FR2864540A1 - New oligonucleotides that hybridize to the tyrosinase-related protein-1 gene, useful for cosmetic or therapeutic depigmentation and whitening of skin and hair - Google Patents

Abstract

Oligonucleotide (ON), containing 7-25, preferably 20, nucleotides, that can hybridize with the gene, or gene product, encoding tyrosine-related protein-1 (TRP1) is new. ACTIVITY : Dermatological. No details of tests for dermatological activity are given. MECHANISM OF ACTION : Inhibiting expression of tyrosinase and/or TRP-1, both of which are involved in the first step of melanin biosynthesis. Pure cultures of normal human foreskin melanocytes were incubated for 7 days in presence of various concentrations of the oligonucleotide (2) 5'-AGACCTGGTTTGCAGCTCTT (2) then tyrosine activity determined by adding L-dopa and monitoring formation of dopachrome at 450 nm. At a concentration of 100 nM, (2) caused a 28.8% reduction in reaction rate, relative to a (2)-free control.

Description

The present invention relates to novel oligonucleotide sequences

as well as their derivatives.

  These novel oligonucleotide sequences are capable of hybridizing with the genes or products of the genes encoding tyrosinase and / or tyrosinase relatecl-protein 1 (TRP-1).

  The present invention also relates to the use of these novel oligonucleotide sequences as a depigmenting or whitening agent in a cosmetic composition or in a dermatological composition.

  In humans, pigmentation results from the synthesis and distribution of melanin pigments in the skin, 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, will condition the color of human skin. Some people have areas and / or spots that are darker and / or more colored on the skin of the face or hands, giving the skin a degree of heterogeneity. These spots are due to a significant concentration of melanin in the keratinocytes of the epidermis.

  The mechanism of formation of cutaneous pigmentation involves the synthesis of melanins. This mechanism is particularly complex and involves schematically the following main steps: Tyrosine * Dopa Dopaquinone 1 * Dopachrome ^ * Melanins Tyrosinase and tyrosinase-related-protein 1 (TRP-1) are the essential enzymes involved in this series of reactions. They catalyze, in particular, the transformation reaction of tyrosine into Dopa (Dihydroxyphenylalanine) and the conversion reaction of Dopa into Dopaquinone leading to the formation of melanin pigments.

  A molecule is recognized as depigmenting if it acts directly on the epidermal melanocytes where the melanogenesis takes place and / or if it interferes with one of the stages of melamine biosynthesis. This is particularly the case when the molecule inhibits one of the enzymes involved in melanogenesis or when it interferes with the chemical compounds of the melanin synthesis chain.

  Known depigmenting substances include hydroquinone and its derivatives, ascorbic acid and its derivatives, placental extracts, kojic acid, arbutin, iminophenols (WO 99/22707), the combination of carnitine and quinone (DE 19806947), amide derivatives of amino phenol (FR 2 772 607), and benzothiazole derivatives (WO 99/24035). These substances may have certain disadvantages. They may be unstable, require use at high concentrations, lack specificity as to their mode of action, or be cytotoxic or irritant.

  The topical use of effective and harmless depigmenting substances is particularly sought after in cosmetics and dermatology. In particular, these substances are used to treat regional hyperpigmentations by melanocytic hyperactivity such as idiopathic melasmas, hyperpigmentations localized by hyperactivity and benign melanocytic proliferation such as senile pigmentary spots (actinic lentigo), accidental hyperpigmentations such as photosensitization or post-lesion healing, as well as some leukoderma such as vitiligo. In these latter cases, failing to repigment the skin, the depigmentation of the residual areas is completed to give the skin a homogeneous color.

  The problem posed to professionals is thus the design, manufacture or isolation of new whitening agents for human skin, hair and / or hair that do not have the disadvantages of known substances, that is to say which are non-irritating, non-toxic and / or non-allergenic for the skin and stable in a composition.

  The use of an antisense oligonucleotide to treat diseases due to melanocyte dysfunction, particularly Vitiligo and other depigmenting diseases, has been described in WO 99/25819. In these cutaneous pathologies, hypopigmentation results from an abnormally high tenascine content. The oligonucleotides described herein act against hypopigmentation by regulating the expression of tenascin.

  On the contrary, the object of the present invention is to provide a depigmenting agent for combating cutaneous hyperpigmentation.

  The inventors of the present invention have found quite unexpectedly that oligonucleotides that can hybridize with genes or gene products (such as RNAs) encoding tyrosinase and / or genes or gene products coding for tyrosinase-related-protein 1 (TRP-1) exhibit depigmenting activity. This activity exists even at very low concentration, which increases the interest of these oligonucleotides. In addition, the oligonucleotides according to the invention exhibit no cytotoxicity.

  The oligonucleotides according to the invention intervene upstream of the mechanisms of melanogenesis by modulating the expression of tyrosinase and / or TRP-1 in melanocytes. The oligonucleotides according to the invention offer an ideal solution to the problems posed by the substances conventionally used. Known substances which inhibit the activity of tyrosinase or TRP-1 (especially hydroquinone and its derivatives, ascorbic acid and its derivatives, placental extracts, kojic acid, arbutin) have multiple unacceptable side effects due to their low specificity. The present invention thus solves the problems encountered by previous researchers by modulating the production of enzymes instead of directly inhibiting the enzymes to achieve the depigmenting effect.

  The oligonucleotides according to the invention are novel as such and novel as medicaments.

  The present invention relates to an oligonucleotide having a number of nucleotides between 7 and 25, for example between 9 and 25, between 12 and 25, between 15 and 25 or between 18 and 25, preferably equal to 20, capable of hybridizing with the genes or products of the genes encoding tyrosinase, and / or with the genes or products of the genes encoding tyrosinase-related-protelin 1 (TRP1), in particular an oligonucleotide whose sequence is chosen from among the SEQ's ID NO.1 to SEQ ID NO.11 having the following meaning: SEQ ID NO.1 SEQ ID NO.2 - SEQ ID NO.3 SEQ ID NO.4 - SEQ ID NO.5 - SEQ ID NO.6 - SEQ ID NO. 7 - SEQ ID NO: SEQ ID NO: 9 - SEQ ID NO. 10 - SEQ ID NO. 15 '-G CAAAACAAAGAC C TG GTTT-3' 5'-AGACCTGGTTTGCAGCTCTT-3 '5'-TGCTTGAAATAAGAGTGCAA-3 5'-AAAATC.CAGCTCACAATCCT-3 '5'-AG GAG CACTCATTCTG CTTG-3' 5'-AGGAACTGGCTAATTGGAGT-3 '5'-CAAGGTCTGCAGGAACTGGC-3' 5'-CCTCAC: AAGGTCTGCAGGAA-3 '5'-CTACAGACAATCTGCCAAGA-3' 5'GCATTCTTCCTCTAGTCCTC-3 '5'-TTCCAGTACCTCACAATC CT- 3 'According to an embodiment of the present invention, the oligonucleotides of sequence SEQ ID NO.1 to 6, in particular SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5 and SEQ ID NO. .6 are preferred.

  The subject of the present invention is also an oligonucleotide as a novel product whose sequence is one of the sequences SEQ ID NO.1 to NO.11 described above, in particular SEQ ID Nos. 1 to 6, and preferentially SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6.

  In the context of the present invention, the term "gene encoding tyrosinase" means the genomic sequence of the tyrosinase gene. Similarly, the term "gene encoding TRP1" refers to the genomic sequence of the TRP1 gene.

  The key roles of tyrosinase and TRP-1 in melanogenesis are known. The use of oligonucleotides directed against a messenger RNA encoding an enzyme or protein to modulate its expression is also known. However, the technique developed by the inventors of the present invention had never been used as a means of depigmentation.

  The oligonucleotides according to the invention are determined to hybridize directly to the messenger RNA or the gene. They thus make it possible to achieve an ultimate modulation of the amount of tyrosinase or TRP-1 produced by the genes.

  As used herein, the term "Hybridization" is used to refer to the formation of hydrogen bonds, also known as Watson-Crick pairing between complementary bases, usually on two nucleic acid strands to form a double helix duplex.

  The degree of complementarity between two nucleic acid sequences of identical length is determined by comparing, after alignment, the first sequence with the complementary sequence of the second sequence. The degree of complementarity is calculated by determining the number of identical positions for which the nucleotide is identical between the two sequences thus compared, by dividing this number of identical positions by the total number of positions and multiplying the result obtained by 100 to obtain the degree of complementarity between these two sequences.

  The term "specific hybridization" means in particular that there is a degree of complementarity sufficient to avoid nonspecific binding of the oligonucleotide to a non-targeted sequence under conditions where specific binding is desired. It is understood that the oligonucleotide need not be 100% complementary to its target nucleic acid sequence to specifically hybridize. In particular, an oligonucleotide having a degree of complementarity of at least about 80% is able to hybridize specifically with the target nucleic acid.

  The oligonucleotides according to the invention preferably hybridize specifically with the genes or products of the genes coding for tyrosinase and / or genes or products of genes encoding TRP1. In particular, the oligonucleotides of the invention are capable of hybridizing with the DNA of genes that encode tyrosinase or DNA genes that encode TRP-1, and / or mRNA derived from these genes. Genoa. The oligonucleotides according to the invention comprise a number of nucleotides sufficient in identity and in number to hybridize specifically. This property is commonly called "antisense".

  The subject of the present invention is therefore oligonucleotides which hybridise specifically upstream of and / or with the region having the codon for initiation of gene translation.

  The subject of the present invention is also oligonucleotides which specifically hybridize with the 5 'non-coding region and / or the region presenting the gene initiation codon, or the coding region and / or the 3' non-coding region of the genes. The present invention also relates to oligonucleotides that specifically hybridize with the DNA and / or messenger RNA that encode tyrosinase or TRP-1.

  In the context of the present invention, the term "DNA" which codes for tyrosinase, both exons and introns, in particular exons.

  The subject of the present invention is also oligonucleotides, comprising one or more chemical modifications at their sugar moieties, their nucleobase moieties or their internucleotide backbone which confer desirable physicochemical characteristics on the oligonucleotides according to the invention such as increased bioavailability. , increasing the affinity for target sequences, increasing cell internalisation or better biological stability or increasing stability in the presence of cellular nucleases.

  By way of example, the modifications that can confer these characteristics are the 2 '. O-alkyl and 2'-O-fluoro derivatives on the sugar part of the nucleoside, and the phosphorothioate derivatives or the methylphosphonate derivatives at the level of the internucleotide backbone.

  In the present document, the term "oligonucleotide" refers to polynucleotides formed from natural nucleobases and pentafuranosyl (sugar) groups forming nucleosides which are linked together by native phosphodiester bonds. The term "oligonucleotides" therefore refers to natural species or synthetic species formed from natural subunits or their close homologues.

  The term "oligonucleotides" may also refer to those parts which have functions similar to naturally occurring oligonucleotides but which may have unnatural moieties. The oligonucleotides may have sugar moieties, nucleobase moieties, or modified internucleotide linkages. Among the possible modifications, the preferred modifications are 2'-O-alkyl derivatives on the sugar moiety, in particular 2'-O-ethyloxymethyl or 2'-O-methyl derivatives, and / or phosphorothioates or methylphosphonates for the internucleotide backbone.

  The chimeric oligonucleotides are included in the preferred modifications of the invention. The oligonucleotides contain at least two chemically different regions, each comprising at least one nucleotide. Typically, one or more regions comprising a modified nucleotide which confers one or more beneficial properties such as, for example, better biological stability, increased bioavailability, increased cellular internalization or increased RNA affinity. target.

  Preferably, the internucleotide backbone may be in whole or in part phosphodiesters, or phosphorothioates, or methylphosphonates or combinations of phosphodiester and / or phosphorothioate and / or methylphosphonate linkages.

  The term "oligonucleotides" may also refer to oligonucleotides to which a plasmid-type circular delivery vector or a nucleic or peptide-type linear delivery vector has been grafted.

  The oligonucleotides of the invention are novel as drugs.

  The present invention also relates to a cosmetic or dermatological composition containing at least one oligonucleotide described above and a cosmetically or dermatologically acceptable medium. Such a composition may contain, in addition, one or more assets to enhance the desired effects.

  The present invention also relates to the use of an oligonucleotide sequence directed against the transcription products of genes coding for tyrosinase and / or TRP-1 in and / or for the manufacture of a cosmetic or dermatological composition for depigmenting and / or whiten human skin and / or remove pigment spots from human skin.

  The present invention also relates to the use of oligonucleotides capable of hybridizing specifically with the genes coding for tyrosinase and / or tyrosinase-related-protein 1 as a cosmetic agent, in particular for depigmenting or whitening the skin, the hairs. and / or human hair.

  The present invention also relates to the use of an oligonucleotide sequence directed against the transcription products of the genes coding for tyrosinase and / or TRP-1 in and / or for the manufacture of a cosmetic or dermatological composition such as io inhibitor of melanin synthesis.

  The present invention also relates to the use of the oligonucleotides described above for the manufacture of a medicament for the treatment or prevention of diseases resulting in overexpression of tyrosinase or tyrosinase-related-protein 1, by topical. This drug may be intended to inhibit the synthesis of melanin, in particular to depigment or whiten the skin, but also for the treatment or prevention of regional hyperpigmentations by hyper-melanocytic activity such as idiopathic melasmas, hyperpigmentations localized by hyper-activity and benign melanocyte proliferation such as senile pigmentary spots (actinic lentigo), accidental hyperpigmentations such as photosensitization or post-lesion scarring, and for the treatment of certain leukoderma such as vitiligo.

  The present invention also relates to the use of an oligonucleotide sequence directed against the transcription products of genes coding for tyrosinase and / or TRP-1 in a depigmenting and / or whitening cosmetic composition of human skin.

  The present invention also relates to the use of the oligonucleotides described above for modulating the expression of tyrosinase and / or tyrosinase-related-protein 1. The present invention: (a further object is the use of a directed oligonucleotide sequence against the transcription products of genes coding for tyrosinase and / or TRP-1 for the manufacture of a dermatological composition depigmenting and / or whitening of human skin.

  The present invention also relates to a cosmetic or dermatological process for the depigmentation and / or bleaching of human skin by applying to the pigmented skin a cosmetic composition comprising an oligonucleotide sequence directed against the transcription products of the genes encoding tyrosinase. and / or TRP-1.

  The subject of the present invention is also a depigmenting composition, characterized in that it contains, in a cosmetically and / or dermatologically acceptable medium, an oligonucleotide sequence directed against the transcription products of the genes coding for tyrosinase and / or TRP-1. .

  The cosmetic or dermatological composition according to the invention is suitable for topical use and therefore contains a cosmetically or dermatologically acceptable medium, that is to say compatible with the skin. The oligonucleotide sequence according to the invention may be present in an amount ranging from 0.00001% to 10% and preferably from 0.0003% to 3% of the total weight of the composition.

  The composition of the invention may be in any of the galenical forms normally used for topical application, especially in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water or water-in-oil or multiple emulsion, a aqueous or oily gel, a liquid anhydrous product, pasty or solid, an oil dispersion in a polymeric phase such as nanospheres and nanocapsules or better lipid vesicles of ionic and / or nonionic type as described in French patent FR 2534487. i1

  This composition may be more or less fluid and have the appearance of a white or colored cream, an ointment, a milk, a lotion, a serum, a paste or a mousse . It can optionally be applied to the skin in the form of an aerosol. It may also be in pulverulent solid form or not, for example in stick form. It can still be in the form of patches, pencils, brushes and applicators allowing a localized application on the spots of the face or hands. It can be used as a care product and / or as a makeup product.

  In a known manner, the composition of the invention may also contain the usual adjuvants in the cosmetic and dermatological fields, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, perfumes, fillers, filters, pigments, odor absorbers and dyestuffs. The amounts of these various adjuvants are those conventionally used in the fields under consideration. These adjuvants, depending on their nature, can be introduced into the fatty phase, into the aqueous phase, into the lipid vesicles and / or into the nanoparticles.

  When the cosmetic or dermatological composition of the invention is an emulsion, the proportion of the fatty phase can range from 5 to 80% by weight, and preferably from 5 to 50% by weight relative to the total weight of the composition. The oils, emulsifiers and coemulsifiers used in the composition in emulsion form are chosen from those conventionally used in the field under consideration. The emulsifier and the co-emulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5% to 20% by weight relative to the total weight of the composition. composition.

  As oils that can be used in combination with the oligonucleotides 3o according to the invention, mention may be made of mineral oils (vaseline oil), oils of vegetable origin (avocado oil, soybean oil), oils of animal origin ( lanolin), synthetic oils (perhydrosqualene), silicone oils (cyclomethicone) and fluorinated oils (perfluoropolyethers). Fatty alcohols (cetyl alcohol), fatty acids, waxes (Carnauba wax, ozokerite) can also be used as fat.

  As emulsifiers and coemulsifiers that can be used in combination with the oligonucleotides according to the invention, mention may be made, for example, of fatty acid and polyethylene glycol esters such as PEG-20 stearate and esters of fatty acid and of glycerol such as glyceryl stearate.

  As hydrophilic gelling agents that may be used in combination with the oligonucleotides according to the invention, mention may be made in particular of carboxyvinyl polymers (carbomer), acrylic copolymers such as acrylate / alkylacrylate copolymers, polyacrylamides, polysaccharides, natural gums and clays. Lipophilic gelling agents that may be mentioned are modified clays such as bentones, metal salts of fatty acids, hydrophobic silica and polyethylenes.

  The present invention relates to a cosmetic or dermatological composition containing at least one oligonucleotide described above and one or more active agents.

  The present invention also relates to the use of an oligonucleotide as described above for the manufacture of a medicament for simultaneous administration, separate or spread over time in combination with one or more active agents.

  Said active agents that can be used in combination with the oligonucleotides according to the invention, used pure or from extracts containing these molecules, are in particular the following compounds: ellagic acid and its derivatives; hydroquinone; arbutin; resorcinol and its derivatives; vitamin C and its derivatives; pantothenate sulfonate and its derivatives; kojic acid; placental extracts; molecules interfering directly or indirectly with the alpha-melanocyte stimulating hormone (α-MSH) or its receptor or adrenocorticotropic hormone (ACTH); polyols such as glycerine, glycol or propylene glycol; vitamins; keratolytic and / or desquamating agents such as salicylic acid and its derivatives; alpha-hydroxy acids such as lactic acid or malic acid, alone or grafted; ascorbic acid and its derivatives; retinoic acid; retinaldehyde; retinol and its derivatives such as palmitate, propionate or acetate, in liposomal preparation or not; anti-glycation agents and / or antioxidants, taken alone or in combination, such as tocopherol and its derivatives, thiotaurine, hypotaurine, aminoguanidine, thiamine pyrophosphate, pyridoxamine, lysine, histidine, arginine, phenylalanine, pyridoxine, adenosine triphosphate; anti-inflammatory agents such as stearyl glycyrrhetinate; soothing agents and mixtures thereof, chemical or physical sunscreens such as octyl methoxycinnamate, butyl-methoxydibenzoylmethane, titanium oxide and zinc oxide; and deoxyribonucleic and / or nucleic acids. In the event of incompatibility, these active agents and / or the oligonucleotides may be incorporated into spherules, in particular ionic or nonionic vesicles as described in French patent FR 2534487 and / or nanoparticles.

  The following examples illustrate the present invention without limiting it.

  Example 1 Synthesis of Oligonucleotides Oligonucleotides were synthesized with an automatic synthesizer (Perseptive Biosystems Expedite Model 8909) using standard phosphoramidite derivative chemistry using the manufacturer's protocols. 13cyanoethyldiisopropylphosphoramidites were supplied by Perseptive Biosystems. For unmodified oligonucleotides, the phosphite oxidation step was performed with an iodine solution. For phosphorothioate oligonucleotides, the phosphite oxidation step was performed using a 0.05 M solution of 3H-1,2benzodithiol-3-one 1,1-dioxide in anhydrous acetonitrile. After cleavage of the column (Controlled Pore Glass, Perseptive Biosystems) and total deprotection of the sequence by a treatment of 18h at 55 ° C. with a 33% ammonia solution, the oligonucleotides were purified by precipitation in ethanol in the presence of sodium acetate. Controls by high pressure liquid chromatography were then carried out by ion exchange chromatography with elution by a sodium chloride gradient and by C18 reversed phase chromatography with elution with a gradient of acetonitrile in the presence of triethylammonium acetate.

  The oligonucleotides studied are described in Table 1.

  In Table 1, the numbers mentioned under the sequences indicate the position of the oligonucleotide in the original sequence.

  The sequences come from the sequence of the cDNA published by Cohen et al., Nucl. Res acid 18: 2807 (1990); Genbank accession number X 51420, of the cDNA sequence published by Shibahara et al., Tohoku J. Exp. Med. 156: 403 (1988) Genbank accession number M 27160, of the DNA sequence published by Box et al., Mamm. Genome 9: 50 (1998), Genbank accession number AF 001295.

TABLE 1

  SEQ ID SEQUENCE OLIGONUCLEOTIDE 92 LOCUS NO. 5'-GCAAAACAAAGACCTGGTTT-3 'HSTYRRP 1.

  2. 5'-AGACCTGGTTTGCAGCTCTT-3 '83 HSTYRRP 3. 5'-TGCTTGAAATAAGAGTGCAA-3' 108 HSTYRRP 4. 5'-AAAATCCAGCTCACAATCCT-3 '34 HSTYRRP 5. 5'-AGGAGCACTCATTCTGCTTG-3' 122 HSTYRRP 6. 5'-AGGAACTGGCTAATTGGAGT 457 HUMTYRA 7. 5'-CAAGGTCTGCAGGAACTGGC-3 '466 HUMTYRA 8. 5'-CCTCACAAGGTCTGCAGGAA-3' 471 HUMTYRA 9. 5'-CTACAGACAATCTGCCAAGA-3 'HUMTYRA 1332 1313 5'-GCATTCTTCCTCTA GTCCTC-3' 487 HUMTYRA 11 5'-TTCCAGTACCTCACAATCCT-3 'AF001295 5875 5856 12. 5'-TTCTCGACGTTTGGTCCAGA-3' CONTROL SENS SEQ ID NO.2 13. 5'-ACGTTTCTCGCCTAGTGATG-3 'CONTROL

BROUILLE

  SEQ ID NO: 2 1.6 Example 2: Depigmentary effect of oligonucleotides on normal human melanocytes of children Melanocytes are obtained from foreskin skin of a white donor. The skin fragments are rinsed with PBS (Gibco, Paisley, GB) and then with 70% ethanol. After a last wash with PBS, the skin is cut into thin strips of 1 mm with a minimum of dermis. The dermis and epidermis are dissociated by incubation of the strips in a 0.25% trypsin solution (Gibco, Paisley, GB) overnight at 4 C. After incubation, the epidermal cells are recovered by scraping the cells with a scalpel. The action of trypsin is stopped by placing the cells in E-199 medium (Gibco, Paisley, GB) containing 10% (VN) of fetal calf serum (Gibco, Paisley, UK). After homogenization and removal of corneocytes floating on the surface, the cell suspension is filtered, centrifuged and the pellet is taken up in an adhesion medium (Medium 1), the composition of which is shown in the following table.

  ENVIRONMENTAL COMPOSITION 1 Compound Manufacturer, reference Final Concentration in Medium - E-199 Gibco, 31150-022 - Gibco Fetal Calf Serum 10% - Hydrocortisone Sigma, H-0135 0.4 μg / ml - L-GIN Gibco, 2 mM - EGF Sigma, E-4127 10 ng / ml After counting the cell population, the cells are seeded into flasks at a rate of 200,000 / cm 2. The cultures are maintained at 37 ° C., in a saturated atmosphere with humidity and with 5% CO 2.

  After adhesion of the cells, the adhesion medium is replaced by KSFM medium (Gibco, Paisley, GB), which will be renewed every 48 hours.

  The epidermal cells thus cultured will make it possible to obtain a keratinocyte-melanocyte co-culture. As soon as this coculture is approximately 70% confluent, the KSFM medium is replaced by a selective medium (medium 2) promoting the growth of melanocytes.

  ENVIRONMENTAL COMPOSITION 2 Compound Manufacturer, reference Final Concentration in Medium - E-199 Gibco, 31150-022 - Gibco Fetal Calf Serum 10% - Gibco L-GIN, 25C) 30-024 0.4 μg / ml - IBMX Sigma , 1-5379 2 μM-PMA Sigma, P-8139 10 μg / ml - Geneticin Gibco, 066-01811Y 100 μg / ml 48 to 72 h later, the cells are detached from the flask using a trypsin mixture 0.1% -EDTA 0.05% (Gibco, Paisley, GB) for 1 to 2 minutes at room temperature. The cell suspension obtained is centrifuged and put back into a selective medium. The cells are again treated 24 to 48 hours later with the trypsin-EDTA mixture (0.1% -0.05%), for 1 to 2 minutes at room temperature, and then resown in a proliferation medium specific for melanocytes (Medium 3).

  Medium 3 is composed of 10% of Medium 2 and 90% of Medium A. Medium A is the SFM keratinocyte medium (Gibco, 17005-034).

  At this stage, the cell population obtained is pure, consisting exclusively of normal human melanocytes.

  Before the study, normal human melanocytes (MHN) are placed for one week in a medium containing no potent metabolic activators such as phorbol esters or IBMX (Medium 4).

  Medium 4 is composed of 10% Medium B and 90% Medium A as previously described. The composition of the medium B is described in the

table below.

COMPOSITION OF ENVIRONMENT B

  Compound Manufacturer, reference Final Concentration in Medium - E-199 Gibco, 31150-022 - Gibco 10% fetal calf serum - Gibco L-GIN, 25030-024 2 mM MHNs are seeded in 96-well microplates (Falcon, 10 Franklin Lakes, NJ, USA), 10,000 per well, in 200 μl of medium 4.

  The oligonucleotides to be tested are prepared extemporaneously in the study medium, at concentrations of 10 nM, 100 nM, 250 nM, 500 nM, 1 μM. The treatments with the different oligonucleotides are carried out daily for 7 days. For each condition, 3 tests are performed.

  At the end of treatment, measurements of the tyrosinase or dopaoxidase activity are carried out. Tyrosinase is also called dopaoxidase because of its multifunctional nature. It performs the same function as dopa-oxidase during the oxidation of tyrosine to dopa.

  Measurement of reaction rates of the dopa-oxidase activity: The cells are rinsed with PBS and then 50 μl of lysis buffer (Triton 100X (Sigma, T9284) at 0.5% in PBS (Gibco, 14190-094)) The reaction is started by adding 50 μl of substrate (10 mM L-DOPA, Sigma) to each well. The appearance of the dopachrome is measured every 2 minutes at 450 nm for one hour and at 37 ° C., with regular stirring. The reaction rates thus obtained are expressed in 10 -4 units OD / min. (a: standard deviation) and presented in Table 2.

TABLE 2

  Identification Numbers Velocity Rates of% Change by Reaction Reaction (Control versus Control without ODN) SEQ ID NO.1 10 nM 21.8 a = 1.0 26.4 a = 1.8 -17.4 nM 23.3 a = 0.4 26.4 a = 1.8 -11.7 250 nM 20.3 a = 0.3 26.4 a = 1.8 -23.1 500 nM 19.5 a = 2 , 26.4 a = 1.8 -26.1 1 μM 23.9 a = 1.4 26.4 a = 1.8 -9.4 SEQ ID NO.2 10 nM 20.5 a = 1, 7 26.4 = 1.8 -22.3 nM 18.8 a = 1.5 26.4 a = 1.8 -28.8 250 nM 19.2 a = 0.7 26.4 a = 1, 8 -27.3 500 nM 19.7 a = 1.7 26.4 a = 1.8 -25.4 1 pM 22.9 a = 1.1 26.4 a = 1.8 -13.3 SEQ ID NO.3 10 nM 23.7 a = 3.1 26, 4 a = 1.8 -10.2 nM 20 a = 0.7 26.4 a = 1.8 -24.2 250 nM 21.1 a = 0.6 26.4 a = 1.8 -20.1 500 nM 19.9 a = 1.7 26.4 a = 1.8 -24.6 1 pM 23.6 a = 0.6 26 , 4 a = 1.8 -10.6 SEQ ID NO.4 10 nM 22.9 a = 1.2 26, 4 a = 1.8 -13.3 nM 19.9 a = 1.6 26.4 a = 1.8 -24.6 250 nM 20.9 a = 1.4 26.4 a = 1.8 -20.8 500 nM 21.2 a = 1.7 26.4 a = 1.8 - 19.7 1 pM 20 a = 1.2 26.4 a = 1.8 -24.2 SEQ ID NO.5 10 nM 23.5 a = 0.7 26.4 a = 1.8 -10.9 nM 19.3 a = 0.9 26.4 a = 1.8 -26.9 250 nM 17.9 a = 1.7 26.4 G = 1.8 -32.2 500 nM 20.5 a = 0.6 26.4 a = 1.8 -22.4 1 μM 21.3 a = 0.8 26.4 a = 1.8 -19.3 SEQ ID NO.6 10 nM 20.7 a = 1.8 26.4 a = 1.8 -21.6 nM 22 0 a = 0.6 26.4 a = 1.8 -16.7 250 nM 20.1 a = 2.5 26.4 = 1.8 -23.9 500 nM 18.7 a = 2.6 26 , 4a = 1.8-29.2 NM 20.5a = 1.1 26.4a = 1.8-22.4 SEQ ID NO.7 10 nM 26.6a = 2.225, 0 a = 0.9 + 6.4 nM 25.3 a = 1.1 25.0 a = 0.9 +1.2 250 nM 23.8 a = 1.3 25.0 a = 0.9 4.8 500 nM 21.7 a = 0.9 25.0 G = 0.9 -13.2 1 NM 22.6 a = 0.7 25.0 a = 0.9 -9.6 SEQ ID NO .8 10 nM 24.1 a = 0.1 25.0 a = 0.9 -3.6 nM 23.7 a = 0.6 25.0 a = 0.9 -2.5 250 nM 25.1 a = 1.0 25.0 a = 0.9 +0.4 500 nM 23.4 a = 0.6 25.0 = 0.9 -6.4 1 = NM 23.7 a = 0.2 25 , 0 a = 0.9 -5.2 SEQ ID NO.9 10 nM 27.1 a = 0.7 25.0 a = 0.9 +8.4 nM 22.3 a = 0.6 25.0 a = 0.9 -10.8 250 nM 22.6 a = 0.2 25.0 = 0.9 -9.6 500 nM 23.2 a = 0.5 25.0 a = 0.9 -7 , 2 1 NM 25.0 a = 0.7 25.0 a = 0.9 0 SEQ ID NO.10 10 nM 26.4 a = 0.9 25.0 a = 0.9 +5.6 nM 24 , 9 a = 0.5 25.0 a = 0.9 -2.4 250 nM 23.1 a = 0, 3 25.0 a = 0.9 -7.6 500 nM 23.3 a = 0.6 25.0 a = 0.9 -6.8 1 NM 26.9 a = 0.7 25.0 a = 0.9 +7.6 SEQ ID NO.11 10 nM 25.7 a = 0.9 25, 0 a = 0.9 +2.8 nM 24.8 a = 0.3 25.0 a = 0, 9 -0.8 250 nM 25.8 a = 0.7 25.0 a = 0.9 +3.2 500 nM 22.1 a = 4.4 25.0 a = 0.9 -11.6 1 PM 23.9 a = 0.8 25.0 a = 0.9 -4.4 Example 3: Detecting effect of the oligonucleotide SEQ ID NO.2 on human normal melanocytes of adults The melanocytes used here are obtained from from thigh skin of an adult Métis donor. The skin fragments are rinsed with PBS (Gibco, Paisley, GB) and then washed with 70% ethanol (VN) diluted to 50% in PBS. After washing with PBS, the skin fragments are scraped using a cutter corns. The resulting pieces were scraped in 0.05% trypsin (Difco Laboratories, West Molesy, UK) for one hour at 37 ° C to allow their dissociation. The epidermis is then collected in E-199 medium (Gibco, Paisley, GB) containing 10% fetal calf serum (FCS, Gibco, Faisley, GB). After homogenization and removal of the stratum corneum fragments floating on the surface, the cell suspension is filtered and then centrifuged. The cell pellet obtained is taken up in medium 1. The cells are counted and a measurement of the cell viability is carried out on a Thoma cell by the trypan blue exclusion test. The epidermal cells are finally seeded in medium 1, in flasks at a rate of 80000 cells / cm 2. The cultures are maintained at 37 ° C., in a saturated humidity atmosphere and with 5% CO 2.

  Medium 1 is replaced by a medium 6 whose composition is specified below.

  ENVIRONMENTAL COMPOSITION 6 Compound Manufacturer, reference Final concentration in media - Gibco E-199, 31150-022 - Sigma Insulin, H-0888 0.5 μg / ml - EGF Sigma, E-4127 10 ng / ml - B-FGF Gibco, 13256-029 10 ng / ml PdBu Sigma, P-1269 0.25 μg / ml - Ca ++ Prolabo, 22317297 1.8 mM This medium is renewed every 48 hours for one to two weeks.

  After this selection of melanocytes, the proliferation of the MHN is favored, replacing the medium 6 with the medium 7.

  Medium 7 is composed of 10% medium C and 90% medium A described above, supplemented with PdBu (Sigma, P-1269), the final concentration in the medium is 0.25 μg / ml.

COMPOSITION OF THE ENVIRONMENT C

  Compound Manufacturer, reference Final Concentration in Medium - E-199 Gibco, 31'150-022 - Gibco Fetal Calf Serum 10% - Gibco L-GIN, 25030-024 2mM - IBMX Sigma, I-5879 10-4M Genecitin Gibco, 06E5-01811Y 100 μg / ml At this stage, the cell population obtained is pure, consisting exclusively of normal adult human melanocytes.

  The MHN are seeded in 96-well microplates (Falcon, Franklin Lakes, NJ, USA) in medium 6. After 24 hours, the medium is replaced by medium 8 which will be used throughout the end of this study.

  The composition of Medium 8 is described below.

  ENVIRONMENTAL COMPOSITION 8 Compound Manufacturer, reference Final concentration in medium - MCDB-153 Sigma, M-7403 2% - Gibco Insulin Sigma fetal calf serum, 1-6634 5 μg / ml - Sigma transferrin 10 μg / ml α-Tocopherol Sigma 1 μg / ml - Penicillin / Sigma 0.4% Streptomycin Oligonucleotide SEQ ID NO.2 is added extemporaneously in the study medium, at concentrations of 50 nM, 100 nM, 250 nM, 500 nM, 1 NM . Treatments are performed daily for 7 days. For each condition, 6 tests are performed.

  At the end of the treatment, measurements of the dopa-oxidase activity are carried out.

  Measurement of reaction rates of dopa-oxidase: The reaction rates thus obtained are expressed in 10-4 unit OD / min. (o: standard deviation) and presented in Table 3.

TABLE 3

  Reaction Rate Variations Speed 0/0 Reaction Identification Variation (Control Without ODN) Compared to Control SEQ ID NO.2 33.8 a = 3.4 33.0 a = 3.5 + 2.4 nM nM 29.8 a = 1.3 33.0 a = 3.5 -9.7 250 nM 29.6 a = 2.2 33.0 a = 3.5 -10.3 500 nM 28.3 a = 1.5 33.0 c = 3.5 -14.2 1 pM 27.6 a = 2.9 33.0 a = 3.5 -16.3 Example 4: Study of the inhibition of UV-melanoqenesis induced, on normal adult human melanocytes by oligonucleotide SEQ ID NO.2.

  The melanocytes used here are obtained from mammoplasty skin of a white adult donor. The technique for obtaining these adult melanocytes is the same as that used and described in Example 3.

  The experiment is carried out in 35 mm boxes. The melanocytes are seeded in 35 mm dishes (Falcon, Franklin Lakes, NJ, USA) at the rate of 250000 MHN and 2 ml of medium 8 per dish. The treatments with the oligonucleotide SEQ ID N0.2 as well as the UVB irradiations (Bio-Energie, Vilbert-Lourmat, Marne-la-Vallee, France) at 8 mJ / cm 2 begin 24 hours after seeding.

  Before each UVB irradiation, the MHNs are placed in PBS (2 ml / dish). After irradiation, the cells are replaced in 2 ml of medium 8 containing or not the oligonucleotide SEQ ID NO.2.

  The oligonucleotide SEQ ID NO: 2 is added extemporaneously in the study medium (medium 7 of Example 3), at concentrations of 50 nM, 100 nM, 250 nM, 500 nM, 1 μM. Treatments are performed daily for 9 days. The irradiations take place every day, except days 5 and 6. For each condition, 3 tests are carried out.

  At the end of treatment, ie after 9 days of treatment with oligonucleotide SEQ ID No. 2 and 7 irradiations, each of 8 mJ / cm 2 in UVB, measurements of the dopa-oxidase activity are carried out.

  Measurement of dopa-oxidase reaction rates: At the end of the treatment, the NHNs are detached from the 35 mm dishes using 500 μl of 0.1% trypsin-0.05% EDTA and then 1.5 ml of E-199 medium without phenol red (Gibco) and supplemented with 10% fetal calf serum. An aliquot of this cell suspension is used for one cell count, another is centrifuged and the resulting melanocyte pellet is taken up in 50 μl of lysis buffer (Appendix 5) for measurement of the reaction rate of dopa-oxidase as describes example 2.

  Measurement of the Dopa-oxidase Activity: The Dopa-oxidase activity is expressed in 10-4 DO Unit / min / 106 MHN. (a: standard deviation) and presented in Table 4.

27 TABLE 4

  Identification numbers Activity Dopa-% of variation variation versus oxidase relative to control to irradiated non-irradiated control Non-irradiated control 368.2 a = 19.9 0 Irradiated control (8 mJ / cm2, 7-fold) 409, 1 32.6 + 11.1% 0 Irradiation + SEQ ID NO.2 328.1 a = 44.8 - 19.8% nM Irradiation + SEQ ID NO.2 325.3 a = 43.1 - 20.5 % 250 nM Irradiation + SEQ ID NO.2 327.4 a = 49.5 - 20% 500 nM Example 5: Study of the inhibition of melanoqenesis by SEQ ID NO.12 and SEQ ID NO.13 on human melanocytes normal of children. The melanocytes used here are obtained from foreskin skin of a white donor. The technique for obtaining these melanocytes is the same as that used and described in Example 2.

  The oligonucleotides SEQ ID NO.12 and SEQ ID NO.13 are added extemporaneously in the study medium, at concentrations of 10 nM, 100 nM, 250 nM, 500 nM, 1 μM. Treatments are performed daily for 7 days.

  At the end of the treatment, the measurements of the dopa-oxidase activity are carried out.

  Measurement of dopa-oxidase reaction rates: This measurement is carried out as described in the previous examples. The results are shown in Table 5. 5 TABLE 5 Speed Numbers of Variation% by ID Reaction Reaction (Control vs. Control Without ODN) SEQ ID NO.12 34.6 a = 1.0 32.5 a = 1.4 + 6.5 nM nM 33, 2 a = 0.7 32.5 a = 1.4 + 2.2 nM 33.9 a = 0.7 32.5 a = 1.4 + 4 , 3 SEQ ID NO. 13 33.5 a = 1.2 32.5 a = 1.4 + 3.1 nM - nM 36.3 a = 0.6 32.5 a = 1.4 + 11.7 nM 34.0 a = 1.2 32.5 a = 1.4 + 4.6 Example 6: Bleaching powder io Concentrations are given in percent by weight Microcellulose 20.00% Sodium lauryl sulfoacetate 15.00% Oligonucleotides (SEQ ID NO.5) 1 , 00% Perfume, dyes, preservatives qs.

  Talc qsp. 100% Example 7: Emulsion-qel face after sun Concentrations are given in percent by weight Glycerin 5.00% triglycerides caprylic / capric / succinic 5.00% Methoxycinnamate octyl 1.00% Dimethicone copolyol 0.50% Acrylates / C10-30 alkyl acrylate crosspolymer 0.50% Oligonucleotides SEQ ID NO.3 0.01% Neutralizer qs.

  Preservatives, perfume, dyes qs.

  Water qs. 100% EXAMPLE 8 Protective Fluid SPF 30 Concentrations are given in percent by weight volatile pentacyclomethicone 49.00% titanium dioxide 15.00% octyl methoxycinnamate 7.50% glycerin 5.00% phenyltrimethicone 5.00% dimethicone copolyol 3.00% Polymethylmethacrylate 2.50% Butyl methoxydibenzoyl methane 1.00% Oligonucleotides SEQ ID NO.2 0, 1% Neutralizer, Fragrance, Preservatives, antioxidants qs.

  Water qs. 100%: Example 9: Crème visaqe dépiqmentante The concentrations are given in percent by weight Glyceryl stearate + Peg-100 stearate 5.00% Polyisobutene hydrogenated 4.00% Magnesium ascorbyl phosphate 3.30% Tricaprylate / glycerol caprate 3.00 % Squalane 3.00% Glycerin 2.00% Beeswax 1.50% Cetearyl octanoate 1.50% Cetyl alcohol 1.00% Stearyl alcohol 1.00% Dimethicone 1.00% Xanthan gum 0.30% Ethylene acid diamine tetracetic 0.20% Citric acid 0.10% Sodium citrate 0.10% Oligonucleotides SEQ ID NO.6 0.10% Neutralizer, Fragrance, Preservatives qs.

Water qs. 100% i0

Example 10 Lotion

  The concentrations are given as percentage by weight Denatured alcohol (Ethanol) 30.00% PPG-3 Myristyl ether 5.00% Glycerin 2.00% Carbomer 0.25% Polysorbate 20.0% Oligonucleotides SEQ ID NO. 01% Neutralizer, Fragrance, Preservatives qs.

Water qs. 100%

Claims (1)

  An oligonucleotide comprising a number of nucleotides comprised between 7 and 25, preferably equal to 20, capable of hybridizing with the gene or a product of the gene coding for one of the essential enzymes involved in the synthesis pathway of the melanin characterized in that said enzyme is tyrosinase related protein 1 (TRP 1).   2. Oligonucleotide according to claim 1 characterized in that the sequence of the oligonucleotide is one of the sequences SEQ ID N 1 to N 5 and SEQ ID N 11 having the following meaning: SEQ ID N 1 5'-GCAAAACAAAGACCTGGTTT- ## STR2 ## 5. The oligonucleotide according to claim 1, wherein the sequence is the sequence SEQ ID No. 2.   6. Oligonucleotide according to one of the preceding claims, comprising one or more chemical modifications in its sugar parts, its nucleobase parts or its internucleotide backbone, said modifications conferring desirable physicochemical characteristics such as increased bioavailability, the increased affinity for target sequences, increased cell internalization or better biological stability, or increased stability in the presence of cellular nucleases.   7. Oligonucleotide according to claim 4, characterized in that its sugar part comprises a 2'-O-fluoro or 2'-O-alkyl substituent, preferably a 2'-O-ethyloxymethyl or 2'-O-methyl substituent. .   6. An oligonucleotide according to claim 4 or 5, characterized in that a part of the phosphodiester groups of its internucleotide backbone is replaced by phophorothioate groups.   7. Oligonucleotide according to claim 4 or 5, characterized in that part of the phosphodiester groups of its internucleotide backbone is replaced by methylphosphonate groups.   8. Oligonucleotide according to claim 6, characterized in that all phosphodiester groups are replaced by phophorothioate groups.   9. Oligonucleotide according to claim 7, characterized in that all the phosphodiester groups are replaced by methylphosphonate groups.   10. An oligonucleotide according to claims 6 and 7, characterized in that the phosphodiester groups are replaced in whole or in part by phosphorothioate groups and / or by methylphosphonate groups.   11. Oligonucleotide according to one of claims 1 to 10 which has been grafted a linear administration vector of nucleic acid or peptide type, or a plasmid-type circular administration vector.   12. Cosmetic or dermatological composition containing at least one oligonucleotide according to one of claims 1 to 11 and a cosmetically or dermatologically acceptable medium.   13. Cosmetic or dermatological composition according to claim 12 containing one or more active agents chosen from ellagic acid and its derivatives; hydroquinone; arbutin; resorcinol and its derivatives; vitamin C and its derivatives; pantothenate sulfonate and its derivatives; kojic acid; placental extracts; molecules interfering directly or indirectly with the alpha-melanocyte stimulating hormone (α-MSH) or its receptor or adrenocorticotropic hormone (ACTH); polyols such as glycerine, glycol or propylene glycol; vitamins; keratolytic and / or desquamating agents such as salicylic acid and its derivatives; alpha-hydroxy acids such as lactic acid or malic acid, alone or grafted; ascorbic acid and its derivatives; retinoic acid; retinaldehyde; retinol and its derivatives such as palmitate, propionate or acetate, in liposomal preparation or not; anti-glycation agents and / or antioxidants, taken alone or in combination, such as tocopherol and its derivatives, thiotaurine, hypotaurine, aminoguanidine, thiamine pyrophosphate, pyridoxamine, lysine, histidine, arginine, phenylalanine, pyridoxine, adenosine triphosphate; anti-inflammatory agents such as stearyl glycyrrhetinate; soothing agents and mixtures thereof, chemical or physical sunscreens such as octyl methoxycinnamate, butyl-methoxydibenzoylmethane, titanium oxide and zinc oxide; and deoxyribonucleic or nucleic acids.   14. Composition according to claim 12 or 13, characterized in that the oligonucleotides represent 0.00001% to 10%, preferably 0.0003% to 3% of the total weight of the composition.   15. Cosmetic or dermatological composition according to one of claims 12 to 14 being in the form of an emulsion containing an oil, an emulsifier chosen from fatty acid esters and polyethylene glycol, such as PEG-stearate. 20, and fatty acid esters of glycerin, such as glycerin stearate, and a co-emulsifier.   16. Use of an olignonucleotide according to one of claims 1 to 11 as a cosmetic agent.   17. Use of an oligonucleotide according to claim 16 for the preparation of a cosmetic or dermatological composition for depigmenting or whitening skin, hair or human hair.   18. Use of an oligonucleotide according to one of claims 1 to 11 for the manufacture of a cosmetic or dermatological composition for the treatment or prevention of diseases resulting in overexpression of tyrosinase, topically.   19. Use of an oligonucleotide according to one of claims 1 to 11 for the manufacture of a medicament for inhibiting melanin synthesis.   20. Use of an oligonucleotide according to claim 19 for the treatment or prevention of regional hyperpigmentations by melanocyte hyperactivity such as idiophatic melasmas, hyperpigmentations localized by hyper-activity and benign melanocyte proliferation such as senile pigmentary spots (actinic lentigo). ), accidental hyperpigmentations such as photosensitization or post-injury healing, and for the treatment of certain leukoderma such as vitiligo.   21. Use of an oligonucleotide according to claim 18 or 19 for the manufacture of a medicament for depigmenting or whitening the skin.   22. Use of an oligonucleotide according to one of claims 1 to 11 for the preparation of a medicament for modulating the expression of tyrosinase.   23. Use of an oligonucleotide according to one of claims 18 and 22 for the manufacture of a medicament intended to be administered simultaneously, separately or spread over time in combination with one or more active agents chosen from the acid ellagic and its derivatives; hydroquinone; arbutin; resorcinol and its derivatives; vitamin C and its derivatives; kojic acid; placental extracts; molecules interfering directly or indirectly with the alpha-melanocyte stimulating hormone (α-MSH) or its receptor or adrenocorticotropic hormone (ACTH); polyols such as glycerine, glycol or propylene glycol; vitamins; keratolytic or desquamating agents such as salicylic acid and its derivatives; alphahydroxy acids such as lactic acid or malic acid, alone or grafted; ascorbic acid and its derivatives; retinoic acid, retinaldehyde, retinol and its derivatives such as palmitate, propionate or acetate, in liposomal preparation or not; anti-glycation agents or antioxidants, taken alone or in combination, such as tocopherol and its derivatives, thiotaurine, hypotaurine, aminoguanidine, thiamine pyrophosphate, pyridoxamine, lysine, histidine, arginine, phenylalanine, pyridoxine, adenosine triphosphate; anti-inflammatory agents such as stearyl glycyrrhetinate; soothing agents and mixtures thereof, chemical or physical sunscreens such as octyl methoxycinnamate, butylmethoxydibenzoylmethane, titanium oxide and zinc oxide; and deoxyribonucleic and / or nucleic acids.