Use of urolithin to prevent / slow down hair thinning

Abstract

Use of a urolithin to prevent / slow down hair thinning. The present invention relates to the cosmetic use of at least one compound of formula (I) and / or one of its salts and / or solvates and / or one of its glucuronide derivatives to prevent / slow down hair thinning, and / or promote increased hair thickness. Figure for the abstract: none

Description

Title of the invention: Use of a urolithin to prevent / slow down hair thinning

[0001] The present invention relates to the cosmetic use of at least one compound of formula (I) and / or one of its salts and / or solvates and / or one of its glucurono-conjugated derivatives to prevent and / or slow down the thinning of the hair, or to promote the increase in the thickness of the hair.

[0002] Increasingly, in the hair care field, users are looking for products that improve the quality of their hair.

[0003] In particular, there is a growing need for the long-lasting thickening of fine hair, mainly in areas with an aging population, such as Europe, the United States and Japan.

[0004] Indeed, several studies show that after 40 years, the heterogeneity of the diameters of hair present on the same scalp increases, particularly in women, and that the average thickness tends to decrease.

[0005] It is therefore necessary today to seek a technical solution making it possible to obtain new compounds as well as compositions for the hair field making it possible to improve the quality of the hair, and in particular by increasing the diameter of the hair, thus increasing the hair mass.

[0006] Hair fibers are composed of a cortex covered by thin, overlapping cuticles. The cortical cells are needle-shaped, tightly packed, and aligned along the hair shaft. They contain macrofibrils, which are a composite material of keratin intermediate filaments (IFs) embedded in a matrix composed of keratin-associated proteins (KAPs) and largely stabilized by disulfide bonds. Within macrofibrils, keratinous IFs exhibit a double-twist architecture as they are axially oriented but also follow a helical shape around the axis. The helical pitch in macrofibrils (so-called double-twist intensity) in human scalp hair varies between macrofibrils and is negatively correlated with macrofibril diameter, allowing us to classify macrofibrils as “small diameter and high twist” or “large diameter and low twist.”Human hair may also contain a medulla composed of large vacuolated cells in the core of the cortex with a unique keratin expression profile. The presence and size of the medulla vary among individuals and body sites, and the presence of a medulla in human scalp hair fibers correlates with hair fiber diameter.

[0007] A hair fiber gradually forms in a hair follicle from matrix keratinocytes at the base of the follicle by keratinization leading to an increase in the stiffness of the hair fiber. This process occurs during the anagen growth phase of the hair and relies on complex molecular interactions between dermal papilla fibroblasts and hair keratinocytes. Interactions within the hair follicle largely govern the shape and diameter of the hair fiber. Scalp hair exhibits notable variable morphology within an individual's scalp, for example, with the coexistence of small-diameter and large-diameter hairs. Small-diameter hair is notably characterized by an increase in the expression of keratin 38 relative to other hair cortex keratins and KAPs (Baltenneck et al.(2022), Age-associated thin hair displays molecular, structural and mechanical characteristic changes, J. Struc. Biol., vol. 214, issue 4, doi: 107908). .

[0008] However, the characteristics of small diameter hair remain poorly understood.

[0009] For obvious reasons, it would be advantageous to have compositions and / or active ingredients capable of preventing and / or slowing down hair thinning, or of promoting an increase in hair thickness. This would make it possible to meet consumer expectations in terms of improving hair thickness, particularly consumers with fine hair.

[0010] The present invention aims precisely to meet this need.

[0011] The Applicant has now surprisingly discovered that natural urolithins are capable of modulating the expression of K38 in hair to promote the expression of cortical keratins, characteristic of thick hair.

[0012] The present invention thus relates to the cosmetic use of at least one compound of the following formula (I), and / or one of its salts, and / or its glucuronoconjugated and / or solvated derivatives:

[0013] [Chem.l] O

[0014] in which: RI = -OH, and - R2 to R5 are identical or different and are independently chosen from H and -OH,

[0015] to prevent / slow down hair thinning, or promote an increase in hair thickness.

[0016] The present invention also relates to a cosmetic treatment method for increasing the diameter of hair fibers. This method is obviously non-therapeutic. The cosmetic treatment method comprises at least one step of applying at least one compound of formula (I) and / or one of its salts and / or one of its glucuronoconjugated and / or solvated derivatives to fine or thinning hair.

[0017] The present invention also relates to a cosmetic composition, in particular for topical application to the scalp and / or hair fibers, comprising at least one compound of formula (I) and / or one of its salts and / or one of its glucuronoconjugated and / or solvated derivatives, and vitamin C or one of its derivatives.

[0018] The present invention also relates to a method for evaluating the effectiveness of a compound for preventing and / or slowing down hair thinning, or for promoting an increase in hair thickness, comprising at least the following steps: a. Providing at least one hair follicle from a scalp sample; b. Incubating said hair follicle in a culture medium in the presence of a compound to be tested, in particular at a temperature of between 30°C and 38°C, preferably 37°C, and under an atmosphere of 2% to 7% CO2, preferably 5% CO2, and for a period of time of at least 1 day, preferably at least 2 days, in particular at least 5 days; c. Measure the expression levels of keratins K38 and K35 in the incubated sample from step b; d. Calculate the ratio of K38 / K35 keratin expression levels from the expression levels measured in step c; and e. Identify the compound as being effective in preventing and / or slowing down hair thinning, or in promoting an increase in hair thickness, when the K38 / K35 ratio is significantly reduced compared to the ratio obtained with a hair follicle not treated with said compound.

[0019] First of all, the present invention relates to the cosmetic use of at least one compound of the following formula (I), and / or one of its salts and / or its conjugated and / or solvated glucurono derivatives:

[0020] [Chem.l] O

[0021] in which: RI = -OH, and - R2 to R5 are the same or different and are independently chosen from H and -OH,

[0022] to prevent / slow down hair thinning, or promote an increase in hair thickness.

[0023] Preferably, the compounds of formula (I) are natural urolithins.

[0024] More preferably, the compound of formula (I) is chosen from urolithins A, B, C, D, E, iso-urolithin A and mixtures thereof.

[0025] According to a preferred embodiment, the compound(s) of formula (I) are such that they contain a hydroxy radical (-OH) such that RI = OH, and R2=R3=R4=R5= H.

[0026] According to another preferred embodiment, the compound(s) of formula (I) are such that they contain 2 hydroxy radicals (-OH), such that RI = OH, R2, R3, R4, R5 = OH or H, it being understood that one of the radicals R2 to R5 denotes a hydroxy (-OH), preferably RI = R2 = OH and R3=R4=R5 = H.

[0027] According to another preferred embodiment, the compound(s) of formula (I) are such that they contain 3 hydroxy radicals (-OH), such that RI = OH, R2, R3, R4, R5 = OH or H, it being understood that two of the radicals R2 to R5 denote a hydroxy (-OH), preferably R 1=R2=R3=OH and R4=R5=H.

[0028] According to another preferred embodiment, the compound(s) of formula (I) are such that they contain 4 hydroxy radicals (-OH), such that RI = OH, R2, R3, R4, R5 = OH or H, it being understood that three of the radicals R2 to R5 denote a hydroxy (-OH), preferably R 1 = R2 = R4 = OH.

[0029] According to a preferred embodiment, the compound(s) of formula (I) are chosen from urolithins A, BC, D, E, iso-urolithin A and their salts and / or solvates.

[0031] Urolithin A is marketed in particular under the name SML1791-5MG by Sigma-Aldrich.

[0032] Urolithin B has the CAS number and formula: => Urolithin B (1139-83-9) cçr O

[0033] Urolithin B is marketed in particular by the company Sigma-Aldrich under the commercial reference SML1649-50mg.

[0034] Urolithin C has the CAS number and formula: => Urolithin C (165393-06-6) HO JL Y. .-O HO y O

[0035] Urolithin C is marketed in particular by the company Sigma-Aldrich under the commercial reference SML3047-50mg.

[0036] Urolithin D has the CAS number and the formula: => Urolithin D (131086-98-1) O HO Il YY î ho^yYYYx^,oh ^^OH

[0037] Urolithin E has the CAS number and the formula: => Urolithin E (1453297-45-4) HO. .OH V HO" ''O'^O OH

[0038] Iso-urolithin A is an isomer of urolithin A. Its chemical name is 3,9-dihydroxy-6H-dibenzo[c]chromen-6-one.Iso-urolithin A is notably marketed by the company Astatech under the commercial reference ATE638637155.

[0039] Preferably, the compound of formula (I) is urolithin A and / or its salts and / or one of its glucuronoconjugated derivatives and / or its solvates.

[0040] By “salt” we mean in particular basic salts.

[0041] By “basic salt” is meant an organic or mineral basic salt, in particular chosen from a salt derived from alkali hydroxides such as sodium hydroxide which leads to sodium salts or potash which leads to potassium salts.

[0042] Urolithins are metabolites produced by the intestinal microbiota from ellagic acid and ellagitannins. Studies have demonstrated positive biological effects of urolithins. Several mechanisms of action of urolithins have been proposed so far.

[0043] By "thinning of the hair" is meant a reduction in the diameter of the hair, in particular characterized by an expression of keratin K38 in at least 50% of the cells of the cortex of a fine hair. By "increase in the thickness of the hair" is meant an increase in the diameter of the hair characterized by an expression of keratin K38 in less than 50% of the cells of the cortex of a thick hair.

[0044] For example, the average diameter of a fine Caucasian hair, in particular in women, is less than or equal to 70 μm, preferably approximately 60 μm. Preferably, the average diameter of a thick Caucasian hair, in particular in women, is approximately 80 μm to approximately 90 μm.

[0045] Hair thinning increases with age and is particularly relevant for people over 40 and the elderly. Thus, preferably, hair thinning is linked to the increase in the ratio of the number of small diameter hairs to the total number of hairs with age.

[0046] In particular, the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives is formulated in a cosmetic composition.

[0047] In particular, the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives is used to increase the volume of hair or hair mass.

[0048] Preferably, the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives is administered topically, in particular to the scalp and / or hair fibers.

[0049] The present invention also relates to a method for evaluating the effectiveness of a compound for preventing and / or slowing down hair thinning, or for promoting an increase in hair thickness, comprising at least the following steps: a. Providing at least one hair follicle from a scalp sample; b. Incubating said hair follicle in a culture medium in the presence of a compound to be tested, in particular at a temperature of between 30°C and 38°C, preferably 37°C, and under an atmosphere of 2% to 7% CO2, preferably 5% CO2, and for a period of time of at least 1 day, preferably at least 2 days, in particular at least 5 days; c. Measure the expression levels of keratins K38 and K35 in the incubated sample from step b; d. Calculate the ratio of K38 / K35 keratin expression levels from the expression levels measured in step c; and e. Identify the compound as being effective in preventing and / or slowing down hair thinning, or in promoting an increase in hair thickness, when the K38 / K35 ratio is significantly reduced compared to the ratio obtained with a hair follicle not treated with said compound.

[0050] Preferably, the culture medium is William's complete medium. This culture medium (William's Medium E) is typically phenol red-free and comprises 40ng / mL (i.e. 2 mM) hydrocortisone, 10g / mL insulin, 2 mM L-glutamine and 1% antibiotics / antimycotics.

[0051] By "expression level" is meant the quantity of mRNA produced, or the quantity of protein produced by the expression of this gene. This quantity can for example be expressed as such, in the form of concentration or ratio. The expression level can be measured by general techniques well known to those skilled in the art such as RT-qPCR, Western blot or immunolabeling.

[0052] By "scalp sample" is meant a fragment of scalp, in particular taken by biopsy from the scalp of a subject or obtained from a reconstructed scalp model.

[0053] As demonstrated in examples, the Applicant has demonstrated that the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives, is capable of reducing the increase in the level of expression of the K38 gene in hair follicles (this increase in the level of expression of K38 being characteristic of fine hair), and promotes the expression of keratin profiles characteristic of thick hair. Preferably, the level of expression of K38 of a hair sample treated with urolithin A is reduced by at least 20%, preferably by at least 40%, preferably by at least 50% compared to the level of expression of K38 of an untreated hair sample.

[0054] Preferably, the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives has no effect on the level of expression of the K35 gene in the hair follicles.

[0055] The present invention also relates to a non-therapeutic, cosmetic treatment method for increasing the diameter of hair fibers comprising the application of at least one compound of formula (I) or one of its salts and / or its glucuronoconjugated and / or solvated derivatives.

[0056] The invention also relates to a cosmetic treatment process comprising at least one step of applying at least one compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives to fine or thinning hair.

[0057] This method preferably comprises a first step of evaluating the level of expression of the K38 and K35 proteins in a sample of hair follicles, then comparing the K38 / K35 ratio obtained with the K38 / K35 ratio characteristic of fine hair.

[0058] By "K38 / K35 ratio" is meant the ratio between the level of expression of K38 and the level of expression of K35 in a sample of hair follicles. As an example for Caucasian hair, particularly in women, a K38 / K35 ratio characteristic of fine hair corresponds to a ratio of the level of expression in RNA of approximately 0.070. Unlike thick hair, particularly in women, where the K38 / K35 ratio characteristic of thick hair corresponds to a ratio of the level of expression in RNA of approximately 0.045.

[0059] The cosmetic treatment method of the invention is implemented in particular by administering at least one compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives topically to fine or thinning hair. For example, the compound of formula (I) or one of its salts or solvates may be formulated in the form of a suspension, a gel, an emulsion, a shampoo, a leave-in lotion or a mousse.

[0060] The cosmetic treatment method according to the invention can thus be implemented by daily topical administration of the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives.

[0061] The method according to the invention may comprise a single administration. According to another embodiment, the administration is repeated for example 2 to 3 times daily over one day or more, and generally over a prolonged period of at least 4 weeks, or even 4 to 15 weeks, with, where appropriate, one or more periods of interruption.

[0062] The present invention relates to a cosmetic composition, in particular for topical application to the scalp and / or hair fibers, comprising, in particular in a physiologically acceptable medium, at least one compound of formula (I), and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives, and vitamin C and / or one of its derivatives. Preferably, the compound of formula (I) is urolithin A.

[0063] The composition according to the invention also comprises vitamin C (i.e. ascorbic acid) or one of its derivatives.

[0064] Ascorbic acid within the meaning of the invention preferably corresponds to L- ascorbic, or vitamin C. It has the structure formula (II):

[0065] [Chem.2] (II).

[0066] L-ascorbic acid is notably marketed under the name Ascorbic Acid (L) by Reckon Organics.

[0067] By "ascorbic acid derivative" is preferably meant a compound chosen from 5,6-di-O-dimethylsilylascorbate (notably sold by the company Exsymol under the reference PRO-AA), the potassium salt of DL-alpha-tocopheryl-DL-ascorbyl-phosphate also called Potassium Ascorbyl Tocopheryl Phosphate (sold by the company SEPPIC under the reference SEPIVITAL EPC), magnesium ascorbyl phosphate, sodium ascorbyl phosphate (sold by the company DSM under the reference Stay-C 50), disodium ascorbyl sulfate, sulfinyl-L-ascorbic acid, glucopyranosyl-L-ascorbic acid and ascorbyl glucoside.

[0068] By "ascorbyl glucoside" is meant the condensation product of glucose, in D form, that is to say in the form of glucopyranose a or [3 or furanose a or [3, or in L form, with ascorbic acid, preferably in L form. Preferably, the ascorbyl glucoside is L-ascorbic acid 2-OaD-glucopyranoside, notably marketed under the name VG2 by Macrocare Tech.

[0069] Preferably, the composition according to the invention comprises ascorbic acid, preferably L-ascorbic acid.

[0070] Preferably, the composition comprises at least 1% by weight, preferably at least 2% by weight of ascorbic acid or one of its derivatives relative to the total weight of the composition, preferably at least 5% by weight, more preferably at least 7% by weight, even better at least 10% by weight.

[0071] Preferably, the composition comprises from 2% to 20% by weight of ascorbic acid or one of its derivatives, relative to the total weight of the composition, more preferably from 5% to 15% by weight, even more preferably from 7% to 12% by weight.

[0072] Preferably, the composition, in particular cosmetic, is intended for topical application in particular to the scalp and / or hair fibers, in particular the hair.

[0073] By "physiologically acceptable medium" is meant a compatible medium with hair fibers and in particular the scalp and / or hair. The physiologically acceptable medium is generally adapted to the nature of the support on which the composition is to be applied, as well as to the form in which the composition is to be packaged.

[0074] The physiologically acceptable medium of the composition used may be an aqueous medium, for example consisting of water or a mixture of water and at least one physiologically acceptable organic solvent.

[0075] As organic solvent, mention may be made, for example, of (C2-C4) alcohols such as ethanol and isopropanol; polyols, in particular those having from 2 to 6 carbon atoms such as glycerin, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, dipropylene glycol, diethylene glycol; polyol ethers such as 2-butoxyethanol, propylene glycol monomethyl ether, monoethyl ether and diethylene glycol monomethyl ether; and mixtures thereof. Preferably, the organic solvent is a monoalcohol, in particular C2 to C6, such as ethanol.

[0076] Preferably, the composition according to the invention comprising, in a physiologically acceptable medium, at least one compound of formula (I) or one of its salts and / or its glucuronoconjugated and / or solvated derivatives, may also comprise one or more usual cosmetic additives, in particular chosen from non-ionic, anionic, cationic and amphoteric surfactants, vitamins and provitamins including panthenol, anti-hair loss active agents such as stemoxydine and 2,4-diamino pyrimidine-3-oxide, sunscreens, fillers, coloring matters, pearlescent agents, opacifying agents, sequestering agents, polymers, in particular film-forming polymers, conditioning agents, plasticizing agents, thickening agents, oils, antioxidant agents, anti-foaming agents, moisturizing agents, emollient agents, penetrating agents, perfumes and preservatives.

[0077] According to one embodiment of the invention, the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives is present in the cosmetic composition, in an amount of at least 0.00001% by weight, in particular in an amount of approximately 0.0001 to approximately 20% by weight relative to the total weight of said composition, and in particular from approximately 0.001% to approximately 2% by weight of the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives relative to the total weight of the composition.

[0078] The cosmetic compositions suitable for implementing the invention may be in the form of a suspension, a gel, an emulsion, a shampoo, a leave-in lotion or a mousse.

[0079] The compositions may be presented in any form suitable for the care of hair fibers, in particular hair and / or the scalp, in particular in form of hair care lotion, for example for daily or bi-weekly application, of a hair shampoo or conditioner, in particular for weekly or bi-weekly application, of a liquid or solid scalp cleansing soap for daily application, of a hair styling product (hairspray, hair gel), of a treatment mask, of a foaming cream or gel for cleansing the hair.

[0080] A composition suitable for implementing the invention may have the form of an aqueous, alcoholic or hydro-alcoholic solution or suspension, or an oily suspension, an emulsion of more or less fluid consistency and in particular liquid, semi-liquid or solid, obtained by dispersion of a fatty phase in an aqueous phase (O / W) or vice versa (W / O) or a multiple emulsion, an aqueous, hydro-alcoholic or oily gel, a free or compacted powder to be used as is or to be incorporated into a physiologically acceptable medium, microcapsules or microparticles, ionic or non-ionic dispersions.

[0081] According to a particular embodiment of the invention, the composition used in the context of the invention is anhydrous. By "anhydrous" is meant a composition comprising less than 5% by weight of water relative to the total weight of the composition, preferably less than 3% by weight, even better less than 1% by weight relative to the total weight of the composition, or even completely free of water (0%).

[0082] Advantageously, the compound of formula (I) and / or one of its salts and / or its glucuronoconjugated and / or solvated derivatives according to the invention is applied in the form of a formulation promoting the penetration of the agent into the hair follicle. By way of example, mention may be made of non-cyclic mono- and di-alcohols, ethyl acetate, butyl acetate, isopropyl myristate, fatty acids, phospholipids, terpenes, azone and derivatives, propylene glycol and glycol derivatives, cyclodextrins, octylsalicylate, cyclopentadecanolide, polysorbates, polyvinylpyrrolidone and derivatives, this list not being limiting.

[0083] According to one embodiment, the compositions contain a surfactant of non-ionic, anionic, cationic or amphoteric type and among these, mention may be made of alkyl sulfates, alkylbenzenesulfates, alkyl ether sulfates, alkyl sulfonates, quaternary ammonium salts, alkyl betaines, oxyethylenated alkylphenols, fatty acid alkanolamides, oxyethylenated fatty acid esters as well as other non-ionic surfactants of the hydroxypropyl ether type.

[0084] When the compositions contain at least one surfactant, the latter is generally present at a maximum concentration of 30% by weight, and preferably between 0.1% and 5% by weight relative to the total weight of the composition.

[0085] In order to improve the cosmetic properties of the hair or to reduce or prevent its degradation, the composition may also contain an agent dealing with cationic, anionic, non-ionic or amphoteric nature.

[0086] Among the treating agents, mention may in particular be made of volatile or non-volatile, linear or cyclic silicones and their mixtures, polydimethylsiloxanes, quaternized polyorganosiloxanes such as those described in French patent application No. 2,535,730, polyorganosiloxanes with aminoalkyl groups modified by alkoxycarbonylalkyl groups such as those described in US patent No. 4,749,732, polyorganosiloxanes such as the polydimethylsiloxane-poly-oxyalkyl copolymer of the Dimethicone Copolyol type, a polydimethylsiloxane with stearoxy end groups (stearoxydimethicone), a polydimethylsiloxanedial-kylammonium acetate copolymer or a polydimethylsiloxane polyalkylbetaine copolymer described in GB application 2 197 352, organo-modified polysiloxanes with mercapto or mercaptoalkyl groups such as those described in French patent No. 1,530,369 and in European patent application No. 0,295,780,as well as silanes such as stearoxytrimethylsilane. ,

[0087] The composition according to the invention may also contain other ingredients such as cationic polymers, basic amino acids (such as lysine, arginine) or acids (such as glutamic acid, aspartic acid), peptides and their derivatives, protein hydrolysates, waxes, as well as other compounds such as fatty alcohols, lanolin derivatives, active ingredients such as panthothenic acid, anti-hair loss agents, anti-dandruff agents, thickeners, suspending agents, sequestering or complexing agents, opacifying agents, sunscreens, antioxidants as well as perfumes and preservatives.

[0088] The invention is now illustrated by the following figures and example. Figures

[0089] [Fig-1] [Fig.l] is the result of measuring K35 and K38 proteins on extracts of hair follicles ex vivo and in vitro.

[0090] [Fig.2] [Fig.2] is an assessment of the relative expression of KRT38 and KRT35 in hair follicles ex vivo and in vitro.

[0091] [Fig.3] [Fig.3] is the measurement of K38 labeling in the cortex of hair follicles ex vivo and in vitro.

[0092] [Fig.4] [Fig.4] is the measurement of K38 labeling in the cortex of hair follicles of human subjects. Examples

[0093] Example 1 - Model of modulation of K38 expression in the human hair follicle

[0094] Materials and methods 1. Obtaining samples

[0095] Biopsies obtained by scalp surgery were shaved and cut into small pieces. The hypodermal dermal junction was cut with a scalpel. Hair follicles were plucked from the hypodermis and their integrity was checked under a microscope before placing them in a 24-well plate in 500 pL of William's complete medium (Williams Medium E without phenol red - hydrocortisone 40ng / mL - 2mM - insulin 100g / mL - L-glutamine 2mM - 1% antibiotics / antimycotics). Plates with follicles were placed at 37°C under 5% CO2 for 5 days with a change in the entire culture medium after 2-3 days. Treatments with 5 pM urolithin A (CAS N: 1143-70-0, SML1791-5MG, Sigma-Aldrich) were performed when culture was initiated and when the culture medium was changed. A 24-well plate, i.e., 24 follicles per condition, was produced for each experiment.Cultured follicles were observed daily with the Zeiss microscope and pictures were taken with Archimed software (Microvision) to measure the viability and elongation of each follicle.

[0096] On day 0 and day 5, follicles were collected to analyze the expression of K38 and K35 in Western blot, RT-qPCR or immunofluorescence. 1. Immunofluorescence on transverse and longitudinal sections of hair follicles

[0097] Ex vivo follicles and in vitro follicles (5 days of culture) with and without treatment were embedded in Tissue-Tek ® OCT ™ compound, frozen in dry ice / ethanol and cryo-sectioned at 10 pm for transverse sections and at 5 pm for longitudinal sections. All sections were preserved and placed each time on 3 consecutive slides. The first slide was used to label K35, the second to label K38 and the third was kept as a backup.

[0098] The sections were permeabilized for 15 minutes in PBS-0.05% Tween 20 and blocked for 15 minutes in PBS-10% Goat Serum-0.05% Tween 20. The sections were then incubated for 30 minutes with anti-K35 (1:500) or anti-K38 (1:500) antibody. After 3 washes in PBS-0.05% Tween 20, the secondary antibody (anti-guinea pig Cy3 1:200) was incubated for 30 minutes. The sections were then washed again and counterstained with Hoechst (1:5000) for nuclear staining for 1 minute and mounted with Prolong Gold Antifade reagent (Life Technologies). Photos of longitudinal sections of hair follicles were taken using Nikon microscope and NIS Element software. For quantitative assessment on the cross-section, photographs were taken at the same distance from the bulb for each hair follicle using the Leica microscope. Area labeling analysis K38 was created using Image J software. 1. Western blot of hair follicle proteins

[0099] The follicles collected ex vivo and in vitro were placed in a tube containing 100 pl of TP3 buffer (Ready Prep Sequential Extraction Kit Reagent 3, Biorad) and stored at -80°C. Then, the samples were crushed for a few minutes with a pestle and then vortexed a few times during 30 minutes of incubation at room temperature. After centrifugation at 10,000 g for 10 minutes, the supernatant was collected and the protein concentration was determined by the Pierce 660 nm method.

[0100] For Western blotting, samples were prepared in NuPAGE LDS Sample Buffer (4X) and NuPAGE Reducing Agent (10X), then heated at 95°C for 1 minute. 10 pg of total protein was separated by 4-15% SDS-PAGE (Criterion™ TGX Stain-Free™ Precast Gels Bio-rad) with 1X Tris / Glycine / SDS running buffer and transferred to a nitrocellulose membrane (Trans-Blot Turbo Midi Format Bio-rad) by semi-dry blotting. Subsequently, the membranes were blocked in PBS-1% nonfat dry milk-0.05%Tween20 for 1 hour at room temperature. The membranes were then probed with the primary antibody (anti-K35 or anti-K38) diluted in blocking buffer for 1 hour at room temperature. After washing in PBS-1%-0.05% Tween 20, the membranes were incubated with the horseradish peroxidase-labeled secondary antibody for 30 minutes at room temperature.Targeted proteins were detected using SuperSignal West Dura Extended Duration Substrate (Thermofisher). Peroxidase activity was detected with the CDD camera imager (ChemiDoc Bio-Rad). Quantification and normalization were performed with ImageLab software (Bio-Rad). Expression levels of K35 and K38 were normalized to total proteins in the electrophoretic gel lane. 1. Purification and extraction of total RNA

[0101] The follicles collected ex vivo and in vitro were placed in a tube containing 200 μl of RLT-1% mercaptoethanol buffer[3 and stored at -80 °C. For the ex vivo condition, 24 follicles were collected and for the in vitro condition, only live follicles were collected for gene expression study (about 20 follicles depending on the conditions). A homogenization step was required before RNA extraction on the hair follicles. The follicles were first disrupted with a pestle, then the lysate was passed through a 20-gauge (0.9 mm) needle attached to a sterile plastic syringe at least 10 times. Finally, the lysate was loaded onto a QIAshredder spin column and spun for 2 min at maximum speed in a microcentrifuge.

[0102] Purification and extraction of total RNA were performed using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol and with a DNase digestion step using the RNase-free DNase kit. RNAs were eluted with 30 μL of RNase-free water.

[0103] RNA concentration was determined by Nanodrop and RNA quality using the RNA 6000 Nano kit (Agilent) on the Agilent 2100 Bioanalyzer according to the manufacturer's protocol. 1. cDNA Synthesis and Real-Time Quantitative PCR (RT-qPCR)

[0104] Reverse transcription was performed with the iScript cDNA Synthesis Kit (Bio-Rad) in a volume of 50 qL and the cDNA was stored at -80 °C until use. qPCR was performed on the CFX384 system (BioRad) and CFX Maestro software with a total volume of 10 qL containing 5 ng of cDNA, 5 qL of SYBER Green Mix (Sso Advanced SYBR Green Supermix Biorad), 1 pL of 10X primers (QuantiTect Primer Assay, Qiagen, Table 3) and 1.5 pL of water. Reactions were seeded in a 384-well plate, and PCR cycles included an initial predenaturation at 95 °C for 30 s and 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s. At the end of the PCR, a melting curve was performed to check that only one PCR product was amplified.The relative mRNA expression level was calculated using the 2-(AACt) method after normalization with the geometric mean of RPL13A, RRN18S, and TBP as the reference gene. Therefore, the relative gene expressions of the treated groups were reported as a fold change from the control mean value. Statistical analyses were performed using the MyStat application and the Wilcoxon test.

[0105] Introductory reference for qRT-PCR

[0106] Hs_RPL13A_l_SG QuantiTect Primer AssayQT00089915Qiagen

[0107] Hs_RRN18S_l_SG QuantiTect Primer AssayQT00199367Qiagen

[0108] Hs_TBP_l_SG QuantiTect Primer AssayQT00000721Qiagen

[0109] Hs_KRT35_l_SG QuantiTect Primer AssayQT00012397Qiagen

[0110] Hs_KRT38_l_SG QuantiTect Primer AssayQT00042000Qiagen [YES] Results 1. Measurement of K35 and K38 proteins in hair follicle extracts ex vivo and in vitro.

[0112] Hair follicles were maintained in vitro for 5 days before measuring the expression level at the keratin protein level. Under these conditions, K38 expression increased while K35 expression decreased (Figure 1a and b). Therefore, the K38 / K35 ratio increased in follicles maintained in vitro (Figure 1c) in a similar manner to what is observed in fine hair, e.g. compared to thick hair. 1. Evaluation of the relative expression of KRT38 and KRT35 in hair follicles ex vivo and in vitro.

[0113] This shift in hair keratin expression was confirmed at the mRNA level by qRT-PCR. The results showed that K38 expression was significantly increased in in vitro hair follicles compared to ex vivo hair follicles (Figures 2a and 2b). An increase of at least 100% was consistently observed, but variable magnitudes were observed, ranging from 2- to 10-fold, depending on the samples. In parallel, K35 expression was significantly decreased when follicles were maintained in vitro (Figures 2c and 2d). For the latter, the decrease was also variable, with 6 out of 9 experiments showing a reduction of more than 50%. 1. Evaluation of the distribution of K38 and K35 in hair follicles in vitro.

[0114] To monitor the distribution of both keratins in hair follicles maintained in vitro, immunostaining of K38 and K35 was performed on ex vivo follicles and on control follicles in vitro. K38 was labeled only in the cortex with a discontinuous pattern of positive cells in both ex vivo hair follicles and in vitro control hair follicles. K35 was labeled throughout the surface of the human hair follicle cortex and then gradually decreased both ex vivo and in vitro. Therefore, the distribution of K35 and K38 in hair follicles maintained in vitro was still restricted to the hair cortex and did not display a deregulated pattern. 1. Measurement of K38 labeling in the cortex of hair follicles ex vivo and in vitro.

[0115] The percentage of hair follicle cortex area labeled for K38 was assessed with ImageJ software on one subject. The analysis showed that the percentage of K38 labeling was significantly higher in the cortex of follicles maintained in vitro for 5 days ([Fig.3]). 1. Measurement of K38 labeling in the cortex of hair follicles of human subjects.

[0116] The percentage of K38 labeling in the cortex was also evaluated on 129 follicles from 5 subjects ([Fig.4] “set 1”) as well as on ex vivo and in vitro follicles obtained for one subject. All values ​​for ex vivo follicles were within the normal distribution of “set 1”. In contrast, in vitro follicles appeared outside the normal distribution for a given hair diameter with an increased percentage of K38 labeling. The percentage of their cortex labeled for K38 (80-90%) was similar to the percentage evaluated in fine hair. 1. Conclusion

[0117] The results presented above show a model using human hair follicles in vitro that recapitulates the shift in hair keratin expression characteristic of fine hair. The model may be useful for evaluating the property of a given product to limit the development of small diameter hair, and to promote the expression of thick hair keratins, and thus to increase hair thickness.

[0118] Example 2 - Evaluation of the effect of urolithin A on the expression of K38 and K35 in the in vitro hair follicle assay

[0119] Materials and methods

[0120] Urolithin A at 5qM (CAS No.: 1143-70-0, SML1791-5MG, Sigma-Aldrich) was added to the culture from the first day of in vitro maintenance and renewed with the culture medium. A 24-well plate, i.e., 24 follicles per condition, was produced for each experimental condition.

[0121] At day 0 and day 5 for in vitro conditions, follicles were collected to analyze K38 and K35 expression by qRT-PCR. Keratin expressions were normalized to the ex vivo (“native”) state, and no treatment (“control”) and urolithin A (“Uro-A”) conditions were compared by Wilcoxon test.

[0122] Results 1. Evaluation of K38 expression by qRT-PCR

[0123] Data represent the mean expression level relative to expression in ex-vivo hair follicles (native, normalized mean equal to 1).

[0124] [Tables 1] Type N (replicates) Fold Change* 1-Native 5 1,000 2-Control 5 8,536 3-Uro-A 5 4,404

[0125] ^ratio of the expression level of 2-Control or 3-Uro-A compared to the expression level of 1-Native

[0126] The results presented above show that urolithin A reduces the increase in K38 expression in treated human hair follicles by approximately 50% compared to the untreated control. 1. Evaluation of K35 expression by qRT-PCR

[0127] Data represent the mean expression level relative to expression in ex-vivo hair follicles (native, normalized mean equal to 1).

[0128] [Tables2] Type N (replicates) Fold Change* 1-Native 5 1.000 2-Control 5 0.698 3-Uro-A 5 0.820

[0129] ^ratio of the expression level of 2-Control or 3-Uro-A compared to the expression level of 1-Native

[0130] The results presented above show that rurolithin A does not significantly modify the expression of K35 in the treated human hair follicle compared to the untreated control. 1. Conclusion

[0131] Urolithin A therefore reduces the increase in K38 expression observed in human hair follicles in vitro while it has no significant effect on K35 expression. Therefore, treatment with urolithin A counteracts the change in K38 keratin expression in hair cortex cells, characteristic of small diameter hair, and promotes the expression of keratins associated with thick hair. Urolithin A can be used to promote the production of large diameter hair.

Claims

Claims

1. Cosmetic use of at least one compound of formula (I) and / or one of its salts and / or one of its glucuronoconjugated and / or solvated derivatives, [Chem.l] in which: - RI = -OH, and

2.

3.

4.

5.

6. - R2 to R5 are identical or different and are independently chosen from H and -OH, to prevent and / or slow down the thinning of the hair, or to promote an increase in the thickness of the hair. Cosmetic use according to claim 1, wherein the compound of formula (I) is chosen from urolithin A, urolithin B, urolithin C, urolithin D, urolithin E, iso-urolithin A and mixtures thereof. Cosmetic use according to one of the preceding claims, in which the compound of formula (I) is urolithin A. Use according to one of the preceding claims, in which the thinning of the hair is linked to the increase in the ratio of the number of small diameter hairs compared to the total number of hairs with age. Use according to any one of the preceding claims, wherein the compound of formula (I) and / or one of its salts and / or one of its glucuronoconjugated and / or solvated derivatives is administered topically, in particular to the scalp and / or hair fibers. Cosmetic treatment method comprising at least one step of applying at least one compound of formula (I) and / or one of its salts and / or one of its glucuronoconjugated and / or solvated derivatives: [Chem.l] O

7. in which: - RI = -OH, and - R2 to R5 are the same or different and are independently chosen from H and -OH, on fine or thinning hair. Cosmetic composition, in particular for topical application to the scalp and / or hair fibers, comprising: -at least one compound of formula (I) and / or one of its salts and / or one of its glucuronoconjugated and / or solvated derivatives: [Chem.l] O

8. in which: - RI = -OH, and - R2 to R5 are the same or different and are independently chosen from H and -OH, and -vitamin C or one of its derivatives. Cosmetic composition according to claim 7, in which vitamin C or one of its derivatives is present in a content of at least 1% by weight, preferably at least 2% by weight relative to the total weight of the composition, preferably at least 5% by weight, more preferably at least 7% by weight, even better at least 10% by weight; preferably vitamin C or one of its derivatives is present in a content of 2% to 20% by weight relative to the total weight of the composition, in particular 5% to 15% by weight, preferably 7% to 12% by weight relative to the total weight of the composition.

9. Cosmetic composition according to any one of claims 7 or 8, in which the compound of formula (I) and / or one of its salts and / or one of its glucuronoconjugated and / or solvated derivatives is present in a content of at least 0.00001% by weight, in particular in an amount of from about 0.0001 to about 20% by weight relative to the total weight of said composition, and in particular from about 0.001% to about 2% by weight.

10. Cosmetic composition according to any one of claims 7 to 9, characterized in that the composition is in the form of a suspension, a gel, an emulsion, a shampoo, a leave-in lotion or a mousse.

11. A method for evaluating the effectiveness of a compound for preventing and / or slowing down hair thinning, or for promoting an increase in hair thickness, comprising at least the following steps: a. Have at least one hair follicle from a scalp sample; b. Incubating said hair follicle in a culture medium in the presence of a compound to be tested, in particular at a temperature between 30°C and 38°C and under an atmosphere of 2% to 7% CO2 and for a period of time of at least 1 day; c. Measure the expression levels of keratins K38 and K35 in the incubated sample from step b; d. Calculate the ratio of K38 / K35 keratin expression levels from the expression levels measured in step c e. Identify the compound as being effective in preventing and / or slowing down hair thinning, or in promoting an increase in hair thickness, when the K38 / K35 ratio is significantly reduced compared to the ratio obtained with a hair follicle not treated with said compound.

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