KR101578901B1 - Peptides for promoting hair growth, method for preparing thereof, and, composition comprising thereof for preventing alopecia or promoting hair growth - Google Patents

Abstract

The present invention relates to a hair-loss-preventing or hair-growth promoting composition containing a hair-growth promoting peptide that has been proved as a hair-growth promoting effect, and the peptide of the present invention exhibits an excellent hair-growth effect. In particular, the peptides of the present invention prepared from the dimer form and the copper metal complex have higher safety and stability in use than existing peptide compositions, and thus can be easily used as a composition for preventing hair loss or promoting hair growth.

Description

FIELD OF THE INVENTION The present invention relates to a hair growth promoting peptide, a method for producing the hair growth promoting peptide, and a composition for preventing hair loss or promoting hair growth,

The present invention relates to a hair growth promoting peptide, a method for producing the same, and a composition for promoting hair loss prevention or hair growth containing the same.

The human hair has about 100,000 to 150,000 hairs, and each hair has a different period and grows and falls through anagen, catagen, and telogen. This cycle repeats over 36 years and results in an average of 50 to 100 hairs per day dropping normally.

In general, alopecia or hair loss means that the number of abnormal hair loss is increased due to the shortening of hair growth rate during the growing period and the increase of hair in the retrograde period or resting period among these cycles. Especially, In the case of male hair loss, there is no decrease in hair density, but it is revealed that hair thickness is extremely reduced.

The most widely used formulation to treat or prevent such alopecia is Minoxidil, one of the two FDA approved hair growth agents. Minoxidil was a remedy for hypertension, but it became a more popular drug as a hair growth drug nowadays with hair growth due to side effects.

Minoxidil's hair growth mechanism has not been clarified precisely, but it is supposed that nutrient supply to the hair follicle promotes hair growth by increasing blood flow through vasodilation effect.

This model of blood flow increase has been shown indirectly by a recent report that minoxidil increases the expression of vascular endothelial growth factor (VEGF), a growth factor related to vasodilation in the dermal papilla, (Br. J. of Dermatol., 1998, 138, 407-411). In addition, in addition to the vasodilating effect of minoxidil, hair follicle cell activation (Skin Pharmacol., 1996, 9, 3-8) and hair follicle tissue culture promoted hair follicle growth in vivo. Invest. Dermatol., 1989, 92, 315-320) suggest that minoxidil acts as a direct growth factor in hair follicles.

Finasteride, a major component of Merck's recently launched propecia, has also been shown to inhibit the conversion of testosterone to the more potent male hormone dihydrotestosterone (DHT) .

In December 1997, a 1 mg tablet was approved for use by the FDA for the treatment of male alopecia, which is currently on the market and has been shown to have significant effects on clinical outcome, but some male functional inhibitory side effects have also been reported (J. Am. Acad. Dermatol , 1998, 39, 578-589). However, the drug is not as excellent as minoxidil, and clinical research is actively pursued for better hair growth due to concerns about side effects.

Although active ingredients that are biased toward each of these effects are reported to be effective in enhancing hair growth in animal experiments or limited clinical trials, they do not meet complex hair growth mechanisms, and the individual hair loss characteristics There are many cases in which the majority of users do not notice a noticeable effect because they are not considered. Therefore, it is necessary to develop new hair growth promoting products that have competitiveness with overseas competitiveness, conceptual competitiveness, and commercialization of original technology.

The inventors of the present invention have completed the present invention by confirming that the present peptides have excellent hair growth or hair loss suppressing effects while carrying out studies using functional peptides.

On the other hand, as the hair growth peptide, peptides for promoting hair growth (Korean Patent Laid-Open Publication No. 2013-0032788), EDAR ligand-derived peptide (Korean Patent Laid-Open Publication No. 2013-0015531), X ₁ -X ₂ -AGX ₃ -SC form Derived peptide (Korea Patent No. 1209117), FGF10-derived peptide (Korean Patent No. 1187871), Noggin-derived peptide (Korean Patent No. 1163171), fibroblast growth factor-9 peptide Patent Document No. 2011-0092301). However, the peptides disclosed in the above prior arts have completely different structures from the peptides contained in the composition of the present invention.

Korean Patent Laid-Open Publication No. 2013-0032788 (peptide for promoting hair growth and use thereof, published on Apr. 04, 2013) Korean Patent Laid-Open Publication No. 2013-0015531 (peptide derived from EDAR ligand and its use, published on March 23, 2013) Korean Patent No. 1209117 (modified WNT10-derived peptides and uses thereof) Korean Patent No. 1187871 (FGF10-derived peptides and uses thereof, registered on September 26, 2012) Korean Registered Patent No. 1163171 (Nogin-derived peptides and uses thereof, registered on June 29, 2012) Korean Patent Publication No. 2011-0092301 (fibroblast growth factor promoting regeneration of injured hair follicles-9, published on Aug. 17, 2011)

Lachgar, S. et al., Minoxidil upregulates the expression of vascular endothelial growth factor in human hair dermal papilla cells. Br. J. of Dermatol., 1998, 138, 407-411. Kurata. S. et al., Effects of hypertrichotic agents on follicular and nonfollicular cells in vitro. Skin Pharmacol., 1996, 9, 3-8. Buhl, A.E. et al., Minoxidil stimulates mouse vibrissae follicles in organ culture. J. Invest. Dermatol., 1989, 92, 315-320. Kaufman K.D. et al., Finasteride in the treatment of men with androgenetic alopecia. Finasteride Male Pattern Hair Loss Study Group. J. Am. Acad. Dermatol., 1998,39, 578-589. E. Kaiser et al. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides., Anal. Biochem., 1970, 34 (2), 595-598.

It is an object of the present invention to provide a hair growth promoting peptide, a method for producing the same, and a composition for preventing hair loss or promoting hair growth containing the same.

The present invention relates to peptides for preventing hair loss or promoting hair growth, which are represented by the following Chemical Formulas (1) to (8).

[Chemical Formula 1]

[Cys- X- His-Lys]

(2)

[ X- His-Lys-Cys]

(3)

[Cys- X- His-Lys] ₂

[Chemical Formula 4]

[ X- His-Lys-Cys] ₂

[Chemical Formula 5]

[Cys- X- His-Lys] Cu

[Chemical Formula 6]

[ X- His-Lys-Cys] Cu

(7)

[Cys- X- His-Lys] ₂ [Cu] ₂

[Chemical Formula 8]

[ X- His-Lys-Cys] ₂ [Cu] ₂

In the above Chemical Formulas 1 to 8, X represents a nucleotide sequence selected from the group consisting of glutamic acid, aspartic acid, histidine, phenylalanine, alanine, cysteine, glycine, glutamine, asparagine, arginine, leucine, methionine, isoleucine, serine, tyrosine, threonine, Selected from proline and valine,

[] ₂ represents a dimer obtained by modifying a thiol group (-SH), which is a cysteine residue, to a reducing form (-SS-).

Preferably, the peptides of the above formulas (1) to (8)

[Cys-Phe-His-Lys] (Compound 1),

[Cys-Lys-His-Lys] (Compound 2),

[Cys-Ala-His-Lys] (Compound 3),

[Cys-Glu-His-Lys] (Compound 4),

[Phe-His-Lys-Cys] (Compound 5),

[Lys-His-Lys-Cys] (Compound 6),

[Ala-His-Lys-Cys] (Compound 7),

[Glu-His-Lys-Cys] (Compound 8),

[Cys-Phe-His-Lys] ₂ (Compound 9),

[Cys-Lys-His-Lys] ₂ (Compound 10),

[Cys-Ala-His-Lys] ₂ (Compound 11),

[Cys-Glu-His-Lys] ₂ (Compound 12),

[Phe-His-Lys-Cys] ₂ (Compound 13),

[Lys-His-Lys-Cys] ₂ (Compound 14),

[Ala-His-Lys-Cys] ₂ (Compound 15),

[Glu-His-Lys-Cys] ₂ (Compound 16),

[Cys-Phe-His-Lys] Cu (Compound 17),

[Cys-Lys-His-Lys] Cu (Compound 18),

[Cys-Ala-His-Lys] Cu (Compound 19),

[Cys-Glu-His-Lys] Cu (Compound 20),

[Phe-His-Lys-Cys] Cu (Compound 21),

[Lys-His-Lys-Cys] Cu (Compound 22),

[Ala-His-Lys-Cys] Cu (Compound 23),

[Glu-His-Lys-Cys] Cu (Compound 24),

[Cys-Phe-His-Lys] ₂ [Cu] ₂ (Compound 25)

[Cys-Lys-His-Lys] ₂ [Cu] ₂ (Compound 26),

[Cys-Ala-His-Lys] ₂ [Cu] ₂ (Compound 27),

[Cys-Glu-His-Lys] ₂ [Cu] ₂ (Compound 28),

[Phe-His-Lys-Cys] ₂ [Cu] ₂ (Compound 29),

[Lys-His-Lys-Cys] ₂ [Cu] ₂ (Compound 30),

[Ala-His-Lys-Cys] ₂ [Cu] ₂ (Compound 31)

[Glu-His-Lys-Cys] ₂ [Cu] ₂ (Compound 32)

[ ₂ ] represents a dimer in which a thiol group (-SH), which is a cysteine residue, is modified to a reduced form (-SS-).

The present invention also provides a composition for promoting hair loss prevention or hair growth comprising the peptides of the above formulas 1 to 8 or the peptides selected from the peptides of the above compounds 1 to 32.

Accordingly, the present invention provides a pharmaceutical composition for preventing hair loss or promoting hair growth comprising the peptide of the above formulas 1 to 8 or the peptide selected from at least one of the peptides of the above compounds 1 to 32. The peptide may be included in the pharmaceutical composition in an amount of 0.0001 to 1.0% by weight.

The present invention also provides a cosmetic composition for promoting hair loss prevention or hair growth comprising the peptide of the above formulas 1 to 8, or the peptide selected from at least one of the peptides of the above compounds 1 to 32. The peptide may be contained in the cosmetic composition in an amount of 0.0001 to 1.0% by weight. The cosmetic may be selected from hair cosmetics selected from hair tonic, hair cream, hair lotion, hair shampoo, hair conditioner, hair conditioner, hair spray, hair aerosol, pomade, powder, gel, sol gel, emulsion, oil, wax and aerosol. .

Hereinafter, the present invention will be described in detail.

Among the peptides of the present invention, the peptides of the compounds 9 to 16 are dimers obtained by modifying the thiol group (-SH), which is the cysteine residue of the peptide of each of the compounds 1 to 8, into the reducing form (-S-S-). That is, the peptides of the compounds 9 to 16 are dimeric peptides in which a cysteine terminal of a peptide in the form of a monomer is bonded. For example, the compound obtained by modifying the thiol group, which is the cysteine residue of Compound 1, into a reducing form is Compound 9, and the compound obtained by modifying the thiol group, which is the cysteine residue of Compound 2, Compounds 3 to 8 can be used to prepare a complex of compounds 11 to 16, respectively. Further, the thus prepared compounds 1 to 16 can be used to prepare the copper complexes of the compounds 17 to 32. [

The peptide of the present invention can be produced by, but not limited to, forming one or more amino acids or a suitably protected amino acid continuously in an amino acid skeleton constantly bonded to a solid phase. In addition, the peptide can be inserted, substituted or deleted with other amino acids within a range that does not significantly deteriorate the stability, and this also falls within the scope of the present invention.

In addition, the peptide of the present invention may be prepared by binding a cell permeable peptide that promotes intracellular movement to C-terminal or N-terminal. For example, the cell permeable peptide includes a TAT peptide (Arg-Lys-Lys-Arg-Arg-Arg-Arg-Arg) Arg-Arg-Arg: Tat PTD). However, the present invention is not limited thereto. Any cell permeable peptide known in the art may be used as long as it does not inhibit the activity of the peptide according to the present invention. Do.

The compounds of the present invention may be prepared in the form of metal complexes and the metal may be selected from among copper, magnesium, calcium, iron, zinc, nickel, silver, germanium and gallium, It is best to use copper.

Meanwhile, the peptide of the present invention may exist in the form of a salt. The salt forms which can be used in the present invention may be those which are made during the final isolation and purification of the compound or by reacting the amino group with an appropriate acid. For example, an acid addition salt can be prepared by reacting an acid addition salt such as acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, Sulfates, heptanoates, hexanoates, formates, fumarates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, mesitylenesulfonates, methanesulfonates, Naphthalene sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, Trichloroate, trifluoroacetate, phosphate, glutamate, bicarbonate, para -Toluenesulfonate and undecanoate, but are not limited thereto. In addition, examples of acids that can be used to form acid addition salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. Preferably, the peptide of the present invention can be prepared in the form of a triploloacetate salt or an acetate salt.

The amino group or carboxyl group of the amino acid used for preparing the peptide contained in the composition of the present invention can be protected by a suitable protecting group. The protected amino acid can be reacted in solution by adding the following amino acid under conditions suitable for attachment to a solid support or formation of an amide bond. In addition, the protecting group may be completely removed prior to addition of the protected amino acid with a suitable protecting group. After all of the amino acids are linked as desired, the final desired peptide can be obtained by sequential or simultaneous separation from the free residual protecting group and the free solid support.

In the present invention, it is preferable that the chiral center is condensed with another appropriately protected dipeptide under a condition that the chiral center is not racemized to form an amide bond and deprotection to synthesize the desired hexapeptide, Technology to produce peptides.

As the most preferable synthesis method for producing the peptide compound of the present invention, there can be used a method of synthesizing a solid phase peptide synthesized by using a solid polymer conjugate. The α-amino group of the peptide prepared by the above method has an acid or base- Lt; / RTI > The protecting group of the amino acid at this time should have a stable property in the peptide condensation reaction condition and should have a property that it can be easily removed without destroying the extended peptide chain or without any racemization of any chiral center contained therein. Accordingly, suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyl- oxycarbonyl, t- N-butyl, isobornyloxycarbonyl, (α, α) -dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulphenyl, 2-cyano-t-butyloxycarbonyl, Other suitable protecting groups known in the art for this purpose are also within the scope of the present invention.

The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group can be used as the most preferable protecting group of the amino acid used in the peptide synthesis of the present invention.

In particular, the protecting group of the amino acid residue used in the peptide synthesis of the present invention is t-butyl (t-Bu) in the case of N-methylglutamic acid; For lysine, t-butoxycarbonyl (Boc); For serine, 7t-butyl (t-Bu); For threonine and allotreonine, t-butyl (t-Bu); In the case of cysteine, trityl (Trt) is preferable, but the present invention is not limited thereto.

In the solid phase peptide synthesis method, the C-terminal amino acid can be attached to a suitable solid support or resin. Suitable solid supports useful for this synthesis include reagents of the staged condensation-deprotection reaction and those which are inert to the reaction conditions and which are insoluble in the medium used, for example rink amid or link amide 4- Methylbenzylhydryl amine resin (rink amid MBHA resin).

In particular, the preferred solid support for the C-terminal amide peptide may be the linkamide 4-methylbenzylhydrylamine resin available from Novabiochem Corporation.

The C-terminal amide is reacted in a solvent such as dichloromethane, N-methylpyridone (NMP) or DMF at a temperature of 10 ° C to 50 ° C, preferably at a temperature of 30 ° C, for 4 to 24 hours -Dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), benzotriazol-1-yloxy-tris (dimethylamino) phosphonium-hexafluorophosphate N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DCC) in the presence or absence of boron trifluoromethanesulfonate (BOP) or bis (DIC), [O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU) or O-benzotriazole- (Bonded, coupled) to the resin or solid support via condensation by activating the carboxylic acid on N, N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU).

When the solid support is a linkamide 4-methylbenzylhydrylamine resin, the Fmoc functional group, as a preferred protecting group, is used in excess of a solution of a secondary amine, preferably 20% piperidine DMF, before condensation with the C-terminal amino acid Cut it. Preferred reagents for use in condensing the desired amino acid in the deprotected 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxyacetamidoethyl resin include, but are not limited to, (NMM), 1-hydroxybenzotriazole (HOBt) and O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluoro (HATU), O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU), N, N'-dicyclohexylcarbodiimide (DCC) or N, N'-diisopropylcarbodiimide (DIC).

Condensation of consecutive amino acids carried out in the present invention can be carried out directly using an automatic peptide synthesizer, which is well known in the related art, or manually. Preferred synthetic reaction conditions include deprotection of the alpha -amino acid protected with the Fmoc group by treatment with a secondary amine solution, preferably piperidine, followed by washing with a sufficient excess of the solvent, The amino acid can then be added in an excess amount of 3 to 7 times by mole, preferably in a DMF solvent.

In the final step of synthesizing the peptide using the solid phase resin of the present invention, the peptides to be obtained from the resin by continuous or one-time operation of the peptides can be removed and the protective groups protecting the amino acid residues respectively can be deprotected. The removal of the peptide from the resin and the deprotection conditions of the protecting groups present in the residue generally include a cleavage reagent cocktail that cleaves the bond between the resin and the peptide, for example, trifluoroacetic acid (TFA), triisopropylsilane (TIS) , Thioanisole, water or ethanedithiol (EDT), and the like. The resulting mixed solution can be precipitated by treating excess refrigerated diethyl ether solvent. The precipitate thus obtained was centrifuged to complete precipitation, excess trifluoroacetic acid, triisopropylsilane, thioanisole, water and ethanedithiol were firstly removed, and the above procedure was repeated twice or more to solidify the precipitate Can be obtained. At this time, the completely deprotected peptide salt can be separated and purified using a mixed solvent composed of water and an acetonitrile solvent and reverse phase high performance liquid chromatography (HPLC). The separated and purified peptide solution can be completely concentrated and dried using lyophilization to obtain a solid peptide.

According to a preferred embodiment of the present invention, the composition of the present invention may be provided as a pharmaceutical composition or a cosmetic composition.

Examples of the hair to which the pharmaceutical composition or cosmetic composition of the present invention is applied include head hair, eyebrows, eyelashes, pubic hair, underarm hair, chest hair, nose hair, Hair follicles and the like may be included.

The dose of the peptide as an active ingredient contained in the pharmaceutical composition of the present invention is 0.001 to 100 mg / kg, preferably 0.1 to 100 mg / kg, more preferably 1 to 50 mg / kg on an adult basis, The dose may be administered once a day or divided into several times. The peptide of the present invention may be contained in an amount of preferably 0.0001 to 1.0% by weight, more preferably 0.001 to 1.0% by weight, and most preferably 0.001 to 0.01% by weight, based on the total weight of the pharmaceutical composition, It is not.

When the composition of the present invention is prepared from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, purified water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. The pharmaceutical composition of the present invention may further include additives commonly used such as lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives and the like in addition to the above components. The pharmaceutical composition of the present invention is preferably parenterally administered, and more preferably, is applied by a local administration method by application.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, .

The dose of the peptide as an active ingredient contained in the pharmaceutical composition of the present invention is 0.001 to 100 mg / kg, preferably 0.1 to 100 mg / kg, more preferably 1 to 50 mg / kg on an adult basis, The dose may be administered once a day or divided into several times.

The cosmetic composition of the present invention contains not only peptides as active ingredients thereof, but also ingredients conventionally used in cosmetic compositions, such as conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, . The composition of the present invention can be used in combination with a hair growth inhibitor or hair loss inhibitor that has been used in the past, insofar as it does not impair hair loss prevention or hair growth promoting action by the active ingredient of the present invention, in addition to the above-mentioned effective ingredients. As the carrier, there can be used water, purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butylene glycol or propylene glycol), higher fatty acids (palmitic acid or linolenic acid) But are not limited to, oil, camellia oil, jojoba oil, olive oil, squalane, sunflower oil, macadamia peanut oil, avocado oil, or fatty acid glyceride). If necessary, surfactants, moisturizers, preservatives, antioxidants and the like may be added.

Examples of the surfactant that can be used in the cosmetic composition of the present invention include anionic surfactants such as alkylbenzenesulfonates, polyoxyalkylene alkylsulfate ester salts, alkylsulfate ester salts, olefin sulfonates, alkyl phosphates, polyoxyalkylene Alkyl ether phosphates, dialkyl sulfosuccinates, fatty acid salts, and the like. Nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyhydric alcohol fatty acid partial esters, polyoxyethylene polyhydric alcohol fatty acid moieties Esters, polyglycerin fatty acid esters, polyoxyethylene hydrogenated castor oil derivatives, and fatty acid diethanolamides. Examples of the cationic surfactant include tertiary aliphatic amine salts, alkyltrimethylammonium halides and dialkyldimethylammonium halides. Examples of the amphoteric surfactants include amide betaine, imidazolinium betaine, sulfobetaine, And the like. Examples of the moisturizing agent include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, and sorbitol. Examples of the preservative include benzoic acid, dehydroacetic acid, paraoxybenzoic acid esters (such as methyl parahydroxybenzoate, butyl parahydroxybenzoate), and phenoxyethanol. Examples of the antioxidant include ascorbic acid and BHA. In addition, an ultraviolet absorber, an anti-inflammatory agent, and a refreshing agent may be added.

The formulations of the cosmetic composition of the present invention may be any formulations that can be combined with the active ingredients of the hair cosmetic composition. Examples of the hair cosmetic composition include hair tonic, hair cream, hair lotion, hair shampoo, hair conditioner, hair conditioner, Hair gels, emulsions, oils, waxes, aerosols, and the like, such as hair, hair, aerosols, pomades, powders and gels.

The peptide of the present invention may be contained in an amount of preferably 0.0001 to 1.0% by weight, more preferably 0.001 to 1.0% by weight, and most preferably 0.001 to 0.01% by weight based on the total weight of the cosmetic composition, but is not limited thereto.

The cosmetic composition of the present invention can be used everyday or can be used for an unspecified period. Preferably, the amount of usage, the number of times of use, and the period of time can be adjusted according to the user's age, skin condition or skin type, and the concentration of the peptide.

The present invention relates to a hair-loss-preventing or hair-growth promoting composition containing a hair-growth promoting peptide that has been proved as a hair-growth promoting effect, and the peptide of the present invention exhibits an excellent hair-growth effect. In particular, the peptides of the present invention prepared from the dimer form and the copper metal complex have higher safety and stability in use than existing peptide compositions, and thus can be easily used as a composition for preventing hair loss or promoting hair growth.

FIG. 1 is a graphical representation of the growth rate (growth rate) of human dermal papilla cells of the peptide compound of the present invention.
2 is a photograph showing the hair growth effect of the compound 25 of the present invention in a mouse.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1 > Synthesis of compounds 1 to 32 >

Nomenclature and abbreviations of amino acids used in the present invention are expressed as follows.

Ala: alanine / Cys: cysteine / Glu: glutamic acid / Phe: phenylalanine / His: histidine / Lys: lysine.

Example 1-1. Synthesis of compounds 1 and 9

71.4 mg (0.10 mmol) of 2-chlorotrityl chloride resin (resin loaded with 1.4 mmol per gram) purchased from Novabiochem corporation was weighed into a reaction vessel. The resin was solubilized with 3 ml of DMF and sufficiently swelled for 5 minutes. Then, 3 ml of a 20% (w / v) piperidine DMF solution was added, shaking for 10 minutes, and a piperidine DMF solution And then washed five times with 10 ml of DMF solvent (5 times in 10 ml portions). * DMF: Dimethylformamide.

Fmoc-Lys (Boc) -OH (374.8 mg, 0.80 mmol), HOBt (108.1 mg, 0.80 mmol) and DIC (0.124 mL, 0.80 mmol) were completely dissolved in 2 mL of DMF solvent and then added to the resin. The reaction solution was shaken at room temperature for 8 hours and then washed 5 times with 10 ml of DMF solvent. 3 ml of a 20% (w / v) piperidine DMF solution was added and shaken for 10 minutes to remove the piperidine solution. Then, 20% (w / v) piperidine DMF solution was added thereto, After the reaction, the Fmoc protecting group protected by the resin was completely removed and washed 5 times with 10 ml of DMF solvent (5 times in 10 ml portions). At this stage, the deprotection of the Fmoc protecting group was carried out using the Kaiser test [E. Kaiser et al. Anal. Biochem., 1970, 34 (2), 595-598). * Fmoc: 9-fluorenylmethyloxycarbonyl / HOBt: 1-hydroxybenzotriazole / DIC: N, N'-diisopropylcarbodiimide.

Next, condensation (coupling) was carried out continuously under the same synthesis cycle as shown below.

(1) washing 5 times with DMF solvent (10 ml);

(2) deprotection twice with 10% (w / v) piperidine DMF solution (3 ml) for 10 minutes;

(3) washing 5 times with DMF solvent (10 ml);

(4) Fmoc-amino acid addition;

(5) Amino acid activation and 2 hour condensation by addition of condensation reagent;

(6) washed 5 times with DMF solvent (10 ml);

The above steps (1) to (6) were repeated, and the Fmoc-protected amino acid (0.80 mmol) after Fmoc-Lys (Boc) -OH was added to the resin reaction vessel in the sequence described below and condensed.

(i) Fmoc-His (Trt) -OH;

(ii) Fmoc-Phe-OH;

(iii) Fmoc-Cys (Trt) -OH;

After (6) after Fmoc-Cys (Trt) -OH condensation, finally, 20% piperidine DMF solution (3 ml) was treated.

Immediately after such synthesis termination, the peptide condensed resin was treated with a mixture of trifluoroacetic acid / thioanisole / ethanediol / triisopropylsilane / water (95: 5: 2.5: 2.5: 2.5) 10 ml), the peptide was cleaved from the resin. The thus obtained mixed solution was treated with 100 ml of refrigerated diethyl ether solvent to produce a precipitate. The obtained precipitate was centrifuged to completely precipitate, and then trifluoroacetic acid, thioanisole and ethanedithiol were firstly removed, and 100 ml of a diethyl ether solvent was added to wash the precipitate, followed by centrifugation, , A process for removing trifluoroacetic acid, thioanisole and ethanedithiol) was repeated twice to obtain a solidified precipitate. The precipitate (peptide) was purified by HPLC using a 5% to 100% acetonitrile / water gradient solvent system containing 0.01% trifluoroacetic acid over 50 minutes using a C-18 column. The purified pure fractions were lyophilized to obtain Compound 1: H ₂ N- [Cys-Phe-His-Lys] -CO ₂ H (50 mg) as a white powder type trifluoroacetate salt.

20 mg of Compound 1: H ₂ N- [Cys-Phe-His-Lys] -CO ₂ H was dissolved in 2 ml of dimethylsulfoxide (DMSO), and then 10 ml of water was added. To obtain the following compound 9 (17 mg) in the form of a dimer in which the thiol group (-SH) as a residue was modified to a reducing form (-SS-).

Compound 1 : H ₂ N- [Cys-Phe-His-Lys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 534.7; (50 mg)

Compound 9 : H ₂ N- [Cys-Phe-His-Lys] ₂ -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 1066.4; (17 mg)

Examples 1-2. Synthesis of compounds 2 to 8 and 10 to 16

Peptides of the following compounds 2 to 8 and 10 to 16 were prepared in the same manner as in the above compound 1 except that the order of amino acids protected by Fmoc was changed.

Compound 2 : H ₂ N- [Cys-Lys-His-Lys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 515.7; (49 mg)

Compound 10 : H ₂ N- [Cys-Lys-His-Lys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1028.4; (21 mg)

Compound 3 : H ₂ N- [Cys-Ala-His-Lys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 458.6; (55 mg)

Compound 11 : H ₂ N- [Cys-Ala-His-Lys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 914.2; (24 mg)

Compound 4 : H ₂ N- [Cys-Glu-His-Lys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 516.6; (53 mg)

Compound 12 : H ₂ N- [Cys-Glu-His-Lys] ₂ -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 1030.3; (22 mg)

Compound 5 : H ₂ N- [Phe-His-Lys-Cys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 534.7; (58 mg)

Compound 13 : H ₂ N- [Phe-His-Lys-Cys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1066.4; (18 mg)

Compound 6 : H ₂ N- [Lys-His-Lys-Cys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 515.7; (51 mg)

Compound 14 : H ₂ N- [Lys-His-Lys-Cys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1028.4; (20 mg)

Compound 7 : H ₂ N- [Ala-His-Lys-Cys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 458.6; (48 mg)

Compound 15 : H ₂ N- [Ala-His-Lys-Cys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 914.2; (21 mg)

Compound 8 : H ₂ N- [Glu-His-Lys-Cys] -CO ₂ H: MS (ESI) m / e, [M + H] ⁺ = 516.6; (52 mg)

Compound 16 : H ₂ N- [Glu-His-Lys-Cys] ₂ -CO ₂ H : MS (ESI) m / e, [M + H] < ⁺ > = 1030.3; (18 mg)

Examples 1-3. Synthesis of compounds 17 to 32

Of the compounds synthesized through Examples 1-1 and 1-2, Compounds 17 to 24 were synthesized on the basis of Compounds 1 to 8 by the following methods, and Compounds 25 to 32 were synthesized based on Compounds 9 to 16 Were synthesized.

Compound 9 (100 mg, 0.94 mol) was dissolved in ₂ ml of H ₂ O and an aqueous solution of 0.72 mg (4 eq.) Of cooper chloride (CuCl ₂ ) was added, followed by stirring at room temperature for 10 hours. To the stirred aqueous solution was added 10 mL of 1.0 N sodium hydroxide (NaOH) aqueous solution, and the mixture was concentrated under reduced pressure and lyophilized to obtain Compound 25 ([H ₂ N- [Cys-Phe-His-Lys] ₂ -CO ₂ H ] - [Cu] ₂ ).

Namely, the following compounds were obtained by using such a method.

Compound _{17: [H 2 N- [Cys} -Phe-His-Lys] -CO 2 H] · Cu ( copper complexes);

Compound 25 : [H ₂ N- [Cys-Phe-His-Lys] ₂ -CO ₂ H] 揃 [Cu] ₂ (copper complex compound);

Compound _{18: [H 2 N- [Cys} -Lys-His-Lys] -CO 2 H] · Cu ( copper complexes);

Compound 26 : [H ₂ N- [Cys-Lys-His-Lys] ₂ -CO ₂ H] 揃 [Cu] ₂ (copper complex compound);

Compound _{19: [H 2 N- [Cys} -Ala-His-Lys] -CO 2 H] · Cu ( copper complexes);

Compound _{27: [H 2 N- [Cys} -Ala-His-Lys] 2 -CO 2 H] · [Cu] 2 ( copper complexes);

Compound _{20: [H 2 N- [Cys} -Glu-His-Lys] -CO 2 H] · Cu ( copper complexes);

Compound 28 : [H ₂ N- [Cys-Glu-His-Lys] ₂ -CO ₂ H] 揃 [Cu] ₂ (copper complex compound);

Compound _{21: [H 2 N- [Phe} -His-Lys-Cys] -CO 2 H] · Cu ( copper complexes);

Compound 29 : [H ₂ N- [Phe-His-Lys-Cys] ₂ -CO ₂ H] 揃 [Cu] ₂ (copper complex compound);

Compound _{22: [H 2 N- [Lys} -His-Lys-Cys] -CO 2 H] · Cu ( copper complexes);

Compound _{30: [H 2 N- [Lys} -His-Lys-Cys] 2 -CO 2 H] · [Cu] 2 ( copper complexes);

Compound _{23: [H 2 N- [Ala} -His-Lys-Cys] -CO 2 H] · Cu ( copper complexes);

Compound 31 : [H ₂ N- [Ala-His-Lys-Cys] ₂ -CO ₂ H] 揃 [Cu] ₂ (copper complex compound);

Compound _{24: [H 2 N- [Glu} -His-Lys-Cys] -CO 2 H] · Cu ( copper complexes);

Compound _{32: [H 2 N- [Glu} -His-Lys-Cys] 2 -CO 2 H] · [Cu] 2 ( copper complexes);

EXPERIMENTAL EXAMPLE 1 Growth and Cleavage Promoting Effect of Human Dermal Papilla Cells [

Human dermal papilla cells (HFDPC) use dermal papilla cells isolated and cultured from human scalp, which is a byproduct of plastic surgery. Human dermal papilla cells were incubated in DMEM (Cambrex) medium containing 10% FBS (Gibco BRL) at 37 ° C and 5% CO ₂ in an incubator.

To treat the compounds of the present invention, human dermal papilla cells were seeded at a constant density of 3,000 cells in 96 well plates for cell culture and cultured in DMEM medium containing 0.1% FBS for 5 hours at 37 ° C under 5% CO ₂ And incubated in an incubator. The test substances were dissolved in serum-free DMEM at a concentration of 100 mM to prepare stocks. The cells were diluted in DMEM medium containing 1 μCi / μl [ ³ H] Thymidine and 0.1% FBS, and each compound was treated with 10 μM final concentration. 37 ℃ days, and cultured in an incubator with 5% CO ₂ condition. After washing the cells with PBS and 5% cold TCA, cells were disrupted with 0.1 N NaOH, 1% SDS (sodium dodecyl sulfate) solution, and the radioactivity contained in the cell lysate solution was measured using a liquid scintillation counter (Beckman) And the results are shown in Table 1 and FIG.

Condition Compared to the control group,
Human dermal papilla cell growth rate (%) Compound 1 120 Compound 2 125 Compound 3 124 Compound 4 123 Compound 5 120 Compound 6 131 Compound 7 125 Compound 8 119 Compound 9 210 Compound 10 201 Compound 11 202 Compound 12 195 Compound 13 208 Compound 14 209 Compound 15 210 Compound 16 205 Compound 17 170 Compound 18 178 Compound 19 168 Compound 20 159 Compound 21 168 Compound 22 167 Compound 23 173 Compound 24 174 Compound 25 230 Compound 26 225 Compound 27 222 Compound 28 229 Compound 29 221 Compound 30 222 Compound 31 225 Compound 32 222 Control group 100

Referring to Table 1 and FIG. 1, it can be seen that the compounds of the present invention are superior to those of the control group (untreated group) in the growth rate of human dermal papilla cells, and thus are more excellent in preventing hair growth or hair loss.

<Experimental Example 2> Hair growth test>

In C57BL / 6 mice, hair growth and decay are comparable after 6-7 weeks of age, which is an effective model for hair growth experiments.

Six-week old female C57BL / 6 rats were adapted to the breeding environment for one week (12 hours / 12 hours, 24 hours, 40-50% humidity, free food and water) The epidermis was evenly depilated using an epilator. Over the next 5 days, Compound 25 was made into 50 ppm aqueous solution and applied twice daily to the rat epidermis, and the control was applied only with water. After about 14 days of observation, changes in the hair growth pattern were confirmed. The results are shown in FIG.

Referring to FIG. 2, it can be seen that when Compound 25 of the present invention was treated, hair growth was excellent in all individuals (n = 10) as compared to the control.

Therefore, it can be seen from the results of Experimental Examples 1 and 2 that the compounds of the present invention have excellent hair growth enhancement and hair loss prevention effect.

On the other hand, in general, it is very important to slow down the degradation rate of the peptide because the peptide is rapidly degraded by various hydrolytic enzymes when it enters the living body and its ability is rapidly reduced. In general, various hydrolytic enzymes decompose an amide bond (-C (= O) NH-) in a peptide structure into a carboxylic acid (-CO ₂ H) and an amine (-NH ₂ ) The cysteine dimer structure has a (-SS-) skeleton, which functions to lower or disturb the decomposition ability of common hydrolytic enzymes. That is, among the above-mentioned peptide compounds, dimer-type compounds are less decomposed by hydrolytic enzymes in vivo than compounds in the form of monomers. Therefore, among the peptide compounds of the present invention, the compounds having cysteine dimer are considered to have excellent stability in the body. In addition, it is considered that the peptide of the present invention is excellent in the hair growth effect of the peptides prepared from the copper complex, and the peptide has an increased stability in the body as in the case of the dimeric peptide.

&Lt; Formulation Example 1: Preparation of external ointment for skin >

The ointment for external use on skin was prepared according to a conventional method with the composition shown in Table 2 below.

Ingredients Addition amount (% by weight) Compound 25 1.0 S-nitrosoglutathione 0.04 Hyaluronic acid 3.0 lecithin 3.0 Diethyl sebacate 8.0 Nonsense 5.0 Polyoxyethylene oleyl ether phosphate 6.0 Paraoxybenzoic acid ester Suitable amount vaseline up to 100

&Lt; Formulation Example 2: Preparation of shampoo &

A shampoo was prepared according to a conventional method with the composition shown in Table 3 below.

Ingredients Addition amount (% by weight) Compound 25 0.01 S-nitrosoglutathione 0.04 Hyaluronic acid 1.5 lecithin 3.0 30% sodium lauryl sulfate 20.0 30% sodium polyoxyethylene lauryl sulfate 20.0 Coconut oil fatty acid diethanolamide 2.00 Propylene glycol 2.00 Cocamidopropyl betaine 3.00 Carboxyvinyl polymer 0.30 Polyquaternium-10 0.50 Paraoxybenzoic acid ester 0.20 Citric acid Suitable amount Spices Suitable amount Purified water Balance

&Lt; Formulation Example 3: Production of hair tonic &

doing A hair tonic was prepared by the usual method with the composition as shown in Table 4.

Ingredients Addition amount (% by weight) Compound 25 0.1 S-nitrosoglutathione 0.04 Hyaluronic acid 1.5 lecithin 3.0 ethanol 50.00 L-menthol 0.10 D-Panthenol 0.20 glycerin 3.00 Salicylic acid 0.10 Topoceryl acetate 0.10 Polyoxyethylene hardened castor oil 0.50 Spices Suitable amount Purified water Balance

Claims (10)

delete A peptide for preventing hair loss or promoting hair growth, which is represented by the following formula (3), (4), (7) or (8)
(3)
[Cys- X- His-Lys] ₂
[Chemical Formula 4]
[ X- His-Lys-Cys] ₂
(7)
[Cys- X- His-Lys] ₂ [Cu] ₂
[Chemical Formula 8]
[ X- His-Lys-Cys] ₂ [Cu] ₂
Wherein X is selected from the group consisting of glutamic acid, aspartic acid, histidine, phenylalanine, alanine, cysteine, glycine, glutamine, asparagine, arginine, leucine, methionine, isoleucine, serine, tyrosine , Threonine, lysine, tryptophan, proline and valine,
[] ₂ represents a dimer obtained by modifying a cysteine residue, thiol group (-SH), to a reduced form (-SS-). delete 3. The method of claim 2,
The peptide of the above formula (3), (4), (7) or (8)
[Cys-Phe-His-Lys] ₂ (Compound 9),
[Cys-Lys-His-Lys] ₂ (Compound 10),
[Cys-Ala-His-Lys] ₂ (Compound 11),
[Cys-Glu-His-Lys] ₂ (Compound 12),
[Phe-His-Lys-Cys] ₂ (Compound 13),
[Lys-His-Lys-Cys] ₂ (Compound 14),
[Ala-His-Lys-Cys] ₂ (Compound 15),
[Glu-His-Lys-Cys] ₂ (Compound 16),
[Cys-Phe-His-Lys] ₂ [Cu] ₂ (Compound 25)
[Cys-Lys-His-Lys] ₂ [Cu] ₂ (Compound 26),
[Cys-Ala-His-Lys] ₂ [Cu] ₂ (Compound 27),
[Cys-Glu-His-Lys] ₂ [Cu] ₂ (Compound 28),
[Phe-His-Lys-Cys] ₂ [Cu] ₂ (Compound 29),
[Lys-His-Lys-Cys] ₂ [Cu] ₂ (Compound 30),
[Ala-His-Lys-Cys] ₂ [Cu] ₂ (Compound 31)
[Glu-His-Lys-Cys] ₂ [Cu] ₂ (Compound 32)
, Wherein [] ₂ in the compound is a dimer obtained by modifying a thiol group (-SH), which is a cysteine residue, with a reducing form (-SS-). . A composition for promoting hair loss prevention or hair growth, characterized by containing at least one compound selected from the peptides according to claims 2 or 4. A pharmaceutical composition for preventing hair loss or promoting hair growth, which comprises a compound selected from at least one peptide selected from the group consisting of peptides according to claims 2 and 4. The method according to claim 6,
Wherein the peptide is contained in the pharmaceutical composition in an amount of 0.0001 to 1.0% by weight. 5. A cosmetic composition for preventing hair loss or promoting hair growth characterized by containing at least one compound selected from the peptides according to claim 2 or 4. 9. The method of claim 8,
Wherein the peptide is contained in the cosmetic composition in an amount of 0.0001 to 1.0% by weight. 9. The method of claim 8,
The cosmetic composition is selected from cosmetic compositions selected from hair tonics, hair creams, hair lotions, hair shampoos, hair rinses, hair conditioners, hair sprays, hair aerosols, pomades, powders, gels, sols, emulsions, oils, waxes and aerosols Wherein the cosmetic composition is a composition for preventing hair loss or promoting hair growth.

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