EP1372697A2

EP1372697A2 - Use of compounds modulating rxr-ppar heterodimer activity as medicine for treating hepatitis c, and corresponding screening method

Info

Publication number: EP1372697A2
Application number: EP02730347A
Authority: EP
Inventors: Pierre Desreumaux; Sébastien DHARANCY; Johan Auwerx
Original assignee: Auwerx Yohan; Universite Lille 2 Droit et Sante
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Lille 2 Droit et Sante; Centre Hospitalier Universitaire de Lille
Priority date: 2001-04-04
Filing date: 2002-04-04
Publication date: 2004-01-02
Also published as: WO2002080956A2; AU2002302685A1; FR2823225B1; FR2823225A1; US20040171689A1; WO2002080956A3

Abstract

The invention concerns a screening method of a compound modulating the activity of a nuclear RXR-receptor heterodimer, preferably RXR-PPAR. The invention also concerns the compound capable of being selectively hybridised with the gene or a product of the gene coding for the RXR and PPAR subunits of said heterodimer, for preparing a medicine for preventive and/or curative treatment of an infection by the hepatitis C virus, or of fatty liver, of liver inflammation, liver lesions, liver cirrhosis, post-hepatic cancer whether or not associated with a hepatitis C virus infection.

Description

Use of compounds modulating the activity of the heterodimer RXR-PPAR as a medicament for the treatment of hepatitis C, and corresponding screening method

The present invention relates to the field of medical biology and more particularly to preventive and curative treatments for viral infection with the hepatitis C virus. The invention relates more particularly to a method of screening for a compound modulating the activity of a RXR-nuclear receptor heterodimer, preferably RXR-PPAR. The invention also relates to the compound capable of being obtained by the screening method of the invention, in particular the agonist compounds of the RXR-PPAR hetarodimere as well as a compound capable of hybridizing selectively with the gene or a product of the gene coding for the RXR and PPAR subunits of said heterodimer, for the preparation of a medicament intended for the preventive and / or curative treatment of an infection by the hepatitis C virus, associated hepatic steatosis or no to hepatitis C virus infection of hepatocytes, hepatic inflammation and liver damage associated or not with hepatitis C virus hepatocyte infection, as well as hepatic cirrhosis and / or to post-hepatic cancer associated or not with an infection of the hepatocytes by the hepatitis C virus.

Hepatitis C virus (HCV) has been identified as the primary agent responsible for hepatitis post-transfusion and related to drug addiction (Kuo G, Science 1989). HCV infection represents a major public health problem since its frequency in France is estimated at 1.3% (Roudot-Thoraval F, Hepatology 1998). More than 600,000 people are therefore affected by this infection in our country. HCV is characterized by a strong affinity for liver cells (hepatocytes) resulting in acute inflammation of this organ or acute hepatitis. This acute hepatitis is most often asymptomatic clinically and turns in 80% of cases into chronic hepatitis which can be complicated by cirrhosis and liver cancer (Poynard T, Lancet 1997). It is estimated worldwide that 3 million patients have chronic viral hepatitis C, 20% of which will progress to cirrhosis over a period of 10 years; these cirrhosis patients will then have an annual risk of liver cancer of 3%. Another complication of the disease would be the appearance of diabetes since recent epidemiological studies show that patients with HCV have an increased risk of type II diabetes (relative risk 2 to 6) (Mehta SH, Ann Intern Med 2000; Sangiorgio L, Diabetes Res Clin Pract 2000). The treatment of chronic hepatitis C is currently based on a combination of two molecules: interferon alpha and ribavirin (McHutchison J, N Engl J Med 1998). These drugs have many contraindications and are generally poorly tolerated by patients. In addition, this combination of drugs is ineffective in about one in two patients despite prolonged use (6 to 12 months). A new therapeutic protocol by delayed Interferon (Schering Plow Laboratory) having obtained MA in March 2001 will begin in our country. The expected results allow an increase in the response to treatment by 10% at a cost of 100 KF / year / patient. Faced with the lack of available vaccine and the difficulty of effectively treating patients with hepatitis C, there is an urgent and real need to develop new preventive and therapeutic strategies for hepatitis C.

This is precisely the object of the present invention. The inventors have thus identified a new therapeutic target constituted by the receptors for PPAR peroxisome proliferators.

(Peroxysome Proliferator-Activated Receptor) for the treatment of hepatitis C virus infections

The identification of this new target is based on recent data from the literature and on results obtained by the inventors. The hepatitis C virus enters hepatocytes using, in part, the receptor for low density lipoproteins (LDLR or CD36) (Monazahian M, J Med Virol 1999), then multiplies in the cell and can then invade others. This infection induces an immune response which is most often incapable of spontaneously eliminating the virus; this leads to chronic hepatitis in around 80% of cases (Alter M, N Engl J Med 1992). The quality of the immune response necessary for the elimination of the virus remains poorly understood but the inventors have shown that the production of interleu ine 4 (IL-4) constitutes a factor of poor prognosis for the course of the infection (Dharancy et al.). Other cytokines are also involved in the inflammatory mechanisms linked to infection with the hepatitis C virus, in particular by the production of TNFα, IL-lβ and IL-18 (Nelson DR, Dig Dis Sci 1997; McGuinness PH, Gut 2000) leading to the activation of the NFKB complex and to the production of molecules involved in the inflammatory reaction. These studies on the production of cytokines suggest that nuclear receptors may be involved in the regulation of the immune response during viral infections C. Thus, IL-4 has been shown to be the main cytokine regulating the expression of PPARγ which plays an essential role in the regulation of the production of in lammatory cytokines such as TNFα and IL-lβ via an inhibition of NFkB. Hepatitis C infection is also the source of a fatty overload characteristic of hepatocytes called fatty liver (Goodman ZD, Semin Liver Dis 1995; Czaja A, J Hepatol 1998; Clouston AD, J Hepatol 2001) the first clinical manifestation of infection with the hepatitis C virus. The origin of this fatty liver disease is unknown but could be secondary to a modulation of the activation of nuclear receptors which represent one of the largest families of transcription some of which can be activated by small lipophilic molecules such as hormones and nutrients for example. An important subfamily of these nuclear receptors corresponds to factors having the property of forming a heterodimer with the receptor X for retinoids (RXR). This subfamily is composed in particular of the receptor for vitamin D (VDR), the receptor for retinoic acid (RAR), the receptor for thyroid hormones (TRs), proliferators of peroxisomes (PPARs), receptors for bile acids (BAR also called FXR), receptors for oxysterols (LXRs), steroids and xenobiotics (SXR also called PXR).

Peroxisome-activated receptors (PPAR) are nuclear receptors capable of forming a heterodimer with RXR. There are 4 types of PPAR: α, β, γ, δ. The expression of PPAR α is mainly localized in the liver, the muscle and in a certain form of adipose tissue corresponding to brown fat. The expression of PPAR δ or β is ubiquitous whereas PPAR γ is produced essentially at the level of adipose tissue, macrophages and colonic epithelial cells. The production of PPARγ in the liver in humans remains unknown. These PPAR receptors are involved in the transcription of many target genes after attachment to a specific DNA response element (PPRE). Different synthetic and natural ligands can fix and activate PPAR. Activation of PPAR leads to various biological effects including activation, differentiation and proliferation of fat cells (adipocytes), regulation of lipid metabolism, regulation of inflammation and regulation of insulin response (Lehmann JM, J Biol Chem 1995) (Shoonjans K, Biochim Biophys Acta 1996 ). Other receptors which heterodimerize with RXR such as LXR, TRs, BAR / FXR, and SXR / PXR are strongly expressed by hepatocytes. The LXRs and FXR / BAR receptors probably play an important role in hepatic homeostasis since they control the metabolism of cholesterol and bile acids and that there is regulation between these receptors and PPAR. Thus, PPARs are capable of inducing the expression of LXRα in the liver (Tobin & Auwerx, Mol. Endo 2000) and macrophages (Tontonoz & Evans, Mol. Cell, 2001).

The inventors therefore propose to use PPAR α, β, δ, γ the modulators of PPAR as well as the nuclear receptors capable of forming a heterodimer with RXR (LXR, TRs, BAR / FXR, and SXR / PXR) as a new therapeutic target and / or new therapeutic proteins for the treatment of viral hepatitis C, and this by having an action on the penetration of the virus into the cells, by reducing the inflammatory reaction and the fibrosis and / or by reducing the risk of cancer.

More particularly, the present invention relates to a method for screening a compound modulating the activity of an RXR heterodimer-nuclear receptor characterized in that said method comprises the steps of (1) bringing into contact in the presence of the reagents necessary for carrying out at least one transcription reaction of said heterodimer RXR-nuclear receptor with at least one reporter gene having all the genetic information necessary for the expression of a reporter protein, said gene having at least one RE regulatory sequence; (2) contacting in the presence of the reagents necessary for carrying out at least one transcription reaction of the said heterodimer RXR-nuclear receptor and of the said compound with at least one reporter gene having all the genetic information necessary for the expression of a reporter protein which has at least one RE regulatory sequence; (3) qualitative, optionally quantitative determination of the expression of said protein triggered in steps 1) and 2) then comparison of said expressions; finally (4) selection, optionally of identification, of the compound if it is capable of selectively modulating the expression of said protein.

The term “heterodimer activity” is intended to denote the biological activity or the natural biological function performed by the heterodimer in a living cell, that is to say an activity of transcription factors. A transcription factor within the meaning of the present invention is a diffusible protein factor capable of modulating positively or negatively the expression of one or more genes by interacting with their regulatory sequence (s). Preferably, said heterodimer RXR-receptor nuclear is selected from the group consisting of RXR-PPAR, RXR-LXR, RXR-TR, RXR-BAR / FXR and RXR-SXR / PXR. Among the SXR steroid receptors, it is particularly worth mentioning the glucocorticoid (GR), estrogen (ER), mineralocorticoid (MR), progesterone (PR) receptors. The term “nuclear receptors” is intended to denote receptors which have an identical overall structure, namely: (1) an NH2 terminal end of variable length, not conserved and specific for the receptor (AB domain of the gene); (2) a region of approximately 65 amino acids, very conserved (domain C of the gene) which interacts with DNA; this region contains two glove finger patterns; (3) a non-conserved region of variable length (domain D); and (4) a domain of variable length which corresponds to the zone where the hormone or the ligand is fixed (domain E).

Within the meaning of the present invention, the term RE regulatory sequence (for “responsive element”) is intended to denote nucleic sequences necessary for the regulation of genes in eukaryotes. These sequences are distinguished from the promoter sequence stricto sensu, which corresponds to the sequence in which RNA polymerase II binds. The RE regulatory sequences are generally located in the 5 ′ region upstream of the genes, upstream of the promoter sequence. They constitute CIS-regulatory sequences on which are fixed transcription factors which act in TRANS. In general, these RE sequences confer tissue specificity on the genes which possess them. Of preferably, said RE regulatory sequence is selected from the group consisting of the PPRE regulatory response sequence PPAR, the LX-RE regulatory sequence LXR response, the regulatory sequence TRE of thyroid hormone receptor response (TR) , the regulatory sequence BAR / FXR-RE in response to BAR / FXR and the regulatory sequence SXR / PXR-RE in response to SXR / PXR. Among the REs to steroid receptors (SXR-RE), it is worth mentioning in particular the regulatory sequence RE to glucocorticoid receptors (GRE), the regulatory sequence RE to estrogen receptors (ERE), the regulatory sequence RE to mineralocorticoid receptors (MRE), the regulatory sequence RE to progesterone receptors (PRE).

More preferably, the heterodimer according to the invention is RXR-PPAR and the regulatory sequence RE (responsive element) is PPRE.

All of the genetic information necessary for a transcription reaction is known to those skilled in the art; they are at least one promoter, transcription initiation and termination signals, as well as suitable regions for transcription regulation. Optionally, the transcribed reporter gene can be translated. For this it is essential that the mRNA has the initiation and termination signals of the translation.

The reporter gene may be present in linear form, preferably it is cloned into an expression vector, allowing expression of the protein reporter in a cell host. The vector may or may not be maintained in a stable manner in the cell and may optionally have specific signals specifying the secretion of the translated polypeptide. The different control signals are chosen according to the cell host used. Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods such as, for example, calcium phosphate precipitation transfection, lipofection, electroporation, thermal shock.

The compound obtained by the screening process activates or increases the expression of said reporter gene. Alternatively, the compound obtained by the screening process, preferably abolishes or inhibits the expression of said reporter gene.

According to a preferred embodiment of the invention, the screening method is characterized in that at least one step is carried out in a living cell. According to a preferred embodiment, at least steps 1 and 2 of the method are carried out in a living cell. Among the cells in which the screening method according to the invention is carried out, mention should be made of prokaryotic cells, such as bacteria and in particular Escherichia coli, yeasts, in particular Saccharomyces cerivisiae, animal cells. Preferably, these are mammalian cells preferably chosen from human, mouse, rat, rabbit, hamster cells. It is also within the scope of the invention to screen the compounds of the invention on laboratory animals or from cells derived from laboratory animals. These laboratory animals, preferably the mouse, the rat or the rabbit, are preferably genetically modified to express a transgene which has all of the genetic information necessary for the expression of a reporter protein, the said gene having at minus one RE regulatory sequence, preferably PPRE. This transgene can be present in the genome of at least one cell of the transgenic animal, this integration being carried out either by homologous recombination or by random integration. Alternatively, the transgene is present in an episomal form.

Alternatively, the method can be an in vitro screening method characterized in that the steps are carried out in an acellular system. In this case, this process is carried out in the presence of reagents necessary for carrying out at least one transcription reaction, and optionally in the presence of reagents necessary for carrying out at least one translation reaction of the reporter protein such as for example rabbit reticulocyte lysate. These various in vitro methods are well known to those skilled in the art (Sambrook et al., 1989).

In the screening method according to the present invention, any conventional procedure or test can be implemented to result in step 3 of said method in obtaining a detectable and / or quantifiable signal representative of the quantity of said expression product present in the reaction medium, depending on the expression product sought. Preferably, the expression product is a polypeptide corresponding to the translation product of the mRNA corresponding to said reporter gene; in this case, this expression product is highlighted and optionally quantified by the so-called “Western blot” method or by immunohistochemistry well known to those skilled in the art. When it is a transcription product (mRNA) coding for said reporter gene, it can be demonstrated, optionally quantified by RT-PCR, by Northern blotting, or by the so-called hybridization method. in situ also well known to those skilled in the art.

According to a preferred embodiment, said reporter protein is selected from the group of autofluorescent proteins and enzymes detectable by a histochemical process. Preferably, the autofluorescent protein is chosen from the group composed of green fluorescence protein (GFP), augmented green fluorescence protein (EGFP), protein of red fluorescence protein (RFP), protein blue fluorescence protein (BFP), yellow fluorescence protein (YFP) and fluorescent variants of these proteins. Alternatively, said reporter gene codes for an enzyme detectable by a histochemical method chosen from the group consisting of β-galactosidase, β-glucoronidase, alkaline phosphatase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholine esterase, lyzozyme, malate dehydrogenase, glucose-6 phosphate dehydrogenase, alcoholic dehydrogenase, luciferase, chloramphenicol-acetyl transferase, growth hormone. More generally, the said reporter gene can correspond to any gene which has at least one PPRE upstream regulatory sequence. It can be a gene present in its natural environment or a gene genetically engineered in order to place a coding sequence under the control of a functional promoter sequence comprising at least the RE sequence, preferably PPRE. The present invention also relates to the compound capable of being obtained by the screening method according to the invention as well as all compounds modulating the activity of a nuclear receptor RXR heterodimer.

According to a first embodiment, this compound activates or increases the activity of the heterodimer RXR-nuclear receptor, in particular RXR-PPAR.

Preferably, the compound is selected from the group consisting of PPARα, PPARβ, PPARγ, PPARδ, RXR, a natural or synthetic agonist ligand of PPARα, PPARγ, PPARβ, PPARδ, RXR, RXR-nuclear receptor, preferably of RXR-PPAR and in particular RXR-PPARα, RXR- PPARβ, RXR-PPARγ, RXR-PPARδ. This agonist ligand of RXR-PPAR is preferably chosen from the group composed of prostaglandins J2, polyunsaturated fatty acids, thiazolinediones, non-inflammatory anti-inflammatories steroids, fibrates, including fenofibrate, pioglitazone. Among the agonist compounds, it is also worth mentioning the anti-ideotypic antibodies corresponding to the compounds PPARα, PPARβ, PPARγ, PPARδ, RXR.

According to a second embodiment, the compound capable of being obtained by the screening method according to the invention abolishes or inhibits the activity of the nuclear receptor RXR heterodimer. Preferably, it is a natural or synthetic antagonist ligand. The term “antagonist ligand” is intended to denote the compounds capable of reducing or abolishing the level of expression and / or the activity of RXR-nuclear receptor, in particular RXR-PPAR. The term "antagonist" refers to a molecule which, when it binds to the polypeptide according to the invention, reduces the quantity or the duration of the effects of the biological activity of the RXR-PPAR polypeptide. Among the compounds capable of being obtained by the screening method according to the invention which abolish or inhibit the activity of the heterodimer RXR-nuclear receptor, mention may be made of the polynucleotides capable of hybridizing specifically with the RE, preferably PCERA. Thus, for example, the said polynucleotide enters into competition with the heterodimer RXR-PPAR for attachment to the response element PPRE. The polynucleotide can also correspond to the RE sequence, preferably PPRE in excess, in order to fix by excess of substrate the heterodimer RXR-nuclear receptor, preferably RXR-PPAR, on the RE sequence, preferably PPRE of the polynucleotide. The polynucleotide can also correspond to an antisense oligonucleotide. In this case, the polynucleotide is capable of selectively hybridizing with the gene or a product of the gene coding for the RXR and PPAR subunits. The oligonucleotides according to the invention have a minimum size of 9 bases, preferably at least 10, 12, 15, 17, 20, 25, 30, 40, 50 bases. According to yet another embodiment, this compound can be an antibody, or antibody fragment, monoclonal or polyclonal directed specifically against RXR-nuclear receptor, preferably RXR-PPAR; preferably, the antibody according to the invention is directed against the PPAR subunit of the heterodimer, preferably it is a PPARγ type subunit. Among the antibodies according to the invention, there may be mentioned rabbit polyclonal antibodies directed against PPARγ and marketed by WAK-CHEMIE (Bad Soden, Germany) or by TEBU (Le Perray en Yvelines, France). In general, for the preparation of monoclonal antibodies or their fragments directed against the PPARγ receptor, reference may be made to the techniques which are in particular described in the “Antibodies” manual (Harlow et al., 1988) or to the preparation technique. from hybridomas described by Kohler and Milstein in 1975. The monoclonal or polyclonal antibodies according to the invention can be obtained for example from the cell of an animal immunized against the PPARγ protein, or one of its fragments comprising the epitope specific (determinant of the protein responsible for the specific interaction with the antibody). Said PPARγ receptor protein, or a fragment thereof, may in particular be produced, according to the usual procedures, by genetic recombination from a nucleic acid sequence contained in the cDNA sequence coding for the PPARγ receptor protein, or by peptide synthesis. The monoclonal or polyclonal antibody fragments according to the invention comprise any fragment of said monoclonal antibody capable of binding to the epitope of the PPARγ protein on which the monoclonal or polyclonal antibody from which said fragment is derived is attached. Examples of such fragments include in particular monoclonal or polyclonal single chain antibodies or monovalent fragments Fab or Fab 'and divalent fragments such as F (ab') 2, which have the same binding specificity as the monoclonal antibody of which they are from. A fragment according to the invention may also be a single chain Fv fragment produced by methods known to those skilled in the art and as described for example by Skerra et al., 1988 and King et al., 1991. A fragment according to the invention may also be an Fc fragment. The monoclonal or polyclonal antibody fragments of the invention can be obtained from the monoclonal or polyclonal antibodies as described above by methods such as digestion with enzymes, such as pepsin or papain and / or by cleavage of the bridges disulfides by chemical reduction. Alternatively, the monoclonal or polyclonal antibody fragments can be synthesized by synthesizers automatic peptides such as those supplied by the company Applied Biosystems, etc., or can be prepared manually using techniques known to those skilled in the art and as described for example by Geysen et al. (1978).

Agonist and antagonist ligands include proteins, nucleic acids, carbohydrates, lipids, chemical compounds. Among the agonist and antagonist ligands of a protein nature, it is appropriate to designate all of the natural polypeptides capable of interacting with the heterodimer RXR-nuclear receptors, preferably RXR-PPAR.

The term “ligand or compound” is intended to define all of the compounds capable of interacting directly or indirectly with the binding of the heterodimer RXR-nuclear receptor, preferably RXR-PPAR to the RE sequence, preferably PPRE; a ligand within the meaning of the present invention forms a complex which affects transcriptional activity, that is to say increases, decreases, modulates or invalidates the transcription of a gene under the control of a promoter containing a sequence of RE DNA to which the nuclear receptor RXR heterodimer binds.

It is also one of the objects of the invention to provide a kit or a kit for the screening of ligands capable of affecting the transcriptional activity of a reporter gene whose promoter sequence comprises at least one RE sequence, preferably PPRE capable of binding a nuclear RXR-receptor heterodimer, preferably RXR-PPAR, and characterized in that it comprises the following elements: (i) a gene, optionally cloned in an expression vector, and optionally present in a living cell; (ii) a ligand; (iii) the reagents necessary for the implementation of a transcription and / or translation reaction.

According to another aspect, the compound or ligand according to the invention is used as a medicament and in particular as active principles of medicament for humans or animals. The compound is preferably in soluble form, associated with a pharmaceutically acceptable vehicle. The term “pharmaceutically acceptable vehicle” is intended to denote any type of vehicle usually used in the preparation of injectable compositions, that is to say a diluent, a suspending agent such as an isotonic or buffered saline solution. Preferably, the compound is administered systemically, in particular intravenously, intramuscularly, intradermally or orally. Their optimal methods of administration, dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a treatment adapted to a patient such as for example the patient's age or body weight, the severity of his general condition, tolerance to treatment and side effects observed, etc.

The present invention also relates to the use of a compound modulating the activity of a nuclear receptor RXR heterodimer for the preparation of a medicament intended for the preventive and / or curative treatment of an infection by a virus which uses to carry out its viral cycle at least one cellular protein coded by a gene which presents at least one regulatory sequence RE. Preferably, said cellular protein is the receptor for low density lipoproteins (LDLR or CD36) and said virus is hepatitis C virus.

Preferably, the said heterodimer RXR-nuclear receptor is selected from the group consisting of RXR-PPAR, RXR-LXR, RXR-TR, RXR-BAR / FXR and RXR-SXR / PXR. According to a preferred embodiment, said regulatory sequence RE is selected from the group composed of the regulatory sequence PPRE in response to PPAR, the regulatory sequence LX-RE in response to LXR, the regulatory sequence TRE of response to the receptor to thyroid hormones (TR), of the BAR / FXR-RE regulatory sequence of response to BAR / FXR and of the SXR / PXR-RE regulatory sequence of response to SXR / PXR; preferably it is PPRE. Preferably, said heterodimer RXR-nuclear receptor is RXR-PPAR and said regulatory sequence RE is PPRE.

The invention also relates to the use of a compound modulating the activity of a nuclear receptor RXR-heterodimer for the preparation of a medicament intended for the preventive and / or curative treatment of an infection by the virus of the hepatitis C, from cells selected from hepatocytes, bile cells, parenchymal liver cells, circulating blood cells. The invention also relates to the use of a compound modulating the activity of a nuclear receptor RXR-heterodimer for the preparation of a medicament intended for the preventive and / or curative treatment of chronic hepatitis.

The invention also relates to the use of a compound modulating the activity of a nuclear receptor RXR-heterodimer for the preparation of a medicament intended for the preventive and / or curative treatment of hepatic steatosis associated or not with an infection of the hepatocytes with hepatitis C virus.

The invention also relates to the use of a compound modulating the activity of a nuclear receptor RXR-heterodimer for the preparation of a medicament intended for the preventive and / or curative treatment of hepatic inflammation and associated hepatic lesions or no to infection of hepatocytes with the hepatitis C virus

The invention also relates to the use of a compound modulating the activity of a nuclear receptor RXR-heterodimer for the preparation of a medicament intended for the preventive and / or curative treatment of hepatic cirrhosis and / or of cancer. post-hepatic or not associated with an infection of the hepatocytes by the hepatitis C virus.

The invention also relates to the use of a compound modulating the activity of a nuclear receptor RXR-heterodimer for the preparation of a medicament intended for controlling carbohydrate metabolism and / or for preventive and / or curative treatment of type II diabetes in patients carrying the hepatitis C virus. The present invention also relates to the use of a compound according to the invention or of a compound capable of hybridizing selectively with the gene or a product of the gene coding for RXR and / or PPAR for the preparation of a medicament intended for the preventive and / or curative treatment of an infection by a virus which uses to carry out its viral cycle at least one cellular protein encoded by a gene which has at least one RE regulatory sequence, preferably PPRE. Preferably, said cellular protein is the receptor for low density lipoproteins (LDLR or CD36) and said virus is hepatitis C.

Also, the present invention relates to the use of a compound according to the invention or of a compound capable of hybridizing selectively with the gene or a product of the gene coding for RXR and / or PPAR for the preparation of a medicament. intended for the preventive and / or curative treatment of an infection of hepatocytes by the hepatitis C virus.

The present invention also relates to the use of a compound according to the invention or of a compound capable of selectively hybridizing with the gene or a product of the gene coding for RXR and / or PPAR for the preparation of a medicament intended preventive and / or curative treatment of fatty liver disease whether or not associated with hepatocyte infection by the hepatitis C virus. The present invention also relates to the use of a compound according to the invention or of a compound capable of selectively hybridizing with the gene or a product of the gene coding for RXR and / or PPAR for the preparation of a medicament intended to the preventive and / or curative treatment of hepatic inflammation and hepatic lesions associated or not with an infection of the hepatocytes by the hepatitis C virus. The present invention also relates to the use of a compound according to the invention or of a compound capable of hybridizing selectively with the gene or a product of the gene coding for RXR and / or PPAR for the preparation of a medicament intended for the preventive and / or curative treatment of hepatic cirrhosis and / or of a post-hepatic cancer associated or not with an infection of the hepatocytes by the hepatitis C virus. The invention also relates to a product comprising at least one compound according to the invention or a compound capable of hybridizing se lectively with the gene or a product of the gene coding for RXR and / or PPAR and at least one anticancer agent as a combination product for simultaneous, separate or spread over time in anticancer therapy. The present invention also relates to the use of a compound according to the invention or of a compound capable of selectively hybridizing with the gene or a product of the gene coding for RXR and / or PPAR for the preparation of a medicament intended carbohydrate metabolism control and / or preventive treatment and / or curative of type II diabetes in patients with the hepatitis C virus.

Finally, the present invention relates to the use of a composition comprising a compound according to the invention modulating the activity of an RXR-nuclear receptor heterodimer, preferably RXR-PPAR, and a pharmaceutically acceptable vehicle as a medicament for the preventive and / or curative treatment of a man or an animal infected with the hepatitis C virus, characterized in that the ability of said compound to selectively modulate the activity of said heterodimer is determined by (a) the contacting in the presence of the reagents necessary for carrying out at least one transcription reaction of said heterodimer with at least one gene having all of the genetic information necessary for the expression of a protein, said gene having at least a RE regulatory sequence, preferably PPRE; (b) bringing into contact in the presence of the reagents necessary for carrying out at least one transcription reaction of said heterodimer and of said compound with at least one gene possessing all of the genetic information necessary for the expression of a protein and which has at least one RE regulatory sequence, preferably PPRE; (c) the qualitative determination, optionally quantitative, of the expression of said protein triggered in a) and b) then comparison of said expressions; (d) finally the identification of the compound which selectively modulates the expression of said protein. The invention also relates to a pharmaceutical composition for the preventive and / or curative treatment of an infection with the hepatitis C virus, characterized in that it contains a therapeutically effective amount of a compound according to the invention or of a compound capable of hybridizing selectively with the gene or a product of the gene coding for RXR and / or PPAR. This composition may also contain at least one antiviral agent as a combination product for simultaneous, separate or spread over time in antiviral therapy linked to an infection with the hepatitis C virus; this antiviral agent is preferably selected from the group composed of interferon alpha (IFNα), ribavirin, delay interferon.

Other characteristics and advantages of the invention appear in the following description with the examples shown below. In these examples, reference is made to the following figures.

FIGURES

Figure 1: Demonstration of the expression of nuclear PPAR receptors in the liver cells of healthy individuals (controls) and of patients infected with the hepatitis C virus (HCV). Quantification by competitive RT-PCR of PPARγ expressed in number of PPARγ Arnm molecules per molecule of β-actin mRNA in the liver cells of 10 controls and 21 patients with chronic hepatitis C. The average is shown and deviations from the average are shown.

Figure 2a: Hepatic expression of the messenger RNAs of RXRα, VDR, LXRα, PPARα, PPARδ and PPARγ in samples of human livers taken from patients with chronic hepatitis C and control subjects using the semi-quantitative RT-PCR technique . Results are expressed as means ± standard deviation from healthy control livers.

Figure 2b: Liver concentration of mRNA

PPARα in samples of human livers taken from patients with chronic hepatitis C and in healthy controls using the competitive RT-PCR technique. Results are expressed as means ± standard deviation from healthy control livers.

Figure 3: Hepatic expression of PPARα in human liver samples taken from patients with HCV-induced cirrhosis (n = 3), HBV-induced cirrhosis (n = 2), alcoholic cirrhosis (n = 5) and healthy controls (n = 5) using the Western blot technique. The results are expressed as means ± standard deviation.

Figure 4: Expression of PPARα in human circulating mononuclear blood cells (PBMC) collected from patients with chronic hepatitis C and healthy controls using the competitive RT-PCR technique (4a) and the Western technique blot (4b). Results are expressed as mean ± standard deviation from healthy control livers.

Figure 5: Susceptibility of the PPARγ ^{+ "} mouse to acute CC1-induced hepatitis _4. The necrotico-inflammatory scores (A) are expressed as mean ± standard deviation. The intensity of the lesions is evaluated by histological analysis of the livers of mice killed two days after induction of hepatitis by administration of CC1 ₄ (lμm / kg) using the Ishak score Mortality (B) is expressed as a percentage of lethality two days after administration of CC1 ₄ .

Figure 6: Susceptibility of PPARα ^"_ mice to acute CC1-induced hepatitis _4. The necrotico-inflammatory scores (A) are expressed as mean ± standard deviation. The intensity of the lesions was evaluated by histological analysis using the score of Ishak on the livers of mice killed 5 days later induction of hepatitis by administration of CC1 (lμm / kg). Mortality (B) is expressed as a percentage of lethality 5 days after the administration of CC1 ₄ .

Figure 7: Quantification of PPARα messenger RNA in clones N3, N4 and SWX using competitive RT-PCR. The results show an inhibition rate of the expression of PPARα of 90% for the clone N4 (a) and 23% for the clone N3 (b). The results are represented as the mean ± standard deviation.

EXAMPLES

1_. Materials and methods

1.1 Quantification of PPARs and other Rs forming a heterodimer with RXR in the liver by PCR:

The RT-PCR quantification of PPARs and other nuclear receptors forming a heterodimer with RXR was carried out on liver biopsies. All biopsies were equivalent in size and the mean weight was 8 ± 4 mg. Biopsies were immediately frozen and stored at -80 ° C. After incubation of the samples in Trizol, the nucleic acids were extracted using chloroform. Following a 30 min treatment at 37 ° C with 20-50 units of DNase I RNase-free, the total RNAs were back-transcribed into cDNA as previously described. The RT reaction mixture was amplified by PCR using sense and antisense primers specific for these receptors and for β-actin. The samples were subjected to 20-40 cycles of PCR and the quantification of the cDNA was carried out by electrophoresis on 3% agarose gel. As the precise quantification of RNA in samples is often difficult and imprecise, the number of mRNA molecules of nuclear receptors was expressed in comparison with the number of mRNA molecules of an internal control, β-actin, in the same sample.

1.2 Quantification of PPARs and other nuclear receptors (RN) forming a heterodimer with RXR in the liver by estern-blot: Total protein extracts are obtained by homogenization of liver biopsies in a lysis buffer consisting of PBS with 1% NP -40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and a classic cocktail of protease inhibitors. Separation of total proteins (50 μ), transfer to PVDF membrane and immunodetection of PPAR and other RNs forming a heterodimer with RXR by incubation of the membrane with a polyclonal rabbit antiserum for 12 hours (dilution l / 500e, TEBU, Le Perray en Yvelines, France) were carried out as previously described. The complex is revealed by chemiluminescence (ECL, Amersham, UK). The results are expressed in optical density unit (OD) for 50 ng of total proteins. 1.3 Immunolabelling of PPARs and other RNs forming a heterodimer with RXR in the liver: The liver biopsies are fixed in 4% paraformaldehyde, included in the paraffin and cut to 4 micrometers for immunohistochemistry and immunofluorescence. The sections are preincubated for 30 minutes at room temperature in a blocking solution containing avidin D and biotin (Blocking Kit, SP2001, Vector Laboratories, Burlingame, CA, USA). They are then exposed to a specific antibody directed against PPARs or other RNs forming with a heterodimer with RXR for 2 hours at room temperature. The sections are washed in PBS containing 0.05% of Triton X 100 and incubated with a secondary goat anti-rabbit biotinylated antibody (dilution 1/500 for 30 minutes, Dako, Trappes, France). The immune complex is detected using avidin-biotin coupled to peroxidase (ABCOMPLEX / HRP, Dako, Trappes, France) and revealed using 3, 3 '-diaminobenzidine (DAB, Dako, Trappes, France). As a negative control, the primary antibody is replaced by an irrelevant rabbit serum.

1.4 Patient and collection of liver and blood specimens.

The study was approved by the local Ethics Committee and all subjects gave their informed consent. Liver biopsies are performed on 20 patients with chronic hepatitis C (7 women, 13 men; average age 43 ± 12 years, range 21 to 70 years).

Chronic hepatitis C is defined by the presence of biochemical abnormalities (mean level of AST (amino transferase) in the serum: 94 ± 61 U / l), the positivity to the enzyme immunoassay of the hepatitis C virus (EIA), the presence of HCV RNA in the serum detected by PCR (average viral load: 603,330 ± 480,000 copies / ml), and by consistent histological data (average histological activity index: 6.5 ± 2.4). As inflammatory controls, liver biopsies are treated under the same conditions in patients with chronic hepatitis B (n = 3), and patients with alcoholic cirrhosis (n = 5). Surgical biopsies of histologically healthy livers are obtained by hepatectomy from 14 patients operated on for metastases of the liver. The patients and controls received no treatment (anti-viral therapy, hepatotoxic drugs, corticosteroids, immunosuppressive therapy) before or during the study, and none of them consumed more than 20g of alcohol per day.

All biopsy specimens are treated under the same conditions and all samples have a similar size and an average weight of 4 ± 1 mg. Biopsies are cut into two parts. They are immediately frozen in liquid nitrogen and stored at -80 ° C for protein and messenger RNA analyzes of nuclear receptors. Cells peripheral blood mononuclear cells (PBMC, for peripheral blood mononuclear cells) are isolated from fresh heparin blood obtained from 10 informed healthy volunteers and 10 patients with chronic hepatitis C. After elimination of platelet-rich plasma, the blood is centrifuged on Ficoll-Hypaque for 20 minutes at 4 ° C, the interphase mononuclear cells are collected, washed twice then resuspended and stored at -80 ° C for protein and mRNA analyzes of nuclear receptors.

1.5 Cells expressing the body protein of the hepatitis C virus (HCV). The transfected human hepatocellular carcinoma (HepG2) cell line is used to analyze the impact of the HCV body protein on the expression of PPARα. The HepG2 cells are transfected as described above (Barba et al. Proc. Natl. Acad. Sci. USA 1997, 94: 1200-1205) with the vector pEF352 neo comprising, under the control of the elongation factor promoter, HCV cDNA including HCV lb sequences from the body to the NS3 region. Two independent clones stably expressing the HCV body protein were analyzed (clone N3 and N4). The clone transfected with the empty vector is used as a negative control (clone SWX). These different clones are cultured at 37 ° C. under an atmosphere comprising 5% of CO ₂ and maintained in an Eagle's culture medium modified by Dulbecco (DMEM) supplemented with 10% fetal calf serum and comprising penicillin-streptomycin. The cells are cultured to confluence then the culture dishes are scraped off and the cells are harvested to prepare the cell extracts.

1.6 Induction of hepatitis induced by CC1 ₄ in mice.

Experiments with animals are carried out in an accredited establishment (No. B59-108 and B67-218-5) according to government directives No. 86/609 / EEC. The animals are grouped in cages (5 to 6 animals per cage) and have free access to water and rodent food. For a study of an acute pathology induced by CC1, the mice are anesthetized for 10 minutes and an intraperitoneal injection of a 1: 1 solution of CC1 ₄ and sterile mineral oil at a dose of lml / kg d animal is realized. Control mice receive a dose of mineral oil using the same technique. First, the PPARα - / - and PPARγ +/- mice both on a 129 / Sv genetic background are used (the homozygous mouse PPARγ - / - is not viable) to verify the potential susceptibility to the development of hepatitis in these disabled animals. In a second type of experiment, wild Balb / c mice are used in intervention studies with the PPARγ agonist, pioglitazone (50 mg / kg). This compound is administered once a day by oral gavage starting 3 days before the induction of hepatitis. The animals are killed by cervical dislocation between days 2 to 5 after the administration of CC1. The livers are recovered and then fixed overnight in 4% paraformaldehyde and then included in paraffin. The sections are stained with hematoxylin and eosin then examined blind by a pathologist and then evaluated according to the score of Ishak (Ishak, et al. J. Hepatol. 1995, 22: 696-699).

1.7 Quantification of messenger RNAs of nuclear receptors and of β-actin by RT-PCR.

The RNA is isolated from a sample of liver and mononuclear cells with the TRIzol® reagent (Life Technologies, Cergy Pontoise, France) as described by the manufacturer. After treatment at 37 ° C for 30 minutes with 20 to 50 units of DNase I RNAse-free (Roche Diagnostics Corporation, Indianapolis, USA) the total RNA (10 μg) is reverse transcribed into cDNA. The reverse transcription reaction mixture is amplified by PCR using sense and antisense primers specific for nuclear receptors (RXRα, LXRα, VDR, PPARα, PPARβ, PPARγ) and β-actin. The samples are subjected to 40 PCR cycles (Perkin-Elmer Corporation, Foster City, California, USA). The quantification of the cDNA is carried out by electrophoresis on agarose gel 2 to 3% using an image analyzer (Gel Analyst, Clara Vision, Paris, France). Accurate quantification of RNA in the sample is often difficult and imprecise, the number of messenger RNA molecules in PPARα in the livers is expressed in comparison with the number of 10 ⁶ molecules of mRNA of an internal control, in this case β-actin in the same sample.

1.8 Quantification of PPARα by Western blot analysis.

The protein preparation is carried out in liver and mononuclear cell biopsies as previously described. The total protein extracts are obtained by homogenization of tissues and cells in an extraction buffer composed of PBS (Phosphate Buffered Saline) with 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and a classic cocktail of protease inhibitors. The total proteins are then separated by electrophoresis on polyacrylamide gel and then electrotransferred. Immunosensing with a second antibody conjugated to peroxidase and with chemiluminescence is carried out according to the manufacturer's instructions (ECL, Hamersham, UK). The results are expressed as optical density units for 50 μg of total proteins.

1.9 Statistical methods. The comparison of the mean ± standard deviation of messenger RNA and PPARγ proteins between patients with ulcerative colitis (UC, ulcerative colitis) and Crohn's disease (CD) and controls is analyzed by the test ANOVA single no parametric Kruskal-Wallis. The differences are statistically significant if p is <0.05.

2. Demonstration of the expression of nuclear receptors in the liver.

By PCR techniques, the inventors have demonstrated, in 10 controls and 21 patients with chronic viral hepatitis C, comparable and significant levels of PPARγ in the hepatic level (Figure No. 1).

The Western blot and immunohistochemistry studies give comparable results highlighting the expression of the PPARγ protein, in particular in the hepatocytes. The same type of study is underway to show the expression of PPAR α, β, δ and of LXR, TRs, BAR / FXR, and SXR / PXR. The presence in large quantities of these receptors in the liver suggests that the modulators of PPAR α, β, δ, γ and LXR, TRs, BAR / FXR, and SXR / PXR will be active in the liver.

3. Modulation of hepatocyte expression of CD36 by nuclear receptors.

Thanks to the use of hepatocyte lines (HepG2, HuH7) maintained in culture with agonists and / or antagonists of PPAR α, β, δ, and of LXR, TRs, BAR / FXR, and SXR / PXR, we demonstrated by immunohistochemistry respectively an increase and a decrease in the expression of CD36 by hepatocytes. These results are being confirmation by other PCR techniques, western blot and flow cytometry.

At the same time, liver samples were taken from wild mice and invalidated mice for the different PPAR isoforms and for LXR, TRs, BAR / FXR, SXR / PXR. The quantification of CD36 in these different models also highlights the in vivo involvement of these nuclear receptors in the expression of CD36 in the liver. Similarly, the use of modulators for these nuclear receptors will allow the inventors to control, in wild animals, their roles in the expression of CD36 in the liver.

4. Anti-inflammatory and anti-fibrotic roles of nuclear receptors in the liver.

Since no animal model outside the monkey can be infected with the virus C and a fortiori develop a viral hepatitis C, hepatitis induced by chemical agents (carbon tetrachloride) are used to demonstrate the effect anti-inflammatory and anti-fibrotic modulators of nuclear receptors PPARs, LXR, TRs, BAR / FXR, SXR / PXR. The first gross results show the anti-inflammatory effect of these different modulators. 5. Activation of PPRE by the hepatitis C virus or the blood of patients infected with the hepatitis C virus

Hepatocytes are transfected with a reporter gene (luciferase) under the control of a response element (PPRE, LXRE, ...) for the receptors described above. Visualization of the activation of the reporter gene by the virus will highlight the involvement of PPAR or other receptors during infection with the virus C.

6. Role of fenofibrates, PPARα agonist, on the severity of viral hepatitis C.

The role of fenofibrates on the severity of liver damage will be analyzed in the cohort of patients followed by the inventors at the Lille University Hospital. The role of these PPARα agonists will be evaluated on biological and histological criteria.

7. Expression of nuclear receptors during HCV infection.

Using the RT-PCR technique, the inventors have quantified the messenger RNAs of RXRα,

VDR, LXRα, PPARα, PPARδ and PPARγ in liver samples taken from patients with chronic hepatitis C and healthy control individuals. While similar concentrations of messenger RNA of β-actin are observed everywhere in the two groups tested, the level of expression of PPARα mRNA is particularly lowered (15 ± 1.8 versus 198 ± 35.5, p = 0.0003), similarly for PPARβ (6 ± 2.25 versus 40 ± 14, p = 0.0002) and a downward trend is also observed for the rate of expression of PPARγ mRNA in the liver of HCV infected patients compared to healthy controls (Figure 2A). The hepatic deficit in PPARα concentration is confirmed using the competitive RT-PCR technique (51 ± 25.7 versus 304 ± 110, p = 0.0006) (Figure 2B).

To further analyze the abnormal level of expression of PPARα mRNA in the liver of patients infected with HCV, the inventors used a Western blot analysis to quantify the PPARα protein in the same samples. The inventors carried out a Western blot using an antibody directed against PPARα in the liver of control individuals and of patients infected with HCV. In view of the ethical considerations, the inventors used explanted livers taken from patients who underwent liver transplantation because they were suffering from cirrhosis associated with an HCV infection. Using this analysis, the optical density (OD) of livers infected with HCV appears less intense than that of control livers (FIG. 3) suggesting that the expression of both proteins and mRNAs of PPARα is affected in the liver of patients infected with HCV.

Conversely, the expression of PPARα in PBMC is not different in patients infected with HCV compared to healthy controls in terms of mRNA levels (12.36 ± 6.18 versus 13.15 ± 6.57) (Figure 4a) and at the protein level (421 210 ± 6 899 versus 270 504 ± 82 252) (Figure 4b). This result suggests that an abnormal expression of PPARα could be confined to the liver only and not extend to other cells classically expressing PPARα.

8. Increased susceptibility of PPARγ ^ ^" and PPARα ^{~ / -} mice to hepatitis induced by CC1 ₄ .

As demonstrated in the previous study, the PPAR / RXR heterodimer is involved in the regulation of the inflammatory process; the inventors have therefore hypothesized that the lack of expression of PPARα in the liver is capable of influencing the development of inflammation after liver injury.

To this end, the inventors sought to determine whether a mouse heterozygous for deficiency in PPARγ (PPARγ ^{+ ~} ) or homozygous for deficiency in PPARγ (PPARγ ^"_ ) was more likely to develop acute hepatitis induced by CC1 ₄ . These mice as well as wild mice of the same litter have a genetic background of 129 / Sv. compared to wild PPARγ ^{+ + individuals} of the same litter, PPARγ ^{+ "} mice have more pronounced microscopic liver lesions evaluated according to Ishak (10.6 ± 0.22 versus 4 ± 0.8) (Figure 5a). are characterized by a confluent necrosis in certain places with multiple bridges linking different vascular structures. The inflammatory infiltra is moderate, from some to all of the portal areas. This susceptibility to acute hepatitis is associated with a mortality rate dramatically increased (40% compared to 0%) (Figure 5b) only 2 days after the injection of CC1 ₄ .

Compared with wild PPARα ^{+ +} mice, PPARα ^{~ _} mice develop microscopic liver lesions much more pronounced 5 days after injection with CC1 (9.2 ± 0.7 versus 4.2 ± 0.13) (Figure 6a). These lesions are characterized by confluent necrosis in most regions and slight portal inflammatory infiltration. However, these lesions appear to be less severe than for PPARγ ^{+ / "} mice. This increase in hepatic lesions is also associated with a higher mortality rate (50% versus 0%) (Figure 6b). 9. Disturbed expressions of PPARα mRNA in HepG2 cells transfected by the core.

As numerous studies have demonstrated the regulation of the transcription of several immunoregulatory cell genes by the HCV core, the inventors tested whether the HCV core protein could influence the expression of PPARα in the cell lines of human hepatocytes transfected. Using competitive RT-PCR, the inventors quantified the cellular concentration of PPARα mRNA in two clones independent of HepG2 stably expressing (clone N3 and N4) or not (clone SWX) the core protein of HCV. While similar concentrations of β-actin mRNA are observed in the two clones tested, a lower level of PPARα messenger RNA expression is observed in the transfected clones compared to the SWX clones. The average inhibition rates of the expression of PPARα is 90% for the clone N4 (FIG. 7a) and 23% for the clone N3 (FIG. 7b) compared with the corresponding control SWX cells.

Thus, these results suggest that the abnormal expression of PPARα mRNA observed during chronic hepatitis C may be the result of a direct or indirect interaction between the body protein of HCV and PPARα. 10. Acute hepatitis induced by CC1 ₄ is improved by PPARγ agonists.

The inventors characterized the development of acute hepatitis in Balb / c mice subjected to treatment with CC1 ₄ whereas the control mice killed three days after the administration of mineral oil did not show microscopic lesions in the liver. Three days after the induction of hepatitis, the inventors evaluated the activation of PPARγ on hepatitis induced by CC1 ₄ using pioglitazone (50 μg / kg / day) as a preventive measure. Compared to mice without a preventive diet, the mortality rate linked to hepatitis is reduced (40% compared to 0%) in animals which received pioglitazone three days after treatment with CC1 ₄ .

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Claims

1. Use of a compound modulating the activity of a heterodimer RXR-nuclear receptor for the preparation of a medicament intended for the preventive and / or curative treatment of an infection by a virus which uses to carry out its viral cycle at least a cellular protein encoded by a gene which has at least one RE regulatory sequence.

2. Use according to claim 1 characterized in that said heterodimer RXR-nuclear receptor is selected from the group consisting of RXR-PPAR, RXR-LXR, RXR-TR, RXR-BAR / FXR and RXR-SXR / PXR.

3. Use according to claim 2 characterized in that said heterodimer is RXR-PPAR.

4. Use according to claims 1 to 3 characterized in that the said regulatory sequence RE is selected from the group composed of the regulatory sequence PPRE of response to PPAR, of the regulatory sequence LX-RE of response to LXR, of the regulatory sequence ERT of response of the thyroid hormone receptor (TR), of the regulatory sequence BAR / FXR-RE of response to BAR / FXR and of the regulatory sequence SXR / PXR-RE of response to SXR / PXR.

5. Use according to claim 4 characterized in that said RE regulatory sequence is PPRE.

6. Use according to claims 3 to 5 characterized in that said heterodimer RXR-nuclear receptor is RXR-PPAR and said regulatory sequence RE is PPRE.

7. Use according to claims 1 to 6 characterized in that said cellular protein is the receptor for low density lipoproteins (LDLR or CD36).

8. Use according to claims 1 to 7 characterized in that said compound activates or increases the activity of the heterodimer RXR-nuclear receptor.

9. Use according to claim 8, characterized in that said compound is selected from the group consisting of PPARα, PPARβ, PPARγ, PPARδ, RXR, a natural or synthetic agonist ligand of PPARα, PPARβ, PPARγ, PPARδ, RXR, RXR-PPARα, RXR-PPARβ, RXR-PPARγ, RXR-PPARδ.

10. Use according to claim 9 characterized in that the said agonist ligand of RXR- PPARα, RXR-PPARβ, RXR-PPARγ, RXR-PPARδ is chosen from the group consisting of prostaglandins J2, polyunsaturated fatty acids, thiazolinediones, non-steroidal anti-inflammatory drugs, fibrates, pioglitazone.

11. Use according to claims 1 to 7 characterized in that the said compound abolishes or inhibits the activity of the heterodimer RXR-nuclear receptor.

12. Use according to claims 1 to 11 characterized in that the said virus is the hepatitis C virus.

13. Use according to claim 12 for the preparation of a medicament intended for the preventive and / or curative treatment of an infection with the hepatitis C virus of cells selected from hepatocytes, biliary cells, parenchymal liver cells , the circulating blood cells.

14. Use according to claims 1 to 13 for the preparation of a medicament intended for the preventive and / or curative treatment of chronic hepatitis.

15. Use according to claims 1 to 13 for the preparation of a medicament intended for the preventive and / or curative treatment of steatosis hepatic or not associated with an infection of the hepatocytes by the hepatitis C virus.

16. Use of a compound according to claims 1 to 13 for the preparation of a medicament intended for the preventive and / or curative treatment of hepatic inflammation and hepatic lesions associated or not with an infection of the hepatocytes by the l virus 'Hepatitis C.

17. Use of a compound according to claims 1 to 13 for the preparation of a medicament intended for the preventive and / or curative treatment of hepatic cirrhosis and / or of post-hepatic cancer associated or not with an infection of hepatocytes by the hepatitis C virus.

18. Use of a compound according to claim 1 to 13 for the preparation of a medicament intended for the control of carbohydrate metabolism and / or for the preventive and / or curative treatment of type II diabetes in patients carrying the virus Hepatitis C.

19. Pharmaceutical composition for the preventive and / or curative treatment of an infection with the hepatitis C virus, characterized in that it contains a therapeutically effective amount of a compound as defined in one of claims 9 or 10.

20. Composition according to claim 19 characterized in that it also contains at least one antiviral agent as a combination product for simultaneous, separate or spread over time in antiviral therapy linked to an infection with the hepatitis virus vs.

21. Pharmaceutical composition according to claim 20, characterized in that said antiviral agent is selected from the group composed of interferon alpha (IFNα), ribavirin, retarding interferon.