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  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/16—Emollients or protectives, e.g. against radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/74—Biological properties of particular ingredients
  • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
  • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
• • 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
  • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

• the present invention relates to novel double-stranded RNA oligonucleotides for decreasing tyrosinase expression, to the use thereof for cosmetic and/or pharmaceutical purposes and also to the association thereof with cationic particles less than or equal to 1 ⁇ m in size, with a zeta potential ranging from 10 to 80 mV.
• Tyrosinase (monophenol dihydroxylphenylalanine: oxygen oxidoreductase EC 1.14.18.1) is the essential enzyme involved in the mechanism of skin pigmentation. It catalyzes in particular the reaction for conversion of tyrosine to Dopa (dihydroxyphenylalanine) by virtue of its hydroxylase activity and the reaction for conversion of Dopa to dopaquinone by virtue of its oxidase activity. The tyrosinase acts only when it is in the maturation state under the action of certain biological factors.
• This enzyme can also be advantageous in the treatment of pathologies such as melanoma (Riley et al., J. Immunother., 2001, 21, 212-220) or Vogt-Koyanagi-Harada disease (Read et al., Curr. Opin. Ophthalmol., 2000, 11, 437-442).
• the mechanism of formation of skin pigmentation i.e., the formation of melanin, is particularly complex and involves schematically the following main steps:
• melanocytes The latter contain organelles called melanosomes, which are the site of melanin biosynthesis. It is the melanosomes which, after migration along the dendrites, are transferred from the melanocytes to the keratinocytes.
• the keratinocytes are then transported to the surface of the skin during the epidermal differentiation process (Gilchrest B A, Park H Y, Eller M S, Yaar M, Mechanisms of ultraviolet light-induced pigmentation, Photochem Photobiol., 1996; 63: 1-10; Hearing V J, Tsukamodo K, Enzymatic control of pigmentation in mammals, FASEB J., 1991; 5: 2902-2909).
• tyrosinase is a key enzyme which catalyses the first two steps of melanin synthesis.
• Homozygous mutations for tyrosinase cause oculocutaneous albinism type I characterized by a complete absence of melanin synthesis (Toyofuku K, Wada I, Spritz R A, Hearing V J, The molecular basis of oculocutaneous albinism type 1 (OCA1): sorting failure and degradation of mutant tyrosinases results in a lack of pigmentation, Biochem J., 2001; 355: 259-269).
• RNA double-stranded RNA, dsRNA, oligonucleotides, and more particularly of siRNA oligonucleotides (of 12 to 40 nucleotides), would make it possible to obtain a specific activity in the cosmetic field, such as skin care or hair care, but also in the dermatological and pharmaceutical fields.
• siRNAs in vivo is known to present various difficulties.
• siRNAs could bring about the triggering of an interferon response reported by numerous publications (Sledz C A et al., Activation of the interferon system by short-interfering RNA, Nat Cell Biol., 2003; 9:834-9. Katalin Karikó et al., Small Interfering RNAs Mediate Sequence-independent Gene Suppression and Induce Immune Activation by Signaling through Toll-Like Receptor 31, J. Immunol., 2004; 172: 6545-6549.
• RNA oligonucleotides such as siRNAs
• anti-sense single-stranded RNA oligonucleotides have different targets and that the activity of an anti-sense RNA cannot be extrapolated to the siRNA of the same sequence
• Xu et al. Effective small interfering RNAs and phosphorothioate anti-sense DNAs have different preferences for target sites in the luciferase mRNAs, BBRC 2003; 306:712-717.
• the concentrations of siRNA proposed are from 50 and 200 nM.
• Jackson et al. have described a strong positive and negative regulation of genes not targeted by the siRNAs used at concentrations of 100 nM.
• WO 2005/060536 which describes siRNAs that specifically inhibit tyrosinase
• concentration ranges proposed are very wide and can result in compositions comprising a very large amount of siRNAs for which a positive and negative regulation of genes not targeted by the siRNAs may be observed.
• Novel siRNAs have now been developed that specifically inhibit tyrosinase expression from which siRNAs have been selected which exhibit an activity such that the siRNAs can be used at a dose of less than or equal to 1 nM.
• OAS-1 and IFIT-1 interferon-inducible tetratricopeptide repeat domain genes known to be induced by interferon (Stefan F. Wieland et al., Searching for Interferon-Induced Genes that inhibit Hepatitis B Virus Replication in Transgenic Mouse Hepatocytes, J. of Virology 2003, 77:1227-1236) according to the protocol described in the manual of the “BLOCK-iTTM RNAi Stress Response Control Kit (Human) for monitoring interferon-mediated stress response to double-stranded RNA in human cells” marketed by Invitrogen.
• OAS-1 and IFIT-1 interferon-inducible tetratricopeptide repeat domain
• tyrosinase-specific siRNAs with cationic particles less than or equal to 1 ⁇ m in size, with a zeta potential of 10 to 80 mV, makes it possible to very significantly improve their penetration into the target cells of a three-dimensional model such as the skin. Once penetrated, the tyrosinase-specific siRNA becomes active.
• the penetration can be evaluated by means of a fluorescent label attached to the siRNA and its activity can be evaluated by quantifying the targeted messenger by quantitative PCR or by assaying the protein corresponding to the targeted messenger.
• the cationic particles of the invention may be surfactant micelles, micelles of block or non-block polymers, cationic liposomes and niosomes, cationic oleosomes, cationic nanoemulsions, and also cationic organic or inorganic particles and nanocapsules.
• the present invention features a double-stranded RNA oligonucleotide (also called dsRNA or siRNA) selected from among the oligonucleotides of sequence SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48, optionally modified.
• dsRNA or siRNA double-stranded RNA oligonucleotide
• dsRNA and, more particularly, of siRNA makes it possible to obtain an activity of specific inhibition of the synthesis of a target protein by degradation of the mRNA encoding the protein.
• the degradation of the target mRNA is obtained through the activation of the RISC complex (RNA Induced Silencing Complex) which has its effect through the binding of the anti-sense strand of the dsRNA to the mRNA (see Tuschl T. Chem. Biochem., 2001; 2:239-245; Nykanen A & al, Cell 2001; 107:309-321; Dorsett Y. and Tuschl T. Nat Rev Drug Discov., 2004; 3:318-329; Downward J. BMJ. 2004; 328:1245-1248; Shanker P. et al., JAMA 2005; 293:1367-1373).
• RISC complex RNA Induced Silencing Complex
• RNA fragments consisting of 12 to 40 nucleotides, preferably of 23 to 27 nucleotides.
• RNA oligonucleotides can preferably be composed of a homologous sense strand and of an anti-sense strand complementary to the sequence of the mRNA of human tyrosinase (GenBank accession number NM — 000372).
• the double-stranded RNA oligonucleotide has blunt ends or unpaired ends of 2 to 6 nucleotides.
• RNA oligonucleotides can be synthesized according to numerous manual or automatic, in vivo or in vitro synthesis methods.
• the in vitro synthesis methods may be chemical or enzymatic, for example using an RNA polymerase (by way of example, T3, T7 or SP6) which will carry out the transcription of a selected DNA (or cDNA) sequence model.
• an RNA polymerase by way of example, T3, T7 or SP6 which will carry out the transcription of a selected DNA (or cDNA) sequence model.
• the double-stranded RNA oligonucleotides according to the invention are modified through the addition of an -O-methyl group in the 2′-position.
• RNA oligonucleotides can be subjected to various modifications, they can in particular be modified as described in Published U.S. Application No. 2004/014956.
• the modifications will result in double-stranded RNA oligonucleotides which are more stable, and, more preferably, double-stranded RNA oligonucleotides which have been rendered furtive with respect to the interferon response; this property is essential for use in vivo.
• the double-stranded RNA oligonucleotides may also have a sense sequence modified in such a way that it cannot be incorporated into the RISC and therefore cannot induce side effects.
• the double-stranded RNA oligonucleotides of SEQ ID NOS. 1, 2, 4, 13, 16, 35, 37, 40 and 42, having a tyrosinase mRNA degradation efficiency of greater than 50% at 0.016 nM, measured according to the protocol of Example 2 hereinafter, will be used.
• the subject of the present invention also relates to a composition
• a composition comprising at least one double-stranded RNA oligonucleotide selected from among the oligonucleotides of sequence SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48.
• the double-stranded RNA oligonucleotide is present at a concentration of less than or equal to 100 ⁇ M, preferably less than or equal to 10 ⁇ M, and more preferably less than or equal to 1 ⁇ M.
• compositions according to the invention are preferably suitable for topical administration to the surface of the body, i.e., the skin, the integuments, the mucous membranes and the eyes.
• the double-stranded RNA oligonucleotides are associated with a cationic particle less than or equal to 1 ⁇ m in size, with a zeta potential of from 10 to 80 mV.
• the double-stranded RNA oligonucleotides will adhere to the surface of the particle, the latter serving as a carrier to cause it to penetrate into the structures of the skin and into the target cells of the skin, of the mucous membranes or else of the eyes for cosmetic, dermatological and/or ophthalmic applications.
• the cationic particles according to the invention are particles that are less than or equal to 1 ⁇ m, preferably less than or equal to 500 nm, more preferably less than or equal to 300 nm, in size, it being possible to measure said size with, for example, a laser particle sizer type BI90 plus from the company Brookhaven, and that have a zeta potential of from 10 to 80 mV which can be measured with a zetameter type DELSA 440 from the company Coultronics.
• the cationic particle can be selected from surfactant micelles, in particular micelles of nonionic amphiphilic surfactants and of cationic surfactants, block polymer micelles, in particular micelles of a cationic amphiphilic block polymer, micelles of a nonionic amphiphilic block polymer and of a cationic amphiphilic block polymer and micelles of a nonionic amphiphilic block polymer and of a cationic surfactant, liposomes of nonionic and cationic surfactants, niosomes, oleosomes, particles of nanoemulsions, nanocapsules, organic particles or inorganic particles.
• cationic particles which can be used according to the invention.
• micelles are aggregates that are spontaneously formed by amphiphilic molecules when they are solubilized in water or oil, beyond a certain concentration referred to as critical: the CMC.
• the micelles which can be used in the context of the invention include at least one cationic surfactant.
• This cationic surfactant can be associated with one or more nonionic amphiphilic surfactants.
• cationic surfactants which can be used in the context of the invention are listed hereinafter.
• nonionic surfactants which can be used are: alkyl and polyalkyl (C6 to C30, saturated or unsaturated, branched or unbranched) esters or ethers of PEO, of glycerol and of polyglycerol, of sorbitan which may or may not be oxyethylenated, of sucrose, of glucose which may or may not be oxyethylenated, of maltose, of PPO-PEO.
• nonionic surfactants in the case of a mixture between nonionic surfactants and cationic surfactants, the respective proportions thereof, by weight, will be from 99/1 and 1/99.
• the amount of surfactants forming the micelles will be dependent on the CMC of the latter. However, in the context of the invention, the concentration of micellar surfactants will range from 0.1 to 10%, and preferably from 0.2 to 5%, by weight relative to the total weight of the composition.
• the micelles of amphiphilic block polymers can be prepared according to the method described in WO 04/035013.
• the block copolymers used for the preparation of the micelles associated with the dsRNAs according to the invention are in particular amphiphilic block polymers which are preferably nonionic, diblock or triblock, and which can form micelles on contact with water. They are in particular of diblock (A-B) or triblock (A-B-A) type, A corresponding to a nonionic hydrophilic polymeric block and B to a hydrophobic polymeric block.
• the molecular weight of the polymers can be from 1000 and 100 000 and the A/B ratio can be from 1/100 and 50/1.
• the nonionic hydrophilic polymeric block can be selected from polyethylene oxide (PEO) and polyvinylpyrrolidone (PVP).
• the hydrophobic polymeric block can be selected from polystyrene, poly(tert-butylstyrene), poly(methyl methacrylate), poly(ethyl acrylate), poly(butyl acrylate), poly(butyl methacrylate), poly(vinyl acetate), polycaprolactones, polycaprolactams, polydimethylsiloxanes, poly(C 3 -C 6 alkylene oxide)s, poly(aspartic acid), poly(lactic acid), poly(glycolic acid), polyleucine, polybutadienes, polyethylenes, polypropylenes, polybutylenes.
• the block copolymer is preferably selected from among the following block copolymers:
• micellar composition In the context of the invention, it is necessary to add to the micellar composition:
• association of a nonionic amphiphilic block polymer with a cationic amphiphilic block polymer is such that the ratio between the two will range from 99/1 to 1/99; and/or
• the respective ratio of the nonionic amphiphilic block polymer to the cationic surfactant will range from 50/50 to 99/1.
• the concentration of micellar block polymers which may or may not be associated with a cationic surfactant, will range from 0.1 to 10%, and preferably from 0.2 to 5%, by weight relative to the total weight of the composition.
• micelles consisting of cationic amphiphilic block polymers as described above.
• nonionic amphiphilic lipids capable of forming nonionic liposomes are in particular those described in EP-0-582,503.
• the nonionic amphiphilic lipids can be a mixture of esters of at least one polyol selected from the group consisting of polyethylene glycol having from 1 to 60 ethylene oxide units, sorbitan, sorbitan bearing 2 to 60 ethylene oxide units, glycerol bearing 2 to 30 ethylene oxide units, polyglycerols containing 2 to 15 glycerol units, sucroses, glucoses bearing 2 to 30 ethylene oxide units, and of at least one fatty acid containing a linear or branched, saturated or unsaturated C 5 -C 17 alkyl chain, the number of alkyl chains per polyol group ranging from 1 to 10.
• mixture of esters covers not only mixtures of pure esters of different chemical families, but also covers any product which contains several chemically pure esters of a polyol of the same family in variable proportions.
• esters which can be used alone according to the invention, because they are in reality mixtures of esters, are, for example, as follows:
• the cationic surfactants are advantageously selected from the list hereinafter and such that they confer on the dispersion a pH of from 5 to 8; the weight ratio of the amount of nonionic amphiphilic lipids to the amount of cationic surfactants in the lipid phase being from 50/1 to 50/25, and the weight ratio of the lipid phase to the aqueous phase of a dispersion being from 1/1000 to 300/1000.
• the oleosomes relating to the invention are described in EP-0-705,593. They involve an emulsion of the oil-in-water type made up of oily globules provided with a lamellar liquid crystal coating, dispersed in an aqueous phase, characterized in that each oily globule is, as a unit, coated with a monolamellar or oligolamellar layer (1 to 10 sheets that can be visualized by transmission electron microscopy after cryofracture) obtained from at least one lipophilic surfactant, at least one hydrophilic surfactant and at least one cationic surfactant conferring on the emulsion a pH ranging from 5 to 8, the coated oily globules having an average diameter of less than 500 nanometers.
• the lipophilic surfactant and the hydrophilic surfactant each contain at least one saturated fatty chain having more than approximately 12 carbon atoms. Even more preferably, this fatty chain contains from 16 to 22 carbon atoms.
• the lipophilic surfactant has an HLB of from approximately 2 to approximately 5.
• HLB Hydrophilic-Lipophilic Balance
• lipophilic surfactants are sucrose distearate, diglyceryl distearate, tetraglyceryl tristearate, decaglyceryl decastearate, diglyceryl monostearate, hexaglyceryl tristearate, decaglyceryl pentastearate, sorbitan monostearate, sorbitan tristearate, diethylene glycol monostearate, the glyceryl ester of palmitic acid and stearic acid, polyoxyethylenated monostearate 2 OE (comprising 2 oxyethylene units), glyceryl monobehenate and dibehenate, and pentaerythritol tetrastearate.
• sucrose distearate diglyceryl distearate, tetraglyceryl tristearate, decaglyceryl decastearate, diglyceryl monostearate, hexaglyceryl tristearate, decaglyceryl pentastearate
• the hydrophilic surfactant preferably has an HLB of from approximately 8 to approximately 12.
• hydrophilic surfactants examples include polyoxyethylenated sorbitan monostearate 4 OE, polyoxyethylenated sorbitan tristearate 20 OE, polyoxyethylenated monostearate 8 OE, hexaglyceryl monostearate, polyoxyethylenated monostearate 10 OE, polyoxyethylenated distearate 12 OE and polyoxyethylenated methylglucose distearate 20 OE.
• the cationic surfactants can advantageously be selected from among the compounds mentioned hereinafter.
• the cationic particles can also be selected from oil-in-water nanoemulsions comprising an oily phase dispersed in an aqueous phase, the oil globules of which have a number-average size of less than 100 nm, characterized in that they comprise at least one amphiphilic lipid comprising at least one nonionic amphiphilic lipid and a cationic amphiphilic lipid, the oily phase and the amphiphilic lipids being present at a content such that the oily phase/amphiphilic lipid weight ratio ranges from 3 to 10.
• the nanoemulsions generally have a transparent to bluish appearance.
• the transparency of said nanoemulsions is measured by means of a coefficient of transmittance at 600 nm ranging from 10 to 90%, or else by means of turbidity.
• the turbidity of the compositions of the invention ranges from 60 to 400 NTU, and preferably from 70 to 300 NTU, which turbidity is measured using a HACH portable turbidity meter—model 2100 P at approximately 25° C.
• the oil globules of the nanoemulsions of the invention have a number-average size of less than 100 nm, and preferably ranging from 20 to 80 nm, and more preferably from 40 to 60 nm. Decreasing the size of the globules makes it possible to promote penetration of the active agents into the superficial layers of the skin (carrier effect).
• the nanoemulsions in accordance with the invention are preferably prepared at temperatures ranging from 4 to 45° C. and are thus compatible with thermosensitive active agents.
• one or more cationic surfactants will be exclusively used as ionic surfactant.
• the nonionic surfactants preferably water-soluble or water-dispersible, contain at least one hydrophobic block and at least one hydrophilic block.
• nonionic amphiphilic lipids of the invention are preferably selected from among:
• the silicone surfactant used according to the present invention is a compound of formula (II): in which:
• R 1 , R 2 and R 3 independently of one another, represent a C 1 -C 6 alkyl radical or a —(CH 2 ) x —(OCH 2 CH 2 ) y (OCH 2 CH 2 CH 2 ) z —OR 4 radical, at least one radical R 1 , R 2 or R 3 not being an alkyl radical; R 4 being hydrogen, an alkyl radical or an acyl radical;
• A is an integer ranging from 0 to 200;
• B is an integer ranging from 0 to 50; with the proviso that A and B are not equal to zero at the same time;
• x is an integer ranging from 1 to 6;
• y is an integer ranging from 1 to 30;
• z is an integer ranging from 0 to 5.
• the alkyl radical is a methyl radical
• x is an integer ranging from 2 to 6
• y is an integer ranging from 4 to 30.
• silicone surfactants of formula (II) representative are the compounds of formula (III): in which A is an integer ranging from 20 to 105, B is an integer ranging from 2 to 10 and y is an integer ranging from 10 to 20.
• silicone surfactants of formula (II) By way of example of silicone surfactants of formula (II), mention may also be made of the compounds of formula (IV): H—(OCH 2 CH 2 ) y —(CH 2 ) 3 —[(CH 3 ) 2 SiO] A —(CH 2 ) 3 —(OCH 2 CH 2 ) y —OH (IV) in which A′ and y are integers ranging from 10 to 20.
• silicone surfactants use may particularly be made of those marketed by Dow Corning under the trademarks DC 5329, DC 7439-146, DC 2-5695 and Q4-3667.
• the compounds DC 5329, DC 7439-146 and DC 2-5695 are compounds of formula (XI) where, respectively, A is 22, B is 2 and y is 12; A is 103, B is 10 and y is 12; A is 27, B is 3 and y is 12.
• the compound Q4-3667 is a compound of formula (IV) where A is 15 and y is 13.
• the C 8 -C 22 or C 14 -C 22 fatty acids which form the fatty unit of the esters which can be used in the nanoemulsion of the invention contain a saturated or unsaturated linear alkyl chain having, respectively, from 8 to 22 or from 14 to 22 carbon atoms.
• the fatty unit of the esters can in particular be selected from stearates, behenates, arachidonates, palmitates, myristates, laurates, caprates and mixtures thereof. Stearates are preferably used.
• esters or of mixtures of esters of a fatty acid and of sucrose, of maltose, of glucose or of fructose representative are sucrose monostearate, sucrose distearate, sucrose tristearate and mixtures thereof, such as the products marketed by Croda under the trademark Crodesta F50, F70, F110 and F160 having, respectively, an HLB (Hydrophilic-Lipophilic Balance) of 5, 7, 11 and 16; and by way of example of esters or of mixtures of esters of a fatty acid and of methylglucose, mention may be made of polyglyceryl-3 methylglucose distearate, marketed by Goldschmidt under the trademark Tego-care 450. Mention may also be made of monoesters of glucose or of maltose such as methyl o-hexadecanoyl-6-D-glucoside and o-hexadecanoyl-6-D-maltoside.
• the ethers of a fatty alcohol and of a sugar which can be used as nonionic amphiphilic lipids in the nanoemulsion according to the invention, are solid at a temperature of less than or equal to 45° C. and can be selected in particular from among the group comprising ethers or mixtures of ethers of a C 8 -C 22 fatty alcohol and of glucose, of maltose, of sucrose or of fructose, and ethers or mixtures of ethers of a C 14 -C 22 fatty alcohol and of methylglucose. They are in particular alkylpolyglucosides.
• the C 8 -C 22 or C 14 -C 22 fatty alcohols which form the fatty unit of the ethers which can be used in the nanoemulsion of the invention contain a saturated or unsaturated linear alkyl chain having, respectively, from 8 to 22 or from 14 to 22 carbon atoms.
• the fatty unit of the ethers can in particular be selected from decyl, cetyl, behenyl, arachidyl, stearyl, palmityl, myristyl, lauryl, capryl and hexadecanoyl units, and mixtures thereof such as cetearyl.
• alkylpolyglucosides such as decylglucoside and laurylglucoside, marketed, for example, by Henkel under the respective trademarks Plantaren 2000 and Plantaren 1200, cetostearylglucoside optionally as a mixture with cetostearyl alcohol, marketed, for example, under the trademark Montanov 68 by Seppic, under the trademark Tego-care CG90 by Goldschmidt and under the trademark Emulgade KE3302 by Henkel, and arachidylglucoside, for example in the form of the mixture of arachidyl and behenyl alcohols and of arachidylglucoside, marketed under the trademark Montanov 202 by Seppic.
• alkylpolyglucosides such as decylglucoside and laurylglucoside
• cetostearylglucoside optionally as a mixture with cetostearyl alcohol
• arachidylglucoside for example in the form of the mixture of arachidy
• nonionic amphiphilic lipid of this type use is more particularly made of sucrose monostearate, sucrose distearate, sucrose tristearate and mixtures thereof, polyglyceryl-3 methylglucose distearate and alkylpolyglucosides.
• esters can in particular be selected from among stearates, behenates, arachidates, palmitates and mixtures thereof. Stearates and palmitates are preferably used.
• CTFA names Polyglyceryl-10 stearate, Polyglyceryl-10 distearate, Polyglyceryl-10 tristearate, Polyglyceryl-10 pentastearate
• CTFA name Polyglyceryl-2 stearate
• the sorbitan fatty esters which can be used as nonionic amphiphilic lipids in the nanoemulsion according to the invention which are solid at a temperature of less than or equal to 45° C., are selected in particular from among the group comprising esters of a C 16 -C 22 fatty acid and of sorbitan and oxyethylenated esters of a C 16 -C 22 fatty acid and of sorbitan. They are formed from at least one fatty acid containing at least one saturated linear alkyl chain having respectively from 16 to 22 carbon atoms, and from sorbitol or ethoxylated sorbitol.
• the oxyethylenated esters generally contain from 1 to 100 ethylene oxide units, and preferably from 2 to 40 ethylene oxide (EO) units.
• esters can in particular be selected from among stearates, behenates, arachidates, palmitates, and mixtures thereof. Stearates and palmitates are preferably used.
• sorbitan fatty ester and of an oxyethylenated sorbitan fatty ester which can be used in the nanoemulsion of the invention
• representative are the sorbitan monostearate (CTFA name: Sorbitan stearate) marketed by ICI under the trademark Span 60, the sorbitan monopalmitate (CTFA name: Sorbitan palmitate) marketed by ICI under the trademark Span 40, and the sorbitan tristearate 20 EO (CTFA name: Polysorbate 65) marketed by ICI under the trademark Tween 65.
• the ethoxylated fatty ethers which are solid at a temperature of less than or equal to 45° C. which can be used as nonionic amphiphilic lipids in the nanoemulsion according to the invention, are preferably ethers formed from 1 to 100 ethylene oxide units and from at least one fatty alcohol chain having from 16 to 22 carbon atoms.
• the fatty chain of the ethers can in particular be selected from among behenyl, arachidyl, stearyl and cetyl units, and mixtures thereof such as cetearyl.
• ethoxylated fatty ethers By way of example of ethoxylated fatty ethers, mention may be made of ethers of behenyl alcohol comprising 5, 10, 20 and 30 ethylene oxide units (CTFA names: Beheneth-5, Beheneth-10, Beheneth-20, Beheneth-30), such as the products marketed under the trademarks Nikkol BB5, BB10, BB20 and BB30 by Nikko, and the ether of stearyl alcohol comprising 2 ethylene oxide units (CTFA name: Steareth-2), such as the product marketed under the trademark Brij 72 by ICI.
• CTFA names: Beheneth-5, Beheneth-10, Beheneth-20, Beheneth-30 such as the products marketed under the trademarks Nikkol BB5, BB10, BB20 and BB30 by Nikko
• CTFA name: Steareth-2 the ether of stearyl alcohol comprising 2 ethylene oxide units
• the ethoxylated fatty esters which are solid at a temperature of less than or equal to 45° C. which can be used as nonionic amphiphilic lipids in a nanoemulsion according to the invention, are esters formed from 1 to 100 ethylene oxide units and from at least one fatty acid chain having from 16 to 22 carbon atoms.
• the fatty chain of the esters can in particular be selected from among stearate, behenate, arachidate and palmitate units, and mixtures thereof.
• ester of stearic acid comprising 40 ethylene oxide units, such as the product marketed under the trademark Myrj 52 (CTFA name: PEG-40 stearate) by ICI and also the ester of behenic acid comprising 8 ethylene oxide units (CTFA name: PEG-8 behenate), such as the product marketed under the trademark Compritol HD5 ATO by Gattefosse.
• Poloxamer 124 such
• nonionic amphiphilic lipids representative are the mixtures of nonionic surfactants described in EP-A-705593, incorporated herein for reference.
• nonionic amphiphilic lipids use may in particular be made of:
• the nonionic amphiphilic lipids can be present in the nanoemulsion according to the invention at a content ranging from 0.2% to 12% by weight, relative to the total weight of the composition, and preferably ranging from 0.2% to 8% by weight, and preferentially ranging from 0.2% to 6% by weight.
• the cationic amphiphilic lipids are selected from the list given hereinafter.
• nanoemulsions of the invention are present in the nanoemulsions of the invention, preferably, in concentrations ranging from 0.01 to 6% by weight relative to the total weight of the nanoemulsion, and more particularly from 0.2 to 4% by weight.
• the oily phase of the nanoemulsions according to the invention comprise at least one oil.
• the oils which can be used in the nanoemulsions of the invention are preferably selected from the group consisting of:
• polyolefins which can be used as synthetic oils are in particular poly- ⁇ -oleflns, and more particularly those of hydrogenated or non-hydrogenated polybutene type, and preferably hydrogenated or non-hydrogenated polyisobutene type.
• the liquid carboxylic acid esters which can be used as synthetic oils may be monocarboxylic, dicarboxylic, tricarboxylic, or tetracarboxylic acid esters.
• the total number of carbons in the esters is generally greater than or equal to 10, and preferably less than 100, and more particularly less than 80. They are in particular monoesters of saturated or unsaturated, linear or branched C 1 -C 26 aliphatic acids and of saturated or unsaturated, linear or branched C 1 -C 26 aliphatic alcohols, the total number of carbons in the esters generally being greater than or equal to 10.
• esters of C 4 -C 22 dicarboxylic or tricarboxylic acids and of C 1 -C 22 alcohols and esters of monocarboxylic, dicarboxylic or tricarboxylic acids and of C 2 -C 26 dihydroxy, trihydroxy, tetrahydroxy or pentahydroxy alcohols.
• alkyl palmitates such as ethyl palmitate, isopropyl palmitate, 2-ethylhexyl palmitate, 2-octyldecyl palmitate; alkyl myristates such as isopropyl myristate, butyl myristate, cetyl myristate, 2-octyidodecyl myristate; alkyl stearates such as hexyl stearate, butyl stearate, isobutyl stearate; alkyl malates such as dioctyl malate; alkyl laurates such as hexyl laurate and 2-hexyldecyl laurate; isononyl isononanoate; cetyl octanoate.
• alkyl palmitates such as ethyl palmitate, isopropyl palmitate, 2-ethylhexyl palmitate, 2-octyldec
• the nanoemulsions according to the invention contain at least one oil of molecular weight greater than or equal to 400, in particular ranging from 400 to 10 000, better still ranging from 400 to 5000, or even ranging from 400 to 2500.
• the oils of molecular weight greater than or equal to 400 can be selected from oils of animal or plant origin, mineral oils, synthetic oils and silicone oils, and mixtures thereof.
• oils of this type mention may, for example, be made of isocetyl palmitate, isocetyl stearate, avocado oil and jojoba oil.
• the nanoemulsions in accordance with the invention comprise an amount of oily phase (oil and other fatty substances besides the amphiphilic lipid) preferably ranging from 2 to 40% by weight relative to the total weight of the nanoemulsion, and more particularly from 4 to 30% by weight, and preferably from 4 to 20% by weight.
• oily phase oil and other fatty substances besides the amphiphilic lipid
• the oily phase and the amphiphilic lipids are preferably present in the nanoemulsions according to the invention according to a weight ratio of the amount of oily phase to the amount of amphiphilic lipid ranging from 3 to 10, and preferably ranging from 3 to 6.
• the term “amount of oily phase” means the total amount of the constituents of this oily phase without including the amount of amphiphilic lipid.
• the nanoemulsions in accordance with the present invention can contain, in addition to the urea derivatives of formula (I) described above, solvents, in particular for improving, if necessary, the transparency of the composition.
• solvents are preferably selected from the group consisting of:
• solvents can be used as a mixture.
• they can be used at concentrations preferably ranging from 0.01 to 30% by weight relative to the total weight of the nanoemulsion, and better still from 5 to 20% by weight relative to the total weight of the nanoemulsion.
• the amount of alcohol(s) and/or of sugar(s) preferably ranges from 5 to 20% by weight relative to the total weight of the nanoemulsion and the amount of glycol(s) preferably ranges from 5 to 15% by weight relative to the total weight of the nanoemulsion.
• the method of preparing a nanoemulsion as defined above entails mixing the aqueous phase containing the urea derivative and the oily phase, with vigorous stirring, at a temperature ranging from 10° C. to 80° C., and in carrying out a high-pressure homogenization step at a pressure of greater than 5 ⁇ 10 7 Pa and in optionally adding the polymer used.
• another high-pressure homogenization step is subsequently carried out at a pressure of greater than 5 ⁇ 10 7 Pa.
• the high-pressure homogenization is preferably carried out at a pressure ranging from 6 ⁇ 10 7 Pa to 18 ⁇ 10 7 Pa.
• the shear preferably ranges from 2 ⁇ 10 6 s ⁇ 1 to 5 ⁇ 10 8 s ⁇ 1 , and better still from 1 ⁇ 10 8 s ⁇ 1 to 3 ⁇ 10 8 s 1 (s ⁇ 1 signifies second ⁇ 1 ).
• thermosensitive active compounds which may contain oils and in particular fragrances which contain fatty substances, without denaturing them.
• nanocapsules relating to the invention are those described in EP-0-447,318, EP-0-557,489, EP-0-780,115, EP-1-025,901, EP-1-029,587, EP-1-034,839, EP-1-414,390, FR-2,830,776, EP-1-342,471, FR-2,848,879 and FR 04/50057.
• the nanocapsules are Core-Shell particles having an oily core and a polymeric shell.
• the various applications mentioned above relate to various families of polymers and various methods for obtaining them.
• the size of the capsules is always less than 1 ⁇ m and it is possible to have sizes of less than 80 nm.
• These particles can be coated with a lamellar liquid crystal phase most commonly consisting of a lecithin or of a dimethicone copolyol.
• the coating must be an amphiphilic lipid capable of spontaneously forming a lamellar liquid crystal phase on contact with water.
• amphiphilic lipid capable of forming a lamellar phase that the cationic surfactant which will confer on the particles (the nanocapsule) a positive zeta potential will be added.
• the weight ratio of the amphiphilic lipid forming the lamellar phase to the cationic surfactant will be from 99/1 and 75/25.
• the cationic surfactants which can be used are those listed hereinafter.
• the organic particles of the invention are solid nanospheres, which do not have an internal cavity, formed by various methods (dispersion in water, nanoprecipitation, microemulsion, etc.) and composed of at least one polymer or of at least one copolymer, or of a mixture thereof.
• the particle is cationic, with the zeta potential defined above, either because the polymer(s) or copolymer(s) is (are) cationic, or because it (they) is (are) nonionic and a cationic surfactant as described hereinafter is used.
• the amount of cationic surfactant will be from 0 and 25%.
• the cationic inorganic particles of the invention may, by way of example, be based on silica, TiO 2 , ZnO, alumina, etc.
• representative are alumina particles in a colloidal dispersion in water, such as the Nanomer 2 particles from Nalco. Clariant and Grace also provide particles of this type.
• cationic surfactants which can be used according to the invention are listed hereinafter, this list being non-limiting.
• the cationic amphiphilic lipids are preferably selected from the group consisting of quaternary ammonium salts and fatty amines and their salts.
• the quaternary ammonium salts are, for example:
• quaternary ammonium salts containing at least one ester function which can be used according to the invention are, for example, those having the following formula (VII): in which:
• the alkyl radicals R 15 may be linear or branched, and more particularly linear.
• R 15 denotes a methyl, ethyl, hydroxyethyl or dihydroxypropyl radical, and more particularly a methyl or ethyl radical.
• the sum x+y+z is from 1 to 10.
• R 16 is a hydrocarbon-based radical R 20 , it may be long and may contain from 12 to 22 carbon atoms, or may be short and may contain from 1 to 3 carbon atoms.
• R 18 is hydrocarbon-based radical R 22 , it preferably contains 1 to 3 carbon atoms.
• R 17 , R 19 and R 21 are selected from among linear or branched, saturated or unsaturated C 11 -C 21 hydrocarbon-based radicals, and more particularly from linear or branched, saturated or unsaturated C 11 -C 21 alkyl or alkenyl radicals.
• x and z which may be identical or different, are 0 or 1.
• y is equal to 1.
• n, p, and r which may be identical or different, are 2 or 3, and even more particularly are equal to 2.
• the anion is preferably a halide (chloride, bromide or iodide) or an alkyl sulfate, more particularly methyl sulfate.
• Use may, however, be made of methanesulfonate, phosphate, nitrate, tosylate, an anion derived from an organic acid such as acetate or lactate, or any other anion compatible with the ammonium containing an ester function.
• the anion X ⁇ 1 is even more particularly chloride or methyl sulfate.
• ammonium salts having the formula (VII) in which:
• hydrocarbon-based radicals are linear.
• acyl radicals preferably contain 14 to 18 carbon atoms, and originate more particularly from a plant oil such as palm oil or sunflower oil.
• the compound contains several acyl radicals, the latter may be identical or different.
• This esterification is followed by a quaternization using an alkylating agent such as an alkyl halide (preferably, methyl or ethyl halide), a dialkyl sulfate (preferably, methyl or ethyl sulfate), methyl methanesulfonate, methyl para-toluenesulfonate, glycol chlorohydrin or glycerol chlorohydrin.
• an alkylating agent such as an alkyl halide (preferably, methyl or ethyl halide), a dialkyl sulfate (preferably, methyl or ethyl sulfate), methyl methanesulfonate, methyl para-toluenesulfonate, glycol chlorohydrin or glycerol chlorohydrin.
• Such compounds are, for example, marketed under the trademarks DEHYQUART by HENKEL, STEPANQUAT by STEPAN, NOXAMIUM by CECA and REWOQUAT WE 18 by REWOWITCO.
• compositions according to the invention preferably contain a mixture of quaternary ammonium monoester, diester and triester salts with a majority, by weight, being diester salts.
• ammonium salts use may, for example, be made of the mixture containing 15 to 30% by weight of acyloxyethyldihydroxyethylmethylammonium methyl sulfate, 45 to 60% of diacyloxyethylhydroxyethylmethylammonium methyl sulfate and 15 to 30% of triacyloxyethylmethylammonium methyl sulfate, the acyl radicals having from 14 to 18 carbon atoms and originating from optionally partially hydrogenated palm oil.
• Use may also be made of the ammonium salts containing at least one ester function described in U.S. Pat. Nos. 4,874,554 and 4,137,180.
• quaternary ammonium salts having the formula (IV) preference is given, firstly, to tetraalkylammonium chlorides, for instance dialkyldimethylammonium or alkyltrimethylammonium chlorides, in which the alkyl radical contains approximately from 12 to 22 carbon atoms, in particular behenyltrimethylammonium, distearyidimethylammonium, cetyltrimethylammonium or benzyldimethylstearylammonium chlorides, or else, secondly, the stearamidopropyldimethyl (myristyl acetate) ammonium chloride marketed under the trademark “CERAPHYL 70” by VAN DYK.
• tetraalkylammonium chlorides for instance dialkyldimethylammonium or alkyltrimethylammonium chlorides, in which the alkyl radical contains approximately from 12 to 22 carbon atoms, in particular behenyltrimethylammonium, distearyidimethylammoni
• behenyltrimethylammonium chloride or behenyltrimethylammonium bromide and CTAB are the quaternary ammonium salts most particularly preferred.
• fatty amines of the invention correspond to the general formula: wherein:
• stearylamine stearate aminoethylethanolamide, stearyl diethanolamide, stearate diethylenetriamine
• stearamidopropyldimethylamine stearamidopropyldiethylamine
• stearamidoethyldiethylamine stearamidoethyldimethylamine
• palmitoamidopropyldimethylamine palmitoamidopropyldiethylamine
• palmitoamidoethyldiethylamine palmitoamidoethyldimethylamine
• behenamidopropyldimethylamine behenamidopropyldiethylamine
• behenamidoethyldiethylamine behenamidoethyldimethylamine
• arachidamidopropyldimethylamine arachidamidopropyldimethylamine
• arachidamidopropyldiethylamine arachidamid
• fatty amine which is commercially available, mention may be made of Incromine BB from Croda, Amidoamine MSP from Nikkol, and Lexamine from Inolex.
• fatty amines mention will, by way of example, be made of stearylamine, stearate aminoethylethanolamide, stearyl diethanolamide and stearate diethylenetriamine that, inter alia, Sabo sells with the Sabomina series.
• fatty amine acetates such as the Acetamine series from Kao Corp.
• fatty amines can also be ethoxylated, such as Berol 380, 390, 453 and 455, the Ethomeens from Akzo Nobel or Marlazin L10, OL2, OL20, T15/2, T50 from Condea Chemie.
• the particles of the present invention can be introduced into any pharmaceutical carrier for cosmetic, dermatological or ophthalmic purposes.
• any pharmaceutical carrier for cosmetic, dermatological or ophthalmic purposes.
• compositions according to the invention can be in any of the pharmaceutical forms normally used for topical application, for example in the form of solutions, gels, dispersions of the lotion or serum type, emulsions which have a liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or conversely (W/O), or suspensions or emulsions which have a soft, semi-solid or solid consistency of the cream or gel type, or else microemulsions, microcapsules, microparticles or vesicular dispersions of ionic and/or nonionic type.
• These compositions are prepared according to the usual methods.
• compositions according to the invention can also comprise any additive normally used in the cosmetics or pharmaceutical field.
• care will be taken to ensure that this introduction does not harm the stability of the cationic particles associated with the siRNA.
• compositions according to the invention may in particular contain cosmetic or pharmaceutical active agents. This will preferably involve depigmenting agents, sunscreens.
• the depigmenting agents which can be incorporated into the composition comprise, for example, the following compounds: kojic acid; ellagic acid; arbutin and derivatives thereof such as those described in EP-895,779 and EP-524,109; hydroquinone; aminophenol derivatives such as those described in WO 99/10318 and WO 99/32077, and in particular N-cholesteryloxycarbonyl-para-aminophenol and N-ethyloxycarbonyl-para-aminophenol; iminophenol derivatives, in particular those described in WO 99/22707; L-2-oxothiazolidine-4-carboxylic acid or procysteine, and its salts and esters; ascorbic acid and its derivatives, in particular ascorbyl glucoside; and plant extracts, in particular extracts of liquorice, mulberry and sculicap, without this list being limiting.
• the ultraviolet-radiation-screening agents can be selected from organic UV-screening agents or inorganic UV-radiation-screening agents.
• organic UV-screening agents in accordance with the invention may be water-soluble, liposoluble or insoluble in the usual cosmetic solvents. They are selected in particular from anthranilates; cinnamic derivatives; dibenzoylmethane derivatives; salicylic derivatives, camphor derivatives; triazine derivatives such as those described in U.S. Pat. No.
• the inorganic screening agents are generally pigments or else nanopigments (average size of the primary particles: generally from 5 nm and 100 nm, preferably from 10 nm and 50 nm) of metal oxides which may or may not be coated, such as, for example, nanopigments of titanium oxide (amorphous or crystalline in rutile and/or anatase form), of iron oxide, of zinc oxide, of zirconium oxide or of cerium oxide, which are all UV photoprotective agents well known per se.
• Conventional coating agents are, moreover, alumina and/or aluminium stearate.
• Such coated or uncoated metal oxide nanopigments are in particular described in EP-0-518,772 and EP-0-518,773.
• the radiation-screening agents in accordance with the invention are generally present in the compositions according to the invention in proportions ranging from 0.1 to 20% by weight relative to the total weight of the composition, and preferably ranging from 0.2 to 15% by weight relative to the total weight of the composition.
• the present invention also features the administration of at least one double-stranded RNA oligonucleotide of a sequence selected from the group consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48 (Table I), for inhibiting tyrosinase expression.
• the oligonucleotides according to the invention are useful as a bleaching agent and/or as an anti-browning agent for the skin.
• the double-stranded RNA oligonucleotides according to the invention can also be used for the formulation of topical, cosmetic or dermatological compositions suited to decrease melanin synthesis, in particular with a view to treating hyperpigmentation, and pigmentation marks and dyschromia, or for bleaching hair follicles, regional hyperpigmentations due to melanocyte hyperactivity, such as idiopathic melasmas, occurring during pregnancy (“pregnancy mask” or chloasma) or oestro-progestin contraception, localized hyperpigmentations due to benign melanocyte hyperactivity and proliferation, such as senile pigmentation marks referred to as actinic lentigo, accidental hyperpigmentations, possibly due to post-lesional wound healing or photosensitization, and also certain forms of leukoderma, such as vitiligo.
• melanocyte hyperactivity such as idiopathic melasmas, occurring during pregnancy (“pregnancy mask” or chloas
• the process is completed by depigmenting the areas of residual normal skin so as to impart to the skin as a whole a homogeneous white tint.
• the present invention also features a cosmetic method (regime or regimen) for bleaching and/or lightening the complexion and/or making the color of a browned skin uniform, comprising the topical application of at least one double-stranded RNA oligonucleotide of a sequence selected from the group consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48.
• the oligonucleotide is formulated in a topical composition at a concentration of less than or equal to 1 nM.
• the oligonucleotide is associated with a cationic particle less than or equal to 1 ⁇ m in size, with a zeta potential of from 10 to 80 mV, selected from among surfactant micelles, block polymer micelles, liposomes of nonionic and cationic surfactants, niosomes, oleosomes, particles of nanoemulsions, nanocapsules, organic particles or inorganic particles, as described above.
• cationic particles associated with siRNA according to the present invention can be applied topically to the skin or the mucous membranes or in the eye, in suspension or incorporated into an acceptable cosmocentric carrier, such as lotions, sera, emulsified or non-emulsified gels, oil/water, water/oil or multiple emulsions, microemulsions, etc.
• the water permeability may be modified; thus, it is possible to control the water permeability by measuring the IWL (insensible water loss) with a Tewameter TM210 or a Dermalab—Cortex technology.
• compositions of the present invention After application of the compositions of the present invention, methods may be employed known to those skilled in the art which promote the penetration of molecules into the skin, such as, for example, iontophoresis or electroporation.
• the 2 surfactants are solubilized in distilled water.
• a suspension of the siRNA of SEQ ID NO. 1 (20 ⁇ M) is added at from 1:1 and 1:9 volume (siRNA: micelles), thus reducing the micellar concentration in proportion.
• Exemplar 2 Micelles of decyl- ⁇ -glucoside/behenyltriammonium chloride at the molar ratio of 5/1 in distilled water. The same concentrations as in the preceding exemplar are prepared.
• These 2 suspensions can be applied to the skin for the treatment of pigmentation marks and dyschromia or for the bleaching of hair follicles.
• Exemplar 1 “Fluid” Vesicles: PEG 400 isostearate 5.5% Behenyltriammonium chloride 0.5% Distilled water qs 100
• liposome suspensions are realized by dialysis.
• the lipids constituting the vesicles are solubilized in an aqueous solution of octyl- ⁇ -glucoside. This solution is then dialyzed against water for 72 h.
• the suspension of siRNA SEQ ID NO. 4 is then added, bringing the concentration with respect to the vesicle to around 3% of lipid.
• This suspension of nonionic liposomes can be applied to the skin for the treatment of pigmentation marks and dyschromia or for the bleaching of hair follicles.
• Aqueous Phase Distilled water qs 100 Preservative 0.1%
• This dispersion is obtained by high-pressure homogenization in order to have a particle size of approximately 170 nm.
• a suspension of siRNA SEQ ID NO. 16 (20 ⁇ M) at ratios ranging from 1:1 to 1:20 (siRNA/oleosomes) is then added.
• the siRNA will then complex with the surface of the particles.
• Aqueous Phase Pluronic F68 0.5% Distilled water 200 ml
• the organic phase is introduced with stirring into the aqueous phase.
• the acetone and 100 ml of aqueous phase are then evaporated off so as to obtain the suspension of nanocapsules, the size of which is 220 nm.
• a suspension of siRNA SEQ ID NO. 37 (20 ⁇ M) at ratios ranging from 1:1 to 1:20 (siRNA/nanocapsules) is then added.
• the siRNA will then complex with the surface of the particles.
• Aqueous Phase Pluronic F68 0.5% Distilled water 200 ml
• the organic phase is introduced with stirring into the aqueous phase.
• the acetone and 100 ml of aqueous phase are then evaporated off so as to obtain the suspension of nanoparticles, the size of which is 180 nm.
• a suspension of siRNA SEQ ID NO. 40 (20 ⁇ M) at ratios ranging from 1:1 to 1:20 (siRNA/nanoparticles) is then added.
• the siRNA will then complex with the surface of the particles.
• Aqueous Phase Distilled water 30% Dipropylene glycol 10%
• An emulsion is prepared by dispersing the oily phase in the aqueous phase with very vigorous stirring.
• the suspension obtained is then homogenized several times using a very-high-pressure homogenizer, at a pressure of approximately 1200 b.
• the particle size is of the order of 50 nm and the suspension is transparent.
• the dilution phase is then added.
• siRNA SEQ ID NO. 42 (20 ⁇ M) at ratios ranging from 1:1 to 1:20 (siRNA/nanoemulsion) is then added.
• the siRNA will then complex with the surface of the particles.
• Oligonucleotide (SEQ ID NO.) 1 nM 0.25 nM 0.063 nM 0.016 nM 1 97.37% 94.34% 89.47% 72.07% 2 97.98% 95.64% 89.34% 66.68% 4 97.93% 95.22% 90.24% 57.92% 5 91.33% 79.88% 61.47% 18.40% 7 96.86% 88.97% 76.45% 38.34% 8 97.84% 90.76% 81.12% 38.57% 13 98.13% 95.73% 91.11% 62.56% 14 97.59% 87.22% 68.77% 33.87% 16 97.95% 94.92% 83.40% 56.25% 23 96.05% 87.42% 62.32% 29.97% 25 93.45% 90.50% 78.36% 46.80% 29 96.42% 88.24% 70.64% 31.15% 32 96.88% 90.69% 80.33% 35.04% 35 97.42% 94.84% 88.34% 5
• siRNAs according to the present invention show an effectiveness of greater than 94% at 1 nM for the 20 sequences most effective.
• the lowest dose tested is 0.016 nM for an effectiveness of 72% on degradation of the mRNA encoding tyrosinase.
• siRNAs which are subjects of the present invention did not induce an interferon-type response.
• OAS-1 and IFIT-1 interferon-inducible tetratricopeptide repeat domain
• Patz inches R et al. Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization. J. Virol. 2001; 75: 1332-1338.), was measured by quantitative PCR (qPCR) according to the protocol described in the “BLOCK-iTTM RNAi Stress Response Control Kit (Human) for monitoring interferon-mediated stress response to double-stranded RNA in human cells” marketed by Invitrogen.
• qPCR quantitative PCR
• the level of expression of the hOAS-1 gene was related to that of the GAPDH reference gene.

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