Thymus-derived peptide derivatives for preventing hair loss or promoting hair growth, and use thereof

Abstract

The present invention relates to thymus-derived peptide derivatives for preventing hair loss or promoting hair growth. By using an expression cassette for producing thymus-derived peptide derivatives, an expression vector, and a transformant comprising same, of the present invention, high-purity thymus-derived peptide derivatives can be mass-produced at low cost. In addition, the thymus-derived peptide derivatives produced according to the present invention exhibit excellent hair loss prevention effects, and thus can be widely used throughout industries such as cosmetic materials, quasi-drugs and medicines for related uses.

Description

Thymus-derived peptide derivatives for preventing hair loss or promoting hair growth and their use

[0001] The present invention relates to a thymus-derived peptide derivative for preventing hair loss or promoting hair growth and its use.

[0002] Hair loss, once considered a problem exclusive to middle-aged men, is now appearing in young people in their 20s, including teenagers, due to the stress and obsession caused by schoolwork, job hunting, fierce competition, and uncertainty about the future. The number of people suffering from hair loss in Korea is rapidly increasing. As of 2018, the number of people suffering from hair loss exceeded 10 million. Of the approximately 1.03 million patients who received hair loss treatment between 2012 and 2016, 210,000, or 20% of the total, were in their 20s. Approximately 220,000 to 250,000 hair loss patients are receiving treatment each year.

[0003] It's estimated that over 50% of the population suffering from hair loss are women. In the case of female hair loss, obsessive dieting and subsequent malnutrition are major causes. Hair dyeing, perms, and hair dryers are also common causes. Hair loss sufferers experience heightened stress levels due to hair loss, including depression and shame, and experience social and emotional difficulties, including difficulties in interpersonal and romantic relationships.

[0004] A variety of functional substances, including organic and inorganic materials, oils, natural extracts, antimicrobial agents, and peptides, are being developed to prevent hair loss and promote hair growth. In particular, among the various hormones secreted by the human thymus, peptides associated with hair loss have been identified. Thymulin, Thymosin α1, and β4 peptides are currently being studied.

[0005] Thymosin is mainly composed of thymosin α1 consisting of 28 amino acids, thymosin β4 consisting of 43 amino acids, and thymulin consisting of 9 amino acids. Instead of obtaining it from the thymus by slaughtering young calves, it is necessary to develop a technology that can manufacture it economically and efficiently.

[0006] The present inventors have devoted extensive research efforts to developing an economical, mass-producible method for thymus-derived peptide derivatives. As a result, they produced thymus-derived peptide derivatives through a fermentation process using a safe yeast strain (GRAS strain) and confirmed their excellent dermal papilla cell growth activity, thereby completing the present invention.

[0007] Accordingly, an object of the present invention is to provide an expression cassette for producing a thymus-derived peptide derivative.

[0008] Another object of the present invention is to provide an expression vector for producing a thymus-derived peptide derivative.

[0009] Another object of the present invention is to provide a transformant comprising an expression vector for producing a thymus-derived peptide derivative.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hair loss comprising a thymus-derived peptide derivative.

[0011] Another problem to be solved by the present invention is to provide a cosmetic composition for preventing hair loss or promoting hair growth, which comprises a thymus-derived peptide derivative.

[0012] Another problem to be solved by the present invention is to provide a pharmaceutical composition for preventing hair loss or promoting hair growth, which comprises a thymus-derived peptide derivative.

[0013] Another problem to be solved by the present invention is to provide a health functional food composition for preventing hair loss or promoting hair growth, which comprises a thymus-derived peptide derivative.

[0014] The present inventors have conducted extensive research to develop an economical, mass-production method for thymus-derived peptide derivatives. As a result, we produced thymus-derived peptide derivatives through a fermentation process using a safe yeast strain (GRAS strain) and confirmed their excellent dermal papilla cell growth activity.

[0015] The present invention relates to an expression cassette for producing a thymus-derived peptide derivative, an expression vector for producing a thymus-derived peptide derivative, a transformant comprising an expression vector for producing a thymus-derived peptide derivative, a pharmaceutical composition for preventing or treating hair loss comprising a thymus-derived peptide derivative, a cosmetic composition for preventing hair loss or promoting hair growth comprising a thymus-derived peptide derivative, a quasi-drug composition for preventing hair loss or promoting hair growth comprising a thymus-derived peptide derivative, and a health functional food composition for preventing hair loss or promoting hair growth comprising a thymus-derived peptide derivative.

[0016] Hereinafter, the present invention will be described in more detail.

[0017]

[0018] One aspect of the present invention relates to an expression cassette for producing a thymus-derived peptide derivative, comprising a polynucleotide sequence represented by SEQ ID NO: 1 encoding a thymus-derived peptide derivative, wherein the polynucleotide sequence is operably linked to a promoter sequence capable of being expressed in yeast.

[0019] In the present invention, the term "thymus-derived peptide" refers to a peptide secreted from the human thymus, including Thymulin, Thymosin α1, and Thymosin β4. Conventional thymus-derived peptides are obtained from the thymus of slaughtered young calves or produced through chemical synthesis, which has the disadvantages of being high in unit price and containing various chemical substances. Therefore, in order to supplement this, the inventors of the present invention have created a strain that produces Thymulin, Thymosin α1, and Thymosin β4 through gene work, and have produced thymus-derived peptides through yeast fermentation.

[0020] In the present invention, the term "thymus-derived peptide derivative" means a peptide or a combination thereof in which P(Pro) and D(Asp) are additionally attached before and after the thymus-derived peptide sequence described above, and the peptide in which P and D are additionally attached before and after is manufactured using a cleavage method (DP formic acid cleavage) using formic acid during the separation and purification process of a thymus-derived peptide produced through yeast fermentation, and when this method is used, more efficient and economical mass production of thymus-derived peptide derivatives is possible.

[0021] Specifically, the thymus-derived peptide derivative may be a single peptide derivative (each Thymulin derivative, Thymosin α1 derivative, or Thymosin β4 derivative), or some or all of each derivative may be combined in a certain ratio.

[0022] More specifically, the thymus-derived peptide derivative may be a mixture of Thymulin derivative: Thymosin α1 derivative: Thymosin β4 derivative in a content ratio of 0.1 to 3: 0.1 to 3: 0.1 to 3, but is not limited thereto.

[0023] More specifically, the thymus-derived peptide derivative may be a mixture of Thymulin derivative: Thymosin α1 derivative: Thymosin β4 derivative in a content ratio of 1:1:2, 1:2:2, 1:2:3, 2:1:1, 2:1:2, 2:2:1, 2:3:2, 2:3:3, 3:2:1, 3:1:2, 3:2:2, 3:2:3, or 3:3:2, but is not limited thereto.

[0024] According to one embodiment of the present invention, among the thymus-derived peptide derivatives described above, a three-type mixed peptide derivative (Thymulin derivative, Thymosin α1 derivative, and Thymosin β4 derivative) exhibited a superior effect on activating hair follicle cells compared to a single peptide derivative, and in particular, the highest effect was exhibited when the ratio of Thymulin: Thymosin α1: Thymosin β4 was 3:2:1. (See Fig. 7)

[0025] The term "expression cassette for producing a thymus-derived peptide derivative" of the present invention means an expression construct capable of expressing a thymus-derived peptide derivative.

[0026] In the present invention, the expression cassette may include a polynucleotide sequence encoding the thymus-derived peptide derivative, and specifically, the polynucleotide sequence encoding the thymus-derived peptide derivative included in the expression cassette may be composed of an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0027] The amino acid sequence used in the present invention is interpreted to include a sequence that shows substantial identity with the sequence listed in the sequence list, considering a mutation that has biologically equivalent activity. The term, 'substantial identity', means a sequence that shows at least 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0028] Therefore, it should be interpreted that a sequence having a high homology with the amino acid sequence represented by the above sequence number 1, for example, an amino acid sequence having a high homology of 70% or more, specifically 80% or more, and more specifically 90% or more, is also included in the scope of the present invention.

[0029] The expression cassette for producing the thymus-derived peptide derivative may be one in which the polynucleotide sequence represented by the above sequence number 1 is operably linked to a promoter sequence capable of being expressed in yeast and a high-expression / high-secretion signal sequence.

[0030] As a specific example, the promoter may be AOX1, TEF, GAP, or a combination thereof, but is not particularly limited thereto as long as it is a promoter suitable for expression in yeast. The sequence of the promoter exemplified above may be a sequence known in the art.

[0031] As another specific example, the high-expression / high-secretion signal sequence may be a fusion partner, but is not particularly limited thereto as long as it can increase efficiency. The exemplified high-expression / high-secretion signal sequence may use a sequence known in the art.

[0032] As used herein, the term "operably linked" refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or peptide are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or peptide are operably linked to affect the expression of the coding sequence. The operably linked sequence with the expression vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0033] In addition, the expression cassette for producing the thymus-derived peptide derivative may additionally include a polynucleotide encoding His upstream or downstream of the polynucleotide sequence in the expression cassette to facilitate purification of the expressed thymus-derived peptide derivative.

[0034] In addition, the expression cassette for producing the thymus-derived peptide derivative may further include components that regulate the expression of the thymus-derived peptide derivative, such as a transcription enhancer, a terminator, an initiator, and other genetic regulatory factors, in addition to a promoter, a sequence encoding the thymus-derived peptide derivative, and a sequence such as His.

[0035] In one embodiment of the present invention, an expression cassette for producing a thymus-derived peptide derivative was constructed by sequentially inserting a TEF promoter sequence, an α-factor sequence, a His-tag sequence, a fusion partner sequence, a D sequence, a polynucleotide sequence encoding a thymus-derived peptide derivative, and a CYC1 terminator sequence (see FIG. 1b).

[0036]

[0037] Another aspect of the present invention relates to an expression vector for producing a thymus-derived peptide derivative, comprising the expression cassette for producing the thymus-derived peptide derivative described above.

[0038] The term "expression vector" of the present invention is a means for efficiently inducing the expression of a target gene by introducing DNA into a host cell, and specifically may mean a genetic construct including essential regulatory elements operably linked to express a target peptide, which is a thymus-derived peptide derivative.

[0039] Specific examples of the above expression vector include a plasmid vector, a cosmid vector, a bacteriophage vector, or a viral vector. More specific examples include a plasmid derived from Escherichia coli (pBR322, pBR325, pUC118, pUC119, pET30a, pET30c, or pGEX-GST), a plasmid derived from Bacillus subtilis (pUB110 or pTP5), a plasmid derived from yeast (YEp13, YEp24, YCp50, pPINKα-HC, pPink-HC, or pPink-LC), or a Ti plasmid, and animal viruses such as retrovirus, adenovirus, or vaccinia virus, insect viruses such as baculovirus, or plant viruses, and binary vectors such as the pPZP, pGA, and pCAMBIA series can be used, but are not limited thereto as long as the expression cassette of the present invention can be introduced into a host cell.

[0040] In addition, the expression vector may be functionally linked to an expression control sequence. As a specific example, the vector may include, but is not limited to, a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, and may be manufactured in various ways depending on the purpose of the invention. In addition, the vector may include a selectable marker, and may be self-replicating or integrated into host DNA. The vector of the present invention may be manufactured using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation may be performed using enzymes generally known in the art.

[0041] In one embodiment of the present invention, the expression cassette for producing the thymus-derived peptide derivative was inserted into the expression vector pPinkα-HC vector using a conventional method to produce an expression vector for producing the thymus-derived peptide derivative (see FIGS. 2a to 2c, FIGS. 3a to 3c, and FIG. 4).

[0042]

[0043] Another aspect of the present invention relates to a transformant comprising the expression vector for producing the thymus-derived peptide derivative described above.

[0044] The term "transformant" of the present invention refers to an organism whose genetic traits have been changed by the introduction of foreign genetic material.

[0045] As a specific example, the transformant may be a strain of the genus Pichia, Escherichia, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Schizosaccharomyces, Issachenkia, Yarrowia or Hansenula, but is not particularly limited thereto as long as it can express the thymus-derived peptide derivative.

[0046] More specifically, the transformant may be Pichia pastoris, but is not particularly limited thereto as long as it can express the thymus-derived peptide derivative.

[0047] In the present invention, Pichia pastoris refers to the yeast strain most widely used for recombinant protein production. This yeast strain has the advantages of being easy to genetically manipulate, various expression systems have been developed, and easy to mass-culture. Furthermore, when producing recombinant proteins derived from higher cells, such as human proteins, it offers the advantage of being able to secrete proteins outside the cell, as well as perform post-translational modifications such as sugar chain addition. Secretory production of recombinant proteins is achieved by artificially fusing a protein secretion signal with a target protein to enable extracellular secretion. Since protein folding, disulfide bond formation, and sugar chain addition processes occur through the protein secretion process, it has the advantage of producing recombinant proteins with complete biological activity. Furthermore, since biologically active proteins can be obtained directly from the medium, the method is highly economical, eliminating the need for economically inefficient cell disruption or refolding steps.

[0048] In one embodiment of the present invention, the expression vector for producing the thymus-derived peptide derivative was introduced (transfected) into a P. pastoris strain using a conventional method to produce a transformant (see Manufacturing Example).

[0049]

[0050] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating hair loss comprising a thymus-derived peptide derivative.

[0051] The above thymus-derived peptide derivative can be produced through a step of culturing a transformant including the expression vector for producing the above-described thymus-derived peptide derivative; and a step of isolating and purifying the expressed peptide from the cultured transformant.

[0052] The step of culturing the transformant can be performed taking into account the nutritional requirements of the transformant.

[0053] As a specific example, P. pastoris is a methylotrophic yeast cell, and for its cultivation, buffered complex methanol medium (BMMY), buffered minimal methanol medium (BMM), etc. can be used, but are not limited thereto, and can be appropriately selected by a person skilled in the art according to the purpose of the invention.

[0054] In addition, the recovery of the thymus-derived peptide derivative from the culture of the transformant can be performed by a method known in the art. Specifically, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but are not particularly limited thereto as long as the thymus-derived peptide derivative of the present invention can be recovered.

[0055] The thymus-derived peptide derivative of the present invention can be produced by utilizing a DP formic acid cleavage system using formic acid in the step of isolating and purifying the thymus-derived peptide derivative expressed from the cultured transformant, but is not limited thereto, and can be appropriately selected by a person skilled in the art according to the purpose of the invention.

[0056] The pharmaceutical composition of the present invention has a use for preventing or treating alopecia.

[0057] The above-mentioned alopecia (or hair loss disease) may be one or more selected from alopecia areata, hereditary androgenic alopecia, telogen effluvium, traumatic alopecia, trichotillomania alopecia, compression alopecia, anagen alopecia, pityriasis alopecia, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, non-scarring alopecia, scarring alopecia, and congenital alopecia, depending on the form, symptoms, or cause.

[0058] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition, the pharmaceutical composition includes diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0059] The above pharmaceutical composition can be formulated in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, parenteral preparations, injection preparations, and topical administration preparations according to conventional methods.

[0060] Among oral preparations, oral solid preparations include tablets, pills, powders, granules, capsules, etc., and these solid preparations may include at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may include lubricants, such as magnesium stearate and talc. Among oral preparations, oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and may include diluents, such as water and liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, etc. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized preparations. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable esters, such as ethyl oleate.

[0061] Additionally, the pharmaceutical composition may be administered to mammals, including or not including humans, by various routes, for example, orally, intravenously, intramuscularly, or subcutaneously.

[0062] The pharmaceutical composition of the present invention may be administered topically. Topical administration refers to the direct application of an active ingredient or composition (e.g., a composition for external use) to the skin and / or hair. The topical composition of the present invention may be in the form of a solution, lotion, wax, cream, ointment, liposome, spray, gel, foam, roller stick, or any other formulation commonly used in dermatology.

[0063] In the pharmaceutical composition, use, and method of the present invention, the dosage of the active ingredient may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, the period, etc., but may be appropriately selected depending on the case. The above administration may be administered once a day or divided into several times.

[0064]

[0065] Another aspect of the present invention relates to a cosmetic composition for preventing hair loss or promoting hair growth, comprising a thymus-derived peptide derivative prepared by the above-described method.

[0066] The cosmetic composition of the present invention is not limited to a specific form (formulation), and may be prepared in a form selected from the group consisting of a solution, an external ointment, a cream, a foam, a nourishing toner, an emollient toner, a pack, an emollient, an emulsion, a makeup base, an essence, a soap, a liquid cleanser, a bath agent, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray, but is not limited thereto.

[0067] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate conventional ingredients such as oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., but is not limited thereto.

[0068] The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

[0069] When the formulation of the present invention is an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in combination of two or more.

[0070] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as a carrier component, and especially in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether can be additionally included, but is not limited thereto. These can be used alone or in a mixture of two or more.

[0071] When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc. may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty acid ester, polyethylene glycol or fatty acid ester of sorbitan may be used, but is not limited thereto. These may be used alone or in a mixture of two or more.

[0072] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components, but are not limited thereto. These may be used alone or in combination of two or more.

[0073] When the formulation of the present invention is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in combination of two or more.

[0074] When the formulation of the present invention is a surfactant-containing cleansing agent, a carrier component may be used, such as, but not limited to, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcositate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester. These may be used alone or in combination of two or more.

[0075] In addition, the cosmetic composition of the present invention may contain conventional auxiliary agents in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blocking agents, pigments, deodorants, and dyes.

[0076]

[0077] Another aspect of the present invention relates to a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising a thymus-derived peptide derivative prepared by the method described above.

[0078] The pharmaceutical composition of the present invention can be prepared in a formulation selected from the group consisting of body cleanser, soap, and hand wash, but is not limited thereto.

[0079]

[0080] Another aspect of the present invention relates to a health functional food composition for preventing hair loss or promoting hair growth, comprising a thymus-derived peptide derivative prepared by the method described above.

[0081] The health functional food composition of the present invention can be used alone or in combination with the thymus-derived peptide derivative, or with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of active ingredients mixed can be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present specification can be added in an amount of 15 parts by weight or less relative to the raw material. There are no particular restrictions on the type of the health functional food.

[0082] Among the types of health functional foods, the beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, just like regular beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame may be used. The health food composition may also contain nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, or a combination thereof.

[0083] The above health functional food composition may also contain fruit pulp for the production of natural fruit juice, fruit juice beverage, vegetable beverage, or a combination thereof.

[0084]

[0085] Meanwhile, the terms 'peptide' and 'protein' used in this specification are defined to have the same meaning and are used interchangeably when necessary.

[0086] The overlapping contents of the thymus-derived peptide derivatives included in each of the above compositions are omitted in consideration of the complexity of this specification.

[0087] The present invention relates to a thymus-derived peptide derivative for preventing hair loss or promoting hair growth. By using the expression cassette for producing the thymus-derived peptide derivative, the expression vector, and the transformant containing the same of the present invention, a high-purity thymus-derived peptide derivative can be produced in large quantities at low cost. In addition, the thymus-derived peptide derivative produced according to the present invention has an excellent hair loss prevention effect, and thus can be widely used in various industries, such as cosmetic materials, quasi-drugs, and pharmaceuticals for related purposes.

[0088] Figures 1a and 1b are schematic diagrams of a DNA cassette manufactured according to one embodiment of the present invention.

[0089] FIGS. 2A to 2C are vector maps manufactured according to one embodiment of the present invention, wherein FIG. 2A is a vector map of Thymulin monomer, FIG. 2B is a vector map of Thymosin alpha1 monomer, and FIG. 2C is a vector map of Thymosin beta4 monomer.

[0090] FIGS. 3A to 3C are vector maps manufactured according to one embodiment of the present invention, wherein FIG. 3A is a vector map of P_Thymulin_D multimer, FIG. 3B is a vector map of P_Thymosin alpha1_D multimer, and FIG. 3C is a vector map of P_Thymosin beta4_D multimer.

[0091] Figure 4 is a vector map of a thymus-derived peptide derivative (hybrid multimer) manufactured according to one embodiment of the present invention.

[0092] FIG. 5 is an SDS-PAGE result of a single peptide (Thymulin derivative, Thymosin α1 derivative, Thymosin β4 derivative) produced by fermentation in a yeast strain according to one embodiment of the present invention.

[0093] FIGS. 6A to 6C are the results of evaluating the activity of mammary papilla cells of a single peptide (Thymulin, Thymulin derivative, Thymosin α1, Thymosin α1 derivative, Thymosin β4, Thymosin β4 derivative) produced by fermentation in a yeast strain according to one embodiment of the present invention. FIG. 6A shows the results of evaluating Thymulin and Thymulin derivative, FIG. 6B shows the results of evaluating Thymosin α1 and Thymosin α1 derivative, and FIG. 6C shows the results of evaluating Thymosin β4 and Thymosin β4 derivative.

[0094] Figure 7 is a result of confirming the mixing ratio of a single peptide (Thymulin derivative, Thymosin α1 derivative, Thymosin β4 derivative) that exhibits excellent mammary gland cell activity according to one embodiment of the present invention.

[0095] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0096]

[0097] Manufacturing example. Manufacturing of peptides

[0098] The synthesized peptides (hybrid multimer) and target peptides (Thymulin, Thymulin derivatives, Thymosin α1, Thymosin α1 derivatives, Thymosin β4, Thymosin β4 derivatives) are shown in Table 1 below.

[0099] Peptide sequence Sequence number Name Sequence (5'-3') 1 Hybrid multimer (3:2:1) PEAKSQGGSNDPEAKSQGGSNDPEAKSQGGSNDPSDAAVDYSSEITTKDLKEKKEVVEEAENDPSDAAVDYSSEITTKDLKEKKEVVEEAENDPSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESD2 ThymulinEAKSQGGSN3 Thymulin derivative PEAKSQGGSND4 Thymosin α1 SDAAVDTSSEITTKDLKEKKEVVEEAEN5 Thymosin α1 derivative PSDAAVDTSSEITTKDLKEKKEVVEEAEND6 Thymosin β4 SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES7 Thymosin β4 DerivativePSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESD

[0100] 1) Strain, DNA cassette, vector

[0101] A DNA cassette was prepared through gene work (see Figs. 1a and 1b and Table 2 below), and a vector containing the same (see Figs. 2a to 2c, Figs. 3a to 3c, and Fig. 4) was introduced (transfected) into a P. pastoris strain to produce a transformant. At this time, a fusion partner was inserted to induce stable expression.

[0102] Cassette 서열서열(5'-3')TEF Promoter(서열번호 8)ATAACTGTCGCCTCTTTTATCTGCCGCACTGCATGAGGTGTCCCCTTAGTGGGAAAGAGTACTGAGCCAACCCTGGAGGACAGCAAGGGAAAAATACCTACAACTTGCTTCATAATGGTCGTAAAAACAATCCTTGTCGGATATAAGTGTTGTAGACTGTCCCTTATCCTCTGCGATGTTCTTCCTCTCAAAGTTTGCGATTTCTCTCTATCAGAATTGCCATCAAGAGACTCAGGACTAATTTCGCAGTCCCACACGCACTCGTACATGATTGGCTGAAATTTCCCTAAAGAATTTTTTTTTCACGAAAATTTTTTTTTTACACAAGATTTTCAGCAGATATAAAATGGAGAGCAGGACCTCCGCTGTGACTCTTCTTTTTTTTCTTTTATTCTCACTACATACATTTTAGTT ATTCGCCAACα-Factor(서열번호 9)MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTT IASIAAKEEGVSLEKRHis-tagHHHHHHFusion partner SUMO3(서열번호 10)GAGGAGAAGCCCAAGGAGGGTGTGAAGACAGAGAATGACCACATCAACCTGAAGGTGGCCGGGCAGGACGGCTCCGTGGTGCAGTTCAAGATCAAGAGGCACACGCCGCTGAGCAAGCTGATGAAGGCCTACTGCGAGAGGCAGGGCTTGTCAATGAGGCAGATCAGATTCAGGTTCGACGGGCAGCCAATCAATGAAACTGACACTCCAGCACAGCTGGAGATGGAGGACGAGGACACCATCGACGTGTTCCAGCAGCAGACGGGAGGTDGACCYC1 Terminator(서열번호11)TCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCTCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTTAATAGTTATGTT AGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGC

[0103] 2) Cultivation of strains into which thymus-derived peptide derivative expression vectors have been introduced

[0104] According to the conditions in Tables 3 and 4 below, a medium suitable for the strain for hybrid multimer production was selected, and the strain was cultured in the medium at a scale of 10 L per flask.

[0105] Flask testPromoterAOX1TEFGAPVolumeBaffled flask 30 ml / 250 mlBaffled flask 50 ml / 250 mlFed-batch0.5% Methanol, 1 ml / day2% Glucose, 1 ml / day2% Glucose, 1 ml / dayInitial OD 600 25~30Temperature30℃After methanol induction 24℃30℃Agitation247 rpmTime96 hrMedia conditionBMGYorBMMY1% Yeast extract2% Peptone2% Glucose or 0.5% Methanol100mM Potassium phosphate pH 6.0Biotin 12 ppmBMGY1% Yeast extract2% Peptone2% Glucose100mM Potassium phosphate pH 6.0Biotin 12 ppmYPDZ1% Yeast extract2% Peptone2% Glucose25 ㎍ / L Zeocin

[0106] 10 L testVolume2 LMedia condition1% Yeast extract2% Peptone2% Glycerol100 mM Potassium phosphate pH 6.0Biotin 40 ppmFed-batchGlycerol batch phaseGlycerol fed-batch phaseGlucose fed-batch phaseUntil wet cell 90~150 g / L2% Glycerol+PTM1, 25 ㎖ / hruntil wet cell 150~220 g / L4 % Glucose+PTM13.6 ㎖ / hr / L 4hr7.3 ㎖ / hr / L 2hr10.9 ㎖ / hr / L 66hrTemperature30℃28℃24℃Time24 hr24 hr72 hrpH6.0Agitation600~800 rpmAeration1~2 vvm

[0107] PTM1, pichiaTrace Metrals

[0108] 3) Expression of recombinant strains

[0109] A single colony of the strain was dissolved in 10 ml of BMGY in a 125 ml flask and cultured in a shaking incubator set at 30°C and 250-300 rpm for 1-2 days. The culture was then transferred to a 50 ml tube and centrifuged (1,500 g, 5 min). The pellet was resuspended in 1 ml of BMMY and cultured overnight in a shaking incubator at 30°C. The following day, 100 μl of 40% methanol was added to a 100 μl sample and cultured overnight in a shaking incubator at 300 rpm. The supernatant and pellet were separated by centrifugation (1,500 g, 10 min) the next day and stored at -80°C. Subsequently, they were analyzed by SDS-PAGE.

[0110]

[0111] Experimental Example 1. Evaluation of mammary papilla cell activity

[0112] In addition to the peptides manufactured in the above manufacturing example, the activity of hair follicle cells was evaluated according to the treatment of peptide samples of three types (Thymulin, Thymulin derivative, Thymosin α1, Thymosin α1 derivative, Thymosin β4, Thymosin β4 derivative).

[0113] 1-1. Cell culture (based on 75 T flask)

[0114] Dermal papilla cells were removed from the LN2 tank and thawed in a water bath for 1–2 minutes until the solid particles were 50% and the liquid particles were 50%. 3 ml of Expansion Media was added to 1 ml of the thawed cells, transferred to a 15 ml conical tube, and centrifuged at 2,500 rpm for 5 minutes. After aspirating the supernatant, 1 ml of Expansion Media was injected and pipetted thoroughly. After pipetting 10 μl of suspended cells and 10 μl of trypan blue, the cells were counted using the following method:

[0115] - Use of Hemocytometer C-Chip

[0116] - Calculate the average value by counting each cell

[0117] After cell counting, the flasks were transferred to 75 T flasks, 13 ml of Expansion Media was added, and the cells were placed in a CO2 incubator and monitored under a microscope. Cells were subcultured when they grew to more than 80% of the flask bottom. During subculture, the walls were washed twice with 10 ml of PBS. 1 ml of trypsin was applied to the entire bottom, incubated for 3 minutes, and the bottom was gently tapped to detach the cells. 3 ml of inactivation media was added, and the cells attached to the bottom were collected and pipetted. The cells and media in the T flask were transferred to a 15 ml conical tube and centrifuged at 2,500 rpm for 5 minutes. Only the media, excluding the cells, was suctioned off, and 10 ml of media was added to the cell pellet and pipetted. After pipetting 10 μl of suspended cells and 10 μl of trypan blue, the cells were counted. After injecting 13 ml of expansion media into a 75 T flask, the suspended cells were mixed and cultured.

[0118]

[0119] 1-2. CCK assay

[0120] 5,000 cells / 100 ㎕ of dermal papilla cells were dispensed into a 96-well plate and cultured for 24 hours. (DMEM + 10% FBS + 1% P / S) After washing twice with DMEM, each well was treated with peptides at various concentrations. After the culture time (1 day to 4 days), all media was removed, and 100 ㎕ of CCK-8 diluted in DMEM to 10% was added to each well. After 2 hours of culture, the absorbance was measured at 450 nm to obtain the results.

[0121] As a result, as can be seen in Figures 6a to 6c, it was confirmed that the derivative peptide showed 13 to 27% higher breast papilla cell growth activity than the existing peptide.

[0122]

[0123] Experimental Example 2. Confirmation of the Optimal Mixing Ratio

[0124] In addition, in order to confirm the synergistic effect between each peptide and to produce a high-efficiency production strain, efficacy analysis (CCK assay) was performed by varying the mixing ratio of three types of Thymulin, Thymosin α1, and Thymosin β at a concentration of 100 pg / ml, where significant dermal papilla cell activation efficacy was confirmed when each peptide was treated alone.

[0125] As a result, it was confirmed that the three types of mixed peptides showed a synergistic effect of about 2.5 times compared to a single peptide. On the first day of treatment, the growth effect of hair papilla cells was confirmed to be more than 120% at all ratios. In particular, the highest effect was shown when the ratio of Thymulin: Thymosin α1: Thymosin β4 was 3:2:1.

Claims

1. Containing a polynucleotide sequence encoding a thymus-derived peptide derivative, The above polynucleotide sequence is operably linked to a promoter sequence capable of being expressed in yeast. Expression cassette for the production of thymus-derived peptide derivatives.

2. In paragraph 1, An expression cassette for producing a thymus-derived peptide derivative, wherein the polynucleotide sequence encoding the thymus-derived peptide derivative is a polynucleotide sequence represented by SEQ ID NO:

1.

3. An expression vector for producing a thymus-derived peptide derivative, comprising the expression cassette of claim 1 or 2.

4. A transformant comprising the expression vector of clause 3.

5. In paragraph 4, A transformant, wherein the transformant is selected from the group consisting of strains of the genera Pichia, Escherichia, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Schizosaccharomyces, Issachenkia, Yarrowia and Hansenula.

6. A pharmaceutical composition for preventing or treating hair loss comprising a thymus-derived peptide derivative represented by sequence number 1.

7. A cosmetic composition for preventing hair loss or promoting hair growth, comprising a thymus-derived peptide derivative represented by sequence number 1.

8. A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising a thymus-derived peptide derivative represented by sequence number 1.

9. A health functional food composition for preventing hair loss or promoting hair growth, comprising a thymus-derived peptide derivative represented by sequence number 1.

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