WO2019039630A1 - Follistatin-derived bioactive peptide and use thereof - Google Patents

Abstract

The present invention relates to a growth factor-derived bioactive peptide and provides a bioactive peptide composed of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 7.

Description

Physiologically active peptides derived from polystatin, and uses thereof

The present invention relates to novel uses of bioactive peptides derived from growth factors.

The animals have a molting period in which the hairs are totally replaced by the seasons, but in humans, some of the whole hairs are constantly replaced every day, so that a similar number is always maintained.

The hair cycle can be divided into three periods of growth period, regenerative period and pausing period. Among them, anagen is a period when active division of cells occurs in the dermal papilla and new hair growth is accelerated. During this period, hair grows .

The period of hair appearance is approximately 3 to 5 years for males and 4 to 6 years for females, and about 80 to 85% of hair as a whole is the growth period. The catagen is about 3 to 4 weeks when cell division is gradually stopped.

Finally, the taloge is a period when the hair is separated from the capillaries by the atrophy of the papillary hair and is simply put on the scalp. It is about 3 months. The hair in the pauses is easily separated from the fur by physical stimulation.

In general, hair loss refers to the shortening of hair growth rate in the growing period and the increase of hair in the retrograde period or rest period in such a cycle, resulting in abnormal abundance of hair.

Although human hair is irrelevant to life, it has a decisive influence on human impressions and appearances, so much attention has been socially attracted, and in recent years, the awareness that the hair loss has been recognized as a symptom is increasing.

Especially, as the recent upsurge and industrialization accelerate, excessive stress and lifestyle habits of socio-cultural factors are causing the hair loss population to increase rapidly. In recent years, hair loss has increased not only in middle-aged men but also in the elderly, and various studies for analyzing the various causes of hair loss have been actively conducted.

Regular hair growth requires hair regeneration and stem cell activation, which is known to be caused by the production and activation of Wnt / β-catenin. In addition, the DKK-1 mentioned above is known to be an antagonist of Wnt / β-catenin, and the above signal system is very important for controlling hair growth.

On the other hand, the transforming growth factor-β (TGF-β) superfamily is a multifunctional growth differentiation factor that regulates the development and differentiation of organs, and is essential for hair follicle formation, hair circulation and growth regulation to be.

Follistatin binds strongly to the transforming growth factor family, such as bone morphogenic protein and activin (Nakamura, Science. 1990, 247; 4944, p836-838) Dissociation constants Kd = 540-680 pmil / L for the kinase) neutralize their activity. Polystatin also directly or indirectly participates in the control of various physiological activities of actibin in vivo.

It has been known through amino acid sequence analysis of polystatin that it consists of four domains: an N-terminal domain, a pol statin domain I, a pol statin domain II, and a pol statin domain III (Shimasaki, Proc. Natl. 1988, 85; 12, p4218-4222). The double pol statin domain is known to be involved in the binding of actin.

In the process of hair growth and degeneration, many factors are linked to each other. The present inventors have found that the most important hair follicle production is promoted by human dermal papilla (HDP) cell proliferation, The growth factors were used in this study.

In particular, various growth factors that regulate hair growth and regression are produced and regulated from the dermal papilla cells of the hair follicles.

The above-mentioned actin is also expressed in dermal papilla cells, inducing the formation of hair follicles and activating the hair growth cycle (Sung, Experimental dermatology. 2017, 26, 2, p108-115).

The activity of actin inhibits the entry of hair into the systolic phase, and the proteins expressed by actinin-pol statin control play an important role in the growth and differentiation of hair, and new cells can be produced. In addition, the regulation of actibin regulates epidermal proliferation and cleavage, and plays an important signaling role in the regulation of epidermal homeostasis.

The herbal remedy for the treatment or prevention of alopecia that has been developed so far includes female hormone-based preparations such as minoxidil, finasteride, tricosaccharide, etc. for promoting blood circulation, enhancing hair follicle function, scalp moisturizing and male hormone suppression, (trichosaccharide), etc., but there are many limitations in use due to adverse effects such as sexual dysfunction, possibility of birth of deformed baby, and general hair growth.

In addition, the above-mentioned medicines are expensive and are required to be continuously used, and when they are discontinued, hair loss is again caused. There is also a disadvantage that individual differences in efficacy are severe and side effects vary from person to person.

The present inventors newly identified that a specific site of polystin acts as an actin binding motif, and the peptide containing the motif competitively binds at binding sites such as actin and inhibits the binding of natural polystin and actin Thereby promoting the growth of hair.

The present inventors also intend to provide the possibility of utilization of the peptide for treating alopecia as well as various diseases associated with the signal system.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide a physiologically active peptide having excellent biostability and excellent hair growth promoting effect and skin condition improving effect.

According to one aspect of the present invention, there is provided a physiologically active peptide consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7.

In one embodiment, the peptide is capable of promoting the growth of fibroblasts, keratinocytes, or hair follicle cells.

In one embodiment, the peptide may activate growth factors of dermal papilla cells.

According to another aspect of the present invention, there is provided a physiologically active peptide structure represented by the following formula (1).

[Chemical Formula 1]

R ₁ - A - R ₂

Wherein A is a linker, R ₁ is a peptide consisting of one or more amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 3, and R ₂ is a peptide consisting of the amino acid sequence of SEQ ID NO: 4.

In one embodiment, A may be a compound represented by the following formula (2).

(2)

H ₂ N (CH ₂ ) n CO ₂ H

In the above formula, n is an integer of 1? N? 18.

In one embodiment, A may be 6-aminohexanoic acid.

In one embodiment, the physiologically active peptide construct may facilitate the growth of fibroblasts, keratinocytes, or hair follicle cells.

In one embodiment, the physiologically active peptide construct can activate growth factors of dermal papilla cells.

According to another aspect of the present invention, there is provided a composition for promoting hair loss prevention and hair growth comprising the peptide or the peptide structure as an active ingredient.

According to another aspect of the present invention, there is provided a composition comprising the peptide or the peptide structure as an active ingredient for improving wrinkles, improving skin elasticity, improving skin moisturization, or regenerating skin.

According to another aspect of the present invention, there is provided a cosmetic composition comprising the peptide or the peptide structure as an active ingredient.

According to another aspect of the present invention, there is provided an external preparation for skin comprising the peptide or the peptide structure as an active ingredient.

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

According to one aspect of the present invention, an excellent skin penetration rate can be realized by using a small-sized peptide, thereby improving the hair and skin condition improving effect.

Due to its physiological activity, the peptides can be used for various purposes such as medicines, quasi-drugs, health functional foods, and cosmetics.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

Figure 1 shows the results of HPLC analysis of peptides according to one embodiment of the present invention.

2A is a graph showing the effect of promoting cell growth in keratinocytes treated with a peptide according to an embodiment of the present invention.

FIG. 2B is a graph showing a cell growth promoting effect in a fibroblast treated with a peptide according to an embodiment of the present invention. FIG.

FIG. 2C is a graph showing a cell growth promoting effect in a hair follicle cell treated with a peptide according to an embodiment of the present invention. FIG.

FIG. 3A is a microscopic examination of the effect of promoting cell growth in a hair follicular cell treated with a peptide according to an embodiment of the present invention through a three-dimensional solid cell culture method.

FIG. 3B shows the sizes of hair follicle cell spheroids in 3-dimensional solid cells treated with peptides according to an embodiment of the present invention.

Figure 4 shows the increase of SMAD2 phosphorylation according to the peptide treatment according to an embodiment of the present invention.

FIG. 5 is a graph showing an increase in signal transduction for activating hair follicles according to the peptide treatment according to an embodiment of the present invention.

FIG. 6 is a graph showing an effect of promoting self-assembly of a papilla according to an embodiment of the present invention and an increase in the expression of a hair growth factor through immunostaining.

FIG. 7 shows hair growth observed at a site of a mouse treated with a peptide according to an embodiment of the present invention.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated.

Hereinafter, embodiments of the present invention will be described in detail, but it should be apparent that the present invention is not limited by the following examples.

One aspect of the present invention provides a physiologically active peptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7.

As a result of intensive efforts to discover peptides having biologically active activities, the present inventors have identified the in vivo physiological activities of the peptides, and in particular, they have clarified the skin condition improving effect and the hair growth promoting effect of the peptides.

In addition, the above-mentioned physiologically active peptide is a concept that collectively refers to a peptide having a predetermined physiological action in vivo, and it is a concept that regulates genetic expression and physiological function, It can play a role of correcting it.

The peptide may be composed of the amino acid sequence of SEQ ID NOS: 1 to 7, or may include the amino acid sequence as a part thereof.

The peptide refers to a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds). For the purpose of the present invention, the peptide may refer to peptides or fragments thereof that exhibit wrinkle-improving and skin elasticity-improving effects.

The general rules for naming the peptides may be based on three letter or single letter amino acid codes, unless specifically indicated otherwise. For example, the center of the amino acid structure is represented by a three-letter code (e.g., Ala, Lys), and a D-stereotype (e.g., D-Ala, D-Lys) It is possible to assume an L-steric form. The amino acid residue constituting the peptide may be a natural or non-natural amino acid residue.

The peptide randomly partially synthesizes various sites of the pol statin protein to first search for a binding site for the receptor protein, and then optimizes the amino acid sequence of the predicted site. Screening for the peptide having the highest activity among the candidate peptides It was completed.

The peptide comprises an actin binding motif of native polystin and can promote the activity of actin by binding to actin and interfering with its binding to native pol statin.

The peptide has fibroblast, keratinocyte, or hair follicle cell growth promoting ability, and can transmit an actin signal even in the presence of native polystin.

The peptides can increase the expression of Wnt5, FGF7, Versican, Lef1, the major genes of hair follicles, and increase the size of the dermal papilla cells through three dimensional solid cell culture.

In particular, it is known that the Wnt ligand inhibits hair loss by activating the beta-catenin signaling mechanism in cells. The Wnt binds to the follicular receptors of hair follicle cells to activate dermal papilla cells and inhibit hair follicle cell destruction materials including TGF-b, DKK-1 and BMP, thereby inhibiting hair loss. That is, the peptide significantly increases the amount of Wnt produced and promotes the growth of hair follicle cells, thereby achieving excellent hair loss prevention and hair growth promoting effect.

The peptide competes with the natural polystin protein to inhibit the function of the natural pol statin and is superior in stability and skin permeability as compared to natural pol statin protein.

The peptide promotes the growth of fibroblasts or keratinocytes to impart elasticity to the skin, thereby suppressing the formation of wrinkles. Furthermore, the peptide may promote hair follicle growth and induce hair growth.

The keratinocytes and fibroblasts in the skin may be damaged and degenerated due to various causes such as mental stress, external harmful factors, and endogenous aging. In particular, the damage of keratinocytes and fibroblasts due to internal and external stress may lower the density of skin cells and interfere with the synthesis of collagen, which can deepen the formation of skin wrinkles.

The collagen is a major component of the connective tissue and is a component mainly distributed in the bones and skin. It is a light protein that maintains the structure and shape of the skin and provides firm strength and elasticity. When the skin is aged, exposed to ultraviolet rays, heat, etc., collagen synthesis ability decreases and collagen amount can be significantly reduced. When the amount of the collagen decreases, the shape of the muscle can not be maintained, so that a skin wrinkle can be generated.

The peptides originate from the body of polystatin and are high in skin permeability due to their small molecular weight and size, and are excellent in promoting skin growth.

Since the peptide promotes the growth of fibroblast and keratinocyte and promotes collagen synthesis, it can grow the stratum corneum, epithelial layer and dermal layer of the skin and improve wrinkles, skin elasticity, skin aging, skin moisturization, .

When the peptide is locally applied to the skin, overall effects such as improvement of skin condition such as wrinkle improvement, improvement of hair loss, and promotion of hair growth can be simultaneously realized.

The peptide can be produced biologically by extracting a protein in vivo and treating it with proteolytic enzymes to lower molecular weight or by using a gene recombination and protein expression system or by a chemical synthesis method using a peptide synthesizer or the like .

For example, the peptides can be prepared according to chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. 1963, Soc. 85: 2149- 54; Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. 1984, Co .: Rockford, 111).

For example, a gene encoding a fusion partner and a fusion protein composed of the peptide may be prepared through gene manipulation, and the host microorganism may be transformed with the gene and expressed in the form of a fusion protein in the host microorganism.

The fusion protein can be cleaved and separated using proteolytic enzymes or compounds, and the peptide can be obtained. Specifically, a DNA sequence encoding an amino acid residue that can be cleaved by a proteolytic enzyme, such as Factor Xa or an enterokinase, a compound such as CNBr or hydroxylamine, can be inserted between the fusion partner and the gene of the peptide .

The N or C-terminal of the peptide may be bonded with an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG). The stability of the peptide can be remarkably improved by the modification. The stability is a concept including not only in vivo stability but also storage stability. The protecting group can protect the peptide of the present invention from attack of a protein cleaving enzyme in vivo.

The peptide may comprise a functional equivalent of a peptide comprising the amino acid sequence. Said "functional equivalent" means that at least 40%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% sequence identity with said amino acid sequence as a result of addition, substitution or deletion of amino acids Means a protein having homology and having a physiological activity substantially equivalent to the above amino acid sequence.

The " substantially homogenous physiological activity " means a skin condition improving activity such as anti-inflammatory activity due to the structural and functional homology of the peptide. The sequence homology is determined by comparing the two optimally arranged sequences with the comparison region, and some nucleic acid sequences in the comparison region may be added or deleted.

According to another aspect of the present invention, there is provided a physiologically active peptide structure represented by the following formula (1).

[Chemical Formula 1]

R ₁ - A - R ₂

Wherein A is a linker, R ₁ is a peptide consisting of one or more amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 3, and R ₂ is a peptide consisting of the amino acid sequence of SEQ ID NO: 4.

The peptide construct comprises a peptide consisting of the amino acid sequence of one of SEQ ID NOS: 1 to 3, and a peptide consisting of the amino acid sequence of SEQ ID NO: 4, wherein the peptide can be linked by a linker.

The linker may be a flexible linker, specifically a peptide linker or a non-peptide linker.

The " non-peptide linker " means a biocompatible linker in which two or more repeating units are bonded, and the repeating units may be connected to each other through any covalent bond other than a peptide bond.

The non-peptide linker may be selected from the group consisting of polyethylene glycol (PEG) homopolymer, polypropylene glycol homopolymer, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl Ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, or a combination thereof, but is not limited thereto.

Specifically, the A may be a compound represented by the following formula (2), preferably 6-aminohexanoic acid.

(2)

H ₂ N (CH ₂ ) n CO ₂ H

In the above formula, n is an integer of 1? N? 18.

The 6-aminohexanoic acid may be represented by the following general formula (3), and may further serve as a linker for the peptide and further increase the physiological activity of the peptide.

(3)

According to another aspect of the present invention, there is provided a composition for promoting hair loss prevention and hair growth comprising the peptide or the peptide structure as an active ingredient.

The term " hair loss " refers to a state in which hair is normally broken at a site where hair should be present, and generally means that the hair of the scalp is missing. Unlike colorless and thick skin, the Virgin Mary can cause cosmetic problems when it is missing.

The hair loss can be classified into two types, that is, a case where the hair is clinically formed and a case where the hair is not formed. The hair loss (scarring) in which a scar is formed can not be regenerated due to the destruction of the hair follicle, Scarring) maintains the hair follicles so that the hair can be regenerated after the symptoms disappear.

The hair loss may include alopecia areata, alopecia totalis, alopecia universalis, androgenic alopecia, telogen effluvium, anagen effluvium, or chemotherapy- But is not limited to, chemotherapy-induced alopecia.

Since the peptide can promote the growth of hair follicle cells, hair growth can be fundamentally promoted and hair growing can be effectively protected.

The above-mentioned " preventing hair loss " or " promoting hair growth " can be used in a similar sense, which may have the same meaning as another term used in the art, hair loss improvement, wool promotion and hair growth promoting.

According to another aspect of the present invention, there is provided a composition comprising the peptide or the peptide structure as an active ingredient for improving wrinkles, improving skin elasticity, improving skin moisturization, or regenerating skin.

The " wrinkles " refers to the scarring caused by degeneration of the skin, and may be caused by a cause of genes, a reduction of collagen and elastin present in the skin dermis, and an external environment.

The " wrinkle improvement " refers to a phenomenon that inhibits or inhibits the generation of wrinkles on the skin, or alleviates the already generated wrinkles.

The " skin elasticity " means elasticity realized by elastic fibers composed of elastin existing in the dermal layer. Since the elastic fibers have a low elastic modulus like rubber, they are easily deformed by external force, It can be easily restored to its original state.

The elastic fibers are embedded in microfibrils in an amorphous matrix called elastin and the elastin is a protein composed of a unique amino acid found only in elastic fibers such as desmosine and isodesmosine derived from lysine . The desmosine and isodesmosin form cross-links within the long peptide chain, and the structure can confer rubber-like properties to elastin.

The " improvement of skin elasticity " means that elastic fibers composed of elastin are present together with collagen, and elastic elasticity is maintained or improved in a state where elastin and collagen are sufficiently present.

Such " comprising as active ingredient " may exhibit effects of preventing hair loss, promoting hair growth, or improving skin, such as promoting Wnt activity associated with hair growth efficacy, collagen synthesis associated with wrinkle- ≪ RTI ID = 0.0 > and / or < / RTI >

According to another aspect of the present invention, there is provided a cosmetic composition or a composition for external application for skin comprising the peptide or the peptide structure as an active ingredient.

The external preparation for skin or the cosmetic composition may be formulated by a conventional method. In the formulation of the external preparation for skin, Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can refer to, and in the formulation of the cosmetic composition, International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995, which is incorporated herein by reference.

The external preparation for skin or the composition for cosmetics may contain a conventional acceptable carrier such as oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative and a flavoring agent. But is not limited thereto.

In addition, the composition may further include a base component that can be compounded with general external scalp and hair treatment agents. Specifically, the composition may contain a solubilizer, a surfactant, a moisturizer, a thickener, a pH adjuster, an antiseptic, an antioxidant, , A bactericide, an anti-inflammatory agent, an antimicrobial agent, a solvent, a coloring agent, or a flavoring agent.

The composition may be formulated as a composition for treating hair, such as an emulsion, a cream, a paste, a gel, a lotion, a pack, a lotion, a powder, a spray or a soap, , A hair mousse, a hair gel, a hair soap, a hair shampoo, a hair rinse, a hair pack, and a hair treatment, and may be specifically formulated into a liquid hair shampoo.

In the above-mentioned formulations, components other than the above-mentioned essential ingredients can be appropriately selected and blended by those skilled in the art depending on the use or purpose of the other external preparation.

For example, when the composition of the composition is a paste, cream or gel, an animal fiber, a plant fiber, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, Can be used.

When the formulation of the composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component. In particular, chlorofluorohydrocarbons, propane, And may further include propellants such as dimethyl ether.

When the formulation of the composition is a solution or an emulsion, a solvent, a solvent or an emulsifier may be used as the carrier component. Examples of the solvent include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan can be used.

When the formulation of the composition is in the form of a suspension, the carrier component may be a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like.

However, the auxiliary agent and the mixing ratio thereof can be appropriately selected so as not to affect the desirable properties of the product according to the present invention.

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

The " prevention " means any action that reduces the frequency or extent of the pathological event. Prevention can be complete or partial. In this case, hair loss symptoms in the subject may be reduced as compared with the case where the composition is not used.

The term " treatment " means any clinical intervention to alter the natural course of a subject or cell to be treated, and may be performed during or during the course of a clinical pathology. The desired therapeutic effect may be to prevent the occurrence or recurrence of the disease, to alleviate the symptoms, to reduce all direct or indirect pathological consequences of the disease, to prevent metastasis, to reduce the rate of disease progression, Relieving or temporarily alleviating the condition, or improving the prognosis.

The compositions can be administered in the form of oral delivery, parenteral delivery. The composition may be administered systemically or topically, and the administration may include oral administration and parenteral administration. The composition may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

The composition may further comprise a carrier, an excipient and a diluent used in the preparation of the pharmaceutical composition. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, But are not limited to, cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution.

The solid preparation for oral administration may be a tablet, a pill, a powder, a granule, a capsule, etc. The solid preparation may be prepared by adding to the compound and its fractions at least one or more excipients such as starch, calcium carbonate, sucrose, lactose , Or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

The liquid preparation for oral administration may be a suspension, a solution, an emulsion, a syrup, etc. In addition to water and liquid paraffin which are simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be used.

The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Examples of the non-aqueous solutions and suspensions may include injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin.

The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The " pharmaceutically effective amount " means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dosage level will depend on the type of disease, severity, activity of the drug, Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is desirable to administer an amount that allows for all of the above factors to be maximally effected in a minimal amount without side effects, which can be readily determined by one skilled in the art.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Preparation Example: Preparation of peptide

Link amide methylbenzhydrylamine resin (147 mg, 0.1 mmol) was added to 3 mL of dimethylformamide (DMF) anhydrous organic solvent and swelled for about 30 minutes.

N-Diisopropyl carbodiimde (DIC, 48 μl) as a carboxyl group activator and N-hydrobenzotriazole (HOBt, 40 mg) as an additive additive were added to 1.5 mL of anhydrous DMF organic solvent, ) Was added and mixed and activated for about 20 minutes.

The activated solution and the resin were reacted for 2 hours with stirring, and then the reaction solution was filtered. The resin was washed several times with DMF and methanol, and the kaiser test was performed.

When the kaiser test was positive, the above-mentioned activated solution was made and participated. If the result of the test is negative, it is mixed with 3 mL of piperidine solution dissolved at a concentration of 20 wt% in DMF solvent and reacted for about 15 minutes to remove the Fmoc group at the N-terminus. Then, the amino acid of the following sequence is activated by the same method, ≪ / RTI >

After the synthesis of the amino acid portion of the last sequence was completed, the N-terminal Fmoc group was removed using 20 wt% piperidine / DMF. The resin was washed several times with DMF and methanol and then dried.

The peptide bound to the resin was reacted with cleavage solution (trifluoroacetic acid (80%), triisopropylsilane (5%), thioanisole (5%), 1,2-ethanedithiol (5% ) The side chain protecting group of the amino acid was removed and the peptide was isolated from the resin.

Trifluoroacetic acid of the peptide solution was removed by passing through nitrogen gas, diethyl ether cooled to -20 ° C was added, and the precipitated peptide was centrifuged (3,000 rpm, 20 min ).

The obtained peptide was purified using reverse phase HPLC (C-18 column C18-120A, 50 mm * 250 mm) (Eluent: water / acetonitrile in 0.1% TFA (gradient): Flow Rate 50 mL / min). A substance of 1308 molecular weight was identified using an ESI-Mass mass spectrometer.

Peptides synthesized according to the above method are shown in Table 1 below.

division	order	Remarks
Example 1	Gly-Asn-Cys-Trp-Leu	SEQ ID NO: 1
Example 2	Gly-Asn-Met-Trp-Leu	SEQ ID NO: 2
Example 3	Gly-Asn-Ser-Trp-Leu	SEQ ID NO: 3
Example 4	Asn-Thr-Leu-Phe-Lys	SEQ ID NO: 4
Example 5	Gly-Asn-Cys-Trp-Leu-Asn-Thr-Leu-Phe-Lys	SEQ ID NO: 5
Example 6	Gly-Asn-Met-Trp-Leu-Asn-Thr-Leu-Phe-Lys	SEQ ID NO: 6
Example 7	Gly-Asn-Ser-Trp-Leu-Asn-Thr-Leu-Phe-Lys	SEQ ID NO: 7
Example 8	Gly-Asn-Cys-Trp-Leu-Linker-Asn-Thr-Leu-Phe-Lys	6-Aminohexanoic acid linker

Experimental Example 1: Assessment of promoting effect of proliferation of dermal papilla cells

Human epidermal keratinocytes (HEK), human dermal fibroblasts (nHDF), human dermal attenuate, and human dermal papillae were used to analyze the natural polystatin inhibitory activity andactivin activity of synthetic peptides synthesized in the synthetic examples. Cell growth potential of human dermal papilla (HDP) was measured by MTT staining.

Human epidermal keratinocytes (Lonza, Switzerland) and human dermal fibroblasts, and human dermal papilla cells with DMEM (Dulbecco's modified Eagle's medium , gibco, USA) , each containing 10% FBS to the medium, 37C, 5% CO ₂ conditions In an incubator.

The cultured cell lines were separated from the bottom of the culture dish with 1% trypsin solution and centrifuged to obtain cell precipitate only.

The cells were resuspended in DMEM containing no FBS and cultured in a 96-well tissue culture plate at 3 x 10 ³ cells / well for 24 hours at 37 ° C and 5% CO ₂ .

After 24 hours, the medium was exchanged with the same culture medium except for the serum completely. Then, a blank sample and a synthetic peptide for standardization were dissolved in 10% DMSO in a sterilized state. Then, 1 ng / mL, 10 ng / mL, mL, 10 μg / mL and 100 μg / mL for 48 hours under the same conditions as above. Viability was measured using MTT after the culture was completed.

The cell proliferation of each of the cells treated with the peptide of the above example (10 / / mL) and the cells not treated (negative control) were compared, and the relative values thereof are shown in Table 2 below.

division		HEK	nHDF	HDP
Cell proliferation (%)	Example 1	116	155	215
	Example 2	117	153	221
	Example 3	118	160	218
	Example 4	117	156	212
	Example 5	120	154	224
	Example 6	117	156	213
	Example 7	118	155	216
	Example 8	119	158	223
	Control group	100	100	100

Referring to FIG. 2, the peptide of Example 8 promoted the growth of keratinocytes, fibroblasts, and hair follicle cells in a concentration-dependent manner.

Referring to FIG. 3, the parental spermoid was formed through three-dimensional stereoculture of dermal papilla cells (Sung, J. Invest. Dermatol. 2012, 132; 237) Thereby effectively promoting spironoid autogenesis.

These results suggest that the peptides of the Examples promote the proliferation of human dermal papilla cells and have excellent hair growth or hair loss prevention effect.

In addition, the peptide not only has excellent stability to the skin or human body, but also promotes the growth of fibroblasts that synthesize collagen effectively, thus making it possible to utilize the peptide as a cosmetic, especially for improving skin wrinkles.

Experimental Example 2: Evaluation of gene expression level

The cultured human dermal papilla cells were treated with the peptides of the example, and after 48 hours, the amount of phosphorylation of SMAD2, which is a signal of activin regulated by polystatin, and the expression level of hair growth factor were measured.

Western blotting was performed using the Phospho-SMAD2 antibody (Abcam) and the β-catenin antibody (Abcam), and the expression of FGF7, WNT5a, LEF1 and VCAN, The expression levels of growth factors were evaluated by PCR and the results are shown in Table 3 below.

division	VCAN mRNA	FGF7 mRNA	WNT5a mRNA	LEF1 mRNA
Example 1	2.0	2.5	1.4	2.7
Example 2	2.1	2.4	1.5	2.6
Example 3	2.0	2.5	1.4	2.8
Example 4	1.9	2.6	1.5	2.7
Example 5	2.1	2.7	1.3	3.0
Example 6	1.9	2.6	1.4	2.7
Example 7	2.2	2.5	1.5	2.9
Example 8	2.3	3.0	1.6	3.3

Referring to FIG. 4, when the peptide construct of Example 8 was treated with the expression of intracellular native polystin, the phosphorylation of SMAD2 was increased through activation of actibin. The expression of β-catenin, a major factor, was increased.

5, when the peptide construct of Example 8 was treated, the expression of FGF7, WNT5a, LEF1, and VCAN, which are hair growth factors produced in the dermal papilla, was increased.

These results suggest that the peptides of the Examples have excellent hair growth promoting and hair loss suppressing functions as well as excellent anti-aging effects.

Experimental Example 3: Evaluation of Versican Expression Level

The peptides of the Examples were evaluated to increase the expression of versican, a papillary cell spe- land self-assembly activity factor.

The dermal papilla cells were plated in 96-well low-adherent tissue culture plates at 1 × 10 ⁴ cells per well and cultured at 37 ° C. and 5% CO ₂ for 48 hours.

To insert the cells, a blank for capturing the standard and the peptide construct of Example 8 were added to the medium in sterilized condition in 10% DMSO.

Referring to FIG. 6, after 48 hours, dermal papilloma cell sphere was immobilized in 2% formaldehyde, PBS, and immunofluorescence staining was performed.

The results showed that the self-assembly activity factor activated by the peptide structure of Example 8 promoted the self-assembly of the papillary spheredoids, and the peptides of the Examples increased the hair growth promoting activity of the papillary cells, May be implemented.

Experimental Example 4: Estimation of hair growth promoting activity

The hair growth promoting effect of the Examples and Comparative Examples was evaluated after removing the hair of the mouse (C57BL / 6) whose hair was in the resting period and about 50 days old.

In this experiment, mice with the characteristics of body weight, size, etc. were divided into 9 groups of 10 rats and individually cultured to measure the degree of hair growth.

After applying the solution containing the peptides of the examples, the hair was raised for 30 days and the hair grown at the hair removal area was separated and weighed. Phosphorylation buffer solution-coated mice were set as control groups to further evaluate the hair growth promoting effect by the peptides. The measurement results are shown in Table 4 below.

division	Hair Weight (mg)
Example 1	194.1 ± 10.1
Example 2	195.7 ± 11.2
Example 3	193.2 ± 11.5
Example 4	188.3 ± 10.6
Example 5	193.4 ± 11.4
Example 6	196.5 ± 12.1
Example 7	194.9 ± 11.4
Example 8	212.1 ± 12.2
Control group	69.5 ± 4.5

Referring to FIG. 7, when the back region of the mouse with the peptide structure of Example 8 was observed, the back region changed to black after 10 days. After 15 days from the application, a larger amount of hair was grown Was visually confirmed.

That is, it is evaluated that the hair growth promoting effect by the peptides of the Examples is very excellent, so that hair health can be improved and hair loss can be suppressed.

Experimental Example 5: Assay for inducing collagen biosynthesis

It was confirmed that collagen synthesis was promoted by the growth of fibroblasts. Human dermal fibroblasts at a concentration of 1.0 x 10 ⁴ cells / mL were dispensed in a volume of 100 μL into a 96-well plate and cultured for 3 days at 37 ° C, 5% CO ₂ , and humidified conditions.

Medium 100 S (manufactured by Kurashiki Boshiki) was used a medium containing 10% by weight of LSGS (Low Serum Growth Supplement, Kurashiki Boshiki Co., Ltd.) per well.

The medium was replaced with DMEM (Dulbecco's Modified Eagle's Medium, Sigma), and the peptide was treated at a concentration of 50 mg / L and then cultured. The control group was prepared by adding purified water without treating the above peptides.

After the cells were cultured for 7 days, the culture solution was collected and the concentration of secreted type I collagen in the culture solution was quantified by enzyme-linked immunosorbent assay (Procollagen type I c-peptide EIA Kit, Takara Bio).

The amount of collagen in each test sample culture fluid was calculated based on the amount of type I collagen in the control group (100%). The measurement results are shown in Table 5 below.

division	Collagen production (%)
Example 1	185.1
Example 2	179.5
Example 3	182.2
Example 4	184.8
Example 5	182.4
Example 6	185.7
Example 7	183.1
Example 8	189.5
Control group	100

[% of control]

The above results suggest that the peptide composed of the amino acid sequence of the above Example is excellent in not only the hair growth promoting effect but also the skin elasticity improvement and skin wrinkle improving effect.

Experimental Example 6: Test for promoting the production of laminin and hyaluronic acid

HaCaT cells cultured for 48 hours were treated with the peptides of Examples 1 and 2, and after 72 hours, the concentrations of laminin and hyaluronic acid, which are markers of skin wrinkle improvement, were measured. At this time, the concentration was measured using a Laminin ELISA kit and a Hyaluronic acid ELISA kit.

That is, the concentrations of laminin and hyaluronic acid in HaCaT cells treated with the peptides of the Example and HaCaT cells (negative control) without any treatment were compared, and the relative values thereof are shown in Table 6 below.

division		density(%)
Laminin	Example 1	161
	Example 2	158
	Example 3	160
	Example 4	161
	Example 5	161
	Example 6	160
	Example 7	159
	Example 8	165
	Control group	100
Hyaluronic acid	Example 1	145
	Example 2	143
	Example 3	147
	Example 4	145
	Example 5	148
	Example 6	150
	Example 7	151
	Example 8	153
	Control group	100

Referring to Table 6, when the peptides of the examples were treated, the concentrations of laminin and hyaluronic acid were significantly increased compared with the negative control.

These results suggest that the peptides of the Examples are also excellent in wrinkle-improving effect and can exhibit an overall skin condition improving effect.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

SEQ ID NO: 1: Physiologically active peptide 1 derived from polystatin

SEQ ID NO: 2: Physiologically active peptide 2 derived from polystatin

SEQ ID NO: 3: Physiologically active peptide 3 derived from polystatin

SEQ ID NO: 4: Physiologically active peptide derived from polystatin 4

SEQ ID NO: 5: Polystatin-derived physiologically active peptide 5

SEQ ID NO: 6: Polystatin-derived physiologically active peptide 6

SEQ ID NO: 7: Polystatin-derived physiologically active peptide 7

Claims (13)

1. A physiologically active peptide consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7.
2. The method according to claim 1,

   A peptide that promotes the growth of fibroblasts, keratinocytes, or hair follicle cells.
3. The method according to claim 1,

   Peptides that activate growth factors of dermal papilla cells.
4. 1. A physiologically active peptide structure represented by the following formula (1).

   [Chemical Formula 1]

   R ₁ - A - R ₂

   Wherein A is a linker, R ₁ is a peptide consisting of one or more amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 3, and R ₂ is a peptide consisting of the amino acid sequence of SEQ ID NO: 4.
5. 5. The method of claim 4,

   Wherein A is a compound represented by the following formula (2).

   (2)

   H ₂ N (CH ₂ ) n CO ₂ H

   In the above formula, n is an integer of 1? N? 18.
6. 5. The method of claim 4,

   Wherein A is a 6-aminohexanoic acid.
7. 7. The method according to any one of claims 4 to 6,

   A peptide structure that promotes the growth of fibroblasts, keratinocytes, or hair follicle cells.
8. 7. The method according to any one of claims 4 to 6,

   Peptide constructs that activate growth factors of dermal papilla cells.
9. A composition for promoting hair loss prevention and hair growth comprising the peptide of any one of claims 1 to 3 or the peptide structure of any one of claims 4 to 8 as an active ingredient.
10. A composition for improving wrinkles, skin elasticity, skin moisturizing, or skin regeneration comprising the peptide of any one of claims 1 to 3 or the peptide structure of any one of claims 4 to 8 as an active ingredient.
11. A cosmetic composition comprising the peptide of any one of claims 1 to 3 or the peptide structure of any one of claims 4 to 8 as an active ingredient.
12. A composition for external application for skin comprising the peptide of any one of claims 1 to 3 or the peptide structure of any one of claims 4 to 8 as an active ingredient.
13. A pharmaceutical composition for preventing or treating hair loss comprising the peptide of any one of claims 1 to 3 or the peptide structure of any one of claims 4 to 8 as an active ingredient.

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