KR20130032788A

KR20130032788A - Peptides for promotion of hair growth and the use thereof

Info

Publication number: KR20130032788A
Application number: KR1020110096592A
Authority: KR
Inventors: 박정민; 이진영; 김상윤; 박승범; 손주아; 김희종; 조대호; 정민경; 하수경; 방사익
Original assignee: 일양약품주식회사; 사회복지법인 삼성생명공익재단; 숙명여자대학교산학협력단
Priority date: 2011-09-23
Filing date: 2011-09-23
Publication date: 2013-04-02

Abstract

The present invention relates to a peptide for promoting hair growth and its use, and more particularly to a pharmaceutical composition for preventing and treating hair loss or alopecia and a composition for promoting hair growth comprising a peptide for promoting hair growth as an active ingredient. Compared to Minoxildil, the peptide of the present invention increases the proliferation and VEGF production of the dermal papilla cells at a lower concentration, thereby providing excellent blood circulation as well as the nutritional supply function of the hair, which is excellent in preventing hair loss and promoting wool. Therefore, the peptide of the present invention can be very advantageously used in medicine, quasi-drugs and cosmetics for the purpose of preventing hair loss and promoting wool.

Description

Peptides for promotion of hair growth and use thereof {Peptides for promotion of hair growth and the use}

The present invention relates to a peptide for promoting hair growth and its use, and more particularly to a pharmaceutical composition for preventing and treating hair loss or alopecia and a composition for promoting hair growth comprising a peptide for promoting hair growth as an active ingredient.

Hair is a thin, keratinized structure produced on the surface of the skin. Hair protects and protects the body from external stimuli such as buffering and external stimuli such as ultraviolet rays, temperature, and friction, and absorbs heavy metals such as arsenic, mercury, and zinc that are unnecessary to the body and releases them to the body. In modern times, the beauty aspect of decoration is also emphasized.

Human hair is about 100,000-150,000 pieces, and each hair has a different growth cycle. The hair growth cycle consists of three stages: the anagen stage in which hair is most actively growing, the catagen stage in which hair degeneration begins, and the telogen stage in which hair growth stops or enters rest. Classified as This cycle is repeated over three to six years, with about 88% of the hair on the scalp in the growth phase, only about 1% in the retrogression period, and the rest in the resting phase. As hair loss progresses, a sharply high rate of hair belongs to the resting phase, and accordingly, the proportion of hair belonging to an active growth phase is lower.

Hair loss is generally characterized by androgenetic alopecia (AGA), which is characterized by scalp hair loss in men and women as they age. Other hair loss includes alopecia areata (AA), which is usually microscopic inflammatory and can be restored and called local alopecia, and alopecia (secondary alopecia) associated with chemotherapy or radiation therapy.

In general, hair loss is more frequent in men than in women. The cause of hair loss, roughness, and malnutrition has not yet been scientifically clearly identified, but this is known to have the largest genetic predisposition, and other disorders of the endocrine system, abnormalities of the autonomic nervous system, allergies, and malnutrition of the hair roots. , Bacterial infections, and aging. Recently, the incidence rate of alopecia is increasing due to external environmental factors such as environmental pollution and excessive mental stress.

Currently, the most widely used method for the treatment of hair loss is self hair transplantation (self hair transplantation), which is performed by transplanting hair using its own hair, and vasodilators (capronium chloride, minoxidil and Extracts of various plants, etc.) or hormones (female hormones such as estrogen, estradiol, progesterone, etc.) to inhibit the action of male hormones that fall off the hair by attaching it to the nipples or pentadecane Drug treatment using acid, fenasteride) and the like are widely used. Among them, two drugs (Minoxidil and Propecia), which are approved by the US Food and Drug Administration (FDA) for hair loss treatment, are minoxidil, which is a drug applied by vasodilator action, and oral propecia is a male hormone based on finasteride. It is widely used as an agent which is excellent in the hair loss prevention effect by activation suppression effect. However, the effects of drug treatment show the hair growth efficacy at the time of administration, but hair loss resumes when the treatment is stopped, and Minoxidil, which has the highest market rate, has a large variation in the effects of each person and should not be used. If the effect does not last and return to the state before use and there is irritation on the skin such as erythema, inflammation, infection, irritation or pain of the scalp, and it has a hypotensive effect, so it is prudent for hypertensive patients, including patients who are taking hypotensives. Side effects such as sexual dysfunction are known as disadvantages (Bouhanna, P., Dermatol Surg., 29: 1130, 2003; Thiboutot, D., Arch Dermatol., 135: 1417, 1999; Messenger, AG and Rundegren J., Br J Dermatol., 150: 186, 2004). In addition, compounds or natural products that are reported to have a hair growth effect have a weak or no effect, and many have not been scientifically or medically verified, and many may cause problems.

In addition, recently, stem cell treatment methods for inducing stem cells to induce differentiation into hair follicle cells have been proposed by using the multipotency of stem cells. Fats and adipocytes are closely related to adipocyte formation and hair follicle formation, and have been shown to be effective in hair growth. Recently, it has been reported that injection of Adipose stem cells is more effective. (Am. J. Anat. 161: 85-100, 1981). However, in order to induce the hair growth effect using stem cells, a large amount of stem cells are required, but there is still insufficient culture technology to obtain sufficient amount to apply the stem cells isolated from the tissue to the clinical practice. It is difficult to apply.

Therefore, the present inventors have conducted a search for a peptide having an effect that can replace Minoxdil, which is used as an existing hair regrowth agent, and synthesized several new peptides, substituted, added, removed, and selected effective derivatives thereof. The present invention was completed by repeatedly synthesizing a novel peptide having hair growth promoting effect.

Accordingly, an object of the present invention is a peptide comprising an amino acid sequence represented by the general formula [N-terminal-X ₁ -X ₂ -AGX ₃ -S-C-terminal], wherein X ₁ is a nonpolar or basic amino acid, X ₂ is a polar amino acid, and X ₃ is a charged amino acid.) It is to provide a pharmaceutical composition for preventing and treating hair loss or alopecia comprising as an active ingredient.

Another object of the present invention to provide a composition for promoting hair growth comprising a peptide of the present invention as an active ingredient.

In order to achieve the above object, the present invention provides a peptide comprising an amino acid sequence represented by the general formula [N-terminal-X ₁ -X ₂ -AGX ₃ -S-C-terminal], wherein X ₁ is a non-polar Or a basic amino acid, X ₂ is a polar amino acid, X ₃ is a charged amino acid), and provides a pharmaceutical composition for preventing and treating hair loss or alopecia.

In order to achieve another object of the present invention, it provides a composition for promoting hair growth comprising the peptide of the present invention as an active ingredient.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following reference provides one of the skills having a general definition of several terms used in the specification of the present invention: Singleton et al ., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); And Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

Hereinafter, the content of the present invention will be described in more detail.

Amino acid sequences used in the present invention are described as follows according to the IUPAC-IUB nomenclature.

Arginine: R, Histidine: H

Lysine: K, Aspartic acid: D

Glutamix acid: E, Serine: S

Threonine: T, Asparagine: N

Glutamine: Q, Cysteine: C

Glycine: G, Proline: P

Alanine: A, Isoleucine: I

Leucine: L, Methionine: M

Phenylalanine: F, Tryptophan: W

Tyrosine: Y, Valine: V

The present invention provides a peptide for promoting hair growth comprising an amino acid sequence represented by the following general formula (I).

(I) [N-terminal-X ₁ -X ₂ -AGX ₃ -S-C-terminal]

In the general formula (I) of the present invention

X ₁ is a nonpolar or basic amino acid,

X ₂ is a polar amino acid,

X ₃ is a charged amino acid.

Nonpolar amino acids represent a group of amino acids whose side chains do not have polarity due to their chemical structure. The nonpolar amino acids may be alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, proline, preferably proline.

Basic amino acids are polar amino acids that represent amino acids with side chains of positive charge at neutral pH. The basic amino acid may be lysine (K), arginine (R), histidine (H), preferably lysine.

Polar amino acids represent a group of amino acids whose side chains are polar in their chemical structure. Polar amino acids include groups of amino acids with no charge side chain at neutral pH, amino acids with positive charge side chain (basic amino acids) and amino acids with negative charge side chain (acidic amino acids). The polar amino acid may be lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, preferably asparagine or lysine.

Charged amino acids are amino acids having a charged side chain at neutral pH, including acidic or basic amino acids, and may be lysine, arginine, histidine, aspartic acid, glutamic acid, preferably histidine, glutamic acid or aspartic acid.

The peptide of the present invention may be any one including the amino acid sequence represented by the general formula (I), but preferably any one amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3 It may be a peptide comprising a.

The peptide of the present invention exhibits the effect of promoting hair growth, and the length thereof is not limited as long as it includes the amino acid sequence represented by the general formula (I), but preferably includes the amino acid sequence represented by the general formula (I). And 6 to 50 amino acids, more preferably 6 to 30 amino acids, more preferably 6 to 25 (or 24, 23, 22, 21, 20) , 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7), most preferably It may consist of an amino acid sequence represented by the general formula (I). Most preferably, the peptide of the present invention may be a peptide composed of an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3.

In addition, the peptides of the present invention may be appropriately modified, preferably C-terminally amidated peptides to protect against protein cleavage enzymes in vivo and increase stability.

The peptide is protected from protein cleavage enzymes in vivo and the amino terminus and carboxy terminus are modified or protected with several organic groups to increase stability.

That is, if the C-terminus of the peptide is a form that can be modified to increase stability, there is no particular limitation, but may be preferably modified with a hydroxy group (-OH) or an amino group (-NH ₂ ). The C-terminus amidated peptide refers to a modification of the C-terminus of the peptide with an amino group (-NH ₂ ).

In addition, if the N-terminus of the peptide is a form that can be modified to increase stability, there is no particular limitation, but preferably an acetyl (Acetyl) group, fluorenyl methoxy carbonyl (Fmoc) group, formyl (Formyl) Group, Palmitoyl (Palmitoyl) group, Myristyl (Myristyl), Stearyl (Stearyl) group and polyethylene glycol (PEG) may be modified to a group selected from the group consisting of.

The peptides of the present invention can be synthesized by methods well known in the art, for example by automated peptide synthesizers, and can also be produced by genetic engineering techniques. For example, a fusion gene encoding a fusion partner of the fusion partner and the peptide of the present invention may be produced by genetic engineering, transformed into a host microorganism, expressed in the form of a fusion protein in the host microorganism, and then a protease or compound. The peptide of the present invention may be cleaved and separated from the fusion protein to produce a desired peptide. To this end, for example, a DNA sequence encoding an amino acid residue that can be cleaved by a protease such as Factor Xa or enterokinase, a compound such as CNBr or hydroxylamine is inserted between the fusion partner and the peptide gene of the invention. Can be.

For example, to confer sites cleaved by CNBr, restriction enzyme sites (eg, AflIII, BsmI, BspHI, BspLU11I, NcoI, NdeI, NsiI, Ppu10I, SphI, StyI) or their isoschizomer sites are inserted and cleaved with restriction enzymes, and the restriction enzyme sites linked to the reading frame are generated while generating ends corresponding to these restriction enzymes. The peptide may be inserted at the 5 'end of the peptide gene of the invention and cleaved with the enzyme to link the fusion partner with the peptide gene of the invention.

As another example, a DNA sequence capable of encoding asparagine-glycine may be inserted between the fusion partner gene and the peptide gene of the present invention to confer sites cleaved by hydroxylamine. For example, in the case of fusion of two genes, a restriction enzyme site containing an asparagine codon with a reading frame or an isoskizomer site thereof is inserted at the 3 'end of the fusion partner, and then cleaved with this enzyme and corresponding to this enzyme. A restriction enzyme site containing a codon of glycine to which the reading frame is linked while generating a compatible or blunt end is inserted into the 5 'end of the peptide gene of the present invention and cleaved with the restriction enzyme, followed by fusion partner By connecting the peptide gene of the present invention and the fusion partner gene and the peptide of the present invention can be fused. The gene construct according to the present invention can be cloned into a host cell by inserting it into a vehicle capable of inserting or incorporating an expression vector, eg, plasmid, virus or other structural genes commonly used in the art. Can be.

In an embodiment of the present invention, a peptide having an amino acid sequence of SEQ ID NOS: 1 to 3 was synthesized using an automatic peptide synthesizer, and purified purely using C18 reversed phase high performance liquid chromatography.

The peptide of the present invention has the effect of promoting hair growth in humans or animals. The hair growth promoting effect of the peptide having the amino acid sequence of the present invention was first found in the present invention.

The hair growth promoting effect of the present invention is well shown in cell line experiments using human dermal papillae cells (HDPCs) and hair loss animal model experiments using C57 / BL6J mice.

In one embodiment of the present invention it was tested whether the proliferation of HDPCs by promoting the peptide of the present invention to HDPCs. As a result, it was confirmed that the proliferation of HDPCs was promoted when the peptide of the present invention was treated (see Example 2).

In another embodiment of the present invention, the effect of the peptide of the present invention on VEGF production of HDPCs was tested. 48 hours after the medium containing the peptide was treated with HDPCs, the cultures were collected to measure the amount of VEGF produced by the ELISA method. As a result, it was confirmed that the peptide of the present invention increased VEGF expression of HDPCs (see Example 3).

In another embodiment of the present invention, the effect of the peptide of the present invention on the killing phenomenon of HDPCs appearing when exposed to DHT. After 48 hours of treatment with HDPCs of a medium containing DHT and this peptide, which promotes cell death, cells were collected and measured for cell death through Annexin V / 7-AAD staining. As a result, it was confirmed that the peptide of the present invention reduced the death of HDPCs by DHT (see Example 3).

In another embodiment of the present invention, the peptide of the present invention was tested to induce hair growth in mice in which hair loss occurred. A hair loss mouse model was constructed by depilating the back using mice inverted during the resting period. After injecting the peptide of the present invention into depilated mice, hair growth was observed. As a result, it was confirmed that the peptide of the present invention promotes the hair growth of the mouse (see Example 4).

As described above, the peptide of the present invention promotes hair growth by increasing proliferation of papillary cells and VEGF production, and inhibits hair loss by DHT.

Accordingly, the present invention provides a pharmaceutical composition for preventing and treating hair loss or alopecia comprising the peptide of the present invention as an active ingredient.

The composition of the present invention is characterized by containing the peptide of the present invention as an active ingredient.

The compositions of the present invention can be administered with other biologically active agents, such as biologically active compounds, other peptides, and the like. In addition, the composition of the present invention may further comprise a cell having the ability to differentiate into hair forming hair cells, the cell having the ability to differentiate into vascular endothelial cells is preferably embryonic stem cells, medium It may be lobe stem cells or hematopoietic stem cells.

The pharmaceutical compositions according to the invention may contain the peptides of the invention alone or may comprise one or more pharmaceutically acceptable carriers. In addition, the compositions of the present invention may further contain excipients or diluents.

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 compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation 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. .

In addition, the pharmaceutical composition of the present invention may add a base component that can be blended in general external preparations for scalp and hair as needed. Specific examples include dissolution aids, surfactants, moisturizers, thickeners, pH adjusters, preservatives, antioxidants, metal ion sequestrants, fungicides, anti-inflammatory agents, antimicrobial agents, solvents, colorants, or flavoring agents.

Here, the dissolution aid may be specifically isopropyl myristate, polyethylene glycol, medium chain fatty acid triglycerides, hydrocarbons, glycols and the like.

Among the surfactants, anionic surfactants are specifically ammonium lauryl sulfosuccinate, ammonium lauryl sulfate, sodium cocoyl isionate, sodium lauryl isceionate, sodium lauryl sulfate, triethanolamine lauryl sulfate, sodium Lauryl ether sulfate (1 to 3 ethylene oxide) and the like. Among the surfactants, nonionic surfactants may specifically be polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyoxyethylene, hardened castor oil derivatives, fatty acid diethanolamides, glyceryl stearate, and the like. The cationic surfactant in the surfactant may specifically be a tertiary aliphatic amine salt, alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, or the like. The amphoteric surfactant in the surfactant may be specifically betaine, amide betaine, sulfobetaine, stear trimonium chloride, and the like.

Moisturizing agent may be specifically glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, sorbitol and the like.

The thickener may specifically be a water-soluble high molecular compound such as methyl cellulose, hydroxyethyl cellulose, carrageenan, carboxymethyl cellulose, hydroxymethyl cellulose and the like.

The pH adjuster may specifically be citric acid, sodium hydroxide, triethanolamine, sodium citrate, phosphoric acid, sodium phosphate, lactic acid, and the like.

Preservatives may specifically be benzoic acid, paraoxybenzoic acid esters, mixtures of methylchloroisothiazolinone, phenoxyethanol, DMDM, and the like.

The antioxidant may specifically be dibutylhydroxytoluene, ascorbic acid, and the like.

Specifically, the metal ion blocking agent may be ethylenediaminetetraacetate acetate, ethylenediaminetetraacetate tetrasodium, or the like.

Fungicides may specifically be chlorhexidine gluconate, quaternary ammonium salts, pyroxtonolamines, zinc pyrithione suspensions, iodopropynyl butylcarbamate, salicylic acid and the like.

The anti-inflammatory component may specifically be monoammonium glycyrrhinate, dipotassium glycyrizine, stearyl glycyrizinate, chamomile, alpha bisabolol, allantoin and mixtures thereof.

The antimicrobial component is specifically phenoxyethanol, chlorhexidine, chlorhexidine gluconate, pyroxtonolamine, ketoconazole, arnica extract, iodopropynylbutylcarbamate, benzalkonium chloride, benzenetonium chloride, benzoic acid and salts thereof, benzyl alcohol, lavender, Rosemary, salicylic acid, triclocarban, zinc pyrithione suspensions and mixtures thereof and the like.

The solvent may be specifically ethanol, purified water, Tween 20, cyclomethicone, mineral oil, dimethicone, and the like.

Colorants and flavoring agents can use the bases used in general scalp and hair preparations.

The pharmaceutical composition of the present invention may be prepared in any formulation that can be applied to the scalp and hair in a liquid, cream, paste, or solid form, and the like, and shampoos and hair conditioners for promoting hair growth by adding conventional additives. , Hair lotions, hair tonics, hair packs, combinations of shampoos and rinses, liquid wool types, and the like, including aerosol types of these formulations. The composition for preventing hair loss and promoting wool is preferably used in a method of transdermal administration such as direct application or spraying to the scalp or hair.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol which is administered in multiple doses for a long time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease. Preferably the preferred total dose of peptide of the invention may be from about 0.0001 μg to 500 mg, most preferably 0.01 μg to 100 mg per kg of patient body weight per day. However, the dose of the peptide is determined by taking into consideration various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the pharmaceutical composition. In view of this, one of ordinary skill in the art will be able to determine appropriate effective dosages for the particular use of the compositions of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown.

In addition, the present invention provides a composition for promoting hair growth comprising the peptide of the present invention as an active ingredient. The hair growth promoting composition of the present invention may be a cosmetic composition.

The cosmetic composition according to the present invention contains all of the forms suitable for topical administration (application, application) according to methods known in the art by further adding cosmetically and / or dermatologically acceptable excipients to the peptides of the present invention as active ingredients. It may be prepared in a dosage form. For example, but not limited to, the cosmetic composition according to the invention may be a solution, gel, solid, emulsion, suspension, microemulsion, microcapsules, microgranules, ionic and / or nonionic vesicle dispersants, creams, skins, It may be prepared in the form of a lotion, powder, ointment, spray or stick.

Such cosmetically and / or dermatologically acceptable excipients may include emollients, emulsifiers, thickening agents and solvents.

The emollients are used to soften, sooth, coat, smooth or moisturize the skin. In the present invention, the emollient may use all kinds known in the art and may be, for example, petroleum based, fatty acid esters, alkyl ethoxylates, fatty acid ester ethoxylates, fatty alcohols, polysiloxanes or mixtures thereof. In addition to propylene glycol, butylene glycol, glycerin, triethylene glycol, mercury, waxes, fatty acids, fatty alcohol ethers, glycerides, acetoglycerides, ethoxylated glycerides, other fatty acid esters of polyhydroxy alcohols, lanolin and derivatives thereof Such conventional emollients.

The emulsifier serves to mix the water phase and the oil phase of the cosmetic composition. In the present invention, the emulsifier may optionally contain one or more, and may include both nonionic, anionic or cationic. The type of the emulsifier may be determined depending on whether the emulsion is in water-in-oil dispersion or oil-in-water dispersion. Examples of suitable emulsifiers include, but are not limited to, sorbitan trioleate, sorbitan tristearate, glycerol monooleate, glycerol monostearate, glycerol monolaurate, sorbitan sesquioleate, sorbitan monooleate, Sorbitan monostearate, polyoxyethylene stearyl ether, polyoxyethylene sorbitol beeswax derivative, PEG 200 dilaurate, PEG 200 monostearate, PEG 400 dioleate, sorbican monopalmitate, polyoxyethylene monostearate and Polyoxyethylene sorbitan monostearate and the like.

Thickeners include crosslinked carboxypolymethylene polymers, ethyl cellulose, polyethylene glycol, tracant gum, karaya gum, xanthan gum, bentonite, hydroxyethyl cellulose and hydroxypropyl cellulose.

As the solvent, purified water, alcohol or a mixture of purified water and alcohol may be used.

Examples of other optional cosmetic additives that may optionally be used include, but are not limited to, preservatives such as para-hydroxybenzoate esters, butyl hydroxy toluene, ascorbic acid and its derivatives and antioxidants such as tocopherol and its derivatives, glycerol, Wetting agents such as sorbitol, 2-pyrrolidone-5-carboxylate, dibutylphthalate, gelatin, polyethylene glycol, pH buffers such as acetates, phosphates, citrate, triethanolamine and carbonates, waxes such as beeswax, paraffin Types, thickeners, activity enhancers, colorants and flavorings can be used.

The preferred content of the peptide of the present invention in the cosmetic composition of the present invention may include about 0.001 to 1% by weight based on the total weight of the cosmetic.

Accordingly, the present invention provides a pharmaceutical composition for preventing and treating hair loss or alopecia and a composition for promoting hair growth, comprising the peptide of the present invention having an hair growth promoting action as an active ingredient. Compared to Minoxildil, the peptide of the present invention increases the proliferation and VEGF production of the dermal papilla cells at a lower concentration, thereby providing excellent blood circulation as well as the nutritional supply function of the hair, which is excellent in preventing hair loss and promoting wool. Therefore, the peptide of the present invention can be very advantageously used in medicine, quasi-drugs and cosmetics for the purpose of preventing hair loss and promoting wool.

1 is a comparative graph measuring the growth promoting effect of the dermal papilla cells of the peptide of the present invention (control: negative control group, Minoxidil: positive control group, Pep_1 to 3: peptide of the present invention represented by SEQ ID NO: 1 to 3).
Figure 2 is a comparison graph measuring the effect of increasing the VEGF production of dermal papilla cells of the peptide of the present invention (control: negative control group, Minoxidil: positive control group, Pep_1 to 3: peptide of the present invention represented by SEQ ID NO: 1 to 3).
3 is a result confirming that the peptide of the present invention inhibits the death of dermal papilla cells caused by DHT (control: negative control, Pep_1 to 3: treated with DHT of the present invention represented by SEQ ID NOs: 1 to 3) Peptide, Minoxidil: efficacy comparison group).
Figure 4 is a comparison graph that quantifies the hair growth rate and the photograph taken that the mouse's hair growth is promoted by the peptide of the present invention (control: negative control, Minoxidil: positive control, Pep_1 to 3: shown in SEQ ID NOs: 1 to 3) Peptide of the present invention).

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

&Lt; Example 1 >

Peptide Production

A peptide having the amino acid sequence shown in Table 1 below was synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and these were analyzed using C18 reversed phase high performance liquid chromatography (HPLC) (Waters Associates, USA). The synthesized peptides were isolated pure. The column used ACQUITY UPLC BEH300 C18 (2.1 × 100 mm, 1.7, Waters Co, USA).

Amino acid sequence of the prepared novel peptide SEQ ID NO: Peptide name order One Pep_1 PKAGES 2 Pep_2 KNAGDS 3 Pep_3 KNAGHS

<Example 2>

Peptide Effect of Human Peptides

In order to examine the effects on the hair growth of the three peptides prepared in Example 1, human hair papilla cells (HDPCs, Human Dermal Papilla Cells), which are cells responsible for hair growth by secreting hair growth regulators from hair follicles To the peptide of the present invention was treated and cell proliferation was measured.

HDPCs (Promocell, Heidelberg, Germany) were cultured using Follicle Dermal papilla Cell medium (Promocell, Heidelberg, Germany) containing 4% fetal bovine serum. Cultured HDPCs were dispensed to inject 1000 cells per well into a 96-well plate and incubated for 24 hours. Three peptides of the present invention were added to each well and incubated for 96 hours. Instead of the peptide of the present invention, the positive control group was cultured under the same conditions as Minoxidil and the negative control group, which were known to induce proliferation of human dermal papilla cells as the main component of a representative hair loss treatment agent. After incubation, the degree of cell proliferation was measured using CyQUANTkit (invitrogen, USA).

As a result, as shown in [FIG. 1], the peptide of the present invention was confirmed to have excellent HDPCs proliferation effect, and some peptides were confirmed to have better effect than Minoxidil which is a positive control group.

<Example 3>

Confirmation of VEGF Production in Human Papillae Cells by Peptides

In order to examine the effect on the hair growth of the three peptides prepared in Example 1, the production of vascular epithelial growth factor (VEGF) in HDPCs by peptides was measured. VEGF is the most common and effective growth factor known to stimulate angiogenesis and is known to increase hair permeability in hair follicles and promote hair growth.

After dispensing HDPCs in a 6-well plate to inject 50000 cells per well and incubating for 24 hours, three peptides of the present invention were added to each well. Instead of the peptide of the present invention, a positive control group was treated with the same condition as Minoxidil and a negative control group, which were known to induce proliferation of human dermal papilla cells as a main component of a representative hair loss treatment agent. After 48 hours of incubation, cell medium was collected. Quantitative determination of VEGF present in the medium was performed according to the protocol provided by purchasing an Enzyme-Linked Immunosorbent Assay (ELISA) kit manufactured and sold by R & D Systems (Minneapolis, MN, USA).

As a result, as shown in FIG. 2, it was confirmed that the production of VEGF was increased in HDPCs through the treatment of Pep_2, 3 (peptide of the present invention represented by SEQ ID NOs: 2,3).

< Example 4>

Peptide DHT Induced by Human dermal papilla cells Death Inhibitory ability Confirm

DHT (Dihydrotestosterone) is a metabolite of testosterone, a male hormone, and is known as a representative substance for inducing male hair loss. DHT induces apoptosis of cells of hair follicles, leading to hair follicle degeneration and leading to hair loss. Therefore, the degree of apoptosis was measured by annexin V / 7-AAD staining to determine whether the three peptides prepared in Example 1 could inhibit cell death caused by DHT.

After dispensing HDPCs in a 6-well plate to inject 50000 cells per well and incubating for 24 hours, three peptides of the present invention and Minoxidil were mixed in a medium containing 100 μM of DHT and treated in wells. The group treated with nothing as a negative control group was cultured under the same conditions. After 48 hours treatment, only cells were collected and transferred to FACS tubes and washed with PBS. The cells were suspended in annexin V binding buffer and stained with annexin V and 7-AAD for 15 minutes at room temperature. Stained cells were analyzed by flow cytometry FACS Calibur (BD Biosciences).

FIG. 3 is an image analyzed by FACS Calibur (BD Biosciences), which is a cell counterpart, and cells corresponding to the second quadrant are undergoing late apoptosis, and cells corresponding to the fourth quadrant are the initial cell death process ( early apoptosis). Therefore, the degree of cell death (apoptosis) was confirmed by quantifying the distribution of cells corresponding to quadrant 2 and quadrant 4.

As a result, when analyzing DPC-treated HDPCs, about 27.1% of all cells developed apoptosis, which is about 6 times higher than the control group (4.4%) which did not process anything. In contrast, mixing treatment with DHT and Pep_1 to 3 (peptides of the present invention represented by SEQ ID NOS: 1 to 3) reduced death occurring in DHT. In particular, the efficacy of Pep_1 (peptide of the present invention represented by SEQ ID NO: 1) was very excellent. On the other hand, Minoxidil confirmed that the ability of DHT to inhibit HDPCs cell death is insignificant.

< Example 5>

Peptides Hair Growth Effect of Treated Mice

In order to verify the efficacy of the three peptides of the present invention obtained in Example 1, animal experiments were evaluated for hair growth efficacy.

As an animal test model for evaluating the effect of promoting hair growth, a C57BL / 6 mouse model was used. The C57BL / 6 mouse has black hair, is characterized by spontaneous alopecia, and also melanocytes. Is present only in hair follicles, and the melanin synthesis matches well with the hair growth cycle, which has the advantage of determining the hair growth cycle by the color of the skin. It is used.

42 ~ 56 days old mice (C57BL / 6, mal, Central Experimental Animal, Korea) were removed with an electric razor and then completely removed with hair removal cream. The experimental animal group was negative control group and positive control group, Pep_1 (peptide of the present invention represented by SEQ ID NO: 1) treated group, Pep_2 (peptide of the present invention represented by SEQ ID NO: 2) treated group, Pep_3 (expressed by SEQ ID NO: 3) The peptide group of the present invention was divided into a total of five groups.

After the adaptation period for one day, three peptides were endothelially injected into the hair removal site once daily for one week from the following day, and the negative control group was endothelially injected with PBS. Positive controls received 3% Minoxidil daily. On the 8th, 15th and 21st day of starting the material treatment, the hair growth site was photographed using a digital camera with visual observation.

As a result of the 15 days after the peptide treatment, as shown in [FIG. 5], all hairs were treated in the group treated with Pep_1 to 3 (peptide of the present invention represented by SEQ ID NOS: 1 to 3) compared to the control group in which the hair growth hardly occurred. Hair growth was observed in all or part of the cut. As such, in the case of the control group, hair growth did not occur as long as there was no artificial stimulus due to entering the resting phase, while a distinct hair growth phenomenon was observed in the mice treated with peptides, thereby confirming the hair growth promoting effect of the peptide of the present invention.

As described above, the present invention provides a pharmaceutical composition for preventing and treating hair loss or alopecia and a composition for promoting hair growth, comprising the peptide of the present invention having a hair growth promoting effect as an active ingredient. Compared to Minoxildil, the peptide of the present invention increases the proliferation and VEGF production of the dermal papilla cells at a lower concentration, thereby providing excellent blood circulation as well as the nutritional supply function of the hair, which is excellent in preventing hair loss and promoting wool. Therefore, the peptide of the present invention can be very advantageously used in medicine, quasi-drugs and cosmetics for the purpose of preventing hair loss and promoting wool.

<110> IL-YANG PHARM. CO., LTD SAMSUNG LIFE PUBLIC WELFARE FOUNDATION Industry-Academic Cooperation Foundation, Sookmyung Women's University <120> Peptides for promotion of hair growth and the use <130> NP11-0054 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> P223: Artificial Peptide for Promotion of HDPCs <400> 1 Pro Lys Ala Gly Glu Ser 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> Pep_2: Artificial Peptide for Promotion of HDPCs <400> 2 Lys Asn Ala Gly Asp Ser 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> Pep_3: Artificial Peptide for Promotion of HDPCs <400> 3 Lys Asn Ala Gly His Ser 1 5

Claims

A pharmaceutical composition for preventing and treating hair loss or alopecia comprising as an active ingredient a peptide having an amino acid sequence represented by the following general formula (I):
(I) [N-terminal-X ₁ -X ₂ -AGX ₃ -S-C-terminal]
only,
X ₁ is a nonpolar or basic amino acid,
X ₂ is a polar amino acid,
X ₃ is a charged amino acid.

The composition of claim 1, wherein X ₁ is proline or lysine.

The composition of claim 1, wherein X ₂ is lysine or asparagine.

The composition of claim 1, wherein X ₃ is selected from the group consisting of glutamic acid, aspartic acid and histidine.

The composition of claim 1, wherein the peptide is any one selected from the group consisting of peptides of SEQ ID NOs: 1 to 3.

The composition according to any one of claims 1 to 5, wherein the peptide is a C-terminally amidated peptide.

A cosmetic composition for promoting hair growth comprising a peptide having an amino acid sequence represented by the following general formula (I) as an active ingredient:
(I) [N-terminal-X ₁ -X ₂ -AGX ₃ -S-C-terminal]
only,
X ₁ is a nonpolar or basic amino acid,
X ₂ is a polar amino acid,
X ₃ is a charged amino acid.