Non-invasive monitoring of NASH treatment with lanifibranor

Abstract

The present disclosure relates to a method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient; b) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in step a).

Description

[0001] NON-INVASIVE MONITORING OF LIVER DISEASE TREATMENT

[0002] Field of the invention

[0003] The present disclosure relates to a method of assessing the effectiveness of a treatment with the investigational drug lanifibranor in a patient with a liver disease. The present disclosure also relates to a method of treating a liver disease comprising a step of assessing the effectiveness of the lanifibranor treatment.

[0004] Background of the invention

[0005] Metabolic dysfunction-associated steatohepatitis (MASH) is a major cause of chronic liver disease. Its diagnosis and characterization currently rely on histological investigations, and liver biopsies are still required for the diagnosis and assessment of treatment response. The identification of biomarker signatures for non-invasive assessment of the histological response would be an important step to overcome the shortcomings of currently used diagnostic methods and facilitate MASH therapies. Further, ongoing biomarker research in MASH focuses on identifying non- invasive biomarkers to detect and monitor disease which may also contribute to the implementation of precision medicine concepts in the diagnosis and management of MASH (Adv Ther 2021;38(5):2130-2158).

[0006] The pan-peroxisome proliferator-activated receptor (pan-PPAR) agonist lanifibranor modulates key metabolic, inflammatory, and fibrogenic pathways by specifically targeting all PPAR alpha, delta and gamma nuclear receptors. Lanifibranor has demonstrated therapeutic efficacy on both MASH resolution and fibrosis improvement in the phase 2b NATIVE trial. The percentage of patients with active MASH who had a decrease of at least 2 points in the steatosis activity fibrosis (SAF)-A score (Hepatology 2014;60(2):565-575) without worsening of fibrosis was significantly higher after treatment with a daily dose of 1200 mg lanifibranor than with placebo (N Engl J Med 2021 ;385(17): 1547-1558). Secondary endpoints of this study included MASH resolution and fibrosis improvement, MASH resolution without worsening of fibrosis and improvement of fibrosis without worsening of MASH, according to MASH Clinical Research Network (MASH-CRN) (Hepatology 2005;41(6): 1313-1321). Results of this study were more favorable than placebo in the lanifibranor groups.

[0007] In this context, the aim of the inventors was to identify biological signatures of histological responders among MASH patients treated with lanifibranor. The identification and development of a biomarker signature can indeed aid in assessing the response to a treatment and help identifying patients most likely to experience clinical benefit from the treatment.

[0008] Summary of the invention

[0009] The present disclosure relates to a method of assessing the effectiveness of treatment with lanifibranor in a patient with a liver disease, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase (hereafter also referred to as “gamma-GT”), AST, insulin and urea, in a biological sample from the patient, and b) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in step a).

[0010] In some embodiments, the level of CK18M65 is measured before the start of the treatment with lanifibranor.

[0011] In some embodiments, the level of gamma-GT is measured before the start of the treatment with lanifibranor.

[0012] In some embodiments, levels of AST, insulin and urea are measured before the start of the treatment with lanifibranor and after at least 3 months of treatment with lanifibranor.

[0013] In some embodiments, the levels measured in step a) are used to obtain a score which falls within a determined or undetermined prognosis class. In some embodiments, the determined prognosis class includes a class of predicted responders to the lanifibranor treatment and a class of predicted nonresponders to the lanifibranor treatment. In some embodiments, the score is obtained by combining the levels measured in step a) in a mathematical function. In some embodiments, the mathematical function is a binary logistic regression. In some embodiments, the method comprises comparing the score with a first calculated cutoff value below which no response to the lanifibranor treatment is predicted. In some embodiments, the method comprises comparing the score with a second calculated cutoff value above which a response to the lanifibranor treatment is predicted.

[0014] In some embodiments, the biological sample is a sample of biological fluid. In some embodiments, the biological fluid is blood, serum or plasma.

[0015] In some embodiments, the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis, and a combination of these diseases. In some embodiments, the liver disease is advantageously selected from non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), and metabolic-associated steatohepatitis. In some embodiments, the liver disease can be associated with diabetes, and in particular with type 2 diabetes.

[0016] The present disclosure also relates to a system for assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the system comprising: a) means for measuring or receiving measurement data of levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient; and b) means for processing the data configured to assess the effectiveness of the treatment with lanifibranor in the patient as a function of the levels measured for the combination of biomarkers.

[0017] The present disclosure also relates to the use of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea in a method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease.

[0018] The present disclosure also relates to a method of treating a liver disease in a subject, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient before initiating a treatment; b) daily administering an effective amount of lanifibranor to the subject for at least 3 months; c) in vitro measuring levels of AST, insulin and urea in a biological sample of the subject after 3 months of treatment; d) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in steps a) and c). e) continuing administering an effective amount of lanifibranor to the subject for at least another 3 months provided the assessment made in step d) is predictive of a response to the lanifibranor treatment, or that no prognosis can be made.

[0019] Description of the figures

[0020] Figure 1 shows the sensitivity, specificity, NPV and PPV as a function of an E3-score.

[0021] Detailed description of the invention

[0022] The present disclosure relates to a method of assessing the effectiveness of treatment with lanifibranor in a patient with a liver disease, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient, and b) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in step a).

[0023] In the context of the present disclosure, CK18M65 stands for cytokeratin 18 (antigen M65); AST stands for aspartate aminotransaminase.

[0024] In the context of the present disclosure, “combination of biomarkers”, “biomarker signature” and “biomarkers signature” are used interchangeably.

[0025] As used herein “assessing the effectiveness / efficacy of a treatment” herein refers to the determination of the clinical condition of a patient subjected to a treatment. The treatment may be preventive, for example in the case of predisposition to a disease, or it may be curative, for example in the case of a diagnosed disease. The effectiveness of the treatment may for example be evaluated by determining the condition of the patient at different time intervals. The condition of the patient may notably be evaluated before the first taking of the treatment then at regular (or irregular) time intervals after this first taking (for example after each new taking of the treatment). A comparison of the condition of the patient evaluated at these different intervals may then be carried out in order to identify a potential change. The condition of the patient may be evaluated on the basis of observations and / or measurements, carried out using different tools.

[0026] As used herein, “treatment” refers to any process, action, application, therapy, or the like, wherein the patient is under aid, in particular, medical, or veterinarian aid with the object of improving the patient's condition, either directly or indirectly.

[0027] As used herein, “patient” or “subject” refers to a human individual or an animal different from a human. The patient is for example a human or an animal liable to have a liver disease or suffering from such a disease. The patient is advantageously a human being. The patient may be a child (human patient 18 years old or less) or an adult (human patient more than 18 years old). In the context of the present disclosure the terms “patient” and “subject” are used interchangeably.

[0028] As used herein, “measuring” or “measurement” is understood as quantitative characterization of a physical object or entity or a multitude (population or plurality) thereof, or their function or quantitative characterization of a physical or chemical process, comprising the assignment of a quantity, value, e.g. a numerical value or a number characteristic of the object or entity or multitude or function or process, by comparison with units and, in comparison with another object or entity or multitude or function or process. Preferably a measurement is consistent with methods known in the art or the international guidelines of metrology.

[0029] As used herein, "quantifying" or "quantification" or "quantitation" is understood herein as an assignment of a physical quantity to a physical object or entity or a multitude (population or plurality) thereof, or their function or quantitative characterization of a physical or chemical process, expressed in a numerical value or number and units, and, in comparison with another object or entity. Advantageously, "quantifying" or "quantification" is a measurement or an essential part of a measurement. The measurement has an uncertainty which may represent the random and systemic errors of the measurement procedure. The skilled person is aware of this and can handle this error in view of the measurement or quantification applied.

[0030] As used herein, lanifibranor, or l-(6-benzothiazolylsulfonyl)-5-chloro-lH-indole-2-butanoic acid, is a pan-PPAR agonist. The chemical formula of lanifibranor is Ci9H15ClN2O4S2, its molecular weight is 434.92 and its CAS Number is 927961-18-0. Advantageously, the term “lanifibranor” also includes any deuterated form of lanifibranor, any pharmaceutical salt thereof, and any crystalline form thereof. Deuterated forms of lanifibranor include those described in international application WO 2020 / 021215, the disclosure of which is incorporated by reference. Crystalline forms of lanifibranor include those described in international applications WO2023 / 194339, WO2023 / 016319, WO2022 / 122014, WO2022 / 261410 or W02022 / 258060, the disclosure of which is incorporated by reference.

[0031] In some embodiments, biomarkers the level of which is measured at step a) are protein biomarkers. Advantageously, biomarkers are serum biomarkers.

[0032] In some embodiments, the expression level of CK18M65 is measured before the start of the treatment with lanifibranor.

[0033] In some embodiments, the level of gamma-GT is measured before the start of the treatment with lanifibranor.

[0034] In some embodiments, levels of AST, insulin and urea are measured before the start of the treatment with lanifibranor and after at least 3 months of treatment with lanifibranor. Advantageously, levels of AST, insulin and urea are measured after at least 4 months, advantageously after at least 5 months, advantageously after at least 6 months, advantageously after at least 7 months, advantageously after at least 8 months, advantageously after at least 9 months, advantageously after at least 10 months, advantageously after at least 11 months, advantageously after at least 12 months, advantageously after at least 13 months, advantageously after at least 14 months, advantageously after at least 15 months, advantageously after at least 16 months, advantageously after at least 17 months, advantageously after at least 18 months, advantageously after at least 19 months, advantageously after at least 20 months, advantageously after at least 21 months, advantageously after at least 22 months, advantageously after at least 23 months, advantageously after at least 24 months, or more of treatment with lanifibranor.

[0035] As used herein, the term “3 months” is equivalent to 12 weeks. As used herein, the term “4 months” is equivalent to 16 weeks. As used herein, the term “5 months” is equivalent to 20 weeks. As used herein, the term “6 months” is equivalent to 24 weeks. As used herein, the term “7 months” is equivalent to 28 weeks. As used herein, the term “8 months” is equivalent to 32 weeks. As used herein, the term “9 months” is equivalent to 36 weeks. As used herein, the term “10 months” is equivalent to 40 weeks. As used herein, the term “11 months” is equivalent to 44 weeks. As used herein, the term “12 months” is equivalent to 48 weeks. As used herein, the term “13 months” is equivalent to 52 weeks. As used herein, the term “14 months” is equivalent to 56 weeks. As used herein, the term “15 months” is equivalent to 60 weeks. As used herein, the term “16 months” is equivalent to 64 weeks. As used herein, the term “17 months” is equivalent to 68 weeks. As used herein, the term “18 months” is equivalent to 72 weeks. As used herein, the term “19 months” is equivalent to 76 weeks. As used herein, the term “20 months” is equivalent to 80 weeks. As used herein, the term “21 months” is equivalent to 84 weeks. As used herein, the term “22 months” is equivalent to 88 weeks. As used herein, the term “23 months” is equivalent to 92 weeks. As used herein, the term “24 months” is equivalent to 96 weeks.

[0036] In some embodiments, the levels measured in step a) are used to obtain a score which falls within a determined or undetermined prognosis class. In some embodiments, the determined prognosis class includes a class of predicted responders to the lanifibranor treatment and a class of predicted non-responders to the lanifibranor treatment.

[0037] In some embodiments, the score is obtained by combining the levels measured in step a) in a mathematical function. In some embodiments, the mathematical function is a binary logistic regression.

[0038] In some embodiments, the method comprises comparing the score with a first calculated cutoff value below which no response to the lanifibranor treatment is predicted. In an advantageous embodiment, the first calculated cutoff value is equal to about 0.39.

[0039] In some embodiments, the method comprises comparing the score with a second calculated cutoff value above which a response to the lanifibranor treatment is predicted. More particularly, when the score obtained (also hereafter “E3-score”) is above the second calculated cutoff value, improvement of fibrosis of at least 1 stage without worsening of MASH can be predicted. In an advantageous embodiment, the second calculated cutoff value is equal to about 0.73.

[0040] In some embodiments, the biological sample is a sample of biological fluid. In some embodiments, the biological fluid is blood, serum or plasma. Advantageously, the biological fluid is blood.

[0041] In some embodiments, the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFFD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFFD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

[0042] As used herein, the term “Acute Fiver Failure” (ALF) is defined as a syndrome of rapid decline in liver function characterized by jaundice, coagulopathy (INR>1.5) and hepatic encephalopathy in patients with no evidence of prior liver disease. Acute liver failure is a further condition where it is thought that non-apoptotic forms of cell death play an important role in disease progression and development, and thus present likely therapeutic targets. There are many causes of ALF including drug toxicity, drug overdose, paracetamol overdose, autoimmune hepatitis, viral hepatitis, Wilson's disease, etc.

[0043] As used herein, the term “acute -on-chronic liver failure” (ACLF) refers to a distinct clinical entity encompassing an acute deterioration of the liver function in patients with cirrhosis, often decompensated cirrhosis, which is usually associated with a precipitating event and results in the failure of one or more organs and high short-term mortality. Unregulated inflammation is thought to be a major contributing factor. A characteristic feature of ACLF is its rapid progression, the requirement for multiple organ supports and a high incidence of short- and medium-term mortality of 50-90%.

[0044] As used herein, the term “cirrhosis” includes cirrhosis caused by alcohol use disorder, such as early stage alcoholism, chronic alcoholism or end-stage alcoholism; cirrhosis caused by chronic viral hepatitis; cirrhosis caused by Non-Alcoholic Fatty Liver Disease (NAFLD), and / or Non-Alcoholic SteatoHepatitis (NASH), and / or metabolic-associated fatty liver disease (MAFLD), and / or metabolic-associated steatohepatitis (MASH); cirrhosis caused by primary biliary cirrhosis and / or primary sclerosing cholangitis or even cirrhosis caused by medication. In some embodiments, cirrhosis is compensated cirrhosis or decompensated cirrhosis. In some embodiments, decompensated cirrhosis includes decompensated cirrhosis caused by alcohol use disorder, such as early stage alcoholism, chronic alcoholism or end-stage alcoholism; decompensated cirrhosis caused by chronic viral hepatitis; decompensated cirrhosis caused by Non-Alcoholic Fatty Liver Disease (NAFLD), and / or Non-Alcoholic Steatohepatitis (NASH), and / or metabolic-associated fatty liver disease (MAFLD), and / or metabolic-associated steatohepatitis (MASH); decompensated cirrhosis caused by primary biliary cirrhosis and / or primary sclerosing cholangitis or even decompensated cirrhosis caused by medication.

[0045] The liver disease is advantageously selected from non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), and metabolic-associated steatohepatitis (MASH). In some embodiments, the liver disease can be associated with diabetes, and in particular with type 2 diabetes. Examples of liver disease associated with diabetes include: non-alcoholic fatty liver disease (NAFLD) associated with diabetes, non-alcoholic steatohepatitis (NASH) associated with diabetes, metabolic-associated fatty liver disease (MAFLD) associated with diabetes, metabolic-associated steatohepatitis (MASH) associated with diabetes, the list being not limitative.

[0046] The present disclosure also relates to a system for assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the system comprising: a) means for measuring or receiving measurement data of levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient; and b) means for processing the data configured to assess the effectiveness of the treatment with lanifibranor in the patient as a function of the levels measured for the combination of biomarkers. The present disclosure also relates to the use of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea in a method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease.

[0047] In some embodiments, the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases. The liver disease is advantageously selected from NAFLD, NASH, MAFLD and MASH. In some embodiments, the liver disease can be associated with diabetes, and in particular with type 2 diabetes. Examples of liver disease associated with diabetes include: non-alcoholic fatty liver disease (NAFLD) associated with diabetes, non-alcoholic steatohepatitis (NASH) associated with diabetes, metabolic-associated fatty liver disease (MAFLD) associated with diabetes, metabolic- associated steatohepatitis (MASH) associated with diabetes, the list being not limitative.

[0048] In some embodiments, the treatment with lanifibranor allows improvement of fibrosis of at least 1 stage without worsening of MASH.

[0049] The present disclosure also relates to a method of treating a liver disease in a subject, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient before initiating a treatment; b) daily administering an effective amount of lanifibranor to the subject for at least 3 months; c) in vitro measuring levels of AST, insulin and urea in a biological sample of the subject after 3 months of treatment; d) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in steps a) and c), whereby a response or a non-response to the lanifibranor treatment can be predicted; e) continuing administering an effective amount of lanifibranor to the subject for at least another 3 months provided the assessment made in step d) is predictive of a response to the lanifibranor treatment, or that no diagnostic can be made.

[0050] According to embodiments that involve administering to a patient in need of treatment a (therapeutically) effective amount of lanifibranor, "therapeutically effective" or “effective amount” or "an amount effective to treat" or “pharmaceutically effective” denotes the amount of lanifibranor needed to inhibit or reverse a disease condition (e.g., to treat liver disease). Determining an effective amount specifically depends on such factors as safety and efficacy of the medicament. These factors will differ depending on other factors such as potency, relative bioavailability, patient body weight, severity of adverse side -effects and preferred mode of administration. Safety may be determined using methods well known in the art. Efficacy may be determined utilizing the same guidance. A pharmaceutically effective amount, therefore, is an amount that is deemed by the clinician to be safe, and efficacious.

[0051] Dosage may be adjusted appropriately to achieve desired level, local or systemic, depending upon the mode of administration. In the event that the response in a patient is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day may also be employed to achieve appropriate systemic levels of lanifibranor. Appropriate systemic levels can be determined by, for example, measurement of the patient's peak or sustained plasma level of the drug. "Dose" and "dosage" are used interchangeably herein.

[0052] In some embodiments, according to step b) of a method of treating a liver disease in a subject, lanifibranor is administered at a daily dosage of from about 400 mg to about 1,200 mg. Advantageously, lanifibranor is administered at a daily dosage of about 400 mg, advantageously at a daily dosage of about 500 mg, advantageously at a daily dosage of about 600 mg, advantageously at a daily dosage of about 700 mg, advantageously at a daily dosage of about 800 mg, advantageously at a daily dosage of about 900 mg, advantageously at a daily dosage of about 1 ,000 mg, advantageously at a daily dosage of about 1,100 mg, advantageously at a daily dosage of about 1,200 mg. In some embodiments, lanifibranor is administered to a patient with a meal. In some embodiments, lanifibranor is administered to a patient under fasted conditions.

[0053] In some embodiments, lanifibranor is employed for in vivo applications. Depending on the intended mode of administration in vivo, lanifibranor may be administered as a solid, semi-solid or liquid dosage form. In some embodiments, lanifibranor is administered in a solid dosage form. Exemplary solid dosage forms include tablets, capsules, stick-packs, sachets, lozenges, powders, pills, or granules. Preferred solid dosage forms include tablets, capsules and stick-packs, tablets being especially preferred. Advantageously, the lanifibranor is administered in unit dosage forms suitable for single administration of precise dosage amounts. Depending on the formulation desired, lanifibranor can be formulated into a pharmaceutical composition comprising lanifibranor and one or more pharmaceutically acceptable excipient(s). The choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. Pharmaceutical compositions of the invention can be prepared by conventional methods, as described e.g. in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995), incorporated herein by reference. In some embodiments, the pharmaceutically acceptable excipients include two or more of a binder, a disintegrant, a filler, a glidant, a lubricant, and a surfactant. In some embodiments, the pharmaceutically acceptable excipients include a binder, a disintegrant, a filler, a glidant, a lubricant and a surfactant.

[0054] In some embodiments, the pharmaceutical composition comprises from 200 mg to 1,200 mg of lanifibranor. Exemplary pharmaceutical compositions comprise 200 mg, 400 mg, 600 mg, 800 mg, 1,000 mg or 1,200 mg of lanifibranor.

[0055] Administration during in vivo treatment may be by any routes, including oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal. The skilled person in the art will recognize that the route of administration varies depending on the disorder to be treated. Advantageously, lanifibranor or the pharmaceutical composition comprising lanifibranor may be administered to a patient via oral, parenteral or topical administration. In one embodiment, the pharmaceutical composition comprising lanifibranor is administered by oral administration.

[0056] In some embodiments, according to step b) of the method, lanifibranor is daily administered in an effective amount to the subject for at least 3 months, advantageously at least 4 months, advantageously at least 5 months, advantageously at least 6 months, advantageously at least 7 months, advantageously at least 8 months, advantageously at least 9 months, advantageously at least 10 months, advantageously at least 11 months, advantageously at least 12 months, advantageously after at least 13 months, advantageously after at least 14 months, advantageously after at least 15 months, advantageously after at least 16 months, advantageously after at least 17 months, advantageously after at least 18 months, advantageously after at least 19 months, advantageously after at least 20 months, advantageously after at least 21 months, advantageously after at least 22 months, advantageously after at least 23 months, advantageously after at least 24 months, or more.

[0057] In some embodiments, according to step c) of the method, levels of AST, insulin and urea are measured in a biological sample of the subject after at least 3 months, advantageously at least 4 months, advantageously at least 5 months, advantageously at least 6 months, advantageously at least 7 months, advantageously at least 8 months, advantageously at least 9 months, advantageously at least 10 months, advantageously at least 11 months, advantageously at least 12 months, advantageously after at least 13 months, advantageously after at least 14 months, advantageously after at least 15 months, advantageously after at least 16 months, advantageously after at least 17 months, advantageously after at least 18 months, advantageously after at least 19 months, advantageously after at least 20 months, advantageously after at least 21 months, advantageously after at least 22 months, advantageously after at least 23 months, advantageously after at least 24 months, or more of treatment. In some embodiments, the assessment in step d) is made by obtaining a score (also called E3-score) from the levels measured in steps a) and c), and comparing the score (i) with a first calculated cutoff value below which no response to the lanifibranor treatment can be predicted, and (ii) with a second calculated cutoff value above which a response to the lanifibranor treatment can be predicted. When the score obtained is below the first calculated cutoff value, no response (i.e. negative response) to the lanifibranor treatment can be predicted. When the score obtained is above the second calculated cutoff value, a response (i.e. positive response) to the lanifibranor treatment can be predicted. In particular, when the score obtained is above the second calculated cutoff value, improvement of fibrosis of at least 1 stage without worsening of MASH can be predicted. When the score obtained is between the first calculated cutoff value and the second calculated cutoff value, no prognosis can be made as to the effectiveness of the lanifibranor treatment.

[0058] In an advantageous embodiment, the first calculated cutoff value is equal to about 0.39. In an advantageous embodiment, the second calculated cutoff value is equal to about 0.73.

[0059] In some embodiments, according to step e) of the method, lanifibranor is administered in an effective amount to the subject for at least another 3 months, advantageously at least another 4 months, advantageously at least another 5 months, advantageously at least another 6 months, advantageously at least another 7 months, advantageously at least another 8 months, advantageously at least another 9 months, advantageously at least another 10 months, advantageously at least another 11 months, advantageously at least another 12 months, advantageously after at least 13 months, advantageously after at least 14 months, advantageously after at least 15 months, advantageously after at least 16 months, advantageously after at least 17 months, advantageously after at least 18 months, advantageously after at least 19 months, advantageously after at least 20 months, advantageously after at least 21 months, advantageously after at least 22 months, advantageously after at least 23 months, advantageously after at least 24 months, or more.

[0060] In some embodiments, the duration of the treatment with lanifibranor as mentioned in step e) is dependent on how long lanifibranor has been administered in step b). In some embodiments, the measurement in step c) is performed after the treatment with lanifibranor. If in step b), the treatment with lanifibranor is of 3 months, the measurement in step c) is performed after the 3 months treatment.

[0061] In some embodiments, the biological sample is a sample of biological fluid. In some embodiments, the biological fluid is blood, serum or plasma. Advantageously, the biological fluid is blood.

[0062] In some embodiments, the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFED), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases. The liver disease is advantageously selected from NAFLD, NASH, MAFLD and MASH. In some embodiments, the liver disease can be associated with diabetes, and in particular with type 2 diabetes. Examples of liver disease associated with diabetes include: non-alcoholic fatty liver disease (NAFLD) associated with diabetes, non-alcoholic steatohepatitis (NASH) associated with diabetes, metabolic-associated fatty liver disease (MAFLD) associated with diabetes, metabolic- associated steatohepatitis (MASH) associated with diabetes, the list being not limitative.

[0063] In some embodiments, the present disclosure also includes kits for evaluating the effectiveness of the treatment with lanifibranor as well as kits for determining CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea levels, for example, from a biological sample. As used herein, “kit” is intended to mean a package, collection, or container of materials intended to aid one in use of the assay of the invention.

[0064] Kits of the present disclosure will typically comprise one or more containers containing one or more reagents useful to practice the invention. Reagents useful to practice the invention include, but are not limited to, buffers, buffer salts, metal ions, chromogenic compounds, antibodies, enzymes, fluorescent compounds and the like. Kits of the present disclosure may comprise one or more containers containing CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, or other compounds that may be used as a reference standard. Kits of the invention may comprise containers containing one or more antibodies wherein the antibodies are conjugated to a detectable moiety. Detectable moieties may be any known to those skilled in the art, for example, enzymes (e.g., peroxidase, luciferase), other proteins (e.g., green fluorescent protein), optically detectable compounds (e.g., fluorophores, chromophores), members of a binding pair (e.g., biotin / streptavidin), or any other detectable moiety known to those skilled in the art.

[0065] The present disclosure is illustrated by the examples below.

[0066] Examples

[0067] Example 1: materials and methods

[0068] Patients

[0069] NATIVE was a multicenter, randomized, placebo-controlled phase 2b study investigating the safety and efficacy of a treatment with lanifibranor in adult patients diagnosed with highly active, non-cirrhotic NASH. Patients were eligible for inclusion if they were 18 years of age or older and had non-cirrhotic NASH (the diagnosis of which required a Steatosis, Activity, Fibrosis [SAF] grade of 1 or higher for steatosis, hepatocellular ballooning and lobular inflammation on liver biopsy). A score of 3 or higher on the SAF-A (the activity part of the SAF scoring system that incorporates the scores for hepatocellular ballooning and lobular inflammation) was also a criterion for eligibility. Patients with stage F4 fibrosis, classified according to the criteria of both the SAF and NASH Clinical Research Network (NASH CRN) were excluded from the study.

[0070] Patients were randomly assigned in a 1:1:1 ratio to receive 800 or 1200 mg lanifibranor or placebo once daily for 24 weeks. Biomarkers

[0071] A total of 71 biomarkers of interest including 65 laboratory parameters and six diagnosis scores already published (FIB4, NAFLD fibrosis score (NFS), FIBC3, ABC3D, ELF and MACK3) were measured at baseline and at the end of treatment (EOT), i.e. at Week 24. The 65 laboratory biomarkers were mainly related to liver enzymes, lipid and glucose metabolisms, inflammation, and liver fibrosis, as shown in Table 1.

[0072] Table 1

[0073]

[0074] The accuracy of the six diagnostic scores for the prediction of treatment response was evaluated first. Then, the baseline value and the evolution under treatment of the 65 laboratory biomarkers were included to derive a new combined biomarker signature.

[0075] Endpoint

[0076] The following histological endpoint according to NASH-CRN criteria was considered: improvement of fibrosis of at least 1 stage without worsening of MASH (E3).

[0077] Statistical methods

[0078] 1 / Evaluation of the available diagnostic scores

[0079] We evaluated the ability to assess the treatment response of the six published diagnostic scores available in the dataset: FIB4, NFS, FIBC3, ABC3D, ELF and MACK3. For E3-score, a multivariate model was derived including each the baseline value of the diagnostic score, its absolute change between baseline and EOT, and its relative change between baseline and EOT. The discriminatory ability of the models was assessed through the Area Under the Receiver Operating Characteristics (AUROC) curve. AUROC ranges from 0 to 1 and is interpreted as follows: 0.90- 1.00 = excellent, 0.80-0.90 = good, 0.70-0.80 = fair, and < 0.70 = poor discriminant abilities. Tests showing an AUROC > 0.8 are considered to be of clinical interest.

[0080] 2 / Development of the new signature for treatment response assessment

[0081] Overall, biomarker selection was done using classical univariate analysis, Principal Component Analysis (PCA) and sparse Partial Least Square Discriminant Analysis (sPLS-DA), and finally combined in scores by logistic regression. The 65 biomarkers were considered in three different ways: baseline value, absolute and relative changes between baseline and EOT, leading to 195 variables considered for analysis. First shortlisting -

[0082] Univariate analysis using Student’s t test, Welch’s t test or Wilcoxon-Mann-Whitney test when appropriate was performed to identify the biomarkers significantly associated with the endpoint evaluated. Biomarkers with p-values below 10% were retained in the first shortlist. Three additional runs of PCA and sPLS-DA were performed: one including all baseline biomarkers, one including all absolute changes between baseline and EOT, and the last including all relative changes between baseline and EOT. For PCA and sPLS-DA analyses, Box-Cox and Yeo-Johnson transformations were applied to normalize biomarkers. PCA was used to evaluate the association between data variation and response status. In case of association, the best represented biomarkers on the PCA graphs (with cosine squared greater than 0.35) were selected. sPLS-DA aimed to identify discriminating biomarkers between responders and non-responders in a supervised way. Finally, biomarkers selected by the seven analyses (univariate analysis, three PCA and three sPLS- DA) constituted the first shortlist of candidates for discrimination between responders and non- responders.

[0083] Second shortlisting

[0084] During this second phase, all candidate biomarkers from the first shortlist were introduced in a “final PCA” and a “final sPLS-DA”, finally leading to a second shortlist of up to 11 biomarkers. Correlation between biomarkers in this second shortlist was controlled, and only biomarkers with variance inflation factors lower than 4 were kept in the final biomarker shortlist.

[0085] Logistic regression model

[0086] The study signature was constructed using logistic regression including the biomarkers of the second shortlist. Model selection was conducted through Akaike information criterion stepwise procedure while controlling for interactions. Finally, a regression formula was retrieved from the logistic regression to compute the probability of being a responder.

[0087] 3 / Evaluation of accuracy of the signature

[0088] Performance evaluation

[0089] The discriminatory ability of the signature obtained for the E3 endpoint was assessed through the AUROC. The calibration (statistical consistency between the predicted probability and the observations, i.e. predicted probabilities being on average close to 1 for responders and close to 0 for non-responders) was assessed through the Brier Score (BS). The BS ranges from 0 to 1, the lower the BS, the better the calibration, i.e. BS of 0 means perfect calibration Use in clinical practice

[0090] In clinical practice, a signature predicting the histological response based on non-invasive tests would be useful to assess lanifibranor treatment efficacy. Thresholds at 80% negative / positive predictive values (NPV / PPV) could be utilized to rule out / in treatment response, providing two cutoff values indicating the likelihood to be a non-responder (i.e. lower than the lowest cut-off value), a responder (i.e. higher than the highest cut-off value) or in the grey zone (i.e. between the 2 cut-off values). A practicable decision rule based on this approach is depicted in Figure lErreur ! Source du renvoi introuvable.. We chose to calculate the thresholds for NPV / PPV because the assessment of treatment response is at the individual level, and not at the level of a population (for which sensitivity and specificity are better dedicated).

[0091] The diagnostic performance of the thresholds calculated was assessed through sensitivity, specificity, negative and positive predictive values, the size of the grey zone (the smaller, the better), and the non-invasive diagnostic effectiveness which is the rate of well classified patients among those outside the grey zone (the higher, the better).

[0092] Software

[0093] R software was used, including the following packages: FactoMiner, MixOmics, CAR, stats, pROC, DescTools, ModelGood.

[0094] Example 2; results

[0095] Study population

[0096] In the NATIVE trial, from February 2017 through July 2019, 247 patients were randomly assigned to receive 1200 mg of lanifibranor (N=83), 800 mg of lanifibranor (N=83), or placebo (N=81) orally once daily for 6 months. A total of 228 patients completed the trial, 77 in each lanifibranor group and 74 in the placebo group. Reasons for discontinuation of the trial regimen have been reported elsewhere. Finally, among the 154 patients treated with lanifibranor at both doses, 142 patients (N=70 and N=72 patients treated with 800 or 1200 mg per day, respectively) presented with pre- and post-treatment liver biopsies within the required time frame (at the latest within 14 days following last lanifibranor intake) and were included in the analysis population for signature development.

[0097] A summary of baseline characteristics of the 142 patients retained in the analysis population for signature development is provided in table 2.

[0098] Table

[0099] MASLD metabolic dysfunction-associated steatotic liver disease, SAF steatosis activity fibrosis

[0100] * To convert the values for high-density lipoprotein cholesterol to mg / dL, divide by 0.02586. To convert the values for triglycerides to mg / dL, divide by 0.01129. To convert the values for glucose to mg / dL, divide by 0.01129. To convert the values for insulin to pg / L, divide by 172.2. 1 Steatosis was assessed as the percentage of hepatocytes containing large and medium-sized intracytoplasmic lipid droplets (but not foamy microvesicles) and graded as 0 (<5%), 1 (5 to 33%), 2 (34 to 66%), or 3 (>67%), according to the Steatosis, Activity, Fibrosis (SAF) scoring system. Patients with grade 0 steatosis were excluded from the trial.

[0101] 2 Lobular inflammation was classified as grade 1 (two small foci of inflammatory cells) or grade 2 (more than two foci of inflammatory cells), according to the SAF scoring system.

[0102] 3 Ballooning was classified as grade 1 (round hepatocytes with pale cytoplasm and size similar to that of normal hepatocytes) or grade 2 (presence of enlarged hepatocytes with a diameter at least twice that of normal hepatocytes in a background of clear and round hepatocytes), according to the Nonalcoholic Steatohepatitis Clinical Research Network (MASH CRN) grading system.

[0103] 4 Fibrosis was classified as stage F0 (no fibrosis), stage Fl (mild fibrosis), stage F2 (significant [moderate] fibrosis), stage F3 (advanced fibrosis), or stage F4 (cirrhosis), according to the SAF-MASH CRN staging system. Patients with stage F4 fibrosis were excluded from the trial.

[0104] 5 The SAF-Activity score ranges from 0 to 4; with higher scores indicating more severe disease activity.

[0105] 6 The NAS ranges from 0 to 8. A score of 2 or less indicates “not MASH”; a score of 3 or 4, “borderline MASH,” and a score of 5 to 8, “definite MASH”.

[0106] 7 The ABC3D score comprises: A = Age>50 years, B = BMI>30 kg / m2, C = platelet Count<200xl0A9 / L, 3 = PRO-C3>15.5 ng / ml, D = Diabetes = present. The presence of each factor scores 1 point, except for T2DM that scores 2 points, yielding to a maximum of 6 (See Boyle M, Tiniakos D, Schattenberg JM, et al. Performance of the PRO-C3 collagen neoepitope biomarker in non-alcoholic fatty liver disease, JHEP Rep 2019;l(3): 188-198).

[0107] 8 FIBC3 score is calculated using the following formula: -5.939 + (0.053*age [years]) +(0.076*BMI [kg / m2]) + (1.614*T2DM |ycs= L no=0|) - (0.009*platelets [10A9 / L]) + (0.071*PRO-C3 [ng / ml]) (See Boyle M, Tiniakos D, Schattenberg JM, et al. Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease, JHEP Rep 2019;l(3):188-198.).

[0108] 9 FIB4 score is calculated using the following formula: (age [years] * AST [U / L]) / (platelets [xlO9 / L] * ALT [U / L]) (See Sterling RK, Lissen E, Clumeck N, et al. Development of a simple noninvasive index to predict significant fibrosis in patients with HIV / HCV coinfection. Hepatology 2006;43(6):1317-1325.).

[0109] 10 MACK-3 score is based on the following parameters: AST (IU / L), Glycemia (mmol / L), Insulin (pU / mL) and Cytokeratin M30 (IU / L) (see Boursier J, Anty R, Vonghia L, et al. Screening for therapeutic trials and treatment indication in clinical practice: MACK-3, a new blood test for the diagnosis of fibrotic NASH. Aliment Pharmacol Ther 2018;47(10):1387- 1396.).

[0110] 11 ELF score is calculated using the following formula: 2.494 + 0.846*ln(Hyaluronic acid [ng / mL]) + 0.735*ln(P3NP [pg / L]) + 0.391*ln(TIMP-l [ng / mL]) (See Lichtinghagen R, Pietsch D, Bantel H, et al. The Enhanced Liver Fibrosis (ELF) score: normal values, influence factors and proposed cut-off values. J Hepatol 2013;59(2):236-242.).

[0111] 12 NAFLD Fibrosis Score is calculated using the following formula: -1.675 + 0.037*age (years) + 0.094*BMI (kg / m2) + 1.13*(Impaired Fasting Glucose / diabetes |ycs= L no=0|) +

[0112] O.99*(AST / ALT ratio (no unit)) - 0.013*platelets (xlO9 / L) - 0.66*albumin (g / dl) (See Angulo

[0113] P, Hui JM, Marchesini G, et al. The NAFLD fibrosis score: a non-invasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 2007;45(4): 846-854).

[0114] As can be seen from Table 2, the mean age of the patients was 55 years, and the mean body mass index (BMI) was 33; 89 patients (63%) were female, and 60 (42%) had type 2 diabetes mellitus. Significant or advanced fibrosis (stages F2 and F3, respectively) was present in 111 patients (78%), and most patients had highly active MASH (the mean [+SD] SAF-A score was 3.3 ± 0.5, and 73% had a NAS of > 6, which indicates high disease activity).

[0115] Fibrosis improvement of at least 1 stage without worsening of MASH (E3) was reached in 56 patients (39%) in the pooled lanifibranor group (i.e. 22 patients (31%) treated with 800 mg and 34 patients (47%) treated with 1200 mg).

[0116] Available diagnostic scores

[0117] In a first step, the performance of currently available diagnostic scores FIB4, FIBC3, ABC3D, NFS, ELF and MACK3 in predicting histological responders with regard to the endpoint E3 was assessed. For the prediction of E3, MACK-3 model (baseline value combined with absolute change) provided the highest AUROC at 0.70+0.09. FIBC3 provided the only other model combining baseline value with evolution under treatment, with AUROC at 0.61+0.11.

[0118] The accuracy of published diagnostic score for the prediction of E3 is presented in table 3.

[0119] Table 3

[0120] None of the models reached AUROC at 0.80 for E3, indicating that existing scores are not sufficiently accurate to predict these endpoints. Therefore, further analyses focused on the development of new and specific signature for E3. Example 3

[0121] Development of a specific non-invasive biomarker signature for E3 (improvement of fibrosis for at least 1 stage without worsening of MASH)

[0122] The signature was built considering the 65 laboratory biomarkers available in the study, in their three different ways (baseline raw values, absolute changes at EOT and relative changes at EOT), providing a total of 195 biomarkers. Of these, 11 biomarkers were selected (*) for the final shortlist (see Table 4).

[0123] Table 4

[0124] Among these 11 biomarkers, 5 were finally selected by logistic regression as independent predictors of E3: baseline CK18M65, baseline gamma GT, relative change of AST, relative change of insulin and relative change of urea. Combining these 5 parameters into an E3-score provided AUROC of 0.81 ± 0.08 for predicting E3 response. Calibration of E3 was good with a Brier score at 0.18 (see Table 5).

[0125] Table 5 Sensitivity (descending curve, top left to bottom right), specificity (ascending curve, bottom left to top right), NPV (descending curve, top left to middle right) and PPV (ascending curve, middle left to top right) curves as a function of E3-score results are depicted in Figure 1. The predictive value thresholds were 0.39 (80% negative predictive value with corresponding 76% sensitivity) and 0.73 (80% positive predictive value with corresponding 94% specificity). Using these thresholds, 67% of patients were identified as biomarker E3 non-responders or responders, with 80% of them confirmed histologically, leaving 33% patients in the grey zone.

[0126] These results suggested that the retrieved biomarker signature consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea can be considered as a good classifier of endpoint E3. It thus results that the specific combination of biomarkers (CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea) allowed the assessing the effectiveness of a treatment with lanifibranor in non-cirrhotic MASH, with good diagnostic performance in particular for improvement of fibrosis of at least 1 stage without worsening of MASH.

[0127] ***

[0128] Aspects of the present disclosure are further illustrated by reference to the following, non-limiting embodiments.

[0129] 1. A method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the method comprising: a) an in vitro measurement of a level of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient, and b) an assessment of the effectiveness of a treatment with lanifibranor in the patient as a function of the level measured for the combination of biomarkers.

[0130] 2. A method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the method comprising: al) an in vitro measurement of a level of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient; bl) a comparison of the level measured in step a) compared to that measured in a plurality of samples of patients with a liver disease and having received a treatment with lanifibranor for which the effectiveness of treatment is known; the comparison being carried out by means of a statistical learning model using as input data the levels of the combination of biomarkers measured at step a); and cl) an assessment of the effectiveness of a treatment with lanifibranor in the patient as a function of the results determined by the model defined at step bl). 3. The method of item 1 or 2, wherein the levels of CK18M65 and Gamma Glutamyl Transferase are measured before the start of the treatment with lanifibranor and the levels of AST, insulin and urea are measured before the start of the treatment and after at least 3 months of treatment with lanifibranor.

[0131] 4. The method of item 3, wherein the levels of the combination of biomarkers measured in step al) are used to obtain a score linked to the assessment of the effectiveness of the treatment in the patient, the score being compared with at least one predetermined cutoff value so as to classify the prognosis among a plurality of classes.

[0132] 5. The method of item 4, wherein the plurality of classes comprises at least two classes of which one class of non-response to the treatment with lanifibranor.

[0133] 6. The method of item 4 or 5, wherein the assessment of the effectiveness of the treatment in the patient comprises a comparison of the score with a first calculated cutoff value below which no response to the lanifibranor treatment can be predicted and a second calculated cutoff value above which a response to the lanifibranor treatment can be predicted.

[0134] 7. The method of any of items 2 to 6, wherein the learning model is based on a prior analysis of samples of a cohort comprising patients treated with lanifibranor presenting a response to the treatment and patients treated with lanifibranor presenting no response to the treatment.

[0135] 8. The method of item 7, wherein said prior analysis comprises the application of a method for learning and for selecting variables.

[0136] 9. The method of item 8, wherein said method for learning and for selecting variables is a logistic regression.

[0137] 10. The method of any of items 7 to 9, wherein the levels are weighted as a function of the prior analysis of said cohort to derive the score.

[0138] 11. The method of item 6, wherein the first calculated cutoff value is equal to about 0.39.

[0139] 12. The method of item 6, wherein the second calculated cutoff value is equal to about 0.73.

[0140] 13. The method of item 6, wherein improvement of fibrosis of at least 1 stage without worsening of MASH can be predicted if the score obtained is above the second calculated cutoff value.

[0141] 14. The method of item 1 or 2, wherein the biological sample is a sample of biological fluid.

[0142] 15. The method of item 14, wherein the sample of biological fluid is a sample of blood, serum or plasma.

[0143] 16. The method of item 1 or 2, wherein the biomarker(s) the level of which is measured in step a) is / are protein biomarker (s).

[0144] 17. The method of item 1 or 2, wherein the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

[0145] 18. The method of item 17, wherein the liver disease is selected from: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH).

[0146] 19. The method of any one of items 17 to 18, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

[0147] 20. A system for assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the system comprising: a) means for measuring or receiving measurement data of a level of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient; and b) means for processing measurement data configured to assess an effectiveness of the treatment with lanifibranor in the patient as a function of the level measured for the combination of biomarkers.

[0148] 21. The system of item 20, wherein the liver disease is at least one selected cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

[0149] 22. The system of item 21, wherein the liver disease is selected from : non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH).

[0150] 23. The system of any one of items 20 to 22, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

[0151] 24. Use of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea in a method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease.

[0152] 25. The use of item 24, wherein the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

[0153] 26. The use of item 25, wherein the liver disease is selected from: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH).

[0154] 27. The use of any one of items 24 to 26, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

[0155] 28. A method of treating a liver disease in a subject, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the subject before initiating a treatment; b) daily administering an effective amount of lanifibranor to the subject for at least 3 months; c) in vitro measuring levels of AST, insulin and urea in a biological sample of the subject after 3 months of treatment; d) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in steps a) and c), whereby a response or a non-response to the lanifibranor treatment can be predicted; e) continuing administering an effective amount of lanifibranor to the subject for at least another 3 months provided the assessment made in step d) is predictive of a response to the lanifibranor treatment, or that no diagnostic can be made.

[0156] 29. The method of item 28, wherein the assessment in step d) is made by obtaining a score from the levels measured in steps a) and c), and comparing the score (i) with a first calculated cutoff value below which no response to the lanifibranor treatment can be predicted, and (ii) with a second calculated cutoff value above which a response to the lanifibranor treatment can be predicted.

[0157] 30. The method of item 29, wherein improvement of fibrosis of at least 1 stage without worsening of MASH can be predicted if the score obtained is above the second calculated cutoff value.

[0158] 31. The method of any one of items 28 to 30, wherein the biological sample is a sample of biological fluid.

[0159] 32. The method of item 31, wherein the biological fluid is blood, serum or plasma.

[0160] 33. The method of any one of items 28 to 32, wherein the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic- associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

[0161] 34. The method of item 33, wherein the liver disease is selected from: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH).

[0162] 35. The method of any one of items 28 to 33, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

[0163] ***

[0164] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, compositions of matter, means, methods, or steps.

[0165] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having any other possible combination of the features disclosed and claimed herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. Thus, the foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0166] It will be apparent to those skilled in the art that various modifications and variations can be made in the device, method, and system of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter include modifications and variations that are within the scope of the appended claims and their equivalents. For any patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of all of which are incorporated herein by reference in their entireties for all purposes.

Claims

CLAIMS1. A method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient; b) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in step a).

2. The method of claim 1, wherein the expression levels of CK18M65 and Gamma Glutamyl Transferase are measured before the start of the treatment with lanifibranor.

3. The method of claim 1 or claim 2, wherein the levels of AST, insulin and urea are measured before the start of the treatment with lanifibranor and after at least 3 months of treatment with lanifibranor.

4. The method of any one of claims 1 to 3, wherein the levels measured in step a) are used to obtain a score which falls within a determined or undetermined prognosis class.

5. The method of claim 4, wherein the score is obtained by combining the levels measured in step a) in a mathematical function.

6. The method of claim 5, wherein the mathematical function is a binary logistic regression.

7. The method of any one of claims 4 to 6, which comprises comparing the score with a first calculated cutoff value below which no response to the lanifibranor treatment can be predicted.

8. The method of any one of claims 4 to 6, which comprises comparing the score with a second calculated cutoff value above which a response to the lanifibranor treatment can be predicted.

9. The method of any one of claims 1 to 8, wherein the biological sample is a sample of biological fluid.

10. The method of claim 9, wherein the biological fluid is blood, serum or plasma.

11. The method of any one of claims 1 to 10, wherein, the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic- associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

12. The method of claim 11, wherein the liver disease is selected from: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), and metabolic-associated steatohepatitis (MASH).

13. The method of any one of claims 11 to 12, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

14. A system for assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease, the system comprising: a) means for measuring or receiving measurement data of levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the patient; and b) means for processing the data configured to assess the effectiveness of the treatment with lanifibranor in the patient as a function of the levels measured for the combination of biomarkers.

15. The system of claim 14, wherein the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

16. The system of claim 15, wherein the liver disease is selected from: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), and metabolic-associated steatohepatitis (MASH).

17. The system of any one of claims 15 to 16, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

18. Use of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea in a method of assessing the effectiveness of a treatment with lanifibranor in a patient with a liver disease.19 The use of claim 18, wherein the liver disease is at least one selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

20. The use of claim 19, wherein the liver disease is selected from: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), and metabolic-associated steatohepatitis (MASH).

21. The use of claims 19 to 20, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

22. A method of treating a liver disease in a subject, the method comprising: a) in vitro measuring levels of a combination of biomarkers consisting of CK18M65, Gamma Glutamyl Transferase, AST, insulin and urea, in a biological sample from the subject before initiating a treatment; b) daily administering an effective amount of lanifibranor to the subject for at least 3 months; c) in vitro measuring levels of AST, insulin and urea in a biological sample of the subject after 3 months of treatment; d) assessing the effectiveness of the treatment with lanifibranor as a function of the levels measured in steps a) and c), whereby a response or a non-response to the lanifibranor treatment can be predicted; e) continuing administering an effective amount of lanifibranor to the subject for at least another 3 months provided the assessment made in step d) is predictive of a response to the lanifibranor treatment, or that no diagnostic can be made.

23. The method of claim 22, wherein the assessment in step d) is made by obtaining a score from the levels measured in steps a) and c), and comparing the score (i) with a first calculated cutoff value below which no response to the lanifibranor treatment can be predicted, and (ii) with a second calculated cutoff value above which a response to the lanifibranor treatment can be predicted.

24. The method of claim 23, wherein improvement of fibrosis of at least 1 stage without worsening of MASH can be predicted if the score obtained is above the second calculated cutoff value.

25. The method of any one of claims 22 to 24, wherein the biological sample is a sample of biological fluid.

26. The method of claim 25, wherein the biological fluid is blood, serum or plasma.

27. The method of any one claims 22 to 26, wherein, the liver disease is selected from cirrhosis, liver fibrosis, liver steatosis, fatty liver disease, acute decompensation, acute decompensation of cirrhosis, acute on chronic liver failure (ACLF), acute liver failure (ALF), decompensated cirrhosis, compensated cirrhosis, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), metabolic- associated steatohepatitis (MASH), autoimmune cholangitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), autoimmune cholangiopathy, hepatocarcinoma (HCC), liver cirrhosis and a combination of these diseases.

28. The method of claim 27, wherein the liver disease is selected from: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), and metabolic-associated steatohepatitis (MASH).

29. The method of any one of claims 22 to 28, wherein the liver disease is associated with diabetes, and in particular with type 2 diabetes.

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