KR102083978B1 - Cosmetic composition for skin care comprising fusion protein with PDGFa - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin comprising a fusion protein of platelet-derived growth factor a subunit (PDGFa), and more specifically, the present invention relates to a fusion protein in which PDGFa is coupled to a peptide for promoting skin permeation, the fusion protein. The present invention relates to a cosmetic composition for improving skin, a cosmetic composition for improving hair loss, and a quasi-drug composition comprising the fusion protein.

Description

Cosmetic composition for skin improvement comprising platelet-derived growth factor a subunit fusion protein {Cosmetic composition for skin care comprising fusion protein with PDGFa}

The present invention relates to a cosmetic composition for improving skin comprising a fusion protein of platelet-derived growth factor a subunit (PDGFa), and more specifically, the present invention relates to a fusion protein in which PDGFa is coupled to a peptide for promoting skin permeation, the fusion protein. The present invention relates to a cosmetic composition for improving skin, a cosmetic composition for improving hair loss, and a quasi-drug composition comprising the fusion protein.

Drugs that pass through the skin are used in pain relief patches, smoking cessation patches, pregnancy control agents, etc. due to the ease of use, and has the purpose of delivering to the systemic circulation through the skin. In addition, atopic dermatitis, whitening, wrinkles cosmetics, such as the purpose of delivering to the skin itself. Despite the goal of convenience and functionality, there is a difficulty in delivering drugs due to the structure of the skin, which can be understood by looking at the structure of the skin. The stratum corneum of the skin, consisting of about 10 to 15 layers of corneocytes, has a brick structure consisting of keratin-rich keratinocytes, and a ceramide, fatty acid, or fatty acid between these keratinocytes. It consists of a mortar structure filled with lipids such as wax, and forms a thickness of about 10 to 45 um. This structure prevents the loss of moisture inside and prevents intrusion from the outside. Thus, faithfully fulfilling its role, material permeability is very low, and only the low molecular structure components of 500 Da or less are delivered into the skin by the diffusion method (Exp. Dermatol., 2000, 9, 165-9.). This is achieved through the intracellular lipid layer of the mortar structure or the water-soluble structure between the lipid layers, and the substance permeability greatly depends on the nature of the drug (Current Drug Delivery, 2005, 2, 23-3). In addition to the direct way through the skin surface is also delivered using the structure of the skin glands, pores, sebaceous glands.

For this reason, studies have been actively conducted to develop a method capable of penetrating the skin regardless of the size and nature of the molecule and evenly transmitting the whole skin.

For example, US Pat. No. 7,659,252 discloses a skin permeable peptide that can be used for the treatment of skin diseases and for promoting skin permeation of a pharmaceutically active agent. The peptide has the advantage that it can be used as a carrier for transdermal delivery of other drugs as well as showing excellent skin permeability, but after penetrating the skin is consumed through the circulatory system of the body, in the case of drugs targeting the skin There was a drawback that it does not work much.

These drawbacks are known to be due to the fact that a variety of active ingredients that promote wrinkles by promoting collagen synthesis, endothelial cell growth or hyaluronic acid production are not normally absorbed through the skin, thereby promoting the absorption of the active ingredients. The research was actively conducted. For example, Korean Patent No. 1054519 discloses a human growth hormone-derived peptide having excellent stability and skin permeability compared to natural human growth hormones and a composition comprising the same, and Korean Patent No. 1104223 discloses human IL. Although IL-10-derived peptides and compositions containing the same have been disclosed, which have the same function as the function of -10 and are excellent in stability and skin permeability as compared to natural IL-10, these peptides only exhibit functionality by themselves, There was a disadvantage that it could not be used as a carrier for delivery of other drugs. This drawback was analyzed to imply that the superior skin permeability alone cannot be used for drug delivery. Therefore, the development of a new material that includes two properties that not only have excellent skin permeability but also improves skin retention is required. Until now, very little research has been reported.

Under these circumstances, the present inventors have diligently researched to develop a new material including two properties not only excellent in skin permeability but also excellent in skin retention, and thus can be used as a carrier for transdermal delivery of a drug and remain on the skin for a long time. The present invention has developed a peptide for promoting skin permeation, and confirmed that the fusion protein fused with PDGFa to the peptide for promoting skin permeation exhibited not only excellent skin permeability but also exhibited properties for improving skin retention.

An object of the present invention is to provide a fusion protein in which the peptide for promoting skin permeation composed of the amino acid sequence of SEQ ID NO: 1 is bound to platelet-derived growth factor a subunit (PDGFa).

Another object of the present invention is to provide a polynucleotide encoding the fusion protein.

Another object of the present invention is to provide a cosmetic composition for skin improvement comprising the fusion protein as an active ingredient.

Another object of the present invention to provide a cosmetic composition for improving hair loss comprising the fusion protein as an active ingredient.

Another object of the present invention to provide a quasi-drug composition for skin improvement comprising the fusion protein as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for improving hair loss comprising the fusion protein as an active ingredient.

As one aspect for achieving the object of the present invention, a skin permeation promoting peptide consisting of the amino acid sequence of SEQ ID NO: 1 provides a fusion protein coupled to platelet-derived growth factor a subunit (PDGFa).

The amino acid sequence of SEQ ID NO: 1 used in the present invention is abbreviated as follows according to the IUPAC-IUB nomenclature.

Peptides for Skin Permeation: NGSLNTHLAPIL (SEQ ID NO: 1)

Specifically, Asparagine Asn N-Glycine Gly G- Serine Ser S- Leucine Leu L- Asparagine Asn N- Threonine Thr T- Histidine His H-Leucine Leu L- Alanine Ala A- Proline Pro P- Isoleucine Ile I- Leucine Leu L

The platelet-derived growth factor a subunit (PDGFa) bound to the peptide is permeable to the skin and may be used as having skin persistence.

In the present invention, the term "skin permeation promoting peptide" refers to a peptide that can penetrate the skin regardless of the size and nature of the molecule, can be evenly delivered to the entire skin, and has excellent skin permeability and skin persistence.

In the present invention, "skin permeability" means the ability or property of the peptide to penetrate the skin and penetrate into the skin. The peptide for promoting skin permeation of the present invention exhibits significantly superior skin permeability than other peptides. .

In the present invention, "skin persistence" means the ability of peptides that penetrate the skin to remain in the skin by binding to tissues in the skin without passing through the skin tissue to the circulation. In the case of pharmaceutical preparations or cosmetics which target skin tissues, it is preferable to use a carrier having excellent properties of remaining in the skin tissues so that the components bound to the peptides can act on the skin tissues or skin cells for a long time. Since the peptide for promoting skin permeation of the present invention is remarkably excellent in skin permeability as well as skin permeability, it can be used as a carrier for pharmaceutical preparations or cosmetics.

Peptides for promoting skin permeation of the present invention may include a peptide excellent in skin permeability and skin residuals discovered by performing a phage display method combining the phage library and the dissolution test method of transdermal agent, specifically SEQ ID NO: 1 It may include a peptide comprising the amino acid sequence of. In one embodiment of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1 was prepared as a peptide for promoting skin permeation through a phage display method (Example 1).

In the present invention, "platelet-derived growth factor (PDGF)" is a low-molecular-weight basic protein consisting of two peptide chains, proliferation of mesenchymal-derived cells such as smooth muscle cells, fibroblasts and blood vessel walls It means to promote.

“Platelet-derived growth factor a subunit (PDGFa)” is one of the proteins belonging to the platelet-derived growth factor (PDGFa) family, and refers to a protein contained in platelets in the blood and having a size of about 18 kDa. PDGF present in platelets consists of a subunit b having a size of about 14 kDa and the subunit a. These disulfide bonds form a homodimer, PDGF-AA or PDGF-BB, or a heterodimer, PDGF-AB. It is known to form.

In the present invention, the amino acid sequence of PDGFa is not particularly limited as long as PDGFa exhibits an effect of promoting the regeneration of damaged skin, hair regeneration and growth through the effect of enhancing collagen production or elastin production, and the like. An amino acid sequence may be used, a mutated amino acid sequence thereof may be used, and some fragments thereof may be used. Specific amino acid sequence of PDGFa or nucleotide sequence information of the gene encoding the same can be obtained from a known database such as GenBank of NCBI. The PDGFa may specifically be a peptide represented by the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

In the present invention, the "fusion protein" is a peptide artificially synthesized to bind the skin penetration promoting peptide to another protein or peptide, and specifically, may include the skin penetration promoting peptide and the PDGFa. More specifically, the skin penetration promoting peptide may be a peptide including the amino acid sequence of SEQ ID NO: 1, the PDGFa may be a peptide consisting of SEQ ID NO: 2, but is not limited thereto.

The fusion protein may include a peptide having a sequence in which at least one amino acid residue differs from the wild type amino acid sequence of each domain included therein. Amino acid exchange in peptides that do not alter the activity of the molecule as a whole is known in the art. The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity against heat, pH, etc., due to variation or modification on the amino acid sequence.

The fusion protein or the protein constituting the fusion protein may be prepared by a chemical protein synthesis method known in the art, or the gene encoding the fusion protein may be amplified by PCR (polymerase chain reaction) or synthesized by a known method. It can be prepared by cloning the expression vector (cloning).

The fusion protein of the present invention may include a linker peptide between the skin penetration promoting peptide and the PDGFa. In detail, the fusion protein may be directly linked to the N-terminus of the PDGFa or the skin penetration promoting peptide, or may be fusion-linked through a linker.

The linker may be specifically linked using amino acids such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, arginine acid, and more specifically. By using a plurality of amino acids such as valine, leucine, aspartic acid, glycine, alanine, proline, and more specifically, amino acids such as glycine, valine, leucine, aspartic acid in consideration of the ease of genetic manipulation It can be used by connecting one to five. For example, the fusion protein may be prepared by connecting the C-terminus of the peptide for promoting skin permeation and the N-terminus of PDGFa through a linker composed of two peptides (GG).

Specifically, the fusion protein of the present invention may be a peptide consisting of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

In another aspect of the present invention, there is provided a polynucleotide encoding the fusion protein.

The polynucleotide is at least 70%, in particular at least 80%, more specifically at least 90%, even more specifically, a nucleotide sequence set forth in SEQ ID NO: 3, or as long as it has similar activity as the fusion protein. May include, but is not limited to, a polynucleotide encoding a protein exhibiting at least 95%, most specifically at least 99% homology. In addition, it is obvious that a polynucleotide which can be translated into a protein consisting of the nucleotide sequence of SEQ ID NO: 3 or a protein having homology thereto by codon degeneracy may also be included. Or a protein having the activity of a protein consisting of the nucleotide sequence of SEQ ID NO: 3 by hybridizing under strict conditions with a probe that can be prepared from a known gene sequence, for example, the complementary sequence to all or part of the nucleotide sequence. Any coding sequence can be included without limitation.

Specifically, the polynucleotide of the present invention may be composed of the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto.

The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are described specifically in the literature (eg, J. Sambrook et al., Homology). For example, genes having high homology, 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more homologous genes 60 ° C., 1 × SSC, 0.1% SDS, specifically 60 ° C., 0.1 × SSC, 0.1, which is a condition that hybridizes to each other and does not hybridize genes having a lower homology, or washing conditions of ordinary Southern hybridization. At a salt concentration and temperature corresponding to% SDS, more specifically, 68 ° C., 0.1 × SSC, and 0.1% SDS, the conditions of washing once, specifically, two to three times can be enumerated.

Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the invention may also include isolated nucleic acid fragments that are complementary to the entire sequence as well as substantially similar nucleic acid sequences.

Specifically, polynucleotides having homology can be detected using hybridization conditions including hybridization steps at Tm values of 55 ° C. and using the conditions described above. In addition, the Tm value may be 60 ° C, 63 ° C or 65 ° C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

Appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides and variables are well known in the art. (See Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).

 As used herein, the term “homology” refers to the percent identity between two polynucleotide or polypeptide moieties. It means the degree of coincidence with a given amino acid sequence or nucleotide sequence and can be expressed as a percentage. In this specification, homologous sequences thereof having the same or similar activity as a given amino acid sequence or base sequence are designated as "% homology". Homology between sequences from one moiety to another can be determined by known art. For example, using standard software that calculates parameters such as score, identity and similarity, in particular BLAST 2.0, or by hybridization experiments used under defined stringent conditions Appropriate hybridization conditions, which are defined within the scope of the art, are well known to those skilled in the art and are well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York.

As used herein, the term "vector" refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding said target polypeptide operably linked to a suitable regulatory sequence so as to be able to express said polypeptide of interest in a suitable host. . The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. After being transformed into a suitable host cell, the vector can be replicated or function independent of the host genome and integrated into the genome itself.

The vector to be used in the present invention is not particularly limited as long as it can replicate in a host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or cosmid vector, and pBR-based, pUC-based, pBluescriptII-based, , pGEM system, pTZ system, pCL system and pET system can be used. The vector usable in the present invention is not particularly limited and known expression vectors can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pPIC, pGAP vectors and the like can be used, and include all vectors expressed in bacteria and yeasts such as Escherichia coli, lactic acid bacteria, Bacillus bacteria, etc. can do.

For example, a polynucleotide encoding a target polypeptide in a chromosome can be replaced with a mutated polynucleotide through an intracellular chromosome insertion vector. Insertion of the polynucleotide into the chromosome can be made by any method known in the art, such as, but not limited to, homologous recombination.

As used herein, the term "transformation" means introducing a vector comprising a polynucleotide encoding a target polypeptide into a host cell so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotides may include all of them, as long as they can be expressed in the host cell, either inserted into the chromosome of the host cell or located outside the chromosome. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate -DMSO method, but is not limited thereto. The polynucleotide also includes DNA and RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosomal binding site, and a translation termination signal, which are typically operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked with a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" means that the gene sequence is functionally linked with a promoter sequence for initiating and mediating the transcription of a polynucleotide encoding a polypeptide of the present invention.

As another aspect of the present invention, there is provided a cosmetic composition for skin improvement comprising the fusion protein as an active ingredient.

In addition, specifically, it may provide a cosmetic composition for improving skin wrinkles or skin elasticity comprising the fusion protein of the present invention as an active ingredient.

In the present invention, "enhance skin elasticity" or "improve skin wrinkles" is to alleviate the extent to which the skin sags or sags, inhibits or inhibits the formation of wrinkles on the skin, or alleviates wrinkles already formed. As the amount of collagen or hyaluronic acid in the intercellular intercellular intercalation of dermal epidermis and the connective tissue of dermis increases, skin elasticity is maintained and thus wrinkle formation is alleviated.

The term "collagen" is a collection of more than a thousand amino acid molecules, it refers to a protein with a high content of hydroproline. The collagen fibers, twisted with three collagen molecules in a triple helix form, keep the skin firm and elastic. In addition, collagen is a major protein of all connective tissues such as skin, blood vessels, bones, teeth, muscles, and collagen is known to be involved in skin elasticity.

The term "hyaluronic acid" is a type of glycosaminoglycans and is a chain-like polymer polysaccharide material in which glucuronic acid and N-acetylglucosamine residues are repeatedly connected. It has high viscosity and elasticity because it combines with a large amount of water to make gel. In addition, hyaluronic acid is a major component of the extracellular matrix and is known to be involved in water retention, intercellular spacing, storage and diffusion of cell growth factors and nutrients, as well as cell division, differentiation and migration. have.

According to a specific embodiment, in order to confirm the effects of skin elasticity and skin wrinkle improvement of the present invention, the effect of the fusion protein on the production of collagen and hyaluronic acid was confirmed. As a result of culturing PDGFa and fusion protein (T-PDGFa) in human-derived dermal fibroblasts, it was confirmed that T-PDGFa also had the same level of collagen and hyaluronic acid production as that of PDGFa (Table 1 and 2).

Through this, it can be seen that even if the peptide for promoting the skin permeation to PDGFa has the skin wrinkle and elasticity improving effect of PDGFa as it is.

In addition, in the embodiment of the present invention by combining the peptide for promoting skin permeation including the amino acid sequence of SEQ ID NO: 1 to the PDGFa to prepare a fusion protein (SEQ ID NO: 3), the effect of the prepared fusion protein conventional PDGFa As a result, it was confirmed that the skin permeability and skin residual property is significantly improved and the wrinkle improvement effect is also excellent (Tables 4 and 5).

Therefore, the fusion protein provided in the present invention is coupled to the PDGFa peptide for promoting skin penetration, while maintaining the efficacy of PDGFa itself to increase the regeneration of damaged skin and hair, while significantly improving skin permeability and skin residuals It can be seen that it can be usefully used as an active ingredient of cosmetic compositions and quasi-drug compositions.

Fusion protein of the present invention may be included in 0.0001 to 50% by weight relative to the total weight of the cosmetic composition. When the content of the fusion protein is less than 0.0001% by weight relative to the total weight of the cosmetic composition, it is difficult to expect a substantial skin improvement effect, and when it is 50% by weight or more, problems such as unstable formulation may occur.

The cosmetic composition according to the present invention is a solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, latex, makeup base, essence, soap, liquid cleanser, bath, sunscreen cream, sun oil, suspension, Emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays can be prepared in a formulation selected from the group consisting of, but not limited to Do not.

In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as conventional components, for example, oil, water, surfactants, moisturizers, lower alcohols, Thickeners, chelating agents, colorants, preservatives, flavoring agents, and the like may be appropriately blended, but are not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the dosage form.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these may be used.

If the formulation of the invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or mixtures thereof may be used, in particular in the case of a spray additionally chloro Propellants such as fluorohydrocarbons, propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, solvents, solubilizers or emulsions are used as carrier components, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil may be used, in particular cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used. have.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, micro Crystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars and the like may be used as carrier components. Can be.

As another aspect of the present invention, there is provided a cosmetic composition for improving hair loss comprising the fusion protein as an active ingredient.

As used herein, the term "hair loss improvement" is a site where hair should normally exist due to various habits and environmental influences such as genetic causes, hormonal imbalances, social stress, exposure to air pollution, and ingestion of processed foods. Improves the absence of hair in the hair, prevents the progression of hair loss and hair.

According to a specific embodiment, the result of culturing the dermal papilla cells treated with the fusion protein of the present invention and quantitatively analyzing the difference in cell proliferation amount using Cell Counter Kit-8 (Dojindo), Compared to T-PDGFa also has the same level of cell proliferation efficacy as PDGFa (Table 3).

Through this, it can be seen that even if the peptide for promoting skin permeation is fused to PDGFa, the hair loss improvement effect of PDGFa remains intact.

The cosmetic composition is a hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisturizing cream, hair massage cream, hair wax, hair aerosol , Hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservative, hair colorant, hair wave agent, hair bleach, hair gel, hair glaze, hair dresser, hair lacquer, hair moisturizer, It may be included in the formulation of hair mousse or hair spray, but is not limited thereto.

Specifically, the compositions of the present invention can be used by methods such as direct application or scattering on hair or scalp. Hair to which the composition of the present invention is applied includes hair follicles and hair follicles of the head, hair and eyelashes, and eyelashes, beards, armpits, pubic hair, and all parts of the hair root and hair follicles throughout the body.

As another aspect of the present invention, there is provided a quasi-drug composition for skin improvement comprising the fusion protein as an active ingredient.

In addition, in particular, it may provide a quasi-drug composition for improving skin wrinkles or skin elasticity comprising the fusion protein of the present invention as an active ingredient.

As another aspect of the present invention, there is provided a quasi-drug composition for hair loss improvement comprising the fusion protein as an active ingredient.

The term fusion protein, skin wrinkle improvement, skin elasticity improvement and hair loss improvement are as described above.

The quasi-drug composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent as necessary in addition to the above components. The pharmaceutically acceptable carriers, excipients or diluents are not limited so long as they do not impair the effects of the present invention and include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, and the like. It may include.

Representative examples of pharmaceutically acceptable carriers, excipients or diluents of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia rubber, alginate, calcium phosphate, calcium Carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil , Injectable esters, witepsol, macrogol, tween 61, cacao butter, lauridge and the like.

In addition, when using a composition containing the fusion protein of the present invention as an active ingredient, it may further contain one or more active ingredients exhibiting the same or similar functions. For example, it may include skin damage protection, elasticity enhancement, wrinkle improvement and moisturizing ingredients. When the ingredient is added, skin safety, ease of formulation, and stability of the active ingredients may be considered. The quasi-drug composition is a whitening component known in the art, a substance that inhibits tyrosinase enzyme activity, such as kojic acid (Kojic acid), arbutin (Arbutin), hydroquinone (Hydroquinone), vitamin-C (L-Ascorbic acid); As skin elasticity, wrinkle improvement or moisturizing ingredients known in the art, retinoic acid, TGF, protein from animal placenta, betulinic acid and chlorella extracts; And one or two or more components selected from the group consisting of derivatives and various plant extracts thereof may be further included. Additional components may be included in 0.0001% to 5% by weight relative to the total composition weight, the content range may be adjusted according to requirements such as skin safety, ease.

The quasi-drug composition of the present invention may be exemplified by disinfecting detergents, shower foams, ointments, wet tissues, coatings, etc., but is not limited thereto. Formulation methods, dosages, methods of use, components, etc. of the quasi-drugs are well known in the art. It may be appropriately selected from the description of.

In addition, the quasi-drug composition comprising the fusion protein of the present invention as an active ingredient may be used for enhancing skin elasticity, improving skin wrinkles, or improving hair loss, including applying to the skin of an individual. The subject includes, without limitation, mammals including rats, livestock, humans and the like.

The fusion protein of the present invention is combined with a peptide for promoting skin penetration to PDGFa, while maintaining or improving the ability of the peptide to exhibit useful effects such as wrinkle improvement, and at the same time significantly improves skin permeability and skin residue, thereby improving skin cosmetics. It may be widely used as an active ingredient of a composition, a cosmetic composition for improving hair loss, a quasi-drug composition for improving skin, or a quasi-drug composition for improving hair loss.

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

Example  1: for promoting skin penetration Peptide  Selection

In order to select a peptide for promoting skin permeation, a phage display method combining a phage library and a dissolution test method of a transdermal agent was performed.

First, 109 phages derived from 500 μL Ph.D-12 Phage Library Kit (New England Biolab) solution of TBS (50 mM Tris pH 7.5, 150 mM NaCl) solution containing 1% BSA were added to obtain a phage solution.

Next, a pig skin (0.7 mm thick, Medikinetics) was mounted between the top and bottom of a Franz glass cell (Standard diameter 9 mm, Receiver 5 mL, Permgear), and the phage solution was added to the top and reacted for 16 hours. Phage that penetrated the porcine skin to reach a receiver at the bottom was obtained and amplified.

The amplification was performed using E. coli ER2738 (New England Biolab) as host cells. Specifically, 5 mL of the phage solution was added to E. coli ER2738 strain shaken in 25 mL of LB medium and cultured for 4 hours. Subsequently, the culture solution was centrifuged at 8,000 G to obtain a supernatant containing a phage fraction. The supernatant was treated with 6 mL of precipitate (20% PEG6000, 2.5 M NaCl) to react and precipitated phage, the reaction solution was centrifuged at 8,000 G to precipitate the phage, and the precipitate was suspended in TBS solution. To obtain an amplified phage solution.

In this way, it was defined as 1 Round to obtain a phage that penetrated the skin by applying the phage to the pig skin, and to amplify it once, and proceeds again with 2 rounds with amplified phage in 1 Round to improve skin permeability Phages with good skin permeability were selected in a competitive manner and a total of 3 rounds were performed.

In order to confirm the sequence of the peptide contained in the phage finally obtained as a result of the three rounds, the TBS solution containing the phage was added to the E. coli ER2738 strain, suspended and mixed by adding TOP agar to the suspension, and then the mixture was mixed. Solidified on top of LB / X-gal / IPTG plate media. Then, the solidified medium was incubated for 16 hours, and blue colonies were selected. The strains derived from the selected colonies were incubated for 6 hours, DNA was obtained therefrom, and the nucleotide sequences derived from phage were analyzed to obtain a skin permeation promoting peptide (SEQ ID NO: 1) that exhibits characteristics that penetrate porcine skin. Screened.

Example  2: Platelet derived  Growth factor a to the subunit (PDGFa)  For skin permeation Pepty Preparation of De-linked Fusion Proteins

The C-terminus of the skin penetration promoting peptide having the amino acid sequence of SEQ ID NO: 1 obtained in Example 1 and the N-terminus of platelet-derived growth factor a subunit (PDGFa) having the amino acid sequence of SEQ ID NO: 2 are two T-PDGFa (SEQ ID NO: 3), a fusion protein in the form of a linker composed of amino acids (GG), was produced.

Specifically, the polynucleotides encoding the amino acid sequence of SEQ ID NO: 1 and the polynucleotides encoding the amino acid sequence of SEQ ID NO: 2 are synthesized, respectively, and linked by nucleotide sequences encoding two amino acids (GG). A polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 was prepared. The obtained polynucleotide was introduced into a pPIC expression vector to prepare an expression vector.

The produced expression vector was introduced into Pichia pastoris to obtain a transformant, the obtained transformant was cultured, and the culture solution was filtered to recover a fusion protein composed of peptide-VEGF for promoting skin permeation. By applying to GPC column chromatography, T-PDGFa (SEQ ID NO: 3), which is finally a fusion protein composed of peptide-PDGFa for promoting skin permeation, was produced.

Example  3: Validation of fusion proteins

In order to confirm the skin improvement use of the T-PDGFa prepared in Example 2, experiments were performed to confirm the skin elasticity improvement, skin wrinkle improvement, hair loss improvement, skin permeability and skin residual effect.

Example  3-1: Collagen Production Efficacy Assay

The T-PDGFa synthesized in Example 2 was compared with PDGFa to verify the effect on collagen production.

Specifically, dermal fibroblasts were seeded in 24-well plates and incubated for 24 hours to obtain cultures exhibiting a saturation of 70-80%. The cultures were washed with PBS and incubated for 2 days in serum-free DMEM medium containing 10 ng / ml T-PDGFa or PDGFa. The supernatant of the culture was obtained, and the content of collagen produced and secreted into the culture was quantitatively analyzed using an ELISA kit (R & D Systems, Inc.) (Table 1).

Effect of Fusion Proteins on Collagen Production Treated Protein Generation level (%) Control 100 ± 3.7 PDGFa 264 ± 17 T-PDGFa 255 ± 15

As shown in Table 1, it was confirmed that T-PDGFa fused with PDGFa-derived peptide for skin permeation also had collagen production efficiency equivalent to that of PDGFa.

Through this, it can be seen that even if the peptide for promoting skin penetration to PDGFa has the skin wrinkle and elasticity improving effect of PDGFa as it is.

Experimental Example  3-2: Hyaluronic Acid (HA) Production Efficacy Assay

To compare the T-PDGFa synthesized in Example 2 with PDGFa to verify the effect on the production of hyaluronic acid (HA).

Specifically, dermal fibroblasts were seeded in 24-well plates and incubated for 24 hours to obtain cultures exhibiting a saturation of 70-80%. The cultures were washed with PBS and incubated for 2 days in serum-free DMEM medium containing 10 ng / ml T-PDGFa or PDGFa. The supernatant of the culture was obtained and the content of hyaluronic acid produced and secreted into the culture was quantitatively analyzed using an ELISA kit (R & D Systems, Inc.) (Table 2).

Effect of Fusion Proteins on Hyaluronic Acid (HA) Production Treated Protein Generation level (%) Control 100 ± 4.9 PDGFa 897 ± 30 T-PDGFa 920 ± 29

As shown in Table 2, it was confirmed that T-PDGFa fused with PDGFa-derived peptide for skin permeation also has the same level of hyaluronic acid production as PDGFa.

Through this, it can be seen that even if the peptide for promoting the skin permeation to PDGFa has the skin wrinkle and elasticity improving effect of PDGFa as it is.

Example  3-3: Hair follicle stem cell proliferation  Efficacy Verification

The T-PDGFa synthesized in Example 2 was compared with PDGFa to verify the effect on skin cell proliferation.

Specifically, dermal papilla cells were seeded in 96-well plates and incubated for 24 hours to obtain a culture showing 6,000 cells per well. The culture was incubated in serum-free DMEM medium for 1 day after washing with PBS. After washing with PBS again, it was incubated for 1 day in serum-free DMEM medium containing 1ng / ml T-PDGFa or PDGFa. Cultures were obtained and quantitatively analyzed for differences in cell proliferation using Cell Counter Kit-8 (Dojindo). At this time, as a control, the result of culturing in serum-free DMEM medium containing no T-PDGFa or PDGFa was used (Table 3).

Effect of Fusion Proteins on Hair Follicle Stem Cell Proliferation Treated Protein Proliferation level (%) Control 100 ± 11 PDGFa 1030 ± 19 T-PDGFa 1016 ± 7

As shown in Table 3, it was confirmed that T-PDGFa fused with PDGFa-derived peptide for skin permeation also had the same level of cell proliferation effect as PDGFa.

Through this, it can be seen that even if the peptide for promoting skin permeation is fused to PDGFa, the hair loss improvement effect of PDGFa remains intact.

Example  3-4: Skin Permeability Verification

The fusion protein T-PDGFa synthesized in Example 2 was compared with PDGFa to verify skin permeability.

Specifically, pig skin (0.7 mm thick, Medikinetics) is mounted between the top and bottom of the Franz glass cell (Standard diameter 9mm, Receiver 5ml, Permegear), TBS (50mM Tris) containing 1% BSA and 0.01% Tween 20 pH 7.5, 150mM NaCl) was prepared, and then 500 µl of TBS was added to the top of the glass cell, and 5 ml of TBS was added to the bottom of the glass cell. Subsequently, 2 μg of PDGFa or T-PDGFa was added at the top and reacted for 16 hours, and then the concentration of PDGFa or T-PDGFa at the bottom was quantitatively analyzed using an ELISA kit (R & D Systems). The relative content of the amount of T-PDGFa to the content was calculated as the permeation amount (Table 4).

Skin Permeability of Fusion Proteins Treated Protein Permeation amount (%) PDGFa 100 ± 29 T-PDGFa 360 ± 45

As shown in Table 4, in the case of T-PDGFa it was confirmed that the skin permeability increased about three times than PDGFa.

Through this, it can be seen that the skin permeability is significantly increased by using the fusion protein of the present invention.

Example  3-5: Residual Skin Verification

The fusion protein T-PDGFa synthesized in Example 2 was compared with PDGFa to verify skin persistence.

Specifically, after recovering the remaining pig skin after the experiment in Example 3-4, liquid nitrogen frozen and pulverized, the content of PDGFa or T-PDGFa contained therein was quantitatively analyzed using an ELISA kit (R & D Systems). . In addition, the relative content of the amount of T-PDGFa to the content of PDGFa was calculated as the residual amount (Table 5).

Dermal Residuals of Fusion Proteins Treated Protein Residual amount (%) PDGFa 100 ± 20 T-PDGFa 9,500 ± 630

As shown in Table 5, in the case of T-PDGFa it was confirmed that the skin residual property increased about 100 times than PDGFa.

Through this, it can be seen that the use of the fusion protein of the present invention significantly increases skin retention.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

<110> LG HOUSEHOLD & HEALTH CARE LTD. <120> Cosmetic composition for skin care comprising fusion protein with          PDGFa <130> KPA170149-KR <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptides for skin permeation promotion <400> 1 Asn Gly Ser Leu Asn Thr His Leu Ala Pro Ile Leu   1 5 10 <210> 2 <211> 125 <212> PRT <213> Unknown <220> <223> Human PDGFa <400> 2 Ser Ile Glu Glu Ala Val Pro Ala Val Cys Lys Thr Arg Thr Val Ile   1 5 10 15 Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro Thr Ser Ala Asn Phe Leu              20 25 30 Ile Trp Pro Pro Cys Val Glu Val Lys Arg Cys Thr Gly Cys Cys Asn          35 40 45 Thr Ser Ser Val Lys Cys Gln Pro Ser Arg Val His His Arg Ser Val      50 55 60 Lys Val Ala Lys Val Glu Tyr Val Arg Lys Lys Pro Lys Leu Lys Glu  65 70 75 80 Val Gln Val Arg Leu Glu Glu His Leu Glu Cys Ala Cys Ala Thr Thr                  85 90 95 Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp Thr Gly Arg Pro Arg Glu             100 105 110 Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu Lys Pro Thr         115 120 125 <210> 3 <211> 139 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein of T-PDGFa <400> 3 Asn Gly Ser Leu Asn Thr His Leu Ala Pro Ile Leu Gly Gly Ser Ile   1 5 10 15 Glu Glu Ala Val Pro Ala Val Cys Lys Thr Arg Thr Val Ile Tyr Glu              20 25 30 Ile Pro Arg Ser Gln Val Asp Pro Thr Ser Ala Asn Phe Leu Ile Trp          35 40 45 Pro Pro Cys Val Glu Val Lys Arg Cys Thr Gly Cys Cys Asn Thr Ser      50 55 60 Ser Val Lys Cys Gln Pro Ser Arg Val His His Arg Ser Val Lys Val  65 70 75 80 Ala Lys Val Glu Tyr Val Arg Lys Lys Pro Lys Leu Lys Glu Val Gln                  85 90 95 Val Arg Leu Glu Glu His Leu Glu Cys Ala Cys Ala Thr Thr Ser Leu             100 105 110 Asn Pro Asp Tyr Arg Glu Glu Asp Thr Gly Arg Pro Arg Glu Ser Gly         115 120 125 Lys Lys Arg Lys Arg Lys Arg Leu Lys Pro Thr     130 135 <210> 4 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> T-PDGFa <400> 4 aacggctccc ttaataccca tttggctcca atattgggag gttcaatcga agaagctgtc 60 cctgcagttt gcaagacacg tactgtaatt tatgagattc ctcgaagtca ggttgatccg 120 acctctgcaa attttttaat ttggccccca tgcgttgaag tgaaaagatg tactggttgt 180 tgcaatacat ccagcgtgaa gtgtcaacca tcgcgtgttc atcacaggtc tgttaaggta 240 gccaaagttg aatacgtcag aaaaaagcca aaactgaagg aagtccaagt tagacttgaa 300 gaacacttag agtgtgcttg tgccactact tcattgaacc ctgattacag agaggaagac 360 acgggaagac caagagagtc tggtaaaaaa agaaagagga aaagactaaa acctacataa 420                                                                          420

Claims (11)

As a fusion protein in which the peptide for promoting skin permeation composed of the amino acid sequence of SEQ ID NO: 1 is bound to platelet-derived growth factor a subunit (PDGFa),
The fusion protein is composed of the amino acid sequence of SEQ ID NO: 3, and the skin permeability and skin persistence of PDGFa, fusion protein.
The method of claim 1,
The skin penetration promoting peptide is a fusion protein that is bound to the N-terminus of PDGFa.
The fusion protein according to claim 1, wherein the PDGFa is composed of the amino acid sequence of SEQ ID NO.
delete delete A polynucleotide encoding the fusion protein of any one of claims 1 to 3.
A cosmetic composition for improving skin wrinkles or enhancing skin elasticity, comprising the fusion protein of claim 1 as an active ingredient.
delete A cosmetic composition for improving hair loss comprising the fusion protein of any one of claims 1 to 3 as an active ingredient.
A quasi-drug composition for improving skin wrinkles or enhancing skin elasticity, comprising the fusion protein of any one of claims 1 to 3 as an active ingredient.
A quasi-drug composition for hair loss improvement comprising the fusion protein of any one of claims 1 to 3 as an active ingredient.