METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF FIBROSIS

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of fibrosis. In particular, the present invention relates to a method of treating fibrosis in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one monoacylglycerol lipase (MGL) inhibitor.

Claims

1. A method of treating fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one monoacylglycerol lipase (MGL) inhibitor.

2. The method of claim 1 wherein the fibrosis affects at least one organ selected from the group consisting of skin, heart, liver, lung, and kidney.

3. The method of claim 1 wherein the fibrosis is selected from the group consisting of dermal scar formation, keloids, liver fibrosis, lung fibrosis, kidney fibrosis, glomerulosclerosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, intestinal fibrosis, interstitial fibrosis, cystic fibrosis of the pancreas and lungs, injection fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, and nephrogenic systemic fibrosis.

4. The method of claim 1 wherein the fibrosis is liver fibrosis.

5. The method of claim 1 wherein the fibrosis is inflammation-induced fibrosis.

Description

FIGURES

[0701] FIG. 1: Experimental protocol of the model of liver fibrosis regression. C57BL/6J mice received bi-weekly i.p. injections of CCl4 for 6 weeks. Injections of MGL inhibitors or vehicle started 2 hours prior the last injection of CCl4 and were administered daily until the end of the experiment. Mice were sacrificed either at day 1 (24 hrs), 4 or 7.

[0702] FIG. 2: Pharmacological inhibitors of MGL accelerates liver fibrosis regression. (a) picrosirius red staining. Representative image of picrosirius-red stained liver tissue sections from mice treated with MGL inhibitors or vehicle for 4 days (left). Morphometric analysis. Results are expressed as mean±SEM of % stained area (n=7-9 CCl4-treated animals and n=5 for mineral oil and vehicle-treated group, * p<0.002). (B) mRNA EXPRESSION OF FIBROGENIC GENES. All data are expressed as mean±SEM of n=7-9 animals for each treatment and n=5 for control groups.

[0703] FIG. 3: MGL inhibition does not modify liver injury. Serum levels of ALAT and ASAT was determined in control mice and in mice after the stated time points after the final CCL4 dose and in the presence or absence of MGL inhibitors daily administered. All data are expressed as mean±SEM, of n=7-9 animals for each treatment and n=5 for control groups.

[0704] FIG. 4: MGL inhibitors reduce proinflammatory cytokines during fibrosis regression—mRNA expression of inflammatory genes. Total liver RNA was extracted at different point post CCL4 last injection and Q-PCR was performed. All data are expressed as mean±SEM, of n=7-9 animals for each treatment and 5 for control groups.

[0705] FIG. 5: MGL inhibitors inhibit DNA synthesis of human and murine hepatic myofibroblasts.

[0706] FIG. 6: MGL expression is induced in PBMC from patients with alcoholic cirrhosis.

[0707] FIG. 7: Mice bearing a global invalidation for MGL show decreased fibrosis. A. picrosirius red staining. B. mRNA expression of fibrogenic gene αSMA.

[0708] FIG. 8: Knock-Down of MGL in myeloid cells decreased liver fibrosis. A. picrosirius red staining. B. mRNA expression of fibrogenic gene αSMA.

EXAMPLE

[0709] Material & Methods

[0710] Mice: C57B1/6J mice were purchased from Janvier and kept under pathogen-free. Experiments were approved by the Paris-Nord Committee 121 for the Care and Use of Laboratory Animals.

[0711] Liver fibrosis model: Hepatic fibrosis was induced in C57BL/6J mice (male, 11 weeks) by i.p. injection of carbon tetrachloride (CCl.sub.4, Sigma 87030) 0.6 ml/kg body weight, 1/10 dilution in mineral oil (MO, Sigma, M-5310) twice a week for six weeks (total 13 injections). Control animals (n=5) received mineral oil.

[0712] Administration of Monoacylglycerol lipase (MGL) inhibitors: MGL inhibitors (JZL 184, Cayman {Long, 2009 #6} and MJN 110 (kind gift of B. Cravatt, The Scripps Institute, {Niphakis, 2013 #7}), were diluted in Emulphor:ethanol:PBS; 1:1:18 and injected ip at dose of 15 mg/kg in and 10 mg/kg for JZL 184 (n=27) and MJN 110 (n=15), respectively. Mice were injected MGL inhibitors or vehicle 2 hours prior to the last CCL4 injection and daily until sacrifice. Animals were sacrificed 1, 4 or 7 days after the final injection of CCl.sub.4.

[0713] Serum analysis: At the time of harvest, whole blood was collected from the inferior veina cava and serum was isolated by centrifugation at 7000 g for 5 min. Alanine aminotransferase and aspartate aminotransferase levels were measured at the Plateforme de Biochimie, Centre de Recherche sur l′Inflammation, INSERM U 1149, Bichat.

[0714] Histochemistry and immunohistochemistry: Liver tissue samples were fixed overnight in 10% formalin and further embedded in paraffin. Picrosirius red and Hematoxilin Eosin staining were performed on 4 gm thick tissue sections, according to standard protocols. Morphometric pixel analysis was performed on 10 non overlapping fields per mice (n=8-10 mice per group) at 100× magnification, using Image J software (NIH, USA).

[0715] Immunodetection of alpha-SMA was performed on 4 gm thick paraffin-embedded liver tissue sections. Antigen retrieval was performed by boiling 10 min in 10 mM sodium citrate buffer, pH 6. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide and non-specific binding was performed after incubation with horse serum, biotin, avidin and mouse IgG using protein block kit (Vector). Anti-α-SMA primary antibody (Sigma, A-2547, clone 1A4) was incubated for 30 min at 1/3000. Biotinylated secondary goat anti-mouse antibody was used at 1/300 dilution and incubated 30 min at room temperature. Immunostaining was developped using 3,3′-diaminobenzidine (Dako), and slides were counterstained with hematoxylin eosin.

[0716] RNA extraction and Q-PCR: For each mouse, 2 samples from the median and left lobs were snap frozen in liquid nitrogen and stored at −80° C. until use. RNA was extracted using RNAsol (Qiagen) and RNAeasy mini columns (Qiagen) as previously described. Reverse transcription was performed on 2 gg RNA using High Capacity cDNA reverse transcriptase kit (Applied Biosystem) and Q-PCR was performed with ABsolute Blue QPCR SYBR Green Low ROX Mix (Thermo Scientific). Gene expression was calculated using AACT method relative to housekeeping gene 18S.

[0717] Isolation and culture of murine hepatic myofibroblasts: Murine hepatic myofibroblasts were isolated from C57BL/6J mice and human hepatic myofibroblasts were isolated from normal human liver, as previously described (Teixeira-Clerc, F., Julien, et al, Nat. Med. 12,671-676, 2006; Mallat et al, JCI, 1996). Human cells were cultured in DMEM medium containing 5% FBS and 5% normal human serum (NHS) and mouse cells in DMEM medium containing 10% FBS. Cells were used between the fourth and ninth passage. DNA synthesis was measured using a Brdu proliferation assay kit (Roche) and 3-6 replicates per point, according to the manufacturer's instructions. Confluent mouse myofibroblasts were serum-starved for 24 h in a 0.02% BSA-containing medium, and human myofibroblasts for 3 days in serum free media. Cells were further incubated with different concentrations of JZL 184 or MJN 110 in the presence of 5% NHS (human) or FBS (mouse) for 24 hours. Brdu was added for the last 18 hours of incubation.

[0718] Statistical analysis: All data are expressed as mean±SEM. Statistical analysis were performed using unpaired t-test followed by Mann-Whitney GraphPad Prism version 5.00 Mac (GraphPad Software, San Diego). A p<0.05 was considered statistically significant.

[0719] Results

[0720] Results are depicted in FIGS. 1-8. The inventors show that MGL inhibitors accelerate liver fibrosis regression (FIG. 2). Moreover, the inventors show that MGL inhibitors reduce proinflammatory cytokines during fibrosis regression (FIG. 4) and inhibit DNA synthesis of human and murine hepatic myofibroblasts (FIG. 5).

[0721] The inventors used peripheral blood mononuclear cells (PBMC) from healthy controls and cirrhotic patients and demonstrated that MGL expression is induced in PBMC from patients with alcoholic cirrhosis (FIG. 6).

[0722] The inventors also demonstrated that mice bearing a global invalidation for MGL show decreased fibrosis (FIG. 7A and B) and the Knock-Down of MGL in myeloid cells decreased liver fibrosis (FIG. 8A and B).

REFERENCES

[0723] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.