Novel skin-permeable peptide, composition for skin and mucous membranes containing the same, and use thereof

Abstract

The skin-penetrating peptide, its derivative or fragment thereof of the present invention effectively delivers skin bioactive molecules deep into skin tissue through an assembly or fusion method, thereby exhibiting not only excellent skin penetration activity but also excellent skin residual effect to maximize bioactivity. Therefore, the skin-penetrating peptide, its derivative or fragment thereof can be widely used as an active ingredient in pharmaceutical compositions for external skin applications and functional cosmetic compositions targeting skin tissue including mucous membranes.

Description

[Technical field]

[0001] The present invention relates to a composition for transdermal delivery of a physiologically active substance comprising a skin-permeable peptide, a derivative thereof, or a fragment thereof; a composition for skin permeation; a carrier for transdermal delivery; a carrier for skin permeation; a pharmaceutical composition; a quasi-drug composition; a cosmetic composition; a kit comprising the composition for transdermal delivery; methods for producing the composition for transdermal delivery, cosmetic composition, and quasi-drug composition; a method for transdermal delivery of a physiologically active peptide; and uses of transdermal delivery of a physiologically active peptide. [Background technology]

[0002] The skin, which occupies the largest part of the human body, is composed of the epidermis, dermis, and subcutaneous tissue, and performs various functions in response to the external environment. In particular, it is known that the molecular weight transmitted through the skin is about 500 Da or less due to the inherent properties of the stratum corneum that constitutes the skin barrier. However, it is known that even low molecular weight substances have low skin permeation efficiency, and the permeation efficiency of high molecular weight substances such as growth factors is even lower. Therefore, there is a great need for a safe method for the effective delivery of bioactive substances in the cosmetics and pharmaceutical industries.

[0003] Physical delivery systems such as microneedles can cause irreversible damage to the skin, and existing skin permeation substances are often lipid-soluble substances or polymers (Korean Patent Registration No. 10-2274435), which are likely to induce various side effects and unknown toxicities in the human body. Therefore, it is necessary to safely deliver physiologically active substances to the dermis using peptides, which are fragments of proteins present in the human body. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent No. 10-2274435 Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors have developed a skin-permeable peptide and completed the present invention. The biomembrane-permeable peptide technology developed in the present invention is a cutting-edge biotechnology that significantly improves the body's absorption rate of raw materials and increases their bioavailability, and is a next-generation new bio material platform technology that can fully exert its efficacy even when added to functional cosmetics and advanced pharmaceuticals in very small amounts. Therefore, we intend to apply this to the development of skin-permeable cosmetics and pharmaceuticals. [Means for solving the problem]

[0006] One object of the present invention is to provide a composition for transdermal delivery of a physiologically active substance, which comprises a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0007] Another object of the present invention is to provide a composition for transdermal penetration of a physiologically active substance, which comprises the above-mentioned skin-penetrating peptide, a derivative thereof, or a fragment thereof.

[0008] Another object of the present invention is to provide a carrier for transdermal delivery of a physiologically active substance, which comprises the above-mentioned skin-penetrating peptide, a derivative thereof, or a fragment thereof.

[0009] Another object of the present invention is to provide a carrier for transdermal penetration of a physiologically active substance, which comprises the above-mentioned skin-penetrating peptide, a derivative thereof, or a fragment thereof.

[0010] Another object of the present invention is to provide a cosmetic composition containing the above-mentioned composition for transdermal delivery.

[0011] Another object of the present invention is to provide a quasi-drug composition containing the above-mentioned composition for transdermal delivery.

[0012] Another object of the present invention is to provide a pharmaceutical composition for external application to the skin or mucosa, which contains the above-mentioned composition for transdermal delivery.

[0013] Another object of the present invention is to provide a kit comprising the above-mentioned composition for transdermal delivery and a physiologically active substance.

[0014] Another object of the present invention is to provide a method for producing the above-mentioned composition for transdermal delivery.

[0015] Another object of the present invention is to provide a method for improving skin using the above-mentioned composition for transdermal delivery.

[0016] Another object of the present invention is to provide a method for preventing or treating skin or mucosal diseases using the above pharmaceutical composition.

[0017] Another object of the present invention is to provide a method for producing the above-mentioned cosmetic composition and quasi-drug composition.

[0018] Another object of the present invention is to provide a method for transdermal delivery of a physiologically active peptide using a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0019] Another object of the present invention is to provide an application for transdermal delivery of a physiologically active peptide using a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0020] Another object of the present invention is to provide an application for skin permeation of a physiologically active peptide using a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof. Effect of the Invention

[0021] The skin-penetrating peptide, its derivative or fragment thereof of the present invention effectively delivers skin bioactive molecules deep into skin tissue through an assembly or fusion method, thereby exhibiting not only excellent skin penetration activity but also excellent skin residual effect to maximize bioactivity. Therefore, the skin-penetrating peptide, its derivative or fragment thereof can be widely used as an active ingredient in pharmaceutical compositions for external skin applications and functional cosmetic compositions targeting skin tissue including mucous membranes. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows the results of a Franz diffusion cell assay performed to analyze the skin permeation effect of the peptides of SEQ ID NOs: 1 to 8 on physiologically active substances, as compared with known peptides, and quantified by HPLC. [Diagram 2] FIG. 2 is a diagram showing the results of Franz diffusion cell assay at different time points, quantified by HPLC, to analyze the skin permeation effect of the skin-permeable peptide of the present invention on a physiologically active substance. [Diagram 3] FIG. 1 shows the results of a Franz diffusion cell assay performed to analyze the skin permeation effect of the skin-penetrating peptide of the present invention on bioactive substances with various molecular weights, quantified using a Fluorescence Microplate Reader. [Figure 4] FIG. 1 shows the results of a Franz diffusion cell assay performed to analyze the skin permeation effect of the skin-penetrating peptide of the present invention on Liposomes, quantified using a Fluorescence Microplate Reader. [Diagram 5] FIG. 1 shows the results of an evaluation of efficacy improvement based on the skin permeation effect of the skin-permeable peptide of the present invention on a physiologically active substance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will be described in detail below. Meanwhile, the description and embodiment of one aspect disclosed in the present invention can be applied to the description and embodiment of other aspects for common matters. Furthermore, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, the following specific description is not considered to limit the scope of the present invention.

[0024] One aspect of the present invention provides a composition for transdermal delivery of a physiologically active substance, comprising a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0025] In the present invention, "skin permeation" means biological membrane permeation, and "skin-permeable peptide" means a peptide having biological membrane permeability. The skin-permeable peptide may have cell permeability and biological membrane permeability, specifically, skin and / or mucous membrane permeability, but is not limited thereto.

[0026] The skin-penetrating peptide of the present invention may include a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0027] The present invention can efficiently deliver skin bioactive molecules that are difficult to transmit through the skin due to their molecular weight or the inherent properties of the stratum corneum into cells and into the skin.

[0028] The skin-penetrating peptide of the present invention is capable of delivering a bioactive substance into skin cells or the dermis layer of the skin through a method of assembling or fusing a transdermal delivery composition, a transdermal delivery system, and a skin-penetrating peptide, which contain a skin bioactive molecule having improved cell and skin permeability compared to existing skin-penetrating peptides.

[0029] The skin-penetrating peptide, its derivative or its fragment of the present invention effectively delivers skin bioactive molecules deep into skin tissue through an assembly or fusion method, thereby exhibiting not only excellent skin penetration activity but also excellent skin residual effect, maximizing bioactivity, and therefore the present invention is used as a component of a composition targeting skin tissue, including mucous membranes.

[0030] In the examples of the present invention, it was confirmed that SEQ ID NO:5 to SEQ ID NO:8 and their derivatives have remarkable skin permeability (Examples 1 to 6).

[0031] In the present invention, the term "derivative" refers to a derivative of a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, and specifically may be a peptide having an amino acid sequence in which a functional group has been added to the amino acid sequence of SEQ ID NO:5 to SEQ ID NO:8, or in which a portion of the amino acid sequence has been deleted, modified, substituted or added, but is not limited thereto.

[0032] Specifically, the derivative in the present invention may be any one or more selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, but is not limited thereto.

[0033] Specifically, the skin-penetrating peptides and derivatives thereof of the present invention consisting of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 may have a sequence as shown in Chemical Formula 1 below and have skin-penetrating properties, but are not limited thereto.

[0034] [C1] [X1 - X2 - Leu - Phe - X3 - X4 - Ile - Leu - X5 - Tyr - Leu - X6]

[0035] In the above formula 1, X1 to X6 may be selected from basic amino acids, and may be selected from arginine, lysine, leucine, and phenylalanine. Preferably, the skin-penetrating peptide and its derivatives composed of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 may have the sequence of the above formula 1, and the derivative of the present invention may be composed of the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:4, but is not limited thereto.

[0036] SEQ ID NO: 1: H2N-[Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Lys]-CO2H, SEQ ID NO: 2: H2N-[Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H, SEQ ID NO: 3: H2N-[Arg-Phe-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H, SEQ ID NO: 4: H2N-[Arg-Arg-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Phe]-CO2H, SEQ ID NO: 5: H2N-[Arg-Lys-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H, SEQ ID NO: 6: H2N-[Lys-Arg-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H, SEQ ID NO: 7: H2N-[Lys-Arg-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H, and SEQ ID NO: 8: H2N-[Arg-Lys-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H

[0037] Specifically, the above-mentioned derivative may be one in which an amino acid corresponding to any one position selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th, and 12th positions from the N-terminus of a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 is substituted with another basic amino acid.

[0038] Amino acid substitutions may generally occur based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residue. Specifically, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine (R), lysine (K), and histidine (H), and negatively charged (acidic) amino acids include glutamic acid (E) and aspartic acid (D); among amino acids having uncharged side chains, nonpolar amino acids include arginine (R), lysine (K), and histidine (H). The polar or hydrophilic amino acids include serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N) and glutamine (Q). Among the above non-polar amino acids, aromatic amino acids include phenylalanine, tryptophan and tyrosine.

[0039] In the present invention, "substitution with another basic amino acid" is not limited as long as it is a substitution of the amino acid before substitution with another basic amino acid. That is, it may be a substitution of the amino acid before substitution at any one position selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th, and 12th positions from the N-terminus of the skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8 with another basic amino acid, specifically, it may be a substitution of the amino acid at any one position selected from the group consisting of the above-mentioned X1, X2, X3, X4, X5, and X6 positions with another basic amino acid. Specifically, the basic amino acid may be selected from histidine, lysine, and arginine, and may be lysine or arginine, but is not limited thereto.

[0040] For the purposes of the present invention, the derivative of the skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 may be any one or more selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:4, but is not limited thereto.

[0041] In the present invention, the term "fragment" refers to a partial sequence of a peptide having a specific sequence. The fragment of the present invention may be a partial sequence of a skin-penetrating peptide or a derivative thereof consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, and may have skin permeability similar to the above-mentioned skin-penetrating peptide or a derivative thereof.

[0042] The skin-penetrating peptide, its derivative, or its fragment, which is composed of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, may be derived from a natural product, but is not limited thereto, and may include, without limitation, any sequence having the same activity as the above-mentioned skin-penetrating peptide, its derivative, or its fragment.

[0043] Although the skin-penetrating peptide of the present invention is described as a skin-penetrating peptide, a derivative thereof, or a fragment thereof, which is composed of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, it is obvious to those skilled in the art that the skin-penetrating peptide of the present invention corresponds to the skin-penetrating peptide of the present invention if it has the same or corresponding activity as the skin-penetrating peptide, a derivative thereof, or a fragment thereof, which is composed of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8. As a specific example, the skin-penetrating peptide of the present invention may be a skin-penetrating peptide, a derivative thereof, or a fragment thereof, which is composed of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, or a peptide composed of an amino acid sequence having 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto.

[0044] In the present invention, even if a peptide is described as "a peptide containing an amino acid sequence represented by a specific SEQ ID NO," "a peptide consisting of an amino acid sequence represented by a specific SEQ ID NO," or "a peptide having an amino acid sequence represented by a specific SEQ ID NO," it is obvious that a peptide having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added can also be used in the present invention, so long as it has the same or corresponding activity as a peptide consisting of the amino acid sequence represented by the SEQ ID NO. For example, a peptide having an addition of a sequence that does not change the function of the peptide, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus and / or C-terminus of the above amino acid sequence.

[0045] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of the peptide.

[0046] The peptide of the present invention can be prepared by, but is not limited to, forming successive amide bonds between one or more amino acids or appropriately protected amino acids in an amino acid backbone fixedly attached to a solid phase. In addition, the peptide can be inserted, substituted, or deleted with other amino acids within the scope of the present invention, so long as the stability is not significantly reduced.

[0047] In addition, the skin-permeable peptide of the present invention can be prepared by further binding a known cell permeable peptide that promotes intracellular transport to the C-terminus or N-terminus. For example, the known cell permeable peptide may be TAT peptide (Arg-Lys-Lys-Arg-Arg-Tyr-Arg-Arg-Arg) and Tat-PTD peptide (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg: Tat PTD), but the present invention is not limited thereto, and any cell permeable peptide known in the art may be used as long as it does not inhibit the activity of the present invention.

[0048] The peptides and compounds of the present invention may also be prepared in the form of metal complexes, the metal being selected from, but not limited to, copper, magnesium, calcium, iron, zinc, nickel, silver, germanium and gallium, preferably, but not limited to, copper.

[0049] On the other hand, the peptide of the present invention may exist in the form of a salt. The salt form usable in the present invention may be one that is prepared during the final isolation and purification of the compound or by reacting an amino group with a suitable acid. For example, the acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, sucrose, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesitylene, etc. The acid addition salt may be, but is not limited to, sulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate and undecanoate. Examples of acids used to form acid addition salts may be, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. Preferably, the peptide of the present invention can be prepared in the form of a trichloroacetate or acetate.

[0050] The amino or carboxyl groups of the amino acids used to prepare the peptides contained in the compositions of the present invention may be protected by a suitable protecting group. The protected amino acid may be attached to a solid support, and the reaction may be carried out in solution by adding the next amino acid under suitable conditions to form an amide bond. Alternatively, the protecting group may be completely removed before adding the amino acid protected with a suitable protecting group. After all amino acids have been linked by the desired method, they may be separated successively or simultaneously from the free remaining protecting groups and the free solid support to obtain the final peptide of interest.

[0051] The most preferred synthesis method for preparing the peptide compound of the present invention is a solid-phase peptide synthesis method using a solid-phase polymer lipid, and the α-amino group of the peptide prepared by the above method can be protected by an acid- or base-sensitive functional group. The protecting group of the amino acid must be stable under peptide condensation reaction conditions and easily removable without breaking the extending peptide chain or without racemizing any chiral center contained therein. Suitable protecting groups may therefore be 9-fluorenylmethyloxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (α,α)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, etc., and other suitable protecting groups known in the art for such purposes may also be used within the scope of the present invention.

[0052] The most preferred protecting group for the amino acids used in the peptide synthesis of the present invention is the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.

[0053] In particular, the protecting groups for the amino acid residues used in the peptide synthesis of the present invention are t-butyl (t-Bu) for N-methylglutamic acid; t-butoxycarbonyl (Boc) for lysine; and t-Butyl (t-Bu) for threonine and allothreonine, t-butyl (t-Bu); and for cysteine, trityl (Trt), although the invention is not limited thereto.

[0054] In solid phase peptide synthesis methods, the C-terminal amino acid can be attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are preferably materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions and insoluble in the media used, and may be, for example, 2-chlorotrityl chloride resin, rink amid or rink amid MBHA resin.

[0055] In particular, preferred solid supports for C-terminal peptides may be 2-chlorotrityl chloride, rink amid or rink amide 4-methylbenzylhydrylamine resin available from Novabiochem Corporation.

[0056] C-terminal amides can be prepared by cleaving the amides with 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOP) in a solvent such as dichloromethane, N-methylpyrrolidone (NMP) or DMF at a temperature of 10° C. to 50° C., preferably at 30° C., for 1 to 24 hours. The carboxylic acid can be activated with N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU) or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of OPCI, and then condensed (bound, coupled) to a resin or solid support through condensation.

[0057] When the solid support is Rink Amide 4-methylbenzylhydrylamine resin, the Fmoc functionality, as a preferred protecting group, is cleaved using an excess of a secondary amine solution, preferably 20% piperidine in DMF, prior to condensation with the C-terminal amino acid. Preferred reagents used to condense the desired amino acid to the deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin include condensation reaction reagents such as N-methylmorpholine (NMM), 1-hydroxybenzotriazole (HOBt) and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC) in DMF solvent for a suitably protected amino acid.

[0058] The condensation of consecutive amino acids in the present invention can be carried out directly by using an automatic peptide synthesizer widely known in the related technical field or manually. As a preferred synthetic reaction condition, an α-amino acid protected with an Fmoc group is deprotected by treating it with a secondary amine solution, preferably piperidine, and then sufficiently washed with an excess amount of solvent, and each of the other protected amino acids to be condensed is subsequently added in a 3- to 7-fold molar excess, and the reaction can be carried out preferably in a DMF solvent.

[0059] In the final step of the peptide synthesis using the solid phase resin of the present invention, the peptide to be obtained can be removed from the resin continuously or in one operation, and the protecting groups protecting the amino acid residues can be deprotected. The conditions for removing the peptide from the resin and deprotecting the protecting groups present in the residues can generally be obtained by treating a dichloromethane mixed cocktail solution composed of a cleavage reagent cocktail that cleaves the bond between the resin and the peptide, for example, trifluoroacetic acid (TFA), triisopropylsilane (TIS), thioanisole, water, or ethanedithiol (EDT). The mixed solution thus obtained can be treated with an excess of diethyl ether solvent stored in a refrigerator to produce a precipitate. The precipitate thus obtained is centrifuged to completely precipitate, and excess trifluoroacetic acid, triisopropylsilane, thioanisole, water, and ethanedithiol are primarily removed, and the above procedure is repeated two or more times to obtain a solidified precipitate. At that time, the completely deprotected peptide salt can be separated and purified using a mixed solvent composed of water and acetonitrile solvent and reversed-phase high performance liquid chromatography (HPLC). The separated and purified peptide solution can be completely concentrated and dried using freeze drying to obtain a solid peptide.

[0060] The present invention provides a composition for transdermal delivery of a biologically active substance.

[0061] In the present invention, the term "biologically active substance" is a general term for substances that have a physiological effect in the body. The above-mentioned bioactive substances may be hydrophilic, hydrophobic, or poorly soluble, and include, without limitation, natural products, chemical compounds, proteins, peptides, amino acids, nucleic acids, lipids, liposomes, and polymers, as long as they have a physiological effect. In addition, the bioactive substances of the present invention may include, without limitation, a single substance used alone, a combination with other bioactive substances, a combination with a carrier, and a combination with a known skin-penetrating peptide.

[0062] In one embodiment, the bioactive substance of the present invention may be a major component of a drug, quasi-drug, and / or cosmetic that is applied to the skin, transdermal, and / or mucous membranes.

[0063] The physiologically active substances of the present invention can perform one or more physiological actions, such as improving skin, promoting health, and preventing or improving disease, and are included without limitation as long as they are used together with the skin-penetrating peptide of the present invention, its derivative, or its fragment.

[0064] In one embodiment, the bioactive substance may be at least one selected from the group consisting of lidocaine, caffeine, collagen, hyaluronic acid, and liposomes, but is not limited thereto.

[0065] The skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 5 to 8, its derivative, or its fragment may be directly or indirectly linked to a physiologically active substance, but is not limited thereto.

[0066] In one embodiment, the skin-penetrating peptide, derivative or fragment thereof, and the bioactive substance, which are composed of any one of the amino acid sequences of SEQ ID NOs: 5 to 8 of the present invention, may be directly linked to each other, linked via a linker, or may further include another protein moiety, but is not limited thereto. The linking method of the present invention may be any method practiced in the art, as long as it does not change the structure or activity of the skin-penetrating peptide, derivative or fragment thereof, which are composed of any one of the amino acid sequences of SEQ ID NOs: 5 to 8, and the bioactive substance to be linked. The linker may be, but is not limited to, a peptidic linker or a non-peptidic linker composed of 1 to 20 amino acids.

[0067] In one embodiment, the skin-penetrating peptide of the present invention, which is composed of any one of the amino acid sequences of SEQ ID NOs: 5 to 8, its derivative, or its fragment may be linked to a physiologically active substance via a non-covalent bond (e.g., ionic bond, hydrogen bond, van der Waals bond, etc.), but is not limited thereto.

[0068] In one embodiment, the skin-penetrating peptide, derivative or fragment thereof, which is composed of any one of the amino acid sequences of SEQ ID NOs: 5 to 8 of the present invention, may be linked (e.g., covalently bonded) to a bioactive substance, but is not limited thereto. The skin-penetrating peptide, derivative or fragment thereof and the bioactive substance may be directly linked to each other, linked via a linker, or may further include another protein moiety, but is not limited thereto.

[0069] In one embodiment, the skin-penetrating peptide may self-assemble or self-fuse with a bioactive substance.

[0070] In one embodiment, the skin-penetrating peptide may be, but is not limited to, one or more molecules surrounding or fused to a bioactive substance.

[0071] In the present invention, "transdermal delivery" means delivery of a physiologically active substance through a biological membrane. The composition for transdermal delivery of the present invention may deliver a physiologically active substance through cell delivery and biological membrane delivery, specifically, through the skin and / or mucosa, but is not limited thereto.

[0072] The composition for transdermal delivery of the present invention may increase the permeability of a physiologically active substance through at least one selected from the group consisting of skin and mucous membrane, but is not limited thereto.

[0073] The composition for transdermal delivery of the present invention may further contain, if necessary, a carrier, an excipient, a diluent, an antioxidant, and / or a buffer, but is not limited thereto.

[0074] In one embodiment, the carrier may be, but is not limited to, a liposome.

[0075] In the present invention, the term "liposome" refers to a hollow structure formed by a phospholipid bilayer in an aqueous solution. The liposome may contain a physiologically active substance in the hollow structure, but is not limited thereto.

[0076] In one embodiment, the liposome may be one that increases the skin permeability and / or transdermal delivery ability of a physiologically active substance, which is a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof, but is not limited thereto.

[0077] In the examples of the present invention, it was confirmed that the present invention increases the skin permeability and transdermal delivery of bioactive substances, regardless of their properties, sizes, and types, including lidocaine, caffeine, 3, 30, and 300 kda collagen, and 800 kda hyaluronic acid (HA). In addition, in one example of the present invention, it was confirmed that the skin-permeable peptide of the present invention increases the skin permeability and transdermal delivery activity of liposomes (Examples 1 to 6). This suggests that the skin-permeable peptide of the present invention increases the skin permeability and transdermal delivery activity of liposomes containing bioactive substances.

[0078] Another aspect of the present invention provides a composition for skin permeation of a physiologically active substance, comprising a skin-permeable peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0079] Any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, the skin-penetrating peptides, derivatives, fragments, physiologically active substances, and skin permeation are as described above.

[0080] In one embodiment, the composition for skin permeation of the present invention may increase the permeability of a biologically active substance through at least one selected from the group consisting of skin and mucous membrane, but is not limited thereto.

[0081] The skin permeation composition of the present invention may further contain, if necessary, a carrier, an excipient, a diluent, an antioxidant, and / or a buffer, but is not limited thereto.

[0082] Another aspect of the present invention provides a carrier for transdermal delivery of a physiologically active substance, comprising a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0083] Another aspect of the present invention provides a skin-penetrating carrier for a physiologically active substance, comprising a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof.

[0084] Any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, the skin-penetrating peptides, derivatives, fragments, physiologically active substances, transdermal delivery, and skin permeation are as described above.

[0085] In the present invention, the term "carrier" means that any substance or component, such as a composition, can be carried, and may be mixed with a carrier, an impregnating agent, a medium, etc. In addition, the composition, which is an example of the carrier, may be applied and delivered to the skin through an application means such as hands, a puff, a tip, or a brush.

[0086] The above-mentioned compositions for transdermal delivery, compositions for skin penetration, carriers for transdermal delivery, and carriers for skin penetration may be those contained in pharmaceutical compositions, quasi-drug compositions, cosmetic compositions, etc., but are not limited thereto.

[0087] For the purposes of the present invention, the above-mentioned carriers for transdermal delivery and carriers for skin permeation of physiologically active substances may contain skin-permeable peptides consisting of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, derivatives thereof, or fragments thereof.

[0088] In one embodiment, the composition for transdermal delivery, the composition for skin penetration, the carrier for transdermal delivery, and the carrier for skin penetration of the present invention may be for use as an external preparation for skin.

[0089] In the present invention, the term "external skin preparation" refers to a preparation that is applied to the skin or mucosa. In the present invention, the external preparation is not limited in shape, etc., as long as it is a preparation that is applied to the skin or mucosa, and the external preparation can be classified into ointments, creams, lotions, gels, etc., but is not limited thereto.

[0090] Specifically, the ointment may be a semi-solid external preparation in which the main ingredient to be applied to the skin or mucosa is dissolved or dispersed in a base. The base is divided into an oily ointment or a water-soluble ointment depending on the ingredient. An ointment using an oily base can soften the skin or mucosa, has excellent sealing properties, and is suitable for application to wounds, but has the disadvantage of being difficult to remove. Due to its fat-soluble property similar to that of the skin or mucosa and its skin softening effect, it is an advantageous dosage form when locally permeating ingredients that are difficult to permeate through the skin.

[0091] The ointment is mainly formed of an oil phase such as oil, and has excellent moisturizing properties and good permeability of fat-soluble ingredients, but has a drawback of being sticky. The cream may be a semi-solid external preparation emulsified into water-in-oil, or may be a dosage form in which the oil phase and the aqueous phase are mixed to improve the stickiness of the ointment. In the emulsification process, the oil phase and the aqueous phase can be mixed using a surfactant or through a special process. In addition, the cream may be a dosage form that is easier to apply and remove than the ointment, and the lotion may be a dosage form that has properties more similar to water than the cream, and may be an external preparation in which the main ingredient is dissolved, emulsified, or finely dispersed in an aqueous liquid. The main ingredient, additives, and purified water may be used to form a solution, suspension, or emulsion to make the whole homogeneous. The gel agent may be a semi-solid external preparation made of organic molecules with a large molecular weight that have been permeated with a liquid. The gel agent may be a dispersion of a gelling agent in an aqueous solution, and a gelling agent such as carbomer or synthetic polymer may be used. Various types of topical preparations are used depending on the active ingredient, purpose of use, method of use, etc., and the topical preparation of the present invention is not limited thereto.

[0092] Another aspect of the present invention provides a cosmetic composition comprising the transdermal delivery composition of the present invention.

[0093] The composition for transdermal delivery is as described above.

[0094] The cosmetic composition may be for improving skin, preventing or improving diseases by utilizing the physiological functions of the physiologically active substances, but is not limited thereto.

[0095] In one embodiment, the cosmetic composition may be for improving skin, but is not limited thereto.

[0096] In one embodiment, the cosmetic composition may be for, but is not limited to, skin regeneration, wound healing, anti-aging, wrinkle improvement, whitening, inflammation relief, antibacterial, moisturizing, moisture increase, and antioxidant purposes.

[0097] In one embodiment, the cosmetic composition may be for preventing or improving a skin disease, but is not limited thereto.

[0098] The skin disease refers to a disease in which abnormal symptoms such as inflammation, erythema, thickening, fibrosis, keratinization, etc. appear on the skin due to genetic factors, physiological factors, environmental factors, etc. Examples of the skin disease include, but are not limited to, atopic dermatitis, allergies, psoriasis, seborrheic dermatitis, contact dermatitis, erythematous lupus, acne, warts, viral infection, papular urticaria, etc.

[0099] In this application, "prevention" refers to any action that inhibits or delays the onset or progression of a disease (eg, a skin disease).

[0100] In the present invention, "improvement" refers to any action that at least reduces a parameter associated with the alleviation or treatment of a condition, for example, the severity of a symptom. In the present invention, the cosmetic composition may be prepared in a dosage form selected from the group consisting of a solution, an ointment for external use, a cream, a foam, a nutritious lotion, a softening lotion, a perfume, a pack, a softening lotion, a milky lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray, but is not limited thereto.

[0101] The cosmetic composition of the present invention further contains one or more cosmetically acceptable carriers that are commonly incorporated into skin cosmetics, and may appropriately contain, as typical ingredients, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., but is not limited thereto.

[0102] The cosmetically acceptable carrier contained in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

[0103] When the dosage form of the present invention is an ointment, paste, cream or gel, the carrier component may be, but is not limited to, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. These may be used alone or in combination of two or more kinds.

[0104] When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. are used as carrier components, and particularly when the dosage form is a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto. These may be used alone or in combination of two or more kinds.

[0105] When the dosage form of the present invention is a solution or emulsion, a solvent, solubilizer, or emulsifier is used as a carrier component, such as water, glycerin, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc., and particularly, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol aliphatic esters, polyethylene glycol, or fatty acid esters of sorbitan are used, but are not limited thereto. These may be used alone or in combination of two or more kinds.

[0106] When the dosage form of the present invention is a suspension, the carrier components include, but are not limited to, liquid diluents such as water, glycerin, ethanol, or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters, and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc. These may be used alone or in combination of two or more kinds.

[0107] When the dosage form of the present invention is a soap, the carrier components include, but are not limited to, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. These may be used alone or in combination of two or more kinds.

[0108] Another aspect of the present invention provides a quasi-drug composition comprising the composition for transdermal delivery of the present invention.

[0109] The quasi-drug composition may be for improving skin or preventing or improving skin diseases by utilizing the physiological functions of the physiologically active substances, but is not limited thereto.

[0110] The composition for transdermal delivery, the physiologically active substance, the skin improvement, the skin disease, the prevention and improvement, etc. are as described above.

[0111] In the present invention, the term "quasi-drug" refers to an article that is used for the purpose of diagnosing, curing, improving, mitigating, treating or preventing a disease in humans or animals, and has a milder effect than a drug. For example, according to the Pharmaceutical Affairs Law, quasi-drugs exclude articles used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products that have a mild effect on the human body or do not have a direct effect.

[0112] The quasi-drug composition of the present invention can be prepared in the form selected from the group consisting of body cleansers, foams, soaps, masks, ointments, creams, lotions, essences and sprays, but is not limited thereto.

[0113] In the cosmetic composition or quasi-drug composition of the present invention, the skin-penetrating peptide, its derivative, or its fragment consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 may be contained in an amount of 0.01 wt% to 100.0 wt%, or 0.1 wt% to 10 wt% of the total composition.

[0114] Another aspect of the present invention provides a pharmaceutical composition for external application to the skin, which comprises the composition for transdermal delivery of the present invention.

[0115] The pharmaceutical composition may be for preventing or treating a skin disease depending on the physiological function of the physiologically active substance, but is not limited thereto. The composition for transdermal delivery, the topical skin preparation, the physiologically active substance, the skin disease, and the prevention thereof are as described above.

[0116] In this application, "treatment" refers to any action that improves or benefits the symptoms of a disease (e.g., a skin disease). In this application, treatment can include any action that prevents recurrence of a disease, alleviates symptoms, reduces the rate of progression, relieves the disease state, temporarily or permanently alleviates the disease state, improves the condition, or improves prognosis.

[0117] The pharmaceutical composition of the present invention may contain a skin-penetrating peptide consisting of the amino acid sequence of SEQ ID NO:5 to SEQ ID NO:8, a derivative thereof, or a fragment thereof, and may further contain a physiologically active substance and a pharma- ceutically acceptable carrier.

[0118] In the present invention, the term "pharmaceutical acceptable" means that it does not stimulate the organism when administered, does not inhibit the biological activity and properties of the administered compound, and is commonly used in the pharmaceutical field. The pharmaceutical composition of the present invention can be formulated with the carrier and used as a food, medicine, feed additive, drinking water additive, etc.

[0119] The type of the carrier is not particularly limited, and any carrier commonly used in the art can be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. These can be used alone or in combination of two or more, but are not limited thereto.

[0120] In addition, the pharmaceutical composition of the present invention may be used by adding other pharma- ceutically acceptable additives, such as excipients, diluents, antioxidants, buffers, or bacteriostatic agents, if necessary, and may be used by additionally adding fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, binders, or lubricants, but is not limited thereto.

[0121] The pharmaceutical composition of the present invention can be formulated and used in a variety of dosage forms suitable for external application to the skin.

[0122] Non-limiting examples of the skin topical agent include aerosol agents, spray agents, cleansing agents, ointments, powders for application, oils, creams, etc., and are not limited thereto as long as they function as skin topical agents.

[0123] In order to formulate the pharmaceutical composition of the present invention for use as an external preparation for skin, a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, etc. may be used. The non-aqueous solvent and suspension may include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, esters such as ethyl oleate, etc.

[0124] More specifically, when the pharmaceutical composition of the present invention is formulated, the pharmaceutical composition of the present invention may be mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which may then be formulated in units such as ampoules. When the pharmaceutical composition of the present invention is formulated into an aerosol, a propellant or the like may be mixed with an additive so that the water-dispersed concentrate or wet powder can be dispersed, but is not limited thereto.

[0125] Furthermore, when the pharmaceutical composition of the present invention is formulated into an ointment, cream or the like, it can be formulated using animal oils, vegetable oils, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or the like as a carrier, but is not limited thereto.

[0126] The pharmaceutical composition of the present invention is formulated for external application to the skin, and more preferably has a dosage form selected from the group consisting of gel, patch, spray, ointment, plaster, lotion, liniment, paste and cataplasm, but is not limited thereto.

[0127] The pharmacologic effective amount of the pharmaceutical composition of the present invention, and the effective dosage for external preparations, may vary depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, and may vary depending on various factors including the type and degree of reaction to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms and degree of disease, sex, diet, excretion, drugs and other composition components used simultaneously or at different times in the individual, and similar factors well known in the medical field, and a person having ordinary skill in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or in several divided doses. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

[0128] In the present invention, administration may be as an external preparation, and the preferred dose of the pharmaceutical composition of the present invention may be 1 mg / kg to 1,000 mg / kg per day.

[0129] The administration route and administration method of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in the method, and can follow any administration route and administration method as long as the pharmaceutical composition can reach the target site. The pharmaceutical composition can be administered in the form of a skin topical agent. Specifically, the composition can be applied or sprayed on the diseased site, but is not limited thereto.

[0130] The pharmaceutical composition of the present invention may be preferably administered by application or spraying, specifically, by topical application to an area where improvement of skin condition or prevention, improvement or treatment of skin disease is required.

[0131] Another aspect of the present invention provides a kit comprising the transdermal delivery composition of the present invention; and a biologically active agent.

[0132] The kit may be for transdermal delivery, skin penetration, skin improvement, or prevention or treatment of skin diseases, but is not limited thereto. The kit may be provided by storing the composition for transdermal delivery and the physiologically active substance in separate containers and mixing them when used, but is not limited thereto.

[0133] The kit of the present application may also include, but is not limited to, one or more other component compositions, solutions, or devices for transdermal delivery, skin penetration, skin improvement, and prevention or treatment of skin diseases. Examples of the other components include, but are not limited to, suitable carriers, solubilizers, buffers, stabilizers, etc. The carriers include soluble carriers and insoluble carriers, and examples of soluble carriers include physiologically acceptable buffer solutions known in the art, such as PBS, and examples of insoluble carriers include, but are not limited to, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesins, cross-linked dextran, polysaccharides, polymers such as magnetic particles formed by plating metal on latex, other paper, glass, metal, agarose, and combinations thereof.

[0134] The kit of the present application may further include a user guidebook describing optimal reaction carrying out conditions. The guidebook is a printed matter that explains how to use the kit, e.g., reaction conditions, etc. The guidebook includes an information booklet in the form of a pamphlet or flyer, a label attached to the kit, and an explanation on the surface of a package including the kit. The guidebook also includes information that is published or provided through an electronic medium such as the Internet.

[0135] Another aspect of the present invention provides a method for preparing the transdermal delivery composition of the present invention.

[0136] Another aspect of the present invention provides a method for transdermal delivery of a biologically active substance, comprising the step of applying the transdermal delivery composition of the present invention and the biologically active substance to the skin or mucosa.

[0137] Another aspect of the present invention provides a method for improving skin, comprising the step of applying the transdermal delivery composition of the present invention and a physiologically active substance to the skin or mucosa.

[0138] Another aspect of the present invention provides a method for preventing or treating a skin or mucosal disease comprising the step of applying a pharmaceutical composition of the present invention to the skin or mucosa of a non-human individual.

[0139] Another aspect of the present invention provides a method for preventing or ameliorating a skin or mucosal disease, which comprises the step of applying the cosmetic composition or quasi-drug composition of the present invention to the skin.

[0140] Another aspect of the present invention provides a method for producing a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition, comprising the step of mixing the composition for transdermal delivery of the present invention and a physiologically active substance.

[0141] Another aspect of the present invention provides use of a physiologically active substance, which is a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 of the present invention, a derivative thereof, or a fragment thereof, for transdermal application.

[0142] Another aspect of the present invention provides an use of a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 of the present invention, a derivative thereof, or a fragment thereof for transdermal delivery of a physiologically active substance.

[0143] Another aspect of the present invention provides use of a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 of the present invention, a derivative thereof, or a fragment thereof, and a physiologically active substance; a cosmetic composition containing the same; or a quasi-drug composition for improving skin or preventing or improving a skin disease.

[0144] Another aspect of the present invention provides a use of a pharmaceutical composition comprising a peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 of the present invention, a derivative thereof, or a fragment thereof, and a physiologically active substance for preventing or treating a skin disease. The above-mentioned compositions for transdermal delivery, physiologically active substances, skin improvement, pharmaceutical compositions, cosmetic compositions, quasi-drug compositions, skin diseases, prevention, improvement, treatment, etc. are as described above.

[0145] As used herein, the term "individual" refers to all animals, including but not limited to mammals, including rats, livestock, humans, etc.

[0146] In the present invention, the term "application" means contacting the above-mentioned transdermal delivery composition and physiologically active substance of the present invention with the skin of an individual by any appropriate method, and includes any action aimed at absorbing the transdermal delivery composition and physiologically active substance into the skin through this, but is not limited to this.

[0147] The present invention will be described in more detail with reference to the following examples. However, these examples and experimental examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0148] Production Example 1: Production of skin-penetrating peptides In order to confirm the function of the skin-penetrating peptides, SEQ ID NO: 5 to SEQ ID NO: 8 and their derivative peptides were produced.

[0149] 1-1: Synthesis of peptide of SEQ ID NO:1 71.4 mg (0.10 mmol) of 2-chlorotrityl chloride resin (1.4 mmol / g loaded resin) purchased from Novabiochem Corporation was weighed out, and the resin was solvated with 7.5 ml of methyl chloride and reacted for 5 minutes, after which the methyl chloride was removed.

[0150] Then, Fmoc-Lys(Boc)-OH (281.1 mg, 0.60 mmol) was completely dissolved in 7.5 ml of MC solvent, and then 0.21 ml, 1.2 mmol of N,N'-diisopropylethylamine (DIPEA) was added to the resin. The reaction solution was shaken at room temperature for 12 hours and then washed four times with 10 ml of DMF solvent.

[0151] Then, 7.5 ml of 20% (w / v) piperidine DMF solution was added and shaken for 15 minutes to completely remove the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group with the substance added to the resin described above, and the resin was washed four times with 10 ml of DMF solvent (four times with 10 ml each). At this stage, the presence or absence of deprotection reaction of the Fmoc protecting group was confirmed by performing the Kaiser test [E. Kaiser et al. Anal.Biochem., 1970,34(2), 595-598.].

[0152] Meanwhile, Fmoc-Leu-OH (212.0mg, 0.60mmol), hexafluorophosphate benzotriazole tetramethyluronium (HBTU) (227.6mg, 0.60mmol), and hydroxybenzotriazole (HOBt) (81.0mg, 0.6mmol) were completely dissolved in 7.5ml of DMF solvent, and then DIPEA (0.21ml, 1.2mmol) was added to the resin. The reaction solution was shaken at room temperature for 3 hours, and then washed four times with 10ml of DMF solvent. At this stage, the Kaiser test was performed to confirm the completion of the reaction.

[0153] Next, peptides were successively condensed (coupled) by the same synthesis cycle as follows.

[0154] (1) Wash with DMF solvent (10 ml) four times; (2) deprotection using 20% ​​(w / v) piperidine in DMF (7.5 ml) for 15 minutes; (3) Washing with DMF solvent (10 ml) four times; (4) Addition of Fmoc-amino acid, HBTU, HOBt, and DIPEA; (5) HBTU, HOBt, and DIPEA condensation reagents were added to activate amino acids and condense for 3 hours; (6) Washing with DMF solvent (10 ml) four times;

[0155] The above (1) to (6) were successively repeated, and the Fmoc-protected amino acids (0.60 mmol) after Fmoc-Leu-OH were then added to the resin reaction vessel in the order described below for condensation.

[0156] (i) Fmoc-Tyr(tBu)-OH; (ii) Fmoc-Lys(Boc)-OH; (iii) Fmoc-Leu-OH; (iv) Fmoc-Ile-OH; (v) Fmoc-Lys(Boc)-OH; (vi) Fmoc-Lys(Boc)-OH; (vii) Fmoc-Phe-OH; (viii) Fmoc-Leu-OH; (ix) Fmoc-Lys(Boc)-OH; (x) Fmoc-Lys(Boc)-OH;

[0157] After Fmoc-Lys(Boc)-OH condensation (6), the reaction mixture was finally treated with 20% piperidine in DMF (7.5 ml), and then washed with 10 ml portions of DMF solvent three times, MC solvent three times, and diethyl ether solvent three times.

[0158] Immediately after the completion of the synthesis, the peptide-condensed resin was treated for 3 hours with a mixture of trifluoroacetic acid / triisopropylsilane / water (95:2.5:2.5) (10 ml) to cleave the peptide from the resin. The resulting mixture was treated with 100 ml of refrigerated diethyl ether solvent to produce a precipitate. The resulting precipitate was centrifuged to completely precipitate, and trifluoroacetic acid was removed for the first time. The above procedure (the step of adding 100 ml of diethyl ether solvent to wash the precipitate and centrifuging - the work of removing the trifluoroacetic acid that was removed for the first time) was repeated twice to obtain a solidified precipitate.

[0159] The above precipitate (peptide) was purified by HPLC using a C-18 column with a concentration gradient solvent system of 5% to 100% acetonitrile / water containing 0.001% trifluoroacetic acid over 50 minutes. The pure and purified fraction was freeze-dried to obtain 155 mg of the skin-penetrating peptide of SEQ ID NO: 1 below as a white powder type trifluoroacetate salt.

[0160] SEQ ID NO: 1: H2N-[Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Lys]-CO2H MS(ESI)m / e, [M+H]+= 1550.17;(155mg)

[0161] 1-2: Synthesis of SEQ ID NO:2 to SEQ ID NO:8 The peptides shown in SEQ ID NO: 2 to SEQ ID NO: 8 were prepared by using the same production process as in Production Example 1-1 above, but by changing the order of the amino acids protected with Fmoc.

[0162] SEQ ID NO:2:H2N-[Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H MS(ESI)m / e, [M+H]+= 1718.23;(171mg) SEQ ID NO: 3: H2N-[Arg-Phe-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H MS(ESI)m / e, [M+H]+= 1666.18;(166mg) SEQ ID NO: 4: H2N-[Arg-Arg-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Phe]-CO2H MS(ESI)m / e, [M+H]+= 1666.18;(166mg) SEQ ID NO: 5: H2N-[Arg-Lys-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H MS(ESI)m / e, [M+H]+= 1634.2;(153mg) SEQ ID NO: 6: H2N-[Lys-Arg-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H MS(ESI)m / e, [M+H]+= 1634.2;(143mg) SEQ ID NO: 7: H2N-[Lys-Arg-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H MS(ESI)m / e, [M+H]+= 1634.2;(132mg) SEQ ID NO: 8: H2N-[Arg-Lys-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H MS(ESI)m / e, [M+H]+= 1634.2;(172mg)

[0163] Example 1: Evaluation of skin permeation activity of SEQ ID NO:5 to SEQ ID NO:8 and their derivatives against physiologically active substances To confirm the function as a skin-penetrating peptide, the skin-penetrating activity against lidocaine was evaluated in comparison with known peptides. The skin-penetrating peptides composed of any one of the amino acid sequences selected from SEQ ID NO:5 to SEQ ID NO:8 and their derivatives SEQ ID NO:1 to SEQ ID NO:4 or known peptides were mixed with lidocaine, or lidocaine was treated alone, and the skin-penetrating activity against this was evaluated.

[0164] Specifically, to evaluate the skin permeation activity, Franz diffusion cell assay was performed using actual human skin, and the amount of lidocaine permeated was measured by HPLC and compared. The known peptide sequences are as follows:

[0165] -Known peptide sequence (TAT): GRKKRRQRRRPQ

[0166] Franz diffusion cell assay The human skin used in this experiment was purchased from UMC Science Co., Ltd., Korea, and PBS (phosphate buffered saline, Gibco, USA) was purchased from Sigma-Aldrich.

[0167] To measure the skin permeation activity of the skin-permeable peptides, a Franz diffusion cell assay was performed according to the Guidance Document for the Conduct of Skin Absorption Studies of the OECD (2004, Paris) and the Ministry of Food and Drug Safety (2009) guidelines for cosmetic animal alternative testing (skin absorption test methods for living organisms).

[0168] First, the skin area is 3cm 2 After that, the stratum corneum of the skin was placed on the receptor chamber with the stratum corneum facing up. The donor chamber was then placed on the stratum corneum and fixed with a clamp. With the magnetic bar still in the receptor chamber, which is the lower part of the Franz diffusion cell, the buffer was filled through the sampling arm. The buffer was PBS (pH 7.4) when the test substance was water-soluble, and ethanol:water = 1:1 when the test substance was lipid-soluble.

[0169] The temperature of the receptor fluid was kept constant at 32°C~±1°C, and the humidity was kept at 30~70% and then stabilized for 30 minutes. Then, the stirrer was set at 600 rpm. Each group was treated with 1 mL of the sample at a concentration of 2 mM on the skin. Then, the stirrer was kept at 600 rpm with the magnetic stirrer bar still in the receptor chamber to ensure that the aqueous solution was mixed well. After 6 hours, the receptor chamber was sampled through the sampling arm, and the STD (standard sample 100uM) and the sample were analyzed through HPLC for quantification, and the transmittance compared to the STD was expressed as a percentage (%).

[0170] As a result, it was confirmed that when SEQ ID NOs: 5 to 8 and their derivatives were used, the amount of lidocaine permeated through the skin was significantly superior compared to when known peptides were used or when no peptide was used (Figure 1).

[0171] Example 2: Evaluation of skin permeation activity of skin-permeable peptides against physiologically active substances The skin permeation activity of the most effective skin permeable peptide, SEQ ID NO: 8, among the peptides SEQ ID NO: 1 to SEQ ID NO: 8, against caffeine was measured by time period. The skin permeation activity was evaluated by mixing the skin permeable peptide, SEQ ID NO: 8, and caffeine or treating caffeine alone.

[0172] Specifically, a Franz diffusion cell assay was performed using actual human skin in the same manner as in Example 1, and the amount of caffeine permeated was measured by HPLC and then compared (Figure 2).

[0173] As a result, it was confirmed that the amount of caffeine permeated through the skin was significantly higher in the case of using SEQ ID NO:8 than in the case of using a known peptide or no peptide at each time point.

[0174] Example 3: Evaluation of the skin permeation activity of skin-permeable peptides against bioactive substances of various molecular weights In order to evaluate whether the skin-penetrating peptide has skin-penetrating activity for bioactive substances of various molecular weights, 3, 30, 300 kda collagen (COLA) and 800 kda hyaluronic acid (HA) conjugated with FITC were mixed with the peptide of SEQ ID NO: 8 or treated alone, and aqueous solutions were obtained through Franz diffusion cell assay, and the FITC fluorescence value was analyzed using a Fluorescence Microplate Reader (SpectraMax i3, Molecular Devices, USA).

[0175] For the FITC conjugation method, a FITC conjugation kit (Lightning-Link (registered trademark) (ab188285)) was purchased from Abcam (Cambridge, MA, USA) and prepared according to the manufacturer's instructions.

[0176] As a result, when the peptide of SEQ ID NO:8 was mixed and treated, collagens of various molecular weights (3 kda, 30 kda, 300 kda) and hyaluronic acid showed a strong skin permeation activity effect (Figure 3).

[0177] Example 4: Evaluation of skin permeation activity of skin-permeable peptides on liposomes In order to closely examine the effect of the skin-penetrating peptide on the skin-penetrating effect of liposome, a type of bioactive substance, human skin was treated with the peptide of SEQ ID NO:8 and liposomes mixed or alone for 3 hours, and then an aqueous solution was obtained through Franz diffusion cell assay, and the FITC fluorescence value was analyzed using a Fluorescence Microplate Reader (SpectraMax i3, Molecular Devices, USA).

[0178] The liposomes were prepared by purchasing a Liposome kit from Sigma Aldrich (Mo., USA) and preparing liposomes from fluorescein. The size of the liposomes was measured by SEM and found to be 180 nm.

[0179] As a result, it was confirmed that when the peptide of SEQ ID NO:8 was mixed with liposomes and treated, the skin permeation effect of liposomes was significantly increased (FIG. 4).

[0180] Example 5: Evaluation of improvement in efficacy of physiologically active substances by skin permeation effect of skin-permeable peptides on physiologically active substances In order to confirm the improvement of efficacy of bioactive substances due to the skin permeation effect of the active ingredient of the peptide of sequence number 8, HDFs were treated with a mixture of the bioactive substances KTTKS peptide and the peptide of sequence number 8 or alone, and the amount of collagen produced was analyzed using an ELISA assay.

[0181] HDF cells used in this experiment were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HDF cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin 100 U / ml, streptomycin 100 μg / ml ( Gibco , Life Technologies, Grand Island, NY, USA) and cultured at 37°C in a 5% CO2 incubator.

[0182] 2x10 HDF cells 5250μl of each sample was dispensed into 48 wells at a concentration of 100 cells / ml and stabilized for 24 hours. The samples were treated according to the concentration and cultured in a CO2 incubator for 48 hours, after which culture medium was obtained. According to the manufacturer's instructions (Collagen type I ELISA Kit, R&D Biosystems), the antibody was diluted in coating buffer solution and coated on the microwells, and then cultured overnight at 4℃. Each well was washed three times with washing buffer solution, and 100μl of culture medium was dispensed into each well. This was left at room temperature for 1 hour, washed three times with washing buffer solution, treated with 100μl of avidin-HRP conjugated antibody, left at room temperature for 1 hour, and washed three times again. 100μl of TMB substrate was dispensed into each well, left in the dark for 30 minutes, treated with 50μl of stop solution, and the absorbance was measured at 450nm using a microspectrophotometer (Molecular Device, Sunnyvale, CA, USA).

[0183] As a result, it was confirmed that when the peptide of SEQ ID NO:8 was mixed with KTTKS and treated, the amount of collagen produced was significantly increased due to an increase in the skin permeation effect of the KTTKS peptide (FIG. 5).

[0184] Example 6: Evaluation of efficacy improvement by skin permeation effect of skin-permeable peptide for biologically active substances in human application test In order to confirm the improvement of efficacy due to the skin permeation effect of the peptide of sequence number 8 on the active ingredient in a human application test, we requested SkinMed Clinical Trial Center Co., Ltd. to conduct a human application test on 20 subjects. The test group was prescribed a mixture of the peptide of sequence number 8 and 1% ultra-low molecular weight hyaluronic acid, and the control group was prescribed 1% ultra-low molecular weight hyaluronic acid.

[0185] The subjects were assigned to use the product on the forearm, with the amount of product used being approximately 0.5g±0.1g per application, and the product use period and method were twice a day (other life patterns were the same as usual). This test was conducted after the subjects had rested their skin for at least 30 minutes under constant temperature and humidity conditions (room temperature 20-25°C, humidity 40-60%) without air movement or direct sunlight, and in order to accurately evaluate the effects of the present invention, the application site was kept as dry as possible during the test period to prevent water from getting on the application site or sweating. In addition, to reduce measurement errors, each measurement was repeated three times by the same tester, and the average value was evaluated.

[0186] To check for improvement in skin texture, a Visioscan was used to distribute light evenly onto a reflector and capture an image using a video sensor chip and objective lens. A non-glossy image of the skin surface (approximately 10 x 8 mm in size) was generated, displayed in 255 gray levels, and the SEr (Roughness) value, which indicates the roughness of the skin, was calculated from the measured values. A certain area was measured, and the same area was remeasured 24 hours after product use, compared to the measurements taken before use, to confirm the roughness of the skin. An increase in SEr indicates a decrease in skin roughness and an improvement in skin texture.

[0187] The improvement of skin elasticity was measured with Cutometer dual MPA 580. The measurement principle of Cutometer is based on the suction method, where the skin is mechanically deformed by negative pressure, and the pressure is generated by the device, and the skin is sucked into the hole of the probe again after a certain time. The penetration depth inside the probe is determined by a non-contact optical measurement system, which consists of a light source and a photoreceptor as well as two prisms facing each other, which project light from the transmitter to the receptor. The light intensity varies depending on the skin penetration depth, and during the measurement, the negative pressure (determinacy) and the skin resistance to return to its original position (elasticity) are displayed in real time as a curve (mm / time unit). In these curves, the R2 value is recalculated to match the measurement parameters to the elasticity and viscoelasticity characteristics of the skin surface and skin aging. The closer the value is to 1 (100%), the more elastic the curve is. In human subject tests, the R2 (R2=Ua / Uf; maximum amplitude ratio) value, which indicates the skin elasticity of the test area, was measured 24 hours after product application.

[0188] Gloss was measured on the forehead using a SkinGlossMeter (SGM, Delfin). When light emitted from a 635nm red diode laser is reflected off the skin surface, the amount of reflection is detected. The light that passes through the diffracting microstructure inside the device is used to calculate the gloss value using the SGU (Skin Gloss Unit) parameter. The higher the shininess of the skin, the higher the measured value. In the human subject application test, the SGU value, which indicates the skin gloss of the test area, was measured before and 24 hours after product use.

[0189] The improvement rate was calculated using the following formula:

[0190] -Improvement rate (%) calculation formula = {(Before use - After use) / Before use} x 100

[0191] The results showed that after 24 hours, the average improvement rates of skin texture, skin elasticity, and gloss in the test group were approximately 40 times, 4 times, and 5 times higher than in the control group (Table 1).

[0192] [Table 1]

[0193] From the above results, it was confirmed that the skin-penetrating peptide of the present invention, its derivative, or its fragment has excellent skin-penetrating activity and is therefore significantly effective as a skin-penetrating peptide.

[0194] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A composition for transdermal delivery of a physiologically active substance, comprising a skin-penetrating peptide or a derivative thereof, the peptide being composed of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, The derivative is an amino acid corresponding to any one position selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th, and 12th positions from the N-terminus of the skin-penetrating peptide consisting of any one of SEQ ID NOs: 5 to 8, substituted with another amino acid; the amino acids corresponding to the first, fifth, and ninth positions are substituted with lysine or arginine; the amino acid at the sixth position is substituted with lysine, arginine, or leucine; A composition, wherein the amino acids corresponding to the second and twelfth positions are substituted with lysine, arginine, or phenylalanine.

2. The composition for transdermal delivery of a physiologically active substance according to claim 1, wherein the derivative is at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

4.

3. The composition for transdermal delivery of a physiologically active substance according to claim 1, which increases the permeability of the physiologically active substance through at least one selected from the group consisting of skin and mucous membrane.

4. The composition for transdermal delivery of a physiologically active substance according to claim 1 , further comprising a carrier.

5. The composition for transdermal delivery of a physiologically active substance according to claim 4, wherein the carrier is a liposome.

6. The composition for transdermal delivery of a physiologically active substance according to claim 1 , wherein the physiologically active substance is a substance having a physiological effect in a living body.

7. A composition for skin permeation of a physiologically active substance, comprising a skin permeation peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, or a derivative thereof, The derivative is an amino acid corresponding to any one position selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th, and 12th positions from the N-terminus of the skin-penetrating peptide consisting of any one of SEQ ID NOs: 5 to 8, substituted with another amino acid; the amino acids corresponding to the first, fifth, and ninth positions are substituted with lysine or arginine; the amino acid at the sixth position is substituted with lysine, arginine, or leucine; A composition, wherein the amino acids corresponding to the second and twelfth positions are substituted with lysine, arginine, or phenylalanine.

8. A carrier for transdermal delivery of a physiologically active substance, comprising a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, or a derivative thereof, The derivative is an amino acid corresponding to any one position selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th, and 12th positions from the N-terminus of the skin-penetrating peptide consisting of any one of SEQ ID NOs: 5 to 8, substituted with another amino acid; the amino acids corresponding to the first, fifth, and ninth positions are substituted with lysine or arginine; the amino acid at the sixth position is substituted with lysine, arginine, or leucine; A carrier in which the amino acids corresponding to the second and twelfth positions are substituted with lysine, arginine, or phenylalanine.

9. A skin-penetrating carrier for a physiologically active substance, comprising a skin-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, or a derivative thereof, The derivative is an amino acid corresponding to any one position selected from the group consisting of the 1st, 2nd, 5th, 6th, 9th, and 12th positions from the N-terminus of the skin-penetrating peptide consisting of any one of SEQ ID NOs: 5 to 8, substituted with another amino acid; the amino acids corresponding to the first, fifth, and ninth positions are substituted with lysine or arginine; the amino acid at the sixth position is substituted with lysine, arginine, or leucine; A carrier in which the amino acids corresponding to the second and twelfth positions are substituted with lysine, arginine, or phenylalanine.

10. A cosmetic composition comprising the composition for transdermal delivery according to claim 1.

11. A quasi-drug composition comprising the composition for transdermal delivery according to claim 1.

12. A pharmaceutical composition for external application to the skin or mucosa, comprising the composition for transdermal delivery according to claim 1.

13. A kit comprising the composition for transdermal delivery according to claim 1; and a physiologically active substance.

14. A method for producing the composition for transdermal delivery according to claim 1.

15. A method for producing a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition, comprising the step of mixing the composition for transdermal delivery according to claim 1 with a physiologically active substance.

16. A method for transdermal delivery of a physiologically active substance, comprising the step of applying the composition for transdermal delivery according to claim 1 and a physiologically active substance to the skin or mucosa of a non-human individual.

17. A method for improving skin, comprising the step of applying the composition for transdermal delivery according to claim 1 and a physiologically active substance to the skin or mucosa of a non-human individual.

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