Combination Therapy Comprising a Compound of Formula (I) and a GLP-1 Receptor Agonist

Abstract

The present invention relates to combination therapies comprising a PPAR agonist and a GLP-1 receptor agonist.

Description

[Technical field]

[0001] The present invention relates to combination therapies comprising a PPAR agonist and a GLP-1 receptor agonist. [Background technology]

[0002] Glucagon-like peptide-1 receptor agonists (i.e., GLP-1R agonists) are a class of compounds proposed for the treatment of metabolic diseases such as obesity and type 2 diabetes. Some compounds of this class have already been approved as medicines, such as semaglutide, liraglutide, exenatide, lixisenatide, albiglutide, and dulaglutide. Despite these approvals, this class of compounds could benefit from combination with other classes of compounds to improve their therapeutic effects. In addition, GLP-1R agonists cause adverse effects, such as gastrointestinal adverse effects. For example, common side effects of semaglutide and liraglutide include nausea, vomiting, diarrhea, abdominal pain, and constipation. A method for reducing these adverse effects would be advantageous. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2012080471 [Non-patent literature]

[0004] [Non-Patent Document 1] NASH Clinical Research Network (Kleiner, 2005; Brunt, 1999) Summary of the Invention [Means for solving the problem]

[0005] The present invention is based on the observation that the combination of GLP-1R agonist with PPAR agonist such as elafibranor can provide several beneficial effects, such as synergistic effects on adiposity, insulin levels and blood glucose, body weight regulation, and reduction of adverse effects.In addition, this combination is shown herein to open up the possibility of reducing the amount of GLP-1R agonist administered to a subject in need thereof, thereby reducing the adverse effects associated with GLP-1R agonist.

[0006] Thus, the present invention provides (i) PPAR agonists, in particular those of formula (I)

[0007] [ka]

[0008] (In the formula, Y1 represents a halogen atom, Ra, or a 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 each represents an oxygen or sulfur atom; Ra represents a hydrogen atom, an unsubstituted (C1-C6) alkyl group, a (C6-C14) aryl group or a (C1-C6) alkyl group substituted by one or more halogen atoms, a (C1-C6) alkoxy group 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), a (C1-C6) alkyl group which may or may not be substituted with one or more halogen atoms, a (C3-C14) cycloalkyl group, or a heterocyclic group; Y4 and Y5 are the same or different and each represents 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, and (ii) Glucagon-like peptide-1 (GLP-1) receptor agonists The present invention relates to a combination product comprising:

[0009] In a particular embodiment of the invention, Y1 represents a Ga-Ra group.

[0010] In another specific embodiment, Ra represents a (C1-C6) alkyl group or a (C3-C14) cycloalkyl group. In yet another embodiment, Ra represents a (C1-C6) alkyl group which may or may not be substituted by one or more halogen atoms, or Ra represents a (C3-C14) cycloalkyl group which may or may not be substituted by one or more halogen atoms.

[0011] In a further embodiment of the invention, Rb represents a (C1-C6) alkyl group substituted by a -COORc group, where Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. In another embodiment, Rc represents a hydrogen atom.

[0012] In certain embodiments of the compounds of formula (I), A represents a CH=CH group; Ra represents a (C1-C6) alkyl group or a (C3-C14) cycloalkyl group, in particular a (C1-C6) alkyl group or a (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 -COORc group (wherein Rc represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); Y4 and Y5 each independently represent a (C1-C4) alkyl group.

[0013] In certain embodiments of the compounds of formula (I), A represents a CH2-CH2 group, Ga represents an oxygen or sulfur atom, and Gb represents an oxygen atom; Ra represents a (C1-C6) alkyl group or a (C3-C14) cycloalkyl group; Rb represents a (C1-C6) alkyl group substituted with at least one -COORc group (wherein Rc represents a hydrogen atom or a (C1-C4) alkyl group); Y4 and Y5 each independently represent a (C1-C4) alkyl group.

[0014] In certain embodiments of the compounds of formula (I), A represents a CH2-CH2 group, Ga represents an oxygen or sulfur atom, and Gb represents an oxygen atom; Ra represents a (C1-C6) alkyl group or a (C3-C14) cycloalkyl group substituted by one or more halogen atoms; Rb represents a (C1-C6) alkyl group which is not substituted or substituted by one or more halogen atoms and which is substituted by at least one -COORc group (wherein Rc represents a hydrogen atom or a (C1-C4) alkyl group); Y4 and Y5 each represent a (C1-C4) alkyl group.

[0015] In a particular embodiment of the compound of formula (I), 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. In a particular variation of this embodiment, Rc represents a hydrogen atom.

[0016] In certain embodiments of the compounds of formula (I), Y1 is a (C1-C6)alkylthio group, including a (C1-C6)alkyl group that is linear or branched and unsubstituted or substituted with one or more halogen atoms. In one variation of this embodiment, Y1 is a methylthio group.

[0017] In certain embodiments, the compound of formula (I) is selected from the group consisting of 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one (elafibranor, ELA, or GFT505), 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-carboxydimethylmethyloxyphenyl]prop-2-en-1-one, 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, 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic acid, 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methyl-propanoic acid isopropyl ester, and pharma- ceutically acceptable salts thereof.

[0018] In a particular embodiment of the invention, component (i) is elafibranor or a pharma- ceutically acceptable salt thereof.

[0019] In the context of the present invention, the expression "glucagon-like peptide-1 (GLP-1) receptor agonist" refers to GLP-1 analogues and non-GLP-1 receptor agonists.GLP-1 receptor agonists (also described as GLP-1R agonists) are compounds that bind to and activate GLP-1 receptors.Exemplary GLP-1 receptor agonists include, but are not limited to, semaglutide, liraglutide, exenatide, albiglutide, dulaglutide, lixisenatide, loxenatide, efpeglenatide, taspoglutide, MKC-253, DLP-205, ORMD-0901, LY-3305677, long-acting oxyntomodulin, and pharmaceutically acceptable salts thereof.

[0020] In certain embodiments, component (ii) is semaglutide, liraglutide, exenatide, lixisenatide, albiglutide, dulaglutide, or a pharma- ceutically acceptable salt of one of these compounds.

[0021] In certain embodiments, component (ii) is semaglutide or a pharma- ceutically acceptable salt thereof.

[0022] In another particular embodiment, component (ii) is liraglutide or a pharma- ceutically acceptable salt thereof.

[0023] According to the present invention, components (i) and (ii) can be selected so that the combination of the compounds produces a synergistic effect. Such synergistic effect can be determined according to methods well known in the art, such as by using the Excess Over Bliss (EOB, i.e., Excess over Highest Single Agent) method. This method, adopted by the FDA for the approval of combination drug products, assumes that the predicted combination effect exceeds the effect obtained when the best components of the combination are administered individually. As demonstrated in the examples, the combination of elafibranor with a GLP-1R agonist, such as semaglutide or liraglutide, produces a synergistic effect on fatty liver (liver fat), triglyceride levels, weight loss, insulin levels, and blood glucose.

[0024] Thus, in a particular embodiment, component (i) is elafibranor or a pharma- ceutically acceptable salt thereof and component (ii) is semaglutide or a pharma- ceutically acceptable salt thereof.

[0025] In another particular embodiment, component (i) is elafibranor or a pharma- ceutically acceptable salt thereof and component (ii) is liraglutide or a pharma- ceutically acceptable salt thereof.

[0026] In certain embodiments, the combination product of the invention is a pharmaceutical composition comprising both components (i) and (ii) in a pharma- ceutically acceptable carrier.

[0027] In another embodiment, the combination product of the invention is a kit of parts comprising component (i) and component (ii) for sequential, separate or simultaneous use. In this embodiment, each of the components may be formulated as a different pharmaceutical composition.

[0028] The pharmaceutical compositions used in the present invention may contain one or several excipients or vehicles acceptable within the pharmaceutical context (e.g. physiological saline, physiological solutions, isotonic solutions, etc., compatible with pharmaceutical use and well known to the skilled artisan). These compositions may also contain one or several agents or vehicles selected from among dispersants, solubilizers, stabilizers, preservatives, etc. Useful agents or vehicles for these formulations (liquid and / or injectable and / or solid) are, inter alia, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, gum acacia, liposomes, etc. The compounds used in the present invention may be formulated for enteral or parenteral administration. For example, the compounds may be formulated for oral, intravascular (e.g. intravenous or intraarterial), intramuscular, intraperitoneal, subcutaneous, transdermal, or intranasal administration. The formulations may be in solid or liquid dosage form. Exemplary formulations include, but are not limited to, suspensions for injection or ingestion, gels, oils, pills, tablets, suppositories, powders, capsules, aerosols, ointments, creams, patches, or galenic forms of means for sustained and / or controlled release. For this type of formulation, agents such as cellulose, carbonates, or starches may be advantageously used.

[0029] The compounds embodied herein can be formulated as pharmaceutically acceptable salts, particularly salts of acids or bases compatible with pharmaceutical use. Salts of the compounds embodied herein include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium salts, and alkylated ammonium salts. These salts are obtained during the final purification step of the compound or by incorporating the salt into a previously purified compound.

[0030] As set out above, the active ingredients (i.e., components (i) and (ii) of the combination product) may be intended to be administered in one or more pharmaceutical compositions, such as in the form of a pill or tablet intended for oral ingestion.

[0031] In another embodiment, the active ingredient is for administration in one or more pharmaceutical compositions, such as in the form of an injectable solution.

[0032] In additional specific embodiments, the active ingredients are for administration as separate compositions.

[0033] In another embodiment, component (i) is in the form of an oral composition and component (ii) is in the form of an oral composition.

[0034] In certain embodiments of the present invention, - component (i) is in the form of a solid composition for oral administration of elafibranor or a pharma- ceutically acceptable salt thereof, in particular in the form of a pill or tablet, more particularly in the form of a tablet; - component (ii) is a solid dosage form for oral administration of a GLP-1R agonist or a pharma- ceutically acceptable salt thereof, in particular in the form of a pill or tablet, more particularly in the form of a tablet.

[0035] In a particular embodiment of the invention, component (ii) is a solid dosage form for oral administration comprising a GLP-1R agonist and a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid, as described in application WO2012080471. In another embodiment, component (ii) is a solid dosage form for oral administration comprising a GLP-1R agonist and sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (also known as SNAC or sarcaprozate sodium). In yet another embodiment, component (ii) is a solid dosage form for oral administration comprising semaglutide and a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid, such as SNAC. In a particular embodiment, component (ii) is in the form of a tablet.

[0036] In an additional embodiment, component (i) is in the form of an oral composition and component (ii) is in the form of an injectable solution.

[0037] In yet another embodiment, component (i) is in the form of an injectable solution and component (ii) is in the form of an oral composition.

[0038] In an additional embodiment, component (i) is in the form of an injectable solution and component (ii) is in the form of an injectable solution.

[0039] The number of doses and / or dosages involved in the administration can be adapted by those skilled in the art in relation to the subject to be treated, the disease to be treated, the stage of the disease, the form of administration, etc. Typically, component (i), such as ELA or a pharma- ceutically acceptable salt thereof, can be administered at a dose comprised between 0.01 mg / day and 4000 mg / day for elafibranor, for example, between 1 mg / day and 2000 mg / day for elafibranor, in particular, between 25 and 1000 mg / day, specifically, between 50 and 200 mg / day, and more specifically, between 80 and 120 mg / day. In another particular embodiment, component (ii), such as semaglutide or liraglutide, in particular, semaglutide or a pharma-ceutically acceptable salt thereof, can be administered at a dose comprised between 0.001 mg / day and 200 mg / day, for example, between 0.01 mg / day and 150 mg / day, in particular, between 0.1 mg / day and 100 mg / day.

[0040] In yet another specific embodiment, the dosages of the components of the combination product of the invention are as follows: component (i) is a solid dosage form of elafibranor for oral administration at a dose comprised between 0.01 mg / day and 4000 mg / day, for example between 1 mg / day and 2000 mg / day, in particular between 25 and 1000 mg / day, specifically between 50 and 200 mg / day, more specifically between 80 and 120 mg / day, - component (ii) is a solid dosage form of semaglutide for oral administration at a dose of 1 to 20 mg / day, for example 3 to 14 mg / day.

[0041] In another embodiment of the invention, the dosages of the components of the combination product of the invention are as follows: - component (i) is a solid dosage form of elafibranor for oral administration in a dose comprised between 80 and 120 mg / day; - component (ii) is a solid dosage form of semaglutide for oral administration in a dose comprised between 3 and 14 mg / day.

[0042] In yet another particular embodiment of the invention, the dosages of the components of the combination product of the invention are as follows: - component (i) is a solid dosage form of elafibranor for oral administration in a dose comprised between 80 and 120 mg / day; - component (ii) is a solid dosage form of semaglutide for oral administration in doses of 3 mg / day, 7 mg / day or 14 mg / day.

[0043] In an additional embodiment, the dosages of the components of the combination product of the present invention are as follows: - component (i) is a solid dosage form of elafibranor for oral administration in a dose comprised between 80 and 120 mg / day; - component (ii) is a solid dosage form of semaglutide for oral administration in doses of 3 mg / day, 7 mg / day or 14 mg / day.

[0044] In an additional embodiment, the dosages of the components of the combination product of the present invention are as follows: - Tablets containing 80-120 mg of elafibranor are administered once a day. - Tablets containing 3mg, 7mg or 14mg of semaglutide taken once daily.

[0045] In an additional embodiment, the dosages of the components of the combination product of the present invention are as follows: - A tablet containing 80 mg of elafibranor is administered once daily, - Tablets containing 3mg, 7mg or 14mg of semaglutide taken once daily.

[0046] In another specific embodiment, the dosages of the components of the combination product of the invention are as follows: - A tablet containing 120 mg of elafibranor is administered once daily, - Tablets containing 3mg, 7mg or 14mg of semaglutide taken once daily.

[0047] The dosage of oral semaglutide can be adapted by those skilled in the art. An oral dosage form of semaglutide has recently been approved by the US Food and Drug Administration and the European Medicines Agency under the trademark Rybelsus®. Therefore, those skilled in the art can determine the dose of semaglutide to be administered based on the recommended dosage disclosed in the prescribing information provided with this medicine, which is indicated for improving glycemic control in adults with type 2 diabetes. In brief: - Oral semaglutide can be initiated at 3mg once daily for 30 days, - After 30 days at a dose of 3 mg, the dose is increased to 7 mg once daily, - After at least 30 days on the 7 mg dose, if additional benefit such as further glycemic control is required in terms of dosing for improved glycemic control, the dose may be increased to 14 mg once daily.

[0048] In another aspect the present invention relates to a COMBINATION OF THE PRESENT DISCLOSURE for use as a medicament.

[0049] The combinations of the invention may be used in methods for reducing body weight. For example, the invention relates to the combinations of the invention for use in methods for treating obesity.

[0050] The combination of the present invention can also be used to reduce liver fat (also called "fatty liver").Therefore, the present invention relates to the combination of the present invention for use in a method for treating non-alcoholic fatty liver disease (NAFLD).In a further specific embodiment, NAFLD is fibrotic NAFLD, i.e., the subject has NAFLD together with liver fibrosis.In another specific embodiment, NAFLD is NASH.NASH can also be fibrotic NASH, i.e., the subject has NASH together with liver fibrosis.

[0051] The combination of the present invention may also be used to lower blood glucose.Accordingly, the present invention also relates to the combination of the present invention for use in a method for lowering blood glucose.The combination of the present invention may also be used to control insulin levels.Accordingly, the present invention also relates to the combination of the present invention for use in a method for controlling insulin levels.

[0052] In certain embodiments, the combination of the present invention is for use in a method for treating diabetes, in particular type 2 diabetes.

[0053] In an additional embodiment, the combination of the invention is for use in a method for improving glycemic control in a subject with type 2 diabetes.

[0054] It is also shown herein that food intake is not affected by the administration of the combination of the present invention. A reduction in food intake is an indication of the adverse effects of GLP-1R agonists.Therefore, it is shown herein that the combination of the present invention can be advantageously used to reduce or suppress the adverse effects of GLP-1R agonists.

[0055] In certain embodiments, the combination of the present invention is for use in a method for reducing body weight or body fat mass while reducing or suppressing adverse effects.In the context of the present invention, the phrase "while reducing or suppressing adverse effects" refers to reducing or suppressing at least one or all of the adverse effects, such as gastrointestinal adverse effects, such as nausea, vomiting, diarrhea, abdominal pain, and / or constipation, that are generally associated with the intake of GLP-1R agonists alone.

[0056] In another embodiment, the combination of the present invention is for use in a method for treating obesity while reducing or inhibiting adverse effects.

[0057] In a further particular embodiment, the combination of the present invention is for use in a method for treating NAFLD while reducing or inhibiting adverse effects. In a particular variation, the combination is used for the treatment of fibrotic NAFLD, NASH, or fibrotic NASH.

[0058] In yet another embodiment, the combination of the present invention is for use in a method for lowering blood glucose while reducing or inhibiting adverse effects.

[0059] In another embodiment, the combination of the present invention is for use in a method for lowering insulin levels while reducing or preventing adverse effects.

[0060] In a further embodiment, the combination of the invention is for use in a method for treating diabetes while reducing or inhibiting the adverse effects. More particularly, the invention relates to the combination of the invention for use in a method for treating type 2 diabetes while reducing or inhibiting the adverse effects.

[0061] It is also shown herein that the combination herein advantageously allows for the regulation of body weight. The present invention therefore also relates to the combination of the present invention for use in a method for regulating body weight.

[0062] In a particular embodiment, the combination of the present invention is for use in a method for regulating body weight in the context of a method for treating obesity.

[0063] In a further specific embodiment, the combination of the present invention is for use in a method for regulating body weight in the context of a method for reducing liver fat, such as in the context of a method for treating NAFLD.In a specific variation, the combination is used to regulate body weight in the context of a method for treating fibrotic NAFLD, NASH, or fibrotic NASH.

[0064] In yet another embodiment the combination of the present invention is for use in a method for regulating body weight in view of the method for lowering blood glucose.

[0065] In another embodiment the combination of the present invention is for use in a method for regulating body weight in view of the method for lowering insulin levels.

[0066] In a further embodiment the combination of the invention is for use in a method for regulating body weight in the context of a method for treating diabetes, such as type 2 diabetes.

[0067] Surprisingly, in addition to the synergistic effect that the combination of the present invention provides, it is also shown herein that the combination of components (i) and (ii) of the combination product leads to the beneficial effect observed at a reduced dose of component (ii). In other words, component (i) can be used to reduce the amount of component (ii) while still benefiting from the desired effects of both components (i) and (ii). This results in a reduction or suppression of the adverse effects commonly observed with GLP-1R agonists without affecting or significantly affecting the desired therapeutic effect. Thus, the present invention also relates to a PPAR agonist, such as a compound of formula (I), for use in combination with a GLP-1 receptor agonist in a method for the treatment of a condition in which administration of a GLP-1 receptor agonist is required, thereby reducing the side effects of the GLP-1 receptor agonist. In this embodiment, the GLP-1 receptor agonist can be administered in a reduced amount compared to the amount administered when the GLP-1 receptor agonist is administered alone. For example, the amount of GLP-1 receptor agonist administered to a subject can be reduced by at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, or even at least 10 fold.

[0068] Thus, the present invention also relates to a combination of the present invention for use in a method for treating a condition in which administration of a GLP-1 receptor agonist is required, whereby the side effects associated with the GLP-1 receptor agonist are reduced. Exemplary side effects that are reduced with the benefit of the present invention may be at least one or all of the following side effects: nausea, vomiting, diarrhea, abdominal pain, and constipation.

[0069] The present invention also relates to the combination of the present invention for use in a method for treating a condition requiring administration of a GLP-1 receptor agonist, wherein the amount of GLP-1 receptor agonist administered to a subject is reduced compared to the amount of GLP-1 receptor agonist required when administered alone.For example, the amount of GLP-1 receptor agonist administered to a subject can be reduced by at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, or even at least 10 times.

[0070] In yet another embodiment, the present invention relates to a combination product disclosed herein for use in a method for treating a condition requiring administration of a GLP-1 receptor agonist, wherein component (i) is administered to enhance the activity of component (ii). Such enhancement may result in an advantageous reduction in the amount of GLP-1 receptor agonist administered compared to the amount of GLP-1 receptor agonist required when administered alone. For example, the amount of GLP-1 receptor agonist administered to the subject may be reduced by at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, or even at least 10-fold.

[0071] Of course, all the above-mentioned dose reductions of GLP-1 receptor agonists can also be expressed as a percentage of the dose that is normally used when the GLP-1 receptor agonist is used alone. For example, by benefit of the present invention, the reduction rate can be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or even more than 50% of the dose that is normally used when the GLP-1 receptor agonist is used alone.

[0072] An exemplary implementation of said reduced amount of GLP-1 receptor agonist may be, for example, a reduced dose of semaglutide administered orally according to the regimen disclosed in the prescribing information for Ryblesus®, as disclosed above. An exemplary non-limiting dosing regimen, particularly for the treatment of NASH (such as NASH with fibrosis) or in improving glycemic control in subjects with type 2 diabetes, is provided below, in which the dose of the GLP-1 receptor agonist is reduced by 50%: - Oral semaglutide can be initiated at 1.5mg once daily for 30 days, - after 30 days at a dose of 3 mg, said dose is increased to 3.5 mg once daily; - After at least 30 days on the 3.5 mg dose, if additional benefit is required such as further glycemic control in terms of dosing for improved glycemic control, the dose may be increased to 7 mg once daily.

[0073] In the context of the present invention, the expression "conditions for which a GLP-1 receptor agonist is indicated" includes, but is not limited to, conditions or diseases whose treatment would benefit from the administration of a GLP-1 receptor agonist, including, but not limited to, diabetes, such as type 2 diabetes, obesity, NAFLD, and NASH.

[0074] In the context of the present invention, the abbreviation ELA refers to elafibranor.

[0075] In the context of the present invention the abbreviation SEMA refers to semaglutide.

[0076] In the context of the present invention, the abbreviation LIRA refers to liraglutide. The term "treatment" or "treating" refers to the curative or prophylactic treatment of a disease in a subject in need thereof. Treatment involves the administration of the combination of the present invention to a subject with a declared disease to prevent, cure, delay, reverse or slow the progression of the disease, thereby improving the condition of the subject. The combination product may also be administered to subjects who are healthy or at risk of developing the disease. The subject to be treated is a mammal, preferably a human. The subject to be treated according to the present invention may be selected based on several criteria related to the specific disease for which the treatment is sought, such as past drug treatment, associated medical condition, genotype, exposure to risk factors, viral infection, and further based on the detection of any biomarkers related to the disease.

[0077] If necessary, administration can be performed once a day or even multiple times a day. The duration of treatment will vary depending on the specific disease being treated. For example, administration can be performed for one or several days, such as at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days. Alternatively, administration can be performed for at least one week, at least two weeks, or at least four weeks. Administration for chronic diseases can be considered to be more than four weeks, such as more than six months, such as at least one month, two months, three months, four months, five months, six months, or at least one year or several years. In some cases, the combination product of the present invention can be administered for the lifetime of the subject.

[0078] The invention will now be further described with reference to the following non-limiting examples. [Brief description of the drawings]

[0079] [Figure 1] Figure 2 shows the effect of a combination of 10 mg / kg / day elafibranor (ELA) and 10 nmol / kg / day semaglutide (SEMA) on body weight after 40 days of treatment. [Diagram 2] Figure 2 shows the effect on body weight, expressed as relative weight gain compared to day 0, after 40 days of treatment with a combination of 10 mg / kg / day ELA and 10 nmol / kg / day semaglutide. [Diagram 3] Figure 1 shows the effect of a combination of 10 mg / kg / day ELA and 10 nmol / kg / day semaglutide on hepatic triglyceride content after 40 days of treatment. [Figure 4] Figure 2 shows the effect on blood glucose after 40 days of treatment with a combination of 10 mg / kg / day ELA and 10 nmol / kg / day semaglutide. [Diagram 5] Figure 1 shows the effect on insulinemia after 40 days of treatment with a combination of 10 mg / kg / day ELA and 10 nmol / kg / day semaglutide. [Figure 6] Figure 1 shows the effect of a combination of 10 mg / kg / day ELA and 10 nmol / kg / day semaglutide on fatty liver after 40 days of treatment. [Figure 7A] Figure 2 shows the effect on food intake after 28 days of treatment with a combination of 10 mg / kg / day ELA and 0.3 or 1 nmol / kg / day semaglutide. [Figure 7B] Figure 2 shows the effect on body weight after 28 days of treatment with a combination of 10 mg / kg / day ELA and 0.3 or 1 nmol / kg / day semaglutide. [Figure 8A] Figure 1 shows the effect of a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide on NAFLD activity score after 12 weeks of treatment in DIO-NASH mice. [Figure 8B] Figure 1 shows the effect of a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide on hepatic steatosis after 12 weeks of treatment in DIO-NASH mice. [Figure 8C]Figure 1 shows the effect on activity index (sum of ballooning score and inflammation score) in DIO-NASH mice after 12 weeks of treatment with a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide. [Figure 8D] Figure 1 shows the effect of a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide on lobular inflammation after 12 weeks of treatment in DIO-NASH mice. [Figure 9A] Figure 1 shows the effect on hepatic triglyceride content (TG) after 12 weeks of treatment with a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide in DIO-NASH mice. [Figure 9B] Figure 1 shows the effect on plasma transaminases (ALT) after 12 weeks of treatment with a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide in DIO-NASH mice. [Figure 9C] Figure 1 shows the effect on plasma transaminases (AST) after 12 weeks of treatment with a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide in DIO-NASH mice. [Figure 9D] Figure 1 shows the effect on CRP after 12 weeks of treatment with a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide in DIO-NASH mice. [Figure 9E] Figure 1 shows the effect on hepatic hydroxyproline after 12 weeks of treatment with a combination of 10 mg / kg / day ELA and 0.3 nmol / kg / day semaglutide in DIO-NASH mice. [Figure 10A] FIG. 1 shows the algorithm used in RNAseq analysis to identify pathological features, compound effects, and altered pathways in liver transcriptome analysis after 12 weeks of treatment with ELA (10 mg / kg / day), SEMA (0.3 nmol / kg / day), and their combination in DIO-NASH mice. [Figure 10B]Venn diagram showing genes involved in ELA / SEMA combination in liver transcriptome analysis after 12 weeks of treatment with ELA (10 mg / kg / day), SEMA (0.3 nmol / kg / day), and their combination in DIO-NASH mice. [Figure 10C] FIG. 1 shows a list of the top 10 pathways enriched by the ELA / SEMA combination in liver transcriptome analysis after 12 weeks of treatment with ELA (10 mg / kg / day), SEMA (0.3 nmol / kg / day), and their combination in DIO-NASH mice. [Figure 10D] Dot plots showing the synergistic effect of ELA / SEMA combination on genes involved in inflammatory pathways in liver transcriptome analysis after 12 weeks of treatment with ELA (10 mg / kg / day), SEMA (0.3 nmol / kg / day), and their combination in DIO-NASH mice. [Figure 10E] FIG. 1 shows expression of fibrotic genes in liver transcriptome analysis after 12 weeks of treatment with ELA (10 mg / kg / day), SEMA (0.3 nmol / kg / day), and their combination in DIO-NASH mice. [Figure 11A] Combination of elafibranor and GLP-1 receptor agonists synergistically inhibits TNFα secretion in LPS-activated macrophages. Dose-response matrix for percent inhibition of TNFα secretion in representative synergistic combinations between elafibranor and semaglutide. Data are presented as mean (quadratures) ± standard deviation (SD). [Figure 11B] Combination of elafibranor and GLP-1 receptor agonists synergistically inhibits TNFα secretion in LPS-activated macrophages. Dose-response matrix for percent inhibition of TNFα secretion in representative synergistic combinations between elafibranor and liraglutide. Data are presented as mean (quadratures) ± standard deviation (SD). [Figure 11C]Combination of elafibranor and GLP-1 receptor agonists synergistically suppresses TNFα secretion in LPS-activated macrophages. Figure 1 shows analysis of representative synergistic combinations between elafibranor and semaglutide according to the excess of Bliss (EOB) additivism model. Data are presented as mean (quadratures) ± standard deviation (SD). [Figure 11D] Combination of elafibranor and GLP-1 receptor agonists synergistically suppresses TNFα secretion in LPS-activated macrophages. Figure 1 shows an analysis of representative synergistic combinations between elafibranor and liraglutide according to the excess of Bliss (EOB) additivity model. Data are presented as mean (quadratures) ± standard deviation (SD). [Figure 11E] Combination of elafibranor and GLP-1 receptor agonists synergistically inhibits TNFα secretion in LPS-activated macrophages. Bar graph plotting TNFα secretion in representative synergistic combinations between elafibranor and semaglutide. Data are presented as mean (quadratures) ± standard deviation (SD). [Figure 11F] Combination of elafibranor and GLP-1 receptor agonists synergistically inhibits TNFα secretion in LPS-activated macrophages. Bar graph plotting TNFα secretion in representative synergistic combinations between elafibranor and liraglutide. Data are presented as mean (quadratures) ± standard deviation (SD). [Figure 12] Combinations of elafibranor and GLP-1 receptor agonists synergistically inhibit αSMA production in TGFβ-stimulated hepatic stellate cells. Combinations were tested in a dose-response matrix fashion and analyzed according to the excess of Bliss (EOB) additivity model. αSMA production in representative synergistic combinations is plotted in bar graph representation. Data are shown as mean (quadratures) ± standard deviation (SD). [Figure 13A]Figure 1 shows the effect of a combination of 3 mg / kg / day ELA and 0.02 mg / kg / day liraglutide on steatosis after 8 weeks of treatment in NASH-B6 mice. The weight of epididymal adipose tissue (EAT) in each mouse is expressed as a percentage of body weight (BW). [Figure 13B] FIG. 1 shows the effect of a combination of 3 mg / kg / day ELA and 0.02 mg / kg / day liraglutide on energy expenditure after 8 weeks of treatment in NASH-B6 mice. [Figure 14] FIG. 1 shows the effect of a combination of 3 mg / kg / day ELA and 0.02 mg / kg / day liraglutide on liver collagen content after 8 weeks of treatment in NASH-B6 mice. [Figure 15] FIG. 1 shows the effect of a combination of 3 mg / kg / day ELA and 0.02 mg / kg / day liraglutide on fibrotic gene expression in the liver of NASH-B6 mice after 8 weeks of treatment. [Figure 16] FIG. 1 shows the effect of a combination of 3 mg / kg / day ELA and 0.02 mg / kg / day liraglutide on blood glucose output before and after 8 weeks of treatment in NASH-B6 mice. [Figure 17] Elafibranor boosts the effect of semaglutide to reduce plasma ALT concentrations to levels similar to high-dose semaglutide. ALT levels in treatment groups compared to AMLN-fed vehicle groups. [Figure 18] Elafibranor boosts the effect of semaglutide to reduce plasma CRP concentrations to levels similar to high-dose semaglutide. Figure 1. CRP levels in treatment groups compared to AMLN-fed vehicle group. [Figure 19]Elafibranor boosts the effect of semaglutide to the point where the liver reproduces the high-dose semaglutide hepatic gene signature. Figure 1 shows the -log10 (adjusted p-value) of gene ontology (GO) category enrichment for each treatment. High values ​​indicate high enrichment of the GO category with treatment, i.e. many genes in the GO category are affected by the treatment. A value of "0" means that only a few genes in the GO category are deregulated by the treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0080] compound Elafibranor was synthesised by Genfit Inc. Semaglutide and liraglutide were commercially available. Elafibranor was dissolved in 1% CMC+0.1% Tween80. Semaglutide was dissolved in 1% CMC + 50 mM Na2HPO4 + 70 mM NaCl + 0.05% Tween 80. Liraglutide was dissolved in 0.1% BSA in phosphate buffered saline (PBS 1×).

[0081] statistical analysis As a statistical method, the Shapiro-Wilk normality test was applied to all raw data. In case of normal distribution, comparisons between chow (Chow) and vehicle (Veh) are performed with one-tailed Student's t-test (#). If necessary, the Welch (○) correction can be applied. For multiple comparison tests, one-way ANOVA and Fisher's least significant difference (LSD) post-hoc test (*) were applied. In other cases, comparisons between chow and vehicle (Veh) were performed with one-tailed Mann-Whitney ($) and, for multiple comparison tests, the Kruskall Wallis uncorrected Dunn's post hoc test (§) was applied. Distributions (£) were compared using Fisher's exact test. Therapeutic synergy of drug combinations was assessed using the excess of maximum single agent effect (HSA) model (one-sided statistical test). # / ○ / * / $ / § / ￡ / °:p<0.05 ## / ○○ / ** / $$ / §§ / ￡￡ / °°:p<0.01 ### / ○○○ / *** / $$$ / §§§ / ￡￡￡ / °°°:p<0.001

[0082] Example 1 Effects of 40 days of treatment with elafibranor alone or in combination with semaglutide on food intake and body weight regulation in male DIO-NASH mice Animal models The effects of elafibranor alone, semaglutide alone, and the combination of both were evaluated in DIO-NASH mice (C57BL6JRj mice fed an Amylin Liver NASH Model Diet (AMLN) (Research Diet, 40% fat (18% trans fat), 40% carbohydrate (20% fructose), and 2% cholesterol) (starting 36 weeks before the start of the study). 41-week-old male DIO-NASH mice were given vehicle (n=12), or vehicle supplemented with elafibranor (10 mg / kg / day), or semaglutide alone (10 nmol / kg) (tited up to the final dose over 5 days using 0.08 mg / mL increments), or the combination (n=12 per group).

[0083] Body weight was monitored daily. Food and water intake was monitored daily for weeks 1-2 and then weekly for the following weeks.

[0084] Tail vein sampling was performed throughout the protocol for the measurement of blood glucose, plasma insulin, alanine transaminase, aspartate transaminase, triglycerides, and total cholesterol.

[0085] On the last day of treatment, plasma samples were obtained after cardiac puncture blood collection and the mice were sacrificed. The livers were rapidly removed for biochemical and histological analysis.

[0086] All animal procedures were performed according to standard protocols and in accordance with standard recommendations for the proper care and use of laboratory animals.

[0087] Body weight and food intake Body weight was monitored daily. Food and water intake was monitored daily for weeks 1-2 and then weekly for the following weeks.

[0088] Final absolute body weights on day 40 are shown in Figure 1. Relative body weights on day 40 compared to day 0 are also shown in Figure 2. Body weight was significantly reduced by the combination of elafibranor and semaglutide.

[0089] biochemical analysis Measurement of liver triglyceride content Approximately 100 mg of frozen liver tissue was homogenized in 150 mM NaCl buffer containing 15.4 mM NaN3 using a tissue homogenizer (Precellys® 24, Bertin Technologies, France). The lipid fraction in the homogenate was extracted with chloroform-methanol (2:1, v / v), followed by triglyceride measurement (Biolabo, Cat. No. 80019).

[0090] Treatment with semaglutide (10 nmol / kg / day) had a significant beneficial effect on liver fat in this model (Figure 3). The combination of semaglutide and elafibranor produced a significant effect on liver triglyceride content.

[0091] Measurement of blood glucose Plasma glucose levels were measured using the Randox kit for the Daytona automate (Randox, Cat. No. GL3815) according to the manufacturer's recommendations. The method was based on a colorimetric assay without any deproteinization of the samples. Briefly, glucose oxidase digestion of the samples led to the synthesis of hydrogen peroxide. In the presence of phenol and 4-aminophenazone, this hydrogen peroxide was catalyzed by peroxidase to form a dye: quinoneimine. The color intensity was directly proportional to the glucose concentration and was measured at 505 nm. Results were expressed in mg / dL.

[0092] Treatment with elafibranor (10 mg / kg / day) and semaglutide (10 nmol / kg / day) alone had no beneficial effect on blood glucose in this model (Figure 4). The combination of semaglutide and elafibranor produced a significant effect on blood glucose (Figure 4).

[0093] Measurement of insulinemia Plasma insulin was measured using a solid-phase two-site enzyme immunoassay from Crystal Chem Inc. (cat. no. 90010). Briefly, 95 μL of guinea pig anti-insulin serum and 5 μL of diluted sample (or standard) were dispensed per well of a 96-well plate. After overnight incubation at 4° C. and washing, 100 μL of anti-guinea pig antibody conjugated to peroxidase was added and the reaction was developed by further incubation at room temperature for 1 h. Then, 100 μL of enzyme substrate was dispensed, followed by further incubation at room temperature in the dark for 30 min. The reaction was then stopped by adding 100 μL of stop solution and the absorbance was immediately measured at 450 nm (subtracting the wavelength of 630 nm). The intensity of this color development is proportional to the amount of insulin present in the sample at the start of the test and was calculated using a standard serum. The results were expressed in ng / mL.

[0094] Treatment with semaglutide (10 nmol / kg / day) had a significant beneficial effect on insulinemia in this model (Figure 5). The combination of semaglutide and elafibranor produced a significant effect on insulinemia (Figure 5).

[0095] histology Tissue embedding and sectioning: The liver slices were first fixed in a 4% solution of formalin for 12 hours. The liver pieces were then washed in PBS for 30 minutes and dehydrated in ethanol solutions (sequential 70, 80, 95, and 100% ethanol baths). The liver pieces were incubated in three different xylene baths (Sigma-Aldrich, Cat. No. 534056) followed by two liquid paraffin baths (56°C). The liver pieces were then placed in a frame and the frame was gently filled with Histowax® to completely cover the tissue. The paraffin block containing the tissue was removed from the frame and stored at room temperature. The liver block was sliced ​​at 3 μm.

[0096] Hematoxylin / eosin / safranin staining Liver sections were deparaffinized, rehydrated, and incubated in Mayer's hematoxylin (Microm, Cat. No. F / C0303) for 3 min. Liver sections were then rinsed in water, incubated in 0.5% Eosin Y alcohol solution (VWR, Cat. No. 1.02439.0500) and 0.5% Erythrosine solution (VWR, Cat. No. 1.15936.0010) for 1 min, and rinsed with ethanol. Sections were then incubated in Safranin for 2 min, and finally dehydrated and mounted using CV Mount medium (Leica, Cat. No. 046430011).

[0097] Histological examination Histological examination was performed by a technician blinded to the source of each liver specimen. Virtual slides were generated using a Pannoramic250 scanner from 3D Histech. A score summarizing the main histological lesions of NASH in each animal was classified according to the NASH Clinical Research Network (Kleiner, 2005; Brunt, 1999). Hepatic steatosis was scored (0–3).

[0098] NASH was induced in DIO-NASH mice. Intervention animals received elafibranor, semaglutide, or both compounds for the entire study period. The development of NASH was assessed by histology. Additional biochemical and molecular analyses for various relevant biomarkers were also performed.

[0099] DIO-NASH mice developed NASH-associated histology with high penetrance of severe disease. All animals had advanced hepatic steatosis.

[0100] The combination of semaglutide and elafibranor led to a significant reduction in hepatic steatosis (Figure 6).

[0101] Example 2 Effects of 4 weeks of treatment with elafibranor alone and in combination with semaglutide on food intake and body weight regulation in male DIO-NASH mice Animal models The effects of elafibranor alone, semaglutide alone, and the combination of both were evaluated in DIO-NASH mice. Male DIO-NASH mice aged 42-44 weeks were given vehicle (n=6), or elafibranor (10 mg / kg / day), or semaglutide alone, or vehicle plus combination (n=6 per group) for 4 weeks. Combinations were evaluated with two doses of semaglutide: - Semaglutide 0.3nmol / kg - Semaglutide 1nmol / kg

[0102] Body weight, food and water intake were monitored once a day. On the last day of treatment, plasma samples were obtained by retro-orbital blood sampling, and mice were sacrificed after a 6-hour fasting period. Livers were rapidly removed for biochemical and histological analysis.

[0103] All animal procedures were performed according to standard protocols and in accordance with standard recommendations for the proper care and use of laboratory animals.

[0104] Body weight and food intake Body weight was monitored daily. Food and water intake was monitored daily for 28 days.

[0105] The mean cumulative food intake is shown in Figure 7A. Food intake was found to be stable over time in all treatment groups, especially in the combination of elafibranor and semaglutide at doses of 0.3 or 1 nmol / kg. This effect demonstrates that the combination of elafibranor and semaglutide reduces the adverse effects of semaglutide.

[0106] Final absolute body weights at the end of the 28 day period are shown in Figure 7B. Body weight was significantly reduced by the combination of elafibranor and semaglutide at both doses tested.

[0107] "We found a significant effect of the combination of elafibranor and semaglutide on weight loss at doses 3-10 times lower than that of semaglutide. Furthermore, the combination of elafibranor and semaglutide may be of therapeutic interest in many diseases, including NASH, and may reduce the adverse effects of semaglutide."

[0108] Example 3 Effect of 12 weeks of treatment with elafibranor alone and in combination with semaglutide on NASH in male DIO-NASH mice Animal models Male C57BL / 6JRj mice were fed the AMLN diet for 35 weeks prior to the start of the study (DIO-NASH model). Mice with biopsy-proven fatty liver (score ≥2) and fibrosis (score ≥1) were randomly assigned to treatment groups (n=12–14 per group). Mice were treated with either vehicle, elafibranor (10 mg / kg / day, orally), semaglutide (0.3 nmol / kg / day, subcutaneously), or both elafibranor and semaglutide for 12 weeks. Suboptimal drug doses were used in this study. A titration protocol was applied during the first 3 weeks of treatment to avoid SEMA-induced hypophagia.

[0109] On the last day of treatment, plasma samples were obtained after cardiac puncture blood collection and mice were sacrificed. Livers were rapidly removed for histological and biochemical analyses, as well as transcriptomic analysis.

[0110] All animal procedures were performed according to standard protocols and in accordance with standard recommendations for the proper care and use of laboratory animals.

[0111] Histological assessment of NASH Liver sections were prepared and stained with hematoxylin, eosin, and safranin (HES) as described in Example 1. Histological features of NASH (fatty liver, ballooning, lobular inflammation, and fibrosis) were assessed using the NASH Clinical Research Network Scoring System (Kleiner, 2005; Brunt, 1999) by a technician blinded to the experimental group. Fatty liver score, activity index (hepatocyte ballooning + lobular inflammation), and NAFLD activity score (NAS) were calculated. The number of inflammatory foci per field was counted using 200x magnification.

[0112] At enrollment, mice had a severe NASH phenotype (NAFLD activity score (NAS) between 5 and 7, and fibrosis stage at least 2). After 12 weeks of treatment with the ELA / SEMA combination, NAS was reduced by 3 stages in 14% of mice and by 2 stages in 44% of mice (Figure 8A). None of the mice receiving either low-dose ELA or low-dose SEMA had a reduction in NAS by more than 1 stage. This reduction was explained by a decrease in both hepatic steatosis (Figure 8B) and activity index (Figure 8C). The number of inflammatory foci in the liver was notably reduced by the combination treatment (-59%) (Figure 8D).

[0113] biochemical analysis Liver samples were homogenized and triglycerides (TG) were extracted in 5% NP-40 by heating. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, as well as liver TG content, were measured using a commercial kit (Roche Diagnostics) and a Cobas™ C-501 automated analyzer according to the manufacturer's instructions. Plasma C-reactive protein (CRP) concentrations were measured by ELISA (Mouse C-Reactive Protein / CRP Immunoassay MCRP00, R&D Systems). Liver collagen was assessed using colorimetric detection of hydroxyproline, a major component of collagen (QZBhypro, Quickzyme).

[0114] ALT and AST are hepatic enzymes whose concentrations increase in plasma when hepatocytes are damaged. Plasma CRP is a marker of liver inflammation that is typically elevated in metabolic diseases with low-grade inflammation such as NASH, and correlates with cardiovascular risk in clinical practice. As expected, plasma ALT, AST, and CRP levels were elevated after 47 weeks of AMLN diet. Interestingly, in addition to the dramatic reductions in plasma ALT (-60%), AST (-56%), and CRP (-56%), a dramatic reduction in hepatic triglycerides (-56%) was also observed in the ELA / SEMA group, confirming the effect in liver histology (Figures 9A-D). Amelioration of fibrosis was observed in animals treated with ELA, and the combination with SEMA tended to further reduce hepatic collagen content (Figure 9E).

[0115] Transcriptome analysis Total RNA was isolated from mouse liver using Nucleospin® 96 RNA kit (Macherey Nagel) according to the manufacturer's instructions. RNAseq data in liver was generated (n=5 per group) using Illumina NexSeq 500 sequencing technology. Raw FASTQ files were trimmed at the 3' end in function of their quality score (Phred score). The parameters used were a minimum end quality level of 25 and a minimum read length of 50. Unaligned reads were aligned to the Mus musculus mm10 reference genome using the software STAR version 2.5.3. Default parameters were used. Count tables were generated using featureCounts v1.5.3 with default parameters. To identify differentially expressed genes (DE genes), R (version 3.5.3) and the DESEq2 library (v. 1.22.2) were used. Briefly, the count matrix generated by FeatureCounts was analyzed by the DESeqDataSetFromMatrix() function followed by the DEseq() function from the DESeq2 library. The results() function from DESeq2 was used to obtain the fold change and p-value in each condition (i.e., ELA / SEMA vs. AMLN). Genes with adjusted p-values ​​<0.01 and |fold change|>1.5 were determined as DE genes. The different tables were merged using Ensembl ID as key. Gene annotation was obtained using the biomaRt library (v. 2.38.0). For the selection of DE genes per condition, gene ontology analysis was performed using Metascape.

[0116] A complete liver transcriptome remodeling was demonstrated. A set of 2194 genes was differentially regulated in the ELA / SEMA combination group, whereas a set of 1140 and 55 genes were differentially regulated in the ELA and SEMA monotherapy groups, respectively (Figure 10A-B). Only SEMA treatment had some effect on the expression of genes related to lipid metabolism. Surprisingly, the transcriptomic signature of the ELA / SEMA combination in the liver was particularly enriched for inflammatory pathways, and in particular, myeloid cell markers were selectively reduced in animals receiving the combination (Figure 10C-D). Furthermore, several fibrotic genes were selectively reduced by the combination treatment, suggesting a therapeutic benefit of the combination in slowing fibrotic progression (Figure 10E).

[0117] The combination of low-dose elafibranor and semaglutide attenuates the severe NASH phenotype and reduces markers of liver injury in an AMLN diet-induced disease model.Transcriptomic analysis revealed that ELA and SEMA exhibited synergistic effects in specifically reducing inflammatory infiltration and attenuating fibrogenesis in the liver.

[0118] Example 4 Combination of elafibranor and a GLP-1 receptor agonist synergistically suppresses immune cell activation Combination treatment in LPS-activated macrophages Human THP-1 monocytes were seeded at a density of 25,000 cells per well of a 384-well plate in RPMI 1640 (Gibco, 21875) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco, 15140122), and 25 mM Hepes (Gibco, 15630080) and differentiated into macrophages with PMA (phorbol 12-myristate 13-acetate, Sigma, P8139) at a final concentration of 100 ng / mL for 24 hours.

[0119] A combination matrix of the two components was prepared. Stock solutions of ELA and GLP-1 receptor agonists were serially diluted in DMSO in 3 consecutive rows (ELA) and 8 consecutive columns (GLP1RA) of a 96-well plate. A 3x8 combination matrix was then made by mixing all single agent concentrations 1:1.

[0120] 24 hours after addition of PMA, the medium was removed and replaced with serum-free RPMI. Serum-starved THP1 macrophages were then pre-incubated with compounds for 24 hours, followed by incubation with lipopolysaccharide (LPS) (E. coli 055 B5) (Sigma, L6529) for an additional 6 hours.

[0121] Human TNFα was quantified in the supernatants using HTRF (Homogenoeus Time Resolved Fluorescence) technology (Cisbio, 62HTNFAPEG), which is based on FRET (Fluorescence Resonance Energy Transfer) technology. For detection with HTRF® reagents, cell supernatants, samples, and standards were directly dispensed into the assay plate. HTRF donor and acceptor labeled antibodies were premixed and added in a single dispensing step. The signal intensity detected at 665 nm is proportional to the number of antigen-antibody complexes formed and thus to the TNFα concentration. A 7-point calibration curve (from 39 pg / mL to 2500 pg / mL of supplied human TNFα) was obtained by fitting the data with a 4-parameter logistic model.

[0122] The excess of Bliss (EOB) method was used to determine synergy between compounds. The values ​​obtained in the HTRF assay for TNFα were first converted to percent inhibition relative to the LPS control. These percent inhibitions were then used to calculate the EOB.

[0123] First, the expected Bliss additive score (E) was calculated using the formula: E = (A + B) - (A × B) where A and B are the percent inhibition of elafibranor (A) and GLP-1 analog (B) at a given dose. The difference between the observed inhibition of ELA / GLP-1 analog combined with the expected Bliss value at the same dose is the "exceeding Bliss" score. - Bliss excess score = 0 suggests that the combination treatment is additive (as expected for independent pathway effects); - A Bliss excess score >0 indicates greater than additive activity (synergy); - A Bliss excess score <0 indicates that the combination is less than additive (antagonism). All EOBs were added together to calculate a total Bliss score.

[0124] Metabolic diseases such as NAFLD / NASH are associated with low-grade inflammation. Activation of immune cells results in the production of cytokines that alter the metabolic function of the liver and peripheral organs (adipose tissue, pancreas, etc.). Intestinal permeability, which is mentioned in metabolic and liver diseases, results in an increase in bacterial components in the blood, such as LPS, which activates macrophages in the liver and peripheral organs (adipose tissue). In light of the synergistic effect between elafibranor and semaglutide on inflammatory pathways in Example 3, we investigated whether elafibranor and various GLP-1 receptor agonists may show a synergistic effect in inhibiting macrophage activation by LPS. LPS treatment activates macrophages, as measured by TNFα secretion, which is increased by about 4-fold by LPS in THP1 monocytes differentiated into macrophages (Figures 11E-11F). The maximum effect of elafibranor alone was observed at a dose of 1 μM, reaching 85% inhibition of TNFα secretion (Figure 11B). In contrast, the maximum effect of GLP-1 receptor agonists was 29% and 38% TNFα inhibition for semaglutide and liraglutide, respectively (Figures 11A-11B). As observed in vivo (Example 3), the combination of elafibranor and semaglutide shows synergistic effect in reducing inflammation at low doses: 0.07 μM elafibranor synergized with 0.7 nM semaglutide to reduce TNFα secretion by 56% (Figure 11E). Surprisingly, 0.1 μM elafibranor also synergized with liraglutide (0.003 nM) to reduce TNFα secretion by 83% (Figure 11F).

[0125] These results demonstrate the ability of elafibranor to synergize with various GLP-1 receptor agonists to reduce the inflammatory tendencies observed in many diseases, including NASH and metabolic diseases.

[0126] Example 5 Combination of elafibranor and a GLP-1 receptor agonist synergistically inhibits hepatic stellate cell (HSC) activation Combination therapy in TGFβ-stimulated HSCs 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) to improve attachment.

[0127] A combination matrix of the two components was prepared. Stock solutions of ELA and GLP-1 receptor agonist were serially diluted in DMSO in 96-well plates in rows of 3 wells (ELA) and columns of 7 wells (GLP-1 receptor agonist). A 3x7 combination matrix was then made by mixing all single agent concentrations 1:1.

[0128] Cells were then seeded at a density of 6500 cells / well in 384-well plates. The next day, cell culture medium was removed and cells were washed with PBS (Invitrogen, Cat. No. 14190). hHSCs were starved in serum-free and SteCGS-free medium for 24 hours. For treatment with ELA, GLP-1 receptor agonist, and their respective combinations, serum-starved hHSCs in serum-free and SteCGS-free medium were pre-incubated with compounds for 1 hour, followed by addition of the pro-fibrotic stimuli TGF-β1 (PeproTech, Cat. No. 100-21, 3 ng / mL) for an additional 48 hours.

[0129] Human α-smooth muscle actin (αSMA) was quantified in cell lysates using HTRF (homogeneous time-resolved fluorescence) technology (Cisbio, 62HTNFAPEG), which is based on FRET (fluorescence resonance energy transfer) technology. Diluted cell lysates, samples, and standards were directly dispensed into the assay plate for detection with HTRF® reagents. HTRF donor and acceptor labeled antibodies were premixed and added in a single dispensing step. The HTRF ratio of the signal intensity detected at 665 nm to that detected at 620 nm is proportional to the number of antigen-antibody complexes formed and therefore to the αSMA concentration.

[0130] After liver injury, quiescent HSCs undergo a process of activation characterized by differentiation into α-SMA-positive myofibroblasts. Elafibranor has antifibrotic activity in hHSCs activated by the profibrotic cytokine TGFβ1. 3 μM ELA reduced α-SMA production by 61% (not shown). Semaglutide and liraglutide alone only slightly reduced α-SMA production (maximum of semaglutide -12%, maximum of liraglutide -30%). However, elafibranor synergized with semaglutide and liraglutide to reduce α-SMA production in activated HSCs (Figure 12). One of the most striking examples of synergy is shown in Figure 12E, where inhibition of α-SMA production reached 84% with 0.3 μM ELA and 0.01 μM semaglutide. Similarly, 0.003 μM liraglutide alone did not show any antifibrotic activity, but when added to 0.1 μM elafibranor, it synergistically reduced αSMA production, reaching 62% inhibition (Figure S12F).

[0131] These results demonstrate that elafibranor and GLP-1 receptor agonists have synergistic effects in reducing fibrogenesis and offer a potential therapeutic opportunity for fibrotic NASH.

[0132] Example 6 Effects of 8 weeks of treatment with elafibranor alone and in combination with liraglutide on steatosis, blood glucose, and liver in a mouse model of diet-induced NASH Animal models Male C57BL / 6N Tac mice (Taconic, USA) were fed a GAN diet (D09100310 Research Diet) for 31 weeks prior to the start of the study to induce NASH pathology (NASH-B6 model). Prior to treatment, blood was collected by retro-orbital sinus puncture after a 6-h fasting period to measure plasma ALT, AST, and TIMP metallopeptidase inhibitor 1 (TIMP1, a surrogate marker of liver fibrosis). After a 6-h fasting period, blood glucose was measured directly from the tail vein using a WellionVet glucometer (CALEA). Ketone bodies (KB) were also measured directly from the tail vein using a FreeStyle Optium Neo ketometer. Mice were randomly assigned to treatment groups (n=10 / group) based on body weight, blood glucose, ketonemia, and plasma ALT, AST, and TIMP1. These mice were treated with vehicle, elafibranor (3 mg / kg / day, orally), liraglutide (0.01 mg / kg, twice daily, subcutaneously), or both elafibranor and liraglutide for 8 weeks. To avoid GLP-1 receptor agonist-induced hypophagia, a titration protocol was applied for liraglutide (starting at 0.001 mg / kg, twice daily) during the first 3 weeks of treatment. Vehicle-treated chow-fed mice served as additional controls (R03-10 U8211G10R, SAFE).

[0133] Body weight and food intake were monitored daily during the treatment period. On the last day of treatment, serum samples were obtained after retro-orbital sinus puncture blood sampling and mice were sacrificed. Livers were rapidly removed for histological, biochemical, and transcriptomic analysis. Epididymal adipose tissue was weighed.

[0134] All animal procedures were performed according to standard protocols and in accordance with standard recommendations for the proper care and use of laboratory animals.

[0135] After 39 weeks of GAN feeding, NASH-B6 mice became obese (48 g). Eight weeks of treatment with elafibranor, either in combination with liraglutide or without liraglutide, reduced body weight by 15% (not shown). Adiposity was reduced by elafibranor and further reduced by combination treatment (Figure 13A). Due to the suboptimal drug doses used in this study, there was no effect of underfeeding by liraglutide (Figure 13B).

[0136] biochemical analysis Liver collagen content was determined using the appropriate Quickzyme kit (total collagen assay, catalog number QZB-totcol2). The assay is based on the detection of hydroxyproline, a non-proteinogenic amino acid found mainly in the triple helix of collagen. Hydroxyproline in tissue hydrolysates can therefore be used as a direct measure of the amount of collagen present in the tissue (without distinguishing between procollagen, mature collagen, and collagen degradation products). Before adding hydroxyproline, the tissue samples must be completely hydrolyzed in 6M HCl at 95°C. The assay produces a chromogen with a maximum absorbance at 570 nm. Results are expressed as μg collagen / mg liver.

[0137] NASH-B6 mice developed liver lesions characterized by a maximum fatty liver score of 3 in all mice, lobular inflammation, and collagen deposition (not shown). Whereas monotherapy had little effect, the combination treatment significantly reduced collagen content (FIG. 14), suggesting that at these low doses, only the combination treatment was able to reduce liver damage.

[0138] Liver gene expression analysis Total RNA was isolated from mouse liver using Nucleospin® 96 RNA kit (Macherey Nagel) 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), 10 mM DTT (Invitrogen), 10 mM dNTPs (Promega), 200 ng pdN6 (Amersham), and 40 U of RNase inhibitor (Promega) in 5× RT buffer (Invitrogen). Quantitative PCR was then performed using the CFX96 Touch™ Real-Time PCR Detection System (Biorad). Briefly, PCR reactions were carried out in a 96-WP format in a total volume of 25 μL containing 5 μL of reverse transcription reaction solution, 0.5 μL of reverse and forward primers (10 mmol each), and 12.5 μL of 2× iQ SYBR Green Supermix (BioRad) using the following primer sequences:

[0139] [Table 1]

[0140] The expression level was normalized using the expression of NONO gene as a housekeeping gene in the samples. A standard curve was drawn by selecting the best points (at least 3 points) to make the PCR reaction efficiency close to 100% and the correlation coefficient close to 1. The equation of the standard curve for both the housekeeping gene and the target gene (taking into account the specific PCR efficiency of each target gene) was used to determine the expression level.

[0141] As expected, expression of the fibrotic gene Col1a1 was induced in NASH-B6 mice fed GAN (Figure 15). Col1a1 expression was reduced by elafibranor treatment and further reduced by combination with liraglutide.

[0142] Before and after 8 weeks of treatment, blood glucose was assessed directly from the tail vein using a WellionVet glucometer (CALEA) after a 6-hour fasting period. Figure 16 shows the blood glucose release of the treatment group compared with that of the vehicle group fed with GAN diet. In this study, ELA (3 mg / kg / day) and liraglutide (0.02 mg / kg / day) had no effect on blood glucose. However, the combination treatment dramatically reduced blood glucose by 66%.

[0143] These results also show that elafibranor synergizes with liraglutide to reduce steatosis, attenuate liver pathology, decrease fibrogenesis, and improve glucose homeostasis, highlighting the therapeutic benefit of combination therapy between elafibranor and a GLP-1 receptor agonist for treating patients with type 2 diabetes and NASH. This synergy allows for a reduction in the therapeutic dose of the GLP-1 receptor agonist, thereby limiting their adverse effects.

[0144] Example 7 Elafibranor enhances the effects of low doses of GLP1 receptor agonists Animal models The effects of elafibranor alone, semaglutide (low and high doses) alone, and in combination were evaluated in DIO-NASH mice (C57BL6JRj mice fed Amylin Liver NASH Model Diet (AMLN) (Research Diet, 40% fat (18% trans fat), 40% carbohydrate (20% fructose), and 2% cholesterol) 35 weeks prior to study initiation. Mice with biopsy-proven hepatic steatosis (score ≥ 2) and fibrosis (score ≥ 1) were randomly assigned to treatment groups (n = 12 per group). Mice were administered either vehicle, elafibranor (10 mg / kg / day, orally), low-dose semaglutide (0.3 nmol / kg / day, subcutaneously), high-dose semaglutide (10 nmol / kg / day, subcutaneously), or a combination of elafibranor (10 mg / kg / day) and semaglutide (0.3 nmol / kg / day).

[0145] On the last day of treatment, plasma samples were obtained after cardiac puncture blood collection and mice were sacrificed. Livers were rapidly removed for transcriptome analysis. All animal procedures were performed according to standard protocols and in accordance with standard recommendations for the proper care and use of laboratory animals.

[0146] Plasma marker evaluation Alanine aminotransferase (ALT) levels were measured using a Cobas™ C-501 automated analyzer according to the manufacturer's instructions. C-reactive protein (CRP) concentrations were measured by ELISA (Mouse C-reactive protein / CRP Immunoassay MCRP00, R&D Systems).

[0147] Transcriptome and gene ontology analysis Total RNA was isolated from mouse liver using Nucleospin® 96 RNA kit (Macherey Nagel) according to the manufacturer's instructions. RNAseq data in liver was generated (n=5 per group) using Illumina NexSeq 500 sequencing technology. Raw FASTQ files were trimmed at the 3' end in function of their quality score (Phred score). The parameters used were a minimum end quality level of 25 and a minimum read length of 50. Unaligned reads were aligned to the Mus musculus mm10 reference genome using the software STAR version 2.5.3. Default parameters were used. Count tables were generated using featureCounts v1.5.3 with default parameters. To identify differentially expressed genes (DE genes), R (version 3.5.3) and the DESEq2 library (v. 1.22.2) were used. Briefly, the count matrices generated by FeatureCounts were analyzed by the DESeqDataSetFromMatrix() function followed by the DEseq() function from the DESeq2 library. A list of differentially expressed (DE) genes was established for each comparison (i.e., SEMA-10 vs. AMLN, SEMA-0.3 vs. AMLN, ELA vs. AMLN, ELA+SEMA-0.3 vs. AMLN). For this, the lfcshrink() function of DESeq2 was used to select DE genes using the following cutoffs: |fold change|>1.5 and adjusted p-value<0.05. These gene lists were subjected to metascape analysis using default parameters (https: / / metascape.org / gp / index.html# / main / step1).

[0148] Each metascape analysis was exported as an Excel file. The files were imported in RV using the read_excel function from the readxl library (version 1.3.1). The different files were combined together to generate a table containing the -log10 (adjusted p-value) of the Gene Ontology (GO) category enrichment for each comparison. Furthermore, for each category, the number of DE genes found in the RNA-seq analysis was compared with the total number of genes in this GO category. Only GO categories with an adjusted p-value <0.01 in at least one comparison were retrieved.

[0149] As expected, the GAN diet increased plasma ALT levels, a marker of liver damage used clinically as a liver function test. High doses (10 nmol / kg / day) of semaglutide dramatically reduced ALT by 74%, whereas low doses of semaglutide (0.3 nmol / kg / day) were less efficient (Figure 17). Surprisingly, combination treatments containing elafibranor and low doses of semaglutide reduced ALT levels to the same extent as treatments with high doses of semaglutide (-77% vs. -83%, p>0.05), suggesting that elafibranor enhances the effect of semaglutide when semaglutide is used at low doses in combination (Figure 17).

[0150] Similarly, levels of CRP, an inflammatory marker also known to be associated with cardiovascular disease risk in humans, were reduced by 43% with high-dose semaglutide, whereas low-dose semaglutide was less effective (Figure 18). Also, the combination of low-dose semaglutide and elafibranor reduced CRP levels to the same extent as high-dose semaglutide (-47% vs -43%, p>0.05).

[0151] To understand this synergistic effect, we compared the hepatic gene signatures between elafibranor, high-dose semaglutide, low-dose semaglutide, and the combination of elafibranor and low-dose semaglutide. Liver RNAseq and pathway analysis was performed using Metascape. Gene ontology analysis shows that high-dose semaglutide (10 nmol / kg / day) reversed the pathological features induced by AMLN, especially inflammatory response, cytokine (IL1, IL6) production, immune cell activation and migration (Figure 19). Low-dose SEMA was less efficient at reversing these pathways. Elafibranor (10 mg / kg / day) had a moderate effect on gene ontology categories linked to inflammation, but rather affected the lipid metabolism category, a known effect of PPAR agonists. However, in combination with low-dose semaglutide, elafibranor reverted the anti-inflammatory gene signature of high-dose semaglutide, in addition to its beneficial effects on lipid metabolism.

[0152] These results indicate that elafibranor exhibits interesting therapeutic benefits, such as enhancing the effects of GLP-1 receptor agonists on liver injury and inflammation and allowing the dose of GLP1 receptor agonists to be reduced, thereby limiting their side effects (nausea, diarrhea) that may lead to treatment cessation.

Claims

1. (i) elafibranor or a pharma- ceutically acceptable salt thereof. and (ii) a glucagon-like peptide-1 (GLP-1) receptor agonist selected from the group consisting of semaglutide, liraglutide, and pharma- ceutically acceptable salts thereof; Including, A combination product for use in a method for treating non-alcoholic fatty liver disease, diabetes or obesity or for use in a method for reducing weight.

2. 2. The combination product of claim 1, wherein component (i) is elafibranor.

3. 3. A combination product according to claim 1 or 2, wherein component (ii) is semaglutide or a pharma- ceutically acceptable salt thereof.

4. 3. The combination product according to claim 1 or 2, wherein component (ii) is liraglutide or a pharma- ceutically acceptable salt thereof.

5. 5. The combination product according to any one of claims 1 to 4, wherein the combination product is a composition comprising components (i) and (ii) and a pharma- ceutically acceptable carrier.

6. 6. The combination product according to any one of claims 1 to 5, wherein components (i) and (ii) are formulated by means of suspensions, gels, oils, pills, tablets, suppositories, powders, capsules, aerosols, ointments, creams, patches or galenic forms for sustained and / or slow release.

7. 5. The combination product according to any one of claims 1 to 4, wherein the combination product is a kit-of-parts comprising components (i) and (ii) for sequential, separate or simultaneous use.

8. 8. The combination product of claim 7, wherein components (i) and (ii) are in oral dosage form.

9. 9. The combination product of claim 8, wherein the oral dosage form is a pill or tablet.

10. 8. The combination product of claim 7, wherein component (i) is an oral dosage form and component (ii) is an injectable solution.

11. 11. A combination product according to any one of claims 1 to 10 for use in a method for treating non-alcoholic steatohepatitis.

12. 11. A combination product according to any one of claims 1 to 10 for use in a method for reducing body weight.

13. 11. A combination product according to any one of claims 1 to 10, in which the amount of GLP-1 receptor agonist administered is reduced compared to the amount of GLP-1 receptor agonist required when administered alone.

14. 12. The combination product of claim 11, wherein at least one side effect of the GLP-1 receptor agonist is thereby reduced.

15. 11. The combination product of any one of claims 1 to 10, wherein at least one adverse effect associated with the GLP-1 receptor agonist is reduced compared to administration of the GLP-1 receptor agonist alone.

Images