WO2011154639A2

WO2011154639A2 - Edn3 and/or ednrb modulators in the treatment of melasma

Info

Publication number: WO2011154639A2
Application number: PCT/FR2011/051217
Authority: WO
Inventors: Itaru Suzuki
Original assignee: Galderma Research & Development
Priority date: 2010-06-11
Filing date: 2011-05-27
Publication date: 2011-12-15
Also published as: WO2011154639A3

Abstract

The invention relates to an in vitro method of screening for candidate compounds for the preventive or curative treatment of melasma, which comprises determining the capacity of a compound to modulate EDN3 and/or EDNRB expression or activity, and also to the use of modulators of EDN3 and/or EDNRB expression or activity for the treatment of melasma. The invention also relates to methods for in vitro diagnosis or prognosis of this pathological condition.

Description

Modulators of EDN3 and / or EDNRB in the treatment of melasma

The invention relates to the identification and use of modulator compounds of EDN3 and / or EDNRB for the treatment of melasma. It also relates to methods of in vitro diagnosis or prognosis in vitro of this pathology.

Prior art and description of the situation

In humans, pigmentation results from the synthesis and distribution of melanin pigments in the skin, hair follicles or eyes.

Pigmentation is genetically predefined but it is regulated by many internal or external factors.

The melanins produced by melanocytes as well as the number of melanocytes, their tyrosinasic activity and their ability to export melanins to keratinocytes, the size of melanosomes that contain melanin grains, will condition the color of human skin. Among the pathologies related to excessive pigmentation include melasma.

Melasma is an acquired hyperpigmentation that is generally symmetrical with the photo-exposed areas of the face (forehead, cheeks, temples) and sometimes the neck. Elemental lesions are small, irregularly-shaped macules, ranging in color from light brown to black, and increasing after sun exposure.

Melasma affects mainly women (90% of cases), especially during pregnancy. Some consider it to be a physiological modification in pregnant women as well as the hyperpigmentation of areoles, genital mucosa and the white line, or linea fusca, whose treatment is similar to that of melasma. The clinical and pathological features are identical in both sexes. There are three different clinical presentations: the most common centrofacial melasma, the malar melasma and the mandibular. On pathological examination, we find a hyperpigmentation of melanic origin that may be at the epidermal and / or dermal level. The number of melanocytes is not modified, but melanin production by the basal cell melanocytes is increased.

The light examination of Wood is very useful, it allows to distinguish three types of melasma according to the pigment distribution: - the epidermal type (70% of cases) where hyperpigmentation is accentuated;

- the dermal type (10% of cases) where the hyperpigmentation is faded;

- the mixed type (20% of cases) where there is a mixture of the two images.

In phototype VI patients, this examination is non-contributory. This distinction is important in the therapeutic decision because the dermal pigment is resistant to all classic depigmenting treatments.

Hydroquinone, tretinoin and azelaic acid are the three agents with scientifically recognized activity in melasma. This therapeutic arsenal is limited, therefore for many years, in the cosmetic or pharmaceutical industry, compositions for treating melasma have been sought, and in particular for modulating the amount of melanin in the skin.

The applicant has now found that the EDN3 and / or EDNRB gene is expressed in YYY (cells in which EDN3 and / or EDNRB are expressed preferentially) and that its expression is EDN3 and / or EDNRB-induced. gene or its expression product, to treat melasma.

EDN3 gene Description

The protein encoded by this gene is a member of the endothelin family. Endothelins are derived from endothelial vasoactive peptides involved in a variety of biological functions. The active form of this protein is a 21 amino acid peptide transformed based on the precursor protein. The active peptide is an endothelin B receptor ligand (EDNRB). The interaction of this endothelin with EDNRB is essential for the development of derived peak neuron cell lines, such as enteric melanocytes and neurons. Mutations in this gene and EDNRB have been associated with Hirschsprung's disease (HSCR) and Waardenburg syndrome (WS), which are congenital disorders involving neural crest cells. Four variants coding for three distinct isoforms were observed.

The human and protein DNA sequences of EDN3 are reproduced in the appendix, respectively SEQ ID No.2 (sequence NM 0001 14 (Genbank)), and SEQ ID No. 1. EDNRB gene description

The protein encoded by this gene is a G-protein coupled receptor that activates a secondary phosphatidylinositol-calcium messenger system. Its ligand, endothelin, consists of a family of three potent vasoactive peptides: ET1, ET2, ET3 and. Studies suggest that multigene disease, Hirschsprung type 2 diseases, is due to mutations in the endothelin-type B receptor gene. Three transcription variants encoding two different isoforms have been found for this gene. Although both isoforms bind to ET1, they exhibit different responses on binding, suggesting that they may be functionally distinct.

The human DNA and protein sequences of EDRNB are reproduced in the appendix (SEQ ID No.4 (sequence NM 0001 (Genbank)) and SEQ ID No.3, respectively.

Diagnostic applications

An object of the invention relates to an in vitro method for diagnosing or monitoring the evolution of melasma in a subject, comprising comparing the expression or the activity of the EDN3 and / or EDNRB protein, the expressing its gene or the activity of at least one of its promoters in a biological sample of a subject relative to a control subject.

The expression of the protein can be determined by an assay of this EDN3 protein and / or EDNRB by immunohistochemical assay, 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 EDN3 and / or EDNRB can also be envisaged.

In the context of a diagnosis, the subject "control" is a "healthy" subject.

As part of a follow-up of the evolution of melasma, the "control subject" refers to the same subject at a different time, which preferably corresponds to the beginning of treatment (To). This measurement of the difference in the expression or activity of EDN3 and / or EDNRB, the expression of its gene or the activity of at least one of its promoters makes it possible in particular to monitor the efficacy of treatment, in particular treatment with a modulator of EDN3 and / or EDNRB, as envisaged above or by another treatment of melasma. Such follow-up can reassure the patient as to the appropriateness, or necessity, of continuing this treatment.

Another aspect of the present invention relates to an in vitro method for determining susceptibility of a subject to develop melasma, comprising comparing the expression or activity of EDN3 and / or EDNRB, expression of of its gene or the activity of at least one of its promoters, in a biological sample of a subject relative to a control subject. Here again, the expression of the protein can be determined by an EDN3 and / or EDNRB assay, by an immunohistochemical test or an immunoassay, 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 EDN3 and / or EDNRB factor activity may also be considered.

The tested subject is here an asymptomatic subject, having no hair disorder related to alopecia. 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 the signs related to alopecia.

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, however, the sample may be a preparation of skin cells, obtained for example by hair removal or biopsy.

Screening methods

Another object of the invention is an in vitro method for screening candidate compounds for the preventive and / or curative treatment of melasma, comprising determining the ability of a compound to modulate the expression or activity of EDN3 and or EDNRB or the expression of its gene or the activity of at least one of its promoters, said modulation indicating the utility of the compound for the preventive or curative treatment of melasma. The method therefore makes it possible to select the compounds capable of modulating the expression or the activity of EDN3 and / or EDNRB, or the expression of its gene, or the activity of at least one of its promoters. More particularly, the invention relates to an in vitro method for screening candidate compounds for the preventive and / or curative treatment of melasma, comprising the following steps:

at. 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 EDN3 and / or EDNRB, the expression of its gene or the activity of at least one of its promoters, in the biological samples or reaction mixtures; d. selection of compounds for which modulation of the expression or activity of EDN3 and EDNRB, the expression of its gene or the activity of at least one of its promoters is measured in the sample or the mixture treated in b), relative to the untreated sample or mixture. "Modulation" means any effect on the level of expression or activity of EDN3 and / or EDNRB, the expression of its gene or the activity of at least one of its promoters, that is, possibly inhibition, but preferably stimulation, partial or complete.

Thus, the compounds tested in step d) above preferably induce the expression or the activity of EDN3 and / or EDNRB, the expression of its gene or the activity of at least one of its promoters.

Throughout this text, unless otherwise specified, "expression of a protein" means the amount of that protein;

By "protein activity" is meant its biological activity; By "promoter activity" is meant the ability of this promoter to trigger the transcription of the coded DNA sequence downstream of this promoter (and thus indirectly the synthesis of the corresponding protein).

The compounds tested can be of any type. They can be of natural origin or have been produced by chemical synthesis. It can be a library of structurally defined chemical compounds, compounds or uncharacterized substances, or a mixture of compounds.

Various techniques can be implemented to test these compounds and identify the compounds of therapeutic interest, modulators of the expression or activity of EDN3 and / or EDNRB.

According to a first embodiment, the biological samples are cells transfected with a reporter gene operably linked to all or part of the promoter of the DE EDN3 gene and / or EDNRB, and the step c) described above consists in measuring the expression of said reporter gene.

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

According to a second embodiment, the biological samples are cells expressing the gene encoding the transcription factor EDN3 and / or EDNRB, and the step c) described above consists in measuring the expression of said gene.

The cell used here can be of any type. It can be a cell expressing the EDN3 gene and / or EDNRB endogenously, for example a liver cell, a cell of prostate, or even better, a skin cell. Organs of human or animal origin can also be used.

It may also be a cell transformed with a heterologous nucleic acid, encoding the EDN3 gene and / or EDNRB, preferably human or mammalian.

A wide variety of host cell systems can be used, such as, for example, Cos-7, CHO, BHK, 3T3, HEK293 cells. The nucleic acid can be stably or transiently transfected by any method known to those skilled in the art, for example by calcium phosphate, DEAE-dextran, liposome, virus, electroporation, or microinjection. In these methods, expression of the EDN3 and / or EDNRB gene can be determined by measuring the rate of transcription of said gene, or its rate of translation.

By transcription rate of a gene is meant the amount of corresponding mRNA produced. By translation rate of a gene is meant the amount of corresponding protein produced.

Those skilled in the art are familiar with the techniques allowing the quantitative or semi-quantitative detection of the mRNA of a gene of interest. Techniques based on the hybridization of mRNA with specific nucleotide probes are the most common (Northern Blot, RT-PCR, Rnase protection). It may be advantageous to use detection markers, such as fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin).

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

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-Early growth response antibody 1 can, inter alia, be obtained by immunizing an animal such as a rabbit or a mouse, using the entire protein. The antiserum is removed and then exhausted according to methods known to those skilled in the art. A monoclonal antibody can, among other things, to be obtained by the classical method of Kohler and Milstein (Nature (London), 256: 495-497 (1975)). Other methods of preparing monoclonal antibodies are also known. For example, monoclonal antibodies can be produced by expression of a cloned nucleic acid from a hybridoma. Antibodies can also be produced by the phage display technique, by introducing antibody cDNAs into vectors, which are typically filamentous phages that have V gene libraries on the surface of the phage. (for example fUSE5 for E.coli).

The immunoassay can be carried out in solid phase or in homogeneous phase; in a time or in two stages; sandwich method or competitive method, by way of non-limiting examples. According to a preferred embodiment, the capture antibody is immobilized on a solid phase. As non-limiting examples of solid phase, it is possible to use microplates, in particular polystyrene microplates, or particles or solid beads, paramagnetic beads.

ELISA assays, immunoassays, or any other detection technique can be used to reveal the presence of the antigen-antibody complexes formed.

The characterization of antigen / antibody complexes, and more generally isolated or purified but also recombinant proteins (obtained in vitro and in vivo) can be performed by mass spectrometry analysis. This identification is made possible thanks to the analysis (determination of the mass) of the peptides generated by the enzymatic hydrolysis of the proteins (trypsin in general). In general, the proteins are isolated according to methods known to those skilled in the art, prior to enzymatic digestion. Peptide analysis (in the form of a hydrolyzate) is carried out by separation of the peptides by HPLC (nano-HPLC) based on their physico-chemical properties (reverse phase). The determination of the mass of the peptides thus separated is carried out by ionization of the peptides and either by direct coupling to the mass spectrometer (electrospray mode ESI) or after deposition and crystallization in the presence of a matrix known to those skilled in the art ( analysis in MALDI mode). The proteins are then identified through the use of appropriate software (eg Mascot).

The EDN3 and / or EDNRB gene can be produced according to standard techniques using Cos-7, CHO, BHK, 3T3 and HEK293 cells. It can also be produced using microorganisms such as bacteria (e.g. E. coli or B. subtilis), yeasts (e.g. Saccharomyces, Pichia) or insect cells, such as Sf9 or Sf21.

Transcription factor modulators

The subject of the invention is also the use of an EDN3 and / or EDNRB modulator obtainable according to one of the methods described above for the preparation of a medicinal product intended for preventive and / or curative treatment. melasma. Thus, a method of preventive and / or curative treatment of melasma is described herein comprising administering a therapeutically effective amount of an EDN3 and / or EDNRB modulator to a patient in need of such treatment. Preferably, such modulators are activators (or inducers) of EDN3 and / or EDNRB.

The invention comprises the use of EDN3 and EDNRB inducing compounds, such as those identified by the screening method described above, for the preventive and / or curative treatment of melasma.

The modulator compounds are formulated in pharmaceutical compositions, in association with a pharmaceutically acceptable carrier. These compositions may be administered, for example, enterally, parenterally, or topically. Preferably, the pharmaceutical composition is applied topically. Orally, the pharmaceutical composition can be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid vesicles or polymers for controlled release. Parenterally, the pharmaceutical composition may be in the form of solutions or suspensions for infusion or for injection.

Topically, the pharmaceutical composition is more particularly intended for the treatment of the skin, the mucous membranes and the scalp and may be in the form of ointments, creams, milks, ointments, powders, soaked swabs, solutions, gels, sprays, lotions or suspensions. It may also be in the form of suspensions of microspheres or nanospheres or lipid or polymeric vesicles or polymeric patches or hydrogels allowing controlled release. This composition for topical application may be in anhydrous form, in aqueous form or in the form of an emulsion. In a preferred variant, the pharmaceutical composition is in the form of a gel, a cream or a lotion.

The composition may comprise a modulator content of EDN3 and / or EDNRB 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:

- wetting agents;

- flavor enhancers;

preserving agents such as esters of parahydroxybenzoic acid; stabilizing agents;

humidity regulating agents;

pH regulating agents;

osmotic pressure modifying agents;

emulsifying agents;

UV-A and UV-B filters;

and antioxidants, such as alpha-tocopherol, butylhydroxyanisole or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelators.

The following figures and examples illustrate the invention without limiting its scope.

Experimental data

ProbeSet 208399_s_at 204271_s_at

GENE_SYMBOL EDN3 EDNRB

UNIGENE Hs.1408 Hs.82002

patient 1 (fold

0.907636293 0.89932481

induction)

patient 3 (fold

1, 625863288 1, 350419408

induction)

patient 4 (fold

1, 312722518 1, 204278623

induction)

patient 5 (fold

2,230254991 2.002457573

induction)

patient 10 (fold

1, 2416189, 08014464

induction) patient 12 (fold

1, 442050763, 2,142297017

induction)

mean geometry

of

6, 406313134, 375644419

patients (fold

induction)

Claims

An in vitro method for screening candidate compounds for the preventive and / or curative treatment of melasma, comprising determining the ability of a compound to modulate the expression or activity of EDN3 and / or EDNRB or expression of its gene or the activity of at least one of its promoters.

2. In vitro method for screening candidate compounds for the preventive and / or curative treatment of melasma according to claim 1, comprising the following steps:

at. Preparation of at least two biological samples or reaction mixtures;

vs. Measuring the expression or the activity of the EDN3 and / or EDNRB protein, the expression of its gene or the activity of at least one of its promoters, in the biological samples or reaction mixtures;

d. Selection of compounds for which a modulation of the expression or activity of the EDN3 and / or EDNRB protein, or a modulation of the expression of its gene or a modulation of the activity of at least one of its promoters , is measured in the sample or the mixture treated in (b) relative to the untreated sample or mixture.

3. Method according to claim 2, characterized in that the compounds selected in step d) activate the expression or the activity of the protein EDN3 and / or EDNRB, or the expression of its gene or the activity of at least one of its promoters.

4. Method according to claim 2 or 3, characterized in that the biological samples are cells transfected with a reporter gene operably linked to all or part of the promoter of the EDN3 gene and / or EDNRB, and in that the step c) 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 EDN3 and / or EDNRB gene, and in that step c) consists in measuring the expression of said gene.

The method of claim 4 or 5, wherein the cells are cells transformed with a heterologous nucleic acid encoding EDN3 and / or EDNRB.

Method according to one of claims 2 to 7, wherein the expression of the gene is determined by measuring the transcription rate of said gene.

Method according to one of claims 2 to 7, wherein the expression of the gene is determined by measuring the translation rate of said gene.

9. Use of a modulator of EDN3 and / or EDNRB obtainable according to one of claims 1 to 9 for the preparation of a medicament for the preventive and / or curative treatment of melasma.

10. Use according to claim 10, characterized in that the modulator is an activator of the EDN3 gene and / or EDNRB.

11. In vitro method for diagnosing or monitoring the course of melasma in a subject, comprising comparing the expression or activity of the EDN3 protein and / or EDNRB, or the expression of its gene or the activity of at least one of its promoters in a biological sample of a subject relative to a biological sample of a control subject.

The method according to claim 13, wherein the expression of the protein is determined by an assay of this protein by an immunoassay.

The method of claim 14, wherein the immunoassay is an ELISA assay.

The method of claim 13, wherein the expression of the gene is determined by measuring the amount of corresponding mRNA.

15. In vitro method for determining susceptibility of a subject to develop alopecia, comprising comparing the expression or activity of the protein

EDN3 and / or EDNRB, 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.