Peptide with activity of promoting hair growth and inhibiting hair loss and application thereof

Abstract

The present invention relates to a peptide having an activity of promoting hair growth or preventing or inhibiting hair loss. The peptide according to the present invention promotes the proliferation of hair follicle dermal papilla cells (HFDPC) and human hair root sheath cells (HHORSC), up-regulates the expression of beta-catenin secondary factors in the hair follicle dermal papilla cells (HFDPC), and up-regulates the expression of growth factors.

Description

Technical Field

[0001] The present invention relates to peptides having the activities of promoting hair growth and inhibiting hair loss and uses thereof. Background Art

[0002] Alopecia is a phenomenon of excessive hair shedding due to the resting phase becoming longer than normal or the number of hair follicles in the resting phase increasing, and means that excessive hair shedding occurs due to stress, nutritional deficiency, local blood flow disorders, hormones, genetic factors, etc., and it cannot be restored.

[0003] As a therapeutic agent for hair loss, 5α-reductase inhibitors convert the male hormone testosterone into the androgenic sex hormone dihydrotestosterone (DHT), for example, drugs such as finasteride or dutasteride have been developed and approved for commercial sale, and minoxidil, a drug that promotes hair growth by stimulating hair follicles and increasing blood flow, has also been approved and used.

[0004] However, for 5α-reductase inhibitors, side effects such as sexual dysfunction, depression, anxiety, and breast enlargement are reported to occur relatively frequently; and for minoxidil, side effects such as electrocardiogram abnormalities, tachycardia, and endocarditis are reported to occur; in addition, it is reported that hair loss begins again once minoxidil administration is stopped.

[0005] Peptides have high biocompatibility and, when used as a single active ingredient in medicine or cosmetics, have fewer side effects than extracts including various ingredients. After inhibiting local hair loss, these peptides are easily decomposed and cleared by proteases in the body; therefore, there are fewer concerns about the side effects of these peptide drugs than existing therapeutic agents.

[0006] PCT International Patent Application WO 2005-082395 discloses a peptide having a hair growth promoting effect by promoting the progression from the telogen phase to the growth phase in the hair growth cycle, and Korean Patent No. 10-2387764 discloses a peptide derivative in the form of a dimer that promotes the proliferation of hair follicle cells.

[0007] [Prior art literature]

[0008] [Patent Document]

[0009] WO 2005-082395

[0010] Korean Patent No.10-2387764 Summary of the invention

[0011] Technical issues

[0012] The present inventors have conducted research work to develop a peptide that has improved hair growth promotion and hair loss inhibition activities, while effectively promoting hair growth and having fewer side effects. As a result, the present inventors experimentally confirmed that the new peptide they developed has the ability to promote the proliferation of human hair follicle dermal papilla cells (HHFDPCs) and human hair outer root sheath cells (HHORSCs), improve the expression levels of downstream factors of β-catenin (i.e., LEF-1 and cyclin D1), down-regulate the expression level of alopecia protein DKK-1 in HHFDPCs, and increase the expression levels of growth factors bFGF, HGF, and VEGF, thereby completing the present invention.

[0013] Therefore, an object of the present invention is to provide novel peptides having the activity of promoting hair growth and inhibiting hair loss.

[0014] Another object of the present invention is to provide a composition for promoting hair growth, or inhibiting, preventing or improving hair loss, which composition comprises a peptide having the above-mentioned activity as an active ingredient.

[0015] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hair loss.

[0016] Another object of the present invention is to provide a cosmetic composition for promoting hair growth, or inhibiting or improving hair loss.

[0017] Technical Solution

[0018] In order to achieve the above objectives,

[0019] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0020] Another aspect of the present invention provides a composition for promoting hair growth, or inhibiting, preventing or improving hair loss, which comprises the peptide as an active ingredient.

[0021] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating hair loss, which comprises the peptide as an active ingredient.

[0022] Another aspect of the present invention provides a cosmetic composition for promoting hair growth, or preventing or improving hair loss, which comprises the peptide as an active ingredient.

[0023] Hereinafter, the present invention will be described in detail.

[0024] 1. Peptides and their activities

[0025] According to one aspect of the present invention, a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0026] As used herein, the term "peptide" refers to a linear molecule formed by amino acid residues linked to each other by peptide bonds.

[0027] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 1 can be used without modification, but within the range not affecting the original activity of the peptide (such as the activity of promoting hair growth or inhibiting hair loss), variants having different amino acid sequences or fragments thereof by deletion, insertion, substitution or a combination thereof of amino acid residues can also be used.

[0028] The peptides of the present invention may be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc., as long as the activity of the peptide is not altered.

[0029] The peptides of the present invention include: peptides comprising an amino acid sequence substantially identical to a peptide comprising the amino acid sequence of SEQ ID NO: 1, and variants or active fragments thereof. Substantially identical amino acid sequences refer to amino acid sequences having at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 1, respectively. In addition, the peptides may additionally include targeting sequences, tags, labeled residues, and amino acid sequences prepared for the specific purpose of increasing half-life or peptide stability.

[0030] The peptides of the present invention may have induced N-terminal and / or C-terminal modifications to select a portion of the amino acid sequence and increase its activity. By these N-terminal and / or C-terminal modifications, the stability of the peptides of the present invention can be significantly improved, for example, when administered in vivo, the half-life of the peptide can be increased. The term "stability" encompasses not only in vivo stability (which protects the peptides of the present invention from attacks by protein cleavage enzymes in vivo), but also storage stability (e.g., storage stability at room temperature).

[0031] The N-terminal modification may be a modification in which a protective group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) is bound to the N-terminal of the peptide. The C-terminal modification may be a modification in which a hydroxyl group (-OH), an amino group (-NH 2 ), azido (-NHNH 2 ) etc. are attached to the C-terminus of the peptide, but are not limited thereto.

[0032] The peptides of the present invention can be prepared by various methods widely known in the art to which the present invention belongs. For example, the peptides of the present invention can be prepared using chemical synthesis methods known in the art, especially solid-phase synthesis techniques (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 (U.S. Patent No. 5516891).

[0033] The peptide of the present invention has the activity of promoting hair growth, or preventing or inhibiting hair loss.

[0034] In an embodiment, the peptides of the invention promote the proliferation of hair follicle dermal papilla cells (HFDPCs).

[0035] In an embodiment, the peptides of the present invention promote the proliferation of human hair outer root sheath cells (HHORSC).

[0036] In an embodiment, the peptide of the present invention upregulates gene expression of one or more factors selected from the group consisting of LEF-1 and Cyclin D1, which are downstream factors of β-catenin, in hair follicle dermal papilla cells (HFDPCs).

[0037] In an embodiment, the peptide of the present invention upregulates the expression of growth factors 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) and vascular endothelial growth factor (VEGF).

[0038] Through the above-mentioned activities, the peptides of the present invention can exhibit the effects of promoting hair growth, or inhibiting, preventing or improving hair loss.

[0039] 2. Compositions for promoting hair growth, or preventing, treating or improving hair loss

[0040] In another aspect of the present invention, a composition for promoting hair growth, or preventing, treating or improving hair loss is provided, the composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0041] As described above, the peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention has the activity of promoting hair growth, or inhibiting or preventing hair loss.

[0042] In another aspect of the present invention, the composition of the present invention may be a pharmaceutical composition for preventing or treating hair loss, the pharmaceutical composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0043] As used herein, the term "hair loss" or "hair loss" refers to a condition in which hair is absent in areas where hair should normally be present, or the number of hairs is reduced compared to a normal state, due to abnormal hair loss. In an embodiment, hair loss or a symptom of hair loss in the present invention includes hair loss on the scalp.

[0044] In the present invention, hair loss includes hereditary androgenic 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 by 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, hair loss caused by drug treatment (anticoagulants, antidepressants, discontinuation of oral contraceptives, chemotherapy), toxic folliculitis, lichen planopilaris, hair loss caused by burns or trauma, hair loss caused by ultraviolet rays, and hair loss caused by contact with fine dust in the air, but is not limited thereto.

[0045] In an embodiment, a pharmaceutical composition comprising the peptide of the present invention as an active ingredient promotes the proliferation of hair follicle dermal papilla cells (HFDPCs).

[0046] In an embodiment, a pharmaceutical composition comprising the peptide of the present invention as an active ingredient promotes the proliferation of human hair outer root sheath cells (HHORSC).

[0047] In an embodiment, the 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 and cyclin D1, which are downstream factors of β-catenin, in hair follicle dermal papilla cells (HFDPCs).

[0048] In an embodiment, the pharmaceutical composition comprising the peptide of the present invention as an active ingredient upregulates the expression of growth factors 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) and vascular endothelial growth factor (VEGF).

[0049] The pharmaceutical composition of the present invention may include a therapeutically effective amount of the peptide of the present invention.

[0050] The term "therapeutically effective amount" refers to an amount sufficient for the peptide (active ingredient of the pharmaceutical composition of the present invention) to achieve its activity or efficacy. For example, a therapeutically effective amount refers to an amount sufficient to achieve the effect of treating or preventing hair loss.

[0051] As used herein, the term "prevent" or "preventing" refers to reducing the risk of developing a disease or disorder, 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 symptoms.

[0052] As used herein, the term "treat" refers to ameliorating a disease or disorder and includes all actions that improve or beneficially alter the symptoms of a disease by arresting or reducing the progression of the disease or one or more of its clinical symptoms.

[0053] In the present invention, the prevention or treatment of hair loss may be to eliminate the cause of hair loss or inhibit the progress of hair loss, or may be to promote hair growth by inhibiting hair loss or promoting hair formation.

[0054] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0055] Pharmaceutically acceptable carriers commonly used in preparations include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc.; but the carrier is not limited to this.

[0056] In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but the ingredients are not limited thereto.

[0057] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0058] The pharmaceutical composition of the present invention can be administered by any route suitable for treating alopecia (e.g., oral or parenteral). In the case of parenteral administration, the composition can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical application, or transdermal administration. Since the pharmaceutical composition of the present invention has the activity of preventing or treating alopecia, it can be applied topically, such as applying it to the scalp.

[0059] The dosage of the pharmaceutical composition may be 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, but is not limited thereto. The pharmaceutical composition may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food intake, administration time, administration route, excretion rate and reaction sensitivity.

[0060] The pharmaceutical composition of the present invention can be formulated into a unit dosage form using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily implemented by a person skilled in the art, or can be formulated by placing it in a multi-dose container. Specifically, the dosage form can be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or can be in the form of an extract, powder, granule, tablet or capsule, and can additionally include a dispersant or stabilizer.

[0061] The pharmaceutical composition of the present invention can be an external skin preparation. An external skin preparation is a preparation that can be used by applying it to the outside of the skin. When the pharmaceutical composition of the present invention is used as an external skin preparation, it can be applied to the scalp, particularly the scalp in the area where hair loss has occurred or the scalp in the area where hair growth is to be promoted. The external skin preparation can be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal delivery patch, a medicated bandage, a lotion or a combination thereof. The external skin preparation can be appropriately mixed with the ingredients commonly used in the external skin preparation (e.g., cosmetics and medicines) as needed, such as aqueous ingredients, oil-based ingredients, powdery ingredients, alcohols, humectants, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients or a combination thereof. Skin topical preparations can be appropriately mixed with metal chelators (such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.), caffeine, tannin, grass extract, glycyrrhizin, various herbs, drugs (such as tocopherol acetate, glycyrrhizin, tranexamic acid and its derivatives or salts, etc.), vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid and sugars (such as glucose, fructose, trehalose, etc.).

[0062] 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 hair loss, the cosmetic composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0063] As used herein, the term "ameliorate" refers to any action that improves or benefits the symptoms of a disease or disorder.

[0064] In an embodiment, a cosmetic composition including the peptide of the present invention as an active ingredient promotes the proliferation of hair follicle dermal papilla cells (HFDPCs).

[0065] In an embodiment, a cosmetic composition including the peptide of the present invention as an active ingredient promotes proliferation of human hair outer root sheath cells (HHORSC).

[0066] In an embodiment, a cosmetic composition including 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 and cyclin D1, which are downstream factors of β-catenin, in hair follicle dermal papilla cells (HFDPCs).

[0067] In an embodiment, the cosmetic composition comprising the peptide of the present invention as an active ingredient upregulates the expression of growth factors 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) and vascular endothelial growth factor (VEGF).

[0068] The cosmetic composition can be prepared into any dosage form commonly prepared in the technical field to which the present invention belongs, and can be an external skin preparation. For example, the cosmetic composition can be formulated into a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a spray, etc., but is not limited thereto.

[0069] The cosmetic composition can be prepared in various forms, such as solutions (e.g., skin softening lotions, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, skin cleansing water, facial packs, sprays, loose powders, hair tonics, hair creams, shampoos, shampoos, hair dyes, conditioners, spray-type hair sprays, aerosol-type hair sprays, hair waxes, gels, etc.), sol-gels, emulsions, oils, waxes, aerosols, etc., but are not limited thereto.

[0070] The cosmetic composition of the present invention may include other additives such as excipients and carriers, and can be applied and mixed with common ingredients mixed in general skin cosmetics as needed.

[0071] When the 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, silicon oxide, talc, zinc oxide and the like may be used as carrier components.

[0072] When the dosage form of the cosmetic composition is a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component, and specifically, when the cosmetic composition is a spray, it can further include a propellant such as chlorofluorocarbons, propane / butane, or dimethyl ether, but is not limited thereto.

[0073] When the cosmetic composition is in the form of a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used, and for example, water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty acid ester, polyethylene glycol, sorbitan fatty acid ester may be used as a carrier component.

[0074] When the dosage form of the cosmetic composition is a suspension, liquid diluents (e.g., water, ethanol and propylene glycol), suspending agents (e.g., ethoxylated isostearyl alcohol, polyoxyethyl sorbitan ester, polyoxyethylene sorbitan ester, etc.), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth gum, etc. can be used as carrier components.

[0075] When the dosage form of the cosmetic composition is a surfactant-containing cleanser, fatty alcohol sulfates / esters, fatty alcohol ether sulfates / esters, sulfosuccinic acid monoesters, isethionates, imidazoline derivatives, methyl taurates / esters, sarcosinates / esters, fatty acid amide ether sulfates / esters, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glyceryl fatty acid esters, etc. can be used as carrier components.

[0076] When the dosage form of the cosmetic composition is shampoo, the peptide of the present invention can be mixed with basic ingredients for forming shampoo, such as thickeners, surfactants, viscosity modifiers, humectants, pH modifiers, preservatives and essential oils. CDE can be used as a thickener; LES (an anionic surfactant) and cocamidopropyl betaine (an amphoteric surfactant) can be used as surfactants; polyquaternium salts can be used as viscosity modifiers; glycerin can be used as a humectant; citric acid and sodium hydroxide can be used as pH modifiers. Grapefruit extract and the like can be used as preservatives; in addition, essential oils of cedar, mint and rosemary, as well as silk acid, pentaol or vitamin E can be added.

[0077] Ingredients included in the cosmetic composition in addition to the peptide of the present invention and the carrier component may include auxiliary ingredients generally used in cosmetic compositions such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, but are not limited thereto.

[0078] The peptide of the present invention can be included in the above composition at a concentration of 0.01 μM to 1000 μM, and specifically, the peptide of the present invention can be included in the above 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. , 0.1μM to 500μM, 0.1μM to 300μM, 0.1μM to 200μM; 1μM to 800μM, 1μM to 700μM, 1μM to 600μM, 1μM to 500μM, 1μM to 300μM, 1μM to 200μM; 5μM to 800μM, 5μM to 700μM, 5μM to 600μM, 5μM to 500μM, 5μM to 300μM or 5μM to 200μM are included in the above-mentioned composition; but are not limited thereto.

[0079] 3. Uses of the peptides of the present invention

[0080] In another aspect of the present invention, provided is a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for promoting hair growth, or inhibiting, preventing, treating or improving hair loss.

[0081] In another aspect of the present invention, a method for promoting hair growth, or inhibiting, preventing, treating or improving hair loss is provided, the method comprising administering a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide to a subject in need of promoting hair growth, or preventing, inhibiting, treating or improving hair loss.

[0082] In another aspect of the present invention, provided is use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the preparation of a medicament or cosmetic for promoting hair growth, or preventing, treating or improving hair loss.

[0083] Beneficial effects

[0084] The peptides of the present invention have the activity of promoting hair growth, or preventing or inhibiting hair loss. The peptides of the present invention promote the proliferation of hair follicle dermal papilla cells (HFDPCs) and human hair outer root sheath cells (HHORSCs), upregulate the expression of LEF-1 and cyclin D1, which are downstream genes of β-catenin in hair follicle dermal papilla cells (HFDPCs), and upregulate the expression of growth factors such as bFGF, HGF and VEGF.

[0085] However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The experimental results showing that the peptide of the present invention promoted the proliferation of HHFDPC cells when the HHFDPC cells were treated at 10 μM and 100 μM.

[0087] Figure 2 The experimental results showing that the peptide of the present invention promoted the proliferation of HHORSC cells when the HHORSC cells were treated at 50 μM and 100 μM.

[0088] Figure 3a The experimental results show that when HHFDPC cells were treated with the peptide of the present invention at 10 μM, 50 μM, and 100 μM, the expression of LEF-1 and cyclin D1, which are downstream factors of β-catenin, increased.

[0089] Figure 3b To measure Figure 3a The intensity of the bands in the graph shows the relative intensity values.

[0090] Figure 4a The experimental results show that when HHFDPC cells were treated with the peptide of the present invention at 10 μM, 50 μM and 100 μM, the activity of HHFDPC increased, thereby increasing the expression of growth factors such as bFGF, HGF and VEGF.

[0091] Figure 4b To measure Figure 4a The intensity of the bands in the graph shows the relative intensity values. DETAILED DESCRIPTION

[0092] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are used to specifically illustrate the present invention, and the content of the present invention is not limited to the following examples.

[0093] Preparation Example 1: Preparation of peptides and peptide complexes

[0094] An automatic peptide synthesizer (Milligen 9050, Millipore, USA) was used to synthesize a peptide having an amino acid sequence of SEQ ID NO: 1 shown in Table 1 below, and the synthesized peptides were purified using C18 reverse phase high performance liquid chromatography (HPLC) (Waters Associates, USA). The chromatographic column used was ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA).

[0095] [Table 1]

[0096] SEQ ID NO: Amino acid sequence of peptide 1 ASKVKVSMKF(Ala-Ser-Lys-Val-Lys-Val-Ser-Met-Lys-Phe)

[0097] The efficacy of the prepared peptide of SEQ ID NO: 1 was evaluated by the following experiment.

[0098] Experimental Example 1: Promoting the proliferation of human hair follicle dermal papilla cells (HHFDPC)

[0099] The effect of the peptide prepared in Preparation Example 1 above on the proliferation of human hair follicle dermal papilla cells (HHFDPC) was tested.

[0100] HHFDPC was added at 4 × 10 3 The cells were inoculated in a 96-well cell culture plate at a density of 10 cells / well and cultured for 24 hours under the condition of mesenchymal stem cell complete medium. Then, the medium was replaced with serum-free mesenchymal stem cell medium and cultured for another 24 hours. The cultured cell cultures were treated with the peptides prepared in Preparation Example 1 above at 10 μM and 100 μM, respectively, and treated with 1 μM minoxidil (MNX) as a positive control, and treated with no substance as a negative control (con), and the cultures were cultured at 37 ° C for another 72 hours. Thereafter, 10 μL of 5 mg / mL MTT solution was added thereto, and the mixture was incubated at 37 ° C in CO 2 After incubation in an incubator for 4 hours, the medium was removed, 100 μL of DMSO was added thereto, and the mixture was shaken for 10 minutes. Finally, the absorbance was measured at a wavelength of 540 nm using a spectrophotometer (Molecular Devices (SpectraMax iD3, CA, USA)).

[0101] The experimental results are as follows Figure 1 As shown, it was confirmed that the treatment of HHFDPCs with the peptide of Preparation Example 1 at 10 μM and 100 μM promoted the proliferation of HHFDPCs compared with the negative control (con).

[0102] Experimental Example 2: Promoting the proliferation of human hair outer root sheath cells (HHORSC)

[0103] The effect of the peptide prepared in Preparation Example 1 above on the proliferation of human hair outer root sheath cells (HHORSC) was tested.

[0104] HHORSC was divided into 4 × 10 3The cells were inoculated in a 96-well cell culture plate at a density of 10 cells / well and cultured for 24 hours under the condition of mesenchymal stem cell complete medium. Then, the medium was replaced with serum-free mesenchymal stem cell medium and cultured for another 24 hours. The cultured cell cultures were treated with 50 μM and 100 μM of the peptide prepared in Preparation Example 1 above, respectively, and 100 nM of epidermal growth factor (EGF) was used as a positive control, and no substance was used as a negative control (con), and the cultures were cultured for another 72 hours at 37 ° C. Subsequently, 10 μL of 5 mg / mL MTT solution was added thereto, and the mixture was incubated at 37 ° C in CO 2 After incubation in an incubator for 4 hours, the medium was removed, 100 μL of DMSO was added, and the mixture was shaken for 10 minutes. Finally, the absorbance was measured at a wavelength of 540 nm using a spectrophotometer (Molecular Devices (SpectraMax iD3, CA, USA)).

[0105] The experimental results are as follows Figure 2 As shown, it was confirmed that the treatment of HHORSC with the peptide of Preparation Example 1 at 50 μM and 100 μM promoted the proliferation of HHORSC, compared with the negative control group (con).

[0106] Experimental Example 3: Promoting the expression of downstream genes of β-catenin in human hair follicle dermal papilla cells

[0107] The effect of the peptide prepared in Preparation Example 1 above on the expression levels of lymphoid enhancer binding factor 1 (LEF-1) and cyclin D1, which are downstream genes of β-catenin, in human hair follicle dermal papilla cells (HHFDPCs) was confirmed by RT-PCR.

[0108] HHFDPC was added at 4 × 10 5The density of cells / well was inoculated in 6-well cell culture plates and cultured for 24 hours under the conditions of mesenchymal stem cell complete medium. Then, the culture medium was replaced with serum-free mesenchymal stem cell culture medium and cultured for another 24 hours. The cultured cell cultures were treated with 10 μM, 50 μM and 100 μM of the peptide prepared in the above preparation example 1, respectively, and treated with 10 ng / mL of recombinant human Wnt-3a (rhWnt-3a protein, R&D Systems (MN, USA)) as a positive control, and treated with no substance as a negative control (con), and these cultures were each cultured for another 24 hours at 37 ° C. After washing each culture with PBS, RNA was isolated by treating with 300 μL of eaay blue (Intron, South Korea). The isolated RNA was quantified using Nanodrop, and then cDNA synthesis was performed using a cDNA synthesis kit (Enzynomics, South Korea). Then, PCR reaction was performed using a PCR premix (Enzynomics, South Korea) and the primers shown in Table 2 below. The PCR products were electrophoresed on 1.5% agarose gel, and the bands were detected using the Bio-Rad gel imaging system.

[0109] [Table 2]

[0110]

[0111] The experimental results are as follows Figure 3a and Figure 3b As shown, it was confirmed that when HHFDPCs were treated with the peptide of Preparation Example 1 above at 10 μM, 50 μM and 100 μM, β-catenin affecting cell proliferation and differentiation was activated, thereby increasing the expression of LEF-1 and cyclin D1 which are downstream factors of β-catenin.

[0112] Experimental Example 4: Promoting the expression of growth factors in human hair follicle dermal papilla cells

[0113] The effect of the peptide prepared in Preparation Example 1 above on the expression level of growth factors in human hair follicle dermal papilla cells (HHFDPC) was determined by RT-PCR.

[0114] HHFDPC was added at 4 × 10 5The density of cells / well was inoculated in 6-well cell culture plates and cultured for 24 hours under the conditions of mesenchymal stem cell complete medium. Then, the culture medium was replaced with serum-free mesenchymal stem cell culture medium and cultured for another 24 hours. The cultured cell cultures were treated with 500nM, 5μM, and 50μM of the peptides prepared in the above preparation example 1, respectively, minoxidil (MNX) was used as a positive control, and no substance was used as a negative control (con), and each was cultured for another 24 hours at 37°C. After washing each culture with PBS, RNA was isolated by treating the culture with 300μL of eaay blue (Intron, South Korea). Nano drop was used to quantify the isolated RNA, and cDNA synthesis was then performed using a cDNA synthesis kit (Enzynomics, South Korea). Then, PCR reaction was performed using a PCR premix (Enzynomics, South Korea) and the primers shown in Table 3 below. The PCR products were electrophoresed on 1.5% agarose gel, and the bands were detected using the Bio-Rad gel imaging system.

[0115] [Table 3]

[0116]

[0117] The experimental results are as follows Figure 4a and Figure 4b As shown, it was confirmed that when HHFDPCs were treated with the peptide of Preparation Example 1 above at 10 μM, 50 μM and 100 μM, the activity of HHFDPCs was increased, thereby increasing the expression of growth factors such as bFGF, HGF and VEGF.

[0118] Preparation Example 2: Preparation of pharmaceutical composition

[0119] 2-1. Preparation of powder

[0120] Peptide 2g of the present invention

[0121] Lactose 1g

[0122] The above ingredients are mixed and filled into airtight bubbles to prepare powders.

[0123] 2-2. Preparation of tablets

[0124] Peptide of the present invention 100 mg

[0125] Corn starch 100mg

[0126] Lactose 100mg

[0127] Magnesium stearate 2mg

[0128] The above ingredients are mixed and prepared into tablets by tableting according to a conventional tablet preparation method.

[0129] 2-3. Preparation of capsules

[0130] Peptide of the present invention 100 mg

[0131] Corn starch 100mg

[0132] Lactose 100mg

[0133] Magnesium stearate 2mg

[0134] The above ingredients are mixed and filled into each gelatin capsule according to a conventional capsule preparation method to prepare capsules.

[0135] 2-4. Preparation of pills

[0136] Peptide 1g of the present invention

[0137] Lactose 1.5g

[0138] Glycerin 1g

[0139] Xylitol 0.5g

[0140] After the above ingredients are mixed, they are made into pills in an amount of 4 g per pill according to a conventional method.

[0141] 2-5. Preparation of granules

[0142] Peptide of the present invention 150 mg

[0143] Soybean extract 50mg

[0144] Glucose 200mg

[0145] Starch 600mg

[0146] The above ingredients were mixed, 100 mg of 30% ethanol was added thereto, and the mixture was dried at 60° C. to form granules, which were then filled into bags.

[0147] Preparation Example 3: Preparation of cosmetic composition

[0148] 3-1. Preparation of cream

[0149] Peptide of the present invention: 4.6 parts by weight

[0150] Cetearyl alcohol: 2.8 parts by weight

[0151] Beeswax: 2.6 parts by weight

[0152] Stearic acid: 1.4 parts by weight

[0153] Lipophilic monostearate glyceryl: 2 parts by weight

[0154] PEG-100 stearate: 1 part by weight

[0155] Sorbitol sesquioleate: 1.4 parts by weight

[0156] Jojoba oil: 4 parts by weight

[0157] Squalene: 3.8 parts by weight

[0158] Polysorbate 60: 1.1 parts by weight

[0159] Macadamia nut oil: 2 parts by weight

[0160] Tocopherol acetate: 0.2 parts by weight

[0161] Methyl polysiloxane: 0.4 parts by weight

[0162] Ethyl p-hydroxybenzoate: 0.1 parts by weight

[0163] Propyl paraben: 0.1 parts by weight Euxyl K-400: 0.1 parts by weight 1,3-Butanediol: 7 parts by weight

[0164] Methyl paraben: 0.05 parts by weight

[0165] Glycerol: 6 parts by weight

[0166] d-Panthenol: 0.2 parts by weight

[0167] Triethanolamine: 0.2 parts by weight pt 41891: 0.2 parts by weight pH 2 O: 46.05 parts by weight

[0168] 3-2. Preparation of lotion Peptide of the present invention: 3.5 parts by weight

[0169] Cetearyl alcohol: 1.6 parts by weight

[0170] Stearic acid: 1.4 parts by weight

[0171] Lipophilic monostearate glyceryl: 1.8 parts by weight PEG-100 stearate: 2.6 parts by weight

[0172] Sorbitol sesquioleate: 0.6 parts by weight

[0173] Squalene: 4.8 parts by weight

[0174] Macadamia nut oil: 2 parts by weight

[0175] Jojoba oil: 2 parts by weight

[0176] Tocopherol acetate: 0.4 parts by weight

[0177] Methyl polysiloxane: 0.2 parts by weight

[0178] Ethyl p-hydroxybenzoate: 0.1 parts by weight

[0179] Propyl p-hydroxybenzoate: 0.1 parts by weight 1,3-Butanediol: 4 parts by weight

[0180] Methyl paraben: 0.1 parts by weight

[0181] Xanthan gum: 0.1 parts by weight

[0182] Glycerin: 4 parts by weight

[0183] d-Panthenol: 0.15 parts by weight

[0184] Allantoin: 0.1 parts by weight

[0185] Calcium carbonate (2% aq.Sol): 4 parts by weight

[0186] Triethanolamine: 0.15 parts by weight

[0187] Ethanol: 3 parts by weight

[0188] pt 41891: 0.1 parts by weight

[0189] pH 2 O: 48.3 parts by weight

[0190] 3-3. Preparation of skin softening lotion

[0191] Peptide of the present invention 0.2wt%

[0192] Ethanol 10.0wt%

[0193] Polyoxyethylene sorbitan polylaurate 1.0wt%

[0194] Methyl paraben 0.2wt%

[0195] Glycerol 5.0wt%

[0196] 1,3-Butanediol 6.0wt%

[0197] Spices to taste

[0198] Appropriate amount of pigment

[0199] Purified water

[0200] Total 100

[0201] 3-4. Preparation of nutritional lotion

[0202] Peptide of the present invention 0.1wt%

[0203] Vaseline 2.0wt%

[0204] Sorbitan sesquioleate 0.8wt%

[0205] Polyoxyethylene oleyl ethyl 1.2wt% Methyl paraben Appropriate amount Propylene glycol 5.0wt%

[0206] Ethanol 3.2wt%

[0207] Carboxyvinyl polyol 18.0wt%

[0208] Appropriate amount of pigment

[0209] Spices to taste

[0210] Purified water

[0211] Total 100

[0212] 3-5. Preparation of Essence

[0213] Peptide of the present invention 5.0wt%

[0214] Propylene glycol 10.0wt%

[0215] Glycerol 10.0wt%

[0216] Aqueous solution of sodium hyaluronate (1%) 5.0 wt% Ethanol 3.2 wt%

[0217] Polyoxyethylene hydrogenated castor oil 1.0wt%

[0218] Methyl paraben 0.1wt%

[0219] Spices to taste

[0220] Purified water

[0221] Total 100

[0222] 3-6. Preparation of facial mask

[0223] Peptide of the present invention 0.5wt%

[0224] Glycerol 5.0wt%

[0225] Propylene glycol 4.0wt%

[0226] Polyvinyl alcohol 15.0wt%

[0227] Ethanol 8.0wt%

[0228] Polyoxyethylene oleyl ether 1.0wt%

[0229] Methyl paraben 0.2wt%

[0230] Spices to taste

[0231] Appropriate amount of pigment

[0232] Purified water

[0233] Total 100

[0234] In a preferred embodiment, the composition of the above ratio is a mixture of suitable components, but the components or the mixing ratio thereof may be arbitrarily modified according to regional or national preferences (such as demand segments, national needs and intended use).

[0235] In the above, representative embodiments of the present application are described by way of exemplary embodiments; however, the scope of the present application is not limited to the above specific embodiments, and those skilled in the art may make appropriate changes within the scope described in the claims of the present application.

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 improving hair loss, comprising the peptide according to claim 1 as an active ingredient.

3. A pharmaceutical composition for preventing or treating hair loss, comprising the peptide according to claim 1 as an active ingredient.

4. The pharmaceutical composition according to claim 3, in, The peptides promote the proliferation of hair follicle dermal papilla cells (HFDPCs) or hair outer root sheath cells (HORSCs).

5. The pharmaceutical composition according to claim 3, in, In the hair follicle dermal papilla cells (HFDPCs), the peptide: (i) upregulating gene expression of one or more factors selected from the group consisting of LEF-1 and cyclin D1, which are downstream factors of β-catenin; or (ii) upregulating the expression of one or more growth factors selected from the group consisting of bFGF, HGF and VEGF.

6. The pharmaceutical composition according to claim 3, in, The pharmaceutical composition is a skin external preparation.

7. A cosmetic composition for promoting hair growth, or preventing or improving hair loss, comprising the peptide according to claim 1 as an active ingredient.

8. The cosmetic composition according to claim 7, in, The peptides promote the proliferation of hair follicle dermal papilla cells (HFDPCs) or hair outer root sheath cells (HORSCs).

9. The cosmetic composition according to claim 7, in, In the hair follicle dermal papilla cells (HFDPCs), the peptide: (i) upregulating the gene expression of one or more factors selected from the group consisting of LEF-1 and cyclin D1, which are downstream factors of β-catenin; or (ii) upregulating the expression of one or more growth factors selected from the group consisting of bFGF, HGF and VEGF.

10. The cosmetic composition according to claim 7, in, The cosmetic composition is a skin external preparation.

11. The cosmetic composition according to claim 7, in, The cosmetic composition is in one or more dosage forms selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations and sprays.

12. The cosmetic composition according to claim 7, in, The cosmetic composition is one or more preparations selected from the group consisting of hair tonic, hair cream, hair shampoo, hair water, hair dye, hair conditioner, spray-type hair spray, aerosol-type hair spray, hair wax and gel.

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