Novel skin-permeable peptide, composition including the same for skin and mucous membrane, and use thereof

Abstract

A skin-permeable peptide, a derivative thereof, or a fragment thereof of the present disclosure effectively delivers skin bioactive molecules deep into the skin tissues through assembly or fusion methods, thereby providing outstanding skin permeability as well as excellent skin residual effect of bioactive materials to maximize bioactive efficacy. Accordingly, the skin-permeable peptide, derivative thereof, or fragment thereof may be widely used as an active ingredient of a pharmaceutical composition for external use and a functional cosmetic composition that target skin tissues including mucous membranes.

Description

Novel skin-penetrating peptide, composition for skin and mucous membrane containing the same, and use thereof

[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 transdermal delivery; a carrier for transdermal delivery; a carrier for skin penetration; a pharmaceutical composition; a quasi-drug composition; a cosmetic composition; a kit comprising the composition for transdermal delivery; a method for preparing the composition for transdermal delivery, a cosmetic composition, and a quasi-drug composition; a method for transdermal delivery of a physiologically active peptide; and a use for transdermal delivery of a physiologically active peptide.

[0002]

[0003] The skin, the largest organ of the human body, consists of the epidermis, dermis, and subcutaneous tissue, and performs various functions in response to the external environment. Due to the unique characteristics of the stratum corneum, which constitutes the skin barrier, the molecular weight that can be transmitted through the skin is known to be less than approximately 500 Da. However, even low-molecular-weight substances have low skin permeation efficiency, and high-molecular-weight substances such as growth factors are known to have even lower permeation efficiency. Therefore, there is a critical need for safe methods for the effective delivery of bioactive substances in the cosmetics and pharmaceutical industries.

[0004] Physical delivery systems such as microneedles can cause irreversible damage to the skin, and existing skin-penetrating substances often utilize fat-soluble substances or polymers (Domestic Patent No. 10-2274435), which can cause various side effects and unknown toxicity in the human body. Therefore, it is necessary to safely deliver bioactive substances to the dermis using peptides, which are fragments of proteins existing in the human body.

[0005]

[0006] The present inventors have developed a skin-penetrating peptide and completed the present invention. The biomembrane-penetrating peptide technology developed in the present invention is a cutting-edge biotechnology that significantly improves the absorption rate of raw materials in the body and increases bioavailability. It is a next-generation bio-new material platform technology that can sufficiently exhibit efficacy even when added in very small amounts to functional cosmetics or advanced pharmaceutical raw materials. Therefore, it is intended to be applied to the development of skin-penetrating cosmetics and pharmaceuticals.

[0007]

[0008] One object of the present invention is to provide a composition for transdermal delivery of a physiologically active substance comprising a skin-permeable peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

[0009] Another object of the present invention is to provide a composition for skin penetration of a physiologically active substance comprising the skin-penetrating peptide, a derivative thereof, or a fragment thereof.

[0010] Another object of the present invention is to provide a carrier for transdermal delivery of a physiologically active substance comprising the skin-penetrating peptide, a derivative thereof, or a fragment thereof.

[0011] Another object of the present invention is to provide a skin-penetrating carrier of a physiologically active substance comprising the skin-penetrating peptide, a derivative thereof, or a fragment thereof.

[0012] Another object of the present invention is to provide a cosmetic composition comprising the composition for transdermal delivery.

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising the above composition for transdermal delivery.

[0014] Another object of the present invention is to provide a pharmaceutical composition for external application to the skin or mucosa, comprising the composition for transdermal delivery.

[0015] Another object of the present invention is to provide a kit comprising the composition for transdermal delivery and a physiologically active substance.

[0016] Another object of the present invention is to provide a method for preparing the composition for transdermal delivery.

[0017] Another object of the present invention is to provide a method for improving skin using the composition for transdermal delivery.

[0018] Another object of the present invention is to provide a method for preventing or treating skin or mucous membrane diseases using the pharmaceutical composition.

[0019] Another object of the present invention is to provide a method for producing the cosmetic composition and the pharmaceutical composition.

[0020] Another object of the present invention is to provide a method for transdermal delivery of a physiologically active peptide using a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof.

[0021] Another object of the present invention is to provide a use for transdermal delivery of a physiologically active peptide using a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof.

[0022] Another object of the present invention is to provide a skin-penetrating use 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.

[0023]

[0024] The skin-penetrating peptide of the present invention, its derivative or its fragment effectively delivers skin physiologically active molecules deep into skin tissue through assembling or fusion methods, thereby exhibiting excellent skin penetration activity as well as excellent skin residual effect, thereby maximizing physiological activity. Therefore, the skin-penetrating peptide, its derivative or its fragment can be widely utilized as an active ingredient of a pharmaceutical composition for external skin application and a functional cosmetic composition targeting skin tissue including a mucous membrane as a target tissue.

[0025]

[0026] Figure 1 is a diagram showing the results of the Franz diffusion cell assay to analyze the skin penetration effect of the physiologically active substances of the peptides of sequence numbers 1 to 8 in comparison with the known peptides, and quantitatively analyzed by HPLC.

[0027] Figure 2 is a diagram showing the results of the Franz diffusion cell assay confirmed by time zone to analyze the skin penetration effect of the skin-penetrating peptide of the present invention on the physiologically active substance, and quantitatively analyzed by HPLC.

[0028] Figure 3 is a diagram showing the results of the Franz diffusion cell assay to analyze the skin penetration effect of various molecular weight physiologically active substances of the skin-penetrating peptide of the present invention, quantified using a Fluorescence Microplate Reader.

[0029] Figure 4 is a diagram showing the results of the Franz diffusion cell assay to analyze the skin penetration effect of the skin-penetrating peptide of the present invention on liposomes, quantified using a Fluorescence Microplate Reader.

[0030] Figure 5 is a diagram showing the results of an evaluation of the efficacy improvement according to the skin penetration effect of the physiologically active substance of the skin-penetrating peptide of the present invention.

[0031]

[0032] Hereinafter, the present invention will be described in detail. The description and embodiments of one aspect disclosed in the present invention may also be applied to other aspects and embodiments regarding common elements. Furthermore, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0033]

[0034] One aspect of the present invention provides a composition for transdermal delivery of a physiologically active substance, comprising a skin-permeable peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

[0035] In the present invention, "skin penetration" means biomembrane penetration, and "skin-permeable peptide" means a peptide having biomembrane penetration. The skin-permeable peptide may have cell penetration and biomembrane penetration, specifically, skin and / or mucosal penetration, but is not limited thereto.

[0036] 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.

[0037] The present invention can efficiently introduce skin physiologically active molecules, which are difficult to be delivered through the skin due to the size of their molecular weight or the unique characteristics of the stratum corneum of the skin, into cells and the skin.

[0038] The skin-permeable peptide of the present invention is a transdermal delivery composition including a skin physiologically active molecule having improved cell and skin permeability compared to existing skin-permeable peptides, a transdermal delivery system, and a skin-permeable peptide capable of delivering a physiologically active substance into skin cells or the skin dermal layer through assembling or fusing.

[0039] The skin-penetrating peptide of the present invention, its derivative or its fragment effectively delivers skin physiologically active molecules deep into skin tissue through assembling or fusion, thereby exhibiting excellent skin penetration activity as well as excellent skin residual effect, thereby maximizing physiological activity. Therefore, the present invention can be used as a component of a composition targeting skin tissue including mucous membranes as a target tissue.

[0040] In the examples of the present invention, it was confirmed that sequence numbers 5 to 8 and derivatives thereof have remarkable skin permeability (Examples 1 to 6).

[0041]

[0042] In the present invention, "derivative" means a derivative of a skin-penetrating peptide composed of any one of the amino acid sequences of SEQ ID NOs: 5 to 8, and specifically, may be a peptide having an amino acid sequence in which some functional groups are added to the amino acid sequence of SEQ ID NOs: 5 to 8, or in which some amino acid sequences are deleted, modified, substituted, or added, but is not limited thereto.

[0043] Specifically, the derivative in the present invention may be at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, but is not limited thereto.

[0044] Specifically, the skin-permeable peptide and its derivatives comprising the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8 of the present invention may have a sequence as shown in the following chemical formula 1 and may have skin permeability, but are not limited thereto.

[0045]

[0046] [Chemical Formula 1]

[0047] [X1 - X2 - Lys - Phe - X3 - X4 - Ile - Leu - X5 - Tyr - Leu - X6]

[0048]

[0049] In the above chemical 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-permeable peptide and derivative thereof 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 chemical 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.

[0050]

[0051] SEQ ID NO: 1: H2N-[Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Lys]-CO2H,

[0052] SEQ ID NO: 2: H2N-[Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H,

[0053] SEQ ID NO: 3: H2N-[Arg-Phe-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H,

[0054] SEQ ID NO: 4: H2N-[Arg-Arg-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Phe]-CO2H,

[0055] SEQ ID NO: 5: H2N-[Arg-Lys-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H,

[0056] SEQ ID NO: 6: H2N-[Lys-Arg-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H,

[0057] SEQ ID NO: 7: H2N-[Lys-Arg-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H, and

[0058] SEQ ID NO: 8: H2N-[Arg-Lys-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H

[0059]

[0060] Specifically, the 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-permeable peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8 is substituted with another basic amino acid.

[0061] Amino acid substitutions can generally occur based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residue. Specifically, among the amino acids with electrically charged side chains, positively charged (basic) amino acids include Arginine (R), Lysine (K), and Histidine (H), while negatively charged (acidic) amino acids include Glutamate (E) and Aspartate (D); Among the amino acids having an uncharged side chain, nonpolar amino acids include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), tryptophan (W), and proline (P), and polar or hydrophilic amino acids include serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N), and glutamine (Q), and among the nonpolar amino acids, aromatic amino acids include phenylalanine, tryptophan, and Contains tyrosine.

[0062] In the present invention, 'substitution with another basic amino acid' is not limited as long as it is a substitution with a different basic amino acid than the amino acid before substitution. That is, the amino acid before substitution present 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-permeable peptide consisting of any one amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 8 may be substituted with another basic amino acid, and specifically, the amino acid present at any one position selected from the group consisting of positions X1, X2, X3, X4, X5, and X6 may be substituted 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.

[0063] For the purpose 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 at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, but is not limited thereto.

[0064]

[0065] In the present invention, "fragment" means a partial sequence of a peptide having a specific sequence. The fragment of the present invention may be a partial sequence of a skin-permeable 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 skin-permeable peptide or derivative thereof.

[0066]

[0067] A skin-penetrating peptide, a derivative thereof, or a fragment thereof comprising any one of the amino acid sequences of SEQ ID NOs: 5 to 8 may be of natural origin, but is not limited thereto, and a sequence having the same activity as the skin-penetrating peptide, a derivative thereof, or a fragment thereof may be included without limitation.

[0068] In the present invention, the skin-permeable peptide is described as a skin-permeable peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof. However, this does not exclude meaningless sequence additions before or after the amino acid sequence of the skin-permeable peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof, or potential mutations that maintain the same function as the skin-permeable peptide. It will be apparent to those skilled in the art that if it has the same or corresponding activity as the skin-permeable peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof, it corresponds to the skin-permeable peptide of the present invention. For example, the skin-penetrating peptide of the present invention may be a skin-penetrating peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, a fragment thereof, or a peptide comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity therewith.

[0069] Even if the present invention describes a 'peptide comprising an amino acid sequence described by a specific sequence number', a 'peptide consisting of an amino acid sequence described by a specific sequence number', or a 'peptide having an amino acid sequence described by a specific sequence number', it is obvious that a peptide having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added can also be used in the present invention, as long as it has the same or corresponding activity as the peptide consisting of the amino acid sequence of the corresponding sequence number. For example, this includes cases in which the peptide has an addition of a sequence that does not change the function of the peptide at the N-terminus and / or C-terminus, a mutation that may occur naturally, a silent mutation thereof, or a conservative substitution.

[0070] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the peptide.

[0071]

[0072] The peptide of the present invention can be prepared by sequentially forming amide bonds with one or more amino acids or suitably protected amino acids in an amino acid backbone consistently bonded to a solid phase, but is not limited thereto. Furthermore, the peptide can be prepared by inserting, substituting, or deleting other amino acids as long as it does not significantly reduce stability, and this also falls within the scope of the present invention.

[0073] In addition, the skin-permeable peptide of the present invention may be prepared in a form in which a known cell-permeable peptide that promotes intracellular movement is additionally linked to the C-terminus or N-terminus. For example, the known cell-permeable peptide may be a TAT peptide (Arg-Lys-Lys-Arg-Arg-Tyr-Arg-Arg-Arg) and a 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.

[0074] The peptides and compounds of the present invention may be prepared in the form of a metal complex, and the metal may be selected from copper, magnesium, calcium, iron, zinc, nickel, silver, germanium, and gallium, but is not limited thereto. Copper may be preferably used, but is not limited thereto.

[0075] Meanwhile, the peptide of the present invention may exist in the form of a salt. The salt form usable in the present invention may be prepared during the final isolation and purification of the compound or by reacting the amino group with an appropriate acid. For example, acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroatate, trifluoroacetate, Examples of acids that can be used to form acid addition salts include, but are not limited to, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Examples of acids that can be used to form acid addition salts include, 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 trifluoroacetate salt or an acetate salt.

[0076]

[0077] The amino or carboxyl groups of the amino acids used to prepare the peptides included in the compositions of the present invention can be protected with a suitable protecting group. The protected amino acid can be attached to a solid support or reacted in solution by adding the next amino acid under conditions suitable for forming an amide bond. Furthermore, the protecting group can be completely removed prior to adding the amino acid protected with the suitable protecting group. After all amino acids have been linked as desired, the resulting peptide can be obtained by sequentially or simultaneously separating the remaining protecting groups and the free solid support.

[0078] The most preferred synthetic method for producing the peptide compound of the present invention is a solid-phase peptide synthesis method using a solid-phase polymer support, and the α-amino group of the peptide produced through the method can be protected by an acid or base-sensitive functional group. The protecting group of the amino acid at this time must have a stable property under the peptide condensation reaction conditions, and must have a property that can be easily removed without destroying the extended peptide chain or without racemizing any chiral center contained therein. Accordingly, suitable protecting groups may include 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 this purpose may also be used within the scope of the present invention.

[0079] The most preferred protecting group for the amino acid used in the peptide synthesis of the present invention is a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.

[0080] In particular, the protecting group of the amino acid residue used in the peptide synthesis of the present invention is preferably t-butyl (t-Bu) for N-methylglutamic acid; t-butoxycarbonyl (Boc) for lysine; 7t-butyl (t-Bu) for serine; t-butyl (t-Bu) for threonine and allothreonine; and trityl (Trt) for cysteine, but the present invention is not limited thereto.

[0081] 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 reaction and insoluble in the medium used, such as 2-chlorotrityl chloride resin, rink amide, or rink amide MBHA resin.

[0082] In particular, preferred solid supports for C-terminal peptides may be 2-chlorotrityl chloride, rink amide or rink amide MBHA resin, commercially available from Novabiochem Corporation.

[0083] C-terminal amides are prepared by reacting N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 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 (BOPCI) in a solvent such as dichloromethane, N-methylpyridone (NMP) or DMF at a temperature of 10°C to 50°C, preferably at a temperature of 30°C, for 1 to 24 hours. [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate](HATU) or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) can be condensed (bonded, coupled) to a resin or solid support through condensation by activating a carboxylic acid.

[0084] When the solid support is a rink amide 4-methylbenzylhydrylamine resin, the Fmoc functional group as a preferred protecting group is cleaved using an excess of a secondary amine solution, preferably a 20% piperidine DMF solution, prior to condensation with the C-terminal amino acid. Preferred reagents used for condensing the desired amino acid onto 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 suitably protected amino acids.

[0085] The sequential condensation of amino acids performed in the present invention can be performed using an automated peptide synthesizer widely known in the art or directly by hand. Preferred synthetic reaction conditions include deprotecting an α-amino acid protected with an Fmoc group by treating it with a secondary amine solution, preferably piperidine, followed by washing with a sufficiently excessive amount of solvent, and then adding a 3- to 7-fold molar excess of another protected amino acid to be condensed, preferably in DMF.

[0086] In the final step of peptide synthesis using the solid-state resin of the present invention, the desired peptide can be removed from the resin either continuously or in a single 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 the resin with a cleavage reagent cocktail that cleaves the bond between the resin and the peptide, for example, a dichloromethane mixed cocktail solution composed of trifluoroacetic acid (TFA), triisopropylsilane (TIS), thioanisole, water, or ethanedithiol (EDT). The mixed solution thus obtained can be treated with an excess of refrigerated diethyl ether solvent to produce a precipitate. The precipitate thus obtained is centrifuged to completely precipitate, and the excess trifluoroacetic acid, triisopropylsilane, thioanisole, water, and ethanedithiol are first removed. The above procedure is repeated two or more times to obtain a solidified precipitate. At this time, the completely deprotected peptide salt can be separated and purified using a mixed solvent consisting of water and acetonitrile and reverse-phase high-performance liquid chromatography (HPLC). The separated and purified peptide solution can be completely concentrated and dried using lyophilization to obtain a solid peptide.

[0087]

[0088] The present invention provides a composition for transdermal delivery of a physiologically active substance.

[0089] In the present invention, "physiologically active substance" is a general concept that refers to substances that have a physiological function in the body. The physiologically active substance may be hydrophilic, hydrophobic, or sparingly soluble, and includes, without limitation, natural products, chemical compounds, proteins, peptides, amino acids, nucleic acids, lipids, liposomes, and polymers as long as they have a physiological function. In addition, the physiologically active substance of the present invention may be used alone, or may include, without limitation, combinations with other physiologically active substances, combinations with carriers, and combinations with known skin-penetrating peptides.

[0090] As an example of implementation, the physiologically active substance of the present invention may be a main ingredient of a pharmaceutical, quasi-drug, and / or cosmetic used on the skin, skin percutaneous, and / or mucous membranes.

[0091] The physiologically active substance of the present invention may have one or more physiological effects, and may have physiological effects such as skin improvement, health promotion, disease prevention, improvement, etc., but is included without limitation as long as it can be used together with the skin-penetrating peptide of the present invention, a derivative thereof, or a fragment thereof.

[0092] As an example of implementation, the physiologically active substance may be at least one selected from the group consisting of lidocaine, caffeine, collagen, hyaluronic acid, and liposomes, but is not limited thereto.

[0093]

[0094] A skin-penetrating peptide comprising any one of the amino acid sequences of SEQ ID NOs. 5 to 8, a derivative thereof, or a fragment thereof may be directly or indirectly linked to a physiologically active substance, but is not limited thereto.

[0095] In one embodiment, the skin-permeable peptide comprising any one of the amino acid sequences of SEQ ID NOs: 5 to 8 of the present invention, a derivative thereof, or a fragment thereof and a physiologically active substance may be directly linked to each other, linked via a linker, or may additionally include another protein moiety, but is not limited thereto. The linking method of the present invention may utilize any method practiced in the art without limitation as long as it does not change the structure or activity of the skin-permeable peptide comprising any one of the amino acid sequences of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof and the physiologically active substance to be linked. The linker may be a peptidic linker or a non-peptidic linker comprising 1 to 20 amino acids, but is not limited thereto.

[0096] As an example of implementation, a skin-permeable peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8 of the present invention, a derivative thereof, or a fragment thereof may be linked to a physiologically active substance by a non-covalent bond (e.g., an ionic bond, a hydrogen bond, a van der Waals bond, etc.), but is not limited thereto.

[0097] As an example of an embodiment, a skin-permeable peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8 of the present invention, a derivative thereof, or a fragment thereof may be linked (e.g., covalently bonded) to a physiologically active substance, but is not limited thereto. The skin-permeable peptide, derivative thereof, or fragment thereof and the physiologically active substance may be directly linked to each other, linked via a linker, or may additionally include another protein moiety, but are not limited thereto.

[0098] As an example of implementation, the skin-penetrating peptide may self-assemble or self-fuse with a physiologically active substance.

[0099] As an example of implementation, the skin-penetrating peptide may be one in which one or more molecules surround or fuse with a physiologically active substance, but is not limited thereto.

[0100]

[0101] In the present invention, "transdermal delivery" refers to delivering a bioactive substance by penetrating a biological membrane. The composition for transdermal delivery of the present invention may deliver a bioactive substance by cell delivery and biological membrane delivery, specifically by penetrating the skin and / or mucosa, but is not limited thereto.

[0102] The composition for transdermal delivery of the present invention may increase the permeability of a physiologically active substance to at least one selected from the group consisting of skin and mucous membranes, but is not limited thereto.

[0103] The transdermal delivery composition of the present invention may additionally include, but is not limited to, carriers, excipients, diluents, antioxidants, and / or buffers, if necessary.

[0104] As an example of implementation, the carrier may be, but is not limited to, a liposome.

[0105] In the present invention, "liposome" refers to a hollow structure formed by a phospholipid bilayer in an aqueous solution. The liposome may contain a physiologically active substance within the hollow structure, but is not limited thereto.

[0106] As an example of implementation, the liposome may be, but is not limited to, a skin-permeable peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof that increases the skin permeability and / or transdermal delivery ability of a physiologically active substance.

[0107] In an embodiment of the present invention, it was confirmed that the present invention can increase skin permeability and transdermal delivery regardless of the characteristics, size, and type of bioactive substances, including lidocaine, caffeine, collagen of 3, 30, and 300 kDa, and hyaluronic acid (HA) of 800 kDa. In addition, in one embodiment 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.

[0108]

[0109] Another aspect of the present invention provides a composition for skin penetration of a physiologically active substance comprising a skin-penetrating peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof.

[0110] The amino acid sequence, skin-permeable peptide, derivative, fragment, physiologically active substance, and skin penetration of any one of the above sequence numbers 5 to 8 are as described above.

[0111] As an example of implementation, the skin-penetrating composition of the present invention may increase the permeability of a physiologically active substance to one or more selected from the group consisting of skin and mucous membranes, but is not limited thereto.

[0112] The skin penetration composition of the present invention may additionally include, but is not limited to, carriers, excipients, diluents, antioxidants, and / or buffers, if necessary.

[0113]

[0114] Another aspect of the present invention provides a carrier for transdermal delivery of a physiologically active substance, comprising a skin-permeable peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof.

[0115] Another aspect of the present invention provides a skin-penetrating carrier of a physiologically active substance comprising a skin-penetrating peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8, a derivative thereof, or a fragment thereof.

[0116] The amino acid sequence, skin-permeable peptide, derivative, fragment, physiologically active substance, transdermal delivery, and skin penetration of any one of the above sequence numbers 5 to 8 are as described above.

[0117] In the present invention, "carrier" means something capable of carrying any substance or component, such as a composition, and may be used interchangeably with a carrier, impregnating agent, or medium. 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 a hand, puff, tip, or brush.

[0118] The above transdermal delivery composition, skin penetration composition, transdermal delivery carrier, and skin penetration carrier may be included in a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition, but are not limited thereto.

[0119] For the purpose of the present invention, the transdermal delivery carrier and skin-penetrating carrier of the above-mentioned physiologically active substance may include a skin-penetrating peptide consisting of an amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

[0120]

[0121] As an example of implementation, the transdermal delivery composition, skin penetration composition, transdermal delivery carrier, and skin penetration carrier of the present invention may be for external use on the skin.

[0122] In the present invention, "skin topical preparation" refers to a formulation applied to the skin or mucous membranes. As used herein, the topical preparation is not limited in shape or other aspects, as long as it is a formulation applied to the skin or mucous membranes. Topical preparations can be further categorized into ointments, creams, lotions, gels, etc., but are not limited thereto.

[0123] Specifically, the ointment may be a semi-solid external preparation in which the main ingredient to be applied to the skin or mucous membrane is dissolved or dispersed in a base. The base may be classified into an oil-soluble ointment or a water-soluble ointment depending on the ingredient. Ointments using an oil-soluble base may have a skin or mucous membrane softening effect, have excellent sealing properties, and are suitable for application to wounds, but have the disadvantage of being difficult to remove. Due to the oil-soluble properties and skin softening effect similar to those of the skin or mucous membrane, it may be an advantageous formulation when locally permeating ingredients that are difficult to penetrate through the skin.

[0124] The above ointment is mainly composed of an oily phase such as oil, so it has excellent moisturizing properties and is good for penetration of fat-soluble ingredients, but has the disadvantage of being sticky. The cream may be a semi-solid external preparation emulsified in a water-in-oil type, or may be a formulation that improves the stickiness of the ointment by mixing the oil phase and the water phase. As an emulsification process, the oil phase and the water phase may be mixed through a surfactant or a special process. In addition, the cream may be a formulation that is easy to apply and remove compared to an ointment, and the lotion may be a formulation that has properties more similar to water than a cream, and may be an external preparation in which the main ingredient is dissolved, emulsified, or finely dispersed in an aqueous solution. It may be a solution, suspension, or emulsion made by using the main ingredient, additives, and purified water, etc. to make the whole homogeneous. The gel may be a semi-solid external preparation made of organic molecules with a large molecular weight that have penetrated the liquid. It may be a gelling agent dispersed in water, and a gelling agent such as a carbomer or a synthetic polymer may be used. Various types of external preparations may be used depending on the active ingredient, purpose of use, method of use, etc., and the external preparation of the present invention is not limited thereto.

[0125]

[0126] Another aspect of the present invention provides a cosmetic composition comprising the transdermal delivery composition of the present invention.

[0127] The composition for transdermal delivery, etc. are as described above.

[0128] The above cosmetic composition may be used for skin improvement, disease prevention, or improvement depending on the physiological function of the physiologically active substance, but is not limited thereto.

[0129] As an example of implementation, the cosmetic composition may be for skin improvement, but is not limited thereto.

[0130] As an example of implementation, the cosmetic composition may be used for, but is not limited to, skin regeneration, wound healing, anti-aging, wrinkle improvement, whitening, anti-inflammation, antibacterial, moisturizing, moisture increase, and antioxidant purposes.

[0131] As an example of implementation, the cosmetic composition may be used for preventing or improving skin diseases, but is not limited thereto.

[0132] The above skin disease refers to a disease in which abnormal symptoms such as inflammation, erythema, hypertrophy, fibrosis, and keratinization appear on the skin due to genetic, physiological, and environmental factors. As examples, the above skin disease may include, but is not limited to, atopic dermatitis, allergies, psoriasis, seborrheic dermatitis, contact dermatitis, lupus erythematosus, acne, warts, viral infections, and papular urticaria.

[0133] In this application, “prevention” means any act of inhibiting or delaying the onset or progression of a disease (e.g., a skin disease).

[0134] In the present invention, "improvement" means any action that at least reduces a parameter related to alleviation or treatment of a condition, for example, the degree of symptoms. In the present invention, the cosmetic composition may be prepared in a formulation selected from the group consisting of a solution, an external ointment, a cream, a foam, a nourishing toner, an emollient toner, a perfume, a pack, an emollient, an emulsion, a makeup base, an essence, a soap, a liquid cleanser, a bath agent, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, 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.

[0135] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate conventional ingredients such as oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., but is not limited thereto.

[0136] The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

[0137] When the formulation of the present invention is an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in combination of two or more.

[0138] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as carrier components, and especially in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether can be additionally included, but is not limited thereto. These can be used alone or in combination of two or more.

[0139] When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component, for example, water, glycerin, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc. may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty acid ester, polyethylene glycol or fatty acid ester of sorbitan may be used, but is not limited thereto. These may be used alone or in a mixture of two or more.

[0140] When the formulation of the present invention is a suspension, liquid diluents such as water, glycerin, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components, but are not limited thereto. These may be used alone or in combination of two or more.

[0141] When the formulation of the present invention is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in combination of two or more.

[0142]

[0143] Another aspect of the present invention provides a pharmaceutical composition comprising the transdermal delivery composition of the present invention.

[0144] The above-mentioned pharmaceutical composition may be used for skin improvement, prevention or improvement of skin diseases depending on the physiological function of the physiologically active substance, but is not limited thereto.

[0145] The composition for transdermal delivery, physiologically active substances, skin improvement, skin disease, prevention, and improvement, etc. are as described above.

[0146] In the present invention, "quasi-drug" means a product that has a milder effect than a pharmaceutical product among products used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals. For example, according to the Pharmaceutical Affairs Act, a quasi-drug is a product excluding products used for the purpose of pharmaceutical products, and includes products used for treating or preventing diseases of humans or animals, products that have a mild effect on the human body or do not act directly, etc.

[0147] The above-described pharmaceutical composition of the present invention can be manufactured as a product selected from the group consisting of body cleanser, foam, soap, mask, ointment, cream, lotion, essence, and spray, but is not limited thereto.

[0148] In the cosmetic composition or quasi-drug composition of the present invention, the 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 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.

[0149]

[0150] Another aspect of the present invention provides a pharmaceutical composition for external application to the skin comprising the composition for transdermal delivery of the present invention.

[0151] The above pharmaceutical composition may be used for preventing or treating skin diseases depending on the physiological function of the physiologically active substance, but is not limited thereto. The above transdermal delivery composition, skin external preparation, physiologically active substance, skin disease, and prevention, etc. are as described above.

[0152] As used herein, "treatment" refers to any action that improves or benefits the symptoms of a disease (e.g., a skin disease). Treatment may include any action that prevents recurrence of the disease, alleviates symptoms, slows progression, alleviates the disease, temporarily or permanently alleviates or reverses the disease, or improves the prognosis.

[0153] The pharmaceutical composition of the present invention may further comprise a physiologically active substance and a pharmaceutically acceptable carrier, in addition to comprising a skin-permeable peptide consisting of an amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

[0154] In the present invention, the term "pharmaceutically acceptable" means that it can be commonly used in the pharmaceutical field without stimulating the organism when administered and without inhibiting the biological activity and properties of the administered compound. The pharmaceutical composition of the present invention can be formulated with the carrier and utilized as a food, pharmaceutical, feed additive, drinking water additive, etc.

[0155] The type of the carrier is not particularly limited, and any carrier commonly used in the relevant technical field can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. These may be used alone or in combination of two or more, but are not limited thereto.

[0156] In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, and may be used by additionally adding fillers, bulking agents, wetting agents, disintegrating agents, dispersing agents, surfactants, binders or lubricants, but is not limited thereto.

[0157]

[0158] The pharmaceutical composition of the present invention can be formulated into various suitable formulations for external skin application and used.

[0159] Non-limiting examples of the above skin external preparations include aerosols, sprays, cleansers, ointments, powders for application, oils, creams, etc., and are not limited thereto as long as they can function as skin external preparations.

[0160] In order to formulate the pharmaceutical composition of the present invention for external use on the skin, a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, etc. can be used. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate can be used, but are not limited thereto.

[0161] In addition, more specifically, when formulating the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which may be formulated into units such as ampoules. In addition, when formulating the pharmaceutical composition of the present invention into an aerosol, a propellant or the like may be mixed with additives to disperse the water-dispersed concentrate or wet powder, but is not limited thereto.

[0162] In addition, when the pharmaceutical composition of the present invention is formulated into an ointment, cream, etc., it may be formulated using animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. as a carrier, but is not limited thereto.

[0163] The pharmaceutical composition of the present invention may be formulated for external application to the skin, and more preferably may have a formulation selected from the group consisting of gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.

[0164]

[0165] The pharmaceutically effective amount of the pharmaceutical composition of the present invention, the effective dosage for external use, 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 the response to be achieved by administration of the pharmaceutical composition, the type, age, body weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field. A person having ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0166] In addition, in the present invention, administration may be as an external agent, and a preferred dosage of the pharmaceutical composition of the present invention may be 1 mg / kg to 1,000 mg / kg per day.

[0167]

[0168] The route and method of administration of the pharmaceutical composition of the present invention may be independent and are not particularly limited in their methods. Any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area. The pharmaceutical composition may be administered in the form of a topical skin preparation. Specifically, the composition may be applied or sprayed onto the affected area, but is not limited thereto.

[0169] The pharmaceutical composition of the present invention can preferably be administered by application or spraying, and specifically, can be administered by applying it locally to an area where improvement in skin condition or prevention, improvement or treatment of a skin disease is required.

[0170] Another aspect of the present invention provides a kit comprising the composition for transdermal delivery of the present invention; and a physiologically active substance.

[0171] The kit may be, but is not limited to, for transdermal delivery, skin penetration, skin improvement, or the prevention or treatment of skin diseases. The kit may be provided so that the transdermal delivery composition and the bioactive substance are stored in separate containers and can be mixed for use, but is not limited thereto.

[0172] In addition, the kit of the present application may include, but is not limited to, one or more other component compositions, solutions or devices for transdermal delivery, skin penetration, skin improvement, skin disease prevention or treatment. Examples of the other components may include, but are not limited to, suitable carriers, solubilizers, buffers, stabilizers, etc. The carrier may include, but is not limited to, soluble carriers, insoluble carriers, and examples of soluble carriers include physiologically acceptable buffers known in the art, such as PBS, and examples of insoluble carriers include, but are not limited to, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharides, magnetic microparticles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

[0173] Additionally, the kit of the present application may additionally include a user guide describing optimal reaction conditions. The guide is a printed document explaining how to use the kit, such as reaction conditions. The guide includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and descriptions on the surface of the package containing the kit. Furthermore, the guide includes information disclosed or provided through electronic media, such as the Internet.

[0174]

[0175] Another aspect of the present invention provides a method for preparing a transdermal delivery composition of the present invention.

[0176] Another aspect of the present invention provides a method for delivering a bioactive substance to the skin, comprising the step of applying the composition for transdermal delivery of the present invention and the bioactive substance to the skin or mucous membrane.

[0177] Another aspect of the present invention provides a method for improving skin, comprising applying the composition for transdermal delivery of the present invention and a physiologically active substance to the skin or mucous membrane.

[0178] Another aspect of the present invention provides a method for preventing or treating a skin or mucous membrane disease, comprising applying the pharmaceutical composition of the present invention to the skin or mucous membrane of a non-human subject.

[0179] Another aspect of the present invention provides a method for preventing or improving a skin or mucous membrane disease, comprising a step of applying a cosmetic composition or a pharmaceutical composition of the present invention to the skin.

[0180] Another aspect of the present invention provides a method for producing a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition, comprising a step of mixing a transdermal delivery composition of the present invention and a physiologically active substance.

[0181] Another aspect of the present invention provides a skin-penetrating use of a physiologically active substance comprising a peptide, a derivative thereof, or a fragment thereof, comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8 of the present invention.

[0182] Another aspect of the present invention provides a use for transdermal delivery of a physiologically active substance comprising a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8 of the present invention, a derivative thereof, or a fragment thereof.

[0183] Another aspect of the present invention provides a use of a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 8 of the present invention, a derivative thereof, or a fragment thereof, and a physiologically active substance; a cosmetic composition comprising the same; or a quasi-drug composition for improving skin or preventing or improving skin diseases.

[0184] 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 NOs: 5 to 8 of the present invention, a derivative thereof, or a fragment thereof, and a physiologically active substance for preventing or treating skin diseases. The composition for transdermal delivery, the physiologically active substance, the skin improvement, the pharmaceutical composition, the cosmetic composition, the quasi-drug composition, the skin disease, the prevention, the improvement, and the treatment are as described above.

[0185] In the present invention, the term “subject” means all animals, including mammals such as rats, livestock, and humans, but is not limited thereto.

[0186] In the present invention, the term "application" means contacting the transdermal delivery composition and physiologically active substance of the present invention with the skin of an individual by any suitable method, and includes, but is not limited to, all acts aimed at allowing the transdermal delivery composition and physiologically active substance to be absorbed into the skin.

[0187]

[0188] Hereinafter, the present invention will be described in more detail through examples. However, these examples and experimental examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0189]

[0190] Manufacturing Example 1: Manufacturing of skin-permeable peptide

[0191] To confirm the function of the skin-penetrating peptide, peptides having sequence numbers 5 to 8 and derivatives thereof were prepared.

[0192]

[0193] 1-1: Peptide synthesis of sequence number 1

[0194] 71.4 mg (0.10 mmol) of 2-chlorotrityl chloride resin (1.4 mmol / g loaded resin) purchased from Novabiochem corporation was weighed, the resin was solvated with 7.5 ml of methyl chloride, reacted for 5 minutes, and then the methyl chloride was removed.

[0195]

[0196] After that, 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 and added to the resin. The reaction solution was stirred at room temperature for 12 hours and then washed four times with 10 mL of DMF solvent.

[0197] Next, 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 from the material added to the aforementioned resin, which was then washed four times with 10 ml of DMF solvent (4 washes of 10 ml each). At this stage, the 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.].

[0198]

[0199] Meanwhile, Fmoc-Leu-OH (212.0 mg, 0.60 mmol), hexafluorophosphate benzotriazole tetramethyluronium (HBTU) (227.6 mg, 0.60 mmol), and hydroxybenzotriazole (HOBt) (81.0 mg, 0.6 mmol) were completely dissolved in 7.5 ml of DMF solvent, and then DIPEA (0.21 ml, 1.2 mmol) was added to the aforementioned resin. The reaction solution was stirred at room temperature for 3 hours and then washed four times with 10 ml each of DMF solvent. The completion of the reaction was confirmed at this stage by performing a Kaiser test.

[0200]

[0201] Next, the peptides were sequentially condensed (coupled) according to the same synthetic cycle as below.

[0202]

[0203] (1) Wash 4 times with DMF solvent (10 ml);

[0204] (2) Deprotection for 15 minutes using 20% ​​(w / v) piperidine DMF solution (7.5 ml);

[0205] (3) Wash 4 times with DMF solvent (10 ml);

[0206] (4) Addition of Fmoc-amino acid, HBTU, HOBt, DIPEA;

[0207] (5) Add HBTU, HOBt, and DIPEA condensation reagents to activate amino acids and condense for 3 hours;

[0208] (6) Wash 4 times with DMF solvent (10 ml);

[0209]

[0210] The above (1) to (6) were repeated continuously, and at this time, the amino acid (0.60 mmol) protected with Fmoc after Fmoc-Leu-OH was added to the resin reaction vessel in the order described below and condensed.

[0211] (i) Fmoc-Tyr(tBu)-OH ;

[0212] (ii) Fmoc-Lys(Boc)-OH ;

[0213] (iii) Fmoc-Leu-OH ;

[0214] (iv) Fmoc-Ile-OH ;

[0215] (v) Fmoc-Lys(Boc)-OH ;

[0216] (vi) Fmoc-Lys(Boc)-OH ;

[0217] (vii) Fmoc-Phe-OH ;

[0218] (viii) Fmoc-Leu-OH ;

[0219] (ix) Fmoc-Lys(Boc)-OH ;

[0220] (x) Fmoc-Lys(Boc)-OH ;

[0221]

[0222] After (6) of Fmoc-Lys(Boc)-OH condensation, the final step was to treat with a 20% piperidine DMF solution (7.5 ml), then wash with 10 ml each of DMF solvent three times, MC solvent three times, and diethyl ether solvent three times.

[0223] Immediately after the completion of the synthesis as described above, the peptide was cleaved from the resin using a mixture of trifluoroacetic acid / triisopropylsilane / water (95:2.5:2.5) (10 ml) for 3 hours. The resulting mixture was treated with 100 ml of refrigerated diethyl ether solvent to produce a precipitate. The obtained precipitate was centrifuged to completely precipitate, and the trifluoroacetic acid was removed once. The above procedure (a step of adding 100 ml of diethyl ether solvent to wash the precipitate and centrifuging - a step to remove the trifluoroacetic acid that was attempted to be removed once) was repeated twice to obtain a solidified precipitate.

[0224] The above precipitate (peptide) was purified by HPLC using a 5% to 100% acetonitrile / water gradient solvent system containing 0.001% trifluoroacetic acid over 50 minutes using a C-18 column. The pure purified fraction was lyophilized to obtain 155 mg of the skin-permeable peptide of the following sequence number 1 as a white powdery trifluoroacetate salt.

[0225]

[0226] SEQ ID NO: 1: H2N-[Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-Lys]-CO2H

[0227] MS(ESI)m / e, [M+H]+= 1550.17; (155 mg)

[0228]

[0229] 1-2: Synthesis of sequence numbers 2 to 8

[0230] Using the same manufacturing process as Manufacturing Example 1-1 above, but changing the order of the amino acids protected with Fmoc, the peptides of SEQ ID NO: 2 to SEQ ID NO: 8 below were manufactured.

[0231]

[0232] SEQ ID NO: 2: H2N-[Arg-Arg-Leu-Phe-Arg-Arg-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H

[0233] MS(ESI)m / e, [M+H]+= 1718.23; (171 mg)

[0234]

[0235] SEQ ID NO: 3: H2N-[Arg-Phe-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Arg]-CO2H

[0236] MS(ESI)m / e, [M+H]+= 1666.18; (166 mg)

[0237]

[0238] SEQ ID NO: 4: H2N-[Arg-Arg-Leu-Phe-Arg-Leu-Ile-Leu-Arg-Tyr-Leu-Phe]-CO2H

[0239] MS(ESI)m / e, [M+H]+= 1666.18; (166 mg)

[0240]

[0241] SEQ ID NO: 5: H2N-[Arg-Lys-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H

[0242] MS(ESI)m / e, [M+H]+= 1634.2; (153 mg)

[0243]

[0244] SEQ ID NO: 6: H2N-[Lys-Arg-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H

[0245] MS(ESI)m / e, [M+H]+= 1634.2; (143 mg)

[0246]

[0247] SEQ ID NO: 7: H2N-[Lys-Arg-Leu-Phe-Lys-Arg-Ile-Leu-Arg-Tyr-Leu-Lys]-CO2H

[0248] MS(ESI)m / e, [M+H]+= 1634.2; (132 mg)

[0249]

[0250] SEQ ID NO: 8: H2N-[Arg-Lys-Leu-Phe-Arg-Lys-Ile-Leu-Lys-Tyr-Leu-Arg]-CO2H

[0251] MS(ESI)m / e, [M+H]+= 1634.2; (172 mg)

[0252]

[0253] Example 1: Evaluation of skin penetration activity of physiologically active substances of SEQ ID NO: 5 to SEQ ID NO: 8 and their derivatives

[0254] To confirm the function as a skin-permeable peptide, the skin permeation activity for lidocaine was evaluated in comparison with known peptides. The skin permeation activity of a skin-permeable peptide composed of any one amino acid sequence selected from SEQ ID NOs: 5 to 8 and its derivatives SEQ ID NOs: 1 to 4 or a known peptide mixed with lidocaine, or treated alone, was evaluated.

[0255] Specifically, to evaluate skin permeation activity, the Franz diffusion cell assay was performed using actual human skin, and the permeation amount of lidocaine was measured by HPLC and then compared. The known peptide sequence is as follows.

[0256]

[0257] - Public peptide sequence (TAT): GRKKRRQRRRPQ

[0258]

[0259] Franz diffusion cell assay

[0260] 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.

[0261] To measure the skin penetration activity of skin-penetrating peptides, the Franz diffusion cell assay was performed according to the OECD (2004, Paris) Guidance Document for the Conduct of Skin Absorption Studies and the Ministry of Food and Drug Safety (2009) Guidelines for Alternative Animal Testing Methods for Cosmetics (in vitro skin absorption test method).

[0262] First, the skin area was determined to be 3㎠, and the stratum corneum of the skin was placed upward on the receptor chamber. Then, the donor chamber was placed on the stratum corneum and fixed with a clamp. A magnetic bar was inserted into the receptor, which is the lower part of the Franz diffusion cell, and a buffer was filled through the sampling arm. The buffer was filled with pH 7.4 PBS if the test substance was water-soluble, and with ethanol:water = 1:1 if the test substance was fat-soluble.

[0263] The temperature of the receptor fluid was maintained constant at 32°C to ±1°C and the humidity was maintained at 30 to 70%, and then stabilized for 30 minutes. After that, the stirrer was set to 600 rpm. 1 mL of the sample at a concentration of 2 mM was applied to the skin of each group. The magnetic stirrer bar was placed in the receptor chamber and maintained at 600 rpm to ensure good mixing of the solution. After 6 hours, samples were taken through the sampling arm of the receptor chamber, and the samples were analyzed by HPLC along with the STD (standard sample 100 uM) for quantification. The transmittance was expressed as a percentage (%) compared to the STD.

[0264]

[0265] As a result, it was confirmed that when using sequence numbers 5 to 8 and derivatives thereof, the skin permeation amount of lidocaine was significantly superior compared to when using known peptides and when no peptides were used (Fig. 1).

[0266]

[0267] Example 2: Evaluation of skin penetration activity of bioactive substances in skin-penetrating peptides

[0268] Among the peptides of SEQ ID NOs. 1 to 8 described above, the skin permeation activity of the skin-permeable peptide of SEQ ID NO. 8, which has the highest efficacy, against caffeine was measured at different time points. The skin permeation activity of the skin-permeable peptide of SEQ ID NO. 8 and caffeine was mixed or treated alone, and its skin permeation activity was evaluated.

[0269] Specifically, the Franz diffusion cell assay was performed using actual human skin as in Example 1, and the amount of caffeine permeation was measured using HPLC and then compared (Fig. 2).

[0270]

[0271] As a result, it was confirmed that when using sequence number 8, the skin penetration amount of caffeine was significantly superior by time zone compared to when using a known peptide or when no peptide was used.

[0272]

[0273] Example 3: Evaluation of skin penetration activity of skin-penetrating peptides for bioactive substances of various molecular weights.

[0274] To evaluate whether the skin-penetrating peptide has skin-penetrating activity for bioactive substances of various molecular weights, FITC-conjugated 3, 30, and 300 kDa collagen (COLA) and 800 kDa hyaluronic acid (HA) were mixed with or treated alone with the peptide of sequence number 8 to obtain aqueous solutions using the Franz diffusion cell assay, and the FITC fluorescence value was analyzed using a Fluorescence Microplate Reader (SpectraMax i3, Molecular Devices, USA).

[0275] The FITC conjugation method was performed by purchasing a FITC conjugation kit (Lightning-Link® (ab188285)) from Abcam (Cambridge, MA, USA) and preparing it according to the manufacturer's instructions.

[0276]

[0277] As a result, when the peptide of sequence number 8 was mixed and treated, it exhibited a strong skin penetration activity effect of collagen of various molecular weights (3 kda, 30 kda, 300 kda) and hyaluronic acid (Fig. 3).

[0278]

[0279] Example 4: Evaluation of skin penetration activity of skin-penetrating peptides in liposomes

[0280] To closely examine the effect of skin-penetrating peptides on the skin penetration effect of liposomes, a type of bioactive substance, human skin was treated with the peptide of sequence number 8 and liposomes mixed or alone for 3 hours, and then an aqueous solution was obtained through a Franz diffusion cell assay. The FITC fluorescence value was analyzed using a Fluorescence Microplate Reader (SpectraMax i3, Molecular Devices, USA).

[0281] Fluorescein was liposomeized using a liposome kit purchased from Sigma Aldrich (MO, USA), and the size of the liposome was confirmed to be 180 nm as measured by SEM.

[0282]

[0283] As a result, it was confirmed that when the peptide of sequence number 8 was mixed with liposomes and treated, the skin penetration effect for liposomes was significantly increased (Fig. 4).

[0284]

[0285] Example 5: Evaluation of the Enhancement of the Efficacy of Bioactive Substances According to the Skin Penetration Effect of Skin-Permeable Peptides on Bioactive Substances

[0286] In order to confirm the enhancement of the efficacy of a physiologically active substance according to the skin penetration effect of the active ingredient of the peptide of sequence number 8, HDF was treated with a mixture of the physiologically active substance KTTKS peptide and the peptide of sequence number 8 or alone, and the amount of collagen production was analyzed using an ELISA assay.

[0287] 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), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gipco, Life Technologies, Grand Island, NY, USA) at 37°C in a 5% CO2 incubator.

[0288] 2x10 HDF cells 5 250 ㎕ was dispensed into 48 wells at a concentration of 10 cells / ㎖ and stabilized for 24 hours. Samples were treated by concentration and cultured in a CO2 incubator for 48 hours to obtain culture medium. Antibodies were diluted in coating buffer according to the manufacturer's instructions (Collagen type I ELISA Kit, R&D Biosystems), coated on microwells, and incubated overnight at 4℃. Each well was washed three times with washing buffer, and 100 ㎕ of culture medium was dispensed. This was left at room temperature for 1 hour, washed three times with washing buffer, treated with 100 ㎕ of avidin-HRP conjugated antibody, left at room temperature for 1 hour, and washed three times again. Here, 100 μl of TMB substrate was dispensed and left in the dark for 30 minutes, then 50 μl of stop solution was treated, and the absorbance was measured at 450 nm using a microspectrophotometer (Molecular Device, Sunnyvale, CA, USA).

[0289]

[0290] As a result, when the peptide of sequence number 8 was mixed with KTTKS and treated, it was confirmed that the amount of collagen production increased significantly as the skin penetration effect of the KTTKS peptide increased (Fig. 5).

[0291]

[0292] Example 6: Evaluation of the efficacy enhancement of skin-penetrating peptides for bioactive substances through human application tests

[0293] To confirm the enhanced efficacy of the active ingredient of the peptide of sequence number 8 through skin penetration through a human application test, a human application test was conducted on 20 test subjects by requesting the Clinical Trial Center of SkinMed Co., Ltd. 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.

[0294] For the test subjects, the product application area was the forearm, the product usage amount was approximately 0.5g±0.1g / time, and the product usage cycle and method were twice a day (other lifestyle patterns were the same as usual). This test was conducted under constant temperature and humidity conditions (indoor temperature 20-25℃, humidity 40-60%) without air movement and direct sunlight after the subjects had rested their skin for at least 30 minutes. In order to accurately judge the effect of the present invention, the application area was kept as preserved as possible by preventing water from coming into contact with the application area or sweating during the test period. In addition, in order to reduce measurement errors, each measurement was repeated three times by the same tester, and the average value was evaluated.

[0295] To determine whether skin texture is improved, Visioscan was used to evenly distribute light to a reflector and capture the image with a video sensor chip and objective lens. A non-glossy image of the skin surface (approximately 10 x 8 mm in size) expressed in 255 gray levels was derived, and the SEr (Roughness) value indicating skin roughness was reflected among the derived measurements. A fixed area was measured, and the same area was re-measured 24 hours after using the product to check the skin roughness compared to before using the product. As SEr increases, skin roughness decreases, indicating an improvement in skin texture.

[0296] Improvement in skin elasticity was measured using the Cutometer dual MPA 580. The Cutometer's measurement principle is based on a suction method, whereby the skin is mechanically deformed by negative pressure. Pressure is generated within the device, and the skin is then sucked back into the probe's aperture after a predetermined time. The penetration depth inside the probe is determined by a non-contact optical measurement system consisting of a light source, a photoreceptor, and two opposing prisms that project light from the transmitter to the receptor. The luminous intensity varies with the skin penetration depth. During the measurement, the negative pressure (determinacy) and the skin's resistance to return to its original position (elasticity) are displayed in real-time as a curve (in mm / hour). From this curve, the measured parameters are recalculated to the R2 value, which reflects the elasticity and viscoelasticity characteristics of the skin surface and aging skin. The closer the value is to 1 (100%), the more elastic the curve is, and in this human application test, the R2 (R2 = Ua / Uf; ratio of maximum amplitudes) value indicating skin elasticity of the test area was measured 24 hours after product use.

[0297] Glossiness was measured on the forearm using a SkinGlossMeter (SGM, Delfin). When light from a 635nm red diode laser reflects off the skin surface, the amount of reflection is detected. The light passing through the device's internal diffractive microstructures is then converted into a Skin Gloss Unit (SGU) parameter, which calculates the gloss value. The higher the skin gloss, the higher the measured value. In this human application test, the SGU value, which represents skin glossiness, was measured before and 24 hours after product use.

[0298] Additionally, the improvement rate was calculated as follows:

[0299]

[0300] - Improvement rate (%) calculation formula = {(before use - after use) / before use} X 100

[0301]

[0302] As a result, after 24 hours, the test group showed an average improvement rate of approximately 40 times, 4 times, and 5 times in skin texture, skin elasticity, and gloss compared to the control group (Table 1).

[0303]

[0304] Efficacy classification Average improvement rate after 24 hours of product use Control group Test group Skin texture (SEr value) 1.0872 % 43.0538 % Skin elasticity (R2 value) 3.4389 % 12.3372 % Gloss (SGU value) 1.6963 % 8.3013 %

[0305] From the above results, it was confirmed that the skin-permeable peptide of the present invention, its derivative, or its fragment has excellent skin permeation activity and thus has a remarkable effect as a skin-permeable peptide.

[0306]

[0307] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition for transdermal delivery of a physiologically active substance comprising a skin-permeable peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

2. A composition for transdermal delivery of a physiologically active substance, wherein the derivative is a composition 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-permeable peptide composed of any one of SEQ ID NOs: 5 to 8 is substituted with another basic amino acid.

3. A composition for transdermal delivery of a physiologically active substance, wherein the basic amino acid in the second paragraph is lysine or arginine.

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

4.

5. A composition for transdermal delivery of a physiologically active substance, wherein the composition for transdermal delivery in the first paragraph increases the permeability of the physiologically active substance to at least one selected from the group consisting of skin and mucous membranes.

6. A composition for transdermal delivery of a physiologically active substance in accordance with claim 1, wherein the composition for transdermal delivery of the physiologically active substance further comprises a carrier.

7. A composition for transdermal delivery of a physiologically active substance, wherein the carrier is a liposome in the sixth paragraph.

8. A composition for transdermal delivery of a physiologically active substance, wherein the physiologically active substance in the first paragraph is a substance having a physiological effect in a living body.

9. A composition for skin penetration of a physiologically active substance comprising a skin-penetrating peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

10. A carrier for transdermal delivery of a physiologically active substance comprising a skin-permeable peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

11. A skin-penetrating carrier of a physiologically active substance comprising a skin-penetrating peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof.

12. A cosmetic composition comprising the transdermal delivery composition of paragraph 1.

13. A pharmaceutical composition comprising the composition for transdermal delivery of paragraph 1.

14. A pharmaceutical composition for external application to the skin or mucosa, comprising the composition for transdermal delivery of paragraph 1.

15. A kit comprising the composition for transdermal delivery of paragraph 1 and a physiologically active substance.

16. A method for preparing a composition for transdermal delivery of paragraph 1.

17. A method for manufacturing a cosmetic composition, a pharmaceutical composition, or a pharmaceutical composition, comprising a step of mixing the composition for transdermal delivery of paragraph 1 and a physiologically active substance.

18. A method for transdermal delivery of a physiologically active substance, comprising a step of applying the composition for transdermal delivery of paragraph 1 and the physiologically active substance to the skin or mucous membrane.

19. A method for improving skin, comprising a step of applying the composition for transdermal delivery and the physiologically active substance of paragraph 1 to the skin or mucous membrane.

20. Use of a physiologically active substance comprising a peptide, a derivative thereof, or a fragment thereof, comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8 for skin penetration.

21. Use of a peptide comprising any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 8, a derivative thereof, or a fragment thereof for transdermal delivery of a physiologically active substance.