WO2021204755A1

WO2021204755A1 - Pharmaceutical combination for the treatment of liver diseases

Info

Publication number: WO2021204755A1
Application number: PCT/EP2021/058856
Authority: WO
Inventors: Sébastien BOLZE; Pascale Fouqueray; Sophie Hallakou-Bozec
Original assignee: Poxel
Priority date: 2020-04-06
Filing date: 2021-04-06
Publication date: 2021-10-14

Abstract

The invention relates to a pharmaceutical combination comprising a specific thienopyridone derivative, namely 2-chloro-4-hydroxy-3-(5-hydroxytetralin-6-yl)-5-phenyl-7H-thieno[2,3- b]pyridin-6-one or one of its pharmaceutically acceptable salts and/or solvates, and at least one incretin agonist. This combination may be used for treating a liver disease such as NASH, including cirrhotic and non-cirrhotic NASH.

Description

PHARMACEUTICAL COMBINATION FOR THE TREATMENT OF LIVER

DISEASES

TECHNICAL FIELD

TECHNICAL BACKGROUND

Non-alcoholic fatty liver disease (NAFLD) consists of a spectrum of conditions ranging from relatively benign steatosis to more severe non-alcoholic steatohepatitis (NASH). NASH is characterized by hepatic lipid accumulation coming mainly from adipose tissue (AT) lipolysis (60%) and hepatic de novo lipogenesis (25%), causing liver inflammation, hepatocyte ballooning and fibrosis. Liver injury is reflected by dramatically increased plasma levels of the transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In several diet-induced murine models of NAFLD, an increase in liver weight has also been reported (Stephenson et al. Gene Expr. 2018). In hepatocytes, steatosis notably results in an excess storage of lipids in the form of particular vesicles called lipid droplets (LD). Accordingly, the formation of very large LDs in hepatocytes is the hallmark of steatosis (Gluchowski et al. Nat Rev Gastroenterol Hepatol. 2017).

White adipose tissue (WAT) is the primary site of energy storage. This storage function involves expansion of WAT through adipocyte hyperplasia (increase in cell number) and adipocyte hypertrophy (increase in cell size). Moreover, it is known that adipocyte hypertrophy is closely associated with WAT inflammation (Muder et al. Sci Rep. 2016).

The disease can be silent for a long period of time, but once it accelerates, severe damage and usually also liver cirrhosis can occur, which can significantly impact liver function or can even result in liver failure or liver cancer. Typical risk factors for NASH include obesity, elevated levels of blood lipids (such as cholesterol and triglycerides) and diabetes. The frequency of this disease has become increasingly common due to consumption of carbohydrate-rich and high- fat diets. However, no efficient and safe curative or specific therapies are currently available (G. C. Farrell and C. Z. Larter, Hepatology , 2006, 43, 99-112). The current treatment for NASH patients with end-stage disease is liver transplant. NASH has become the second indication for liver transplantation in the US and should become the first one in the short term (Wong et al., Gastro 2015). In addition, cirrhosis due to NASH increases the risk of hepatocellular carcinoma and hepatocellular cancer, although these have also been observed among patients having non cirrhotic NASH. Cardiovascular mortality is also a major cause of death in NASH patients.

The NAS is a score that was developed as a tool to measure changes in NASH during therapeutic trials. This score is calculated as the underweighted sum of the score of steatosis (0- 3), lobular inflammation (0-3) and ballooning (0-2). In particular, steatosis score can be evaluated to monitor the progression of NASH.

The underlying pathophysiologic mechanisms that contribute to the development and progression of NAFLD and NASH are complex and reflected by the myriad of therapies, with different targets, currently under investigation.

Specifically, numerous drugs targeting key-steps in NAFLD pathogenesis are under investigation. These compounds can be grouped in medications targeting (1) metabolic derangements including excess bodyweight, (2) inflammation and oxidative stress, and (3) dysregulation of the gut-liver axis. In this regard incretin agonists, including GIP agonists and especially GLP-1 agonists, exhibit potent metabolic effects, although they might also affect other of the proposed targets.

Incretins are indeed a group of gastrointestinal hormones that are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake. It has been suggested that patients with NAFLD exhibit a reduced incretin effect (reduced beta cell sensitivity to GIP and/or GLP- 1) which may play a role in the pathophysiology of NAFLD. Thus, targeting of the GLP-1 and/or GIP receptors with suitable agonists offers an attractive approach for treatment of NAFLD/NASH.

However, it is well-known that GLP1 treatments are associated with nausea, vomiting, and/or diarrhea. For example, one study reported that GLP-1 receptor agonist dosing regimens significantly increased the incidence of gastrointestinal adverse events ( Diabetes Technol Ther. 2015 Jan;17(l):35-42). Also, previous clinical trials of a GIP/GLP1 co-agonist compound have been performed and found that tolerability at high doses was limited by gastrointestinal adverse events (Schmitt, C. et al. , Diabetes Obes. Metab. 2017; 19: 1436- 1445 and also Portron, A. et al “, 2390-PUB, A624, ADA-2017). The dose limitation associated with gastrointestinal adverse events may prevent dosing to the desired effective dose, may compromise patient compliance with treatment, and may limit the effectiveness of the treatment regimen.

Therefore, there remains the need for improved treatment of liver disease, such as improved efficacy and/or better tolerance of the treatment.

SUMMARY OF THE INVENTION

It has now been found that the combination of a specific thienopyridone derivative with an incretin agonist led to an improved treatment of liver diseases, such as NASH.

More specifically, it has now been found that the combination of a specific thienopyridone derivative with an incretin agonist led to higher efficacy in the treatment of NASH, which is reflected by reduced liver steatosis, including reduced lipid droplets in hepatocytes, reduced liver steatosis score, reduced liver weight, reduced WAT weight and reduced plasma ALT and AST levels of the combination compared to each of these compounds alone.

Such drug combination allows reducing the effective amount of at least one of said compounds while obtaining a satisfactory therapeutic effect and/or obtaining a higher therapeutic effect of the liver disease.

Thus, the invention relates to a pharmaceutical combination comprising:

(a) a thienopyridone derivative which is the compound of formula (I):

(I), or one of its pharmaceutically acceptable salts and/or solvates and

(b) at least one incretin agonist.

It also relates to this combination for use as a medicament and more specifically for use in the treatment of a liver disease such as diabetes, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steato-hepatitis (NASH) and liver fibrosis, preferably non-alcoholic steatohepatitis (NASH), including cirrhotic and non-cirrhotic NASH.

This invention further pertains to the use of this combination in the manufacture of a pharmaceutical composition intended for the treatment of a liver disease.

It is also directed to a method for treating a liver disease, comprising administering to a patient an effective amount of this combination.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Steatosis score improvement in DIO-NASH mice treated with different compounds. Percentage of animals with improved steatosis score was assessed for each group of treated mice, by comparing pre-treatment and post-treatment (or termination) samples. Figure 2: Steatosis quantification in DIO-NASH mice treated with different compounds. The mean area fraction associated with steatosis, relevant to liver lipid content, was assessed for each group of treated mice, from termination samples.

Figure 3: Lipid droplets-containing hepatocytes in DIO-NASH mice treated with different compounds. Percentage of LD-containing hepatocytes was assessed for each group of treated mice, from termination sample.

Figure 4: Liver weight of DIO-NASH mice treated with different compounds. The liver weight was measured for each group of treated mice, from termination dissection.

Figure 5: Epididymal White Adipose Tissue (eWAT) weight of DIO-NASH mice treated with different compounds. The eWAT weight was measured for each group of treated mice, from termination dissection.

Figure 6: Plasma ALT levels of DIO-NASH mice treated with different compounds. Figure 7: Plasma AST levels of DIO-NASH mice treated with different compounds. DETAILED DESCRIPTION OF THE INVENTION

The combination of this invention comprises as a first ingredient a thienopyridone derivative which is the compound of formula (I):

or one of its pharmaceutically acceptable salts and/or solvates.

These compounds, which are direct activators of AMPK (AMP-activated protein kinase), as well as their preparation process, have been described in WO 2014/001554, the content of which is incorporated herein by reference.

Examples of pharmaceutically acceptable salts of the compound of formula (I) can be obtained by reacting the compound of formula (I) with various organic and inorganic bases by procedures usually known in the art to give the corresponding base-addition salt. Such bases are, for example, alkali metal hydroxides, including potassium hydroxide, sodium hydroxide and lithium hydroxide; alkali metal carbonates, including potassium carbonate and sodium carbonate; alkaline earth metal hydroxides, such as barium hydroxide and calcium hydroxide; alkaline earth metal carbonates; alkali metal alkoxides, for example potassium ethoxide and sodium propoxide; and various organic bases, such as piperidine, diethanolamine and N-methylglutamine. The aluminium salts of the compounds of formula (I) are likewise included.

The salts of the compound of formula (I) thus include aluminum, ammonium, calcium, copper, iron(III), iron(II), lithium, magnesium, manganese(III), manganese(II), potassium, sodium and zinc salts, but this is not intended to represent a restriction. Of the above-mentioned salts, preference is given to the mono-, di- and tri- sodium or potassium salts and most preferably to the potassium salts. Any of the pharmaceutically acceptable salts of the compound of formula (I), or this compound itself, may be used in this invention in the form of one of its solvates. “Solvates” of the compounds are taken in the present invention to mean adductions of inert solvent molecules onto the compounds which form owing to their mutual attractive force. The nature of the solvate thus depends on the solvent used during the reaction of the base with the compound of formula (I). Examples of solvates include alcohol solvates, for instance methanol or ethanol solvates, and hydrates, including mono-, di-, tri- or tetrahydrates, but this is not intended to represent a restriction. In a most preferred embodiment of this invention, the compound used in this invention is the monohydrate monopotassium salt of the compound of formula (I), corresponding to the following structure (la):

This compound, also referred to as PXL770, may be prepared according to a process comprising the steps of:

(A) reacting a compound of formula (I) with potassium carbonate in a solution comprising water and a solvent selected from /7-butyl acetate and isopropanol:

(B) forming a precipitate; and (C) recovering the precipitate obtained in step (B), preferably by filtration.

The compound of formula (I) and a preparation process thereof have been disclosed in patent application WO 2014/001554.

Alternatively, said compound of formula (I) may be obtained by an improved process comprising the steps of:

(a) reacting 6-acetyl-5-hydroxytetralin with an electrophilic benzyl source, preferably benzyl bromide, in the presence of a base;

(b) reacting the compound obtained in step (a) with ethyl cyanoacetate in the presence of hexamethyldisilazane and acetic acid;

(c) reacting the compound obtained in step (b) with sulfur in the presence of a base;

(d) optionally forming a salt of the compound obtained in step (c), preferably a hydrochloride salt;

(e) reacting the compound obtained in step (c) or (d) with an electrophilic chlorine source, preferably N-chlorosuccinimide;

(f) reacting the compound obtained in step (e) with phenylacetyl chloride;

(g) reacting the compound obtained in step (f) with a base;

(h) reacting the compound obtained in step (g) with boron tribromide or trichloride, preferably boron trichloride; and

(i) optionally recovering the compound obtained in step (h).

Typically, step (B) can comprise a substep (bl) of heating the mixture obtained in step (A), preferably at a temperature close to reflux of the mixture, followed by a sub step (b2) of cooling the resulting mixture, for instance at a temperature comprised between -15 °C and 35 °C. The expression “close to reflux of the mixture” refers typically to a temperature comprised between 90% and 100 % of the boiling point of the solvent system in step (A) (for instance, water/isopropanol or water/n-butyl acetate).

A distillation step, preferably under reduced pressure, can be carried out between the heating substep and substep (b2).

Step (B) allows a crystalline precipitate to form, which formation may be favored or triggered by adding seeds to steps (b2). In a preferred embodiment, said precipitate is recovered by filtration in step (C). It may then be washed successively with one or more solvents, preferably water, /7-butyl acetate and/or tert- butyl methyl ether. The compound of formula (la) is thus obtained in the form of a solid, such as a powder, having the following XRPD (X-Ray Powder Diffraction) peaks, as measured by means of a diffractometer, using Cu K(alpha) radiation:

In the following description, the wording "the thienopyridone derivative" refers to the compound of formula (I) or to one of its pharmaceutically acceptable salts and/or solvates.

In this invention, the thienopyridone derivative is combined with at least one incretin agonist. As used herein, “incretin agonist” refers to any agent that binds to and activates downstream signaling of the GLP-1 and/or GIP receptor. The incretin agonist used in this invention is thus selected from GLP-1 agonists, GIP agonists and GLP-1 / GIP co-agonists.

In some embodiments of the present disclosures, the incretin agonist exhibits at least or about 0.1% activity of native GIP and/or native GLP-1 at the GIP and/or GLP-1 receptor, respectively.

In exemplary embodiments, the incretin agonist exhibits at least or about 0.2%, at least or about 0.3%, at least or about 0.4%, at least or about 0.5%, at least or about 0.6%, at least or about 0.7%, at least or about 0.8%, at least or about 0.9%, at least or about 1%, at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 100% of the activity of native GIP and/or native GLP-1 at the GIP and/or GLP-1 receptor, respectively.

In some embodiments of the present disclosures, the incretin agonist exhibits activity at the GIP and/or GLP-1 receptor which is greater than that of native GIP and/or GLP-1, respectively. In exemplary embodiments, the incretin agonist exhibits at least or about 101%, at least or about 105%, at least or about 110%, at least or about 125%, at least or about 150%, at least or about 175% at least or about 200%, at least or about 300%, at least or about 400%, at least or about 500% or higher % of the activity of native GIP and/or GLP-1 at the GIP and/or GLP-1 receptor, respectively. A compound's activity at the GIP and/or GLP-1 receptor relative to native GIP and/or GLP-1, respectively, is calculated as the inverse ratio of EC50s for the GIP and/or GLP- 1 agonist vs. native GIP and/or GLP-1, respectively. In some embodiments, the incretin agonist exhibits an EC50 for GIP and/or GLP-1 receptor activation which is in the nanomolar range. In exemplary embodiments, the EC50 of the GIP and/or GLP-1 agonist at the GIP and/or GLP-1 receptor is less than 1000 nM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM. In some embodiments, the EC50 of the incretin agonist at the GIP and/or GLP-1 receptor is about 100 nM or less, e.g., about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less. In some embodiments, the incretin agonist exhibits an EC50 for GIP and/or GLP-1 receptor activation which is in the picomolar range. In exemplary embodiments, the EC50 of the incretin agonist at the GIP and/or GLP-1 receptor is less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM. In some embodiments, the EC50 of the incretin agonist at the GIP and/or GLP-1 receptor is about 100 pM or less, e.g., about 75 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 8 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less, or about 1 pM or less. Receptor activation can be measured by in vitro assays measuring cAMP induction in HEK293 cells over- expressing the GIP and/or GLP-1 receptor, e.g. assaying HEK293 cells co-transfected with DNA encoding the receptor and a luciferase gene linked to cAMP responsive element. In some embodiments of the present disclosures, the incretin agonist is a co-agonist insofar as it activates both the GIP and the GLP-1 receptors. In some embodiments, the EC50 of the incretin agonist at the GIP receptor is within about 50- or less fold (higher or lower) than the EC50 of the incretin agonist at the GLP-1 receptor. In some embodiments, the EC50 of the incretin agonist at the GIP receptor is within about 40-fold, about 30-fold, about 20-fold (higher or lower) from its EC50 at the GLP-1 receptor. In some embodiments, the GIP potency of the incretin agonist is less than or about 25-, 20-, 15-, 10-, or 5-fold different (higher or lower) from its GLP-1 potency. In some embodiments, the ratio of the EC50 of the incretin agonist at the GIP receptor divided by the EC50 of the incretin agonist at the GLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the GIP potency of the incretin agonist compared to the GLP- 1 potency of the incretin agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the EC50 of the incretin agonist at the GLP-1 receptor divided by the EC50 of the incretin agonist at the GIP receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the ratio of the GLP-1 potency of the incretin agonist compared to the GIP potency of the incretin agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments, the selectivity of the incretin agonist does not have at least 100- fold selectivity for the human GLP-1 receptor versus the GIP receptor. In exemplary embodiments, the selectivity of the incretin agonist for the human GLP-1 receptor versus the GIP receptor is less than 100-fold (e.g., less than or about 90-fold, less than or about 80- fold, less than or about 70-fold, less than or about 60-fold, less than or about 50-fold, less than or about 40-fold, less than or about 30-fold, less than or about 20-fold, less than or about 10-fold, less than or about 5-fold).

The incretin agonist may be selected for example from albiglutide (GSK-716155), dulaglutide, liraglutide (NN-2211), semaglutide, taspoglutide (R-1583), exenatide, lixisenatide, loxenatide, epfeglenatide, peptides such as those disclosed in WO 2019/030268, tirzepatide (LY-3298176), MKC-253, CJC-1134PC and AVE-0010, preferably the incretin agonist is semaglutide; without any limitation.

A preferred incretin agonist for use in this invention is semaglutide.

The pharmaceutical combination comprising the incretin agonist and the thienopyridone derivative may be used as a medicament and more specifically in the treatment of a liver disease, such as diabetes, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and liver fibrosis, preferably non-alcoholic steatohepatitis (NASH), including cirrhotic and non-cirrhotic NASH.

The term "treatment" or “treating” refers to therapy, prevention and prophylaxis of a liver disease. The treatment involves the administration of the pharmaceutical combination to a subject having a declared liver disorder to cure the disease or to delay its outcome or slow down its progress, thus improving the condition of patient. More specifically, the term treatment includes preventing or reducing the risk of developing liver disease, such NAFLD or NASH, i.e., causing the clinical symptoms of NAFLD or NASH not to develop in a subject who may be predisposed to NAFLD or NASH but who does not yet experience or display symptoms of the NAFLD or NASH (i.e. prophylaxis). In another embodiment, the term treatment includes inhibiting liver disease, such as NAFLD or NASH, i.e., arresting or reducing the development of NAFLD or NASH or its clinical symptoms. In another embodiment, the term treatment includes relieving liver disease, such as NAFLD or NASH, i.e., causing regression, reversal, or amelioration of the NAFLD or NASH or reducing the number, frequency, duration or severity of its clinical symptoms. This term thus broadly includes “preventing, delaying or treating.” Within the context of the invention, the term “subject” or “patient” means a mammal and more particularly a human.

According to an embodiment of the invention, (i) the incretin agonist and (ii) the thienopyridone derivative are administered simultaneously or sequentially, in the form of separate pharmaceutical compositions, one comprising the incretin agonist in a pharmaceutically acceptable vehicle, the other comprising the thienopyridone derivative in a pharmaceutically acceptable vehicle. In another embodiment, the incretin agonist and the thienopyridone derivative are combined and administered in the same pharmaceutical composition. In the context of the present invention, the terms “pharmaceutical combination” and “combined administration” refers to one or the other of these aspects.

The term “pharmaceutically acceptable support” refers to carrier, adjuvant, or excipient acceptable to the subject from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding to composition, formulation, stability, subject acceptance and bioavailability. The term "carrier", “adjuvant”, or "excipient" refers to any substance, not itself a therapeutic agent, that is added to a pharmaceutical composition to be used as a carrier, adjuvant, and/or diluent for the delivery of a therapeutic agent to a subject in order to improve its handling or storage properties or to enable or facilitate formation of a dosage unit of the composition into a discrete article. The pharmaceutical compositions used in this invention, either individually or in combination, can comprise one or several agents or vehicles chosen among dispersants, solubilizers, stabilisers, preservatives, etc.

In more detail, “combined administration” means, for the purpose of the present invention, fixed and, in particular, free combinations, i.e. either the incretin agonist and the thienopyridone derivative are present together in one dosage unit, or the incretin agonist and said compound, which are present in separate dosage units, are administered successively, either immediately or at a relatively large time interval; a relatively large time interval means a time span up to a maximum of 24 hours. A “dosage unit” means, in particular, a medicinal dosage form in which the release of the active ingredient(s) is achieved with as few problems as possible, such that the two active-ingredient components (the incretin agonist on the one hand and said thienopyridone derivative on the other hand) are released, or made available effectively for the body, in such a way that an optimal active ingredient profile, and thus action profile, is achieved.

The separate dosage units are preferably made available together in one pack and either mixed prior to administration or sequentially administered. For example, the two dosage units may be packed together in blister packs that are designed with regard to the relative arrangement of the two dosage units with respect to one another, the inscription and/or coloring in a manner known per se so that the times for taking the individual components (dosage regimen) of the two dosage units are evident to a patient. This free combination is of benefit by individually allotting an effective amount of both compounds to the patient. Another possibility is the provision of single preparations of both compounds, i.e. being independent medicaments. The single preparations are converted to contain the required amounts of ingredient for the inventive combination. Corresponding instructions are given at the package insert concerning the combined administration of the respective medicament.

As evident from the foregoing, the invention may be practiced as a pharmaceutical package comprising as active ingredients an effective amount of the incretin agonist, together with one or more pharmaceutically acceptable adjuvants, in a first dosage unit, and an effective amount of the thienopyridone derivative as defined above, together with one or more pharmaceutically acceptable adjuvants, in a second dosage unit. This package may include an article that comprises written instructions or directs the user to written instructions for how to practice the method of the invention. The prior teaching of the present specification concerning the composition and its administration is considered as valid and applicable without restrictions to the pharmaceutical package if expedient.

The dosage units mentioned above can comprise, for example, 0.5 mg to 1000 mg, preferably 20 mg to 1000 mg, more preferably 60 mg to 500 mg, of the thienopyridone derivative, and 0.01 mg to 1000 mg, preferably 0.05 mg to 1000 mg, more preferably 0.1 mg to 500 mg, of the incretin agonist, depending on the disease condition treated, the method of administration and the age, weight and condition of the patient. Preferred dosage unit formulations are those which comprise a daily dose or a corresponding fraction thereof of an active ingredient. Furthermore, pharmaceutical compositions of this type can be prepared using a process which is generally known in the pharmaceutical art.

For simultaneous administration as fixed composition, a single pharmaceutical formulation may also be prepared which includes both ingredients. This invention is thus also directed to the use of (i) the incretin agonist, and (ii) the thienopyridone derivative as defined above in the manufacture of a pharmaceutical composition intended for the treatment of a liver disease.

The ratio between each of the thienopyridone derivative, the incretin agonist and the pharmaceutically acceptable support may be comprised in a wide range. In particular, this ratio may be comprised between 1/99 (w/w) and 99/1 (w/w), preferably between 10/90 (w/w) and 90/10 (w/w).

This invention is further directed to a method for treating a liver disease, comprising administering to a patient an effective amount of a combination of (i) an incretin agonist, and (ii) the thienopyridone derivative as defined above.

The pharmaceutical combination of this invention may be suitable for administration via any desired appropriate route, for example by oral (including buccal or sublingual), topical (including buccal, sublingual or transdermal), or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) methods. Preferably, the pharmaceutical combination according to the invention is suited for oral administration.

Pharmaceutical compositions and dosage units suitable for oral administration of the pharmaceutical combination include capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam foods; or emulsions, such as oil-in-water liquid emulsions or water-in-oil liquid emulsions.

The therapeutically effective amount of the compounds included in the pharmaceutical combination of this invention depends on a number of factors, including, for example, the age and weight of the human or animal, the precise disease condition which requires treatment, and its severity, the nature of the formulation and the method of administration, and is ultimately determined by the treating doctor. For instance, the thienopyridone derivative may be administered once or twice a day at a daily dose of 0.5 mg to 300 mg for a human patient, preferably from 20 mg to 1000 mg, more preferably from 60 mg to 500 mg. It can be administered 4, 5, 6 or 7 days a week as a long-life medication. In addition, the incretin agonist may be administered orally at a daily dose in the range of from 0.1 to 100 mg, preferably from 1 to 50 mg and still preferably from 5 to 20 mg, where this amount can be administered as an individual dose per day or in a series of part-doses (such as, for example, two, three, four, five or six) per day, so that the total daily dose is the same. Alternatively, the incretin agonist may be administered subcutaneously at a weekly dose in the range of from 0.01 to 50 mg, preferably from 0.05 to 10 mg, still preferably from 0.1 to 5 mg, where this amount may be administered as an individual dose per week or usually in a series of part-doses (such as, for example, two, three, four, five, six or seven) per week, so that the total weekly dose is the same.

The invention will also be described in further detail in the following examples, which are not intended to limit the scope of this invention, as defined by the attached claims.

EXAMPLES

Abbreviations a/a : ratio of the peak area of a given compound to the total of the peak areas on a spectrum or a chromatogram. eq Analytical methods

XRPD

X-Ray Powder Diffraction (XRPD) analyses were performed using a Panalytical Xpert Pro diffractometer equipped with a Cu (K alpha radiation) X-ray tube and a Pixcel detector system. The samples were analysed in transmission mode and held between low density polyethylene films. XRPD patterns were sorted, manipulated and indexed using HighScore Plus 2.2c software.

TG/DTA

Thermogravimetric (TG) analyses were carried out on a Perkin Elmer Diamond Thermogravimetric/Differential Temperature Analyser (TG/DTA). The calibration standards were indium and tin. Samples were placed in an aluminium sample pan, inserted into the TG furnace and accurately weighed. The samples were heated from 30-300°C in a stream of nitrogen at a rate of 10°C/minute. The temperature of the furnace was equilibrated at 30°C prior to the analysis of the samples.

6-Acetyl-5-hydroxytetralin (100 g, 1 eq.) was dissolved in acetonitrile (300 mL). After addition of K2CO3 (1.1 eq.) and benzyl bromide (1.05 eq.), the suspension was heated (76°C). After 48 hours, benzyl bromide (0.1 eq) was added. After overall 74 hours, the solid was filtered off and washed with acetonitrile (200 mL), and the combined filtrates were evaporated.

Compound 1 was obtained as a syrup: m = 148.6 g, quantitative yield, 96.6% a/a purity. lb) Synthesis of ethyl 2-amino-4-(5-benz.yloxytetralin-6-yl)thiophene-3-carboxylate (2)

Afterwards, a solution of compound 1 (69.5 g, 1 eq.) and ethyl cyanoacetate (1.5 eq.) in acetic acid (140 mL) was added. The resulting mixture was stirred at T = 65°C for 24 h.

After cooling to room temperature, aqueous NaOH (1 M, 140 mL) and TBME (210 mL) were added. The layers were separated. The organic layer was washed with aqueous NaOH (1 M, 4 x 140 mL) until the pH of the aqueous phase was basic (pH = 13). The organic layer was washed with aqueous HC1 (1M, 140 mL) and H2O (2 x 140 mL).

EtOH (240 mL), NaHC03 (1.3 eq.) and sulfur (1.0 atom eq.) were added. After heating to reflux for 180 min, the reaction mixture was concentrated to 210 mL and co-evaporated with TBME (3 x 140 mL). After cooling to room temperature, the suspension was filtered and the solid was washed with TBME (70 mL). The combined filtrates were concentrated to 210 mL and HC1 in dioxane (1.1 eq.) was added dropwise at room temperature. After seeding, precipitation was observed. Heptane (350 mL) was added dropwise at room temperature. After stirring for 14 h, the suspension was filtered. After washing with heptane (3 x 70 mL) and drying, compound 2 was recovered as a solid m = 83.2 g, 71% yield, 93.7% a/a purity. lc) Synthesis of ethyl 4-(5-benz.yloxytetralin-6-yl)-5-chloro-2-f(2-phenylacetyl)amino] thiophene-3 -carboxylate ( 3 )

Compound 2 (17.69 g, 1 eq.) was dissolved in dichloromethane (140 mL). The resulting solution was cooled with ice/water. Under stirring, N-chlorosuccinimide (1.05 eq.) was added. The mixture became dark over a few minutes. After 1 h, phenylacetyl chloride (1.25 eq.) was added.

After 1 hour at 0 °C and 2 hours at room temperature, the mixture was evaporated down to ca. 35 mL and EtOH (2 x 70 mL) was added, and evaporated down again. The mixture was diluted with EtOH (35 mL) and cooled with ice/water. The product precipitated. The solid was filtrated and washed with cold EtOH (3 x 18 mL).

Compound 3 was obtained as a solid: m = 20.99 g, 94.2 % yield, 99.3 % a/a purity.

Id) Synthesis of 3-(5-benzyloxytetralin-6-yl)-2-chloro-4-hvdroxy-5-phenyl-7H-thieno[2,3- blpyridin-6-one (4)

reaction mixture was cooled to a temperature between -16°C and -10 °C (NaCl/Ice). Potassium tert-butoxide (5 eq.) was added in four portions. Then, the reaction mixture was warmed up to room temperature, and stirred for 65 min at room temperature. A dropwise addition of 2N HC1 (5 eq.) was carried out at T = 0-5°C (water/ice) and the resulting mixture was stirred vigorously. The organic phase was washed with NaCl(aq) (11%, 1 x 50 mL) and water (2 x 50 mL). The organic phase was concentrated to -50% solution. Methyltetrahydrofuran (80 mL) was added, and the resulting solution was concentrated to -50% solution. TBME (100 mL) was added, and the resulting solution was concentrated to -50% solution (this step was repeated 3 times). Then, TBME (25 mL), seeds of compound 4 and n-Heptane (20 mL) were added and the resulting solution was stirred at room temperature overnight. The mixture was concentrated to ca. 50 mL, filtrated, rinsed with mother liquor and washed with n-Heptane (2 x 40 mL) and dried. Compound 4 was obtained as a granular solid. Yield 88 %, 99.5 % a/a purity. le) Synthesis of 2-chloro-4-hydroxy-3-(5-hydroxytetralin-6-yl)-5-phenyl-7H-thieno[2,3- blpyridin-6-one (I)

Compound 4 (15 g, 1 eq.) was dissolved in 75 mL of dichloromethane and was cooled to T = -10°C/-15°C (with ice/NaCl). BCb (1.5 eq., solution: 1 mol/L in dichloromethane) was added dropwise and the resulting mixture was stirred at room temperature for 15 hours. The resulting mixture was cooled with ice/water, and water (75 mL) was added. The resulting mixture was stirred vigorously and the organic phase was extracted with water/MeOH (9: 1 v/v, 5 x 45 mL.). The organic phase was concentrated, a solvent swap was carried out with toluene (3 x 90 mL) and diluted with toluene to reach a final volume of 90 mL of toluene. The resulting mixture was heated to reflux and 15 mL of methanol was added. A brownish solution with few particles was obtained. Seeds were added at T = 40 °C, warmed to T = 52°C and cooled to room temperature. The resulting mixture was stirred overnight, and then was cooled with ice/NaCl (T = -10°C/-15°C) for 100 minutes. The precipitated product was filtrated, washed with toluene/heptane 1 :2 v/v (15 mL) and heptane (15 mL) and dried. Crystals of compound (I) were obtained: 87 % yield, 99.0 % a/a purity.

If) Synthesis of the monohydrate potassium salt of2-chloro-4-hydroxy-3-(5-hydroxytetralin-6- yl)-5-phenyl-7H-thieno[2,3-b]pyridin-6-one (la)

Compound (I) was suspended in water/isopropanol mix (1/1, 5 parts of each solvents) then 0.50 to 0.55 eq of potassium carbonate was added. The pH was about 12 (pH indicator paper) at the end of the addition of potassium carbonate. After 3 hours of stirring at 50°C, the suspension was thicker and the pH was about 8 (pH indicator paper). The temperature was raised to 80 °C until a solution was obtained (10-15 minutes). A clarification can be done at this point of the process if required. 7 parts of water were added and the reaction mixture was then cooled to 40°C (turbid solution observed). The solvent was distilled under reduce pressure (from 180mbar to 40mbar) at 40°C until 7 parts of solvents remained in the reactor. Crystallization of potassium salt monohydrate may occur here. 4.2 parts of water were added and the mixture was seeded with compound (I) (1 to 2% of seeds). The suspension was then cooled down from 40°C to 5°C in 7 hours (5°C/hour) and kept at 5°C for several hours. The suspension was filtered. The cake was washed twice by 1.42 parts of water. The collected solid was dried at 40°C under vacuum given minimum 80% yield of Compound (la), at required chemical purity (i.e. 98%+).

Example 2: Characterization of compound (la) a) X-ray powder diffraction (XRPD) data of compound (la) indicated that it was composed of a crystalline material. The XRPD description of compound (la) is shown in Table 1.

Table 1

b) TG/DTA analysis showed an initial weight loss of 1.1% from 30-100°C, followed by larger weight loss of 3% from 117-160°C due to loss of bound water. The second weight loss was accompanied by a large endotherm and the combined weight losses of 4% approximate the theoretical weight loss for a monohydrate (3.75% w/w). The compound decomposed above

240°C.

Example 3 : Biological effects of compound (la) in combination with an incretin agonist on DIO-NASH mice.

Abbreviations

BID (bis in die) : twice a day DAB : 3,3'-Diaminobenzidine eq. : equivalent eWAT : epididymal white adipose tissue H&E : hematoxylin & eosin HRP : horseradish peroxidase IHC : immunohistochemistry LD : lipid droplet PO : per os

QD ( quaque die) : once a day SC : subcutaneous ALT : alanine aminotransferase AST : aspartate aminotransferase

Material and Methods

Study on DIO-NASH mice

DIO-NASH mice model Mice (C57BL/6J, male) were feed with a High Fat High Fructose diet (40% fat, 20% fructose and 2% cholesterol - D09100301, Research diets, New Jersey) for 34 weeks to induce a NASH phenotype. They were then treated for 8 weeks while kept on high fat diet.

Liver pre-biopsy

Before the administration of the different compounds, and after 30 weeks of specific diet (week -4), mice were anesthetized by inhalation anesthesia using isoflurane (2-3%). A small abdominal incision was made in the midline and the left lateral lobe of the liver was exposed. A cone shaped wedge of liver tissue (approximately 50 mg) was excised from the distal portion of the lobe and fixated in 10% neutral buffered formalin (10% NBF) for histology. The cut surface of the liver was instantly electrocoagulated using bipolar coagulation (ERBE VIO 100 electrosurgical unit). The liver was returned to the abdominal cavity, the abdominal wall is sutured, and the skin was closed with staplers. For post-operative recovery mice received carprofen (5mg/kg) administered subcutaneously on OP day and post-OP day 1 and 2.

Randomization and division into four groups

Mice with fibrosis stage >1 and steatosis score >2 (see Table 4) were selected for randomization. A stratified randomization into treatment groups was performed according to liver Collagen lal quantification of pre-biopsies taken at week -4, i.e. 4 weeks before the beginning of compounds administration. Mice were then divided into four groups to receive different administration of compounds (as detailed in Table 2), each diluted in the same vehicle solution (Carboxy methylcellulose 0.5% / Tween 80 (98/2 in volume).

Table 2 : DIO-NASH mice groups

Termination and tissue sampling

After the animal has been terminated by heart puncture under isoflurane anesthesia, the liver was collected and weighed. Specific liver samples were dissected and processed as specified in table 3 and further described below. The left lateral lobe was used for the pre-biopsy (not applicable at termination) and a termination biopsy. The liver termination biopsy (-200 mg, less than 0.7 x 0.5 cm) was cut 4 mm from the prebiopsy site and with an edge. The tissue was collected in paraformaldehyde (4%).

Table 3 : Tissue sampling

Serological analysis

Blood samples were collected in heparinized tubes and plasma was separated and stored at - 80°C until analysis. Liver injury markers, i.e. ALT and AST, were measured using commercial kits (Roche Diagnostics) on the Cobas c 501 Autoanalyzer, according to the manufacturer's instructions.

Histological Methods Histological staining procedures

In brief, slides with paraffin embedded sections are de-paraffmated in xylene and rehydrated in series of graded ethanol.

• H&E staining

The slides are incubated in Mayer’s Hematoxylin (Dako), washed in tap water, stained in Eosin Y solution (Sigma-Aldrich), hydrated, mounted with Pertex and then allowed to dry before scanning.

• Type I collagen IHC staining

Type I collagen (Southern Biotech, Cat. 1310-01) IHC are performed using standard procedures. Briefly, after antigen retrieval and blocking of endogenous peroxidase activity, slides are incubated with primary antibody. The primary antibody is detected using a polymeric HRP -linker antibody conjugate. Next, the primary antibody is visualized with DAB as chromogen. Finally, sections are counterstained in hematoxylin and cover-slipped. Steatosis score

Liver samples were fixed in formalin, paraffin embedded, and sections were stained with H&E. Steatosis percentage was evaluated in samples by a specialist using of the clinical criteria outlined by Kleiner et al. 2005. The steatosis score then corresponds to a range of percentage of steatosis and ranges from 0 to 3 (see Table, steatosis line). Steatosis score was assessed for pre-biopsy samples and termination biopsy samples.

Table 4 : Evaluation of steatosis score

(adapted from Kleiner et al. ; Hepatology, 41 ; 2005)

Quantitative assessment of steatosis

Steatosis was quantified on H&E stained slides by image analysis using the VIS software (Visiopharm, Denmark). VIS protocols are designed to analyze the virtual slides in two steps: Firstly, crude tissue is detected at low magnification (1 x objective). Then, steatosis (blue) and tissue (black) are detected at high magnification (20 x objective). The quantitative estimates of steatosis were calculated as an area fraction in the following way:

Area fraction steatosis = Areasteatosis / Areanssue + Areasteatosh

Quantification of hepatocytes with lipid droplets

Deep learning-based image analysis was used to quantify the percent of hepatocytes containing lipid droplets, at termination.

Statistical analysis

Comparison of data and significance different for multiple testing will be visualized on the graph when p<0.05.

A one-way ANOVA with Tukey ’ s multiple comparisons test was performed to assess the effect of the combination, except for the serological analysis for which Student t-test was used for the comparison of the combination therapy with the monotherapies. Results:

The effects of compound (la) (or “PXL770”) alone, or combined with Semaglutide, on histological parameters of the liver of DIO-NASH mice, are reported here.

After 34 weeks, only DIO-NASH mice with biopsy-confirmed steatosis (score >2) and fibrosis (stage >1) were selected. Mice with NASH phenotype were then divided into four groups and treated during 8 weeks with different compound(s) administrations : PXL770 (n=12), Semaglutide (n=13), PXL770 and Semaglutide at the same doses as for mice treated with a single compound (n=12), or a vehicle (n=13), for control.

In a first set of experiments, steatosis was quantified (Fig. 1-2) and LDs detected in hepatocytes (Fig. 3), using histological methods for each group of mice. Liver samples from the four mice groups were fixed in formalin, paraffin embedded, and sections were stained with H&E. Steatosis score was assessed using histological method (see Material and Methods section) and quantification was performed by image analysis using the VIS software. The improvement in the measured steatosis score was determined by comparing samples taken before (pre-biopsy) and after (termination dissection) administration of the different compounds. For each group of treated mice, percentage of animals with improved score was assessed and represented on a bar plot (Fig. 1). Compared to control mice treated with vehicle, percentage of animals with improved score was significantly higher for mice treated with PXL770 or Semaglutide. Among these two compounds, a higher proportion of mice exhibited a steatosis score improvement in the Semaglutide group. Interestingly, steatosis score improvement was even higher in mice treated with PXL770 and Semaglutide, as 100% of these mice exhibited such an improvement. In addition, histological method coupled with morphometry allowed to determine the steatosis associated-area fraction in termination samples, for each group of treated mice (Fig. 2). The quantitative estimates of steatosis, corresponding to liver lipid content, were calculated as an area fraction (% fractional area, see Material and Method section). Compared to Vehicle group lipid content, a significant lipid content decrease was observed for the PXL770 group of mice. Furthermore, liver lipid content decrease was higher when PXL770 and Semaglutide were administered in combination than with the drugs administered alone.

Using deep learning-based image analysis coupled to histological method, the percentage of hepatocytes containing LDs was determined for each group of treated mice, from termination samples (Fig. 3). Compared to Vehicle group, the percentage of LD-containing hepatocytes was slightly but significantly reduced in the PXL770 group of mice. Importantly, the LD-containing hepatocytes percentage decrease was higher when PXL770 and Semaglutide were administered in combination than with the drugs administered alone. Surprisingly, the decrease observed for the PXL770 + Semaglutide group seems to be the result of a synergistic effect between the two compounds.

In a second set of experiments, liver and eWAT were dissected to be weighed at termination. After an 8 weeks period of treatment (i.e. at termination), livers from each treated DIO-NASH mouse were weighed, and individual liver weights expressed as a percentage of the animal's weight. Then, the mean percentages of the different mice groups were represented on a bar plot (Fig. 4). Compared to Vehicle group, a significant decrease in relative liver weight was observed for the PXL770 group of mice. Moreover, relative liver weight decrease was higher when PXL770 and Semaglutide were administered in combination than with the drugs administered alone. It should be noted that the comparison of the Semaglutide group vs PXL770

+ Semaglutide group only tends to be significant (p= 0.0563).

The weight of the eWAT taken from each 8 weeks treated mouse was measured and expressed as a percentage of the animal's weight. For each group of mice, the mean weight value was represented on a bar plot (Fig. 5). Compared to Vehicle group, a significant decrease in eWAT weight was observed for the PXL770 group of mice. Also, the eWAT weight decrease was higher when PXL770 and Semaglutide were administered in combination than when the drugs were administered alone.

Finally, the analysis of liver injury markers showed that plasma ALT and AST levels were significantly reduced for mice treated with the combination of PXL770 and semaglutide, compared to mice treated with these drugs administered alone (see Figures 6 and 7, respectively).

Claims

1. A pharmaceutical combination comprising:

(a) a thienopyridone derivative, which is the compound of formula (I):

or one of its pharmaceutically acceptable salts and/or solvates, and (b) at least one incretin agonist.

2. The pharmaceutical combination of claim 1, wherein the thienopyridone derivative has the following structure:

3. The pharmaceutical combination of claim 1 or 2, wherein the incretin agonist is selected from the group consisting of GLP-1 agonists, GIP agonists and GLP-1 / GIP co-agonists.

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein the incretin agonist is selected from albiglutide (GSK-716155), dulaglutide, liraglutide (NN-2211), semaglutide, taspoglutide (R-1583), exenatide, lixisenatide, loxenatide, epfeglenatide, peptides, tirzepatide (LY-3298176), MKC-253, CJC-1134PC and AVE-0010, preferably the incretin agonist is semaglutide.

5. The combination according to any one of claims 1 to 4 for use as a medicament.

6. The combination according to any one of claims 1 to 4 for use in the treatment of a liver disease such as diabetes, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non alcoholic steato-hepatitis (NASH) and liver fibrosis, preferably non-alcoholic steatohepatitis (NASH), including cirrhotic and non-cirrhotic NASH.

7. The combination according to any one of claims 1 to 4 for use according to claim 5 or 6, wherein the thienopyridone derivative and the incretin agonist are administered simultaneously or sequentially, in the form of separate pharmaceutical compositions, one comprising the incretin agonist in a pharmaceutically acceptable vehicle, the other comprising the thienopyridone derivative in a pharmaceutically acceptable vehicle.

8. The combination according to any one of claims 1 to 4 for use according to claim 5 or 6, wherein the incretin agonist and the thienopyridone derivative are combined and administered in the same pharmaceutical composition.

9. Use of the combination according to any one of claims 1 to 4 in the manufacture of a pharmaceutical composition intended for the treatment of a liver disease, such as diabetes, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steato-hepatitis (NASH) and liver fibrosis, preferably non-alcoholic steatohepatitis (NASH), including cirrhotic and non-cirrhotic NASH.