EP1830825A1

EP1830825A1 - Use of a compound capable of increasing glutathione level in melanocytes for treating canities

Info

Publication number: EP1830825A1
Application number: EP05850545A
Authority: EP
Inventors: Stéphane COMMO
Original assignee: LOreal SA
Current assignee: LOreal SA
Priority date: 2004-12-22
Filing date: 2005-12-20
Publication date: 2007-09-12
Also published as: US20080014229A1; FR2879444A1; US8481762B2; WO2006070101A1; JP2008525387A; FR2879444B1; KR101165648B1; JP5730462B2; KR20120116900A; CN101087584B; KR101078635B1; KR20070086593A; KR20100127890A; CN101087584A

Abstract

The invention concerns the use of compounds capable of increasing glutathione (GSH) level in hair follicle melanocytes for treating canities

Description

USE OF A COMPOUND CAPABLE OF INCREASING THE GLUTATHION RATE IN MELANOCYTES FOR THE TREATMENT OF CANITIS

The present invention relates to the cosmetic use of a compound capable of increasing the level of glutathione (GSH) in the melanocytes of the hair follicle to treat canities.

The hair follicle is a tubular invagination of the epidermis that penetrates to the deep layers of the dermis. The lower part, or hairy bulb, itself contains an intussusception in which is the dermal papilla. The lower part of the bulb is a zone of cellular proliferation where the precursors of the keratinized cells constituting the hair are found. Ascending cells from these precursors keratinize progressively in the upper part of the bulb, and this set of keratinized cells will form the hair shaft. The color of hair and hair is based in particular on the presence in quantities and variable ratios of two groups of melanins: eumelanins (brown and black pigments) and pheomelanines (red and yellow pigments). The pigmentation of hair and hair requires the presence of melanocytes in the bulb of the hair follicle. These melanocytes are in an active state, i.e. they synthesize melanins. These pigments are transmitted to the keratinocytes intended to form the hair shaft which will lead to the growth of a pigmented hair or hair. This structure is hereinafter called "follicular unit of pigmentation".

In mammals, melanogenesis involves at least three enzymes: tyrosinase, DOPAchrome tautomerase (TRP-2, for Tyrosinase Related Protein 2) and DHICAoxidase (TRP-1, for Tyrosinase Related Protein 1). Tyrosinase is the enzyme that initiates the biosynthesis of melanins. It is also described as the limiting enzyme of melanogenesis.

TRP-2 catalyzes the tautomerization of DOPAchrome to 5,6-Dihydroxyindole-2-carboxylic acid (DHICA). In the absence of TRP-2, DOPAchrome undergoes spontaneous decarboxylation to form 5,6-dihydroxyindole (DHI). DHICA and DHI are both precursors of pigments, TRP-1 oxidizes DHICA molecules to form quinone derivatives (Pawelek JM and Chakraborty AK.) The enzymology of melanogenesis In: Nordlund JJ, Boissy RE, Hearing VJ, King RA , Orton, J P. The Pigmentary System: Physiology and Pathophysiology, New York: Oxford University Press, 1998, pp. 391-400). The three enzymes, tyrosinase, TRP-2 and TRP-1, appear specifically involved in melanogenesis. In addition, the activity of these three enzymes has been described as necessary for maximal activity of eumelanin biosynthesis.

Hair and hair undergo a cycle. This cycle comprises a growth phase (anagen phase), a degeneration phase (catagen phase) and a rest phase (telogen phase) following which a new anagen phase will develop. Because of this hair cycle, and unlike the epidermal pigmentation unit, the follicular unit of pigmentation must also be cyclically renewed.

Canitia (natural whitening of the hair) is related to a specific and progressive thinning of the hair melanocytes affecting both the hair bulb melanocytes and the precursor cells of melanocytes (Commo et al., Br J Dermatol 2004; 150: 435-443 ). Other cell types present in hair follicles are unaffected. In addition, this rarefaction of melanocytes is not observed in the epidermis. The cause of this progressive and specific rarefaction of melanocytes and precursors of melanocytes in the hair follicle is not yet identified.

It therefore seems necessary to fight against the disappearance of melanocytes from human hair follicles, a process that affects both the active melanocytes of the bulbs and the quiescent melanocytes of the upper region of the hair follicles, to fight against canities.

The Applicant has already described a means of combating the whitening of the hair by acting on the enzyme TRP-2 (WO 03/103568).

Surprisingly and unexpectedly, the Applicant has now discovered that the expression of the TRP-2 enzyme is correlated with the expression of GSH, indeed, the expression of TRP-2 induces an increase in the level of GSH in the melanocytes. Thus, it has been demonstrated that in melanocytes that do not express TRP-2 (for example, hair precursors of melanocytes), there is a low level of GSH compared to melanocytes that express the TRP enzyme. -2 (for example, all melanocytes in the skin). Thus, the Applicant has now identified a new target for the treatment of canities, it has shown that the compounds capable of increasing the level of GSH in melanocytes led, contrary to their depigmenting effect described in the literature, to the restoration of hair pigmentation.

This use is particularly surprising in view of the fact that the compounds known to increase the level of GSH in melanocytes, such as lipoic acid and hydrocoumarins (Yamamura et al., 2002, Lin et al., 2002), are described. as agents limiting the synthesis of melanins and thus decreasing skin pigmentation in accordance with the demonstration that GSH is unfavorable to melanin synthesis (DeI Marmol et al., 1993, Jara et al., 1988).

Thus the object of the present invention relates to the use of compounds capable of increasing the level of GSH in hair melanocytes as an agent for preventing, limiting or stopping the progression of canities, and maintaining and / or promoting natural repigmentation of hair and / or hair.

In particular, the subject of the invention relates to the use of a compound capable of increasing the level of GSH to prevent and / or limit and / or stop the development of canities.

Compounds capable of increasing the level of GSH are, for example, compounds inducing the synthesis of GSH or compounds limiting its consumption, they may in particular be identified by the following method: a- culturing melanocytes, for example a primary culture of skin or hair melanocytes obtained as described in the article by Commo et al; Pigment CeII Res

2004; 17: 488-497. b-addition to the culture medium of a compound for which it is desired to test the property of increasing the level of GSH. incubating the melanocytes for a time long enough to allow the compound to be active. d- measure of the GSH rate. e-selection of compounds that increase the level of GSH. In a particular embodiment of the method for identifying a compound which increases the level of GSH, the cell cultures are carried out in an oven, at 37 ^° C., 5% CO ₂ .

In particular, step (a) can be carried out according to the following protocol: the melanocytes are seeded at 0 with medium M2 (PromoCell, Heidelberg, D). It may be for example a primary culture of hair melanocytes or skin obtained as described in the article by Commo et al. Pigment CeII Res 2004; 17: 488-497. The cells are maintained in this culture medium between 12 and 72 hours before the treatment.

Step (b) may be carried out according to the following protocol: the melanocytes are treated in culture with the compound for which it is desired to test the property of increasing the GSH level for the time necessary to reveal this property, this time is generally between 12 and 72 hours.

The step (d) of measuring the GSH level may be carried out for example by HPLC method. In a particular embodiment of this measurement, the free amino acids (AA) extracted from the cultured and treated cells are analyzed using the HITACHI L-8500 amino acid auto-analyzer. In this way the free amino acids are separated on an ion exchange column with eluents based on lithium salts, then assayed by colorimetry after reaction with ninhydrin.

The GSH assay can also be performed by a fluorescence method using an intracellular GSH fluorescent probe, for example such as Monochlorobimane (Molecular Probes, USA).

Alternatively the GSH assay can be performed using commercial kits such as the Bioxytech Kit GSH-400 colorimetry assay (Calbiochem, USA).

The subject of the invention also relates to the use of a compound capable of increasing the level of GSH to maintain the natural pigmentation of hair and / or gray hairs.

The compound capable of increasing the level of GSH may in particular be chosen from lipoic acid, oltipraz, kahweol, cafestol, angelicalactone, diallyl sulfide and bensyl. isothyocyanate (Scharf G et al Nutr Cancer 2003, 45 (1): 74-83, Huber WW et al., Mol Mol about 2004, 44 (4): 265-276, Sheen LY et al., Food Chem Toxicol 1996 34 (10): 971-978, Gupta E et al., Clin Cancer Res., 1995; 1 (10): 1133-1138).

Preferably, the compound capable of increasing the level of GSH will not be N-acetyl cysteine or quercitin.

An object of the invention is also a composition for combating canities, comprising in a cosmetically acceptable medium, at least one compound capable of increasing the level of GSH as defined above, with the exception of N-acetyl cysteine and quercitin, associated with another active agent selected from desquamating agents of the scalp and / or plant extracts with pro-pigmenting activity. Preferably, the compound capable of increasing the level of GSH is chosen from lipoic acid, oltipraz, kahweol, cafestol, angelicactone, diallyl sulfide and bensyl isothyocyanate.

The present invention also relates to a composition for combating canities comprising, in a cosmetically acceptable medium, at least one compound capable of increasing the level of GSH chosen from oltipraz, kahweol, cafestol, angelicalactone, bensyl isothyocyanate, combined with a hair regrowth agent.

The composition according to the invention comprises an amount of compound capable of increasing the level of GSH between 0.001% and 10% by weight per volume, preferably between 0.01 and 5% by weight by volume and even more preferably between 0. , 1 and 1% by weight by volume.

The composition according to the invention may be administered orally or applied topically to the skin (on any cutaneous area of the body covered with hair) and / or the scalp.

Orally, the composition according to the invention may contain the compound or compounds capable of increasing the level of GSH in solution in a food liquid such as an aqueous or aqueous-alcoholic solution, optionally flavored. They can also be incorporated in an unmanageable solid excipient and be presented for example in the form of granules, pills, tablets or dragees. They can also be placed in solution in a food liquid conditioned itself possibly in unmanageable capsules.

Depending on the mode of administration, the composition of the invention may be in any galenical form normally used, particularly in cosmetology. A preferred composition of the invention is a cosmetic composition suitable for topical application to the scalp and / or the skin.

For topical application, the composition that may be used according to the invention may especially be in the form of an aqueous, aqueous-alcoholic or oily solution or of the lotion-type or serum-type dispersion, of emulsions of 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 of soft consistency of the cream or gel type, aqueous or anhydrous, or else microcapsules or microparticles, or vesicular dispersions of ionic and / or nonionic type. It can thus be in the form of ointment, tincture, cream, ointment, powder, patch, impregnated buffer, solution, emulsion or vesicular dispersion, lotion, gel, spray, suspension, shampoo, aerosol or foam. They can be anhydrous or aqueous. It can also consist of solid preparations constituting soaps or cleaning rolls. These compositions are prepared according to the usual methods.

The composition that may be used according to the invention may in particular be a composition for hair care, and in particular a shampoo, a setting lotion, a treatment lotion, a cream or a styling gel, a dye composition (in particular oxidation dyes). ) optionally in the form of coloring shampoos, restructuring lotions for the hair, mask.

The cosmetic composition according to the invention will preferably be a cream, a hair lotion, a shampoo or a conditioner.

The amounts of the various constituents of the compositions that can be used according to the invention are those conventionally used in the fields under consideration. When the composition that can be used according to the invention is an emulsion, the proportion of the fatty phase can range from 5% to 80% by weight, and preferably from 5% to 50% by weight relative to the total weight of the composition. The oils, the waxes, the emulsifiers and the co-emulsifiers used in the composition in emulsion form are chosen from those conventionally used in the cosmetics field. The emulsifier and the coemulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5% to 20% by weight relative to the total weight of the composition. . The emulsion may further contain lipid vesicles.

When the composition that can be used according to the invention is an oily solution or gel, the fatty phase may represent more than 90% of the total weight of the composition.

In one variant of the invention, the composition will be such that the compound capable of increasing the level of GSH is encapsulated in a coating such as microspheres, nanospheres, oleosomes or nanocapsules, the coating will be chosen according to the nature chemical compound capable of increasing the GSH level.

By way of example, the microspheres may be prepared according to the method described in the patent application EP 0 375 520.

The nanospheres may be in the form of an aqueous suspension and be prepared according to the methods described in patent applications FR 0015686 and FR 0101438.

The oleosomes consist of an oil-in-water emulsion formed by oily globules provided with a lamellar liquid crystal coating dispersed in an aqueous phase (see patent applications EP 0 641 557 and EP 0 705 593).

The compound capable of increasing the level of GSH may also be encapsulated in nanocapsules consisting of a lamellar coating obtained from a silicone surfactant (see patent application EP 0 780 115), the nanocapsules may also be prepared based on water-dispersible sulphonic polyesters (see patent application FR 0113337). The compound capable of increasing the level of GSH may also be complexed on the surface of cationic oily globules, whatever their size (see patent applications EP 1 010413, EP 1 010 414, EP 1 010 415, EP 1 010416, EP 1 013 338, EP 1 016453, EP 1 018 363, EP 1 020219, EP 1 025 898, EP 1 120 101, EP 1 120 102, EP 1 129 684, EP 1 160 005 and EP 1 172 077).

The compound capable of increasing the level of GSH can finally be complexed on the surface of nanocapsules or nanoparticles provided with a lamellar coating (see EP 0 447 318 and EP 0 557 489) and containing a cationic surfactant on the surface (see references cited above for cationic surfactants).

In particular, preference will be given to a composition such that the coating containing the compound capable of increasing the level of GSH has a diameter of less than or equal to 10 μm. When the coating does not form a spherical vesicle, diameter is understood to mean the largest dimension of the vesicle.

In a known manner, the composition according to the invention may also contain adjuvants customary in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillers, filters, odor absorbers and dyes. The amounts of these various adjuvants are those conventionally used in the cosmetics field, and for example from 0.01% to 10% of the total weight of the composition. These adjuvants, depending on their nature, can be introduced into the fatty phase, into the aqueous phase and / or into the lipid spherules.

As oils or waxes that can be used in the invention, mention may be made of mineral oils (vaseline oil), vegetable oils (liquid fraction of shea butter, sunflower oil), animal oils (perhydrosqualene), synthetic oils ( Purcellin oil), silicone oils or waxes (cyclomethicone) and fluorinated oils (perfluoropolyethers), beeswax, camauba or paraffin waxes. To these oils can be added fatty alcohols and fatty acids (stearic acid).

As emulsifiers used in the invention there may be mentioned for example glycerol stearate, polysorbate 60 and the PEG-6 / PEG-32 / glycol stearate sold under the name Tefose ^® 63 by the company Gattefosse. As solvents that can be used in the invention, mention may be made of lower alcohols, in particular ethanol and isopropanol, and propylene glycol.

As hydrophilic gelling agents that may be used in the invention, mention may be made of carboxyvinyl polymers (carbomer), acrylic copolymers such as acrylate / alkylacrylate copolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural gums and clays, and, as lipophilic gelling agents, mention may be made of modified clays such as bentones, metal salts of fatty acids such as aluminum stearates and hydrophobic silica, ethylcellulose and polyethylene.

The compositions that may be used according to the invention may combine at least one compound capable of increasing the level of GSH with other active agents. Among these active agents, there may be mentioned by way of example: agents that modulate the differentiation and / or proliferation and / or pigmentation of skin cells such as retinol and its esters, vitamin D and its derivatives, estrogens such as estradiol, modulators of cAMP such as I ¹ POMC derivatives, adenosine, or forskolin and its derivatives, prostaglandins and derivatives thereof, triiodotrionine and its derivatives; extracts of plants such as Iridaceae or soybean extracts, which may then contain isoflavones or not;

extracts of microorganisms;

anti-free radical agents such as α-tocopherol or its esters, superoxide dismutases or its mimetics, certain metal chelators or ascorbic acid and its esters;

anti-seborrheic agents such as certain sulfur amino acids, 13-cis retinoic acid, cyproterone acetate;

other desquamating agents of the scalp such as zinc pyrithione, selenium disulphide, climbazole, undecylenic acid, ketoconazole, piroctone olamine (octopirox) or ciclopirocton (ciclopirox); in particular, it may be active stimulating regrowth and / or promoting the slowing down of hair loss, it may more particularly be mentioned without limitation:

esters of nicotinic acid, including in particular tocopherol nicotinate, benzyl nicotinate and C- ₎ -Cg alkyl nicotinates, such as methyl nicotinates or hexyl;

pyrimidine derivatives, such as 2,4-diamino-6-piperidinopyrimidine 3-oxide or "Minoxidil" described in US Pat. Nos. 4,139,619 and 4,596,812; Aminexil or 2,4 diamino pyrimidine 3 oxide described in WO96 / 09048; the lipoxygenase or cyclooxidase inducing agents promoting hair regrowth, such as those described by the Applicant in the European patent application EP 0 648 488;

antibacterial agents such as macrolides, pyranosides and tetracyclines, and in particular Erythromycin; calcium antagonists, such as Cinnarizine, Nimodipine and Nifedipine;

hormones, such as estriol or analogues, or thyroxine and its salts;

antiandrogens, such as oxendolone, spironolactone, diethylstilbestrol and flutamide;

steroidal or nonsteroidal inhibitors of 5-α-reductases such as those described by the Applicant in the European patent applications EP 0 964 852 and

EP 1 068 858 or alternatively iminasteride;

- agonists dependent potassium channels of I ¹ ATP such as cromakalim and nicorandil;

plant extracts with pro-pigmenting activity such as chrysanthemum extracts as described in FR 2768343 and extracts of Sanguisorba described in FR 2782920A1.

Preferably, the compound capable of increasing the level of GSH is associated with another active agent selected from the desquamative states scalp control agents, agents promoting hair regrowth, plant extracts with propigmenting activity.

Another subject of the present invention relates to a process for the cosmetic treatment of canities, characterized in that a composition as defined above, comprising at least one compound capable of increasing, is administered or applied to the zone to be treated. the GSH rate.

The invention also relates to a cosmetic treatment method intended to maintain the natural pigmentation of hair and / or gray or white hairs, characterized in that a composition as defined is administered or applied to the zone to be treated. previously comprising at least one compound capable of increasing the GSH level. The methods of treating canities and pigmentation of hair and / or gray or white hairs may also consist of the ingestion of a composition comprising at least one compound capable of increasing the level of GSH.

The areas to be treated may be, for example and without any limitation, the scalp, the eyebrows, the mustache and / or the beard and any area of the skin covered with hair.

More particularly, the methods of cosmetic treatment of canities and natural pigmentation of hair and / or gray or white hairs consist in applying a composition comprising at least one compound capable of increasing the GSH level.

Cosmetic treatment methods for combating canities and / or for maintaining the natural pigmentation of hair and / or gray or white hairs may for example consist in applying the composition to the hair and the scalp, in the evening, keeping the composition all night and possibly shampoo in the morning or wash the hair with this composition and leave it in contact for a few minutes before rinsing. The composition according to the invention has proved particularly advantageous when it is applied in the form of a hair lotion, optionally rinsed or even in the form of a shampoo.

Figures:

Figure 1: Figure 1 shows a Western blot with anti-TRP-2 and anti-vimentin antibodies on melanocyte lines WM35-wt (wild type), C2, C4, C7 and C8 (transfected and selected clones) as described in Example 1. It follows from this Western Blot that only clones C2, C4 and C8 express TRP-2.

Figure 2:

FIG. 2 is a histogram showing the GSH ratio on the sum of a selection of amino acids assayed (see Example 1, Part C) in melanocyte lines WM35-wt (wild type), C2, C4, C7 and C8 . Figures 3 and 4:

Figures 3 and 4 show the effect of oxidative stress (H ₂ O ₂ ) on cell viability of wild-type melanocytes expressing little or no TRP-2 (rhombus), TRP2 (+) melanocytes (square) and melanocytes wild-type xprimant little or no TRP-2 treated with the combination of Cafestol + Kahweol (Figure 3) or with oitipraz (Figure 4) (triangle).

Example 1 Assay of GSH in melanocytes as a function of TRP-2 expression. A- obtaining TRP-2 positive and TRP-2 negative melanocyte lines. A1: Cloning of the TRP-2 protein.

The entire region of the messenger RNA encoding the TRP-2 protein is cloned, from messenger RNA extracted from a primary skin melanocyte culture, by the RT-PCR technique using the δ probes. -GGAGATGGTGAGAAGCGCTAC-S 'and 5'-GCGGAAACTACAGCTAAGCAT-3', according to Genebank D17547 sequence. The PCR product obtained is cloned again using probes containing nucleotide sequences corresponding to the restriction sites for cloning in a eukaryotic expression vector: δ'-AGGGATCCATGAGCCCCCTTTGGTGGGGGTTT-S 'and 5'-GGAATTCAGCACCCTAGGCTTC-3' . The expression vector used is pCDNA3.1 (+). The vector containing the region coding for the TRP-2 protein is then produced from competent bacteria and then purified, this vector is then named pCDNA-TRP2. A2: Obtaining TRP-2 Positive Melanoma Lines.

WM35 melanoma cells (Pak BJ et al., Melanoma Res., 2000: 10: 499) that express little of the TRP-2 protein constitutively in vitro (wild-type) are transfected with plasmid pCDNA-TRP2. The stably transfected cells are then selected by a geneticin (G418) treatment. The clones obtained are then isolated and amplified.

B: Verification of the differential expression of TRP-2 in the different clones selected by the western blot method (see Commo et al; Pigment CeII Res 2004; 17: 488-497)

Cultures of each of the clones are lysed with the same lysis buffer suitable for protein extraction and analysis in western blot The rate of TRP-2 in the various extracts is determined by the Western blot method from 8 μg of protein extracts . The Western blot (see protocol in Maniatis et al.) Is carried out with the following antibodies: αPEPδh, polyclonal antibody donated by Dr. VJ Hearing (NIH, Bethesda, USA), and αvimentin, monoclonal antibody Vim3B4 (Cymbus, UK).

Figure 1 shows that clones C2, C4 and C8 express more TRP-2 protein compared to clone 7 and the wild line (wt).

C: GSH assay in TRP-2 positive and TRP-2 negative clones.

For each of the clones studied, the cells are seeded at a rate of 2 × 10 ⁴ cells / cm ² . The cell cultures are then lysed using a lysis buffer pH 2.

The extracted free amino acids (AA) are analyzed using the HITACHI L-8500 amino acid auto-analyzer. In this way the free AAs are separated on an ion exchange column by eluents based on lithium salts, and then assayed by colorimetry after reaction with ninhydrin. In each extract the measured GSH level is reported as the sum of AA (praline (P), glycine (G), alanine (A), valine (V), cysteine (C), methionine (M), isoleucine (I) , leucine (L), tyrosine (Y), phenylalanine (F), lysine (K), histidine (H), arginine (R)) to normalize the sample.

Figure 2 shows that clones C2, C4, and C8 have a higher GSH level than clone C7 and that the wild line.

Example 2 - Example of the effect of compounds which increase the level of GSH or limit its decrease on the viability of TRP-2M melanocytes.

The principle of this test is as follows: a- culture of two cell types expressing TRP-2 and not expressing TRP-2, developed according to a method similar to that used in Example 1A respectively Cell-TRP2 (+) and Cell-TRP2 (-), it can be any cell type, preferably melanocytes. b-addition to the Cell-TRP2 (-) cell culture medium of a compound capable of promoting the accumulation of GSH. c) Incubation of the cultures for a long enough time to allow the increase of the level of GSH in the cells. exposure of cells to a condition inducing apoptosis or senescence. e-measure of apoptosis or senescence. f- selection of compounds promoting GSH accumulation and making it possible to protect TRP2 (-) cells.

In a particular embodiment of the method, the cell cultures are carried out in an oven, at 37 ° C., 5% CO ₂ .

In particular, step (a) may be carried out according to the following protocol: the cells are seeded at a density of 5 × 10 ⁴ cells / cm ² , at 0 ° C. with M2 medium (PromoCell, Heidelberg, D). The cells are maintained in this culture medium between 12 and 72 hours before the treatment.

Step (b) may be carried out according to the following protocol: the cells are treated in culture with the test compound for the time necessary to reveal this property, this time is generally between 12 and 72 hours.

For carrying out step (d), the Cell-TRP2 (+) and Cell-TRP2 (-) populations are exposed to a condition inducing apoptosis or senescence in culture, it may be, for example, treatment with cis-platinum (Pak BJ et al., 2000, Melanoma Res., 10: 499-505) or oxaliplatin, with a toxic agent or a precursor compound for toxic molecules such as adriamycin, dihydroxyphenylalanine, paraquat, paracetamol, 4-hydroxyoestradiol, or 4-hydroxyanisol, exposure to ultraviolet radiation, oxidative stress (H ₂ O ₂ ,, diethylmaleate) (see Vaux DL & Strasser A. 1996, Proc.Natl.Acad.Sci 93: 2239-2244).

For the realization of step (e), the methods of revelation of apoptosis or senescence can be used:

the apoptotic response can be determined by any method to reveal cellular apoptosis, for example, identification of DNA fragmentation after agarose gel electrophoresis, labeling of DNA fragments by the "TUNEL" method (Gavrieli Y et al, J CeII Biol 1992: 119: 493-501), revelation of anexin V (ApoAlert Annexin V Apoptosis Kit (1996) CLONTECHniques Xl (3): 9-11 (BD Biosciences, Belgium)), determination of cytosolic nucleosomes (CeII Death Detection ElisaPlus kit (1-774-425, Roche, Germany), measuring cell viability. the senescent response can be determined by any method making it possible to reveal cellular senescence, for example, determination of a shortening of the telomeres, measurement of telomerase activity (TRAPeze kit, Intergen), determination of the decrease in the rate of cyclin E, determination of the decrease in the level of phosphorylated Rb protein (Bandyopadhyay D et al., Experimental Gerontology 2001: 36: 1265-1275), measurement of beta-galactosidase activity (Dimri GP et al., PNAS 1995; 92: 9363 -9367)

In the present case, the ability of both the Cafestol / Kahweol mixture and the Oltipraz to compensate for the weak expression of TRP-2 in the wild-type WM35 strain was measured by protecting this cell line when these cells are exposed to H ₂ O ₂ stress.

The study was performed using the human melanocyte line WM35, which expresses TRP-2 very weakly (Pak BJ et al., Melanoma Res., 2000; 10: 499), referred to as the "wild" line in study and a cell line derived from the WM35 line that strongly expresses TRP-2 called the "clone-2" in the study, obtained by transfection (see points A and B of Example 1).

For carrying out the test, the cells are seeded at the density of 2.5 × 1 OM cells / well then treated with the compounds to be evaluated: a- a 1: 1 mixture at 4 μg / ml of Cafestol and Kahweol; b- Oltipraz at 50 μM.

After 24 hours the cells are exposed to H ₂ O ₂ induced stress.

The cell viability is measured 24h after the stress using 2 ', 7'-dichlorodihydrofluorescein diacetate (H ₂ DCFDA, D399, Molecular Probes).

The results obtained (Tables 1 and 2 and Figures 3 and 4) show that the treatment of the wild line on the one hand by a mixture Cafestol / Kahweol and on the other hand by Oltipraz limits the sensitivity of cells to stress induced by H ₂ O ₂ . The results thus show that after the treatment, the susceptibility of the wild line approaches the susceptibility of the clone-2 line. Table 1: Cafestol + Kahweol treatment (the results are expressed in% viability according to H ₂ O ₂ treatment)

Table 2: Oltipraz treatment (the results are expressed in% viability as a function of H ₂ O ₂ treatment)

Thus we can conclude that the treatment of melanocytes expressing little or no TRP-2, on the one hand, with a Cafestol / Kahweol mixture and, on the other hand, with Oltipraz, compensates for the low expression of TRP-2 with regard to the susceptibility to stress induced by H ₂ O ₂ and thus provides a benefit for melanocytes with a low or no level of TRP-2 expression.

Example 3 - Compositions

- hair lotion

Compound capable of acting on the metabolic pathway of DOPAchrome tautomerase 0.5 g Propylene glycol 20 g Ethanol 95 ° 30 g Water qs 100 g This lotion is applied daily to the areas to be treated and preferably to the entire scalp for at least 10 days and preferably 1 to 2 months. There is a decrease in the appearance of white or gray hair and a repigmentation of gray hair.

- treating shampoo

Compound capable of acting on the metabolic pathway of DOPAchrome tautomerase 1, 5 g

Polyglyceryl 3-hydroxylarylether 26 g Hydroxy propyl cellulose sold under the name Klucell G by the company Hercules 2 g

Conservatives qs

Ethanol 95 ° 50 g

Water qs 100 g

This shampoo is used with each wash with a laying time of about one minute. Prolonged use, of the order of two months, leads to the progressive repigmentation of gray hair. This shampoo can also be used as a preventive measure to delay hair whitening.

- Gel treating

Compound capable of acting on the metabolic pathway of DOPAchrome tautomerase 0.75 g Essential oils of Eucalyptus 1 g

Econozole 0.2 g

Lauryl polyglyceryl 6 cetearyl glycoether 1, 9 g

Preservatives qs Carbopol 934P sold by the company BF Goodrich Corporation 0.3 g Neutralization agent qs pH 7

Water qs 100 g This gel is applied on the areas to be treated twice a day (morning and evening) with a terminal massage. After three months of application, repigmentation of the hairs or hair of the treated area is observed.

Claims

1. Cosmetic use of a compound capable of increasing the level of GSH, with the exception of quercetin, for preventing and / or limiting and / or stopping the development of canities.

2. Use according to claim 1 for maintaining and / or restoring the natural pigmentation of hair and / or gray hairs.

3. Use according to claim 1 or 2, characterized in that the compound capable of increasing the level of GSH is selected from lipoic acid, oltipraz, kahweol, cafestol, angelicalactone, diallyl sulfide, bensyl isothyocyanate.

4. Use according to any one of claims 1 to 3, characterized in that the use of the compound capable of increasing the level of GSH is administered topically or orally.

5. A cosmetic composition for combating canities comprising, in a cosmetically acceptable medium, at least one compound capable of increasing the level of GSH, with the exception of quercetin, combined with another active agent chosen from desquamative control agents. scalp and / or herbal extracts with propigmenting activity.

6. Composition according to claim 5, characterized in that said compound is selected from lipoic acid, oltipraz, kahweol, cafestol, angelicalactone, diallyl sulfide, bensyl isothyocyanate.

7. Cosmetic composition comprising, in a cosmetically acceptable medium, at least one compound capable of increasing the level of GSH chosen from oltipraz, kahweol, cafestol, angelicalactone and bensyl isothyocyanate, combined with an agent promoting the regrowth of hair.

8. Composition according to any one of claims 5 to 7, characterized in that the compound capable of increasing the level of GSH is encapsulated in a coating such as microspheres, nanospheres, oleosomes or nanocapsules.

9. Composition according to any one of claims 5 to 8, characterized in that it is suitable for topical application to the scalp and / or areas of the skin covered with hair.

10. Process for the cosmetic treatment of canities, characterized in that a composition comprising a compound capable of increasing the level of GSH is administered orally or applied topically to the area to be treated.