EP2285987A2 - Modulators of mcam in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea - Google Patents

Modulators of mcam in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea

Info

Publication number
EP2285987A2
EP2285987A2 EP09742128A EP09742128A EP2285987A2 EP 2285987 A2 EP2285987 A2 EP 2285987A2 EP 09742128 A EP09742128 A EP 09742128A EP 09742128 A EP09742128 A EP 09742128A EP 2285987 A2 EP2285987 A2 EP 2285987A2
Authority
EP
European Patent Office
Prior art keywords
expression
gene
activity
mcam
acne
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09742128A
Other languages
German (de)
French (fr)
Inventor
Jérôme AUBERT
Valérie TRINQUET
Johannes Voegel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Galderma Research and Development SNC
Original Assignee
Galderma Research and Development SNC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Galderma Research and Development SNC filed Critical Galderma Research and Development SNC
Publication of EP2285987A2 publication Critical patent/EP2285987A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders

Definitions

  • the invention relates to the identification and the use of compounds which modulate the melanoma cell adhesion molecule (MCAM) for treating acne, seborrhoeic dermatitis, and also skin disorders associated with hyperseborrhoea. It also relates to methods for the in vitro diagnosis of or in vitro prognosis for these pathologies.
  • MCAM melanoma cell adhesion molecule
  • Hyperseborrhoeic greasy skin is characterized by exaggerated secretion and excretion of sebum.
  • a sebum level greater than 200 ⁇ g/cm 2 measured on the forehead is considered to be characteristic of greasy skin.
  • Greasy skin is often associated with a desquamation deficiency, a glistening complexion and a thick skin grain.
  • excess sebum can serve as a support for the anarchical development of saprophytic bacterial flora ⁇ P. acnes in particular), and cause the appearance of comedones and/or acneic lesions.
  • Acne is, in fact, a chronic disease of the pilosebaceous follicle under hormonal control. Hormone therapy against acne is one treatment possibility for women, the objective being to prevent the effects of androgens on the sebaceous gland.
  • oestrogens, anti-androgens or agents which reduce the production of androgens by the ovaries or the adrenal gland are generally used.
  • the anti-androgens used for the treatment of acne include, in particular, spironolactone, cyproterone acetate and flutamide.
  • these agents have potentially severe side effects. Thus, any pregnancy must be absolutely prevented, in particular because of a risk of feminization for the male foetus. These agents are prohibited in male patients.
  • Seborrhoeic dermatitis is a common inflammatory skin dermatosis which presents in the form of red plaques covered with greasy, yellowish squames, which are more or less pruriginous, and are predominant in the seborrhoeic areas.
  • MCAM melanoma cell adhesion molecule
  • MCAM gene or the expression product thereof for preventing and/or improving acne, seborrhoeic dermatitis and also skin disorders associated with hyperseborrhoea, in particular the greasy skin appearance.
  • the target proposed is downstream of the PPAR receptor, it is said target which is responsible for the effects observed on the sebaceous glands and on sebum excretion.
  • the gene identified can be used to identify the compounds which are the most active as PPAR modulators, to classify them and to select them.
  • the MCAM gene or the MCAM protein is also proposed to use the MCAM gene or the MCAM protein, as a marker for screening for candidate PPAR modulators for the treatment of acne, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea .
  • the ability of a PPAR modulator to modulate the expression or the activity of MCAM or the expression of the gene thereof or the activity of at least one of the promoters thereof, can be determined.
  • acne is intended to mean all the forms of acne, i.e. in particular acne vulgaris, comedonal acne, polymorphous acne, nodulocystic acne, acne conglobata, or else secondary acne such as solar acne, acne medicamentosa or occupational acne.
  • the Applicant also proposes methods of in vitro, in vivo and clinical diagnosis or prognosis based on the detection of the level of expression or of activity of MCAM.
  • MCAM denotes the melanoma cell adhesion molecule, also known as CD146, MUC18, gicerin or melanoma adhesion molecule.
  • MCAM is a surface glycoprotein which belongs to the immunoglobulin superfamily (Lehmann et al . , 1989, PNAS, 86:9891-9895) . Its homology to many cell adhesion molecules made it possible, initially, to demonstrate its role in cell adhesion and in cohesion of the endothelial monolayer at the level of the intercellular junctions in vascular tissue. The protein was then associated with tumour progression and with the development of metastases in human malignant melanoma
  • MCAM acts as a surface receptor which triggers FYN and PTK2 tyrosine phosphorylation (Anfosso et al . , 1998, Journal Biol Chem, 273(41) : 26852-26856) and the transient increase in intracellular calcium concentration (Anfosso et al . , 2001, Journal Biol Chem, 276 (2) : 1564-1569) .
  • MCAM gene or "MCAM nucleic acid” signifies the gene or the nucleic acid sequence which encodes the melanoma cell adhesion molecule.
  • target aimed for is preferably the human gene or the expression product thereof, the invention may also call upon cells expressing a heterologous melanoma cell adhesion molecule, by genomic integration or transient expression of an exogenous nucleic acid encoding the protein.
  • a human cDNA sequence of MCAM is reproduced in the annexe (SEQ ID No. 1) . It is the sequence NM_006500 (Genbank) , the open reading frame of which contains 3332 base pairs encoding an amino acid sequence of 603 residues (Lehmann et al . , 1989, PNAS, 86:9891-9895) . The protein thus synthesized is of 113 KDa.
  • a subject of the invention concerns an in vitro method for diagnosing or monitoring the development of acneic lesions, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea in an individual, comprising the comparison of the expression or of activity of the melanoma cell adhesion molecule (MCAM) , of the expression of the gene thereof or of the activity of at least one of the promoters thereof, in a biological sample from an individual, with respect to a biological sample from a control individual.
  • MCAM melanoma cell adhesion molecule
  • the protein expression can be determined by assaying the MCAM protein according to one of the methods such as Western blotting, immunohistochemistry, mass spectrometry analysis (Maldi-TOF and LC/MS analysis), radioimmunoassay (RIA) or 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. Assaying the activity of MCAM can also be envisaged.
  • control individual In the context of a diagnosis, the "control" individual is a "healthy” individual.
  • control individual refers to the same individual at a different time, which preferably corresponds to the beginning of the treatment (TO) .
  • TO the beginning of the treatment
  • This measurement of the difference in expression or in activity of MCAM, or in expression of the gene thereof or in activity of at least one of the promoters thereof makes it possible in particular to monitor the effectiveness of a treatment, in particular a treatment with an MCAM modulator, as envisaged above, or another treatment against acne, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea.
  • Such monitoring can reassure the patient with regard to whether continuing the treatment is well-founded or necessary.
  • Another aspect of the present invention concerns an in vitro method for determining an individual's susceptibility to developing acneic lesions, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea, comprising the comparison of the expression or of the activity of the MCAM protein, of the expression of the gene thereof or of the activity of at least one of the promoters thereof, in a biological sample from an individual, with respect to a biological sample from a control individual.
  • the expression of the MCAM protein can be determined by assaying 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. Assaying of the MCAM activity can also be envisaged.
  • the individual tested is in this case an asymptomatic individual exhibiting no skin condition associated with hyperseborrhoea, seborrhoeic dermatitis or acne.
  • the "control" individual in this method signifies a "healthy” reference population or individual. The detection of this susceptibility makes it possible to set up a preventive treatment and/or increased monitoring of the signs associated with acne, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea .
  • the biological sample tested may be any sample of biological fluid or a sample of a biopsy.
  • the sample may be a preparation of skin cells, obtained for example by desquamation or biopsy. It may also be sebum.
  • a subject of the invention is an in vitro or in vivo method for screening for candidate compounds for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of any skin disorder associated with hyperseborrhoea, comprising the determination of the ability of a compound to modulate the expression or the activity of the melanoma cell adhesion molecule or the expression of the gene thereof or the activity of at least one of the promoters thereof, said modulation indicating the usefulness of the compound for the preventive or curative treatment of acne, seborrhoeic 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 MCAM, or the expression of the gene thereof, or the activity of at least one of the promoters thereof.
  • the subject of the invention is an in vitro method for screening for candidate compounds for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea, comprising the following steps: a. preparing at least two biological samples or reaction mixtures; b. bringing one of the samples or reaction mixtures into contact with one or more of the test compounds; c. measuring the expression or the activity of the melanoma cell adhesion molecule, the expression of the gene thereof or the activity of at least one of the promoters thereof, in the biological samples or reaction mixtures; d.
  • An in vivo screening method can be carried out in any laboratory animal, for example, a rodent.
  • the screening method comprises administering the test compound to the animal preferably by topical application, then optionally sacrificing the animal by euthanasia, and taking a sample of an epidermal split, before evaluating the expression of the gene in the epidermal split, by any method described herein.
  • modulation is intended to mean any effect on the expression or the activity of the enzyme, the expression of the gene or the activity of at least one of the promoters thereof, i.e. optionally a stimulation, but preferably a partial or complete inhibition.
  • the compounds tested in step d) above preferably inhibit the expression or the activity of the melanoma cell adhesion molecule, the expression of the gene thereof or the activity of at least one of the promoters thereof.
  • the difference in expression obtained with the compound tested, compared with a control carried out in the absence of the compound, is significant starting from 25% or more.
  • the term "expression of a gene” is intended to mean the amount of mRNA expressed; the term “expression of a protein” is intended to mean the amount of this protein; the term “activity of the MCAM protein” is intended to mean the ability of the protein to act as a receptor and to induce signal transduction, in particular via FYN and PTK2 protein phosphorylation; the term “activity of a promoter” is intended to mean the ability of this promoter to initiate the transcription of the DNA sequence encoded downstream of this promoter (and therefore indirectly the synthesis of the corresponding protein) .
  • the compounds tested may be of any type. They may be of natural origin or may have been produced by chemical synthesis. This may involve a library of structurally defined chemical compounds, uncharac- terized compounds or substances, or a mixture of compounds .
  • the invention is directed towards the use of the MCAM gene or of the MCAM protein, as a marker for candidate PPAR or AR (androgen receptor) modulators for treating acne, seborrhoeic dermatitis and also skin disorders associated with hyperseborrhoea . More specifically, the ability of a PPAR or AR modulator to modulate the expression or the activity of MCAM or the expression of the gene thereof or the activity of at least one of the promoters thereof is determined.
  • the modulator is a PPAR ⁇ 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 the compounds of therapeutic interest which modulate the expression or the activity of the melanoma cell adhesion molecule.
  • the biological samples are cells transfected with a reporter gene functionally linked to all or part of the promoter of the gene encoding the melanoma cell adhesion molecule, and step c) described above comprises measuring the expression of said reporter gene.
  • the reporter gene may in particular encode an enzyme which, in the presence of a given substrate, results in the formation of coloured products, such as CAT (chloramphenicol acetyltransferase) , GAL (beta- galactosidase) or GUS (beta-glucuronidase) . It may also be the luceriferase gene or the GFP (green fluorescent protein) gene.
  • the assaying of the protein encoded by the reporter gene, or of the activity thereof, is carried out conventionally by colorimetric, fluorometric or chemiluminescence techniques, inter alia.
  • the biological samples are cells expressing the gene encoding the melanoma cell adhesion molecule, and step c) described above comprises measuring the expression of said gene.
  • the cell used herein may be of any type. It may be a cell expressing the MCAM gene endogenously, for instance an endothelial cell, a lymphocyte cell, or better still a sebocyte. Organs of human or animal origin may also be used, for instance the preputial gland, the clitoral gland, or else the sebaceous gland of the skin.
  • It may also be a cell transformed with a heterologous nucleic acid encoding the preferably human, or mammalian, melanoma cell adhesion molecule.
  • a large variety of host-cell systems may be used, such as, for example, Cos-7, CHO, BHK, 3T3 or HEK293 cells.
  • the nucleic acid may be transfected stably or transiently, by any method known to those skilled in the art, for example by calcium phosphate, DEAE- dextran, liposome, virus, electroporation or microinjection .
  • the expression of the MCAM gene or of the reporter gene can be determined by evaluating the level of transcription of said gene, or the level of translation thereof.
  • level of transcription of a gene is intended to mean the amount of corresponding mRNA produced.
  • level of translation of a gene is intended to mean the amount of protein produced.
  • the expression of the gene can be measured by real-time PCR or by RNase protection.
  • RNase protection is intended to mean the detection of a known mRNA among the poly (A) -RNAs of a tissue, which can be carried out using specific hybrid ⁇ ization with a labelled probe.
  • the probe is a labelled (radioactive) RNA complementary to the messenger to be sought. It can be constructed from a known mRNA, the cDNA of which, after RT-PCR, has been cloned into a phage. PoIy(A)-RNA from the tissue in which the sequence is to be sought is incubated with this probe under slow hybridization conditions in a liquid medium.
  • RNAiRNA hybrids form between the mRNA sought and the antisense probe.
  • the hybridized medium is then incubated with a mixture of ribonucleases specific for single-stranded RNA, such that only the hybrids formed with the probe can withstand this digestion.
  • the digestion product is then deproteinated and repurified, before being analysed by electrophoresis.
  • the labelled hybrid RNAs are detected by autoradiography.
  • the level of translation of the gene is evaluated, for example, by immunological assaying of the product of said gene.
  • the antibodies used for this purpose may be of polyclonal or monoclonal type.
  • the production thereof involves conventional techniques.
  • An anti- melanoma cell adhesion molecule polyclonal antibody can, inter alia, be obtained by immunization of an animal, such as a rabbit or a mouse, with the whole protein. The antiserum is taken and then depleted according to methods known per se to those skilled in the art.
  • a monoclonal antibody can, inter alia, be obtained by the conventional method of K ⁇ hler and Milstein (Nature (London), 256: 495-497 (1975)) . Other methods for preparing monoclonal antibodies are also known.
  • Monoclonal antibodies can, for example, be produced by expression of a nucleic acid cloned from a hybridoma.
  • Antibodies can also be produced by the phage display technique, by introducing antibody cDNAs into vectors, which are typically filamentous phages which display V-gene libraries at the surface of the phage
  • the immunological assaying can be carried out in solid phase or in homogeneous phase; in one step or in two steps; in a sandwich method or in a competition method, by way of nonlimiting examples.
  • the capture antibody is immobilized on a solid phase.
  • a solid phase use may be made of microplates, in particular polystyrene microplates, or solid particles or beads, or paramagnetic beads.
  • ELISA assays can be used to reveal the presence of the antigen/antibody complexes formed.
  • the characterization of the antigen/antibody complexes, and more generally of the isolated or purified, but also recombinant, proteins (obtained in vitro and in vivo) can be carried out by mass spectrometry analysis. This identification is made possible by virtue of the analysis (determination of the mass) of the peptides generated by enzymatic hydrolysis of the proteins (in general, trypsin) . In general, the proteins are isolated according to the methods known to those skilled in the art, prior to the enzymatic digestion. The analysis of the peptides (in hydrolysate form) is carried out by separating of the peptides by HPLC (nano-HPLC) based on their physicochemical properties (reverse phase) .
  • HPLC nano-HPLC
  • the deter ⁇ mination of the mass of the peptides thus separated is carried out by ionization of the peptides and either by direct coupling with mass spectrometry (electrospray ESI mode) , or after deposition and crystallization in the presence of a matrix known to those skilled in the art (analysis in MALDI mode) .
  • the proteins are subsequently identified through the use of appropriate software (for example, Mascot) .
  • the screening method comprises bringing a compound into contact with an MCAM receptor protein and determining the ability of the compound to modulate the activity of the MCAM protein, a difference in activity, compared to a control carried out in the absence of the compound, indicating the usefulness of the compound for the preventive or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea .
  • the ability of the compound to bind to the MCAM protein is also evaluated.
  • the determination of the activity of the MCAM protein can be carried out in various ways. It refers in particular to the determination of the cell adhesion activity, of transporter activity, of apoptotic activity, of activity in lipid metabolism, in signal transduction and/or in cytokine secretion.
  • the compounds selected by means of the screening methods defined herein can subsequently be tested on other in vitro models and/or in vivo models (in animals or humans) for their effects on acne, seborrhoeic dermatitis or skin disorders associated with hyperseborrhoea .
  • a subject of the invention is also the use of a modulator of the human MCAM protein, that can be obtained by means of one of the methods above, for the preparation of a medicament for use in the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea .
  • a method for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea comprising the administration of a therapeutically effective amount of a modulator of the human melanoma cell adhesion molecule protein to a patient requiring such a treatment .
  • the invention is directed towards the cosmetic use of a modulator of the human melanoma cell adhesion molecule protein, for the aesthetic treatment of greasy skin.
  • the modulator is an antagonist of the receptor.
  • antagonist refers to a compound or a chemical substance which eliminates or substantially reduces the biological activity of the melanoma cell adhesion molecule.
  • substantially signifies a reduction of at least 25%, preferably of at least 35%, more preferably of at least 50%, and more preferably of 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 0.01 ⁇ M.
  • the modulator compound may be an anti-melanoma cell adhesion molecule, preferably a monoclonal antibody.
  • such an inhibitory antibody is administered in an amount sufficient to obtain a plasma concentration of approximately 0.01 ⁇ g per ml to approximately 100 ⁇ g/ml, preferably of approximately 1 ⁇ g per ml to approximately 5 ⁇ g/ml.
  • the modulator compound may also be a polypeptide, an antisense DNA or RNA polynucleotide, an siRNA or a PNA (peptide nucleic acid, polypeptide chain substituted with purine and pyrimidine bases, the spatial structure of which mimics that of the DNA and enables hybridization thereto) .
  • a polypeptide an antisense DNA or RNA polynucleotide, an siRNA or a PNA (peptide nucleic acid, polypeptide chain substituted with purine and pyrimidine bases, the spatial structure of which mimics that of the DNA and enables hybridization thereto) .
  • the modulator compound may also be an aptamer.
  • the aptamer is a class of molecules representing, in terms of molecular recognition, an alternative to the antibodies. They are oligonucleotide sequences which have the ability to recognize virtually all the classes of target molecules with high affinity and specificity.
  • Such ligands can be isolated by systematic evolution of ligand by exponential enrichment (SELEX) carried out on a random-sequence library, as described by Tuerk and Gold, 1990.
  • the random-sequence library can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear, optionally chemically modified, oligomer of a unique sequence.
  • modulated compounds identified by the screening method described above are also useful.
  • the modulator compounds are formulated within a pharmaceutical composition, in combination with a pharmaceutically acceptable carrier.
  • These compositions may be administered, for example, orally, enterally, parenterally, or topically.
  • the pharmaceutical composition is applied topically.
  • oral administration the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid or polymeric vesicles for controlled release.
  • parenteral administration the pharmaceutical composition may be in the form of solutions or suspensions for a drip or for injection.
  • the pharmaceutical composition is more particularly for use in treating the skin and the mucous membranes and may be in the form of salves, creams, milks, ointments, powders, impregnated pads, 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 for controlled release.
  • This composition for topical application may be in anhydrous form, in aqueous form or in the form of an emulsion.
  • 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, in particular from 0.01% to 5% by weight, relative to the total weight of the composition.
  • the pharmaceutical composition may also contain inert additives or combinations of these additives, such as wetting agents; flavour enhancers;
  • - preservatives such as para-hydroxybenzoic acid esters; - stabilizers; moisture regulators; pH regulators; osmotic pressure modifiers;
  • antioxidants such as alpha-tocopherol, butylhydroxyanisol or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelating agents.
  • Example 1 Expression of the melanoma cell adhesion molecule in the human sebaceous gland and in human epidermis
  • RNA samples were prepared from the sebaceous glands and from the epidermis .
  • the expression of the genes was analysed on an Affymetrix station (microfluidic module; hybridization oven; scanner; computer) according to the protocols supplied by the company. Briefly, the total RNA isolated from the tissues is transcribed into cDNA. A biotin-labelled cRNA is synthesized, from the double- stranded cDNA, using T7 polymerase and a precursor NTP conjugated to biotin. The cRNAs are subsequently fragmented into small fragments. All the molecular biology steps are verified using the Agilent "lab on a chip" system in order to confirm that the enzymatic reactions are very efficient.
  • the Affymetrix chip is hybridized with the biotinylated cRNA, rinsed, and subsequently labelled by fluorescence using a Streptavidin-conjugated fluorophore. After washing, the chip is scanned and the results are calculated using the MAS5 software supplied by Affymetrix. An expression value is obtained for each gene, as is 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 which are obtained following hybridization of the cRNA of a given gene with a perfect match oligonucleotide versus an oligonucleotide which contains a single mismatch in the central region of the oligonucleotide (see Table 1) .
  • Table 1 Measurement of the expression of the melanoma cell adhesion molecule in the epidermis and in the human sebaceous gland by the use of the Affymetrix chip technology
  • Example 2 Expression of the melanoma cell adhesion molecule in the human sebaceous gland and in human epidermis
  • the samples of epidermis and of human sebaceous gland were prepared by laser microdissection from three lifts of healthy human skin (female donors) .
  • the expression of the messenger RNA encoding the MCAM protein was analysed by quantitative RT-PCR (qRT- PCR) using the microfluidics cards technology developed by Applied Biosystems.
  • the Ct corresponds to the number of PCR cycles which makes it possible to choose the same level of fluorescence for all the samples.
  • the level of expression is represented in each tissue by the mean of the Cts and the standard deviation obtained on the three donors .
  • the differential expression between the two tissues is measured via a mean induction factor (I. F) for the sebaceous gland with respect to the epidermis after standardization of the Cts via the expression of the three housekeeping genes (ribosomal 18S RNA, glyceraldehyde 3-phosphate dehydrogenase GAPDH, beta- actin) .
  • I. F mean induction factor
  • Table 2 qRT-PCR measurement of the expression of MCAM in the epidermis and the human sebaceous gland via the use of the microfluidic cards technology (Applied Biosystems)
  • Example 3 Expression of the melanoma cell adhesion molecule in human sebocytes in primary culture
  • Human sebocytes are cultured using lifts from healthy human donors according to the method described by Xia et al . (J Invest Dermatol. 1989 Sep; 93 (3) : 315- 21) after separation of the epidermis from the dermis through the action of dispase and microdissection of the sebaceous glands under binocular magnifying lenses.
  • the sebaceous glands are seeded in 6-well plates on a feeder layer of mitomycin-treated 3T3 fibroblasts in DMEM-Ham' s F12 (3:1) medium supplemented with 10% foetal calf serum (FCS) ; 10 ng/ml of epidermal growth factor (EGF); 10 ⁇ 10 M cholera toxin (CT); 0.5 ⁇ g/ml of hydrocortisone (HC); 5 ⁇ g/ml of insulin (INS); 2 mM
  • FCS foetal calf serum
  • EGF epidermal growth factor
  • CT cholera toxin
  • HC hydrocortisone
  • INS insulin
  • L-glutamine 100 IU/ml of penicillin-streptomycin
  • PS sebocytes
  • the cells are then treated for 6 days with the sebogenic cocktail corresponding to the combination of PPAR ⁇ agonist rosiglitazone (1 ⁇ M) and the androgen R1881 (10 nM) , or with dimethyl sulphoxide (DMSO) used as carrier.
  • the sebogenic cocktail corresponding to the combination of PPAR ⁇ agonist rosiglitazone (1 ⁇ M) and the androgen R1881 (10 nM) , or with dimethyl sulphoxide (DMSO) used as carrier.
  • the expression of the messenger RNA encoding the MCAM protein was analysed by qRT-PCR using the microfluidics cards technology developed by Applied Biosystems, as described above (Example 2), on a culture of human sebocytes corresponding to one donor.
  • the level of expression (Ct) is represented for each treatment condition.
  • the induction of MCAM expression by the sebogenic cocktail is measured via an induction factor (I. F) versus the DMSO control after standardization of the Cts via the expression of the three housekeeping genes (ribosomal 18S RNA, glyceraldehyde 3-phosphate dehydrogenase GAPDH, beta-actin) .
  • Table 3 qRT-PCR measurement of the expression of MCAM in a primary culture of human sebocytes treated for 6 days with the sebogenic cocktail (combination of 1 ⁇ M PPAR ⁇ agonist rosiglitazone; 10 nM androgen R1881) or with DMSO, via the use of the microfluidic cards technology (Applied Biosystems)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

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

Description

Modulators of MCAM in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
The invention relates to the identification and the use of compounds which modulate the melanoma cell adhesion molecule (MCAM) for treating acne, seborrhoeic dermatitis, and also skin disorders associated with hyperseborrhoea. It also relates to methods for the in vitro diagnosis of or in vitro prognosis for these pathologies.
Hyperseborrhoeic greasy skin is characterized by exaggerated secretion and excretion of sebum. Conventionally, a sebum level greater than 200 μg/cm2 measured on the forehead is considered to be characteristic of greasy skin. Greasy skin is often associated with a desquamation deficiency, a glistening complexion and a thick skin grain. In addition to these aesthetic disorders, excess sebum can serve as a support for the anarchical development of saprophytic bacterial flora {P. acnes in particular), and cause the appearance of comedones and/or acneic lesions.
This stimulation of sebaceous gland production is induced by androgens .
Acne is, in fact, a chronic disease of the pilosebaceous follicle under hormonal control. Hormone therapy against acne is one treatment possibility for women, the objective being to prevent the effects of androgens on the sebaceous gland. In this context, oestrogens, anti-androgens or agents which reduce the production of androgens by the ovaries or the adrenal gland are generally used. The anti-androgens used for the treatment of acne include, in particular, spironolactone, cyproterone acetate and flutamide. However, these agents have potentially severe side effects. Thus, any pregnancy must be absolutely prevented, in particular because of a risk of feminization for the male foetus. These agents are prohibited in male patients.
Seborrhoeic dermatitis is a common inflammatory skin dermatosis which presents in the form of red plaques covered with greasy, yellowish squames, which are more or less pruriginous, and are predominant in the seborrhoeic areas. A need therefore exists, for these diseases, to identify mediators downstream of the action of the steroid hormones, and to modulate them, in order to obtain a similar therapeutic profile, but with reduced side effects. The Applicant has now discovered that the gene encoding the melanoma cell adhesion molecule (MCAM) is expressed preferentially in human sebaceous glands in comparison with the epidermis, and that the expression thereof is regulated in vitro by a cocktail which promotes the differentiation of sebocyte precursors, containing an androgen (R1881, also known as methyltrienolone, at 1 nM) and a PPARγ ligand
(Rosiglitazone, which is 6- (2-methoxyethoxy- methoxy) 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 consequently proposes targeting the MCAM gene or the expression product thereof, for preventing and/or improving acne, seborrhoeic dermatitis and also skin disorders associated with hyperseborrhoea, in particular the greasy skin appearance.
It is, moreover, known that treatment with a PPAR agonist induces a large decrease in the size of the sebaceous glands, and a reduction in androgen-induced hyperseborrhoea (WO2007/093747) .
Since the target proposed is downstream of the PPAR receptor, it is said target which is responsible for the effects observed on the sebaceous glands and on sebum excretion.
Thus, the gene identified can be used to identify the compounds which are the most active as PPAR modulators, to classify them and to select them. On this basis, it is also proposed to use the MCAM gene or the MCAM protein, as a marker for screening for candidate PPAR modulators for the treatment of acne, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea . More specifically, the ability of a PPAR modulator to modulate the expression or the activity of MCAM or the expression of the gene thereof or the activity of at least one of the promoters thereof, can be determined.
The term "acne" is intended to mean all the forms of acne, i.e. in particular acne vulgaris, comedonal acne, polymorphous acne, nodulocystic acne, acne conglobata, or else secondary acne such as solar acne, acne medicamentosa or occupational acne. The Applicant also proposes methods of in vitro, in vivo and clinical diagnosis or prognosis based on the detection of the level of expression or of activity of MCAM.
MCAM
The term "MCAM" denotes the melanoma cell adhesion molecule, also known as CD146, MUC18, gicerin or melanoma adhesion molecule.
MCAM is a surface glycoprotein which belongs to the immunoglobulin superfamily (Lehmann et al . , 1989, PNAS, 86:9891-9895) . Its homology to many cell adhesion molecules made it possible, initially, to demonstrate its role in cell adhesion and in cohesion of the endothelial monolayer at the level of the intercellular junctions in vascular tissue. The protein was then associated with tumour progression and with the development of metastases 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 which triggers FYN and PTK2 tyrosine phosphorylation (Anfosso et al . , 1998, Journal Biol Chem, 273(41) : 26852-26856) and the transient increase in intracellular calcium concentration (Anfosso et al . , 2001, Journal Biol Chem, 276 (2) : 1564-1569) . Reference is made herein, without distinction, to "MCAM protein" or "MCAM receptor" . In the context of the invention, the term "MCAM gene" or "MCAM nucleic acid" signifies the gene or the nucleic acid sequence which encodes the melanoma cell adhesion molecule. While the target aimed for is preferably the human gene or the expression product thereof, the invention may also call upon cells expressing a heterologous melanoma cell adhesion molecule, by genomic integration or transient expression of an exogenous nucleic acid encoding the protein.
A human cDNA sequence of MCAM is reproduced in the annexe (SEQ ID No. 1) . It is the sequence NM_006500 (Genbank) , the open reading frame of which contains 3332 base pairs encoding an amino acid sequence of 603 residues (Lehmann et al . , 1989, PNAS, 86:9891-9895) . The protein thus synthesized is of 113 KDa.
Diagnostic applications
A subject of the invention concerns an in vitro method for diagnosing or monitoring the development of acneic lesions, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea in an individual, comprising the comparison of the expression or of activity of the melanoma cell adhesion molecule (MCAM) , of the expression of the gene thereof or of the activity of at least one of the promoters thereof, in a biological sample from an individual, with respect to a biological sample from a control individual.
The protein expression can be determined by assaying the MCAM protein according to one of the methods such as Western blotting, immunohistochemistry, mass spectrometry analysis (Maldi-TOF and LC/MS analysis), radioimmunoassay (RIA) or 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. Assaying the activity of MCAM can also be envisaged.
In the context of a diagnosis, the "control" individual is a "healthy" individual.
In the context of monitoring the development of acneic lesions, of seborrhoeic dermatitis or of a skin disorder associated with hyperseborrhoea, the "control individual" refers to the same individual at a different time, which preferably corresponds to the beginning of the treatment (TO) . This measurement of the difference in expression or in activity of MCAM, or in expression of the gene thereof or in activity of at least one of the promoters thereof, makes it possible in particular to monitor the effectiveness of a treatment, in particular a treatment with an MCAM modulator, as envisaged above, or another treatment against acne, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea. Such monitoring can reassure the patient with regard to whether continuing the treatment is well-founded or necessary.
Another aspect of the present invention concerns an in vitro method for determining an individual's susceptibility to developing acneic lesions, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea, comprising the comparison of the expression or of the activity of the MCAM protein, of the expression of the gene thereof or of the activity of at least one of the promoters thereof, in a biological sample from an individual, with respect to a biological sample from a control individual.
Here again, the expression of the MCAM protein can be determined by assaying 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. Assaying of the MCAM activity can also be envisaged.
The individual tested is in this case an asymptomatic individual exhibiting no skin condition associated with hyperseborrhoea, seborrhoeic dermatitis or acne. The "control" individual in this method signifies a "healthy" reference population or individual. The detection of this susceptibility makes it possible to set up a preventive treatment and/or increased monitoring of the signs associated with acne, seborrhoeic dermatitis or a 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 may also be sebum.
Screening methods A subject of the invention is an in vitro or in vivo method for screening for candidate compounds for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of any skin disorder associated with hyperseborrhoea, comprising the determination of the ability of a compound to modulate the expression or the activity of the melanoma cell adhesion molecule or the expression of the gene thereof or the activity of at least one of the promoters thereof, said modulation indicating the usefulness of the compound for the preventive or curative treatment of acne, seborrhoeic 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 MCAM, or the expression of the gene thereof, or the activity of at least one of the promoters thereof.
More particularly, the subject of the invention is an in vitro method for screening for candidate compounds for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea, comprising the following steps: a. preparing at least two biological samples or reaction mixtures; b. bringing one of the samples or reaction mixtures into contact with one or more of the test compounds; c. measuring the expression or the activity of the melanoma cell adhesion molecule, the expression of the gene thereof or the activity of at least one of the promoters thereof, in the biological samples or reaction mixtures; d. selecting the compounds for which a modulation of the expression or of the activity of the melanoma cell adhesion molecule, of the expression of the gene thereof or of the activity of at least one of the promoters thereof, is measured in the sample or the mixture treated in b) , compared with the untreated sample or with the untreated mixture.
An in vivo screening method can be carried out in any laboratory animal, for example, a rodent. According to one preferred embodiment, the screening method comprises administering the test compound to the animal preferably by topical application, then optionally sacrificing the animal by euthanasia, and taking a sample of an epidermal split, before evaluating the expression of the gene in the epidermal split, by any method described herein.
The term "modulation" is intended to mean any effect on the expression or the activity of the enzyme, the expression of the gene or the activity of at least one of the promoters thereof, i.e. optionally a stimulation, but preferably a partial or complete inhibition. Thus, the compounds tested in step d) above preferably inhibit the expression or the activity of the melanoma cell adhesion molecule, the expression of the gene thereof or the activity of at least one of the promoters thereof. The difference in expression obtained with the compound tested, compared with a control carried out in the absence of the compound, is significant starting from 25% or more.
Throughout the present text, unless otherwise specified, the term "expression of a gene" is intended to mean the amount of mRNA expressed; the term "expression of a protein" is intended to mean the amount of this protein; the term "activity of the MCAM protein" is intended to mean the ability of the protein to act as a receptor and to induce signal transduction, in particular via FYN and PTK2 protein phosphorylation; the term "activity of a promoter" is intended to mean the ability of this promoter to initiate the transcription of the DNA sequence encoded downstream of this promoter (and therefore indirectly the synthesis of the corresponding protein) .
The compounds tested may be of any type. They may be of natural origin or may have been produced by chemical synthesis. This may involve a library of structurally defined chemical compounds, uncharac- terized compounds or substances, or a mixture of compounds . In particular, the invention is directed towards the use of the MCAM gene or of the MCAM protein, as a marker for candidate PPAR or AR (androgen receptor) modulators for treating acne, seborrhoeic dermatitis and also skin disorders associated with hyperseborrhoea . More specifically, the ability of a PPAR or AR modulator to modulate the expression or the activity of MCAM or the expression of the gene thereof or the activity of at least one of the promoters thereof is determined. Preferably, the modulator is a PPARγ 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 the compounds of therapeutic interest which modulate the expression or the activity of the melanoma cell adhesion molecule. According to a first embodiment, the biological samples are cells transfected with a reporter gene functionally linked to all or part of the promoter of the gene encoding the melanoma cell adhesion molecule, and step c) described above comprises measuring the expression of said reporter gene.
The reporter gene may in particular encode an enzyme which, in the presence of a given substrate, results in the formation of coloured products, such as CAT (chloramphenicol acetyltransferase) , GAL (beta- galactosidase) or GUS (beta-glucuronidase) . It may also be the luceriferase gene or the GFP (green fluorescent protein) gene. The assaying of the protein encoded by the reporter gene, or of the activity thereof, is carried out conventionally by colorimetric, fluorometric or chemiluminescence techniques, inter alia.
According to a second embodiment, the biological samples are cells expressing the gene encoding the melanoma cell adhesion molecule, and step c) described above comprises measuring the expression of said gene.
The cell used herein may be of any type. It may be a cell expressing the MCAM gene endogenously, for instance an endothelial cell, a lymphocyte cell, or better still a sebocyte. Organs of human or animal origin may also be used, for instance the preputial gland, the clitoral gland, or else the sebaceous gland of the skin.
It may also be a cell transformed with a heterologous nucleic acid encoding the preferably human, or mammalian, melanoma cell adhesion molecule.
A large variety of host-cell systems may be used, such as, for example, Cos-7, CHO, BHK, 3T3 or HEK293 cells. The nucleic acid may be transfected stably or transiently, 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 of the reporter gene can be determined by evaluating the level of transcription of said gene, or the level of translation thereof.
The expression "level of transcription of a gene" is intended to mean the amount of corresponding mRNA produced. The expression "level of translation of a gene" is intended to mean the amount of protein produced.
Those skilled in the art are familiar with the techniques for quantitatively or semi-quantitatively detecting the mRNA of a gene of interest. Techniques based on hybridization of the mRNA with specific nucleotide probes are the most common (Northern blotting, RT-PCR (reverse transcriptase polymerase chain reaction) , quantitative RT-PCR (qRT-PCR) , RNase protection) . It may be advantageous to use detection labels, such as fluorescent, radioactive or enzymatic agents or other ligands (for example, avidin/biotin) .
In particular, the expression of the gene can be measured by real-time PCR or by RNase protection. The term "RNase protection" is intended to mean the detection of a known mRNA among the poly (A) -RNAs of a tissue, which can be carried out using specific hybrid¬ ization with a labelled probe. The probe is a labelled (radioactive) RNA complementary to the messenger to be sought. It can be constructed from a known mRNA, the cDNA of which, after RT-PCR, has been cloned into a phage. PoIy(A)-RNA from the tissue in which the sequence is to be sought is incubated with this probe under slow hybridization conditions in a liquid medium. RNAiRNA hybrids form between the mRNA sought and the antisense probe. The hybridized medium is then incubated with a mixture of ribonucleases specific for single-stranded RNA, such that only the hybrids formed with the probe can withstand this digestion. The digestion product is then deproteinated and repurified, before being analysed by electrophoresis. The labelled hybrid RNAs are detected by autoradiography.
The level of translation of the gene is evaluated, for example, by immunological assaying of the product of said gene. The antibodies used for this purpose may be of polyclonal or monoclonal type. The production thereof involves conventional techniques. An anti- melanoma cell adhesion molecule polyclonal antibody can, inter alia, be obtained by immunization of an animal, such as a rabbit or a mouse, with the whole protein. The antiserum is taken and then depleted according to methods known per se to those skilled in the art. A monoclonal antibody can, inter alia, be obtained by the conventional method of Kδhler and Milstein (Nature (London), 256: 495-497 (1975)) . Other methods for preparing monoclonal antibodies are also known. Monoclonal antibodies can, for example, be produced by expression of a nucleic acid cloned from a hybridoma. Antibodies can also be produced by the phage display technique, by introducing antibody cDNAs into vectors, which are typically filamentous phages which display V-gene libraries at the surface of the phage
(for example, fUSE5 for E.coli) . The immunological assaying can be carried out in solid phase or in homogeneous phase; in one step or in two steps; in a sandwich method or in a competition method, by way of nonlimiting examples. According to one preferred embodiment, the capture antibody is immobilized on a solid phase. By way of nonlimiting examples of a solid phase, use may be made of microplates, in particular polystyrene microplates, or solid particles or beads, or 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 the antigen/antibody complexes, and more generally of the isolated or purified, but also recombinant, proteins (obtained in vitro and in vivo) can be carried out by mass spectrometry analysis. This identification is made possible by virtue of the analysis (determination of the mass) of the peptides generated by enzymatic hydrolysis of the proteins (in general, trypsin) . In general, the proteins are isolated according to the methods known to those skilled in the art, prior to the enzymatic digestion. The analysis of the peptides (in hydrolysate form) is carried out by separating of the peptides by HPLC (nano-HPLC) based on their physicochemical properties (reverse phase) . The deter¬ mination of the mass of the peptides thus separated is carried out by ionization of the peptides and either by direct coupling with mass spectrometry (electrospray ESI mode) , or after deposition and crystallization in the presence of a matrix known to those skilled in the art (analysis in MALDI mode) . The proteins are subsequently identified through the use of appropriate software (for example, Mascot) . According to a third embodiment, the screening method comprises bringing a compound into contact with an MCAM receptor protein and determining the ability of the compound to modulate the activity of the MCAM protein, a difference in activity, compared to a control carried out in the absence of the compound, indicating the usefulness of the compound for the preventive or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea . Preferably, the ability of the compound to bind to the MCAM protein is also evaluated.
It is subsequently determined whether this binding causes an agonistic or antagonistic effect on the activity of the MCAM protein, by evaluating the impact of the compound on the signalling pathway controlled by the MCAM receptor.
The determination of the activity of the MCAM protein can be carried out in various ways. It refers in particular to the determination of the cell adhesion activity, of transporter activity, of apoptotic activity, of activity in lipid metabolism, in signal transduction and/or in cytokine secretion.
The compounds selected by means of the screening methods defined herein can subsequently be tested on other in vitro models and/or in vivo models (in animals or humans) for their effects on acne, seborrhoeic dermatitis or skin disorders associated with hyperseborrhoea .
Modulators of the receptor
A subject of the invention is also the use of a modulator of the human MCAM protein, that can be obtained by means of one of the methods above, for the preparation of a medicament for use in the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea .
A method for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea is thus described herein, said method comprising the administration of a therapeutically effective amount of a modulator of the human melanoma cell adhesion molecule protein to a patient requiring such a treatment .
Finally, the invention is directed towards the cosmetic use of a modulator of the human melanoma cell adhesion molecule protein, for the aesthetic treatment of greasy skin.
Preferably, the modulator is an antagonist of the receptor. The term "antagonist" refers to a compound or a chemical substance which eliminates or substantially reduces the biological activity of the melanoma cell adhesion molecule. The term "substantially" signifies a reduction of at least 25%, preferably of at least 35%, more preferably of at least 50%, and more preferably of at least 70% or 90%.
A preferred antagonist interacts with the receptor in solution at antagonist concentrations of less than 20 μM, less than 10 μM, less than 5 μM, less than 1 μM, preferably less than 0.1 μM, more preferably less than 0.01 μM. The modulator compound may be an anti-melanoma cell adhesion molecule, preferably a monoclonal antibody. Advantageously, such an inhibitory antibody is administered in an amount sufficient to obtain a plasma concentration of approximately 0.01 μg per ml to approximately 100 μg/ml, preferably of approximately 1 μg per ml to approximately 5 μg/ml.
The modulator compound may also be a polypeptide, an antisense DNA or RNA polynucleotide, an siRNA or a PNA (peptide nucleic acid, polypeptide chain substituted with purine and pyrimidine bases, the spatial structure of which mimics that of the DNA and enables hybridization thereto) .
The modulator compound may also be an aptamer. The aptamer is a class of molecules representing, in terms of molecular recognition, an alternative to the antibodies. They are oligonucleotide sequences which have the ability to recognize virtually all the classes of target molecules with high affinity and specificity. Such ligands can be isolated by systematic evolution of ligand by exponential enrichment (SELEX) carried out on a random-sequence library, as described by Tuerk and Gold, 1990. The random-sequence library can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear, optionally chemically modified, oligomer of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena, 1999, Clinical Chemistry 45(9) : 1628-1650. A number of modulators of the melanoma cell adhesion molecule are known and proposed as treatment for reducing the growth of melana tumours (EP 1382615; Mills et al., 2002, Cancer Research, 62 (17) : 5106-14) . The invention comprises the use of such melanoma cell adhesion molecule antagonist compounds for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea . In a nonlimiting manner, mention may be made of the AP-2 transcription factor (Jean et al., 1998, Journal Biol Chem, 273 (26) : 16501-16508) or of the anti-MCAM ABX-MAl antibody as inhibitors of the melanoma cell adhesion molecule.
Other modulated compounds identified by the screening method described above are also useful.
The modulator compounds are formulated within a pharmaceutical composition, in combination with a pharmaceutically acceptable carrier. These compositions may be administered, for example, orally, enterally, parenterally, or topically. Preferably, the pharmaceutical composition is applied topically. By oral administration, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid or polymeric vesicles for controlled release. By parenteral administration, the pharmaceutical composition may be in the form of solutions or suspensions for a drip or for injection. By topical administration, the pharmaceutical composition is more particularly for use in treating the skin and the mucous membranes and may be in the form of salves, creams, milks, ointments, powders, impregnated pads, 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 for 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, in particular from 0.01% to 5% by weight, relative to the total weight of the composition.
The pharmaceutical composition may also contain inert additives or combinations of these additives, such as wetting agents; flavour enhancers;
- preservatives such as para-hydroxybenzoic acid esters; - stabilizers; moisture regulators; pH regulators; osmotic pressure modifiers;
- emulsifiers; - UV-A and UV-B screens; and antioxidants, such as alpha-tocopherol, butylhydroxyanisol or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelating agents.
The following examples illustrate the invention without limiting the scope thereof.
Examples: EXPERIMENTAL DATA
Example 1: Expression of the melanoma cell adhesion molecule in the human sebaceous gland and in human epidermis
Human sebaceous glands were separated from human epidermis by treatment with dispase and dissection under a binocular magnifying lens. Total RNA samples were prepared from the sebaceous glands and from the epidermis .
The expression of the genes was analysed on an Affymetrix station (microfluidic module; hybridization oven; scanner; computer) according to the protocols supplied by the company. Briefly, the total RNA isolated from the tissues is transcribed into cDNA. A biotin-labelled cRNA is synthesized, from the double- stranded cDNA, using T7 polymerase and a precursor NTP conjugated to biotin. The cRNAs are subsequently fragmented into small fragments. All the molecular biology steps are verified using the Agilent "lab on a chip" system in order to confirm that the enzymatic reactions are very efficient. The Affymetrix chip is hybridized with the biotinylated cRNA, rinsed, and subsequently labelled by fluorescence using a Streptavidin-conjugated fluorophore. After washing, the chip is scanned and the results are calculated using the MAS5 software supplied by Affymetrix. An expression value is obtained for each gene, as is 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 which are obtained following hybridization of the cRNA of a given gene with a perfect match oligonucleotide versus an oligonucleotide which contains a single mismatch in the central region of the oligonucleotide (see Table 1) .
Table 1: Measurement of the expression of the melanoma cell adhesion molecule in the epidermis and in the human sebaceous gland by the use of the Affymetrix chip technology
indicator of the significance of the expression of the gene analysed in the sample indicated: presence (= 1) or absence (= 0) .
Example 2 : Expression of the melanoma cell adhesion molecule in the human sebaceous gland and in human epidermis
The samples of epidermis and of human sebaceous gland were prepared by laser microdissection from three lifts of healthy human skin (female donors) .
The expression of the messenger RNA encoding the MCAM protein was analysed by quantitative RT-PCR (qRT- PCR) using the microfluidics cards technology developed by Applied Biosystems.
The Ct corresponds to the number of PCR cycles which makes it possible to choose the same level of fluorescence for all the samples. The level of expression is represented in each tissue by the mean of the Cts and the standard deviation obtained on the three donors .
The differential expression between the two tissues is measured via a mean induction factor (I. F) for the sebaceous gland with respect to the epidermis after standardization of the Cts via the expression of the three housekeeping genes (ribosomal 18S RNA, glyceraldehyde 3-phosphate dehydrogenase GAPDH, beta- actin) .
Table 2: qRT-PCR measurement of the expression of MCAM in the epidermis and the human sebaceous gland via the use of the microfluidic cards technology (Applied Biosystems)
Example 3: Expression of the melanoma cell adhesion molecule in human sebocytes in primary culture
a. Isolation and culture of human sebocytes
Human sebocytes are cultured using lifts from healthy human donors according to the method described by Xia et al . (J Invest Dermatol. 1989 Sep; 93 (3) : 315- 21) after separation of the epidermis from the dermis through the action of dispase and microdissection of the sebaceous glands under binocular magnifying lenses.
The sebaceous glands are seeded in 6-well plates on a feeder layer of mitomycin-treated 3T3 fibroblasts in DMEM-Ham' s F12 (3:1) medium supplemented with 10% foetal calf serum (FCS) ; 10 ng/ml of epidermal growth factor (EGF); 10~10 M cholera toxin (CT); 0.5 μg/ml of hydrocortisone (HC); 5 μg/ml of insulin (INS); 2 mM
L-glutamine (GIn); 100 IU/ml of 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 agonist rosiglitazone (1 μM) and the androgen R1881 (10 nM) , or with dimethyl sulphoxide (DMSO) used as carrier.
b. PCR expression data
The expression of the messenger RNA encoding the MCAM protein was analysed by qRT-PCR using the microfluidics cards technology developed by Applied Biosystems, as described above (Example 2), on a culture of human sebocytes corresponding to one donor. The level of expression (Ct) is represented for each treatment condition.
The induction of MCAM expression by the sebogenic cocktail is measured via an induction factor (I. F) versus the DMSO control after standardization of the Cts via the expression of the three housekeeping genes (ribosomal 18S RNA, glyceraldehyde 3-phosphate dehydrogenase GAPDH, beta-actin) .
Table 3: qRT-PCR measurement of the expression of MCAM in a primary culture of human sebocytes treated for 6 days with the sebogenic cocktail (combination of 1 μM PPARγ agonist rosiglitazone; 10 nM androgen R1881) or with DMSO, via the use of the microfluidic cards technology (Applied Biosystems)

Claims

1. In vitro or in vivo method for screening for candidate compounds for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea, comprising the determination of the ability of a compound to modulate the expression or the activity of the melanoma cell adhesion molecule (MCAM) or the expression of the gene thereof or the activity of at least one of the promoters thereof.
2. In vitro method for screening for candidate compounds for the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea according to Claim 1, comprising the following steps: a. preparing at least two biological samples or reaction mixtures; b. bringing one of the samples or reaction mixtures into contact with one or more of the test compounds; c. measuring the expression or the activity of the melanoma cell adhesion molecule, the expression of the gene thereof or the activity of at least one of the promoters thereof, in the biological samples or reaction mixtures; d. selecting the compounds for which a modulation of the expression or of the activity of the MCAM protein, or a modulation of the expression of the gene thereof or a modulation of the activity of at least one of the promoters thereof, is measured in the sample or the mixture treated in b) compared with the untreated sample or with the untreated 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 the gene thereof or the activity of at least one of the promoters thereof .
4. Method according to Claim 2 or 3, characterized in that the biological samples are cells transfected with a reporter gene functionally linked to all or part of the promoter of the gene encoding the melanoma cell adhesion molecule, and in that step c) comprises 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 encoding the melanoma cell adhesion molecule, and in that step c) comprises measuring the expression of said gene.
6. Method according to Claim 4 or 5, in which the cells are sebocytes.
7. Method according to Claim 5, in which the cells are cells transformed with a heterologous nucleic acid encoding the melanoma cell adhesion molecule.
8. Method according to one of Claims 2 to 7, in which the expression of the gene is determined by measuring the level of transcription of said gene.
9. Method according to one of Claims 2 to 7, in which the expression of the gene is determined by measuring the level of translation of said gene.
10. Method according to Claim 1, comprising bringing a compound into contact with an MCAM receptor and determining the activity of the MCAM protein, a difference in activity, compared to a control carried out in the absence of the compound, indicating the usefulness of the compound for the preventive or curative treatment of acne, of seborrhoeic dermatitis or of a skin disorder associated with hyperseborrhoea .
11. Use of a modulator of the human MCAM receptor, that can be obtained by means of the method according to one of Claims 2 to 10, for the preparation of a medicament for use in the preventive and/or curative treatment of acne, of seborrhoeic dermatitis or of skin disorders associated with hyperseborrhoea.
12. Use according to Claim 11, in which the modulator is an antagonist of the MCAM receptor.
13. Cosmetic use of a modulator of the human MCAM protein, for the aesthetic treatment of greasy skin.
14. In vitro method for diagnosing or monitoring the development of acne, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea in an individual, comprising the comparison of the expression or of the activity of the melanoma cell adhesion molecule, of the expression of the gene thereof or of the activity of at least one of the promoters thereof, in a biological sample from an individual, with respect to a biological sample from a control individual.
15. Method according to Claim 14, in which the expression of the protein is determined by assaying this protein by immunoassay.
16. Method according to Claim 15, in which the immunoassay is an ELISA assay.
17. Method according to Claim 14, in which the expression of the gene is determined by measuring the amount of corresponding mRNA.
18. In vitro method for determining an individual's susceptibility to developing acne, seborrhoeic dermatitis or a skin disorder associated with hyperseborrhoea, comprising the comparison of the expression or of the activity of the MCAM protein, of the expression of the gene thereof or of the activity of at least one of the promoters thereof, in a biological sample from an individual, with respect to a biological sample from a control individual.
19. Method according to Claim 18, in which the expression of the protein is determined by assaying this protein by means of an immunoassay.
20. Method according to Claim 19, in which the immunoassay is an ELISA assay or a radioimmunoassay.
21. Method according to Claim 18, in which the expression of the gene is determined by measuring the amount of corresponding mRNA.
22. Use of the MCAM gene or of the MCAM protein, as a marker for screening for candidate PPAR modulators for the treatment of acne, of seborrhoeic dermatitis or of a skin disorder associated with hyperseborrhoea .
23. Use according to Claim 22, comprising the determination of the ability of a PPAR modulator to modulate the expression or the activity of MCAM or the expression of the gene thereof or the activity of at least one of the promoters thereof.
24. Use according to Claim 22 or 23, in which the PPAR modulator is a PPARγ modulator.
25. Use according to any one of Claims 22 to 24, in which the modulator is a PPAR receptor agonist.
26. Use of an MCAM gene or of an MCAM protein, as a marker for screening for candidate AR (androgen receptor) modulators for the treatment of acne, of seborrhoeic dermatitis or of a skin disorder associated with hyperseborrhoea.
27. Use according to Claim 26, comprising the determination of the ability of an AR modulator to modulate the expression or the activity of MCAM or the expression of the gene thereof or the activity of at least one of the promoters thereof.
28. Use according to either one of Claims 26 and 27, in which the modulator is an androgen receptor agonist .
EP09742128A 2008-05-07 2009-05-07 Modulators of mcam in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea Withdrawn EP2285987A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US7160208P 2008-05-07 2008-05-07
FR0857706A FR2938337A1 (en) 2008-11-13 2008-11-13 MCAM MODULATORS FOR THE TREATMENT OF ACNE, SEBORRHEA DERMATITIS OR HYPERSEBORRHEA
PCT/EP2009/055555 WO2009135910A2 (en) 2008-05-07 2009-05-07 Modulators of mcam in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea

Publications (1)

Publication Number Publication Date
EP2285987A2 true EP2285987A2 (en) 2011-02-23

Family

ID=40875197

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09742128A Withdrawn EP2285987A2 (en) 2008-05-07 2009-05-07 Modulators of mcam in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea

Country Status (3)

Country Link
EP (1) EP2285987A2 (en)
FR (1) FR2938337A1 (en)
WO (1) WO2009135910A2 (en)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7317091B2 (en) * 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
FR2862870A1 (en) * 2003-12-01 2005-06-03 Galderma Res & Dev USE OF PPAR RECEPTOR ACTIVATORS IN COSMETICS AND DERMATOLOGY.
FR2904003B1 (en) * 2006-07-19 2008-09-05 Galderma Res & Dev S N C Snc ABCD3 TRANSPORTER MODULATORS IN THE TREATMENT OF ACNE OR HYPERSEBORRHEA
FR2904004B1 (en) * 2006-07-19 2008-09-05 Galderma Res & Dev S N C Snc MODULATORS OF SCARB-1 IN THE TREATMENT OF ACNE OR HYPERSEBORRHEA

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2009135910A3 *

Also Published As

Publication number Publication date
WO2009135910A2 (en) 2009-11-12
WO2009135910A3 (en) 2009-12-23
FR2938337A1 (en) 2010-05-14

Similar Documents

Publication Publication Date Title
US20110150773A1 (en) Modulators of acetyl-coenzyme a acyltransferase 1 or 2 in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20110262450A1 (en) Modulators of monoglyceride lipase in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20110165168A1 (en) Cidea modulators in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20100021892A1 (en) Modulators of SC4MOL for treating acne or hyperseborrhea
US20090246776A1 (en) Modulators of scarb-1 for treating acne or hyperseborrhea
US20090258356A1 (en) Modulators of the transporter ABCD3 for treating acne or hyperseborrhea
US20110213009A1 (en) Adfp modulators in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20090298074A1 (en) Modulators of ELOVL5 for treating acne or hyperseborrhea
WO2009135910A2 (en) Modulators of mcam in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
CA2723838A1 (en) Screening for modulators of ces1 and/or ces3 for the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20110263677A1 (en) Gos2 modulators in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20110268742A1 (en) Pctp modulators in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20110150772A1 (en) Modulators of carnitine octanoyltransferase in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20110262451A1 (en) Modulators of isovaleryl-coenzyme a dehydrogenase in the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea
US20110189686A1 (en) Screening for modulators of cyp2b15 and/or gpd1 for the treatment of acne, of seborrhoeic dermatitis or of hyperseborrhoea

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20101207

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA RS

RIN1 Information on inventor provided before grant (corrected)

Inventor name: VOEGEL, JOHANNES

Inventor name: TRINQUET, VALERIE

Inventor name: AUBERT, JEROME

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20120405

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20120817