Inhibitor of CD44 for use in the treatment of alcohol-related liver disease

Abstract

Alcohol-related liver disease (ALD) is one of the leading causes of severe liver disease and death in Europe. There are few pharmacological treatments for hepatic inflammation associated with alcohol abuses (ASH), the main driver of ALD progression. CD44, a glycoprotein mainly expressed in immune cells, has been implicated in multiple inflammatory diseases but has never been studied in the context of ALD. The inventors therefore explored its contribution to ASH development in mice and its expression in ALD patients. Mice with a global (Cd44- / -) or myeloid cell specific (Cd44myel-KO) deficiency of CD44 were subjected to chronic plus one binge (CPB) ethanol feeding or chronic ethanol feeding. Human CD44 expression was assessed in liver biopsies obtained from patients with ALD. In this context, the present invention relates to a method of treatment of alcohol-related liver disease (ALD) in a subject in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms.

Description

[0001] INHIBITOR OF CD44 FOR USE IN THE TREATMENT OF ALCOHOL-

[0002] RELATED LIVER DISEASE

[0003] FIELD OF THE INVENTION:

[0004] The present invention relates to the treatment of alcohol-related liver disease (ALD) in a subject in need thereof.

[0005] BACKGROUND OF THE INVENTION:

[0006] Alcohol consumption is prevalent in US, with over 50% of adults over 18 years old being regular drinkers h In Europe, alcohol consumption also remains high, with 10 liters of pure alcohol consumed per adult each year2. In addition, drinking habits have changed, particularly in terms of binge drinking and heavy drinking. A recent report published in September 2019 by the WHO reveals that 30.4% of people report having consumed more than 60 g of pure alcohol on a single occasion in the last 30 days3. Among the several complications associated with acute and chronic alcohol consumption, alcohol-related liver disease (ALD) is one of the most common chronic liver diseases worldwide and the most prevalent cause of advanced liver disease in Europe. ALD also constitutes the leading cause of death among adults with excessive alcohol consumption4’5.

[0007] ALD encompasses a broad spectrum of disorders ranging from alcohol -related fatty liver to alcohol associated steatohepatitis (ASH), characterized by steatosis, hepatocellular damage and lobular inflammation. ASH represents a progressive disease form that can evolve into fibrosis and cirrhosis. In addition, patients with underlying ASH or cirrhosis and recent excessive alcohol consumption can also develop a form of acute-on-chronic liver failure called alcohol -associated hepatitis (AH)4'6. Despite the constantly increasing (burden of ALD, no targeted therapies are yet available (current therapies are abstinence, nutritional support, and corticosteroids for severe AH)7.

[0008] ASH development is the consequence of aberrant activation of hepatic immune and parenchymal cells in response to inflammatory mediators from the liver, adipose tissue and gut (including PAMPs, DAMPs, metabolites ). Hepatocytes and Kupffer cells (resident macrophages) concomitantly contribute to the massive recruitment and accumulation of bone marrow-derived monocytes and neutrophils into the liver, a hallmark of ASH. The expression of chemokines (CCL2, etc) and adhesion molecule (E-selectin, etc), known to promote monocyte and neutrophil tissue infiltration, is markedly upregulated and correlates with disease severity5’6’8. An imbalance between hepatic anti- and pro-inflammatory resident or recruited macrophages is a key characteristic in ALD development9. Importantly, binge alcohol feeding markedly elevates hepatic and circulating neutrophils in chronically ethanol-fed mice and in heavy alcohol drinkers and this facilitates hepatic neutrophil infiltration and ALD progression 5, io, ii while chronic / excessive alcohol consumption disrupts bone remodeling, injures the hematopoietic tissue and increases the susceptibility to infections12, 13, ethanol could also modify the bone marrow myelopoiesis and subsequently the blood immune cell levels, even at the early stages of the disease.

[0009] While signaling through CD44 impacts inflammation, injury and fibrogenesis, its potential role in ALD pathogenesis has not yet been investigated. CD44 is a cell-surface glycoprotein involved in cell-cell interactions, cell-adhesion and migration and it is expressed on various cell types (mainly leukocytes). Hyaluronan (HA), osteopontin (OPN) and E-selectin, which can all bind to CD44, are involved in ALD progression (inflammation, injury and fibrosis)14'21. In addition, the inventors have previously reported that CD44 targeting (genetically, by neutralization and pharmacologically) in a mouse model of metabolic steatohepatitis, strongly alleviates liver injury, inflammation and fibrosis by downregulating the recruitment of macrophages and neutrophils into the liver22. They also demonstrated that CD44 regulates proinflammatory macrophage polarization mediated by LPS and hepatic DAMPs, pathogenic factors share with ALD22. This led us to speculate that CD44 might also play a role in promoting the development of ALD. They report here that CD44 expression is upregulated in human and mouse livers during ALD and its systemic or myeloid cell-specific deletion prevented inflammation and liver injury in murine model of chronic plus binge ethanol feeding. These beneficial effects are associated with regulating the frequency of circulating immune cells and neutrophil functions. In addition, blocking CD44 blockage with a neutralizing antibody was associated with the amelioration of neutrophil mobilization (in blood and liver) and liver complications in acute-on-chronic alcohol-related liver injury

[0010] SUMMARY OF THE INVENTION:

[0011] The present invention relates to a method of treatment of alcohol -related liver disease (ALD) in a subject in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms. In particular, the invention is defined by its claims.

[0012] DETAILED DESCRIPTION OF THE INVENTION: Alcohol -related liver disease (ALD) is one of the leading causes of severe liver diseases and death in Europe. There are few pharmacological treatments for hepatic inflammation associated with alcohol abuses (ASH), the main driver of ALD progression. CD44, a glycoprotein mainly expressed in immune cells, has been implicated in multiple inflammatory diseases but has never been studied in the context of ALD. The inventors therefore explored its contribution to ASH development in mice and its expression in ALD patients. Mice with a global (Cd44- / -) or myeloid cell specific (Cd44myel-KO) deficiency of CD44 were subjected to chronic plus one binge (CPB) ethanol feeding or chronic ethanol feeding. Human CD44 expression was assessed in liver biopsies obtained from patients with ALD.

[0013] Here, the inventors report that liver CD44 expression is associated with liver injury and inflammation and its deficiency partially protected mice upon CPB ethanol feeding. CD44 deletion in myeloid cells recapitulated the same protective effects associated with a reduction in inflammatory monocyte infiltration and neutrophil activation in the liver and diminished the neutrophil-lymphocyte ratio (NLR) in blood. CD44-deficient neutrophils displayed reduced PMA-induced inflammatory mediator expression and increased phagocytosis of live bacteria. Cd44myel-KO mice were also protected against hepatic steatosis, which appears to be caused by increased fatty acid beta-oxidation. Neutralization of CD44 with antibodies strongly decreased liver injury and inflammation (hepatic neutrophil frequency) and blood NLR upon CPB ethanol feeding. In human samples, the hepatic expression of CD44 increased with ALD severity and positively correlated with liver TNFa and myeloid marker CD11B expression. Thus, human and experimental evidence supports CD44 as a marker of hepatic inflammation in ALD. In addition, CD44 modulates neutrophil mobilization and functions and its targeting partially prevents liver inflammation and injury in the context of acute-on-chronic alcohol drinking.

[0014] Methods of the present invention:

[0015] In a first embodiment, the present invention relates to a method of treatment of alcohol- related liver disease (ALD) in a subject in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms.

[0016] As used herein, the term “subject” or “patient” refer to any mammals, such as a rodent, a feline, a canine, and a primate. In some embodiments, the patient according to the invention is a human. In some embodiments, the patient according to the invention is a girl or a boy. In some embodiments, the patient according to the invention is an adult. In some embodiments, the patient according to the invention is a child (human being between the stages of birth and puberty), a teenager (human being between the stages of puberty to adulthood) or an elderly person (human being after the puberty).

[0017] As used herein, the term “alcohol-associated liver disease” (ALD) also called alcohol- related liver disease (ARLD) refers to a spectrum of liver injury resulting from alcohol use, ranging from hepatic steatosis to more advanced forms including alcohol associated steatohepatitis (ASH), alcohol-associated cirrhosis (AC), and acute alcohol associated hepatitis presenting as acute-on-chronic liver failure. ALD is a major cause of liver disease worldwide, both on its own and as a co-factor in the progression of chronic viral hepatitis, metabolic dysfunction-associated steatotic liver disease (MASLD), iron overload, and other liver diseases. ALD develops through several stages, beginning with hepatic steatosis, and, in some individuals, gradually progressing through ASH (the histological correlate of which is alcohol associated steatohepatitis), culminating in cirrhosis. Progression through these various stages is dependent on continued heavy alcohol use and other risk factors, including female sex, genetic and epigenetic susceptibility, diet, and comorbid liver disease.

[0018] In some embodiment, the alcohol -related liver disease is acute-on-chronic liver failure.

[0019] As used herein, the term “acute on chronic liver failure” is a clinical syndrome of sudden hepatic decompensation observed in patients with pre-existing chronic liver disease. Acute on chronic liver failure is a serious condition with very high morbidity and mortality.

[0020] The three most widely recognised steps of the alcohol related liver disease are alcohol associated fatty liver or alcohol associated hepatic steatosis, alcohol associated steatohepatitis and alcohol associated cirrhosis. At least 80% of heavy drinkers develop steatosis, 10-35% develop alcohol associated steatohepatitis and approximately 10% develop cirrhosis.

[0021] As used herein, the term “alcohol associated hepatic steatosis”, also called alcohol associated fatty liver or alcohol -related fatty liver, consists in the occupation of a large proportion of the cytoplasm of affected hepatocytes by lipid droplet occlusion. This state is reversible if abstinence but may progress to cirrhosis if excess alcohol intake persists.

[0022] In some embodiment, the alcohol -related liver disease is alcohol associated hepatitis (AH).

[0023] As used herein, the term “alcohol associated hepatitis” (AH) is the second main step of alcohol related liver disease and associates steatosis together with inflammation and hepatocyte death, due to excessive intake of alcohol.

[0024] In some embodiment, the alcohol -related liver disease is alcohol-associated cirrhosis

[0025] (AC). As used herein, the term “alcohol associated cirrhosis” also called alcohol-related cirrhosis (AC) is the most severe and stage prior to liver cancer of the alcohol related liver disease. It is characterized by fibrosis, leading to a progressive loss of liver function. Survival for patients affected by alcohol associated cirrhosis is 60%-70% at one year and 35%-50% at five years.

[0026] In some embodiment, the alcohol -related liver disease is alcohol associated steatohepatitis (ASH).

[0027] As used herein, the term “alcohol associated steatohepatitis” (ASH) refers an advanced stage of fatty liver disease in which the liver builds up fat in irregular quantities and which arises due to alcohol consumption. This is associated with liver inflammation and injury.

[0028] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0029] As used herein, the term “inhibitor” or “antagonist” refers to a compound that decreases the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. In some instances, an inhibitor will substantially decrease the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity or expression observed in the absence of the inhibitor. In some instances, an inhibitor will completely diminish the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, aptamers, antisense oligonucleotides, interfering RNA, carbohydrates or small organic molecules.

[0030] In some embodiment, the inhibitor of the present invention is an inhibitor of CD44 variant (CD44v) isoforms.

[0031] The biological diversity of CD44 is partly conferred by distinct CD44 isoforms that are generated through alternative splicing. The terms “inhibitor” or “antagonist” of CD44 also refer to a compound that decreases the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor mediated by standard and / or variants of CD44

[0032] In some embodiment, the present invention relates to an inhibitor of CD44 standard (CD44s) or an inhibitor of CD44 variant (CD44v) isoforms for use in a method of treatment of alcohol -related liver disease (ALD) in a subject in need thereof.

[0033] As used herein, the term “CD44” refers to a type of transmembrane proteoglycan adhesion molecules widely expressed on the cell membrane of cells such as immune cells (lymphocytes, monocytes...), hepatic stellate cells and endothelial cells, consisting of three segments, i.e., an extracellular segment, a transmembrane segment, and an intracellular segment. The CD44 molecule can mediate a variety of interactions between cells, and between cells and extracellular matrix, participating in the transmission of various signals in the organism and thus modifying the biological function of cells. The primary ligand for the CD44 molecule is hyaluronic acid (also called hyaluronan (HA)), and the receptor-ligand binding of the CD44 molecule and the hyaluronic acid determines the adhesion and / or migration of cells in the extracellular matrix. In addition, the CD44 molecule is also involved in the metabolism of the hyaluronic acid. Hyaluronan (HA), osteopontin (OPN) and E-selectin, which can all bind to CD44, are involved in ALD progression (inflammation, injury and fibrosis). Human CD44 is having the following Gene ID: 960 and the following UniProt number: P16070. The smallest isoform, devoid of variants, is called “standard” CD44 (CD44s).

[0034] In a particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a small organic molecule, peptide, peptidomimetic, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

[0035] In a particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a peptide. Example of certain variant of CD44 is the v6 peptide (Gastroenterology. Inhibition of Tumor Growth and Metastasis in Pancreatic Cancer Models by Interference With CD44v6 Signaling, Matzke-Ogi et al. PMID: 26597578. DOI: 10.1053 / j.gastro.2015.10.020. Epub 2015 Oct 24.).

[0036] In a particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. In a particular embodiment, an example of an aptamer is disclosed in Lo CW etal. Development of CD44E / s dual-targeting DNA aptamer as nanoprobe to deliver treatment in hepatocellular carcinoma. Nanotheranostics. 2022 Jan 1;6(2): 161-174. doi: 10.7150 / ntno.62639. PMID: 34976591; PMCID: PMC8671951.

[0037] In some embodiments, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of metabolites involved in CD44 dependent pathway.

[0038] In a particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene.

[0039] In a particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an anti-sense oligonucleotides (ASO). Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector (for example a vector to specifically target immune cells). In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically myeloid cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art (for example modified AAV8 liver-specific and myeloid cell targeting).

[0040] In some embodiments, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

[0041] In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0042] In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 Bl and US 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534 / genetics.l 13.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0043] In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0044] In some embodiments, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv- Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93 / 1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06 / 030220 and WO 06 / 003388.

[0045] In a particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

[0046] In particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an intrabody. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking / localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

[0047] In a particular embodiment, the inhibitor is a neutralizing anti-CD44 antibody. In a particular embodiment, the inhibitor is an anti-CD44 mAb.

[0048] As used herein, the term “a neutralizing monoclonal anti-CD44 antibody” refers to an antibody that blocks or reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. The neutralizing antibody reduces CD44 biological activity in in cellulo and / or in vivo tests.

[0049] In a particular embodiment, the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In a particular embodiment, a topical administration is performed to the subject. In another embodiment, an oral administration is performed to the subject. In a further embodiment, intravenous administration is performed to the subject.

[0050] By a "therapeutically effective amount" is meant a sufficient amount of an inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms for use in a method of treatment of alcohol -related liver disease (ALD) in a subject in need thereof at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic 20 adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0051] Typically, the active ingredient of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0052] Combined preparation:

[0053] As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

[0054] The inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms as described above is also combined with a classical treatment.

[0055] In a second embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) a classical treatment as a combined preparation for treating of alcohol-related liver disease (ALD) in a subject.

[0056] In another embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) a classical treatment as a combined preparation for treating of alcohol-related liver disease (ALD) in a subject.

[0057] In a particular embodiment, the classical treatment refers to corticosteroids.

[0058] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) corticosteroid used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0059] In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) corticosteroid used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0060] As used herein, the term “corticosteroid” is well known in the art and refers to class of steroid hormones that are produced in the adrenal cortex as well as the synthetic analogues of these hormones. Two types of classes of corticosteroid exist in the art: glucocorticoids and mineralocorticoids. The corticosteroid for use in the invention is selected from the group consisting of: Flugestone (flurogestone); FluoromethoIone; Medrysone; Prebediolone acetate; chlormadinone acetate, cyproterone acetate, medrogestone, medroxyprogesterone acetate, megestrol acetate, and segesterone acetate; Chloroprednisone; Cloprednol; Difluprednate; Fludrocortisone; Fluocinolone; Fluperolone; Fluprednisolone; Loteprednol; Methylprednisolone; Prednicarbate; Prednisolone; Prednisone; Tixocortol; Triamcinolone; Alclometasone; Beclometasone; Betamethasone; Clobetasol; Clobetasone; Clocortolone; Desoximetasone; Dexamethasone; Diflorasone; Difluocortolone; Fluclorolone; Flumetasone; Fluocortin; Fluocortolone; Fluprednidene; Fluticasone; Fluticasone furoate; Halometasone; Meprednisone; Mometasone; Mometasone furoate; Paramethasone; Prednylidene; Rimexolone; Ulobetasol (halobetasol); Amcinonide; Budesonide; Ciclesonide; Deflazacort; Desonide; Formocortal (fluoroformylone); Fluclorolone acetonide (flucloronide); Fludroxycortide (flurandrenolone, flurandrenolide); Flunisolide; Fluocinolone acetonide; Fluocinonide; Halcinonide; Triamcinolone acetonide; Cortivazol; RU-28362.

[0061] In a particular embodiment, the classical treatment refers to agonists of glucagon-like peptide 1 (GLP1) receptor, GLP1 base polyagonists, lipid-lowering agents, anti-obesity agents that are used for the treatment of comorbidities (e.g. obesity), antidiabetic agents, anti- hyperglycemic agents, and antihypertensive agents.

[0062] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) agonists of glucagon-like peptide 1 (GLP1) receptor used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0063] In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) agonists of glucagon-like peptide 1 (GLP1) receptor used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0064] Agonists of glucagon-like peptide 1 (GLP1) receptor (also known as GLP-1 receptor agonists, incretin mimetics, or GLP-1 analogs) represent a class of medications used to treat type 2 diabetes mellitus (T2DM) and, in some cases, obesity. Examples of agonists of GLP1 receptor include but are not limited to: Exenatide, Liraglutide, Lixisenatide, Dulaglutide, Albiglutide and Semaglutide.

[0065] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) glucagon-like peptide 1 (GLP1) base polyagonists used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0066] In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) glucagon-like peptide 1 (GLP1) base polyagonists used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0067] Examples of glucagon-like peptide 1 (GLP1) base polyagonists are described in Transforming obesity: The advancement of multi-receptor drugs. Kusminski CM et al. Cell. 2024 Jul 25; 187(15):3829-3853. doi: 10.1016 / j.cell.2024.06.003. The glucagon-like peptide 1 (GLP1) base polyagonists reduce body weight through complementary pharmacology by incorporating the receptors for glucagon (glucagonR) and / or the glucose-dependent insulinotropic polypeptide (GIP).

[0068] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) lipid-lowering agents used as a combined preparation for treating of alcohol-related liver disease (ALD) in a subject.

[0069] In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) lipid-lowering agents used as a combined preparation for treating of alcohol- related liver disease (ALD) in a subject.

[0070] Suitable lipid-lowering agents that can be used in conjunction with a provided compound or composition thereof include, but are not limited to, bile acid sequestrants, Hm G- CoA reductase inhibitors, HMG-CoA synthase inhibitors, cholesterol absorption inhibitors, acyl coenzyme A cholesterol acyltransferase (ACAT) inhibitors, CETP inhibitors, squalene synthetase inhibitors, PPAR-alpha agonists, FXR receptor modulators, modulators LXR receptor, lipoprotein synthesis inhibitors, renin-angiotensin system inhibitors, pan-PPAR agonists, bile acid reabsorption inhibitors, PPAR-gamma agonists, triglyceride synthesis inhibitors, microsomal triglyceride transport inhibitors, transcription modulators, squalene epoxidase inhibitors, low-density lipoprotein receptor inducers, platelet aggregation inhibitors, 5-LO or FLAP inhibitors, niacin and niacin-bound chromium.

[0071] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) antihypertensive agents used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0072] In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) antihypertensive agents used as a combined preparation for treating of alcohol- related liver disease (ALD) in a subject.

[0073] Suitable antihypertensive agents that may be used in conjunction with a provided compound or composition thereof include, but are not limited to, diuretics, beta-adrenergic blockers, calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, neutral endopeptidase inhibitors, endothelin antagonists, vasodilators, angiotensin II receptor antagonists, alpha / beta adrenergic blockers, alpha 1 blockers, alpha 2 agonists, aldosterone inhibitors, mineralocorticoid receptors, renin inhibitors and angiopoietin 2 binding agents.

[0074] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) antidiabetic agents used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject. In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) antidiabetic agents used as a combined preparation for treating of alcohol- related liver disease (ALD) in a subject.

[0075] Suitable antidiabetic agents that may be used in conjunction with a provided compound or composition thereof include, but are not limited to, other acetyl-CoA carboxylase (ACC) inhibitors, DGAT-1 inhibitors, AZD7687, LCQ908, DGAT-1 inhibitors. 2, , PPAR-gamma agonists, monoacylglycerol O-acyltransferase inhibitors, PDE-10 inhibitors, AMPK activators, sulfonylureas (e.g., acetohexamide, chlorpropamide, diabinese, glibenclamide, glipizide, glyburide, blimipiride, gliclazide, glipentide, gliquidone, glisolamide, tolazamide, tolbutamide), meglitinides, alpha-amylase inhibitors (e.g., tendamistat, treastatin, AL-3688), alpha-glucoside hydrolase inhibitors (e.g., acarbose), alpha-glucosidase inhibitors (e.g., adiposin, camiglibose, emiglitate, miglitol, voglibose, pradymycin-Q, sarbostatin), PPAR- gamma agonists (e.g., balaglitazone, ciglitazone, darglitazone, englitazone, isaglitazone, pioglitazone, rosiglitazone, troglitazone), PPAR-alpha / gamma agonists ( e.g. CLX-0940, GW- 1536, GW-1929, GW-2433, KRP-297, L-796449, LR-90, MK-0767, SB-219994), biguanides (e.g. metformin, buformin), modulators of GLP-1 (exendin-3, exendin-4), liraglutide, albiglutide, exenatide (Byetta), taspoglutide, lixisenatide, dulaglutide, semaglutide, N, N-9924, TTP-054, PTP-1B inhibitors (trodusquemin, extract of hyrtiosal), SIRT-1 inhibitors (e.g., resveratrol, GSK2245840, GSK 184072), DP-IV inhibitors (e.g., sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin, saxagliptin), insulin secretagogues, inhibitors of fatty acid oxidation, A2 antagonists, JNK inhibitors, glucokinase activators (e.g. TTP-399, TTP-355, TTP-547, AZD1656, ARRY403, m K-0599, TAK-329, AZD5658, GKM- 001), insulin, insulin mimetics, glycogen phosphorylase inhibitors (e.g., GSK1362885), VPAC2 receptor agonists, SGLT2 inhibitors (dapagliflozin, canagliflozin, BI- 10733, tofogliflozin, A<s>P-1941, THR1474, TS- 071, ISIS388626, LX4211), glucagon receptor modulators, GPR119 modulators (e.g. MBX-2982, GSK1292263, APD597, PSN821), FGF21 derivatives, TGR5 (GPBAR1) receptor agonists (e.g. INT777), GPR40 agonists (e.g., TAK-875), GPR120 agonists, nicotinic acid receptor activators (HM74A), SGLT1 inhibitors (e.g., GSK1614235), carnitine palmitoyl transferase enzyme inhibitors, fructose 1,6-diphosphatase inhibitors, aldose reductase, mineralocorticoid receptor inhibitors, TORC2 inhibitors, CCR2 inhibitors, CCR5 inhibitors, PKC inhibitors (e.g., PKC-alpha, PKC-beta, PKC-gamma), fatty acid synthase inhibitors, inhibitors of serine palmitoyl transferase, GPR81 modulators, GPR39 modulators, GPR43 modulators, GPR41 modulators, GPR105 modulators, Kvl.3 inhibitors, retinol binding protein 4 inhibitors, glucocorticoid receptor modulators, somatostatin receptor inhibitors (for example SSTR1, SSTR2, SSTR5), PDHK2 inhibitors, PDHK4 inhibitors, MAP4K4 inhibitors, IL 1 -beta modulators and RXR-alpha modulators.

[0076] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) anti-obesity agents used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0077] In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) anti-obesity agents used as a combined preparation for treating of alcohol- related liver disease (ALD) in a subject.

[0078] As used herein, the terms “anti-obesity agents” refer to weight-loss medicines used along with diet, exercise and behavior changes, not instead of them. Suitable anti-obesity agents include, but are not limited to, 11 -beta-hydroxy steroid dehydrogenase 1 inhibitors, stearoyl- CoA desaturase (SCD-1) inhibitors, MCR-4 agonists, CCK-A agonists, monoamine reuptake agents (e.g., sibutramine), sympathomimetic agents, beta-3 -adrenergic receptor agonists, dopamine receptor agonists (e.g., bromocriptine), melanocyte-stimulating hormone and analogs thereof, 5-HT2C agonists (e.g., lorcaserin / Belviq), melanin-concentrating hormone antagonists, leptin, leptin analogues, leptin agonists, galanin antagonists, lipase inhibitors (e.g., tetrahydrolipstatin / Orlistat), anorexigenic agents (e.g., agonists bombesin), NPY antagonists (for example, velneperit), PYY3-36 (and analogs thereof), BRS3 modulators, mixed opioid receptor antagonists, thyromimetic agents, dehydroepiandrosterone, glucocorticoid agonists or antagonists, orexin antagonists, GLP-1 agonists, ciliary neurotrophic factors (e.g. Axokine), human agouti -related protein (AGRP) inhibitors, H3 antagonists or inverse agonists, neuromedin U agonists, MTP / ApoB inhibitors (e.g. Gut-selective MTPs such as dirlotapide, JTT130, Usistapide, SLX4090), MetAp2 inhibitors (e.g. ZGN-433), agents with mixed modulatory activity at two or more of the glucagon, GIP and GLP1 receptors (e.g. MAR -701, ZP2929), norepinephrine reuptake inhibitors, opioid antagonists (e.g. naltrexone), CB1 receptor antagonists or inverse agonists, ghrelin agonists or antagonists, oxyntomodulin and analogues thereof, monoamine uptake inhibitors (e.g., tesofensine), and combination agents (e.g., bupropion plus zonisamide (Empatic), pramlintide plus metreleptin, bupropion plus naltrexone (Contrave), phentermine plus topiramate (Qsymia).

[0079] In one embodiment, the invention relates to i) an inhibitor of CD44 standard (CD44s) and ii) a thyroid hormone receptor beta (NR1 A2) agonist used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject. In one embodiment, the invention relates to i) an inhibitor of CD44 variant (CD44v) isoforms and ii) a thyroid hormone receptor beta (NR1A2) agonist used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

[0080] In some embodiment, the hepatic thyroid hormone receptor agonist is the resmetirom.

[0081] As used herein, the term “resmetirom” refers to an oral thyroid hormone receptor beta (NR1 A2) agonist having the following CAS number: 920509-32-6.

[0082] As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

[0083] Pharmaceutical composition:

[0084] An inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms for use according to the invention as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0085] Accordingly, in a further aspect, the invention relates to a pharmaceutical composition comprising an inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms for use in method of treatment of alcohol-related liver disease (ALD) in a subject in need thereof.

[0086] In a particular embodiment, the pharmaceutical composition according the invention, wherein the inhibitor of CD44 or the inhibitor of its CD44 variant (CD44v) is an antibody, a siRNA or an oligonucleotide or a peptide.

[0087] In a particular embodiment, the pharmaceutical composition according to the invention, wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a neutralizing anti-CD44 antibody.

[0088] In a particular embodiment, the pharmaceutical composition according to the invention, wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an anti-CD44 mAb.

[0089] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0090] As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0091] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0092] Method for screening:

[0093] In a further aspect, the invention relates to a method of screening a drug suitable for the treating alcohol -related liver disease (ALD) comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of CD44 standard (CD44s) or CD44 variant (CD44v) isoforms.

[0094] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity or expression of CD44. In some embodiments, the assay first comprises determining the ability of the test compound to bind to CD44. In some embodiments, a population of cells then contacted and activated so as to determine the ability of the test compound to inhibit the activity or expression of CD44. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to inhibit a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity or expression of CD44, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intraantibodies), aptamers or nucleic acids. For example, the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In a particular embodiment, the CD44 expression is determined on circulating immune cells

[0095] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0096] FIGURES:

[0097] Figure 1: CD44 deficiency strongly prevented liver injury and inflammation induced by chronic plus binge ethanol feeding. After acclimation to a semi-liquid diet, Wildtype (Wt) and Cd44- / - mice were allowed free access to the ethanol Lieber-DeCarli diet containing 5% (vol / vol) ethanol or were pair-fed with an isocaloric control diet for 10 days. On day 11, mice received a single dose of ethanol (5 g / kg body weight [b.w.]) (EtOH) or isocaloric dextrin-maltose (Malto) by gavage and sacrificed 9 hours later (5-17 mice / group). (A) Hepatic Cd44 expression was evaluated at the mRNA level. (B) The serum levels of ALT were evaluated. (C) H&E staining of liver tissue section samples from Wt and Cd44- / - after chronic plus binge (CPB) EtOH feeding were performed. Quantification of hepatic steatosis are shown. Results are expressed as means ± SEM and statistically analyzed using a two-way ANOVA test.

[0098] Figure 2: The CD44 deficiency in myeloid cells prevented hepatic expression of CD44, liver injury and hepatic steatosis induced by ethanol feeding. (A-D) After acclimation to a semi-liquid diet, Cd44myel~KOand littermate control Cd44F / Fmice were allowed free access to the ethanol Lieber-DeCarli diet containing 6% (vol / vol) ethanol or were pair-fed with an isocaloric control diet (Malto) for 10 days. On day 11, mice received a single dose of ethanol (5 g / kg body weight [b.w.]) (EtOH) or isocaloric dextrin-maltose (Malto) by gavage and sacrificed 9 hours later (9-16 mice / group). (E-G) After acclimation to a semi-liquid diet, Cd44myel~KOand Cd44F / Fmice were allowed free access to the ethanol Lieber-DeCarli diet containing 5% (vol / vol) ethanol or were pair-fed with an isocaloric control diet (Malto) for 4 weeks (6-14 mice / group). (A) Hepatic Cd44 expression was evaluated at the mRNA (9-16 mice / group). (B, E) H&E staining of liver tissue section samples from Cd44myel~KOand Cd44: :mice after CPB or chronic EtOH feeding were performed. Quantification of hepatic steatosis are shown. (C, F) The serum levels of ALT were evaluated. (D, G) Hepatic mRNA expression levels of Ppara and Cptl were analyzed by real-time quantitative PCR. Data are presented as relative mRNA levels normalized to RplpO mRNA levels. Results are expressed as means ± SEM and statistically analyzed using a two-way ANOVA test. Figure 3: The CD44 deficiency in myeloid cells strongly prevented liver inflammation induced by chronic plus binge ethanol feeding. After acclimation to a semiliquid diet, Cd44myel~KOand littermate control Cd44F / Fmice were subjected to CPB EtOH or Malto feeding (11-18 mice / group). (A) Hepatic non parenchymal cells were stained for CD45, Ly6G, Ly6C, and CD62L and analyzed by flow cytometry (5-6 mice / group). Inflammatory monocytes (Ly6ChlghLy6G' cells), neutrophils (Ly6G+Ly6C' cells) and neutrophil activation levels (Ly6G+Ly6C' CD62LLOWcells) in the liver were assessed by flow cytometric analysis. (B) The serum levels of CCL2 were evaluated (7-14 mice / group). Results are expressed as means ± SEM and statistically analyzed using a two-way ANOVA test.

[0099] Figure 4: CD44 deficiency in myeloid cells altered the proportions of circulating immune cells and partially corrected the elevation of the neutrophil / lymphocyte ratio induced by chronic plus binge ethanol feeding. After acclimation to a semi-liquid diet, Cd44myel~KOand littermate control Cd44: :mice were subjected to CPB EtOH or Malto feeding (9-15 mice / group). The number of white blood cells (WBC) (A), neutrophils, monocytes and lymphocytes (B) were evaluated by blood haematology analyzer. The results were also expressed as % of blood cells (C) and neutrophil to lymphocyte ratio (NLR) (D). (A-D) Results are expressed as means ± SEM and statistically analyzed using a two-way ANOVA test.

[0100] Figure 5: CD44 deficiency in neutrophils reduced their capacity to produce inflammatory mediators. Purified bone marrow neutrophils from Wt and Cd44KOmice were stimulated with PMA (lOnM) for 4h at 37°C. Data are presented as relative mRNA levels normalized to RplpO mRNA levels. Data from three independent experiments are expressed as means ± SEM and statistically analyzed using a Student’s t-test.

[0101] Figure 6: Neutralization of CD44 corrected the liver inflammation and injury induced by the chronic plus binge EtOH feeding. After acclimation to a semi-liquid diet, WT C57BL / 6 female mice (20 weeks of age) were allowed free access to the ethanol Lieber-DeCarli diet containing 5% (vol / vol) ethanol for 10 days. In the evening on day 10 and in the early morning on day 11 mice received one intra-peritoneal injection of either purified rat anti -mouse CD44 mAb (n = 7; IM7) or purified rat IgG2b, isotype control (n = 7; A95-1) (100 pg / mouse). Mice were then immediately gavage-fed with a single dose of ethanol (5 g / kg body weight [b.w.]) and 9 hours later the blood was collected and mice were sacrificed. In blood, white blood cell (WBC) number (A), neutrophil to lymphocyte ratio (NLR) (B) and serum ALT activity (C) were evaluated. In liver, quantification of hepatic steatosis (from H&E staining of sections samples) (D), number of liver neutrophils (Ly6G+Ly6C'), inflammatory monocytes (Ly6ChlghLy6G ) and T cells (CD3+NK1.1 ) (E) (from hepatic non parenchymal cells) and liver expression of CD44 and inflammatory markers at the mRNA level (F) were assessed. (A-F) Results are expressed as means ± SEM and statistically analyzed using a Mann Whitney test.

[0102] Figure 7: Liver CD44 expression increased with liver inflammation in heavy alcohol drinkers. (A) CD44 mRNA levels was first evaluated in the dataset of patients with AH (n=15) versus subject without liver complications (n=5) (GSE28619). (B-G) In 19 consecutive heavy alcohol drinkers, patients were classified into 2 groups: with mild or moderate ALD on the basis of severity of liver injury and steatosis as assessed by serum ALT activity (B) and by histological analysis (C), respectively. (D) Hepatic CD44 mRNA expression levels were analyzed by real-time quantitative PCR. Gene expression was normalized to the mRNA levels of RPLPO and expressed relative to the mild ALD group. Results are expressed as means ± SEM (A, C-D) or medians with 25-95 percentiles (B) and statistically analyzed using a Mann Whitney test. (E, G) Correlations between liver CD44 expression (fold change) and ALT level, liver expression of INF and ITGAM (CD1 IB) and WBC number were analyzed using the Spearman correlation test. (F) In blood, white blood cell (WBC) number were evaluated, expressed as means ± SEM and statistically analyzed using the Mann Whitney test.

[0103] Figure 8 CD44 expression strongly increased in liver neutrophils in patients with severe alcohol-associated hepatitis. We reanalyzed single-cell RNA sequencing data of livers from 5 healthy donors, 5 alcohol-associated cirrhosis (AC), and 6 severe alcohol-associated hepatitis (AH) (GEO database GSE136103 and GSE255772). (A) Bar plot showing the composition of conditions for each group. (B) Histogram showing quantification of CD44- expressing myeloid cells whose CD44 expression is either above average (0.89) (2= “TRUE”) or below (1= “FALSE”). (C) Violin plot showing CD44, CXCL8, and CXCR2 expression in neutrophils from AH or healthy conditions. Abbreviations: DC, dendritic cell; NA, not associated to specific cells; NK, natural killer.

[0104] EXAMPLE:

[0105] Material & Methods

[0106] Human studies

[0107] Alcohol -related hepatitis (AH): CD44 mRNA levels were evaluated in the dataset of patients with AH (GSE28619). Microarray studies with i) 5 liver biopsies from normal livers (optimal cadaveric liver donors (n=3) or resection of liver metastasis (n=4) and ii) 15 liver biopsies of patients with severe AH based on clinical criteria (Maddrey ’ s discriminant functions >32). Alcohol-related liver disease (ALD): From October 1997 to June 1998, 19 consecutive heavy alcohol drinkers (16 men, 3 women, mean age: 44±7 years) admitted to our Liver unit for detoxification and / or in rehabilitation were included in this study. All patients had consumed over 80 g ethanol per day for more than 5 years. All patients were negative for circulating hepatitis B surface antigen, hepatitis C virus, and human immunodeficiency virus. No patient had osteoarthritis or rheumatoid arthritis. A needle liver biopsy was performed on all patients by the transparietal approach. Biopsies were processed routinely and stained with hematoxylin- eosin-safran and Sirius Red. The length of the liver biopsy was over 15 mm. Histopathological features were semi -quantitatively evaluated: grade of steatosis (0, 5%; 1, 5%-30%; 2, .30%- 60%; 3, .60%); hepatocellular ballooning (0, none; 1, a few balloon cells; 2, many cells / prominent ballooning); megamitochondria (0, none to rare; 1, many); Mallory’s hyaline (0, none to rare; 1, many) and fibrosis stage (From 0, none, to 4, cirrhosis). The grading of hepatic activity was done according to the Activity score of Orrego et al.23. The characteristics of the 19 alcoholic patients selected for the hepatic gene expression analysis are described in Table SI. Liver tissues were stored at -80°C until use. All subjects gave their informed written consent to participate in this research study according to French legislation regarding Ethical and Human Research (Huriet-Serusclat law, the “Comite' Consultatif de Protection des Personnes dans la Recherche Biomedicale de Nice” approved this study, Nu03.613).

[0108] Mice and study design

[0109] CD44-deficient mice (Cd44~ / ‘)(B6.Cg-Cd44tmlHbg / J) and LysMCremice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). (4d44x"x x"xmice were kindly provided by Pr. V. Orian-Rousseau (Karlsruhe Institute, Germany)24C57BL / 6J / Rj wild-type, Cd44 LysMcre / +Cd44,!ox ,!oxand littermate control LysM+ / +Cd44,!ox ,!oxmice were acclimated to our animal facilities under a 12 / 12h light / dark cycle at a temperature of 21 ± 2 °C. After acclimation to a liquid diet, mice were subjected to one of two different ethanol feeding protocols (chronic and NIAAA models)25.

[0110] Acclimation protocol

[0111] Female mice (24-30 weeks old for Cd44~ ~m\ Wt mice and 12-17 weeks old for LysMcre / +Cd44flox / floxand LysM+ / +Cd44nox oxmice) were initially fed the control Lieber- DeCarli diet (SSNIFF, Soest, Germany) ad libitum for 5 days to acclimatize them to a semiliquid diet. Chronic plus one binge ethanol feeding

[0112] Then, mice were allowed free access to the ethanol Lieber-DeCarli diet (SSNIFF) containing 5-6% (vol / vol) ethanol for 10 days, and control -fed groups were pair-fed with an isocaloric control diet. On day 11, mice received a single dose of ethanol by gavage (5 g / kg body weight [b.w.]) or isocaloric dextrin-maltose in the early morning and sacrificed 9 hours later.

[0113] Chronic ethanol feeding

[0114] After acclimation to a semi-liquid diet, mice were allowed free access to the ethanol Lieber-DeCarli diet (SSNIFF) containing 5% (vol / vol) ethanol for 4 weeks, and control -fed groups were pair-fed with an isocaloric control diet.

[0115] Anti-Cl)44 treatment

[0116] After acclimation to a semi-liquid diet, WT C57BL / 6 female mice (20 weeks of age) were allowed free access to the ethanol Lieber-DeCarli diet containing 5% (vol / vol) ethanol for 10 days. In the evening on day 10 and in the early morning on day 11 mice received one intraperitoneal injection of either purified rat anti -mouse CD44 mAb (n = 7; IM7, BD Pharmingen, catalog #553131; BD Biosciences) or purified rat IgG2b, isotype control (n = 7; A95-1, BD Pharmingen, catalog #553986; BD Biosciences) (100 pg / mouse). Mice were then immediately gavaged with a single dose of ethanol (5 g / kg body weight [b.w.]) and 9 hours later the blood was collected and mice were immediately sacrificed, after which the liver was removed. One part of the liver was immediately frozen in liquid nitrogen and stored at -80 °C until analysis. A second part was fixed in buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin-eosin. The remainder liver was used for a flow cytometry analysis. The guidelines of laboratory animal care were followed, and the local ethical committee approved the animal experiments (APAFIS#5100-2015121110477413 v6; APAFIS#8495-2017010514042986v5).

[0117] Results

[0118] CD44 deficiency attenuated liver injury and inflammation induced by chronic plus binge ethanol feeding.

[0119] We first evaluated the hepatic expression levels of Cd44 in a dietary mouse model of acute-on-chronic liver injury, the NIAAA model25. This chronic plus one binge alcohol feeding mouse model specifically triggers high levels of alcohol in blood, liver injury, fatty liver and inflammation, which mimics acute-on-chronic alcohol associated liver injury in patients. Upon chronic plus binge ethanol feeding, the mice displayed elevated hepatic Cd44 expression (Figure 1A) associated with liver injury (as evaluated by ALT activity) (Figure IB), liver steatosis (Figure 1C) and inflammation (as evaluated by inflammatory marker expression related to ALD) (Data not shown). The role of CD44 in liver complications induced by the NIAAA model was then investigated using mice deficient for Cd44. The Cd44-I- mice displayed less liver damage (Figure IB) and hepatic inflammation (Data not shown) with a similar grade of hepatic steatosis when compared with the livers of chronic plus acute ethanol- consuming Wt mice (Figure 1C). To gain insight into the impact of CD44 deficiency on liver complications, 3 livers of EtOH Wt and EtOH Cd44~ / ~ mice were randomly selected for RNA sequencing. Following dimension reduction, the samples clustered according to mouse genotype (Data not shown). From gene expression analysis, the Cd44 deficiency had a strong influence on the expression of 244 transcripts with 80 up-regulated and 164 down-regulated compared to Wt liver (Data not shown). Focusing on down regulated genes, the gene ontology analysis highlighted that the absence of CD44 mainly regulated responses related to neutrophil and innate immune cell functions including cytokine production and phagocytosis (Data not shown).

[0120] Myeloid cell specific CD44 deficiency strongly attenuated liver injury and steatosis induced by ethanol feeding

[0121] Since CD44 deficiency preferentially impacted innate immune and neutrophil responses, we then focused on the role of CD44 in myeloid cells. The Cd44mye~Omice ( / - / - / Ilox lloxI.ysM^ mice) displayed a significant decrease in CD44 expression in hepatic inflammatory monocytes (Ly6G‘ Ly6Chlghcells), macrophages (F4 / 80+CDl lb+cells) and neutrophils (Ly6G+Ly6C' cells) without disruption of the CD44 expression in lymphocytes (CD3+NKl.T cells) as evaluated by flow cytometry analysis (Data not shown). In addition, the CD44 deficiency in myeloid cells strongly decreases the total CD44 hepatic expression in the steady state and in response to EtOH (Figure 2A; and at protein level (data not shown)), suggesting that myeloid cells are the main hepatic populations expressing CD44. This marked reduction in liver CD44 expression was associated with the prevention of hepatic steatosis (Figure 2B) and liver injury (Figure 20). The protective effect of CD44 myeloid cell deficiency on liver steatosis was also associated with only a partial reduction of the expression of Ppar-alpha and Cptl, the master regulators of beta-oxidation in response to chronic plus binge EtOH feeding (Figure 2D). This protective effect of CD44 deficiency in myeloid cells on liver steatosis was also associated with the prevention of increased p53 / miR34a pathway that negatively regulated hepatic beta-oxidation via the SIRTl / AMPK-dependent regulation of Ppar-alpha and Cptl (Data not shown). To mimic the chronic drinking patterns in humans, Cd44myel~KOand Cd44F / Fmice were also challenged with semi-liquid diet supplemented with ethanol for 4 weeks. CD44 deletion in myeloid cells mediated the same protective effects on liver steatosis (Figure 2E), injury (Figure 2F) and beta-oxidation regulator expression (Figure 2G).

[0122] Myeloid cell specific CD44 deficiency strongly prevented liver inflammation induced by chronic plus binge ethanol feeding.

[0123] The NIAAA model (CPB ethanol feeding) also produces hepatic inflammation, which is characterized by a higher frequency of inflammatory monocytes and neutrophils and elevated expression of inflammatory markers. Whereas these responses are clearly marked in Cd44F / Fmice upon chronic plus binge EtOH feeding, the Cd44m'F F)mice displayed a substantially lower hepatic frequency of inflammatory monocytes (Ly6ChlghLy6G ) and activated neutrophils (Ly6G+Ly6CintCD62Llow) (Figure 3A), lower hepatic expression of tumor necrosis factor (Tnf), CXC motif chemokine ligand 2 (Cxcl2) and E-selectin (Sele) (Data not shown and lower hepatic (Data not shown) and blood level of chemokine ligand 2 (Ccl2) (Figure 3B). In addition to these markers, bulk RNA-seq analysis from 3 livers of EtOH Cd44mye~Omice versus 3 livers of EtOH Cd44F / Fmice, randomly selected and well clustered according to mouse genotype (Data not shown), revealed that Cd44 deficiency in myeloid cells positively regulated the expression of 645 genes and down-regulated 800 genes compared to EtOH Cd44F / Fliver (Data not shown). Focusing on the preventive effect, the pathway analysis revealed that the absence of CD44 in myeloid cells mainly decreased EtOH stimulated inflammatory pathways such as those related to TNF, inflammatory responses and interleukin (Data not shown).

[0124] Myeloid cell specific CD44 deficiency strongly prevented the elevated neutrophil / lymphocyte ratio in blood induced by the chronic plus binge ethanol feeding.

[0125] Since increased neutrophil levels in peripheral blood and liver tissue is consistently reported in patients with alcohol -related liver disease10, we then investigated the impact of acute-on-chronic EtOH feeding and myeloid cell CD44 deficiency on blood immune cell levels in mice. In response to chronic plus binge EtOH feeding, the number of white blood cells (WBC) decreased in both genotypes (Figure 4A). Neutrophil counts were increased in control ETOH mice and this augmentation was only partial in Cd44m'F F)mice. WBC decrease likely resulted from lower blood monocyte and lymphocyte numbers (Figures 4B and 4C) The deficiency of CD44 in myeloid cells partially prevented these changes in blood immune cell frequency without altering the WBC number (Figures 4A, 4Band 4C). In line with this, the blood neutrophil-lymphocyte ratio (NLR) elevation in response to EtOH feeding is reduced in CtZ 4myel-KOmice (Figure 4D). It is also of interest to highlight that CD44 expression in blood neutrophils is not increased in response to ethanol (Data not shown), unlike in liver neutrophils (Data not shown).

[0126] CD44 deficiency in neutrophils reduced their capacity to produce inflammatory mediators, while enhancing their phagocytosis potential.

[0127] It has been clearly established that the infiltrating neutrophils are the main contributor to alcohol-induced liver damage. In ALD, neutrophils are strongly activated and are important sources of reactive oxygen species (ROS) and cytokines. Although ROS production is a powerful bactericidal process, excessive ROS formation causes adverse tissue damage. We then investigated the potential role of CD44 in the neutrophil production of ROS and inflammatory factors upon phorbol 12-myristate 13 -acetate (PMA) activation. While CD44 deficiency in bone-marrow neutrophils did not alter the frequency of ROS+ neutrophils (Data not shown) or ROS production per cell (Data not shown), it strongly decreased the PMA-mediated Tnf and Cxcll expression (Figure 5). The activation of neutrophils by PMA was also associated with the up-regulation of CD44 (Figure 5). The antimicrobial activities of neutrophils also depend on their ability to phagocytose cellular debris and / or bacteria. Incubation of BM neutrophils with labelled live E-coli bacteria was associated with an increased frequency of CDl lbhlghLy6Ghlghneutrophils and bacteria-loaded neutrophils after Ih at 37°C (Data not shown). The CD44 silencing amplified these responses with a higher frequency of CDl lbhlghLy6Ghlghneutrophils, bacteria-containing neutrophils and phagocytosis intensity (Data not shown). These results indicate that CD44 deficiency modified the properties of neutrophils with higher susceptibility to remove bacteria and lower proinflammatory phenotypes upon activation.

[0128] CD44 neutralization corrected liver inflammation and injury induced by the chronic plus binge EtOH feeding.

[0129] Our data revealed that CD44 is a central player in liver EtOH-driven injury and inflammation. To explore this novel therapeutic avenue, we then investigated if CD44 neutralization by a specific antibody could correct the liver complications induced by chronic plus binge ethanol feeding. After 10 days of chronic EtOH feeding, mice received two intraperitoneal injections (one the evening on day 10 and one early the next morning prior to gavage) of either purified rat anti-CD44 mAh or isotype control before gavage with a single dose of EtOH. The treatment with anti-mouse CD44 mAh strongly decreased WBC (Figure 6A), normalized blood NLR (Figure 6B) and strongly reduced liver injury as evaluated by the ALT activity (Figure 60). No improvement in hepatic steatosis (Figure 6D), liver triglyceride levels (Data not shown), a hepatic expression of p21, Naprt, Nampt, Sirtl, Ppara, and Cptl was observed, likely due to our experimental protocol. Anti-CD44 mAb treatment was also associated with a marked reduction in liver inflammation with less hepatic enrichment in neutrophils (Ly6G+Ly6Cint) (Figure 6E) and a marked reduction in hepatic levels of inflammatory markers Tnf, Ccl2 and Cxcl2) (Figure 6F). Therefore, anti-CD44 mAb treatment efficiently lower EtOH induced liver injury and inflammation.

[0130] Liver CD44 expression increased with local inflammation and injury in heavy alcohol drinkers.

[0131] Then, we examined the relationship between hepatic CD44 expression and human ALD progression. CD44 mRNA levels were first evaluated in the dataset of patients with AH (n=15) versus subjects without liver complications (n=5) (GSE28619). A shown in Figure 7A, the hepatic expression of CD44 was increased with AH. In 19 consecutive heavy alcohol drinkers (16 men, 3 women, mean age: 44±7 years) admitted to our Liver unit, patients were classified into 2 groups: with mild ALD (Figures 7B and 70) and with moderate ALD (Figures 7B and 70) on the basis of the severity of hepatic steatosis and injury as assessed by histological analysis and serum ALT activity, respectively. Liver mRNA levels of CD44 increased with the severity of ALD (Figure 7D) and correlated with liver injury (Figure 7E). We also found that CD44 expression strongly correlated with liver mRNA levels of TNF and myeloid cell maker ITGAM (CD1 IB), markers of hepatic inflammation (Figure 7E). With the aggravation of ALD, WBC counts decreased (Figure 7F) and negatively correlated with liver expression of CD44 (Figure 7G). Consistent with our animal studies, the liver expression of CD44 could be a local marker of hepatic inflammation in ALD patients.

[0132] Alcohol-associated hepatitis (AH) has been associated with CD44 upregulation in liver neutrophils

[0133] To then explore CD44 expression in immune cells in AH, we reanalyzed single-cell RNA sequencing data of livers from 5 healthy donors, 5 alcohol-associated cirrhosis (AC), and 6 severe AH (GEO database GSE136103 and GSE255772). As previously reported43, the main difference between AH and AC was that AH livers had significantly higher numbers of neutrophils than AC livers, while differences in other myeloid cells between AC and AH were less obvious (Figure 8A}. While myeloid cells (DCs, monocytes, macrophages, and neutrophils) express CD44, AH is associated with a marked increase in liver neutrophils with increased CD44 expression (Figures 8B and 8C}. These neutrophils, including those with high CD44 expression, also showed high expression of IL8 (CXCL8) and CXCR2 (Figure 8C), as recently described43. In addition, although there was no obvious difference in the number of blood neutrophils between patients with AH and AC, the number of blood neutrophils expressing CD44 was higher in patients with AH than blood neutrophils in patients with AC (Data not shown . CD44 upregulation in liver neutrophils is associated with severe AH.

[0134] Discussion

[0135] We first described in this study that hepatic inflammation associated with alcohol abuse was linked to hepatic CD44 upregulation in mice and patients. We then reported that myeloid cells are the main contributors to this CD44 upregulation in a mouse model of acute-on-chronic alcohol consumption. The deletion of CD44 in these cells largely protected the mice against hepatic steatosis caused by chronic alcohol consumption, with or without binge drinking, and was associated with an increase in beta-oxidation of fatty acids (PPARa and CPT1). CD44 deficient mice were also largely protected against inflammation and liver damage, which was linked to the prevention of neutrophil mobilization in blood concomitantly with the regulation of its activation and functions in the liver. Our preclinical study also demonstrated that systemic CD44 neutralization with an antibody prior to ethanol exposure (binge drinking) was effective in strongly inhibiting neutrophil mobilization in the blood and liver and, consequently, decreasing liver inflammation and injury.

[0136] The elevated expression of CD44 in liver upon chronic plus binge ethanol feeding is closely related to the frequency and activation level of the myeloid cells in the liver. Liver infiltration of inflammatory monocytes and activation of liver resident neutrophils are reduced in mice with CD44 deficiency in myeloid cells. In addition, CD44 expression is augmented in the activated macrophages (induced by LPS, liver DAMPs and saturated fatty acids as we previously reported21) and activated neutrophils (Data not shown . Deletion of CD44 in these cells greatly reduced its hepatic expression level in baseline conditions and blunted its upregulation following chronic plus binge EtOH consumption (Data not shown}.

[0137] We then assessed the consequences of this poor hepatic expression of CD44 on the development of hepatic steatosis, which is almost universal at early stages of ALD. It has been estimated that steatosis develops in more than 90% of drinkers who consume 4-5 standard drinks per day26. We report here that the CD44 deficiency in myeloid cells reduces liver steatosis upon chronic alcohol drinking and also in chronic plus binge EtOH feeding. Although additional studies to better understand this preventive response are needed, the CD44 deficiency in myeloid cells is associated with the preventing a decreased expression of peroxisome proliferator activated receptor alpha (PPARa) and carnitine palmitoyltransferase 1 (CPT1) upon alcohol consumption (Figures 2D and2G). It is well established that PPARa and CPT1 are key regulators of hepatocyte metabolism including fatty acid uptake, beta-oxidation, and triglyceride turnover27. In addition, inflammatory mediators known to modify lipid metabolism in hepatocytes, such as TNF and CCL26, were strongly reduced in EtOH-treated CD44myel'KOmice. Ethanol consumption also dysregulates lipid metabolism and inflammation (e.g. elevating TNF and osteopontin expression21’28) in adipose tissue. Since CD44 plays a critical role in macrophage recruitment into adipose tissue in the context of obesity29, 30, the impact of CD44 deficiency in myeloid cells on adipose tissue responses after alcohol exposure remains to be examined.

[0138] In addition to regulating monocyte / macrophage infiltration into the liver, we also report that CD44 deficiency alters the frequency of activated hepatic neutrophils in the liver. Key neutrophil properties were altered as we observed reduced susceptibility to produce inflammatory mediators and higher anti-inflammatory phenotypes by phagocytosis of bacteria. We note that these responses in CD44-deficient neutrophils are strongly similar to the effects mediated by neutrophils during the resolution phase of liver disease. Emerging evidence shows the importance of neutrophils in the resolution of inflammation and injury. For example, neutrophils may phagocytose pathogens and cellular debris that would otherwise prolong inflammation, drive the conversion of pro-inflammatory macrophages to reparative macrophages in acute liver injury111 31and provide matrix metalloproteinases to the liver which suppress fibrosis progression in a carbon tetrachloride-induced fibrosis mouse model10, 32. In addition, specialized pro-resolving lipid mediators, such as lipoxins, protectins, resolvins, and maresins, promote tissue repair by inhibiting neutrophil chemotaxis, suppressing cytokine production, and enhancing phagocytosis10, 33. Although it is difficult to assess the relative contribution of CD44-deficent neutrophils in preventing pro-inflammatory pathways or increasing those that resolve inflammation, we report here that CD44 is a novel regulator of neutrophil function and its deficiency dampens liver inflammation and injury.

[0139] The chronic plus binge EtOH feeding is also associated with leukopenia resulting from decreased levels of lymphocytes and monocytes concomitant with the elevation of neutrophils. Leukopenia is common in heavy alcohol drinkers, which can occur along with other haematological abnormalities including anaemia and thrombocytopenia. In addition, chronic drinkers with recent excessive drinking could have higher levels of circulating neutrophils compared to healthy controls or chronic drinkers without recent excessive drinking34Although the deficiency of CD44 in myeloid cells did not completely prevent the WBC number reduction, a partial correction in the frequency of these immune cells was observed. Similarly, the neutrophil-lymphocyte ratio (NLR) was lower in mice deficient for CD44 in the myeloid cells when they were exposed to EtOH. This may relevant because elevated NLR has been associated with higher susceptibility to infection and poor clinical outcome after steroid treatment in patients with alcohol -associated hepatitis35. The same partial correction of the NLR in CD44myel'KOmice is also achieved by the preventive injection of anti-CD44 antibodies prior to binge ethanol exposure. The molecular mechanisms explaining these changes in blood immune cell frequency require future study, but this beneficial effect on the NLR in the absence of CD44 is not the result of a different mobilization of neutrophils from the bone marrow into the bloodstream (data not shown). However, we note that maturation of bone marrow CD44- deficient neutrophils was already modified in the steady state, as assessed by Ly6G and CXCR2 expression levels (Data not shown)36, 37

[0140] Consistent with our animal studies, the hepatic expression of CD44 was upregulated with AH and ALD in humans. In heavy alcohol drinkers, liver CD44 also correlated with liver injury and hepatic expression of inflammatory markers. This upregulation of CD44 has been also reported in patients with alcohol -related cirrhosis compared to healthy individuals, and decreased after abstinence38. Inflammation thus seems to be an important regulator of liver CD44 expression.

[0141] Among CD44 ligands, HA is highly expressed in the liver sinusoids and binds CD44 on neutrophils, providing a potential mechanism of neutrophil recruitment. McDonald et al. demonstrated that the blocking of the HA-CD44 interaction by anti-CD44 antibodies ameliorated liver injury and neutrophil infiltration in a mouse model of LPS-induced liver injury39. While circulating levels of HA correlated with the severity of hepatic fibrosis in heavy alcohol drinkers20and its elevated expression in AH was rapidly reduced after cessation of alcohol consumption40, the contribution of CD44-HA complex to the liver infiltration of neutrophils remains elusive in the context of ALD.

[0142] The development of the hepatic inflammation associated with alcohol consumption shares pathophysiological features with metabolic dysfunction associated steatohepatitis (MASH). We here highlighted that CD44 is a key driver of liver inflammation and injury in an acute-on-chronic drinking pattern by regulating myeloid cell mobilization and / or function (monocytes and neutrophils). We have also previously reported that CD44 regulates the recruitment of monocytes and neutrophils into the liver in a context of metabolic steatohepatitis. In addition, CD44 regulates the macrophage activation mediated by TLR4 ligands including DAMPs, LPS and saturated fatty acids22. However, in the context of binge drinking, CD44- dependent neutrophil responses appear to be particularly crucial in the development of hepatic inflammation and injury. The beneficial effects on liver complications obtained by neutralizing CD44 using a specific antibody only prevented the recruitment of neutrophils into the liver without affecting the liver monocyte / macrophage enrichment. In line with common pathogenic factors, the hepatic expression levels of E-selectin (SELE), which is expressed by activated endothelial cells and interacts with CD44, were upregulated in human and mouse livers with MASH and mouse livers upon chronic plus binge ethanol feeding8’41’42. The deletion of SELE in mice reduced liver complications in both aetiologies by regulating neutrophil recruitments (preferentially into the liver in response to alcohol and more into adipose tissue with obesity)8’42. Expression of E-selectin and markers of inflammation is reduced in ALD livers when CD44 is manipulated (systemic deletion or deletion in myeloid cells). Since obesity is an emerging factor that accelerates ALD progression2, pathogenic factors associated with both aetiologies, such as CD44, could be very promising therapeutic targets.

[0143] Some limitations in our study should be noted. While the in vitro studies have enhanced our understanding of the role of CD44 in neutrophils, the neutrophil functions regulated by CD44 could mediate additional responses in the liver by directly acting with the other liver cells (endothelial cells, hepatocytes, macrophages and T cells for example). In addition, it remains difficult to investigate liver fibrosis associated with alcohol drinking in such preclinical models.

[0144] In summary, the harmful functions of CD44 in ALD occur at different stages of the disease (from steatosis to inflammation and liver injury) and in different liver cells (neutrophils and macrophages). With regard to the development of hepatic steatosis, CD44 in myeloid cells could reinforce the low-grade inflammation that impairs the beta-oxidation of fatty acids. Regarding liver inflammation associated with an acute-on-chronic drinking pattern, CD44 enhances the neutrophil mobilization in blood, monocyte recruitment into the liver and the pro- inflammatory responses of both neutrophils and macrophages. This elevated local inflammation subsequently contributes to liver injury. The targeting of CD44 specifically in myeloid cells could represent a new therapeutic strategy to dampen liver inflammation and injury in chronic liver diseases (ALD and metabolic dysfunction-associated steatotic liver disease). REFERENCES:

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Claims

- 39 -CLAIMS:

1. A method of treatment of alcohol -related liver disease (ALD) in a subject in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms.

2. The method according to claim 1 wherein the alcohol-related liver disease is acute-on- chronic liver failure.

3. The method according to claim 1 wherein the alcohol -related liver disease is alcohol associated steatohepatitis (ASH).

4. The method according to claim 1 wherein the alcohol -related liver disease is alcohol associated hepatitis (AH).

5. The method according to claim 1 wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an antibody.

6. The method according to claim 1 wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a neutralizing anti-CD44 antibody.

7. The method according to claim 1 wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an anti-CD44 mAb.

8. The method according to claim 1 wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is a siRNA or an oligonucleotide or a peptide.

9. A pharmaceutical composition comprising an inhibitor of CD44 standard (CD44s) or an inhibitor of its CD44 variant (CD44v) isoforms for use in method of treatment of alcohol -related liver disease (ALD) in a subject in need thereof.

10. The pharmaceutical composition according to claim 9 wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an antibody, a siRNA or an oligonucleotide or a peptide.

11. The pharmaceutical composition according to claim 9 wherein the inhibitor of CD44 standard (CD44s) or the inhibitor of its CD44 variant (CD44v) isoforms is an anti-CD44 mAb.

12. i) an inhibitor of CD44 standard (CD44s) and ii) corticosteroid used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

13. i) an inhibitor of CD44 standard (CD44s) and ii) agonists of glucagon-like peptide 1 (GLP1) receptor used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

14. i) an inhibitor of CD44 standard (CD44s) and ii) glucagon-like peptide 1 (GLP1) base polyagonists used as a combined preparation for treating of alcohol -related liver disease(ALD) in a subject.

15. i) an inhibitor of CD44 standard (CD44s) and ii) resmetirom used as a combined preparation for treating of alcohol -related liver disease (ALD) in a subject.

16. A method of screening a drug suitable for the treating alcohol -related liver disease (ALD) comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of CD44 standard (CD44s) or CD44 variant (CD44v) isoforms.

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