KR20190036314A - Composition for preventing depilation and improving hair growth - Google Patents

Abstract

According to an aspect of the present invention, a pharmaceutical composition, a health functional food, and a cosmetic composition for preventing hair loss or promoting hair growth can be used to prevent or treat hair loss of an individual and to promote hair growth. According to another aspect of the present invention, a method for preventing or treating hair loss can effectively prevent or treat hair loss of an individual and promote hair growth.

Description

Technical Field [0001] The present invention relates to a composition for preventing depilation and improving hair growth,

A health functional food, a cosmetic composition, and a method for preventing hair loss or promoting hair growth by using the same.

It is estimated that the number of patients suffering from alopecia is estimated to reach to 10 million, and the number of patients suffering from hair loss is estimated to reach 20 million Socioeconomic problems are becoming serious. Currently, treatments for treating hair loss are classified as pharmaceuticals, quasi-drugs, and cosmetics. There are 'Propecia' which is developed and sold by Merck in the US, Finasteride, the main component of Propecia, was approved by the US FDA for hair loss treatment in December 1997. Pinasteride is a 5-α-reductase inhibiting agent that converts testosterone to dihydrotestosterone (DHT), which acts as a thick, long hair. This is effective in improving hair loss in the short term, but it is accompanied by side effects such as erectile dysfunction, sexual dysfunction, and male breast enlargement. Minoxidil is approved for safety and efficacy and can be purchased without a doctor's prescription. It was approved by the FDA in December 1997 as the first treatment for hair loss. This drug has the effect of promoting hair growth by improving blood circulation and opening the potassium channel, but local reactions such as itching, rash, and tachycardia may occur.

Human hair loss cycles are largely divided into anagen, catagen, and telogen. The growing period is the time when the activity of the breast milk is active and the cell division is vigorous and the hair grows at a high speed. The lifespan of the growing period varies depending on the type of hair, but in the case of hair, it is about 3 to 6 years. Growing hair accounts for 80% to 90% of the total hair, and a person with hair loss progresses to a shorter growth period and a longer period of hair rest period, so that the specific gravity of the growing hair decreases from the whole hair. The regressive phase is the period when the growth of hair ends and the production of hair gradually slows down and eventually stops cell division and growth. It lasts for 1 to 1.5 months and about 1% of total hair belongs to this stage. The resting stage is the final stage of growth, in which the hair follicles and hair follicles are completely separated and the hair follicles are contracted, and the hair follicles are further raised upward and hair is removed. The rest period lasts for 3 to 4 months and 4 to 14% of total hairs are in this stage. When the dormant period is over and the activity of the dermal papilla becomes active again, the new dermal papilla is made, and the hair at the dormant pillar is pushed out completely out of the scalp. In general, alopecia refers to an increase in the number of abnormally dropped hair due to shortening of hair growth rate in the growing period and increased number of hair in the retrogressive or resting period among these cycles. Conventional hair loss preventing agents or hair growth agents are known to stimulate blood circulation of the scalp to prevent hair loss or to promote hair growth. However, in many cases, the effect is not proven, is not effective, or even when there is an effect, severe side effects Is known to accompany.

One aspect provides a pharmaceutical composition for preventing hair loss or promoting hair growth.

Another aspect provides a health functional food for preventing hair loss or promoting hair growth.

Another aspect provides a cosmetic composition for preventing hair loss or promoting hair growth.

Another aspect provides a method of preventing hair loss or promoting hair growth using the composition.

One aspect provides a composition for promoting hair loss prevention or hair growth, comprising Lefty-A protein as an active ingredient.

The composition may be one for inhibiting, delaying, improving, preventing or treating hair loss. The composition may be to form hair, hair, or hair shaft on the skin. The composition may be one which promotes the expression of the Krox20 gene.

The " hair loss " means that the hair ratio of the growing period is shortened and the hair of the retrograde period or the resting period is increased, so that the number of abnormal hair dropping is increased. This " hair growth " means that the number of hairs increases.

The Lefty-A protein is a kind of transforming growth factor-beta (TGF-beta) superfamily, which affects symmetry and asymmetry of organs at the developmental stage. The protein comprises an amino acid sequence having at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology with the amino acid sequence of SEQ ID NO: 6 . Wherein said protein comprises an amino acid sequence of SEQ ID NO: 6 and comprising at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98% And may comprise an amino acid sequence having about 99% or more sequence homology. The protein comprises, for example, the amino acid sequence of SEQ ID NO: 6, An amino acid sequence having at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology with the amino acid sequence of NP_003231.1 or NP_003231.2 ≪ / RTI > The protein may comprise the amino acid sequence of SEQ ID NO: 1. The polypeptide having the amino acid sequence of SEQ ID NO: 1 may specifically include the RXXR region in the Lefty-A protein and the amino acid sequence of about 120 AA from the RXXR region. The RXXR region may correspond to amino acids 132 to 135 from the N-terminus in the Lefty-A protein. The polypeptide having the amino acid sequence of SEQ ID NO: 2 may contain an amino acid sequence located after the sequence signal of the Lefty-A protein. When the composition comprises a polypeptide having a sequence homology of at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% with the amino acid sequence of SEQ ID NO: 6 , And is effective in preventing hair loss and promoting hair growth.

The term " polypeptide " means a peptide or protein in which two or more amino acids are bound through a peptide bond or a modified peptide bond. Such modifications include, but are not limited to, acetylation, acylation, lysylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, The formation of cysteine, the formylation, the gamma-carboxylation, the glycosylation, the GPI anchor formation, the covalent attachment, the cross-linking, the cyclization, the disulfide bond formation, the demethylation, For example, include hydroxylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation or ubiquitination.

The term " homology " refers to the percentage of identity between two polynucleotides or polypeptide mimetics. The homology between sequences from one moiety to another can be determined by known techniques. For example, the algorithm BLAST (Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA by Pearson (see Methods Enzymol., 183, 63 (1990)).

 &Quot; Active ingredient " is intended to carry out the function mentioned in the composition and excludes those that do not perform the function because they are contained in small amounts as impurities.

The Lefty-A protein may further comprise a stabilizing protein. The stabilizing protein may be one end or both ends of the Lefty-A protein. The stabilizing protein may be linked to the N-terminus, the C-terminus or both the N-terminus and the C-terminus of the Lefty-A protein. The stabilizing protein may be, for example, an immunoglobulin G, ubiquitin, ecotin, Brazzein, beta-lactoglobulin, Sakacin B, Mxyn 10, Lt; / RTI >

The immunoglobulin G may comprise an Fc fragment of an immunoglobulin G. The Fc fragment of the immunoglobulin G may be one end or both ends of the Letfy-A protein. The Fc fragment of the immunoglobulin G may be linked to the N-terminus, the C-terminus, or the N-terminus and the C-terminus of the Letfy-A protein. The immunoglobulin G (IgG) is the main antibody in blood and extracellular fluid, which is synthesized and secreted in plasma B cells, and activates the complement system pathway producing immune protein. The IgG may be of human origin. The IgG may be IgGl. The Fc fragment refers to a portion of the antibody that is unable to bind to the antigen. An antibody binding site is formed by combining two heavy and light chain variable regions of the antibody. Each of these regions is present in each Y-shaped arm, and a portion capable of binding to the antigen is referred to as a Fab fragment antibody binding fragment, and the part that can not bind to an antigen is called an Fc fragment. Fc fragments can be obtained by cleaving the antibody to pepsin. The Fc fragment includes, for example, SEQ ID NO: 4, NCBI accession No. About 90% or more, about 92% or more amino acid sequence identity with the amino acid sequence of AAC82527.1, AAL96263.1, ABH03478.1, ABG91559.1, AAX09635.2, AAX09634.1, AAX09633.1, CAC20454.1, AAX09628.2 or AAX09632.2. , At least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology.

The ubiquitin is the most conserved protein found in nature and consists of 76 amino acid sequences, and is a protein showing perfect homology between various species. In addition, ubiquitin is known to be stable to changes in pH, not easily denatured at high temperatures, and stable to proteases. The ubiquitin has a sequence homology of at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 5 Lt; / RTI > polypeptide.

The stabilizing protein may be directly linked to the C-terminus or N-terminus of the Lefty-A protein, or may be covalently linked to the terminus of the Lefty-A protein through a linker. That is, the Lefty-A protein may be linked to a stabilizing protein by a linker. The linker can separate the Lefty-A protein and the stabilization protein at a sufficient distance to allow each Lefty-A protein to fold into a suitable secondary and tertiary structure. For example, the Lefty-A protein may be linked to an Fc fragment of an IgG by a linker. The Fc fragment of the IgG may be covalently linked to the end of the Lefty-A protein via a linker or directly to the C-terminus or N-terminus of the Lefty-A protein. The stabilizing protein may be one which forms dimers with the Lefty-A protein. The Fc fragment of the IgG may be one which forms dimers with the Lefty-A protein. By linking the Fc fragment of IgG to the Lefty-A protein, the stability of the Lefty-A protein is improved and the half-life is increased.

The Lefty-A protein may be administered at 1 to 1000 占 퐂 / kg. The Lefty-A protein may be administered at 2 to 500 μg / kg, 5 to 200 μg / kg, 7 to 150 μg / kg, or 8 to 130 μg / kg. When the Lefty-A protein is administered at a dose of 1 to 1000 占 퐂 / kg or less or 1000 占 퐂 / kg or more, it is difficult to prevent hair loss and achieve hair growth promoting effect.

The Lefty-A protein may be administered at 1 to 1000 占 퐂 / kg / day. The Lefty-A protein may be administered at a dose of 2 to 500 μg / kg / day, 5 to 200 μg / kg / day, 7 to 150 μg / kg / day, or 8 to 130 μg / kg / day.

The Lefty-A protein may be administered at least once, at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, or at least eight times.

The Lefty-A protein is more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, Month, six months, or more than one year. The Lefty-A protein may be administered at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 10 days, 15 days, or more than one month.

The Lefty-A protein may be administered orally, intraperitoneally, intramuscularly, subcutaneously, intravenously, or a combination thereof. Administration can be by any method known in the art. The administration can be directly administered to the individual by any means, for example, by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intratracheal, peritoneal or subcutaneous administration. The administration can be systemically or locally administered. The administration may be topical administration to the site where hair loss is present. The administration may be by application, for example. The application refers to any method of contacting the composition to the skin of a subject in any suitable manner, thereby allowing the composition to be absorbed into the skin.

The composition may be present in an amount of from 0.001% to 99% by weight, for example from 0.01% to 80% by weight, from 0.01% by weight to 60% by weight, from 0.01% by weight to 40% %, 0.01% to 15%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% To 10% by weight, or from 0.1% to 5% by weight of the Lefty-A protein.

Another aspect is a composition for promoting hair loss or promoting hair growth comprising a cell comprising a polynucleotide encoding a Lefty-A protein or a cell overexpressing a Lefty-A protein transduced with the vector, or a culture thereof as an active ingredient to provide.

The polynucleotide encoding the Lefty-A protein comprises at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: Or may comprise a nucleotide sequence encoding an amino acid sequence having homology. Wherein the polynucleotide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 and having at least about 90%, at least about 92%, at least about 95%, at least about 97% , At least about 98%, or at least about 99% sequence identity to a nucleotide sequence encoding an amino acid sequence. The polynucleotide includes, for example, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6, Encoding an amino acid sequence having at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology with the amino acid sequence of NP_003231.1 or NP_003231.2 , ≪ / RTI > The NCBI accession No. The nucleotide sequence coding for the amino acid sequence of NP_003231.1 or NP_003231.2 is NCBI accession no. It is disclosed in NM_003240.1 or NM_003240.4. The polynucleotide sequence may comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

The vector may comprise linear DNA, plasmid DNA, recombinant viral vectors, or a combination thereof. The vector containing the polynucleotide encoding the Lefty-A protein may be a vector containing a linear DNA, a plasmid vector, a viral expression vector, or a recombinant retrovirus vector expressed in human or animal cells, a recombinant adenovirus ) Vector, a recombinant viral vector comprising a recombinant adeno-associated virus (AAV) vector, a recombinant herpes simplex virus vector, or a recombinant lentivirus vector Do not.

The cells overexpressing the Lefty-A protein transduced with the vector include a polynucleotide sequence encoding the Lefty-A protein in the genome of the host cell, or a recombinant vector containing the polynucleotide sequence. Any host cell known to those skilled in the art may be used as a host cell capable of cloning or expressing the vector stably and continuously. Examples of the prokaryotic cell include Escherichia coli, insect cells, plant cells and animal cells, For example, Sp2 / 0, Chinese hamster ovary (CHO), CHO K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines, have. The delivery of the polynucleotide or vector containing the polynucleotide into a host cell can be carried out by a method well known in the art. For example, when the host cell is a prokaryotic cell, the CaCl ₂ method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, Liposome-mediated transfection, gene bombardment, and the like, but are not limited thereto.

The composition may be a pharmaceutical composition for preventing hair loss or promoting hair growth comprising Lefty-A protein as an active ingredient.

The composition may comprise a " therapeutically effective amount " of the Lefty-A protein. In this composition, " therapeutically effective amount " means an amount sufficient to exhibit a therapeutic effect when administered to a subject or a cell in need thereof. &Quot; Treatment " means treating a disease or medical condition in a mammal, including a human, including an individual, which includes: (a) preventing the occurrence of the disease or medical condition, cure; (b) relieving the disease or medical condition, i. e., eliminating or ameliorating the disease or medical condition in the patient; (c) inhibiting the disease or medical condition, i.e. slowing or stopping the progression of the disease or medical condition in the individual; Or (d) relieving the disease or medical condition in the subject.

The composition may comprise a pharmaceutically acceptable carrier. In such a composition, an " acceptable carrier " refers to a substance, usually an inert substance, used in combination with the active ingredient to aid in the application of the active ingredient. The carrier may be an excipient, a disintegrant, a binder, a lubricant, a diluent, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low substituted hydroxy cellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, calcium monohydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The composition may be formulated into parenteral or oral dosage forms. The parenteral dosage form may be an injection or an external preparation for skin. The external skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, and the like can be appropriately blended as needed. The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Sugars such as trehalose and the like can also be appropriately compounded. Oral dosage forms may be tablets, capsules, aqueous solutions or suspensions. In the case of tablets, excipients such as lactose and corn starch, and lubricants such as magnesium stearate may be usually added. In the case of capsules, lactose and / or dried corn starch may be used as a diluent. When a suspending agent is required, the active ingredient may be combined with an emulsifying agent and / or a suspending agent. If desired, certain sweetening and / or flavoring agents may be added.

The composition may be a cosmetic composition for preventing hair loss or promoting hair growth comprising Lefty-A protein as an active ingredient.

The above-mentioned " cosmetic composition " means that the effect on the human body is slight as it is an article used in the human body for cleansing and beautifying the human body, adding charm, brightly changing appearance, or maintaining or promoting the health of skin and hair. The cosmetic composition may be a functional cosmetic having an effect of preventing hair loss or promoting hair growth. The composition may comprise a cosmetically acceptable carrier. The cosmetic composition may contain a component that can be used in conventional cosmetic compositions, for example, a moisturizer, a powder component, an ultraviolet absorber, an antioxidant, a cosmetic ingredient, a glycolipid, a plant extract, a preservative, A flavoring agent, a pH adjusting agent, a coloring matter, a viscosity adjusting agent or a gelling agent as an auxiliary component. The cosmetic composition may be at least one selected from the group consisting of lotion, skin, lotion, nutrition lotion, nutritional cream, massage cream, essence, pack, skin lotion, skin softener, skin toner, astringent, milk lotion, moisturizing lotion, , Shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, or loose powders.

Another aspect provides a health functional food for preventing hair loss or promoting hair growth, comprising Lefty-A protein as an active ingredient.

Refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and gums using raw materials and components having useful functions in the human body. The health functional food may be a functional food having an effect of preventing hair loss or promoting hair growth. The composition may comprise a pharmaceutically acceptable carrier. The health functional food may contain a food-acceptable food-aid additive in addition to the active ingredient. The above-mentioned " food-aid additive " refers to a component which can be added to foods in a supplementary manner, and is added to produce a health functional food, and can be appropriately selected and used by an ordinary person skilled in the art. The health functional food may be, for example, a nutrient, a vitamin, a mineral (electrolyte), a flavor such as a synthetic flavor and a natural flavor, a colorant and a filler, a pectic acid and its salt, an alginic acid and its salt, A thickener, a pH adjuster, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated beverage. Unlike the pharmaceutical composition, the health functional food can be manufactured in various forms, and it is advantageous that there is no side effect that may be caused when a medicine is used for a long period of time, and it is excellent in portability, It can be ingested as an adjuvant to promote hair growth promoting effect.

Another aspect provides a method of preventing hair loss or promoting hair growth of an individual comprising administering the composition to a subject.

The subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The subject may be an individual in need of preventing hair loss or promoting hair growth.

According to one aspect of the present invention, a pharmaceutical composition, a health functional food, and a cosmetic composition for preventing hair loss or promoting hair growth can be used to prevent or treat hair loss of an individual and to promote hair growth. According to the method for preventing or treating hair loss according to another aspect, hair loss of an individual can be effectively prevented or treated, and hair growth can be promoted.

Figure 1 shows the results of confirming the expression and size of Lefty-A-Fc protein.
FIGS. 2A to 2I are images obtained by administering Lefty-A-Fc protein to mice in a concentration-dependent manner and confirming the degree of formation of a moraine.
FIG. 3A is an image obtained by administering Lefty-A-Fc protein in a concentration-dependent manner to a mouse and confirming the degree of formation of a moraine. FIG. 3B is a graph showing the length of the hairy root formed by administering the Lefty-A-Fc protein to the mouse at different concentrations.
Fig. 4 is an image obtained by administering Lefty-A protein at a concentration of a mouse and confirming the degree of formation of a moraine.
FIGS. 5A and 5B are graphs showing quantitative images and images obtained by administering phosphate buffered saline, Lefty-A-Fc protein, and the degree of expression of Krox20 gene in mice.
FIG. 6 is a graph showing the extent of expression of Krox20 gene by adding Lefty-A-Fc mutant protein to cells.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Use of a composition containing Lefty-A to confirm hair growth promoting efficacy

1. Preparation and Expression of Lefty-A-Fc Protein

1.1. Preparation of Lefty-A-Fc Protein

A polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, and an expression plasmid of a polynucleotide encoding the Fc fragment of human IgG1 were prepared.

A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 was cloned into a vector expressing the Fc fragment of human IgG1. The vector used for the cloning has a CMV promoter and is a vector comprising a polynucleotide encoding an Fc region of a human antibody. The polynucleotide encoding the polypeptide is located at the 5 'position of the polynucleotide encoding the Fc region of the human antibody To clone such that the polypeptide is expressed at the terminal portion of the polypeptide constituting the Fc region of the human antibody. Specifically, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and a restriction enzyme of Not I (Roche) and Xba I (Roche) were added to the vector and reacted, and the cleaved product was ligated with T4 DNA ligase (NEB) to prepare a vector for expression of a peptibody containing a desired polypeptide region. This vector was confirmed to be a plasmid containing the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 and the Fc fragment of human IgG1 by performing DNA base sequence analysis (Solgent Co., Daejeon, Korea).

Subsequently, CHO-S cells were transfected with the plasmid obtained above.

The CHO-S cells (CD-CHO) (Invitrogen) dulbe course modified Eagle's medium (Dulbecco's Modified Eagle Medium: D -MEM) was dispensed to 0.4 × 10 ⁵ cells on a 96-well plate containing (Invitrogen). 0.2 μg of plasmid DNA was added to 25 μl of Opti-MEM reduced serum medium and mixed. 0.5 μl of Lipofectamine 2000 (Invitrogen) was added to 25 μl of Opti-MEM reduced serum medium and mixed. The medium supplemented with plasmid DNA and the medium supplemented with lipofectamine were mixed and incubated at room temperature for 20 minutes. 50 쨉 l of the plasmid DNA and lipofectamine complex was added to each well containing CHO-S cells and medium, and mixed. The cells were then incubated overnight at 5% CO ₂ , and 37 ° C. The medium in each well was aspirated and 100 μl of DMEM containing serum was added to each well. Forty-eight hours after transduction, the supernatant of the medium in which the cells were cultured was collected. The collected supernatant was loaded onto protein A agarose beads (Invitrogen) to bind the Lefty-A-Fc protein. Next, the beads were washed with a buffer solution of 50 mM Tris-HCl, pH 8.0 and eluted with a gradient of NaCl concentration of 0 to 500 mM to obtain an eluate. The eluate was applied to a Superdex 75 column in a buffer of 50 mM Tris-HCl, pH 8.0 and 0.2 M NaCl to obtain a purified Lefty-A-Fc protein.

1.2. Expression of Lefty-A-Fc Protein

1.1 protein was separated by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. The separated proteins were reacted with Lefty-A antibody (ROCKLAND 200-301-279) to confirm the size of the protein. Figure 1 shows the results of confirming the expression and size of Lefty-A-Fc protein. As shown in Fig. 1, under non-reducing conditions, the protein showed a molecular weight of about 130 kDa (lane 3) and the protein under the reducing conditions was found to have a molecular weight of about 65 kDa (lane 2). Therefore, it was confirmed that the expressed protein forms Lefty-A-Fc protein in which the Fc fragment of human IgG1 is fused to the Lefty-A protein and the end portion of the Lefty-A protein.

2. Assessment of hairy stimulating activity of Lefty-A protein

2.1. Assessment of Hairy Promoting Effectiveness of Lefty-A-Fc Protein 1

BALB / c mice were maintained in a sterile controlled environment (temperature 22 ± 2 ° C., humidity 60 ± 5%, and 12 h light / 12 h dark environment). The weight of male BALB / c mice at 2 months was about 25 g to about 30 g.

The hair of the mouse was shaved with an epilator, hair was secondarily removed using a depilator, and sterilized with alcohol. 0.2 μg, 0.5 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg and 10 μg of Lefty-A-Fc protein of 1.1 were respectively dissolved in phosphate buffered saline and administered by the subcutaneous injection of the mice . The administration was administered twice a day for a total of three times. The control group was administered with phosphate buffered saline. FIG. 2 is an image obtained by administering the Lefty-A-Fc protein to the mouse at a concentration and confirming the degree of formation of the moraine. The image on the left is taken one day after two days, and the image on the right after three doses. As shown in FIG. 2, when the Lefty-A-Fc protein was administered from about 0.2 μg to about 2 μg, the length of the hair was increased in a concentration-dependent manner, and about 3 μg or about 4 μg of Lefty-A-Fc protein The length of the hair was increased. In addition, when the Lefty-A-Fc protein is administered, it can be seen that the moraine is formed around the administered region. On the other hand, when about 5 또는 or about 10 의 of Lefty-A-Fc protein was administered, the hair was scarcely grown and the skin was reddened immediately after administration.

2.2. Assessment of Hairy Promoting Effectiveness of Lefty-A-Fc Protein 2

0.2 μg, 2 μg, 5 μg, and 10 μg of Lefty-A-Fc protein of 1.1 were respectively dissolved in phosphate buffered saline and administered to male BALB / c mice at 6 weeks of age by subcutaneous injection. The administration was administered twice a day for a total of three times. The control group was administered with phosphate buffered saline. Morain length was measured at day 12 from the day of administration. FIG. 3A is an image obtained by administering Lefty-A-Fc protein in a concentration-dependent manner to a mouse and confirming the degree of formation of a moraine. FIG. 3B is a graph showing the length of the hairy root formed by administering the Lefty-A-Fc protein to the mouse at different concentrations. As shown in FIGS. 3A and 3B, when the Lefty-A-Fc protein was administered in an amount of 0.2 to 2 μg, the length of the hairy lobe was increased in a concentration-dependent manner, and 5 μg or 10 μg of Lefty-A-Fc protein In the case of Morgan almost did not grow.

2.3. Assessment of hairy stimulating activity of Lefty-A protein

The Lefty-A protein having the amino acid sequence of SEQ ID NO: 2 was used (R & D system, 746-LF) in place of the Lefty-A-Fc protein of 1.1, and 0.5 μg, 1 μg, The effect of promoting hair growth was confirmed in the same manner as in 2.1, except that 4 ㎍ was administered. Fig. 4 is an image obtained by administering Lefty-A protein at a concentration of a mouse and confirming the degree of formation of a moraine. As shown in Fig. 4, when the Lefty-A protein of 0.5 to 4 占 퐂 was administered, the length of the mammal increased in a concentration-dependent manner.

2.4. Increase of hair growth factor

2.4.1. 1

Transgenic C22 mice (Huxley et al., 1996), an animal model of CMT, were housed in a sterile controlled environment (temperature 22 ± 2 ° C., humidity 60 ± 5%, and 12 h light / 12 h cancer environment) Respectively.

1.1 Lefty-A-Fc protein was dissolved in phosphate-buffered saline, respectively, and administered to male C22 mice at about 6 days of age with 10 ug / kg intraperitoneal injection (IP). The treatment was administered once every two days from day 6 to day 24. The control group used wild-type mice and C22 mice administered with phosphate buffered saline. After the administration, the nerve nerve was collected at day 28 after birth, and the degree of expression of Krox20 gene was confirmed by immunoblotting. Figures 5a and 5b show the results of a C22 mouse treated with phosphate buffered saline, 1.1 Lefty-A-Fc protein (designated Lefty-A-Fc) and Lefty-A-Fc protein -Fc < ^- >), and the amount of expression of Krox20 gene was confirmed and quantified. As shown in FIGS. 5a and 5b, when the Lefty-A-Fc ^- and Lefty-A-Fc proteins were administered, the amount of Krox20 protein was increased (*, P value 0.05, *** P value 0.01 ).

2.4.2. Increase of hair growth factor 2

The same method as in 1.1., Except that the amino acid residue at position 308 in the amino acid sequence of SEQ ID NO: 3 of the Lefty-A-Fc protein of 1.1 was changed to R, A, E, H, I, K, 7 mutant proteins were prepared.

The RT4 cells (ATCC ^® HTB2 ™) was dispensed into a 6-well plate at 3 × 10 ⁶ cells. After cells were attached to the bottom of the plate, 200 ng of the Lefty-A-Fc variant protein was added to the cells at 100 ng / ml and incubated for about 72 minutes. After incubation, cells were harvested and RNA was extracted from the cells using phenol / chloroform. The extracted RNA was reverse transcribed to synthesize cDNA. The degree of gene expression of cDNA was analyzed by real time polymerase chain reaction on an Applied Biosystems 700 sequence detection system (Foster City, CA, USA). At this time, synthesized cDNA, Krox20 specific primer set, 2 x TaqMan master mixture and 20 x premade TaqMan gene expression analysis (Applied Biosystems) kit were used. The primer set used was as follows: forward 5'-CCTGGGTGTGTGTACCATGT-3 ', reverse 5'-GAGAGGAGGTGGAAGTGGTG-3'. MRNA levels of Krox20 were normalized to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. FIG. 6 is a graph quantifying the degree of expression of the Krox20 gene by adding a Lefty-A-Fc mutant protein (denoted as Lefty-A-Fc-mut) to the cells. The altered proteins were designated as Lefty-A-Fc mut 1, 2, 3, 4, 5, 6, 7, respectively. As shown in FIG. 6, even when the Lefty-A-Fc mutant protein was administered, the expression level of the Krox20 gene was increased (* P value: 0.05).

<110> Samsung Life Public Welfare Foundation <120> Composition for preventing depilation and improving hair growth <130> PN118491 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 124 <212> PRT <213> Homo sapiens <400> 1 Arg His Gly Arg Leu Ser Pro Arg Ser Ala Gln Ala Arg Val Thr Val   1 5 10 15 Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr Ser Leu Ile              20 25 30 Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp Lys Ala Phe Asp          35 40 45 Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg Pro Arg Gln      50 55 60 Pro Leu Leu Leu Gln Val Ser Val Gln Arg Glu His Leu Gly Pro Leu  65 70 75 80 Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln Gly Ala Pro                  85 90 95 Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu Asp Leu Arg             100 105 110 Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala         115 120 <210> 2 <211> 345 <212> PRT <213> Homo sapiens <400> 2 Leu Thr Glu Glu Glu Leu Glu Le Leu Glu Leu   1 5 10 15 Ser Glu Val Pro Val Leu Asp Arg Ala Asp Met Glu Lys Leu Val Ile              20 25 30 Pro Ala His Val Arg Ala Gln Tyr Val Val Leu Leu Arg Arg Ser His          35 40 45 Gly Asp Arg Ser Ser Gly Lys Arg Phe Ser Gln Ser Phe Arg Glu Val      50 55 60 Ala Gly Arg Phe Leu Ala Ser Glu Ala Ser Thr His Leu Leu Val Phe  65 70 75 80 Gly Met Glu Gln Arg Leu Pro Pro Asn Ser Glu Leu Val Gln Ala Val                  85 90 95 Leu Arg Leu Phe Gln Glu Pro Val Lys Ala Ala Leu His Arg His             100 105 110 Gly Arg Leu Ser Pro Arg Ser Ala Gln Ala Arg Val Thr Val Glu Trp         115 120 125 Leu Arg Val As Asp Asp Gly Ser Asn Arg Thr Ser Leu Ile Asp Ser     130 135 140 Arg Leu Val Ser Val Glu Ser Gly Trp Lys Ala Phe Asp Val Thr 145 150 155 160 Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg Pro Arg Gln Pro Leu                 165 170 175 Leu Leu Gln Val Ser Val Gln Arg Glu His Leu Gly Pro Leu Ala Ser             180 185 190 Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln Gly Ala Pro Ala Gly         195 200 205 Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu Asp Leu Arg Asp Tyr     210 215 220 Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro Met Thr Glu Gly Thr 225 230 235 240 Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu Gln Gly Met Lys Trp                 245 250 255 Ala Lys Asn Trp Val Leu Glu Pro Pro Gly Phe Leu Ala Tyr Glu Cys             260 265 270 Val Gly Thr Cys Gln Gln Pro Pro Glu Ala Leu Ala Phe Asn Trp Pro         275 280 285 Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser Glu Thr Ala Ser Leu Pro     290 295 300 Met Ile Val Ser Ile Lys Glu Gly Gly Arg Thr Arg Pro Gln Val Val 305 310 315 320 Ser Leu Pro Asn Met Arg Val Gln Lys Cys Ser Cys Ala Ser Asp Gly                 325 330 335 Ala Leu Val Pro Arg Arg Leu Gln Pro             340 345 <210> 3 <211> 366 <212> PRT <213> Homo sapiens <400> 3 Met Trp Pro Leu Trp Leu Cys Trp Ala Leu Trp Val Leu Pro Leu Ala   1 5 10 15 Gly Pro Gly Ala Ala Leu Thr Glu Glu Gln Leu Leu Gly Ser Leu Leu              20 25 30 Arg Gln Leu Gln Leu Ser Glu Val Pro Val Leu Asp Arg Ala Asp Met          35 40 45 Glu Lys Leu Val Ile Pro Ala His Val Arg Ala Gln Tyr Val Val Leu      50 55 60 Leu Arg Arg Ser Ser Gly Asp Arg Ser Ser Gly Lys Arg Phe Ser Gln  65 70 75 80 Ser Phe Arg Glu Val Ala Gly Arg Phe Leu Ala Ser Glu Ala Ser Thr                  85 90 95 His Leu Leu Val Phe Gly Met Glu Gln Arg Leu Pro Pro Asn Ser Glu             100 105 110 Leu Val Gln Ala Val Leu Arg Leu Phe Gln Glu Pro Val Lys Ala         115 120 125 Ala Leu His Arg His Gly Arg Leu Ser Pro Arg Ser Ala Gln Ala Arg     130 135 140 Val Thr Val Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr 145 150 155 160 Ser Leu Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp Lys                 165 170 175 Ala Phe Asp Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg             180 185 190 Pro Arg Gln Pro Leu Leu Leu Gln Val Ser Val Gln Arg Glu His Leu         195 200 205 Gly Pro Leu Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln     210 215 220 Gly Ala Pro Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu 225 230 235 240 Asp Leu Arg Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro                 245 250 255 Met Thr Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu             260 265 270 Gln Gly Met Lys Trp Ala Lys Asn Trp Val Leu Glu Pro Pro Gly Phe         275 280 285 Leu Ala Tyr Glu Cys Val Gly Thr Cys Gln Gln Pro Pro Glu Ala Leu     290 295 300 Ala Phe Asn Trp Pro Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser Glu 305 310 315 320 Thr Ala Ser Leu Pro Met Ile Val Ser Ile Lys Glu Gly Gly Arg Thr                 325 330 335 Arg Pro Gln Val Val Ser Leu Pro Asn Met Arg Val Gln Lys Cys Ser             340 345 350 Cys Ala Ser Asp Gly Ala Leu Val Pro Arg Arg Leu Gln Pro         355 360 365 <210> 4 <211> 329 <212> PRT <213> Homo sapiens <400> 4 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser   1 5 10 15 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe              20 25 30 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly          35 40 45 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu      50 55 60 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr  65 70 75 80 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys                  85 90 95 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro             100 105 110 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys         115 120 125 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val     130 135 140 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 145 150 155 160 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu                 165 170 175 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His             180 185 190 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys         195 200 205 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln     210 215 220 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 225 230 235 240 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro                 245 250 255 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn             260 265 270 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu         275 280 285 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val     290 295 300 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 305 310 315 320 Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 <210> 5 <211> 76 <212> PRT <213> Homo sapiens <400> 5 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu   1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp              20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys          35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu      50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly  65 70 75 <210> 6 <211> 255 <212> PRT <213> Homo sapiens <400> 6 Glu Leu Val Gln Ala Val Leu Arg Leu Phe Gln Glu Pro Val Pro Lys   1 5 10 15 Ala Ala Leu His Arg His Gly Arg Leu Ser Pro Arg Ser Ala Gln Ala              20 25 30 Arg Val Thr Val Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg          35 40 45 Thr Ser Leu Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp      50 55 60 Lys Ala Phe Asp Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser  65 70 75 80 Arg Pro Arg Gln Pro Leu Leu Leu Gln Val Ser Val Gln Arg Glu His                  85 90 95 Leu Gly Pro Leu Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser             100 105 110 Gln Gly Ala Pro Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr         115 120 125 Leu Asp Leu Arg Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala     130 135 140 Pro Met Thr Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp 145 150 155 160 Leu Gln Gly Met Lys Trp Ala Lys Asn Trp Val Leu Glu Pro Pro Gly                 165 170 175 Phe Leu Ala Tyr Glu Cys Val Gly Thr Cys Gln Gln Pro Pro Glu Ala             180 185 190 Leu Ala Phe Asn Trp Pro Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser         195 200 205 Glu Thr Ala Ser Leu Pro Met Ile Val Ser Ile Lys Glu Gly Gly Arg     210 215 220 Thr Arg Pro Gln Val Ser Ser Leu Pro Asn Met Arg Val Gln Lys Cys 225 230 235 240 Ser Cys Ala Ser Asp Gly Ala Leu Val Pro Arg Arg Leu Gln Pro                 245 250 255

Claims (12)

A composition for preventing hair loss or promoting hair growth, which comprises Lefty-A protein as an active ingredient. The composition of claim 1, wherein the protein comprises the amino acid sequence of SEQ ID NO: 6. The composition of claim 1, wherein the protein comprises the amino acid sequence of SEQ ID NO: 6 and has at least 90% sequence homology to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. The method of claim 1, wherein the protein comprises a stabilizing protein and the stabilizing protein is selected from the group consisting of immunoglobulin G, ubiquitin, ecotin, Brazzein,? -Lactoglobulin, Sakacin B, , Mixin 10 (MxynlO), or a combination thereof. 5. The composition of claim 4, wherein the immunoglobulin G is an Fc fragment of an immunoglobulin G. 6. The composition of claim 5, wherein the Fc fragment of the immunoglobulin G is linked to the end of the Letfy-A protein. The composition according to claim 1, wherein the protein is administered at 1 to 1000 μg / kg. The composition of claim 1, wherein the protein is administered more than once. The composition of claim 1, wherein the protein is administered by oral, intraperitoneal, intramuscular, subcutaneous, intravenous, or a combination thereof. A composition for promoting hair loss or hair growth, comprising as an active ingredient a cell comprising a polynucleotide encoding a Lefty-A protein or a cell overexpressing a Lefty-A protein transduced with said vector, or a culture thereof. 11. The composition of claim 10, wherein the vector comprises linear DNA, plasmid DNA, a recombinant viral vector, or a combination thereof. A functional food for preventing hair loss or promoting hair growth, comprising Lefty-A protein as an active ingredient.

Images