Combination of PPAR and FXR agonists

Abstract

The present invention relates to combinations of active ingredients for use in the treatment of disease.

Description

[Background technology]

[0001] According to the Washington Manual of Medical Therapeutics (31st ed., 2004, Lippincott Williams and Wilkins), liver disorders can be classified into various disease groups, in particular into various diseases such as viral diseases, drug- and alcohol-related liver diseases, immune-mediated liver diseases, metabolic liver diseases, non-alcoholic fatty liver disease, and complications of liver dysfunction (such as fulminant hepatic failure or hepatocellular carcinoma).

[0002] In particular, non-alcoholic fatty liver disease (NAFLD) is a common liver disorder with histological features of alcohol-induced fatty liver disease in individuals with little or no alcohol intake (Yeh M et al., 2007; Marchesini et al., 2003). NAFLD is caused by abnormal lipid retention (commonly defined as steatosis) in cells, an event that is more frequent in the liver since this organ is primarily responsible for lipid metabolism. NAFLD has a range of histological forms, including fatty liver and non-alcoholic steatohepatitis (NASH), characterized by inflammation, steatosis, necrosis and fibrosis of the liver due to destruction of hepatocytes. Conditions associated with NAFLD are diverse and include type 2 diabetes, obesity, dyslipidemia, metabolic syndrome, treatment with hepatotoxic drugs, toxins, infectious agents or other exogenous causes.

[0003] While NAFLD typically follows a benign, non-progressive clinical course, NASH is a potentially severe condition and as many as 25% of patients can progress to advanced fibrosis, cirrhosis, and experience complications of portal hypertension, liver failure, and hepatocellular carcinoma, making early and correct evaluation essential (Yeh M et al., 2007).

[0004] Liver imaging systems are also useful for evaluating liver structure and the presence of steatosis. However, liver biopsy remains the gold standard for evaluating liver fibrosis, but this analytical method cannot be performed in one-to-one studies due to its invasiveness. Non-invasive liver biochemistry and metabolic assessments are often used to define liver disease, such as in NAFLD and NASH (Gressner A et al., 2009; Vuppalanchi R and Chalasani N, 2009). By using plasma, elevated 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, truncated or intact cytokeratin-18) are commonly measured in addition to serum glucose and insulin resistance parameters. ALAT activity level is often increased in NASH patients (Angulo P et al., 2002), so this criterion is considered as a surrogate marker for measuring liver damage.In fact, there is no reliable non-invasive method to correctly diagnose NAFLD and NASH, and even histological features are not always sufficient to properly distinguish NAFLD or NASH from other conditions such as alcoholic liver disease (Yeh M et al., 2007; Vuppalanchi R and Chalasani N, 2009).

[0005] There is still insufficient means to effectively treat liver fibrosis, especially NAFLD and NASH. There is no established treatment for patients with NASH, and several treatment options are tested in clinical trials (Vuppalanchi R and Chalasani N, 2009; Dowman JK et al., 2009). These studies involve the use of many different chemical families (fibrates, thiazolidinediones, biguanides, statins, cannabinoids) and therapeutic target families (nuclear receptors, angiotensin receptors, cannabinoid receptors, HMG-CoA reductase). Recently, studies with thiazolidinediones (rosiglitazone and pioglitazone) have shown that these drugs can improve liver status, but treatment with these drugs is not without undesirable effects, such as weight gain with higher risk of congestive heart failure and osteoporosis, and psychological effects on patients (Dowman JK et al., 2009; Shiri-Sverdlov R et al., 2006; Neuschwander-Tetri et al., 2003). Clinical trials with the administration of cannabinoids have raised concerns about neuropsychiatric collapse (Vuppanchi R and Chalasani N, 2009). Other current therapies are exploring antioxidant assessments for NASH drugs, but none of these treatments have shown convincing evidence yet (Nelson A et al., 2009).

[0006] There is currently no approved treatment for NASH, but two compounds with different mechanisms of action - obeticholic acid (OCA, an FXR agonist) and elafibranor - are currently in phase 3 clinical trials.

[0007] The PPAR-alpha / delta dual agonist disclosed in WO2004005233, 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one (elafibranor, or ELA, formerly GFT505), has properties that may be advantageous for the treatment of a number of gastrointestinal and hepatic diseases, in particular cholestatic diseases such as PBC (primary biliary cholangitic) and PSC (primary sclerosing cholangitis), or liver diseases, in particular non-alcoholic fatty liver disease (NAFLD) such as non-alcoholic steatohepatitis (NASH).

[0008] Elafibranor has been tested for clinical efficacy in NASH in a 1-year liver biopsy-based Phase 2b study (GFT505-2127), the largest interventional study ever conducted in NASH. Administered to over 800 patients and healthy volunteers to date, elafibranor has demonstrated beneficial properties in NASH, including in particular improved markers of liver dysfunction, including ALAT, ASAT, γGT, ALP; improved insulin sensitivity and glucose homeostasis; favorable effects on plasma lipids, including lowering plasma triglycerides and LDL-C and increasing HDL-C levels; anti-inflammatory properties; efficacy on histological NASH parameters (steatosis, inflammation, fibrosis) in animal disease models - anti-fibrotic activity, and no safety concerns were identified in a 2-year complete toxicological and carcinogenicity study. Elafibranor is currently being evaluated for the treatment of NASH in a clinical Phase 3 study. Evaluation of this molecule for the treatment of PBC in a clinical Phase 2 study has also been initiated. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2004 / 005233 [Non-patent literature]

[0010] [Non-Patent Document 1] Washington Manual of Medical Therapeutics (31st ed., 2004, Lippincott Williams and Wilkins) [Non-Patent Document 2] Yeh M et al., 2007 [Non-Patent Document 3] Marchesini et al., 2003 [Non-Patent Document 4] Gressner A et al., 2009 [Non-Patent Document 5] Vuppalanchi R and Chalasani N, 2009 [Non-Patent Document 6] Angulo P et al., 2002 [Non-Patent Document 7] Dowman JK et al., 2009 [Non-Patent Document 8] Shiri-Sverdlov R et al., 2006 [Non-Patent Document 9] Neuschwander-Tetri et al., 2003 [Non-Patent Document 10] Nelson A et al., 2009 Summary of the Invention [Means for solving the problem]

[0011] Summary of the Invention The present invention relates to (i) PPAR agonists, and (ii) FXR agonist The present invention relates to a combination comprising:

[0012] In certain embodiments, the PPAR agonist is a PPARα / δ agonist, such as elafibranor (hereinafter ELA), or a pharma- ceutically acceptable salt thereof.

[0013] In certain embodiments, the FXR agonist is a bile acid FXR agonist or a non-bile acid FXR agonist, hi further specific embodiments, the non-bile acid FXR agonist is Tropifexor (or LJN452, hereinafter LJN452).

[0014] In further specific embodiments, the FXR agonist is a bile acid derivative, such as a semi-synthetic bile acid derivative such as obeticholic acid (or OCA, hereinafter OCA), or a pharma- ceutically acceptable salt thereof, or INT-767, or a pharma- ceutically acceptable salt thereof.

[0015] In certain embodiments, the FXR agonist has dual activity towards FXR and TGR5 (which is a G protein-coupled bile acid receptor), i.e., a dual FXR / TGR5 agonist, such as the compound INT-767, or a pharma- ceutically acceptable salt thereof.

[0016] In a further particular embodiment, the combination of the present invention comprises (i) a PPAR, in particular a PPARα / δ agonist such as ELA, or a pharma- ceutically acceptable salt thereof; (ii) an FXR agonist, in particular OCA or a pharma- ceutically acceptable salt thereof, and Pharmaceutically acceptable carriers A composition comprising:

[0017] In another particular embodiment, the combination comprises: (i) a PPAR, in particular a PPARα / δ agonist such as ELA or a pharma- ceutically acceptable salt thereof, and (ii) an FXR agonist, in particular OCA or a pharma- ceutically acceptable salt thereof; It is a kit of parts including:

[0018] In a further particular embodiment, the combination of the present invention comprises (i) a PPAR agonist, in particular a PPAR α / δ agonist such as ELA or a pharma- ceutically acceptable salt thereof; (ii) a dual FXR / TGR5 agonist, in particular INT-767 or a pharma- ceutically acceptable salt thereof, and Pharmaceutically acceptable carriers A composition comprising:

[0019] In another particular embodiment, the combination comprises: (i) a PPAR agonist, in particular a PPAR α / δ agonist such as ELA or a pharma- ceutically acceptable salt thereof, and (ii) A dual FXR / TGR5 agonist, in particular, INT-767 or a pharma- ceutically acceptable salt thereof. It is a kit of parts including:

[0020] In a further particular embodiment, the combination of the present invention comprises (i) a PPAR agonist, in particular a PPAR α / δ agonist such as ELA or a pharma- ceutically acceptable salt thereof; (ii) a non-bile acid FXR agonist, in particular LJN452 or a pharma- ceutically acceptable salt thereof; and a pharma- ceutically acceptable carrier. A composition comprising:

[0021] In another particular embodiment, the combination comprises: (i) a PPAR agonist, in particular a PPAR α / δ agonist such as ELA or a pharma- ceutically acceptable salt thereof, and (ii) a non-bile acid FXR agonist, in particular, LJN452 or a pharma- ceutically acceptable salt thereof; It is a kit of parts including:

[0022] The kit-of-parts of the present invention is for sequential, separate or simultaneous use in the treatment of any of the diseases mentioned herein, in particular for the treatment of NAFLD, NASH, liver fibrosis, cirrhosis, PBC or PSC.

[0023] The present invention also relates to a method for the treatment of a number of diseases, including inflammatory, metabolic, fibrotic and cholestatic diseases, comprising administering to a subject in need thereof a combination of the present invention.

[0024] In certain embodiments, (i) and (ii) are used in synergistically effective amounts, where the combined effect of the amount of (i) and (ii) is greater than the sum of the therapeutic effects of the amount of (i) and (ii) administered individually. Thanks to the present invention, the amount of each of compounds (i) and (ii) administered to a patient can be reduced by at least 1.5 times, at least 2 times, or even at least 3 times, such as at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times, compared to the amount of compounds (i) and (ii) administered individually. For example, the amount of ELA administered to a patient in clinical trials is 80 or 120 mg / day, and the amount of OCA administered to a patient in clinical trials is 10 or 25 mg / day, and the synergistic amount according to the present invention can be a reduction of 3 times, such as an amount of ELA of 27 mg or 40 mg / day and an amount of OCA of 3 and 8 mg / day.

[0025] In certain embodiments, the patient treated with the combination of the invention is a patient with NAFLD, NASH, liver fibrosis, cirrhosis, PBC or PSC.

[0026] In another embodiment, the present invention relates to the combination of the present invention for use in a method for treating a disease such as inflammatory, metabolic, fibrotic or cholestatic disease.In this embodiment, each of the components of the combination can be used in a synergistically effective amount.In a further embodiment, the disease is NAFLD, NASH, liver fibrosis, liver cirrhosis, PBC or PSC.

[0027] In a further aspect, the present invention relates to the use of the combination of the present invention in the manufacture of a medicament for the treatment of a disease, such as an inflammatory, metabolic, fibrotic or cholestatic disease.In this aspect, each of the components of the combination can be used in a synergistically effective amount.In a further embodiment, the disease is NAFLD, NASH, liver fibrosis, liver cirrhosis, PBC or PSC. [Brief description of the drawings]

[0028] [Figure 1] Effect of elafibranor (GFT505), OCA and their combination on liver fibrosis in CDAA / chol-fed rats (n=10 / group). The percentage of fibrotic surface was assessed by morphological quantification of picrosirius-positive area relative to liver cross-sectional area. Data are presented 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 Kruskal-Wallis and uncorrected Dunn's post-hoc tests. HSA, best single agent model. [Diagram 2] Effect of elafibranor (GFT505), OCA and their combination on hepatic collagen content in CDAA / chol-fed rats (n=10 / group). Data are presented 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 uncorrected Fisher post-hoc test. §p<0.05, §§p<0.01, §§§p<0.001, §§§§p<0.0001 using Kruskal-Wallis and uncorrected Dunn post-hoc test. HSA, best single agent model. [Figure 3-1]Effect of elafibranor (GFT505), OCA and their combination on expression of genes involved in tissue remodeling and inflammation in CDAA / chol-fed rat liver (n=10 / group). Expression of αSMA (ACTA2), TIMP1 and TGFβ was assessed by real-time quantitative PCR. Data are presented 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 uncorrected Fisher post-hoc test. §p<0.05, §§p<0.01, §§§p<0.001, §§§§p<0.0001 using Kruskal-Wallis and uncorrected Dunn's post-hoc test. HSA, best single agent model. [Figure 3-2] Effect of elafibranor (GFT505), OCA and their combination on expression of genes involved in tissue remodeling and inflammation in CDAA / chol-fed rat liver (n=10 / group). Expression of αSMA (ACTA2), TIMP1 and TGFβ was assessed by real-time quantitative PCR. Data are presented 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 uncorrected Fisher post-hoc test. §p<0.05, §§p<0.01, §§§p<0.001, §§§§p<0.0001 using Kruskal-Wallis and uncorrected Dunn's post-hoc test. HSA, best single agent model. [Figure 4] Differential antifibrotic effects of elafibranor and INT-767 on TGFβ-induced hHSCs. Serum-starved HSCs were preincubated with elafibranor (A) and INT-767 (B) for 1 h and then activated with the profibrotic cytokine TGFβ1 (1 ng / ml). After 48 h of incubation, expression of α-SMA was measured by ELISA. Values ​​obtained were converted to percentage inhibition relative to TGFβ1 control. Data are presented as mean (four replicates) ± standard deviation (SD). [Diagram 5] Figure 1: Combination of elafibranor and INT-767 synergistically inhibits α-SMA production in TGFβ1-induced hHSCs. Combinations were tested in a dose-response matrix format and analyzed according to the Excess Over Bliss (EOB) additivity model. Dilution series of elafibranor (rows) and INT-767 (columns) were prepared, including their respective DMSO controls. The resulting mixtures were added to serum-starved HSCs and activated 1 h later with the pro-fibrotic cytokine TGFβ1 (1 ng / ml). (A) Percentage of α-SMA inhibition relative to TGFβ1 control for all combination pairs. Data are presented as the average of four replicates. (B) EOB scores were calculated as described in Materials and Methods. Any compound pairs with an EOB value >10 were considered synergistic (colored light grey to black). A total EOB score including all combinations was also calculated. (C) Data values ​​derived from synergistic combination pairs are plotted in bar graph representation. Data are presented as mean (four replicates) ± standard deviation (SD). *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001 using Student's t-test for comparison of combination groups to the best single agent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The term "administering" as used herein includes any method of administration, such as oral, subcutaneous, sublingual, transmucosal, parenteral, intravenous, intraarterial, buccal, sublingual, topical, vaginal, rectal, ophthalmic, otic, nasal, inhalation, intramuscular, intraosseous, intrathecal and transdermal administration or a combination thereof. In a preferred embodiment, the compound is administered orally, particularly in the form of one or more tablets.

[0030] "Administering" can also include prescribing a dosage form containing a particular compound or dispensing a prescription for such a dosage form. "Administering" can also include providing instructions for carrying out a method involving a particular compound or a dosage form containing this compound.

[0031] As used herein, the term "disease" refers to a disease, disorder, condition, symptom, or indication. This term is used synonymously with the phrase "disease or disorder."

[0032] As used above and throughout this disclosure, the term "therapeutically effective amount" refers to an amount effective at the dosage and for the period of time necessary to achieve the desired result in terms of treating the relevant disorder, condition or side effect. It is understood that the effective amount of the components of the present invention will vary from patient to patient, depending not only on the specific compound, component or composition selected, the route of administration, and the ability of the component to elicit the desired response in the individual, but also on factors such as the pathology or severity of the condition to be alleviated, the individual's hormone levels, age, sex, weight, existing condition of the patient, and the severity of the condition being treated, concomitant drug therapy or special diet followed by the particular patient at the time, and other factors recognized by those skilled in the art, and the appropriate dosage is ultimately at the discretion of the attending physician. Dosage regimens can be adjusted to obtain an improved therapeutic response. An effective amount is also an amount in which any toxic or adverse effects of the components are outweighed by the therapeutically beneficial effects.

[0033] The term "synergistic" as used herein means that the effect achieved by the combinations and methods of the present invention is greater than the sum of the effects obtained from the components of the combination or from a method including one of the components separately in the amounts used in the methods and compositions herein. Such synergy can be determined according to methods well known in the art, such as by using the excess over bliss (EOB) method.

[0034] The combination of the present invention can achieve better effects than those achievable for each of its components used separately, especially when used in synergistically effective amounts. A "synergistically effective amount" is an amount for each of the components of the combination that allows the achievement of a synergistic effect when administered to a subject in need thereof. In other words, when components (i) and (ii) are used in synergistically effective amounts, the combined effect of the amounts of (i) and (ii) is greater than the sum of the therapeutic effects of the amounts of (i) and (ii) administered individually. As mentioned above, the amount of each of compounds (i) and (ii) administered to a 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 less, for example at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times. For example, the amount of ELA administered to patients in clinical trials is 80 or 120 mg / day and the amount of OCA administered to patients in clinical trials is 10 or 25 mg / day, and a synergistic amount according to the present invention may be a 3-fold reduction of ELA amounts of 27 mg or 40 mg / day and OCA amounts of 3 or 8 mg / day.

[0035] According to certain embodiments, the PPAR agonist is a PPAR-alpha agonist, a PPAR-gamma agonist, a PPAR-delta agonist, a PPAR-alpha / gamma dual agonist, a PPAR-alpha / delta dual agonist, a PPAR-gamma / delta dual agonist or a PPAR-alpha / gamma / delta pan agonist.

[0036] In certain embodiments, component (ii) of the combination is - at least one PPAR-alpha agonist, at least one PPAR-gamma agonist, at least one PPAR-delta agonist, - at least one PPAR-alpha / delta dual agonist, - at least one PPAR-alpha agonist and at least one PPAR-delta agonist, - at least one PPAR-alpha / gamma dual agonist, - at least one PPAR-alpha agonist and at least one PPAR-gamma agonist, at least one PPAR-gamma / delta dual agonist, - at least one PPAR-gamma agonist and at least one PPAR-delta agonist, at least one PPAR-alpha / gamma / delta pan-agonist, and at least one PPAR-alpha agonist, at least one PPAR-gamma agonist and at least one PPAR-delta agonist It is.

[0037] According to the present invention, the term "PPAR agonist" refers to peroxisome proliferator-activated receptor agonists, a class of drugs that play a central role in lipid and glucose homeostasis. PPARα mainly affects fatty acid metabolism, and its activation reduces lipid levels, while PPARγ is mainly involved in the regulation of adipogenesis, energy balance and lipid biosynthesis. PPARδ is involved in fatty acid oxidation, mainly in skeletal and cardiac muscles, but also regulates blood glucose and cholesterol levels.

[0038] In accordance with the present invention, the term "PPAR alpha agonist" as used herein includes, but is not limited to, fenofibrate, ciprofibrate, pemafibrate, gemfibrozil, clofibrate, binifibrate, clinofibrate, clofibric acid, nicofibrate, pirifibrate, plafibrid, lonifibrate, theofibrate, tocofibrate and SR10171.

[0039] In accordance with the present invention, 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.

[0040] According to the present invention, the term "PPAR delta agonist" as used herein includes, but is not limited to, GW501516 (Endurabol, i.e., ({4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}acetic acid)) or MBX8025 (Seladelpar, i.e., {2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsylfanyl]-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.

[0041] According to the present invention, the term "PPAR alpha / gamma agonists" (also known as glitazars) as used herein includes, but is not limited to, saroglitazar, aleglitazar, muraglitazar, tesaglitazar, DSP-8658.

[0042] According to the present invention, the term "PPAR alpha / delta agonist" as used herein includes, but is not limited to, ELA or T913659.

[0043] In accordance with the present invention, the term "PPAR gamma / delta agonist" as used herein includes, but is not limited to, conjugated linoleic acid (CLA), T3D-959.

[0044] According to the present invention, the term "PPAR alpha / gamma / delta agonist" as used herein includes, but is not limited to, IVA337 (Lanifibranor) or TTA (tetradecylthioacetic acid) or Bavachinin 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)-2-methylphenoxy)-2-methylpropanoic acid (MHY2013).

[0045] The PPAR agonist may be in the form of a salt, hydrate, solvate, polymorph or co-crystal. The PPAR agonist may also be in the form of a salt, hydrate, solvate, polymorph or co-crystal.

[0046] In a more particular embodiment, the PPAR agonist is a compound of formula (I):

[0047] [ka] (In the formula, Y1 represents a halogen, Ra or Ga-Ra group; A represents a CH=CH or CH2-CH2 group, Y2 represents a Gb-Rb group, Ga and Gb are the same or different and represent oxygen or sulfur atoms; Ra represents a hydrogen atom, an unsubstituted (C1-C6) alkyl group, a (C6-C14) aryl group or a (C1-C6) alkyl group substituted with one or more halogen atoms, a (C1-C6) alkoxy or a (C1-C6) alkylthio group, a (C3-C14) cycloalkyl group, a (C3-C14) cycloalkylthio group or a heterocyclic group; Rb represents a (C1-C6) alkyl group substituted with at least one -COORc group (wherein Rc represents a hydrogen atom), or a (C1-C6) alkyl group unsubstituted or substituted with one or more halogen atoms, or a (C3-C14) cycloalkyl group, or a heterocyclic group; Y4 and Y5 are the same or different and represent a (C1-C6) alkyl group, a (C3-C14) cycloalkyl group, or a heterocyclic group, which may or may not be substituted with one or more halogen atoms. or a pharma- ceutically acceptable salt thereof.

[0048] In certain embodiments of the compound of formula (III), Y1 represents a halogen, Ra or Ga-Ra group; A represents a CH=CH group; Y2 represents a Gb-Rb group, Ga and Gb are the same or different and represent oxygen or sulfur atoms; Ra represents a (C1-C6) alkyl or (C3-C14) cycloalkyl group, in particular a (C1-C7) alkyl or (C3-C14) cycloalkyl group which may or may not be substituted by one or more halogen atoms; Rb represents a (C1-C6) alkyl group substituted with a -COOR3 group, where Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; Y4 and Y5 independently represent a (C1-C4) alkyl group.

[0049] In certain embodiments of the compound of formula (III), Y1 represents a Ra or Ga-Ra group; A represents a CH2-CH2 group, Y2 represents a Gb-Rb group, Ga represents an oxygen or sulfur atom, Gb represents an oxygen atom, Ra represents a (C1-C6) alkyl or (C3-C7) cycloalkyl group; Rb represents a (C1-C6) alkyl group substituted with at least one -COORc group, where Rc represents a hydrogen atom or a (C1-C4) alkyl group; Y4 and Y5 independently represent a (C1-C4) alkyl group.

[0050] In certain embodiments of the compound of formula (III), Y1 represents a halogen atom or a Ra or Ga-Ra group; A represents a CH2-CH2 group, Y2 represents a Gb-Rb group, Ga represents an oxygen or sulfur atom, Gb represents an oxygen atom, Ra represents a (C1-C6) alkyl or (C3-C14) cycloalkyl group substituted by one or more halogen atoms; Rb represents a (C1-C6) alkyl group which may or may not be substituted with one or more halogen atoms and which is substituted with at least one -COORc group, where Rc represents a hydrogen atom or a (C1-C4) alkyl group; Y4 and Y5 each represent a (C1-C4) alkyl group.

[0051] In certain embodiments of the compound of formula (III), Gb is an oxygen atom and Rb is a (C1-C6) alkyl group substituted with a -COORc group, where Rc represents a hydrogen atom or an unsubstituted linear or branched (C1-C4) alkyl group.

[0052] In certain embodiments of compounds of Formula (III), Y1 is a (C1-C6) alkylthio group, including a (C1-C6) alkyl group, which may or may not be substituted with one or more halogen atoms, linear or branched.

[0053] In certain embodiments, the compound of formula (III) is selected from the group consisting of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one (ELA), 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-isopropyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one, 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one, 1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one, 1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one, 1-[4-trifluoromethylphenyl]-3-[3,5 1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one, 1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one, 1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one, 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic acid, and 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methyl-propanoic acid isopropyl ester.

[0054] In a more particular embodiment, the PPAR agonist is ELA or a pharma- ceutically acceptable salt thereof. ELA has the following structure:

[0055] [ka]

[0056] ELA can be prepared by the methods described in WO2004 / 005233, WO2005 / 005369 or WO2011 / 144579.

[0057] The FXR agonist used in accordance with the present invention may be a steroidal or a non-steroidal FXR agonist.

[0058] Illustrative, non-limiting FXR agonists useful in the practice of the present invention include those disclosed in WO02072598, WO2005082925, WO03080803, WO04007521, WO04046162, WO04045511, WO04048349, WO05082925, WO07140174, WO08000643, WO08025540, WO08025539, WO07140183, WO08157270, WO09005998 , WO09027264, WO09062874, WO09080555, WO09149795, WO10034649, WO10034657, WO11020615, WO11039130, WO12087519, WO13007387, WO14184271, WO15138986, WO16073767, WO16086115, WO16086134, WO16086169 and WO16086218. In accordance with the present invention, each embodiment and each specific FXR agonist disclosed in these references is individually disclosed herein in combination with a PPAR agonist.

[0059] In certain embodiments, the FXR agonist is selected from the group consisting of obeticholic acid (INT-747), GS-9674, LJN-452 or LJN452, LJN-763, LMB763, EDP-305, AKN-083, INT-767, GNF-5120, LY2562175, INV-33, NTX-023-1, EP-024297, EPY-001, Px-103 and SR-45023. In a further specific embodiment, the PPAR agonist is ELA and the FXR agonist is selected from the group consisting of obeticholic acid (INT-747), GS-9674, LJN-452 or LJN452, LJN-763, LMB-763, EDP-305, AKN-083, INT-767, GNF-5120, LY-2562175, INV-33, NTX-023-1, EP-024297, EPY-001, Px-103 and SR-45023.

[0060] In a particular embodiment, the FXR agonist is INT-767, which has the following structure:

[0061] [ka]

[0062] In a particular embodiment, the FXR agonist is LJN452, which has the following structure:

[0063] [ka]

[0064] In another embodiment, the FXR agonist is LY2562175, which has the following structure:

[0065] [ka]

[0066] In certain embodiments, the FXR agonist is obeticholic acid (OCA; 6α-ethylchenodeoxycholic acid; INT-747) or a pharma- ceutically acceptable salt thereof. OCA has the following chemical structure:

[0067] [ka]

[0068] OCA can be prepared by the method described in WO2006122977.

[0069] The combinations of the present invention may be used to inhibit the proliferation and / or activation of fibroblasts involved in the production of collagen fibres and / or involved in the production of the extracellular matrix.

[0070] The combinations of the invention may be used to treat disorders such as immune (eg autoimmune), inflammatory, metabolic, fibrotic or cholestatic disorders.

[0071] According to the present invention, the term "autoimmune disease" is used to indicate a condition resulting from an abnormal immune response of the body against substances and tissues normally present in the body. The disease may be limited to certain organs (e.g., in the case of type I diabetes or autoimmune thyroiditis) or may affect specific tissues in different locations (e.g., in the case of Goodpasture's disease, basement membrane involvement in the lungs and kidneys).

[0072] The term "inflammation" is used to denote a condition resulting from a protective response involving host cells, blood vessels, and proteins and other mediators that may play a role in removing the cause of cell / tissue damage, in removing necrotic cells / tissue resulting from the initial insult, and in initiating the repair process. Inflammatory responses can be manifested by pain, heat, redness, swelling, vasodilation, increased blood flow, and loss of function.

[0073] The terms "fibrosis", "fibrotic disease", "fibrotic disorder" and their derivations refer to a pathological condition of excessive accumulation of fibrous connective tissue in an organ or tissue. More specifically, fibrosis is a pathological process that involves persistent fibrous scar formation and excessive production of extracellular matrix by connective tissue in response to tissue injury. Physiologically, the accumulation of connective tissue can completely destroy the structure and function of the underlying organ or tissue.

[0074] According to the present invention, fibrosis may be any organ or tissue fibrosis. Illustrative non-limiting examples of specific organ fibrosis include liver, kidney, skin, epidermis, endothelium, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, ovary, adrenal gland, artery, vein, colon, intestine (e.g., small intestine), bile duct, soft tissue (e.g., mediastinal or retroperitoneal), bone marrow, joint, or stomach fibrosis.

[0075] In preferred embodiments, the fibrotic disorder is selected from the group consisting of hepatic, gastrointestinal, pulmonary, cardiac, renal, muscle, skin, soft tissue (e.g., mediastinal or retroperitoneal), bone marrow, intestine, and joint (e.g., knee, shoulder or other joint) fibrosis.

[0076] In a more preferred embodiment, the fibrotic disorder is selected from the group consisting of hepatic, pulmonary, skin, renal and small intestinal fibrosis.

[0077] In a more preferred embodiment of the invention, the fibrotic disorder to be 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, dermal fibrosis, ocular fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (complication of coal worker's pneumoconiosis), proliferative fibrosis, neoplastic fibrosis; secondary to chronic inflammatory airway diseases (COPD, asthma, emphysema, smoker's lung, tuberculosis, IPF). pulmonary fibrosis; 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, keloids, old myocardial infarction, scleroderma / systemic sclerosis, arthrofibrosis; some forms of adhesive capsulitis; chronic fibrosing cholecystitis such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC); biliary atresia, familial intrahepatic cholestasis type 3 (PFIC3), pre-implantation fibrosis and asbestosis.

[0078] Cholestasis is defined as a reduction in bile flow due to failure of hepatocytes to secrete (hepatocellular cholestasis) or due to obstruction of bile flow through the intrahepatic or extrahepatic bile ducts (obstructive cholestasis). In clinical practice, cholestasis is any condition in which the flow of bile from the liver is slowed or blocked.

[0079] Examples of inflammatory, fibrotic, metabolic and cholestatic diseases include metabolic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-induced liver disease, alcohol-induced liver disease, infectious agent-induced liver disease, inflammatory liver disease, immune system dysfunction-mediated liver disease, dyslipidemia, cardiovascular disease, restenosis, syndrome X, metabolic syndrome, diabetes, obesity, hypertension; chronic cholangiopathies, such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC); biliary tract diseases. obstructive pulmonary disease, familial intrahepatic cholestasis type 3 (PFIC3), inflammatory bowel disease, Crohn's disease, ulcerative colitis, keloids, old myocardial infarction, scleroderma / systemic sclerosis, inflammatory diseases, neurodegenerative diseases, cancer, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer; meningiomas associated with neurofibromatosis; pancreatic neuroendocrine tumors, pancreatic exocrine tumors, leukemia, myeloproliferative / myelodysplastic disorders, mastocytosis, dermatofibrosarcoma; solid tumors, including breast, lung, thyroid or colorectal cancer; prostate cancer, hepatic fibrosis of any origin or cirrhosis, 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 agent-induced 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 liver fibrosis or cirrhosis, dual HCV and HIV infection-induced liver fibrosis or cirrhosis , radiation or chemotherapy induced fibrosis or cirrhosis, bile duct fibrosis; liver fibrosis or cirrhosis due to any chronic cholestatic disease; gastrointestinal fibrosis of any cause; Crohn's disease induced fibrosis, ulcerative colitis induced fibrosis, intestinal (e.g. small intestine) fibrosis, colonic fibrosis, gastric fibrosis, dermal fibrosis, epidermal fibrosis, endothelial fibrosis; skin fibrosis due to scleroderma / systemic sclerosis; pulmonary fibrosis; pulmonary fibrosis secondary to chronic inflammatory airway diseases such as COPD, asthma, emphysema, smoker's lung, tuberculosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF);Cardiac fibrosis, renal fibrosis, nephrogenic systemic fibrosis, myofibrosis, soft tissue (e.g., mediastinal or retroperitoneal) fibrosis, bone marrow fibrosis, arthrofibrosis, 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, bone marrow fibrosis, retroperitoneal fibrosis, progressive massive fibrosis (a complication of coal workers' pneumoconiosis), proliferative fibrosis, neoplastic fibrosis, peri-implantation fibrosis, and asbestosis, arthrofibrosis, adhesive capsulitis;

[0080] In certain embodiments, the disease is a metabolic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-induced liver disease, alcohol-induced liver disease, infectious agent-induced liver disease, inflammatory liver disease, immune system dysfunction-mediated liver disease, dyslipidemia, cardiovascular disease, restenosis, syndrome X, metabolic syndrome, diabetes, obesity, hypertension; chronic cholangiopathy, 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, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, colorectal cancer, 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 agent-induced 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 liver fibrosis or cirrhosis, dual HCV and HIV infection-induced liver fibrosis or cirrhosis, radiation or chemotherapy-induced fibrosis or cirrhosis, bile duct fibrosis; liver fibrosis or cirrhosis due to any chronic cholestatic disease; gastrointestinal fibrosis of any cause; Crohn's disease-induced fibrosis, ulcerative colitis-induced fibrosis, intestinal (e.g., small intestine) fibrosis, colonic fibrosis, gastric fibrosis, pulmonary fibrosis; pulmonary fibrosis secondary to chronic inflammatory airway disease such as COPD, asthma, emphysema, smoker's lung, tuberculosis, pulmonary fibrosis, and idiopathic pulmonary fibrosis (IPF).

[0081] In a further embodiment, the disease is NAFLD, NASH, liver fibrosis, cirrhosis, PBC or PSC.

[0082] The term "treatment" or "treating" refers to the treatment, prevention, or prophylaxis of onset of a disorder in a subject in need thereof. Treatment includes the administration of a combination of the present invention to a subject (e.g., patient) having the stated disorder to prevent, cure, delay, reverse, or slow the progression of the disorder, thereby improving the patient's condition. Treatment may be administered to subjects who are healthy or to subjects at risk of developing an immune (e.g., autoimmune), inflammatory, fibrotic, or cholestatic disorder.

[0083] The term "subject" refers to a mammal, more particularly a human. Subjects to be treated according to the present invention can be suitably selected based on several criteria related to immune (e.g. autoimmune), inflammatory, fibrotic and cholestatic pathogenesis processes, such as past and / or current drug treatments, concomitant pathologies, genotype, exposure to risk factors, and based on any other relevant biomarkers that can be assessed by any suitable immunological, biochemical or enzymatic method.

[0084] The present inventors show herein that the combination of PPAR agonist and FXR agonist, in particular the combination of ELA and OCA or INT-767, in particular the combination of ELA and OCA, can benefit a broader patient population and have synergistic effects, thereby allowing for reduced therapeutic doses.The associated reduced therapeutic doses can reduce the incidence of adverse drug effects.Therefore, the present invention also relates to a method for the treatment of diseases as defined above, with reduced incidence of adverse drug effects.

[0085] The frequency and / or amount of administration can be adapted by the skilled person in function of the patient, the pathology, the administration form, etc. Typically, the combination of the present invention can be administered for the treatment of a disease at a dose of 10 mg / day to 1000 mg / day of ELA, such as 50 mg / day to 500 mg / day, in particular 70 mg / day to 150 mg / day. The dose of OCA can be comprised between 5 mg / day to about 100 mg / day, such as a dose of about 10 mg / day to about 50 mg / day. In a particular embodiment of the present invention, OCA is used in combination with ELA at a dose of 10 mg / day to 25 mg / day for OCA and 80 to 120 mg / day for ELA. In a particular embodiment, OCA is used at 10 or 25 mg / day and ELA at 80 or 120 mg / day.

[0086] In another embodiment, the amount of both the PPAR agonist and the FXR agonist is a synergistically effective amount. Certain embodiments include a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 8.5-fold, 10-fold or greater reduction in the PPAR agonist and / or the FXR agonist. In further particular embodiments, incorporating ELA (or a pharma- ceutically effective amount thereof) as the PPAR agonist and OCA or a pharma- ceutically acceptable salt thereof, or INT-767 or a pharma-ceutically acceptable salt thereof, or LJN452 or a pharma-ceutically acceptable salt thereof as the FXR agonist includes a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 8.5-fold, 10-fold, or less than 10-fold reduction of the ELA agonist and / or FXR agonist. Further particular embodiments incorporating ELA (or a pharma-ceutically acceptable salt thereof) as the PPAR agonist and OCA (or a pharma-ceutically acceptable salt thereof) as the FXR agonist include: - A 1.5-fold reduction in the amount of both ingredients, with ELA used in doses of 5-80 mg / day and OCA used in doses of 7-17 mg / day; - A reduction in the amount of both ingredients by half, with ELA used at a dose of 40-60 mg / day and OCA used at a dose of 5-12.5 mg / day; - A 2.5-fold reduction in the amount of both ingredients, with ELA used in a dose of 32-48 mg / day and OCA used in a dose of 4-10 mg / day; - A reduction in the amount of both ingredients by one third, with ELA used in a dose of 27-40 mg / day and OCA used in a dose of 3-8 mg / day; - A 5-fold reduction in the amount of both ingredients, with ELA used in a dose of 16-24 mg / day and OCA used in a dose of 2-5 mg / day; - A tenfold reduction in the amount of both ingredients, with ELA used in a dose of 8-12 mg / day and OCA used in a dose of 1-2.5 mg / day; etc.

[0087] In certain embodiments, the amount of each of the components of the combination of the invention is administered once daily as a single dosage, or by multiple administration of the components, for example, half the daily dose administered twice daily, such as during meals (e.g., during lunch and dinner).

[0088] In certain embodiments, the PPAR agonist and the FXR agonist are administered simultaneously, sequentially, or separately. In certain embodiments, the PPAR agonist and the FXR agonist are administered sequentially, with the PPAR agonist being administered first, followed by the FXR agonist, or the FXR agonist being administered first, followed by the PPAR agonist.

[0089] The duration of treatment can vary widely depending on the condition being treated and the stage of said condition. For example, the combination can be administered for at least 2 consecutive days or more, such as at least 7, 8, 9, or 10 days or more; at least 1 week or more, such as at least 1, 2, 3, 4, 5, 10, 20, 50, 60, 70, or 72 weeks or more; at least 1 month or more, such as at least 1, 2, 3, 4, 5, 10, 15, 20, or 24 months or more; or at least 1 year or more, such as at least 1, 2, 3, 4, or 5 years or more.

[0090] The invention will be further described with reference to the following non-limiting examples. EXAMPLES

[0091] Materials and Methods Evaluation of elafibranor, OCA, and the combination elafibranor+OCA in a chronic CDAA+1% cholesterol model (12 weeks) The preventive effects of elafibranor (GFT505) alone, OCA alone, and the combination of both were evaluated in a fibrotic NASH model in rats fed a CDAA+1% cholesterol diet.Male Wistar rats weighing 150-175 g were fed a control (CSAA) diet, a CDAA+1% cholesterol diet, or a CDAA+1% cholesterol diet supplemented with elafibranor 1, 3, and 10 mg / kg / day, OCA 10 and 30 mg / kg / day, or the combined drugs (elafibranor 1, 3, and 10 mg / kg / day combined with OCA 10 mg / kg / day) for 12 weeks.

[0092] Body weight and food intake were monitored twice weekly. On the last day of treatment, rats were sacrificed after a 6-h fasting period. Livers were rapidly excised for biochemical and histological studies.

[0093] All animal procedures were performed according to standard protocols and in accordance with recommended practices for the proper care and use of laboratory animals.

[0094] Organizational Diagnosis Tissue embedding and sectioning: The liver slices were first fixed in a 4% solution of formalin for 12 hours. The liver slices were then washed with PBS for 30 minutes and dehydrated in ethanol solutions (successive baths of 70, 80, 95 and 100% ethanol). The liver slices were incubated in three different baths of xylene (Sigma-Aldrich Cat. No. 534056) followed by two baths of liquid paraffin (56°C). The liver slices were then placed in a rack and the rack was gently filled with Histowax® to completely cover the tissue.

[0095] The paraffin blocks containing the tissue sections were removed from the rack and stored at room temperature. The liver blocks were cut into 3 μm sections.

[0096] Hematoxylin / eosin staining Liver sections were deparaffinized, rehydrated, and incubated in Mayer's hematoxylin (Microm, Cat. No. F / C0303) for 3 minutes. Liver sections were then rinsed in water and incubated in Eosin G (VWR, Cat. No. 1.09844.1000) for 1 minute. Sections were rinsed in water, then dehydrated and mounted using CV Mount medium (Leica, Cat. No. 14046430011).

[0097] Picrosirius Red Staining Liver sections were deparaffinized, rehydrated, and incubated in a solution of Fast Green FCF 0.1% (Sigma-Aldrich, Cat. No. F7258) for 15 min, followed by rinsing in a 0.5% acetic acid (Panreac, Cat. No. 131008.1611) bath. Liver sections were then rinsed in water and incubated in a solution of 0.1% Sirius Red (Direct Red 80, Fluka Cat. No. 43665) in saturated aqueous picric acid (Sigma-Aldrich Cat. No. P6744) for 30 min. Sections were then dehydrated and mounted using CV Mount medium (Leica, Cat. No. 14046430011).

[0098] Histological examination Histological examination was performed by a technician blinded to the source of each liver section. Virtual slides were generated using a Pannoramic 250 scanner from 3D Histech. For each animal, a score summarizing the major histological lesions of NASH was imputed according to the NASH Clinical Research Network (Kleiner 2005; Brunt 1999). Briefly, steatosis, lobular inflammation and hepatocyte ballooning were scored. A NAFLD activity score (NAS score) was established for each individual as the unweighted sum of steatosis, lobular inflammation and balloon injury grading.

[0099] Collagen stained area was quantified using Quant Center software (3D Histech, which includes Pattern Quant and Histo Quant modules). Briefly, Pattern Quant was used to detect tissue and measure its surface. Histo Quant was then used to detect stained collagen content and measure its surface based on color thresholding. Fibrosis area was then expressed as a percentage of collagen surface to total tissue per animal.

[0100] Measurement of liver collagen content Liver collagen content was determined using the appropriate QuickZyme kit (Total collagen assay, catalog number QZB-totcol2). This assay is based on the detection of hydroxyproline, a non-proteinogenic amino acid found primarily in the triple helix of collagen. Thus, hydroxyproline in tissue hydrolysates can be used as a direct measure of the amount of collagen present in the tissue (without distinction between procollagen, mature collagen and collagen degradation products).

[0101] Complete hydrolysis of tissue samples in 6 M HCl at 95°C is required prior to administration of hydroxyproline. The assay results in the production of a chromogen with a maximum absorbance at 570 nm. Results are expressed as mg of collagen per g of liver.

[0102] Hepatic gene expression analysis Total RNA was isolated from rat liver using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using M-MLV RT (Moloney Murine Leukemia Virus Reverse Transcriptase) (Invitrogen Cat. No. 28025) in 1× RT buffer (Invitrogen), 0.5 mM DTT (Invitrogen), 0.18 mM dNTPs (Promega), 200 ng pdN6 (Amersham) and 30 U of RNase inhibitor (Promega).

[0103] Quantitative PCR was then performed using the CFX96 Touch™ Real-Time PCR Detection System (Biorad). Briefly, PCR reactions were performed in a total volume of 25 μl in 96-WP format, containing 1 μl of reverse transcription reaction, 0.5 μl of reverse and forward primers (10 pmol each) and 12.5 μl of 2× iQ SYBR Green Supermix (BioRad), and used the following primer sequences:

[0104] [Table 1]

[0105] Expression levels were normalized using the expression of Rplp0 gene in the samples as a reference. Standard curves were drawn for each gene by selecting the best points (at least 3 points) to have PCR reaction efficiency close to 100% and correction factor close to 1. Expression levels were determined using the standard curve equations for both housekeeping and target genes (taking into account the specific PCR efficiency of each target gene).

[0106] Evaluation of the combination of elafibranor with other FXR agonists in an a vitro model of liver fibrogenesis hHSC culture Human primary hepatic stellate cells (hHSC) (Innoprot) were cultured in STeCM medium (ScienCell Cat. No. 5301) supplemented with 2% fetal bovine serum (FBS, ScienCell Cat. No. 0010), 1% penicillin / streptomycin (ScienCell Cat. No. 0503) and stellate cell growth supplement (SteCGS; ScienCell Cat. No. 5352). Cell culture flasks were coated with poly-L-lysine (Sigma Cat. No. P4707) for better attachment.

[0107] Preparation of the composition: Binary combination matrix (FXR agonist / elafibranor) FXR agonist INT-767 (CAS number 1000403-03-1, catalog number HY-12434, batch number 19249) was purchased from Haoyuan Chemexpress. For these experiments, a checkerboard matrix was generated. INT-767 and elafibranor were dissolved in dimethyl sulfoxide (DMSO, Fluka catalog number 41640) and serially diluted in a 5-point series in the rows (elafibranor) and an 11-point series in the columns (INT-767) of a 384-well plate. A 5x11 combination matrix was then generated by 1:1 mixing of all single agent concentrations. Test concentrations for each compound were selected based on the literature (Rizzo et al.; McMahan et al.).

[0108] Activation of hHSCs with TGF-β1 and compound treatment hHSC, 6.5 x 10 cells in a 384-well plate 3hHSCs were plated at a density of 1000 cells / well. The next day, cell culture medium was removed and cells were washed with PBS (Invitrogen Cat. No. 14190). hHSCs were starved in serum-free, SteCGS-free medium for 24 hours. For treatment with INT-767 and elafibranor and their combinations, serum-starved hHSCs were pre-incubated with the compounds for 1 hour, followed by addition of the pro-fibrotic stimulant TGF-β1 (PeproTech Cat. No. 100-21, 1 ng / mL) in serum-free, SteCGS-free medium for an additional 48 hours.

[0109] α-SMA ELISA A sandwich ELISA was used to measure α-SMA levels. Briefly, wells of an ELISA plate were first coated with capture antibody (mouse monoclonal anti-ACTA2, Abnova) overnight at 4°C. After washing three times with PBS+0.2% Tween20, a blocking solution consisting of PBS+0.2% BSA was added for 1 hour, followed by another washing cycle. Cell lysates were transferred into the wells for 2 hours at room temperature for binding with the capture antibody. After a washing procedure, detection antibody (biotin-labeled mouse monoclonal anti-ACTA2, Abnova) was added for 2 hours at room temperature, followed by three washings. For detection, streptavidin-HRP conjugate (R&D Systems Cat. No. DY998) was first applied for 30 minutes at room temperature. After washing, HRP substrate TMB (BD#555214) was added and incubated for 7 minutes at room temperature in the dark. Upon oxidation, TMB forms a water-soluble blue reaction product that turns yellow upon addition of sulfuric acid (stopping dissolution), allowing accurate measurement of intensity at 450 nm using a spectrophotometer. The color developed is directly proportional to the amount of α-SMA present in the lysate.

[0110] Determination of synergy by the excess over bliss (EOB) method The values ​​obtained in the αSMA ELISA assay were first converted to a percentage of inhibition relative to the TGF-β1 control. These percentages of inhibition were then used to determine the excess over Bliss (EOB) to define the synergistic effect of the drug combination. The expected Bliss additivity score (E) was calculated by first using the equation: E = (A + B) - (A × B) where A and B are the percentage inhibition of elafibranor (A) and INT-767 (B) at a given dose. The difference between the Bliss expected value and the observed inhibition for the combined INT-767 / elafibranor at the same dose is the "excess over Bliss" score. - An excess-over-Bliss score of 0 indicates that the combination treatment is additive (as expected for the effects of independent pathways); - An Excess-Over-Bliss score >0 indicates greater than additive activity (synergism), - An excess-over-bliss score <0 indicates that the combination is less than additive (antagonism).

[0111] For the combination INT-767 / elafibranor, an additional total BLISS score was calculated by summing all EOBs.

[0112] To verify synergy, the experimental values ​​corresponding to the highest EOB scores for the FXR agonist / elafibranor combination were plotted in a bar graph.

[0113] The significance of observed differences between INT-767 / elafibranor versus the best single agent was determined by Student's t-test. *: p<0.05; **: p<0.01; ***: p<0.001.

[0114] Results and Discussion Evaluation of elafibranor, OCA, and the combination elafibranor+OCA in a chronic CDAA+1% cholesterol model (12 weeks) The results are reported in the following table and in Figures 1-3.

[0115] [Table 2]

[0116] Western lifestyles are always associated with a high incidence of non-alcoholic steatohepatitis (NASH), a chronic liver disease that often progresses to liver fibrosis and cirrhosis, and may eventually lead to hepatocellular carcinoma. Currently, there is no approved treatment for NASH. Drug combinations directed simultaneously at multiple therapeutic targets have the potential to dramatically improve drug response and benefit the broadest patient population. Drug combinations have been previously tested in other systemic diseases, such as hypertension, dyslipidemia, or type 2 diabetes, showing better control of the underlying disease and reducing morbidity and mortality. In a recent Phase 2B study, both elafibranor (a PPARα / δ agonist) and OCA (an FXR agonist) showed efficacy against NASH and fibrosis endpoints. We wanted to compare their effects on meaningful NASH pathology outcomes and to look for the therapeutic benefits of the combination.

[0117] To this end, fibrotic NASH was induced in Wistar rats by feeding a choline-deficient L-amino acid diet supplemented with cholesterol (CDAA / chol diet). Intervention animals received either elafibranor or OCA or both compounds throughout the entire study period. NASH and fibrosis development were assessed by histology. Further biochemical and molecular analyses were also performed using different relevant biomarkers.

[0118] Wistar rats fed the CDAA / chol diet developed NASH-associated histology and fibrosis, with a high penetrance of severe disease. Extensive steatosis, lobular inflammation, and ballooning were present in all animals, with NAS scores [min 0–max 8] ranging from 6 to 8. Liver histology (picrosirius-positive areas) and biochemistry (hepatic collagen concentration) showed, on average, a four-fold increase in liver fibrotic content, with fibrosis scores of either 3 or 4 for all animals that continued on the CDAA / chol diet without drug treatment (Figures 1–2). Expression of genes associated with inflammation, tissue remodeling, and fibrosis was increased, consistent with a gene signature previously reported in NASH patients with severe disease (Figure 3).

[0119] Elafibranor administration alone attenuated fibrosis development, whereas OCA administration alone does not significantly attenuate fibrosis (Figures 1-2). The combination treatment was more potent in reducing fibrosis, which allowed for a reduction in dose, with both GFT505 1 mg / kg and 3 mg / kg combined with OCA 10 mg / kg achieving similar efficacy against fibrosis (Figures 1-2). Administration of either drug candidate alone attenuated the increase in tissue remodeling only to a certain extent, whereas the combination of both compounds was more efficient compared to any single agent (Figure 3).

[0120] Thus, it is shown herein that the synergistic effect of elafibranor and OCA on liver fibrosis in CDAA / chol diet-induced NASH model produces comparable therapeutic benefit at significantly lower doses of both drug candidates compared to any single agent. From this study, it is certainly expected that the dose of both drug candidates can be reduced by at least 1.5, 2, 2.5 or even at least 3 times to obtain similar results as the initial dose of each compound used individually. In addition, elafibranor showed a clear protective effect against liver injury. The effect of OCA on ballooning and lobular inflammation was rather small in this model. From this study, it can be concluded that the combination of elafibranor / OCA benefits a wider patient population, and the associated reduction in therapeutic dose reduces the incidence of adverse side effects.

[0121] Evaluation of the combination of elafibranor and other FXR agonists in an a vitro model of liver fibrogenesis The results are reported in Figures 4-5.

[0122] Abnormal persistence of differentiated myofibroblasts is a hallmark of many fibrotic diseases. Following liver injury, quiescent HSCs undergo an activation process characterized by differentiation into (α-SMA) positive myofibroblasts.

[0123] The PPAR agonist elafibranor demonstrated antifibrotic activity in hHSCs activated with the profibrotic cytokine TGFβ1. α-SMA markers were reduced by 68% with the highest dose of elafibranor tested (5 μM) (Figure 4A). INT-767 alone only inhibited α-SMA production by 13% at the highest dose tested (30 μM) (Figure 4B). To evaluate whether the combination of elafibranor and INT-767 could synergistically reduce fibrosis, combination matrix experiments were performed in TGFβ-induced HSCs. Briefly, INT-767 and elafibranor solutions were serially diluted in a checkerboard format to generate 55 combination matrices encompassing a large panel of INT-767 / elafibranor ratios. Synergy was first determined by calculating the excess-over-Bliss score. These experiments revealed that elafibranor can synergize with INT-767 to reduce α-SMA production in activated HSCs (FIG. 5). One of the best examples of synergy is shown in FIG. 5C, with 2.5 μM elafibranor and 1.9 μM INT-767. While 1.9 μM INT-767 alone does not show any antifibrotic activity, its addition to 2.5 μM elafibranor synergistically increased the activity of elafibranor, reaching up to 69% inhibition (compared to 26% for elafibranor alone at 2.5 μM).

[0124] In conclusion, the synergistic effects of elafibranor and different classes of FXR agonists on fibrogenesis indicate the potential benefit of such combinations in multiple types of fibrotic diseases.

[0125] [Table 3]

Claims

1. A combination for use in a method for treating a fibrotic disease, the combination comprising: (i) elafibranor (ELA) or a pharma- ceutically acceptable salt thereof, and (ii) FXR agonist Including, A combination wherein the FXR agonist is obeticholic acid (OCA) or a pharma- ceutically acceptable salt thereof.

2. A combination for use in a method for treating a fibrotic disease, the combination comprising: (i) elafibranor (ELA) or a pharma- ceutically acceptable salt thereof, and (ii) FXR agonist Including, A combination wherein said FXR agonist is INT-767 or a pharma- ceutically acceptable salt thereof.

3. A combination for use in a method for treating liver fibrosis, the combination comprising: (i) elafibranor (ELA) or a pharma- ceutically acceptable salt thereof, and (ii) FXR agonist Including, A combination wherein the FXR agonist is obeticholic acid (OCA) or a pharma- ceutically acceptable salt thereof.

4. A combination for use in a method for treating liver fibrosis, the combination comprising: (i) elafibranor (ELA) or a pharma- ceutically acceptable salt thereof, and (ii) FXR agonist Including, A combination wherein said FXR agonist is INT-767 or a pharma- ceutically acceptable salt thereof.

5. A combination according to any one of claims 1 to 4, which is a composition of (i), (ii) and a pharma- ceutically acceptable carrier.

6. The combination according to any one of claims 1 to 5, which is a kit-of-parts comprising (i) and (ii).

7. A combination according to any one of claims 1 to 6, wherein (i) and (ii) are present in synergistically effective amounts for the treatment of the disease.

8. ELA is administered at a dose of 5-80 mg / day and OCA is administered at a dose of 7-17 mg / day; ELA is administered at a dose of 40-60 mg / day and OCA is administered at a dose of 5-12.5 mg / day; ELA is administered at a dose of 32-48 mg / day and OCA is administered at a dose of 4-10 mg / day; ELA is administered at a dose of 27-40 mg / day and OCA is administered at a dose of 3-8 mg / day; ELA is administered at a dose of 16-24 mg / day and OCA is administered at a dose of 2-5 mg / day; or ELA is administered at a dose of 8-12 mg / day and OCA is administered at a dose of 1-2.5 mg / day; A combination according to any one of claims 1 to 7.

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