KR20190073146A - Cosmetic composition for skin care comprising fusion protein with VEGF - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin care comprising a fusion protein with vascular endothelial growth factor (VEGF). More specifically, the present invention relates to a fusion protein in which VEGF is bound to a peptide for promoting skin permeation, a cosmetic composition for skin care comprising the fusion protein, a cosmetic composition for hair loss prevention, and a quasi-drug composition comprising the fusion protein.

Description

[0001] The present invention relates to a cosmetic composition for skin care comprising a fusion protein of vascular endothelial cell growth factor (VEGF)

The present invention relates to a cosmetic composition for improving skin comprising a fusion protein of vascular endothelial growth factor (VEGF). More specifically, the present invention relates to a skin permeation enhancing peptide comprising a fusion protein in which VEGF is bound, A cosmetic composition for skin improvement, a cosmetic composition for improving hair loss, and a quasi-drug composition containing the fusion protein.

Drugs that pass through the skin are used in analgesic patches, smoking cessation patches, pregnancy regulators, and the like due to their ease of use and have the purpose of delivering them to the systemic circulation through the skin. At the same time, it may have the purpose of delivering it to the skin's own organs such as atopic treatments, whitening, and cosmetics for improving wrinkles. Despite the goal of having such convenience and functionality, it is difficult to deliver 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, which is composed of about 10-15 layers of corneocytes, has a brick structure composed of keratin-rich keratinocytes, and a ceramide, a fatty acid, It has a mortar structure filled with lipids such as wax and has a thickness of about 10 to 45 um. This structure prevents internal moisture loss and prevents external intrusion. Thus, the material permeability is very low in accordance with its role. Only low-molecular-weight components below 500 Da are delivered into the skin by the diffusion method (Exp. Dermatol., 2000, 9, 165-9). This is done through the water-soluble structure between the lipid layer in the cell and the lipid layer in the mortar structure, and the substance permeability is highly dependent on the nature of the drug (Current Drug Delivery, 2005, 2, 23-3). In addition to passing directly through the surface of the skin, it can also be delivered using structures such as sweat glands, pores, and sebaceous glands of the skin.

For this reason, research has been actively conducted to develop a method that can permeate the skin regardless of the size or nature of the molecule, and can evenly transmit the whole skin.

 For example, U.S. Patent No. 7,659,252 discloses skin permeable peptides that can be used to treat skin diseases and promote skin penetration of pharmaceutical active agents. The peptide not only exhibits excellent skin permeability but also can be used as a carrier for transdermal delivery of other drugs. However, since the peptide is consumed through the circulatory system in the body after permeating through the skin, in the case of a drug targeting the skin It has a disadvantage that it can not exert any significant effect.

This disadvantage is known as a cause of promoting collagen synthesis, endothelial cell growth or hyaluronic acid production, and various effective ingredients showing wrinkle-reducing effects are not normally absorbed through the skin. Therefore, a method for promoting absorption of the active ingredient has been developed Research was actively pursued. For example, Korean Patent Registration No. 1054519 discloses a human growth hormone-derived peptide and a composition containing the human growth hormone-derived peptide, which are superior in stability and skin permeability to natural human growth hormone. In Korean Patent No. 1104223, 10-derived peptides having the same functions as those of natural IL-10 and having excellent stability and skin permeability as compared with natural IL-10, and compositions comprising the same. However, these peptides are merely functional, It can not be used as a delivery carrier for other drugs. This disadvantage has been analyzed as suggesting that the property of excellent skin permeability can not be used for drug delivery. Therefore, it has been required to develop a new substance including two properties that not only have excellent skin permeability but also improve skin retention, There is no report on the research results.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a novel substance that has both excellent skin permeability and excellent skin durability. As a result, they have found that they can be used as a carrier for transdermal delivery of drugs, And a fusion protein prepared by fusing VEGF to the skin permeation promoting peptide exhibits both skin permeability and skin retention properties. The present invention has been completed based on this finding.

The object of the present invention is to provide a fusion protein in which the peptide for promoting skin permeation, which is composed of the amino acid sequence of SEQ ID NO: 1, is bound to Vascular Endothelial Growth Factor (VEGF).

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

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

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

Another object of the present invention is 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.

In one embodiment of the present invention, a peptide for promoting skin permeation, which is composed of the amino acid sequence of SEQ ID NO: 1, is conjugated to a vascular endothelial growth factor (VEGF).

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

Skin permeation promoting peptide: (SEQ ID NO: 1)

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

The peptide-associated vascular endothelial growth factor (VEGF) is skin permeable and can be used to have skin retention.

In the present invention, "peptides for promoting skin permeation" means peptides capable of permeating through the skin regardless of the size or nature of the molecules, uniformly transmitting the same throughout the skin, and having excellent skin permeability and skin retention.

In the present invention, the term "skin permeability" means the ability or property of the peptide to permeate through the skin to penetrate into the skin, and the peptide for skin permeation promotion of the present invention shows remarkably superior skin permeability than other peptides .

In the present invention, "skin persistence" means the ability of the peptides that permeate through the skin to pass through the skin tissue and not to be transmitted to the circulatory system, but to bind to the tissues in the skin and remain in the skin. In the case of pharmaceutical preparations or cosmetics containing the skin tissue as a target tissue, it is preferable to use a carrier having excellent properties to remain in the skin tissue so that the component bound to the peptide can act on the skin tissue or skin cells for a long time. The skin penetration enhancing peptide of the present invention is remarkably excellent in skin permeability as well as skin retention, and thus can be used as a carrier for pharmaceutical preparations or cosmetics.

The peptide for skin permeation promotion of the present invention may include a peptide having excellent skin permeability and skin retention derived by performing a phage display method combining a phage library and a dissolution test method of a transdermal preparation, Lt; RTI ID = 0.0 > of: < / RTI > In one embodiment of the present invention, a peptide containing the amino acid sequence of SEQ ID NO: 1 was prepared as a skin permeation promoting peptide through a phage display method (Example 1).

In the present invention, " vascular endothelial growth factor (VEGF) " is a kind of signal transduction protein and plays an important role in vasculogenesis and angiogenesis. VEGF functions as part of a system to store and supply oxygen to tissues when blood circulation is inadequate. The general function of VEGF is to inhibit the development of embryonic development, injury or post-exercise muscle, and the formation of blood vessels bypassing infarcted blood vessels To create new blood vessels. However, increased amounts of VEGF may cause abnormal angiogenesis.

VEGF, which plays an important role in angiogenesis, mainly affects cells constituting the vascular endothelium. According to in vitro results, VEGF stimulates vascular endothelial cell division and migration and enhances microvascular permeability. VEGF is classified into five types of VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF (placenta growth factor) in mammals. VEGF-A promotes angiogenesis, migration of vascular endothelial cells, division, lumen formation, chemotaxis of macrophages and granulocytes, vasodilatation, and VEGF-B promotes angiogenesis of embryo, especially myocardial tissue . VEGF-C promotes lymphangiogenesis, and VEGF-D is necessary for the development of the lymphatic vessels surrounding the lungs. PlGF plays an important role in vasculogenesis, ischemia, inflammation, wound healing, and angiogenesis in cancer.

In the present invention, as long as VEGF exhibits an effect of promoting regeneration of damaged skin, regeneration and growth of hair through induction of angiogenesis, epidermal cell proliferation, cell migration promotion, microvascular permeability increase and the like, its amino acid sequence is specifically limited The entire amino acid sequence of the VEGF may be used, the mutated amino acid sequence thereof may be used, or a fragment thereof may be used. The specific amino acid sequence of the VEGF or the nucleotide sequence information of the gene encoding the VEGF can be obtained from a known database such as NCBI's GenBank. The VEGF may specifically be a peptide represented by the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

In addition, VEGF can be present in various forms (isoforms) such as VEGF-189, VEGF-165 and VEGF-121 due to splicing at various positions. In the case of VEGF-165, About 19.2 kDa. VEGF may exist as a homodimer through a disulfide bond or may exist as a heterodimer with another growth factor protein, PIGF.

In the present invention, the term "fusion protein" refers to a peptide artificially synthesized to bind the skin permeation-promoting peptide to another protein or peptide, and specifically includes the skin permeation-promoting peptide and the VEGF. More specifically, the skin permeation promoting peptide may be a peptide comprising the amino acid sequence of SEQ ID NO: 1, and the VEGF 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 that differs from a wild-type amino acid sequence of each domain included therein by one or more amino acid residues. Amino acid exchange in peptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

The fusion protein or the protein constituting the fusion protein may be prepared by a chemical protein synthesis method known in the art, or a gene encoding the fusion protein may be amplified by PCR (polymerase chain reaction) or synthesized by a known method Cloning into an expression vector and expressing it.

The fusion protein of the present invention may comprise a linker peptide between the skin permeation promoting peptide and the VEGF. Specifically, the fusion protein may be directly linked to the N-terminal of the VEGF, or may be fusion-linked through a linker.

The linker may be specifically linked with amino acids such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine and arginic acid, Amino acids such as valine, lysine, aspartic acid, glycine, alanine and proline can be used in combination, and more specifically, amino acids such as glycine, valine, leucine and aspartic acid One to five of them may be connected and used. For example, a fusion protein can be prepared by linking the C-terminal of the skin permeation-promoting peptide and the N-terminal of VEGF 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.

As long as the polynucleotide has an activity similar to that of the fusion protein, the polynucleotide may have a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having 70% or more, specifically 80% or more, more specifically 90% or more, May comprise, but is not limited to, a polynucleotide encoding a protein that exhibits at least 95%, and most specifically at least 99% homology. Also, it is apparent that a polynucleotide that can be translated into a protein consisting of the nucleotide sequence of SEQ ID NO: 3 or a protein having the same homology can be also included by codon degeneracy. Or a probe that can be prepared from a known gene sequence, for example, a protein having the activity of a protein consisting of the nucleotide sequence of SEQ ID NO: 3 by hybridizing under stringent conditions with a complementary sequence to all or a part of the nucleotide sequence Encoding sequences may be included without limitation. Specifically, the polynucleotide of the present invention may comprise the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto.

The term " stringent conditions " means conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., J. Sambrook et al., Sangdong). For example, a gene having a homology of at least 80%, specifically at least 90%, more specifically at least 95%, more specifically at least 97%, particularly at least 99% 1 × SSC, 0.1% SDS, specifically 60 ° C., 0.1 × SSC, 0.1 ° C., or 0.1 × SSC, which is a condition for hybridization of genes having lower homology to each other or hybridization with normal hybridization, More specifically, two times to three times at a salt concentration and a temperature corresponding to 0.1% SDS, more specifically, 68 占 폚, 0.1 占 SSC, and 0.1% SDS.

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

Specifically, polynucleotides having homology can be detected using hybridization conditions including the hybridization step at a Tm value 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 suitably adjusted by those skilled in the art according to the purpose.

The appropriate stringency of hybridizing the polynucleotide depends on the length and complementarity of the polynucleotide, and the 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 percentage of identity between two polynucleotide or polypeptide moieties. Means the degree of agreement with a given amino acid sequence or base sequence and can be expressed as a percentage. In the present specification, its homologous sequence having the same or similar activity as a given amino acid sequence or base sequence is referred to as "% homology ". The homology between sequences from one moiety to another may be determined by known techniques. For example, standard software for calculating parameters such as score, identity and similarity, specifically BLAST 2.0, or by sequential hybridization experiments under defined stringent conditions, And the appropriate hybridization conditions to be defined are within the skill of the art and can be determined by methods well known to those skilled in the art (for example, 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).

The term "vector" as used herein refers to a DNA construct containing a nucleotide sequence of a polynucleotide encoding the desired polypeptide operably linked to a suitable regulatory sequence so as to be capable of expressing the polypeptide of interest in the appropriate host . The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The vector may be transcribed into an appropriate host cell and then cloned or functioned independently of the host genome and integrated into the genome itself.

The vector used in the present invention is not particularly limited as long as it is replicable in the host cell, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A can be used as the phage vector or cosmid vector, and pBR, pUC, pBluescriptII , pGEM-based, pTZ-based, pCL-based, pET-based, and the like. 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 and pGAP vectors can be used and include vectors expressed in bacteria and yeast such as Escherichia coli, can do.

For example, a polynucleotide encoding a desired polypeptide in a chromosome can be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto.

The term "transformation" in the present invention means introducing a vector comprising a polynucleotide encoding a target polypeptide into a host cell so that the polynucleotide encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides may include all of these, whether inserted into the chromosome of the host cell or located outside the chromosome, provided that the polynucleotide can be expressed in the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, -DMSO method, and the like. The polynucleotide also includes DNA and RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as far as it is capable of being introduced into a host cell and expressed. 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 the elements necessary for its expression. The expression cassette can typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Also, the polynucleotide may be introduced into the host cell in its own form and operatively linked to the sequence necessary for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" in the above refers to a functional linkage of the gene sequence to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the polypeptide of the present invention.

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

Specifically, a cosmetic composition for improving skin wrinkles or skin elasticity comprising the fusion protein of the present invention as an active ingredient can be provided.

The term " skin elasticity enhancement " or " skin wrinkle improvement " in the present invention means to alleviate the degree to which the skin is stuck or stretched, or to suppress or inhibit the generation of wrinkles on the skin, , The intercellular shorts of the skin epidermis and the amount of hyaluronic acid distributed in the connective tissues of the dermis, the skin elasticity is maintained and the wrinkle formation is thereby alleviated.

The term " hyaluronic acid " is a type of glycosaminoglycans, and is a chain-like polymer polysaccharide substance in which glucuronic acid and N-acetylglucosamine residues are repeatedly connected. It has high viscosity and elasticity because it has a property of making gel by combining with a large amount of water. In addition, hyaluronic acid is a major component of the extracellular matrix and is involved not only in the retention of moisture, maintaining intercellular space, the storage and spread of cell growth factors and nutrients, but also the division and differentiation and migration of cells have.

According to one embodiment, the effect of the fusion protein on hyaluronic acid production was confirmed in order to confirm the effect of the skin elasticity and skin wrinkle improvement of the present invention. VEGF and a fusion protein (T-VEGF) were added to the human-derived dermal fibroblast and cultured. As a result, it was confirmed that T-VEGF had a hyaluronic acid-producing effect equivalent to that of VEGF.

As a result, it can be seen that even if the peptide for skin permeation promotion is fused to VEGF, the effect of improving the wrinkles and elasticity of the skin of VEGF is maintained.

According to a specific embodiment, the effect of the fusion protein on the umbilical vein endothelial cell proliferation was confirmed in order to confirm the effects of the skin elasticity and skin wrinkle improvement of the present invention. When VEGF and fusion protein (T-VEGF) were added to the umbilical vein endothelial cells, T-VEGF was found to be equivalent to VEGF in comparison to the control group (Table 2).

As a result, it can be seen that even if the peptide for skin permeation promotion is fused to VEGF, the effect of improving the wrinkles and elasticity of the skin of VEGF is maintained.

In addition, in the example of the present invention, a fusion protein was prepared by binding a peptide for promoting skin permeation containing the amino acid sequence of SEQ ID NO: 1 to VEGF (SEQ ID NO: 3), and the effect of the fusion protein prepared was compared with the conventional VEGF , Skin permeability and skin retention were remarkably improved and wrinkle-reducing effect was also excellent (Table 4 and Table 5).

Therefore, the fusion protein provided by the present invention can enhance the skin permeability and skin retention while maintaining the efficacy of VEGF itself, which increases the regeneration of damaged skin and hair, by binding the peptide for promoting skin permeation to VEGF , A cosmetic composition and a quasi-drug composition.

The fusion protein of the present invention may be contained in an amount of 0.0001 to 50% by weight based on the weight of the whole cosmetic composition. When the content of the fusion protein is less than 0.0001% by weight of the total cosmetic composition, it is difficult to substantially improve the skin. If the content of the fusion protein is more than 50% by weight, the formulation may become unstable.

The cosmetic composition according to the present invention can be used as a cosmetic composition in the form of a solution, an ointment for external use, a cream, a foam, a nutritional lotion, a softening water, a pack, a soft water, an emulsion, a makeup base, But are not limited to, those selected from the group consisting of emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays Do not.

In addition, the cosmetic composition of the present invention may further comprise at least one cosmetically acceptable carrier to be incorporated in a general skin cosmetic composition, and examples thereof include oil, water, a surfactant, a moisturizer, A thickener, a chelating agent, a coloring matter, an antiseptic, a flavoring, and the like may be appropriately compounded, but the present invention is not limited thereto.

The cosmetically acceptable carrier to be contained in the cosmetic composition of the present invention varies depending on the formulations.

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

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as the carrier component, Propellants such as fluorohydrocarbons, propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil may be used, in particular fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycols or sorbitan may be used have.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, glycerol, .

As another embodiment 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 " refers to the area where hair normally should be present due to various habits and environmental influences such as genetic causes, hormone imbalance, mental stress of social life, exposure to air pollution, To improve hairless state, to prevent hair loss from progressing, and to be hairy.

According to one specific embodiment, the dermal papilla cells treated with the fusion protein of the present invention were cultured, and the difference in the amount of cell proliferation was quantitatively analyzed using Cell Counter Kit-8 (Dojindo) And T-VEGF had the same level of cell proliferation as VEGF (Table 3).

Thus, it can be seen that even when the peptide for skin permeation promotion is fused to VEGF, the effect of improving VEGF is still maintained.

The cosmetic composition may be at least one selected from the group consisting of hair tonic, hair conditioner, hair essence, hair lotion, hair nourishing lotion, hair shampoo, hair conditioner, hair treatment, hair cream, hair nourishing cream, hair moisturizing cream, Hair shampoo, hair shampoo, hair shampoo, hair shampoo, hair shampoo, hair shampoo, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, A hair mousse or a hair spray.

Specifically, the composition of the present invention can be used by a method of directly applying or dispersing the hair or scalp. The hair to which the composition of the present invention is applied includes hair follicles and hair follicles, hair and eyelashes and eyelashes, beard, armpits, pubic hair, hair follicles and hair follicles.

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

Specifically, a quasi-drug composition for improving skin wrinkles or skin elasticity comprising the fusion protein of the present invention as an active ingredient can be provided.

As another embodiment of the present invention, there is provided a quasi-drug composition for improving hair loss 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 contain a pharmaceutically acceptable carrier, excipient or diluent as necessary in addition to the above components. The pharmaceutically acceptable carrier, excipient or diluent is not limited as long as the effect of the present invention is not impaired, and examples thereof include fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, .

Representative examples of the pharmaceutically acceptable carrier, excipient or diluent of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia rubber, alginate, calcium phosphate, calcium Methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oils such as sodium carboxymethylcellulose, , Injectable ester, witepsol, macrogol, tween 61, cacao paper, and laurie paper.

When the composition containing the fusion protein of the present invention as an active ingredient is used as a quasi-drug, it may further contain one or more active ingredients exhibiting the same or similar functions. For example, it may include skin barrier protection, elasticity enhancement, wrinkle enhancement, and moisturizing ingredients. When the above ingredients are added, skin safety, easiness of formulation, and stability of effective ingredients can be considered according to the combined use. The quasi-drug composition is a whitening ingredient known in the art, and includes a substance inhibiting tyrosinase enzyme activity such as kojic acid and arbutin, hydroquinone, L-ascorbic acid; Retinoic acid, TGF, proteins from animal placenta, betulinic acid and chlorella extract, as skin elasticity, wrinkle or moisturizing ingredients known in the art; And derivatives thereof, and various plant extracts. ≪ Desc / Clms Page number 2 > The additional ingredient may be included in an amount of 0.0001% to 5% by weight based on the total weight of the composition, and the content range may be adjusted according to requirements such as skin safety, ease of use and the like.

The quasi-drug composition of the present invention can be exemplified by a disinfectant cleaner, a shower foam, a softener solution, a wet tissue, a coating agent, and the like. The formulation method, dosage, usage method, And the like.

In addition, the quasi-drug composition comprising the fusion protein of the present invention as an active ingredient can be used for skin elasticity enhancement, skin wrinkle improvement, or hair loss improvement, including a step of 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 binds VEGF to the skin permeation enhancing peptide, thereby maintaining or improving the ability of the peptide to exhibit useful effects such as wrinkle improvement, and at the same time, significantly improving skin permeability and skin retention, A composition for improving hair loss, a quasi-drug composition for skin improvement, or a quasi-drug composition for hair loss improvement.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

Example  1: For skin permeation promotion Of peptide  Selection

In order to select peptides for skin permeation promotion, a phage display method was performed in which a phage library and a dissolution test method of a transdermal preparation were combined.

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

Next, pig skin (0.7 mm thick, Medikinetics) was mounted between the upper and lower ends of a Franz glass cell (standard diameter 9 mm, Receiver 5 mL, Permgear), and the phage solution was added to the upper end thereof. A phage permeating the pig skin and reaching the lower receiver was obtained and amplified.

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

As described above, it is defined as 1 Round that the process of amplifying the phage obtained by passing the skin through the skin by applying the phage to the pig skin is carried out once, and 2 rounds are carried out again with the phage amplified in the 1 round, In order to compete with each other, phages with good permeability were selected and 3 rounds were performed.

In order to confirm the sequence of the peptide contained in the phage finally obtained as a result of the 3 round, the TBS solution containing the phage was added to E. coli strain ER2738 to suspend, TOP agar was added to the suspension, LB / X-gal / IPTG plate media. Then, the solidified medium was cultured for 16 hours, and a blue colony was selected. The strain derived from the selected colonies was cultured for 6 hours, DNA was obtained therefrom, and the base sequence derived from the phage was analyzed to obtain a skin permeation promoting peptide (SEQ ID NO: 1) exhibiting the property of penetrating pig skin Respectively.

Example  2: Vascular endothelial growth factor VEGF ) For skin permeation promotion The peptide Combined  Preparation of fusion protein

The N-terminus of the VEGF having the amino acid sequence of SEQ ID NO: 2 and the C-terminal of the peptide for skin permeation promotion having the amino acid sequence of SEQ ID NO: 1 obtained in Example 1 was replaced with two amino acids ( GG) to produce T-VEGF (SEQ ID NO: 3), a fusion protein in the form of a linker.

Specifically, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 and a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 are synthesized, and a nucleotide sequence encoding two amino acids (GG) is linked at the center 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 prepared expression vector was introduced into Pichia pastoris to obtain a transformant. The obtained transformant was cultured, and then the culture solution was filtered to recover the fusion protein composed of peptide-VEGF for skin permeation promotion. GPC column chromatography to finally produce T-VEGF (SEQ ID NO: 3), a fusion protein composed of peptide-VEGF for skin permeation promotion.

Example  3: Verification of effect of fusion protein

In order to confirm the skin improvement application of T-VEGF prepared in Example 2, an experiment for improving skin elasticity, improving wrinkles of skin, improving hair loss, skin permeability and residual skin effect was conducted as described below.

Example  3-1: Test for hyaluronic acid (HA) production

The effect of T-VEGF synthesized in Example 2 on VEGF production and hyaluronic acid production was examined.

Specifically, dermal fibroblast was inoculated into a 24-well plate and cultured for 24 hours to obtain a culture showing a degree of saturation of 70 to 80%. The cultures were washed with PBS and then cultured in serum-free DMEM medium containing 10 ng / ml of T-VEGF or VEGF for 2 days. The supernatant of the culture was obtained and the content of hyaluronic acid (HA) produced and secreted in the culture medium was quantitatively analyzed using an ELISA kit (R & D Systems) (Table 1).

Effects of Fusion Proteins on the Production of Hyaluronic Acid (HA) Treated protein Generation level (%) Control group 100 ± 4.9 VEGF 220 ± 20 T-VEGF 230 ± 18

As shown in Table 1, it was confirmed that T-VEGF, in which VEGF was fused with a skin permeation enhancing peptide, had a hyaluronic acid-producing effect equivalent to that of VEGF.

As a result, it can be seen that even if the peptide for skin permeation promotion is fused to VEGF, the effect of improving the wrinkles and elasticity of the skin of VEGF is maintained.

Experimental Example  3-2: Endothelial cell proliferation assay

The effect of T-VEGF synthesized in Example 2 on VEGF proliferation was examined.

Specifically, a human Umbilical Vein Endothelial Cell was inoculated into a 96-well plate and cultured for 24 hours to obtain a culture showing 5,000 cells per well. The cultures were washed with PBS and then cultured in serum-free M199 medium for 16 hours. After washing with PBS, the cells were cultured in serum-free M199 medium containing 100 ng / ml of T-VEGF or VEGF for 1 day. Cultures were obtained and quantified by Cell Counter Kit-8 (Dojindo). At this time, as a control group, the result of culturing in serum-free M199 medium without T-VEGF or VEGF was used (Table 2).

Effects of Fusion Proteins on Endothelial Cell Proliferation Treated protein Growth level (%) Control group 100 ± 9.9 VEGF 170 ± 10 T-VEGF 185 ± 15

As shown in Table 2, it was confirmed that T-VEGF, in which VEGF was fused with a skin permeation enhancing peptide, also had cell proliferation efficiency equivalent to that of VEGF.

As a result, it can be seen that even if the peptide for skin permeation promotion is fused to VEGF, the effect of improving the wrinkles and elasticity of the skin of VEGF is maintained.

Example  3-3: Follicular stem cell proliferation  Efficacy verification

The effect of T-VEGF synthesized in Example 2 on VEGF and skin cell proliferation was verified.

Specifically, dermal papilla cells were inoculated into 96-well plates and cultured for 24 hours to obtain cultures representing 6,000 cells per well. The cultures were washed with PBS and then cultured in serum-free DMEM medium for 1 day. After washing with PBS, the cells were cultured in serum-free DMEM medium containing 1 ng / ml of T-VEGF or VEGF for 1 day. The culture was obtained, and the difference in the amount of cell proliferation was quantitatively analyzed using Cell Counter Kit-8 (Dojindo). As a control, the resultant product was used in a serum-free DMEM medium containing no T-VEGF or VEGF (Table 3).

Effects of Fusion Proteins on Follicular Stem Cell Proliferation Treated protein Growth level (%) Control group 100 ± 4.9 VEGF 340 ± 50 T-VEGF 310 ± 20

As shown in Table 3, it was confirmed that T-VEGF, in which VEGF was fused with skin permeation enhancing peptide, had cell proliferation efficiency equivalent to that of VEGF.

Thus, it can be seen that even when the peptide for skin permeation promotion is fused to VEGF, the effect of improving VEGF is still maintained.

Example  3-4: Verification of skin permeability

The skin permeability was verified by comparing the fusion protein T-VEGF synthesized in Example 2 with VEGF.

Specifically, 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, Permegear), and TBS (50 mM Tris pH 7.5, 150 mM NaCl) was prepared. To the top of the glass cell, 500 μl of TBS was added to the donor chamber, and 5 ml of TBS was added to the bottom of the glass cell (receiver chamber). Then, 2 μg of VEGF or T-VEGF was added to the top and allowed to react for 16 hours. Then, the concentration of VEGF or T-VEGF present at the bottom was quantitatively analyzed using an ELISA kit (R & D Systems) The relative content of the amount of T-VEGF to the content was calculated as the amount of permeation (Table 4).

Skin permeability of fusion protein Treated protein Permeability (%) VEGF 100 ± 16 T-VEGF 295 ± 21

As shown in Table 4, it was confirmed that the skin permeability of T-VEGF was about 3 times higher than that of VEGF.

Thus, it can be seen that the skin permeability is remarkably increased when the fusion protein of the present invention is used.

Example  3-5: Verification of skin persistence

Skin fusion was verified by comparing the fusion protein T-VEGF synthesized in Example 2 with VEGF.

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

Skin persistence of fusion protein Treated protein Residual amount (%) VEGF 100 ± 24 T-VEGF 9,800 ± 790

As shown in Table 5, it was confirmed that the skin residual was increased about 100 times as compared with VEGF in the case of T-VEGF.

Thus, it can be seen that skin retention remarkably increases when the fusion protein of the present invention is used.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> LG HOUSEHOLD & HEALTH CARE LTD. <120> Cosmetic composition for skin care containing fusion protein with          VEGF <130> KPA170150-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> 165 <212> PRT <213> Unknown <220> <223> Human VEGF <400> 2 Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys   1 5 10 15 Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu              20 25 30 Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys          35 40 45 Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu      50 55 60 Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile  65 70 75 80 Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe                  85 90 95 Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg             100 105 110 Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe         115 120 125 Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser     130 135 140 Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys 145 150 155 160 Asp Lys Pro Arg Arg                 165 <210> 3 <211> 179 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein of T-VEGF <400> 3 Asn Gly Ser Leu Asn Thr His Leu Ala Pro Ile Leu Gly Gly Ala Pro   1 5 10 15 Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met              20 25 30 Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp          35 40 45 Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser      50 55 60 Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu  65 70 75 80 Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg                  85 90 95 Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln             100 105 110 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu         115 120 125 Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln     130 135 140 Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys 145 150 155 160 Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys                 165 170 175 Pro Arg Arg             <210> 4 <211> 550 <212> DNA <213> Artificial Sequence <220> <223> T-VEGF <400> 4 aatggaagtt tgaacaccca tctggcccct atacttggag gtgcaccaat ggctgagggt 60 ggcggtcaga atcaccatga agttgtcaaa tttatggatg tttaccagag atcatattgc 120 catcccatcg aaaccctcgt ggatatattt caggaatacc ctgatgaaat tgagtatatc 180 ttcaagccct cttgtgtccc attaatgaga tgtggtggat gttgtaatga tgaaggactt 240 gaatgcgtac caactgagga atccaatatt acaatgcaaa tcatgaggat taagccacac 300 cagggtcaac atattgggga gatgtctttc ctacaacaca acaagtgtga atgcagacca 360 aaaaaggata gagctagaca agagaaccct tgcggtccgt gctctgagag aagaaaacat 420 ttgtttgttc aagacccaca aacatgtaag tgctcatgta aaaacactga ctctcgatgt 480 aaggctagac aattggaatt gaacgagaga acttgtcgtt gtgacaaacc taggcgttaa 540 tctagaataa 550

Claims (11)

A fusion protein comprising a peptide for promoting skin permeation, which is composed of the amino acid sequence of SEQ ID NO: 1, is bound to a vascular endothelial growth factor (VEGF).
The method according to claim 1,
Wherein the skin permeation enhancing peptide is bound to the N-terminus of VEGF.
2. The fusion protein of claim 1, wherein the VEGF is comprised of the amino acid sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 3.
The method according to claim 1,
Wherein said fusion protein is enhanced in skin permeability and skin retention of VEGF.
A polynucleotide encoding the fusion protein of any one of claims 1 to 5.
A cosmetic composition for improving skin comprising the fusion protein of any one of claims 1 to 5 as an active ingredient.
The cosmetic composition for skin improvement according to claim 7, wherein the skin improvement is skin wrinkle improvement or skin elasticity enhancement.
A cosmetic composition for improving hair loss, comprising the fusion protein of any one of claims 1 to 5 as an active ingredient.
A quasi-drug composition for improving skin comprising the fusion protein of any one of claims 1 to 5 as an active ingredient.
A quasi-drug composition for improving hair loss comprising the fusion protein of any one of claims 1 to 5 as an active ingredient.