EA044618B1 - COMBINED PRODUCT FOR THE TREATMENT OF FIBROUS OR CHOLESTATIC DISEASE - Google Patents

Description

BACKGROUND OF THE INVENTION

According to the Washington Manual of Medical Therapeutics (31st ed., 2004, Lippincott Williams & Wilkins), liver disorders can be divided into various groups of diseases, in particular viral diseases, drug- or alcohol-related liver diseases, immune-mediated liver diseases, metabolic liver diseases, mixed diseases such as non-alcoholic fatty liver and complications of liver failure (such as fulminant liver failure or hepatocellular carcinoma).

In particular, non-alcoholic fatty liver disease (NAFLD) is a common liver disorder with histological evidence of alcohol-induced fatty liver disease in individuals who consume little or no alcohol (Yeh M. et al., 2007; Marchesini et al., 2003 ). NAFLD is caused by abnormal cellular lipid retention (usually defined as steatosis), an event more common in the liver since this organ is primarily responsible for lipid metabolism. NAFLD has a spectrum of histologic forms, including hepatic steatosis and nonalcoholic steatohepatitis (NASH), which is characterized by liver inflammation, steatosis, necrosis, and fibrosis resulting from liver cell destruction. Conditions associated with NAFLD are variable and include type 2 diabetes, obesity, dyslipidemia, metabolic syndrome, treatment with hepatotoxic drugs, exposure to toxins, infectious agents, or other exogenous causes.

Although NAFLD typically follows a benign, nonprogressive clinical course, NASH is a potentially serious disease; in as many as 25% of patients, it may progress to advanced fibrosis, cirrhosis, and they may experience complications of portal hypertension, liver failure, and hepatocellular carcinoma, making early and correct evaluation imperative (Yeh M. et al., 2007).

Liver imaging systems can also be used to evaluate liver structure and the presence of steatosis. However, liver biopsy remains the gold standard for assessing liver fibrosis, but this method of analysis cannot be performed on a single study basis due to its invasiveness. Non-invasive assessment of liver biochemistry and metabolism is often used to identify liver diseases such as NAFLD and NASH (Gressner A. et al., 2009; Vuppalanchi R., Chalasani N., 2009). When plasma is used, high levels of enzymes such as alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (AP) and/or gamma-glutamyl transpeptidase (GGT), as well as the presence of other liver-derived proteins (including haptoglobin, total bilirubin, alpha -2-microglobulin, resistin, cleaved or intact cytokeratin-18), are usually measured in addition to serum glucose and insulin resistance parameters. Since ALAT activity levels are often increased in patients with NASH (Angulo P. et al., 2002), this criterion is considered a surrogate marker for assessing liver damage. In fact, reliable non-invasive methods are not available to correctly diagnose NAFLD or NASH, and even histological features are not always sufficient to correctly distinguish NAFLD or NASH from other conditions such as alcoholic liver disease (Yen M. et al., 2007; Vuppalanchi R. , Chalasani N., 2009).

Methods for effectively treating fibrotic liver diseases and, in particular, NAFLD and NASH are still insufficient. No treatment has been developed for patients with NASH, and several possible treatments are being tested in clinical trials (Vuppalanchi R., Chalasani N., 2009; Dowman J.K. et al., 2009). These studies include the use of many different families of chemical compounds (fibrates, thiazolidinediones, biguanides, statins, cannabinoids) and therapeutic targets (nuclear receptors, angiotensin receptors, cannabinoid receptors, HMG-CoA reductase). Recently, studies using thiazolidinediones (rosiglitazone and pioglitazone) have shown that these drugs can improve liver health, but treatment with these drugs is not without side effects, such as a high risk of congestive heart failure and osteoporosis, as well as weight gain with physiological effects per patient (Dowman J.K. et al., 2009; Shiri-Sverdlov R. et al., 2006; Neuschwander-Tetri et al., 2003). Clinical studies involving the administration of cannabinoids have identified the problem of neuropsychiatric impairment (Vuppanchi R., Chalasani N., 2009). Other therapies currently in development are seeking to evaluate anti-NASH drugs as antioxidants, but none of these therapies have yet produced conclusive results (Nelson A. et al., 2009).

There are currently no approved treatments for NASH, but two compounds with different mechanisms of action are currently in phase 3 clinical trials: obeticholic acid (OCA, an FXR agonist) and elafibranor.

1-[4-Methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one (elafibranor or ELA, formerly called GFT505), a dual PPAR alpha/delta agonist described in WO 2004005233, has properties that may provide benefits for the treatment of a number of gastroenterological diseases and liver diseases, in particular cholestatic diseases such as PBC (primary biliary cholangitis) and PSC (primary sclerosing cholangitis), or liver diseases, in particular types of non-alcoholic fatty infiltration liver (NAFLD), such as non-alcohol

- 1 044618 naked steatohepatitis (NASH).

Elafibranor was tested for clinical efficacy in NASH in a 1-year liver biopsy-based phase 2b study (GFT505-2127), one of the largest intervention studies ever conducted for NASH. Administered to over 800 patients and healthy volunteers to date, elafibranor has NASH-promoting properties including, but not limited to, improvement in markers of liver dysfunction including ALAT, ASAT, yGT, ALP; improving insulin sensitivity and glucose homeostasis; beneficial effects on plasma lipids, including reductions in plasma levels of triglycerides and LDL-C, and increases in HDL-C levels; anti-inflammatory properties; effectiveness in relation to NASH histological parameters (steatosis, inflammation, fibrosis) in animal models of the disease - types of antifibrotic activity; and the absence of safety concerns was confirmed in a full battery of toxicology tests up to and including 2-year carcinogenicity studies. Elafibranor is currently being evaluated in a phase 3 clinical trial for the treatment of NASH.

Evaluation of this molecule for the treatment of RVS in a phase 2 clinical trial has also begun.

The essence of the invention

The present invention relates to a combination product for the treatment of fibrotic or cholestatic disease containing (i) elafibranor (ELA) or a pharmaceutically acceptable salt thereof; and (ii) an FXR agonist selected from obeticholic acid (OCA) or a pharmaceutically acceptable salt thereof and a compound of the structural formula

(INT-767) or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the present invention, the combination product is a composition of ELA or a pharmaceutically acceptable salt thereof, an FXR agonist selected from OCA or a pharmaceutically acceptable salt thereof and compound INT-767 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In another preferred embodiment of the present invention, the combination product is a kit of parts containing ELA or a pharmaceutically acceptable salt thereof and an FXR agonist selected from OCA or a pharmaceutically acceptable salt thereof and the compound INT-767 or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the present invention, ELA or a pharmaceutically acceptable salt thereof and an FXR agonist selected from OCA or a pharmaceutically acceptable salt thereof and compound INT-767 or a pharmaceutically acceptable salt thereof are present in a synergistically effective amount for treating the disease.

The present invention also relates to a method of treating a fibrotic or cholestatic disease, comprising administering the combination product as provided above to a subject having a fibrotic or cholestatic disease.

The disease is non-alcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC), preferably PBC.

In another embodiment of the present invention, the disease is non-alcoholic fatty liver disease (NAFLD)-induced liver fibrosis or cirrhosis or non-alcoholic steatohepatitis (NASH)-induced fibrosis or cirrhosis.

In another preferred embodiment of the present invention

ELA is administered at a dose ranging from 5 to 80 mg/day, and OCA is administered at a dose ranging from 7 to 17 mg/day;

ELA is administered at a dose ranging from 40 to 60 mg/day, and OCA is administered at a dose ranging from 5 to 12.5 mg/day;

ELA is administered at a dose ranging from 32 to 48 mg/day, and OCA is administered at a dose ranging from 4 to 10 mg/day;

ELA is administered at a dose ranging from 27 to 40 mg/day, and OCA is administered at a dose ranging from 3 to 8 mg/day;

ELA is administered at a dose ranging from 16 to 24 mg/day, and OCA is administered at a dose ranging from 2 to 5 mg/day; or

ELA is administered at a dose ranging from 8 to 12 mg/day, and OCA is administered at a dose ranging from 1 to 2.5 mg/day.

- 2 044618

The present invention also relates to the use of the above combination product for the treatment of fibrotic or cholestatic disease. The disease is liver fibrosis, liver cirrhosis, primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC), preferably PBC.

In yet another preferred embodiment of the present invention, the disease is fibrosis or cirrhosis of the liver induced by non-alcoholic fatty liver disease, or fibrosis or cirrhosis induced by non-alcoholic steatohepatitis.

In another preferred embodiment of the present invention, the above combination product is used in the following dosage:

ELA is administered at a dose ranging from 5 to 80 mg/day, and OCA is administered at a dose ranging from 7 to 17 mg/day;

ELA is administered at a dose ranging from 40 to 60 mg/day, and OCA is administered at a dose ranging from 5 to 12.5 mg/day;

ELA is administered at a dose ranging from 32 to 48 mg/day, and OCA is administered at a dose ranging from 4 to 10 mg/day;

ELA is administered at a dose ranging from 27 to 40 mg/day, and OCA is administered at a dose ranging from 3 to 8 mg/day;

ELA is administered at a dose ranging from 16 to 24 mg/day, and OCA is administered at a dose ranging from 2 to 5 mg/day; or

ELA is administered at a dose ranging from 8 to 12 mg/day, and OCA is administered at a dose ranging from 1 to 2.5 mg/day.

Description of the figures

In fig. Figure 1 shows the effect of elafibranor (GFT505), OCA and their combination on liver fibrosis in rats fed CDAA/chol. (n=10/group).

The percentage of surface area affected by fibrosis was assessed by morphometric quantification of the picrosirius positive area relative to the area of the liver section. Data are expressed as mean±SD. # p < 0.05, ## p < 0.01, ### p < 0.001 using Student's t test. § p < 0.05, §§ p < 0.01, §§§ p < 0.001, §§§ p < 0.0001 using the Kruskal-Wallis test and Dunn's post hoc test without correction. HSA, best single means model.

In fig. Figure 2 shows the effect of elafibranor (GFT505), OCA and their combination on liver collagen content in rats fed CDAA/chol. (n=10/group).

Data are expressed as mean±SD. # p < 0.05, ## p < 0.01, ### p < 0.001 using Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001 using one-way ANOVA and Fisher's post hoc test without correction. § p < 0.05, §§ p < 0.01, §§§ p < 0.001, §§§ p < 0.0001 using the Kruskal-Wallis test and Dunn's post hoc test without correction. HSA, best single means model.

In fig. Figure 3 shows the effect of elafibranor (GFT505), OCA, and their combination on the expression of genes involved in tissue remodeling and inflammation in the liver in rats fed CDAA/chol. (n=10/group).

The expression of aSMA (ACTA2), TIMP1 and TGFe was assessed by semi-quantitative real-time PCR. Data are expressed as mean±SD. # p < 0.05, ## p < 0.01, ### p < 0.001 using Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001 using one-way ANOVA and Fisher's post hoc test without correction. § p < 0.05, §§ p < 0.01, §§§ p < 0.001, §§§ p < 0.0001 using the Kruskal-Wallis test and Dunn's post hoc test without correction. HSA, best single means model.

In fig. Figure 4 shows the differential antifibrotic effect of elafibranor and INT-767 on TGFe-induced hHSCs.

Serum-free HSCs were preincubated for 1 h with elafibranor (A) and INT-767 (B) before activation with the profibrogenic cytokine TGFe1 (1 ng/ml). After 48 h of incubation, α-SMA expression was measured by ELISA. The obtained values were transformed into the percentage of inhibition compared to the control with TGFe1. Data are presented as mean (of four replicates)±standard deviation (SD).

In fig. Figure 5 shows the combination of elafibranor with INT-767 synergistically inhibits α-SMA production in TGFe1-induced hHSCs

Combinations were tested in dose response matrix format and analyzed according to the incremental Bliss additivity (IOB) model. Dilution series of elafibranor (row) and INT-767 (column) were prepared, including their corresponding DMSO control. The resulting mixtures were added to serum-free HSCs 1 h before activation using the profibrogenic cytokine TGFe1 (1 ng/ml). (A) Percentage of α-SMA inhibition compared to TGFe1 control for all pair combinations. Data are presented as the average of four replicates. (B) EOB scores were calculated as described in Materials and Methods. Any pair of connections with

- 3044618 EOB values>10 were considered synergistic (colored from light gray to black). The total EOB indicator, including all combinations, was also calculated. (C) Data values derived for synergistic pair combinations were plotted in bar graph representation. Data are presented as mean (of four replicates)±standard deviation (SD). * p<0.05; ** p<0.01; *** p<0.001; **** p < 0.0001 using Student's t test to compare the combination group with the best single agent.

Detailed Description of the Invention

As used above, the term administration includes any route of administration such as oral, subcutaneous, sublingual, transmucosal, parenteral, intravenous, intra-arterial, buccal, sublingual, topical, vaginal, rectal, ocular, auricular, nasal, inhalation, intramuscular , intraosseous, intrathecal and percutaneous administration or a combination thereof. In preferred embodiments, the compounds are administered orally, in particular in the form of one or more tablets.

Administration may also include issuing a prescription or dispensing a dosage form containing a particular compound. The introduction may also include providing instructions for carrying out a method involving a particular compound or dosage form containing the compound.

As used herein, the term disease refers to a disease, disorder, condition, symptom or indication. The term is used interchangeably with the phrase disease or disorder.

As used above and throughout this specification, the term therapeutically effective amount refers to an amount effective, at the required dosages and for the required periods of time, to achieve the desired result in relation to the treatment of the corresponding disorder, condition or side effect. It is understood that the effective amount of the components of the present invention may vary from patient to patient not only due to the specific compound, component or composition selected, the route of administration and the ability of the components to produce the desired response in the individual, but also due to factors such as disease state or the severity of the condition being treated, hormone levels, age, sex, weight of the individual, the general health of the patient and the severity of the condition being treated, concomitant medications or special diets followed by a particular patient, and other factors known to one of ordinary skill in the art. , with the decision on the appropriate dose ultimately made by the attending physician. Dosage regimens may be adjusted to provide improved therapeutic response. An effective amount is also an amount at which the therapeutically beneficial effects of the components exceed any toxic or adverse effects.

The term synergistic within the meaning of the invention means that the effect achieved using the combination product and methods of this invention exceeds the sum of the effects resulting from the effects of the components of the combination or methods including one of the components, individually and in the quantities used in these methods and their compositions . Such synergy can be determined in accordance with methods well known in the art, for example, using the incremental Bliss (EB) method.

The combination product of the invention may provide better effects than those achieved by each of its components used separately, particularly when used in a synergistically effective amount. A synergistically effective amount is an amount of each component of the combination product that produces a synergistic effect when administered to a subject in need thereof. In other words, when components (i) and (ii) are used in a synergistically effective amount, the combined effect of amounts (i) and (ii) exceeds the sum of the therapeutic effects of amounts (i) and (ii) administered individually. As mentioned above, the amount of each of compounds (i) and (ii) administered to the patient can be reduced compared to the amount of said compounds (i) and (ii) administered individually. For example, the amount of each component can be reduced by at least 1.5 times, at least 2 times, or even at least 3 times or more, such as at least 4, 5, 6, 7, 8, 9 or 10 times. For example, the amount of ELA administered to patients in clinical studies is 80 or 120 mg/day, and the amount of OCA administered to patients in clinical studies is 10 or 25 mg/day, and the synergistic amount of the invention with a 3-fold reduction may be 27 or 40 mg/day ELA and 3 and 8 mg/day OCA.

In accordance with the present invention, the term PPAR agonists (multiple PPARs) refers to peroxisome proliferator-activated receptor agonists, which are a class of drugs that play a central role in lipid and glucose homeostasis. PPARa mainly affects fatty acid metabolism and its activation reduces lipid levels, while PPARy is mainly involved in the regulation of adipogenesis, energy balance and lipid biosynthesis. PPARδ is involved in the oxidation of fatty acids mainly in skeletal and cardiac muscles, but it also regulates blood glucose and cholesterol levels.

As used herein, the term PPAR-alpha agonist as used herein includes, but is not limited to, fenofibrate, ciprofibrate, pemafibrate, gemfibrozil, clofibrate, binifibrate, clinofibrate, clofibric acid, nicofibrate, pyrifibrate, plafibride, ronifibrate, theofibrate, tocofibrate, and SR10171.

As used herein, the term PPAR-gamma agonist as used herein includes, but is not limited to, rosiglitazone, pioglitazone, deuterated pioglitazone, efatutazone, ATx08-001, OMS-405, CHS-131, THR-0921, SER-150-DN, KDT-501 , GED-0507-34-Levo, CLC-3001 and ALL-4.

As used herein, the term PPAR-delta agonist as used herein includes, but is not limited to, GW501516 (endurabol or ({4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5- yl}methyl)sulfanyl]2-methylphenoxy}acetic acid)) or MBX8025 (seladelpar or {2-methyl-4-[5-methyl-2-(4trifluoromethylphenyl)-2H-[1,2,3]triazol-4- ylmethylsulfanyl]phenoxy}acetic acid), or GW0742 ([4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]acetic acid), or L165041, or HPP-593, or NCP-1046.

As used herein, the term PPAR-alpha/gamma agonist (also referred to as glitasars) as used herein includes, but is not limited to, saroglitazar, aleglitazar, muraglitazar, tezaglitazar, DSP-8658.

As used herein, the term PPAR-alpha/delta agonist as used herein includes, but is not limited to, ELA or T913659.

As used herein, the term PPAR gamma/delta agonist as used herein includes, but is not limited to, conjugated linoleic acid (CLA), T3D-959.

As used herein, the term PPAR-alpha/gamma/delta agonist as used herein includes, but is not limited to, IVA337 (lanifibranor) or TTA (tetradecylthioacetic acid) or bavacinin or GW4148 or GW9135 or bezafibrate or lobeglitazone or CS038. In a further embodiment, the PPAR-alpha/gamma/delta agonist is 2-(4-(5,6-methylenedioxybenzo[d]thiazol-2-yl)-2methylphenoxy)-2-methylpropionic acid (MHY2 013).

The PPAR agonist may be in the form of a salt, hydrate, solvate, polymorph, or cocrystal. The PPAR agonist may also be in the form of a hydrate, solvate, polymorph, or salt cocrystal.

The combination product of the invention can be used to treat fibrotic or cholestatic disease.

The terms fibrosis, fibrotic disease, fibrotic disorder and their declinations denote a pathological condition of excessive accumulation of fibrous connective tissue in an organ or tissue. More specifically, fibrosis is a pathological process involving the persistent formation of a fibrotic scar and excess production of extracellular matrix by connective tissue in response to tissue damage. Physiologically, the accumulation of connective tissue can blunt the architecture and function of the underlying organ or tissue.

In accordance with the present invention, fibrosis can be fibrosis of any organ or tissue. Illustrative, non-limiting examples of specific organ fibrosis include fibrosis of the liver, kidney, skin, epidermis, endoderm, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, ovary, adrenal gland, artery, vein, colon, intestine (eg, small intestine), bile ducts, soft tissue (eg, mediastinum or retroperitoneum), bone marrow, joint, or stomach.

In a preferred embodiment, the fibrotic disorder is selected from the group consisting of fibrosis of the liver, intestine, lung, heart, kidney, muscle, skin, soft tissue (eg, mediastinum or retroperitoneum), bone marrow, intestine, and joint (eg, knee, shoulder). or other joints).

More preferably, the fibrotic disorder is selected from the group consisting of liver, lung, skin, kidney and intestinal fibrosis.

More preferably, the fibrotic disorder being treated is selected from the group consisting of the following non-exhaustive list of fibrotic disorders: non-alcoholic steatohepatitis (NASH), pulmonary fibrosis, idiopathic pulmonary fibrosis, cutaneous fibrosis, ocular fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis , massive progressive fibrosis (complication of pneumoconiosis of coal industry workers), proliferative fibrosis, neoplastic fibrosis, pulmonary fibrosis following chronic inflammatory airway disease (COPD, asthma, emphysema, smoker's lung, tuberculosis, IPF), alcohol- or drug-induced liver fibrosis , liver cirrhosis, infection-induced liver fibrosis, radiation- or chemotherapy-induced fibrosis, nephrogenic systemic fibrosis, Crohn's disease, ulcerative colitis, keloid, previous myocardial infarction, scleroderma/systemic scleroderma, arthrofibrosis, some forms of adhesive capsulitis, chronic fibrosing cholangiopathies, such such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), biliary atresia, familial intrahepatic cholestasis type 3 (PFIC3), peri-implant fibrosis and asbestosis.

Cholestasis is defined as a decrease in bile secretion due to impaired secretion by hepatocytes (hepatic cholestasis) or obstruction of bile secretion through intra- or extrahepatic

- 5 044618 bile ducts (obstructive cholestasis). In clinical practice, cholestasis is any condition in which the release of bile from the liver is slowed or blocked.

Examples of inflammatory diseases, fibrotic diseases, metabolic diseases and cholestatic diseases include metabolic liver diseases, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-induced liver disease, alcohol-induced liver disease, infectious agent-induced liver disease, inflammatory diseases liver, immune dysfunction-mediated liver diseases, dyslipidemia, cardiovascular diseases, restenosis, syndrome X, metabolic syndrome, diabetes, obesity, hypertension, chronic cholangiopathies such as primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), biliary atresia, familial intrahepatic cholestasis type 3 (PFIC3), inflammatory bowel disease, Crohn's disease, ulcerative colitis, keloid, previous myocardial infarction, scleroderma/systemic scleroderma, inflammatory diseases, neurodegenerative diseases, malignant tumors, liver cancer, hepatocellular carcinoma, gastrointestinal malignancy -intestinal tract, gastric cancer, neurofibromatosis-associated meningioma, pancreatic neuroendocrine tumors, pancreatic exocrine tumors, leukemia, myeloproliferative/myelodysplastic diseases, mastocytosis, dermatofibrosarcoma, solid tumors including malignancies of the breast, lung, thyroid or colorectal cancer , prostate cancer, liver fibrosis or cirrhosis of any origin, metabolic disease-induced liver fibrosis or cirrhosis, NAFLD-induced fibrosis or cirrhosis, NASH-induced fibrosis or cirrhosis, alcohol-induced liver fibrosis or cirrhosis, drug-induced liver fibrosis or cirrhosis, infectious agents-induced liver fibrosis or cirrhosis liver fibrosis or cirrhosis, parasitic infection-induced liver fibrosis or cirrhosis, bacterial infection-induced liver fibrosis or cirrhosis, viral infection-induced fibrosis or cirrhosis, HBV infection-induced liver fibrosis or cirrhosis, HCV infection-induced liver fibrosis or cirrhosis, HIV infection-induced fibrosis or cirrhosis liver, HCV and HIV dual infection-induced liver fibrosis or cirrhosis, radiation- or chemotherapy-induced fibrosis or cirrhosis, bile duct fibrosis, liver fibrosis or cirrhosis caused by any chronic cholestatic disease, intestinal fibrosis of any etiology, Crohn's disease-induced fibrosis, ulcerative colitis-induced fibrosis, intestinal fibrosis (eg small intestine), colonic fibrosis, gastric fibrosis, skin fibrosis, epidermal fibrosis, endodermal fibrosis, skin fibrosis due to scleroderma/systemic scleroderma, pulmonary fibrosis, pulmonary fibrosis following chronic inflammatory diseases of the airways, such as COPD, asthma, emphysema, smoker's lung, tuberculosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cardiac fibrosis, renal fibrosis, nephrogenic systemic fibrosis, muscle fibrosis, soft tissue fibrosis (eg, mediastinum or retroperitoneum), bone fibrosis brain, joint fibrosis, tendon fibrosis, cartilage fibrosis, pancreatic fibrosis, uterine fibrosis, nervous system fibrosis, testicular fibrosis, ovarian fibrosis, adrenal fibrosis, arterial fibrosis, venous fibrosis, ocular fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis , massive progressive fibrosis (a complication of pneumoconiosis of coal industry workers), proliferative fibrosis, neoplastic fibrosis, peri-implant fibrosis and asbestosis, arthrofibrosis, adhesive capsulitis.

The term treat or treat refers to the treatment, prevention or prophylaxis of a disorder in a subject in need thereof. Treatment involves administering a combination product of the invention to subjects (eg, patients) having a specified disorder to prevent, cure, delay, reverse, or slow the progression of the disorder, thereby improving the condition of the patients. Treatment may also be administered to subjects who are either healthy or at risk of developing a disorder, such as an immune (eg, autoimmune), inflammatory, fibrotic, or cholestatic disorder.

The term subject refers to a mammal and, more specifically, a human. Subjects to be treated according to the invention can be suitably selected based on several criteria associated with immune (eg, autoimmune), inflammatory, fibrotic and cholestatic pathological processes, such as previous and/or current drug treatment, associated pathologies, genotype, exposure factors risk, as well as any other relevant biomarker, which can be assessed by any suitable immunological, biochemical or enzymatic method.

The inventors herein demonstrate that the combination of a PPAR agonist and an FXR agonist, particularly the combination of ELA and OCA, or INT-767, particularly the combination of ELA and OCA, may provide benefit to a broader patient population and may have a synergistic effect, thus allowing a reduction in the therapeutic dose. An associated reduction in therapeutic dose may reduce the incidence of adverse drug effects.

- 6 044618 funds. Accordingly, the invention also provides a method of treating a disease as defined above with a reduced incidence of adverse drug effects.

The frequency and/or amount of administration may be adjusted by one skilled in the art depending on the patient, pathology, form of administration, etc. In general, the combination product of the present invention can be administered for the treatment of a disease in the following dosages:

ELA is administered at a dose ranging from 5 to 80 mg/day, and OCA is administered at a dose ranging from 7 to 17 mg/day;

ELA is administered at a dose ranging from 40 to 60 mg/day, and OCA is administered at a dose ranging from 5 to 12.5 mg/day;

ELA is administered at a dose ranging from 32 to 48 mg/day, and OCA is administered at a dose ranging from 4 to 10 mg/day;

ELA is administered at a dose ranging from 27 to 40 mg/day, and OCA is administered at a dose ranging from 3 to 8 mg/day;

ELA is administered at a dose ranging from 16 to 24 mg/day, and OCA is administered at a dose ranging from 2 to 5 mg/day; or

ELA is administered at a dose ranging from 8 to 12 mg/day, and OCA is administered at a dose ranging from 1 to 2.5 mg/day.

The treatment time can vary greatly depending on the condition being treated and the stage of said condition. For example, the combination product can be administered over at least two or more consecutive days, such as at least 7, 8, 9 or 10 days or more; at least one week or more, such as at least 1, 2, 3, 4, 5, 10, 20, 50, 60, 70 or 72 weeks or more; at least one month or more, such as at least 1, 2, 3, 4, 5, 10, 15, 20 or 24 months or more; or at least one year or more, such as at least 1, 2, 3, 4 or 5 years or more.

The invention is further described with reference to the following non-limiting examples.

Examples

Materials and methods.

Evaluation of elafibranor, OCA and the combination of elafibranor+OSA in a chronic CDAA+1% cholesterol model (12 weeks).

The preventive effects of elafibranor (GFT505) alone, OCA alone, and a combination of both were evaluated in a rat model of fibrosing NASH fed a CDAA+1% cholesterol diet. 150-175 g male Wistar rats were fed a control (CSAA) diet, either a CDAA+1% cholesterol diet or a CDAA+1% cholesterol diet supplemented with 1, 3 and 10 mg/kg/day elafibranor, OCA 10 and 30 mg/kg/day or combination drugs (elafibranor 1, 3 and 10 mg/kg/day in combination with OSA 10 mg/kg/day) for 12 weeks.

Body weight and feed intake were monitored twice weekly. On the last day of treatment, rats were sacrificed after a 6-hour fasting period. The liver was quickly excised for biochemical and histological studies.

All animal procedures were performed in accordance with standard protocols and in accordance with standard recommendations for the proper care and use of laboratory animals.

Histology.

Immersion of tissue and obtaining sections.

Liver fragments were first fixed for 12 hours in a solution of 4% formalin. The liver fragments were then washed for 30 min in PBS and dehydrated in ethanol solutions (in sequential baths of 70, 80, 95 and 100% ethanol). Liver fragments were incubated in three different xylene baths (SigmaAldrich, cat. #534056), then in two vaseline oil baths (56°C). The liver fragments were then placed in trays, which were carefully filled with Histowax® to completely cover the tissue.

Paraffin blocks containing tissue fragments were removed from the tray and stored at room temperature. Liver blocks were cut into 3-μm sections.

Hematoxylin/eosin staining.

Liver sections were deparaffinized, rehydrated, and incubated for 3 min in Mayer's hematoxylin (Microm, cat. #F/C0303). Liver sections were then rinsed with water and incubated for 1 min in eosin G (VWR, cat. #1.09844.1000). Sections were rinsed with water, then dehydrated and mounted using CV Mount medium (Leica, cat. #14046430011).

Picrosirius red staining.

Liver sections were deparaffinized, rehydrated, and incubated for 15 min in Fast Green FCF 0.1% (Sigma-Aldrich, cat. #F7258) before rinsing in a 0.5% acetic acid bath (Panreac, cat. #131008,1611 ). Then the liver sections were rinsed with water and incubated for 30 min in a solution of 0.1% Sirius red (Direct Red 80, Fluka, cat. #43665) in saturated aqueous picric acid (Sigma-Aldrich, cat. #P6744). The sections were then dehydrated and fixed using CV medium

Mount (Leica, cat. #14046430011).

Histological studies.

A technician blinded to the source of each liver sample performed histological examinations. Virtual sections were obtained using a Pannoramic 250 scanner from 3D Histech. Each animal was assigned a score summarizing the major NASH histological lesions according to the NASH Clinical Research Network (Kleiner 2005, Brunt 1999). Steatosis, lobular inflammation and hepatocyte ballooning were briefly scored. The NAFLD activity score (NAS score) was determined for each individual as the unweighted sum of steatosis, lobular inflammation, and ballooning lesion scores.

Using Quant Center software (3D Histech, including Pattern Quant and Histo Quant modules), areas of stained collagen were quantified. Briefly, Pattern Quant was used to detect tissue and measure its surface. Histo Quant was then used to detect the dyed collagen content and measure its surface area based on the color threshold method. The area of fibrosis was then expressed as the percentage of collagen surface area of total tissue for the animal.

Measurement of hepatic collagen content.

Hepatic collagen content was determined using the appropriate QuickZyme kit (total collagen assay, cat. #QZB-totcol2). The assay is based on the detection of hydroxyproline, which is a non-proteinogenic amino acid primarily found in the triple helix of collagen. Thus, hydroxyproline in tissue hydrolysates can be used as a direct indicator of the amount of collagen present in the tissue (without distinguishing between procollagen, mature collagen, and collagen degradation products).

Complete hydrolysis of tissue samples in 6 M HCl at 95 °C is necessary before dosing hydroxyproline. The analysis results in the formation of a chromogen with a maximum optical density at 570 nm. Results are expressed as mg collagen/g liver.

Analysis of liver gene expression.

Total RNA was isolated from rat liver using the RNeasy Mini kit (Qiagen) following the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using M-MLV RT (Moloney murine leukemia virus reverse transcriptase) (Invitrogen, cat. #28025) in 1x RT buffer (Invitrogen), 0.5 mM DTT (Invitrogen), 0.18 mM dNTP (Promega), 200 ng pdN6 (Amersham) and 30 units. RNase inhibitor (Promega).

Quantitative PCR was then performed using the CFX96 Touch™ Real-Time PCR System (Biorad). Briefly, PCR reactions were performed in 96-WP format in a total volume of 25 μl containing 1 μl of the reverse transcription reaction product, 0.5 μl of reverse and forward primers (10 pmol each), and 12.5 μl of 2X iQ SYBR Green Supermix (BioRad ), using the following primer sequences.

Gene Straight Back RPLP0 CATGCTCAACATCTCCCCCTTCT CC (SEQ ID NO:1) GGGAAGGTGTAATCCGTCTCCAC AG (SEQ ID NO:2) aSMA (ASTA2) AC T GGGACGACAT GGAAAAG (SEQ ID NO:3) CAT C T C CAGAG T C GAG GAGA (SEQ ID NO:4) TIMP1 T C C C C AGAAAT CATC GAGAC (SEQ ID NO:5) TCAGATTATGCCAGGGAACC (SEQ ID NO:6) TGFB1 TGAGTGGCTGTCTTTTGACG (SEQ ID NO:7) TGGGACTGATCCCATTGATT (SEQ ID NO:8) CCR5 CAGAACAGTCAACTTTGGGG (SEQ ID NO:9) ACGTGGAAAATGAGGACTGC (SEQ ID NO:10)

Expression levels were normalized using Rplp0 gene expression as a reference across samples.

For each gene, standard curves were generated by selecting the best points (at least three points) to obtain a PCR reaction efficiency close to 100% and a correlation coefficient close to 1. Expression levels were determined using the standard curve equation as for the housekeeping gene , and for the target gene (taking into account the specific PCR efficiency for each target gene).

Evaluation of the combination of elafibranor and another FXR agonist in an in vitro model of hepatic fibrogenesis.

Cultivation of hHSCs.

Primary human hepatic stellate cells (hHSC) (Innoprot) were cultured in STeCM medium (ScienCell, cat. #5301) supplemented with 2% fetal bovine serum (FBS, ScienCell, cat. #0010),

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1% penicillin/streptomycin (ScienCell, cat. #0503) and stellate cell growth supplement (SteCGS; ScienCell, cat. #5352). Cell culture flasks were coated with poly-L-lysine (Sigma, cat. # P4707) for better adhesion.

Preparation of compositions: matrix of combinations of 2 components (FXR agonist/elafibranor).

The FXR agonist INT-767 (CAS#1000403-03-1, Cat #HY-12434, Lot #19249) was commercially obtained from Haoyuan Chemexpress. For these experiments, a matrix was prepared in a checkerboard pattern. INT-767 and elafibranor were dissolved in dimethyl sulfoxide (DMSO, Fluka, cat. #41640) and serial dilutions were made in a series of 5 points in a row (elafibranor) and in a series of 11 points in a column (INT-767) of a 384-well plate. A 5X11 combination matrix was then prepared by mixing 1:1 of all concentrations of the individual agents. Test concentrations for each compound were selected based on the literature (Rizzo et al.; McMahan et al.).

hHSC activation using TGF-e1 and compound treatment.

hHSCs were seeded at a density of 6.5 x 103 cells/well in 384-well plates. The next day, the culture medium was removed and the cells were washed using PBS (Invitrogen, cat. #14190). hHSCs were starved for 24 h in serum-free and SteCGS-free medium. For treatments using INT-767 and elafibranor, as well as their pairwise combinations, serum-free hHSCs were preincubated for 1 h with compounds followed by the addition of the profibrogenic stimulator TGF-e1 (PeproTech, cat. #100-21, 1 ng/ml ) in serum-free and SteCGS-free medium for an additional 48-hour period.

ELISA a-SMA.

α-SMA levels were measured using sandwich ELISA. Briefly, ELISA plate wells were first coated with binding antibody (mouse monoclonal anti-ACTA2 antibody, Abnova) at 4°C overnight. After 3 washes in PBS+0.2% Tween 20, a blocking solution consisting of PBS+0.2% BSA was added for 1 hour followed by another wash cycle. Cell lysates were transferred to binding wells with binding antibody for a period of 2 h at room temperature. After the washing procedure, detection antibody (biotinylated mouse monoclonal anti-ACTA2 antibody, Abnova) was added for 2 h at room temperature, followed by 3 washes. For detection, HRP-conjugated streptavidin (R&D Systems, cat. #DY998) was first added for 30 min at room temperature. After washing, HRP TMB substrate (BD#555214) was added and incubated for 7 min at room temperature in the dark. When oxidized, TMB produces a water-soluble blue reaction product that turns yellow when sulfuric acid (stop solution) is added, allowing accurate intensity measurement at 450 nm using a spectrophotometer. The color developed is directly proportional to the amount of α-SMA present in the lysate.

Determination of synergy using the Bliss incremental method (EB).

The values obtained from the aSMA ELISA assays were first transformed into percent inhibition compared to the TGF-e1 control. The Bliss increment was then determined using these inhibition percentages to determine the synergistic effects of the drug combinations. The expected Bliss additivity exponent (E) was first determined using Eq.

E=(A+B)-(AxB), where A and B represent the percentage inhibition for elafibranor (A) and INT-767 (B) at a given dose.

The difference between the Bliss expected and observed inhibition for INT-767/elafibranor combinations at the same dose is the Bliss increment score.

A Bliss increment score of 0 indicates that the combination treatment is additive (as expected for effects on independent pathways);

Bliss increment index>0 indicates activity exceeding additive (synergism);

a Bliss increment index <0 indicates that the combination is less active than the additive one (antagonism).

For INT-767/elafibranor combinations, an additional overall Bliss score was calculated by summing all EOBs.

To confirm synergy, the experimental values corresponding to the highest EOB for the FXR agonist/elafibranor combinations were plotted on a bar graph.

The significance of the observed differences between INT-767/elafibranor and the highest value for an individual agent was determined by Student's t test. * p<0.05; ** p<0.01; ***p<0.001.

Results and discussion.

Evaluation of elafibranor, OCA and the combination of elafibranor+OSA in a chronic model with CPAA+1% cholesterol (12 weeks).

The results are presented in the following table and Fig. 1-3.

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GFT505 WASP GFT505+OCA Zmg/kg/day 10 mg/kg/day Surface fibrosis 34%+17% *** 74%+45% 19%±4% # Content liver collagen 45%±12% *** 67%±23%** 34%±5% # a-SMA mRNA level 66%±27% 109%±68% 39%±18% # TIMP1 mRNA level 78%±23% 110%±43% 46%±13% ## TGFpl mRNA level 94%±20% 110%±23% 67%±16% ## CCR5 mRNA level 103%±51% 81%±28% 56%±17% #

Percentage relative to rats with CDAA+1% cholesterol without treatment.

** p<0.01, *** p<0.001 compared with the CDAA+1% cholesterol group (AN OV A+Bonferroni).

# p < 0.05, ## p < 0.01 compared with the best individual means (Student's t test).

(+ inflammation marker).

Western lifestyles are consistently associated with a high incidence of nonalcoholic steatohepatitis (NASH), a chronic liver disease that often progresses to liver fibrosis and cirrhosis and can ultimately lead to hepatocellular carcinoma. There is currently no approved therapy for NASH. Drug combinations that target multiple therapeutic targets simultaneously have the potential to greatly improve drug response and provide benefits to the broadest patient population. Drug combinations have previously been tested in other systemic diseases, such as hypertension, dyslipidemia, or type 2 diabetes, and have been shown to provide better control of underlying diseases and reduced morbidity and mortality. Recent phase 2B studies of both elafibranor (a PPARa/δ agonist) and OCA (an FXR agonist) have shown efficacy for the endpoints NASH and fibrosis. It was the intention of the present inventors to compare their effects on the outcomes of the respective NASH pathologies and to search for the therapeutic benefits of the combination.

To achieve this goal, fibrosing NASH was induced by feeding Wistar rats a choline-deficient diet with a specific L-amino acid composition supplemented with cholesterol (CDAA/cholesterol diet). Animals in the exposure groups were administered either elafibranor, OCA, or both compounds throughout the study period. The development of NASH and fibrosis was assessed by histology. Additional biochemical and molecular analyzes were also performed for various relevant biomarkers.

Wistar rats fed a CDAA/cold diet developed NASH-related histological features and fibrosis with high incidence of severe disease. Advanced steatosis, lobular inflammation and ballooning were present in all animals, and NAS [min. 0-max. 8] varied between 6 and 8. Histology (picrosirius positive area) and biochemistry (liver collagen concentration) of the liver showed an average fourfold increase in liver fibrosis, and the fibrosis score was either 3 or 4, for all animals on the CDAA/chol diet ., who did not receive drug treatment (Fig. 1, 2). Expression of genes associated with inflammation, tissue remodeling, and fibrosis was increased and consistent with gene profiles previously published for NASH patients with severe disease ( Fig. 3 ).

Administration of elafibranor alone attenuated the development of fibrosis, while administration of OCA alone did not significantly attenuate fibrosis (Figs. 1, 2). Combination treatments were more active in reducing fibrosis, allowing dose reductions, with similar antifibrosis efficacy achieved using GFT505 1 and 3 mg/kg in combination with OCA 10 mg/kg (Figs. 1, 2). Administration of either drug candidate alone only partially attenuated the increase in tissue remodeling, while the combination of both compounds was more effective than either drug alone (Figure 3).

In summary, the present report demonstrates that the synergistic effect of elafibranor and OCA on liver fibrosis in a diet-induced CDAA/chol model. NASH provided comparable therapeutic benefit at significantly lower doses of both drug candidates compared to either agent alone. From this study, it was realistic to expect that the doses of both drug candidates could be reduced by at least a factor of 1.5, 2, 2.5, or even at least 3 to obtain results similar to those for the initial dose of each compound , used individually. In addition, elafibranor showed a clear protective effect on liver damage. The effects of OCA on ballooning and lobular inflammation were quite modest in this model. From this study, it can be concluded that the elafibranor/OCA combination may provide benefits to a broader patient population, and the associated reduction in therapeutic dose may reduce the incidence of adverse drug effects.

Evaluation of the combination of elafibranor and another FXR agonist in an in vitro model of hepatic fibrogenesis.

The results are shown in Fig. 4, 5.

Abnormal persistence of differentiated myofibroblasts is a characteristic of many fibrotic diseases.

Following liver injury, quiescent HSCs undergo an activation process characterized by differentiation into (α-SMA) positive myofibroblasts.

The PPAR agonist elafibranor exhibits antifibrotic activity in hHSCs activated using the profibrogenic cytokine TGFe1. α-SMA marker levels were reduced by up to 68% using the highest dose of elafibranor tested (5 μM) (Fig. 4A). INT-767 alone only slightly inhibited α-SMA production by 13% at the highest dose tested (30 μM) (Fig. 4B). To evaluate whether the combination of elafibranor with INT-767 could reduce fibrosis in a synergistic manner, combination matrix experiments were performed in TGFe-induced HSCs. Dilutions of INT-767 and elafibranor solutions were briefly prepared in a checkerboard format, yielding a matrix of 55 combinations overlapping a large panel of INT-767/elafibranor ratios. Synergy was first determined by calculating Bliss increment scores. These experiments revealed that elafibranor could synergize with INT-767 to reduce α-SMA production in activated HSCs (Figure 5). One of the best examples of synergy is shown in FIG. 5C with 2.5 µM elafibranor and 1.9 µM INT-767. Although 1.9 µM INT-767 alone did not show any antifibrotic activity, when added to 2.5 µM elafibranor, it synergistically increased the activity of elafibranor and achieved up to 69% inhibition (compared to 26% for 2. 5 µM elafibranor alone).

In conclusion, the synergistic effects of elafibranor and different classes of FXR agonists on fibrogenesis demonstrate the potential benefit of such combinations in multiple types of fibrotic diseases.

Claims (14)

CLAIM 1. A combination product for the treatment of a fibrotic or cholestatic disease containing (i) elafibranor (ELA) or a pharmaceutically acceptable salt thereof; and (ii) an FXR agonist selected from obeticholic acid (OCA) or a pharmaceutically acceptable salt thereof and a compound of structural formula (INT-767) or a pharmaceutically acceptable salt thereof. 2. The combination product of claim 1, wherein the combination product is a composition of ELA or a pharmaceutically acceptable salt thereof, an FXR agonist selected from OCA or a pharmaceutically acceptable salt thereof and compound INT-767 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 3. The combination product of claim 1 or 2, wherein the combination product is a kit of parts comprising ELA or a pharmaceutically acceptable salt thereof and an FXR agonist selected from OCA or a pharmaceutically acceptable salt thereof and compound INT-767 or a pharmaceutically acceptable salt thereof. 4. The combination product according to any one of claims 1 to 3, wherein ELA or a pharmaceutically acceptable salt thereof and an FXR agonist selected from OCA or a pharmaceutically acceptable salt thereof and compound INT-767 or a pharmaceutically acceptable salt thereof are present in a synergistically effective amount for the treatment the specified disease. 5. A method of treating a fibrotic or cholestatic disease, comprising administering the combination product according to any one of claims 1 to 4 to a subject having a fibrotic or cholestatic disease. 6. The method of claim 5, wherein the disease is non-alcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC). 7. The method according to claim 6, where the disease is RVS. 8. The method of claim 6, wherein the disease is non-alcoholic fatty liver disease (NAFLD)-induced liver fibrosis or cirrhosis, or non-alcohol-induced fibrosis or cirrhosis. - 11 044618 naked steatohepatitis (NASH). 9. Method according to any one of claims 5-8, where ELA is administered at a dose ranging from 5 to 80 mg/day, and OCA is administered at a dose ranging from 7 to 17 mg/day; ELA is administered at a dose ranging from 40 to 60 mg/day, and OCA is administered at a dose ranging from 5 to 12.5 mg/day; ELA is administered at a dose ranging from 32 to 48 mg/day, and OCA is administered at a dose ranging from 4 to 10 mg/day; ELA is administered at a dose ranging from 27 to 40 mg/day, and OCA is administered at a dose ranging from 3 to 8 mg/day; ELA is administered at a dose ranging from 16 to 24 mg/day, and OCA is administered at a dose ranging from 2 to 5 mg/day; or ELA is administered at a dose ranging from 8 to 12 mg/day, and OCA is administered at a dose ranging from 1 to 2.5 mg/day. 10. Use of a combination product according to any one of claims 1 to 4 for the treatment of fibrotic or cholestatic disease. 11. Use according to claim 10, wherein the disease is liver fibrosis, liver cirrhosis, primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC). 12. Use according to claim 11, where the disease is RVS. 13. Use according to claim 10, wherein the disease is non-alcoholic fatty liver disease (NAFLD)-induced liver fibrosis or cirrhosis or non-alcoholic steatohepatitis (NASH)-induced liver fibrosis or cirrhosis. 14. Application according to any one of claims 10-13, where ELA is administered at a dose ranging from 5 to 80 mg/day, and OCA is administered at a dose ranging from 7 to 17 mg/day; ELA is administered at a dose ranging from 40 to 60 mg/day, and OCA is administered at a dose ranging from 5 to 12.5 mg/day; ELA is administered at a dose ranging from 32 to 48 mg/day, and OCA is administered at a dose ranging from 4 to 10 mg/day; ELA is administered at a dose ranging from 27 to 40 mg/day, and OCA is administered at a dose ranging from 3 to 8 mg/day; ELA is administered at a dose ranging from 16 to 24 mg/day, and OCA is administered at a dose ranging from 2 to 5 mg/day; or ELA is administered at a dose ranging from 8 to 12 mg/day, and OCA is administered at a dose ranging from 1 to 2.5 mg/day.