Peptide having activity of promoting hair growth and inhibiting alopecia and use thereof

Abstract

The invention relates to a peptide capable of promoting hair growth and preventing and inhibiting alopecia and application thereof. The peptide provided by the invention has the following activities: promoting proliferation of hair follicle dermal papilla cells (HFDPC), activating cell proliferation-related signaling proteins and beta-catenin in human dermal papilla cells, enhancing the expression level of growth factor-related genes, enhancing the expression level of beta-catenin downstream genes, reducing the expression level of alopecia protein DKK-1, and promoting the growth of the hair follicle dermal papilla cells. Increasing the expression of keratin in hair outer root sheath cells (HORSC), and increasing the expression of transcription factors involved in cell activation in hair growth stromal cells (GMC); and promoting differentiation of hair follicle stem cells (HFSC).

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Korean Patent Application No. 10-2023-0122858, filed on September 15, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to peptides that have the activity of promoting hair growth and inhibiting hair loss, and their uses. Background Technology

[0004] Hair loss refers to excessive hair loss caused by an extended resting phase in the hair growth cycle or an increase in the number of resting hair follicles. It can also be caused by factors such as stress, nutritional deficiencies, local blood flow disorders, hormones, and genetics, resulting in excessive and irreversible hair loss.

[0005] Besides endogenous alopecia (such as androgenetic alopecia and seborrheic alopecia), extrinsic alopecia caused by external factors (such as ultraviolet radiation or fine particulate matter) is also known. People with alopecia complain strongly of the psychological stress caused by changes in appearance, rather than the loss of hair's function (such as protection against external stimuli).

[0006] As treatments for alopecia, 5α-reductase inhibitors that convert the male hormone testosterone into the androgen dihydrotestosterone (DHT), such as finasteride or dutasteride, have been developed and approved. Minoxidil, a drug that promotes hair growth by stimulating hair follicles and increasing blood flow, has also been approved and used. However, 5α-reductase inhibitors have been reported to cause relatively frequent side effects, such as sexual dysfunction, depression, anxiety, and gynecomastia; moreover, minoxidil has also been reported to cause cardiovascular side effects, such as electrocardiogram abnormalities, tachycardia, and endocarditis; furthermore, minoxidil is known to cause relapse of hair loss upon discontinuation of the drug.

[0007] Peptides are highly biocompatible and, when used as a single active ingredient in pharmaceutical or cosmetic compositions, have fewer side effects compared to extracts containing various components. Furthermore, after exerting their effect of inhibiting hair loss topically, peptides are readily degraded and eliminated by proteolytic enzymes in the body; therefore, unlike conventional therapeutic agents, peptides are less likely to cause concerns about side effects.

[0008] International patent application WO2005082395 discloses a peptide that promotes hair growth by facilitating the transition from the resting phase to the anagen phase of the hair growth cycle. Korean patent number 10-2387764 discloses a dimer peptide derivative that promotes the proliferation of hair follicle cells.

[0009] [Related Technical Documents]

[0010] [Patent Literature]

[0011] WO2005-082395

[0012] Korean Patent No. 10-2387764 Summary of the Invention

[0013] Technical issues

[0014] The inventors have conducted research and developed a peptide with improved activity in promoting hair growth and inhibiting hair loss. This peptide effectively promotes hair growth and reduces side effects. As a result, the inventors confirmed that the novel peptide developed by the inventors promotes the proliferation of human dermal papillary cells (HHFDPC), activates cell proliferation-related signaling proteins in HHFDPC, upregulates the gene expression of growth factors, activates β-catenin, upregulates the expression levels of downstream genes of β-catenin, and downregulates the expression level of the hair loss-inducing protein DKK-1. In addition, experiments have shown that the peptide of the present invention increases the expression of keratin in outer root sheath cells (HORSC), increases the expression of cell activity-related transcription factors MSX2, HOXC13, and FOXN1 in human hair stromal cells (HHGMC), promotes the proliferation of hair follicle stem cells (HFSC), and induces their differentiation, thereby completing the present invention.

[0015] The purpose of this invention is to provide a novel peptide that promotes hair growth and inhibits hair loss.

[0016] Another object of the present invention is to provide a composition for promoting hair growth or inhibiting, preventing or alleviating alopecia, the composition containing the peptide as an active ingredient.

[0017] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of alopecia, the pharmaceutical composition containing the peptide as an active ingredient.

[0018] Another object of the present invention is to provide a cosmetic composition for promoting hair growth or preventing or alleviating alopecia, the cosmetic composition containing the peptide as an active ingredient.

[0019] Another object of the present invention is to provide a method for promoting hair growth or inhibiting, preventing, alleviating or treating alopecia, the method comprising applying the peptide or a composition containing the peptide as an active ingredient to a subject.

[0020] Technical solution

[0021] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0022] Another aspect of the present invention provides a composition for promoting hair growth or inhibiting, preventing or alleviating alopecia, the composition containing a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0023] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of alopecia, the pharmaceutical composition comprising a peptide including the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0024] Another aspect of the present invention provides a cosmetic composition for promoting hair growth or preventing or alleviating hair loss, the cosmetic composition containing a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0025] Another object of the present invention is to provide a method for promoting hair growth or inhibiting, preventing, alleviating or treating alopecia, the method comprising applying a peptide comprising the amino acid sequence of SEQ ID NO:1 or a composition containing the peptide as an active ingredient to a subject.

[0026] The present invention will now be described in detail.

[0027] 1. Peptides and their activities

[0028] According to one aspect of the present invention, the present invention provides a peptide comprising the amino acid sequence shown in SEQ ID NO:1.

[0029] As used in this article, the term "peptide" refers to a linear molecule composed of amino acid residues linked together by peptide bonds.

[0030] The peptide comprising the amino acid sequence SEQ ID NO:1 of the present invention can be used without modification; however, amino acid variants or fragments with different sequences can also be used, provided that such variants or fragments are formed by the deletion, insertion, substitution or combination of amino acid residues within a range that does not affect the inherent activity of the peptide (e.g., the activity of promoting hair growth or inhibiting hair loss).

[0031] The peptides of the present invention can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc., without changing their activity.

[0032] The peptides of the present invention include peptides containing the amino acid sequence of SEQ ID NO:1, peptides containing substantially the same amino acid sequence, variants thereof, or active fragments thereof. "Substantially the same amino acid sequence" means an amino acid sequence having 75% or higher, for example 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, or 99% or higher sequence identity with the amino acid sequence of SEQ ID NO:1. Furthermore, the peptides may further include targeting sequences, tags, labeled residues, or amino acid sequences prepared for a specific purpose of increasing half-life or peptide stability.

[0033] In one embodiment, the peptide of the present invention may be a peptide composed of the amino acid sequence of SEQ ID NO:1.

[0034] The peptides of the present invention can be peptides whose activity is increased by selecting partial regions of the amino acid sequence to induce N-terminal and / or C-terminal modifications. Through such N-terminal and / or C-terminal modifications, the stability of the peptides of the present invention can be significantly improved, and, for example, the half-life of the peptide can be increased after in vivo administration. The term "stability" includes not only in vivo stability that protects the peptides of the present invention from attack by in vivo proteases, but also storage stability (e.g., stability during storage at room temperature).

[0035] N-terminal modification can be a modification in which a protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearoyl, and polyethylene glycol (PEG) is attached to the N-terminus of the peptide. C-terminal modification can be a modification in which a group such as hydroxyl (-OH), amino (-NH2), or azide (-NHNH2) is attached to the C-terminus of the peptide, but is not limited thereto.

[0036] The peptides of the present invention can be prepared by various methods known in the art to which this invention pertains. For example, the peptides of the present invention can be prepared according to chemical synthesis methods known in the art, particularly solid-phase synthesis (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963)) or liquid-phase synthesis (US Patent No. 5,516,891).

[0037] The peptides of this invention have the activity of promoting hair growth or preventing or inhibiting alopecia.

[0038] In one embodiment, the peptide of the present invention promotes the proliferation of follicular dermal papillary cells (HFDPC).

[0039] In one embodiment, the peptide of the present invention promotes the proliferation of hair follicle outer root sheath cells (HORSC).

[0040] In one embodiment, the peptide of the present invention promotes the proliferation of hair follicle stem cells (HFSC).

[0041] In one embodiment, the peptides of the present invention activate Akt or ERK in follicular dermal papillary cells (HFDPC), which are proteins involved in signal transduction.

[0042] In one embodiment, the peptide of the present invention upregulates the expression of growth factors in follicular dermal papillary cells (HFDPC). The growth factors may be one or more selected from the group consisting of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and vascular endothelial growth factor (VEGF).

[0043] In one embodiment, the peptide of the present invention activates β-catenin in follicular dermal papillary cells (HFDPC) to promote its nuclear translocation.

[0044] In one embodiment, the peptide of the present invention upregulates the gene expression of one or more downstream factors of β-catenin selected from the group consisting of LEF-1, c-Myc and cyclin D1 in follicular dermal papillary cells (HFDPC).

[0045] In one embodiment, the peptide of the present invention downregulates the expression level of the hair loss-inducing protein DKK-1 (Dickkopf-1) in dermal papillary cells of the hair follicle (HFDPC). It is known that in hair follicles, dihydrotestosterone (DHT) promotes the expression of DKK-1 (Dickkopf-1), and DHT-induced and generated DKK-1 inhibits the proliferation of keratinocytes in the hair follicle and induces their apoptosis, thereby promoting hair loss (J of Invest Dermatolo, 2008, Feb., 128(2), pp. 262-269). The hair loss-inducing protein DKK-1 is a protein whose expression level is increased by dihydrotestosterone (DHT), and the peptide of the present invention can downregulate the DHT-increased expression level of DKK-1.

[0046] In one embodiment, the peptide of the present invention increases gene expression of one or more proteins selected from the group consisting of Ha3-II, keratin 5, keratin 14 and keratin 19 in human hair outer root sheath cells (HHORSC).

[0047] In one embodiment, the peptide of the present invention increases the expression of one or more transcription factors selected from the group consisting of MSX2, HOXC13 and FOXN1 in human hair stromal cells (HHGMC).

[0048] In one embodiment, the peptide of the present invention downregulates the expression of one or more stem cell markers selected from the group consisting of CD34, CD133, keratin 15 and LHX2 in hair follicle stem cells (HFSC).

[0049] In one embodiment, the peptide of the present invention upregulates the expression of one or more stem cell differentiation markers selected from the group consisting of α-SMA, keratin-75, vimentin, versican, and Nexin-1 in hair follicle stem cells (HFSC).

[0050] Through the above-mentioned activities, the peptides of the present invention can exhibit the effects of promoting hair growth or inhibiting, preventing or alleviating hair loss.

[0051] 2. Compositions for promoting hair growth or for preventing, alleviating or treating alopecia.

[0052] In another aspect of the invention, a method for promoting hair growth or The A composition for the prevention, relief or treatment of alopecia, the composition comprising a peptide including the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0053] In another aspect of the invention, a method for promoting hair growth or The A composition for the prevention, relief or treatment of alopecia, the composition comprising a peptide including the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0054] As described above, the peptide of the present invention, including the amino acid sequence of SEQ ID NO:1, has the activity of promoting hair growth or inhibiting or preventing alopecia.

[0055] In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of alopecia, the pharmaceutical composition comprising a peptide including the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0056] In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of alopecia, the pharmaceutical composition comprising a peptide including the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0057] Peptides including the amino acid sequence of SEQ ID NO:1 as described in the “1. Peptides and their activities” section.

[0058] As used herein, the term "alopecia" refers to a condition or disease in which hair is absent from areas where it should normally be present, or a condition or disease in which the number of hairs is reduced compared to a normal state, due to abnormal hair loss. In this invention, the term "alopecia" may be used interchangeably with the term "hair loss." In one embodiment, hair loss or symptoms of hair loss in this invention include hair loss on the scalp.

[0059] In this invention, hair loss includes, but is not limited to, hereditary androgenetic alopecia (male pattern baldness), female pattern baldness, telogen effluvium, anagen effluvium, alopecia areata, tinea capitis caused by fungal infection, hair loss caused by hypothyroidism or hyperthyroidism, hair loss caused by inflammation (seborrheic dermatitis), hair loss caused by malnutrition, hair loss caused by diabetes, hair loss caused by lupus, hair loss caused by hair formation disorders, hair loss caused by drug administration (anticoagulants, antidepressants, discontinuation of oral contraceptives or chemotherapy), folliculitis alopecia, lichen planus of the hair follicle, hair loss caused by burns or trauma, hair loss caused by ultraviolet radiation, or hair loss caused by contact with airborne fine particulate matter.

[0060] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient promotes the proliferation of follicular dermal papillary cells (HFDPC).

[0061] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient promotes the proliferation of hair follicle outer root sheath cells (HORSC).

[0062] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient promotes the proliferation of hair follicle stem cells (HFSC).

[0063] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient activates Akt or ERK in follicular dermal papillary cells (HFDPC), which are proteins involved in signal transduction.

[0064] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient upregulates the expression of growth factors in follicular dermal papillary cells (HFDPC). The growth factors may be one or more selected from the group consisting of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and vascular endothelial growth factor (VEGF).

[0065] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient activates β-catenin in follicular dermal papillary cells (HFDPC) to promote its nuclear translocation.

[0066] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient upregulates the gene expression of one or more factors selected from the group consisting of LEF-1, c-Myc, and cyclin D1 (all of which are downstream factors of β-catenin) in follicular dermal papillary cells (HFDPC).

[0067] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient downregulates the expression level of DKK-1 (Dickkopf-1), a hair loss inducing protein, in follicular dermal papillary cells (HFDPC). The hair loss inducing protein DKK-1 is a protein whose expression level is increased by dihydrotestosterone (DHT), and the peptide of the present invention can downregulate the DHT-increased expression level of DKK-1.

[0068] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient increases gene expression of one or more proteins selected from the group consisting of Ha3-II, keratin 5, keratin 14 and keratin 19 in human hair follicle outer root sheath cells (HHORSC).

[0069] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient increases the expression of one or more transcription factors selected from the group consisting of MSX2, HOXC13 and FOXN1 in human hair stromal cells (HHGMC).

[0070] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient downregulates the expression of one or more stem cell markers selected from the group consisting of CD34, CD133, keratin 15, and LHX2 in hair follicle stem cells (HFSC).

[0071] In one embodiment, a composition or pharmaceutical composition containing the peptide of the present invention as an active ingredient upregulates the expression of one or more stem cell differentiation markers selected from the group consisting of α-SMA, keratin-75, vimentin, multifunctional proteoglycan, and microtubule connexin-1 in hair follicle stem cells (HFSC).

[0072] Compositions or pharmaceutical compositions containing the peptides of the present invention as active ingredients may include therapeutically effective amounts of the peptides of the present invention.

[0073] The term "therapeutic effective amount" refers to the amount of peptide contained in a composition as an active ingredient sufficient to achieve its activity or efficacy, for example, sufficient to achieve the efficacy of treating or preventing hair loss.

[0074] As used in this article, the term “prevention” means reducing the risk of the development of a disease or condition, and refers to any action that inhibits or delays the onset of a disease by preventing the progression of the disease or one or more of its clinical signs.

[0075] As used in this article, the term "treatment" refers to the relief of a disease or symptom, including any action that improves or beneficially alters the symptoms of a disease by halting or reducing the progression of the disease or one or more of its clinical signs.

[0076] In this invention, preventing or treating hair loss may include eliminating the cause of hair loss or inhibiting the development of hair loss, and may include inhibiting hair loss or promoting hair formation to promote hair growth.

[0077] The compositions or pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers.

[0078] Pharmaceutically acceptable carriers may be those commonly used in formulations, including but not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0079] In addition to the above-mentioned components, the compositions or pharmaceutical compositions of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but are not limited thereto.

[0080] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: Pharmaceutical Science and Practice (19th edition, 1995, Williams & Wilkins).

[0081] The compositions or pharmaceutical compositions of the present invention can be administered via any route suitable for treating alopecia, and can be administered, for example, orally or parenterally, and in the case of parenterally administration, via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical application, transdermal application, etc. Because the compositions or pharmaceutical compositions of the present invention have activity in preventing or treating alopecia, they can also be administered via topical application (e.g., to the scalp).

[0082] The dosage of the composition or pharmaceutical composition may be from 0.0001 μg to 100 mg daily, from 0.001 μg to 100 mg daily, from 0.01 μg to 100 mg daily, from 0.1 μg to 100 mg daily, or from 1.0 μg to 1,000 mg daily, but is not limited thereto, and may be prescribed differently based on factors such as preparation method, administration pattern, age, weight, sex, pathological condition, food intake, administration time, route of administration, excretion rate and patient responsiveness.

[0083] The compositions or pharmaceutical compositions of the present invention can be prepared by methods readily practiced by those skilled in the art, using pharmaceutically acceptable carriers and / or excipients, either by formulation into unit dosage forms or by filling into multi-dose containers. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granules, tablet, or capsule, and may further include dispersants or stabilizers.

[0084] The compositions or pharmaceutical compositions of the present invention can be topical formulations. These topical formulations are suitable for external skin application. When used as topical formulations, the compositions or pharmaceutical compositions of the present invention can be applied to the scalp, particularly to areas of the scalp experiencing hair loss or areas of the scalp where hair growth needs to be promoted. Topical formulations can be in the form of creams, gels, ointments, skin emulsions, skin suspensions, transdermal delivery patches, medicated bandages, lotions, or combinations thereof. As needed, the topical formulations can be appropriately formulated with ingredients commonly used in cosmetic or pharmaceutical topical formulations, such as aqueous components, oily components, powder components, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. Topical formulations may be appropriately formulated with metal chelating agents such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; pharmaceuticals such as caffeine, tannic acid, verapamil, licorice extract, glycyrrhizin, hot water extract of kale fruit, various herbs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts; vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid; and sugars such as glucose, fructose, and trehalose, but are not limited to these.

[0085] In another aspect of the invention, the composition of the invention may be a cosmetic composition for promoting hair growth or preventing or alleviating alopecia, the cosmetic composition containing a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0086] In another aspect of the invention, the composition of the invention can be a cosmetic composition for promoting hair growth or preventing or alleviating alopecia, the cosmetic composition containing a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0087] As used in this article, the term “relief” refers to any action that reduces or alleviates the symptoms of a disease or condition.

[0088] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient promotes the proliferation of follicular dermal papillary cells (HFDPC).

[0089] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient promotes the proliferation of hair follicle outer root sheath cells (HORSC).

[0090] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient promotes the proliferation of hair follicle stem cells (HFSC).

[0091] In one embodiment, a cosmetic composition containing the peptides of the present invention as active ingredients activates Akt or ERK in follicular dermal papillary cells (HFDPCs), which are proteins involved in signal transduction.

[0092] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient upregulates the expression of growth factors in follicular dermal papillary cells (HFDPC). The growth factors may be one or more selected from the group consisting of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and vascular endothelial growth factor (VEGF).

[0093] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient activates β-catenin (HFDPC) in dermal papillary cells of hair follicles to promote their nuclear translocation.

[0094] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient upregulates the gene expression of one or more factors selected from the group consisting of LEF-1, c-Myc, and cyclin D1 (all of which are downstream factors of β-catenin) in follicular dermal papillary cells (HFDPC).

[0095] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient downregulates the expression level of DKK-1 (Dickkopf-1), a hair loss inducing protein, in follicular dermal papillary cells (HFDPC). The hair loss inducing protein DKK-1 is a protein whose expression level is increased by dihydrotestosterone (DHT), and the peptide of the present invention can downregulate the DHT-increased expression level of DKK-1.

[0096] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient increases the expression of one or more genes selected from the group consisting of Ha3-II, keratin 5, keratin 14 and keratin 19 in human hair follicle outer root sheath cells (HHORSC).

[0097] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient increases the expression of one or more transcription factors selected from the group consisting of MSX2, HOXC13 and FOXN1 in human hair stromal cells (HHGMC).

[0098] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient downregulates the expression of one or more stem cell markers selected from the group consisting of CD34, CD133, keratin 15, and LHX2 in hair follicle stem cells (HFSC).

[0099] In one embodiment, a cosmetic combination containing the peptide of the present invention as an active ingredient upregulates the expression of one or more stem cell differentiation markers selected from the group consisting of α-SMA, keratin-75, vimentin, multifunctional proteoglycan, and microtubule connexin-1 in hair follicle stem cells (HFSC).

[0100] Cosmetic compositions can be formulated into any formulation commonly manufactured in the technical field to which this invention pertains, and can be topical formulations. For example, cosmetic compositions can be formulated as solutions, suspensions, emulsions, creams, gels, lotions, lotions, powders, soaps, surfactant-containing cleansing compositions, oils, powder foundations, emulsion foundations, wax-based foundations, or sprays, but are not limited thereto.

[0101] Cosmetic compositions can be prepared in various forms, such as solutions, sol-gels, emulsions, oils, waxes, and aerosols, including but not limited to toners, moisturizing lotions, moisturizing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, masks, sprays, loose powders, hair growth oils, hair lotions, shampoos, hair conditioners, hair sprays, hairsprays, hair oils, and gels.

[0102] The cosmetic compositions of the present invention may contain other additives, such as excipients and carriers, and may be formulated as needed by appropriately incorporating conventional ingredients commonly used in ordinary cosmetic compositions.

[0103] When a cosmetic composition is in the form of an ointment, cream, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc., can be used as carrier components.

[0104] When the cosmetic composition is in powder or spray form, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in the case of sprays, propellants such as chlorofluorocarbons, propane / butane, or dimethyl ether may be additionally included, but are not limited thereto.

[0105] When a cosmetic composition is in the form of a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, examples of which include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glyceryl fatty acid esters, polyethylene glycol, or fatty acid esters of sorbitol.

[0106] When the cosmetic composition is in suspension form, liquid diluents such as water, ethanol, or propylene glycol; suspending agents such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester and polyoxyethylene dehydrated sorbitol ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, tragacanth gum, etc., can be used as carrier components.

[0107] When a cosmetic composition is a cleansing composition containing surfactants, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurate, sarcosinates, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, etc., can be used as carrier components.

[0108] When the cosmetic composition is in the form of a shampoo, the basic ingredients used to formulate the shampoo, such as thickeners, surfactants, viscosity control agents, moisturizers, pH control agents, preservatives, and essential oils, can be mixed with the peptides of this invention. As a thickener, CDE can be used; as a surfactant, anionic surfactant LES and amphoteric surfactant cocobetaine can be used; as a viscosity control agent, polyquaternium salts can be used; as a moisturizer, glycerin can be used; and as a pH control agent, citric acid or sodium hydroxide can be used. As a preservative, grapefruit extract, etc., can be used. In addition, essential oils such as cedarwood oil, peppermint, and rosemary, as well as serine, pentanol, or vitamin E, can be added.

[0109] In addition to the peptides and carrier components of the present invention as active ingredients, the ingredients included in the cosmetic composition may further include, but are not limited to, ingredients commonly used in cosmetic compositions, such as conventional adjuvants, antioxidants, stabilizers, solubilizers, vitamins, pigments or fragrances.

[0110] The peptides of the present invention can be included in the above composition at a concentration of 0.01 μM to 1,000 μM. Specifically, the peptides of the present invention may be included in the above compositions at the following concentrations: 0.01 μM to 1,000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, or 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, or 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, or 1 μM to 200 μM; or 5 μM to 600 μM. μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM or 5 μM to 200 μM, but not limited to these.

[0111] The terms or elements mentioned in section “2. Compositions for promoting hair growth or for preventing, alleviating or treating alopecia” that are the same as those mentioned in section “1. Peptides and their activities” should be understood as described in section “1. Peptides and their activities”.

[0112] 3. Uses of the peptides of the present invention

[0113] In another aspect of the invention, a peptide comprising the amino acid sequence SEQ ID NO:1 is provided for use in promoting hair growth or inhibiting, preventing, alleviating or treating alopecia.

[0114] In another aspect of the invention, a method for promoting hair growth or inhibiting, preventing, alleviating or treating alopecia is provided, the method comprising applying a peptide comprising the amino acid sequence of SEQ ID NO:1 or a composition containing said peptide as an active ingredient to a subject who requires promotion of hair growth or inhibition, prevention, alleviation or treatment of alopecia.

[0115] In this method, application may include, but is not limited to, administration, injection, infusion, inoculation, or application (e.g., to the skin). The skin includes the scalp.

[0116] Subjects are those who need to promote hair growth or inhibit, prevent, alleviate or treat hair loss, and more specifically, they can be mammals such as humans or non-human primates, mice, rats, dogs, cats, horses or cattle.

[0117] In another aspect, the use of a peptide comprising the amino acid sequence SEQ ID NO:1 is provided in the preparation of a medicament or cosmetic for promoting hair growth or inhibiting, preventing, alleviating or treating alopecia.

[0118] In “3. Use of the peptides of the present invention”, any terms or elements that are the same as those mentioned in “1. Peptides and their activities” or “2. Compositions for promoting hair growth or for preventing, alleviating or treating alopecia” shall be understood to be the same as those described in the above-mentioned “1. Peptides and their activities” or “2. Compositions for promoting hair growth or for preventing, alleviating or treating alopecia”.

[0119] Beneficial effects

[0120] The peptides of this invention have activities that promote hair growth, or prevent, inhibit, or treat alopecia. The peptides of this invention promote the proliferation of human follicular dermal papilla cells (HFDPC), follicular outer root sheath cells (HORSC), and follicular stem cells (HFSC), upregulate the expression of growth factors (such as bFGF, KGF, and VEGF) in HFDPC, and downregulate the expression level of DKK-1 (Dickkopf-1), a hair loss-inducing protein. Furthermore, the peptides of this invention increase keratin protein expression in HORSC, increase the expression of transcription factors involved in cell activation in hair follicle stromal cells (HGMC), and promote the differentiation of follicular stem cells (HFSC).

[0121] However, the effects of the present invention are not limited to those described above, and other effects not mentioned will be readily apparent to those skilled in the art from the following description. Attached Figure Description

[0122] Figure 1A and 1B The experimental results shown indicate that treatment of HHFDPC cells with the peptides of the present invention at 10 μM, 50 μM, and 100 μM increased phosphorylation of Akt, a cell proliferation-related signaling molecule, and extracellular regulated kinase (ERK). Figure 1A and 1B In this context, p-Akt and p-ERK represent phosphorylated Akt and ERK proteins, respectively, while t-Akt and t-ERK represent the total expressed Akt and ERK proteins, respectively.

[0123] Figure 2The experimental results shown indicate that treatment of HHFDPC cells with the peptides of this invention at 10 μM, 50 μM, and 100 μM increased the activity of HHFDPC cells, thereby increasing the expression of growth factors such as bFGF, HGF, KGF, and VEGF. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each gene, obtained by calculating the band intensity value of each gene relative to the band intensity value of GAPDH, and normalizing the control to 1.

[0124] Figure 3 The experimental results shown indicate that β-catenin is activated and its nuclear translocation increases when HHFDPC cells are treated with 10 μM, 50 μM, and 100 μM of the peptide of the present invention. The values ​​shown above the bands in the figure are relative values ​​of the β-catenin band intensity, obtained by calculating the β-catenin band intensity value relative to the HDAC1 band intensity value and normalizing the control to 1.

[0125] Figure 4 The experimental results shown indicate that treatment of HHFDPC cells with the peptides of this invention at 10 μM, 50 μM, and 100 μM increased the expression of genes LEF-1, cyclin D1, and c-Myc, which are downstream factors of β-catenin. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each gene, obtained by calculating the band intensity value of each gene relative to the band intensity value of GAPDH, and normalizing the control to 1.

[0126] Figure 5 The experimental results shown indicate that when HHFDPC cells were treated with 10 μM, 50 μM, and 100 μM of the peptides of this invention, the expression of the DHT-induced hair loss protein DKK-1 decreased in a concentration-dependent manner. The values ​​shown above the bands in the figure are relative values ​​of the DKK-1 band intensity, obtained by calculating the DKK-1 band intensity relative to the α-tubulin band intensity and normalizing the control to 1.

[0127] Figure 6 The experimental results shown indicate that treatment of HHORSC cells with the peptides of the present invention at 10 μM, 50 μM, and 100 μM increased cell viability, thereby increasing the gene expression of cytokeratins Ha3-II, keratin 19, keratin 14, and keratin 5. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each gene, obtained by calculating the band intensity value of each gene relative to the band intensity value of GAPDH, and normalizing the control to 1.

[0128] Figure 7The experimental results shown indicate that treatment of HHGMC cells with the peptides of this invention at 10 μM, 50 μM, and 100 μM increased the gene expression of transcription factors MSX2, HOXC13, and FOXN1, which are involved in cell activity. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each gene, obtained by calculating the band intensity value of each gene relative to the band intensity value of GAPDH, and normalizing the control to 1.

[0129] Figure 8 The experimental results shown indicate that treatment of HHFSC cells with the peptides of the present invention at 10 μM, 50 μM, and 100 μM promoted the proliferation of HHFSCs.

[0130] Figure 9 The experimental results shown indicate that when HHFSC cells were treated with 10 μM, 50 μM, and 100 μM of the peptides of this invention, the gene expression of the stem cell markers CD133, keratin 15, and LHX2 in HHFSC cells decreased. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each gene, obtained by calculating the band intensity value of each gene relative to the band intensity value of GAPDH, and normalizing the control to 1.

[0131] Figure 10 The experimental results shown indicate that treatment of HHFSC cells with the peptides of this invention at 10 μM, 50 μM, and 100 μM increased the gene expression of differentiation markers α-SMA, vimentin, multifunctional proteoglycan, and microtubule connexin-1. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each gene, obtained by calculating the band intensity value of each gene relative to the band intensity value of GAPDH, and normalizing the control to 1.

[0132] Figure 11 The experimental results shown indicate that treatment of HHFSC cells with the peptides of this invention at 10 μM, 50 μM, and 100 μM reduced the protein expression of HHFSC stem cell markers CD34, CD133, and keratin 15. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each marker, obtained by calculating the band intensity value of each marker relative to the band intensity value of α-tubulin, with the control normalized to 1.

[0133] Figure 12The experimental results shown indicate that treatment of HHFSC cells with the peptides of this invention at 10 μM, 50 μM, and 100 μM increased the protein expression of differentiation markers α-SMA, keratin 75, and multifunctional proteoglycans. The values ​​shown above the bands in the figure are relative values ​​of the band intensity for each marker, obtained by calculating the band intensity value of each marker relative to the band intensity value of α-tubulin, with the control normalized to 1. Detailed Implementation

[0134] The present invention will now be described in detail through embodiments. However, the following embodiments are intended to specifically illustrate the present invention, and the present invention is not limited to the following embodiments.

[0135] [Example]

[0136] Preparation Example 1: Preparation of Peptides

[0137] Peptides having the amino acid sequence of SEQ ID NO:1 shown in Table 1 were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA). The synthesized peptides were separated and purified by C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). An ACQUITY UPLC BEH300C18 column (2.1 mm × 100 mm, 1.7 μm, Waters, USA) was used as the column.

[0138] [Table 1]

[0139] The efficacy of the prepared peptides was evaluated in the following experimental examples.

[0140] Experimental Example 1: Promoting the proliferation of human follicular dermal papillary cells (HHFDPC)

[0141] The effect of the peptide prepared in Preparation Example 1 on activating proliferation-related signaling molecules in human follicular dermal papillary cells (HHFDPC) was confirmed by Western blotting.

[0142] HHFDPC at 4×10 5Cells were seeded at a density of 1 / 2 well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 1 μM minoxidil (MNX) served as a positive control, and untreated cell cultures served as a control. Cell cultures were further cultured at 37°C for 24 hours. After washing the cultures with PBS, cells were lysed using lysis buffer to obtain cell lysates. BCA quantification of the cell lysates was performed, and equal volumes of protein samples were prepared. The prepared protein samples were subjected to 10% SDS-PAGE electrophoresis. The proteins separated by SDS-PAGE were transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk at room temperature for 30 minutes. Antibodies against phosphorylated AKT and phosphorylated ERK (Cell Signaling, USA) were diluted 1:1000 in 3% BSA and reacted with the membrane at 4°C for 16 hours. The membrane was washed three times for 15 minutes each with 0.1% PBS-T (PBS solution containing 0.1% Tween-20), and then reacted with secondary antibody diluted 1:2000 in 5% skim milk at room temperature for 1 hour. The membrane was washed three times for 15 minutes each with 0.1% PBS-T (PBS containing 0.1% Tween-20) and treated with ECL solution (GE Healthcare, USA). Expression levels were analyzed using an ImageQuant 800 (Cytiva, USA).

[0143] As experimental results, such Figure 1A and 1B As shown, treatment of HHFDPC with peptides from Preparation Example 1 at 10 μM, 50 μM, and 100 μM confirmed an increase in phosphorylation of Akt, a cell proliferation-related signaling molecule, and extracellular signal-regulated kinase (ERK).

[0144] Experimental Example 2: Promoting the Gene Expression of Growth Factors in Human Hair Follicle Dermal Papillary Cells

[0145] The effect of the peptide prepared in Preparation Example 1 on the growth factor expression level of human follicular dermal papillary cells (HHFDPC) was confirmed by RT-PCR.

[0146] HHFDPC at 3×10 5Cells were seeded at a density of 10 cells / well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 1 μM minoxidil (MNX) served as a positive control, and untreated cell cultures served as a control. Cell cultures were further cultured at 37°C for 24 hours. After washing the cultures with PBS, RNA was isolated using 300 μL Easy Blue (Intron, Korea). The isolated RNA was quantified using Nano Drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, Korea). PCR was then performed using the primers and PCR premix shown in Table 2 (Enzymomics, Korea). PCR products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad gel imaging system.

[0147] [Table 2]

[0148] As experimental results, such Figure 2 As shown, it was confirmed that when HHFDPC was treated with peptides from Preparation Example 1 at 10 μM, 50 μM, and 100 μM, the activity of HHFDPC increased, thereby promoting the expression of growth factors (such as bFGF, HGF, KGF, and VEGF).

[0149] Experimental Example 3: Activation of β-catenin in human dermal papillary cells of hair follicles

[0150] The effect of the peptide prepared in Preparation Example 1 on activating β-catenin in human follicular dermal papillary cells (HHFDPC) was confirmed by Western blotting.

[0151] HHFDPC at 4×10 5Cells were seeded at a density of 1 / 2 well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for an additional 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 100 ng / mL rhWnt-3a (recombinant human Wnt-3a protein, R & D Systems (USA, MN)) served as a positive control, and untreated cell cultures served as a control. Cell cultures treated with the above substances were cultured for an additional 24 hours at 37°C. After washing the cell cultures with PBS, nucleoproteins were isolated using a nucleoprotein extraction kit (ThermoScientific, USA) and quantified using BCA to prepare equal volumes of protein samples. The prepared protein samples were subjected to electrophoresis with 10% SDS-PAGE. The proteins separated by SDS-PAGE were transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 30 minutes. Antibodies against β-catenin (Cell Signaling, USA) and HDAC1 (Santa Cruz, USA) were diluted 1:1,000 in 3% BSA and reacted with the membrane at 4°C for 16 hours. The membrane was washed three times for 15 minutes with 0.1% PBS-T (PBS solution containing 0.1% Tween-20), and then reacted with secondary antibody diluted 1:2,000 in 5% skim milk at room temperature for 1 hour. The membrane was washed three times for 15 minutes with 0.1% PBS-T (PBS containing 0.1% Tween-20) and treated with ECL solution (GE Healthcare, USA). Expression levels were analyzed using an ImageQuant 800 (Cytiva, USA).

[0152] As experimental results, such Figure 3 As shown, it was confirmed that when HHFDPC was treated with peptides from Preparation Example 1 at 10 μM, 50 μM, and 100 μM, activation of Wnt / β-catenin was observed. Wnt / β-catenin is known to be an important signaling pathway associated with hair growth, and nuclear translocation of β-catenin was significantly increased due to activation of β-catenin.

[0153] Experiment Example 4: Promoting the expression of downstream genes of β-catenin in human dermal papillary cells of hair follicles

[0154] The effects of the peptide prepared in Preparation Example 1 on the expression levels of lymphocyte enhancer-binding factor 1 (LEF-1), cell cycle protein D1, and c-Myc, which are downstream genes of β-catenin, were confirmed by RT-PCR.

[0155] HHFDPC at 4×10 5 Cells were seeded at a density of 1 / 2 well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for an additional 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 100 ng / mL rhWnt-3a (recombinant human Wnt-3a protein, R & D Systems (USA, MN)) served as a positive control, and untreated cell cultures served as a control; both were cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, RNA was isolated using 300 μL of Easy Blue (Intron, Korea). The isolated RNA was quantified using Nano Drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, Korea). PCR was then performed using the primers and PCR premix shown in Table 3 (Enzymomics, Korea). The PCR products were electrophoresed on a 1.5% agarose gel, and the bands were detected using the Bio-Rad gel imaging system.

[0156] [Table 3]

[0157] As experimental results, such Figure 4 As shown, it was confirmed that when HHFDPC was treated with peptides from Preparation Example 1 at 10 μM, 50 μM, and 100 μM, β-catenin, which affects cell proliferation and differentiation, was activated, thereby increasing the gene expression of downstream factors LEF-1, cyclin D1, and c-Myc.

[0158] Experimental Example 5: Inhibition of the expression of hair loss-inducing protein DKK-1 in human dermal papilla cells.

[0159] It is known that dihydrotestosterone (DHT) promotes the expression of DKK-1 (Dickkopf-1) in hair follicles, and that DHT-induced and generated DKK-1 inhibits the proliferation of keratinocytes in hair follicles and induces their apoptosis, thereby promoting hair loss (J of InvestDermatolo, 2008, Feb., 128(2), pp. 262-269). The effect of the peptide prepared in Preparation Example 1 on the expression level of the hair loss-inducing protein DKK-1 in human follicular dermal papillary cells (HHFDPC) was confirmed by Western blotting.

[0160] HHFDPC at 4×10 5Cells were seeded at a density of 1 / well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for an additional 24 hours. Cell cultures were treated with 10 μM, 50 μM, and 100 μM peptides prepared in Preparation Example 1, respectively, with a positive control using 5 μM finasteride. Simultaneously, the cells were treated with 100 nM DHT, an inducer of the hair loss-inducing protein DKK-1. Cell cultures treated with DHT alone served as a negative control, and untreated cell cultures served as a control. Cell cultures treated with the above substances were cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, the cells were lysed using lysis buffer to obtain cell lysates. The cell lysates were quantified using BCA, and an equal volume of protein samples was prepared. The prepared protein samples were subjected to 10% SDS-PAGE electrophoresis. Proteins separated by SDS-PAGE were transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 30 minutes. Anti-DKK-1 antibody (Cell Signaling, USA) was diluted 1:1,000 in 3% BSA and reacted with the membrane at 4°C for 16 hours. The membrane was washed three times with 0.1% PBS-T (PBS solution of 0.1% Tween-20) for 15 minutes each time, and then reacted with secondary antibody diluted 1:2,000 in 5% skim milk at room temperature for 1 hour. The membrane was washed three times with 0.1% PBS-T (PBS containing 0.1% Tween-20) for 15 minutes each time, treated with ECL solution (GE Healthcare, USA), and expression levels were detected using an ImageQuant 800 (Cytiva, USA).

[0161] As experimental results, such Figure 5 As shown, it was confirmed that DHT treatment induced the expression of DKK-1, a hair loss inducing protein, in HHFDPC cells. Furthermore, when treated with the peptides of Preparation Example 1 at 10 μM, 50 μM, and 100 μM, the expression of the hair loss inducing protein DKK-1 induced by DHT treatment decreased again in a concentration-dependent manner.

[0162] Experimental Example 6: Activation of Human Hair Outer Root Sheath Cells (HHORSC)

[0163] The effect of the peptide prepared in Preparation Example 1 on activating human hair outer root sheath cells (HHORSC) was confirmed by measuring the gene expression of cytokeratin.

[0164] HHORSC at 4×10 5Cells were seeded at a density of 1 / 2 well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 50 nM rhEGF (recombinant human EGF protein) served as a positive control, and untreated cell cultures served as a control. Cell cultures were further cultured at 37°C for 24 hours. After washing the cultures with PBS, RNA was isolated using 300 μL of Leasy Blue (Intron, Korea). The isolated RNA was quantified using Nano Drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, Korea). PCR was then performed using the primers and PCR premix shown in Table 4 (Enzymomics, Korea). PCR products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad gel imaging system.

[0165] [Table 4]

[0166] As experimental results, such Figure 6 As shown, treatment of HHORSCs with peptides from Preparation Example 1 at 10 μM, 50 μM, and 100 μM increased cell activity, thereby increasing gene expression of cytokeratins Ha3-II, keratin 19, keratin 14, and keratin 5, which form the cytoskeleton and contribute to hair formation.

[0167] Experiment 7: Activation of human hair follicle stromal cells (HHGMC)

[0168] The efficacy of the peptide prepared in Preparation Example 1 in activating human hair stromal cells (HHGMCs) was confirmed by measuring the gene expression of transcription factors Msh homeobox 2 (MSX2), homeobox C13 (HOXC13), and Forkhead Box N1 (FOXN1), which are associated with cell activation.

[0169] HHGMC at 4×10 5Cells were seeded at a density of 1 / 2 well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 50 nM rhEGF (recombinant human EGF protein) served as a positive control, and untreated cell cultures served as a control. Cell cultures were further cultured at 37°C for 24 hours. After washing the cultures with PBS, RNA was isolated using 300 μL Leasy Blue (Intron, Korea). The isolated RNA was quantified using Nano Drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, Korea). PCR was then performed using the primers and PCR premix shown in Table 5 (Enzymomics, Korea). PCR products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad gel imaging system.

[0170] [Table 5]

[0171] As experimental results, such Figure 7 As shown, treatment of HHGMCs with peptides from Preparation Example 1 at 10 μM, 50 μM, and 100 μM confirmed that the gene expression of transcription factors MSX2, HOXC13, and FOXN1, which are involved in cell activation, was increased.

[0172] Experiment Example 8: Promoting the proliferation of human hair follicle stem cells (HHFSC)

[0173] The effect of the peptide prepared in Preparation Example 1 on the proliferation of human hair follicle stem cells (HHFSC) was investigated.

[0174] HHFSC at 2×10 3Cells were seeded at a density of 1 / well in 96-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 100 ng / ml rhWnt-3a (recombinant human Wnt-3a protein) served as a positive control, and untreated cell cultures served as a control. Cell cultures were further cultured at 37°C for 72 hours. Next, 10 μL of 5 mg / ml MTT solution was added, and the cells were cultured in a CO2 incubator at 37°C for 4 hours. After removing the medium, 100 μL of DMSO was added, and the mixture was shaken for 10 minutes. Finally, the absorbance at 540 nm was measured using a Molecular Devices (USA, CA) SpectraMax iD3 spectrophotometer.

[0175] As experimental results, such Figure 8 As shown, it was confirmed that when HHFSCs were treated with peptides from Preparation Example 1 at 10 μM, 50 μM, and 100 μM, the proliferation of HHFSCs was increased by up to 53.73% in a concentration-dependent manner compared to the control.

[0176] Experiment Example 9: Reducing the expression of stem cell marker-related genes in human hair follicle stem cells

[0177] The effect of the peptide prepared in Preparation Example 1 on differentiation induction of human hair follicle stem cells (HHFSC) was confirmed by measuring the gene expression of stem cell markers CD133, keratin 15 and LIM homeobox 2 (LHX2).

[0178] HHFSC at 1.5×10 5Cells were seeded at a density of 10 cells / well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 50 ng / mL rhWnt-7b (recombinant human Wnt-7b protein) served as a positive control, and untreated cell cultures served as a control. Cell cultures were further cultured at 37°C for 24 hours. After washing the cultures with PBS, RNA was isolated using 300 μL Easy Blue (Intron, Korea). The isolated RNA was quantified using Nano Drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, Korea). PCR was then performed using the primers and PCR premix shown in Table 6 (Enzymomics, Korea). The PCR products were electrophoresed on a 1.5% agarose gel, and the bands were detected using the Bio-Rad gel imaging system.

[0179] [Table 6]

[0180] As experimental results, such Figure 9 As shown, treatment of HHFSCs with the peptides of Preparation Example 1 at 10 μM, 50 μM, and 100 μM significantly reduced the gene expression of HHFSC stem cell markers CD133, keratin 15, and LHX2 compared to the control. These results indicate that the peptides of Preparation Example 1 induced HHFSC differentiation, thereby reducing the expression of stem cell marker genes.

[0181] Experimental Example 10: Increasing the expression of genes related to stem cell differentiation markers in human hair follicle stem cells

[0182] The differentiation-inducing effect of the peptide prepared in Preparation Example 1 on human hair follicle stem cells (HHFSC) was confirmed by measuring the gene expression of differentiation markers α-SMA, vimentin, multifunctional proteoglycan and microtubule connexin-1.

[0183] HHFSC at 1.5×10 5Cells were seeded at a density of 10 cells / well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 50 ng / mL rhWnt-7b (recombinant human Wnt-7b protein) served as a positive control, and untreated cell cultures served as a control. Cell cultures were further cultured at 37°C for 24 hours. After washing the cultures with PBS, RNA was isolated using 300 μL Easy Blue (Intron, Korea). The isolated RNA was quantified using Nano Drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, Korea). PCR was then performed using the primers and PCR premix shown in Table 7 (Enzymomics, Korea). The PCR products were electrophoresed on a 1.5% agarose gel, and the bands were detected using the Bio-Rad gel imaging system.

[0184] [Table 7]

[0185] As experimental results, such Figure 10 As shown, treatment of HHFSCs with the peptides of Preparation Example 1 at 10 μM, 50 μM, and 100 μM significantly increased the gene expression of differentiation markers α-SMA, vimentin, multifunctional proteoglycans, and microtubule connexin-1 compared to the negative control. These results indicate that the peptides of Preparation Example 1 induced HHFSC differentiation, thereby increasing the expression of differentiation marker genes.

[0186] Experimental Example 11: Reducing the expression of stem cell markers in human hair follicle stem cells

[0187] The effect of the peptide prepared in Preparation Example 1 on the differentiation induction of human hair follicle stem cells (HHFSC) was confirmed by measuring the protein expression of stem cell markers CD34, CD133 and keratin 15.

[0188] HHFSC at 3×10 5Cells were seeded at a density of 10 cells / well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 50 ng / mL rhWnt-7b (recombinant human Wnt-7b protein, R&D Systems (USA, MN)) served as a positive control, and untreated cell cultures served as a control. Cell cultures treated with the above substances were further cultured at 37°C for 72 hours. After washing the cultures with PBS, the cells were lysed using lysis buffer to obtain cell lysates. BCA quantification of the cell lysates was performed, and equal volumes of protein samples were prepared. The prepared protein samples were subjected to 10% SDS-PAGE electrophoresis. The proteins separated by SDS-PAGE were transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk at room temperature for 30 minutes. Antibodies against CD34, CD133, and keratin 13 (Cell Signaling, USA) were diluted 1:1,000 in 3% BSA and reacted with the membrane at 4°C for 16 hours. The membrane was washed three times for 15 minutes each with 0.1% PBS-T (PBS solution containing 0.1% Tween-20), and then reacted with secondary antibody diluted 1:2,000 in 5% skim milk at room temperature for 1 hour. The membrane was washed three times for 15 minutes each with 0.1% PBS-T (PBS containing 0.1% Tween-20) and treated with ECL solution (GE Healthcare, USA). Expression levels were analyzed using an ImageQuant 800 (Cytiva, USA).

[0189] As experimental results, such Figure 11 As shown, treatment of HHFSCs with the peptides of Preparation Example 1 at 10 μM, 50 μM, and 100 μM reduced the protein expression of stem cell markers CD34, CD133, and keratin 15. These results indicate that the peptides of Preparation Example 1 induced HHFSC differentiation, thereby reducing the expression of stem cell marker proteins.

[0190] Experimental Example 12: Increasing the expression of stem cell differentiation markers in human hair follicle stem cells

[0191] The effect of the peptide prepared in Preparation Example 1 on the differentiation induction of human hair follicle stem cells (HHFSC) was confirmed by measuring the protein expression of differentiation markers α-SMA, keratin 75 and multifunctional proteoglycan.

[0192] HHFSC at 3×10 5Cells were seeded at a density of 10 cells / well in 6-well cell culture plates and cultured for 24 hours in complete mesenchymal stem cell medium. The medium was then replaced with serum-free mesenchymal stem cell medium, and the cells were cultured for another 24 hours. Cell cultures were treated with peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively. Cell cultures treated with 50 ng / mL rhWnt-7b (recombinant human Wnt-7b protein, R&D Systems (USA, MN)) served as a positive control, and untreated cell cultures served as a control. Cell cultures treated with the above substances were further cultured at 37°C for 72 hours. After washing the cultures with PBS, the cells were lysed using lysis buffer to obtain cell lysates. BCA quantification of the cell lysates was performed, and equal volumes of protein samples were prepared. The prepared protein samples were subjected to 10% SDS-PAGE electrophoresis. The proteins separated by SDS-PAGE were transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk at room temperature for 30 minutes. Antibodies against α-SMA, keratin 75, and multifunctional proteoglycans (Cell Signaling, USA) were diluted 1:1,000 in 3% BSA and reacted with the membrane at 4°C for 16 hours. The membrane was washed three times for 15 minutes each with 0.1% PBS-T (PBS solution containing 0.1% Tween-20), and then reacted with secondary antibody diluted 1:2,000 in 5% skim milk at room temperature for 1 hour. The membrane was washed three times for 15 minutes each with 0.1% PBS-T (PBS containing 0.1% Tween-20) and treated with ECL solution (GE Healthcare, USA). Expression levels were analyzed using an ImageQuant 800 (Cytiva, USA).

[0193] As experimental results, such Figure 12 As shown, treatment of HHFSCs with the peptides of Preparation Example 1 at 10 μM, 50 μM, and 100 μM significantly increased the protein expression of stem cell differentiation markers α-SMA, keratin 75, and multifunctional proteoglycans. These results indicate that the peptides of Preparation Example 1 induce HHFSC differentiation, thereby increasing the expression of differentiation marker proteins.

[0194] Preparation Example 2: Preparation of Pharmaceutical Compositions

[0195] 2-1. Powder Preparation

[0196] The peptide of this invention: 2 g

[0197] Lactose: 1 g

[0198] The above ingredients are mixed and filled into an airtight bag to prepare a powder.

[0199] Preparation of 2-2 tablets

[0200] The peptide of this invention: 100 mg

[0201] Corn starch: 100 mg

[0202] Lactose: 100 mg

[0203] Magnesium stearate: 2 mg

[0204] The above ingredients are mixed and then compressed into tablets according to conventional tablet preparation methods.

[0205] 2-3. Capsule Preparation

[0206] The peptide of this invention: 100 mg

[0207] Corn starch: 100 mg

[0208] Lactose: 100 mg

[0209] Magnesium stearate: 2 mg

[0210] The above ingredients are mixed and then filled into gelatin capsules according to conventional capsule manufacturing methods to prepare capsules.

[0211] 2-4. Preparation of pills

[0212] The peptide of this invention: 1 g

[0213] Lactose: 1.5 g

[0214] Glycerin: 1 g

[0215] Xylitol: 0.5 g

[0216] Mix the above ingredients and shape them according to conventional methods so that each pill weighs 4 g.

[0217] 2-5. Preparation of Granules

[0218] The peptide of this invention: 150 mg

[0219] Soybean extract: 50 mg

[0220] Glucose: 200 mg

[0221] Starch: 600 mg

[0222] Mix the above ingredients, add 100 mg of 30% ethanol, dry the mixture at 60°C to form granules, and then fill them into bags.

[0223] Preparation Example 3: Preparation of Cosmetic Compositions

[0224] 3-1. Preparation of Cream

[0225] The peptide of this invention: 4.6 parts by weight

[0226] Cetearyl alcohol: 2.8 parts by weight

[0227] Beeswax: 2.6 parts by weight

[0228] Stearic acid: 1.4 parts by weight

[0229] Lipophilic glyceryl monostearate: 2 parts by weight

[0230] PEG-100 stearate: 1 part by weight

[0231] Sorbitan sesquioleate: 1.4 parts by weight

[0232] Jojoba oil: 4 parts by weight

[0233] Squalane: 3.8 parts by weight

[0234] Polysorbate 60: 1.1 parts by weight

[0235] Macadamia nut oil: 2 parts by weight

[0236] Tocopherol acetate: 0.2 parts by weight

[0237] Methyl polysiloxane: 0.4 parts by weight

[0238] Ethyl para-hydroxybenzoate: 0.1 parts by weight

[0239] Propylparaben: 0.1 parts by weight

[0240] EUXYL K-400: 0.1 parts by weight

[0241] 1,3-Butanediol: 7 parts by weight

[0242] Methylparaben: 0.05 parts by weight

[0243] Glycerin: 6 parts by weight

[0244] d-Panthenol: 0.2 parts by weight

[0245] Triethanolamine: 0.2 parts by weight

[0246] pt 41891: 0.2 parts by weight

[0247] p-H2O: 46.05 parts by weight

[0249] 3-2. Preparation of the detergent

[0250] The peptide of this invention: 3.5 parts by weight

[0251] Cetearyl alcohol: 1.6 parts by weight

[0252] Stearic acid: 1.4 parts by weight

[0253] Lipophilic glyceryl monostearate: 1.8 parts by weight

[0254] PEG-100 stearate: 2.6 parts by weight

[0255] Sorbitan sesquioleate: 0.6 parts by weight

[0256] Squalane: 4.8 parts by weight

[0257] Macadamia nut oil: 2 parts by weight

[0258] Jojoba oil: 2 parts by weight

[0259] Tocopheryl acetate: 0.4 parts by weight

[0260] Methyl polysiloxane: 0.2 parts by weight

[0261] Ethyl para-hydroxybenzoate: 0.1 parts by weight

[0262] Propylparaben: 0.1 parts by weight

[0263] 1,3-Butanediol: 4 parts by weight

[0264] Methylparaben: 0.1 parts by weight

[0265] Xanthan gum: 0.1 parts by weight

[0266] Glycerin: 4 parts by weight

[0267] d-Panthenol: 0.15 parts by weight

[0268] Allantoin: 0.1 parts by weight

[0269] Carbomer (2% aq. Sol): 4 parts by weight

[0270] Triethanolamine: 0.15 parts by weight

[0271] Ethanol: 3 parts by weight

[0272] pt 41891: 0.1 parts by weight

[0273] p-H2O: 48.3 parts by weight

[0274] 3-3. Preparation of Toner

[0275] The peptide of this invention: 0.2% by weight

[0276] Ethanol: 10.0% by weight

[0277] Polyoxyethylene dehydrated sorbitan polylaurate: 1.0% by weight

[0278] Methylparaben: 0.2% by weight

[0279] Glycerin: 5.0% by weight

[0280] 1,3-Butanediol: 6.0% by weight

[0281] Fragrance: qs (appropriate amount)

[0282] Colorant: qs

[0283] Purified water: qs

[0284] Total 100

[0285] 3-4. Preparation of Nourishing Water

[0286] The peptide of this invention: 0.1% by weight

[0287] Vaseline: 2.0% by weight

[0288] Sorbitan sesquioleate: 0.8% by weight

[0289] Polyoxyethylene oil-based ether: 1.2% by weight

[0290] Methylparaben: qs

[0291] Propylene glycol: 5.0% by weight

[0292] Ethanol: 3.2% by weight

[0293] Carboxyvinyl polymer: 18.0% by weight

[0294] Colorant: qs

[0295] Fragrance: qs

[0296] Purified water: qs

[0297] Total 100

[0298] 3-5. Preparation of the essence

[0299] The peptide of this invention: 5.0% by weight

[0300] Propylene glycol: 10.0% by weight

[0301] Glycerin: 10.0% by weight

[0302] Sodium hyaluronate aqueous solution (1%): 5.0% by weight

[0303] Ethanol: 3.2% by weight

[0304] Polyoxyethylene hydrogenated castor oil: 1.0% by weight

[0305] Methylparaben: 0.1% by weight

[0306] Fragrance: qs

[0307] Purified water: qs

[0308] Total 100

[0309] 3-6. Preparation of Facial Masks

[0310] The peptide of this invention: 0.5% by weight

[0311] Glycerin: 5.0% by weight

[0312] Propylene glycol: 4.0% by weight

[0313] Polyvinyl alcohol: 15.0% by weight

[0314] Ethanol: 8.0% by weight

[0315] Polyoxyethylene oil-based ether: 1.0% by weight

[0316] Methylparaben: 0.2% by weight

[0317] Fragrance: qs

[0318] Colorant: qs

[0319] Purified water: qs

[0320] Total 100

[0321] In a preferred embodiment, the above composition ratio is prepared by mixing suitable ingredients; however, the ingredients or mixing ratio may be arbitrarily changed according to regional or ethnic preferences, such as target consumer groups, target countries or intended uses.

[0322] Although representative embodiments of the invention have been described above by way of example, the scope of the invention is not limited to the specific embodiments described herein. Various modifications can be made by those skilled in the art within the scope of the claims of this invention.

Claims

1. A peptide comprising the amino acid sequence of SEQ ID NO:

1.

2. A composition for promoting hair growth or inhibiting, preventing or alleviating alopecia, said composition comprising the peptide according to claim 1 as an active ingredient.

3. A pharmaceutical composition for the prevention or treatment of alopecia, said pharmaceutical composition comprising the peptide according to claim 1 as an active ingredient.

4. The pharmaceutical composition of claim 3, wherein the peptide promotes the proliferation of one or more cells selected from the group consisting of dermal papilla cells (HFDPC), outer root sheath cells (HORSC), and follicular stem cells (HFSC).

5. The pharmaceutical composition according to claim 3, wherein the peptide is in follicular dermal papillary cells (HFDPC). (i) Activation of Akt or ERK signaling proteins; (ii) Upregulate the gene expression of one or more factors selected from the group consisting of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF). (iii) Activation of β-catenin; (iv) Upregulate the gene expression of one or more downstream factors of β-catenin selected from the group consisting of LEF-1, cyclin D1, and c-Myc; or (v) Downregulates the expression level of hair loss inducing protein DKK-1 (Dickkopf-1).

6. The pharmaceutical composition of claim 3, wherein the peptide increases the expression of one or more proteins selected from the group consisting of Ha3-II, keratin 19, keratin 14 and keratin 5 in hair follicle outer root sheath cells (HORSC).

7. The pharmaceutical composition of claim 3, wherein the peptide increases the expression of one or more transcription factors selected from the group consisting of MSX2, HOXC13 and FOXN1 in germinal stromal cells (HGMC).

8. The pharmaceutical composition according to claim 3, wherein the peptide is in hair follicle stem cells (HFSC), (i) Downregulation of one or more stem cell markers selected from the group consisting of CD34, CD133, keratin 15, and LHX2; or (ii) Upregulate the expression of one or more stem cell differentiation markers selected from the group consisting of α-SMA, keratin 75, vimentin, multifunctional proteoglycan and microtubule connexin-1.

9. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is a topical formulation.

10. A cosmetic composition for promoting hair growth or preventing or alleviating alopecia, said cosmetic composition comprising the peptide according to claim 1 as an active ingredient.

11. The cosmetic composition of claim 10, wherein the peptide promotes the proliferation of one or more cells selected from the group consisting of dermal papilla cells (HFDPC), outer root sheath cells (HORSC), and follicular stem cells (HFSC).

12. The cosmetic composition of claim 10, wherein the peptide is in follicular dermal papillary cells (HFDPC), (i) Activation of Akt or ERK signaling proteins; (ii) Upregulate the gene expression of one or more factors selected from the group consisting of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF). (iii) Activation of β-catenin; (iv) Upregulate the expression of one or more downstream factors of β-catenin selected from the group consisting of LEF-1, c-Myc, and cyclin D1; or (v) Downregulates the expression level of hair loss inducing protein DKK-1 (Dickkopf-1).

13. The cosmetic composition of claim 10, wherein the peptide increases the expression of one or more proteins selected from the group consisting of Ha3-II, keratin 5, keratin 14 and keratin 19 in the outer root sheath cells (HORSC) of hair.

14. The cosmetic composition of claim 10, wherein the peptide increases the expression of one or more transcription factors selected from the group consisting of MSX2, HOXC13 and FOXN1 in germinal stromal cells (HGMC).

15. The cosmetic composition of claim 10, wherein the peptide is in hair follicle stem cells (HFSC), (i) Downregulation of one or more stem cell markers selected from the group consisting of CD34, CD133, keratin 15, and LHX2; or (ii) Upregulate the expression of one or more stem cell differentiation markers selected from the group consisting of α-SMA, keratin 75, vimentin, multifunctional proteoglycan and microtubule connexin-1.

16. The cosmetic composition of claim 10, wherein the cosmetic composition is a topical formulation.

17. The cosmetic composition according to claim 10, wherein the cosmetic composition has a dosage form selected from the group consisting of solutions, suspensions, emulsions, ointments, gels, creams, lotions, powders, soaps, surfactant-containing cleansing compositions, oils, powder foundations, emulsion foundations, wax-based foundations, and sprays.

18. The cosmetic composition according to claim 10, wherein the cosmetic composition has a dosage form selected from the group consisting of hair growth oil, hair lotion, shampoo, hair conditioner, hair spray, hairspray, hair gel, hair oil and gel.

19. A method for promoting hair growth or inhibiting, preventing, alleviating or treating alopecia, the method comprising applying a peptide comprising the amino acid sequence of SEQ ID NO:1 or a composition containing said peptide as an active ingredient to a subject who requires promotion of hair growth or inhibition, prevention, alleviation or treatment of alopecia.

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