Peptide having activities of promoting hair growth and inhibiting hair loss, and uses thereof

Abstract

The present invention relates to a peptide having the activities of promoting hair growth, and preventing and inhibiting alopecia, and uses thereof. The peptide of the present invention has the activities of: promoting the 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 levels of growth factor-related genes, enhancing the expression levels of beta-catenin downstream genes, downregulating the expression level of hair loss protein DKK-1, increasing the expression of keratin proteins in hair outer root sheath cells (HORSC), and increasing the expression of transcription factors involved in cell activation in germinal matrix cells (GMC); and promoting the differentiation of hair follicle stem cells (HFSC).

Description

Peptide having hair growth promotion and hair loss inhibition activity and use thereof

[0001] [Cross-citation with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0122858, filed September 15, 2023, the entire contents of which are incorporated herein by reference.

[0003] [Technical Field]

[0004] The present invention relates to a peptide having hair growth promotion and hair loss inhibition activity and its use.

[0005] Alopecia is a condition in which excessive hair loss occurs due to a longer than normal resting phase in the hair growth cycle or an increase in the number of hair follicles in the resting phase. It refers to hair that falls out excessively and does not recover due to stress, malnutrition, local blood flow disorders, hormones, and genetic factors.

[0006] Hair loss is known to be caused by intrinsic factors such as androgenic alopecia and seborrheic alopecia, as well as extrinsic factors such as ultraviolet rays and fine dust. Patients with hair loss often complain more about the psychological stress caused by external changes than about the functional loss of hair, which serves as a defense against external stimuli.

[0007] As hair loss treatments, drugs such as finasteride or dutasteride, which act as 5-alpha reductase inhibitors that convert testosterone, a male hormone, into dihydrotestosterone (DHT), an androgen sex hormone, have been developed and approved for sale. Minoxidil, a drug that stimulates hair follicles and increases blood flow to promote hair growth, has also been approved and used. However, side effects such as sexual dysfunction, depression, anxiety, and breast enlargement have been reported to occur relatively frequently with 5-alpha reductase inhibitors, and cardiovascular side effects such as electrocardiogram abnormalities, tachycardia, and endocarditis have also been reported with minoxidil. In addition, it is known that hair loss will start again if minoxidil is discontinued.

[0008] Peptides have high biocompatibility, and when used as a single active ingredient in drugs or cosmetics, they have fewer side effects than extracts containing various ingredients. In addition, after exerting a local hair loss suppression effect, they can be easily broken down and eliminated by the body's decomposing proteins, so there are fewer concerns about side effects compared to existing treatments.

[0009] PCT International Patent No. WO2005-082395 discloses a peptide that has the effect of promoting hair growth by promoting the progression from the resting phase to the growth phase in the hair growth cycle, and Korean Patent No. 10-2387764 discloses a dimeric peptide derivative that has the effect of promoting the proliferation of hair follicle cells.

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] WO 2005-082395

[0013] Republic of Korea Patent No. 10-2387764

[0014] The present inventors have conducted research efforts to develop a peptide with improved activity that is effective in promoting hair growth and inhibiting hair loss with fewer side effects. As a result, the novel peptide developed by the present inventors was confirmed to promote the proliferation of human dermal papilla cells (HHFDPCs), activate cell proliferation-related signaling proteins in human dermal papilla cells, upregulate the gene expression of growth factors, activate beta-catenin, upregulate the expression level of beta-catenin downstream genes, and downregulate the expression level of hair loss protein DKK-1. In addition, the present invention was completed by experimentally proving that the peptide increases the expression of keratin proteins in outer root sheath cells (HORSCs), increases the expression of cell activity-related transcription factors MSX2, HOXC13, and FOXN1 in hair follicle stem cells (HHGMCs), and has the activity of promoting the proliferation and inducing differentiation of hair follicle stem cells (HFSCs).

[0015] Therefore, the purpose of the present invention is to provide a novel peptide having hair growth promotion and hair loss inhibition activity.

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

[0017] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating alopecia, which contains 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 improving alopecia, which contains 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, improving or treating alopecia, comprising a step of applying the peptide or a composition containing the peptide as an active ingredient to a subject.

[0020] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0021] Another aspect of the present invention provides a composition for promoting hair growth or inhibiting, preventing or improving alopecia, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0022] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating alopecia, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0023] Another aspect of the present invention provides a cosmetic composition for promoting hair growth or preventing or improving alopecia, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0024] Another aspect of the present invention provides a method for promoting hair growth or inhibiting, preventing, improving or treating alopecia, comprising applying to a subject a peptide comprising an amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide as an active ingredient.

[0025]

[0026] The present invention is described in detail below.

[0027]

[0028] 1. Peptides and their activities

[0029] According to one aspect of the present invention, a peptide comprising an amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0030] The term "peptide" in this specification means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0031] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention may be used without modification, but a variant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination thereof of amino acid residues may be used within a range that does not affect the original activity of the peptide, such as hair growth promotion or alopecia inhibition activity.

[0032] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within a range that does not change its activity.

[0033] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising an amino acid sequence of SEQ ID NO: 1, and a variant or active fragment thereof. The substantially identical amino acid sequence refers to an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more, with the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0034] In one embodiment, the peptide of the present invention may be a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0035] The peptide of the present invention may be modified at the N-terminus and / or C-terminus to select a portion of the amino acid sequence and increase its activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" encompasses not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0036] The above N-terminal modification may be a modification in which a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) is bonded to the N-terminus of the peptide. The above C-terminal modification may be a modification in which a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), or the like is bonded to the C-terminus of the peptide, but is not limited thereto.

[0037] The peptide of the present invention can be prepared by various methods widely known in the art to which the present invention pertains. For example, the peptide of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis techniques (US Patent No. 5,516,891).

[0038] The peptide of the present invention has an activity of promoting hair growth or preventing or suppressing alopecia.

[0039] In one embodiment, the peptide of the present invention promotes proliferation of hair follicle dermal papilla cells (HFDPC).

[0040] In one embodiment, the peptide of the present invention promotes proliferation of hair outer root sheath cells (HORSCs).

[0041] In one embodiment, the peptide of the present invention promotes proliferation of hair follicle stem cells (HFSCs).

[0042] In one embodiment, the peptide of the present invention activates the protein Akt or ERK, which is involved in signal transduction in hair follicle papilla cells (HFDPCs).

[0043] In one embodiment, the peptide of the present invention upregulates the expression of a growth factor in hair follicle dermal papilla cells (HFDPCs). The growth factor 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).

[0044] In one embodiment, the peptide of the present invention activates beta-catenin in hair follicle papilla cells (HFDPCs) to promote its translocation to the nucleus.

[0045] In one embodiment, the peptide of the present invention upregulates gene expression of one or more factors selected from the group consisting of LEF-1, c-Myc and Cyclin D1, which are downstream factors of beta-catenin, in hair follicle dermal papilla cells (HFDPCs).

[0046] In one embodiment, the peptide of the present invention down-regulates the expression level of DKK-1 (Dickkopf-1), a hair loss protein, in hair follicle dermal papilla cells (HFDPCs). Dihydrotestosterone (DHT) promotes the expression of DKK-1 (Dickkopf-1) in hair follicles, and DKK-1 produced by DHT is known to promote hair loss by inhibiting the proliferation of keratinocytes in hair follicles and inducing their apoptosis (J of Invest Dermatolo. 2008, Feb., 128(2), pp. 262-269.). The expression level of the hair loss protein DKK-1 is increased by dihydrotestosterone (DHT), and the peptide of the present invention can down-regulate the expression level of DKK-1 increased by DHT.

[0047] In one embodiment, the peptide of the present invention increases the expression of a gene for one or more proteins selected from the group consisting of Ha3-II, Keratin 5, Keratin 14, and Keratin 19 in hair outer root sheath cells (HHORSC).

[0048] 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 hair germinal matrix cells (HHGMC).

[0049] 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 (HFSCs).

[0050] 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, Keratin75, Vimentin, Versican and Nexin-1 in hair follicle stem cells (HFSCs).

[0051] The peptide of the present invention described above can exhibit the effect of promoting hair growth or inhibiting, preventing, or improving alopecia by having the above-described activity.

[0052]

[0053] 2. Composition for promoting hair growth or preventing, improving or treating alopecia

[0054] In another aspect of the present invention, a composition for promoting hair growth or preventing, improving or treating alopecia is provided, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0055] In another aspect of the present invention, a composition for use in promoting hair growth or preventing, improving or treating alopecia is provided, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0056] The peptide comprising the amino acid sequence of sequence number 1 of the present invention has the activity of promoting hair growth or inhibiting or preventing alopecia as described above.

[0057] In another aspect of the present invention, a pharmaceutical composition for preventing or treating alopecia is provided, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0058] In another aspect of the present invention, a pharmaceutical composition for use in the prevention or treatment of alopecia is provided, comprising a peptide comprising an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0059] The peptide comprising the amino acid sequence of the above sequence number 1 is as described in the above item '1. Peptide and its activity'.

[0060] As used herein, the term "alopecia" refers to a condition or disease in which hair is absent in areas where hair should normally be present, or the number of hairs is reduced compared to the normal state, due to abnormal hair loss. In the present invention, the term "alopecia" may be used interchangeably with the term "hair loss." In one embodiment, hair loss or a symptom of hair loss in the present invention includes hair loss on the scalp.

[0061] In the present 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 growth disorder disease, hair loss caused by drugs (anticoagulants, antidepressants, discontinuation of oral contraceptives, chemotherapy), folliculitis pilaris, lichen planus, hair loss caused by burns or trauma, hair loss caused by ultraviolet rays, or hair loss caused by contact with fine dust in the air.

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

[0063] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient promotes proliferation of hair outer root sheath cells (HORSC).

[0064] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient promotes proliferation of hair follicle stem cells (HFSCs).

[0065] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient activates Akt or ERK, a protein involved in signal transduction in hair follicle dermal papilla cells (HFDPCs).

[0066] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient upregulates the expression of a growth factor in hair follicle dermal papilla cells (HFDPCs). The growth factor 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).

[0067] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient activates beta-catenin in hair follicle papilla cells (HFDPCs) and promotes its migration to the nucleus.

[0068] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient upregulates gene expression of one or more factors selected from the group consisting of LEF-1, c-Myc and Cyclin D1, which are downstream factors of beta-catenin, in hair follicle dermal papilla cells (HFDPCs).

[0069] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient downregulates the expression level of DKK-1 (Dickkopf-1), a hair loss protein, in hair follicle dermal papilla cells (HFDPCs). The expression level of DKK-1, a hair loss protein, is increased by dihydrotestosterone (DHT), and the peptide of the present invention can downregulate the expression level of DKK-1 increased by DHT.

[0070] In one embodiment, a composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient increases the expression of a gene of one or more proteins selected from the group consisting of Ha3-II, Keratin 5, Keratin 14, and Keratin 19 in hair outer root sheath cells (HHORSC).

[0071] In one embodiment, a composition or pharmaceutical composition comprising 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 hair germinal matrix cells (HHGMC).

[0072] In one embodiment, a composition or pharmaceutical composition comprising 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 (HFSCs).

[0073] In one embodiment, a composition or pharmaceutical composition comprising 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, Keratin75, Vimentin, Versican and Nexin-1 in hair follicle stem cells (HFSCs).

[0074] A composition or pharmaceutical composition comprising the peptide of the present invention as an active ingredient may contain a therapeutically effective amount of the peptide of the present invention.

[0075] The above term “therapeutically effective amount” means an amount of the active ingredient peptide contained in the composition sufficient to achieve its activity or efficacy, for example, an amount sufficient to achieve efficacy in treating or preventing alopecia.

[0076] The term "prevention" as used herein means reducing the risk of developing a disease or disorder, and means any action that inhibits or delays the onset of a disease by preventing the disease or one or more of its clinical symptoms from progressing.

[0077] The term "treatment" as used herein means alleviating a disease or disorder, and includes any action that improves or beneficially alters the symptoms of a disease by arresting or reducing the progression of the disease or one or more of its clinical symptoms.

[0078] In the present invention, prevention or treatment of hair loss may be to remove the cause of hair loss or to inhibit the progression of hair loss, or to promote hair growth by inhibiting hair loss or promoting hair formation.

[0079] The composition or pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0080] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0081] The composition or pharmaceutical composition of the present invention may additionally include, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., but is not limited thereto.

[0082] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (19th ed., 1995, Williams & Wilkins).

[0083] The composition or pharmaceutical composition of the present invention may be administered by any suitable route for treating alopecia, for example, orally or parenterally, and in the case of parenteral administration, may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, etc. Since the composition or pharmaceutical composition of the present invention has a preventive or therapeutic activity for alopecia, it may be applied by topical administration, such as application to the scalp.

[0084] The dosage of the composition or pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, and may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0085] The composition or pharmaceutical composition of the present invention can be manufactured in a unit dosage form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersant or stabilizer.

[0086] The composition or pharmaceutical composition of the present invention may be a topical skin preparation. The topical skin preparation is a preparation that can be applied to the outside of the skin and used. When the composition or pharmaceutical composition of the present invention is used as a topical skin preparation, it may be applied to the scalp, specifically, to the scalp where hair loss has occurred or to the scalp where hair growth is to be promoted. The topical skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The topical skin preparation may be appropriately mixed with ingredients commonly used in topical skin preparations such as cosmetics or pharmaceuticals, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or a combination thereof, as needed. The above skin external preparation may also appropriately contain metal sequestrants such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glabridin, hot water extract of the fruit of Calin; various herbal medicines; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0087] In another aspect of the present invention, the composition of the present invention may be a cosmetic composition for promoting hair growth or preventing or improving alopecia, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0088] In another aspect of the present invention, the composition of the present invention may be a cosmetic composition for use in promoting hair growth or preventing or improving alopecia, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0089] As used herein, the term “improvement” means any action that improves or benefits the symptoms of a disease or disorder.

[0090] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient promotes proliferation of hair follicle dermal papilla cells (HFDPC).

[0091] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient promotes proliferation of hair outer root sheath cells (HORSC).

[0092] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient promotes proliferation of hair follicle stem cells (HFSCs).

[0093] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient activates Akt or ERK, a protein involved in signal transduction in hair follicle papilla cells (HFDPCs).

[0094] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient upregulates the expression of a growth factor in hair follicle dermal papilla cells (HFDPCs). The growth factor 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).

[0095] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient activates beta-catenin in hair follicle papilla cells (HFDPC) and promotes its movement to the nucleus.

[0096] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient upregulates gene expression of one or more factors selected from the group consisting of LEF-1, c-Myc, and Cyclin D1, which are downstream factors of beta-catenin, in hair follicle dermal papilla cells (HFDPCs).

[0097] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient downregulates the expression level of DKK-1 (Dickkopf-1), a hair loss protein, in hair follicle dermal papilla cells (HFDPCs). The expression level of DKK-1, a hair loss protein, is increased by dihydrotestosterone (DHT), and the peptide of the present invention can downregulate the expression level of DKK-1 increased by DHT.

[0098] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient increases the expression of a gene of one or more proteins selected from the group consisting of Ha3-II, Keratin 5, Keratin 14, and Keratin 19 in hair outer root sheath cells (HHORSC).

[0099] In one embodiment, a cosmetic composition comprising 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 hair germinal matrix cells (HHGMC).

[0100] In one embodiment, a cosmetic composition comprising 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 (HFSCs).

[0101] In one embodiment, a cosmetic composition comprising 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, Keratin75, Vimentin, Versican and Nexin-1 in hair follicle stem cells (HFSCs).

[0102] The above cosmetic composition may be prepared in any formulation commonly manufactured in the technical field to which the present invention pertains, and may be a topical skin preparation. For example, it may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, or spray, but is not limited thereto.

[0103] The above cosmetic composition can be manufactured in various forms such as a solution, sol-gel, emulsion, oil, wax, aerosol, etc., such as a flexible toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, a powder, a hair tonic, a hair cream, a hair lotion, a hair shampoo, a hair rinse, a hair conditioner, a hair spray, a hair aerosol, a pomade, a gel, etc., but is not limited thereto.

[0104] The cosmetic composition of the present invention may include other additives such as excipients and carriers, and it is possible to apply and mix as much as necessary the usual ingredients mixed in general skin cosmetics.

[0105] When the formulation of the above cosmetic composition is in the form of a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0106] When the formulation of the above cosmetic composition is in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included, but is not limited thereto.

[0107] When the formulation of the above cosmetic composition is in the form of a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0108] When the formulation of the above cosmetic composition is in the form of a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0109] When the formulation of the above cosmetic composition is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0110] When the formulation of the above cosmetic composition is a hair shampoo, base ingredients for forming a shampoo, such as a thickener, a surfactant, a viscosity modifier, a moisturizer, a pH modifier, a preservative, and essential oils, may be mixed with the peptide of the present invention. CDE may be used as the thickener, LES, which is an anionic surfactant, and coco betaine, which is an amphoteric surfactant, polyquat may be used as the viscosity modifier, glycerin may be used as the moisturizer, and citric acid or sodium hydroxide may be used as the pH modifier. Grapefruit extract or the like may be used as the preservative, and in addition, essential oils such as cedarwood, peppermint, and rosemary, as well as silk amino acids, pentaol, or vitamin E may be added.

[0111] The ingredients included in the above cosmetic composition may further include, in addition to the peptide of the present invention and the carrier component as active ingredients, ingredients commonly used in cosmetic compositions, such as conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments or fragrances, but are not limited thereto.

[0112] The peptide of the present invention can be included in the above-described composition at a concentration of 0.01 μM to 1000 μM, specifically, the peptide of the present invention can be included in the above-described composition at a concentration of 0.01 μM to 1000 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, 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, 0.1 μM to 200 μM; It may be included at a concentration of, but is not limited to, 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, 1 μM to 200 μM; 5 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.

[0113] Among the terms or elements mentioned in the above '2. Composition for promoting hair growth or preventing, improving or treating alopecia', those mentioned in the above '1. Peptide and its activity' are understood to be the same as those described in the above '1. Peptide and its activity'.

[0114]

[0115] 3. Uses of the peptide of the present invention

[0116] In another aspect of the present invention, a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for promoting hair growth or inhibiting, preventing, improving or treating alopecia is provided.

[0117] In another aspect of the present invention, a method for promoting hair growth or inhibiting, preventing, improving or treating alopecia is provided, comprising the step of applying a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide as an active ingredient to a subject in need of promoting hair growth or inhibiting, preventing, improving or treating alopecia.

[0118] In the above method, the application may include, but is not limited to, administration, injection, infusion, vaccination, or application (e.g., application to the skin). The skin includes the scalp.

[0119] The above subject refers to a subject in need of hair growth promotion or inhibition, prevention, improvement or treatment of alopecia, and more specifically, may refer to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses and cows.

[0120] In another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament or cosmetic for promoting hair growth or inhibiting, preventing, improving or treating alopecia.

[0121] Among the terms or elements mentioned in the above '3. Use of the peptide of the present invention', those mentioned in the above '1. Peptide and its activity' or '2. Composition for promoting hair growth or preventing, improving or treating alopecia' are understood to be the same as those described in the above '1. Peptide and its activity' or '2. Composition for promoting hair growth or preventing, improving or treating alopecia'.

[0122] The peptide of the present invention has an activity of promoting hair growth or preventing, inhibiting, or treating alopecia. The peptide of the present invention promotes the proliferation of hair follicle dermal papilla cells (HFDPCs), outer root sheath cells (HORSCs), and hair follicle stem cells (HFSCs), upregulates the expression of growth factors such as bFGF, KGF, and VEGF in hair follicle dermal papilla cells (HFDPCs), and downregulates the expression level of the hair loss protein DKK-1 (Dickkopf-1). In addition, it has an activity of increasing the expression of keratin proteins in outer root sheath cells (HORSCs), increasing the expression of cell activation-related transcription factors in hair follicle stem cells (HGMCs), and has an activity of promoting the differentiation of hair follicle stem cells (HFSCs).

[0123] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0124] Figures 1a and 1b are experimental results showing that when the peptide of the present invention was treated in HHFDPC cells at 10 μM, 50 μM, and 100 μM, the phosphorylation of Akt and ERK (extracellular-regulated kinase), which are signaling molecules related to cell proliferation, increased. In Figures 1a and 1b, p-Akt and p-ERK refer to phosphorylated proteins of Akt and ERK, and t-Akt and t-ERK refer to the total expressed proteins of Akt and ERK.

[0125] Figure 2 is an experimental result showing that when the peptide of the present invention was treated in HHFDPC cells at 10 μM, 50 μM, and 100 μM, the activity of HHFDPC increased, thereby increasing the expression of growth factors such as bFGF, HGF, KGF, and VEGF. The numbers above the bands in the figure are relative values ​​of the band intensities of each gene, calculated by comparing the band intensity measurements of each gene to the GAPDH band intensity measurements, with the control set to 1.

[0126] Figure 3 is an experimental result showing that when the peptide of the present invention was treated in HHFDPC cells at 10 μM, 50 μM, and 100 μM, beta-catenin was activated and its movement to the nucleus was increased. The numerical value above the band in the figure is the relative value of the beta-catenin band intensity, calculated by comparing the beta-catenin band intensity measurement value to the HDAC1 band intensity measurement value, with the control being 1.

[0127] Figure 4 is an experimental result showing that when the peptide of the present invention was treated in HHFDPC cells at 10 μM, 50 μM, and 100 μM, the gene expression of LEF-1, Cyclin D1, and c-Myc, which are downstream factors of beta-catenin, increased. The numbers above the bands in the figure are the relative values ​​of the band intensities of each gene, calculated by comparing the band intensity measurements of each gene to the GAPDH band intensity measurements, with the control being 1.

[0128] Figure 5 is an experimental result showing that when the peptide of the present invention was treated in HHFDPC cells at 10 μM, 50 μM, and 100 μM, the expression of DKK-1, a hair loss protein induced by DHT treatment, was reduced in a concentration-dependent manner. The numerical value above the band in the figure is the relative value of the DKK-1 band intensity, calculated by comparing the band intensity measurement value of DKK-1 to the measured α-tubulin band intensity value, with the control being 1.

[0129] Figure 6 is an experimental result showing that when the peptide of the present invention was treated in HHORSC cells at 10 μM, 50 μM, and 100 μM, cell activity increased and gene expression of cytokeratin proteins Ha3-II, Keratin 19, Keratin 14, and Keratin 5 increased. The numbers above the bands in the figure are relative values ​​of the band intensities of each gene, calculated by comparing the band intensity measurements of each gene to the GAPDH band intensity measurements, with the control being 1.

[0130] Figure 7 is an experimental result showing that when the peptide of the present invention was treated in HHGMC cells at 10 μM, 50 μM, and 100 μM, the gene expression of transcription factors MSX2, HOXC13, and FOXN1 involved in cell activity increased. The numbers above the bands in the figure are the relative values ​​of the band intensities of each gene, calculated by comparing the band intensity measurements of each gene to the GAPDH band intensity measurements, with the control set to 1.

[0131] Figure 8 is an experimental result showing that the proliferation of HHFSCs is promoted when the peptide of the present invention is treated to HHFSC cells at 10 μM, 50 μM, and 100 μM.

[0132] Figure 9 is an experimental result showing that when the peptide of the present invention was treated in HHFSC cells at 10 μM, 50 μM, and 100 μM, the gene expression of CD133, Keratin 15, and LHX2, which are stem cell markers of HHFSC, was reduced. The numerical value above the band in the figure is the relative value of the band intensity of each gene, calculated by comparing the band intensity measurement value of each gene to the GAPDH band intensity measurement value, with the control being 1.

[0133] Figure 10 is an experimental result showing that when the peptide of the present invention was treated in HHFSC cells at 10 μM, 50 μM, and 100 μM, the gene expression of HHFSC differentiation markers α-SMA, Vimentin, Versican, and Nexin-1 increased. The numbers above the bands in the figure are the relative values ​​of the band intensities of each gene calculated by comparing the band intensity measurements of each gene to the GAPDH band intensity measurements, and then setting the control to 1.

[0134] Figure 11 is an experimental result showing that when the peptide of the present invention was treated in HHFSC cells at 10 μM, 50 μM, and 100 μM, the expression of CD34, CD133, and Keratin 15 proteins, which are stem cell markers of HHFSC, was reduced. The numbers above the bands in the figure are relative values ​​of the band intensities of each marker, calculated by comparing the band intensity measurements of each marker to the α-tubulin band intensity measurements, with the control being 1.

[0135] Figure 12 is an experimental result showing that when the peptide of the present invention was treated in HHFSC cells at 10 μM, 50 μM, and 100 μM, the expression of HHFSC differentiation markers α-SMA, Keratin75, and Versican proteins increased. The numbers above the bands in the figure are relative values ​​of each marker band intensity, calculated by comparing the band intensity measurement value of each marker to the α-tubulin band intensity measurement value, with the control set to 1.

[0136] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0137]

[0138] [Example]

[0139] Manufacturing Example 1: Manufacturing of peptides

[0140] A peptide having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm Y 100 mm, 1.7 μm, Waters Co, USA).

[0141] Amino acid sequence sequence number DRFF1

[0142]

[0143] The efficacy of the peptides manufactured in the following experimental examples was evaluated.

[0144]

[0145] Experimental Example 1: Promoting the proliferation of human hair follicle papilla cells (HHFDPCs).

[0146] The effect of the peptide manufactured in the above Manufacturing Example 1 on the activation of proliferation-related signaling molecules in human hair follicle dermal papilla cells (HHFDPC) was confirmed using the Western blot method.

[0147] HHFDPC 4 x 10 5Cells were seeded at a density of 10 cells / well in 6-well cell culture plates and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell medium and cultured for another 24 hours. The cultured cell cultures were treated with 10 μM, 50 μM, and 100 μM of the peptides prepared in Preparation Example 1, respectively, and treated with 1 μM of Minoxidil (MNX) as a positive control, and a control group that was not treated with anything was cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, the cells were lysed by treating with lysis buffer to obtain cell lysates. The cell lysates were subjected to BCA quantitation to prepare protein samples of the same amount. The prepared protein samples were electrophoresed using 10% SDS-PAGE. Proteins separated by SDS-PAGE were transferred to PVDF membranes and blocked with 5% skim milk for 30 minutes at room temperature. Antibodies against phospho-AKT and phospho-ERK (Cell signaling, USA) were diluted 1:1000 in 3% BSA and reacted with the membranes at 4°C for 16 hours. The membranes were washed three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), and the secondary antibody was diluted 1:2000 in 5% skim milk and reacted for 1 hour at room temperature. After washing three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), the cells were treated with ECL solution (GE Healthcare, USA), and the expression level was analyzed using ImageQuant 800 (Cytiva, USA).

[0148] As shown in Fig. 1a and Fig. 1b, when HHFDPC was treated with the peptide of Preparation Example 1 at 10 μM, 50 μM, and 100 μM, it was confirmed that the phosphorylation of Akt and ERK (extracellular-regulated kinase), which are signaling molecules related to cell proliferation, increased.

[0149]

[0150] Experimental Example 2: Stimulation of Growth Factor Gene Expression in Human Hair Follicle Dermal Papillary Cells

[0151] The effect of the peptide manufactured in the above Manufacturing Example 1 on the expression level of growth factors in human hair follicle dermal papilla cells (HHFDPC) was confirmed using RT-PCR.

[0152] HHFDPC 3 x 10 5Cells were seeded in 6-well cell culture plates at a density of 10 cells / well and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with 10 μM, 50 μM, and 100 μM of the peptides prepared in Preparation Example 1, respectively, and treated with 1 μM minoxidil (MNX) as a positive control, and untreated as a control, and cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, RNA was isolated by treating them with 300 μL of easy blue (Intron, South Korea). The isolated RNA was quantified using Nano drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, South Korea). Next, PCR was performed using the primers and PCR premix (Enzynomics, South Korea) in Table 2 below. The PCR reaction products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad Gel Image System.

[0153] Gene primer sequence (5' → 3') SEQ ID NO:bFGFForward5'-TGCTGGTGATGGGAGTTGTA -3'2Reverse5'-CCTCCAAGTAGCAGCCAAAG -3'3HGFForward5'-CAATGCCTCTGGTTCCCCTT -3'4Reverse5'-CCCCTCGAGGATTTCGACAG -3'5KGFForward5'-TCTGTCGAACACAGTGGTACCT -3'6Reverse5'-GTGTGTCCATTTAGCTGATGCAT -3'7VEGFForward5'-ATGAACTTTCTGGGTCTTGGGT -3'8Reverse5'-TGGCCTTGGTGAGGTTTGATCC -3'9GAPDHForward5'-GGAGCCAAAAGGGTCATCAT -3'10Reverse5'-GTGATGGCATGGACTGTGGT -3'11

[0154]

[0155] As a result of the experiment, as shown in Fig. 2, when the peptide of Manufacturing Example 1 was treated to HHFDPC at 10 μM, 50 μM, and 100 μM, it was confirmed that the activity of HHFDPC increased, promoting the expression of growth factors such as bFGF, HGF, KGF, and VEGF.

[0156]

[0157] Experimental Example 3: Activation of β-catenin in human hair follicle papilla cells

[0158] The effect of the peptide manufactured in the above Manufacturing Example 1 on the activation of β-catenin in human hair follicle dermal papilla cells (HHFDPC) was confirmed using the Western blot method.

[0159] HHFDPC 4 x 10 5Cells were seeded at a density of 10 cells / well in a 6-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with 10 μM, 50 μM, and 100 μM of the peptides prepared in Preparation Example 1, respectively, and 100 ng / mL of rhWnt-3a (recombinant human Wnt-3a protein, R&D systems (USA, MN)) was treated as a positive control, and the untreated group served as a control. The cell cultures treated with the above substances were cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, nuclear proteins were isolated using a nuclear protein extraction kit (Thermo scientific, USA), and BCA was quantified to prepare equal amounts of protein samples. The prepared protein samples were electrophoresed using 10% SDS-PAGE. The proteins separated by SDS-PAGE were transferred to PVDF membranes and blocked with 5% skim milk for 30 minutes at room temperature. Antibodies against beta-catenin (Cell signaling, USA) and HDAC1 (Santa Cruz, USA) were diluted 1:1000 in 3% BSA and reacted with the membranes at 4°C for 16 hours. The membranes were washed three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), and the secondary antibodies were diluted 1:2000 in 5% skim milk and reacted for 1 hour at room temperature. 0.1% PBS-T (0.The cells were washed three times for 15 minutes each with 1% Tween-20 in PBS, treated with ECL solution (GE Healthcare, USA), and the expression level was analyzed using ImageQuant 800 (Cytiva, USA).

[0160] As a result of the experiment, as shown in Fig. 3, when the peptide of Manufacturing Example 1 was treated to HHFDPC at 10 μM, 50 μM, and 100 μM, activation of Wnt / β-catenin, known as an important signaling pathway related to hair growth, was confirmed, and it was confirmed that β-catenin was activated and its movement to the nucleus was significantly increased.

[0161]

[0162] Experimental Example 4: Promotion of expression of beta-catenin downstream genes in human hair follicle papilla cells.

[0163] The effect of the peptide manufactured in the above Manufacturing Example 1 on the expression levels of LEF-1 (Lymphoid enhancer-binding factor 1), Cyclin D1, and c-Myc, which are downstream genes of β-catenin, in human hair follicle dermal papilla cells (HHFDPC), was confirmed by RT-PCR.

[0164] HHFDPC 4 x 10 5Cells were seeded at a density of 10 cells / well in a 6-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with the peptides prepared in Preparation Example 1 at 10 μM, 50 μM, and 100 μM, respectively, and 100 ng / mL of rhWnt-3a (recombinant human Wnt-3a protein, R&D systems (USA, MN)) as a positive control group, and the untreated control group was used as a control group, and each was cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, RNA was isolated by treating them with 300 μL of easy blue (Intron, South Korea). The isolated RNA was quantified using Nano Drop and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, South Korea). Subsequently, PCR was performed using the primers listed in Table 3 and a PCR premix (Enzynomics, South Korea). The PCR reaction products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad Gel Image System.

[0165] Gene primer sequence SEQ ID NO: LEF-1Forward5'-CCAGCTATTGTAACACCTCA -3'12Reverse5'-TTCAGATGTAGGCAGCTGTC -3'13cyclin-D1Forward5'-ACCTTCGTTGCCCTCTGTGCCACAGATG -3'14Reverse5'-AGGCCCGGAGGGCAGTCCGGGT -3'15c-MycForward5'-ACCAGCAGCGACTCTGAGGAGGAAC -3'16Reverse5'-TGACCCTCTTGGCAGCAGGATAGTCC -3'17GAPDHForward5'-GGAGCCAAAAGGGTCATCAT -3'10Reverse5'-GTGATGGCATGGACTGTGGT -3'11

[0166]

[0167] As a result of the experiment, as shown in Fig. 4, when the peptide of Manufacturing Example 1 was treated to HHFDPC at 10 μM, 50 μM, and 100 μM, it was confirmed that beta-catenin, which affects cell proliferation and differentiation, was activated, and the gene expression of the downstream factors LEF-1, Cyclin D1, and c-Myc increased.

[0168]

[0169] Experimental Example 5: Inhibition of the expression of hair loss protein DKK-1 in human hair follicle papilla cells.

[0170] Dihydrotestosterone (DHT) in hair follicles promotes the expression of DKK-1 (Dickkopf-1), and DKK-1 produced by DHT is known to inhibit the proliferation of keratinocytes in hair follicles and induce their apoptosis, thereby promoting hair loss (J of Invest Dermatolo. 2008, Feb., 128(2), pp. 262-269.). The effect of the peptide produced in Manufacturing Example 1 on the expression level of the hair loss-inducing protein DKK-1 in human hair follicle papilla cells (HHFDPC) was confirmed using the Western blot method.

[0171] HHFDPC 4 x 10 5Cells were seeded at a density of 10 cells / well in a 6-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with the peptides prepared in Manufacturing Example 1 at concentrations of 10 μM, 50 μM, and 100 μM, respectively, and 5 μM finasteride was treated as a positive control. At this time, DHT, an inducer of the hair loss protein DKK-1, was simultaneously treated at a concentration of 100 nM. In addition, the cell culture treated with only DHT was used as a negative control, and the cell culture treated with nothing was used as a control. The cell cultures treated with the above substances were cultured for an additional 24 hours at 37°C. After washing the culture medium with PBS, the cells were lysed using lysis buffer to obtain a cell lysate. The cell lysate was quantified with BCA to prepare an equal amount of protein sample. The prepared protein sample was electrophoresed using 10% SDS-PAGE. The proteins separated by SDS-PAGE were transferred to a PVDF membrane and blocked with 5% skim milk for 30 minutes at room temperature. An antibody against DKK-1 (Cell Signaling, USA) was 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 (0.1% Tween-20 in PBS), and the secondary antibody was diluted 1:2000 in 5% skim milk and reacted at room temperature for 1 hour. 0.1% PBS-T (0.After washing three times for 15 minutes each with 1% Tween-20 in PBS, the cells were treated with ECL solution (GE Healthcare, USA), and the expression level was detected using ImageQuant 800 (Cytiva, USA) equipment.

[0172] As a result of the experiment, as shown in Fig. 5, by treating HHFDPC with DHT, the expression of DKK-1, a hair loss protein, was induced, and when the peptide of Preparation Example 1 was treated at 10 μM, 50 μM, and 100 μM, it was confirmed that the expression of DKK-1, a hair loss protein induced by DHT treatment, was reduced again in a concentration-dependent manner.

[0173]

[0174] Experimental Example 6: Activation of human hair root sheath cells (HHORSCs)

[0175] The effect of the peptide manufactured in the above Manufacturing Example 1 on the activation of human hair outer root sheath cells (HHORSC) was confirmed by measuring the gene expression of cytokeratin proteins.

[0176] HHORSC 4 x 10 5Cells were seeded in 6-well cell culture plates at a density of 10 cells / well and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with the peptides prepared in Preparation Example 1 at concentrations of 10 μM, 50 μM, and 100 μM, respectively, and treated with 50 nM rhEGF (recombinant human EGF protein) as a positive control, and untreated as a control, and cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, RNA was isolated by treating them with 300 μL of easy blue (Intron, South Korea). The isolated RNA was quantified using Nano drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, South Korea). Next, PCR was performed using the primers and PCR premix (Enzynomics, South Korea) in Table 4 below. PCR reaction products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad Gel Image System.

[0177] Gene primer sequence SEQ ID NO: Ha3-IIForward5'-GATCATCGAGCTGAGACGCA -3'18Reverse5'-CTGGCCCCAGGGTATCTAGT -3'19Keratin 19Forward5'-CGCGGCGTATCCGTGTCCTC -3'20Reverse5'-AGCCTGTTCCGTCTCAAACTTGGT -3'21Keratin 14Forward5'-CCAAATCCGCACCAAGGTCA -3'22Reverse5'-GTATTGATTGCCAGGAGGGGG -3'23Keratin 5Forward5'-CCCTCAAGGATGCCAGGAAC -3'24Reverse5'-CACTGCTACTCTCGGCAAG -3'25GAPDHForward5'-GGAGCCAAAAGGGTCATCAT -3'10Reverse5'-GTGATGGCATGGACTGTGGT -3'11

[0178]

[0179] As a result of the experiment, as shown in Fig. 6, when the peptide of Manufacturing Example 1 was treated to HHORSC at 10 μM, 50 μM, and 100 μM, it was confirmed that the activity of the cells increased, and the gene expression of Ha3-II, Keratin 19, Keratin 14, and Keratin 5, which are cytokeratin proteins that form the cell skeleton and constitute hair, all increased.

[0180]

[0181] Experimental Example 7: Activation of human ghrelin-derived macrophages (HHGMC)

[0182] The effect of the peptide manufactured in the above Manufacturing Example 1 on the activation of human hair germinal matrix cells (HHGMC) was confirmed by measuring the gene expression of transcription factors MSX2 (Msh Homeobox2), HOXC13 (Homeobox C13), and FOXN1 (Forkhead Box N1), which are related to cell activation.

[0183] HHGMC 4 x 10 5 Cells were seeded in 6-well cell culture plates at a density of 10 cells / well and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with the peptides prepared in Preparation Example 1 at concentrations of 10 μM, 50 μM, and 100 μM, respectively, and treated with 50 nM rhEGF (recombinant human EGF protein) as a positive control, and untreated as a control, and cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, RNA was isolated by treating them with 300 μL of easy blue (Intron, South Korea). The isolated RNA was quantified using Nano drop, and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, South Korea). Next, PCR was performed using the primers and PCR premix (Enzynomics, South Korea) in Table 5 below. PCR reaction products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad Gel Image System.

[0184] Gene primer sequence sequence number MSX2 Forward 5'-CGGTCAAGTCGGAAAATTCA -3'26 Reverse 5'-GAGGAGCTGGGATGTGGTAA -3'27 HOXC13 Forward 5'-GCCGTCTATACGGACATGCC -3'28 Reverse 5'-GCAGTACACCTGACTGTCCC -3'29 FOXN1 Forward 5'-CAGGACTGGGTGATGGTGTC -3'30 Reverse 5'-TCTGAGGGTGAGAGCCTGAA -3'31 GAPDH Forward 5'-GGAGCCAAAAGGGTCATCAT -3'10 Reverse 5'-GTGATGGCATGGACTGTGGT -3'11

[0185]

[0186] As a result of the experiment, as shown in Fig. 7, when HHGMC was treated with the peptide of Manufacturing Example 1 at 10 μM, 50 μM, and 100 μM, it was confirmed that the gene expression of transcription factors MSX2, HOXC13, and FOXN1 involved in cell activity increased.

[0187]

[0188] Experimental Example 8: Promoting the Proliferation of Human Hair Follicle Stem Cells (HHFSCs)

[0189] The effect of the peptide manufactured in the above Manufacturing Example 1 on the proliferation of human hair follicle stem cells (HHFSC) was tested.

[0190] HHFSC 2 x 10 3Cells were seeded at a density of 10 cells / well in a 96-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with 10 μM, 50 μM, and 100 μM of the peptides prepared in Manufacturing Example 1, respectively, and 100 ng / ml of rhWnt-3a (recombinant human Wnt-3a protein) was treated as a positive control, and a control group that was not treated with anything was used as an untreated control group, and cultured for an additional 72 hours at 37°C. Next, 10 μL of 5 mg / ml MTT solution was added, incubated for 4 hours in a 37°C CO2 incubator, the medium was removed, 100 μL of DMSO was treated, and shaking was performed for 10 minutes. Finally, the absorbance at a wavelength of 540 nm was measured using a spectrophotometer (SpectraMax iD3, Molecular Devices (USA, CA)).

[0191] As shown in Fig. 8, the experimental results confirmed that when the peptide of Manufacturing Example 1 was treated at 10 μM, 50 μM, and 100 μM in HHFSC, the proliferation of HHFSC was promoted up to 53.73% in a concentration-dependent manner compared to the control.

[0192]

[0193] Experimental Example 9: Decreased Expression of Stem Cell Marker-Related Genes in Human Hair Follicle Stem Cells

[0194] The effect of the peptide manufactured in the above Manufacturing Example 1 on inducing differentiation of human hair follicle stem cells (HHFSCs) was confirmed by measuring the gene expression of stem cell markers CD133, Keratin 15, and LHX2 (LIM homeobox 2).

[0195] HHFSC 1.5 x 10 5 Cells were seeded at a density of 10 cells / well in a 6-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with the peptides prepared in Preparation Example 1 at concentrations of 10 μM, 50 μM, and 100 μM, respectively, and 50 ng / mL of rhWnt-7b (recombinant human Wnt-7b protein) was treated as a positive control, and the untreated control group was used as a control group, and cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, RNA was isolated by treating them with 300 μL of easy blue (Intron, South Korea). The isolated RNA was quantified using Nano Drop and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, South Korea). Subsequently, PCR was performed using the primers listed in Table 6 and a PCR premix (Enzynomics, South Korea). The PCR reaction products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad Gel Image System.

[0196] Gene primer sequence sequence number CD133 Forward 5'-GAGACCAAAGAGGCGTTGGA -3'32 Reverse 5'-GGCTAGTTTTCACGCTGGTC -3'33 Keratin 15 Forward 5'-CCAGCAAGACGGAGATCACA -3'34 Reverse 5'-GAAGAGGCTTCCCTGATGGC -3'35 Lhx2 Forward 5'-AAGCTGCAACGAAAACGACG -3'36 Reverse 5'-GGTGGGGCTAGTCAAGTCTG -3'37 GAPDH Forward 5'-GGAGCCAAAAGGGTCATCAT -3'10 Reverse 5'-GTGATGGCATGGACTGTGGT -3'11

[0197]

[0198] As shown in Fig. 9, when the peptide of Manufacturing Example 1 was treated to HHFSCs at 10 μM, 50 μM, and 100 μM, it was confirmed that the gene expression of stem cell markers CD133, Keratin 15, and LHX2 of HHFSCs was significantly reduced compared to the control. These results show that the peptide of Manufacturing Example 1 induces differentiation of HHFSCs, thereby reducing the expression of stem cell marker genes.

[0199]

[0200] Experimental Example 10: Increased Expression of Stem Cell Differentiation-Related Genes in Human Hair Follicle Stem Cells

[0201] The effect of the peptide manufactured in the above Manufacturing Example 1 on inducing differentiation of human hair follicle stem cells (HHFSC) was confirmed by measuring the gene expression of differentiation markers α-SMA, Vimentin, Versican, and Nexin-1.

[0202] HHFSC 1.5 x 10 5Cells were seeded at a density of 10 cells / well in a 6-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with the peptides prepared in Preparation Example 1 at concentrations of 10 μM, 50 μM, and 100 μM, respectively, and 50 ng / mL of rhWnt-7b (recombinant human Wnt-7b protein) was treated as a positive control, and the untreated control group was used as a control group, and cultured for an additional 24 hours at 37°C. After washing the cultures with PBS, RNA was isolated by treating them with 300 μL of easy blue (Intron, South Korea). The isolated RNA was quantified using Nano Drop and cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, South Korea). Subsequently, PCR was performed using the primers listed in Table 7 and a PCR premix (Enzynomics, South Korea). The PCR reaction products were electrophoresed on a 1.5% agarose gel, and bands were detected using a Bio-Rad Gel Image System.

[0203] Gene primer sequence SEQ ID NO: α-SMAForward5'-CCTATCCCCGGGACTAAGAC -3'38Reverse5'-ATGCTCTTCAGGGGCAACAC -3'39VimentinForward5'-TGGACCAGCTAACCAACGAC -3'40Reverse5'-GTCATTGTTCCGGTTGGCAG -3'41VersicanForward5'-CCGTTCTCCCCAGGAAAACTT -3'42Reverse5'-TGCAGCGATCAGGTCGTTTA -3'43Nexin1Forward5'-CCCAGCCAACCTATGAGGAG -3'44Reverse5'-CTTGAGGAGACCAGCACCAC -3'45GAPDHForward5'-GGAGCCAAAAGGGTCATCAT -3'10Reverse5'-GTGATGGCATGGACTGTGGT -3'11

[0204]

[0205] As shown in Fig. 10, when the peptide of Manufacturing Example 1 was treated to HHFSCs at 10 μM, 50 μM, and 100 μM, it was confirmed that the gene expression of differentiation markers of HHFSCs, α-SMA, Vimentin, Versican, and Nexin-1, was significantly increased compared to the negative control group. These results show that the peptide of Manufacturing Example 1 induces differentiation of HHFSCs, thereby increasing the expression of differentiation marker genes.

[0206]

[0207] Experimental Example 11: Decreased Expression of Stem Cell Markers in Human Hair Follicle Stem Cells

[0208] The effect of the peptide manufactured in the above Manufacturing Example 1 on inducing differentiation of human hair follicle stem cells (HHFSC) was confirmed by measuring the expression of stem cell markers CD34, CD133, and Keratin 15 proteins.

[0209] HHFSC 3x10 5Cells were seeded at a density of 10 cells / well in a 6-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with 10 μM, 50 μM, and 100 μM of the peptides prepared in Preparation Example 1, respectively, and 50 ng / mL of rhWnt-7b (recombinant human Wnt-7b protein, R&D systems (USA, MN)) was treated as a positive control, and the untreated group served as a control. The cell cultures treated with the above substances were cultured at 37°C for an additional 72 hours. After washing the cultures with PBS, the cells were lysed by treating with lysis buffer to obtain cell lysates. BCA was quantified for the cell lysates to prepare equal amounts of protein samples. The prepared protein samples were electrophoresed using 10% SDS-PAGE. The proteins separated by SDS-PAGE were transferred to PVDF membranes and blocked with 5% skim milk for 30 minutes at room temperature. Antibodies against CD34, CD133, and Keratin13 (Cell signaling, USA) were diluted 1:1000 in 3% BSA and reacted with the membranes at 4°C for 16 hours. The membranes were washed three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), and the secondary antibody was diluted 1:2000 in 5% skim milk and reacted at room temperature for 1 hour. After washing three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), the cells were treated with ECL solution (GE Healthcare, USA), and the expression level was analyzed using ImageQuant 800 (Cytiva, USA).

[0210] As shown in Fig. 11, when HHFSCs were treated with the peptide of Preparation Example 1 at 10 μM, 50 μM, and 100 μM, it was confirmed that the protein expression of stem cell markers CD34, CD133, and Keratin 15 decreased. These results show that the peptide of Preparation Example 1 induced differentiation of HHFSCs, thereby reducing the expression of stem cell marker proteins.

[0211]

[0212] Experimental Example 12: Increased Expression of Stem Cell Differentiation Markers in Human Hair Follicle Stem Cells

[0213] The effect of the peptide manufactured in the above Manufacturing Example 1 on inducing differentiation of human hair follicle stem cells (HHFSC) was confirmed by measuring the expression of differentiation markers α-SMA, Keratin75, and Versican proteins.

[0214] HHFSC 3x10 5Cells were seeded at a density of 10 cells / well in a 6-well cell culture plate and cultured for 24 hours in mesenchymal stem cell complete media. Subsequently, the medium was replaced with serum-free mesenchymal stem cell media and cultured for an additional 24 hours. The cultured cell cultures were treated with 10 μM, 50 μM, and 100 μM of the peptides prepared in Preparation Example 1, respectively, and 50 ng / mL of rhWnt-7b (recombinant human Wnt-7b protein, R&D systems (USA, MN)) was treated as a positive control, and the untreated group served as a control. The cell cultures treated with the above substances were cultured at 37°C for an additional 72 hours. After washing the cultures with PBS, the cells were lysed by treating with lysis buffer to obtain cell lysates. BCA was quantified for the cell lysates to prepare equal amounts of protein samples. The prepared protein samples were electrophoresed using 10% SDS-PAGE. The proteins separated by SDS-PAGE were transferred to PVDF membranes and blocked with 5% skim milk for 30 minutes at room temperature. Antibodies against α-SMA, Keratin75, and Versican (Cell signaling, USA) were diluted 1:1000 in 3% BSA and reacted with the membranes at 4°C for 16 hours. The membranes were washed three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), and the secondary antibodies were diluted 1:2000 in 5% skim milk and reacted at room temperature for 1 hour. After washing three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), the cells were treated with ECL solution (GE Healthcare, USA), and the expression level was analyzed using ImageQuant 800 (Cytiva, USA).

[0215] As shown in Fig. 12, when HHFSCs were treated with the peptide of Preparation Example 1 at 10 μM, 50 μM, and 100 μM, it was confirmed that the protein expression of stem cell differentiation markers α-SMA, Keratin75, and Versican significantly increased. These results show that the peptide of Preparation Example 1 induces differentiation of HHFSCs, thereby increasing the expression of differentiation marker proteins.

[0216]

[0217] Manufacturing Example 2: Manufacturing of a pharmaceutical composition

[0218] 2-1. Manufacturing of Sanje

[0219] 2 g of the peptide of the present invention

[0220] 1 g lactose

[0221] The above ingredients were mixed and filled into a sealed bag to prepare a powder.

[0222]

[0223] 2-2. Manufacturing of tablets

[0224] 100 mg of the peptide of the present invention

[0225] 100 mg of corn starch

[0226] 100 mg of lactose

[0227] Magnesium stearate 2 mg

[0228] After mixing the above ingredients, tablets were manufactured by pressing them according to a conventional tablet manufacturing method.

[0229]

[0230] 2-3. Manufacturing of capsules

[0231] 100 mg of the peptide of the present invention

[0232] 100 mg of corn starch

[0233] 100 mg of lactose

[0234] Magnesium stearate 2 mg

[0235] After mixing the above ingredients, the mixture was filled into a gelatin capsule according to a conventional capsule manufacturing method to produce a capsule.

[0236]

[0237] 2-4. Manufacturing of rings

[0238] 1 g of the peptide of the present invention

[0239] 1.5 g of lactose

[0240] 1 g of glycerin

[0241] 0.5 g of xylitol

[0242] After mixing the above ingredients, it was manufactured to have 4g per pill according to the usual method.

[0243]

[0244] 2-5. Preparation of granules

[0245] 150 mg of the peptide of the present invention

[0246] 50 mg of soybean extract

[0247] 200 mg of glucose

[0248] 600 mg of starch

[0249] After mixing the above ingredients, 100 mg of 30% ethanol was added, dried at 60°C to form granules, and then filled into a bag.

[0250]

[0251] Manufacturing Example 3: Manufacturing of a cosmetic composition

[0252] 3-1. Preparation of cream

[0253] 4.6 parts by weight of the peptide of the present invention

[0254] 2.8 parts by weight of cetostearyl alcohol

[0255] 2.6 parts by weight of beeswax

[0256] 1.4 parts by weight of stearic acid

[0257] 2 parts by weight of glycerin monostearate

[0258] 1 part by weight of PEG-100 stearate

[0259] 1.4 parts by weight of sorbitol sesquioleate

[0260] 4 parts by weight of jojoba oil

[0261] 3.8 parts by weight of squalane

[0262] Polysorbate 60 1.1 parts by weight

[0263] 2 parts by weight of macadia oil

[0264] 0.2 parts by weight of tocopherol acetate

[0265] 0.4 parts by weight of methylpolysiloxane

[0266] 0.1 part by weight of ethylparaben

[0267] Propylparaben 0.1 part by weight

[0268] Euxyl K-400 0.1 part by weight

[0269] 7 parts by weight of 1,3-butylene glycol

[0270] 0.05 parts by weight of methylparaben

[0271] 6 parts by weight of glycerin

[0272] 0.2 parts by weight of d-panthenol

[0273] 0.2 parts by weight of triethanolamine

[0274] pt 41891 0.2 parts by weight

[0275] p-H2O 46.05 parts by weight

[0276]

[0277] 3-2. Preparation of lotion

[0278] 3.5 parts by weight of the peptide of the present invention

[0279] 1.6 parts by weight of cetostearyl alcohol

[0280] 1.4 parts by weight of stearic acid

[0281] 1.8 parts by weight of glycerin monostearate

[0282] PEG-100 Stearate 2.6 parts by weight

[0283] 0.6 parts by weight of sorbitol sesquioleate

[0284] 4.8 parts by weight of squalene

[0285] 2 parts by weight of macadia oil

[0286] 2 parts by weight of jojoba oil

[0287] 0.4 parts by weight of tocopherol acetate

[0288] 0.2 parts by weight of methylpolysiloxane

[0289] 0.1 part by weight of ethylparaben

[0290] Propylparaben 0.1 part by weight

[0291] 4 parts by weight of 1,3-butylene glycol

[0292] 0.1 part by weight of methylparaben

[0293] 0.1 part by weight of xanthan gum

[0294] 4 parts by weight of glycerin

[0295] d-panthenol 0.15 parts by weight

[0296] Allantoin 0.1 part by weight

[0297] 4 parts by weight of carbonate (2% aq. Sol)

[0298] 0.15 parts by weight of triethanolamine

[0299] 3 parts by weight of ethanol

[0300] pt 41891 0.1 parts by weight

[0301] p-H20 48.3 parts by weight

[0302]

[0303] 3-3. Manufacturing of flexible toner

[0304] 0.2 wt% of the peptide of the present invention

[0305] 10.0 wt% ethanol

[0306] 1.0 wt% polyoxyethylene sorbitan polylaurate

[0307] 0.2 wt% methyl parahydroxybenzoate

[0308] 5.0 wt% glycerin

[0309] 1,3-butyl glycol 6.0 wt%

[0310] Appropriate amount of fragrance

[0311] Appropriate amount of pigment

[0312] Appropriate amount of purified water

[0313] A total of 100

[0314]

[0315] 3-4. Manufacturing of Nutritious Toner

[0316] 0.1 wt% of the peptide of the present invention

[0317] 2.0 wt% Vaseline

[0318] 0.8 wt% sorbitan sesquioleate

[0319] 1.2 wt% polyoxyethylene oleyl ethyl

[0320] Appropriate amount of methyl parahydroxybenzoate

[0321] 5.0 wt% propylene glycol

[0322] 3.2 wt% ethanol

[0323] 18.0 wt% carboxyvinyl polymer

[0324] Appropriate amount of pigment

[0325] Appropriate amount of fragrance

[0326] Appropriate amount of purified water

[0327] A total of 100

[0328]

[0329] 3-5. Preparation of essence

[0330] 5.0 wt% of the peptide of the present invention

[0331] 10.0 wt% propylene glycol

[0332] 10.0 wt% glycerin

[0333] Sodium hyaluronate aqueous solution (1%) 5.0 wt%

[0334] 3.2 wt% ethanol

[0335] 1.0 wt% polyoxyethylene hydrogenated castor oil

[0336] 0.1 wt% methyl parahydroxybenzoate

[0337] Appropriate amount of fragrance

[0338] Appropriate amount of purified water

[0339] A total of 100

[0340]

[0341] 3-6. Pack manufacturing

[0342] 0.5 wt% of the peptide of the present invention

[0343] 5.0 wt% glycerin

[0344] 4.0 wt% propylene glycol

[0345] 15.0 wt% polyvinyl alcohol

[0346] 8.0 wt% ethanol

[0347] 1.0 wt% polyoxyethylene oleyl ethyl

[0348] 0.2 wt% methyl parahydroxybenzoate

[0349] Appropriate amount of fragrance

[0350] Appropriate amount of pigment

[0351] Appropriate amount of purified water

[0352] A total of 100

[0353]

[0354] The above composition ratio is a mixture of suitable ingredients as a preferred example, but the ingredients or mixing ratio may be arbitrarily modified according to regional or national preferences such as demand class, demand country, or use.

[0355]

[0356] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A peptide comprising the amino acid sequence of sequence number 1.

2. A composition for promoting hair growth or inhibiting, preventing or improving alopecia, comprising the peptide of claim 1 as an active ingredient.

3. A pharmaceutical composition for preventing or treating alopecia, comprising the peptide of claim 1 as an active ingredient.

4. In claim 3, A pharmaceutical composition for preventing or treating alopecia, wherein the above peptide promotes proliferation of one or more cells selected from the group consisting of hair follicle papilla cells (HFDPCs), hair root sheath cells (HORSCs), and hair follicle stem cells (HFSCs).

5. In claim 3, The above peptide is expressed in hair follicle dermal papilla cells (HFDPC). (i) activating Akt or ERK signaling proteins, or (ii) upregulates the gene expression of one or more factors selected from the group consisting of growth factors bFGF (basic fibroblast growth factor), HGF (hepatocyte growth factor), KGF (keratinocyte growth factor) and VEGF (vascular endothelial growth factor), or (iii) activating beta-catenin, or (iv) upregulates the gene expression of one or more factors selected from the group consisting of beta-catenin downstream factors LEF-1, Cyclin D1 and c-Myc, or (v) A pharmaceutical composition for preventing or treating alopecia, which downregulates the expression level of DKK-1 (Dickkopf-1), a hair loss protein.

6. In claim 3, The above peptide is expressed in the outer root sheath cells (HORSC). A pharmaceutical composition for preventing or treating alopecia, which increases the expression of one or more proteins selected from the group consisting of Ha3-II, Keratin 19, Keratin 14, and Keratin 5.

7. In claim 3, The above peptide is expressed in human goblet cells (HGMC). A pharmaceutical composition for preventing or treating alopecia, which increases the expression of one or more transcription factors selected from the group consisting of MSX2, HOXC13 and FOXN1.

8. In claim 3, The above peptide is in hair follicle stem cells (HFSC). (i) downregulate the expression of one or more selected from the group consisting of stem cell markers CD34, CD133, Keratin 15 and LHX2, or (ii) A pharmaceutical composition for preventing or treating alopecia, which upregulates the expression of one or more markers selected from the group consisting of α-SMA, Keratin75, Vimentin, Versican and Nexin-1, which are stem cell differentiation markers.

9. In claim 3, The above pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of alopecia, which is an external skin preparation.

10. A cosmetic composition for promoting hair growth or preventing or improving alopecia, comprising the peptide of claim 1 as an active ingredient.

11. In claim 10, A cosmetic composition for promoting hair growth or preventing or improving alopecia, wherein the above peptide promotes the proliferation of one or more cells selected from the group consisting of hair follicle papilla cells (HFDPCs), hair root sheath cells (HORSCs), and hair follicle stem cells (HFSCs).

12. In claim 10, The above peptide is expressed in hair follicle dermal papilla cells (HFDPC). (i) activating Akt or ERK signaling proteins, or (ii) upregulates the gene expression of one or more factors selected from the group consisting of growth factors bFGF (basic fibroblast growth factor), KGF (keratinocyte growth factor) and VEGF (vascular endothelial growth factor), or (iii) activating beta-catenin, or (iv) upregulates the gene expression of one or more factors selected from the group consisting of beta-catenin downstream factors LEF-1, c-Myc and Cyclin D1, or (v) A cosmetic composition for promoting hair growth or preventing or improving alopecia, which downregulates the expression level of DKK-1 (Dickkopf-1), a hair loss protein.

13. In claim 10, The above peptide is expressed in the outer root sheath cells (HORSC). A cosmetic composition for promoting hair growth or preventing or improving alopecia, which increases the expression of one or more proteins selected from the group consisting of Ha3-II, Keratin 5, Keratin 14, and Keratin 19.

14. In claim 10, The above peptide is expressed in human goblet cells (HGMC). A cosmetic composition for promoting hair growth or preventing or improving alopecia, which increases the expression of one or more transcription factors selected from the group consisting of MSX2, HOXC13 and FOXN1.

15. In claim 10, The above peptide is in hair follicle stem cells (HFSC). (i) downregulate the expression of one or more selected from the group consisting of stem cell markers CD34, CD133, Keratin 15 and LHX2, or (ii) A cosmetic composition for promoting hair growth or preventing or improving alopecia, which upregulates the expression of one or more genes selected from the group consisting of stem cell differentiation markers α-SMA, Keratin75, Vimentin, Versican, and Nexin-1.

16. In claim 10, The above cosmetic composition is a cosmetic composition for external use on the skin, for promoting hair growth or preventing or improving alopecia.

17. In claim 10, A cosmetic composition for promoting hair growth or preventing or improving alopecia, wherein the cosmetic composition has any one formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray.

18. In claim 10, A cosmetic composition for promoting hair growth or preventing or improving alopecia, wherein the cosmetic composition has any one formulation selected from the group consisting of hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, and gel.

19. A method for promoting hair growth or inhibiting, preventing, improving or treating alopecia, comprising the step of applying a peptide comprising the amino acid sequence of sequence number 1 or a composition comprising the peptide as an active ingredient to a subject in need of promoting hair growth or inhibiting, preventing, improving or treating alopecia.

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