Peptide-based hair treatment

Abstract

The treatment is suitable for preventing hair bleaching and contains peptide X-(Xaa) n -P * P * -(Xaa) m -Z{where, P * corresponds to proline, an analogue or derivative thereof; Xaa is an amino acid selected from P*, glycine, alanine, valine, leucine, isoleucine, arginine and phenylalanine; n and m are 0, 1, 2, 3 or 4, with n+m≦8; and at the N-terminus, X is H, —CO—R 1 , -SO2-R 1 or a biotinoyl group; at the C-terminus, Z is selected from OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from; R 1 and R 2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, saccharide, and aryloxy groups having 1 to 24 carbon atoms.

Description

[Technical Field]

[0001] The present invention relates to peptide-based hair treatments, including body and head hair (including scalp hair, beard, eyelashes, and eyebrows) of mammals, animals, or humans. It more particularly relates to the treatment of graying hair, i.e., treatments related to hair bleaching. It is relevant to the cosmetics, hygiene, personal care, and dermopharmaceutical industries. [Background technology]

[0002] Hair's life cycle consists of three phases that repeat until the hair dies, known as the hair cycle. Therefore, hair does not grow continuously, but in successive cycles. The genetically programmed number of hair cycles is 25-30 in total during a lifetime.

[0003] The hair cycle begins with a phase called anagen, during which hair grows intensely inside the hair follicle. It lasts an average of 2-5 years. 85-90% of hair is usually in the growth phase. During this phase, the hair bulb, located at the lower end of the hair follicle, regenerates and then gives rise to hair fibers. This allows hair to grow approximately 1 cm per month. It is a highly proliferative phase resulting from the proximity between the dermal papilla, which is in contact with blood vessels (where hair stem cells (melanocytes and keratinocytes) are found), and the bulging part of the sheath called the hair bulge. The genesis of hair fiber production occurs in the hair bulb, due to the intense production of keratin and keratinocytes that form the outer cuticle and cortex of the hair.

[0004] At the end of this growth phase, the hair follicle enters a regression phase, called catagen, and ceases synthesis. Due to the lack of blood vessel nourishment for the dermal papilla, the fiber no longer grows. This phase lasts for only a few weeks and affects about 2-3% of the hair at a time. The hair follicle retracts and gradually involutes. The keratinocytes and melanocytes gradually disappear, and the hair bulb collapses under its own weight, while retaining the dermal papilla.

[0005] At the start, the dermal papilla rises towards the ridge. This is a phase called telogen, which precedes the new growth phase. 8-10% of the hairs on the scalp are in this phase at any one time. For young people, they are in this phase for about 6-7 months, but the older you are, the longer it lasts.

[0006] This hair cycle is similar for body hair, although the duration of the phases differs, in particular the duration of the anagen phase is shorter in body hair.

[0007] The natural coloration of hair is related to the presence of two types of melanin: eumelanin, a dark brown pigment, and pheomelanin, a reddish yellow pigment. The balance between these two pigments regulates all observed shades of hair. However, like all organs, hair ages, which results in a loss of pigmentation.

[0008] The starting point for melanin synthesis is L-tyrosine (the initial substrate of tyrosinase (TYR), the enzyme whose activity produces dopaquinone), the molecule from which both types of melanin are derived. Eumelanin is synthesized through several transformations involving the involvement of other enzymes, such as TYRP1 and TYRP2. Pheomelanin follows a different pathway and uses the sulfur-containing amino acid L-cysteine. Certain proteins also play a crucial role in this synthesis. In addition to the three enzymes mentioned above, the MITF protein triggers the production of TYR and TYRP1, and therefore melanin. There is also the CREB protein, which is used to trigger MITF production.

[0009] Melanin production in hair follicles is cyclical and occurs only during the anagen phase. This production takes place in melanocytes. Melanin is stored in melanosomes, which are then distributed to surrounding keratinocytes by dendrites (a type of tentacle, an extension of the melanocyte). The keratinocytes then phagocytose the melanosomes; the more active the phagocytosis, the better the hair pigmentation.

[0010] Greying or canity of hair is highly age-related. Genetic factors, as well as environmental factors in the broadest sense, are often mentioned in promoting precocious canities.

[0011] In gray hair, there is a gradual decrease in fiber pigmentation. There is a certain consensus around the fact that graying is more or less associated with a significant decrease in active melanocytes in the hair bulb of gray hair under the influence of age and oxidative stress. This leads to a decrease in melanin and melanosome production, as well as a decrease in dendrite size and melanosome transfer to keratinocytes. Because there is also lower tyrosinase activity, melanocytes exhibit greater cytoplasmic vacuolation and oxidative stress. In gray hair, melanocytes are absent from the hair bulb, and the latter are in fact unable to migrate during a new hair cycle. Melanogenesis, which is highly active in hair throughout life, generates many radicals and oxidative species, including H2O2 (hydrogen peroxide), which cause cumulative oxidative damage to melanocytes over months and years. Thus, gray hair follicles are significantly associated with more mitochondrial DNA damage and apoptosis. The BCL-2 protein plays an antioxidant role and protects melanocytes from apoptotic death. It limits greying.

[0012] With age, cellular antioxidant defenses decline, particularly the production of catalase, which is involved in the detoxification of H2O2 to oxygen and water, as well as glutathione production, which has cytotoxic implications for hair follicles and therefore accelerates greying.

[0013] To correct graying, women often use synthetic hair dyes, which last well for a long time but often use chemical fixatives that can be sensitizing. However, this process is absent from men's cultures.

[0014] Therefore, there is a real need for bioactive compounds that slow the bleaching of hairs and also promote their repigmentation, in particular to avoid or delay the need to use synthetic colorants. Summary of the Invention

[0015] To this end, the Applicant has developed a cosmetic non-therapeutic hair treatment suitable for preventing hair bleaching, which comprises at least one compound of the following formula 1: X-(Xaa) n -P * P * -(Xaa) m -Z {In the formula, -P * corresponds to proline, an analogue or derivative thereof; -Xaa is P * glycine, alanine, valine, leucine, isoleucine, arginine, and phenylalanine, analogs and derivatives, wherein each Xaa is independently selected; - n and m are integers selected independently of one another, where n + m ≤ 8 and equal to 0, 1, 2, 3 or 4; and - At the N-terminus, X is H, -CO-R 1 , -SO2-R 1 or a biotinoyl group; - At the C-terminus, Z is OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from; -R 1 and R 2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, saccharide, and aryloxy groups, which may be linear or branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and / or sulfurized, said groups having from 1 to 24 carbon atoms and optionally having one or more O, S, and / or N heteroatoms in their backbone.

[0016] The detailed description of in vitro and in vivo tests given below shows that the peptide according to the invention provides a long-term action, characterized by an anti-aging effect and aimed at improving the general functioning of the hair system, particularly at the level of the hair follicle, since it strengthens melanin and keeps it in a working state, preventing it from decomposing, so that it continues to produce it and optimally mobilizes it, thereby preventing hair bleaching and the appearance of grey hair.

[0017] The peptides according to the invention have an antioxidant effect on the melanocytes present in the hair follicle and / or promote the transfer of melanin to the keratinocytes by the dentrites.

[0018] The studies also show a complementary and beneficial effect on pigmentation stimulation in vitro and especially in vivo, with a long-term prolonged effect in this second case (persistent effect).

[0019] Therefore, the peptides according to the invention are particularly advantageous in addressing the common problem of grey hair by significantly and permanently improving this condition.

[0020] This dual level of action, both preventative (preventing depigmentation, especially by prolonging it) and curative (repigmentation), is particularly advantageous.

[0021] In formula 1 according to the present invention, P * corresponds to proline, a derivative or analogue thereof. Thus, the present invention provides the following: 1) from rings of different sizes (e.g., 4 or 6 bonds); 2) resulting from modification of the relative position (α or β) of the acid functional group (COOH) with respect to the ring nitrogen; 3) resulting from substitution by groups R on the ring that modify its size and / or polarity; 4) resulting from a combination of the preceding items; It encompasses derivatives or analogs of proline.

[0022] They have the following general formula 1: [ka] is expressed by

[0023] In this formula 1, the R group can be in any position, and the COOH can be in the α or β position. The present invention provides analogous compounds presented in Table 1 below, preferably bearing a nitrogen in the ring such as proline (5 atom ring and COOH alpha):

[0024] Table 1: [Table 1]

[0025] Other compounds resulting from the substitution of proline with R groups are also possible. Non-limiting examples are shown in Table 2 below. Table 2: [Table 2]

[0026] Proline can also be replaced by a substituted analogue having a six-membered ring, as shown as examples in Table 3 below: Table 3: [Table 3]

[0027] According to another preferred feature of the present invention: The peptides are modified at their N-terminus and / or C-terminus (except when X=H and Z=OH), but preferably only at their N-terminus. Derivatization of the peptides at the N- and / or C-terminus is intended to improve the bioavailability of the peptides, in particular by improving their penetration through the skin. This effect can also be achieved by vectorization, for example via encapsulation of the peptide(s). -R 1 and / or R 2 is an alkyl chain of 1 to 24 carbon atoms, preferably a lipophilic alkyl chain of 3 to 24 carbon atoms; and / or -X is an acyl group CO-R 1 and Z is selected from OH, OMe, OEt and NH2, preferably OH; X is preferably selected from octanoyl (C8), decanoyl (C10), lauroyl (C12), myristoyl (C14), palmitoyl (C16), stearoyl (C18), biotinoyl, elaidoyl, oleoyl and lipoyl; more preferably selected from lauroyl (C12), myristoyl (C14) and palmitoyl (C16); and / or -Z is OH and X is selected from palmitoyl (C16), myristoyl (C14) and lauroyl (C12); more preferably palmitoyl (C16).

[0028] Peptides that contain N- or C-terminal derivatives of specific acids, such as derivatives of ascorbic acid, retinoic acid, cinnamic acid, oleanolic acid, hyaluronic acid, nicotinic acid, lipoic acid, gallic acid, or pantothenic acid, are encompassed by the present invention.

[0029] As shown in Formula 1, the active peptide sequence P * P * can be part of a peptide having additional amino acids Xaa (which are hydrophobic amino acids) in either of its side chains. Preferably, these Xaa(s) are / are P * , glycine, alanine, valine, leucine and isoleucine, preferably P * , glycine and alanine, more preferably selected from proline, glycine and alanine.

[0030] A preferred peptide according to the invention is XP * P * -Z peptide, more preferably X-PP-Z peptide. A particularly preferred peptide according to the invention is palmitoyl-PP-OH, hereinafter referred to as Pal-PP-OH. The peptides according to the invention may be optically pure or consist of the L or D isomer or a mixture thereof, with the naturally occurring L isomer being preferred.

[0031] The present invention also encompasses derivatives (with modification and / or addition of chemical functions to one or more amino acids but without alteration of the carbon backbone) and analogues (with modification and / or addition of chemical functions to one or more amino acids but with alteration of the carbon backbone upon addition) of amino acid Xaa, as well as complexes with other species such as metal ions (e.g., copper, zinc, manganese, magnesium, etc.). The peptides may optionally be used in the form of a salt, in particular the hydrochloride or acetate salt.

[0032] The present invention also provides a composition, in particular a topical composition, comprising at least one peptide according to the invention and a physiologically acceptable medium. Depending on the concentration of the physiologically acceptable medium and the peptide(s), this composition may constitute a concentrated active ingredient intended for addition to the final composition intended for the consumer, or it may directly constitute said final composition (which is less concentrated in the peptide(s)).

[0033] In the composition according to the invention, at least one peptide is present in an amount of 10% by weight relative to the total weight of the composition. -7 % to 20%, preferably 10 -6 % to 10%, more preferably 10% by weight based on the total weight of the composition. -5 % to 5%, but may be present at higher or lower concentrations depending on the purpose.

[0034] For example, in compositions forming the active ingredient for treatment according to the present invention, at least one peptide is typically present at a high concentration ranging from 100 ppm to 20,000 ppm, preferably from 500 ppm to 15,000 ppm, and more preferably from 1000 ppm to 10,000 ppm. This component is then typically formulated at 0.01 to 10%, preferably 1 to 5%, in the final topical formulation.

[0035] When several peptides according to the invention are present in a composition according to the invention, they may be present in different relative proportions, in equal amounts or even in different ratios. All percentages and ratios used in this application are expressed by weight of the total composition and all measurements are made at 25°C unless otherwise specified.

[0036] By "physiologically acceptable medium" is meant, according to the present invention, without limitation, an aqueous or hydroalcoholic solution, a water-in-oil emulsion, an oil-in-water emulsion, a microemulsion, an aqueous gel, an anhydrous gel, a serum, a vesicle dispersion, or a powder. By "physiologically acceptable" is meant that the composition is suitable for topical or transdermal use in contact with mucous membranes, nails, scalp, hair, body hair and skin of mammals, more particularly humans, for compositions ingested or injected into the skin without risk of toxicity, incompatibility, instability, allergic response, and other risks. This "physiologically acceptable medium" forms what is conventionally referred to as the vehicle of the composition.

[0037] The peptide(s) according to the invention are solubilized in a lipophilic or hydrophilic matrix, optionally using a solubilizing agent, depending on the intended application. The peptide(s) in the treatment according to the invention may be combined with other active ingredients in effective concentrations that can act synergistically or in an enhanced state to achieve the desired effects described in relation to the present invention, such as, for example, the following agents: hair styling agents, moisturizing agents, antioxidants, anti-hair loss agents, anti-dandruff agents, agents for restoring skin flora, agents acting on the hair cuticle, agents acting on the softness and silkiness of the hair, etc. These active ingredients may be obtained from plant materials, such as plant extracts or products of in vitro plant culture or fermentation.

[0038] More specifically, the peptide(s) are combined with at least one of the compounds selected from the following compounds: anti-dandruff active agents acting as antifungals: Zinc Pyrithione, Ketoconazole, Climbazole, Piroctone Olamine, Selenium Disulfide or APISCALP™ (Sederma), etc.; and / or active agents that reduce or inhibit the occurrence of yeasts (of the genus Malassezia) involved in dandruff conditions and that act favorably on the skin barrier, such as Pal-KTTKS peptide (SEQ ID NO: 1) or Pal-KTSKS (SEQ ID NO: 2); and / or moisturizing active agents, such as DuraQuench IQ™ (Croda) or Shea Unsaponifiable™ (Sederma); and / or active agents that rebalance the skin microflora, such as active HAIRSPA™ (Sederma); and / or hair styling active agents, such as the active ingredient PACIFEEL™ (Sederma); and / or active agents that prevent hair loss and stimulate hair growth, such as CAPIGENE™, CAPILECTINE™, PROCAPIL™ (Sederma), etc.; and / or active agents that strengthen the structure of damaged hair, such as CERAMIDE A2™, CERAMIDE HO3™, HELIOGENOL™ (Sederma), etc.; and / or - hair smoothing active agents, such as FRUIT BIO™ (Sederma); and / or - Active agents that protect hair and body hair from UV and IR radiation, such as VENUCEANE™.

[0039] The at least one peptide according to the invention may be used in vectorized form by being bound, entrapped or adsorbed to macro-, micro- or nanoparticles, such as capsules, spheres, liposomes, oleosomes, chylomicrons, sponges, in the form of micro- or nanoemulsions, or by being adsorbed to, for example, powdered organic polymers such as talc, bentonite, spores or exines, and other inorganic or organic supports.

[0040] Hair care formulations may have a variety of forms including hair conditioning, straightening formulations, straightening and repair products, perms, hair shampoos, pre-shampoo conditioners, aftercare shampoos, lotions, leave-on shampoos, styling products, leave-in hair products, waterless products, emulsions, two-in-one foaming emulsions, creams, masks, aerosol or non-aerosol foams, sprayable emulsions, emulsifier-free products, water-based or oil-based sprays, serums, gels, mild sulfate-free formulations, silicone-free formulations, dye-containing products, color cosmetics, dye products, and shower products.

[0041] These hair care formulations comprising at least one peptide according to the invention may contain a variety of other ingredients known to those skilled in the art, for example, and not exhaustively, cleansing agents, skin revitalizers, styling agents, antidandruff agents, sequestering or complexing agents (EDTA and its salts), stabilizers, pearlizing and opacifying agents, plasticizers or coalescing agents, gelling agents, emollients, acidifying or alkalizing agents, hair growth promoters, hair loss prevention agents, essential oils, polymers, proteins or derivatized proteins, silicones, sunscreen compounds (filters), pigments, moisturizers, antioxidants, co-emulsifiers, film-forming agents, alpha-hydroxy acids, hair colorants, clarifying agents, thickeners, sheathing agents, texturizing agents, preservatives, preservatives or surfactants.

[0042] These formulations are prepared by methods known to those skilled in the art. The vehicle or carrier excipient for the formulation can be water, oil, or powder, depending on the final destination of the formulation: for example, for shampoo, it can be water; in other formulations, oil is preferred, such as vegetable oil (coconut, argan, jojoba, olive, sunflower, etc.), mineral oil, animal oil, or synthetic oil. Synthetic esters, such as C12-15 alkyl benzoate, can also be used as excipients. Liquid fatty alcohols, liquid fatty acid esters, solid fatty substances, especially waxes, solid fatty acid esters, and solid alcohols can also be used as supports.

[0043] The present invention therefore provides the use of at least one peptide according to the invention, or a composition comprising it, as defined above, for a non-therapeutic cosmetic treatment for improving the general condition of the hair and treating its shortcomings, the treatment consisting of applying an effective amount of at least one peptide according to the invention to the hair in need thereof. Preferably, treatment according to the present invention is topical.

[0044] The present invention covers a cosmetic, non-therapeutic topical treatment method for maintaining and improving the appearance and general condition of hair, in particular for preventing greying, which method comprises topically applying to the hair of a subject in need thereof an effective amount of a peptide or mixture of peptides according to the present invention, or a composition according to the present invention comprising said peptide or mixture of peptides, wherein the peptide is as defined above. The treatment is intended for all types of hair, including body hair (including scalp hair, beard, eyelashes and eyebrows) and head hair.

[0045] "Non-therapeutic cosmetic treatment" means a treatment directed at hair in a healthy state (as opposed to a pathological state) with the aim of beautifying it or (as a preventative measure) avoiding aesthetic problems.

[0046] "Topical treatment" or "topical use" refers to the application where it is applied, the application where it is intended to act. The peptide or composition according to the invention may be applied topically to the target area. An "effective" amount depends on several factors, such as the age, condition, and severity of the disorder of the patient, how it is administered, etc. An effective amount means a non-toxic amount sufficient to produce the desired effect.

[0047] For example, a cosmetic hair treatment for the scalp is recommended for at least 4 weeks, preferably at least 12 weeks, which can be carried out in the form of at least 3 treatments per week, each time applying a dose of 5 ml of shampoo followed by 5 ml of leave-on lotion after shampooing, each product containing 1.5% of the active ingredient according to the invention (according to the formulation in the Galenic Formulation section below) and 6000 ppm of peptides.

[0048] According to another feature, the cosmetic treatment method according to the invention may be combined with one or more other treatment methods targeting the hair, such as, for example, phototherapy, heat or aromatherapy treatments.

[0049] According to the present invention, it is possible to provide a device or kit with several compartments intended for carrying out the methods described above, which may, by way of example and not limitation, contain in a first compartment a composition containing at least one peptide of the invention and in a second compartment additional excipients and / or active agents, wherein the compositions contained in said first and second compartments are considered combined compositions for simultaneous, separate or temporally separated use, in particular in one of the treatments defined above.

[0050] According to a further aspect, the present invention provides the use of at least one peptide according to the invention as an active ingredient in the preparation of a cosmetic composition for preventing and / or improving the general condition of the hair. DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description The present invention will be better understood in light of the following description of embodiments, in vitro evaluations, in vivo evaluations, and hair formulations for practicing the invention. [Example]

[0052] A - Example of peptide synthesis according to the invention: Pal-PP-OH Pal-PP-OH peptides are prepared by peptide synthesis. N-protected proline is attached to the resin via its terminal acidic functional group. The amine functional group is deprotected, and the proline-resin is then reacted with a proline derivative in the presence of a coupling reagent (e.g., DCC (dicyclohexylcarbodiimide) / NHS (N-hydroxysuccinimide) or HBTU (2-(1H-benzotriazol)-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) / HOBT (1-hydroxybenzotriazole). The same deprotection and coupling procedures are then repeated with palmitic acid. The peptide is then cleaved from the resin in an acidic medium, and after precipitation, washing, and drying, the product palmitoyl-prolyl-proline is obtained in solid form. The peptides according to the invention can also be prepared by biotechnological routes, at least in part by microorganisms capable of producing them.

[0053] B - Example of the preparation of a composition according to the invention containing the dipeptide Pal-PP-OH (from Example A) Starting materials: - pure peptides, synthesized by the synthesis methods described above; - Excipients: Water, glycerin, 30% NaOH, orthophosphoric acid Procedure: A selected amount of peptide is dissolved in water at alkaline pH. Once the peptide is solubilized, the pH is lowered to 6 and gicerin is added. In this example, 0.6% Pal-PP peptide is mixed with excipients to form the active ingredient used in dosing part D) below.

[0054] C - In vitro evaluation The peptides according to the present invention exhibit the following remarkable effects. The peptides prepared according to A) above are dissolved in DMSO, a suitable solvent for cell culture, in advance. The solution of the peptide in DMSO is then added to the culture medium. The final DMSO concentration in the tested medium is 0.1%.

[0055] 1-Anti-aging effect of the peptides according to the invention on hair - Protective effect against oxidative stress Combating hair graying not only requires stimulating melanin production and pigment diffusion in keratinocytes, but it is also advantageous to protect melanocytes from damage induced by age (intrinsic or actual aging), premature aging associated with environmental factors (extrinsic aging), and melanin production itself. Indeed, melanin production generates oxidizing radical species (ROS), which contribute to the effects of aging, leading to the production of ROS. The main radical species is H2O2, which can diffuse into cells and react with essential cellular components (weakening the cell and reducing its production both qualitatively and quantitatively). To control this, cells possess defenses against oxidation, including catalase, which detoxifies H2O2 into oxygen and water, and glutathione, a small peptide that also participates in the detoxification of H2O2 to H2O via GSH-peroxidase. The reduced form of glutathione (GSH) allows this enzyme to be regenerated to resume the detoxification process. Expression of the BCL-2 gene, which encodes a protein that protects cells against oxidative stress, is also crucial for this antioxidant defense.

[0056] 1.1-Reduction of ROS levels protocol: Near-confluent normal human melanocytes (NHM) are contacted with the peptides of the present invention for 24 hours. At the end of this contact, the cells are contacted with a DCFH-DA probe inserted into these cells. After rinsing to remove excess probe, the cells are placed in contact with H2O2 to mimic oxidative stress. A case without oxidative stress is also performed. The reaction of H2O2 with the intracellular probe is measured by fluorescence. An estimation of the number of cells is performed in parallel to adjust the results.

[0057] result: Table 4: Variation in ROS levels in NHM; Effect of the present invention (n=3): [Table 4]

[0058] Catalase activity of 1,2-NHM protocol: Nearly confluent NHMs are contacted with the peptides of the present invention for 24 hours. At the end of this contact, the cells are disrupted. The catalase activity extracted from these cells is measured using resorufin reagent. The latter is combined with the H2O2 remaining in the reaction well after the action of cellular catalase. Protein analysis using the bicinchoninic acid (BCA) method is used to estimate cell number and to homogenize the data obtained.

[0059] result: Table 5: Variation of catalase activity in NHM, effect of peptides of the invention (n=3): [Table 5]

[0060] 1.3-NHM reduces glutathione protocol: Nearly confluent NHMs are contacted with the peptides according to the invention for 24 hours. After this contact, the cells are disrupted in an extraction buffer that preserves glutathione. The amount of GSH is measured by a specific fluorescent probe.

[0061] result: Table 6: Variation of reduced glutathione levels in NHM, effect of peptides according to the invention (n=6): [Table 6]

[0062] BCL-2 marker in 1.4-NHM protocol: NHMs were cultured and contacted with a peptide according to the invention or its solvent. At the end of 72 hours of contact, melanocytes were disrupted and mRNA was collected and converted to cDNA, which was analyzed by qRT-PCR to assess changes in Bcl-2 mRNA expression levels.

[0063] result: Table 7: Variation in the expression of the BCL-2 gene in NHM, effect of peptides according to the invention (n=4): [Table 7]

[0064] These four sets of results show that the peptides of the present invention stimulate the antioxidant defenses of human melanocytes. Catalase enzyme was stimulated by +37% in a dose-dependent manner (p<0.01 vs. control). In parallel, the peptides of the present invention stimulated reduced glutathione production (+38%, p<0.01 vs. control) and overexpressed the gene for BCL-2 protein by +99% (p<0.01 vs. control). Similarly, the peptides of the present invention reduced intracellular peroxide production by 33% during oxidative stress (p<0.01 vs. control).

[0065] 2. Effect of the peptides according to the invention on melanin transfer The first step is the production of melanin by tyrosinase in melanosomes. The latter must then be transferred from melanocytes to keratinocytes via melanocyte dendrites. The size of the dendrites and the transfer capacity of pigmented melanosomes (phagocytosis) are also very important parameters for successful melanization.

[0066] 2.1 - Dendritic level protocol: The cells are contacted with the peptides of the present invention for 48 hours. Then, the melanocyte layer is labeled with an antibody after rinsing. The area occupied by dendrites is obtained by image analysis of the photographs taken, and the parameter is converted into relative dendrite length / 100 cells. At the same time, the cell nuclei are counterstained with Hoescht33258 fluorescent dye to estimate the cell number and adjust the data.

[0067] result: Table 8: Variation in the length of melanocyte dendrites, effect of peptides according to the invention (n=3): [Table 8]

[0068] These results show the effect of the peptides according to the invention on the degree of dendritic formation of NHMs: the peptides stimulate the elongation of these extensions necessary for the contact between melanocytes and keratinocytes.

[0069] 2.2-Level of melanosome phagocytosis protocol: Normal human keratinocytes (NHK) are seeded and cultured until a confluent cell mat is obtained. The cells are contacted with the peptides of the present invention for 24 hours, and then a solution of fluorescent microbeads that mimic melanocyte melanosomes is added for 3 hours. The beads are phagocytosed (ingested) by the keratinocytes and, after rinsing, quantified by image analysis. In parallel, cell nuclei are counterstained using Hoescht33258 fluorescent dye to estimate cell number and adjust the data.

[0070] result: Table 9: Variation of microbead phagocytosis by keratinocytes, effect of peptides according to the invention (n=3): [Table 9]

[0071] These results show the effect of the peptide according to the invention on stimulating the phagocytosis of melanosome-mimicking microbeads, which is dose-dependent and reaches +283% compared to the control (p<0.01). The dendritization data consequently confirm that the peptides according to the invention potently promote the transfer of melanin-laden melanosomes to neighboring keratinocytes.

[0072] 3-Effect of the peptides according to the invention on melanogenesis 3.1-Melanin production protocol: Normal skin melanocytes (NHM), which produce little or moderate amounts of melanin, are used. These cells are cultured and contacted with the peptides of the present invention at various concentrations or with the solvent (control) at an equal concentration for 10 days. The culture medium is changed every 2-3 days. After this contact, the cell mat is disrupted and melanin is extracted from the cells. The amount of melanin is estimated spectrophotometrically at 490 nm using a previously established standard range from the melanin solution. Protein analysis using the bicinchoninic acid (BCA) method is used to estimate the cell number and homogenize the data obtained.

[0073] result: Table 10: Variation of melanin production in moderately (§) and poorly (§§) pigmented NHM after 10 days; effect of peptides according to the invention; n=4: [Table 10]

[0074] These results show that the peptides according to the invention stimulate melanin production in human melanocytes in culture, regardless of whether the cells are derived from a moderate (§) or low-producing (§§) clone. This overproduction is already clearly visible in photographs taken before disruption of the cell layer. The overproduction is dose-dependent and highly significant compared to that observed in the negative control, where pigmentation is only slightly maintained even after 10 days.

[0075] 3.2-Tyrosinase activity protocol: The same culture and contact protocol as above is used with melanocytes from two different donors (medium and low pigmented) regarding their melanin production. After contact, tyrosinase is extracted from the cells and dopa-oxidase activity is assessed using the substrate L-DOPA at 37°C. The absorbance due to the production of dopaquinone is measured at 490 nm and converted to activity units using a preset range. Protein analysis using the bicinchoninic acid (BCA) method is used to estimate cell number and homogenize the data obtained.

[0076] result: Table 11: Variation of tyrosinase activity in moderately (§) and poorly (§§) pigmented NHM after 10 days; effect of peptides according to the invention; n=4: [Table 11]

[0077] These results show that the peptides according to the invention enhance tyrosinase activity in a dose-dependent manner, regardless of whether the cells are derived from low- or medium-producing clones, and therefore may promote more active melanogenesis.

[0078] 3.3 - Effects of genes involved in melanogenesis protocol: NHMs are cultured and contacted with the peptides according to the invention or their solvents. At the end of 72 hours of contact, melanocytes are disrupted and mRNA is collected and converted to cDNA, which is analyzed by qRTPCR to estimate changes in mRNA expression levels. The expression of the following genes was examined: MITF, TYRP1, and CREB.

[0079] result: Table 12: Changes in the expression of genes involved in melanogenesis, effect of the peptide according to the present invention; n=4: [Table 12]

[0080] These results show that the peptide according to the invention dose-dependently upregulates the expression of several protein genes involved in melanogenesis: MITF, TYRP1 and CREB.

[0081] 3.4 - Effect on hair follicles protocol: Hair follicles, containing their bulbs with a small amount of pigment and isolated from scalp plastic surgery (resulting from cosmetic surgery - female donor, dark blond hair; 51 years old), were individually placed in wells of a culture plate containing culture medium and kept viable (37°C; 5% CO2). Twelve hair follicles are reserved for TO to serve as a reference for visual estimation and quantification of pigmentation, but also for subsequent labeling of MC1R and MITF by immunohistology (6 follicles per each marker).

[0082] Twelve hair follicles were placed in contact with the peptides of the present invention at 10 ppm in the solvent, and 12 other hair follicles were placed in contact with the solvent alone (0.1% DMSO in culture medium) for 8 days. At the 8-day point, all hair follicles were arrested, fixed, sectioned (7 μm), labeled with alumated carmine, and photographed under a microscope. Six of the sections (per group) were used for visual and quantitative evaluation of their pigmentation by image analysis (using a specific filter to measure melanin). The other six sections were used for MC1R and MITF scoring (assessed by visual estimation).

[0083] 3.4.1-Pigmentation results: Table 13: Variation of melanin production in the hair follicle bulb after 8 days of contact with the peptide according to the invention (n=6 hair follicles / case; total of 22 / 23 photographs / case): [Table 13]

[0084] Visual estimation shows a very strong pigmentation in the six hair follicles contacted with the peptide according to the invention for 8 days compared to the six control hair follicles. Photo analysis shows 11.6 times more dark pixels in the six hair follicles contacted with the peptide according to the invention, which corresponds exactly to the deposition of melanin. This difference is highly significant.

[0085] 3.4.2 - Results for MITF and MC1R markers After immunohistochemical labeling of sections, MC1R and MITF proteins appear dark. Compared to TO, visual estimation shows that MC1R increases in the hair bulb of cases treated with the peptide according to the invention, while it remains stable in control cases. In parallel, MC1R increases more in the lower sheath with the peptide according to the invention than in controls. MC1R is the α-MSH hormone receptor, which causes melanization. The peptide according to the invention stimulated its production in hair follicles.

[0086] Compared to TO, visual estimation shows that MITF increases in the hair bulbs of cases treated with the peptide according to the invention, whereas it decreases in control cases.

[0087] MITF is a protein that induces the production of tyrosinase and TYRP1, and thus the production of melanin in melanosomes. The peptides according to the invention clearly induce the production of MITF, which leads to the increase in melanin observed in cultured cells and hair bulbs. This is consistent with what was observed by qRT-PCR (see above), which showed an increase in the expression of CREB, MITF, and TRP-1 in melanocytes.

[0088] D-galenic preparations Various formulations are described below. Additional cosmetic active ingredients that are expected to support and / or complement the activity of the active ingredients according to the invention are added in a phase appropriate to their hydrophobic or hydrophilic properties. These ingredients can be of any category depending on their role(s), the application site (head hair, body hair, eyelashes, eyebrows, etc.), the desired final effect, and the consumer. They are mentioned above in the description.

[0089] The formulations described below contain the peptide-based active ingredient according to the invention described in Section B above, containing 6000 ppm of peptide. These formulations are presented as numerical values ​​using the recommended percentage of active ingredient of 1.5%. They may contain higher or lower percentages depending on the efficacy strength of higher or lower strengths.

[0090] 1-Beauty serum Table 14: [Table 14]

[0091] Disperse carbomer in water and allow to swell for 30 minutes. Add Part B to Part A while stirring. Neutralize Part A+B with Part C. Homogenize thoroughly by stirring. Pour Part D into Part A+B+C while stirring. Add Part E and mix thoroughly. A fluid, transparent and colorless gel is obtained which forms a light, non-greasy textured serum and which can be used to treat hair, including body hair (beard and, for example, application to the trunk).

[0092] Examples of additional active ingredients: Neroli Floral Water™: An active ingredient sold by Crodarom that is a bitter orange extract (Citrus Aurantium Amara) with hair styling and regenerating properties.

[0093] 2- Liquid shampoo Table 15: [Table 15]

[0094] Accurately weigh out Part A. Disperse Part B into Part A, stir normally, and mix thoroughly for 1 hour. Heat Part A+B to 55°C in a water bath and mix thoroughly. Weigh and heat Part C to 55°C in a water bath. Add Part C to Part A+B with rapid stirring. Add the ingredients of Part D one at a time to Part A+B with normal stirring. Adjust the pH to pH=5.90+ / -0.10 with Part E. Add Part F and mix thoroughly. A white, opaque, viscous gel is obtained.

[0095] Examples of additional active ingredients: APISCALP™: (added to Part C) An active ingredient marketed by Sederma based on a CO2 supercritical extract of Apium graveolens seeds for the treatment of irritated scalp, helping to reduce dandruff and soothe scalp itching. Zinc Pyrithione: (added to Part A) Anti-dandruff agent.

[0096] 3-Rinse Oil Table 16: [Table 16]

[0097] Weigh and mix Part A. Weigh and mix Part B. Add Part B to Part A with normal stirring. A liquid, clear yellow oil is obtained that is suitable for leaving on and then rinsing, and which can also be used to treat hair, including body hair (e.g., as a massage oil for the torso).

[0098] Examples of additional active ingredients: Crodabond™ CSA: (added to Part B) An active ingredient sold by Croda that is a mixture of hydrogenated castor oil and sebacic acid copolymer that seals the built-up cuticle (outermost layer of the hair shaft) and repairs split ends. Phytolea® Baobab EC: (added to part A) an active ingredient sold by Crodarom, Adansonia digitata seed oil, which is a regenerating and antioxidant agent due to its fatty acid, vitamin E and A content.

[0099] 4-Solid shampoo, e.g. in stick form Table 17: [Table 17]

[0100] Heat Part A in a water bath to 75°C. Heat Part B in a water bath to 75°C. Add Part C to Part A and mix thoroughly, while stirring normally. Add Part B to Part A+C in the water bath with light and rapid stirring. Add Part D to the above parts and mix thoroughly. Pour quickly into molds. An opaque solid shampoo is obtained.

[0101] Examples of additional active ingredients: Matcha Tea Extract™: (added to Part C) An active ingredient sold by Crodarom, which is an antioxidant and purified active ingredient based on Camellia Sinensis leaf extract. Hairspa™: (added to part C) A lactitol and xylitol based active ingredient for styling and moisturizing the scalp sold by Sederma.

[0102] 5 – Energizing wax Table 18: [Table 18]

[0103] Heat Part A in a water bath to 80°C. Dissolve and mix thoroughly until completely homogenized. Add Part B to Part A and mix thoroughly at 55°C. Pour Part C into the above portion and mix. Immediately pour into the conditioning pot. A transparent gel is obtained that can be used on hair and body hair.

[0104] Examples of additional active ingredients: NG Shea Unnsaponifiables™: (Butyrospermum Parkii (Shea) Butter (and) Butyrospermum Parkii (Shea) Butter Unsaponifiables), an active ingredient sold by Sederma that improves hydration (added to part A). Phytolea™ Cranberry EC: (added at the end of Part A) An active scalp moisturizer (Vaccinium Macrocarpon (cranberry) seed oil) sold by Crodarom.

[0105] 6-Regenerating and Nourishing Mask Table 19: [Table 19]

[0106] Heat Part A in a water bath to 85°C. Heat Part B in a water bath to 90°C. Weigh out Part C and mix thoroughly. Pour Part C into Part A and stir normally. Disperse Part B into Part A+C with vigorous stirring and homogenize thoroughly. Subsequently, add Part D to Part F and homogenize thoroughly.

[0107] Cutissential™ Behenyl 18 MEA: An excipient sold by Croda for use in formulations to replenish the lipid layer of the cuticle for a healthier appearance of hair and body hair and to promote superior wet detangling performance. A yellow, opaque, viscous emulsion is obtained which can be used on hair and body hair.

[0108] Examples of additional active ingredients: Procapil™: (Added to part D) An active ingredient marketed by Sederma that prevents hair loss and contains a mixture of apigenin, oleanolic acid and Biot-GHK peptide. Crodarom Manuka Honey™: (added to part D) An active ingredient sold by Crodarom based on honey extract that helps repair damaged hair. Ceramide HO3: (added to Part B) An active ingredient sold by Sederma that helps repair damaged hair and promotes hydration.

[0109] 7-Leave-on protection spray Table 20: [Table 20]

[0110] Pour Part B into Part A with normal stirring. Adjust pH to 5.80 + / - 0.20 with Part C. Add Part D to Parts A+C with stirring. Add Part E to the above parts and mix. An opaque fluid emulsion is obtained.

[0111] Examples of additional active ingredients: Phytessence™ Hazel Leaf: (added to part D) An active ingredient marketed by Crodarom, based on Corylus Avellana leaf extract, that restores vitality and tone to the scalp. Venuceane™: (added to part D) An active ingredient sold by Sederma, based on a fermented extract of Thermus Termophillus Ferment, that prevents damage from UV and IR radiation.

[0112] E - In vivo evaluation 1-General theory In vivo testing was carried out by applying the hair lotion previously described in point 1) of the Galenic formulation section. This lotion contains 90 ppm of the peptide of the present invention. Four independent tests were carried out with a total of 84 volunteers (the average age of the entire panel of selected volunteers was 42 years). All four tests were carried out across a wide range of geographic locations, times, seasons, application sites, phototypes and methods. Details are specified in the table below. Comparison of protocols for the studies performed: Table 21: [Table 21]

[0113] These four studies were not performed against a placebo. A graying study was previously performed against a placebo formulation. Thirty volunteers (mean age 51 [33-62] years; 21 men, 9 women) were enrolled, with application of the lotion at least three times per week for three months. No significant graying reduction effect was observed or measured in this study on either the beard or the temples. This confirms that application of the placebo lotion, even when accompanied by daily massage, has no effect on graying.

[0114] 2 - Method details 2.1 - Standardized photography All tests used a sophisticated photography system, with a camera mounted on the bench to ensure complete repositioning of the volunteer's head during every test. For each volunteer, a profile photograph, and in some tests, photographs of the back and top of the head, were also taken in cross-polarized mode to eliminate any harmful glare that might interfere with the image analysis photo processing. These photographs were used to obtain an overview of gray hair intensity and, depending on their image quality, to work in smaller, isolated areas.

[0115] 2.2 - Photo Processing System Assessment of the area occupied by gray / white hairs was performed photographically using NIH-USA™ Image J software by targeting the area of ​​interest. Generally, except for test site 2, short hair size (eg, 2 cm) is required, and this size is the same for volunteers at various measurement times to facilitate before-and-after comparisons. In addition, each original color photograph was typically converted to a gray-level image, and the same level filter (thresholding) was applied to the same volunteer's photographs before and after to select non-black hairs. Finally, a binarization (black or white) procedure was performed to separate non-black hairs (gray and white) from black hairs and quantify the former.

[0116] 2.3 - Standardization of Micrographs Using TrichoScan® Only one study (at location 2) used this technique (a new tool for analyzing hair growth), which provides data on a very small area and has the advantage of looking at hair individually. Scalp areas were identified and shaved several days before and at various times before the photographs. Due to the high magnification, this technique has been widely used in hair loss studies, but it is also capable of handling dyed hair. The TrichoScan® system includes an epiluminescence microcamera system.

[0117] 2.4 - Evaluation of grey and non-grey hair counts TrichoScan® software was used to determine the number of white hairs, the number of black hairs, and to calculate the black / white ratio, which increases with the number of black hairs when the treatment is working.

[0118] 3-Results No adverse effects were reported in these volunteers, even at 3 or 4 months. There was also no negative feedback 3 months after the last application. This, together with the tolerance studies, demonstrates the safety of the peptides of the invention in large groups and over a long period of time.

[0119] 3. Gray hair density at 1-3 months, comparison of effects across three studies Table 22 compares the quantitative effects obtained in three of the four groups for a common area, the same application time (3 months) and a common parameter, gray / white hair density (although measured separately by each group).

[0120] Table 22: Variation in grey / white hair density measured on one side (profile), effect of peptides according to the invention after 3 months of application; N=3 trials [Table 22]

[0121] After 3 months of application, gray hair density decreased by a mean (3 trials) of 32.4%, with extreme values ​​ranging from 21.1% to 38.7% on the temporal regions. These decreases were highly significant (p<0.01) and involved almost all volunteers (68-100% depending on the trial).

[0122] 3.2 - Comparison of two application areas In one study, measurements were taken on the lateral face and occipital region of the same volunteer, allowing comparison of peptide effects on the two regions on the same volunteer group.

[0123] Table 23: Variation in grey / white hair density measured at the sides and back of the head, effect of peptides according to the invention after 3 months of application; N=1 trial [Table 23]

[0124] After 3 months of application, gray hair density was reduced by 37.5% and 31.9% on the temporal and occipital regions, respectively. These reductions were highly significant (p<0.01) and involved almost all volunteers (100 and 95% depending on region).

[0125] 3.3 Comparison of effects after 3 to 4 months One study (Site 2) was conducted at the apex for a total of 4 months with a time point at 3 months to assess effects over time. Table 24 compares the effects obtained at T3 and T4 months.

[0126] Table 24: Variation in grey / white hair density measured on the crown of the head, effect of peptides according to the invention after 3 and 4 months of application; N=1 study [Table 24]

[0127] The study shows that after 4 months of use, the reduction in gray / white hairs observed on the crown of the head further increases compared to that observed at 3 months (10.9% vs. 8.8%, both highly significant: p<0.01). The percentage of volunteers with improvement also increases slightly (from 79% to 84%).

[0128] 3.4 - TrichoScan® Testing Table 25: Variation in the non-grey / grey hair ratio measured on the temporal region using Trichoscan®, effect of the peptides of the invention after 4 months of application; N=1 study [Table 25]

[0129] An increase in the black / white hair ratio of approximately +27.4% (p<0.05) is calculated at T4 months. This technique, which differs significantly from those using image analysis because it uses a microscope-type probe, therefore confirms the reduction in the number of white hairs after the use of the peptide according to the invention.

[0130] 3.5 - Duration of effect 3 or 4 months after end of study In two separate studies, the durability of the effect of the peptide according to the invention was evaluated over time: for this purpose, all volunteers stopped using the peptide according to the invention after 3 or 4 months of application. In the first study, the durability of the effect without application was evaluated in 10 volunteers after 4 months of application. Measurements were taken on the top of the head. In a second study with 3-month application, the durability of the effect without application was evaluated in 7 volunteers after 3 months. Measurements were taken at the back of the head.

[0131] Table 26: Variation in grey / white hair density measured at the back of the head (N=7) or at the top of the head (N=10) - Effect of peptides according to the invention 3 or 4 months after the last application; N=2 trials [Table 26]

[0132] Two independent studies involving a small number of volunteers show that after 3 or 4 months of daily use, the reduction in gray hair is very clear, even 3 or 4 months after the last application. The reduction values ​​even increase slightly (-22.20% vs. -15.04% and -39.9% vs. -37.2%). This shows that 90 ppm of the peptide according to the invention recolors gray hair and, advantageously, this effect is maintained for several months after the end of application.

[0133] 4 - Conclusions of in vivo studies The peptide of the present invention at 90 ppm in a hair lotion enabled significant hair repigmentation in several dozen individuals included in four independent studies. Repigmentation was independent of the application site. A clear effect was observed in the temporal, vertex, or occipital regions. This effect was independent of the geographical location of the study and the quantification method used. All four independent studies demonstrated a net repigmentation effect in almost all volunteers. Women showed similar repigmentation to men. The phenomenon of persistence of the effect three months after the end of the study is particularly interesting because it reflects the fact that the peptide of the present invention exhibits a "delayed" effect, i.e., an effect that persists long after the study, without the need for further application of the peptide. Advantageously, this indicates that the peptide used in accordance with the present invention acts by reactivating one of the mechanisms that lead to altered pigmentation over time and with the age of the subject.

Claims

1. For non-therapeutic hair treatments for cosmetic use that have anti-aging effects on hair and are suitable for preventing hair discoloration, the following Pal-PP-OH Use of peptides.

2. The use according to claim 1, wherein the treatment is an antioxidant treatment for melanocytes present in the hair follicle.

3. The use according to claim 1 or 2, wherein the treatment is for promoting the migration of melanin to keratinocytes by dendrites.

4. The use according to any one of claims 1 to 3, wherein the treatment is further suitable for the re-pigmentation of hair.

5. The use according to claim 4, wherein the treatment is suitable for sustained hair pigmentation.

6. The use according to any one of claims 1 to 5, wherein the treatment is localized.

7. The use according to any one of claims 1 to 6, wherein the peptide is used in a vectorized form by binding it to, incorporating, or adsorbing it as a micro or nano emulsion to macroparticles, microparticles, or nanoparticles.