Patent Description:
The activation of immune response can be a triggering event for a number of diseases. For example, infiltration of lymphocytes in organs can result in the activation of pathophysiological events. In the liver, the infiltration and activation of an immune response can be the triggering event for the induction of fibrosis, a phenomenon observed in a number of liver diseases such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

<NPL> discloses the in vivo synergistic effect of a combination of nitazoxanide and elafibranor in a disease model of non-alcoholic steatohepatitis (NASH).

<CIT> discloses pharmaceutical combinations of nitazoxanide or tizoxanide, amongst other agents, with PPAR agonist such as elafibranor for use in the treatment of immune, inflammatory, metabolic, fibrotic or cholestatic diseases.

<CIT> discloses pharmaceutical combinations of nitazoxanide and an anti-NASH, anti-fibrotic or anti-cholestatic agent (with the proviso that the anti-NASH, anti-fibrotic or anti-cholestatic agent is not a PPAR agonist), for use in the treatment of immune, inflammatory, metabolic, fibrotic or cholestatic diseases.

<CIT> discloses a combination product comprising elafibranor and an anti-NASH, antifibrotic or anti-cholestatic agent, for use in a method for treating an inflammatory, metabolic, fibrotic or cholestatic disease.

<CIT> discloses combinations comprising tizoxanide and elafibranor for use in the treatment of diseases involving oxidative stress.

There is thus a need to provide new treatment strategies for stopping or decreasing lymphocyte infiltration, or for stopping or decreasing lymphocyte activation in a cell, tissue or organ of interest.

The present invention is as defined in the claims.

The present invention stems from the surprising observation that a combination of (i) nitazoxanide, tizoxanide or a pharmaceutically acceptable salt thereof, with (ii) elafibranor or a pharmaceutically acceptable salt thereof, prevents T cell infiltration into the liver.

Accordingly, the invention relates to a combination product comprising:.

for use in a method for the treatment of primary biliary cholangitis (PBC)-associated inflammation or primary sclerosing cholangitis (PSC)-associated inflammation.

The description (not according to the invention) relates to a combination product comprising:.

for use in a method for the treatment of an immune disease or inflammation.

The description (not according to the invention)further relates to a combination product comprising:.

for use in a method for reducing or stopping immune cell infiltration into a tissue or organ of interest.

Compounds of formula (I), which include NTZ and analogues thereof, are defined as follows:
<CHM>
in which :.

In a particular embodiment of the description, the compound of formula (I) is as follows:.

In a particular embodiment of the description, in the compound of formula (I) of the present invention:.

Nitrogen protecting groups are well known to those skilled in the art, such as those described in the literature, as , for example, in the book "Greene's Protective Groups in Organic Synthesis" (<NPL>).

In a specific embodiment of the description, the compound of formula (I) is a compound of formula (I'):
<CHM>
in which R9 represents a hydrogen atom, a deuterium atom, a O-R8 group (R8 being as defined above), or an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or a moiety of formula (A):
<CHM>
wherein R' represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an (C2-C6)alkynyl group, a (C3-C14)cycloalkyl group, (C3-C14)cycloalkyl(C1-C6)alkyl group, a (C3-C14)cycloalkyl(C1-C6)alkenyl group, a (C3-C14)cycloalkenyl group, a (C3-C14)cycloakenyl(C1-C6)alkyl group, a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a (C3-C14)cycloalkenyl(C2-C6)alkynyl group; R" and R"', independently, represent a hydrogen atom, an (C1-C6)alkyl group, or a nitrogen protecting group or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the description, the compound of formula (I) is selected from: - NTZ:
<CHM>.

In a particular embodiment of the invention, the compound of formula (I) is selected from:.

In another embodiment of the description, the compound of formula (I') is such that.

In a particular embodiment of the description, the compound of formula (I) is [(<NUM>-nitro-<NUM>,<NUM>-thiazol-<NUM>-yl)carbamoyl]phenyl (d3)ethanoate, <NUM>-[(<NUM>-nitro-<NUM>,<NUM>-thiazol-<NUM>-yl)carbamoyl]phenyl (d2) ethanoate; or <NUM>-[(<NUM>-nitro-<NUM>,<NUM>-thiazol-<NUM>-yl)carbamoyl]phenyl (d1) ethanoate.

In another particular embodiment of the description, the compound of formula (I) is <NUM>-(<NUM>-nitrothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>,<NUM>-dimethylbutanoate, in particular (S)-<NUM>-(<NUM>-nitrothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>,<NUM>-dimethylbutanoate, or a pharmaceutically acceptable salt thereof such as its hydrochloride salt (RM5061) of formula:
<CHM>
<CHM>.

In another particular embodiment of the description, the compound of formula (I) is <NUM>-(<NUM>-nitrothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>-methylpentanoate, in particular (<NUM>,<NUM>)-<NUM>-(<NUM>-nitrothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>-methylpentanoate, or a pharmaceutically acceptable salt thereof such as its hydrochloride salt (RM5066) of formula:
<CHM>.

In another particular embodiment of the description, the compound of formula (I) is <NUM>-(<NUM>-chlorothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>,<NUM>-dimethylbutanoate, in particular (S)-<NUM>-(<NUM>-chlorothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>,<NUM>-dimethylbutanoate, or a pharmaceutically acceptable salt thereof such as its hydrochloride salt (RM5064) of formula:
<CHM>
<CHM>.

In another particular embodiment of the description, the compound of formula (I) is -<NUM>-(<NUM>-chlorothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>-methylpentanoate, in particular (<NUM>,<NUM>)-<NUM>-(<NUM>-chlorothiazol-<NUM>-ylcarbamoyl)phenyl <NUM>-amino-<NUM>-methylpentanoate, or a pharmaceutically acceptable salt thereof such as its hydrochloride salt (RM5065) of formula:
<CHM>
<CHM>.

In a particular embodiment of the description, component (i) is NTZ, TZ, TZG or a pharmaceutically acceptable salt thereof. In another particular embodiment of the invention, component (i) is NTZ, TZ or a pharmaceutically acceptable salt thereof. In yet another embodiment of the invention, component (i) is NTZ or a pharmaceutically acceptable salt thereof.

Synthesis of NTZ or analogues can be for example carried out as described in (Rossignol et al. <NUM>-Benzamido-<NUM>-nitrothiazoles, S. Phavic, Belg. ), or by any other way of synthesis known by a person skilled in the art.

According to the description (not part of the invention), compounds of formula (II) are PPAR agonists, and are defined as follows:
<CHM>
in which:.

In a particular embodiment of the compound of formula (II) of the description:.

In a particular embodiment of the compound of formula (II) of the description, Gb is an oxygen atom and Rb is (C1-C6)alkyl group substituted by a -COORc group, wherein Rc represents a hydrogen atom or an unsubstituted linear or branched (C1-C4)alkyl group.

In a particular embodiment of the compound of formula (II) of the description, Y1 is a (C1-C6)alkylthio group that comprises a (C1-C6)alkyl group that is linear or branched that is substituted or not by one or more halogen atoms.

In a particular embodiment of the description, the compound of formula (II) is selected in the group consisting of <NUM>-[<NUM>-methylthiophenyl]-<NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-carboxydimethylmethyloxy phenyl]prop-<NUM>-en-<NUM>-one (Elafibranor, ELA or GFT505), <NUM>-[<NUM>-methylthiophenyl]-<NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-isopropyloxy carbonyldimethylmethyloxyphenyl]prop-<NUM>-en-<NUM>-one, <NUM>-[<NUM>-methylthiophenyl]-<NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-tertbutyloxycarbonyldimethylmethyloxyphenyl] prop-<NUM>-en-<NUM>-one, <NUM>-[<NUM>-trifluoromethylphenyl]-<NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-tertbutyloxycarbonyl dimethylmethyloxyphenyl]prop-<NUM>-en-<NUM>-one, <NUM>-[<NUM>-trifluoromethylphenyl]-<NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-carboxydimethylmethyloxyphenyl]prop-<NUM>-en-<NUM>-one, <NUM>-[<NUM>-trifluoromethyl oxyphenyl]-<NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-tertbutyloxycarbonyldimethylmethyloxy phenyl] prop-<NUM>-en-<NUM>-one, <NUM>-[<NUM>-trifluoromethyloxyphenyl]-<NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-carboxydimethylmethyl oxyphenyl]prop-<NUM>-en-<NUM>-one, <NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-[<NUM>-[<NUM>-(methylthio)phenyl]-<NUM>-oxo-propyl] phenoxy]-<NUM>-methylpropanoic acid, <NUM>-[<NUM>,<NUM>-dimethyl-<NUM>-[<NUM>-[<NUM>-(methylthio) phenyl]-<NUM>-oxo-propyl]phenoxy]-<NUM>-methyl-propanoic acid isopropyl ester, and pharmaceutically acceptable salts thereof.

In a preferred embodiment of the invention, the compound of formula (II) is ELA or a pharmaceutically acceptable salt thereof.

According to the invention the compound of formula (I) and the compound of formula (II) can be selected so that the combination of said compounds provides a synergistic action against immune cell infiltration. Such synergy may be determined according to methods well-known in the art, such as by using the Excess Over Bliss (EOB, or Excess over Highest Single Agent) method. This method, employed by the FDA for approval of combination drug products, assumes that the expected combination effect is superior to the effect obtained with the best component of the combination when taken individually. As demonstrated in the examples, the combination of NTZ and ELA produces a synergistic action against immune infiltration.

Accordingly, in a particular embodiment of the invention, the compound of formula (I) is NTZ, TZ or a pharmaceutically acceptable salt thereof, and the compound of formula (II) is ELA or a pharmaceutically acceptable salt thereof.

In a more preferred embodiment of the invention, the compound of formula (I) is NTZ or a pharmaceutically acceptable salt thereof, and the compound of formula (II) is ELA or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, the compound of formula (I) is NTZ and the compound of formula (II) is ELA.

In a particular embodiment of the invention, the combination product of the invention is a pharmaceutical composition comprising both the compound(i) and the compound (ii), in a pharmaceutically acceptable carrier.

In another embodiment of the invention, the combination product of the invention is a kit of parts comprising the compound (i) and the compound (ii), for sequential, separate or simultaneous use. In this embodiment, each of the compounds can be formulated in different pharmaceutical compositions.

The pharmaceutical compositions used in the invention can comprise one or several excipients or vehicles, acceptable within a pharmaceutical context (e.g. saline solutions, physiological solutions, isotonic solutions, etc., compatible with pharmaceutical usage and well-known by one of ordinary skill in the art). These compositions can also comprise one or several agents or vehicles chosen among dispersants, solubilisers, stabilisers, preservatives, etc. Agents or vehicles useful for these formulations (liquid and/or injectable and/or solid) are particularly methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate <NUM>, mannitol, gelatin, lactose, vegetable oils, acacia, liposomes, etc. The compounds of formula (I) and (II) can be formulated for enteral or parenteral administration. For example, the compounds can be formulated for oral, intravascular (e.g. intravenous or intra-arterial), intramuscular, intraperitoneal, subcutaneous, transdermal or nasal administration. The formulation can be a solid or liquid dosage form. Illustrative formulations include, without limitation, an injectable suspension, or suspension for oral ingestion, a gel, an oil, a pill, a tablet, a suppository, a powder, a capsule, an aerosol, an oinment, a cream, a patch, or means of galenic forms for a prolonged and/or slow release. For this kind of formulation, agents such as cellulose, carbonates or starches can be advantageously used.

The compounds (i) and (ii) can be formulated as pharmaceutically acceptable salts, particularly acid or base salts compatible with pharmaceutical use. Salts of compounds(i) and (ii) 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 product of the invention is for use in a method for the treatment of a disease that involves or is characterized by the infiltration of a tissue or organ with immune cells. Such diseases include, for example, immune diseases and inflammation.

The term "treatment" or "treating" refers to the curative or preventive treatment of a disease in a subject in need thereof. The treatment involves the administration of the combination of the invention to a subject having a declared disease, to prevent, cure, delay, reverse, or slow down the progression of the disease, improving thereby the condition of the subject. The combination product can also be administered to a subject that is healthy or at risk of developing a disease. The subject to be treated is a mammal, preferably a human. The subject to be treated according to the invention can be selected on the basis of several criteria associated to the specific disease the treatment of which is sought such as previous drug treatments, associated pathologies, genotype, exposure to risk factors, viral infection, as well as on the basis of the detection of any biomarker relevant to the disease.

Illustrative tissue or organ of interest include, without limitation, liver, kidney, skin, epidermis, endodermis, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, penis, ovary, adrenal gland, artery, vein, colon, intestine (e.g. small intestine), biliary tract, soft tissue (e.g. mediastinum or retroperitoneum), bone marrow, joint (e.g. knee, shoulder or other joints) and stomach. In a preferred embodiment, the tissue or organ of interest is the liver.

The description (not part of the invention) thus relates, without limitation, to the combination product of the description for use in a method for the treatment of an immune disease or inflammation of the liver, kidney, skin, epidermis, endodermis, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, penis, ovary, adrenal gland, artery, vein, colon, intestine (e.g. small intestine), biliary tract, a soft tissue (e.g. mediastinum or retroperitoneum), bone marrow, joint (e.g. knee, shoulder or other joints) or stomach. In a preferred embodiment of the description, the combination product of the description is for use in a method for the treatment of an immune disease or inflammation of the liver, in particular of an inflammation of the liver. In a further particular embodiment of the description, the combination product of the description is for use in a method for the treatment of an inflammation of the liver associated to NASH, NAFLD, ASH, PBC or PSC. In yet another embodiment of the description, the combination product is for use in a method for the treatment of NASH-associated inflammation.

In a preferred embodiment of the invention, the combination product of the description is for use in a method for the treatment of primary biliary cholangitis (PBC)-associated inflammation or primary sclerosing cholangitis (PSC)-associated inflammation.

Illustrative immune cells whose infiltration can be reduced or stopped include granulocytes or agranulocytes. The immune cells also include myeloid cells or lymphoid cells. Further illustrative immune cell types include, without limitation, neutrophils, eosinophils, basophils, lymphocytes and monocytes. Among the lymphocytes, T cell, B cell and NK cell infiltration can be reduced or stopped, in particular T cell infiltration.

In a particular embodiment, the combination product of the invention is for use in a method for treating a disease involving or characterized by T cell infiltration into a tissue or organ of interest. More specifically, the combination product is for use in a method for treating a disease involving or characterized by T cell infiltration into the liver.

In another aspect of the description (not part of the invention), the combination product is for use in a method for reducing or stopping immune cell infiltration associated to a disease. In a particular embodiment of the description, the combination product is for use in a method for reducing or stopping immune cell infiltration occurring in the liver. Illustrative uses include, without limitation, the reduction or stopping of immune cell infiltration into the liver associated to NASH, ASH, NAFLD, PBC or PSC. In a specific embodiment of the description, the immune cells are T cells. In a further specific embodiment of the description, the combination product is for use in a method for reducing or stopping NASH-associated T cell infiltration into the liver.

The frequency and/or dose relative to the administration can be adapted by one of ordinary skill in the art, in function of the subject to be treated, the disease to be treated, the stage of the disease, the form of administration, etc. Typically, the compound(i), in particular NTZ or a pharmaceutically acceptable salt thereof, can be administered at a dose comprised between <NUM>/day to <NUM>/day, such as from <NUM>/day to <NUM>/day, and particularly from <NUM>/day to <NUM>/day, more particularly from <NUM>/day to <NUM><NUM>/day. The compound (ii), in particular ELA or a pharmaceutically acceptable salt thereof, can be administered at a dose comprised between <NUM>/day to <NUM>/day, such as from <NUM>/day to <NUM>/day, in particular from <NUM> to <NUM>/day, particularly from <NUM> to <NUM>/day, and even more particularly from <NUM>/day to <NUM>/day. In a particular embodiment, the compound (i) and the compound (ii) are orally administered at these doses, e.g. in the form of a pill or tablet. In a further particular embodiment, the compound (i) and the compound (ii) are in the same composition, such as oral compositions (e.g. pills or tablets) and are administered at these doses. In another embodiment, the compound (i) and the compound (ii) are in different compositions, such as oral compositions (e.g. pills or tablets) and are administered at these doses. In another embodiment, the compounds (i) and (ii) are in different compositions and the compound (i) is in the form of a liquid suspension for oral ingestion and the compound (ii) is in the form of a tablet.

Administration can be performed daily or even several times per day, if necessary. The duration of the treatment will depend on the specific disease to be treatment. For example, ghe administration can be performed during one or several days, such as during at least one day, at least two days, at least three days, at least four days, at least five days, at six two days or at least seven days. Alternatively, the administration can be performed for at least one week, at least two weeks, at least four weeks. For chronic diseases, administration can be considered for more than four weeks, such as for at least one month, two months, three months, four months, five months, six months or more than six months, such as for at least one year or several years. In some cases, the combination product of the invention can be administered during the lifetime of the subject.

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

Experimental results were expressed as mean ± SEM and plotted as bar graphs. Statistical analyses were performed using Prism Version <NUM>, as follows:
CSAA vs CDAA + <NUM>% chol groups were compared by a Mann-Whitney test ($: p<<NUM>; $$: p<<NUM>; $$$: p<<NUM>).

Treatment groups were compared to CDAA + <NUM> % chol diet or to other treatment groups by one-way ANOVA and uncorrected Fisher's LSD post-hoc (*: p<<NUM>; **: p<<NUM>; ***: p<<NUM>).

The choline-deficient and L-amino acid-defined (CDAA) diet lacks choline, which is essential for hepatic β-oxidation and very low density lipoprotein production, and is believed to induce hepatocellular steatosis. Subsequently, lipid peroxidation and oxidative stress lead to lobular inflammation, comprehensively resulting in fibrosis.

In the current study, the preventive effects of NTZ <NUM>/kg/day, ELA <NUM>/kg/day and the combination of both were assessed in a murine model. <NUM> week-old male C57BI/6J mice were fed a control (CSAA) diet (n=<NUM>), CDAA + <NUM>% cholesterol diet (n=<NUM>), or CDAA + <NUM>% cholesterol diet supplemented with NTZ <NUM>/kg/day (n=<NUM>), ELA <NUM>/kg/day (n=<NUM>) or combined drugs (NTZ <NUM>/kg/day coadministered with ELA <NUM>/kg/day (n=<NUM>) ) for <NUM> weeks. The food was purchased from Ssniff® company (Soest, Germany). Nitazoxanide (Interchim , Ref #RQ550), Elafibranor (Genfit) or both compounds were incorporated by Ssniff® into CDAA + <NUM>% chol diet in powder form to the required dose.

The body weight and the food intake were monitored twice per week. On the last day of treatment, mice were sacrificed after a <NUM> fasting period. The liver was rapidly excised for transcriptomic and histological studies.

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

Hepatic Total RNA was isolated using Nucleospin® <NUM> Kit (Macherey Nagel) following manufacturer's instructions. <NUM> ng of total RNA were reverse transcribed in cDNA using M-MLV-RT (Moloney Murine Leukemia Virus Reverse Transcriptase) (Invitrogen cat# <NUM>) in presence of RT buffer 1x (Invitrogen cat#P/NY02321), <NUM> DTT (Invitrogen cat#P/NY00147), <NUM> dNTPs (Promega), <NUM> ng pdN6 (Roche cat#<NUM>) and <NUM> U of Ribonuclease inhibitor (Promega cat#N2515).

Upon measurement of RNA samples concentration by nanodrop, the quality was assessed using bioanalyser. Libraries were prepared using the Illumina TruSeq stranded mRNA LT kit and mRNA were sequenced using a NextSeq <NUM> device (paired-end sequence, 2x75 bp), with a High Output flow cell.

Reads were cleaned using Trimmomatic v. <NUM> with the following parameters: SLIDINGWINDOW:<NUM>:<NUM> LEADING:<NUM> TRAILING:<NUM> MINLEN:<NUM>. Then reads were aligned on the genome reference (Mus musculus GRCm38. <NUM>) with rnacocktail using hisat2 v. <NUM> as aligner with default parameters.

A count table was produced using featureCounts v1. <NUM> with default parameters.

To identify differentially expressed genes (DE genes), we used R (version <NUM>. <NUM>) and the DESEq2 library (v. Genes annotation were retrieved using the AnnotationDbi library (v. Briefly, the count matrix produced by FeatureCounts was analysed by the DESeqDataSetFromMatrix() function followed by the DEseq() function from the DESeq2 library. For each condition (i.e. comparison NTZ+CDAA/c vs CDAA/c), the fold change and the p-value were retrieved using the results() function from DESeq2. The different tables were merged using the Ensembl ID as a key.

At sacrifice, liver samples were processed for histological analysis and examined as follows.

The liver slices were first fixed for <NUM> hours in formalin <NUM>% solution followed by several dehydration steps in ethanol (successive baths at <NUM>, <NUM>, <NUM> and <NUM>% ethanol). The liver pieces were subsequently incubated in three xylene baths followed by two baths in liquid paraffin (<NUM>). Liver pieces were then put into small racks that were gently filled with Histowax® to completely cover the tissue. Then, tissue samples were thicked in <NUM> sections. Sections were prepared for immunohistochemistry (IHC).

Immunohistochemistry assay was performed by using an immunoperoxidase protocol. Sections were dewaxed at <NUM> and in xylene baths (<NUM> × <NUM>). The specimens were hydrated ethanol (successive baths at <NUM>%, <NUM>%, <NUM>% and <NUM>%) (<NUM> each) and submerged in 1x PBS (<NUM> × <NUM>). Subsequently, endogenous peroxidase was blocked with H2O2 solution (<NUM>% H2O2 in distilled water) for <NUM>, followed by three washes in 1x PBS for <NUM>. Furthermore, heat mediated antigen retrieval was performed with citrate buffer at pH <NUM> for <NUM> at <NUM>. To block nonspecific binding, 1x PBS solution with <NUM>% normal goat serum and <NUM>% Triton was added for <NUM>. Subsequently, the tissues were incubated with primary CD3 antibody overnight at <NUM> and rinsed with 1x PBS (<NUM> × <NUM>). The tissues were incubated with HRP secondary antibody (Novus Biological) for <NUM> at room temperature and then rinsed with 1x PBS (<NUM> × <NUM>). Slides are then revelated with the peroxidase substrate <NUM>,<NUM>'-diaminobenzidine ((DAB) for <NUM>, and rinsed with tap water. Finally, the stains were counterstained with Mayer hematoxylin for <NUM> and rinsed with tap water (<NUM>) and tissues were dehydrated in ethanol and xylene.

The histological examinations and scoring were performed blindly. Images were acquired using Pannoramic <NUM> Flash II digital slide scanner (3DHistech). Scoring: ten randomly selected fields from each section were examined and analyzed in QuPath software. The positive cells which were stained into brown were presented by the mean of positive cells/selected fields.

Nitazoxanide, elafibranor or a combination of both drugs were evaluated in a fibrosing NASH model. Transcriptomics analyses of liver samples revealed that several markers associated with T cells infiltration and activation were significantly induced by the CDAA/c regimen in comparison with the CSAA control condition (CD3g, Clelc4e, IL12b). Unexpectedly, the ELA/NTZ combination reduces significantly the mRNA levels of these markers.

To confirm those data, immunohistochemistry analyses were performed. Accordingly, a significant increase of CD3 staining reflecting T cells number is observed comparing the CDAA/c group vs CSAA. As for the transcriptomics analyses, only the ELA/NTZ combination reduces significantly the amount of the CD3+ cells.

Claim 1:
A combination product comprising:
(i) nitazoxanide, tizoxanide or a pharmaceutically acceptable salt thereof; and
(ii) elafibranor or a pharmaceutically acceptable salt thereof
for use in a method for the treatment of primary biliary cholangitis (PBC)-associated inflammation or primary sclerosing cholangitis (PSC)-associated inflammation.