FR2938337A1 - MCAM MODULATORS FOR THE TREATMENT OF ACNE, SEBORRHEA DERMATITIS OR HYPERSEBORRHEA - Google Patents

Abstract

The invention relates to an in vitro or in vivo method for screening candidate compounds for the preventive or curative treatment of acne, seborrheic dermatitis or skin disorders associated with hyperseborrhoea comprising determining the capacity of a compound to modulate the expression or the activity of the melanoma cell adhesion molecule (MCAM), as well as the use of modulators of the expression or the activity of this protein for the treatment of acne, seborrheic dermatitis or cutaneous disorders associated with hyperseborrhoea. The invention also relates to methods of diagnosis or prognosis in vitro of these pathologies.

Description

The invention relates to the identification and use of modulating compounds of the melanoma cell adhesion molecule (MCAM) for the treatment of acne, seborrheic dermatitis and cutaneous disorders associated with hyperseborrhoea. It also relates to methods of in vitro diagnosis or prognosis in vitro of these pathologies.

Hyperseborrhoeic oily skin is characterized by excessive secretion and excretion of sebum. Classically, a sebum level greater than 200 g / cm 2 measured at the forehead is considered to be characteristic of oily skin. Oily skin is often associated with a lack of desquamation, a glowing complexion, a thick skin texture. In addition to these aesthetic disorders, the excess of sebum can serve as a support for the anarchic development of the saprophytic bacterial flora (P. acnes in particular), and cause the appearance of comedones and / or acne lesions. This stimulation of sebaceous gland production is induced by androgens.

Acne is, in fact, a chronic disease of the pilosebaceous follicle under hormonal dependence. A hormonal therapy against acne is a possibility of treatment for women, the goal being to oppose the effects of androgens on the sebaceous gland. In this context, estrogens, anti-androgens, or agents that decrease the production of androgens by the ovaries or the adrenal gland are generally used. Antiandrogens used for the treatment of acne include spironolactone, cyproterone acetate, and flutamide. However, these agents have potentially severe side effects. Thus, any pregnancy must be absolutely prevented, particularly because of a risk of feminization for the male fetus. These agents are prohibited in male patients.

Seborrheic dermatitis is a frequent skin inflammatory dermatosis in the form of red patches, covered with fatty and yellowish scales, more or less itchy, predominant in the seborrheic zones.

There is a need for these diseases to identify mediators downstream of the action of steroid hormones, and to modulate them, to obtain a similar therapeutic profile, but with reduced side effects.

The Applicant has now discovered that the gene coding for the melanoma cell adhesion molecule (MCAM) is expressed preferentially in the human sebaceous glands in comparison with the epidermis, and that its expression is regulated in vitro by a cocktail promoting differentiation. of androgen-containing sebocyte precursors (R1881 also called methyltrienolone at 1 nM) and PPARγ ligand (Rosiglitazone, which is 6- (2-Methoxy-ethoxymethoxy) -naphthalene-2-carboxylic acid [4 '- (2 4-dioxothiazolidin-5-ylmethyl) -biphenyl-3-ylmethyl] methyl-amide, at 100 nM), in a primary culture of human sebocytes. It therefore proposes to target the MCAM gene or its expression product, to prevent and / or improve acne, seborrheic dermatitis and skin disorders associated with hyperseborrhoea, including the appearance of oily skin.

It is further known that treatment with a PPAR agonist induces a sharp decrease in the size of the sebaceous glands, and a reduction in androgen-mediated hyperseborrhea (WO2007 / 093747).

Since the proposed target is downstream of the PPAR receptor, it is responsible for the observed effects on the sebaceous glands and sebum excretion.

Thus the identified gene can be used to identify the most active compounds as PPAR modulators, classify them, and select them. On this basis, it is therefore also proposed to use the MCAM gene or the MCAM protein as a marker to screen candidate PPAR modulators for the treatment of acne, seborrheic dermatitis or skin disorder associated with a seborrhoea. More specifically, the ability of a PPAR modulator to modulate the expression or activity of MCAM or the expression of its gene or the activity of at least one of its promoters can be determined.

By acne is meant all forms of acne, namely in particular vulgar, comedonal, polymorphic acne, nodulocystic acne, conglobata, or secondary acne such as solar acne, medicinal or professional. The Applicant also proposes in vitro, in vivo and clinical diagnostic or prognostic methods based on the detection of the level of expression or activity of the MCAM. MCAM The term "MCAM" refers to the cell adhesion molecule of melanoma, also called CD146, MUC18, Gicerin or melanoma adhesion molecule. MCAM is a surface glycoprotein that belongs to the super family of immunoglobulins (Lehmann et al., 1989, PNAS, 86: 9891-9895). Its homology to numerous cell adhesion molecules has first made it possible to highlight its role in cell adhesion and in the cohesion of the endothelial monolayer at intercellular junctions in the vascular tissue. The protein was then associated with tumor progression and metastasis development in human malignant melanoma (Lehmann et al., 1987, Cancer Research, 47: 841-845, Luca et al, 1993, Melanoma research, 3: 35-41). ). MCAM acts as a surface receptor that triggers tyrosine phosphorylation of FYN and PTK2 (Anfosso et al, 1998, Journal Biol Chem, 273 (41): 26852-26856) and the transient increase in intracellular calcium concentration ( Anfosso et al, 2001, Biol Chem Journal, 276 (2): 1564-1569). We speak here indifferently of MCAM protein or MCAM receptor.

In the context of the invention, the term MCAM gene or MCAM nucleic acid means the gene or nucleic acid sequence that encodes the melanoma cell adhesion molecule. If the targeted target is preferably the human gene or its expression product, the invention may also use cells expressing a cell adhesion molecule of the heterologous melanoma, by genomic integration or transient expression of an exogenous nucleic acid. encoding the protein. A human cDNA sequence of the MCAM is reproduced in the appendix (SEQ ID No.1). It is the sequence NM_006500 (Genbank) whose open reading frame contains 3332 base pairs coding for an amino acid sequence of 603 residues (Lehmann et al., 1989, PNAS, 86: 9891-9895). The protein thus synthesized is 113 KDa. Diagnostic applications An object of the invention relates to an in vitro method for diagnosing or monitoring the progression of acne lesions, seborrheic dermatitis or skin disorder associated with hyperseborrhea in a subject, comprising comparing the expression or activity of the melanoma cell adhesion molecule (MCAM), the expression of its gene or the activity of at least one of its promoters, in a biological sample of a subject in relation to to a biological sample of a control subject.

The expression of the protein can be determined by an assay of the MCAM protein according to one of the methods such as Western blot, immunohistochemistry, mass spectrometry analysis (Maldi-TOF and LC / MS analysis), the radioimmunoassay (RIA) or the ELISA or any other method known to those skilled in the art. Another method, in particular for measuring the expression of the MCAM gene, is to measure the amount of corresponding mRNA. An assay of the activity of MCAM can also be envisaged.

As part of a diagnosis, the subject control is a healthy subject.

As part of a follow-up of the evolution of acne lesions, seborrheic dermatitis or skin disorder linked to a hyperseborrhea, the control subject refers to the same subject at a different time, which preferably corresponds to the beginning treatment (To). This measurement of the difference in expression or activity of MCAM, or expression of its gene or activity of at least one of its promoters, makes it possible in particular to monitor the efficacy of a treatment, in particular a treatment with a modulator of MCAM, as envisaged above, or another treatment against acne, seborrheic dermatitis or a skin disorder associated with hyperseborrhoea. Such follow-up can reassure the patient as to the appropriateness, or necessity, of continuing this treatment.

Another aspect of the present invention relates to an in vitro method for determining susceptibility of a subject to develop acne lesions, seborrhoeic dermatitis or skin disorder associated with hyperseborrhoea, comprising comparing the expression or activity of the MCAM protein, the expression of its gene or the activity of at least one of its promoters, in a biological sample of a subject relative to a biological sample of a control subject. Here again, the expression of the MCAM protein can be determined by an assay of this protein by immunoassay, for example by ELISA assay or by any other method mentioned above. Another method, in particular for measuring the expression of the MCAM gene, is to measure the amount of corresponding mRNA by any method as described above. An assay of the activity of MCAM can also be envisaged. The subject tested here is an asymptomatic subject, having no skin disorder associated with hyperseborrhoea, seborrheic dermatitis or acne. The subject controls in this method, signifies a subject or a healthy reference population. The detection of this susceptibility allows the establishment of a preventive treatment and / or increased monitoring of signs related to acne, seborrheic dermatitis or skin disorder associated with hyperseborrhoea.

In these in vitro diagnostic or prognostic methods, the biological sample tested may be any sample of biological fluid or a sample of a biopsy. Preferably, the sample may be a preparation of skin cells, obtained for example by desquamation or biopsy. It can also be sebum.

SCREENING METHODS An object of the invention is an in vitro or in vivo screening method for candidate compounds for the preventive and / or curative treatment of acne, seborrheic dermatitis or any skin disorder associated with a hyperseborrhoea, comprising determining the ability of a compound to modulate the expression or activity of the melanoma cell adhesion molecule or the expression of its gene or the activity of at least one of its promoters, said modulation indicating the utility of the compound for the preventive or curative treatment of acne, seborrheic dermatitis or any skin disorder associated with hyperseborrhoea. The method therefore makes it possible to select the compounds capable of modulating the expression or the activity of the MCAM, or the expression of its gene, or the activity of at least one of its promoters.

More particularly, the subject of the invention is an in vitro method for screening candidate compounds for the preventive and / or curative treatment of acne, seborrheic dermatitis or skin disorders associated with hyperseborrhoea, comprising the steps following: a. preparation of at least two biological samples or reaction mixtures; b. contacting one of the samples or reaction mixtures with one or more of the test compounds; vs. measuring the expression or the activity of the melanoma cell adhesion molecule, the expression of its gene or the activity of at least one of its promoters, in the biological samples or reaction mixtures; d. selection of compounds for which a modulation of the expression or activity of the melanoma cell adhesion molecule, the expression of its gene or the activity of at least one of its promoters, is measured in the sample or mixture treated in (b), relative to the untreated sample or mixture.

An in vivo screening method can be performed in any laboratory animal, for example a rodent. According to a preferred embodiment, the screening method comprises administering to the animal, preferably by topical application, the test compound, then optionally euthanizing the animal, and taking an epidermal leaflet, before evaluation of the expression of the gene in the epidermal sheet, by any method described here.

By modulation is meant any effect on the expression or the activity of the protein, the expression of the gene or the activity of at least one of its promoters, that is to say a stimulation, but preferably a partial inhibition. or complete. Thus, the compounds tested in step d) above preferably inhibit the expression or the activity of the melanoma cell adhesion molecule, the expression of its gene or the activity of at least one of its promoters. The difference in expression obtained with the test compound compared to a control carried out in the absence of the compound is significant from 25% or more. Throughout the present text, unless otherwise specified, expression of a gene means the amount of expressed mRNA; By expression of a protein is meant the amount of this protein; By activity of the MCAM protein is meant the ability of the protein to act as a receptor and to induce signal transduction, in particular via the phosphorylation of the FYN and PTK2 proteins. By promoter activity is meant the ability of this promoter to trigger the transcription of the coded DNA sequence downstream of this promoter (and thus indirectly the synthesis of the corresponding protein). The compounds tested can be of any type. They can be of natural origin or have been produced by chemical synthesis. It can be a library of structurally defined chemical compounds, compounds or uncharacterized substances, or a mixture of compounds.

In particular, the invention relates to the use of the MCAM gene or the MCAM protein as a marker for screening PPAR or AR (androgen receptor) modulators which are candidates for the treatment of acne, seborrheic dermatitis as well as skin disorders associated with hyperseborrhoea. More specifically, the ability of a PPAR or AR modulator to modulate the expression or activity of MCAM or the expression of its gene or the activity of at least one of its promoters is determined. Preferably, the modulator is a PPARy modulator. The PPAR modulator is a PPAR agonist or antagonist, preferably an agonist.

The AR modulator is an AR agonist or antagonist, preferably an agonist.

Various techniques can be used to test these compounds and to identify compounds of therapeutic interest that modulate the expression or the activity of the cell adhesion molecule of melanoma.

According to a first embodiment, the biological samples are cells transfected with a reporter gene operably linked to all or part of the promoter of the gene coding for the melanoma cell adhesion molecule, and the step c) described above. above is to measure the expression of said reporter gene.

The reporter gene may in particular code for an enzyme which, in the presence of a given substrate, leads to the formation of colored products, such as CAT (chloramphenicol acetyltransferase), GAL (beta galactosidase), or GUS (beta glucuronidase). It may also be the gene for luciferase or GFP (Green Fluorescent Protein). The assay of the protein encoded by the reporter gene, or its activity, is carried out conventionally, by colorimetric, fluorometric or chemiluminescent techniques, among others.

According to a second embodiment, the biological samples are cells expressing the gene coding for the cell adhesion molecule of the melanoma, and the step c) described above consists in measuring the expression of said gene. The cell used here can be of any type. It may be a cell expressing the MCAM gene endogenously, such as an endothelial cell, a lymphocyte cell, or more preferably a sebocyte. It is also possible to use organs of human or animal origin, such as, for example, the preputial gland, clitoral gland or the sebaceous gland of the skin. It may also be a cell transformed with a heterologous nucleic acid, encoding the cellular adhesion molecule of melanoma, preferably human, or mammalian. A wide variety of host cell systems can be used, such as, for example, Cos-7, CHO, BHK, 3T3, HEK293 cells. The nucleic acid can be stably or transiently transfected by any method known to those skilled in the art, for example by calcium phosphate, DEAE-dextran, liposome, virus, electroporation, or microinjection.

In these methods, the expression of the MCAM gene or the reporter gene can be determined by evaluating the transcription rate of said gene, or its translation rate. By transcription rate of a gene is meant the amount of corresponding mRNA produced. By translation rate of a gene is meant the amount of protein produced. Those skilled in the art are familiar with techniques for the quantitative or semi-quantitative detection of the mRNA of a gene of interest. Techniques based on the hybridization of mRNA with specific nucleotide probes are the most common (Northern Blot, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), RNase protection). . It may be advantageous to use detection markers, such as fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin).

In particular, the expression of the gene can be measured by real-time PCR or by RNase protection. By RNase protection is meant the detection of a known mRNA from the poly (A) RNAs of a tissue that can be done by means of specific hybridization with a labeled probe. The probe is a complementary RNA labeled (radioactive) messenger to look for. It can be constructed from a known mRNA whose cDNA, after RT-PCR, has been cloned into a phage. RNA-poly (A) of the tissue where the sequence is to be searched is incubated with this probe under slow hybridization conditions in a liquid medium. RNA: RNA hybrids are formed between the desired mRNA and the antisense probe. The hybridized medium is then incubated with a mixture of ribonucleases specific for single-stranded RNA, so that only the hybrids formed with the probe can resist this digestion. The digestion product is then deproteinized and repurified, before being analyzed by electrophoresis. The labeled hybrid RNAs are detected by autoradiography.

The translation rate of the gene is evaluated for example by immunological assay of the product of said gene. The antibodies used for this purpose may be of polyclonal or monoclonal type. Their production is based on conventional techniques. A polyclonal anti-melanoma cell adhesion antibody melanoma can, inter alia, be obtained by immunizing an animal such as a rabbit or a mouse, using the entire protein. The antiserum is removed and then exhausted according to methods known to those skilled in the art. A monoclonal antibody can, inter alia, be obtained by the conventional method of Kohler and Milstein (Nature (London), 256: 495-497 (1975)).

Other methods of preparing monoclonal antibodies are also known. For example, monoclonal antibodies can be produced by expression of a cloned nucleic acid from a hybridoma. Antibodies can also be produced by the phage display technique, by introducing antibody cDNAs into vectors, which are typically filamentous phages that have V gene libraries on the surface of the phage. (for example fUSE5 for E.coli). The immunoassay can be carried out in solid phase or in homogeneous phase; in a time or in two stages; sandwich method or competitive method, by way of non-limiting examples. According to a preferred embodiment, the capture antibody is immobilized on a solid phase. As non-limiting examples of solid phase, it is possible to use microplates, in particular polystyrene microplates, or particles or solid beads, paramagnetic beads. ELISA assays, radioimmunoassays, or any other detection technique can be used to reveal the presence of the antigen-antibody complexes formed. The characterization of antigen / antibody complexes, and more generally isolated or purified but also recombinant proteins (obtained in vitro and in vivo) can be performed by mass spectrometry analysis. This identification is made possible thanks to the analysis (determination of the mass) of the peptides generated by the enzymatic hydrolysis of the proteins (trypsin in general). In general, the proteins are isolated according to methods known to those skilled in the art, prior to enzymatic digestion. Peptide analysis (in the form of a hydrolyzate) is carried out by separation of the peptides by HPLC (nano-HPLC) based on their physico-chemical properties (reverse phase). The determination of the mass of the peptides thus separated is carried out by ionization of the peptides and either by direct coupling to the mass spectrometer (electrospray mode ESI), or after deposition and crystallization in the presence of a matrix known to those skilled in the art (MALDI mode analysis). The proteins are then identified through the use of appropriate software (eg Mascot).

According to a third embodiment, the screening method comprises contacting a compound with a MCAM receptor protein and determining the ability of the compound to modulate the activity of the MCAM protein, a difference in activity, by compared to a control carried out in the absence of the compound, indicating the utility of the compound for the preventive or curative treatment of acne, seborrheic dermatitis or skin disorders associated with hyperseborrhoea.

Preferably, the ability of the compound to bind to the MCAM protein is also evaluated.

It is then determined whether this binding causes an agonist or antagonist effect on the activity of the MCAM protein, by evaluating the impact of the compound on the MCAM-controlled signaling pathway.

The determination of the activity of the MCAM protein can be carried out in different ways. It refers in particular to the determination of cell adhesion activity, carrier activity, apoptotic activity, activity in lipid metabolism, signal transduction and / or secretion of cytokines.

The compounds selected by the screening methods defined herein can then be tested on other models in vitro and / or in vivo (in animals or humans) for their effects on acne, seborrheic dermatitis or disorders. cutaneous associated with a hyperseborrhea.

Modulators of the Receptor The subject of the invention is also the use of a modulator of the human protein MCAM, obtainable by one of the above methods, for the preparation of a medicament intended for preventive treatment and / or curative of acne, seborrheic dermatitis or skin disorders associated with hyperseborrhoea. Thus, a method of preventive and / or curative treatment of acne, of seborrheic dermatitis or of skin disorders associated with hyperseborrhea is described here, a method comprising the administration of a therapeutically effective amount of a modulator of the human protein cellular adhesion molecule of melanoma, to a patient in need of such treatment. The invention finally relates to the cosmetic use of a modulator of the human protein cell adhesion molecule of melanoma, for the aesthetic treatment of oily skin. Preferably, the modulator is a receptor antagonist. The term antagonist refers to a compound or chemical substance that substantially eliminates or reduces the biological activity of the melanoma cell adhesion molecule. The term substantially means a reduction of at least 25%, preferably at least 35%, more preferably at least 50%, and more preferably at least 70% or 90%.

A preferred antagonist interacts with the receptor in solution at antagonist concentrations of less than 20 M, less than 10 M, less than 5 M, less than 1 M, preferably less than 0.1 M, more preferably less than 0 The modulator compound may be an anti-melanoma cell adhesion molecule inhibitory antibody, preferably a monoclonal antibody. Advantageously, such an inhibitory antibody is administered in an amount sufficient to achieve a plasma concentration of about 0.01 g per ml to about 100 g / ml, preferably about 1 g per ml to about 5 g / ml. The modulator compound may also be a polypeptide, an antisense polynucleotide of DNA or RNA, an siRNA, or a PNA ("Peptide nucleic acid", polypeptide chain substituted with purine and pyrimidine bases, whose spatial structure mimics that of DNA and allows hybridization to it). The modulator compound may also be an aptamer. The aptamer is a class of molecules representing in terms of molecular recognition an alternative to antibodies. These are oligonucleotide sequences having the ability to recognize virtually all classes of target molecules with high affinity and specificity. Such ligands can be isolated by systematic evolution of exponential enrichment ligand (SELEX) from a random sequence library as described by Tuerk and Gold, 1990. The library of random sequences can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, optionally chemically modified, of a single sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena,, 1999, Clinical Chemistry 45 (9): 1628-1650.

Several modulators of the cell adhesion molecule of melanoma are known and proposed as a treatment to decrease the growth of melanoma tumors (EP 1382615, Mills et al., 2002, Cancer Research, 62 (17): 5106-14). The invention comprises the use of such antagonistic compounds of the melanoma cell adhesion molecule for the preventive and / or curative treatment of acne, seborrheic dermatitis or skin disorders associated with hyperseborrhoea. Non-limiting mention may be made of the transcription factor AP-2 (Jean et al., 1998, Journal Biol Chem, 273 (26): 16501-16508) or the anti-MCAM antibody ABX-MA1 as inhibitors of the molecule. cell adhesion of melanoma. Other modulator compounds identified by the screening method described above are also useful.

The modulating compounds are formulated within a pharmaceutical composition, in association with a pharmaceutically acceptable vehicle. These compositions may be administered, for example, orally, enterally, parenterally, or topically. Preferably, the pharmaceutical composition is applied topically. Orally, the pharmaceutical composition can be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid vesicles or polymers for controlled release. Parenterally, the pharmaceutical composition may be in the form of solutions or suspensions for infusion or for injection. Topically, the pharmaceutical composition is more particularly intended for the treatment of skin and mucous membranes and may be in the form of ointments, creams, milks, ointments, powders, soaked swabs, solutions, gels , sprays, lotions or suspensions. It may also be in the form of suspensions of microspheres or nanospheres or lipid or polymeric vesicles or polymeric patches or hydrogels allowing controlled release. This composition for topical application may be in anhydrous form, in aqueous form or in the form of an emulsion. In a preferred variant, the pharmaceutical composition is in the form of a gel, a cream or a lotion.

The composition may comprise an MCAM modulator content ranging from 0.001 to 10% by weight, especially from 0.01 to 5% by weight relative to the total weight of the composition.

The pharmaceutical composition may further contain inert additives or combinations thereof, such as wetting agents; - flavor enhancers; preserving agents such as esters of parahydroxybenzoic acid; stabilizing agents; humidity regulating agents; pH regulating agents; osmotic pressure modifying agents; emulsifying agents; UV-A and UV-B filters and antioxidants, such as alpha-tocopherol, butylhydroxyanisole or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelators.

The following examples illustrate the invention without limiting its scope. Examples: EXPERIMENTAL DATA Example 1: Expression of the cellular adhesion molecule of melanoma in the human sebaceous gland and in the human epidermis

Human sebaceous glands were separated from the human epidermis by dispase treatment and dissection under a binocular microscope. Total RNA samples were prepared from the sebaceous glands and from the epidermis. Gene expression was analyzed on an Affymetrix station (microfluidic module, hybridization oven, scanner, computer) following the protocols provided by the company. In short, the total RNA isolated from the tissues is transcribed into cDNA. From the double-stranded cDNA, biotin-labeled cRNA is synthesized using T7 polymerase and a precursor NTP conjugated to biotin. The cRNAs are then fragmented into small fragments. All molecular biology steps are controlled using Agilent's Lab on a chip system to confirm the good efficiencies of enzymatic reactions. The Affymetrix chip is hybridized with the biotinylated cRNA, rinsed and then fluorescently labeled using a streptavidin-conjugated fluorophore.

After washes, the chip is scanned and the results are calculated using the MAS5 software provided by Affymetrix. An expression value is obtained for each gene as well as an indication of the significance of the value obtained. The calculation of the significance of the expression is based on the analysis of the signals that are obtained following the hybridization of the cRNA of a given gene with a perfect match oligonucleotide versus an oligonucleotide that contains a mutation. (single mismatch) in the central region of the oligonucleotide (see Table 1). Table 1: Measurement of melanoma cell adhesion molecule expression in the epidermis and in the human sebaceous gland via the use of the affymetrix flea technology. Identifier Name of the Expression Expression Significativity Significance Affymetrix gene in the gland in the epidermis the expression * the expression * human sebaceous in the human gland human sebaceous epidermis 209087_x_at melanoma 105 30 1 0 cell adhesion molecule * Indicator of the significance of the expression of the analyzed gene in the indicated sample: presence (= 1) or absence (= 0).

EXAMPLE 2 Expression of the Melanoma Cellular Adhesion Molecule in the Human Sebaceous Gland and in the Human Epidermis The epidermis and human sebaceous gland samples were prepared by laser microdissection from three facelifts of healthy human skin ( female donors).

Expression of the messenger RNA encoding the MCAM protein was analyzed by quantitative RTPCR (qRT-PCR) using the Microfluidics Cards technology developed by Applied Biosystems. The Ct corresponds to the number of PCR cycles making it possible to reach the same level of fluorescence for all the samples. The level of expression is represented in each tissue by the mean of the Ct and the standard deviation obtained on the three donors. The differential expression between the two tissues is measured by a mean induction factor (IF) between the sebaceous gland and the epidermis after normalization of Ct by the expression of the three housekeeping genes (18S ribosomal RNA, Glyceraldehyde 3-phosphate GAPDH dehydrogenase, beta-actin).

Table 2: qRT-PCR measurement of the expression of MCAM in the epidermis and the human sebaceous gland via the use of Microfluidic Cards technology (Applied Biosystems). Name of Number of Difference Number of Difference Factor Gene Cycles Type Cycles Induction Type Necessary Required Medium for for Expression Detect Detect It in Level Level Expression Medium Sebaceous Expression Gland in Medium in Versus Gland epidermis human sebaceous epidermis (Ct) human (FI) human (Ct) melanoma cell 26 0.93 33 0.82 121 adhesion molecule Example 3: Expression of the cell adhesion molecule of melanoma in human sebocvlets in primary culture a. Isolation and culture of human sebocytes

The culture of human sebocytes is carried out from facelifts of healthy human donors according to the methodology described by Xia et al. (J Invest Dermatol, 1989 Sep 10, 93 (3): 315-21) after separation of the epidermis from the dermis under the action of dispase and microdissection of the sebaceous glands under binocular loupes. The sebaceous glands are seeded in a 6-well plate on a feeder layer of 3T3 fibroblasts subjected to mitomycin in DMEM-Ham's F12 medium (3: 1) supplemented with 10% fetal calf serum (FCS); 10ng / mI epidermal growth factor (EGF); 10-10M cholera toxin (CT); 0.5 g / ml hydrocortisone (HG); 5 g / ml insulin (INS); 2mM L-Glutamine (GIn); 10001 / ml penicillin-streptomycin (PS). The first foci of human sebocytes appear 3 days after seeding of the glands. The cells are then treated for 6 days with the sebogenic cocktail corresponding to the combination of PPARγ Rosiglitazone agonist (1M) and androgen R1881 (10nM), or Dimethyl sulfoxide (DMSO) as vehicle.

b. PCR expression data

The expression of the messenger RNA encoding the MCAM protein was analyzed by qRTPCR using the Microfluidics Cards technology developed by Applied Biosystems, as previously described (Example 2), on a donor-specific human sebocyte culture. The level of expression (Ct) is represented for each treatment condition.

The induction of the expression of MCAM by the sebogenic cocktail is measured by an induction factor (FI) versus the DMSO control after normalization of the Ct by the expression of the three housekeeping genes (18S ribosomal RNA, Glyceraldehyde 3- GAPDH phosphate dehydrogenase, beta-actin). Table 3: qRT-PCR measurement of MCAM expression in a primoculture of human sebocytes treated for 6 days with the sebogenic cocktail (agonist combination PPARy Rosiglitazone 1M, androgen R1881 10nM) or by DMSO, through the use of Microfluidic Cards technology (Applied Biosystems). Gene Name Number of Cycle Cycles Induction Factor Cycles Required for Expression in Needed to Detect the Level Condition Treated Detect Mean Expression by Level Mix in Human Sebogenic versus Human Sebocytes Treated by the the average DMSO condition in sebogenic cocktail (FI) human sebocytes (Ct) treated with DMSO (Ct) melanoma cell 31 29 3.2 adhesion molecule 5

Claims (25)

REVENDICATIONS1. In vitro or in vivo method for screening candidate compounds for the preventive and / or curative treatment of acne, seborrheic dermatitis or skin disorders associated with hyperseborrhoea, comprising determining the ability of a compound to modulate the expression or the activity of the melanoma cell adhesion molecule (MCAM) or the expression of its gene or the activity of at least one of its promoters. 2. In vitro method for screening candidate compounds for the preventive and / or curative treatment of acne, seborrheic dermatitis or skin disorders associated with hyperseborrhea according to claim 1, comprising the following steps: a. Preparation of at least two biological samples or reaction mixtures; b. Contacting one of the samples or reaction mixtures with one or more of the test compounds; vs. Measuring the expression or activity of the melanoma cell adhesion molecule, the expression of its gene or the activity of at least one of its promoters, in biological samples or reaction mixtures; d. Selection of compounds for which a modulation of the expression or activity of the MCAM protein, or a modulation of the expression of its gene or a modulation of the activity of at least one of its promoters, is measured in the sample or mixture treated in (b) in relation to the untreated sample or mixture. 3. Method according to claim 2, characterized in that the compounds selected in step d) inhibit the expression or the activity of the melanoma cell adhesion molecule, the expression of its gene or the activity at least one of its promoters. 4. Method according to claim 2 or 3, characterized in that the biological samples are cells transfected with a reporter gene operably linked to all or part of the promoter of the gene coding for the melanoma cell adhesion molecule, and what step c) consists in measuring the expression of said reporter gene. 5. Method according to claim 2 or 3, characterized in that the biological samples are cells expressing the gene coding for the cellular adhesion molecule of the melanoma, and in that step c) consists in measuring the expression of said uncomfortable. The method of claim 4 or 5, wherein the cells are sebocytes. The method of claim 5, wherein the cells are cells transformed with a heterologous nucleic acid, encoding the cell adhesion molecule of melanoma. 8. Method according to one of claims 2 to 7, wherein the expression of the gene is determined by measuring the transcription rate of said gene. 9. The method according to one of claims 2 to 7, wherein the expression of the gene is determined by measuring the rate of translation of said gene. 10. The method of claim 1 comprising contacting a compound with an MCAM receptor, and determining the activity of the MCAM protein, a difference in activity, compared to a control performed in the absence of the Compound, indicating the utility of the compound for the preventive or curative treatment of acne, seborrheic dermatitis or skin disorder associated with hyperseborrhoea. 30 11. In vitro method for diagnosing or monitoring the progress of acne, seborrheic dermatitis or skin disorder associated with hyperseborrhea in a subject, including comparing expression or activity of the melanoma cell adhesion molecule, the expression of its gene or the activity of at least one of its promoters, in a biological sample of a subject with respect to a biological sample of a subject control. 15 12. The method of claim 11, wherein the expression of the protein is determined by an assay of this protein by immunoassay. The method of claim 12, wherein the immunoassay is an ELISA assay. The method of claim 11, wherein the expression of the gene is determined by measuring the amount of corresponding mRNA. 10 15. In vitro method for determining susceptibility of a subject to develop acne, seborrhoeic dermatitis or skin disorder associated with hyperseborrhea, comprising comparing the expression or activity of the MCAM protein, the expression of its gene or the activity of at least one of its promoters in a biological sample of a subject relative to a biological sample of a control subject. The method of claim 15, wherein the expression of the protein is determined by assaying for this protein by an immunoassay. 20 17. The method of claim 16, wherein the immunoassay is an ELISA assay or a radioimmunoassay. The method of claim 15, wherein the expression of the gene is determined by measuring the amount of corresponding mRNA. 19. Use of the MCAM gene or MCAM protein as a marker for screening candidate PPAR modulators for the treatment of acne, seborrheic dermatitis or skin disorder associated with hyperseborrhoea. 30 The use of claim 19, comprising determining the ability of a PPAR modulator to modulate the expression or activity of the MCAM or the expression of its gene or the activity of at least one of its promoters. . 21. Use according to claim 19 or 20, wherein the PPAR modulator is a PPARy modulator. 25 22. Use according to any one of claims 19 to 21, wherein the modulator is a PPAR receptor agonist. 23. Use of an MCAM gene or MCAM protein as a marker for screening candidate androgen receptor (AR) modulators for the treatment of acne, seborrhoeic dermatitis or skin disorder associated with hyperseborrhoea . 24. The use according to claim 23, comprising determining the ability of an AR modulator to modulate the expression or activity of the MCAM or the expression of its gene or the activity of at least one of its promoters. Use according to any one of claims 23 or 24, wherein the modulator is an androgen receptor agonist.