Olfactory receptor 2A4 / 7 agonist for promoting hair growth and improving aesthetic aspects

Abstract

The present invention relates to the use of a selected olfactory receptor 2A4 / 7 agonist for improving the aesthetic aspects of hair and preventing or treating hair growth disorders. The olfactory receptor 2A4 / 7 agonist can be formulated as a composition suitable for either systemic administration or topical application to a body area having hair.

Description

Technical Field

[0001] The present invention relates to olfactory receptor 2A4 / 7 agonists for promoting hair growth and improving the aesthetic aspects of hair.

[0002] The present invention originates from the fields of cosmetics and trichology.

[0003] Specifically, the present invention relates to a composition containing an olfactory receptor 2A4 / 7 agonist as an active ingredient for either improving the aesthetic aspects of hair or treating hair loss in mammals, particularly humans.

Background Art

[0004] Hair has a protective role and is considered an appendage of the skin together with the sebaceous glands, sweat glands, and nails.

[0005] The life cycle of the hair follicle includes three main stages: a growth phase known as anagen, a regression phase known as catagen, and a resting phase known as telogen.

[0006] Hair on the scalp is produced by hair follicles, which are skin organs of mammals. Hair follicles do not continuously produce hair. Hair follicles go through a growth stage that can last for more than two years, then regress, have a resting stage of up to two months, and then repeat the cycle of restarting the growth of new hair fibers.

[0007] The biological aspect of this phenomenon lies in the ability of the stem cells of the hair follicle to exit the dormant state in alternating steps. During the growth and hair production stages of the hair follicle, proliferation, differentiation, and survival activities are dominant, and these are regulated by growth factors. In contrast, the catagen phase is characterized by the activation of molecular pathways that lead hair follicle cells to apoptosis.

[0008] During the growth phase, the dermal papilla moves from the hair matrix to the base of the hair follicle, activating the bulge stem cells and generating chemical signals that direct the bulge stem cells. This "movement" creates a "pathway" for cells to rise to the outer root sheath or ORS.

[0009] In response to further signals from the dermal papilla, hair matrix cells derived from the stem cells proliferate, initiate the differentiation process, and move upward to form the hair shaft and the inner root sheath of the hair follicle.

[0010] The onset of the regression phase is characterized by the end of cell proliferation and apoptosis of the hair matrix cells. During the regression phase, the dermal papilla moves towards the bottom of the bulge. This proximity of the dermal papilla to the bulge is thought to be essential to initiate another hair growth cycle. This enables the interaction / activation of bulge cells during rest and a new hair growth cycle.

[0011] During the transition from the regression phase to the resting phase, some bulge cells move to encounter the dermal papilla and generate hair germs.

[0012] Resting-phase hairs contain a cell population at their base, which is actually called a hair germ and is located in close proximity to the dermal papilla. Even before the bulge surrounds the dermal papilla to form the hair matrix of a new hair bulb, the hair germ is activated and begins to proliferate towards the end of the resting phase.

[0013] Also, eyelashes go through the same three individual stages during their hair growth cycle: the growth phase, the regression phase, and the resting phase, but the growth rate and the length of the growth phase are shorter than those observed in scalp hair.

[0014] Various factors, especially stress, malnutrition, and aging, have an adverse effect on the hair bulb life cycle and determine hair loss and thinning.

[0015] Hair growth disorders are very common, especially in the male population. When the role of hair in social relations is considered, hair loss can be difficult for many people to face.

[0016] One of the more well-known hair disorders is represented by alopecia, which is a disorder in hair growth and can be caused by various factors ranging from genetic predisposition to environmental factors. The degree and pattern of alopecia can vary, but male pattern alopecia (AGA) is representative of one of the most common disease types of alopecia. Disease types of alopecia other than male pattern alopecia include telogen effluvium, alopecia areata, tinea capitis, cicatricial alopecia, and alopecia due to excessive cosmetic treatment.

[0017] In an individual suffering from male pattern alopecia (AGA), the hair follicles formed at the start of a new anagen phase gradually decrease in size over time (miniaturization), and as time progresses, the formation of hair that is finer than the previous hair is brought about. As a result, micro hairs are formed.

[0018] Dermatologists classify alopecia by subdividing it into a cicatricial category and a non-cicatricial category. Some types of alopecia, such as lichen planopilaris, discoid lupus erythematosus, and graft-versus-host disease, are associated with the destruction of bulge hair follicle stem cells and permanent alopecia. In treatable types of alopecia such as alopecia areata, inflammation affects the progenitor cells of the hair follicle, but its stem cells remain. In these diseases, regrowth occurs when the inflammation is eliminated and when the hair follicles regenerate starting from the undamaged stem cells that result.

[0019] In an individual troubled by male pattern alopecia (AGA), the hair follicles reformed at the start of a new anagen phase gradually come to shrink in size over time, bringing about the formation of hair with a smaller diameter compared to the initial diameter. As a result, the formation of micro hairs occurs.

[0020] Even when the hair follicles on the scalp atrophy, it has been observed that stem cells that change into progenitor cells are still supplied, although to a lesser extent compared to the scalp under physiological conditions.

[0021] Currently, many options are available for the treatment of hair growth disorders including alopecia.

[0022] The first treatment line involves the topical application and / or administration of formulations for preventing and treating hair disorders. Most of the hair formulations available on the market target the hair bulb, act on scalp metabolism, nutrition, oxygenation, and microcirculation, and improve the conditions contributing to physiological hair growth.

[0023] Typically, these hair products incorporate cosmetic ingredients that restore the skin and conditions suitable for promoting hair growth.

[0024] Numerous hair products contain antioxidants such as vitamin E, glycosaminoglycans such as hyaluronic acid, and vitamins, such as vitamins A and B groups or their derivatives (such as niacin or nicotinamide adenine dinucleotide (NAD)).

[0025] However, in many cases, the application of hair compositions containing the above ingredients and other components to the scalp or the administration of nutritional formulations has shown unsatisfactory effects on hair growth stimulation.

[0026] Hair products containing pharmacologically active substances as hair ingredients have shown excellent activity against cosmetic / hair formulations.

[0027] Among these pharmaceuticals, formulations containing minoxidil, a pharmacologically active ingredient with vasodilatory effects, are well-known. These products are usually formulated as alcohol solutions for topical application.

[0028] Other products used for treating hair disorders include pharmaceutical compositions containing finasteride, a hormonal substance. These products inhibit type II 5-alpha-reductase, an enzyme that converts testosterone to dihydrotestosterone (DHT), which is a hormone that contracts or miniaturizes hair follicles and causes hair loss when its secretion exceeds physiological production.

[0029] However, the use of minoxidil and finasteride is not without drawbacks. Specifically, topical application of minoxidil can result in local side effects such as rash, local inflammation, and general drawbacks such as headache and hirsutism, while oral administration of finasteride can result in hormonal dysfunction.

[0030] There is a need for alternative medicine suitable for treating both aesthetic problems of hair and hair growth disorders, which is effective in topical use and substantially free of side effects.

[0031] One object of the present invention is to provide a novel use in the field of hair science related to olfactory receptor 2A4 / 7 agonists.

[0032] Another object of the present invention is to provide a composition for the cosmetic treatment of either aesthetic problems of hair or hair growth disorders.

Summary of the Invention

[0033] According to a general aspect, the present invention provides a novel use in the field of hair science related to a selected olfactory receptor 2A4 / 7 agonist.

[0034] The present invention originates from the study of the role of olfactory receptor OR2A4 / 7 in human pigment cells and the OR2A4 / 7 protein expression in the hair funnel and hair isthmus (ORS directly above the basal layer) of HF in normal human skin in situ.

[0035] The inventors have discovered that cyclohexyl salicylate (CHS), a specific olfactory receptor 2A4 / 7 agonist, promotes the proliferation and differentiation of epidermal keratinocytes and plays an important role in the process of governing specific steps of hair growth.

[0036] In particular, since CHS induces a transient intracellular Ca2+ signal, OR2A4 / 7 is functionally expressed and active in human pigment cells.

[0037] The selected olfactory receptor 2A4 / 7 agonists disclosed herein are suitable for either cosmetic treatment of a population of individuals having aesthetic problems with hair or therapeutic use in the treatment of a population of individuals suffering from hair growth disorders or diseases.

[0038] Accordingly, in a first aspect, the present invention provides a cosmetic use of an olfactory receptor 2A4 / 7 agonist for improving the aesthetic aspects of hair or treating aesthetic defects of hair in the case of non-treatment, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0039] According to one embodiment, when aiming at treating hair thinning, cyclohexyl salicylate is used for cosmetic purposes.

[0040] The topical application of cyclohexyl salicylate stimulates the olfactory receptor (OR) 2A4 / 7, thereby extending human hair growth and promoting physiological hair growth.

[0041] According to another aspect, the present invention relates to a cosmetic use of a composition comprising an olfactory receptor 2A4 / 7 agonist and a physiologically acceptable carrier for improving the aesthetic aspects of hair, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0042] Typically, the composition is a cosmetic composition for topical application to the skin or scalp.

[0043] According to a particular embodiment, the composition for cosmetic use is a cosmetic composition containing cyclohexyl salicylate which can be used for treating cosmetic or aesthetic defects or hair defects such as hair thickening or discoloration.

[0044] The topical application of cyclohexyl salicylate to a subject troubled by hair thinning makes the thinner hair areas of the scalp progress to hypertrophy.

[0045] According to another aspect, the present invention relates to an olfactory receptor 2A4 / 7 agonist for use in the prevention or treatment of hair growth disorders, which is cyclohexyl salicylate.

[0046] Advantageously, cyclohexyl salicylate is suitable for the treatment of hair follicle (HF) disorders and aging that result in hair loss.

[0047] According to another aspect, there is provided herein a pharmaceutical composition for use in the prevention or treatment of hair growth disorders, comprising an olfactory receptor 2A4 / 7 agonist and a pharmaceutically acceptable carrier, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0048] Preferably, the composition for medical use is for topical application. The topically applicable compositions disclosed herein are suitable as an alternative or adjunctive therapeutic agent to drugs for improving hair density and enhancing the aesthetic aspect.

[0049] According to certain embodiments, the composition of the present invention is a pharmaceutical composition.

[0050] Typically, the composition of the present invention contains a cosmetically or pharmaceutically active amount of the olfactory receptor 2A4 / 7 agonist disclosed herein.

[0051] The compositions described herein can be applied to the skin or scalp of an individual for the intended use.

[0052] Certain advantages and features associated with the cosmetic and medical use of the olfactory receptor 2A4 / 7 agonist are further described with reference to the accompanying drawings, which briefly illustrate the content hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0053]

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BRIEF DESCRIPTION OF THE INVENTION

[0054] According to certain aspects of the present invention, the inventors have discovered that selected olfactory receptor 2A4 / 7 agonists are suitable for both cosmetic and therapeutic uses in the field of trichology.

[0055] Applicants have discovered that cyclohexyl salicylate, an olfactory receptor 2A4 / 7 agonist, stimulates the activity of hair follicle cells and dormant hair bulbs on the scalp. These activities activate the hair follicle life cycle, promote hair growth, and improve the aesthetic aspects of hair.

[0056] Cyclohexyl salicylate, a compound also known as cyclohexyl 2-hydroxybenzoate, has the CAS number 25485-88-5.

[0057] Thus, according to a first aspect, the present invention provides a cosmetic / non-therapeutic use of an olfactory receptor 2A4 / 7 agonist for promoting hair growth, or preventing or slowing hair loss, and / or improving the aesthetic aspects of hair, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0058] The compound cyclohexyl salicylate is useful in the treatment of populations having aesthetic hair problems such as non-physiological hair pigmentation, thinning, or breakage of the hair shaft.

[0059] Typically, topical application of cyclohexyl salicylate to the skin or scalp stimulates physiological hair growth and hair thickening.

[0060] Advantageously, the present invention also provides a cosmetic non-therapeutic use of a composition comprising cyclohexyl salicylate and a physiologically acceptable carrier for stimulating physiological hair growth.

[0061] According to a further aspect, the present invention provides an olfactory receptor 2A4 / 7 agonist for use in the prevention or treatment of hair growth disorders or diseases, which is cyclohexyl salicylate.

[0062] Hair growth disorders or diseases that can be treated according to this aspect of the present invention include alopecia, particularly male pattern alopecia and alopecia induced by chemotherapy and telogen effluvium, and disorders suffered by male and female subjects. The compositions described herein are also suitable for the treatment or prevention of female pattern alopecia.

[0063] According to another aspect, a pharmaceutical composition for the medical use defined in claim 8 is provided herein. The pharmaceutical composition of the present invention is effective in preventing and / or treating the disease types of alopecia or hair thinning as described above.

[0064] Examples of hair growth disorders treated by the composition include alopecia and telogen effluvium. Typically, these compositions are formulated as pharmaceutical compositions.

[0065] The amount of cyclohexyl salicylate contained in the composition for cosmetic / medical / nutritional use is an amount such that a prophylactically or therapeutically effective dose is obtained.

[0066] According to certain embodiments, the composition for both cosmetic and medical uses may contain cyclohexyl salicylate in an amount of 0.001 - 20% by weight, 0.05 - 15% by weight, 0.1 - 10% by weight, 1 - 7% by weight.

[0067] In certain embodiments, the composition of the present invention may further comprise one or more additional components having hair growth activity.

[0068] The dosage and frequency of administration of the composition depend on the type and severity of the hair growth condition being treated.

[0069] In certain embodiments, the composition for both cosmetic and therapeutic uses further comprises additional components.

[0070] In some embodiments, the compositions of the invention for both cosmetic and medical uses comprise a physiologically and / or pharmaceutically acceptable carrier, diluent or excipient.

[0071] Typically, the physiologically acceptable carrier of the compositions of the invention is an excipient, carrier or diluent suitable for topical application and / or systemic administration. The physiological and pharmaceutical carriers may be the same carrier.

[0072] Within the scope of this document, the term "carrier" relates to an excipient, carrier, diluent or adjuvant that may be present in the compositions of the invention. Any carrier and / or excipient suitable for the desired form of preparation for administration is contemplated in the use of plant extracts or the active ingredients described herein present therein.

[0073] The term "hair" as used herein encompasses keratinous structures such as scalp, eyelash, eyebrow and beard hair, as well as common nails.

[0074] Typically, suitable carriers are physiologically, edible or pharmaceutically acceptable carriers.

[0075] The term "treatment of a 'hair disorder' or 'aesthetic aspect'" means the treatment of any condition in which there is a change in the physiological state of the hair, particularly a change in the aesthetic aspect of the hair. The compositions of the invention include any composition produced by mixing an association of a compound of formula (I) with a physiologically acceptable carrier. Such compositions are suitable for topical application and / or use in mammals, particularly humans.

[0076] The carrier can take a variety of suitable forms depending on the form of the preparation desired for topical administration.

[0077] Typically, the topical composition is applied in an effective amount to the skin, particularly to the scalp.

[0078] Compositions for topical application can be in solid, semi-solid or fluid form.

[0079] Suitable formulations in solid form include creams, gels, pastes, ointments.

[0080] In other embodiments, the formulation for topical administration is in fluid form, for example, in the form of lotions, gels, shampoos, suspensions, emulsions.

[0081] In the case of formulations in fluid or semi-fluid form, the compound of formula (I) may be diluted in a carrier in the form of a physiologically acceptable liquid such as water, alcohol, hydroalcoholic or glycerin solution, or may be mixed with other liquids suitable for topical application.

[0082] In the form of solutions, suspensions or dispersions, the compositions of the present invention may optionally contain from about 1 to 99.9% of a liquid such as water mixed with alcohol. In some embodiments, water is present in an amount of 5 to 95%. In other embodiments, water is present in an amount of 10 to 90% by weight.

[0083] A typical composition for topical use is a hydroalcoholic solution containing the compound of formula (I) dissolved therein.

[0084] By way of example, the composition of the present invention in liquid form can be prepared by dissolving the active ingredient and the remaining ingredients in alcohol and then recombining the various fractions with stirring. The resulting mixture is then buffered to reach a conveniently selected pH interval between 5 and 7 so as to be compatible with the pH of the scalp, then filtered and packaged in a suitable container such as a bottle or vial.

[0085] In certain embodiments, the composition of the present invention is in the form of a topical lotion.

[0086] Typically, the active compound of formula (I) of the pharmaceutical composition is formulated in dosage units. The dosage unit may contain 0.1 to 100 mg, 1 to 10 mg of the active ingredient per dosage unit for daily administration.

[0087] In some embodiments, the amount effective for formulation depends on the severity of the disorder or condition to be treated.

[0088] According to certain aspects, the present invention relates to a composition for topical use useful for stimulating hair follicles, which is particularly advantageous as a result of the treatment of hair growth disorders such as female alopecia.

[0089] A method of treating a relevant population is also a feature of the present invention.

[0090] According to another aspect of the present invention, there is provided a method of cosmetic treatment comprising applying an effective amount of a composition of the aforementioned type to the scalp.

[0091] For example, when used topically, the composition of the present invention in a cosmetically or pharmaceutically active amount is conveniently applied directly to the scalp once or multiple times a day during a cycle having a duration of 2 to 3 months, alternating with a drug-free period.

[0092] The following examples are provided only to illustrate certain aspects of the present invention.

[0093] Example 1 Lotion Ingredient (INCI name) Amount (w / w%) Denatured alcohol 15.0 - 35.0 PEG - 40 hydrogenated castor oil 0.5 - 6.0 Ethoxydiglycol 0.1 - 2.5 Propanediol 0.1 - 4.5 Dipropylene glycol 0.1 - 2.5 Cyclohexyl salicylate 0.1 - 5.0 30% sodium hydroxide solution 0.001 - 1.0 Citric acid 0.003 - 1.0 Appropriate amount up to 100 g of Aqua

[0094] Example 2 Restructuring and Conditioning Mask Ingredients (INCI name) Amount (w / w%) Propanediol 0.5 - 6.5 Ammonium acryloyldimethyltaurate / VP copolymer 1.0 - 2.0 Cyclohexyl salicylate 0.3 - 7.0 Tocopherol 0.001 - 0.2 Disiroxane 1.0 - 2.0 Fragrance 0.5 - 1.0 Caprylyl glycol 0.25 - 0.75 Phenyltrimethicone 0.25 - 0.75 Silicone quaternium - 17 0.1 - 0.4 Dimethicone 0.1 - 0.4 Dimethiconol 0.025 - 0.075 Laureth - 4 0.1 - 0.4 Inulin 0.1 - 0.4 Tocopheryl acetate 0.1 - 0.3 Laureth - 23 0.1 - 0.3 Potassium sorbate 0.05 - 0.15 Citric acid 0.04 - 0.08 Trisodium ethylenediaminedisuccinate 0.025 - 0.075 Ethylhexyl methoxycinnamate 0.025 - 0.075 Phenoxyethanol 0.25 - 0.75 Appropriate amount up to 100 g of Aqua

[0095] Example 3 Styling Gel Ingredients (INCI name) Amount (w / w%) Cyclohexyl salicylate 0.1 - 10.0 PEG - 40 hydrogenated castor oil 1.0 - 3.0 PPG - 26 - buteth - 26 0.1 - 1.5 Perfume 0.5 - 1.5 Polyacrylate - 14 0.5 - 1.5 Hydroxypropyl guar 0.5 - 1.5 BHA 0.01 - 0.1 Hydrolyzed hydrogenated starch 0.5 - 1.5 EDTA disodium 0.05 - 0.15 Polyquaternium - 11 0.0025 - 0.025 Aminomethylpropanol 0.05 - 6.0 Phenylpropanol 0.05 - 1.0 Propanediol 0.05 - 3.0 Caprylyl glycol 0.05 - 1.0 Sodium benzoate 0.01 - 0.1 30% Sodium hydroxide solution 0.01 - 1.0 Citric acid 0.03 - 1.0 Hydroxyacetophenone 0.05 - 1.0 O - Cymen - 5 - OL 0.05 - 0.1 Purified water q.s. to 100g

[0096] Example 4 Intensive hair conditioner Ingredient (INCI name) Amount (w / w%) Cetyl alcohol 1.0 - 7.0 Glyceryl stearate 0.5 - 28.0 Coconut (Cocos nucifera) oil 1.0 - 5.0 Cetrimonium chloride 2.5 - 7.5 Ceteareth - 20 0.5 - 7.5 Behentrimonium methosulfate 0.1 - 2.5 Stearate PEG - 100 0.1 - 3.5 Betaine 0.1 - 0.35 Xylitol 2.0 - 6.0 Cyclohexyl salicylate 0.1 - 10.0 Hydroxyethyl cellulose 1.0 - 3.0 Isoamyl Laurate 1.0 - 3.0 Benzyl Alcohol 0.1 - 1.0 Panthenol 1.0 - 3.0 Parfum 1 - 2 Bis - Isobutyl PEG / PPG - 20 / 35 / Amodimethicone Copolymer 0.5 - 1.0 Phytantriol 0.5 - 1.0 Panthenol 0.5 - 1.0 Sodium Benzoate 0.01 - 0.5 Sodium Dehydroacetate 0.5 - 1.0 Cetyl Ethylhexanoate 0.5 - 1.0 Butylene Glycol 0.5 - 1.0 Disodium EDTA 0.2 - 0.6 Polysorbate 80 0.2 - 0.6 Hydroxypropyltrimonium Hyaluronate 0.1 - 0.6 Dehydroacetic Acid 0.1 - 0.3 Glucono - δ - Lactone 0.05 - 0.15 30% Sodium Hydroxide Solution 0.01 - 0.5 80% Lactic Acid 0.02 - 1.0 Aqua q.s. to 100g

[0097] Example 5 Revitalizing Shampoo Ingredient (INCI name) Amount (w / w%) Magnesium Lauryl Sulfate 5.0 - 10.0 Sodium Lauroyl Sarcosinate 2.0 - 3.0 Cyclohexyl Salicylate 0.1 - 10.0 Disodium Lauryl Sulfosuccinate 1.5 - 2.5 PEG - 200 Hydrogenated Glyceryl Palmitate 1.0 - 2.0 Cocamid MIPA 0.5 - 1.5 Parfum 0.5 - 1 Glycol Distearate 0.5 - 1 PEG - 7 Glyceryl Cocoate 0.25 - 0.75 Betaine 0.25 - 0.75 Lauryl methyl gluceth - 10 hydroxypropyl dimonium chloride 0.25 - 0.75 Laureth - 7 0.25 - 0.75 Polyquaternium - 10 0.25 - 0.75 Hydroxyacetophenone 0.05 - 1.0 O - cymene - 5 - OL 0.05 - 0.1 Undecylenoyl hydrolyzed wheat protein potassium 0.2 - 0.4 Panthenol 0.1 - 0.3 EDTA disodium 0.1 - 0.3 Phenyltrimethicone 0.05 - 0.15 Silicone quaternium - 17 0.05 - 0.12 Laureth - 4 0.05 - 0.12 Laureth - 23 0.025 - 0.075 Cocamidopropyl betaine sodium 0.025 - 0.075 BHA 0.005 - 0.015 Sodium benzoate 0.1 - 0.5 Potassium sorbate 0.05 - 0.5 30% Sodium hydroxide solution 0.01 - 0.5 80% Lactic acid 0.02 - 1.0 Purified water q.s. to 100 g

[0098] Example 6 Mousse Ingredient (INCI name) Amount (w / w%) Alcohol 10 - 20 Cyclohexyl salicylate 0.1 - 1.2 PEG - 40 hydrogenated castor oil 1 - 2 Glycerin 1 - 1.5 Olive amphoacetate sodium 0.5 - 1.5 Fragrance 0.5 - 1 Tocopherol 0.05 - 0.15 EDTA disodium 0.025 - 0.075 Polyquaternium-16 0.025 - 0.075 Potassium metabisulfite 0.01 - 0.03 Sodium benzoate 0.1 - 0.5 80% Lactic acid 0.02 - 1.0 Appropriate amount up to 100 g of Aqua

[0099] Example 7 Theoretical basis: Hair follicle (HF) disorders and aging lead to hair thinning and hair loss. Affected individuals are troubled by severe psychological stress. Therefore, it is very important to develop a topically applicable formulation that can serve as an alternative or adjunctive therapeutic agent for drugs to improve hair density.

[0100] Human keratinocytes express an OR newly identified as OR2A4 / 7, which showed similarities but also differences in the physiological effects involved after activation by specific agonists (Tsai et al., Exp Dermatol. 2017). In the literature, OR2A4 / 7 expression has been reported in the epidermis of human skin and in primary and immortalized human keratinocytes (HaCaT) (Tsai et al., Exp Dermatol. 2017).

[0101] In this study, the inventors investigated the functional role of the olfactory receptor OR2A4 / 7 in normal human pigment cells after stimulation with the specific ligand cyclohexyl salicylate (CHS).

[0102] The inventors focused on OR2A4 / 7, which promotes the proliferation and differentiation of epidermal keratinocytes. Since CHS was identified as a specific agonist in a screening method, OR2A4 / 7 is no longer an orphan (Tsai et al., Exp Dermatol. 2017). In particular, since CHS induced a transient intracellular Ca2+ signal, OR2A4 / 7 is functionally expressed and active in human pigment cells.

[0103] The inventors herein for the first time show OR2A4 / 7 protein expression in the hair funnel and the isthmus (ORS immediately above the basal layer) of HF in normal human skin in situ.

[0104] Materials and methods: 1.1 In situ hybridization and immunofluorescence staining on fresh scalp frozen immediately after extraction to characterize OR2A4 / 7 mRNA and protein expression in HF For in situ hybridization, normal human full-thickness scalp (temporal / parietal) containing HF was used. Samples were left untreated and frozen directly at D0. Frozen samples were sectioned on a cryostat (Leica), and 7-μm sections were collected. Skin samples were carefully oriented to obtain full-length HF sections. Serial sections of each full-length HF were collected, and the slides were stored at -80 °C (Haslam, I.S., Hardman, J.A. and Paus, R. Topically applied nicotinamide inhibits human hair follicle growth ex vivo. J. Invest. Dermatol. 138, 1420-1422 (2018)). The sections were then post-fixed by immersion in 4% PFA and then washed in PBS (pH 7.4) for 15 min.

[0105] After the washing step, in situ hybridization of the sections was performed. Specially designed probes (Advanced Cell Diagnostics Srl, Milan, Italy) from RNAScope were used.

[0106] Full-length human HF ex vivo organ cultures derived from the scalp unit (occipital region) were used for immunofluorescence staining. The organ cultures were left untreated and directly frozen on the day of isolation (D0). The frozen samples were sectioned with a cryostat (Leica), and 7-μm sections were collected. The skin samples were carefully oriented to obtain full-length HF sections. Serial sections of each full-length HF were collected, and the slides were stored at -80 °C (Haslam, I.S., Hardman, J.A. and Paus, R. Topically applied nicotinamide inhibits human hair follicle growth ex vivo. J. Invest. Dermatol. 138, 1420-1422 (2018)). Sections obtained from the organ cultures were stained using the primary antibodies and staining protocols. Sections without primary antibodies were used as negative controls. Counterstaining to visualize the nuclei was performed using 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Biochemie GmbH, Hamburg, Germany).

[0107] Images of the evaluation areas were taken using Keyence-Biozero 8100 and 9100 microscopes (Keyence Corporation, Osaka, Japan) at default magnifications of 100× or 200×.

[0108] The staining intensity was evaluated in a well-defined reference area by quantitative (immuno)histomorphometry using NIH ImageJ software (NIH, Bethesda, MD, USA) (Fischer TW, et al. Differential effects of caffeine on hair shaft elongation, matrix and outer root sheath keratinocyte proliferation, and transforming growth factor-β2 / insulin-like growth factor-1-mediated regulation of the hair cycle in male and female human hair follicles in vitro. Br. J. Dermatol. 2014;171:1031-1043. doi:10.1111 / bjd.13114.; Samuelov L, et al. P-cadherin regulates human hair growth and cycling via canonical Wnt signaling and transforming growth factor-β2. J. Invest. Dermatol. 2012;132:2332-2341. doi:10.1038 / jid.2012.171.).

[0109] 1.2 Effects on microdissected human HF organ cultures, and hair follicle stem cells and progeny numbers, in the presence of cyclohexyl salicylate (CHS), an OR2A4 / 7 agonist for beauty purposes [by quantitative (immuno)histomorphometry] Human scalp from the temporal and occipital regions was obtained from healthy donors (38 - 69 years old) who had undergone regular cheek rhytidectomy. Scalp samples were processed for HF microdissection (Edelkamp J, Gherardini J, Bertolini M, Methods to study human hair follicle growth ex vivo: human microdissected hair follicle and human full thickness skin organ culture, Methods Mol. Biol. 2154 (2020) 105 - 119). Microdissected human scalp HFs were cultured at 37 °C in minimum nutrient medium of William’s E Media (WEM, Gibco, Life Technologies) supplemented with 2 mM L - glutamine (Gibco), 10 ng / mL hydrocortisone (Sigma - Aldrich), 10 μg / mL insulin (Sigma - Aldrich), and 1% penicillin / streptomycin mix (Gibco) 25, 26, 62 using 5% CO2. After microdissection, HFs were first incubated in WEM for 24 h for re - equilibration. Quality - controlled HFs (fully pigmented and present in telogen VI) were randomly assigned to various experimental groups.

[0110] After 24 h, the WEM medium was changed and the HFs were treated with vehicle or 50 μM CHS for 6 days for (immuno)histomorphometry.

[0111] OCT - embedded samples were sectioned using a Leica cryostat (6 μm thick for HFs and 7 μm thick for skin). Sections obtained from the organ cultures were stained using OR2A4 / 7 and integrin α - 6 antibodies. Double - staining was also performed, and OR2A4 / 7 co - localizes with CD71 only within selected hair follicle compartments, i.e., on the ORS above the dermal papilla and in the hair matrix. Sections without primary antibody were used as negative controls. Counterstaining to visualize nuclei was performed using 4’,6 - diamidino - 2 - phenylindole (DAPI) (Sigma - Aldrich Biochemie GmbH, Hamburg, Germany).

[0112] Images of the evaluation area were captured at a default magnification of 100× or 200× using a Keyence - Biozero 8100 and 9100 microscope (Keyence Corporation, Osaka, Japan).

[0113] Staining intensity was evaluated quantitatively (immunohistomorphometry) (Fischer TW, et al. Differential effects of caffeine on hair shaft elongation, matrix and outer root sheath keratinocyte proliferation, and transforming growth factor - β2 / insulin - like growth factor - 1 - mediated regulation of the hair cycle in male and female human hair follicles in vitro. Br. J. Dermatol. 2014;171:1031 - 1043. doi:10.1111 / bjd.13114.; Samuelov L, et al. P - cadherin regulates human hair growth and cycling via canonical Wnt signaling and transforming growth factor - β2. J. Invest. Dermatol. 2012;132:2332 - 2341. doi:10.1038 / jid.2012.171.) in a well - defined reference area using NIH ImageJ software (NIH, Bethesda, MD, USA).

[0114] 1.3 Effects of CHS on the organ culture of microdissected HF and on the hair cycle and matrix keratinocyte proliferation in the presence of CHS [by quantitative (immuno)histomorphometry and Masson - Fontana staining] Human scalp from the temporal and occipital regions was obtained from healthy donors (38 - 69 years old) who had undergone regular buccal rhytidectomy. Scalp samples were processed for HF microdissection (Edelkamp J, Gherardini J, Bertolini M, Methods to study human hair follicle growth ex vivo: human microdissected hair follicle and human full thickness skin organ culture, Methods Mol. Biol. 2154 (2020) 105 - 119). Microdissected human scalp HFs were cultured at 37°C in minimum essential medium of William’s E Media (WEM, Gibco, Life Technologies) supplemented with 2 mM L - glutamine (Gibco), 10 ng / mL hydrocortisone (Sigma - Aldrich), 10 μg / mL insulin (Sigma - Aldrich), and 1% penicillin / streptomycin mix (Gibco) 25, 26, 62 using 5% CO2. After microdissection, HFs were first incubated in WEM for 24 h for re - equilibration. Quality - controlled HFs (fully pigmented and present in anagen VI stage) were randomly assigned to various experimental groups.

[0115] After 24 h, the WEM medium was replaced and HFs were treated with vehicle or 50 μM CHS for 6 days for (immuno)histomorphometry.

[0116] For histochemical visualization of melanin, Masson - Fontana staining was performed on frozen sections. Melanin was stained as brown dots (Kloepper JE, et al. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Exp. Dermatol. 2010;19:305 - 312. doi:10.1111 / j.1600 - 0625.2009.00939.x.).

[0117] OCT-embedded samples were sectioned using a Leica cryostat (6 μm thick for HF and 7 μm thick for skin). To stain apoptotic and proliferating cells, the ApopTag kit (Merck Milipore) was used according to the manufacturer's protocol, followed by Ki-67 staining (Langan EA, et al. Human hair follicle organ culture: theory, application and perpsectives. Exp. Dermatol. 2015;24:903-911. doi:10.1111 / exd.12836; Kloepper JE, et al. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Exp. Dermatol. 2010;19:305-312. doi:10.1111 / j.1600-0625.2009.00939.x; Ahn SY, et al. Effect of IGF-I on hair growth is related to the anti-apoptotic effect of IGF-I and up-regulation of PDGF-A and PDGF-B. Ann. Dermatol. 2012;24:26-31. doi:10.5021 / ad.2012.24.1.26.; Purba TS, et al. A primer for studying cell cycle dynamics of the human hair follicle. Exp. Dermatol. 2016;25:663-668. doi:10.1111 / exd.13046). After the TdT-enzyme step, the primary antibody was incubated overnight (Ki-67, clone M7240: MIB-1, DAKO, 1 / 20). After the fluorescent-labeled anti-digoxigenin step of the ApopTag kit, the secondary antibody was incubated at room temperature for 45 minutes. Counterstaining with DAPI was performed to visualize the nuclei. Negative controls were performed by omitting the primary antibody.For further analysis, imaging was performed using a Keyence fluorescence microscope BZ9100 (Osaka, Japan) while maintaining a constant exposure time set through imaging.

[0118] HF was evaluated microscopically for hair cycle analysis using Masson-Fontana histochemistry and Ki-67 / TUNEL immunostaining (Langan EA, et al. Human hair follicle organ culture: theory, application and perpsectives. Exp. Dermatol. 2015;24:903-911. doi:10.1111 / exd.12836; Kloepper JE, et al. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Exp. Dermatol. 2010;19:305-312. doi:10.1111 / j.1600-0625.2009.00939.x;). HCS, which consists of the assignment of arbitrary units to each stage of the hair cycle (anagen VI = 100, catagen (early) = 200, catagen (mid) = 300, and catagen (late) = 400), was also measured (Langan EA, et al. Human hair follicle organ culture: theory, application and perpsectives. Exp. Dermatol. 2015;24:903-911. doi:10.1111 / exd.12836; Fischer TW, et al. Differential effects of caffeine on hair shaft elongation, matrix and outer root sheath keratinocyte proliferation, and transforming growth factor-β2 / insulin-like growth factor-1-mediated regulation of the hair cycle in male and female human hair follicles in vitro. Br. J. Dermatol. 2014;171:1031-1043. doi:10.1111 / bjd.13114.).After classifying each HF according to its hair cycle according to the previously defined objective classification criteria for organ-cultured human HF (Kloepper JE, et al. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Exp. Dermatol. 2010;19:305-312. doi;10.1111 / j.1600-0625.2009.00939.x), the average HCS was calculated for each experimental condition. The closer the average value is to 100, the greater the number of HFs in anagen VI in a given group. HCS provides a comprehensive readout parameter that examines and sums all HFs in a given experimental group into a single number, thereby reflecting how close most HFs are to either anagen VI or catagen, and enabling statistical analysis that would not be possible in the hair cycle. Therefore, the hair cycle and HCS are independent readout parameters that complement each other.The staining intensity was evaluated quantitatively (immunohistomorphometrically) (Fischer TW, et al. Differential effects of caffeine on hair shaft elongation, matrix and outer root sheath keratinocyte proliferation, and transforming growth factor-β2 / insulin-like growth factor-1-mediated regulation of the hair cycle in male and female human hair follicles in vitro. Br. J. Dermatol. 2014;171:1031-1043. doi:10.1111 / bjd.13114.; Samuelov L, et al. P-cadherin regulates human hair growth and cycling via canonical Wnt signaling and transforming growth factor-β2. J. Invest. Dermatol. 2012;132:2332-2341. doi:10.1038 / jid.2012.171.) using NIH ImageJ software (NIH, Bethesda, MD, USA) in well-defined reference areas.

[0119] Results: As reported by Tsai et al., Exp Dermatol, 2017, the OR2A4 / 7 protein is expressed in the epidermis of normal skin in situ, confirming the specificity of our immunostaining. We successfully established RNA in situ hybridization for OR2A4 / 7 and showed OR2A4 / 7 RNA expression in the entire epidermis, hair matrix (HM, ORS, and IRS), and ORS keratinocytes within the bulge region of HF in normal skin in situ (Figure 1).

[0120] Indeed, the inventors detected ubiquitous expression of OR2A4 / 7 mRNA in HF epithelium, namely outer root sheath (ORS) and hair matrix (HM), by in situ hybridization (Figure 1a).

[0121] Analysis of freshly embedded human scalp revealed that expression of OR2A4 / 7 protein was mainly restricted to the hair funnel. However, during HF organ culture (OC), OR2A4 / 7 protein became widely expressed in ORS and HM, suggesting upregulation of OR2A4 / 7 expression in the hair follicle under tissue stress conditions (Figure 1b).

[0122] OR2A4 / 7 expression increases under organ culture conditions, and CHS treatment results in downregulation of the receptor in HF hair bulbs ex vivo (Figure 2).

[0123] Therefore, OR2A4 / 7 stimulation significantly increases ex vivo HF stem cell progeny (Figure 3). CHS did not seem to affect K15+ bulge stem cells (Figure 3a), but expression of CD34+ cells (their immediate progeny) increased significantly in the supra-ORS of the hair bulb (Figure 3b). The same was true for the percentage of CD71+ limited proliferating cells (thought to be derived from CD34+ cells) in HM and supra-ORS of the hair bulb (Figure 3c).

[0124] OR25A4 / 7 stimulation also prolongs the anagen phase (Figure 4a) and induces hair matrix keratinocyte proliferation in ex vivo HF (Figure 4b).

Claims

1. A cosmetic use of an olfactory receptor 2A4 / 7 agonist for improving the aesthetic aspect of hair, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate, the cosmetic use.

2. The cosmetic use according to claim 1, wherein the aesthetic aspect is hair thinning.

3. A cosmetic use of a composition comprising an olfactory receptor 2A4 / 7 agonist and a physiologically acceptable carrier for improving the aesthetic aspect of hair, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate, the cosmetic use.

4. The cosmetic use according to claim 3, wherein the aesthetic aspect is hair thinning.

5. The cosmetic use according to any one of claims 1 to 4, wherein the composition is for topical application.

6. An olfactory receptor 2A4 / 7 agonist for use in the prevention or treatment of hair growth disorders, which is cyclohexyl salicylate.

7. The olfactory receptor 2A4 / 7 agonist for use according to claim 6, wherein the hair growth disorder is selected from the group consisting of alopecia, alopecia areata, telogen effluvium, and male pattern baldness, and alopecia areata induced by chemotherapy.

8. A pharmaceutical composition comprising an olfactory receptor 2A4 / 7 agonist and a pharmaceutically acceptable carrier for use in the prevention or treatment of hair growth disorders, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

9. The pharmaceutical composition for use according to claim 8, wherein the composition is for local / topical application.

10. The pharmaceutical composition for use according to claim 8 or 9, wherein the hair growth disorder is selected from the group consisting of alopecia, alopecia areata, telogen effluvium, and male pattern baldness, and alopecia induced by chemotherapy.

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