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
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • 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
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• • 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 use of hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7) modulator compounds for the treatment of acne, as well as skin disorders associated with hyperseborrhoea. It also relates to methods of in vitro diagnosis or prognosis in vitro of these pathologies.
• HSD17b7 hydroxysteroid (17-beta) dehydrogenase 7
• Hyperseborrhoeic oily skin is characterized by excessive secretion and excretion of sebum.
• a level of sebum 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.
• 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.
• 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 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.
• the Applicant has now discovered that the HSD17b7 gene is expressed in the human sebaceous glands, and that its expression is regulated by androgens, in vivo, in a mouse preputial gland model. It therefore proposes to target this gene, or its expression product, the hydroxysteroid enzyme (17-beta) dehydrogenase 7, to prevent or improve acne phenomena, as well as skin disorders associated with a hyperseborrhoea, especially the oily skin appearance.
• acne we mean all forms of acne, namely in particular vulgar acne, comedonal, polymorphic, nodulocystic acne, conglobata, or secondary acne such as solar acne, drug or professional.
• the Applicant also proposes in vitro diagnostic methods or prognostic in vitro, based on the detection of the expression or activity of the hydroxysteroid (17-beta) dehydrogenase type 7.
• the protein HSD17b7 which refers to the hydroxysteroid (17-beta) dehydrogenase type 7, belongs to the family of hydroxysteroid (17-beta) dehydrogenases, which catalyze the last reaction of the biosynthesis of several estrogens and androgens, such as estradiol, 5 ⁇ -androstane-3 ⁇ , 17 ⁇ -diol, testosterone, and dihydrotestosterone. Twelve isoforms are known to have specific reducing or oxidizing activities (Poirier, Current Medicinal Chemistry, 2003, 10: 453-477, Thiboutot et al, J. Invest Dermatol, 1998, 11: 390-395).
• HSD17b As an androgenic stimulation is responsible for an increase in sebum production, which can induce acne lesions, the inhibition of 17beta- hydroxysteroid dehydrogenases, key enzymes in androgen metabolism, has been indicated as a treatment potential of acne. In these cases, a non-specific inhibition of one or more hydroxysteroid (17-beta) dehydrogenases is sought.
• Members of the HSD17b family have less than 30% primary structure homology to each other. The different types of HSD17b differ in their substrate and cofactor specificities (Labrie et al (2000) Trends Endocrinol Metab., 1: 421-7). In addition, certain types of HSD 17b are involved in the pathogenicity of certain human disorders.
• HSD17b-3 is known to be involved in the development of pseudohermaphroditism
• HSD17b-8 plays a role in multiple cystic kidney disease
• HSD17b-4 is related to the occurrence of bifunctional enzyme deficiency.
• By modulating a particular HSD17b it is possible to influence or control the local and paracrine concentration of estrogens and androgens in different tissues.
• Several reversible and irreversible inhibitors of HSD17b-2 enzymes of steroidal or nonsteroidal origin are already known in the literature (Poirier D. (2003) Curr Med Chem 10: 453-77).
• the application WO 02/26706 which describes inhibitors of HSD17b-2 of non-steroidal origin.
• JP48042271 discloses compounds used as an anti-inflammatory compound.
• US Pat. No. 5,597,823 describes, for its part, adrenergic antagonists that are useful for the treatment of hyperplasia. benign prostate.
• Patent application JP62132884 discloses benzylthienopyrimidinones for the treatment of cardiovascular disorders.
• WO2005032527 relates to the use of thiophenepyrimidinone derivatives, for the treatment or prevention of steroid hormone-dependent diseases by the inhibition of 17 ⁇ -HSD-1. Since each subtype of HSD17b has a selective substrate affinity, a particular tissue distribution, the selectivity of the action can be achieved by targeting a particular HSD17b isozyme.
• HSD17b7 In addition to its ability to synthesize 17 ⁇ -estradiol in vitro for which there is no evidence of physiological relevance to date, the protein HSD17b7 also has 3-ketosteroid reductase activity, which is the aspect of interest. in the present invention: HSD17b7 is capable of complementing a deficiency of 3-ketosteroid reductase activity in Erg27p-deficient yeasts and thus allows cell growth in a sterol-free medium.
• HSD17b7 is localized in the endoplasmatic reticulum, which is the site of the postsqualene part of cholesterol synthesis, and is expressed specifically in tissues involved in congenital cholesterol deficiency disorders (Marijanovic et al, Molecular Endocrinology 2003, 17 (9 ): 1715-1725).
• HSD17b7 gene or "HSD17b7 nucleic acid” means the gene or nucleic acid sequence that encodes the hydroxysteroid (17-beta) dehydrogenase type 7. If the targeted target is preferably the human gene or its expression product, the invention may also use cells expressing a heterologous type 7 hydroxysteroid (17-beta) dehydrogenase, by genomic integration or transient expression of an exogenous nucleic acid coding for the enzyme (for example an enzyme from another organism).
• a human cDNA sequence of HSD17b is reproduced in the appendix (SEQ ID No. 1). ). It is the NM016371 sequence whose coding portion is from nucleic acid 96 to 1121.
• Another subject of the invention relates to an in vitro method for diagnosing or monitoring the progression of acne lesions or a skin disorder associated with a hyperseborrhea in a subject, comprising comparing the expression or the activity hydroxysteroid (17-beta) dehydrogenase type 7 (HSD 17B7), the expression of its gene or activity of at least one of its promoters, in a biological sample of a subject compared to a biological sample of a control subject.
• the expression of the protein can be determined by assaying the protein (HSD17B7) by radioimmunoassay, for example by ELISA assay.
• Another method, especially for measuring the expression of the gene is to measure the amount of corresponding mRNA by any method as described above.
• An assay of the activity of the enzyme can also be envisaged.
• control is a "healthy” subject.
• control subject refers to the same subject at a different time, which preferably corresponds to the beginning of the treatment (To ).
• This measurement of the difference in the expression or activity of the protein or the expression of its gene or the activity of at least one of its promoters notably makes it possible to monitor the efficacy of a treatment, in particular a treatment. by a modulator of the enzyme, as envisaged above or by another treatment against acne or a skin disorder associated with hyperseborrhoea.
• 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 or skin disorder associated with hyperseborrhoea, comprising comparing the expression or activity of the subject.
• HSD17b7 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 expression of the HSD17b7 protein can be determined by assaying this protein by radioimmunoassay, for example by ELISA assay.
• Another method, especially for measuring the expression of its gene is to measure the amount of corresponding mRNA by any method as described above.
• An assay of the activity of the enzyme can also be envisaged.
• the subject tested here is an asymptomatic subject, having no skin disorder associated with hyperseborrhoea or acne.
• the subject "control" in this method means a "healthy" reference subject or population.
• the detection of this susceptibility allows the establishment of a preventive treatment and / or increased monitoring of signs related to acne 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 can also be sebum.
• An object of the invention is an in vitro method for screening candidate compounds for the preventive and / or curative treatment of acne, or skin disorders associated with hyperseborrhoea, comprising determining the ability of a compound to modulate the expression or the activity of the hydroxysteroid (17-beta) dehydrogenase type 7 or the expression of its gene or the activity of at least one of its promoters, a modulation of the expression or of the activity of the enzyme indicating the utility of the compound for the preventive or curative treatment of acne or skin disorders associated with hyperseborrhoea.
• the method therefore makes it possible to select compounds capable of modulating the expression or the activity of HSD17b7, or the expression of its gene, or the activity of at least one of its promoters.
• the invention relates to an in vitro method for screening candidate compounds for the preventive and / or curative treatment of acne and / or skin disorders associated with hyperseborrhoea, 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 the activity of the hydroxysteroid protein
• (17-beta) dehydrogenase type 7 the expression of its gene or the activity of at least one of its promoters
• “Modulation” means any effect on the expression or the activity of the enzyme, the expression of the gene or the activity of at least one of its promoters, that is to say, a stimulation, but preferably a inhibition, partial or complete. 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.
• expression of a protein means the amount of that protein
• protein activity is meant its biological activity
• 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.
• the biological samples are cells transfected with a reporter gene operably linked to all or part of the promoter of the HSD17b7 gene, and the step c) described above consists in measuring the expression of said gene. reporter.
• 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 of its activity is carried out conventionally, by colorimetric, fluorometric or chemiluminescence techniques, among others.
• the biological samples are cells expressing the HSD17b7 gene coding for the hydroxysteroid (17-beta) dehydrogenase type 7, 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 HSD17b7 gene endogenously, such as for example a liver cell, an ovarian cell, or even better 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 a hydroxysteroid (17-beta) dehydrogenase 7, preferably human, or mammalian.
• a heterologous nucleic acid encoding a hydroxysteroid (17-beta) dehydrogenase 7, preferably human, or mammalian.
• 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.
• the level of expression of the HSD17B7 gene can be determined by evaluating the transcription rate of said gene, or its rate of translation.
• transcription rate of a gene is meant the amount of corresponding mRNA produced.
• translation rate of a gene is meant the amount of corresponding protein produced.
• the expression of the gene can be measured by real-time PCR or by RNase protection.
• RNase protection is meant the detection of a known mRNA among 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 hybrid 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-hydroxysteroid antibody (17-beta) dehydrogenase 7 may, inter alia, be obtained by immunizing an animal such as a rabbit or a mouse, using the entire enzyme, sampling and then depleting the antiserum according to methods in themselves 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.
• 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.
• the capture antibody is immobilized on a solid phase.
• 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).
• 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
• HPLC nano-HPLC
• 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 (ESI electrospray 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 then identified through the use of appropriate software (eg Mascot).
• step a) described above consists in preparing reaction mixtures comprising a hydroxysteroid enzyme (17-beta) dehydrogenase 7 and a substrate of the enzyme, and step c) described above. above is to measure enzymatic activity.
• step a) consists of preparing reaction mixtures comprising a hydroxysteroid enzyme (17-beta) dehydrogenase 7 and a substrate of the enzyme, and a reductase system, and step c) consists in measuring the activity enzyme.
• the enzyme can be produced according to standard techniques using Cos-7, CHO, BHK, 3T3, HEK293 cells. It can also be produced using microorganisms such as bacteria (for example E. coli or B. subtilis), yeasts (for example Saccharomyces, Pichia) or insect cells, such as Sf9 or Sf21.
• bacteria for example E. coli or B. subtilis
• yeasts for example Saccharomyces, Pichia
• insect cells such as Sf9 or Sf21.
• An example of measurement of the enzymatic activity can be done according to the classical method of Marijanovic et al. Mol Endocrinol, September 2003, 17 (9): 1715-1725. It consists of measuring the conversion of zymosterone to zymosterol by the hydroxysteroid (17 beta) dehydrogenase.
• the enzyme is incubated for 90 min at 37 ° C. in a reaction medium containing 880 ⁇ l of buffer (10 mM KPI, 0.05% BSA, and 1 mM EDTA pH8), 10 ⁇ L of bacterial lysate, 10 ⁇ L of NADPH (5 mg / ml), 10 ⁇ l of substrate (zymosterone, or 17 ⁇ estradiol, or androsterone).
• the steroids produced during the reaction are extracted by RP18 column chromatography and eluted twice with a 1: 1 solution of methanol: chloroform and once with chloroform alone. After evaporation of the solvent, the steroids are solubilized in chloroform, separated by thin layer chromatography (Merck) using an 80:20 solution of toluene: ethyl acetate as a mobile phase and detected by spraying with a solution of 30% H 2 SO 4 in ethanol and developed at 135 9 C. Identification of substrates and metabolites is done by comparison with reference steroids.
• the subject of the invention is also the use of a modulator of the human hydroxysteroid (17-beta) dehydrogenase 7 enzyme that can be obtained by one of the above methods for the preparation of a medicament intended for preventive and / or curative treatment of acne, or skin disorders associated with hyperseborrhoea. It is thus described here a method of preventive and / or curative treatment of acne, or skin disorders associated with a hyperseborrhea, a method comprising administering a therapeutically effective amount of a modulator of the human hydroxysteroid enzyme (17-beta) dehydrogenase 7, to a patient in need of such treatment.
• the invention finally relates to the cosmetic use of a modulator of the human hydroxysteroid enzyme (17-beta) dehydrogenase 7, for the aesthetic treatment of oily skin.
• the modulator is an inhibitor of the enzyme.
• inhibitor refers to a compound or a chemical substance that substantially eliminates or reduces the enzymatic activity of the hydroxysteroid (17-beta) dehydrogenase 7.
• 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%. More particularly it may be a compound that interacts with, and blocks, the catalytic site of the enzyme, as competitive inhibitory type compounds.
• a preferred inhibitor interacts with the enzyme in solution at inhibitor concentrations of less than 1 ⁇ M, preferably less than 0.1 ⁇ M, more preferably less than 0.01 ⁇ M.
• the modulator compound may be an anti-hydroxysteroid (17-beta) dehydrogenase 7 inhibitory antibody, preferably a monoclonal antibody.
• an inhibitory antibody is administered in an amount sufficient to obtain a plasma concentration of about 0.01 ⁇ g per ml to about 100 ⁇ g / ml, preferably from about 1 ⁇ g per ml to about 5 ⁇ g / ml.
• the modulator compound may also be a polypeptide, an antisense DNA or RNA polynucleotide, an si-RNA, or a PNA ("Peptide nucleic acid", a polypeptide chain substituted with purine and pyrimidine bases, whose spatial structure mimes that of DNA and allows hybridization to it).
• PNA Peptide nucleic acid
• hydroxysteroid (17-beta) dehydrogenase inhibitors are known, but the invention preferably targets the use of specific inhibitors of isoform 7.
• the invention comprises the use of such hydroxysteroid-inhibiting compounds (17-beta ) dehydrogenase 7 for the preventive and / or curative treatment of acne, or skin disorders associated with hyperseborrhoea.
• the modulator compounds are formulated in pharmaceutical compositions, in association with a pharmaceutically acceptable carrier. These compositions may be administered, for example, orally, parenterally, or topically. Preferably, the pharmaceutical composition is applied topically. Orally, the pharmaceutical composition may be in the form of tablets, capsules, lozenges, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid or polymeric vesicles allowing controlled release. Parenterally, the pharmaceutical composition may be in the form of solutions or suspensions for infusion or for injection.
• 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.
• the pharmaceutical composition is in the form of a gel, a cream or a lotion.
• the composition may comprise a modulator content of HSD17b7 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 of these additives, such as:
• osmotic pressure modifying agents emulsifying agents
• antioxidants such as alpha-tocopherol, butylhydroxyanisole or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelators.
• Figs. 1A and 1B are graphs which show the extent of expression of the HSD17B7 gene in male gonadectomized and vehicle-treated mice, DHT, DHEA or the combination of DHEA-Flutamide for a period of 7 days. times a day (long-term treatment).
• the results obtained by the Affymetrix technique (FIG. 1A) were confirmed by the RT-PCR technique in real time (FIG. 1B).
• GDX gonadectomized and vehicle-treated mice
• DHT gonadectomized mice treated with Dihydrotestosterone (androgen receptor agonist)
• DHEA mice gonadectomized and treated with Dihydroepiandrosterone (precursor of steroid hormones, in preputial glands metabolized to active androgen)
• DHEA-FIu gonadectomized mice and treated with a combination of Dihydroepiandrosterone and Flutamide (Androgen receptor antagonists, which block the effects DHT and DHEA agonists).
• Figures 2A and 2B are graphs reporting a kinetic study from 15 minutes to 96 hours using two different sets of primers ("probe sets") hybridizing to different regions of the HSD17B7 gene.
• the set of primers used is 1417871_at
• the set of primers used is 1448865_at.
• Figures 2C and 2D are graphs reporting a 1 hour to 24 hour kinetic study using two different probe sets that hybridize to different regions of the HSD17B7 gene.
• Figure 2C shows the level of expression of HSD17B7 using the primer series 1417871_at
• Figure 2D represents the level of expression of HSD17B7 using the primer series 1448865_at.
• the points Veh-24h-a and Veh-24h-b show the level of HSD17B7 expression of gonadectomized mice not treated with DHT.
• 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 was transcribed into cDNA. From the double-stranded cDNA biotin-labeled cRNA was synthesized using T7 polymerase and a precursor NTP conjugated to biotin. The cRNAs were then fragmented into small fragments.
• 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 perfectly matched oligonucleotide ("perfect match”) versus an oligonucleotide that contains a mutation ("single mismatch") in the central region of the oligonucleotide (see Table 1).
• Table 1 Measurement of the Expression of HSD17B7 in the Epidermis and the Sebaceous Gland by Using Affymetrix Technology
• HSD17B7 is well expressed in both tissues (sebaceous gland, epidermis).
• the differential analysis between the expression in the human sebaceous gland and human repornm shows that the slightly stronger expression in the sebaceous gland does not reach any significance with respect to the value observed in the epidermis.
• mice show differentiation of the sebocyte type and are used as an experimental model of the sebaceous gland. They are of sufficient size to allow isolation of RNA without the use of microdissection technologies.
• the inventors analyzed the expression of HSD17B7 in the preputial glands of mice under conditions of steroid hormone deficiency (especially in androgenic hormones) following gonadectomy.
• the gonadectomized animals were then treated with physiological amounts of Dihydrotestosterone (DHT) or Dihydroepiandrosterone (DHEA) to restore a physiological level of the androgenic hormones, or as a control experiment with a combination of DHEA-Flutamide in which Flutamide, a Androgen receptor antagonist blocks the effect of DHEA.
• DHT Dihydrotestosterone
• DHEA Dihydroepiandrosterone
• the comparison of the gene expression under these experimental conditions makes it possible to unambiguously identify the modulation or not of the gene expression of a gene in question by the androgenic hormones.
• Gene expression was analyzed using the Affymetrix technology described above ( Figure 1A) and the results were then confirmed by the real-time PCR technique ( Figure 1
• RNA isolated from the tissues is transcribed (RT) into the cDNA and this is amplified by PCR (polymerase chain reaction).
• PCR polymerase chain reaction
• the progress of the PCR is monitored in real time using fluorescent TaqMan probes, allowing precise quantification of the amount of mRNA of a given gene present in the biological sample at the start.
• the mRNA of HSD17B7 is induced by chronic treatment for 7 days with androgens in the preputial gland.
• Figures 2A, 2B, 2C and 2D represent the level of relative expression of mRNA as a function of time.

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