KR102206808B1 - Novel cell-penetrating peptiedes and composition for skin improvement including the same - Google Patents

Abstract

The present application relates to novel cell-penetrating multifunctional peptides, a composition for drug delivery comprising the same, and a cosmetic composition for skin improvement. Since the novel peptides according to embodiments of the present application have cell permeability and skin permeability, the peptide itself and a peptide-active ingredient complex can be easily delivered into cells, in particular, into the skin. In addition, since the peptide itself has skin whitening, wrinkle alleviation, anti-inflammatory, antioxidant, atopic dermatitis alleviation, hair loss prevention, and hair growth promoting effects, the peptide can be used for skin improvement. The cell-penetrating multifunctional peptides consist of the amino acid sequence of SEQ ID NO: 1.

Description

A novel cell-penetrating peptide and a composition for improving skin comprising the same {NOVEL CELL-PENETRATING PEPTIEDES AND COMPOSITION FOR SKIN IMPROVEMENT INCLUDING THE SAME}

The present application relates to a novel cell-penetrating multifunctional peptide, a drug delivery composition comprising the same, and a cosmetic composition for improving skin.

In drug delivery, intracellular delivery of therapeutic molecules is very important. In intracellular organelles such as the cytoplasm, nucleus, ribosomes, and mitochondria, drugs must pass through cell membranes to exert their therapeutic effect. However, large hydrophilic therapeutic reagents such as proteins or nucleic acids cannot enter cells.

One of the alternatives to solve such a problem is to use a cell penetrating peptide (CPP), which is a peptide that generally exhibits cell membrane permeability. It is a method of delivery into the body, and such CPP is typically the HIV-1 TAT translocation domain (Green; M. and Loewenstein, PM (1988) Cell 55, 1179-1188), and the homeo domain of the Antennapedia protein (Joliot; A. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 1864-1868), etc., and various types other than these are known, and are being developed through continuous research. These CPPs commonly contain basic amino acids in a relatively high content, exhibit amphiphilicity, and generally consist of a sequence of 50 or less amino acids, and damage or destroy the cell membrane by inhibiting the stability of the cell membrane. It is known to exhibit cytotoxicity.

Intracellular delivery using a cell-permeable peptide allows modification of the peptide sequence than a system using cationic lipids or polyethyleneimine (PEI), so it can be transported to sub-domains within cells at various sites, It has the advantage of being able to transport various types of materials.

Accordingly, the inventors of the present application have developed a new cell-permeable peptide that has excellent skin-improving effect as well as cell permeation effect, and the new peptide and peptide-active ingredient complex is used for cell penetration, skin whitening, wrinkle improvement, antioxidant, anti-aging, atopy improvement. , It was confirmed that there are various skin improvement effects such as preventing hair loss and promoting hair growth.

The present application is to provide a novel cell penetrating multifunctional peptide, a drug delivery composition including the same, and a cosmetic composition for improving skin.

However, the problem to be solved by the present application is not limited to the problem mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The first aspect of the present application provides a cell-penetrating multifunctional peptide consisting of the amino acid sequence of SEQ ID NO: 1.

A second aspect of the present application provides a composition for drug delivery, including the cell-penetrating multifunctional peptide and a target drug.

A third aspect of the present application provides a cosmetic composition for improving skin, comprising the cell-penetrating multifunctional peptide.

A fourth aspect of the present application provides a pharmaceutical composition for improving skin, comprising the cell-penetrating multifunctional peptide.

Since the novel peptides according to the embodiments of the present disclosure have cell permeability, the peptide itself and the peptide-active ingredient complex can be easily delivered intracellularly, particularly intradermally and transdermally. In addition, the peptide itself has skin improvement effects such as skin whitening, wrinkle improvement, anti-inflammatory, antioxidant, atopy improvement, hair loss prevention, and/or hair growth promotion. Can be used for improvement purposes.

1 is a diagram showing information such as the sequence of a new peptide according to an embodiment of the present application.
2 is a view confirmed by a fluorescence microscope and flow cytometry (Flow cytometry) the novel peptide cell penetration effect of an embodiment of the present application.
3A is a diagram showing the structures of active ingredients baicalin, gallic acid, and nurukic acid in an embodiment of the present application, and B and C are a combination of Spep-1 and baicalin, gallic acid, or nurukic acid by HPLC and A diagram showing the results of MALDI analysis, and D is a diagram showing the structure of a complex of Spep-1 and baicalin, gallic acid, or nurukic acid.
4 is a diagram schematically showing the basic structure of a Franz cell device and a method for measuring skin permeability.
5 is a view showing the analysis conditions of baicalin and Spep-1 + baicalin complex using the Franz cell experimental method.
6 is a diagram showing the results of analysis of skin permeability of baicalin using the Franz cell test method.
7 is a view showing the results of skin permeability analysis of the Spep-1 + baicalin complex using the Franz cell test method.
Figure 8 is, in an embodiment of the present application, Spep-1, baicalin, gallic acid and kojiic acid, Spep-1 + baicalin (Spep-1 + B) in HaCaT cells. ), Spep-1+gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) are treated by concentration, and then the LDH assay results are shown.
9 is, in one embodiment of the present application, Spep-1, baicalin, gallic acid and kojiic acid, Spep-1 + baicalin (Spep-1 + B) in HaCaT cells. ), Spep-1+gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) are treated by concentration, and then the MTT assay results are shown.
Figure 10 is, in an embodiment of the present application, Spep-1, baicalin, gallic acid and kojiic acid, Spep-1 + baicalin (Spep-1) in MNT-1 cells. +B), Spep-1+gallic acid (Spep-1+G), and Spep-1+nurukic acid (Spep-1+K) were treated by concentration, and then LDH assay results were shown.
Figure 11 is, in an embodiment of the present application, Spep-1, baicalin, gallic acid and kojiic acid, Spep-1 + baicalin (Spep-1) in MNT-1 cells. +B), Spep-1+gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) were treated by concentration, and then the MTT assay result was shown.
12 is, in one embodiment of the present application, Spep-1, baicalin, gallic acid, and kojiic acid, Spep-1 + baicalin (Spep-1 + B) in B16F10 cells. ), Spep-1+gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) are treated by concentration, and then the MTT assay results are shown.
13 is, in one embodiment of the present application, Spep-1, baicalin, gallic acid and kojiic acid, Spep-1 + baicalin (Spep-1 + B) in B16F10 cells. ), Spep-1+gallic acid (Spep-1+G), and Spep-1+nurukic acid (Spep-1+K) according to concentrations.
14 is a view showing the melanin inhibitory effect according to concentrations of Spep-1, gallic acid, and Spep-1 + gallic acid (Spep-1 + G) in B16F10 cells according to an embodiment of the present application.
15 is, in one embodiment of the present application, Spep-1, baicalin, gallic acid, and kojiic acid, Spep-1 + baicalin (Spep-1 + B) in HDP cells. ), Spep-1+gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) are treated by concentration, and then the MTT assay results are shown.
Figure 16 is, in an embodiment of the present application, in HDP cells minoxidil, Spep-1, baicalin, gallic acid (Gallic acid) Spep-1 + baicalin (Spep-1 + B), Spep-1 + gallic acid A diagram showing the expression levels of β-catenin and KGF after treatment with (Spep-1+G).
17A and B are in an embodiment of the present application, after treatment with minoxidil, Spep-1, baicalin, and Spep-1 + baicalin (Spep-1 + B) by concentration in HDP cells, It is a diagram showing the expression level of VEGF.
FIG. 18 shows the expression level of IL-13 after treatment with Spep-1, Gallic acid, and Spep-1+gallic acid (Spep-1+G) in primary human fibroblasts in an embodiment of the present application It is a figure shown.

Hereinafter, exemplary embodiments of the present application will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present application. However, the present application may be implemented in various different forms and is not limited to the embodiments described herein. In addition, in the drawings, parts not related to the description are omitted in order to clearly describe the present application, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the present specification, when a part is said to be “connected” with another part, this includes not only the case that it is “directly connected”, but also the case that it is “indirectly connected” with another linker interposed therebetween. do.

Throughout the specification of the present application, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise specified. The terms "about", "substantially", etc. of the degree used throughout the present specification are used at or close to the numerical value when manufacturing and material tolerances specific to the stated meaning are presented. To assist, accurate or absolute figures are used to prevent unfair use of the stated disclosure by unscrupulous infringers. As used throughout the specification of the present application, the term "step (to)" or "step of" does not mean "step for".

Throughout the present specification, the term “combination(s) thereof” included in the expression of the Makushi format refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of the Makushi format, It means to include at least one selected from the group consisting of the above components.

Throughout this specification, the description of “A and/or B” means “A or B, or A and B”.

Hereinafter, embodiments and examples of the present application will be described in detail with reference to the accompanying drawings. However, the present application may not be limited to these embodiments and examples and drawings.

The first aspect of the present application provides a cell penetrating multifunctional peptide, consisting of the amino acid sequence of SEQ ID NO: 1.

The term "cell permeability" as used throughout the present specification means the ability or property of a peptide to penetrate the cell membrane and penetrate into the cell.

In one embodiment of the present application, the peptide may exhibit cell permeability or skin permeability.

In one embodiment of the present application, the peptide may be derived from a ribosomal protein, or may be derived from a protein having an rRNA binding function.

In one embodiment of the present application, the peptide may include 10 to 30 amino acids, and specifically may be composed of 11 or 12 amino acids, but is not limited thereto.

In one embodiment of the present application, the net charge of the peptide may be +5 to +10, and specifically, the net charge may be +6, but is not limited thereto.

In one embodiment of the present application, the peptide is prepared by a chemical peptide synthesis method known in the art, or after amplifying the gene encoding the peptide by PCR (polymerase chain reaction) or synthesizing by a known method, the expression vector It can be cloned and expressed to be prepared, but is not limited thereto.

In one embodiment of the present application, the peptide may be prepared using a human-derived peptide, a non-human-derived peptide, or a viral peptide, but may not be limited thereto.

In one embodiment of the present application, the peptide may further include some or all of a sequence derived from a conventionally known cell-penetrating peptide or a skin-penetrating peptide, but may not be limited thereto. For example, the conventionally known cell-penetrating peptide is not limited as long as it exhibits cell-penetrating activity, and specifically, dNP2, Penetratin, Tat, Transpotan, MAP, KALA, P1, MPG, Pep-1, Arg( 7,9,11), hCT, pVEC, SPEH, YARA, WLR, VP22, MTS, FHV coat, and may be selected from the group consisting of a combination thereof, but may not be limited thereto.

In the exemplary embodiment of the present application, the peptide may mean a peptide prepared using cells or an artificially synthesized peptide, but may not be limited thereto. For example, the peptide may be obtained as a recombinant (recombinants) by putting the DNA encoding the peptide into a suitable expression system, or may be artificially synthesized, but may not be limited thereto.

In one embodiment of the present application, the peptide may contain the amino acid sequence of SEQ ID NO: 1 or more times, and specifically, the peptide is the amino acid sequence of SEQ ID NO: 1 twice, three times, five times, seven times or ten times. It may be included, but is not limited thereto.

In one embodiment of the present application, the peptide may exhibit a skin improvement effect, and the skin improvement effect may include skin whitening, wrinkle improvement, anti-inflammatory, antioxidant, atopy improvement/treatment/relief, hair loss prevention, and hair growth promotion, etc. In particular, it may be one or more selected from the group consisting of skin whitening, anti-inflammatory, atopy improvement, hair loss prevention, and hair growth promotion, but is not limited thereto.

The peptide according to an embodiment of the present invention has cell-penetrating ability or skin-penetrating ability, thereby being able to transport or deliver various active substances such as low-molecular substances, and at the same time, itself is multifunctional, specifically skin whitening, wrinkle improvement, anti Characterized in that it exhibits various skin improvement effects such as inflammation, antioxidant, atopy improvement/treatment/mitigation, hair loss prevention, and promotion of hair growth, the peptide is highly useful for drugs, skin external preparations, or cosmetics.

In one embodiment of the present application, the peptide may be included in a pharmaceutical composition for improving skin, a cosmetic composition, a composition for external application for skin, a food composition, etc., but is not limited thereto.

A second aspect of the present application provides a composition for drug delivery, comprising the cell-penetrating multifunctional peptide and a target drug.

With respect to the composition for drug delivery according to the second aspect of the present application, detailed descriptions of parts overlapping with the first aspect of the present application have been omitted, but even if the description is omitted, the content described in the first aspect of the present application is the second aspect of the present application. The same can be applied to the side.

In one embodiment of the present application, the composition may be a composition for intradermal, intradermal or intracellular drug delivery.

In one embodiment of the present application, the composition may be a composition for enhancing cell permeability or a composition for enhancing skin permeability.

In one embodiment of the present application, the composition may be for enhancing cell permeability or skin permeability of the drug, or for delivering the drug intracellularly or intradermally.

In one embodiment of the present application, the drug used in the composition is not particularly limited as long as it is an active ingredient or a physiologically active substance capable of exhibiting pharmacological activity to regulate intracellular or intradermal activity by being delivered intracellularly or intradermally. However, as an example, it may be a compound, a protein, a nucleic acid, and the like, and as another example, a compound such as a charged polymer compound or a fluorescent compound; Proteins such as enzymes, ligands, and antibodies; Nucleic acid such as siRNA, plasmid, gene, genetic material, fat, carbohydrates, dyes, photosensitizers, anticancer agents, antibiotics, chemical compounds, and may include one or more selected from the group consisting of combinations thereof, but is not limited thereto. May not.

In one embodiment of the present application, the drug may be bound to the peptide through a linker, or may be directly linked. The linker is not particularly limited as long as it exhibits an effect of improving the activity of the conjugate, but as an example, when the compound is used as a drug, a compound capable of promoting binding of the drug; Alternatively, when a protein or nucleic acid is used as a drug, it may be a peptide or the like capable of promoting the binding of the drug.

In one embodiment of the present application, the linker may be cleaved or degraded by various biological and chemical actions such as enzyme action in cells or in the skin, and the peptide and the drug according to cleavage or decomposition of the linker They may be separated from each other within the desired cells or within the skin.

In one embodiment of the present application, the drug may be linked to the N-terminus or C-terminus of the peptide, and as long as it can improve cell permeability or skin permeability without inhibiting the pharmacological activity of the drug. It is not limited.

In one embodiment of the present application, the drug may be bound to the peptide to form a conjugate or complex, or may be mixed with each other to form a non-covalent conjugate, and does not inhibit the pharmacological activity of the drug. In addition, it is not limited thereto as long as it can improve cell permeability or skin permeability.

In one embodiment of the present application, the drug may be a substance that cannot be originally introduced into cells or into the skin, or cannot be introduced into cells or into the skin at an inherently useful rate.

In one embodiment of the present application, in the composition, the peptide and the drug are chemically bonded, or physically bonded, for example, covalently or non-covalently bonded to be processed in vivo or in vitro. It can be quickly and safely penetrated into cells. For example, the composition in which the peptide and the drug are combined may be introduced directly into cells without an endocytosis process, which is an existing intracellular absorption method, but may not be limited thereto.

In one embodiment of the present application, the composition may easily deliver the drug intracellularly, particularly, intradermally or transdermally, but may not be limited thereto.

A third aspect of the present application provides a cosmetic composition for improving skin, comprising the cell-penetrating multifunctional peptide.

With respect to the composition according to the third aspect of the present application, detailed descriptions of portions overlapping with the first aspect or the second aspect of the present application have been omitted, but contents described in the first aspect or the second aspect of the present application even if the description is omitted Is equally applicable to the third aspect of the present application.

In one embodiment of the present application, the skin improvement may be skin whitening, wrinkle improvement, anti-inflammatory, antioxidant, atopy improvement/treatment/relief, hair loss prevention, hair growth promotion, etc., specifically skin whitening, anti-inflammatory, atopic improvement It may be one or more selected from the group consisting of preventing hair loss, and promoting hair growth, but is not limited thereto.

In one embodiment of the present application, the composition may further include an active ingredient for skin improvement, and the active ingredient is skin whitening, wrinkle improvement, anti-inflammatory, antioxidant, atopy improvement, hair loss prevention, or hair growth promotion If it is a substance having a skin improvement effect such as an effect, it is not limited thereto, and specifically, Baikalin, Gallic acid. It may be one or more selected from the group consisting of kojic acid and a pharmaceutically acceptable salt thereof.

The term "pharmaceutically acceptable salt" as used herein refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bound by electrostatic attraction, and typically metal salts, organic salts It may be a salt with a base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt, and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine May be salts with the like; Salts with inorganic acids may be salts of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; Salts with organic acids may be salts of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine; Salts with acidic amino acids may be salts with aspartic acid and glutamic acid. Particularly preferred salts include inorganic salts such as alkali metal salts (e.g. sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g. calcium salts, magnesium salts, barium salts, etc.), when the compound has an acidic functional group therein, And organic salts such as ammonium salts, and when the compound has a basic functional group therein, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc., acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, There are salts with organic acids such as succinic acid, methanesulfonic acid, and p-toluenesulfonic acid.

In one embodiment of the present application, the composition may be that the peptide is bound to an active ingredient, and specifically, the active ingredient may be bound to the N-terminus or C-terminus of the peptide, and is directly bound or It may be bonded through a linker, but is not limited thereto.

In one embodiment of the present application, the linker may be cleaved or degraded by various biological and chemical actions such as enzyme action in cells or skin, and the peptide and the active ingredient according to cleavage or decomposition of the linker May be separated from each other within the desired cell.

In one embodiment of the present application, the composition may be that the peptides are mutually bonded to the active ingredient or are not bonded to each other, and are mixed with each other to form a non-covalent conjugate.

In one embodiment of the present application, the composition may be a cosmetic composition, and the formulation of the cosmetic composition is not particularly limited, and, for example, skins, lotions, essences, creams, packs, foundations, patches Types, hair tonics, microneedle patches, and makeup bases may have a formulation selected from, but may not be limited thereto.

In one embodiment of the present application, the cosmetic composition may be added with a material arbitrarily selected according to the formulation or purpose of use of the cosmetic, but may not be limited thereto. For example, purified water, oil, surfactant, moisturizing agent, higher alcohol, thickening agent, chelating agent, colorant, fatty acid, antioxidant, preservative, wax, pH adjuster, fragrance, etc. may be added, but may not be limited thereto. have.

In one embodiment of the present application, when the formulation of the cosmetic composition is a paste, cream, or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica , Talc or zinc oxide may be included, but may not be limited thereto.

In one embodiment of the present application, when the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be included as a carrier component, and in particular, a spray In the case, it may additionally include a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether, but may not be limited thereto.

In one embodiment of the present application, when the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be included as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, Ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan may be included, but the present invention may not be limited thereto.

In one embodiment of the present application, when the formulation of the cosmetic composition is a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorb Suspension agents such as non-ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be included, but may not be limited thereto.

In one embodiment of the present application, when the formulation of the cosmetic composition is a surfactant-containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl Taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester, etc., but are not limited thereto. May not.

A fourth aspect of the present application provides a pharmaceutical composition for improving skin, including the cell-penetrating multifunctional peptide.

With respect to the pharmaceutical composition according to the fourth aspect of the present application, detailed descriptions of parts overlapping with the first to third aspects of the present application have been omitted, but even if the description is omitted, it is described in the first aspect to the second aspect of the present application. The content can be equally applied to the fourth aspect of the present application.

In one embodiment of the present application, the composition may be that the peptide is bound to an active ingredient, and specifically, the active ingredient may be bound to the N-terminus or C-terminus of the peptide, and is directly bound or It may be bonded through a linker, but is not limited thereto.

In one embodiment of the present application, the linker may be cleaved or degraded by various biological and chemical actions such as enzyme action in cells or in the skin, and the cell-permeable peptide and the The active ingredients may be separated from each other within the desired cell.

In one embodiment of the present application, the composition may be that the peptides are not mutually bound to the active ingredient, but are mixed with each other to form a non-covalent conjugate.

In one embodiment of the present application, the active ingredient is not particularly limited as long as it can exhibit pharmacological activity and disease treatment effect to regulate intracellular or skin activity by being delivered intracellularly, and specifically, a drug or a physiologically active substance It may be, for example, nucleic acids, aptamers, genetic materials, fats, carbohydrates, dyes, photosensitizers, anticancer agents, including proteins, compounds, siRNAs, microRNAs, plasmids, genes including enzymes, ligands, antibodies, etc. It may include one or more selected from the group consisting of antibiotics, chemical compounds, and combinations thereof, but may not be limited thereto.

In one embodiment of the present application, the pharmaceutical composition is in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. It may be formulated and used as, but may not be limited thereto.

In one embodiment of the present application, when the pharmaceutical composition is formulated, it may be prepared using a diluent or excipient such as a generally used filler, extender, binder, wetting agent, disintegrant, or surfactant, but is not limited thereto. May not.

In one embodiment of the present application, solid formulations for oral administration include tablets, pills, powders, granules, or capsules, and such solid formulations include at least one excipient, such as starch, calcium carbonate (calcium carbonate), sucrose, lactose, glucose, malto-dextrin, or gelatin. In addition, for example, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used, but may not be limited thereto.

In one embodiment of the present application, the liquid formulation for oral administration includes a suspension, an inner solution, an emulsion, a syrup, etc., and various excipients, such as a wetting agent, in addition to water and liquid paraffin, which are commonly used simple diluents, Sweeteners, fragrances, preservatives, etc. may be included, but may not be limited thereto.

In one embodiment of the present application, the formulation for parenteral administration may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository, but may not be limited thereto. For example, as the non-aqueous solvent or suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used, but may not be limited thereto. For example, as the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used, but may not be limited thereto.

The pharmaceutical composition according to an embodiment of the present application may be a pharmaceutical composition or a quasi-drug composition.

The term “quasi-drug” used throughout the specification of this application refers to items that are less effective than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals. According to the report, quasi-drugs exclude items used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases of humans and animals, and products with minor or no direct action on the human body.

The quasi-drug composition of the present application consists of a body cleanser, a disinfectant cleaner, a cleaner, a kitchen cleaner, a cleaning cleaner, a toothpaste, a gargle, a wet tissue, a detergent, a soap, a hand wash, a hair cleaner, a hair softener, a humidifier filler, a mask, an ointment and a filter filler. It can be prepared in a formulation selected from the group, but is not limited thereto.

The pharmaceutical composition according to an embodiment of the present application may be a composition for external application for skin.

The term "external preparation" used throughout the specification of the present application is a preparation provided for external use, and includes external powders, tablets for external use, liquids for external use, ointments, warnings, suppositories, etc., and the external preparations for skin of the present invention are particularly effective for external use of the skin. Includes without limitation.

The external preparation for skin according to an embodiment of the present disclosure may be a parenteral administration formulation formulated in a solid, semi-solid or liquid form by adding a commercially available inorganic or organic carrier, excipient, and diluent. The preparation for parenteral administration may be a transdermal dosage form selected from the group consisting of drops, ointments, lotions, gels, creams, patches, sprays, suspensions, and emulsions, but is not limited thereto.

Carriers, excipients and diluents that may be included in the external preparation include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The composition for external application for skin by each formulation can be appropriately selected and blended by a person skilled in the art without difficulty depending on the formulation or purpose of use of other external preparations for skin, and in this case, synergistic effect when applied simultaneously with other ingredients. Can happen.

Hereinafter, the present application will be described in more detail by examples, but the present application is not limited thereto.

[Example]

Example 1: Design and selection of novel cell permeable peptides

In order to design a peptide having excellent cell penetration effect, novel peptides were derived and designed from numerous rRNA binding proteins and signaling proteins, and information such as the sequence of the designed peptides is shown in Table 1 and Fig. It was described in 1, and in the following examples and throughout the specification of the present application, Spep-1 was used in combination with Spep1 or SPEP1.

Next, the following experiment was performed to confirm whether the designed new peptide can penetrate cells.

Specifically, a Cellular uptake test was performed on HaCaT (Human Keratinocyte) cells using 5 uM of a novel peptide. At this time, PBS was used as a negative control, TAT peptide was used as a positive control, the nucleus was stained with DAPI, the new peptide was FITC, and the cell membrane was stained with Phalloidin to observe the cellular uptake of the peptide with a fluorescence microscope, and the cell penetration rate of the new peptide was observed. It was confirmed quantitatively through flow cytometry.

As a result, it was observed that Spep-1 was present in the cell membrane, and the result of quantitative analysis also showed that the peptide penetrated the cell membrane and was absorbed into the cell (FIG. 2). However, it was observed that Spep-4 was not excellent in cell membrane penetration and cell absorption effects.

Therefore, based on the above results, it can be seen that Spep-1 among the novel peptides designed based on AMP has remarkably excellent cell permeation effect and skin permeation effect.

Example 2: Confirmation of complex formation by binding of new peptide and active ingredient

In order to confirm that Spep-1, the novel peptide selected in Example 1, forms a complex with the compound, the following experiment was performed.

Specifically, Spep-1 was combined with baicalin, gallic acid, and kojiic acid, respectively, to form a complex, and the complex was analyzed through HPLC and MALDI, and the structure of the expected complex Was confirmed (Fig. 3).

As a result, it can be seen that the carboxy group or alcohol group of Baicalin, Gallic acid, and Kojiic acid can bind to the C-terminus or N-terminus of Spep-1-.

Example 3: Identification of the skin penetration effect of the novel peptide-active ingredient complex

In order to confirm the skin penetration effect of the complex in which the novel peptide Spep-1 selected in Example 1 and the active ingredient are combined, the following experiment was performed.

Specifically, the Franz cell experiment, which tests the permeability of substances using tissue, was used to confirm the skin permeability of the new peptide-active ingredient complex, and the degree of sample permeation according to the structure of the Franz cell was determined in 4 parts (recovery, contact tissue , Non-contact tissue, permeation) were divided into experiments (Fig. 4), and in order to confirm the effect of improving skin penetration of the new peptide, baicalin was used as a representative of the active ingredients. First, for the quantitative analysis of the sample, a calibration curve for the complex of baicalin and Spep-1 + baicalin was measured using the Shimadzu Nexera X2 LCMS-2020 System (FIG. 5). Next, in order to measure the amount of material passing through the skin using a Franz cell device, baicalin and Spep-1 + baicalin complex were treated for 3 hours, and the material contact portion of the tissue treated for 3 hours After dividing and pulverizing the non-contact part, the amount of the substance to be eluted and the amount of substance that penetrated the skin were measured.

As a result, when only baicalin was treated on the skin tissue, it was confirmed that there were almost no substances detected in the contact tissue, non-contact tissue, and the permeable portion, and baicalin was detected only in the recovery portion (Fig. 6), but the Spep-1 + baicalin complex In the case of treatment, it was confirmed that the complex was detected in all of the recovered, contacted, non-contacted, and permeated portions (FIG. 7), but baicalin itself did not penetrate the skin well, but the Spep-1 + baicalin complex It can be seen that the skin permeability is excellent.

In addition, in order to confirm the permeated amount of the Spep-1 + baicalin complex, the amount of the contact tissue eluted material, the amount of the non-contact tissue eluted material, and the amount of the permeated material, which are the amount of the sample permeated at the initial sample usage, were calculated. In addition, the amount of material reduced in the amount of sample used was calculated as 1.4mg, and as a result of calculating based on this, it was confirmed that 0.295mg of material was permeated. In order to calculate the transmittance, it was calculated using the equation {(contact tissue + non-contact tissue + transmission)/(reduction)}X100, and as a result of the calculation, 0.295mg/1.4mgX100=21.07% of the material was calculated to permeate.

Based on the above experimental results, it can be seen that the novel peptide of the present application can improve the skin permeability of the active ingredient and the drug, and Spep-1, the novel peptide of the present application, can deliver the drug or the active ingredient intradermally or intradermally. It can be seen that it can be used as a carrier.

Example 4: Confirmation of cytotoxicity of novel peptides and complexes thereof

The following experiments were performed to confirm the cytotoxicity of the novel peptides disclosed in Examples 1 and 2, active ingredients, baicalin, gallic acid, nurukic acid, and complexes thereof.

Specifically, targeting HaCaT cells and MNT-1 cells, Spep-1, baicalin, gallic acid and kojiic acid, Spep-1 + baicalin (Spep-1 + B), After treatment of Spep-1 + gallic acid (Spep-1 + G) and Spep-1 + nurukic acid (Spep-1 + K) by concentration, cytotoxicity was confirmed through LDH assay and MTT assay (FIG. 8 To Figure 11).

As a result, it can be seen that there is no toxicity to cells even if the peptides, active ingredients and complexes are treated at a concentration of 100 μM, and the novel peptides and complexes containing them of the present invention are not toxic even if treated to cells, skin, etc. , It can be used as an external preparation for the skin, a cosmetic, a microneedle patch, and an internal medicine.

Example 5: Confirmation of whitening effect of novel peptides and complexes thereof

The following experiments were performed to confirm the skin whitening effect of the novel peptides disclosed in Examples 1 and 2, active ingredients, baicalin, gallic acid, and nurukic acid, and their complexes.

First, targeting mouse melanoma cells, B16F10 cells, Spep-1, Baicalin, Gallic acid and Kojiic acid, Spep-1+Baicalin (Spep-1+B), and Spep. After -1+gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) were treated by concentration, cytotoxicity was confirmed through MTT assay, and as a result, at a concentration of 100 μM. Even after treatment, it was confirmed that cytotoxicity did not appear (FIG. 12).

Next, IBMX (3-isobutyl-1-methylxanthine) was treated in B16F10 cells, and Spep-1, baicalin, gallic acid and kojiic acid, Spep-1+ baicalin ( Spep-1+B), Spep-1+gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) were treated with concentrations (1, 12.5, 25, 50 and 100 μM) Thus, the inhibitory effect of melanin produced by IBMX was confirmed.

As a result, it was confirmed that Spep-1 inhibits melanin by itself, and it was confirmed that all of the Spep-1 complexes bound with baicalin, gallic acid, or nurukic acid showed melanin inhibitory effects. In addition, in the case of baicalin and gallic acid, it was confirmed that the melanin inhibitory effect was significantly increased when the complex conjugated with Spep-1 was treated than when the above components were treated alone (FIG. 13).

In addition, in order to confirm whether the above result is displayed even at a lower concentration than the above experiment, representatively, Spep-1, gallic acid, and Spep-1 + gallic acid (Spep-1 + G) are selected for each concentration (1, 2, 5 and 10 μM), the inhibitory effect of melanin produced by IBMX was confirmed. As a result, as in the above experiment, it was confirmed that Spep-1 inhibits melanin by itself, and in the case of gallic acid, it has a melanin inhibitory effect when the complex is treated with Spep-1 than when the component is treated alone. It was confirmed that is rising (Fig. 14).

In summarizing the above experimental results, the novel peptide of the present application has a skin whitening effect as it has a melanin inhibitory effect by itself, and the peptide-active ingredient complex has a remarkably improved skin whitening effect by the cell permeation function and whitening function of the peptide. It can be seen that it represents.

Example 6: Confirmation of the effect of promoting hair growth and inhibiting hair loss of novel peptides and complexes thereof

The following experiments were performed to confirm the hair growth promotion and hair loss inhibitory effects of the novel peptides disclosed in Examples 1 and 2, active ingredients, baicalin, gallic acid, and nurukic acid, and their complexes.

First, Spep-1, Baicalin, Gallic acid and Kojiic acid, Spep-1+Baicalin (Spep-1+B), and Spep-1 target HDP cells, which are dermal papilla cells. After treatment with +gallic acid (Spep-1+G) and Spep-1+nurukic acid (Spep-1+K) by concentration, cytotoxicity was confirmed through MTT assay. As a result, Spep-1 + baicalin It was confirmed that no cytotoxicity was observed except when the complex and gallic acid were treated at a concentration of 100 μM (FIG. 15).

Next, 10 μM of Spep-1, Baicalin, Gallic acid, Spep-1+Baicalin (Spep-1+B), and Spep-1+gallic acid (Spep-1 G) were added to HDP cells. After treatment at the concentration, expression levels of various factors (β-catenin, KGF) related to hair growth or hair loss prevention were confirmed through qPCR (FIG. 16). In addition, 20 μM of minoxidil (MXD) known as a hair loss treatment was used as a positive control.

As a result of the above experiment, when only Spep-1, a novel peptide of the present application was treated, the expression levels of β-catenin and KGF were improved, and the expression level was improved than that of minoxidil, a positive control, and the peptide itself alone could prevent hair growth and hair loss. I can see what it represents. In addition, it was confirmed that the expression levels of β-catenin and KGF were improved when the Spep-1 + baicalin complex and the Spep-1 + gallic acid complex were treated compared to the cases of Spep-1, baicalin, and gallic acid, respectively. It can be seen that the complex exhibits remarkably excellent hair growth and hair loss prevention effects.

In addition, in order to confirm the expression level of VEGF, a factor related to hair growth or hair loss prevention, Spep-1, baicalin, and Spep-1 + baicalin were treated at the same concentration as above, resulting in the Spep-1 + baicalin complex. In the case of treatment, it was confirmed that the expression level of VEGF was improved compared to the case of treatment with Spep-1 and baicalin, respectively (FIG. 17A).

In addition, in order to check whether the above result is displayed even at a lower concentration than the experiment, Spep-1, baicalin, and Spep-1 + baicalin were treated at each concentration (1, 2, 5 and 10 μM). Thus, the expression level of VEGF, a factor related to hair growth or hair loss prevention, was confirmed (FIG. 17B). As a result, as in the above experiment, it was confirmed that Spep-1 alone improves the expression level of VEGF, and in the case of baicalin, the complex treated with Spep-1 is more than when the component is treated alone. In the case, it was confirmed that the expression level of VEGF was further increased.

Taken together, the above experimental results show that the novel peptide of the present application has an effect of improving the expression level of hair growth-related factors by itself, and has an effect of promoting hair growth or preventing hair loss, and by the function of promoting cell permeation and hair growth, the peptide- It can be seen that the active ingredient complex exhibits remarkably improved hair growth promotion or hair loss prevention effect.

Example 7: Confirmation of anti-inflammatory effect of novel peptides and complexes thereof

The following experiments were performed to confirm the anti-inflammatory effect of the novel peptides disclosed in Examples 1 and 2, active ingredients, baicalin, gallic acid, and nurukic acid, and their complexes.

Specifically, primary human fibroblasts were seeded in a 6-well plate at a concentration of 3 x 10 ⁵ cells/well and cultured for 24 hours, followed by Spep-1, Gallic acid, or Spep. After -1 + gallic acid (Spep-1 + G) was treated at a concentration of 10 μM and incubated for 1 hour, TNFα (20 ng/mL) was treated for 4 hours to induce an inflammatory response. Next, the expression level of IL-13 (Interleukin 13), which is an inflammatory response factor, was confirmed through real time PCR (α-tubulin was used as a loading control) (FIG. 18 ).

As a result of the above experiment, it was found that the expression level of IL-13 was reduced when only Spep-1, the novel peptide of the present application was treated, and specifically, it was further reduced than when only gallic acid was treated, and the peptide itself showed an anti-inflammatory effect. I can. In addition, it was confirmed that the expression level of IL-13 was decreased when the Spep-1+gallic acid complex was treated than that of Spep-1 or gallic acid, and the complex showed remarkably excellent anti-inflammatory effect. have.

Therefore, it can be seen that the novel peptide of the present application has an anti-inflammatory effect as it has an effect of suppressing the expression level of an inflammatory response-related factor by itself, and the peptide-effective by the cell permeation function and anti-inflammatory function of the peptide. It can be seen that the component complex exhibits remarkably improved anti-inflammatory effects.

The foregoing description of the present application is for illustrative purposes only, and those of ordinary skill in the art to which the present application pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present application is indicated by the claims to be described later rather than the detailed description, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present application.

<110> S-Skin. Co., Ltd. Research & Business Foundation Sungkyunkwan University <120> NOVEL CELL-PENETRATING PEPTIEDES AND COMPOSITION FOR SKIN IMPROVEMENT INCLUDING THE SAME <130> DP20200002KR <150> KR 10-2019-0110214 <151> 2019-09-05 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Spep-1 <400> 1 Arg Gly Gly Arg Val Ser Arg Phe Lys Lys Lys Phe 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Spep-4 <400> 2 Lys Val Ala Lys Ala Leu Lys Ala Ala Lys 1 5 10

Claims (12)

Consisting of the amino acid sequence of SEQ ID NO: 1, a cell-penetrating multifunctional peptide.
The method of claim 1,
The peptide is a cell-penetrating multifunctional peptide that exhibits cell permeability or skin permeability.
The method of claim 1,
The peptide is a cell-penetrating multifunctional peptide that exhibits skin improvement effects,
The skin improvement is one or more selected from the group consisting of skin whitening, anti-inflammatory, atopy improvement, hair loss prevention, and hair growth promotion.
The method of claim 1,
The peptide will contain the amino acid sequence of SEQ ID NO: 1 more than once, cell penetrating multifunctional peptide.
The peptide of any one of claims 1 to 4; And
A composition for drug delivery containing the desired drug.
The method of claim 5,
The drug will be a compound, protein or nucleic acid, a composition for drug delivery.
The method of claim 5,
The drug is linked to the N-terminus or C-terminus of the peptide, a composition for drug delivery.
The method of claim 5,
The drug is bound through a linker with the peptide, the drug delivery composition.
As a cosmetic composition for improving skin, comprising the peptide of any one of claims 1 to 4,
The skin improvement is one or more selected from the group consisting of skin whitening, anti-inflammatory, atopy improvement, hair loss prevention, and hair growth promotion.
delete The method of claim 9,
The composition further comprises an active ingredient for skin improvement, skin improvement cosmetic composition.
The method of claim 11,
The active ingredient is at least one selected from the group consisting of baicalin, gallic acid, kojic acid, and pharmaceutically acceptable salts thereof. Cosmetic composition for skin improvement.

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