EA042720B1 - PHARMACEUTICAL COMBINATION PRODUCT AND ITS USE AS A MEDICINE - Google Patents

Description

The present invention relates to a pharmaceutical combination product containing nitazoxanide (NTZ) or a derivative thereof and a PPAR agonist for therapeutic use.

[2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate (or nitazoxanide or NTZ), first described in 1975 (Rossignol and Cavier, 1975), showed a high degree of efficacy against anaerobic protozoa, helminths, and a wide range of microbes, including both anaerobic and aerobic bacteria (Rossignol and Maisonneuve, 1984; Dubreuil, Houcke et al., 1996; Megraudd, Occhialini et al., 1998; Fox and Saravolatz, 2005; Pankuch and Appelbaum , 2006; Finegold, Molitoris et al., 2009). It was first studied in humans for the treatment of intestinal cestodes (Rossignol and Maisonneuve, 1984) and is now licensed in the United States (Alinia®, Romark laboratories) for the treatment of diarrhea caused by the protozoan parasites Crystosporidium parvum and Giardia intestinalis. NTZ has also been widely commercialized in Latin America and India, where it is indicated for the treatment of a wide range of intestinal parasitic infections (Hemphill and Mueller et al., 2006). The proposed mechanism of action by which NTZ exerts its antiparasitic activity is through the inhibition of pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reactions that are essential for anaerobic metabolism (Hoffman, Sisson et al., 2007). NTZ also showed activity against Mycobacterium tuberculosis, which lacks a PFOR homologue, suggesting an alternative mechanism of action. Indeed, the authors have shown that NTZ can also act as an uncoupling agent that disrupts membrane potential and pH homeostasis within the body (de Carvalho and Darby et al., 2011).

The pharmacological effects of NTZ are not limited to its antiparasitic or antibacterial activity, and in recent years several studies have found that NTZ may also provide antiviral activity (Di Santo and Ehrisman, 2014; Rossignol, 2014). NTZ interferes with viral replication in various ways, including blockade during maturation of hemagglutinin (influenza) or VP7 (rotavirus) proteins or activation of the PKR protein involved in the innate immune response (for a review, see (Rossignol, 2014)). NTZ has also been shown to have broad antitumor properties, interfering with critical metabolic and death-promoting signaling pathways (Di Santo and Ehrisman, 2014).

PPARs (α, β/δ (hereinafter δ) and γ) belong to the family of hormone-activated nuclear receptors. PPARs or peroxisome proliferator-activated receptors are nuclear receptors of the superfamily of transcription factors activated by the following ligands: steroids/thyroid hormones/retinoids. To date, three PPAR isotypes have been identified in mice and humans: PPARa, PPARδ, and PPARy. While PPAR β/γ expression in humans appears to be ubiquitous, PPARa and γ show differential tissue distribution (Braissant O and Wahli W, 1998). PPARa is expressed in cells with high catabolic activity of fatty acids and in cells with high peroxisomal activity (hepatocytes, cardiomyocytes, renal proximal tubules, intestinal mucosa). PPARe/δ is expressed ubiquitously and in abundance in most tissues. As for PPARy expression, it is limited mainly to adipose tissue, certain cells of the immune system and the retina, and is present only in trace amounts in other organs (Braissant O and Wahli W, 1998).

Taking PPARa as an example, its action is mediated by a class of compounds such as fibrates that have lipid-lowering effects. Natural ligands have also been identified, such as, for example, fatty acids, eicosanoids (leukotriene B4) and 8(S)hydroxyeicosatetraenoic acid (Kliewer SA et al., 1997). PPAR has been associated mainly with lipid and glucose metabolism. PPAR activators such as fibrates allow the regulation of plasma cholesterol and triglyceride concentrations through PPARα activation (Hourton D et al., 2001). Fibrate therapy leads to increased fatty acid oxidation in the liver. Fibrates also reduce triglyceride synthesis (Staels B and Auwerx J, 1998). PPARα activators are also able to correct hyperglycemia and insulin levels. Fibrates also reduce adipose tissue mass through a mechanism that is independent of food intake and leptin gene expression (Guerre-Millo M et al., 2000). The therapeutic utility of PPARy agonists has been extensively investigated in the treatment of type 2 diabetes (Spiegelman BM, 1998). PPARy agonists have been shown to restore insulin sensitivity in target tissues and reduce plasma glucose, lipid and insulin levels in both animal models of type 2 diabetes and in humans (Ram VJ, 2003). Activation of PPAR by ligands also plays a role in the regulation of gene expression that is involved in processes such as inflammation, angiogenesis, cell proliferation and differentiation, apoptosis, and the activity of inducible NO synthetase (iNOS), matrix metalloproteinase (MMPase), and tissue inhibitors of metalloproteinases (TIMP) . Activation of PPARα in keratinocytes leads to the cessation of their proliferation and the cessation of expression of genes involved in differentiation (Komuves LG et al., 2000). PPARs have anti-inflammatory properties because they negatively affect transcriptional mechanisms involving other transcription factors such as NF-kB or transcription activators such as STAT and AP-1 (Desvergne B and Wahli W, 1999). These anti-inflammatory and anti-proliferative properties make PPAR (and especially PPARa)

- 1 042720 interesting therapeutic targets for the treatment of diseases such as occlusive vascular diseases (atherosclerosis, etc.), hypertension, diseases associated with neovascularization (diabetic retinopathy, etc.), inflammatory diseases (inflammatory bowel disease, psoriasis and etc.) and neoplastic diseases (carcinogenesis, etc.)

The inventors have found that NTZ, a synthetic antiprotozoal agent, or derivatives thereof, or its metabolites, in combination with PPAR agonists, exhibit therapeutic activities that are useful in therapy, in particular for the treatment of immune, inflammatory, metabolic, fibrotic or cholestatic diseases.

Therefore, the present invention relates to a pharmaceutical combination product containing:

(i) a compound selected from the group consisting of nitazoxanide, tizoxanide and a pharmaceutically acceptable salt thereof; and (ii) at least one PPAR agonist selected from the group consisting of Elafibranor, Celadelpar, Saroglitazar, Lanifibranor and a pharmaceutically acceptable salt thereof.

In a specific embodiment of the invention, the pharmaceutical combination product contains:

(i) a compound selected from nitazoxanide, tizoxanide and a pharmaceutically acceptable salt thereof; and (ii) Elafibranor or a pharmaceutically acceptable salt thereof.

In another specific embodiment of the invention, the pharmaceutical combination product is a composition containing components i) and ii) and a pharmaceutically acceptable carrier.

In another specific embodiment of the invention, the pharmaceutical combination product is a set of parts containing components i) and ii) for sequential, separate or simultaneous use.

In another specific embodiment, the pharmaceutical combination product further comprises at least one therapeutically active agent with known antifibrotic activity, selected from pirfenidone or receptor tyrosine kinase (RTKI) inhibitors such as Nintedanib, Sorafenib, and other RTKIs, or angiotensin II receptor blockers (AT1 ), or CTGF inhibitors, or any antifibrotic compound sensitive to inhibition of TGFe AND BMP-activated pathways, including TGFe latent complex activators such as MMP2, MMP9, THBS1, or cell surface integrins, type I TGFe receptors (TGFBRI) or type II (TGFBRII) and their ligands such as TGFe, Activin, inhibin, Nodal, anti-Mullerian hormone, GDF or BMP, accessory co-receptors (also known as type III receptors) or components of the SMAD-dependent canonical pathway, including regulatory or inhibitory SMAD proteins or members SMAD-independent or non-canonical pathways, including various branches of MAPK signaling, TAK1, GTPase Rho-like signaling pathways, phosphatidylinositol-3 kinase/AKT pathways, TGFe-induced EMT process, or canonical and non-canonical Hedgehog signaling pathways, including Hh ligands or target genes, or any members of the WNT or Notch pathway that are sensitive to the influence of TGFe.

In another specific embodiment of the invention, the pharmaceutical combination product further comprises at least one therapeutically active agent selected from JAK/STAT inhibitors and other anti-inflammatory and/or immunosuppressant agents.

In a preferred embodiment, the additional therapeutically active agent is selected from glucocorticoids, NSAIDs, cyclophosphamide, nitrosoureas, folic acid analogs, purine analogs, pyrimidine analogs, methotrexate, azathioprine, mercaptopurine, cyclosporine, myriocin, tacrolimus, sirolimus, mycophenolic acid derivatives, fingolimod, and other sphingosine modulators. -1-phosphate receptors, monoclonal and/or polyclonal antibodies against such targets as pro-inflammatory cytokines and pro-inflammatory cytokine receptors, T-cell receptor and integrins.

In another specific embodiment of the invention, components (i) and (ii) of the pharmaceutical combination product are combined into a dosage form in the form of an injectable suspension, gel, oil, pill, tablet, suppository, powder, capsule, aerosol, ointment, cream, patch or means of galenic forms for prolonged and / or slow release.

The present invention also relates to the use of the pharmaceutical combination product described above as a medicine.

In a specific embodiment, the present invention relates to the use of the pharmaceutical combination product described above in a method for the treatment of fibrotic disease.

In particular, the present invention relates to the use of a pharmaceutical combination product as defined above in a method for the treatment of a fibrotic disease, wherein the fibrous

- 2 042720 the disease is selected from the group consisting of fibrosis of the liver, kidneys, skin, epidermis, endoderm, muscles, tendons, cartilage, heart, pancreas, lungs, uterus, nervous system, testicles, ovaries, adrenal glands, arteries, veins, colon intestines, intestines (eg, small intestine), biliary tract, soft tissues (eg, mediastinum or retroperitoneum), bone marrow, joints, and stomach, particularly fibrosis of the liver, gastrointestinal tract, lungs, heart, kidneys, muscles, skin, soft tissues , bone marrow, intestines and joints.

In accordance with the present invention, component (i) of the combination product is selected from:

NTZ:

tizoxanide:

and their pharmaceutically acceptable salts.

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

According to the invention, the term PPAR delta agonist in the context of this document includes MBX8025 (Celadelpar or {2-methyl-4-[5-methyl-2-(4-trifluoromethylphenyl)-2H-[1,2,3]triazol4-ylmethylsulfanyl ]phenoxy}acetic acid).

In accordance with the invention, the term PPAR-alpha/gamma agonist (also referred to as glitazar) in the context of this document includes Saroglitazar.

According to the invention, the term PPAR alpha/delta agonist means Elafibranor (GFT505).

In accordance with the invention, the term PPAR alpha/gamma/delta agonist, IVA337 (Lanifibranor).

The PPAR agonist may be in the form of a pharmaceutically acceptable salt.

The terms fibrosis, fibrotic disease, fibrotic disorder and their declensions mean a pathological condition of excessive deposition of fibrous connective tissue in an organ or tissue. More specifically, fibrosis is a pathological process that involves the continuous formation of a fibrous scar and overproduction of extracellular matrix by connective tissue in response to tissue injury. Physiologically, connective tissue deposition can disrupt the architecture and function of an underlying organ or tissue.

According to the present invention, fibrosis or fibrotic disorder may refer to fibrosis of any organ or tissue. Illustrative, non-limiting examples of specific organ fibrosis include fibrosis of the liver, kidney, skin, epidermis, endoderm, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, penis, ovary, adrenal, arteries, veins , colon, intestines (eg, small intestine), biliary tract, soft tissues (eg, mediastinum or retroperitoneum), bone marrow, joints (eg, knee, shoulder, or other joints), or stomach.

The term treating or treating refers to the treatment or prevention of a fibrotic disorder in a subject in need thereof. Treatment comprises administering a combination of the invention to a subject having the claimed disorder, ie a patient, to prevent, cure, delay, reverse or slow the progression of the disorder, thereby improving the subject's condition. Treatment may also be administered to a subject who is healthy or at risk for developing a fibrotic disorder.

Therefore, the treatment of fibrotic disease comprises administering a pharmaceutical combination product of the present invention, for example in the form of a pharmaceutical composition containing components (i) and (ii) of the combination, to a subject having the claimed disorder in order to cure, delay, reverse or slow the progression of the disorder, such thus improving the condition of the patient, or a healthy subject, in particular a subject who is at risk of developing such a disease.

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The treatment comprises administering a combination of the invention to a patient having a disorder of the subject to cure, delay, reverse or slow progression, thereby improving the patient's condition, or to a healthy subject, in particular a subject who is at risk of developing an inflammatory, metabolic, fibrotic and cholestatic disease.

The subject to be treated is a mammal, preferably a human. The subject to be treated may be selected based on several criteria associated with fibrotic diseases, such as prior drug treatment, comorbidities, genotype, exposure to risk factors, viral infection, as well as the detection of any relevant biomarker that can be assessed with using imaging techniques and immunological, biochemical, enzymatic, chemical detection methods or nucleic acid detection methods.

Subjects to be treated according to the invention may be selected based on several criteria related to inflammatory, metabolic, fibrotic and cholestatic diseases, such as prior drug treatment, comorbidities, genotype, exposure to risk factors, viral infection, as well as any other relevant biomarker that can be assessed by imaging and immunological, biochemical, enzymatic, chemical or nucleic acid detection methods.

The synthesis of NTZ or analogues can, for example, be carried out as described in (Rossignol and Cavier, 1975), or by any other synthetic method known to the person skilled in the art.

The pharmaceutical combination product of the invention is intended for simultaneous, sequential or separate administration in therapy, so it is acceptable that it be included in different compositions. In the case of sequential administration, NTZ and/or tizaxonide may be administered prior to the PPAR agonist(s), or the PPAR agonist(s) may be administered prior to NTZ and/or tizaxonide.

NTZ and tizaxonide may be formulated as pharmaceutically acceptable salts, in particular acid or base salts compatible with pharmaceutical use. NTZ and tizaxonide salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. These salts can be obtained during the final purification step of the compound or by incorporating the salt into the previously purified compound.

The combination of a compound of formula (I) or (II) with one or more PPAR agonist(s) may be formulated as pharmaceutically acceptable non-toxic salts derived from organic or inorganic bases or acids of the compound of formula (I) or (II) or the agonist(s). ) PPAR. These salts can be obtained during the final purification step of the compound or by incorporating the salt into the previously purified compound.

Pharmaceutical compositions of the present invention containing a compound of formula (I) or (II) and one or more PPAR agonist(s) may also contain one or more excipients or carriers acceptable in a pharmaceutical context (e.g. saline solutions, saline solutions, isotonic solutions, etc., compatible with pharmaceutical use and well known to the person skilled in the art).

These compositions may also contain one or more agents or carriers selected from dispersants, solubilizers, stabilizers, preservatives, and the like. The agents or carriers used for these formulations (liquid and/or injectable and/or solid) are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, gum arabic, liposomes, etc. d.

These compositions may be in the form of injectable suspensions, gels, oils, ointments, pills, suppositories, powders, gel capsules, capsules, aerosols, etc., in some cases using galenic forms or devices that provide prolonged and/or slow release. For this type of formulation, agents such as cellulose, carbonates or starches can advantageously be used.

The pharmaceutical compositions of the present invention may be administered in a variety of ways and in various forms. For example, the compound(s) may be administered via a systemic route, orally, parenterally, by inhalation, by nasal spray, by nasal instillation, or by injection, for example, intravenously, intramuscularly, subcutaneously, transdermally. by, by topical application, by intra-arterial route, etc.

Of course, the route of administration will be matched to the form of NTZ and/or tizaxonide in combination with one or more PPAR agonist(s) according to procedures well known to those skilled in the art.

NTZ and/or tizaxonide in combination with one or more PPAR agonist(s) is administered in a therapeutically effective amount. In the context of the invention, the term effective amount refers to an amount of a compound sufficient to produce the desired therapeutic result.

The frequency and/or dose related to administration may be adjusted by one of ordinary skill in the art depending on the patient, pathology, form of administration, etc. Typically, the combination (for example, in the form of a pharmaceutical composition or compound kit) of the present invention can be administered for the treatment of fibrotic disease at a dose for NTZ or an NTZ analog or agonist.

A PPAR such as a compound of formula (I) or (II) or (III) ranging from 0.01 to 4000 mg/day, for example from 50 to 2000 mg/day and in particular from 100 to 1000 mg/day .

The dose of PPAR agonist(s) in said combination may vary depending on the PPAR agonist itself. The dose is adjusted to the effectiveness of the PPAR agonist.

In a preferred embodiment, NTZ is used in combination with Elafibranor at a dose of 100 to 1000 mg/day (such as 1000 mg/day) (particularly 20 to 40 mg/day) for NTZ and 80 to 120 mg /day for Elafibranor.

In another preferred embodiment, the active ingredients are administered as one or more pharmaceutical composition(s) in the form of tablets intended for oral administration.

The introduction can be carried out daily or even several times a day, if necessary.

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

Description of figures and tables

Fig. 1: Antifibrotic effect of Elafibranor and Nitazoxanide in TGFe-induced hHSC

Serum-free HSCs were pre-incubated for 1 h with Elafibranor (A) or Nitazoxanide (B) or Bezafibrate (C) before activation with the profibrogenic cytokine TGFe1 (1 ng/ml). After 48 hours of incubation, α-GMA expression was measured by ELISA. The obtained values were transformed into percent inhibition compared to the TGFe1 control. Data are presented as mean (of three repetitions) ± standard deviation (SD). Curve processing and calculation of half maximum inhibitory concentration (IC50) were performed using XLFit 5.3.1.3 software.

Fig. 2: Combination of Elafibranor with Nitazoxanide synergistically inhibits α-GMA in TGFe1 induced hHSC

Combinations were tested in a dose-response matrix and analyzed according to Bliss' incremental-over-additivism (EOB) model. Dilution series of Elafibranor (row) and Nitazoxanide (column) were prepared, including their respective DMSO controls. The resulting mixtures were added to serum-free HSC 1 h before activation with the profibrogenic cytokine TGFe1 (1 ng/ml). (A) Percent inhibition of α-GMA compared to TGFe1 control for all pairs of combinations. Data are presented as an average of four repetitions. (B) EOB scores were calculated as described in the Materials and Methods section. Any pair of compounds with a positive EOB value was considered synergistic (light gray to black). An overall EOB including all combinations was also calculated. (C) Data values obtained from a pair of synergistic combinations were plotted on a graph presented as a histogram. Data are presented as mean (of four repetitions) ± standard deviation (SD). The EOHSA model was used, as described in the Materials and Methods section, to validate the synergy of selected pairs of NTZ/ELA combinations.

Fig. 3: Combination of PPAR agonists with Nitazoxanide synergistically inhibits α-GMA in TGFe1-induced hHSCs

Combinations were tested in a dose-response matrix and analyzed according to Bliss' incremental-over-additivism (EOB) model. Dilution series of Saroglitazar, Celadelpar and Lanifibranor, and Nitazoxanide were prepared, including their respective DMSO controls. The resulting mixtures were added to serum-free HSC 1 h before activation with the profibrogenic cytokine TGFe1 (1 ng/ml). The percentages of α-GMA inhibition compared to the TGFe1 control were calculated for all pairs of combinations. EOB scores were calculated as described in the Materials and Methods section. Any pair of compounds with a positive EOB value was considered synergistic (light gray to black).

(A) Saroglitazar (SARO) (B) Celadelpar (SELA) (C) Lanifibranor (LANI)

The percentage inhibition of α-GMA compared to the control TGFe1, obtained from a pair of synergistic combinations, was applied to the graph, presented as a histogram. Data are presented as mean (of four repetitions) ± standard deviation (SD). The EOHSA model was used as described in the Materials and Methods section to confirm the synergy of the selected combination pairs.

Fig. 4: Hepatic collagen content

6-week-old C57BL/6 mice received a control diet (CSAA), a CDAA+1% cholesterol diet (CDAAc), or a CDAAc diet supplemented with NTZ (30 or 100 mg/kg/day for 12 weeks or Elafibranor (1 or 3 mg/day). kg/day), or a combination of Elafibranor and NTZ (respectively 1+30 mg/kg/day, 1+100 mg/kg/day, 3+30 mg/kg/day and 3+100 mg/kg/day).

For each graph, the exact number of exposure doses was indicated.

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After sacrifice, the content of hepatic collagen was determined.

Fig. 5: Liver fibrosis percentage

6-week-old C57BL/6 mice received a control diet (CSAA), a CDAA+1% cholesterol diet (CDAAc), or a CDAAc diet supplemented with NTZ (30 or 100 mg/kg/day for 12 weeks or Elafibranor (1 or 3 mg/day). kg/day), or a combination of Elafibranor and NTZ (respectively 1+30 mg/kg/day, 1+100 mg/kg/day, 3+30 mg/kg/day and 3+100 mg/kg/day).

For each graph, the exact number of exposure doses was indicated.

After sacrifice, the area of liver fibrosis was determined.

Fig. 6: Expression of the AGMA gene in the liver

Fig. Figure 7: Expression of the CCR2 gene in the liver

Fig. Figure 8: Expression of the CCR5 gene in the liver

Fig. Figure 9: Expression of the Col1a2 gene in the liver

Fig. Figure 10: Expression of the MMP2 gene in the liver

Fig. 11: Expression of the TIMP2 gene in the liver

Fig. 12: Expression of the TGFe1 gene in the liver

Abbreviations used in figures, tables and text:

AR-1 Activating protein-1 ASBTi Apical sodium codependent inhibitor bile acid transporter ASK1 Signal-regulating kinase 1 ATI Angiotensin 1 COPD Chronic obstructive pulmonary disease CTGF Connective tissue growth factor DGAT Diacylglycerol-O-acyltransferase DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPP4 Dipeptidyl peptidase ELISA Linked immunosorbent assay EOB Bliss Increment FABAC Fatty acid conjugate with bile acids FBS Fetal bovine serum FGF Fibroblast growth factor FXR Farnesoid X receptor GDF Growth and differentiation factor GLP-1 Glucagon-like peptide-1 GPCR G-protein coupled receptor HBV Hepatitis B virus HCV Hepatitis C virus 15-HERE 5-hydroxyeicosapentaenoic acid HIV AIDS virus HSC stellate cell of the liver IC50 Half maximum inhibitory concentration INOS Inducible nitric oxide synthase

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IPF Idiopathic pulmonary fibrosis LANI Lanifibranor LBD Ligand binding domain LPS Lipopolysaccharide LT Leukotriene MARK Mitogen-activated protein kinase MMR-9 Metalloprotease-9 MMPase Metalloprotease NADPH Nicotinamide adenine dinucleotide phosphate NAFLD Non-alcoholic fatty liver disease NASH Non-alcoholic steatohepatitis NF-KB Nuclear factor kappa-B NOx NADPH oxidase NSAIDs non-steroidal anti-inflammatory drugs NTZ Nitazoxanide PAR Protease-activated receptor PBC Primary biliary cholangitis PDE Phosphodiesterase PDGF Platelet growth factor PFIC3 Hereditary intrahepatic cholestasis type 3 PFOR Pyruvate:ferredoxin oxidoreductase PPAR peroxisome proliferator-activated receptor PPRE PPAR response elements PSC Primary sclerosing cholangitis ROCK Rho-associated protein kinase RTK Receptor tyrosine kinase SARO Saroglitazar SD Standard deviation SELA Celadelpar SGLT Sodium-glucose transporter STAT Signal transmitters and transcription activators TGFp Transforming growth factor-β TGFBRI ΤΠΕβ type I receptors TGFBRII Τ6Εβ type II receptors THBS1 Thrombospondin-1 THRβ thyroid β-receptor TIMP Tissue metalloprotease inhibitor TLR-4 Toll-like receptor 4 TZ Tizoxanide TZG Tizoxanide glucuronide VAP-1 Vascular adhesive protein-1

Examples Materials and Methods

Compounds were dissolved in dimethyl sulfoxide (DMSO, Fluka cat. no. 41640). Nitazoxanide (INTERCHIM cat. no. RQ550U), Tizoxanide (INTERCHIM cat. no. RP253), Lanifibranor (ARK PHARM cat. no. AK689102), Celadelpar (ARK PHARM cat. no. AK689146), and Saroglitazar (CHEMEXPRESS cat. no. YY-1997A) were commercially available. way.

Cultivation of hHSC

Primary human liver stellate cells (hHSC) (Innoprot) were cultured in STeCM medium (ScienCell cat. no. 5301) supplemented with 2% fetal bovine serum (FBS, ScienCell cat. no. 0010), 1% penicillin/streptomycin (ScienCell cat. no. 0503) and stellate cell growth supplement (SteCGS; ScienCell cat. no. 5352). Cell culture flasks were coated with poly-L-lysine (Sigma cat. no. P4707) for better adhesion.

Getting compositions

2-component combination matrix (NTZ/Elafibranor)

For these experiments, a matrix was compiled using the checkerboard method. Stock solutions

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NTZ/Elafibranor was serially diluted in DMSO in a 5-dot series in a row (PPAR agonist Elafibranor) and in a 6-dot series in a NTZ column of a 96-well plate or a 384-well plate. Subsequently, a combination matrix 5x6 was obtained by mixing 1:1 all concentrations of a single agent. Test concentrations for each compound were selected based on the respective IC ₅₀ of each compound as a single agent, obtained by measuring the content of αGMA in the HSC model stimulated with TGF-βΓ

Then 2 times and 4 times higher and lower concentrations were chosen.

hHSC activation with TGF-βΙ and compound treatment

Primary human liver stellate cells (hHSC) (Innoprot) were cultured under standard conditions as described above. Cells were then plated at 2x104 cells/well in 96-well plates and 6500 cells/well in 384-well plates for α-GMA measurement by ELISA. The next day, the cell culture medium was removed and the cells were washed with phosphate-buffered saline (PBS) (Invitrogen cat. no. 14190). hHSCs were deprived for 24 h in serum-free and SteCGS-free medium.

For treatments with NTZ, Elafibranor or other PPAR agonists and appropriate combinations of NTZ/Elafibranor or PPAR agonists, serum-free hHSCs were pre-incubated for 1 h with the compounds followed by the addition of profibrogenic TGF-e1 stimuli (PeproTech cat. no. 100-21, 1 ng /ml) in serum-free and SteCGS-free for an additional 48 hours.

Enzyme immunoassay (ELISA) α-GMA

The level of α-GMA was measured using a sandwich version of the ELISA. Briefly, the wells of the ELISA plate were first coated with immobilized antibody (mouse monoclonal anti-ACTA2, Abnova) at 4° C. overnight. After 3 washes in PBS+0.2% Tween 20, a blocking solution consisting of PBS+0.2% BSA was added for one hour, followed by another wash cycle. Cell lysates were transferred to the wells for binding to immobilized antibody for 2 hours at room temperature. After the washing procedure, identification antibody (biotinylated mouse monoclonal anti-ACTA2, Abnova) was added for 2 h at room temperature followed by 3 washes. For detection, HRP-conjugated Streptavidin (R&D Systems cat. no. DY998) was first applied for 30 minutes at room temperature. After washing, TMB substrate for HRP (BD, no. 555214) was added and incubated for 7 min at room temperature in the dark. When oxidized, TMB forms a water-soluble blue reaction product that turns yellow with the addition of sulfuric acid (stop solution), allowing accurate measurement of the intensity at 450 nm using a spectrophotometer. The color developed is directly proportional to the amount of α-GMA present in the lysate.

Incremental Bliss (EOB) Synergy Determination and EOSHA Validation (increment above single agent maximum)

The values obtained in the αGMA ELISA assays were first converted to percent inhibition compared to the TGF-βΓ control. Then, using these percent inhibition, EOB (Bliss increment) was determined to establish the synergistic effect of the drug combinations. The expected Bliss additivism score (E) was first determined by the formula:

E=(A+B)-(AxB), where A and B are the percent inhibition of NTZ (A) and Elafibranor, Saroglitazar, Celadelpar or Lanifibranor (B) at a given dose. The difference between the Bliss expectation and the observed inhibition of combined NTZ/Elafibranor, Saroglitazar, Celadelpar, or Lanifibranor at the same dose is the Bliss gain.

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

A Bliss increment >0 indicates activity that is greater than additive (synergism); And

A Bliss increment <0 indicates that the activity of the combination is less than additive (antagonism).

For the NTZ+Elafibranor combination, an additional total Bliss score was calculated by summing all EOBs.

EOHSA is the standard synergy score used by the US Food and Drug Administration (FDA) to evaluate drug combinations and is calculated as the difference between the effect produced by the drug combination and the greatest effect produced by each of the individual agents in the combination in the same concentrations as when combined (Borisy et al., 2003). For synergistic combinations identified by the EOB method, the experimental % inhibition was plotted on a bar graph and the significance of the observed differences between NTZ/ELA and a single agent was assessed using one-way ANOVA and retrospective unadjusted least significant significant analysis. Fisher difference (*: p<0.05; **: p<0.01;

***: p<0.001).

Evaluation of Elafibranor, Nitazoxanide and the Elafibranor+Nitazoxanide Combination in the Chronic CDAA+1% Cholesterol Model in Fibrous NASH (12 weeks)

Experiment plan

The L-amino acid-restricted choline-deficient diet (CDAA) lacks choline, which is required for liver β-oxidation and VLDL production, and is thought to stimulate hepatocytes to accumulate fat and subsequently cause cell damage. The CDAA diet-induced rodent model develops fibrosis over a relatively short period of time, which is ideal for rapidly studying the reversibility of NASH pathology, especially fibrosis.

Increased cholesterol intake accelerates liver fibrosis in several NASH mouse models. Exacerbation of liver fibrosis mainly involves accumulation of free cholesterol in hepatic stellate cells, which increases the cells' sensitivity to transforming growth factor β (TGFP) and subsequently exacerbates hepatic fibrosis.

In the present study, we investigated the effect of nitazoxanide on hepatic fibrosis in C57Bl/6J mice fed a CDAA diet supplemented with 1% cholesterol.

The prophylactic effects of Elafibranor alone, NTZ alone, and the combination thereof were evaluated in a mouse model of fibrosing NASH fed a CDAA+1% cholesterol diet. Male C57Bl/6J mice at 6 weeks of age received a control diet (CSAA), a CDAA+1% cholesterol diet, or a CDAA+1% cholesterol diet supplemented with Elafibranor 1 and 3 mg/kg/day, NTZ 30 and 100 mg/kg/day or combination drugs (Elafibranor 1 and 3 mg/kg/day in combination with NTZ 30 and 100 mg/kg/day) for 12 weeks.

Body weight and food intake were monitored twice a week. On the last day of treatment, mice were sacrificed after a 6 hour fasting period. The liver was quickly surgically removed for biochemical and histological studies.

All animal manipulations were performed according to standard protocols and in accordance with standard guidelines for the proper care and use of laboratory animals.

Male C57BL/6 mice at 6 weeks of age were fed for 12 weeks according to the experimental design detailed in Table 1. 1.

Table 1. Plan of the experiment

Diet Compound Dose Quantity mg/kg/day mice CSAA 8 12 Elafibranor 1 8 3 8 CDAA+1% NTZ thirty 8 cholesterol 100 8 Elafibranor+NTZ 1+30 8 1+100 8 3+30 8 3+100 8

The CSAA diet served as the control.

Some mice received a CDAAc diet.

Some mice received a CDAAc diet supplemented with Elafibranor at 1 or 3 mg/kg/day.

Some mice received a CDAAc diet supplemented with NTZ at 30 or 100 mg/kg/day.

Some mice received a CDAAc diet supplemented with the Elafibranor+NTZ combination in various ratios: 1+30, 1+100, 3+30 and 3+100 mg/kg/day.

The CDAAc diet supplemented with 30mg/kg NTZ is also referred to as C57Bl/6J mice fed CDAA+0.02% (w/w) nitazoxanide, corresponding to a theoretical dose of 30mg/kg/day.

The CDAAc diet supplemented with 100 mg/kg NTZ is also referred to as C57Bl/6J mice fed a CDAA+0.0667% (w/w) nitazoxanide diet, corresponding to a theoretical dose of 100 mg/kg/day.

The feed was purchased from Ssniff® (Soest, Germany).

Nitazoxanide (see standard sample in Table 1) was included in the Ssniff® diet CDAA + 1% cholesterol in powdered form up to the required dose.

Standard sample and batch number of nitazoxanide are given in table. 2.

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Table 2. Standard samples of nitazoxanide

Connection (INN) Lab code Provider standard sample The consignment External identifier ID in Genfit Nitazoxanide GFE 50455 GSL022597.08 Interchim RQ550 1505

For each dose, accurate dose calculations were performed according to the following example. This makes it possible to account for the exact dose of each product that was accurately consumed by each group of mice.

Calculation of actual therapeutic doses: example with 0.02% (w/w) nitazoxanide

Food intake is expressed in grams of food/gram animal/day:

0.02% nitazoxanide in the diet

0.02 g compound/100 g food = 0.2 mg compound/g food

Actual Compound Dose:

0.2 mg compound/gram food/gram animal/day (0.2 mg compound/gram food/gram animal/day) x 1000=(0.2 mg compound/gram food/kg animal/day)=200 mg compound /gram food/kg animal per day

Therefore, multiplying by 200 the food intake value expressed in grams of food/grams of animal/day; The value obtained corresponds to the actual dose administered, expressed in mg NTZ/kg animal/day.

In the same way:

For a dose of NTZ 0,00667% wt./wt. the actual treatment dose was obtained by multiplying the food intake (grams of food/grams of animal/day) by 66.7.

For a dose of NTZ 0,0667% wt./wt. the actual treatment dose was obtained by multiplying the food intake (grams of food/grams of animal/day) by 667.

According to the calculation, the calculated doses in comparison with the planned doses are given in the following tables. 3 and 4.

Table 3. Planned and calculated doses for each compound at various doses

Groups GFT505 NTZ Planned doses 1 mg/kg 3 mg/kg 30 mg/kg 100 mg/kg Calculated doses 1 mg/kg 2.8 mg/kg 26.3 mg/kg 78.1 mg/kg

Table 4. Planned and calculated doses for . each combination at different doses

Groups Combination 1 Combination 2 Combination 3 Combination 4 GFT505 NTZ GFT505 NTZ GFT50 5 NTZ GFT505 NTZ Planned 1 thirty 1 100 3 thirty 3 100 DOSES mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Calculated 0.9 26, 4 0.9 79.4 2.9 27.4 2.8 77.7 DOSES mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

Body weight and food intake were recorded twice a week throughout the study.

At the end of the treatment period, the animals were anesthetized with isoflurane and blood samples were taken as described below. The animals were then euthanized by dislocation of the cervical vertebrae and decapitated for brain removal and weighing. The liver was also collected and weighed. Part of the liver was fixed in 4% formalin, embedded in paraffin and used for histological analysis. The remaining liver was quickly frozen in liquid nitrogen and stored at -80°C until used for further analysis.

Blood sampling was performed at the stage of killing the experimental animal after a 6-hour fasting period. Blood samples were taken under anesthesia by retro-orbital puncture. Heparin tubes containing blood were rapidly centrifuged (15 min at 4000 o6/muh/4°C) and the plasma fraction was collected. Plasma aliquots were stored at -20°C until further analysis.

Plasma biochemistry Alanine aminotransferase (ALT)

Plasma ALT concentration was determined using the appropriate Randox kit for the Daytona machine (Randox cat. no. AL 3801). Briefly, ALT in a plasma sample enzymatically converts α-oxoglutarate and L-alanine to L-glutamate and pyruvate. In the presence of NADH, the resulting pyruvate is converted by lactate dehydrogenase to form L-lactate and NAD+. The kinetics of the reaction has been studied and allows the plasma ALT level to be calculated. The results are expressed in U/l.

Aspartate aminotransferase (AST)

Plasma AST concentration was determined using the appropriate Randox kit for the Daytona machine (Randox cat. no. AS 3804). Briefly, AST in a plasma sample enzymatically converts α-oxoglutarate and L-aspartate to L-glutamate and oxaloacetate. In the presence of NADH, the resulting oxalo acetate is converted by malate dehydrogenase to form L-malate and NAD+. The reaction kinetics have been studied and allow calculation of plasma AST levels. The results are expressed in U/l.

Histology

When experimental animals were sacrificed, liver samples were processed for histological analysis and examined as follows.

Tissue embedding and cutting

Liver sections were first fixed for 12 hours in 4% formalin solution. Then the liver pieces were washed for 30 min in PBS and dehydrated in ethanol solutions (successive baths with 70, 80, 95, and 100% ethanol). The liver pieces were incubated in three different xylene baths (Sigma-Aldrich cat. no. 534056) and then in two liquid paraffin baths (60° C.). The liver pieces were then placed on racks, which were carefully filled with Histowax® to completely cover the tissue.

Paraffin blocks containing tissue pieces were removed from the racks and stored at room temperature. The liver blocks were cut into 3 µm pieces.

Red staining with picrosirius

Liver sections were deparaffinized, rehydrated, and incubated for 15 minutes in 0.04% Fast Green FCF (Sigma-Aldrich, cat. no. F7258) before washing in a 0.5% acetic acid bath (Sigma-Aldrich, cat. no. 695092). ). Liver sections were then washed in water and incubated for 30 min in a solution of Fast Green FCF 0.04-0.1% Sirius red (Direct Red 80, Fluka cat. no. 43665) in saturated aqueous picric acid (Sigma-Aldrich cat. no. P6744) . The sections were then dehydrated and mounted using CV Mount medium (Leica, cat. no. 14046430011).

Histological studies

The laboratory assistant, who did not see the source of each liver sample, performed histological studies. Virtual slides were created using a Pannoramic 250 scanner from 3D Histech. Using the Quant Center software (3D Histech including Pattern Quant and Histo Quant modules), areas of stained collagen were quantified. Briefly, Pattern Quant was used to detect tissue and measure its surface. Histo Quant was then used to determine the content of colored collagen and measure its surface based on the color threshold method. The area of fibrosis was then expressed as a percentage of collagen surface relative to total tissue for each animal.

Liver fibrosis staging was blinded using the CRN fibrosis criteria.

Detailed information on parameters, scoring/counting, and number of areas considered is provided in the following table. 5.

___________ Table 5. CRN Criteria for Fibrosis______

Parameter Scale points Description (considered the whole section) Fibrosis 0 No fibrosis 1 Centrilobular perisinusoidal/pericellular fibrosis or portal/periportal fibrosis 2 Centrilobular

perisinusoidal/pericellular fibrosis and portal/periportal fibrosis 3 Centrilobular perisinusoidal/pericellular fibrosis and/or portal fibrosis with focal or widespread bridging fibrosis 4 cirrhosis

Statistical analysis

Experimental results were expressed as mean ± standard deviation (SD) and plotted as histograms or curves. Statistical analysis was performed using Prism Version 7 as follows:

For post-mortem measurements, the CSAA and CDAA+1% cholesterol groups were compared by Student's t-test (#: p<0.05; ##: p<0.01; ###: p<0.001) or using the Mann-Whitney test ($: p<0.05; $$: p<0.01; $$$: p<0.001). Treatment groups were compared to those receiving CDAA+1% cholesterol diet using one-way analysis of variance.

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ANOVA and retrospective unadjusted least significant difference analysis by

Fisher (*: p<0.05; **: p<0.01; ***: p<0.001).

Measurement of hepatic collagen content

Liver collagen content was determined using the appropriate QuickZyme kit (Total Collagen Assay, cat. no. QZB-totcol5). The assay is based on the detection of hydroxyproline, which is a non-proteinogenic amino acid found primarily in the collagen triple helix. Thus, hydroxyproline in tissue hydrolysates can be used as a direct measure of the amount of collagen present in tissue (without distinguishing between procollagen, mature collagen, and collagen breakdown products).

Complete hydrolysis of tissue samples in 6 M HCl at 95°C is required prior to dosing with hydroxyproline. As a result of the analysis, a chromogen is formed with an absorption maximum at 570 nm. Results are expressed as mg collagen/g liver.

N-terminal pro-collagen III propeptide (PIIINP)

Plasma PIIINP concentration was determined using an ELISA assay from Cloud-Clone Corp (Cat. No. SEA573Ra) according to the manufacturer's instructions. The microtiter plate is pre-coated with an antibody specific for PIIINP. Standards or samples are added to the appropriate wells of a biotin-conjugated PIIINP-specific biotin-conjugated microtiter plate. Avidin conjugated with horseradish peroxidase (HRP) is then added to each well of the microplate and incubated. After adding the TMB substrate solution, only those wells containing PIIINP, biotin-conjugated antibody, and enzyme-conjugated avidin will have a color change. The enzyme-substrate reaction is terminated by adding a sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 10 nm. The concentration of PIIINP in the samples is then determined by comparing the optical density (OD) of the samples with a standard curve. Results are expressed in pg/ml.

Gene expression RNA extraction

Total liver RNA was isolated using the Nucleospin® 96 kit (Macherey Nagel) according to the manufacturer's instructions. 150 ng total RNA was reverse transcribed into cDNA using M-MLV-RT (Moloney mouse leukemia virus reverse transcriptase) (Invitrogen cat. no. 28025) in the presence of 1x reverse transcriptase (RT) buffer (Invitrogen cat. no. P/NY02321), 1 mM DTT (Invitrogen cat. no. P/NY00147), 0.5 mM dNTP (Promega), 200 ng pdN6 (Roche cat. no. 11034731001), and 40U ribonuclease inhibitor (Promega cat. no. N2515).

Quantitative PCR was then performed using the CFX96 Touch™ real-time PCR detection system (Biorad). Briefly, PCR reactions were performed in 5 µl 5x diluted RT mix in 96-well plates using the iQ SYBR Green Supermix kit (Biorad cat. no. 170887). The experimental conditions were: 20 μl of the bulk reaction and 0.5 μl of each of the reverse and forward primers (10 pmol).

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Primer name Identifier (ID) of the sequence Sequence (5'->3') AGMA straight 1 CTGACAGAGGCACCACTGAA agma reverse 2 CATCTCCAGAGTCCACACA collal straight 3 AGGCGAACAAGGTGACAGAG collal reverse 4 GCCAGGAGAACCAGCAGAG Colla2 straight 5 ATTGGAAGCCGAGGTCCCAG Co11a2 reverse 6 TTTGCCCCCAGGTATGCCAG Τ6Εβ1 straight 7 TTGCTTCAGCTCCACAGAGA Τ6Εβ1 reverse 8 TGGTTGTAGAGGGCAAGGAC TIMP1 straight 9 ATTCAAGGCTGTGGGAAATG TIMP1 reverse 10 CTCAGAGTACGCCAGGGAAC TIMP2 straight eleven GCATCACCCAGAAGAAGAGC TIMP2 reverse 12 GGGTCCTCGATGTCAAGAAA MMP2 straight 13 TCCCTAAGCTCATCGCAGAC MMP2 reverse 14 GCTTCCAAACTTCACGCTCT MMP7 straight 15 TAATTGGCTTCGCAAGGAGA MMP7 reverse 16 AAGGCATGACCTAGAGTGTTCC CCR2 straight 17 TAATATGTTACCCTCAGTTCATCCACGG CCR2 reverse 18 TGCTCTTCAGCTTTTTACAGCCTATC CCR5 straight 19 ATTCCCACACCCTGTTTCG CCR5 reverse 20 GAATTCCTGGAAGGTGGTCA GAPDH direct 21 TATGACTCCACTCACGGCAA GAPDH reverse 22 TCCACGACATACTCAGCACC

Expression levels were normalized using GAPDH gene expression as a reference.

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

Results and conclusions:

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

After liver injury, resting HSCs undergo an activation process characterized by differentiation into (α-GMA)-positive myofibroblasts.

NTZ alone has been shown to provide anti-fibrotic activity in TGFe-induced hGSCs (FIG. 1B). Since NTZ is known to rapidly hydrolyze to its active metabolite thizoxanide (TZ) (Broekhuysen, Stockis et al., 2000), this metabolite was also evaluated for its antifibrotic activity in HSCs. TZ showed a profile similar to the parent drug (data not shown). On the other hand, some PPAR agonists such as Elafibranor also showed an antifibrotic profile in a TGFp-induced HSC model (Fig. 1A). Other PPAR agonists, such as bezafibrate, showed little activity, suggesting that PPAR agonists are not equivalent in terms of their antifibrotic properties (Figure 1C).

To evaluate whether the combination of Elafibranor with NTZ could synergistically alleviate fibrosis, combination matrix experiments were performed in TSCF-induced HSCs. Briefly, solutions of NTZ and Elafibranor were serially diluted in a checkerboard format, making a matrix of 42 combinations covering a large panel of Elafibranor/NTZ ratios. Synergy was first determined by calculating the Bliss increments. These experiments have shown that NTZ can interact synergistically with Elafibranor to reduce α-GMA production in activated HSCs. Several combination pairs showed an EOB greater than 10, indicating synergy (FIG. 2B).

To confirm synergy, the experimental values corresponding to the upper EOB were plotted on a histogram (FIG. 2C). These graphs show that the combination of NTZ with Elafibranor demonstrates a superior anti-fibrotic effect that is statistically significant compared to the greatest effect of the single agent (NTZ or Elafibranor). The most impressive example is provided by the combination pair of NTZ at a concentration of 0.6 μM and Elafibranor at a concentration of 5 μM. Although NTZ exhibits virtually no antifibrotic activity at 0.6 µM, the addition of Elafibranor at 5 µM results in a strong reduction in AGMA by 55%,

- 13 042720 which is much stronger than the effect observed only for individual agents.

To assess whether the combination of other PPAR agonists, Saroglitazar, Celadelpar and Lanifibranor with NTZ, could synergistically alleviate fibrosis, combination matrix experiments were also performed in TSC-β-induced HSCs and the EOB score was determined. To assess synergy, NTZ combinations with Saroglitazar, Celadelpar and Lanifibranor were tested for antifibrotic properties. The combination of NTZ with Saroglitazar, Celadelpar, or Lanifibranor showed an EOB > 0, indicating synergy. The combination of Sarogliatzar at a concentration of 2.5 μM and NTZ at a concentration of 0.625 μM also resulted in a strong reduction in AGMA by 52%, which is much stronger than the effect observed for agents in monotherapy (Fig. 3A). The combination of Celadelpar at a concentration of 20 μM and NTZ at a concentration of 2.5 μM also resulted in a strong decrease in aGMA by 72%, which is much stronger than the effect observed for agents in monotherapy (Fig. 3B). To a lesser extent, the combination of Lanifibranor at 10 μM and NTZ at 1.25 μM tends to be significant (p=0.06) for reducing α-GMA production in activated HSCs compared to NTZ (Fig. 3C).

In conclusion, Applicant has found unexpected antifibrotic activities for the combination of a compound of formula (I) with a specific PPAR agonist(s). These results suggest that the combination of a compound of formula (I) with a PPAR agonist may be synergistic and may provide therapeutic benefits in multiple types of fibrotic diseases.

Application to mice of a choline-deficient diet deficient in L-amino acids (CDAA) + 1% cholesterol causes progressive fibrous steatohepatitis, which is pathologically similar to human non-alcoholic steatohepatitis (NASH).

The CDAA+1% cholesterol diet, in particular, causes a significant increase in hepatic collagen, as shown in FIG. 4.

This figure also demonstrates that administration of Elafibranor or NTZ as monotherapy reduces hepatic collagen content. The decrease in collagen is proportional to the administered dose of Elafibranor or NTZ.

When the combination of Elafibranor and NTZ is administered, the reduction in collagen production is greater than the reduction observed for either compound taken alone.

Therefore, there is a synergistic effect of the combination of Elafibranor and NTZ in reducing collagen production. In other words, there is a marked anti-fibrotic effect with the combination of Elafibranor and NTZ.

The best effect is observed for a combination containing Elafibranor at a concentration of 2.8 mg / kg (mg per kilogram) and NTZ at a concentration of 77.7 mg / kg, expressed in calculated doses.

In FIG. 5 shows the results obtained using histology, that is, the determination of the area of fibrosis, which was expressed as a percentage of the collagen surface of the entire tissue for each animal.

The CDAA+1% cholesterol diet, in particular, causes a significant increase in the percentage of fibrosis.

In FIG. 5 also shows that the administration of Elafibranor or NTZ alone reduces the percentage of fibrosis. The reduction is proportional to the administered dose of Elafibranor or NTZ.

When the combination of Elafibranor or NTZ is administered, the reduction in percentage of fibrosis is greater than the reduction observed for either compound taken alone.

Therefore, there is a synergistic effect of the combination of Elafibranor and NTZ in reducing the percentage of fibrosis. In other words, there is a marked anti-fibrotic effect with the combination of Elafibranor and NTZ.

The best effect is observed for a combination containing Elafibranor at a concentration of 2.8 mg / kg (mg per kilogram) and NTZ at a concentration of 77.7 mg / kg, expressed in calculated doses.

Fig. 6-12 depict gene expression for various hepatic fibrosis markers. For all markers, the CDAA+1% cholesterol diet in particular causes a significant increase in gene expression.

It can also be noted that the introduction of Elafibranor or NTZ in monotherapy reduces the level of expression of various genes. The reduction is proportional to the administered dose of Elafibranor or NTZ.

When the combination of Elafibranor and NTZ is administered, the reduction in gene expression is greater than the reduction observed for each compound taken alone.

Therefore, there is a synergistic effect of the combination of Elafibranor and NTZ on the reduction of gene expression of various liver fibrosis markers. In other words, there is a marked synergistic anti-fibrotic effect with the combination of Elafibranor and NTZ.

In conclusion, Applicant has found an unexpected synergistic antifibrotic activity for the combination of a compound of formula (I) with specific PPAR agonist(s). These results suggest that the combination of a compound of formula (I) with a PPAR agonist may be synergistic and/or may have additional effects, and may also provide therapeutic benefits in multiple types of fibrotic diseases.

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Claims (11)

CLAIM 1. Pharmaceutical combination product containing: (i) a compound selected from the group consisting of nitazoxanide, tizoxanide and a pharmaceutically acceptable salt thereof; and (ii) at least one PPAR agonist selected from the group consisting of elafibranor, celadelpar, saroglitazar, lanifibranor, and a pharmaceutically acceptable salt thereof. 2. Pharmaceutical combination product according to claim 1, containing: (i) a compound selected from nitazoxanide, tizoxanide and a pharmaceutically acceptable salt thereof; and (ii) Elafibranor or a pharmaceutically acceptable salt thereof. 3. A pharmaceutical combination product according to claim 1 or 2, wherein the combination product is a composition containing components i) and ii) and a pharmaceutically acceptable carrier. 4. Pharmaceutical combination product according to claim 1 or 2, where the combination product is a set of parts containing components i) and ii) for sequential, separate or simultaneous use. 5. Pharmaceutical combination product according to any one of claims 1 to 4, further comprising at least one therapeutically active agent with known antifibrotic activity, selected from pirfenidone or receptor tyrosine kinase inhibitors (RTKIs), such as Nintedanib, Sorafenib and other RTKIs, or blockers angiotensin II receptors (AT1), or CTGF inhibitors, or any antifibrotic compound sensitive to inhibition of TGFe and BMPactivated pathways, including TGFe latent complex activators such as MMP2, MMP9, THBS1, or cell surface integrins, TGFe type I receptors (TGFBRI) or type II (TGFBRII) and their ligands such as TGFe, Activin, inhibin, Nodal, anti-Mullerian hormone, GDF or BMP, accessory co-receptors (also known as type III receptors), or components of a SMAD-dependent canonical pathway, including regulatory or inhibitory SMADs -proteins, or members of SMAD-independent or non-canonical pathways, including various branches of MAPK, TAK1 signaling, GTPase Rho-like signaling pathways, phosphatidylinositol-3 kinase/AKT pathways, TGFe-induced EMT process, or canonical and non-canonical Hedgehog signaling pathways, including Hh ligands or target genes, or any members of the WNT, or Notch pathways that are sensitive to the influence of TGFe. 6. A pharmaceutical combination product according to any one of claims 1 to 5, further comprising at least one therapeutically active agent selected from JAK/STAT inhibitors and other anti-inflammatory and/or immunosuppressive agents. 7. The pharmaceutical combination product of claim 6, wherein the therapeutically active agent is selected from glucocorticoids, NSAIDs, cyclophosphamide, nitrosoureas, folic acid analogs, purine analogs, pyrimidine analogs, methotrexate, azathioprine, mercaptopurine, cyclosporine, myriocin, tacrolimus, sirolimus, derivatives mycophenolic acid, fingolimod and other modulators of sphingosine-1-phosphate receptors, monoclonal and/or polyclonal antibodies against such targets as pro-inflammatory cytokines and pro-inflammatory cytokine receptors, T-cell receptor and integrins. 8. Pharmaceutical combination product according to any one of claims 1 to 7, in which components (i) and (ii) are combined into a dosage form in the form of an injectable suspension, gel, oil, pill, tablet, suppository, powder, capsule, aerosol, ointment , cream, patch or galenic formulation for prolonged and/or slow release. 9. The use of a pharmaceutical combination product according to any one of claims 1 to 8 as a medicine. 10. The use of a pharmaceutical combination product according to any one of claims 1 to 8 in a method for the treatment of fibrotic disease. 11. The use of claim 10, wherein the fibrotic disease is selected from the group consisting of fibrosis of the liver, kidney, skin, epidermis, endoderm, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, ovary , adrenal glands, arteries, veins, colon, intestines (for example, small intestine), biliary tract, soft tissues (for example, mediastinum or retroperitoneal space), bone marrow, joints and stomach, in particular fibrosis of the liver, gastrointestinal tract, lungs, heart, kidneys, muscles, skin, soft tissues, bone marrow, intestines and joints. -