WO2004046732A1 - Methods for screening compounds modulating cholesterol flux and uses thereof - Google Patents

Abstract

The invention concerns methods for selecting, identifying or characterizing compounds capable of modulating cholesterol flux which consist in determining modulation of adipophilin activity. The invention also concerns methods for preparing medicines.

Description

 METHODS FOR SCREENING OF COMPOUNDS

MODULATING CHOLESTEROL FLOW AND USES.

The present invention relates to methods for identifying, selecting or characterizing biologically active compounds, in particular compounds active on the vascular system, said methods being based on the modulation of the activity of adipophilin through cells or membrane preparations . The methods of the invention can include in vitro binding tests, binding tests in the cellular system (or on natural or synthetic membrane preparations) or functional tests in the cellular or artificial system. It relates in particular to a method of selection, identification or characterization of compounds capable of modulating the flow of cholesterol comprising bringing into vitro or ex vivo contact, a test compound with a foam cell or a natural or synthetic membrane preparation expressing l adipophilin, and the demonstration of a modulation of the activity of said adipophilin. It also relates to the uses of the compounds thus selected, identified or characterized.

Atherosclerosis is a multifactorial disease of the vessels, of slow evolution, during which numerous genetic and / or environmental risk factors act to favor the development of lesions. It corresponds to a chronic inflammatory reaction of the arterial wall, transformed into an acute clinical event on the occasion of the rupture of an atheroma plaque and the formation of an occlusive thrombus. Atherosclerosis is a major cause of morbidity, mortality, myocardial infarction, cerebral ischemia, cardiovascular disease and peripheral vascularization. Hypercholesterolemia and cholesterol overload of macrophages, involved in vascular inflammation, are major factors that contribute to atherosclerosis. Hypercholesterolemia is currently treated through the combination of diet and drug intervention with, for example, bile acid sequestering agents or statins. The development of new therapeutic strategies is however necessary to overcome the limits of existing therapies.

The reverse transport of cholesterol, implemented by HDLs ("High Density Lipoproteins" or Lipoproteins of High Density), makes it possible to discharge the cholesterol which accumulates in peripheral tissues and ensures its metabolic elimination via the liver. It thus contributes to the protection of the organism against atherosclerosis. Apolipoprotein A-1 is a fundamental constituent of HDL, responsible for their effectiveness. In this regard, the increased expression of apoA-1 has a protective effect against atherosclerosis.

Atherosclerosis is initiated by an infiltration, through the endothelium, of LDL (“Low Density Lipoproteins” or Lipoproteins of Low Density). LDL particles bind to glycosaminoglycans and, once trapped in the extracellular matrix of the sub-endothelium, accumulate in certain preferred sites. In this wall microenvironment, the particles begin to undergo oxidative changes. There is also a massive infiltration by the circulating monocytes of the intima of the artery to which they adhere to the level of the extracellular matrix. They then differentiate into mature macrophages. These cells, thanks to specific receptors (“scavengers” receptors) capture and degrade the modified LDL accumulated in the intimal space of the artery.

Macrophages are present at all stages of atherogenesis. Their participation is predominant in the atheromatous process (as opposed to sclerosis) since they participate in the constitution of the lipido-necrotic nucleus, determining for the vulnerability of the plaque. The importance of macrophagic infiltration seems to be directly correlated to the instability and fragility of the plaque. The functions of macrophages are manifold: presentation of antigens to T lymphocytes, purification of debris and toxic materials, secretion of an extraordinary variety of enzymes, cytokines and growth factors.

In animal models, macrophages are on the front line in the event of "lipid aggression". The first arterial response to dietary hypercholesterolemia, observed within a few days, is a massive adhesion of blood monocytes to the endothelium. Monocytes normally provide regular cleaning of the intima. Become macrophages "scavengers", they rid the subendothelial area of molecular debris (especially lipoproteins) and eventually return to the blood to be captured by the spleen, ganglia or liver. If the burden of purification imposed on them becomes excessive, they accumulate, transform into frothy macrophages, precursors of atheroma plaques. In fact, macrophages and foam cells trapped in the intima are active cells secreting multiple substances which modify the cellular and extracellular components of the wall. This is how a physiological phenomenon of purification is transformed into a local pathological process. The participation of monocyte-macrophages in the development of atherosclerosis is broken down into three major stages: (a) penetration and activation in the intima, (b) interactions with smooth muscle cells and T lymphocytes, and (c) transformation into foam cells.

The activated endothelial cells (EC) produce chemo-attractants which recruit the circulating monocytes, migrating by diapedesis between the endothelial junctions. The main chemotactic factors for monocytes are: "Monocyte Chemotactic Peptide-1" (MCP-1), "Colony Stimulating Factors" (CSFs), "Transforming Growth Factor-β" (TGF-β), and LDL oxidized. The latter in particular induce the synthesis, by the endothelium, of adhesion glycoproteins such as Plntercellular Adhesion Molecule-1 "(ICAM) or the" Vascular Cell Adhesion Molecule-1 "(VCAM-1) which cause the adhesion of monocytes and are abundantly expressed in human atherosclerotic plaques.The expression by endothelial cells of these adhesion molecules is increased by cytokines (IL-1 and TNF-α).

The activation of monocytes exposed to modified lipoproteins, cytokines, chemo-attractive molecules and growth factors, follows their penetration into arterial tissue. It is characterized by the acquisition of an ability to synthesize and express other factors which in turn will stimulate the activity of surrounding cells.

A priori, the capacities of phagocytosis, purification of lipids and unwanted debris, and finally the possibility of returning to the circulation in order to be purified by the liver, give macrophages a protective role. However, the macrophages synthesize multiple proteases, growth factors, active oxygen species, peroxide lipids, chemotactic factors that can be toxic to other cell types and allow them to play an important role in the development of atherosclerosis .

All cells in the body self-regulate their cholesterol needs thanks to the LDL receptors expressed on their cell membranes and finely back-regulated. They are therefore naturally sheltered from an overload of cholesterol. Similarly, macrophages in the presence of native LDL in vitro in high concentration do not transform into foam cells. Macrophages therefore have other receptors, the scavenger receptors, capable of picking up modified LDL (glycosylated or oxidized LDL) and thus accumulating enormous quantities of lipids. The oxidation phenomenon plays the central pathological role within the plaque. All cell types in the arterial wall can oxidize LDL.

The transformation of macrophages into foam cells can be accompanied by significant modifications, for example shifting the balance towards pro-coagulants in the atheroma plaque. In fact, the intracellular accumulation of free and esterified cholesterol induces stimulation of the expression of tissue factor, one of the main molecules initiating the extrinsic pathway of coagulation.

The transition from silent to symptomatic disease is almost always the consequence of a rupture of the plaque, even if, conversely, many ruptures have no clinical expression and simply participate - after incorporation of the thrombus and healing - to the mechanism of plaque growth. The richness in plaque macrophages is a marker for unstable plaques: the coronary plaques of patients with unstable angina contain more macrophages than those of stable coronary patients. In fact, 20% of plaques are responsible for 80% of symptoms.

The concordant results of large secondary prevention studies have shown that the incidence of clinical events decreases very rapidly under treatment with lipid-lowering drugs (in a few months) despite a very modest anatomical regression of the lesions (estimated by angiography or by ultrasound in mode B). Lipid-lowering treatments therefore have other mechanisms of action, in particular the stabilization of plaques making them less vulnerable but not necessarily less bulky. Vulnerability is not a permanent feature of plaque: it depends on its composition much more than its size, and is therefore likely to change faster than the overall volume of the plaque. The components associated with vulnerability (lipids and macrophages) are likely to regress faster than the components associated with stability (collagens and cholesterol crystals).

Human adipophilin is a protein of around 50 kDa (437 amino acids) which is defined as a marker for lipid accumulation in cells. It was first called ADRP (for adipose differentiation-related protein) because its counterpart in mice was isolated from adipocytes in 1992 by Jiang et al. (Jiang and Serrera, 1992). Indeed, its messenger RNA is over-expressed during the differentiation of adipocytes. It has therefore been mainly described in adipocytes.

Human adipophilin has 83.5% identity with mouse ADRP, as well as 12 additional amino acids (Heid et al., 1998). The N-terminal region of adipophilin has homologies with other proteins such as perilipins A / B and TIP47 (Londos et al., 1999). They form a family of proteins associating with lipid particles. Adipophilin, TIP47 and perilipins co-localize in the lipid droplets (Miura et al., 2002). The binding of these proteins is hydrophobic (Wolins et al., 2001). Adipophilin does not have an N-terminal signal sequence and does not appear to be glycosylated, but the presence of two forms having an isoelectric point of 7.0 and 7.2 suggests post-translational modifications. It would fix fatty acids thus increasing its hydrophobicity and its ability to fix lipid particles. Finally, the microsomal form of adipophilin is approximately 5 kDa smaller than the form associated with lipid particles (Heid et al., 1996; Mather, 2000). Adipophilin and perilipins cover the surface of the droplets to protect lipids from hydrolysis by lipases (Brasaemle et al., 2000; Shen et al., 1999; van Meer, 2001). However, adipophilin is not detected in cells that express perilipins. Adipophilin is found on the surface of small lipid droplets of preadipocytes and adipocytes during differentiation but is absent from adipocytes at the end of maturation. Conversely, perilipins are absent at the start of adipocyte differentiation, but are found on small and large droplets at the end of maturation ((Brasaemle et al., 1997). The distribution of perilipins is strictly limited to adipocytes and steroid cells, this transition is not observed in other cell types. Adipophilin, although present in smaller quantities than in adipose tissue, has been detected in many cell lines in culture (epithelial, endothelial, hepatic cells HepG2, fibroblasts, monocytes, macrophages) and tissues (liver, testes, lung, kidney, heart, mammary epithelium, adrenal cortex) where it is associated with lipid particles (Brasaemle et al., 1997; Heid et al., 1998).

This ubiquitous distribution of adipophilin therefore implies an essential role in the metabolism and storage of lipids. In addition, adipophilin is one of the rare proteins associated with lipid particles identified in cells capable of storing lipids. It could be involved in the formation and stabilization of lipid droplets. Adipophilin has thus been shown to promote the uptake of long carbon chain fatty acids in COS-7 cells and that these fatty acids stimulate the expression of its messenger RNA in adipocyte precursors (Gao and Serrera, 1999; Gao et al., 2000; Serrera et al., 2000).

Other recent studies have shown that adipophilin is induced in macrophagic foam cells (Wang et al., 1999). In addition, its messenger RNA is strongly expressed at the level of the atheroma plaque in regions enriched in macrophages and modified low density lipoproteins (LDL) (Shiffman et al., 2000; Wang et al., 1999). However, oxidized LDLox or acetylated LDLac induce both the expression of adipophilin and the transformation of macrophages into foam cells. This induction could result from activation the nuclear hormone receptor PPARγ (peroxisome proliferator-activated receptor γ) which regulates the expression of genes involved, among other things, in lipid metabolism and in the differentiation of monocytes into macrophages (Buechler et al., 2001). PPARγ and δ induce, for example, the expression of “scavengers” receptors at the level of macrophages, which facilitates the fixation of modified LDLs which are then taken up in excess by macrophages. However, among this family of nuclear receptors, it seems that only PPARδ is specifically involved in the accumulation of lipids in human macrophages and THP-1 cells (cells derived from a line of human monocytes) as well as in the control expression of adipophilin (Patel et al., 2002; Vosper et al., 2001).

In macrophages that have not accumulated lipids, adipophilin is present in small quantities. Adipophilin is upregulated during lipid accumulation and negatively during lipolysis. The expression of adipophilin is therefore linked to the metabolism of lipid particles which is regulated by the activity of protein kinase C (PKC) (Chen et al., 2001). However, it has been shown in murines that PKC is not biochemically associated with ADRP and that the activation of PKC by Phorbol 12-Myristate 13-Acetate (PMA) or its inhibition by calphostin C n have no effect on the distribution of ADRP on the surface of lipid droplets (Chen et al., 2002; Fong et al., 2002).

In 1998, Ye H. and Serrero G. showed that expression of ADRP is stimulated by inhibitors of cyclooxygenase in the adipocytes of mice (Ye and Serrero, 1998). However, cyclooxygenase inhibitors are also activators of PPARγ. It may therefore be that the observed effect is not direct, but modulated by PPARγ or that the inhibitors act at the transcriptional level. Since other PPARγ agonists are not good stimulators of ADRP expression and no element of response to PPAR (PPRE) could be identified on the ADRP promoter or adipophilin, PPARγ does not seem to be involved. The mechanism therefore remains to be elucidated. This article shows that ibuprofen and indomethacin increase the expression of ADRP in the adipocytes of mice, but the authors do not study the effect of ADRP and are content to study the regulation of its gene, which is nevertheless a potential target for cyclooxygenase inhibitors.

The regulation of the gene encoding adipophilin was further studied in 1999 by Wang et al. They noted that the amount of adipophilin mRNA was increased in macrophages stimulated by oxidized LDL, but not by native LDL, suggesting a potential role of this protein in the formation of foam cells and in atherogenesis. . They also showed that pro-inflammatory factors did not induce the expression of adipophilin. Once again, the authors suggest an indirect role for LDLox, the overexpression of adipophilin may be the consequence of stimulation of PPARγ. In this article, the authors expand on the regulation of adipophilin by LDLox and its localization at the level of the atheroma plaque but do not study its function in macrophages.

In a more recent article, Vosper et al. (Vosper et al., 2001) show that compound F (PPARδ agonist) induces the expression of receptors scavengers SR-A and CD36 (involved in the uptake of lipids) and negatively regulates genes such as apoE and cyp27 (involved in lipid efflux), which contributes to the accumulation of lipids. On the other hand, the treatment of macrophages with compound F or the overexpression of PPARδ also induces opposite effects on another lipid efflux pathway (independent of apoE and cyp27) by stimulating the expression of ABCAl (involved in the efflux of cholesterol dependent on apoA-1). The total efflux is however much reduced but this induction of ABCAl by the treatment with the agonist of PPARδ or the over-expression of PPARδ in macrophages results in an increase in the efflux of cholesterol dependent on apoA -l (see also in PNAS: (Oliver et al., 2001)).

The inventors investigated whether adipophilin plays a role in the metabolism and the regulation of the flows (influx and efflux) of lipids through these cells. They decided to observe the effect of increased adipophilin in macrophagic cells. For this, two techniques have been developed in parallel:

the use of an expression vector expressing adipophilin under the dependence of a strong promoter (CMV), and - the use of an adenovirus expressing adipophilin.

The effect of adipophilin overexpression in macrophages could be observed by techniques measuring the efflux of cholesterol in the presence of apoA-1, HDL and various inhibitors, such as acyl CoA inhibitors : cholesterol acyltransferase.

ApoA-l is synthesized in the liver and intestine. It is mainly found in HDL (High Density Lipoprotein) but also in small quantities in chylomicrons (Least dense Lipoproteins) and VLDL (Very Low Density Lipoprotein). ApoA-1 promotes the efflux of membrane cholesterol, but is also involved in maintaining the structure of HDL and in the activation of Lecithin Cholesterol Acyltransferase (LCAT). LCAT is found in HDL and realizes, in plasma, the esterification of free cholesterol into cholesterol esters, participating in the maturation of HDL.

Acyl CoA: cholesterol acyltransferase (ACAT) inhibitors prevent the re-esterification of cholesterol in the endoplasmic reticulum and therefore the deposition of lipids in lipid droplets. Indeed, after their uptake, the components of the modified LDL, including the cholesterol esters, are hydrolyzed in the lysosomes. The free cholesterol is then transferred to the endoplasmic reticulum where it is re-esterified into a cholesterol ester by ACAT and then accumulates in the cytosol where it forms the lipid droplets (Figure 1, (Brown and Goldstein, 1983).

The presence in the culture medium of cholesterol acceptors, such as apoA-1 or HDL, allows the efflux of cholesterol. However, the efflux of cholesterol is only possible if the pool of cholesterol ester stored in the lipid particles is hydrolyzed by a neutral hydrolase (Small et al., 1989). FIG. 1 represents the flow of cholesterol at the macrophage level and the techniques used to modulate the activity of adipophilin at the level of the THP-1 macrophage cells.

The inventors therefore transiently transfected macrophages with a plasmid overexpressing adipophilin. The results surprisingly show that if one acts selectively on adipophilin, the efflux of cholesterol dependent on apoA-1 is significantly reduced. This result can be explained by the fact that the quantity of adipophilin present in the cells is directly modulated while the agonists of PPARδ act on many other genes involved at the same time in absorption, transport, metabolism and l efflux of lipids. Although the article de Vosper et al. (Vosper et al., 2001) indicates that compound F increases the expression of adipophilin, none of their experiments demonstrates the implication of this protein in the retention or efflux of cholesterol.

SUMMARY OF THE INVENTION

The present invention is therefore based on the observation of the role of adipophilin in the modulation of the cholesterol efflux.

The present invention thus demonstrates for the first time a modulation of the efflux of cholesterol by adipophilin. The present invention thus provides new targets and new approaches for the search for compounds capable of regulating the efflux of lipids, and in particular the efflux of cholesterol (reverse transport of cholesterol).

The invention also provides methods for increasing the reverse transport of cholesterol based on the use of compounds which decrease or even inhibit the activity of adipophilin.

The invention also provides screening methods intended to identify therapeutic substances capable of modulating the efflux of cholesterol, in particular modulating the reverse transport of cholesterol, by determining in vitro or ex vivo the capacity of the substances tested to modulate the activity. adipophilin.

The present invention relates in particular to a method of selection, identification or characterization of compounds capable of modulating the efflux of cholesterol, in particular the efflux of cholesterol dependent on apoA-1, comprising contacting a test compound with adipophilin and determining the ability of said test compound to modify the activity of adipophilin.

According to a variant of the invention, bringing the test compound into contact with adipophilin consists in bringing the test compound into contact with a foam cell or a natural or synthetic membrane preparation expressing adipophilin. The invention further relates to a method for producing an active compound, in particular a compound capable of modulating the flow of cholesterol, and potentially active on the vascular system, comprising:

the determination of the ability of a compound to modulate the activity of adipophilin in vitro or ex vivo, and

- The synthesis of said compound or of a structural analog thereof.

It also relates to a process for producing a compound which modulates the flow of cholesterol, comprising: - bringing a compound into contact with a cell or a cell membrane expressing adipophilin,

the determination of the capacity of said compound to modulate the flow of cholesterol through said cell or said cell membrane, and

- The synthesis of said compound or of a structural analog thereof.

It further relates to a method for producing a medicament comprising an active compound, in particular on the vascular system, comprising:

- determining the ability of a compound to modulate the flow of cholesterol through foam cells or natural or synthetic membrane preparations expressing adipophilin in vitro or ex vivo, and

- Mixing said compound or a structural analog thereof with a pharmaceutically acceptable vehicle.

It also relates to a method for producing a medicament comprising a compound modulating the flow of cholesterol through foam cells expressing adipophilin, comprising:

- bringing a compound into contact with adipophilin,

- determining an effect of said compound on adipophilin, said effect indicating that the compound is a modulator of the cholesterol flow, and - mixing said compound or a structural analog thereof with an acceptable vehicle on the pharmaceutical plan. Finally, it relates to the use of a compound modulating the activity of adipophilin for the preparation of a medicament intended to modulate the flow of cholesterol and / or the cholesterol concentration of foam cells.

DETAILED DESCRIPTION OF THE INVENTION

Reverse cholesterol transport is a process that allows excess cellular cholesterol to exit into peripheral tissue. This is then esterified to be incorporated into HDL, which can then transfer it to low and very low density lipoproteins which will transport it to the liver where it will be eliminated or recycled. This process contributes to the maintenance of cellular cholesterol homeostasis. Since there is practically no exit of cholesterol esters, the efflux of cholesterol can be defined as the exit of free cholesterol molecules from inside the cell to the outside through the plasma membrane , either by passive diffusion thanks to a concentration gradient between the plasma membrane and acceptor particles containing phospholipids (for example, HDL), or by a second route involving lipoproteins poor in lipids (preβ1-HDL) and rich in apoA- l or finally by a route involving the SR-BI scavenger receptor. At the macrophage level, another efflux pathway results from the hydroxylation of sterols by sterol 27-hydroxylase and is dependent on the endogenous expression of apoE. Sterol 27-hydroxylase converts cholesterol to 27-hydroxycholesterol and to 3β-hydroxy-5-cholestenoic acid. In the absence of cholesterol acceptors in the medium, these oxysterols cross the lipophilic membranes faster than free cholesterol and are transported to the liver to be converted into bile acids (Babiker et al., 1997).

Passive scattering does not necessarily require contact between the cell and an acceptor particle, but can be modulated by the size and composition of the acceptor HDLs. Passive efflux can be increased by LCAT activity which maintains a cholesterol gradient between the cell surface and the acceptor particles.

The SR-BI dependent efflux allows passive diffusion of cholesterol towards HDL but would be limited to regions of the plasma membrane rich in cholesterol. Thus, when the cells placed in the presence of LDLac are loaded with cholesterol via the scavenger SR-A receptor, the intracellular cholesterol pool is not accessible to SR-BI.

The second efflux pathway, dependent on apoA-1, is linked to the activity of an “ATP-binding transporter” named ABCA1 (Owen, 1999). ABCA1 allows the efflux of cholesterol and phospholipids, whether located at the plasma membrane or in the intracellular pools, by facilitating their transfer between the internal and external sheets of cell membranes. An interaction between ABCA1 and apoA-1 is necessary to obtain the efflux of cholesterol and phospholipids. This cholesterol efflux mechanism is different from that dependent on SR-BI which preferentially passes through HDL.

The activity of ABCAl is inhibited by SR-BI which facilitates the return of cholesterol from nascent HDL to the cell. This competitive role of SR-BI would allow the maintenance of a minimal level of cholesterol in the plasma membranes when ABCA1 is active. In fact, macrophages loaded with cholesterol overexpress ABCA1 which leads to an increase in the efflux of cholesterol. Likewise, the expression of ABCAl in THP-1 macrophages can be stimulated by activators of the nuclear receptors PPARα and γ as well as by oxysterols. Conversely, when macrophages are not loaded with cholesterol and the expression of ABCAl is not increased, SR-BI would stimulate the efflux of cholesterol towards HDL (Chen et al., 2000). In summary, SR-BI is involved in the bidirectional flow of free cholesterol between cells and lipoproteins as a function of a concentration gradient, selectively pumps cholesterol esters from HDL, increases the cellular content of free cholesterol and acts on the membrane distribution of cholesterol pools (Bultel-Brienne et al., 2002; de la Llera-Moya et al., 1999; Jian et al., 1998).

The present invention therefore proposes to identify compounds modulating the reverse transport of cholesterol or the efflux of cholesterol and in particular compounds modulating the cholesterol efflux dependent on apoA-1 or HDL.

The invention therefore relates to methods for identifying, selecting or characterizing biologically active compounds, in particular compounds active on the vascular system, said methods being based on the modulation of the activity of adipophilin and / or of the cholesterol flow to through cells or preparations membrane. The methods of the invention can include in vitro binding tests, binding tests in the cellular system (or on membrane preparations, natural or synthetic) or functional tests in the cellular or artificial system.

The present invention relates to a method for selecting, identifying or characterizing compounds capable of modulating the flow of cholesterol comprising bringing into contact, optionally in the presence of cholesterol, in vitro or ex vivo, a test compound with a foam cell or a natural or synthetic membrane preparation expressing adipophilin, and the demonstration of a modulation of the activity of said adipophilin.

A particular method of selection, identification or characterization of compounds capable of modulating the flow of cholesterol comprises demonstrating a modulation of the activity of adipophilin, possibly in the presence of cholesterol, via the demonstration of a modulation of the cholesterol flow through a cell or membrane preparation expressing said adipophilin.

The determination of the modulation of adipophilin activity can be carried out by measuring the activity of adipophilin and comparing the activity with that obtained in the absence of test compound.

The demonstration of a modulation of the activity of adipophilin is preferably accomplished via the demonstration of the binding of the test compound to adipophilin.

By "adipophilin" is meant, in the context of the present invention, the complete protein or a fragment thereof, in particular a fragment retaining the capacity for association with lipids, in particular the capacity for binding to cholesterol.

Thus, the compounds identified, selected or characterized according to the methods of the invention are compounds modulating the efflux of cholesterol, in particular at the level of macrophages or foam cells. The cholesterol efflux corresponds more specifically to that dependent on apoA-1 as defined above. The binding of the test compound can be demonstrated in different ways, such as for example by gel migration or electrophoresis of the complexes formed. Other methods based on luminescence or using the FRET technique (Fluorescence Resonance Energy Transfer) well known to those skilled in the art or the SPA technique (“Scintillation Proximity Assay”), can be implemented, within the framework of the present invention, to determine the possible binding of the test compound to adipophilin. The binding of the test compound to adipophilin can also be demonstrated for example by using a labeled test compound, a ligand labeled with adipophilin or a labeled antibody specific for a synthetic enzyme or for a transporter thereof. , displacement of the binding of the labeled ligand reflecting the binding of the test compound.

The labeled ligand can be any product binding the target molecule (antibody, agonist or antagonist, fragment or derivative of an endogenous ligand, etc.). The ligand can be labeled by any technique known to those skilled in the art, in particular by incorporating a radioactive, fluorescent, luminescent, enzymatic, colorimetric element, etc.

Preferably, the demonstration of the binding of the test compound to adipophilin is carried out in the presence of a ligand labeled with adipophilin by determining the displacement of the binding of the labeled ligand.

In a particular embodiment, the ligand is a labeled antibody, specific for the molecular target considered. The antibody can be polyclonal or monoclonal. It can also be a fragment or an antibody derivative, such as for example Fab, F (ab ') ₂ , CDR fragments, etc. Antibodies can be produced in a conventional manner, by immunization against the molecular target (or an immunogenic part thereof), and recovery of serum (polyclonal) or of spleen cells (to make hybridomas by fusion with an appropriate line), as described for example in Vaitukaitis et al. (Vaitukaitis et al., 1971) or in Harlow et al. (Harlow and Lane, 1988). The Fab or F (ab ') ₂ fragments can be produced for example according to Riechmann et al. (Riechmann et al., 1988).

In another particular embodiment, the adipophilin ligand is an endogenous ligand, a labeled agonist or antagonist. In an implementation mode specific, marked cholesterol, in particular radiolabelled, in particular tritiated, is used.

According to a particular embodiment, the method therefore comprises bringing a test compound into contact with a cell (or a natural or synthetic membrane preparation) expressing adipophilin and demonstrating a bond of the test compound to said adipophilin.

According to another particular mode, the method comprises bringing a test compound into contact with a cell (or a natural or synthetic membrane preparation) expressing adipophilin, in the presence of a labeled ligand of said adipophilin, and setting up evidence of binding of the test compound by determining the displacement of the binding of the labeled ligand.

In another variant, the method of the invention comprises bringing a test compound into contact with a cell or a membrane preparation expressing adipophilin, and demonstrating a modulation of a characteristic biological or pharmacological effect of said adipophilin. The biological or pharmacological effect may be the expression of one or more cellular proteins or of the corresponding mRNAs, the expression of adipophilin or of adipophilin mRNA, the internalization of adipophilin, the activation of a gene, the appearance of an electric current, an efflux or an influx of ions, an efflux or an influx of cholesterol, phospholipids, triglycerides or fatty acids , etc. Demonstration of this effect can be carried out by any appropriate means, such as measuring the expression of a reporter gene, measuring the membrane expression of adipophilin, measuring total cholesterol ( free and esterified) and triglycerides, ions or an electric current, etc.

Conventionally, the effect of the test compound is compared to the effect determined in the absence of said compound. In addition, the effect of the test compound can be determined in the presence of cholesterol, in particular when an activity of modulation or inhibition of the activity of adipophilin is sought. The invention therefore comprises a method of selection, identification or characterization of compounds comprising bringing into vitro or ex vivo contact, a test compound with a cell or a natural or synthetic membrane preparation expressing adipophilin, measuring a biological or pharmacological effect characteristic of adipophilin and the comparison of the effect measured with that obtained in the absence of test compound.

According to a particular variant, the invention comprises a method of selection, identification or characterization of compounds comprising bringing into contact, optionally in the presence of cholesterol, in vitro or ex vivo, a test compound with a foam cell or a membrane preparation natural or synthetic expressing adipophilin, measuring the flow of said cholesterol through said cell or membrane preparation and comparing the measured flow to that obtained in the absence of test compound.

The cells used in the tests can be any cell expressing a target molecule involved in the activity of adipophilin, in particular adipophilin. They can be cells naturally expressing this molecule, or cells genetically modified or treated to overexpress said molecule. It is, in a preferred embodiment of the invention, endothelial cells, endothelial cells HUVEC, epithelial cells, hepatic HepG2, fibroblasts, monocytes, mammalian macrophages. Even more preferably, these cells are human cells. It can also be primary cultures or established lines (THP-1 cells differentiated into macrophages by PMA). In another embodiment, it is also possible to use prokaryotic cells (bacteria), yeast cells (Saccharomyces, Kluyveromyces, etc.), etc. These cells can be isolated, cultured and characterized according to known techniques, in particular those described in the examples.

The discovery of the role of adipophilin in the modulation of cholesterol flows allows, according to the present invention, to highlight new biologically active compounds, capable of modulating the cholesterol flow. The demonstration of the pharmacological role of adipophilin underlines the importance of the availability of such active compounds. In a particular embodiment, for the implementation of the above methods, a membrane preparation expressing adipophilin is used. The membrane preparation can advantageously be of natural origin, that is to say produced from a cell expressing adipophilin. Membrane preparations can be produced by mechanical, chemical, physical, electrical lysis, etc., and in particular by treatment with detergents, ultrasound, freezing / thawing, etc. This membrane preparation is essentially characterized by the presence of membrane fragments, comprising a lipid bilayer in which all or part of the adipophilin is present. These membrane preparations are generally devoid of intact cells. In addition, they can be enriched in membrane debris by appropriate treatments (centrifugation, etc.). It is also possible to use a membrane preparation of synthetic origin, such as for example a liposome into which all or part of the adipophilin has been introduced, or a supported membrane.

For the binding tests or the functional tests mentioned above, the compounds can be brought into contact with the cells (or membrane preparations) at different times, according to their effect (s), their concentration or the nature of the cells. and for various periods, which can be adjusted by a person skilled in the art. The test can be carried out on any suitable support and in particular on a plate, a blade, a box, in a tube or a flange. Generally, the contacting is carried out in a multi-well plate, which makes it possible to conduct, in parallel, numerous and varied tests. Among the typical supports, one finds microtitration plates and more particularly 96 or 384 well plates (or more), easy to handle.

Depending on the support and the nature of the test compound, variable amounts of cells can be used when implementing the methods described. Conventionally, 10 ² to 2 * 10 ⁶ cells are brought into contact with a type of test compound, in an appropriate culture medium, and preferably between 10 ³ and 10 ⁵ cells. When a membrane preparation is used, generally 0.01 to 50 mg of protein is applied per test, more preferably 0.05 to 2 mg of protein per test. The tests can be carried out in any type of environment suitable, such as saline solutions, swabs, etc. Mention may in particular be made of Tris, Pipes, Hepes buffers, etc. The temperature is typically close to room temperature. The pH of the medium is advantageously between 5.5 and 8, more preferably between 7 and 8. It is understood that these parameters can be adjusted by a person skilled in the art, according to the indications provided in the examples.

The amount (or concentration) of test compound can also be adjusted by the user according to the type of compound (its toxicity, its cell penetration capacity, etc.), the length of the incubation period, etc. Generally, cells (or membranes) are exposed to amounts of test compounds which vary from 1 nM to 1 mM. It is of course possible to test other concentrations without deviating from the present invention. Each compound can, moreover, be tested, in parallel, at different concentrations and over different periods. Furthermore, adjuvants and / or vectors and / or products facilitating the penetration of the compounds into cells such as liposomes, cationic lipids, polymers, peptides derived from viruses, etc., can also be used, if necessary. Contact can be maintained for a period of between 1 hour and several hours (72 hours). In the case of transfection, contact is typically maintained from 4 hours to 48 hours. In the case of an infection, contact is typically maintained from 4 hours to 24 hours.

The present invention can be applied to any type of test compound. Thus, the test compound can be any product which is in an isolated form or in admixture with other products. The compound may be defined in terms of structure and / or composition or may not be defined. The compound can, for example, be an isolated and structurally defined product, an isolated product of indefinite structure, a mixture of known and characterized products or an indefinite composition comprising one or more products. Such indefinite compositions can be, for example, tissue samples, biological fluids, cell supernatants, plant preparations, etc.

The test compounds capable of modulating the activity of adipophilin according to the invention can be of varied nature and origin. The test compounds can be inorganic or organic products and in particular a polypeptide (or a protein or a peptide), a nucleic acid, more particularly an antisense nucleic acid or an RNAi (RNA interference), a lipid, a polysaccharide, a chemical or biological compound such as a nuclear factor, a cofactor or any mixture or derivative thereof. The compound can be of natural or synthetic origin and include a combinatorial library, a clone or a library of nucleic acid clones expressing one or more DNA-binding polypeptide (s), etc.

In this context, RNAi type compounds have been identified and synthesized. RNAi and the use of siRNAs (small RNA interference or small RNA interference) in mammalian cells are described in particular in the publication by Brummelkamp et al. (Brummelkamp et al., 2002).

The RNAi of the present invention are adipophilin specific RNAi. They inhibit the activity, in particular the expression, of adipophilin by binding to a target sequence of the mRNA of adipophilin. This target sequence can be located in the coding regions or not of the adipophilin mRNA, the non-coding regions can be located for example in 5 ′ or 3 ′ UTR sequences. The RNAi of the present invention are more specifically siRNA and are advantageously used in double-stranded form (antisense matrix and sense matrix).

The present invention therefore further relates to RNA interference from adipophilin. In particular, the interference RNAs are constructed from the target sequences on the adipophilin mRNA, said target sequences on the adipophilin mRNA are chosen from the following sequences:

- in the 5 'UTR:

5'- AATGGCATCCGTTGCAGTTGA -3 '(SEQ ID 1),

- in the coding sequence:

5'- AAGCTAGAGCCGCAAATTGCA -3 '(SEQ ID 2) 5'- AATTGCCCGCAACCTGACTCA -3' (SEQ ID 3)

- in the 3'UTR:

5'- AAAAGGCGTCTTCACTGCTTT -3 '(SEQ ID 4) Preferably, the siRNAs comprise an antisense matrix and a sense matrix, said antisense matrix comprising at least one sequence chosen from the sequences SEQ ID 1, 2, 3 and 4.

Advantageously, the antisense matrices and the sense matrices of siRNAs comprise at least 21 bases, preferably have a length of 29 bases.

Advantageously, the siRNAs are chosen from the RNAs of the following sequences:

- RNAsil -adipophilin: Antisense matrix:

5'- AATGGCATCCGTTGCAGTTGACCTGTCTC -3 '(SEQ ID 5)

Meaning matrix:

5'- AATCAACTGCAACGGATGCCACCTGTCTC -3 '(SEQ ID 6)

- siR2-adipophilin

Antisense matrix:

5'- AAGCTAGAGCCGCAAATTGCACCTGTCTC -3 '(SEQ ID 7)

Meaning matrix:

5'- AATGCAATTTGCGGCTCTAGCCCTGTCTC -3 '(SEQ ID 8)

- siRNA-adipophilin Matrix antisense:

5'- AATTGCCCGCAACCTGACTCACCTGTCTC -3 '(SEQ ID 9) Meaning matrix: 5'- AATGAGTCAGGTTGCGGGCAACCTGTCTC -3' (SEQ ID 10)

- RNAsi4-adipophilin Matrix antisense:

5 '. AAAAGGCGTCTTCACTGCTTTCCTGTCTC -3 "(SEQ ID 11) Meaning matrix:

5'- AAAAAGCAGTGAAGACGCCTTCCTGTCTC -3 "(SEQ ID 12)

The siRNAs of the present invention which inhibit the expression of adipophilin modulate, and in particular increase, the efflux of cholesterol. As such, they can be used as a medicament, in particular for the preventive or curative treatment of atherosclerosis, hypercholesterolemia or cholesterol overload of macrophages. They can also preventively or curatively treat myocardial infarction, cerebral ischemia, cardiovascular diseases or pathologies of peripheral vascularization.

In general, another subject of the invention therefore relates to a process for the production of an active compound, in particular a compound capable of modulating the flow of cholesterol, and potentially active on the vascular system, comprising: - the determination of the ability of a compound to modulate the activity of adipophilin, in particular to modulate the expression of adipophilin, in vitro or ex vivo, and

- The synthesis of said compound or of a structural analog thereof.

The invention also comprises a method for producing a medicament comprising a compound modulating the flow of cholesterol through foam cells expressing adipophilin, comprising:

- bringing a compound into contact with adipophilin,

- determining an effect of said compound on adipophilin, said effect indicating that the compound is a modulator of the cholesterol flow, and - mixing said compound or a structural analog thereof with an acceptable vehicle on the pharmaceutical plan.

Another subject of the invention relates to a process for producing a compound which modulates the cholesterol flow, comprising: - bringing a compound into contact with a cell or a cell membrane expressing adipophilin,

- the determination of the cholesterol flow through said cell or said cell membrane, the modulation of said flow indicating that the compound is a modulator of the cholesterol flow, and - the synthesis of said compound or of a structural analog thereof.

Another subject of the invention relates to a method for producing a medicament comprising an active compound, in particular on the vascular system, comprising: - determining the ability of a compound to modulate the flow of cholesterol through foam cells or natural or synthetic membrane preparations expressing adipophilin in vitro or ex vivo, and

- Mixing said compound or a structural analog thereof with a pharmaceutically acceptable vehicle.

The adipophilin RNAi defined above were produced according to the methods defined above.

In the context of the invention, the term “structural analog” designates any molecule obtained by molecular modeling or structural variation from a test compound.

The effect of the test compound preferentially measured is the ability of said compound to modulate the activity (i.e., action or expression) of adipophilin.

A particularly advantageous use of a compound modulating the activity of adipophilin consists in preparing a medicament intended to modulate the flow of cholesterol and the cholesterol concentration of the foam cells. Preferably, the compound decreases the activity of adipophilin to increase the efflux of cholesterol and decrease the cholesterol concentration of the foam cells.

Preferably, the drug thus prepared is intended to limit the accumulation of lipids and / or the formation of foam cells and thus to treat atherosclerosis by limiting the formation of atheroma plaque.

The compound modulating the activity of adipophilin is advantageously chosen from an siRNA as defined above.

The compounds, compositions or medicaments according to the invention can be administered in different ways and in different forms. Thus, they can be injected by the systemic or oral route, preferably systemic, such as for example by the intravenous, intramuscular, subcutaneous, trans-dermal, intra-arterial, intra-cerebral, intra-peritoneal, intra-cerebrovascular route. , etc. For injections, the Compounds are usually packaged as liquid suspensions, which can be injected using syringes or infusions, for example. In this regard, the compounds are generally dissolved in saline, physiological, isotonic, buffered solutions, etc., compatible with pharmaceutical use and known to those skilled in the art. Thus, the compositions can contain one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or vehicles which can be used in liquid and / or injectable formulations are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc. The compounds can also be administered in the form of gels, oils, tablets, suppositories, powders, capsules, capsules, etc., optionally by means of dosage forms or of devices ensuring sustained and / or delayed release. For this type of formulation, an agent such as cellulose, carbonates or starches is advantageously used.

It is understood that the flow rate and / or the dose injected can be adapted by a person skilled in the art depending on the patient, the pathology, the mode of administration, etc. Typically, the compounds are administered in doses which can vary between 0.1 μg and 1000 mg / kg of body weight, more generally from 0.01 to 500 mg / kg, typically between 1 and 200 mg / kg. In addition, repeated injections can be given, if necessary. On the other hand, for chronic treatments, delayed or prolonged systems can be advantageous.

The preferred doses for achieving a decrease in atherosclerosis in animals are doses less than 50 mg / kg. A significant effect is advantageously obtained in animals at doses between 100 and 200 mg / kg

The invention also includes tools and kits for implementing these methods.

The feasibility, implementation and other advantages of the invention are illustrated in more detail in the examples which follow, which should be considered as illustrative and not limiting. LEGENDS OF FIGURES

Figure 1: Cholesterol flow at the macrophage level and techniques used to modulate the activity of adipophilin at the level of THP-1 macrophage cells. ACAT, acyl CoA: cholesterol acyltransferase; CL, Free Cholesterol; EC,

Esterified Cholesterol; CEHn, Cholesteryl ester hydrolase neutral; LDLac, acetylated LDL; SR, Receivers scavengers.

Figure 2: Analysis by (A) Western blot of adipophilin expression after transfection of THP-1 macrophages with Effectene and (B) Northern blot of adipophilin mRNA expression after transfection of macrophages

THP-1 by jetPEI-Man.

Figure 3: Accumulation of triglycerides and cholesterol in macrophages

THP-1 after loading with acetylated LDL for 48 hours.

Figure 4: Kinetics of the efflux of ApoA-1 dependent cholesterol in THP-1 macrophages after transfection, expression of the vector pCI-adipophilin then loading with the acetylated LDL tritiated for 48 h (C: control without ApoA-

I) -

Figure 5: Analysis by (A) Western Blot of the expression of adipophilin after infection of THP-1 macrophages by adenoviruses control (ad-CMV-GFP) or overexpressing adipophilin (ad-CMV-adipophilin) and ( B) RT-PCR of the expression of the mRNA of adipophilin after infection of the THP-1 macrophages by the adenoviruses control (ad-CMV-GFP) or overexpressing adipophilin (ad-CMV-adipophilin).

Figure 6: Accumulation of triglycerides and cholesterol in THP-1 macrophages infected with control adenoviruses (ad-CMV-GFP) or overexpressing adipophilin (ad-CMV-adipophilin), after loading with acetylated LDL for 48 hours.

Figure 7: Kinetics of cholesterol efflux dependent on ApoA-1 or HDL in THP-1 macrophages after infection, expression of adenoviruses then loading with tritiated acetylated LDL for 48 h.

Figure 8: Analysis by Western Blot of the inhibition of the expression of adipophilin by siRNA3-adipophilin (siRNA-adip) (transfection of macrophages THP-1 with jetSI) in the presence or absence of acetylated LDL. Figure 9: Accumulation of triglycerides and cholesterol in macrophages

THP-1 transfected with siRNA, after loading with acetylated LDL for

48h.

Figure 10: Kinetics of ApoA-1 dependent cholesterol efflux in THP-1 macrophages after transfection with siRNA, inhibition of adipophilin expression then loading with acetylated tritiated LDL for 48 h.

EXAMPLES

Cellular culture

The macrophages used come from a continuous line (THP-1) of human monocytes. The base medium corresponds to RPMI 1640 medium (Gibco) containing, 4 mM L-glutamine, 20 IU / ml of penicillin and 20 μg / ml of streptomycin. The cells in suspension multiply at 37 ° C in 5% CO ₂ in base medium containing 10% of decomplemented fetal calf serum (complete medium). The differentiation of monocytes into macrophage is obtained by depositing 2.10 ⁶ cells / well (6-well dishes) in complete medium in the presence of 1.6.10 ⁷ M of PMA (Phorbol 12-Myristate 13-acetate) for 72 hours. The differentiated THP-1s adhere to the support.

Macrophage transfection

Macrophages (0.7.10 ^e to 2.10 ⁶ cells / well, 6-well dishes) are transiently transfected with 1 to 3 μg of plasmid using Effectene ™ (Qiagen) or jetPEI ™ -Man (Qbiogene) and / or per 2 to 3 μg of siRNA using jetSI ™

(Qbiogene) as transfection products. Macrophages are incubated from 24 to

72 h then analyzed.

Macrophage infection Macrophages (0.7.10 ^e to 2.10 ^e cells / well, 6-well dishes) are infected with 10 to 2000 pfu (plaque-forming units) of adenovirus containing either the cDNA encoding adipophilin (ad- CMV-adipophilin) or a control adenovirus containing the sequence coding for GFP ("green fluorescent protein") (ad-CMV-GFP) for 4 to 24 h. Plasmid constructs

It was chosen to clone the cDNA of human adipophilin into the expression vector pCI (Promega) under the control of a ubiquitous promoter CMV. After amplification of the coding sequence from macrophagic mRNA by RT-PCR, this vector was sequenced and named pCI-adipophilin. Transfection of THP-1 differentiated into macrophages by PMA was then carried out. The efficiency of the transfection was studied by carrying out co-transfections with a reporter plasmid pEGFP-C3 making it possible to visualize the autofluorescent protein GFP ("green fluorescent protein").

SiRNA design: selection of targets on mRNA

Each AA dinucleotide sequence is scanned along the adipophilin mRNA (coding and non-coding regions). The next 19 nucleotides, adjacent to these two AA nucleotides, are selected as potential targets. Over a hundred sequences were selected, then compared (notably using www.ncbi.nlm.nih.gov/BLAST/) with human genome banks. All the target sequences, selected on the adipophilin mRNA, having significant homology with other coding sequences or a percentage of GC nucleotides not between 35 and 60% were eliminated.

After this selection step, four sequences of 21 nucleotides (see description of the sequences) were retained for the production of siRNA in order to inhibit the expression of adipophilin.

SiRNA in vitro production

The siRNAs are produced with the “Silencer ™ siRNA Construction Kit” (Ambion) according to the supplier's instructions.

Protein extraction and Western Blot analysis Total proteins are extracted from cells grown in 35 mm dishes. The cells are washed three times with PBS (phosphate buffered saline), lysed (Triton X100 1%, Deoxycholate 0.5%, 10 mM Na pyruvate phosphate, 2 mM Na vanadate, NaF 100 mM, Aprotinin, PMSF 0.5 mM, ICN inhibitor, PBS) and then centrifuged for 30 min at 10,000 g, 4 ° C. The protein concentration is determined by Peterson's method (Peterson, 1977). The samples (20 μg of protein) are separated by SDS-PAGE (10% acrylamide) according to the Laemmli method ((Laemmli, 1970)), using the Mini Protean 3 tank system (Bio-Rad). The proteins are then transferred to a nitrocellulose membrane by electrotransfer (10 mA / cm ² ) (Amersham). The membranes are saturated in basic buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween 20) containing 5% milk for 1 h at room temperature. They are incubated with a first antibody directed against the protein of interest, washed again with basic buffer and then incubated with a second antibody directed against the first antibody. After washing, the proteins of interest are revealed by chemiluminescence (ECL, Amersham), then quantified by densitometric analysis. The expression of the β-actin protein being constant under our experimental conditions, the inventors used this protein to normalize the differences between each sample.

RNA extraction, Northern Blot analysis and RT-PCR

The total RNAs were extracted according to the indications in the RNeasy Mini or Midi kits (Qiagen). The RNA samples (15 μg / well) migrated 3 h at 100 V through a 1% formaldehyde-agarose gel and then were transferred to a nylon membrane (Pall Gelman Sciences). The DNA probes were labeled with [α- ³² P] dCTP (3000 Ci / mmol, EASYTIDES ™, PerkinElmer Life Sciences) using the random-priming DNA labeling kit (Boehringer, Mannheim). The pre-hybridization, the hybridization and the washings were carried out according to the conditions described above (Tontonoz et al., 1994). The dried membranes were then exposed on autoradiographic films (Kodak Biomax) at -80 ° C, the signal being quantified by densitometric analysis.

The expression of the GAPDH gene being constant under these experimental conditions, the inventors used it to normalize the differences between each sample.

Lipoprotein preparation

The isolation of LDL (density 1, 03-1, 063) was carried out from human plasmas by sequential ultracentrifugation as described previously (Havel et al., 1955). The isolated LDLs were sterilized by membrane filtration (0.22 μm) and stored at 4 ° C in phosphate buffered saline containing 10 mM ethylenediaminetetraacetate disodium dihydrate (PBS-EDTA).

Modification of LDL LDL has been chemically modified at the protein level by acetic anhydride. This compound reacts on lysine residues to form an amide bond. Acetylation increases the load of LDL. The acetylated LDLs were dialyzed for 24 hours, changing the medium regularly against PBS-EDTA. They were then filtered on a membrane (0.22 μm). The protein concentration was determined by the Peterson method (Peterson, 1977).

The LDLacs were labeled with ³ H by incubating 33.3 μCi (per mg of LDLac proteins) of tritiated free cholesterol (1α, 2α (n) - [ ³ H] cholesterol, Amersham) in the presence of ethanol, for 24 h at 37 ° C.

Measurement of triglycerides, cellular cholesterol and protein content

The amounts of total and free cholesterol were measured according to the conditions of the FC-test kit (Wako Pure Chemicals). The amount of esterified cholesterol was determined by calculating the difference between total cholesterol and free cholesterol. The amount of triglycerides was measured according to the conditions of the PAP 1000 Triglycerides kit (Biomérieux). The concentration of proteins extracted from the cells was determined by the method of Lowry (Lowry et al., 1951).

Cholesterol efflux

The THP-1 macrophages, transfected with the plasmid of interest, were loaded with lipids by incubation for 48 h with 100 μg / ml of LDLac-free cholesterol [ ³ H] in base medium containing 10% serum of fetal calf. The cells were then washed with PBS and then incubated for 24 h in basic medium containing 2 mg / ml of BSA. The efflux of cholesterol (or lipids) from the cells to the culture medium was carried out in base medium over a period of 24 h, by taking 120 μl of sample (efflux medium) at 0, 2, 4 , 8, 12 and 24 h. The samples were centrifuged and 100 μl of these different supernatants were counted in scintillation liquid (β Wallac 1410 counter). RESULTS

The approach is either to overexpress adipophilin or on the contrary to inhibit it. Overexpression is first obtained from an expression vector and then using an adenoviral vector at the level of macrophages in culture. This work makes it possible to evaluate the impact of the overexpression of adipophilin both on the lipid load and on the efflux of cholesterol.

It was chosen to clone the cDNA of human adipophilin into the expression vector pCI (Promega) under the control of a ubiquitous promoter CMV. After amplification of the coding sequence from macrophagic mRNA by RT-PCR, this vector was sequenced and named pCI-adipophilin. A transfection of THP-1 differentiated into macrophages by PMA was carried out. The study of the expression of the vector in these cells is represented in FIG. 2.

The impact of this adipophilin overexpression on the lipid load was then evaluated, by measuring cholesterol and triglycerides (Figure 3).

The assay results show a 40% increase for triglycerides and a 15% increase for total cholesterol in transfected cells overexpressing adipophilin compared to cells transfected with the empty pCI vector.

The accumulation of intracellular cholesterol results in an increase in esterified cholesterol.

The effect of overexpression of adipophilin on the efflux of ApoA-1-dependent cholesterol has also been studied in macrophages in culture (Figure 4). ApoA-1 was used as a cholesterol acceptor and the efflux was monitored for 24 h, taking samples at times 2, 4, 8, 12 and 24 h. The tritiated cholesterol present in cells at time t was also measured. The results were compared with those obtained with THP-1 macrophages having undergone the same treatments but incubated in medium without ApoA-1 (RPMI control). After incubation of the cells loaded with lipids, a significant decrease in efflux is observed in the cells transfected with the vector expressing adipophilin in comparison with the control cells (pCI). The efflux of cholesterol is in fact reduced by 47% after 24 h of incubation with apoA-1 (FIG. 4) confirming the role of adipophilin in the storage and retention of lipids. All these adipophilin overexpression experiments were also reproduced and verified using an adenovirus (ad-CMV-adipophilin) making it possible to increase the cellular expression of adipophilin up to 40 times (FIG. 5). The impact of this overexpression was evaluated on the lipid load by measuring total cholesterol, free cholesterol and triglycerides (Figure 6). The assay results show a 40% increase in both triglycerides and esterified cholesterol as well as a 60% decrease in free cholesterol in infected cells overexpressing adipophilin compared to cells infected with the ad-CMV vector. - GFP. It has also been studied the effect of overexpression of adipophilin on the efflux of cholesterol dependent on ApoA-1 and HDL on the macrophages in culture (FIG. 7). The results obtained with the ad-CMV-adipophilin adenovirus are similar to those obtained with the expression vector pCI-adipophilin.

The results of the Western Blot analysis of the inhibition of the expression of adipophilin by siRNA-adipophilin (transfection of THP-1 macrophages with jetSI) in the presence or absence of acetylated LDL are given in FIG. 8 .

The impact of the inhibition of adipophilin expression was evaluated on the lipid load by measuring total cholesterol, free cholesterol and triglycerides (Figure 9). Inhibition of adipophilin expression results in a 50% decrease for triglycerides and a 44% decrease for total cholesterol in cells transfected with siRNA-adipophilin compared to cells transfected with siRNA-GAPDH. The decrease in intracellular cholesterol results in a significant drop in esterified cholesterol.

The effect of the inhibition of adipophilin expression on the efflux of ApoA-1-dependent cholesterol at the level of macrophages in culture has also been studied (FIG. 10). After incubation of the cells loaded with lipids, a significant reduction in efflux is observed in the cells transfected with siRNA-adipophilin in comparison with the control cells. This decrease in the efflux of cholesterol compared to the control cells, can be explained by the strong decrease in the total cholesterol accumulated in the cells no longer expressing adipophilin (FIG. 9). REFERENCES

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Claims

1. Method of selection, identification or characterization of compounds capable of modulating the efflux of cholesterol comprising the determination in vitro or ex Vo of the capacity of the substances tested to modulate the activity of adipophilin. 2. Method according to claim 1, comprising contacting, optionally in the presence of cholesterol, a test compound with adipophilin. 3. Method according to claim 1 or 2, comprising bringing a test compound into contact with a foam cell or a natural or synthetic membrane preparation expressing adipophilin, and demonstrating a modulation of the activity of said adipophilin. 4. Method according to one of claims 1 to 3, characterized in that the demonstration of a modulation of the activity of said adipophilin is carried out by the demonstration of a modulation of the flow of cholesterol through a cell or membrane preparation expressing said adipophilin. 5. Method according to one of claims 1 to 3, characterized in that the demonstration of a modulation of the activity of adipophilin comprises the demonstration of the binding of the test compound to adipophilin. 6. Method according to claim 5, characterized in that the binding of the test compound is demonstrated by migration on gel, electrophoresis, FRET, SPA or by determination of the displacement of a ligand labeled with the test compound. 7. Method according to claim 5, characterized in that the demonstration of the binding of the test compound to adipophilin is carried out in the presence of a labeled ligand of adipophilin by determining the displacement of the binding of the labeled ligand. 8. Method according to claim 1, characterized in that the demonstration of a modulation of the activity of adipophilin comprises the demonstration of a modulation of a biological or pharmacological effect characteristic of adipophilin. 9. Method for selecting, identifying or characterizing compounds comprising bringing into contact, optionally in the presence of cholesterol, in vitro or ex vivo, a test compound with a foam cell or a natural or synthetic membrane preparation expressing adipophilin, measuring the flow of said cholesterol through said cell or membrane preparation and comparing the measured flow with that obtained in the absence of test compound. 10. Method for producing an active compound, in particular a compound capable of modulating the flow of cholesterol, comprising: determining the capacity of a compound to modulate the activity of adipophilin, in particular to modulate the expression of adipophilin, in vitro or ex wVo, and - The synthesis of said compound or of a structural analog thereof. 11. Method for producing a cholesterol flow modulating compound, comprising: - bringing a compound into contact with a cell or a cell membrane expressing adipophilin, the determination of the cholesterol flow through said cell or said cell membrane, the modulation of said flow indicating that the compound is a modulator of the cholesterol flow, and - The synthesis of said compound or of a structural analog thereof. 12. Method for producing a medicament comprising an active compound, in particular on the vascular system, comprising: - the determination of the ability of a compound to modulate the flow of cholesterol through foam cells or natural or synthetic membrane preparations expressing adipophilin in vitro or ex vivo, and - the mixture of said compound or of a structural analog thereof with a pharmaceutically acceptable carrier. 13. A method of producing a medicament comprising a compound modulating the flow of cholesterol through foam cells expressing adipophilin, comprising: - bringing a compound into contact with adipophilin, determining an effect of said compound on adipophilin, said effect indicating that the compound is a modulator of the cholesterol flow, and - Mixing said compound or a structural analog thereof with a pharmaceutically acceptable vehicle. 14. The method of claim 13, characterized in that the effect of the compound is the ability of said compound to modulate the activity of adipophilin. 15. Use of a compound modulating the activity of adipophilin for the preparation of a medicament intended to modulate the flow of cholesterol and / or the cholesterol concentration of the foam cells. 16. Use according to claim 15, characterized in that the compound decreases the activity of adipophilin to increase the efflux of cholesterol and / or decrease the cholesterol concentration of the foam cells. 17. Use according to claim 15 or 16, characterized in that the medicament is intended to treat atherosclerosis. 18. RNA interference from adipophilin. 19. Adipophilin interference RNA comprising an antisense matrix and a sense matrix, said antisense matrix comprising at least one sequence chosen from the sequences SEQ ID 1, 2, 3 and 4. 20. Adipophilin interference RNA comprising an antisense matrix and a sense matrix which are at least 21 bases long, preferably which are 29 bases long. 21. RNAi chosen from double-strands of RNAs of the following sequences: - mRNAs -adipophilin: Antisense matrix: 5'- AATGGCATCCGTTGCAGTTGACCTGTCTC -3 '(SEQ ID 5) Meaning matrix: 5'- AATCAACTGCAACGGATGCCACCTGTCTC -3 '(SEQ ID 6) - RNAsi2-adipophilin Matrix antisense: 5'- AAGCTAGAGCCGCAAATTGCACCTGTCTC -3 '(SEQ ID 7) Meaning matrix: 5'- AATGCAATTTGCGGCTCTAGCCCTGTCTC -3' (SEQ ID 8) - siRNA-adipophilin Matrix antisense: 5'- AATTGCCCGCAACCTGACTCACCTGTCTC -3 '(SEQ ID 9) Meaning matrix: 5'- AATGAGTCAGGTTGCGGGCAACCTGTCTC -3 '(SEQ ID 10) - siRNA-adipophilin Antisense matrix: 5'- AAAAGGCGTCTTCACTGCTTTCCTGTCTC -3 '(SEQ ID 11) Meaning matrix: 5-. AAAAAGCAGTGAAGACGCCTTCCTGTCTC -3 "(SEQ ID 12) 22. Use according to one of claims 15 to 17, characterized in that the compound modulating the activity of adipophilin is an RNAi as defined in one of claims 18 to 21.