Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C233/00—Carboxylic acid amides
  • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
  • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/51—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings
• • 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
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • 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
  • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
  • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
• • 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
  • 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
• • 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
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C233/00—Carboxylic acid amides
  • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
  • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/49—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a carbon atom of an acyclic unsaturated carbon skeleton
• • 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

Definitions

• the present invention relates to a novel use of cosmetic active agents for lightening the skin.
• depigmenting cosmetic formulations are based on kojic acid, arbutin or magnesium ascorbyl phosphate.
• the inventors became interested in the development of novel depigmenting active agents that have better compatibility with the skin than those of the prior art. They demonstrated that molecules that inactivate protein kinase A give rise to a skin depigmentation that was attributed hitherto only to inhibition of the enzyme phosphorylated tyrosinase.
• the subject of the invention is the use of a compound that inactivates protein kinase A in a composition containing a cosmetically acceptable medium, for lightening the skin.
• PKA protein kinase A
• the inhibition of protein kinase A induces reduced activation of tyrosinase, as a result of the reduced conversion of the latter enzyme into phosphorylated tyrosinase; this reduced activation of tyrosinase results in a reduction in melanin synthesis, giving rise to skin depigmentation.
• compound that inactivates protein kinase A especially denotes any compound which, by incubating protein kinase A in the presence of adenosine triphosphate and a protein that may be phosphorylated, for instance the histone H1, inhibits its phosphorylation, with a percentage of inhibition of greater than or equal to 10%, more particularly with a percentage of inhibition of greater than or equal to 25% and preferably greater than or equal to 50%.
• a subject of the invention is, more particularly, the use of a compound of formula (I): or salts thereof, in which R 1 represents the characterizing chain of a saturated or unsaturated, linear or branched fatty acid containing from 3 to 30 carbon atoms, R 2 represents the characterizing chain of an amino acid and m is between 1 and 50, or a mixture of said compounds of formula (I) or salts thereof, in a composition containing a cosmetically acceptable medium, for lightening the skin.
• the compound of formula (I) as defined above may be in free acid form or in partially or totally salified form.
• the salts are especially alkali metal salts such as the sodium, potassium or lithium salts, alkaline-earth metal salts such as the calcium, magnesium or strontium salts; the ammonium salt or the salt of an amino alcohol, for instance the (2-hydroxyethyl)-ammonium salt. They may also be metal salts such as divalent zinc or manganese salts or trivalent iron, lanthanum, cerium or aluminum salts.
• compound of formula (I) means the compound of formula (I) in free form or in partially or totally salified form.
• characterizing chain used to define the radicals R 1 and R 2 denotes the nonfunctional main chain of the fatty acid or of the amino acid under consideration.
• the subject of the invention is, mainly, the use of a compound of formula (I) as defined above, in which the group R 1 —C( ⁇ O)— contains from 7 to 22 carbon atoms.
• R 1 —C( ⁇ O)— especially represents an octanoyl, decanoyl, undecylenoyl dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, docosanoyl, 8-octadecenoyl, eicosenoyl, 13-docosenoyl, 9,12-octadecadienoyl or 9,12,15-octadecatrienoyl radical.
• a subject of the invention is, more particularly, the use of a compound of formula (I) as defined above, in which the fragment R 1 —C( ⁇ O) is chosen from octanoyl, ⁇ -undecylenoyl, dodecanoyl, hexadecanoyl, 8-octa-decenoyl, 13-docosenoyl, 9,12-octadecadienoyl and 9,12,15-octadecatrienoyl radicals.
• R 2 especially represents the characterizing chain of an amino acid chosen from glycine, alanine, serine, aspartic acid, glutamic acid, valine, threonine, arginine, lysine, proline leucine, phenylalanine, isoleucine, histidine, tyrosine, tryptophan, asparagine, glutamine, cysteine, cystine, methionine, hydroxyproline, hydroxylysine, sarcosine and ornithine.
• an amino acid chosen from glycine, alanine, serine, aspartic acid, glutamic acid, valine, threonine, arginine, lysine, proline leucine, phenylalanine, isoleucine, histidine, tyrosine, tryptophan, asparagine, glutamine, cysteine, cystine, methionine, hydroxyproline, hydroxylysine, sarcosine
• the subject of the invention is, mainly, the use of a compound of formula (I) as defined above, in which, in at least one of the residues
• R 2 represents the characterizing chain of phenyl-alanine, tyrosine, histidine, methionine, cysteine or tryptophan.
• a subject of the invention is, more particularly, the use of a compound of formula (I) as defined above, in which m is a decimal number between 1 and 10 and is preferably less than 5.
• m is less than or equal to 2 and is more particularly less than or equal to 1.4.
• n is equal to 1.
• only one compound of formula (I), as defined above, is used in the composition containing the cosmetically acceptable medium.
• the compounds of formula (I) are generally obtained by N-acylation of compounds of formula (IIIa) or (IIIb) as defined above, or salts thereof.
• proteins may be of animal origin, for instance collagen, elastin, fish flesh protein, fish gelatin, keratin or casein, of plant origin, for instance, proteins from cereals, flowers or fruit, for instance proteins derived from soybean, sunflower, oat, wheat, maize, barley, potato, lupin, bean, sweet almond, kiwi, mango or apple; they may also be proteins obtained from chorellae (unicellular algae), pink algae, yeasts or silk.
• chorellae unicellular algae
• This hydrolysis is performed, for example, by heating a protein placed in an acidic or alkaline medium to temperatures of between 60 and 130° C.
• This hydrolysis may also be performed enzymatically with a protease, optionally coupled to an alkaline or acidic posthydrolysis.
• a protease optionally coupled to an alkaline or acidic posthydrolysis.
• R 2 represents one and the same chain or several chains characterizing different amino acids, depending on the protein hydrolyzed and the degree of hydrolysis.
• the acylation reaction is known to those skilled in the art. It is described, for example, in the international patent application published under the number WO 98/09611. It is performed either on an amino acid or on an amino acid mixture.
• the acylating agent generally consists of an activated derivative of a carboxylic acid of formula R 1 C( ⁇ O)—OH, such as a symmetrical anhydride of this acid or an acid halide, for instance the acid chloride or acid bromide.
• It may also consist of a mixture of activated derivatives of carboxylic acids derived from natural oils or fats of animal or plant origin, such as coconut oil, palm kernel oil, palm oil, soybean oil, rapeseed oil, maize oil, beef tallow, spermaceti oil or herring oil.
• a subject of the invention is also a nontherapeutic process for treating the skin to lighten it, characterized in that a composition containing a cosmetically acceptable medium and an effective amount of at least one compound that inactivates protein kinase A is applied thereto.
• a subject of the invention is also a pharmaceutical composition for performing a therapeutic skin treatment to lighten it, characterized in that it contains a pharmaceutically acceptable medium and an effective amount of at least one compound that inactivates protein kinase A.
• the compound that inactivates protein kinase A is generally used in an amount of between 0.01% and 10% of their weight, more particularly between 0.1% and 5% of their weight and most particularly between 1% and 5% of their weight.
• a subject of the invention is the use as defined above, characterized in that the compound that inactivates protein kinase A also inactivates adenylate cyclase.
• adenylate cyclase results in reduced conversion of intracellular ATP into cyclic AMP; the reduction in the level of cyclic AMP results in inhibition of protein kinase A (PKA); the inhibition of protein kinase A induces reduced activation of tyrosinase as a result of the reduced conversion of said enzyme into phosphorylated tyrosinase; this reduced activation of tyrosinase results in a reduction in melanin synthesis, giving rise to the skin depigmentation.
• PKA protein kinase A
• compound that inactivates adenylate cyclase especially denotes, in the context of the present invention, any compound which, by incubation of this enzyme in the presence of adenosine triphosphate, inhibits its conversion into cyclic adenosine mono-phosphate, with a percentage of inhibition of greater than or equal to 10%, more particularly with a percentage of inhibition of greater than or equal to 25% and preferably greater than or equal to 50%.
• the compounds that inactivate adenylate cyclase contained in said composition are more particularly chosen from the compounds of formula (I) as defined above or salts thereof, and most particularly from the compounds of formula (I) as defined above in which R 1 —C( ⁇ O) is chosen from octanoyl and ⁇ -undecylenoyl radicals and in which, in at least one of the residues R 2 represents the characterizing chain of phenylalanine.
• a subject of the invention is also a process as defined above, characterized in that a composition containing a cosmetically acceptable medium and an effective amount of at least one compound that inactivates protein kinase A and adenylate cyclase, and also a pharmaceutical composition as defined above, characterized in that it contains an effective amount of at least one compound that inactivates protein kinase A and adenylate cyclase, are applied to the skin.
• a subject of the invention is the use as defined above, characterized in that the compound that inactivates protein kinase A and adenylate cyclase is a compound with affinity for the melanocyte specific hormone ( ⁇ -MSH) receptor.
• ⁇ -MSH melanocyte specific hormone
• the competition between the hormone ⁇ -MSH and the molecule with affinity for the ⁇ -MSH receptor results in a reduced level of binding of said hormone to the cell receptors; the consequence of this competition is to inhibit the activity of adenylate cyclase, which results in reduced conversion of intracellular ATP into cyclic AMP; the reduction in the level of cyclic AMP results in inhibition of the enzyme protein kinase A (PKA); the inhibition of protein kinase A induces reduced activation of tyrosinase as a result of the reduced conversion of said enzyme into phosphorylated tyrosinase; this reduced activation of tyrosinase results in a decrease in melanin synthesis, giving rise to skin depigmentation. It is this set of successive inhibitions that bears witness to the ⁇ -MSH-antagonist nature of the compounds of the invention.
• PKA protein kinase A
• compound with affinity for the melanocyte specific hormone, ⁇ -MSH, receptor denotes any compound which displaces the specific binding of a radioactive ligand, for instance nucleoside diphosphate- ⁇ -melanocyte specific hormone ([ 125 I]NDP- ⁇ -MSH) to the ⁇ -melanocyte specific hormone ( ⁇ -MSH) type 1 receptor, known as the MC1R receptor, with a percentage of inhibition of greater than or equal to 10%, more particularly with a percentage of inhibition of greater than or equal to 25% and preferably greater than or equal to 50%.
• a radioactive ligand for instance nucleoside diphosphate- ⁇ -melanocyte specific hormone ([ 125 I]NDP- ⁇ -MSH) to the ⁇ -melanocyte specific hormone ( ⁇ -MSH) type 1 receptor, known as the MC1R receptor, with a percentage of inhibition of greater than or equal to 10%, more particularly with a percentage of inhibition of greater than or equal to 25% and preferably greater than or equal to 50%.
• the melanocyte specific hormone antagonists contained in said composition are more particularly chosen from the compounds of formula (I) as defined above, or salts thereof.
• a subject of the invention is also a process as defined above, characterized in that a composition containing a cosmetically acceptable medium and an effective amount of at least one compound that inactivates protein kinase A and adenylate cyclase, which is a melanocyte specific hormone antagonist, and also a pharmaceutical composition as defined above, characterized in that it contains an effective amount of at least one compound that inactivates protein kinase A and adenylate cyclase, which is a melanocyte specific hormone antagonist, are applied to the skin.
• the compounds used in the cosmetic or therapeutic treatments defined above are characterized, unexpectedly, by skin-lightening activity that is higher than that of the compositions of the prior art. They are thus generally suitable for treatments for lightening the skin, especially by depigmentation, and more particularly for removing or attenuating colored marks appearing on elderly skin.
• compositions used in said treatments are generally in the form of dilute aqueous or aqueous-alcoholic solutions, in the form of simple or multiple emulsions, such as water-in-oil (W/O), oil-in-water (O/W) or water-in-oil-in-water (W/O/W) emulsions, in which the oil is of plant or mineral nature, or in the form of powder. They may also be dispersed or impregnated onto fabric or nonwoven materials, whether they are wipes, paper towels or clothing.
• compositions used in said treatments are administered to the individual in the conventional forms used in cosmetics and pharmacy; these are more particularly topical, oral or parenteral administrations.
• the compounds of formula (I) that inactivate protein kinase A, possibly adenylate cyclase and possibly melanocyte specific hormone antagonists, which are used in the invention that is the subject of the present patent application, as defined above, are combined with numerous types of adjuvants or active principles used in cosmetic formulations, whether they are fatty substances, organic solvents, thickeners, gelling agents, softeners, antioxidants, opacifiers, stabilizers, foaming agents, fragrances, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preserving agents, chemical screening agents or mineral screening agents, essential oils, dyestuffs, pigments, hydrophilic or lipophilic active agents, humectants, for instance glycerol, preserving agents, dyes, fragrances, cosmetic active agents, mineral or organic sunscreens, mineral fillers, for instance iron oxides, titanium oxides and talc, synthetic fillers, for instance Nylons and crosslinked or noncrosslinked
• oils that may be combined with the compound of formula (I)
• fatty materials that may be combined with this active agent, mention may be made of fatty alcohols or fatty acids.
• thickening and/or emulsifying polymers used in the present invention there are, for example, homopolymers or copolymers of acrylic acid or of acrylic acid derivatives, acrylamide homopolymers or copolymers, homopolymers or copolymers derived from acrylamide, homopolymers or copolymers of acrylamidomethylpropanesulfonic acid, of vinyl monomer or of trimethylaminoethyl acrylate chloride, sold under the names CarbopolTM, UltrezTM 10, PermulenTM TR1, PermulenTM TR2, SimulgelTM A, SimulgelTM NS, SimulgelTM EPG, SimulgelTM EG, Luvigel ⁇ EM, SalcareTM SC91, SalcareTM SC92, SalcareTM SC95, SalcareTM SC96, FlocareTM ET100, HispagelTM, SepigelTM 305, SepigelTM 501, SepigelTM 502, FlocareTM ET58 and StabilezeTM 06; hydrocolloids
• waxes that may be used in the context of the present invention, examples that may be mentioned include beeswax; carnauba wax; candelilla wax; ouricury wax; Japan wax; cork fiber wax or sugarcane wax; paraffin waxes; lignite waxes; microcrystalline waxes; lanolin wax; ozokerite; polyethylene wax; hydrogenated oils; silicone oils; plant waxes; fatty alcohols and fatty acids that are solid at room temperature; glycerides that are solid at room temperature.
• emulsifiers that may be used in the context of the present invention, examples that may be mentioned include fatty acids; ethoxylated fatty acids; fatty acid esters of sorbitol; ethoxylated fatty acid esters; polysorbates; polyglycerol esters; ethoxylated fatty alcohols; sucrose esters; alkylpolyglycosides; sulfated and phosphated fatty alcohols or mixtures of alkylpolyglycosides and of fatty alcohols described in French patent applications 2 668 080, 2 734 496, 2 756 195, 2 762 317, 2 784 680, 2 784 904, 2 791 565, 2 790 977, 2 807 435 and 2 804 432.
• active principles that may be combined with the compound of formula (I) in order to synergistically potentiate its properties
• compounds with lightening or depigmenting activity for instance arbutin, kojic acid, hydroquinone, ellagic acid, vitamin C, magnesium ascorbyl phosphate, polyphenol extracts, grape extracts, pine extracts, wine extracts, olive extracts, pond extracts, N-acyl proteins, N-acyl peptides, N-acylamino acids, partial hydrolyzates of N-acyl proteins, amino acids, peptides, total protein hydrolyzates, partial protein hydrolyzates, polyols (for instance glycerol, butylene glycol, etc.), urea, pyrrolidonecarboxylic acid or derivatives of this acid, glycyrrhetinic acid, ⁇ -bisabolol, sugars or sugar derivatives, polysaccharides or derivatives thereof, hydroxy acids, for instance arbutin, k
• sunscreens that may be incorporated into the composition according to the invention, mention may be made of any of those featured in the Cosmetic Directive 76/768/EEC amended appendix VII.
• a subject thereof is N-(- ⁇ -undecylenoyl)phenylalanine of formula: its cosmetic use, pharmaceutical compositions containing it and emulsions characterized in that they have a content thereof of between 0.01% and 10% of their weight, more particularly between 0.1% and 5% of their weight and most particularly between 1% and 5% of their weight.
• the object of this study was to demonstrate the depigmenting activity of N-undecylenoylphenylalanine, according to a mechanism involving the antagonist effect of the molecule on the a-melanocyte specific hormone ( ⁇ -MSH) type 1 receptor, known as the MC1R receptor.
• ⁇ -MSH a-melanocyte specific hormone
• FIG. 1 The control of melanogenesis using this receptor is shown in FIG. 1. It especially involves adenylate cyclase, cAMP, protein kinase A and tyrosinase.
• ⁇ -MSH stimulates the ⁇ subunit of the stimulating protein G (Prot G ⁇ S).
• This protein activates the enzyme adenylate cyclase, which converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP).
• cAMP activates the A protein kinases (PK A), which convert tyrosinase into phosphorylated tyrosinase, which stimulates melanogenesis.
• PK A A protein kinases
• the depigmenting activity of N-undecylenoylphenylalanine on melanocyte cultures of the B16/F1 line was determined in vitro by measuring the intracellular and extracellular melanin contents and by measuring the tyrosinase activity.
• a fourth step the depigmenting activity of N-undecylenoylphenylalanine was evaluated in a model of pigmented reconstructed human epidermides (photo-type IV) in order to test the efficacy of the product under real application conditions (topical application of the formulated product).
• MC1R receptors are isolated from cell membranes of mouse melanocytes of the B16/F1 line via the method described in: Siegrist W., Oestreicher M., Stutz M., Girard J. and Eberle A. E.; J. Recep. Res., 8, 1988, 323-343”.
• N-Undecylenoylphenylalanine, arbutin and kojic acid are diluted to a concentration of 10 mg/ml in decinormal aqueous sodium hydroxide solution. They are each tested separately at concentrations of 0.1 mg/ml and 1 mg/ml. Sodium hydroxide has no effect on the parameter studied.
• the MC1R receptors are incubated, in the presence or absence of these products, with an iodine-125 labeled radioactive ligand, the nucleoside diphosphate- ⁇ -melanocyte specific hormone [ 125 I]NDP- ⁇ -MSH at a concentration of 0.05 nM, for 90 minutes at 22° C.
• Control cultures are incubated, in the absence of product, and in the presence of the radioactive ligand. Each test is performed in triplicate.
• the cell membranes are rapidly filtered and the filters are washed several times with cold buffer.
• the amount of radioactive ligand bound to the MC1R receptors is measured using a scintillation counter (Topcount, Packard).
• Adenylate cyclase which converts ATP into cAMP, is extracted from rat brains via the method described in “Salamon Y., Londos C. and Rodbell M.; Anal. Biochem., 58, 1974, 541-548”; it is then activated with 10 ⁇ M of forskolin.
• N-Undecylenoylphenylalanine, arbutin and kojic acid are diluted to a concentration of 10 mg/ml in decinormal aqueous sodium hydroxide solution. They are each tested separately at a concentration of 1 mg/ml. Sodium hydroxide has no effect on the parameter studied.
• the activated enzyme is incubated, in the presence or absence of these products, and in the presence of 0.5 mM of ATP, for 30 minutes at 30° C.
• Control cultures are incubated, in the absence of product, and in the presence of ATP. Each test is performed in triplicate.
• the amount of cAMP produced is evaluated via a radioimmunological assay performed using a commercial kit; the radioactivity is measured with a scintillation counter (Topcount, Packard), a small radioactivity count reflecting small activation of adenylate cyclase.
• a scintillation counter Topcount, Packard
• PK A protein kinase A
• Protein kinase A is extracted from bovine brains via the method described in: “Chijiwa T., Mishima A., Hagiwara M., Sano M., Hayashi K., Inoue T., Naito K., Shioka T., Hidaka H.; J. Biol. Chem., 265, 1990, 5267-5272”. It is then activated with 3 ⁇ M of cAMP.
• N-Undecylenoylphenylalanine, arbutin and kojic acid are diluted to a concentration of 10 mg/ml in decinormal aqueous sodium hydroxide solution. They are each tested separately at a concentration of 1 mg/ml. Sodium hydroxide has no effect on the parameter studied.
• the activated enzyme is incubated, in the presence or absence of these products, and in the presence of 33 P-labeled radioactive ATP ([ ⁇ - 33 P]ATP) and 200 ⁇ g/ml of histone H 1 , for 20 minutes at 30° C.
• 33 P-labeled radioactive ATP [ ⁇ - 33 P]ATP
• 200 ⁇ g/ml of histone H 1 for 20 minutes at 30° C.
• Control cultures are incubated, in the absence of product, and in the presence of radioactive ATP and histone H 1 . Each test is performed in triplicate.
• the amount of 33 P-labeled phosphorylated histone H 1 is measured using a scintillation counter (Topcount, Packard), a small radioactivity count reflecting small activation of the protein kinase A.
• the tyrosinase used is a commercial product extracted from fungi.
• N-Undecylenoylphenylalanine, hydroquinone, arbutin and kojic acid are diluted to a concentration of 10 mg/ml in decinormal aqueous sodium hydroxide solution. They are each tested separately at concentrations of 0.1 mg/ml and 1 mg/ml. Sodium hydroxide has no effect on the parameter studied.
• Tyrosinase at 66.66 IU/ml is incubated, in the presence or absence of these products, and in the presence of 0.2 mM of tyrosine, for 10 minutes at 37° C.
• Control cultures are incubated, in the absence of product, and in the presence of tyrosinase and L-tyrosine. Each test is performed in triplicate.
• the amount of dopaquinone histone formed is measured using a spectro-photometer at 490 nm.
• Mouse melanocytes of the B16/F1 line are inoculated in 96-well culture plates at a density of 1500 cells/well.
• the cells are cultured in a culture medium (MCM medium) at 37° C. under a humid atmosphere containing 5% CO 2 .
• MCM medium culture medium
• the cells are used at 60% of confluence, i.e. 4 days after inoculation.
• the MCM medium has the following composition: DMEM medium (Dulbecco's Modified Eagle's Medium) containing 4.5 g/l of glucose supplemented with L-glutamine (2 mM), penicillin (50 IU/ml), streptomycin (50 ⁇ g/ml) and fetal calf serum (10% v/v).
• DMEM medium Dulbecco's Modified Eagle's Medium
• penicillin 50 IU/ml
• streptomycin 50 ⁇ g/ml
• fetal calf serum 10% v/v.
• N-Undecylenoylphenylalanine is diluted to 4 mg/ml in decinormal aqueous sodium hydroxide solution. It is tested at 40 ⁇ g/ml in the MCM medium. Sodium hydroxide has no effect on the parameters analyzed.
• Hydroquinone is tested at 5 ⁇ g/ml in the MCM medium. Given its toxicity, it is not tested at 40 ⁇ g/ml.
• Arbutin and kojic acid are tested at 40 ⁇ g/ml in the MCM medium.
• the melanocyte cultures are incubated in the presence of the test product or of the reference products for 72 hours at 37° C., under a humid atmosphere containing 5% CO 2 .
• Control cultures are incubated, in the absence of product, in the MCM medium. These control cultures are prepared on each culture plate.
• the extracellular melanin is quantified by spectrophotometry at 450 nm.
• a melanin calibration range is prepared in parallel.
• PBS phosphate-buffered saline
• the intracellular melanin is quantified by spectrophotometry at 450 nm.
• a melanin calibration range is prepared in parallel.
• the cells are lyzed with TritonTM X100 at a concentration of 0.1% (w/v) for 30 minutes at room temperature.
• the activity of the endogenous tyrosinase is evaluated by adding 0.1% (w/v) of L-dopa, followed by incubation for 3 hours at 37° C. in the absence of air and light.
• the dopaquinone formed by the reaction between the tyrosinase and the L-dopa is measured by spectrophotometry at 450 nm.
• a calibration range of purified tyrosinase is prepared in parallel.
• This assay allows the cytotoxicity of the test products to be evaluated. It is performed in cell lysates prepared as described in the preceding paragraph.
• the protein assay is performed according to the Coomassie blue method described by: “Bradford M.; Anal. Biochem., 72, 1976, 248-254”. The measurement is performed by spectrophotometry at 640 nm. A bovine serum albumin (BSA) calibration range is prepared in parallel.
• BSA bovine serum albumin
• hydroquinone tested at 5 ⁇ g/ml, inhibits the extracellular melanin content by 85%, the intracellular melanin content by 100% and the tyrosinase activity by 69%, respectively.
• the depigmenting effect of hydroquinone is partly derived from its cytotoxic effect, since a 38% decrease in the total protein quantity is observed.
• Arbutin tested at 40 ⁇ g/ml, inhibits the extracellular melanin content by 47%, the intracellular melanin content by 19% and the tyrosinase activity by 23%, respectively. At this concentration, arbutin has no effect on the total protein content.
• Kojic acid tested at 40 ⁇ g/ml, inhibits the extracellular melanin content by 70%, the intracellular melanin content by 17% and the tyrosinase activity by 31%, respectively. At this concentration, kojic acid has no significant effect on the total protein content.
• N-Undecylenoylphenylalanine tested at 40 ⁇ g/ml, inhibits the extracellular melanin content by 72%, the intracellular melanin content by 66% and the tyrosinase activity by 67%, respectively. At this concentration, N-undecylenoylphenylalanine decreases the total protein content by 17%.
• N-Undecylenoylphenylalanine thus has depigmenting activity, demonstrated by a concommitant reduction of the intracellular and extracellular melanin contents and of tyrosinase activity. Unlike that of hydroquinone, its depigmenting activity is not linked to a cytotoxic effect. It has higher depigmenting activity than arbutin and kojic acid.
• Pigmented human epidermides supplied by Skinethic, of 0.63 cm 2 , are reconstructed from a coculture of normal human keratinocytes (skin of the forearm, 3 year old donor, 2nd passage) and from normal human melanocytes (skin of the forearm, 4 year old donor of phototype IV, 3rd passage).
• the keratinocyte/melanocyte ratio is 10:1.
• the cocultures are inoculated onto inert polycarbonate filters. They are cultured for 10 days in the medium supplied by Skinethic, consisting of MCDB 153 medium supplemented with 5 ⁇ g/ml of insulin, 1.5 mM of calcium and growth factors.
• the products are tested after having been incorporated into a cosmetic formulation consisting of an emulsion comprising an aqueous phase, 10% by weight of a fatty phase (LanolTM 1688), 2% by weight of an emulsifier (SimugelTM EG), 0.5% by weight of preserving agents (0.3% of SepicideTM HB+0.2% of SepicideTM CI).
• a cosmetic formulation consisting of an emulsion comprising an aqueous phase, 10% by weight of a fatty phase (LanolTM 1688), 2% by weight of an emulsifier (SimugelTM EG), 0.5% by weight of preserving agents (0.3% of SepicideTM HB+0.2% of SepicideTM CI).
• N-Undecylenoylphenylalanine, arbutin and kojic acid are incorporated therein at elevated temperature (75° C.), in a proportion of 1% or 3% by weight per unit volume (w/v).
• hydroquinone is incorporated therein at a concentration of 0.1% by weight per unit volume (w/v).
• the epidermides are cultured in 6-well plates containing 1 ml of the medium described above. They are incubated at 37° C. under a humid atmosphere containing 5% CO 2 .
• the formulations containing the various active principles are applied to the surface of the epidermides, at a rate of 2 ⁇ l/epidermis, using a sterile bacteriological inoculator.
• the application is performed every day for 4 consecutive days.
• the incubation medium of the reconstructed epidermides is renewed every day for 4 consecutive days.
• Control epidermides are treated with a formulation free of active principle. Each test is performed in duplicate.
• the color of the epidermides is evaluated using a chromameter (Minolta) by measuring the following parameters L*, a* and b*:
• the intracellular melanin is extracted from the epidermides by incubation for 45 minutes at 100° C. in SolueneTM 350 (200 ⁇ l/epidermis), as described in “Ozeki H., Ito S., Wakamatsu K., Hirobe T.; J. Invest. Dermatol., 105, 1995, 361-366. The samples are centrifuged for 10 minutes at 10 000 rpm.
• the extracted intracellular melamin is measured by spectrophotometry at 500 nm.
• a melanin calibration range is prepared in parallel.
• Hydroquinone tested in topical application at a concentration of 0.1% (w/v) in an emulsion, has no significant effect either on the chromametric parameters L*, a* and b* or on the melanin content of the reconstructed human epidermides.
• the absence of a depigmenting effect of hydroquinone is due either to the low test concentration, which was deliberately selected as noncytotoxic, or to the short duration of the treatment.
• Arbutin tested in topical application at 1% and 3% (w/v) in an emulsion, has no significant effect on the chromametric parameters L*, a* and b*. However, it inhibits the melanin content of the reconstructed human epidermides by 28% and 24%, respectively.
• Kojic acid tested in topical application at 1% and 3% (w/v) in an emulsion, has no significant effect on the chromametric parameters L*, a* and b*. However, it inhibits the melanin content of the reconstructed human epidermides by 21% and 33%, respectively.
• N-Undecylenoylphenylalanine tested in topical application at 1% (w/v) in an emulsion, inhibits the b* color parameter by 15% and the melanin content of the reconstructed human epidermides by 24%.
• N-undecylenoylphenylalanine increases the L* parameter by 9% and concomitantly reduces the a* color parameter by 14%, the b* color parameter by 29% and the melanin content of the reconstructed epidermides by 24%.
• N-undecylenoylphenylalanine The results obtained in this study together demonstrate strong depigmenting activity of N-undecylenoylphenylalanine. This activity is quantified both in melanocyte cultures and in a 3D model composed of reconstructed human epidermides. In contrast to the reference products, the depigmenting activity of N-undecylenoylphenylalanine involves the MC1R receptors. N-Undecylenoylphenylalanine is an MC1R receptor antagonist and inhibits all the steps of the ⁇ -MSH cycle involved in melanogenesis.
• This lotion may be sold in bottles or impregnated into wipes.
• SepiliftTM DHP (INCI name: dipalmitoyl hydroxyproline), sold by the company SEPPIC.
• SepicideTM HB is a preserving mixture comprising phenoxyethanol, methyl paraben, ethyl paraben, propyl paraben and butyl paraben, sold by the company SEPPIC.
• SepicideTM CI is imidazolidinylurea, sold by the company SEPPIC.
• SepicalmTM VG (INCI name: sodium palmitoyl proline and extract of water lily flower), sold by the company SEPPIC.
• KathonTM CG (INCI name: methylisothiazolinone/methylchloroisothiazolinone).
• SimulgelTM EG is a copolymer inverse latex (INCI name: sodium acrylate/sodium acryloyldimethyltaurate copolymer and isohexadecane and Polysorbate 80) sold by the company SEPPIC.
• SimulgelTM NS is a copolymer inverse latex (INCI name: hydroxyethyl acrylate/sodium acryloyldimethyltaurate copolymer and squalane and Polysorbate 60) sold by the company SEPPIC.
• LanolTM 1688 is cetearyl ethylhexanoate, sold by the company SEPPIC.
• SepigelTM 305 is a polymer inverse latex (INCI name: polyacrylamide and C13-C14 isoparaffin and Laureth 7).
• MontanovTM L is an emulsifier based on C14-C22 alcohol and on C12-C20 alkyl polyglucoside.
• MontanovTM 68 is an emulsifier based on cetearyl alcohol and cetearyl polyglucoside.
• MontanovTM 202 is an emulsifier based on arachidyl alcohol, behenyl alcohol and arachidyl polyglucoside.

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• 2002
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