Olfactory receptor 2A4 / 7 agonists for promoting hair growth and improving aesthetics

Abstract

The present invention relates to the use of a selected olfactory receptor 2A4 / 7 agonist for improving the aesthetics of hair and for preventing or treating hair growth disorders. The olfactory receptor 2A4 / 7 agonist may be formulated as a composition suitable for systemic or topical application to a body region with hair.

Description

Technical Field

[0001] The present invention relates to an olfactory receptor 2A4 / 7 agonist for promoting hair growth and improving hair beauty.

[0002] The present invention originates in the field of cosmetology and trichology.

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

[0004] Hair has a protective function and is considered a skin appendage along with sebaceous glands, sweat glands, and nails.

[0005] The life cycle of a hair bulb consists of three main phases: a growing phase called anagen, a catagen phase called telogen, and a resting phase called telogen. The hair on your scalp is produced by follicles in the mammalian skin organ. Follicles do not produce hair continuously. They go through a growing phase that can last two years or more, then return to a resting phase that can last up to two months before they start growing new hair fibers again.

[0006] The biological phase of this phenomenon lies in the ability of hair bulb stem cells to leave their quiescent state in alternating steps. During the bulb growth and hair production phase, proliferation, differentiation and survival activities predominate, and they are regulated by growth factors. In contrast, the catagen phase is characterized by the activation of molecular pathways that induce apoptosis of hair bulb cells.

[0007] During the growth phase, the dermal papilla produces chemical signals that activate and instruct the bulge stem cells to form the hair matrix by migrating to the base of the hair follicle. Through this "migration", the bulge stem cells form a cellular "pathway" to form the outer root sheath or ORS.

[0008] In response to further signals from the dermal papilla, stromal cells derived from the stem cells proliferate and begin a differentiation process to form the shaft and inner sheath of the hair follicle by moving upward.

[0009] The onset of the catagen phase is characterized by the end of cell proliferation and apoptosis of matrix cells. During catagen, the dermal papilla migrates to the lowest part of the bulge. This close proximity of the papilla to the bulge is thought to be critical for the initiation of another hair growth cycle. This allows the quiescent bulge cells to interact / activate and initiate a new hair growth cycle.

[0010] During the catagen / telogen transition, some cells of the bulge migrate to the papilla and form the hair germ.

[0011] The hair in the resting phase contains a cell group at its base, actually called the hair germ, which is located in close proximity to the dermal papilla. The hair germ is activated to proliferate at the end of the resting phase, even before the bulge, forming a new hair bulb matrix by surrounding the papilla.

[0012] Eyelashes also go through the same three distinct phases in their growth cycle: anagen, catagen, and telogen, but the growth rate and duration of the anagen phase are shorter than those observed for scalp hair.

[0013] Different factors, among which stress, nutritional deficiencies, and aging can negatively impact the life cycle of the hair bulb, leading to a decrease in hair quantity and thinning of hair.

[0014] Hair growth disorders are particularly common in men, and considering the role hair plays in social relationships, hair loss can be a difficult issue for many people to deal with.

[0015] Alopecia is a more widespread hair disease, a disorder of hair growth caused by different factors such as genetics and environment. The degree and pattern of hair loss can vary, but one of the most common forms of hair loss is androgenic alopecia (AGA). In addition to androgenic alopecia, other forms of hair loss include telogen effluvium, alopecia areata, tinea, scarring alopecia, and hair loss caused by excessive cosmetic processing.

[0016] In individuals affected by androgenetic alopecia (AGA), the hair follicles that form at the onset of a new growth phase decrease in size (miniaturize) over time, resulting in the formation of hairs that are smaller than the previous hairs over time. The result is the formation of microhairs.

[0017] Dermatologists subdivide hair loss into scarring and non-scarring categories. Some types of hair loss, such as lichen planus, discoid lupus erythematosus, and graft-versus-host disease, are associated with destruction of the bulge hair follicle stem cells and permanent hair loss. In reversible types of hair loss, such as alopecia areata, inflammation affects the hair follicle progenitor cells but not their stem cells. In these diseases, regeneration occurs upon resolution of inflammation and subsequent regeneration of the hair follicle from undamaged stem cells.

[0018] In individuals with androgenetic alopecia (AGA), over time, the size of the hair follicles that are formed again at the onset of a new growth phase decreases, resulting in the formation of hairs of a smaller diameter than the initial diameter. As a result, the formation of microhairs occurs.

[0019] It has been observed that although the scalp hair follicles shrink, there is still a supply of stem cells that can be converted into progenitor cells, albeit to a lesser extent compared to the scalp under physiological conditions.

[0020] There are many options available for treating hair growth disorders, including alopecia.

[0021] First-line treatment involves topical use and / or administration of formulations for the prevention and treatment of hair growth disorders. Most hair growth formulations available on the market target the hair bulb and act on the metabolism, nourishment, oxygenation and microcirculation of the scalp to improve conditions conducive to physiological hair growth.

[0022] Typically, these hair products include cosmetic ingredients designed to repair the skin and conditions suitable for promoting hair growth.

[0023] A large number of trichological products contain antioxidants such as vitamin E, glycosaminoglycans such as hyaluronic acid and vitamins such as vitamin A and B vitamins or their derivatives such as niacin or nicotinamide adenine dinucleotide (NAD).

[0024] However, in many cases, use of a trichological composition containing the above-mentioned ingredients and other ingredients or application of a nutritional formula to the scalp has been shown to be insufficiently effective in stimulating hair growth.

[0025] Trichological products containing pharmacologically active substances as trichological ingredients have shown superior activity compared to cosmetic / trichological formulations.

[0026] Among these pharmaceutical products, formulations containing minoxidil, a pharmacologically active ingredient with vasodilatory activity, are well known. These products are usually formulated as alcohol solutions for topical use.

[0027] Other products used to treat 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, a hormone that causes hair follicles to shrink or miniaturize, causing baldness when secreted in excess of physiological levels).

[0028] However, the use of minoxidil and finasteride is not without disadvantages. Specifically, topical use of minoxidil may cause local side effects such as rash, local inflammation, and general disadvantages such as headache and hirsutism, while oral finasteride may cause hormonal dysfunction.

[0029] There is a need for an alternative treatment for aesthetic hair problems and hair growth disorders that is effective for topical use and has substantially no side effects.

[0030] One of the purposes of the present invention includes providing a new use of olfactory receptor 2A4 / 7 agonist in the field of trichology.

[0031] Another object of the present invention is to provide a composition for the cosmetic treatment of hair aesthetic problems and / or hair growth disorders. Summary of the invention

[0032] According to one general aspect, the present invention provides novel uses of selected olfactory receptor 2A4 / 7 agonists in the field of trichology.

[0033] The present invention originates from the study of the role of olfactory receptor OR2A4 / 7 in human melanocytes and the in situ expression of OR2A4 / 7 protein in the infundibulum and isthmus (superior basal ORS) of HF in normal human skin.

[0034] The inventors have found that cyclohexyl salicylate (CHS) is a specific olfactory receptor 2A4 / 7 agonist that can promote the proliferation and differentiation of epidermal keratinocytes and play an important role in controlling certain steps of hair growth.

[0035] In particular, OR2A4 / 7 are functionally expressed and active in human melanocytes, as CHS induces a transient intracellular Ca 2+ Signal.

[0036] Selected olfactory receptor 2A4 / 7 agonists disclosed herein are suitable for cosmetic treatment of individual populations with hair aesthetic problems, and for therapeutic use in individual populations affected by hair growth disorders or diseases.

[0037] Thus, in a first aspect, the present invention provides the non-therapeutic, cosmetic use of an olfactory receptor 2A4 / 7 agonist for improving aesthetic aspects of hair, or for treating aesthetic imperfections of hair, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0038] According to one embodiment, the cosmetic use of cyclohexyl salicylate may be for the treatment of thinning hair.

[0039] Topical application of cyclohexyl salicylate stimulates olfactory receptor (OR) 2A4 / 7, thereby prolonging human hair growth and promoting physiological hair growth.

[0040] According to another aspect, the present invention relates to the 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.

[0041] Typically, the composition is a cosmetic composition for topical use on the skin or scalp.

[0042] According to certain embodiments, the composition for cosmetic use is a cosmetic composition containing cyclohexyl salicylate, which can be used to treat cosmetic or aesthetic defects or imperfections of hair, such as hair thickening or hair discoloration.

[0043] Topical application of cyclohexyl salicylate to subjects with thinning hair resulted in gradual thickening of thinning areas of the scalp.

[0044] According to another aspect, the present invention relates to an olfactory receptor 2A4 / 7 agonist for use in preventing or treating hair growth disorders, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0045] Advantageously, cyclohexyl salicylate is useful in the treatment of hair follicle (HF) diseases and hair loss associated with aging.

[0046] According to another aspect, provided herein is a pharmaceutical composition comprising an olfactory receptor 2A4 / 7 agonist and a pharmaceutically acceptable carrier for preventing or treating hair growth disorders, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0047] Preferably, the composition for medical use is for topical use.The topical compositions disclosed herein are suitable as an alternative to drugs or as a therapeutic additive to improve hair density and improve aesthetic aspects.

[0048] According to certain embodiments, the composition of the present invention is a pharmaceutical composition.

[0049] Typically, the compositions of the present invention contain a cosmetically or pharmaceutically active amount of an olfactory receptor 2A4 / 7 agonist disclosed herein.

[0050] The compositions described herein may be applied to the skin or scalp of a subject for the intended use.

[0051] Certain advantages and features associated with the cosmetic and medical uses of olfactory receptor 2A4 / 7 agonists will be further described with reference to the accompanying drawings, which are briefly described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 .A shows images of OR2A4 / 7 mRNA expression in the olfactory epithelium (upper panel), hair follicle (HF) bulb (middle panel), and bulge (lower panel) (pink arrowheads are dark grey in the black and white version). Figure 1 B) Representative images showing OR2A4 / 7 protein expression in the olfactory epithelium (positive control; upper panel) and fresh human scalp epidermis (lower panel). Figure 1 C) shows different HF regions in fresh human scalp skin. n=3 independent donors. CTS: connective tissue sheath; DP: dermal papilla; IRS: inner root sheath; ORS: outer root sheath.

[0053] Figure 2.A) Images showing OR2A4 / 7 (green, light grey in black and white version) protein expression in different regions of isolated organ-cultured HF hair bulbs (left); bar graphs showing quantitative analysis of pooled data for OR2A4 / 7 expression in dermal papilla, hair matrix (germ + pre-cortex) and outer root sheath when treated with vehicle or 50 μM CHS (right). Figure 2 B) Bar graph showing summary data quantitative analysis of OR2A4 / 7 expression in the basal and suprabasal layers of ridges upon treatment with vehicle or 50 μM CHS (left panel); Representative images of ridges following vehicle and 50 μM CHS treatment of HFs showing expression of OR2A4 / 7 (green, white in black and white version) and integrin α-6 (red, light grey in black and white version) (right panel). Nuclei were counterstained with DAPI (blue, dark grey in black and white version). All data are presented as individual normalizations to vehicle-treated controls; mean ± SEM; n = 28-49 HFs from n = 5-8 independent donors, *p < 0.05, ***p < 0.001.

[0054] Figure 3 A) Shows a bar graph showing the elevation of basal K15 in response to vehicle or 50 μM CHS treatment. + Quantitative analysis of stem cells. No differences were observed. B) Representative images of CD34 (red, light grey in black and white version) protein expression in isolated HF (left); bar graph shows CD34 in the basal layer of the outer root sheath (ORS) under vehicle or 50 μM CHS treatment. + Quantification of cells (right). C) Representative images of co-expression of CD71 (red, light grey in black and white version) and OR2A4 / 7 (green, white in black and white version) proteins in isolated HF (left); bar graphs show CD71 in the hair matrix and proximal ORS + Quantitative analysis of cells (middle); bar graphs show the percentage of CD71 / OR2A4 / 7 double positive cells in the proximal outer root sheath (right). Nuclei were counterstained with DAPI (blue, dark grey in black and white version). All data are presented as individual normalizations to vehicle-treated controls; mean ± SEM; n = 36-44 HFs from 8 independent donors, *p < 0.05, *p < 0.01, ***p < 0.001.

[0055] Figure 4.A) shows the percentage of HFs in each hair cycle phase incubated with vehicle or 50 μM CHS. Green arrows (grey in black and white version) indicate the increase in HFs in the growth phase under 50 μM CHS treatment. Panel B) shows representative images of Ki-76 (red, light grey in black and white version) and TUNEL (green) protein expression in isolated HF hair bulbs for analysis of cell proliferation (left); representative images of Masson Fontana staining in isolated HF hair bulbs for melanin staining (right); bar graphs show quantitative analysis of Ki-67 protein expression in hair matrix keratinocytes under vehicle or 50M CHS treatment. Cell nuclei were counterstained with DAPI (blue, dark grey in black and white version). All data are expressed as individual normalization to vehicle-treated control group; mean ± SEM; n = 45-47 HFs from n = 8 independent donors, *p < 0.05, ***p < 0.001. DETAILED DESCRIPTION

[0056] According to certain aspects of the present invention, the inventors have discovered that a selected olfactory receptor 2A4 / 7 agonist is suitable for cosmetic and therapeutic applications in the field of trichology.

[0057] Applicants have discovered that cyclohexyl salicylate is an olfactory receptor 2A4 / 7 agonist that stimulates the activity of scalp hair follicle cells and resting hair bulbs. These activities activate the life cycle of hair follicles, promote hair growth, and improve the aesthetic aspects of hair.

[0058] The compound cyclohexyl salicylate, also known as cyclohexyl 2-hydroxybenzoate, has a CAS number of 25485-88-5.

[0059] Therefore, according to a first aspect, the present invention provides a cosmetic / non-therapeutic use of an olfactory receptor 2A4 / 7 agonist in promoting hair growth or preventing or slowing hair loss and / or improving hair beauty, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0060] The compound cyclohexyl salicylate can be used in the treatment of people with aesthetic hair problems, such as non-physiological hair pigmentation or hair thinning or hair shaft breakage.

[0061] Typically, cyclohexyl salicylate is applied topically to the skin or scalp to stimulate physiological hair growth and hair thickening.

[0062] Advantageously, the present invention also provides for the cosmetic, non-therapeutic use of a composition comprising cyclohexyl salicylate and a physiologically acceptable carrier for stimulating the physiological growth of hair.

[0063] According to another aspect, the present invention provides an olfactory receptor 2A4 / 7 agonist for preventing or treating hair growth disorders or diseases, wherein the olfactory receptor 2A4 / 7 agonist is cyclohexyl salicylate.

[0064] According to this aspect of the invention, hair growth disorders or diseases that may be treated include alopecia, particularly androgenic alopecia and chemotherapy-induced alopecia and telogen effluvium, and diseases affecting both male and female subjects. The compositions described herein are also suitable for treating or preventing female pattern baldness.

[0065] According to another aspect, there is provided herein a pharmaceutical composition for medical use as defined in claim 8. The pharmaceutical composition of the present invention is effective in preventing and / or treating the above-mentioned forms of baldness or hair thinning.

[0066] Examples of hair growth disorders treated by the compositions include alopecia and telogen effluvium.Typically, these compositions are formulated as pharmaceutical compositions.

[0067] The amount of cyclohexyl salicylate contained in the composition for cosmetic / pharmaceutical / nutraceutical use should be such that an effective dose for prevention or treatment is achieved.

[0068] According to certain embodiments, the composition for cosmetic and medical use may contain cyclohexyl salicylate in an amount of 0.001% to 20% by weight, 0.05% to 15% by weight, 0.1% to 10% by weight, 1-7% by weight.

[0069] In certain embodiments, the compositions of the present invention may further include one or more additional trichologically active ingredients.

[0070] The amount of composition applied and the frequency of application will depend on the type and severity of the hair disorder to be treated.

[0071] In certain embodiments, the compositions for cosmetic and therapeutic uses further comprise additional ingredients.

[0072] In some embodiments, the compositions of the present invention for cosmetic and medical use include a physiologically and / or pharmaceutically acceptable carrier, diluent or excipient.

[0073] Typically, the physiologically acceptable carrier of the composition of the present invention is an excipient, carrier or diluent suitable for topical use and / or systemic administration. The physiological carrier and the pharmaceutical carrier may be the same carrier.

[0074] In the present context, the term "carrier" refers to an excipient, carrier, diluent or adjuvant that may be present in the composition of the present invention. In the use of plant extracts or the active ingredients described herein present therein, any carrier and / or excipient suitable for the desired form of administration formulation may be considered.

[0075] As used herein, the term "hair" includes hair of the scalp, eyelashes, eyebrows and beard, as well as generally keratin-based structures such as nails.

[0076] Typically, suitable carriers are physiologically, edible or pharmaceutically acceptable carriers.

[0077] The term "trichological disorder" or "aesthetic" treatment refers to the treatment of any disease in which the physiological condition of hair is changed, especially the aesthetic aspect of hair. The composition of the present invention includes any composition prepared by combining and mixing a compound of formula (I) with a physiologically acceptable carrier. Such compositions are suitable for topical application and / or use in mammals, especially humans.

[0078] The carrier may take a wide variety of suitable forms depending on the form of preparation desired for topical administration.

[0079] Typically, topical compositions are applied to the skin, particularly the scalp in an effective amount.

[0080] Compositions for topical application may be in solid, semisolid or liquid form.

[0081] Suitable formulations in solid form include creams, gels, pastes and ointments.

[0082] In other embodiments, the formulation for topical administration is in liquid form, such as in the form of a lotion, gel, shampoo, suspension, emulsion.

[0083] In the case of liquid or semi-liquid formulations, the compounds of formula (I) may be diluted in a physiologically acceptable carrier in the form of a liquid, such as water, alcohol, hydroalcoholic or glycerol solution, or mixed with other liquids suitable for topical application.

[0084] In the form of a solution, suspension or dispersion, the composition of the present invention may contain about 1-99.9% of a liquid, such as water, optionally mixed with an alcohol. In some embodiments, water is present in an amount between 5-95%. In other embodiments, water is present in an amount between 10-90% by weight.

[0085] A typical composition for topical application is a hydroalcoholic solution containing a compound of formula (I) dissolved therein.

[0086] For example, a composition of the invention in liquid form may be prepared by dissolving the active ingredient and the remaining ingredients in alcohol and then remixing the various components under stirring. The resulting mixture may then be buffered to a conveniently selected pH interval between 5 and 7 to be compatible with the pH of the scalp, then filtered and packaged in a suitable container, such as a bottle or vial.

[0087] In certain embodiments, the compositions of the present invention are in the form of a topical emulsion.

[0088] Typically, the active compound of formula (I) of the pharmaceutical composition is formulated in dosage units. The dosage unit may contain 0.1 mg to 100 mg, 1 mg to 10 mg of active ingredient per dosage unit for daily administration.

[0089] In some embodiments, the effective amount of the formulation will depend on the severity of the disease or condition to be treated.

[0090] According to certain aspects, the present invention is directed to compositions for topical use useful for stimulating hair follicles, thereby facilitating the treatment of hair growth disorders, such as hair loss, particularly female pattern baldness.

[0091] Methods of treating related populations are also a feature of the invention.

[0092] According to another aspect of the present invention, there is provided a method of cosmetic treatment comprising applying to the scalp an effective amount of a composition of the aforementioned type.

[0093] For example, for topical use, a cosmetically or pharmaceutically active amount of the composition of the invention is applied directly to the scalp once or more daily, conveniently for a duration of 2-3 months, alternating with rest periods.

[0094] The following examples are provided merely to illustrate certain aspects of the present invention.

[0095] Example 1

[0096] Lotion

[0097]

[0098]

[0099] Water (up to 100g)

[0100] Example 2 Reconstruction-Conditioning Mask

[0101]

[0102] Water (up to 100 g)

[0103] Example 3 Styling gel

[0104] Water (up to 100 g)

[0105] Example 4 Strengthening Conditioner

[0106]

[0107] Water (up to 100 g)

[0108] Example 5 Repairing and nourishing shampoo

[0109]

[0110] Water (up to 100 g)

[0111] Example 6 mousse

[0112]

[0113] Water (up to 100 g)

[0114] Example 7

[0115] Basic principle:

[0116] Hair follicle (HF) diseases and aging lead to hair thinning and hair loss. Affected individuals suffer from severe psychological stress. Therefore, it is of great importance to develop topical formulations that can serve as an alternative or as a therapeutic additive to drugs used to improve hair density.

[0117] Human keratinocytes express ORs, recently identified as OR2A4 / 7, which show similarities and differences in their physiological effects after activation by specific agonists (Tsai et al., Exp Dermatol. 2017). In the literature, OR2A4 / 7 expression has been reported in human skin epidermis and in primary and immortalized human keratinocytes (HaCaT) (Tsai et al., Exp Dermatol. 2017).

[0118] In this study, the inventors investigated the functional role of the olfactory receptor OR2A4 / 7 in normal human melanocytes after stimulation by the specific ligand cyclohexyl salicylate (CHS).

[0119] The inventors focused on OR2A4 / 7, which promotes proliferation and differentiation of epidermal keratinocytes. OR2A4 / 7 has been de-orphaned because CHS was identified as a specific agonist in a screening method (Tsai et al., Exp Dermatol. 2017). In particular, OR2A4 / 7 is functionally expressed and active in human melanocytes because CHS triggers a transient intracellular Ca 2+ Signal.

[0120] The inventors demonstrated for the first time the in situ expression of OR2A4 / 7 protein in the infundibulum and isthmus (superobasal ORS) of HF in normal human skin.

[0121] Materials and methods:

[0122] 1.1 In situ hybridization and immunofluorescence staining on fresh scalp skin frozen immediately after extraction to characterize OR2A4 / 7 mRNA and protein expression in HF

[0123] Normal human full-thickness scalp skin (temporal / occipital) with HF was used for in situ hybridization. The samples were untreated and directly frozen at D0. The frozen samples were sectioned with a cryostat (Leica) and 7 μm sections were collected. The orientation of the skin samples was carefully adjusted to obtain full-length HF sections. Serial sections of each full-length HF were collected and stored at -80°C (Haslam, IS, Hardman, JA and Paus, R. Topically applied nicotinamide inhibits human hair follicle growth ex vivo. J. Invest. Dermatol. 138, 1420–1422 (2018)). Then, the sections were fixed by immersion in 4% PFA and rinsed in PBS (pH 7.4) for 15 min.

[0124] After a washing step, sections were subjected to in situ hybridization using custom-made probes using RNAScope (Advanced Cell Diagnostics Srl, Milan, Italy).

[0125] Full-length human HF ex vivo organotypic cultures from the hair follicle unit (occipital) were used for immunofluorescence staining. Organotypic cultures were not treated and frozen directly on the day of isolation (D0). Frozen samples were sectioned using a cryostat (Leica) and 7 μm sections were collected. The orientation of the skin samples was carefully adjusted to obtain full-length HF sections. Serial sections of each full-length HF were collected and the sections were stored at -80 °C (Haslam, IS, Hardman, JA and Paus, R. Topically applied nicotinamide inhibits human hair follicle growth exvivo. J. Invest. Dermatol. 138, 1420–1422 (2018)). Sections obtained from organotypic cultures were stained using dedicated antibodies and staining protocols. Sections without primary antibodies were used as negative controls. Counterstaining was performed with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Biochemie GmbH, Hamburg, Germany) to visualize cell nuclei.

[0126] Images of the evaluation areas were taken with Keyence-Biozero 8100 and 9100 microscopes (Keyence Corporation, Osaka, Japan) at an original magnification of 100× or 200×.

[0127] Staining intensity was assessed by quantitative (immuno)histomorphometry in well-defined reference areas 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 Wntsignaling and transforming growth factor-β2. J. Invest. Dermatol. 2012;132:2332–2341. doi:10.1038 / jid.2012.171.).

[0128] 1.2 Organotypic culture of microdissected human HF and the effects on the number of hair follicle stem cells and progeny in the presence of the cosmetic OR2A4 / 7 agonist cyclohexyl salicylate (CHS) by quantitative (immuno)histoporesis

[0129] Temporal and occipital human scalp skin was obtained from healthy donors (38-69 years old) undergoing routine facelift surgery. Scalp skin samples were processed by 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 HF were cultured at 37°C and 5% CO2 in William's E medium (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 mixture (Gibco) 25,26,62 After microdissection, HFs were first incubated in WEM for 24 h to re-equilibrate. HFs were quality controlled (fully pigmented and at growth phase VI) and then randomly assigned to different experimental groups.

[0130] After 24 h, WEM medium was changed and HF were treated with vehicle or 50 μM CHS for 6 days for (immuno)histomorphometry.

[0131] OCT-embedded samples were sectioned with a Leica cryostat (6 μm thickness for HF and 7 μm for skin). Sections obtained from organ cultures were stained with antibodies against OR2A4 / 7 and integrin α-6. Double staining was also performed, and colocalization of OR2A4 / 7 with CD71 was only present in selected areas of the hair follicle, namely the upper basal ORS and the hair matrix. Sections without primary antibodies were used as negative controls. Counterstaining was performed with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Biochemie GmbH, Hamburg, Germany) to visualize cell nuclei.

[0132] Images of the evaluation areas were taken with Keyence-Biozero 8100 and 9100 microscopes (Keyence Corporation, Osaka, Japan) at an original magnification of 100× or 200×.

[0133] Staining intensity was assessed by quantitative (immuno)histomorphometry in well-defined reference areas 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 Wntsignaling and transforming growth factor-β2. J. Invest. Dermatol. 2012;132:2332–2341. doi:10.1038 / jid.2012.171.).

[0134] 1.3 Organotypic cultures of microdissected HF in the presence of CHS and effects on the hair cycle and proliferation of hair matrix keratinocytes by quantitative (immuno)histomorphometry and Masson Fontana staining

[0135] Temporal and occipital human scalp skin was obtained from healthy donors (38-69 years old) undergoing routine facelift surgery. Scalp skin samples were processed by 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 HF were cultured at 37°C and 5% CO2 in William's E medium (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 mixture (Gibco) 25,26,62 After microdissection, HFs were first incubated in WEM for 24 h to re-equilibrate. HFs were quality controlled (fully pigmented and at growth phase VI) and then randomly assigned to different experimental groups.

[0136] After 24 h, WEM medium was changed and HF were treated with vehicle or 50 μM CHS for 6 days for (immuno)histomorphometry.

[0137] Cryosections were stained with Masson–Fontana for histochemical visualization of melanin, which is 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.).

[0138] OCT-embedded samples were sectioned using a Leica cryostat (6 μm thickness for HF and 7 μm thickness for skin). To stain apoptotic and proliferating cells, we used the Apoptag kit (Merck Milipore) and performed Ki-67 staining according to the manufacturer's protocol (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, M7240 Clone: ​​MIB-1, DAKO, 1 / 20). After the fluorescent anti-Digoxigenin step of the apoptag kit, the secondary antibody was incubated at RT for 45 min. DAPI counterstaining was performed to observe the cell nucleus. Negative control was performed by omitting the primary antibody. Images were captured using a Keyence fluorescence microscope BZ9100 (Osaka, Japan) for further analysis, with a constant exposure time throughout the imaging process.

[0139] HF was assessed microscopically using Masson–Fontana histochemistry and Ki-67 / TUNEL immunostaining for hair cycle staging analysis (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 was also determined (Langan EA et al. Humanhair 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 outerroot 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 in vitro. Br. J. Dermatol. 2014; 171: 1031–1043. doi: 10.1111 / bjd.13114.), which included assigning an arbitrary unit to each phase of the hair cycle (anagen VI = 100; early catagen = 200; mid catagen = 300; late catagen = 400).After each HF had been classified according to its hair cycle phase, the mean HCS was calculated for each experimental condition following previously defined criteria for objective classification of human HFs in organ culture (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 closer the mean is to 100, the greater the number of anagen VI HFs in a given group. HCS provides a global readout parameter that looks at all HFs in a given experimental group and combines them into a single number that reflects how close most HFs are to anagen VI or catagen and allows for statistical analysis that is not possible with hair cycle staging. Therefore, hair cycle staging and HCS are independent readout parameters that complement each other. Staining intensity was assessed by quantitative (immuno)histoporesis in well-defined reference areas using NIH ImageJ software (NIH, Bethesda, MD, USA) (Fischer TW et al. Differential effects of caffeine on hairshaft 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 invitro. 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 Wntsignaling and transforming growth factor-β2. J. Invest. Dermatol. 2012;132:2332–2341. doi:10.1038 / jid.2012.171.).

[0140] result:

[0141] As reported by Tsai et al., Exp Dermatol, 2017, OR2A4 / 7 protein is expressed in situ in the epidermis of normal skin, confirming the specificity of the inventors' immunostaining. The inventors successfully established RNA in situ hybridization for OR2A4 / 7 and showed that OR2A4 / 7 RNA is expressed in situ in the entire epidermis of normal skin, hair bulbs (HM, ORS and IRS), and ORS keratinocytes in the HF bulge area ( Figure 1 ).

[0142] In fact, the inventors detected ubiquitous expression of OR2A4 / 7 mRNA in the HF epithelium, termed the outer root sheath (ORS) and the hair matrix (HM) by in situ hybridization ( Figure 1 a).

[0143] Analysis of freshly embedded human scalp skin revealed that OR2A4 / 7 protein expression was primarily restricted to the infundibulum. However, during HF organ culture (OC), OR2A4 / 7 protein was widely expressed in the ORS and HM, suggesting that OR2A4 / 7 expression is upregulated in hair follicles under conditions of tissue stress ( Figure 1 b).

[0144] OR2A4 / 7 expression was increased under organ culture conditions, and CHS treatment resulted in receptor downregulation in isolated HF hair bulbs ( Figure 2 ).

[0145] Therefore, OR2A4 / 7 stimulation significantly increased the number of HF stem cell progeny in vitro ( Figure 3 ). Although CHS seems to be + The bulge stem cells had no effect ( Figure 3 a), but in the upper basal ORS, CD34 + cells (and their immediate progeny) showed a significant increase in expression ( Figure 3 b) In HM and upper basal ORS, CD71 + Transitional amplifying cells (thought to be derived from CD34 + The same is true for the percentage of cells Figure 3 c).

[0146] OR25A4 / 7 stimulation also prolonged the growth period ( Figure 4 a), and induced the proliferation of hair matrix keratinocytes in isolated HF ( Figure 4 b).

Claims

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

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

3. 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.

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

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 preventing or treating hair growth disorders, wherein the olfactory receptor 2A4 / 7 agonist 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 androgenic alopecia and chemotherapy-induced alopecia.

8. A pharmaceutical composition comprising an olfactory receptor 2A4 / 7 agonist and a pharmaceutically acceptable carrier for preventing or treating 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 site-specific / topical application.

10. A pharmaceutical composition for use according to claim 8 or 9, wherein the growth disorder is selected from the group consisting of alopecia, alopecia areata, telogen effluvium, and androgenic alopecia and chemotherapy-induced alopecia.

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