KR102503729B1 - Composition for improving skin condition comprising cell-penetrating peptide and epidermal growth factor fusion protein - Google Patents

Abstract

The present invention relates to a composition for improving skin condition comprising a fusion protein of a cell-penetrating peptide and an epithelial cell growth factor, and specifically to a fusion protein containing cell-penetrating growth factor and epithelial cell growth factor; and a composition for cell or skin regeneration, improvement of skin condition, or prevention or treatment of skin diseases including the same as an active ingredient. The fusion protein of the cell-penetrating peptide and epithelial cell growth factor of the present invention can surpass safety and penetration efficiency of conventional cell-penetrating growth factors, and has high penetration efficiency, and has the effects of healing or regenerating wounds in cells or the skin and moisturizing the cells or skin, enabling it to be used as a material for cosmetics.

Description

Composition for improving skin condition comprising cell-penetrating peptide and epidermal growth factor fusion protein {Composition for improving skin condition comprising cell-penetrating peptide and epidermal growth factor fusion protein}

The present invention relates to a composition for improving skin condition comprising a fusion protein in which a cell permeable growth factor and an epidermal growth factor are fused. Specifically, a fusion protein in which cell permeable growth factor and epidermal growth factor are fused; And it relates to a composition for cell or skin regeneration, skin condition improvement, or skin disease prevention or treatment comprising the same as an active ingredient.

Recombinant protein technology is a technology that uses genetic engineering technology to mass-express proteins derived from higher organisms in microorganisms and animal cells. Depending on the functional characteristics of proteins, processing technologies produced by microorganisms and animal cells are being applied, and proteins produced in this way are widely used in pharmaceutical, cosmetic, food, analysis and diagnostic industries. In the case of new cosmetic materials based on recombinant proteins, R&D is active mainly for trouble skin diseases. It is expected that the side effects of external skin products can be minimized by applying the growth factors produced based on this recombinant protein technology to skin regeneration, anti-aging, and wrinkle improvement.

EGF (Epidermal Growth Factor) is a polypeptide with a molecular weight of about 6Ka composed of 53 amino acids with 3 disulfide bonds. EGF is a growth factor that specifically binds to the epidermal growth factor receptor (EGFR) present on the cell surface to promote cell growth, proliferation, and differentiation. It plays a key role in skin regeneration, such as promoting angiogenesis, inducing the secretion of other regeneration promoting factors, and promoting the synthesis of fibronectin.

However, these recombinant proteins, such as epidermal growth factor (EGF), are generally hydrophilic, and it is very difficult for large molecules to enter cells due to the barrier of the cell membrane. The cell membrane prevents macromolecules such as peptides, proteins, and nucleic acids from entering the cell, and even if they enter the cell through a physiological mechanism called endocytosis by cell membrane receptors, they fuse with the cell's lysosomal compartment and eventually disintegrate. Therefore, there are many limitations in delivering these macromolecular bioactive materials outside the cell. In order to increase the skin absorption of such a high molecular weight protein, a method of physically puncturing the skin such as a laser or mesoroller or a method of using a carrier such as a surfactant and nanostructure is usually applied, but these methods There is a risk of damaging the skin barrier, there are still limitations in absorbing macromolecules, and a separate device treatment is required.

In addition, since it is difficult to penetrate the skin due to the protective function of the skin, epidermal growth factor is used in excess for a therapeutic effect, and excessive use causes various side effects. Therefore, attempts have been made to more efficiently permeate growth factors into skin and cells.

In order to overcome these limitations, new alternatives have recently been proposed. Among them, cell penetrating peptides (CPPs), which have been difficult to use as drugs due to their low cell membrane permeability and fast in vivo half-life, are used to treat large molecules such as therapeutic proteins and genes. Research is under way to more safely and effectively deliver bio-active materials into the vagina or nucleus of living cells using the peptides, since the value of the molecules can be increased.

Under this background, the present inventors combined some sequences of human PRM1, a cell and skin penetrating peptide with higher penetration efficiency than previously discovered cell penetrating peptides, to epidermal growth factor (EGF) to improve the stability of existing cell penetrating growth factors. By developing human-derived cells and skin-penetrating epithelial growth factor capable of overcoming hyper-permeation efficiency, it was confirmed that skin permeability was improved and there were effects such as wound regeneration and moisturizing, and the present invention was completed.

Republic of Korea Patent No. 10-2333650

An object of the present invention is to provide a fusion protein in which a cell penetrating peptide and an epidermal growth factor are fused.

Another object of the present invention is to provide a polynucleotide encoding the fusion protein and a recombinant vector containing the polynucleotide.

Another object of the present invention is to provide a transformant transformed by the recombinant vector.

Another object of the present invention is culturing the transformant in a culture medium; and recovering the fusion protein from the culture medium.

Another object of the present invention is to provide a cosmetic composition for cell or skin regeneration or skin condition improvement containing the fusion protein.

Another object of the present invention is to provide a quasi-drug composition for preventing or treating skin diseases comprising the fusion protein.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin diseases comprising the fusion protein.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and another problem not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

As one aspect for achieving the above object, the present invention provides a fusion protein in which a cell penetrating peptide and an epidermal growth factor are fused.

In the present invention, the fusion protein may consist of all or part of the amino acid sequence of SEQ ID NO: 2.

SEQ ID NO: 2:

RSRRRRRRSCQTRRRNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

The term 'Cell Penetrating Peptides (CPP)' of the present invention is a cell membrane penetrating peptide consisting of about 10 to 60 short peptides that move into cells without damaging cell membranes and DNA that does not pass through cell membranes. Peptides capable of delivering proteins into cells. The type of cell penetrating peptide is not particularly limited, but is preferably a group consisting of TAT, penetratin, Antp (antennapedia protein), low molecular weight protamine (LMWP) or PRM1 (protamine 1) It may be one or more selected from. As an example, the 'cell penetrating peptide' may include a sequence of human-derived protamine 1 (PRM1) and may be a peptide composed of all or part of the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1: RSRRRRRRSCQTRRR

The term 'cell penetrating type' of the present invention refers to a state in which a cell penetrating peptide and a substance having biological or pharmaceutical activity are chemically, physically, covalently or non-covalently linked.

The term 'fusion protein' of the present invention includes a transport domain and at least one portion of a target protein, and refers to a complex formed by genetic fusion or chemical bonding between a transport domain and a target protein. In addition, the 'genetic fusion' refers to a linear, covalent linkage formed through genetic expression of a DNA sequence encoding a protein. The 'fusion protein' of the present invention refers to a substance in which a cell-penetrating peptide and a substance having biological or pharmaceutical activity are linked by a chemical or physical covalent or non-covalent bond.

In one embodiment of the present invention, the substance having biological or pharmaceutical activity means a substance capable of regulating physiological phenomena in vivo, and includes DNA, RNA, proteins, peptides, fats, carbohydrates and chemical compounds, Alternatively, a fluorescent label may be included, and epidermal growth factor (EGF) may be used as a protein.

In the present invention, the production of the fusion protein can be prepared by genetic recombination method fusion protein of cell penetrating peptide and epidermal growth factor.

The term 'Epithermal Growth Factor (EGF)' of the present invention is a protein with a molecular weight of 6 kDa and composed of 53 amino acids. The EGF is involved in the skin regeneration process by acting on the cell nucleus to promote the division and proliferation of epidermal cells. The EGF is used in a variety of ways, such as cosmetics for improving skin conditions and wound healing, but has problems such as relatively low transmission and durability to the wound.

As another aspect for achieving the above object, the present invention provides a polynucleotide encoding the fusion protein.

The 'polynucleotide' is a polymer of deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form. It encompasses RNA genome sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified. The polynucleotide includes not only the nucleotide sequence encoding the fusion protein, but also a sequence complementary to the sequence. The complementary sequences include sequences that are substantially complementary as well as sequences that are perfectly complementary. This means a sequence capable of hybridizing with the nucleotide sequence encoding the fusion protein under stringent conditions known in the art. Also, the polynucleotide may be modified. Such modifications include additions, deletions or non-conservative or conservative substitutions of nucleotides. A polynucleotide encoding the amino acid sequence is also construed to include a polynucleotide sequence exhibiting substantial identity to the nucleotide sequence. The substantial identity is at least 80% homology when the nucleotide sequence and any other sequence are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. It may be a sequence exhibiting 90% homology or at least 99% homology.

As another aspect for achieving the above object, the present invention provides a recombinant vector comprising the polynucleotide.

The 'vector' refers to a means for expressing a target gene in a host cell. In the present invention, after fusing the cell-penetrating peptide and the epidermal growth factor, it can be obtained in the form of a fusion protein by inserting it into an expression vector. As an expression vector that can be used here, for example, an E. coli expression vector In pBF-01(+), pET21 vectors, etc. are possible, but conventionally used expression vectors can be used without significant limitations.

As another aspect for achieving the above object, the present invention provides a transformant into which the recombinant vector is introduced.

In the present invention, the term "transformant" refers to an organism whose genetic material has been changed by introducing foreign genetic material.

The 'vector' is as described above.

In the present invention, the transformant can be obtained by introducing the expression vector into a host cell. That is, such an introduction method can be performed by a method known in the art, and the transformation method is not limited as long as the recombinant vector for expressing the fusion protein can be efficiently introduced into a host cell, but, for example, electroporation It can be performed by methods such as electroporation, protoplast method, Li+ ion method, lithium acetate method, and transformation method using yeast cell wall lysis enzyme.

The host cell may be Escherichia coli ( E.coli ), and is not limited to a specific type as long as it is a cell suitable for efficient expression of the fusion protein of the present invention.

The method for selecting the transformant can be easily performed according to a method known in the art using a phenotype expressed by a selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

As another aspect for achieving the above object, culturing the transformant in a culture medium; and recovering the fusion protein from the culture medium.

In the present invention, the culture may be a large-scale cell culture, and the cell culture method may use a commonly used cell culture method. For example, the cell culture method is, but is not limited to, batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture , It may be any one or more selected from the group consisting of continuous culture and perfusion culture.

In the present invention, the step of recovering the fusion protein from the culture medium can be performed through various separation and purification methods known in the art. Usually, after centrifugation of the cell lysate to remove cell debris, culture impurities, etc., precipitation, e.g., salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (acetone, ethanol, iso protein fraction precipitation using propyl alcohol, etc.), etc., dialysis, affinity chromatography, etc. can be performed.

As another aspect for achieving the above object, the present invention provides a cosmetic composition for cell or skin regeneration or skin condition improvement comprising the fusion protein.

The term 'fusion protein' of the present invention refers to a fusion protein of a cell penetrating peptide and epidermal growth factor (EGF). As an example, the fusion protein may consist of all or part of the amino acid sequence of SEQ ID NO: 2, and the cell-penetrating peptide of the fusion protein may consist of all or part of the amino acid sequence of SEQ ID NO: 1.

In the present invention, the cells may be dermal papilla cells, keratinocytes, skin fibroblasts, melanocytes or hair follicle stem cells.

The 'fusion protein', 'cell penetrating peptide' and 'epithelial cell growth factor' are as described above.

The term 'skin regeneration' of the present invention refers to a process of recovering skin tissue from damage caused by external and internal skin causes. Damage caused by external causes may include ultraviolet rays, external contaminants, wounds, trauma, and the like, and damage caused by internal causes may include stress, but is not limited thereto.

The term 'skin condition improvement' of the present invention may include skin moisturizing, wrinkle improvement, skin aging prevention, skin damage recovery by ultraviolet rays, skin protection from ultraviolet rays, skin elasticity maintenance, or wound improvement, but is not limited thereto.

The term 'moisturizing the skin' of the present invention means increasing the feeling of moisture in the skin and maintaining a moist state.

The term 'wrinkle improvement' of the present invention means maintaining or enhancing wrinkles and elasticity of the skin.

The term 'prevention of skin aging' of the present invention is used to prevent chronological aging (endogenous aging), which is natural aging caused by physiological changes in the body over time, and photoaging (photogenic aging) that occurs in areas exposed to sunlight. or delay all of them. Specifically, skin aging of the present invention may be photoaging caused by UV-B ultraviolet rays, but is not limited thereto. Specifically, photoaging of skin cells may be progressed by light of a wavelength of 280 nm to 40 nm included in sunlight. Among them, damage to skin or fibers may occur due to irradiation of ultraviolet rays such as UV-B having a wavelength in the range of 280 nm to 320 nm, and a soot phenomenon in which the skin is charred may occur. When UV-B is irradiated, the accumulation of active oxygen (eg H ₂ O ₂ ) and free radicals in skin cells is promoted, and the intracellular signaling system is stimulated by the radicals to oxidize biomolecules such as DNA, proteins, and lipids. It can cause enemy stress, which can cause damage to skin tissue. The decrease in hyaluronic acid due to aging can atrophy the epidermis due to defects in the skin moisturizing barrier, increase skin wrinkles, and reduce elasticity and moisture content, thereby improving moisturizing power and anti-aging by controlling the synthesis of hyaluronic acid. there is.

The cosmetic composition of the present invention may contain the fusion protein in a large amount as long as it has effects such as skin regeneration, skin moisturizing, wrinkle improvement or wound improvement.

In addition to the fusion protein, the cosmetic composition according to the present invention includes various components commonly used in cosmetic compositions, for example, water-soluble components, powder components, oils, surfactants, and moisturizers within a range that does not reduce the effect of the present invention, if necessary. , It may be composed of a viscosity modifier, preservatives, antioxidants, fragrances, pigments, and the like.

Non-limiting examples of the usable surfactant include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants. More specifically, the anionic surfactants include alkylbenzene sulfonates, polyoxyalkylene alkyl sulfate ester salts, alkyl sulfate ester salts, olefin sulfonates, alkyl phosphates, polyoxyalkylene alkyl ether phosphates, and dialkyl sulfosuccines. acid salts, fatty acid salts, and the like, and as nonionic surfactants, polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyhydric alcohol fatty acid partial esters, polyoxyethylene polyhydric alcohol fatty acid partial esters, polyglycerin fatty acid esters, polyoxyethylene Ethylene hydrogenated castor oil derivatives, fatty acid diethanolamide, etc. are mentioned. Examples of cationic surfactants include tertiary aliphatic amine salts, alkyltrimethylammonium halides, and dialkyldimethylammonium halides. Examples of amphoteric surfactants include amide betaine type, imidazolinium betaine type, and sulfobetaine type. etc. can be mentioned.

Examples of the moisturizing agent include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, and sorbitol. Benzoic acid, dehydroacetic acid, paraoxybenzoic acid ester (methyl paraoxybenzoate, butyl paraoxybenzoate, etc.), phenoxyethanol etc. are mentioned as said preservative. In addition, as the antioxidant, ascorbic acid, BHA, and the like may be mentioned, and in addition, an ultraviolet absorber, an anti-inflammatory agent, and a cooling agent may be added.

The cosmetic composition of the present invention is a solution, external ointment, cream, foam, nutrient lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath additive, sunscreen cream, sun oil, suspension, emulsion Liquids, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays may be prepared in formulations selected from the group consisting of, but are not limited thereto no.

The cosmetic composition of the present invention may further include one or more acceptable carriers in cosmetic formulations, and as typical ingredients, for example, oil, water, surfactants, humectants, lower alcohols, thickeners, chelating agents, pigments, Preservatives, flavorings, etc. may be appropriately blended, but are not limited thereto. Here, "acceptable carrier in cosmetic preparation" means a compound or composition that is already known and used, or a compound or composition to be developed in the future, which can be included in a cosmetic preparation, and has toxicity, instability or irritation more than the human body can adapt to when in contact with the skin. say nothing The carrier may be included in the composition of the present invention in an amount of about 1 wt % to about 99.99 wt %, preferably about 90 wt % to about 99.99 wt %, based on the total weight of the composition. However, since the ratio varies depending on the formulation of the composition of the present invention and its specific application area (face, neck, etc.) or its preferred application amount, the ratio does not limit the scope of the present invention in any aspect. should not be understood as

Acceptable carriers in cosmetic preparations included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

When the dosage form of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. This may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohard It may include, but is not limited to, propellants such as low carbon, propane/butane or dimethyl ether, and these may be used alone or in combination of two or more.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil and the like may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan Can be used, but is not limited thereto, and they may be used alone or in combination of two or more.

When the formulation of the present invention is a suspension, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used, but is not limited thereto, and these may be used alone or in combination of two or more.

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, sugar, etc. are used as carrier components. It may be, but is not limited thereto, and these may be used alone or in combination of two or more.

As one aspect for achieving the above object, the present invention provides a quasi-drug composition for preventing or treating skin diseases comprising the fusion protein.

The 'fusion protein' is as described above.

The term 'skin disease' of the present invention may be selected from the group consisting of skin wounds, skin scars and skin pigmentation, but is not limited thereto.

Non-limiting examples of the terms 'skin wound' and 'skin scar' of the present invention include abrasions, scars caused by abrasions, and the like.

The term 'skin pigmentation' of the present invention refers to all diseases caused by skin pigmentation due to increased production of melanin. Non-limiting examples of the skin pigmentation disease include melasma, freckles, spots, solar pigmentation spots, pigmentation after drug use, pigmentation after inflammation, and pregnancy pigmentation.

The term 'prevention' of the present invention refers to all activities that suppress or delay skin diseases such as skin wounds, skin scars, and skin pigmentation by administering or applying the fusion protein of the present invention to a subject.

The term 'treatment' of the present invention refers to all activities that improve or alleviate or benefit skin diseases such as skin wounds, skin scars, and skin pigmentation by administering or applying the fusion protein of the present invention to a subject.

In the present invention, 'quasi-drug' refers to items that are not instruments, machines or devices among items used for the purpose of diagnosing, treating, mitigating, treating or preventing diseases of humans or animals and that have pharmacological effects on the structure and function of humans or animals. Refers to items other than instruments, machines, or devices used for the purpose, and may include, but is not limited to, preparations for internal use as one specific example, formulation method, dosage, method of use, and components of quasi-drugs and the like can be appropriately selected from conventional techniques known in the art.

The quasi-drug composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent as needed 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, aromatics, preservatives, and the like. can include

As one aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating skin diseases comprising the fusion protein.

The 'fusion protein', 'skin disease', 'prevention' and 'treatment' are as described above.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, for example, a carrier for oral administration or a carrier for parenteral administration may be further included. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like.

In addition, carriers for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like. In addition, a stabilizer and a preservative may be further included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include intravenous, intramuscular, intraarterial, intracentral, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal , It may be enteral, topical, sublingual or rectal administration, preferably centrally administered, but not limited thereto.

The pharmaceutical composition of the present invention may be formulated into a formulation for oral administration or parenteral administration according to the administration route as described above. When formulated, one or more buffers (e.g. saline or phosphate buffered saline (PBS)), antioxidants, bacteriostats, chelating agents (e.g. EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g. eg, aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations contain the pharmaceutical composition of the present invention and at least one or more excipients, e.g. For example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol , Cellulose, methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropylmethyl-cellulose or gelatin may be mixed and prepared. Tablets or dragees may be obtained, for example, by combining the active ingredient with a solid excipient which is then milled and processed into a mixture of granules after adding suitable auxiliaries.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions for oral use, emulsions, or syrups. In addition to water or liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, or preservatives may be included. there is. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and may further include anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives. .

In the case of parenteral administration, the pharmaceutical composition of the present invention may be formulated according to a method known in the art in the form of injection, transdermal administration, and nasal inhalation with a suitable parenteral carrier. In the case of the injection, it must be sterilized and must be protected from contamination by microorganisms such as bacteria and fungi. In the case of injections, examples of suitable carriers may include water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof, and/or solvents or dispersion media containing vegetable oil, but are not limited thereto. It is not. More preferably, suitable carriers include Hanks' solution, Ringer's solution, PBS containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. . In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, and thimerosal may be further included. Also, in most cases, the injection may further include an isotonic agent such as sugar or sodium chloride.

Transdermal preparations include ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. Here, 'transdermal administration' means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.

For administration by inhalation, the compositions used according to the present invention may be prepared in pressurized packs or with a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. In the case of pressurized aerosols, dosage units may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in all pharmaceutical chemistry generally known prescriptions, Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour.

In the pharmaceutical composition of the present invention, the amount of the active compound in a unit dose formulation may vary, and may be specifically adjusted to about 0.01 mg to about 1 g per dose based on an average human weight of 70 kg. However, the dosage may vary depending on the requirements of humans and mammals, the severity of the disease to be treated, and the final composition of the compound used. Determination of the appropriate dosage for a particular situation is within the skill of the art.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers.

The fusion protein of the present invention, in which the cell-penetrating peptide and the epidermal growth factor are fused, can overcome the safety and penetration efficiency of existing cell-penetrating growth factors, has high penetration efficiency, heals or regenerates cell or skin wounds, and It has a moisturizing effect, so it can be used as a material for cosmetics and medicines.

1 is a result of confirming the purification of human-derived cells and skin permeable epidermal growth factor fusion protein. (lane 1: protein size marker, lane 2: fusion protein (lot No. 01), lane 3: fusion protein (lot No. 02))
2 is a result of comparing cell safety when fusion proteins were added at different concentrations.
3 is a result of confirming the cytotoxicity and cell proliferation effect when the fusion protein was treated.
Figure 4a is a result of confirming the cell penetrating ability of the fusion protein by fluorescence transmittance. (A: untreated control, B: epidermal growth factor 0.5 ppm, C: LMWP epidermal growth factor 0.5 ppm D: fusion protein 0.5 ppm)
4B is a graph showing the relative permeability of fusion proteins.
Figure 5a is a result of confirming the wound regeneration effect on human keratinocytes through cell staining when the fusion protein was treated. (A: untreated control, B: 0.25 ppm, C: 0.5 ppm, D: 1 ppm)
Figure 5b is a graph showing the relative wound regeneration effect when the fusion protein is treated.
Figure 6a is a result of confirming the expression level of the moisturizing factor HAS in human keratinocytes by the fusion protein.
Figure 6b is a graph showing the relative HAS expression level by the fusion protein.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited only to these examples.

Example 1. Preparation of skin-penetrating peptide and epidermal growth factor (EGF) fusion protein

1.1. Preparation of cDNA and expression vectors

Human-derived cells and skin-penetrating peptides were fused with EGF (Epidermal Growth Factor) to produce cell and dermal epidermal growth factor fusion proteins. A human having restriction sites of restriction enzymes NdeI and XhoI at both ends for cloning into an expression vector by fusing a gene encoding a cell- and skin-penetrating peptide to the N-terminus of an epidermal growth factor (EGF) cDNA. cDNAs of the derived cells and skin permeable epidermal growth factor fusion proteins were obtained. The insert cDNA thus obtained was inserted into an expression vector. E.coli expression vector, pBF-01(+), was double digested with NdeI and XhoI to open the cloning site, and after double digestion with NdeI and XhoI, insert cDNA was purified. Then, the expression vector and the insert cDNA of the target protein were ligated and fused. After fusing the expression vector pBF-01(+)- thus prepared at the gene level of human-derived cells and skin-penetrating peptide epidermal growth factor, transforming the BL21 series of Escherichia coli used for the expression of foreign recombinant proteins to obtain an expression clone manufactured. Table 1 below shows amino acid compositions of human-derived cells and skin-penetrating peptides and human-derived cells and skin-penetrating epidermal growth factor fusion proteins.

division amino acid composition SEQ ID NO: 1 Human-derived cells and skin-penetrating peptides N-terminal-RSRRRRRRSCQTRRR-C-terminal SEQ ID NO: 2 Human-derived cell and skin permeable epidermal growth factor fusion protein N-terminal-RSRRRRRRSCQTRRRNSDSECPLSHDGYCL
HDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR-C-terminus

1.2. Expression and purification of human-derived cell and skin permeable epidermal growth factor fusion protein

An expression strain was constructed by transforming the expression vector of the human-derived cell and skin permeable epidermal growth factor fusion protein prepared in Example 1.1 into an E.coli strain for recombinant protein expression. The obtained expression strain was expressed and purified as follows using a glycerol stock for expression. The glycerol stock for expression was inoculated into L-broth (LB) culture medium at a ratio of 1/200 and pre-cultured at 37° C. and 150 rpm. The main culture was inoculated at a ratio of 1/100 to the previous culture and cultured at 37 ° C. at 100 rpm until the optical density was reached. For expression, isopropyl-β-D-thiogalactopyranoside (IPTG) was treated at a final concentration of 0.5 mM in the culture medium to induce induction of the target protein, followed by induction at 37°C and 100 rpm. Conditions were incubated for 3 hours. The culture solution was centrifuged to obtain cultured cells, and the obtained cells were suspended by adding a lysis buffer. When completely suspended, the cells were disrupted by sonication at 4 ° C. The disrupted bacterial body fluid was centrifuged to remove non-disrupted bacterial cells and insoluble fractions, and only the supernatant was taken and filtered through a 0.45 μm filter. The centrifuged supernatant containing the filtered human-derived cells and the skin-permeable epidermal growth factor fusion protein is passed through an affinity column pre-equilibrated with the same lysis buffer to induce resin binding, and then bind. Contaminant proteins that were not identified were removed by elution. After washing thoroughly with washing buffer 1 corresponding to 10 times the amount of resin, human-derived cells and skin-permeable epithelial cells were eluted from the column with elution buffer, followed by ultra filtration. It was concentrated 20 times compared to the volume. The concentrated solution is subjected to gel filtration (Sephadez G-100, Buffer GF (20mM sodium phosphate buffer, pH7.5, NaCl 500mM)), and the target protein, human-derived cells and skin permeable epidermal growth factor fusion protein Only fractions were recovered. The recovered fraction was subjected to dialysis with 200-fold amount of NaCl-free buffer. 15% SDS-PAGE purification was most effectively separated from the elution fraction of the elution buffer containing 200 mM imidazole, and about 90-95% purification was secured (FIG. 1). As a result of the purification, the size of the human-derived cell and skin permeable epidermal growth factor fusion protein composed of 68 amino acids obtained by the above method was confirmed to be about 8.1 kDa as a result of SDS-PAGE analysis, confirming that the target protein was expressed and purified.

In addition, it was confirmed that the fusion protein of human-derived cells and skin permeable epidermal growth factor was expressed and purified with high purity even through a standard production process established through repetitive purification (FIG. 1).

The standard production process consisted of a pre-purification sample treatment protocol, a purification protocol based on affinity chromatography, and post-treatment processes such as dialysis and ultrafiltration of the sample fraction eluted through the column.

Experimental Example 1. Confirmation of cell stability of human-derived cell and skin-penetrating peptide and epidermal growth factor fusion protein

1.1. Human keratinocyte culture

The HaCaT cell line, a human keratinocyte, was cultured in a 37°C, 5% CO ₂ incubator for this study. When the degree of culture as much as 85 to 90% of the area of the culture vessel was shown, the cells were detached by trypsin treatment, counted, and subcultured at 5 × 10 ³ cells/cm ² . For cell culture, Dulbecco's Modified Eagle Medium (DMEM, GIBCO, Cat. No., 26140-079, USA), 100 U/ml penicillin, and 100 ug/ml streptomycin were added to Dulbecco's No. 11995-065, USA) was used. A 75 T-flask (NUNC, Cat. No. 156499, Danmark) was used for subculture, and a 96 well plate (NUNC, Cat. No., 142475, Danmark) was used for the cytotoxicity test.

1.2. Cell stability test of fusion protein using MTT test method

In order to confirm whether the human-derived cells and skin permeable epidermal growth factor fusion protein used in this study can show improvement effects without skin irritation even when used for a long time, cytotoxicity to human keratinocytes was tested. Human keratinocytes cultured in the manner described in Experimental Example 1.1 were counted using a heamacytometer at the same rate of 5 × 10 ³ cells/well in a 96 well plate and then dispensed. After culturing in DMEM containing 10% FBS for 24 hours and culturing up to 60 to 70% of the surface area of the culture vessel, replace it with FBS-free DMEM containing human-derived cells and skin permeable epidermal growth factor fusion protein and further culture for 24 hours. did After culturing, 20 ul of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) solution (2.5 mg/ml) was added and incubated for an additional 3 hours. Thereafter, the cell culture medium was completely discarded, and 200 ul of dimethyl sulfoxide (DMSO, Sigma D2650, USA) was added to each well, stirred, and then absorbance was measured at 540 nm by Enzyme-Linked Immunosorbent Assay (ELISA). The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the control group treated with pure water instead of the sample.

Cell proliferation rate (%) = (absorbance of test group/absorbance of control group) × 100

At the time when the initial culture 5 × 10 ³ cells/well cells were cultured by 60 to 70% of the surface area of the culture container, the human-derived cells and skin permeable epidermal growth factor fusion protein used in this study were added at different concentrations to control ( The cell stability compared to the untreated group) is shown in FIG. 2. As a result of treating human-derived cells and skin permeable epidermal growth factor fusion protein up to 2 ppm, it was confirmed that there was no cytotoxicity, and it was confirmed that there was a cell proliferation effect in a concentration-dependent manner (FIG. 3).

Experimental Example 2. Skin cell permeation enhancement effect of human-derived cell and skin penetrating peptide and epidermal growth factor fusion protein

Using human keratinocytes (HaCaT cells), the cell membrane permeability of the human-derived cells prepared in Example 1 and the skin permeable epidermal growth factor fusion protein of skin constituent cells was evaluated.

Human keratinocytes (HaCaT cells) were dispensed on chamber slides by 5×10 ³ cells and cultured in 10% FBS/DMEM medium for 24 hours. After treatment with epidermal growth factor (EGF), human-derived cells, and skin permeable epidermal growth factor fusion protein, cultured for 48 hours, washed with phosphate buffer (pH 7.4), and then fixed with 4% paraformaldehyde solution at room temperature for 15 minutes.

After washing twice with phosphate buffer solution (pH 7.4), a 0.25% Triton X-100 solution was added, reacted for 10 minutes, and washed once with phosphate buffer solution (pH 7.4). After blocking with 1% BSA solution for 1 hour, the cells were treated with EGF primary antibody diluted in 1% BSA solution and then reacted overnight at 4 °C. After washing once with a phosphate buffer solution (pH 7.4), a secondary antibody (Alexa 647) was attached. After mounting with mounting media, cell permeability was confirmed and quantified using EVOS ™ M5000.

As a result of measuring the fluorescence image, epidermal growth factor (EGF) showed a cell permeability of 12.36% compared to the control group (untreated group), whereas human-derived cells and skin permeable epidermal growth factor prepared in Example 1 In the fusion protein, a high cell permeability of 84.21% was confirmed (FIG. 4a).

Compared to epidermal growth factor (EGF), human-derived cell and skin permeable epidermal growth factor fusion protein showed about 6.8 times higher fluorescence intensity (FIG. 4b).

Accordingly, it was found that the human-derived cells and the skin permeable epidermal growth factor fusion protein showed very good skin cell permeability in human keratinocytes.

Experimental Example 3. Human keratinocyte wound regeneration effect of human-derived cells and skin-penetrating peptide and epidermal growth factor fusion protein

The degree of wound regeneration of the human-derived cells and the skin permeable epidermal growth factor fusion protein used in this study was investigated. As a simple test method, human keratinocytes were counted using a hemocytometer at the same rate of 2×10 ⁵ cells/well in a 6-well plate and then dispensed. After culturing in DMEM containing 10% FBS for 48 hours and culturing 90 to 100% of the surface area of the culture vessel, cross-shaped wounds are made on each cell using a sharp tool, and then human-derived cells and skin-penetrating type are applied to FBS-free DMEM. It was replaced with a culture medium containing epidermal growth factor fusion protein and further cultured for 24 hours. After that, data was obtained by comparing the increase in regeneration after wounding with the control group (untreated group) through a microscope.

The culture medium was replaced with a culture medium containing 1 ppm, 0.5 ppm, and 0.25 ppm of human-derived cells and skin permeable epidermal growth factor fusion protein, and cultured for 24 hours, and the degree of wound regeneration was confirmed through a microscope.

As a result, as shown in FIG. 5, it was confirmed that the wound regeneration ability significantly increased in a concentration-dependent manner compared to the control group (untreated group) in both the experimental groups treated with the human-derived cells and the skin permeable epidermal growth factor fusion protein. From these results, it was confirmed that the fusion protein of human-derived cells and skin permeable epidermal growth factor can be effectively used as a functional material capable of improving skin regeneration and skin condition.

Experimental Example 4. Moisturizing effect on human keratinocytes of human-derived cells and skin-penetrating peptide and epidermal growth factor fusion protein

Hyaluronic acid (HA) is a water-soluble polysaccharide biomolecular substance with high viscosity, moisturizing, and biocompatibility, and is involved in tissue water retention, storage and diffusion of growth factors and nutrients, and immune regulation. The decrease in the amount of hyaluronic acid with aging is considered to be one of the direct causes of wrinkle increase, decrease in elasticity, and decrease in moisture content of the skin by atrophy of the epidermis due to defects in the skin moisturizing barrier.

In human keratinocytes, it was confirmed by RT-PCR whether moisturizing power and anti-aging were improved by regulating the synthesis of hyaluronic acid by increasing the expression of hyaluronan synthase (HAS) gene.

4.1. RNA extraction and cDNA synthesis from human keratinocytes

The cultured cells were equally counted by 1×10 ⁵ cells/well in a 6 well plate using a hemacytometer and then dispensed. When the cells are cultured in DMEM containing 10% FBS for 24 hours and cultured as much as 60 to 70% of the surface area of the culture vessel, human-derived cells and skin permeable epithelium at concentrations of 2, 1.5, 1, 0.5, and 0.25 ppm used in this experiment It was cultured for 24 hours by replacing it with FBS-free DMEM containing cell growth factor fusion protein.

The cells after cell culture were lysed using QIAzol Lysis Reagent (QIAGEN, 79306, USA), and then 0.2 mL chloroform (Duksan, 67-66-3, Korea) was added and left at room temperature. Centrifugation at 12,000 rpm and 4℃ for 20 min separated the protein-containing supernatant from the mRNA-containing supernatant, and the supernatant was left at room temperature for 10 min after adding 0.5 mL isopropanol (Merck, 109634, Germany). Next, RNA was precipitated by centrifugation at 12,000 rpm and 4°C, washed with 75% ethanol, ethanol was removed, and dried at room temperature. The dried mRNA was dissolved in Diethylpyrocarbonate (DEPC, Biopure) water and used in the experiment, and the extracted RNA was used in the experiment only with a purity of 260 nm/280 nm ratio of 1.8 or higher using Nanodrop (Nanodrop, USA). For cDNA synthesis, Takara PrimeScript™ RT Master Mix (RR036A) was used. 1 μg RNA, 4 μl of 5×PrimeScript RT Master Mix, and RNase Free dH ₂ O were prepared in a total of 20 μl in a PCR tube, and then reacted at 37 ° C for 15 min and 85 ° C for 5 sec to synthesize cDNA.

4.2. RT-PCR

Using the cDNA synthesized in 4.1, expression of moisturizing factor-related genes in the fractions was confirmed through RT-PCR. The conditions of RT-PCR were as follows.

After reacting at 94°C for 5 minutes, denaturing at 94°C for 30 seconds, annealing at an appropriate temperature (55 ~ 65°C) for 30 seconds, and then extending at 72°C for 1 minute. After repeating 30 times, the final extension was performed in a PCR machine (BIO-GENER) at 72°C for 10 minutes. Each PCR product was loaded on a 1.5% agarose gel and analyzed through electrophoresis at 100 V for 30 minutes. After analysis, it was quantified as a graph through ImageJ program.

Primer base sequences are shown in Table 2. The moisturizing related gene HAS2 was used, and the housekeeping gene GAPDH was used as a control.

Gene Length
(bp) Forward Sequences (5’→3’) Reverse Sequences (5’→3’) HAS2 1004 GCT ACC AGT TTA TCC AAA CG
(SEQ ID NO: 3) GTG ACT CAT CTG TCT CAC CG
(SEQ ID NO: 4) GAPDH 226 GAA GGT GAA GGT CGG AGT
(SEQ ID NO: 5) GAA GAT GGT GAT GGG ATT TC
(SEQ ID NO: 6)

4.3. Confirmation of expression of moisturizing factor in human keratinocytes

In human keratinocytes, it was confirmed that the human-derived cell and skin permeable epidermal growth factor fusion proteins used in this study increased the expression of HAS2 at all concentrations (FIG. 6a). In particular, at 0.25 ppm and 0.5 ppm, the expression was increased by about 1.5 times compared to the control group (untreated group) (FIG. 6b). As a result, it was confirmed that the fusion protein of human-derived cells and skin permeable epidermal growth factor was effective in moisturizing.

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

Claims (15)

A fusion protein in which a cell-penetrating peptide and an epidermal growth factor are fused,
The fusion protein is composed of the sequence of SEQ ID NO: 2, the fusion protein. According to claim 1,
The cell-penetrating peptide is composed of the sequence of SEQ ID NO: 1, the fusion protein. delete A polynucleotide encoding the fusion protein according to claim 1 or 2. A recombinant vector comprising the polynucleotide of claim 4. As a transformant transformed by the recombinant vector of claim 5,
Wherein the transformants are excluding humans. Culturing the transformant of claim 6 in a culture medium; and recovering the fusion protein from the culture medium. A cosmetic composition for skin regeneration or skin condition improvement comprising the fusion protein according to claim 1,
The skin condition improvement is skin moisturizing, wrinkle improvement, skin aging prevention, skin damage recovery by ultraviolet rays, skin protection from ultraviolet rays or skin elasticity maintenance or wound improvement, cosmetic composition. delete delete A quasi-drug composition for preventing or treating skin diseases comprising the fusion protein according to claim 1,
The skin disease is one or more selected from the group consisting of skin wounds, skin scars and skin pigmentation, quasi-drug composition. delete delete A pharmaceutical composition for preventing or treating skin diseases comprising the fusion protein according to claim 1,
The skin disease is one or more selected from the group consisting of skin wounds, skin scars and skin pigmentation, the pharmaceutical composition. delete

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