KR20110032587A - Fgf10-derived peptides and uses thereof - Google Patents

Abstract

PURPOSE: A composition containing FGF-10-peptide for treating alopecia and improving skin condition is provided to ensure stability and skin permeability. CONSTITUTION: A peptide with growth factor activation contains an amino acid sequence of sequence number 1. The peptide is derived from FGF-10(fibroblast growth factor). A composition for treating or relieving alopecia contains the peptide as an active ingredient. A composition for improving skin condition contains the peptide as an active ingredient. The improvement of skin condition includes anti-wrinkling, anti-aging, wound healing, or skin regeneration.

Description

FGF10-derived peptide and its use {FGF10-derived Peptides and Uses Thereof}

The present invention relates to growth factor-related peptides that promote hair growth and skin regeneration and hair growth and skin improvement peptides comprising the same.

Hair follicles are unique organs of mammalian skin, the organs of which the lower part of the primitive epidermis grows and extends into a deeper skin layer. At the base of the hair follicle is a plug of cells known as follicular or dermal papilla cells (Stenn and Paus, Physiol. Rev. , 81: 449 (2002)). The papilla is essential for normal circulation of hair follicles (Oliver, Embryol. Exp. Morph. , 15: 331 (1966); Oliver, Embryol. Exp. Morph. , 16: 231 (1966)) and hair shaft growth. The hair shaft is a tread-shaped structure made of tightly adherent epithelial cells filled with keratin filaments and filament aggregate proteins.

Human hair periodically undergoes a process of growing, degenerating, and resting, losing hair and regenerating it. The hair cycle is achieved through hormonal control and many growth factors. On the other hand, due to severe stress or malnutrition, the hair enters the resting stage early and causes severe hair loss (American Journal of Pathology, Vol, 162. No. 3, Stress and the Hair Follicle: Exploring the Connections (2003). )). In male baldness, the front and upper hair follicles of the scalp are susceptible to androgens, and in susceptible individuals they are transformed from large vesicles into microvesicles and appear clinically as hair loss. In addition, 20% of women with hair loss often show thinning hair on the top of the scalp, which is estimated to undergo some type of hair loss in their lifetime. As you age, hair loss spreads. In addition, different disease states such as, for example, scarring alopecia, burns, or scarring conditions associated with compression injuries can cause significant hair loss. Regardless of the cause, hair loss can ultimately result in a significant mental, social and sexual influence with loss of self-esteem and self-esteem. To treat this hair loss phenomenon, various substances have been used as a medicine until now, but there are disadvantages that the price is too expensive or the individual difference in efficacy is too great.

In addition, cosmetic products have been used for plant extracts, such as low price but less effective, but the effect was insignificant. Therefore, in order to compensate for these shortcomings, human-derived keratinocyte growth factor was reduced by using fermentation and purification method using Escherichia coli, and by using nanosome technology to easily penetrate into the skin. It has developed a product that shows great effects due to its high penetration into the skin. In addition, the effect was maximized by adding together with the keratinocyte growth factor-10-derived peptide. Keratinocyte growth factor-10 suppresses the death of hair follicle cells due to UV or stress, and shows the effect of inhibiting hair loss by allowing hair cycle to stay in the growing season due to various environmental factors. In addition, it plays a role in promoting growth period, which is the time at which hair is produced and grown. In normal hair, hair growth and nourish the hair greatly affect healthy hair.

Treatment and solutions for hair loss have changed significantly over the long term. Although wigs, partial wigs, and hair enlargements can hide bald parts, they do not cause new growth, and the two available drugs known to date (minoxidil and finasteride) can delay further hair loss, but in fact new hair follicles Did not play any role in inducing the regeneration of. In addition, many hair cosmetic products have been released to prevent hair loss using plant extracts, in particular, extracts such as red ginseng, red pepper, medicinal herbs, lettuce, green leaf, ginseng, licorice, peony, turmeric, fennel, cornus, garlic, etc. Addition of a composition containing a product, xanthine and growth hormone improves the inhibition of cell metabolism caused by the excess of dihydrotestosterone (DHT), while growth hormone promotes hair growth, thereby preventing hair loss and regenerating hair, thereby promoting hair growth. To develop effective products, products containing minerals and vitamins, green tea, rosemary, mugwort and licorice extract to promote hair growth and hair growth, nourish scalp and hair, prevent hair loss and promote hair growth Effective hair growth products, such as vitamin B, vitamin C, vitamin D, vitamin E, nicotinic acid, pantothenic acid, biotin and folic acid The plant extracts were mixed to inhibit 5-alpha reductase in the human body to prevent the formation of dihydrotestosterone during the metabolism of male hormones and to help the metabolism of the hair. Male hair loss products were developed. The product affecting was hard to find. On the other hand, we developed a product using corosolic acid, which has been determined to be effective in diabetes, by a clinical team from the Tokyo Self-Consciousness Association and a research group at the University Health and Medical Center, Japan, to inhibit 5-alpha reductase in the human body and improve hair growth. Products with excellent functions have been developed.

On the other hand, many factors are linked to the process of hair growth and degeneration. Korean Patent No. 1007968170000 has been developed to a variety of human-derived cytokines (e.g., KGF, αFGF, FGF-10, etc.) as a cosmetic containing growth factors by mass fermentation and purification method using E. coli To grow hair and prevent hair loss. However, while the growth factors described above are very effective, additional processes and time called re-folding are required in order to obtain natural growth factors, and also to remove contaminants derived from E. coli during the purification process. The complex purification process is required, and due to its stability and high molecular weight, it is difficult to easily jump over the protective film of the hair.

In order to solve the problem of expression of growth factors described above, attempts to obtain similar functions by producing only a part of some growth factors using the method of solid phase synthesis have been reported. For example, in US Pat. No. 5,473,054, Jameson et al. Name fragments 29-38 and 61-70 of IGF-1 as JB2 and JB1, respectively, and the cell growth effects of the peptide fragment and IGF-1 of JB3, the enantiomer of JB1. Inhibitory effect was reported. Teruo et al. Also report on the complementary action in wound healing with 33-37 fragments of IGF-1 and Substance P-derived tetrapeptides in WO 03/048192. In addition, Kodama et al. Reported in Autoimmunity 37: 481-487 (2004) that 50-70 fragments of IGF-1 help treat diabetes in mice.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a substance having the same or similar function or function as the natural fibroblast growth factor (FGF) -10, but having better activity, skin permeability and stability than the FGF-10 protein. As a result, among many peptide candidates synthesized based on the amino acid sequence of natural growth factors, not only are they excellent in physiological activity (e.g., hair loss improvement, cell growth promoting ability, cell migration promoting ability, etc.) but also stability and skin permeability. By synthesizing an excellent FGF-10-derived peptide, the present invention was completed.

Accordingly, an object of the present invention is to provide a peptide of SEQ ID NO: 1 sequence.

Another object of the present invention to provide a composition for improving hair loss.

Still another object of the present invention is to provide a composition for improving skin conditions.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, a peptide having an amino acid sequence of SEQ ID NO: 1 is provided.

According to another aspect of the present invention, there is provided a composition for treating or improving hair loss comprising the peptide as an active ingredient.

The present inventors have tried to develop a substance having the same or similar function or function as the natural fibroblast growth factor (FGF) -10, but having better activity, skin permeability and stability than the FGF-10 protein. As a result, among many peptide candidates synthesized based on the amino acid sequence of natural growth factors, not only are they excellent in physiological activity (e.g., hair loss improvement, cell growth promoting ability, cell migration promoting ability, etc.) but also stability and skin permeability. Excellent FGF-10-derived peptides were synthesized.

Fibroblast growth factor (FGF) -10 is a human gene. FGF-10 protein is a member of the fibroblast growth factor family, which has a wide range of mitogenic and cell viability, and has a variety of biological processes (eg embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion, Etc.). This protein has the activity of keratinizing epidermal cells but does not show activity against fibroblasts similarly to FGF-7. Although the efficacy of the FGF-10 protein is very good, obtaining an active FGF-10 protein requires an additional process and time of refolding, and requires a complicated purification process to remove contaminants derived from E. coli during the purification process. In order to compensate for the disadvantages of not easily jumping over the protective film of the hair due to the stability and high molecular weight and the disadvantage of lowering the utilization due to the high price, the present inventors have developed a peptide having a similar function to that of the FGF-10 protein and having excellent stability. The application of this product to a new, economically competitive analogue has been developed.

Peptides of the invention comprise an amino acid sequence selected from the group consisting of FGF-10-derived amino acid sequences. Preferably, the peptide in the present invention consists essentially of the amino acid sequence of SEQ ID NO: 1 sequence. As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds.

Peptides of the invention can be prepared according to chemical synthesis methods known in the art, in particular 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)).

Peptides of the present invention by randomly partial synthesis of several sites present in the FGF-10 protein, the first search for a binding site for the receptor protein, and then optimized the amino acid sequence of the predicted site to the peptide of the present invention The peptides of the present invention are provided by screening peptides having the highest activity among these candidate peptides.

The first sequence peptide of SEQ ID NO: 1 is a peptide that performs a function similar to native FGF-10 and binds to a receptor to perform a function such as a growth factor.

According to a preferred embodiment of the present invention, the peptide of the present invention is derived from human fibroblast growth factor (FGF) -10 protein.

According to a preferred embodiment of the present invention, the peptide of the present invention has a growth promoting ability of cells, more preferably keratinocytes, fibroblasts or hair follicle cells.

According to a preferred embodiment of the present invention, the peptide of the present invention promotes the migration of keratinocytes.

According to a preferred embodiment of the present invention, the peptides of the present invention are characterized by the level of phospho-ERK (P-ERK), phospho-JAK / SAPK (P-JNK / SAPK) and phospho-p38 (P-p38). Increase.

The peptide of the present invention is superior in stability to the natural FGF-10 protein by itself, but may be further improved in stability by modification of amino acids.

According to a preferred embodiment of the invention, the N-terminus of the peptide is from the group consisting of acetyl group, fluorenyl methoxy carbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group and polyethylene glycol (PEG) The protecting group selected is combined.

According to a preferred embodiment of the present invention, the peptide of the present invention is selected from the group consisting of acetyl group, fluorenyl methoxy carbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group and polyethylene glycol (PEG) Protecting groups can be bound to provide peptides that are more stable than naturally occurring modified growth factors.

Modification of the above-mentioned amino acid serves to greatly improve the stability of the peptide of the present invention. The term "stability" herein means not only " in vivo" stability but also storage stability (eg, room temperature storage stability). The above protecting groups serve to protect the peptides of the present invention from the attack of protein cleavage enzymes in vivo.

According to another aspect of the present invention, the present invention provides a hair growth composition comprising the growth factor-related peptide of the present invention as an active ingredient.

According to another aspect of the present invention, the present invention provides a composition for treating skin and wounds comprising the growth factor-related peptide of the present invention as an active ingredient.

Since the composition of the present invention includes the growth factor-related peptide of the present invention described above as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

The inventors of the present invention conducted a study utilizing a series of growth factors that promote keratinocyte growth factor, promote vascular endothelial growth factor, and promote hair production, which are the most important targets for hair growth. Was developed.

Human hair has about 100,000 to 150,000 pieces, and each hair has a different cycle and grows and drops out through anagen, catagen, and telogen. This cycle is repeated over three to six years, with the result that on average 50 to 100 hairs fall out normally. The mechanism of hair loss is not yet known precisely, but it is generally known to be due to unbalanced diet, excessive drinking and smoking, drug use, lack of sleep, excessive stress, childbirth, menopausal disorders, frequent perm and pigmentation, and hormone imbalance. . Human hair grows and grows and falls out of the human hair repeatedly, and maintains a constant density.However, when the hair loss proceeds due to the various causes described above, the hair on the crown or forehead starts to become thinner and loses strength. The number will increase. In other words, hair is rooted in the scalp and supplied with nutrients from the blood. When the capillaries shrink and the scalp becomes thin for a long time, the hair becomes incapable of developing normally. This shortened, shortened life of the hair is not only easy to fall out, but also in the hair follicles as described above, the hair does not come out again and eventually will show the symptoms of alopecia.

According to a preferred embodiment of the present invention, the treatment or amelioration of hair loss by the peptides of the present invention is hair growth promotion or hair production.

As demonstrated in the Examples below, the growth factor-related peptides of the present invention have growth promoting ability of keratinocytes and fibroblasts and promote migration of keratinocytes. In addition, it performs a function of promoting hair growth very high through animal experiments. Therefore, the composition of the present invention is very effective for improving hair growth and skin condition.

According to a preferred embodiment of the present invention, the improvement of the skin condition by the peptide of the present invention is wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, wound removal or skin regeneration.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the growth factor-related peptide of the present invention described above; And (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the growth factor-associated peptide described above.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, granules, tablets, capsules, or gels (eg, hydrogels), and may further include dispersants or stabilizers. .

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a cosmetically effective amount of the growth factor-related peptide of the present invention described above; And (b) a cosmetically acceptable carrier.

As used herein, the term “cosmetic effective amount” means an amount sufficient to achieve the skin improving efficacy of the composition of the present invention described above.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions, in addition to peptides and carrier components as active ingredients, and include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Phosphorus adjuvant.

The features and advantages of the present invention are summarized as follows:

(Iii) The FGF-10-derived peptides of the present invention may function identical or similar to natural human growth factors.

(Ii) The peptide of the present invention is very excellent in stability compared to the natural FGF-10 protein, and very excellent in skin permeability.

(Iii) Therefore, the composition comprising the peptide of the present invention can exhibit very excellent efficacy in treating or improving hair loss (for example, promoting hair growth or generating hair).

(Iii) The excellent activity and stability of the peptides of the present invention described above make it possible to be very advantageously applied to medicines, quasi-drugs and cosmetics.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1

Synthesis Example 1 Synthesis of Leu-Gln-Asp-Gly-Val-Arg (SEQ ID NO: 1)

700 mg of chloro trityl chloride resin (CTL resin, Nova biochem Cat No. 01-64-0021) was added to a reaction vessel, and 10 ml of methylene chloride (MC) was added thereto, followed by stirring for 3 minutes. The solution was removed, 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of dichloromethane solution was added to the reactor, 200 mmole of Fmoc-Arg (pbf) -OH (Bachem, Swiss) and 400 mmole of diisopropyl ethylamine (DIEA) were added thereto, stirred, and dissolved. The reaction was stirred for 1 hour. I was. After the reaction, the mixture was washed with methanol and DIEA (2: 1) in dichloromethane (DCM) for 10 minutes and washed with excess DCM / DMF (1: 1). The solution was removed, 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of deprotection solution (20% of piperidine / DMF) was added to the reaction vessel and stirred at room temperature for 10 minutes to remove the solution. After adding the same amount of deprotection solution and maintaining the reaction again for 10 minutes, the solution was removed and washed 3 times with DMF twice, once with MC, once with DMF to prepare Arg (pbf) -CTL Resin. 10 ml of DMF solution was added to the new reactor, 200 mmole of Fmoc-Val-OH (Bachem, Swiss), 200 mmole of HoBt, and 200 mmole of Bop were stirred, and the mixture was dissolved well. 400 mmole DIEA was added to the reactor twice in fractions and stirred for at least 5 minutes until all solids dissolved. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin and reacted with stirring at room temperature for 1 hour. The reaction solution was removed and stirred with a DMF solution three times for 5 minutes and then removed. A small amount of the reaction resin was taken and the degree of reaction was checked using a Nihydrin Test. The deprotection reaction was performed twice in the same manner as above with the deprotection solution to prepare Val-Arg (pbf) -CTL resin. After sufficient washing with DMF and MC, once again Kaiser test was performed, and the following amino acid attachment experiment was performed as above. Based on the selected amino acid sequence, Fmoc-Gly, Fmoc-Asp (tBu), Fmoc-Gln (trt), and Fmoc-Leu were sequentially chained. The Fmoc-protector was reacted twice with a deprotection solution twice for 10 minutes and then washed well. Acetylation of acetic anhydride, DIEA, and HoBt was carried out for one hour, and then the prepared peptidyl resin was washed three times with DMF, MC, and methanol, and dried slowly with nitrogen air, followed by vacuum under P ₂ O ₅ . After drying completely under reduced pressure, 30 ml of a fugitive solution (Trifluroacetic acid (95%, Trifluroacetic acid), 2.5%, distilled water 2.5%, Thianisole) 2.5%) was added thereto, and the reaction was kept at room temperature for 2 hours. The resin was filtered off and the resin was washed with a small amount of TFA solution and then combined with the mother liquor. Distillation was carried out using reduced pressure to half the total volume and 50 ml of cold ether was added to induce precipitation, followed by centrifugation to collect the precipitate and washing with two more cold ethers. The mother liquor was removed and dried sufficiently under nitrogen to synthesize 0.65 g of NH ₂ -Leu-Gln-Asp-Gly-Val-Arg-OH peptide 1 before purification (yield: 92.6%). The molecular weight 687.2 (theoretical value: 686.8) was obtained when measured using a molecular weight meter.

number Amino acid sequence Analytical Values (Mass Spectrometer) Analytical value Theoretical value One NH ₂ -Leu-Gln-Asp-Gly-Val-Arg-OH 687.2 686.8

Test Example 1 Identification of Cell Growth Effect Using Synthetic Peptides

In order to analyze the similar and inhibitory effects of growth factors on the sequence peptide synthesized in Synthesis Example 1, the HaCaT keratinocyte line was described with reference to Rizino et al. (Rizzino, et al. , Cancer Res. 48: 4266 (1988)). (Sulforhodamine B, Sigma) colorimetric method using (Korea Cell Line Bank) and NIH3T3 fibroblasts (Korea Cell Line Bank).

HaCaT keratinocyte line and NIH3T3 fibroblasts were cultured in Dulbecco's modied Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Sigma) using a 250 ml tissue culture flask. It was. The cultured cell lines were removed from the bottom of the culture vessel with 1% trypsin solution and centrifuged to collect only cell precipitates. The cultured cell lines were removed from the bottom of the culture vessel with 1% trypsin solution and centrifuged to collect only cell precipitates. After resuspending in DMEM culture medium containing no FBS, it was put in a 96-well tissue culture plate to be 3 X 10 ³ cells per well and incubated for 24 hours at 37 ℃, 5% CO ₂ conditions. After 24 hours, the medium was changed to the same culture except for the serum completely, and the blank and synthetic peptides were sterilely dissolved in 10% DMSO for standardization. 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 72 hours were incubated under the same conditions as above at concentrations of μg / ml and 10 μg / ml. After the incubation was completed, the culture supernatant was removed, the cells were immobilized with ethanol, and the cells were fixed and washed three times with PBS (phosphate buffer saline). After removing the washing solution and treated with colorimetric SRB solution and washed with 1% acetic acid sufficiently, the cells were observed under a microscope to observe the state of the viable cells, the absorbance was measured at 590 nm ultraviolet light to measure the viability of the cells.

Figure 2 is a result showing the growth of keratinocyte (fibreblast) and fibroblast (fibroblast) after the peptide treatment of the present invention, Figure 3 shows the keratinocytes by observing the survival state of the cell 72 hours after peptide treatment to the cells The growth of fibroblasts was confirmed.

As can be seen in Figure 2, SEQ ID NO: 1 sequence peptide of the present invention greatly promoted the concentration-dependent growth of keratinocytes and fibroblasts. In addition, as shown in Figure 3, the treatment of the first sequence peptide of the sequence listing of the present invention can be confirmed through the microscope that the shape or shape of the cell significantly changed by inducing concentration-dependent growth of keratinocytes and fibroblasts. there was.

Test Example 2: Analysis of cell migration promoting effect of the synthetic peptide

After culturing keratinocytes sufficiently in a 60 mm culture dish, the cells are damaged using a tip, and the peptides of the present invention are treated at a concentration of 100 ng / ml and 1 ㎍ / ml and cultured for 48 hours. After the incubation was completed, the cells were fixed with ethanol and stained with SRP to observe the movement of cells through the microscope.

As can be seen in Figure 4, the sequence of the first sequence peptide of the present invention was confirmed that the concentration-dependently promote the migration of keratinocytes.

Test Example 3: Increase of Signaling Molecules by Synthetic Peptides

Incubate keratinocytes sufficiently in a 60 mm culture dish and replace the medium with serum-free medium. Subsequently, the first sequence peptide of SEQ ID NO: 100 ng / ml, 1 μg / ml, 5 μg / ml was treated for 5 hours and lysis buffer (0.4% SDS, 100 mM NaCl, 1% Tritonx-100, 50 mM Tris ( pH 7.4), 1mM EDTA-Sigma) to separate the cells and then reacted for 1 hour on ice and centrifuged (15,000 rpm, 20 minutes). After obtaining only the supernatant protein, the protein value was quantified using the Bio-red Protein Assay Kit I (500-0001), and 30 μg of total protein was loaded onto the SDS-PAGE, followed by PVDF membrane (Amersham). ). Western blot was performed using antibodies of the target proteins, phospho-ERK (SantaCruz), phospho-JAK / SAPK (SantaCruz), and phospho-p38 (SantaCruz) to observe the effect of SEQ ID NO: 1 peptide. (FIG. 5).

As shown in FIG. 5, the first sequence peptide of the present invention is phospho-ERK (P-ERK), phospho-JAK / SAPK (P-JNK / SAPK) and phospho-p38 (P-p38). It was observed that the signal of increased concentration-dependently.

Test Example 4: Confirmation of hair root cell growth effect using synthetic peptide

Primary hair cells were used to investigate the growth promoting ability of the hair root cells by the sequence peptide synthesized in Synthesis Example 1. Primary hair cells were isolated from the hair follicles of the mouse nose, and cultured through three passages and used for the experiment. The first sequence peptide of the present invention was incubated for 72 hours at the concentration of 100 ng / ml, 1 μg / ml and 10 μg / ml in the same manner as in Test Example 1, to observe the growth of hair root cells. After the incubation was completed, the culture supernatant was removed, the cells were immobilized with ethanol, and the cells were fixed and washed three times with PBS (phosphate buffer saline). After removing the washing solution and treated with colorimetric SRB solution and washed with 1% acetic acid sufficiently, the cells were observed under a microscope to observe the state of the viable cells, the absorbance was measured at ultraviolet light 590 nm to measure the cell viability.

72 hours after the cells were treated with the peptide sequence 1 sequence of the present invention, as shown in FIG. 6, the survival state of the cells was observed under a microscope, the concentration-dependent growth of the hair root cells extracted from the mouse of the peptide of the present invention. The effect was obvious even at low concentrations.

Test Example 5: Confirming the effect of inhibiting cell death of the synthetic peptide

In order to observe the inhibitory function against apoptosis of the sequence peptide synthesized in Synthesis Example 1, it was measured using the SRB colorimetric method using HaCaT keratinocyte line.

HaCaT keratinocyte line (Korea Cell Line Bank) was put in a 96-well tissue culture plate to be 3 X 10 ³ cells per well and incubated for 24 hours at 37 ℃, 5% CO ₂ conditions. After 24 hours, the medium was exchanged with the same culture except for the serum completely, and the cells were treated with 100 μM H ₂ O ₂ for 30 minutes to induce cell death. Thereafter, the empty sample and the synthetic peptide for standardization were dissolved in 10% DMSO in a sterile state and treated at concentrations of 10 ng / ml, 100 ng / ml, 1 ug / ml and 5 ug / ml for 72 hours. Incubated. After the incubation was completed, the culture supernatant was removed and the cells were fixed with ethanol and washed three times with PBS. After the washing solution was removed, treated with colorimetric SRB solution, washed well with 1% acetic acid, and then observed the cells under a microscope to observe the state of the viable cells, the absorbance was measured at 590 nm ultraviolet light to measure the viability of the cells. As a result of the experiment, it was observed that the killing of keratinocytes induced by H ₂ O ₂ is suppressed by treating the peptide of the present invention.

7 is a result showing that the first sequence peptide of the present invention inhibits the killing of keratinocytes induced by H ₂ O ₂ .

Test Example 6: Thermal Stability of the Prepared Peptides

The standard growth factor (FGF-10) obtained from the peptide of the present invention and NIBSC (UK) prepared through Synthesis Example 1 was prepared as a phosphate buffer solution at a concentration of 0.11 mg / ml. The prepared solution was placed in a glass vial of 1 ml each and left at 37 ° C. The solution left at 37 ° C. was sampled at 0, 5, 10, 20, 30, 50, and 70 days and centrifuged by date to remove denatured peptide or protein, and the supernatant was taken and quantified using HPLC. As can be seen in Figure 8, it was confirmed that the thermal stability of the peptide of the present invention is very excellent compared to the natural growth factor.

Test Example 7 hair growth effect of the mouse treated with the prepared peptide

Nanopeptide peptide of the present invention prepared in Synthesis Example 1 was applied twice a day to C57BL / 6 mice depilating the back skin for 15 days. As a control, phosphate buffer solution was applied twice a day. On the 9th day of application, it was confirmed that the back skin of the mouse came up black, and on the 15th day of application, it was confirmed that the hair grew much more than the control group (FIG. 9).

Example 1: Preparation of Nanonized Peptides

50 mg of the peptide obtained in the above synthesis example was weighed accurately, and then dissolved by stirring sufficiently with 500 ml of distilled water. The blend solution is mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol and a little oil phase, then the amount is adjusted to 1 L with distilled water and then with a microfluidizer. Emulsification using a high pressure to prepare a nanosome of size 100 nm. The prepared nanosomes were used for cosmetic preparation alone or in combination with a final concentration of about 50 ppm.

Formulation Example 1: Softening Longevity

Including the peptide nanosomes prepared in the above Example, a flexible cosmetic made of the following composition was prepared according to a general lotion manufacturing method.

ingredient Content (% by weight) Peptide Nanosomes 0.001 1,3-butylene glycol 6.0 glycerin 4.0 PEG 1500 1.0 Sodium hyaluronate 1.0 Polysorbate 20 0.5 ethanol 8.0 Preservative, pigment Quantity Benzophenone-9 0.05 incense a very small amount Purified water Balance Sum 100

Formulation Example 2: Nutrition Cream

Peptide nanosomes prepared in the above Example, the nutrition cream consisting of the following composition was prepared according to the general nutrition cream manufacturing method.

ingredient Content (% by weight) Peptide Nanosomes 0.001 Meadowfoam oil 3.0 Cetearyl alcohol 1.5 Stearic acid 1.5 Glyceryl Stearate 1.5 Floating paraffin 10.0 Beeswax 2.0 Polysorbate 60 0.6 Solbitan Sesquioleate 2.5 Squalane 3.0 1,3-butylene glycol 3.0 glycerin 5.0 Triethanolamine 0.5 Tocopheryl acetate 0.5 Preservative, pigment Quantity incense Quantity Purified water Balance Sum 100

Formulation Example 3: Nutrients

Including the peptide nanosomes prepared in the above Example, nutrient lotion consisting of the following composition was prepared according to a general lotion manufacturing method.

ingredient Content (% by weight) Peptide Nanosomes 0.002 1,3-butylene glycol 4.0 glycerin 4.0 Cetearyl alcohol 0.8 Glyceryl Stearate 1.0 Triethanolamine 0.13 Tocopheryl acetate 0.3 Liquid paraffin 5.0 Squalane 3.0 Macadamia Nut Oil 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Carboxy Vinyl Polymer 1.0 Preservative, pigment Quantity incense Quantity Purified water Balance Sum 100

Formulation Example 4: Essence

Including the peptide nanosomes prepared in the above example, the essence consisting of the following composition was prepared according to the general essence preparation method.

ingredient Content (% by weight) Peptide Nanosomes 0.005 glycerin 10.0 1,3-butylene glycol 5.0 PEG 1500 2.0 Allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Hydroxyethylcellulose 0.1 Sodium hyaluronate 8.0 Carboxy Vinyl Polymer 0.2 Triethanolamine 0.18 Octyldodec-16 0.4 ethanol 6.0 Incense, preservative, coloring Quantity Purified water Balance Sum 100

Formulation Example 5: Hair Serum

Including the peptide nanosomes prepared in the above example, a hair serum having the following composition was prepared according to a general hair serum preparation method.

ingredient Content (% by weight) Peptide Nanosomes 0.005 glycerin 7 1,3-butylene glycol 5.0 PEG 1500 2.0 Allantoin 0.2 DL-Panthenol 0.3 EDTA-2Na 0.02 Hydroxyethylcellulose 0.2 Sodium hyaluronate 3.0 Carboxy Vinyl Polymer 0.5 Triethanolamine 0.18 Octyldodec-16 0.4 ethanol 4.0 Incense, preservative, coloring Quantity Purified water Balance Sum 100

1 is a graph showing the results of high performance liquid chromatography analysis of the peptide of SEQ ID NO: 1 prepared by the synthesis example of the present invention.

Figure 2 is a graph showing the growth promoting effect of keratinocytes and fibroblasts treated with peptide 1 prepared by the synthesis example of the present invention.

Figure 3 is a photograph confirming the effect of the cell growth promoting effect of the keratinocytes treated with the peptide of the present invention under a microscope.

4 is a graph showing that the movement of keratinocytes is increased by treating the peptide of the present invention.

5 is a western blot showing that the peptides of the present invention increase the signals of phospho-ERK (P-ERK), phospho-JAK / SAPK (P-JNK / SAPK) and phospho-p38 (P-p38). The result is.

Figure 6 is a graph showing the growth promoting effect of the hair root cells treated with the peptide of the present invention.

7 is a graph showing that the peptide of the present invention inhibits cell death when a substance that promotes cell death (eg, H ₂ O ₂ ) is added.

Figure 8 is a graph comparing the thermal stability of the peptides and natural peptides of the present invention.

9 is a result showing the hair growth effect of the skin, such as a mouse treated with the peptide of the present invention.

<110> Caregen <120> FGF10-derived Peptides and Uses Thereof <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> FGF-10 <400> 1 Leu Gln Asp Gly Val Arg   1 5  

Claims (12)

Peptide showing growth factor activity having the amino acid sequence of SEQ ID NO: 1. The peptide of claim 1, wherein the peptide is derived from human fibroblast growth factor (FGF) -10 protein. According to claim 1, wherein the peptide is a peptide, characterized in that having a growth promoting ability of the cell. The peptide of claim 3, wherein the cells are keratinocytes, fibroblasts or hair follicle cells. The peptide of claim 1, wherein the peptide promotes the migration of keratinocytes. The method of claim 1, wherein the peptide increases the levels of phospho-ERK (P-ERK), phospho-JAK / SAPK (P-JNK / SAPK) and phospho-p38 (P-p38). Peptide. The peptide of claim 1, wherein the peptide inhibits apoptosis when a substance that induces apoptosis is added. 8. The peptide according to claim 7, wherein the substance inducing apoptosis is H ₂ O ₂ . A composition for treating or improving hair loss comprising the peptide of any one of claims 1 to 8 as an active ingredient. 10. The composition of claim 9, wherein said treating or improving hair loss is promoting hair growth or hair production.  Composition for improving skin conditions comprising the peptide of any one of claims 1 to 8 as an active ingredient. The composition of claim 11, wherein the improvement of the skin condition is wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, wound removal and skin regeneration.

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