Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/60—Sugars; Derivatives thereof
  • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/18—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with another compound as one donor, and incorporation of one atom of oxygen (1.14.18)
  • C12Y114/18001—Tyrosinase (1.14.18.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
  • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/323—Chemical structure of the sugar modified ring structure
  • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/33—Chemical structure of the base
  • C12N2310/334—Modified C
  • C12N2310/3341—5-Methylcytosine

Definitions

• the present invention relates to an anti-gene oligonucleotide hybridizing specifically with the gene encoding human tyrosinase by Hoogsteen pairing between complementary bases, said oligonucleotide forming a triple helix structure with the human tyrosinase gene.
• the invention also relates to the use of said oligonucleotide as a depigmenting or whitening agent for the skin in a cosmetic composition or in a dermatological composition.
• 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. For each individual, the color of the skin varies mainly according to the more or less significant irradiation of ultraviolet (UV) rays.
• UV ultraviolet
• the difference of shade of the skin between the basic pigmentation and the maximum pigmentation is more or less important.
• people belonging to certain populations with light skin (basic pigmentation) react quickly and / or important to the action of the UV and can thus easily present a skin of dark shade, even when these people did not have exposed themselves voluntarily and prolonged to the sun.
• these people will be referred to as "people who are very reactive to UV”. This is particularly true of populations of Asian origin or of certain so-called mixed-race populations.
• the mechanism of formation of cutaneous pigmentation involves the synthesis of melanins. This mechanism is particularly complex and schematically involves the following main steps: Tyrosine "* • Dopa” * • Dopaquinone "* • Dopachrome” * • melanins
• TRP-I tyrosinase-related-protein 1
• a molecule is recognized as depigmenting if it acts directly on the epidermal melanocytes by inhibiting the activity of these cells and / or if it blocks one of the stages of melanin biosynthesis or if it degrades the melanin formed. This is particularly the case when the molecule inhibits one of the enzymes involved in melanogenesis or when it reacts with the chemical compounds of the melanin synthesis chain.
• the known depigmenting substances are in particular hydroquinone and its derivatives, ascorbic acid and its derivatives, placental extracts, kojic acid, arbutin, iminophenols, the combination of carnitine and quinone, amide derivatives of amino-phenol, and benzothiazole derivatives. 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.
• these substances are used to treat regional hyperpigmentations by melanocytic hyperactivity such as idiopathic melasmas, hyperpigmentations localized by melanocytic hyper-activity such as pigmentary spots called solar lentigo and senile lentigo, accidental hyperpigmentations such as photosensitization or post-injury healing, as well as some leukoderma such as vitiligo.
• melanocytic hyperactivity such as idiopathic melasmas
• hyperpigmentations localized by melanocytic hyper-activity such as pigmentary spots called solar lentigo and senile lentigo
• accidental hyperpigmentations such as photosensitization or post-injury healing
• some leukoderma such as vitiligo.
• Depigrating substances are also used as skin whitening agents by some people, in particular those mentioned above, which are very reactive with UV, to lighten their complexion, especially that of their face and their hands, so to keep skin color as clear as possible or at least to reduce the pigmenting effects of UV rays.
• oligonucleotides for treating melanocyte dysfunction has been described in application US 20040014700. Furthermore, certain strategies for obtaining a depigmentation consist in blocking the synthesis of tyrosinase by the use of an oligonucleotide. Double-stranded RNA (FR 2840217). In previous applications, the biological target considered is messenger RNA.
• the object of the present invention is to provide a depigmenting agent acting on the process of melanogenesis, intended firstly, in the case of a substantially homogeneous pigmentation, the bleaching of the skin and / or hair, it that is to say to reduce their pigmentation, and secondly, to fight against cutaneous hyperpigmentation, ie when the skin has a heterogeneity of pigmentation.
• the inventors of the present invention have found that oligonucleotides capable of hybridizing with the gene coding for tyrosinase and only with this gene exhibit depigmenting activity.
• that of the present invention therefore consists in using an oligonucleotide capable of hybridizing with the gene itself.
• An advantage of this is that the oligonucleotides of the present invention can hybridize with the gene as a double-stranded DNA, whereas the oligonucleotides described in particular in the applications US 20040014700 or FR 2840217 do not have this capacity.
• Another advantage of this new strategy is that the gene is a less abundant entity than messenger RNA or the protein for which it codes, since most genes have two alleles in diploid cells. As a result, this activity exists even at very low concentration, which increases the interest of these oligonucleotides.
• the oligonucleotides according to the invention have no cytotoxicity and can be synthesized, characterized, isolated with a high degree of purity and this industrially.
• the oligonucleotides according to the invention therefore intervene very far upstream of the mechanisms of melanogenesis, interacting directly with the tyrosinase gene to modulate its expression and consequently inhibit the expression of the messenger RNA coding for tyrosinase. This results in a decrease in tyrosinase levels in melanocytes.
• the oligonucleotides according to the invention offer an ideal solution to the problems posed by the substances conventionally used. Known substances that inhibit the activity of tyrosinase have multiple unacceptable side effects due to their low specificity. Among the various sequences tested, we have found that the sequence SEQ ID No.
• the invention relates to an anti-gene oligonucleotide comprising a sequence of 15 to 25 nucleotides comprising the sequence 5'- C * TTC * TC * TC * TTTTTC * C * TTTTTC * -3 '(SEQ ID No. 1, C * designating a 5-methyl-cytosine) specifically hybridizing with the gene encoding human tyrosinase by Hoogsteen pairing between complementary bases, said oligonucleotide forming a triple helix structure with the human tyrosinase gene.
• this anti-gene oligonucleotide comprises between 18 and 21 nucleotides or between 21 and 25 nucleotides, preferably 21 nucleotides.
• oligonucleotides according to the invention hybridize directly to the gene consisting of a DNA double helix in the cell. They thus make it possible to achieve an ultimate modulation of the amount of messenger RNA coding for tyrosinase and therefore of this enzyme produced by the gene.
• Hybridization is used to designate the formation of hydrogen bonds, also known as Hoogsteen or reverse-Hoogsteen pairing, between on the one hand the complementary bases, usually distributed on two strands of nucleic acid forming a double helix according to the Watson scheme.
• - Crick on the other hand a third strand to form a triplex, triple helix structure.
• the degree of complementarity between the nucleic acid sequences 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 complementary positions for which the nucleotide is complementary between the two sequences thus compared, by dividing this number of complementary positions by the total number of positions and multiplying the result. obtained by 100 to obtain the degree of complementarity between these two sequences expressed as a percentage.
• 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.
• the oligonucleotides according to the invention preferably hybridize specifically with the gene coding for tyrosinase.
• the oligonucleotides of the invention are capable of hybridizing only with the DNA of the genes that encode tyrosinase.
• the oligonucleotides according to the invention comprise a number of nucleotides sufficient in identity and in number to hybridize specifically.
• 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.
• DNA which codes for tyrosinase both exons and introns, especially 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 cellular internalisation or better biological stability or increasing stability in the presence of cellular nucleases.
• LNAs Locked Nucleic Acids
• PNAs Peptide Nucleic Acids
• PNA protein kinase
• backbone is composed of repeating units of N- (2-aminoethyl) -glycine connected by linkages. peptide.
• the different purine bases and pyrimidines are linked to the backbone by methylene carbonyl bonds. The structure is presented below:
• LNA LNA
• a nucleic acid analogue containing a 2'-O, 4'-C methylene layer This pond blocks the flexibility of the ribofuranose cycle by forming a rigid bicyclic structure. The structure is presented below:
• oligonucleotide refers to polynucleotides formed from natural nucleobases and pentafuranosyl (sugar) groups forming nucleosides which are linked together by native phosphodiester bonds.
• oligonucleotides therefore refers to natural species or synthetic species formed from natural subunits or their close homologues.
• oligonucleotides may also refer to those parts which have functions similar to natural oligonucleotides but which may exhibit unnatural portions.
• the oligonucleotides may have sugar moieties, nucleobase moieties, or modified internucleotide linkages.
• the preferred modifications are Locked Nucleic Acids (LNAs) as described by Braasch DA and Corey DR (Chem Biol.2001, 8, 1-7), N3'-derivatives.
• Phosphoramidates as described by Faria M and Giovannangeli C (J.
• PNA Peptide Nucleic Acids
• the 2'-O-alkyl derivatives on the sugar part in particular the 2'-O-ethyloxymethyl or 2'-O-methyl derivatives, and / or the phosphorothioates or the methylphosphonates for the internucleotide backbone or derivatives thereof.
• 5 'and / or 3' carriers of intercorptive type reactive group such as acridine or bridging with the target sequence such as psoralen or an azide group.
• 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. It is in particular one or more regions comprising a modified nucleotide which confers one or more beneficial properties such as for example a better biological stability, an increased bioavailability, the increase of the cellular internalisation or the increase of the affinity for the target DNA.
• the chimeric oligonucleotides according to the invention are the locked nucleic acids (LNA), the N3'-P5 'phosphoramidate derivatives, the peptide nucleic acids or molecules with an internucleotide backbone which may be wholly or partly phosphodiesters, or phosphorothioates, or methylphosphonates or combinations of phosphodiester and / or phosphorothioate and / or methylphosphonate linkages.
• LNA locked nucleic acids
• N3'-P5 'phosphoramidate derivatives the peptide nucleic acids or molecules with an internucleotide backbone which may be wholly or partly phosphodiesters, or phosphorothioates, or methylphosphonates or combinations of phosphodiester and / or phosphorothioate and / or methylphosphonate linkages.
• 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 present invention also relates to a cosmetic or dermatological composition containing at least one oligonucleotide described above and at least one excipient or vehicle cosmetically or dermatologically acceptable.
• a cosmetic or dermatological composition containing at least one oligonucleotide described above and at least one excipient or vehicle cosmetically or dermatologically acceptable.
• Such a composition may contain, in addition, one or more active ingredients. This or these active ingredients are intended to supplement or reinforce the desired depigmenting effects.
• the present invention also relates to the use of oligonucleotides capable of hybridizing specifically with the genes coding for tyrosinase as cosmetic agents, in particular for depigmenting and / or bleaching the skin or hair, and / or for reducing stains. pigmentary skin of human skin.
• the present invention comprises cosmetic compositions using these oligonucleotides as cosmetic agents. They are intended in particular to lighten the complexion, prevent or treat the formation of pigmentary spots due to the action of solar radiation, to reduce pigmentary spots of senescence (senile lentigo), especially on the hands, or to lighten the hairiness of the skin. body or limbs.
• the present invention also relates to the use of the oligonucleotides described above for the manufacture of a dermatological medicament for the treatment or prevention of diseases resulting in overexpression and overactivity of tyrosinase, topically.
• This drug may be intended to treat or prevent regional hyperpigmentations by hyper-active melanocytic such as idiopathic melasmas, accidental hyperpigmentations such as photosensitization or in some cases post-injury healing, and for the attenuation of pigmentary contrasts in some leukoderma such as vitiligo.
• the present invention also relates to a depigmenting composition characterized in that it contains, in a cosmetically and / or dermatologically acceptable medium, an oligonucleotide sequence directed against the gene coding for tyrosinase.
• the cosmetic composition and the drug according to the invention are suitable for topical use and therefore contain 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 these compositions.
• compositions of the invention may be in any form suitable for topical application, especially in the form of an aqueous solution, hydroalcoholic or oily, an oil-in-water or water-in-oil emulsion, single or multiple, an aqueous or oily gel, a liquid, pasty or solid anhydrous product, an aqueous or oily dispersion of solid particles, such as nanospheres or polymeric nanocapsules, or an aqueous dispersion of lipid vesicles of ionic or nonionic type, as described in French patent FR 2534487.
• compositions 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 . They can optionally be applied to the skin in the form of an aerosol. They may also be in pulverulent solid form or not, for example in stick form. They may also be in the form of single doses, patches, pencils, or applicators allowing a localized application on the spots of the face or hands.
• the cosmetic composition in particular can be formulated for use as a care product and / or as a make-up product.
• compositions 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, sunscreens, pigments, odor absorbers and dyestuffs.
• adjuvants in the cosmetic and dermatological fields, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, perfumes, fillers, sunscreens, 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 and / or nanospheres.
• the proportion of the fatty phase may 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 co-emulsifiers 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.
• oils that can be used in combination with the oligonucleotides according to the invention mention may be made of mineral oils (vaseline oil), oils of vegetable origin (avocado oil, soya oil), oils of animal origin (lanolin ), synthetic oils (perhydrosqualene), silicone oils (cyclomethicone) and fluorinated oils (perfluoropolyethers).
• mineral oils vaeline oil
• oils of vegetable origin oils of vegetable origin
• oils of animal origin lanolin
• synthetic oils perhydrosqualene
• silicone oils cyclomethicone
• fluorinated oils perfluoropolyethers
• Fatty alcohols cetyl alcohol
• fatty acids fatty acids
• waxes can also be used as fat.
• 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 fatty acid and glycerin esters, such as glyceryl stearate.
• hydrophilic gelling agents that may be used in combination with the oligonucleotides according to the invention, mention may in particular be made of carboxyvinyl polymers (carbomer), acrylic copolymers such as copolymers of acrylates / alkylacrylates, 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 other active agents.
• the present invention also relates to a cosmetic or dermatological process for depigmenting and / or bleaching human skin comprising applying to the pigmented skin a cosmetic composition comprising an oligonucleotide sequence directed against the gene coding for tyrosinase. More particularly, the invention relates to a cosmetic treatment method for depigmenting or bleaching the skin, characterized in that it comprises the application of a composition defined above to one or more pigmented area (s) of the skin. skin for a fixed period. Optionally it is possible to repeat the operation until the appearance of the depigmenting effect.
• the oligonucleotide content is preferably between 0.0003% and 3% of the total weight of the composition.
• 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.
• the cosmetic composition of the invention may further comprise one or more active ingredients that can be used in cosmetics. Said active agents, in combination with the oligonucleotides according to the invention, are used pure or from extracts containing these active ingredients.
• ellagic acid and its derivatives are especially selected from the following compounds: ellagic acid and its derivatives; resorcinol and its derivatives; arbutin and its derivatives, vitamin C and its derivatives; pantothenate sulfonate and its derivatives; molecules interfering directly or indirectly with the alpha-melanocyte stimulating hormone ( ⁇ -MSH) or its receptor or adrenocorticotropic hormone (ACTH); polyols such as glycerine, glycol or propylene glycol; 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; 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, ergothi
• one and / or the other may be incorporated into spherules, in particular liposomes or vesicles formed from ionic or nonionic amphiphilic lipids as described in the patent.
• spherules in particular liposomes or vesicles formed from ionic or nonionic amphiphilic lipids as described in the patent.
• FR 2534487 and / or nanoparticles and / or nanospheres and / or nanocapsules The following examples illustrate the present invention without limiting it.
• Figure 1 Effect of oligonucleotides SEQ ID No. 1 composed of LNA bases and SEQ ID 6 on the expression of the tyrosinase gene in normal human melanocytes treated with 500 nM oligonucleotides for 8 hours.
• the symbol * represents a statistically significant difference (p ⁇ 0.05) compared to untreated cells.
• Figure 2 Effect of oligonucleotide SEQ ID No. 1 composed of DNA bases on the expression of the tyrosinase gene in normal human melanocytes treated with 500 nM oligonucleotides for 8 hours.
• the symbol * represents a statistically significant difference (p ⁇ 0.05) compared to untreated cells.
• Figure 3 Effect of oligonucleotide SEQ ID No. 1 on DOPA oxidase activity of tyrosinase in normal human melanocytes.
• the DOPA oxidase activity is determined in terms of OD per minute and normalized with respect to the viability obtained using the XTT test.
• the symbol * represents a statistically significant difference (p ⁇ 0.05) compared to untreated cells.
• Oligonucleotides with natural bases were synthesized with an automatic synthesizer (Perseptive Biosystems Expedite Model 8909) using standard phosphoramidite derivative chemistry using the manufacturer's protocols.
• the ⁇ -cyanoethyldiisopropylphosphoramidite derivatives of the nucleosides have been used.
• the oxidation step of the phosphite was carried out with an iodine solution. After cleavage of the column (Controlled Pore Glass, Perseptive Biosystems) and total deprotection of the sequence by a treatment of 18 h at 55 ° C.
• the oligonucleotides were purified by precipitation in ethanol in 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 reverse phase chromatography. Cl 8 eluting with a gradient of acetonitrile in the presence of triethylammonium acetate.
• the LNA oligonucleotides were synthesized by Proligo or Eurogentec. By way of examples, oligonucleotides have been synthesized. They are described in Table 1. The stars mentioned next to the cytosines indicate that these bases are methylated in position 5. The first 5 sequences of this table, numbered from SEQ ID No. 1 to SEQ ID No. 5 were subjected to 'studies. Their hybridization property with their target sequence on the tyrosinase gene and their depigmenting activity are reported in the following examples.
• SEQ ID No. 7 A so-called "scrambled control" sequence, designated SEQ ID No. 7, also in Table 1, comprising the same bases, in kind and in number, as those of the sequence SEQ ID No. 1, but placed in any order. TABLE 1
• the gel delay technique makes it possible to demonstrate the hybridization of an oligonucleotide with its target DNA sequence.
• the presence of the target DNA with the oligonucleotide of the radiolabeled invention generates a DNA-o ligonucleotide complex whose migration will be slowed compared with that of the free oligonucleotide. Determining the amount of delayed oligonucleotide makes it possible to measure the formation efficiency of the triple helix.
• SEQ ID No. 1 and SEQ ID No. 6 are labeled with ⁇ - [ 32 P] ATP by T4 polynucleotide kinase. Radiolabeled SEQ ID No. 1 and SEQ ID No. 6 are isolated on an exclusion column.
• the purity of the oligonucleotides is controlled by electrophoresis on a 15% acrylamide-urea gel with a migration at 7OW for 1h30.
• the gel is then dried under vacuum at 70 0 C and the radioactive imprint is read using a Phospholmager (Storm 860, Molecular Dynamics).
• Hybridization between the labeled oligonucleotides and the DNA fragment comprising a portion of the human tyrosinase gene sequence is performed overnight at 4 ° C at different values of the target DNA / oligonucleotide concentration ratio.
• the conditions are set forth in Table 2.
• the radiolabeled sequences SEQ ID No. 1 and SEQ ID No. 6 are tested in two forms: in modified form (LNA) and in unmodified form (DNA).
• Electrophoresis on acrylamide-Bis 8% non-denaturing gel is carried out during Ih, at a power of 3 W, at 37 ° C.
• the gel is then dried under vacuum at 70 ° C. and brought into contact one night with an exposure plate.
• the efficiency of formation of the complex between the oligonucleotide and its target sequence (expressed as a percentage) can be determined by LumiAnalyst software (Roche, Meylan, France).
• Table 3 Quantification by LumiAnalyst software of the hybridization of SEQ ID 1 with the fragment of the tyrosinase gene.
• Example 3 Real-Time Quantitative RT-PCR Study of Inhibition of Human Tyrosinase Gene Expression on Normal Human Melanocytes
• Normal human melanocytes are inoculated into 60 mm diameter Petri dishes at 710,000 cells per dish in Medium 1 (Table 4).
• an extemporaneous solution is prepared consisting of medium 1 to which was added the complexed sequence with Superfect (Qiagen, Courtab ⁇ uf, France), at a concentration of 500 nanomolar. Twenty four hours after seeding, the cells are treated once with these different solutions and then recovered after 8 hours of treatment in order to extract the total RNA.
• RNAs all the cellular RNAs thus comprising the messenger RNAs of tyrosinase
• the total RNAs are extracted (Kit Rneasy, Qiagen, Courtaboeuf, France), assayed, and their quality verified using Bioanalyzer 2100 (Agilent Technologies, Massy, France). ). They are then treated with DNAse, in order to eliminate any residual genomic DNA.
• a reverse transcription reaction by the enzyme Superscript II (Qiagen) makes it possible to generate the complementary DNAs (cDNA) from the RNAs. .
• the real-time quantitative PCR on LightCycler (Roche, Meylan, France) is then carried out according to the reaction mixture described in Table 5 in order to quantify the messenger RNAs of interest present in the initial cell population.
• Table 5 Typical reaction mixture used for quantitative real-time PCR.
• sequences - SEQ ID No 2 to 5 - do not exhibit any significant activity on the inhibition of the expression of the tyrosinase gene, nor even the sequences SEQ ID No 6 and SEQ ID No 7 which are respectively the control inverted and scrambled control of the sequence SEQ ID No. 1, which alone has this activity.
• Table 6 Summary of the effect of the sequences SEQ ID No. 1 to SEQ ID No. 7 on the rate of transcripts encoding the tyrosinase gene.
• the oligonucleotides according to the invention appearing in Table 1 of Example 1 are studied for their action on melanogenesis, and therefore on their ability to modulate the production of melanin pigments by melanocytes.
• the oligonucleotides "reverse control” SEQ ID No. 6
• "scrambled control” SEQ ID No. 7
• tyrosinase and TRP-I form an enzymatic complex that catalyzes the transformation of L-dopa into dopaquinone and then dopachrome (Kobayashi T.
• tyrosinase is also referred to as "dopa-oxidase" because of its multi-functional nature. It fulfills the same function as dopa-oxyidase during the oxidation of tyrosine to dopa. The principle of this test is based on the measurement of the rate of reaction catalyzed by dopa-oxidase in the transformation of L-dopa, used as a substrate, into dopachrome.
• dopachrome is quantified by measurement of optical density at determined time intervals, which makes it possible to calculate the transformation reaction rate of L-dopa for each of the oligonucleotides tested and for the control test. Knowing that the speed of this reaction depends in particular on the amount of enzyme available, it will be possible, from the results obtained, to verify the inhibitory action of the oligonucleotides tested of the invention on the expression of the tyrosinase gene. , action already observed by the tests of the previous example.
• a decrease in the reaction rate compared to the control test will correspond to a decrease in the amount of enzyme formed, and therefore a decrease in the enzymatic activity, and consequently, in the reduction of the formation of melanin, and therefore to a depigmenting effect.
• Table 7 Summary of the effect of sequences SEQ ID No. 1 to SEQ ID No. 7 on tyrosinase dopa oxidase activity of normal human melanocytes.
• Table 7 shows that DOPA oxidase activity is significantly inhibited by 14% in normal human melanocytes treated with SEQ ID No. 1 at 500 nanomolar.
• SEQ ID No. 6 and SEQ ID No. 7 have no activity on the DOPA oxidase activity. This shows that the inhibitory effect observed with the sequence SEQ ID No. 1 composed of the LNA bases is specific to the targeted sequence.
• Oligonucleotide SEQ ID No. 1 1.00%
• This powder has a double action. It allows a cleansing of the skin, and moreover it allows, by a regular use for a few days, to lighten the complexion. It can be applied to the skin of the face once or twice a day.
• Titanium dioxide 15.00%
• This composition is to be used before exposure to intense sunlight. It prevents the appearance of pigment spots, in people predisposed to this phenomenon. It should be noted that the presence of a high concentration of sunscreen makes it possible to compensate for the decrease in natural protection, as a consequence of the drop in the melanin content.
• EXAMPLE 8 Dermatological cream for the treatment of cutaneous hyperpigmentations of pathological or traumatic origin
• Oligonucleotide SEQ ID No. 1 0.10%
• This lotion for lightening complexion is used after cleansing and cleansing the skin.
• This lotion is applied on the hairy areas to be lightened, especially the arms, for the duration sufficient to obtain a progressive lightening of the hairs.
• Example 12 Cosmetic cream anti-stains for the hands
• Purified water qs 100% This cream must be applied directly on the spots (solar lentigo and / or senile) hands, to reduce the color.

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