMP-3 (stromelysin-1) is a protease known to primarily degrade the portion non-collagenous components of the ECM, such as fibronectin, proteoglycans, and laminin (Kahari and Saarialho-Kere 1997). Increases in both MMP1 and MMP-3 expression and released by fibroblasts can initiate degradation of nearly all major components of the ECM (Saus et al., 1988). It is now accepted that MMPs produced by keratinocytes facilitate epithelial migration, whereas MMPs expressed by fibroblasts promote tissue remodeling (Saio et al., 1991).

[0069] "Fibrosis" is a general form involving 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 component of an organ or fabric. Scar is the 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: keloids, hypertrophic scars, pulmonary fibrosis, renal fibrosis, cirrhosis of the liver, chronic inflammation of the tunica albuginia (CITA), endomyocardial fibrosis, fibrosis mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's disease, age-related myocardial infarction, scleroderma, and systemic sclerosis.

[0070] A number of compounds are provided herein 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 the treatment of existing fibrosis or fibrotic disease, or alternatively may refer to treatment that takes place before or during the fibrotic process, so as to prevent the development or progression of fibrosis. The compounds described herein can be isolated, or can be linked to or in combination with marker compounds, liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one skilled in the art. In an alternative embodiment, such compounds may contain 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, in view of the fact that the subject may benefit from the prophylaxis or treatment of fibrosis or fibrotic disease. The compounds can also include tautomers or stereoisomers.

[0071] As used herein "FS" refers to FibroStops (e.g., 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 Kynurenine Acid ) . L-kynurenine can be represented herein as L-Kyn and D-kynurenine can be represented herein as D-Kyn. Similarly, xanthurenic acid can be represented here as XA.

[0072] The term "medicament" as used herein refers to a composition that can 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 a human or non-human animal, such as a transgenic rodent or mouse, or a dog, cat, cow, sheep, horse, hamster, rabbit or pork. The medicament may be composed of the effective chemical entity alone or in combination with a pharmaceutically acceptable excipient.

[0073] The term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial, antimicrobial or antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. An excipient may be suitable for intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, topical or oral administration. An excipient can include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media for the preparation of medicaments is known in the art.

[0074] The compositions or compounds according to some embodiments can be administered in any of a variety of known ways. Examples of methods that may be suitable for administering a compound include orally, intravenously, inhalation, intramuscularly, subcutaneously, topically, intraperitoneally, intrarectally or intravaginally suppository, sublingually, and the like. The compounds described herein can be administered as a sterile aqueous solution, or can be administered in a fat-soluble excipient, or in other solution, suspension, plaster, tablet or paste format as appropriate. A composition comprising the compounds described herein can be formulated for administration by inhalation. For example, a compound can be combined with an excipient to allow for aerosol dispersion. Examples of formulations for inhalation are known to those skilled in the art. Other agents may be included in combination with the compounds described herein to aid absorption or metabolism, or delayed dispersal within the host, such as in a controlled release formulation. Examples of controlled release formulations are known to those skilled in the art, and can include microencapsulation, embolism within a carbohydrate or polymer matrix, and the like. Other methods known in the art for making formulations are found, for example, "Remington's Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.

[0075] The dosage of the compounds or compositions 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, delivery controlled or similar). Determination of appropriate dosages is well within the ability of one skilled in the art. As used herein, an "effective amount", a "therapeutically effective amount", or a "pharmacologically effective amount" of a drug refers to an amount of a drug present in such a concentration that results in a therapeutic level of the drug released over time. the period the drug is used. This may be dependent on the mode of administration, the time period of dosing, age, weight, general health, sex and diet of the individual receiving the drug. Methods for determining effective amounts are known in the art.

[0076] In one embodiment, there is provided a method of treating 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 the formula I, II, or III. Fibrotic disease can be any of the following: keloid, hypertrophic scarring, pulmonary fibrosis, renal fibrosis, cirrhosis of the liver, chronic inflammation of the tunica albuginea (CITA), endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, systemic fibrosis nephrogenic, Crohn's disease, age-related myocardial infarction, scleroderma, systemic sclerosis, uterine fibroids, restenosis.

MATERIALS AND METHODS cell cultures

[0077] Neonatal foreskin and joints used as the sources of fibroblasts, keratinocytes and synoviocytes. The procedures were done based on the approval of the Human Ethics Committee of the University of British Columbia. Cultures of human foreskin fibroblasts were established as previously described (Li et al., 2006). Briefly, foreskin was collected and washed three times with Dulbecco's Modified Eagle's Medium (DMEM; GIBCO™, Grand Island, NY) supplemented with the 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 smaller than 2.0 mm in diameter, washed six times with DMEM, distributed in 60 x 15 mm Petri dishes and incubated at 37 °C in a humidified incubator lined with water in an atmosphere of 5% CO2. The medium was replaced twice a week. Upon reaching confluence, 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 in 75 cm2 flasks. Fibroblasts from passages 3-7 were used for this study.

[0078] 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 in passages 2-5.

[0079] 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 seeded in synoviocyte growth medium (Cell Applications™ Inc., San Diego, CA) 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 homogeneous fibroblast-like cells and were used in passages 2-5.

[0080] Squamous cell carcinoma cell line (UMSCC) derived from head and neck cancer patients (ATCC, Manassas, VA) were maintained in RPMI-1640 medium with 10% FBS. Human HACAT Keratinocyte Cell Line (ATCC) and A549 Human Basal Epithelial Alveolar Carcinoma Cell Line (ATCC™) were grown in DMEM with 10% FBS. IMR-90 diploid lung fibroblasts (ATCC™) were maintained in Minimal Essential Medium (MEM, Invitrogen™) with 10% FBS.

Gene transfection by adenoviral vector

[0081] 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 a multiplicity of infection (MOI) of 100. Free virus particles were removed from the culture medium 30 hours after infection. Infection success was determined by fluorescence microscopy using a Motic™ inverted microscope equipped with a fluorescein isothiocyanate (FITC) filter (Motic Instruments™, Richmond, BC, Canada) to visualize the GFP reporter gene. IDO expression was assessed by Western blot using anti-human IDO antibody as previously described (Li et al., 2004). The biological activity of IDO was evaluated by measuring the levels of the tryptophan degradation product, kynurenine, present in conditioned medium.

Kynurenine measurement in conditioned medium

[0082] Kynurenine levels were measured by a previously described method (Tokikawa et al., 1988). In summary, about 2 mL of conditioned medium was collected from the same number of cells in culture started 3 days after transfection. Proteins from conditioned media were precipitated by trichloroacetic acid. After centrifugation to remove precipitated proteins, approximately 0.5 ml of supernatant was transferred to a new 1.5 ml tube and incubated with an equal volume of Ehrich's reagent (Sigma™) for 10 minutes at room temperature. The absorption of the resulting solution was measured at 490 nm with a spectrophotometer within 2 hours. Kynurenine values in conditioned medium were calculated by a standard curve with the defined kynurenine concentration (020 μg/mL).

Cell treatments

[0083] For collection of conditioned medium, fibroblasts were transduced by either no mock vector control or IDO adenovirus for 30 hours. Viruses were removed by washing with PBS. Fresh DMEM containing 10% FBS and antibiotics were added and the cells were continued to be cultured for an additional 48 hours. Conditioned medium from either untreated medium, mock vector, or IDO adenovirus transduced fibroblasts were then collected. Fibroblasts at 80% confluence were treated with medium containing 90% conditioned medium plus 10% fresh medium, in the presence of 10% FBS. Cells were then harvested after 48 hours and Western blot analysis was performed.

[0084] In another set of experiments, fibroblasts at 80% confluence were treated with 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.

[0085] 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 medium for each cell type as described above for 48 hours. Cells were then harvested for Western blot analysis.

Western Blot Analysis

[0086] Cells were harvested by trypsin/EDTA and lysed with cell lysis buffer containing 50 mM Tris HCl (pH 7.40), 150 mM NaCl, 10 mM EDTA, 5 mM EGTA, 1% Triton X-100™, 0.5% Igepal CA-630, 0.025% NaN3 and protease inhibitor cocktail (Sigma™). Cell debris was removed by centrifugation at 20,000 x g for 10 minutes. Protein concentration in the supernatant was determined using the MicroBCA™ method (Pierce™, Rockford, IL). Proteins in the supernatant were mixed with protein sample loading buffer (final concentration: 60 mM Tris-HCl (pH 6.80), 2% SDS, 10% glycerol, 1.5% β-mercaptoethanol, 0.002 % bromophenol blue) and size fractionated by 10% SDS-polyacrylamide gel. After proteins were transferred to a nitrocellulose membrane by iBlot™ (Invitrogen Life Technologies™), non-specific binding was blocked with twenty-20 phosphate buffered saline (PBS-T) containing 5% skim milk for 1 hour. The membrane was then incubated overnight with primary antibody. After incubation with a secondary antibody for 1 hour, protein bands were visualized by an enhanced chemiluminescence detection system (ECL™) (Santa Cruz Biotechnology™, Santa Cruz, CA). The primary antibodies used in this study were: mouse monoclonal anti-human MMP-1 (R&D Systems™, Minneapolis, MN), mouse monoclonal anti-human MMP-3 (R&D System™), rabbit monoclonal anti-human MMP- 2 (Epitomics™, Burlingame, CA), rabbit polyclonal anti-phospho-MEK1/2 (Ser217/221™) (Cell Signaling Technology™, Danvers, MA), 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, IA). 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.

MMP activity assay

[0087] The activity of MMPs was evaluated using a fluorescence resonance energy transfer peptide (FRET) M-FAM/QXL™ 520 as the MMP substrate (SensoLyte 520™ Generic MMP Assay Kit, AnaSpec, Inc .™, Fremont, CA) according to the manufacturer's protocol. Briefly, cells were treated with or without 50 µg/ml kynurenine for 48 hours. Conditioned media were collected and incubated with 1 mM APMA (4-aminophenyl-mercuric acetate, in component 0, AnaSpect™) at 37°C for 3 hours. After activating the MMPs with APMA, 50 µg/well of 96-well plate of conditioned medium was mixed with 50 µL of MMP Substrate Solution. After incubating 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 the Infinito F500™ fluorescence microplate reader (Tecan Group Ltd™, Morrisville, NC).

Phosphorylation of protein array

[0088] Human fibroblasts at 90% confluence were starved in DMEM without FBS overnight, followed by treatment with or without 100 µg/mL kynurenine for 2 hours. Protein phosphorylation was assessed using the Human Phospho-Kinase Array™ (R&D System™) according to the manufacturer's instructions. Briefly, capture and control antibodies were seen in duplicate on nitrocellulose membranes (total of 46 kinase phosphorylation sites). Cell lyses (300 µg of total protein per matrix) were incubated with matrix overnight. The matrix was washed to remove unbound proteins, followed by incubation with the biotinylated detection antibody cocktail. After incubation with streptavidin-HPR for 30 minutes, signals were visualized by the ECL detection system (Santa Cruz™). Blots were analyzed by densitometry and protein phosphorylation was normalized to a positive control, which was plotted on each blot.

Rabbit ear hypertrophic scar model and topical application of kynurenine

[0089] Female rabbits (New Zealand White) weighing 4.5-5 kg were used for this study. The protocol was reviewed and approved by the Animal Care Committees at the University of British Columbia. The hypertrophic scar rabbit ear model was created as previously described (Neishaboor Rahman, et al., 2010). Briefly, 2 female rabbits were anesthetized by intramuscular injection of ketamine (22.5 mq/kg) and xylazine (2.5 mg/kg) followed by isoflurane gas via tracheal intubation. Four wounds were created downward to uncover the cartilage on the ventral side of each ear using an 8mm dermal biopsy punch to remove full thickness sections of skin. Antibiotics were applied to the wounds daily until kynurenine treatment was started.

[0090] Kynurenine in CMC gel (Rahman-Neishaboor et al., 2010) with a concentration of 500 μg/mL was topically applied to wounds of the experimental group (0.1 mL per wound) daily for 3 weeks from one week after the injury. Wounds in the control group were treated with an equal amount of cream daily only.

[0091] The animals were euthanized at 3 weeks after treatments. Scars (10 mm biopsies) were taken. Each scar was sectioned in two along its longitudinal axis and half of which was processed for routine histological analysis and the other half was kept at -80°C for future use.

[0092] Scar lift was quantified by measuring the scar lift index (SEI) from tissue section stained with H&E. The SEI is a ratio of the total height in the wound tissue to the normal tissue below the hypertrophic scar. An SEI of 1 indicates that the height of the scar is equal to the surrounding intact dermis; an SEI > 1 indicates a raised hypertrophic scar.

MTT test

[0093] The effect of kynurenine on human dermal fibroblast proliferation was detected by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Briefly, 10,000 cells were seeded in a 24-well plate, and incubated with different concentrations of kynurenine for 48 hours. The media was removed and 0.2 ml of MTT (5 mg/ml in DMEM containing 2% FBS) was added. Cells were incubated with MTT 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.

Measurement of the hydroxyproline content of the skin sample

[0094] According to a previously reported method (Gawronskao-Kozak B. et al. 2006), half of the 8 mm diameter skin punctures were weighed and frozen at -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. Hydroxyproline concentration was calculated using a standard curve.

RNA extraction, cDNA synthesis and quantitative RT-PCR

[0095] The 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 left at room temperature for 5 minutes. Upper aqueous phase was transferred to a new eppendorf tube after centrifugation for 10 minutes at 20,000 x g. Equal volume of 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 H2O and its concentration was measured by Nanodrop2000™. cDNA was synthesized by the Roche cDNA synthesis kit according to the manufacturing introduction using 1 µg of total RNA in each sample. Quantitative real-time PCR for rabbit type-1 and rabbit collagen, MMP-1 and the employed β-actin gene were performed on ViiA7 (Invitrogen™). The cDNA samples were added to a PCR reaction master mix containing STBR Green Master Mix™ (ROX) (Roche™, Indianapolis, IN). All reactions were performed in duplicate using the following cycling 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 collagen type-1 al and MMP-1 in each sample was normalized to β-actin. RT-PCR primers: rabbit type-1 al 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'-CTCTGCATGCGGTCCGCGA-3'(reverse).

EXAMPLES Example 1- Expression of indoleamine 2,3-dioxygenase (IDO) upregulates MMP-1 expression in human dermal fibroblasts

[0096] To assess the effect of IDO on MMP-1 expression, a recombinant human IDO adenoviral vector was used for gene transduction in human dermal fibroblasts by a previously reported procedure (Li et al., 2004). Transfection efficiency was assessed by detecting protein expression with IDO and its activity by Western blot analysis and measurement of kynurenine in conditioned media, respectively. As shown in the left panel of Figure 1A, IDO protein was expressed in IDO transduced adenovirus fibroblasts, but not detectable in control and mock adenovirus transduced fibroblasts. Kynurenine level, 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 non-transduced or mock-transduced controls (Figure 1, right panel).

[0097] MMP-1 expression in control, mock-transduced, IDO-expressing fibroblasts was examined using Western blot analysis. As shown in Figure 1B, there was more than a nine-fold increase in MMP-1 expression in IDO-expressing fibroblasts (12.56 ± 2.37, n=3), as compared to that in untreated control fibroblasts (1 ± 0, n=3) and mock transduced (1.37 ± 0.59, n=3). This finding suggests that the upregulation of MMP-1 expression in IDO-expressing fibroblasts is not due to adenovirus infection, since mock-transduced fibroblasts did not show a significant difference in MMP-1 expression from untreated fibroblasts.

[0098] IDO is an intracellular enzyme that converts tryptophan to kynurenine. Therefore, it remains to be clarified whether the effect of MMP-1 stimulation on 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 medium and 10% fresh medium was then used to treat dermal fibroblasts. Cells were harvested 48 hours after treatment. As shown in Figure 1C, a significant increase in MMP-1 expression was observed in cells treated with conditioned medium from IDO transduced fibroblasts (2.06 ± 0.62, n=3), compared to those in any transduced fibroblasts (1.16 ± 0.31, n=3) or untreated control (1 ± 0, n=3). This result suggests that the factor or factors in adenovirus-infected fibroblast conditioned medium with IDO rather than intracellular IDO protein is responsible for an increase in the level of MMP-1 expression in fibroblasts.

Example 2 - Kynurenine but not tryptophan depletion induces MMP-1 expression in human dermal fibroblasts

[0099] IDO is an enzyme converting tryptophan to kynurenine. To examine which factor (either tryptophan depletion or kynurenine increase) is responsible for IDO upregulation of MMP-1 expression. To examine which factor is responsible for IDO upregulation of MMP-1 expression, fibroblasts were cultured in either tryptophan-depleted culture medium or regular medium with various concentrations of kynurenine. Cells were then evaluated for MMP-1 expression by Western blotting. As shown in Figure 2C, there was no significant difference in MMP-1 expression, between fibroblasts cultured in the presence of 25 μg/mL of tryptophan or in cultured medium with tryptophan depletion. However, MMP-1 expression was significantly increased in response to different doses (25-150 μg/mL) of kynurenine (Figure 2A and Figure 2B). These results suggest that the presence of kynurenine, but not tryptophan depletion, contributes to the upregulation of MMP-1 in cells expressing IDO. Furthermore, it was found that as little as 12.5 μg/mL of kynurenine could stimulate MMP-1 expression in dermal fibroblasts (data not shown). This kynurenine concentration is similar to that detected in the conditioned medium of fibroblasts expressing IDO (Figure 1A right panel). Stimulation of MMP-1 in fibroblasts is thus clearly specific for kynurenine as the addition of various concentrations of tryptophan with a similar structure fails to increase MMP-1 expression in dermal fibroblasts (Figure 2D).

Example 3 - Effects of kynurenine on MMP-2 and -3 expression in dermal fibroblasts

[00100] To investigate whether kynurenine also affects the expression of other MMPs, we treated dermal fibroblasts with kynurenine at concentrations similar to those used in Figure 2. Western blotting was used to detect MMP-2 and -3 expression using untreated cells as controls. As shown in Figure 3A, 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 (Figure 3B/3C). Furthermore, to test whether increased levels of MMPs in kynurenine-treated fibroblasts were followed by an increase in MMP activity, conditioned media from fibroblasts in the presence or absence of 50 μg/mL kynurenine was collected 48 hours after treatment. . MMP activity in the conditioned medium was detected by a SensoLyte 520™ generic MMP assay kit using a 5-FAM/QXL™520 fluorescence resonance energy transfer (FRET) peptide: as an MMP substrate. As shown in Figure 4, the mean activity of MMPs in conditioned media from kynurenine-treated fibroblasts was significantly higher than the control media. This indicates that the increase in MMPs in fibroblasts treated with kynurenine has enzymatic activity.

Example 4 - Epithelial and mesenchymal cells respond differently to kynurenine treatment

[00101] To determine which cell types are sensitive to kynurenine-induced MMP-1 expression, both mesenchymal cells (such as an immobilized cell line IMR-90 of lung fibroblast and fibroblast-like synoviocytes) and epithelial cells (such as A549 epithelial lung carcinoma cell line, primary dermal keratinocytes, HACAT immobilized human keratinocyte cell line, and UMSCC head and neck squamous cell carcinoma cell line) were used. As with dermal fibroblasts, MMP-1 expression in synoviocytes and IMR-90 were upregulated by kynurenine treatments at concentrations from 12.5 μg/mL to 150 μg/mL, as shown in Figure 5. However, the MMP-1 expression in all epithelial cells tested, including dermal keratinocytes, HACAT, A549 and UMSCC, did not differ significantly from untreated controls in response to various concentrations of kynurenine (Figure 6). These results suggest that there is a difference between epithelial and mesenchymal cells in response to kynurenine-stimulating MMP-1 expression.

Example 5 - Identification of phosphorylated signal molecules by phosphokinase array in kynurenine-treated cells

[00102] To determine the possible mechanism of kynurenine upregulated MMP-1 expression in dermal fibroblasts, the activation of multiple serine, threonine or tyrosine kinases using a phosphorus kinase array was analyzed. This arrangement gives the possibility to simultaneously detect the activation status of 46 different protein kinases and their downstream transcription factors. As shown in Figure 7, after 1 hour of treatment in dermal fibroblasts with kynurenine, extracellular signal-regulated kinases 1/2 (ERK1/2) were activated.

[00103] To confirm these results from matrix phosphokinase, dermal fibroblasts were treated with 100 μg/mL of kynurenine at different times. Immunoblot analysis, using a different antibody than those placed on the matrix, was then used to detect phosphorylation of ERKtl/2 and its extracellular signal-regulated mitogen-activated protein/kinase kinase (MEK) upstream molecule. As shown in Figure 8, ERKtl/2 was phosphorylated in kynurenine-treated cells. The result was further confirmed by detection of MEK phosphorylation of the upstream signal molecule ERKtl/2 in kynurenine-treated cells ( Figure 8 ). Both ERKl/2 and MEK showed similar patterns of activation, with a peak at 8 hours following kynurenine treatments (Figure 8).

Example 6 - Addition of inhibitors to MEK-ERK1/2 phosphorylation negates the effects of kynurenine-stimulated MMP-1 expression in dermal fibroblasts

[00104] In another set of experiments, we tested whether activation of the MEK-ERK1/2 MAPK pathway by kynurenine is associated with kynurenine-stimulating MMP-1 expression in dermal fibroblasts. To do this, the effects of inhibitors of either MEK or ERK1/2 phosphorylation on kynurenine-stimulating MMP-1 expression were examined. As shown in Figure 9A, 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. Likewise, treatment of cells with 10 µM of U0126, a specific inhibitor for MEK activation, also significantly reduced the upregulation of MMP-1 expression by kynurenine ( Figure 9B ). These results demonstrate that activation of the MEK-ERK1/2 signaling pathway contributes to kynurenine-induced upregulation of MMP-1 expression in dermal fibroblasts.

Example 7 - Effects of kynurenine on collagen expression in dermal fibroblasts and fibroblast proliferation

[00105] Before studying its anti-fibrotic role in vivo, kynurenine was tested for its effect on collagen expression and cell proliferation. As shown in Figure 10 (top), the addition of 25 to 150 μg/mL kynurenine remarkably decreased the expression of type 1 procollagen. However, this did not have any significant effect on fibroblast proliferation, even when the cells were cultured at concentrations of up to 150 μg/mL of kynurenine (Figure 11). Also, testing of kynurenine analogues/metabolites, kynurenic acid and xanthurenic acid, demonstrate that these compounds are also effective in inhibiting type 1 procollagen expression (Figure 10 (bottom)).

Example 8 - Topical application of kynurenine to rabbit ear wounds reduces scarring

[00106] Since treatment of dermal fibroblasts with kynurenine showed an increase in both MMP-1 and -3 expression, as well as a decrease in procollagen type-1 expression, we were interested in knowing whether kynurenine can be used as a an anti-fibrotic agent for the treatment or prevention of hypertrophic scars. To achieve this goal, as previously described (Rahman-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 μg of kynurenine for three weeks starting on day 8 after wounding. The dose of 50 μg of kynurenine per wound was combined with that used in an in vitro system, with an optimal result. The result showed no significant difference for wound closure in wounds treated with kynurenine as compared to that of one of these untreated or CMC gel treated controls (data not shown). However, as shown in Figure 12A, significantly less scarring was observed in kynurenine-treated wounds than either the untreated wounds or the vehicle-only control wounds after three weeks. The mean scar lift index (SEI) was significantly reduced in the kynurenine-treated group (1.172 ± 0.156, n=8) compared to the vehicle-only control group (1.978 ± 0.442, n=4, p <0 .01) and the untreated group (2.098 ± 0.324, n=4, p<0.01) (Figure 12B). Massons trichrome for collagen revealed a significant reduction in collagen content in wounds treated with kynurenine, compared to wounds that received either no treatment or gel alone (Figure 12C). Consistent with this finding, the hydroxyproline content (used as an index for tissue collagen content) was significantly lower in wounds treated with kynurenine compared to those wounds that received either no treatment or gel only (Figure 12D).

[00107] Finally, it was demonstrated that the topical application of kynurenine in a fibrotic rabbit ear model decreased the expression of type 1 al collagen and increased the expression of MMP-1, compared to those in wounds that did not receive treatment or gel only (Figure 13). These results further support the assumption that kynurenine could potentially be used as an anti-fibrotic factor for the treatment of hypertrophic scars and even keloids so often seen in patients with burns or surgical incisions.

Example 9 - Effect of kynurenine isoforms on MMP-1 expression in human dermal fibroblasts

[00108] Different kynurenine isoforms were tested for their ability to affect MMP-1 expression. The tested isoforms were: DL-kynurenine (DL-Kyn) or D-kynurenine (D-Kyn) and L-kynurenine (L-Kyn). The result showed that all isoforms increase MMP-1 expression in dermal fibroblasts, however, L-kynurenine appears to have more activity compared to the other two isoforms - see Figure 14.

Example 10 - Effects of different kynurenine isoforms/analogs on collagen expression in human dermal fibroblasts

[00109] Dermal fibroblasts were treated with either FS-1 (DL-kynurenine) or D-kynurenine or L-kynurenine or FS-2 (kynurenine acid) as shown in Figure 15. Type-1 collagen expression was detected by Real-time PCR. The results indicate that these isoforms/analogs have similar efficacy in reducing collagen expression.

Example 11 - Kynurenine and its metabolites down-regulate fibronectin expression in cultured fibroblasts

[00110] Dermal fibroblasts were treated with various concentrations of either DL-kynurenine (FS1), L-kynurenine, D-kynurenine or kynurenic acid (FS2) as shown in Figure 16 . Fibronectin expression was detected by real-time PCR. The results demonstrate that kynurenine, DL-kynurenine, and L-kynurenine are all capable of down-regulating fibronectin expression, indicating that kynurenine metabolites may also be suitable for the prevention or treatment of fibroproliferative disorders.

Example 12 - Kynurenine and metabolites/analogs have significant effects on splenocytes

[00111] The results in Figure 17 showed that there was almost a 5-fold reduction in conA-induced splenocyte proliferation after treatment with 100 and 150 μg/mL of D-kynurenine, L-kynurenine or DL-kynurenine after 96 hours ( P<0.05), although splenocyte proliferation was 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 the proliferation of other metabolites. The results in Figure 18 showed that FS1 has immune suppressive effect on some of the pro-inflammatory chemokine and chitokine production, such as IL-1, IL-2, CXCL9, and CXCL10. Furthermore, it can significantly decrease the production of IL-17, which is thought to play an important role in inflammation.

Example 13 - Long lasting effect of kynurenic acid and kynurenine on MMP1 expression in fibroblasts

[00112] 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. After 48 hours of treatment, the medium was changed to fresh medium and the cells were then harvested at 0, 12, 24 or 48 hours after removal of treatment. There was a significant increase in MMP1 expression in fibroblasts in response to either KynA or Kyn treatment within 48 hours of treatment. After removal of Kyn and KynA, MMP1 expression remained significantly higher than untreated cells for an additional 24 hours (Figure 19A). Interestingly, while MMP1 protein expression gradually reduced to normal levels within 48 hours of Kyn removal, MMP1 expression in response to KynA remained higher than controls (Figure 19A). Figure 19B represents the quantitative analysis of the data from Figure 19A (*P value <0.05, **P value <0.01, n=4). From these results, it appears that KynA has a longer lasting effect on MMP-1 expression than Kyn in treated fibroblasts.

[00113] Although several embodiments are described herein, many adaptations and modifications can be made within the scope of the invention according to the common general knowledge of those skilled in the art. Such modifications include substituting known equivalents for any aspect of the invention in order to achieve the same result in substantially the same manner. Numeric ranges are inclusive of the numbers that define 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/the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the 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 described hereinbefore and with reference to the examples and drawings. REFERENCES Gawronskao-Kozak B. et al. Wound Repair Regeneration (2006) Scarless skin repair in immunodeficient mice 14: 265-276. Ghahacy A, Tredget EE, Chang LJ, et al. (1998) Genetically modified dermal keratinocytes express high levels of transforming growth factor-βl. J Invest Dermatol 110: 800-805. Kahari, V.M. and U. Saarialho-Kere (1997). "Matrix metalloproteinases in skin." Exp Dermatol 6(5): 199-213. Kloeters 0, Tandara A, Mustoe T (2007) Hypertrophic scar model in the rabbit ear: the reproducible model for studying scar tissue behavior with new observation on silicone gel sheeting for scar reduction. Wound Repair Regen 15: S40-S45. Li Y, Tredget EE, Ghaffari A, et al. (2006) Local expression of indoleamine 2,3-dioxygenase protects engraftment of xenogeneic skin substitutes. J Invest Dermatol 126: 128-136. Li Y, Tredget EE, Kilani RT, et al. (2004) Expression of indoleamine 2,3-dioxygenase in dermal fibroblasts functions the local immunosuppressive factor. J Invest Dermatol 122: 953-964. Rahmani-Neishaboor E, Yau FM, Jalili R, et al. Improvement of hypertrophic scarring by using topical anti-fibrogenic anti-inflammatory factors in the rabbit ear model. Wound Repair Regen 18: 401-408. Salo, T., J.G. Lyons, et al. (1991). "Transforming growth factor-beta 1 up-regulates type IV collagenase expression in cultured human keratinocytes" J Biol Chem 266 (18):11436-41. Saus, J., S. Quinones, et al. (1988). "The complete primary structure of human matrix metalloproteinase-3. Identity with stromelysin" J Biol Chem 263 (14): 6742-5. Scott, P.G., A. Ghahacy, et al. (1994). In: Advances in Structural Biology 3: 157-201. Tokikawa 0, Kuroiwat, Yamazaki M, et al. (1988) Mechanism of interferon-y action: characterization of indoleamine 2,3-dioxygenase in cultured human cells by interferon-y and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity. J Biol Chem 263: 2041-2048. Xie JL, Bian HN, Qi SH, et al. (2008) Basic fibroblast growth factor (bFGF) alleviates the scar of the rabbit ear model in wound healing. Wound Repair Regen 16: 576-581.

Claims (6)

1. Use of a compound, the compound having the structure of one or more of: the use being characterized by the fact that it is for the manufacture of a medicine for the treatment of fibrotic disease, in which the fibrotic disease is selected from keloids and hypertrophic scarring. 2. Use, according to claim 1, characterized in that the treatment is keloids. 3. Use, according to claim 1, characterized in that the treatment is for hypertrophic scarring. 4. Use according to any one of claims 1 to 3, characterized in that the compound is selected from one or more of the following: 5. Use according to any one of claims 1 to 3, characterized in that the compound is selected from one or more of the following: 6. Use according to any one of claims 1 to 5, characterized in that the medicine additionally comprises a pharmaceutically acceptable excipient to form a pharmaceutical composition.