阅读说明：本技术 包含脂联素衍生肽的促进毛发生长用组合物 (Hair growth promoting composition comprising adiponectin-derived peptide ) 是由 郑振镐 权五祥 金恩珠 金镇用 李东勋 于 2018-09-03 设计创作，主要内容包括：本发明涉及一种包含脂联素衍生肽片段的组合物,并且更具体地,涉及一种促进毛发生长用化妆品组合物和药物组合物,其包含一种或多种脂联素衍生肽作为活性成分,该一种或多种脂联素衍生肽选自由SEQ ID NO:1至21所示的氨基酸序列组成的组。该脂联素衍生肽能够激活毛囊毛乳头细胞和外根鞘细胞的细胞分裂、促进毛囊中毛发的生长、并使毛乳头细胞中毛发生长因子的表达增加,因此,包含脂联素衍生肽的本发明组合物可以有效地用于促进毛发生长并抑制脱发。(The present invention relates to a composition comprising adiponectin-derived peptide fragments, and more particularly, to a cosmetic composition and a pharmaceutical composition for promoting hair growth, which comprise one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences represented by SEQ ID NOs 1 to 21 as an active ingredient. The adiponectin-derived peptide can activate cell division of hair follicle papilla cells and outer root sheath cells, promote hair growth in hair follicles, and increase expression of hair growth factors in hair follicle papilla cells, and therefore, the composition of the present invention comprising the adiponectin-derived peptide can be effectively used for promoting hair growth and inhibiting hair loss.)

1. A cosmetic composition for promoting hair growth, comprising: one or more adiponectin-derived peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 21 as an active ingredient.

2. The cosmetic composition of claim 1, comprising: one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences shown in SEQ ID NOs 16 and 21 as an active ingredient.

3. The cosmetic composition according to claim 1, wherein the concentration of the adiponectin derived peptide is between 0.001mM and 20 mM.

4. The cosmetic composition according to claim 1, wherein the cosmetic composition is selected from any one of the group consisting of: a scalp care agent, soap, hair tonic, shampoo, hair dye, hair film, hair gel, lotion, hair conditioner, hair oil, mousse, cream, solid, solution, emulsion, dispersion, micelle, liposome, ointment, toner, essence, patch, or spray.

5. A pharmaceutical composition for promoting hair growth, comprising: one or more adiponectin-derived peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 21 as an active ingredient.

6. The pharmaceutical composition of claim 5, comprising: one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences shown in SEQ ID NOs 16 and 21 as an active ingredient.

7. The pharmaceutical composition according to claim 5, wherein the concentration of the adiponectin derived peptide is between 0.001mM and 20 mM.

8. A method of promoting hair growth comprising: treating or administering to a subject a cosmetically effective amount of one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs 1 through 21.

9. The method for promoting hair growth according to claim 8, wherein the subject is a human.

Technical Field

The present invention relates to a hair growth promoting composition comprising a peptide fragment (or small peptide) derived from adiponectin.

Background

The amount of human hair is about 130 tens of thousands or more, the amount of hair on the scalp is 100,000 to 150,000, each hair has a different cycle, and is grown, maintained and shed through three stages of anagen (active growth phase), catagen (apoptotic decay phase) and telogen (resting phase). These cycles are repeated for 3 to 6 years, as a result of which, on average, 50 to 100 hairs are typically shed per day. Generally, the term "alopecia" refers to a condition in which the amount of hair is less than normal or absent for some reason.

Nowadays, causes of alopecia include internal causes such as action of male hormones, and external causes such as mental stress in daily life and lipid peroxidation accumulation in the scalp, and these factors are known to cause alopecia symptoms in a complicated manner. In recent years, the number of people suffering from alopecia in women as well as men has increased due to increased stress caused by changes in dietary life and social environment, and the number of people suffering from such scalp and hair abnormality has gradually increased and has become less aged.

When discussing the effects of androgens on hair loss, among many factors, the following play a major role in inhibiting the production of hair follicles in the hairy roots: testosterone in humans binds to 5 α -reductase to produce dihydrotestosterone (hereinafter referred to as "DHT"), which binds to androgen receptors in hair roots. Therefore, research is being actively conducted to develop a substance for preventing hair loss by using a 5 α -reductase inhibitor. In addition to the action of enzymes, hair loss may also result from nutritional deficiencies, dry scalp, stress, and the like. If it is alopecia caused by these causes, sufficient nutrition, scalp management, and intake or application of antioxidants may prevent alopecia and promote hair growth (Korean patent laid-open No. 10-2016-.

As hair growth agents currently on the market, preparations containing, as main ingredients, minoxidil from Upzone, usa, mucopolysaccharide auxin from cris, italy, and the like, for example, have been used, but there are problems in that the effects are not remarkable and side effects occur. In addition, Propecia (component name: finasteride, developed by merck as a therapeutic agent for prostatic hyperplasia, which is then introduced into the treatment of alopecia) is known as a substance inhibiting the activity of 5 α -reductase, an enzyme acting on testosterone metabolism of male hormone in hair follicles, but it requires continuous administration and has problems of causing side effects such as decreased sexual function, allergy, depression, and the like.

Further, since female hair loss is known to be caused by various causes, as one of the known causes, for example, blood disorders of the scalp occur with the increase of age because the scalp development stops faster than the skull, there is a problem that narrow treatment (narrow treatment) such as hormone inhibition as described above is only a short-sight treatment regimen.

In view of these problems, studies have been made on a composition for preventing hair loss or promoting hair growth, which is nontoxic even when used for a long time and does not cause side effects, by containing an extract derived from natural substances as an active ingredient. However, in the case of related products containing extracts derived from natural substances, there are many cases where it is not sufficient to exhibit desired effects of preventing hair loss, promoting hair growth and improving hair (Korean patent laid-open No. 10-2016-.

On the other hand, adiponectin, a fat factor (adipocaine), is a protein hormone secreted specifically by adipocytes and plays an important role in the regulation of cardiovascular diseases such as hyperglycemia, hyperinsulinemia, obesity, and arteriosclerosis by enhancing the function of insulin and inducing insulin resistance. In addition, adiponectin has a function of inhibiting metastasis of cancer cells and inflammatory reaction. Adiponectin can not only proliferate keratinocytes but also promote the expression of filaggrin, hyaluronic acid and extracellular matrix in the skin, thereby exerting functions such as wound healing, inhibition of fibrosis, improvement of skin wrinkles and moisture retention (korean patent laid-open No. 10-2015-. In addition, adiponectin is disclosed to have an effect of promoting hair growth in vitro.

Adiponectin consists of 244 amino acids and consists of a signal sequence, a collagen-like domain at the N-terminus, and a C1 q-like globular domain at the C-terminus. The hexamer and the 400kDa high molecular complex (HMW complex) are known to be the major oligomers, and the HMW complex is known to be more active than the low molecular complex (LMW complex).

Many researchers and pharmaceutical companies at home and abroad have targeted the development of adiponectin-derived peptides having various physiological activities in the related fields as research targets, but the probability of ultimate success is relatively low because they have difficulty in forming polymers in vivo. Therefore, there is a need to develop short adiponectin-derived peptides that can be applied to the skin and have excellent physiological activity.

Disclosure of Invention

[ problem ] to

The present invention aims to provide a hair growth promoting composition comprising an adiponectin-derived peptide.

[ solution ]

In order to solve the above problems, the present invention provides a cosmetic or pharmaceutical composition for promoting hair growth, comprising one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences represented by SEQ ID NOs 1 to 21 as an active ingredient.

According to one embodiment of the present invention, the composition of the present invention preferably comprises one or more adiponectin-derived peptides selected from the group consisting of SEQ ID NOs 16 and 21 as an active ingredient, but is not limited thereto.

According to one embodiment of the present invention, the adiponectin-derived peptide may be included in the cosmetic or pharmaceutical composition in a concentration of 0.001mM to 20mM, but is not limited thereto.

According to another embodiment of the present invention, the cosmetic composition may be selected from the group consisting of formulations such as scalp conditioner, soap, hair tonic, shampoo, hair dye, hair mask, hair gel, lotion, hair conditioner, hair oil, mousse, cream, solid, solution, emulsion, dispersion, micelle, liposome, ointment, toner, essence, patch or spray, but is not limited thereto.

Further, the present invention provides a method of promoting hair growth, comprising: treating or administering to a subject a pharmaceutically or cosmetically effective amount of one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs 1 through 21.

According to an embodiment of the present invention, the subject may be a human, but is not limited thereto.

[ advantageous effects ]

The present invention relates to a composition comprising an adiponectin-derived peptide having a hair growth promoting effect. The adiponectin-derived peptide can activate cell division of hair follicle papilla cells and outer root sheath cells, promote growth of hair in hair follicles, and increase expression of hair growth factors in hair follicle papilla cells, and thus the composition of the present invention comprising the adiponectin-derived peptide can be effectively used for promoting hair growth and inhibiting hair loss.

Drawings

Fig. 1 is a photograph showing the results of a skin permeability test of the adiponectin-derived peptide of the present invention.

Fig. 2 is a graph showing the effect of the adiponectin-derived peptides of the present invention on cell division and activation in human hair follicle-derived hair papilla cells and outer root sheath cells, wherein Con represents a negative control, Mnx represents minoxidil used as a positive control, P1 and P2 represent adiponectin peptide positive controls, and P3 to P10 represent adiponectin-derived peptides prepared in the present invention.

Fig. 3 is a graph and photograph showing the effect of adiponectin-derived peptides of the present invention on human hair follicle growth.

FIG. 4 is a photograph of fluorescent staining of Ki-67 positive cells expressed around hair follicles treated with an adiponectin-derived peptide of the present invention.

Fig. 5 is a graph showing the effect of the adiponectin-derived peptides of the present invention on the expression of hair growth factor in hair papilla cells of human hair follicles.

FIG. 6 is an electrophoretogram showing the effect of the adiponectin-derived peptide of the present invention on phosphorylation of a subsignal protein of an adiponectin receptor in human outer root sheath cells.

Fig. 7 is a photograph showing the effect of adiponectin derived peptides of the present invention on the induction of hair growth stage.

Figure 8 shows the crystal structure of the structural position where the adiponectin peptide of the present invention is present in adiponectin protein.

Detailed Description

In order to solve the above objects, the present invention provides a cosmetic or pharmaceutical composition for promoting hair growth, comprising one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences represented by SEQ ID NOs 1 to 21 as an active ingredient.

The adiponectin-derived peptides of the present invention are peptide fragments derived from adiponectin having a length of 4 amino acids (4-mer) or a length of 5 amino acids (5-mer), and have the amino acid sequences shown in table 1 below.

[ Table 1]

According to one embodiment of the present invention, the adiponectin-derived peptide is preferably contained in the cosmetic composition at a concentration of 0.001mM to 20mM, more preferably 0.01mM to 10mM, but not limited thereto. When the peptide concentration is less than 0.001mM, it is difficult to obtain the effect of promoting hair growth or inhibiting hair loss, while when the peptide concentration exceeds 20mM, there is a problem in that the production economy is lowered because no significant increase in the effect due to an increase in the content is exhibited.

The peptides of the invention can be prepared by conventional chemical synthesis, such as solid phase peptide synthesis, and can also be prepared in such a way that: the method is not limited thereto, but includes culturing a microorganism transformed with a recombinant vector containing a nucleic acid encoding a peptide, expressing the peptide, and then purifying the peptide by a conventional method.

The cosmetic composition according to the present invention may additionally contain other ingredients having a synergistic effect on the effect of the peptide, in addition to the peptide, within a range that does not impair the desired effect of the present invention. For example, the cosmetic composition of the present invention may contain not only any other substance known to promote hair growth, but also conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and perfumes, or carriers.

The formulation of the cosmetic composition of the present invention is not particularly limited, and may be prepared in any formulation that is generally prepared in the art, for example, may be formulated as 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, a milky foundation, a waxy foundation, a spray, and the like. Meanwhile, the cosmetic composition may also be prepared in the form of, for example, scalp care agents, soaps, hair tonics, shampoos, hair dyes, hair masks, hair sprays, lotions, hair conditioners, hair oils, mousses, creams, solids, solutions, emulsions, dispersions, micelles, liposomes, ointments, lotions, serums, patches or sprays.

When the cosmetic composition is in the form of a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier ingredient. In the case of powders or sprays, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as carrier component. In particular, in the case of sprays, propellants such as chlorofluorocarbons, propane/butane or dimethyl ether may also be included.

When the dosage form of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier may be used as a carrier ingredient. For example, the carrier component includes water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol oil, aliphatic glycerides, polyethylene glycol, or sorbitan fatty acid esters.

When the dosage form of the present invention is a suspension, a liquid diluent (e.g., water, ethanol or propylene glycol), a suspending agent (e.g., ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, or the like may be used as a carrier ingredient. In the case of a surfactant-containing detergent, fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, ethoxylated glycerin fatty acid ester, and the like can be used as the carrier component.

According to a specific embodiment of the present invention, the adiponectin-derived peptide of the present invention is excellent in skin permeability (see fig. 1), promotes cell division of hair papilla cells and outer root sheath cells (see fig. 2), and promotes growth of hair follicles (see fig. 3).

Furthermore, according to another embodiment of the present invention, the adiponectin-derived peptides of the present invention increase the expression of hair growth factors such as FGF7, FGF10, HGF, IGF, etc. in hair papilla cells of human hair follicles (see fig. 4), increase the phosphorylation of the semaphorin protein of adiponectin receptor in human outer root sheath cells (see fig. 5), and can significantly induce the hair growth stage (see fig. 6).

Therefore, the adiponectin-derived peptide of the present invention activates cell division of hair papilla cells and outer root sheath cells of hair follicles, promotes hair growth in hair follicles, and increases expression of hair growth factors in hair papilla cells, and thus, the composition of the present invention comprising the adiponectin-derived peptide can be effectively used as an active ingredient in a cosmetic composition to promote hair growth and inhibit hair loss.

On the other hand, the administration route of the pharmaceutical composition for promoting hair growth according to the present invention includes skin, oral, intravenous, intramuscular, intraarterial, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal route, preferably oral or parenteral administration, but is not limited thereto. "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intralesional injection or infusion techniques.

In addition to the adiponectin-derived peptides of the present invention, the pharmaceutical compositions of the present invention may also comprise one or more active ingredients that exhibit the same or similar function. The composition can be formulated according to conventional methods, and can be used in the form of oral preparations (such as powder, granule, tablet, capsule, suspension, emulsion, syrup, etc.), external preparations, suppositories, sterile injections, etc.

The oral solid preparation comprises powder, granules, tablets, capsules, soft capsules, pills and the like. Liquid preparations for oral administration may correspond to suspensions, oral liquids, emulsions, syrups and the like, and may contain various excipients in addition to water and liquid paraffin, which are generally used as simple diluents, for example, preparations for parenteral administration such as wetting agents, sweeteners, aromatics, preservatives and the like, may be formulated according to conventional methods, and used in the form of external preparations (such as powders, granules, tablets, capsules, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols and the like) and sterile injections and the like, respectively. Preferably, a pharmaceutical composition for external application to the skin, such as a cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, or cataplasm, may be prepared and used, but is not limited thereto. As non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), and the like may be used. As the base of the suppository, there can be used vicisol (witepsol), polyethylene glycol, tween 61, cacao butter, lauric acid glyceride (laurinum), glycerogelatin, and the like.

The composition may additionally comprise adjuvants such as preservatives, stabilisers, humectants or emulsification promoters, salts and/or buffers for controlling osmotic pressure, and other therapeutic substances, and may be formulated according to conventional methods, for example mixing, granulating or coating.

The pharmaceutical composition of the present invention may vary depending on various factors, including age, body weight, general health, sex of the subject, administration time, administration route, excretion rate, drug combination, and severity of specific diseases.

When a pharmaceutical composition containing the adiponectin-derived peptide of the present invention as an active ingredient is formulated into a unit dosage form, the amount of the adiponectin-derived peptide as an active ingredient contained in the unit dosage is preferably about 0.01mg to 1,500mg, and the dose required for adult treatment is generally in the range of about 1mg to 500mg per day, depending on the frequency and intensity of administration, but is not limited thereto. When administered intramuscularly or intravenously to adults, a total dose of about 5mg to 300mg per day is generally sufficient to meet the needs of a single dose, but for some patients, higher daily doses may be more desirable. The compositions of the present invention may be used alone or in combination with surgery, radiation therapy, hormonal therapy, chemotherapy and methods of using biological response modifiers.

Further, the present invention provides a method of promoting hair growth, comprising: treating or administering to a subject a pharmaceutically or cosmetically effective amount of one or more adiponectin-derived peptides selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs 1 through 21.

The pharmaceutically or cosmetically effective dose is 0.0001mg/kg to 100mg/kg, preferably 0.001mg/kg to 10mg/kg, but is not limited thereto. The amount of treatment or administration can be adjusted depending on the weight, age, sex and health condition of the particular patient, diet, administration period, administration method, elimination rate, severity of disease, etc. The treatment or administration may be once or several times per day.

The subject is a vertebrate, preferably a mammal, more preferably a human or experimental animal, such as a mouse, rabbit, guinea pig, hamster, dog and cat, most preferably a human, but is not limited thereto.

In the method of treatment or administration, the composition may be administered orally or parenterally, and in the case of parenteral administration, the composition may be administered by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, injection of the dura mater of the uterine cavity, cerebrovascular injection or intrathoracic injection.

Hereinafter, the present invention will be described in more detail by examples.

However, the following examples are merely illustrations of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1 preparation of adiponectin-derived peptides

For the synthesis of the peptides of the invention, the Solid Phase Peptide Synthesis (SPPS) method of Merrifield (J.Am.chem.Soc.,85 (14)), 2149-. First, to synthesize the peptide of the present invention, the synthesis was started using 2-chlorotrityl chloride (CTC) resin and in the first step, the 9-fluorenylmethoxycarbonyl (Fmoc) amino acid was coupled using N, N-Diisopropylethylamine (DIEA) in Dichloromethane (DCM) solvent. In the next step, N-Diisopropylethylamine (DIEA) and N- [ (dimethylamino-) -1H-1,2, 3-triazolo [4,5-b ] are used]Pyridin-1-yl-methylene]N-Methylmethylammonium hexafluorophosphate N-oxide (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt) or N- [ (1H-benzotriazol-1-yl) (dimethylamino) methylene]-N-Methylmethylammonium hexafluorophosphate N-oxide (HBTU) and hydroxybenzotriazole (HOBt) coupling of Fmoc amino acids to extend the peptide chain. At this time, the Synthesis was performed from C-terminus to N-terminus, and after Fmoc-amino acid coupling to the amino terminus, the Fmoc group was removed with a 20% piperidine solution (Solid-Phase Synthesis: A Practical Guide) (1 st Guide)Version), CRC press, 848) and washed several times with Dimethylformamide (DMF) before coupling. Finally, after coupling the Fmoc-amino acid, the Fmoc group was removed with 20% piperidine solution and coupled with the acyl group by using carboxylic acid and N, N' -Diisopropylcarbodiimide (DIC), 1-hydroxy-7-azabenzotriazole (HOAt), followed by washing several times with DMF and drying. Here, trifluoroacetic acid (TFA) -H was added₂O-triisopropylsilane (95:2.5:2.5, v/v) solution, reacted for 2 to 3 hours to remove the protecting group and separate the peptide from the resin, and then the peptide was immersed in ether to synthesize peptides having the amino acid sequences shown in Table 1 above, respectively.

The purity of the crude peptide obtained by the above procedure was confirmed using RP-HPLC (2996 detector, 515 pump, Waters) with a gradient solvent composition of acetonitrile and water containing 0.1% TFA. As a result, the peptide having a purity of 95% or less was isolated and purified by Prep-LC to obtain a peptide having a purity of 97% or more. After elemental analysis using an elemental analyzer (EA1112, CE Instrument, italy) to confirm the exact composition of the peptide, it was confirmed that the element content of the peptide was consistent with the element content calculated from the amino acid sequence, and thus the peptide had the correct amino acid sequence.

Example 2 skin Permeability assay of adiponectin-derived peptides

Adiponectin is broken down into smaller units comprising globular domains, subunits with globular domains are more active than adiponectin with full length, and these adiponectin receptors are known as adiponectin receptor 1(AdipoR1) and adiponectin receptor 2(AdipoR2) (Crystal structures of the human adiponectin receptor), Nature, 2015, 4/16/520 (7547):312-6, doi: 10.1038/Nature 14301). Therefore, the skin permeability of the adiponectin-derived peptide of the present invention prepared in example 1 was most similar to that of human skin, and was verified using pig skin commonly used in the permeability test. After cutting the pigskin into sizes of 2 cm in width and height, the corresponding peptide stock with FITC fluorescence (positive control 8-mer length of peptide 2, 4-mer peptide 4) and DMSO were prepared by concentration and coated in sufficient quantity on the pigskin. Shading and placing the pigskin in an incubator at 37 ℃ for 2 hours. After flash freezing with LN2, cryosections were performed. Treated with DAPI (nuclear staining) for 5 minutes, then washed, and observed with a confocal microscope.

As a result, it was confirmed that the adiponectin derived peptide of the present invention was very excellent in permeability to the skin as compared with the 8 amino acid length peptide used as a control (fig. 1).

Example 3 cell division and differentiation of adiponectin-derived peptides into human Hair follicle-derived Hair papilla cells and outer root sheath cells Effect of activation

Primary cultured human hair follicle-derived hair papilla cells and outer root sheath cells were subcultured to 2 to 3 passages, and then treated at a concentration of 1mM with minoxidil (minoxidil is generally used as a drug for preventing hair loss and treating hair growth) as a positive control, and further treated at concentrations of 0.5 μ g/ml and 5 μ g/ml with adiponectin-derived peptides of the present invention prepared in example 1 alone. After 48 hours, the activated living cells were stained with MTT solution and compared with a negative control group to confirm the division and activation degree of the cells.

As a result, it was confirmed that the adiponectin-derived peptides of the present invention promote cell division of hair papilla cells and outer root sheath cells, and in particular, the adiponectin-derived peptides have more excellent effects of peptides P5 and P10 and a combination of the two peptides than minoxidil used as a positive control (fig. 2).

Example 4 Effect of adiponectin-derived peptides on human Hair follicle growth

In vitro hair follicle organ culture tests were performed using human hair follicles obtained from volunteers. Minoxidil, treated at a concentration of 1mM using minoxidil (minoxidil is generally used as a drug for preventing hair loss and treating hair growth) as a positive control, and additionally treated at a concentration of 5 μ g/ml with adiponectin-derived peptides of the present invention prepared in example 1 alone, and effects on hair follicle growth of humans were confirmed. After the human hair follicle culture experiment, the hair lengths on days 3 and 6 were measured and compared with the negative control group, thereby confirming the effect of promoting the hair growth of human hair follicles.

As a result, the adiponectin-derived peptides of the present invention promoted the growth of hair follicles, and in particular, adiponectin peptides combined with peptides P5 and P10 significantly promoted the growth of hair in adiponectin-derived peptides as compared with minoxidil used as a positive control (fig. 3). In addition, the hair follicles were rapidly frozen and then sectioned into tissue sections, cells in the tissue were labeled with a Ki-67 antibody (which is a marker of cell division and activation), and fluorescently labeled with a fluorescent secondary antibody capable of detecting the Ki-67 antibody, and then the number of Ki-67 positive cells expressed around the hair follicles was examined in a dark room using a fluorescence microscope. As a result, it could be confirmed that the number of Ki-67 positive cells was significantly increased in the adiponectin peptide group combined with P5 and P10, compared to the negative control group (fig. 4).

Example 5 Effect of adiponectin-derived peptides on Hair growth factor expression in Hair papilla cells of human Hair follicles

Primary cultured human hair follicle-derived hair papilla cells were subcultured to 2 to 3 passages, and then treated with minoxidil (minoxidil is conventionally used as a drug for preventing hair loss and treating hair growth) as a positive control at a concentration of 1mM, and further treated with adiponectin-derived peptides of the present invention prepared in example 1 alone at concentrations of 0.5 μ g/ml and 5 μ g/ml. After 3 hours, mRNA was extracted from papilla cells, synthesized again into cDNA, and the effect on the expression of FGF7, FGF10, HGF, and IGF, which are called hair growth factors, was confirmed using a real-time quantitative PCR method, as compared to the negative control group.

As a result, the adiponectin derived peptides of the present invention increased the expression of FGF7, FGF10, HGF and IGF, and in particular, the expression of FGF7 and FGF10, which are the most important hair growth factors, significantly increased compared to minoxidil used as a positive control (fig. 5).

Example 6 phosphorylation of adiponectin-derived peptides on the semaphorin protein of adiponectin receptor in human outer root sheath cells Function of

To confirm whether adiponectin peptides actually act through adiponectin receptors, primary-cultured human hair follicle-derived outer root sheath cells were divided into adiponectin receptor inhibitor-treated groups and untreated groups, which were treated at concentrations of 0 μ g/ml, 0.05 μ g/ml, 0.1 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 5 μ g/ml and 10 μ g/ml using adiponectin-derived peptides of the combination of P5 and P10 prepared in example 1, respectively. As a positive control, after treatment with adipon (an agonist of adiponectin, which is a selective agonist of adiponectin receptor, such as adipoR1 and adipoR 2.10 minutes, proteins were extracted only from cells, purified, and then the amount of the proteins was uniformly quantified.thereafter, each protein was separated by size using Western blotting, and detected using an antibody specific to the target protein, and then stained using a peroxidase method.thus, the effect of phosphorylation of acetyl-CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK) as adiponectin receptors (sub-signal proteins of adipoR1 and adipoR2) was confirmed.

As a result, it was confirmed that the adiponectin derived peptide of the present invention significantly increased the phosphorylation of ACC and AMPK (signaling protein of adiponectin receptor), which were increased at the same level as adipon used as a positive control (fig. 6).

Example 7 adiponectin derived peptide pairs for inducing hair growthPeriod of timeFunction of

It is known that in 7 to 8-week-old mice, all hairs enter the resting stage, and then the hairs shift to the anagen stage. The effect of the adiponectin derived peptides of the invention on the induction of the growth phase was then confirmed using C57BL/6 mice. 3% minoxidil (conventionally used as a drug for preventing hair loss and treating hair growth) and a combination of adiponectin-derived peptides P5 and P10 prepared in example 1 at a concentration of 0.1mM were applied to the lower back skin of mice once a day, and then hair growth was confirmed.

As a result, in the group treated with the adiponectin-derived peptide of the present invention, the proportion of the area of anagen hair growth significantly increased in the area where hair was initially removed after about 3 weeks. Thereafter, when biopsies of the corresponding regions were performed to assess the formation of hair follicles in anagen phase, it was confirmed that similar to the group treated with minoxidil (used as a positive control), the formation of hair follicles was also promoted in the group coated with the adiponectin-derived peptide (fig. 7).

Example 8 structural confirmation of adiponectin peptide in adiponectin and Effect by Forming Polymer in vivo Performance of

The structural position of the adiponectin peptide prepared by the present invention and the possibility of acting with adiponectin receptors in vivo were analyzed by analyzing the three-dimensional structure of adiponectin protein present in the human body.

As a result, with regard to the adiponectin peptide, it was found that the adiponectin peptide exists in the form of a trimer since the globular domain (known as the action site of adiponectin) is repeated 3 times in total. In particular, since the site of action is distributed on the outermost side of the globular structure of the globular domain, the probability of actually acting on adiponectin receptors is high. In particular, in the case of peptide P5, it was confirmed that a peptide sequence repeated 3 times in adiponectin protein forms a polymer when the three-dimensional protein structure was examined. When the polymer is composed of lipophilic amino acids inside the polymer and hydrophilic amino acids outside the polymer, and many hydrophilic substances actually permeate into the human body, the polymer is formed again, and the possibility of acting on adiponectin receptors can be confirmed (fig. 8).

Example 9 search for sequence overlap of adiponectin peptides with other proteins

In the case of the adiponectin peptide produced by the present invention, since the adiponectin peptide is a short amino acid sequence consisting of a 5-mer, it may actually overlap with an amino acid sequence contained in other proteins in the human body. In this case, it is difficult to display a sequence that is specifically present only in adiponectin, and therefore, there is a problem that the possibility of specifically acting on adiponectin receptors is low. Therefore, using the UniProt database provided by NCBI, usa, sequences overlapping with the adiponectin peptide sequence were searched for in all protein sequences present in humans.

As a result, particularly in the case of the peptide P5 of the present invention, it was found that the peptide P5 was present only in the C1q complement protein superfamily to which adiponectin protein belongs, and it could be confirmed that the peptide P5 was a peptide sequence having very high specificity for adiponectin protein (fig. 9).

Preparation example 1 preparation of cosmetics

<1-1>Preparation of smoothing toner

As shown in table 2 below, a smoothing toner containing the adiponectin-derived peptide of the present invention as an active ingredient was prepared.

[ Table 2]

Raw materials	Content (wt%)
		Adiponectin-derived peptide of example 1	10.00
1, 3-butanediol	1.00
		Ethylenediaminetetraacetic acid disodium salt	0.05
Allantoin	0.10
		Glycyrrhizic acid dipotassium salt	0.05
Citric acid	0.01
		Citric acid sodium salt	0.02
Glycerol polyether-26	1.00
		Arbutin	2.00
Hydrogenated castor oil	1.00
		Ethanol	30.00
Preservative	Trace amount of
		Coloring agent	Trace amount of
Flavouring agent	Trace amount of
		Purified water	Balance of

<1-2>Preparation of nourishing cream

As shown in table 3 below, nourishing creams containing the adiponectin-derived peptides of the present invention as an active ingredient were prepared.

[ Table 3]

Raw materials	Content (wt%)
		Adiponectin-derived peptide of example 1	10.0
1, 3-butanediol	7.0
		Glycerol	1.0
D-panthenol	0.1
		Plant extracts	3.2
Magnesium aluminum silicate	0.3
		PEG-40 stearate	1.2
Stearic acid	2.0
		Polysorbate 60	1.5
Lipophilic glyceryl stearate	2.0
		Sorbitan sesquioleate	1.5
Cetostearyl alcohol	3.0
		Mineral oil	4.0
Squalane	3.8
		Caprylic/capric triglyceride	2.8
Vegetable oil	1.8
		Dimethyl silicone oil	0.4
Glycyrrhizic acid dipotassium salt	Trace amount of
		Allantoin	Trace amount of
Hyaluronic acid sodium salt	Trace amount of
		Tocopherol acetate	Proper amount of
Triethanolamine	Proper amount of
		Preservative	Proper amount of
Flavouring agent	Proper amount of
		Purified water	Balance of

<1-3>Preparation of lotions

As shown in table 4 below, a lotion containing the adiponectin-derived peptide of the present invention as an active ingredient was prepared.

[ Table 4]

Raw materials	Content (wt%)
		Adiponectin-derived peptide of example 1	3.5
Octadecanol	1.6
		Stearate salt	1.4
Lipophilic glyceryl monostearate	1.8
		PEG-100 stearinAcid esters	2.6
Sorbitan sesquioleate	0.6
		Squalene	4.8
Macadamia nut oil	2
		Jojoba oil	2
Tocopheryl acetate	0.4
		Methyl polysiloxane	0.2
P-hydroxybenzoic acid ethyl ester	0.1
		Tocopheryl acetate	0.4
Methyl polysiloxane	0.2
		P-hydroxybenzoic acid ethyl ester	0.1
Propyl p-hydroxybenzoate	0.1
		1, 3-butanediol	4
P-hydroxybenzoic acid methyl ester	0.1
		Xanthan gum	0.1
Glycerol	4
		D-panthenol	0.15
Allantoin	0.1
		Carbomer (2% aqueous solution)	4
Triethanolamine	0.15
		Ethanol	3
pt 41891	0.1
		p-H20	48.3

<1-4>Preparation of hair growth agentPrepare for

A hair restorer having the composition shown in table 5 below was prepared using the same method as the conventional preparation method of a hair restorer.

[ Table 5]

Raw materials	Content (wt%)
		Adiponectin-derived peptide of example 1	5.0
Panthenol	0.2
		Menthol	0.3
Salicylic acid	0.25
		Citric acid sodium salt	0.65
PEG-40 hydrogenated Castor oil	0.6
		C12-14Alkanol polyether-12	0.6
Pigment	Proper amount of
		Flavouring agent	Proper amount of
Ethanol	Proper amount of
		Purified water	Balance of

<1-5>Preparation of shampoo

A shampoo having the composition shown in table 6 below was prepared using the same method as the conventional preparation method of a shampoo.

[ Table 6]

Raw materials	Content (wt%)
		Adiponectin-derived peptide of example 1	5.0
Panthenol	0.5
		Nicotinic acid amides	0.3
Tocopheryl acetate	0.1
		Biotin	0.06
Citric acid	0.3
		Sodium lauryl sulfate solution (28%)	40.0
Cocoiloyl amphodiacetic acid disodium salt	5.0
		Cocoamidopropyl betaine	3.0
Cocoamide DEA	1.2
		Cetroronium chloride	1.0
Dimethyl silicone oil	1.2
		Polyquaternium-10	0.5
Triethanolamine	0.55
		Glycerol	0.5
Carbomer	Proper amount of
		Pigment	Proper amount of
Flavouring agent	Proper amount of
		Ethanol	Proper amount of
Purified water	Balance of

<1-6>Preparation of hair conditioner

The same method as the conventional method for preparing a hair conditioner was used to prepare a hair conditioner having the composition shown in table 7 below.

[ Table 7]

PREPARATION EXAMPLE 2 preparation of pharmaceutical preparation

<2-1>Preparation of the powder

Adiponectin-derived peptide 2g of the present invention

Lactose 1g

Mixing the above components, and packaging in a sealed package to obtain powder.

<2-2>Preparation of tablets

The above ingredients are mixed and then compressed according to a conventional tablet preparation method to prepare a tablet.

<2-3>Preparation of capsules

Mixing the above components, and filling into gelatin capsule according to conventional capsule preparation method to obtain capsule.

<2-4>Preparation of pills

Mixing the above components, and making into 4 g/pill by conventional method.

<2-5>Preparation of granules

Mixing the above components, adding 100mg of 30% ethanol, drying at 60 deg.C to form granule, and packaging the granule.

<2-6>Preparation of injection

Adiponectin-derived peptide of the present invention is 10. mu.g/ml

Dilute hydrochloric acid BP until pH 3.5

Maximum amount of sodium chloride BP for injection is 1ml

The adiponectin derived peptide of the present invention is dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the prepared solution is adjusted to 3.5 with dilute hydrochloric acid BP, then the volume is adjusted with sodium chloride BP for injection, and the mixture is thoroughly mixed. The solution was filled into a 5ml type I ampoule made of clear glass, the glass was dissolved and sealed under an upper air grid, and sterilized by autoclaving at 120 ℃ for 15 minutes or more to prepare an injection.

<110> Seoul national university Hospital

<120> composition for promoting hair growth comprising adiponectin-derived peptide

<130> 2018-OPA-1532/PCT

<160> 23

<170> KoPatentIn 3.0

<210> 1

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-1 (IPGL) derived from adiponectin

<400> 1

Ile Pro Gly Leu

1

<210> 2

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-2 (PGLY) derived from adiponectin

<400> 2

Pro Gly Leu Tyr

1

<210> 3

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide 4-3 (GLYY) from adiponectin

<400> 3

Gly Leu Tyr Tyr

1

<210> 4

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-4 (LYYF) from adiponectin

<400> 4

Leu Tyr Tyr Phe

1

<210> 5

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-5 (YYFA) from adiponectin

<400> 5

Tyr Tyr Phe Ala

1

<210> 6

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-6 (YFAY) from adiponectin

<400> 6

Tyr Phe Ala Tyr

1

<210> 7

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-7 (FAYH) derived from adiponectin

<400> 7

Phe Ala Tyr His

1

<210> 8

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-8 (AYHI) from adiponectin

<400> 8

Ala Tyr His Ile

1

<210> 9

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-9 (YHIT) from adiponectin

<400> 9

Tyr His Ile Thr

1

<210> 10

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-10 (NIPG) derived from adiponectin

<400> 10

Asn Ile Pro Gly

1

<210> 11

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-11 (CNIP) from adiponectin

<400> 11

Cys Asn Ile Pro

1

<210> 12

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-12 (HCNI) derived from adiponectin

<400> 12

His Cys Asn Ile

1

<210> 13

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4-13 (palmitoyl l-LYYF) from adiponectin

<400> 13

Leu Tyr Tyr Phe

1

<210> 14

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P3 (IPGLY) derived from adiponectin

<400> 14

Ile Pro Gly Leu Tyr

1 5

<210> 15

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P4 (PGLYY) derived from adiponectin

<400> 15

Pro Gly Leu Tyr Tyr

1 5

<210> 16

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P5 (GLYYYF) from adiponectin

<400> 16

Gly Leu Tyr Tyr Phe

1 5

<210> 17

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P6 (LYYFA) from adiponectin

<400> 17

Leu Tyr Tyr Phe Ala

1 5

<210> 18

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P7 (CNIPG) from adiponectin

<400> 18

Cys Asn Ile Pro Gly

1 5

<210> 19

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P8 (KFHCN) derived from adiponectin

<400> 19

Lys Phe His Cys Asn

1 5

<210> 20

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P9 (HCNIP) derived from adiponectin

<400> 20

His Cys Asn Ile Pro

1 5

<210> 21

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P10 (NIPGL) derived from adiponectin

<400> 21

Asn Ile Pro Gly Leu

1 5

<210> 22

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P1 from adiponectin (KFHCNIPGLYYFAYHITV)

<400> 22

Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe Ala Tyr His Ile

1 5 10 15

Thr Val

<210> 23

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide P2 (IPGLYYYFA) from adiponectin

<400> 23

Ile Pro Gly Leu Tyr Tyr Phe Ala

1 5