Novel skin permeable peptides for skin and mucosa, compositions comprising the same and uses thereof

Abstract

The skin-permeable peptide, the derivative thereof, or the fragment thereof of the present disclosure effectively delivers a skin bioactive molecule to a deep skin tissue by an assembly or fusion method, thereby providing excellent skin permeability of the bioactive substance and a very good skin residual effect to maximize the bioactive efficacy. Thus, the skin-permeable peptide, the derivative thereof, or the fragment thereof can be widely used as an active ingredient of an external pharmaceutical composition and a functional cosmetic composition targeting skin tissues including mucosa.

Description

Technical Field

[0001] This disclosure relates to compositions for the transdermal delivery of bioactive substances, said compositions comprising skin-permeable peptides, derivatives thereof, or fragments thereof; skin-permeable compositions; carriers for transdermal delivery; skin-permeable carriers; pharmaceutical compositions; quasi-pharmaceutical compositions; cosmetic compositions; kits comprising compositions for transdermal delivery; methods for preparing compositions, cosmetic compositions, and quasi-pharmaceutical compositions for transdermal delivery; methods for transdermal delivery of bioactive substances; and uses for transdermal delivery of bioactive substances. Background Technology

[0002] The skin, comprising the largest part of the human body, is composed of the epidermis, dermis, and subcutaneous tissue, and performs various functions in the external environment. In particular, due to the unique properties of the stratum corneum, which forms the skin barrier, substances known to penetrate the skin have molecular weights of approximately 500 Da or less. However, low molecular weight substances are known to have low skin penetration efficiency, and high molecular weight substances (such as growth factors) have even lower penetration efficiency. Therefore, in the cosmetics and pharmaceutical industries, there is a strong need for safe methods to effectively deliver bioactive substances.

[0003] Physical delivery systems (such as microneedles) can cause irreversible damage to the skin. Existing skin-permeable materials often use lipophilic substances or polymers (Korean Patent No. 10-2274435), which can cause various side effects and unknown toxicities in the human body. Therefore, it is necessary to use peptides, which are fragments of proteins present in the human body, to safely deliver bioactive substances to the dermis.

[0004] public

[0005] Technical issues

[0006] The inventors of this application have accomplished this invention by developing skin-permeable peptides. The biomembrane-penetrating peptide technology developed in this disclosure is a state-of-the-art biotechnology that significantly improves the absorption rate and bioavailability of raw materials in the body. It is also a next-generation novel biomaterial platform technology that can fully realize the efficacy even when injected in trace amounts into the raw materials of functional cosmetics or advanced pharmaceuticals. Therefore, this technology is intended for application in the development of skin-permeable cosmetics and pharmaceuticals.

[0007] Technical solution

[0008] The objective of this disclosure is to provide compositions for the transdermal delivery of bioactive substances, said compositions comprising skin-permeable peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8.

[0009] Another objective of this disclosure is to provide compositions for the skin penetration of bioactive substances, said compositions comprising skin-permeable peptides, derivatives thereof, or fragments thereof.

[0010] Another objective of this disclosure is to provide a carrier for the transdermal delivery of bioactive substances, said carrier comprising skin-permeable peptides, derivatives thereof, or fragments thereof.

[0011] Another objective of this disclosure is to provide a skin-permeable carrier for bioactive substances, said carrier comprising skin-permeable peptides, their derivatives, or fragments thereof.

[0012] Another objective of this disclosure is to provide cosmetic compositions comprising compositions for transdermal delivery.

[0013] Another objective of this disclosure is to provide quasi-pharmaceutical compositions comprising compositions for transdermal delivery.

[0014] Another objective of this disclosure is to provide pharmaceutical compositions for external application to the skin or mucous membranes, said pharmaceutical compositions including compositions for transdermal delivery.

[0015] Another objective of this disclosure is to provide a kit comprising a composition for transdermal delivery and a bioactive substance.

[0016] Another objective of this disclosure is to provide a method for preparing compositions for transdermal delivery.

[0017] Another objective of this disclosure is to provide a method for improving skin using a composition for transdermal delivery.

[0018] Another objective of this disclosure is to provide a method for preventing or treating skin or mucous membrane diseases using a pharmaceutical composition.

[0019] Another objective of this disclosure is to provide methods for preparing cosmetic compositions and quasi-pharmaceutical compositions.

[0020] Another objective of this disclosure is to provide a method for transdermal delivery of bioactive substances using peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8.

[0021] Another objective of this disclosure is to provide the use of peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 for the transdermal delivery of bioactive substances.

[0022] Another objective of this disclosure is to provide the use of peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 for the skin penetration of bioactive substances.

[0023] Beneficial effects

[0024] The skin-permeable peptides, their derivatives, or fragments disclosed herein effectively deliver skin-active molecules deep into skin tissue via assembly or fusion methods, thereby providing excellent skin permeability and good skin residue to maximize bioactive efficacy. Therefore, skin-permeable peptides, their derivatives, or fragments can be widely used as active ingredients in topical pharmaceutical compositions and functional cosmetic compositions targeting skin tissues, including mucous membranes. Brief description of the attached diagram

[0026] Figure 1 The results of Franz diffusion cell assays used to analyze the skin penetration of the peptides of SEQ ID NO:1-8 for bioactive substances compared with known peptides are presented by HPLC quantification.

[0027] Figure 2 HPLC quantification of the Franz diffusion cell determination over time, used to analyze the skin penetration effect of the skin-permeable peptides of this disclosure on bioactive substances;

[0028] Figure 3 The results of a Franz diffusion cell assay for analyzing the skin penetration effect of the skin-permeable peptides of this disclosure on bioactive substances of different molecular weights are shown using a fluorescence microplate reader.

[0029] Figure 4 The results of a Franz diffusion cell assay for analyzing the skin penetration effect of the skin-permeable peptides of this disclosure on liposomes are shown using a fluorescence microplate reader; and

[0030] Figure 5 The results show the efficacy improvement of the skin penetration effect of skin-permeable peptides according to this disclosure on the skin penetration of bioactive substances. Invention Details

[0032] The present disclosure will be described in detail below as follows. Furthermore, each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. Moreover, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not limited to the specific descriptions described below.

[0033] One aspect of this disclosure provides a composition for transdermal delivery of a bioactive substance, the composition comprising a skin-permeable peptide, a derivative thereof, or a fragment thereof consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8.

[0034] As used herein, the term "skin permeation" refers to biomembrane permeation, and "skin-permeable peptide" refers to a peptide that is permeable to biomembranes. Skin-permeable peptides may be permeable to cells and biomembranes, specifically, to skin and / or mucous membranes, but are not limited thereto.

[0035] The skin-permeable peptides disclosed herein may include skin-permeable peptides, their derivatives, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8.

[0036] This disclosure can effectively introduce bioactive molecules into cells and skin that are difficult to deliver through the skin due to their molecular weight or the unique characteristics of the stratum corneum.

[0037] Compared to existing skin-permeable peptides, the skin-permeable peptides of this disclosure can deliver bioactive substances to skin cells or the dermis of the skin via compositions, transdermal delivery systems, and skin-permeable peptide assembly or fusion methods that have improved cell and skin permeability, said compositions comprising skin bioactive molecules.

[0038] The skin-permeable peptides, derivatives thereof, or fragments thereof disclosed herein can efficiently deliver skin-active molecules deep into skin tissue via assembly and fusion methods, exhibiting excellent skin permeability and superior skin residue, thereby maximizing physiological activity. Therefore, this disclosure can be used as a component of compositions targeting skin tissues including mucous membranes.

[0039] In exemplary embodiments of this disclosure, SEQ ID NO:5 to SEQ ID NO:8 and their derivatives have been confirmed to have significant skin permeability (Examples 1-6).

[0040] As used herein, the term "derivative" means a derivative of a skin-permeable peptide consisting of any of the amino acid sequences in SEQ ID NO:5-SEQ ID NO:8. Specifically, it can be, but is not limited to, a peptide having an amino acid sequence prepared by adding functional groups to the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8 or by deleting, modifying, substituting or adding amino acid sequences.

[0041] Specifically, in this disclosure, the derivative may be any one or more selected from SEQ ID NO:1 to SEQ ID NO:4, but is not limited thereto.

[0042] Specifically, the skin-permeable peptides and their derivatives comprising any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 disclosed herein may have sequences as shown in the following chemical formula 1, and may also have skin permeability, but are not limited thereto.

[0043] [Chemical Formula 1]

[0044] [X1–X2–Leu–Phe–X3–X4–Ile–Leu–X5–Tyr–Leu–X6]

[0045] In Formula 1, X1 to X6 can be selected from basic amino acids, and can be selected from arginine, lysine, leucine, and phenylalanine. Preferably, the skin-permeable peptide and its derivatives comprising any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 disclosed herein can have the sequence of Formula 1, and the derivatives disclosed herein can be composed of the amino acid sequences in SEQ ID NO:1 to SEQ ID NO:4, but are not limited thereto.

[0046] SEQ ID NO: 1: H2N–[Lys–Lys–Leu–Phe–Lys–Lys–Ile–Leu–Lys–Tyr–Leu–Lys]–CO2H,

[0047] SEQ ID NO:2: H2N–[Arg–Arg–Leu–Phe–Arg–Arg–Ile–Leu–Arg–Tyr–Leu–Arg]–CO2H,

[0048] SEQ ID NO:3: H2N–[Arg–Phe–Leu–Phe–Arg–Leu–Ile–Leu–Arg–Tyr–Leu–Arg]–CO2H,

[0049] SEQ ID NO:4: H2N–[Arg–Arg–Leu–Phe–Arg–Leu–Ile–Leu–Arg–Tyr–Leu–Phe]–CO2H,

[0050] SEQ ID NO:5: H2N–[Arg–Lys–Leu–Phe–Lys–Arg–Ile–Leu–Arg–Tyr–Leu–Lys]–CO2H,

[0051] SEQ ID NO: 6: H2N–[Lys–Arg–Leu–Phe–Arg–Lys–Ile–Leu–Lys–Tyr–Leu–Arg]–CO2H,

[0052] SEQ ID NO:7: H2N–[Lys–Arg–Leu–Phe–Lys–Arg–Ile–Leu–Arg–Tyr–Leu–Lys]–CO2H, and

[0053] SEQ ID NO: 8: H2N–[Arg–Lys–Leu–Phe–Arg–Lys–Ile–Leu–Lys–Tyr–Leu–Arg]–CO2H.

[0054] Specifically, the derivative may be a derivative in which the amino acid corresponding to any of the positions 1, 2, 5, 6, 9 and 12 from the N-terminus of the skin-permeable peptide composed of any amino acid sequence selected from SEQ ID NO:5 to SEQ ID NO:8 is replaced by another basic amino acid.

[0055] Amino acid substitutions can typically occur based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Specifically, among amino acids with 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 amino acids with uncharged side chains, nonpolar amino acids... The amino acids include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), tryptophan (W), and proline (P). Polar or hydrophilic amino acids include serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N), and glutamine (Q). Among the nonpolar amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0056] In this disclosure, "replaced by another basic amino acid" is not limited, as long as it is replaced by a basic amino acid different from the amino acid before substitution. In other words, any amino acid at positions 1, 2, 5, 6, 9, and 12 from the N-terminus of the skin-permeable peptide composed of any amino acid sequence selected from SEQ ID NO:5 to SEQ ID NO:8 can be replaced by another basic amino acid. Specifically, any amino acid selected from positions X1, X2, X3, X4, X5, and X6 can be replaced by another basic amino acid. Specifically, the basic amino acid can be selected from histidine, lysine, and arginine, and can be lysine or arginine, but is not limited thereto.

[0057] Regarding the objectives of this disclosure, the skin-permeable peptide derivative consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 may be any one or more selected from SEQ ID NO:1 to SEQ ID NO:4, but is not limited thereto.

[0058] As used herein, a “fragment” refers to a partial sequence of a peptide having a specific sequence. The fragments disclosed herein may be partial sequences of a skin-permeable peptide or a derivative thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8, wherein said partial sequence may also have skin permeability similar to that of the skin-permeable peptide or a derivative thereof.

[0059] Skin-permeable peptides, their derivatives, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 may be derived from natural products, but are not limited thereto, and may include any sequence as long as it has the same activity as skin-permeable peptides, their derivatives, or fragments thereof.

[0060] Although the skin-permeable peptides of this disclosure are described as skin-permeable peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5-SEQ ID NO:8, this does not preclude the addition of nonsense sequences upstream or downstream of the amino acid sequences of the skin-permeable peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5-SEQ ID NO:8, or allow for silent mutations that retain the same function as the skin-permeable peptide. It will be apparent to those skilled in the art that a peptide possesses the same or corresponding activity as the skin-permeable peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5-SEQ ID NO:8, it belongs to the category of skin-permeable peptides of this disclosure. For specific examples, the skin-permeable peptides of this disclosure may be skin-permeable peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5-SEQ ID NO:8, or peptides having 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity of the amino acid sequences.

[0061] Even though this disclosure describes "a peptide comprising an amino acid sequence listed with a specific sequence number", "a peptide composed of an amino acid sequence listed with a specific sequence number" or "a peptide having an amino acid sequence listed with a specific sequence number", it is obvious that peptides having partially deleted, modified, substituted, conservedly substituted or added amino acid sequences may also be used in this disclosure, provided that the peptide has the same or equivalent activity as the peptide composed of the amino acid sequence with the corresponding sequence number. For example, the addition of sequences that do not change the function of the peptide, naturally occurring mutations, their silent mutations, or conserved substitutions at the N-terminus and / or C-terminus of the amino acid sequence.

[0062] "Conservative substitution" refers to the replacement of one amino acid with another amino acid that has similar structure and / or chemical properties. Such substitutions typically occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Generally, conservative substitutions have little or no effect on the activity of the peptide.

[0063] The peptides disclosed herein can be prepared by, but are not limited to, the continuous formation of one or more amino acid amide bonds or suitably protected amino acid bonds on an amino acid backbone bound to a solid phase. Insertion, substitution, or deletion of another amino acid into the peptide is also possible without significantly impairing stability, and this also falls within the scope of this disclosure.

[0064] Additionally, the skin-permeable peptides of this disclosure can be prepared by further conjugating known cell-permeable peptides that promote intracellular migration to the C-terminus or N-terminus. For example, known cell-permeable peptides may be TAT peptides (Arg–Lys–Lys–Arg–Arg–Tyr–Arg–Arg–Arg) and Tat-PTD peptides (Gly–Arg–Lys–Lys–Arg–Arg–Gln–Arg–Arg–Arg:Tat PTD), but this disclosure is not limited thereto, and any cell-permeable peptide known in the art may be used, provided that the permeable peptide does not inhibit the activity of this disclosure.

[0065] The peptides and compounds disclosed herein can be prepared in the form of metal complex compounds, wherein the metal may be selected from, but is not limited to, copper, magnesium, calcium, iron, zinc, nickel, silver, germanium, and gallium. Preferably, copper may be used, but is not limited thereto.

[0066] Furthermore, the peptides disclosed herein can exist in the form of salts. The salt forms available for use in this disclosure can be prepared during the final separation and purification of the compound, or by reacting the amino group with a suitable acid. For example, acid addition salts may include, but are not limited to: acetates, adipates, alginates, citrates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butyrates, camphorates, camphorsulfonates, diglucuronides, glycerol phosphates, hemisulfates, heptanates, hexanoates, formates, fumarates, hydrochlorides, hydrobromide, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, mesitylenesulfonates, methanesulfonates, naphthalenesulfonates, nicotinates, 2-naphthalenesulfonates, oxalates, palmitates, pectinates, persulfates, 3-phenylpropionates, picrates, neopentanoates, propionates, succinates, tartrates, trichloroacetates, trifluoroacetates, phosphates, glutamates, bicarbonates, p-toluenesulfonates, and undecanoates. Additionally, 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 peptides of this disclosure can be prepared in the form of trifluoroacetate or acetate.

[0067] The amino or carboxyl groups of the amino acids used to prepare peptides included in the compositions of this disclosure can be protected by suitable protecting groups. The protected amino acids 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. Optionally, the protecting groups can be completely removed before adding the amino acid protected with a suitable protecting group. After all amino acids have been linked as needed, the final target peptide can be obtained by sequentially or simultaneously separating it from the free residual protecting groups and the free solid support.

[0068] As the most preferred synthetic method for preparing the peptide compounds of this disclosure, a solid-phase peptide synthesis method using a solid-phase polymer support can be employed, and the α-amino group of the peptide prepared by the above method can be protected by an acid- or basic-sensitive functional group. In this case, the protecting group of the amino acid must be stable under peptide condensation reaction conditions and must be easily removable without disrupting the grown peptide chain or causing any chiral centers contained therein to racemic. Therefore, suitable protecting groups may include 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropoxycarbonyl, tert-pentoxycarbonyl, isobornyloxycarbonyl, (α,α)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulfinyl, 2-cyano-tert-butoxycarbonyl, etc. Other suitable protecting groups known in the art for this purpose may also be used within the scope of this disclosure.

[0069] The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group can be used as the most preferred protecting group for amino acids used in the peptide synthesis of this disclosure.

[0070] In particular, as protecting groups for amino acid residues used in the peptide synthesis of this disclosure, tert-butyl (t-Bu) for N-methylglutamic acid; tert-butyloxycarbonyl (Boc) for lysine; t-butyl (t-Bu) for serine; tert-butyl (t-Bu) for threonine and allothreonine; and triphenylmethyl (Trt) for cysteine ​​are preferred, but this disclosure is not limited thereto.

[0071] In solid-phase peptide synthesis methods, C-terminal amino acids can be attached to a suitable solid support or resin. Suitable solid supports for the above synthesis are those that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and insoluble in the medium used, such as 2-chlorotriphenylmethyl chlorideresin, rink amide, or rink amide 4-methylbenzylhydrylamine resin (rink amide MBHA resin).

[0072] In particular, the preferred solid support for the C-terminal peptide can be 2-chlorotriphenylmethyl chloride, rink amide, or rink amide 4-methylbenzylhydroamine resin (rink amide MBHA resin), which is available from Novabichchem.

[0073] The process can be carried out at temperatures ranging from 10°C to 50°C, preferably at 30°C, in solvents such as dichloromethane, N-methylpyrrolidone (NMP), or DMF, with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate (BOP), or bis(2-oxo-3-oxazolyl)phosphine chloride (BOPCl), by... N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC), [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate] (HATU), or O-benzotriazol-1-yl-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) activate carboxylic acids for approximately 1 hour to approximately 24 hours, thereby condensing (binding, coupling) the C-terminal amide with the resin or solid support via a condensation reaction.

[0074] When the solid support is rink amide 4-methylbenzylhydroamine resin, the Fmoc functional group, which is the preferred protecting group, is cleaved with a secondary amine, preferably an excess of 20% piperidine DMF solution, before coupling with the C-terminal amino acid. Preferred reagents for coupling the target amino acid to the deprotected 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin are coupling agents in a DMF solvent for the suitably protected amino acid, such as N-methylmorpholine (NMM), 1-hydroxybenzotriazole (HOBt), [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate] (HATU), O-benzotriazol-1-yl-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU), N,N′-dicyclohexylcarbodiimide (DCC), or N,N′-diisopropylcarbodiimide (DIC).

[0075] The coupling of consecutive amino acids as described in this disclosure can be performed using automated peptide synthesizers widely known in the relevant art, or it can be performed directly by hand. Preferred conditions for the synthetic reaction include treatment with a secondary amine solution (preferably piperidine), followed by washing with a sufficient excess of solvent, and then introducing each protected amino acid to be coupled in a molar excess of about 3 to 7 times. This reaction can preferably be carried out in DMF solvent to deprotect the α-amino acids protected with the Fmoc group.

[0076] In the final step of peptide synthesis using the solid-phase resin of this disclosure, the peptide to be obtained can be removed from the resin by continuous or single manipulation, and the protecting groups protecting the amino acid residues can be deprotected. As conditions for removing the peptide from the resin and deprotecting the protecting groups present in the residues, a mixture of cleaving reagents that cleave the peptide bonds in the resin can typically be used, for example, a solution of dichloromethane containing trifluoroacetic acid (TFA), triisopropylsilane (TIS), anisole, water, or ethylenedithiol (EDT). The resulting solution can be treated with an excess of refrigerated diethyl ether solvent to form a precipitate. The resulting precipitate is completely precipitated by centrifugation, primarily removing excess trifluoroacetic acid, triisopropylsilane, anisole, water, and ethylenedithiol, and the above procedure is repeated two or more times to obtain a solidified precipitate. At this point, the completely deprotected peptide salt can be separated and purified using a mixed solvent consisting of water and acetonitrile 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.

[0077] This disclosure provides compositions for the transdermal delivery of bioactive substances.

[0078] As used herein, "bioactive substance" is a concept that generally refers to a substance that has physiological effects in the body. Bioactive substances can be hydrophilic, hydrophobic, or poorly soluble, and include, but are not limited to, any of these, as long as they can have physiological effects, such as natural products, chemical compounds, proteins, peptides, amino acids, nucleic acids, lipids, liposomes, polymers, etc. Furthermore, the bioactive substances disclosed herein can be used alone, and can be combined with, but are not limited to, combinations with other bioactive substances, combinations with carriers, and combinations with known skin-permeable peptides.

[0079] In one embodiment, the bioactive substance disclosed herein may be a major component of a medicament, quasi-medicine, and / or cosmetic for use on the skin, transdermal, and / or mucous membranes.

[0080] The bioactive substances disclosed herein may have one or more physiological effects, such as skin improvement, health promotion, disease prevention, and improvement, but may include, but are not limited to, any one of these effects, as long as they can be used with the skin-permeable peptides, their derivatives, or fragments thereof disclosed herein.

[0081] In one embodiment, the bioactive substance may be any one or more selected from lidocaine, caffeine, collagen, hyaluronic acid, and liposomes, but is not limited thereto.

[0082] Skin-permeable peptides, their derivatives, or fragments thereof, consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8, can be directly or indirectly linked to bioactive substances, but are not limited thereto.

[0083] In one embodiment, a skin-permeable peptide, its derivative or fragment thereof, consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8, and a bioactive substance are directly linked to each other or linked via a linker, or may additionally include other protein moieties, but are not limited thereto. As a linking method of this disclosure, any method performed in the art can be used without limitation, as long as the method does not alter the structure or activity of the skin-permeable peptide, its derivative or fragment thereof, consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8, and the bioactive substance to be linked. The linker can be a peptide linker consisting of 1 to 20 amino acids, or a non-peptide linker, but is not limited thereto.

[0084] In one embodiment, the skin-permeable peptide, its derivatives or fragments thereof, composed of any amino acid sequence from SEQ ID NO:5 to SEQ ID NO:8 disclosed herein may be linked to a bioactive substance via non-covalent bonds (e.g., ionic bonds, hydrogen bonds, van der Waals forces, etc.), but are not limited thereto.

[0085] In one embodiment, the skin-permeable peptide, its derivatives, or fragments thereof comprising any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 disclosed herein may be linked to a bioactive substance (e.g., via a covalent bond), but is not limited thereto. The skin-permeable peptide, its derivatives, or fragments thereof and the bioactive substance may be directly linked to each other, or may be linked via a linker, or may include other protein moieties, but is not limited thereto.

[0086] In one embodiment, the skin-permeable peptide can be self-assembled or self-fused using bioactive substances.

[0087] In one embodiment, one or more molecules of the skin-permeable peptide may surround or be fused with the bioactive substance, but are not limited thereto.

[0088] As used herein, “transdermal delivery” means delivery of a bioactive substance through a biological membrane. The compositions disclosed herein for transdermal delivery can be used to deliver bioactive substances to cells and biological membranes, specifically, for delivery through the skin and / or mucous membranes, but are not limited thereto.

[0089] The compositions disclosed herein for transdermal delivery can increase the permeability of bioactive substances to any one or more selected from skin and mucous membranes, but are not limited thereto.

[0090] The compositions disclosed herein for transdermal delivery may be used, as needed, by further including, but not limited to, carriers, excipients, diluents, antioxidants and / or buffers.

[0091] In one embodiment, the carrier may be a liposome, but is not limited thereto.

[0092] As used herein, "liposome" refers to a hollow structure formed by a phospholipid bilayer in aqueous solution. Liposomes may include, but are not limited to, bioactive substances within the hollow structure.

[0093] In one embodiment, liposomes may increase the skin permeability and / or transdermal delivery of bioactive substances through skin-permeable peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 of this disclosure, but are not limited thereto.

[0094] In one embodiment of this disclosure, it is demonstrated that bioactive substances such as lidocaine (3 kDa), caffeine (30 kDa), collagen (300 kDa), and hyaluronic acid (HA) (800 kDa) can increase skin permeability and transdermal delivery, regardless of their properties, size, or type. Furthermore, in one embodiment of this disclosure, it is also demonstrated that the skin-permeable peptides of this disclosure increase the skin permeability and transdermal delivery activity of liposomes (Examples 1-6). This indicates that the skin-permeable peptides of this disclosure increase the skin permeability and transdermal delivery activity of liposomes containing bioactive substances.

[0095] Another aspect of this disclosure provides a composition for skin penetration of a bioactive substance, said composition comprising a skin-permeable peptide, a derivative thereof, or a fragment thereof consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8.

[0096] Any amino acid sequence in SEQ ID NO:5 to SEQ ID NO:8, skin-permeable peptide, derivative or fragment, bioactive substance and skin permeability as described above.

[0097] In one embodiment, the skin penetration composition disclosed herein can increase the permeability of a bioactive substance to any one or more selected from skin and mucous membranes, but is not limited thereto.

[0098] The skin-penetrating compositions of this disclosure may be used, as needed, by further including, but not limited to, carriers, excipients, diluents, antioxidants and / or buffers.

[0099] Another aspect of this disclosure provides a carrier for transdermal delivery of bioactive substances, said carrier comprising a skin-permeable peptide, a derivative thereof, or a fragment thereof consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8.

[0100] Another aspect of this disclosure provides a skin-permeable carrier for a bioactive substance, the carrier comprising a skin-permeable peptide, a derivative thereof, or a fragment thereof consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8.

[0101] Any amino acid sequence in SEQ ID NO:5 to SEQ ID NO:8, skin-permeable peptide, derivative, fragment, bioactive substance, transdermal delivery and skin penetration as described above.

[0102] As used herein, taking a composition as an example, "carrier" means a support capable of supporting any substance or component and can be used interchangeably with supports, impregnating agents, or media. Furthermore, compositions that serve as examples of carriers can be applied and delivered to the skin by methods such as hand, suction, pouring, or brushing.

[0103] Compositions for transdermal delivery, compositions for skin penetration, carriers for transdermal delivery, and skin-permeable carriers may be included in pharmaceutical compositions, quasi-pharmaceutical compositions, or cosmetic compositions, but are not limited thereto.

[0104] Regarding the objectives of this disclosure, carriers and skin-permeable carriers for transdermal delivery of bioactive substances may include skin-permeable peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8.

[0105] In one embodiment, the composition for transdermal delivery, the composition for skin penetration, the carrier for transdermal delivery, and the skin-permeable carrier disclosed herein may be a topical skin preparation.

[0106] As used herein, "topical skin preparation" means a preparation applied to the skin or mucous membranes. In this disclosure, topical preparations are not limited to any particular form, as long as they are applied to the skin or mucous membranes, and topical preparations can be further classified as ointments, creams, lotions, gels, etc., but are not limited thereto.

[0107] Specifically, ointments can be semi-solid preparations for external use, in which the main components applied to the skin or mucous membranes are dissolved or dispersed in a matrix. Matrixes can be classified into lipophilic ointments and water-soluble ointments based on their composition. Ointments using lipophilic matrices can soften the skin or mucous membranes, have excellent sealing properties, and are suitable for application to wounds, but have the disadvantage of being difficult to remove. Ointments can also be preparations that favor locally penetrating components that are difficult to penetrate the skin due to their lipophilic properties and skin-softening effects, similar to those of the skin or mucous membranes.

[0108] Because ointments are primarily composed of an oily phase (such as oil), they possess excellent moisturizing properties and facilitate the penetration of lipophilic components, but suffer from the disadvantage of being sticky. Creams can be semi-solid topical preparations emulsified in a water-in-oil emulsion, and can be formulations whose viscosity is increased by mixing the oil and aqueous phases. As an emulsification process, surfactants or special processes can be used to mix the oil and aqueous phases. Furthermore, creams can be formulations that are easier to apply and remove than ointments, and emulsions are formulations with properties more similar to water than creams, and are topical preparations in which the main components are dissolved, emulsified, or finely dispersed in an aqueous liquid formulation. The main components, additives, and purified water are used, and all are homogenized to prepare solutions, suspensions, or emulsions. Gels can be topical semi-solid preparations composed of high-molecular-weight organic molecules that penetrate a liquid. Gels can be dispersions of gelling agents in water, and gelling agents (such as carbomer) or synthetic polymers can be used. Depending on the active ingredient, the purpose of use, and the method of application, various types of topical preparations can be used, and the topical preparations according to this disclosure are not limited thereto.

[0109] Another aspect of this disclosure provides cosmetic compositions comprising the compositions of this disclosure for transdermal delivery.

[0110] Compositions for transdermal delivery, etc., are as described above.

[0111] Cosmetic compositions may be used for skin improvement or disease prevention or improvement based on the physiological functions of bioactive substances, but are not limited thereto.

[0112] In one embodiment, the cosmetic composition may be used for skin improvement, but is not limited thereto.

[0113] In one embodiment, the cosmetic composition may be used for skin regeneration, wound healing, anti-aging, wrinkle improvement, whitening, inflammation relief, antibacterial effects, moisturizing, humectant and antioxidant effects, but is not limited thereto.

[0114] In one embodiment, the cosmetic composition may be used to prevent or improve skin conditions, but is not limited thereto.

[0115] Skin diseases are those conditions that present with abnormal symptoms on the skin caused by genetic, physiological, and environmental factors (such as inflammation, erythema, thickening, fibrosis, and dead skin cells). Examples of skin diseases include, but are not limited to, atopic dermatitis, allergies, psoriasis, seborrheic dermatitis, contact dermatitis, lupus erythematosus, acne, warts, viral infections, and papular urticaria.

[0116] As used in this article, “prevention” refers to any action taken to suppress or delay the onset or progression of a disease (e.g., a skin condition).

[0117] As used herein, “improvement” means any action that at least reduces parameters related to the relief or treatment of a condition (e.g., the severity of symptoms). In this disclosure, cosmetic compositions may be prepared in formulations selected from: solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, perfumes, face masks, softening lotions, emulsions, cosmetic bases, fragrances, soaps, liquid cleansers, bath preparations, sunscreens, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, lotion foundations, wax foundations, patches, and sprays, but are not limited thereto.

[0118] The cosmetic compositions disclosed herein may also include one or more cosmetically acceptable carriers, which are mixed in general skin cosmetics and may be suitably mixed with, but not limited to, common ingredients such as oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc.

[0119] The cosmetically acceptable carriers included in the cosmetic compositions disclosed herein vary depending on the formulation of the cosmetic composition.

[0120] When the formulations disclosed herein are ointments, pastes, creams, or gels, animal fats, vegetable oils, waxes, paraffin waxes, starches, tragacanth gum, cellulose derivatives, polyethylene glycol, silicones, bentonite, silica, talc, zinc oxide, etc., may be used as carrier components, but are not limited thereto. These substances may be used alone or in mixtures of two or more of them.

[0121] When the formulation disclosed herein is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc., may be used as a carrier component. In particular, when the formulation is a spray, a propellant (such as chlorofluorocarbon, propane / butane, or dimethyl ether) may be additionally included, but is not limited thereto. These substances may be used alone or in mixtures of two or more of them.

[0122] When the formulations disclosed herein are solutions or emulsions, solvents, solubilizers, or emulsifiers may be used as carrier components. For example, water, glycerol, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, etc., may be used. In particular, cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol, or sorbitol fatty acid esters may be used, but are not limited thereto. These substances may be used alone or in mixtures of two or more of them.

[0123] When the formulation disclosed herein is a suspension, liquid phase diluents (such as water, glycerol, ethanol, or propylene glycol), suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester, polyoxyethylene sorbitan acid ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, or astragalus gum, etc.) may be used as carrier components, but are not limited thereto. These substances may be used alone or in mixtures of two or more of them.

[0124] When the formulation disclosed herein is soap, fatty acid alkali metal salts, fatty acid half-ester salts, fatty acid protein hydrolysates, hydroxyethyl sulfonates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerin, sugars, etc., may be used as carrier components, but are not limited thereto. These substances may be used alone or in mixtures of two or more of them.

[0125] Another aspect of this disclosure provides quasi-pharmaceutical compositions comprising the compositions of this disclosure for transdermal delivery.

[0126] The quasi-pharmaceutical composition may be used for skin improvement or for the prevention or improvement of skin diseases based on the physiological function of the bioactive substance, but is not limited thereto.

[0127] Compositions for transdermal delivery, bioactive substances, skin improvement, dermatology, prevention and improvement, etc., as described above.

[0128] As used herein, "quasi-medicine" refers to an article that has a milder effect than a medicine among articles used for the diagnosis, treatment, improvement, relief, management, or prevention of human or animal diseases. For example, under the Pharmaceutical Affairs Law, quasi-medicine refers to articles that have a slight effect or no direct effect on the human body, excluding articles used as medicines, but including articles used for the purpose of treating or preventing human or animal diseases.

[0129] The quasi-pharmaceutical compositions disclosed herein can be prepared in formulations selected from, but are not limited to, body cleansers, foams, soaps, face masks, ointments, creams, lotions, serums, and sprays.

[0130] In the cosmetic or quasi-pharmaceutical compositions disclosed herein, skin-permeable peptides, their derivatives or fragments thereof, consisting of any amino acid sequence from SEQ ID NO:5 to SEQ ID NO:8 may be included in amounts from 0.01% to 100.0% by weight or from 0.1% to 10% by weight, based on the total amount of the composition.

[0131] Another aspect of this disclosure provides pharmaceutical compositions for external use on the skin, said pharmaceutical compositions comprising the compositions of this disclosure for transdermal delivery.

[0132] Based on the physiological functions of bioactive substances, pharmaceutical compositions can be used for the prevention or treatment of skin diseases, but are not limited to this. Compositions for transdermal delivery, preparations for topical application to the skin, bioactive substances, skin diseases, and prevention are as described above.

[0133] As used herein, “treatment” means any action by which the symptoms of a disease (e.g., a skin condition) improve or are beneficially enhanced. In this disclosure, treatment may include all actions that prevent disease recurrence, relieve symptoms, reduce the rate of progression, alleviate the disease state, provide temporary or sustained relief from the disease state, or achieve reduced or improved prognosis.

[0134] In addition to skin-permeable peptides, their derivatives or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8, the pharmaceutical compositions disclosed herein may also include bioactive substances and pharmaceutically acceptable carriers.

[0135] As used herein, "pharmaceutical acceptable" means that, during administration, it does not interfere with the biological activity and properties of the compound that is generally used in the pharmaceutical field without irritating the organism. The pharmaceutical compositions disclosed herein can be formulated with a carrier for use as food, pharmaceutical, feed additive, drinking water additive, etc.

[0136] There are no particular restrictions on the type of carrier, and any carrier commonly used in the art can be used. Non-limiting examples of carriers may include saline, sterile water, Ringer's solution, buffered saline, albumin, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. These may be used alone or in mixtures of two or more, but are not limited thereto.

[0137] In addition, the pharmaceutical compositions disclosed herein may be used by adding other pharmaceutically acceptable additives (such as excipients, diluents, antioxidants, buffers or antibacterial agents), and additionally by adding fillers, supplements, wetting agents, disintegrants, dispersants, surfactants, binders or lubricants as needed, but are not limited thereto.

[0138] The pharmaceutical compositions disclosed herein can be used after being formulated into various preparations suitable for topical application to the skin.

[0139] Non-limiting examples of preparations for external use on the skin may include, but are not limited to, aerosols, sprays, cleansers, ointments, coated powders, oils, creams, etc., as long as they can function as preparations for external use on the skin.

[0140] To formulate the pharmaceutical compositions disclosed herein for external application to the skin, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, topical formulations, etc., can be used. As non-aqueous solvents and suspensions, propylene glycol and polyethylene glycol, vegetable oils (such as olive oil), esters (such as ethyl oleate), etc., can be used, but are not limited to these.

[0141] Furthermore, more specifically, when formulating the pharmaceutical compositions of this disclosure, the pharmaceutical compositions of this disclosure are mixed with a stabilizer or buffer in water to prepare a solution or suspension, which can then be formulated into units such as ampoules. Additionally, when the pharmaceutical compositions of this disclosure are formulated into aerosols, propellants, etc., can be mixed with additives to disperse water-dispersible concentrates or wet powders, but are not limited thereto.

[0142] In addition, when the pharmaceutical compositions disclosed herein are formulated into ointments, creams, etc., animal fats, vegetable oils, waxes, paraffin waxes, starches, tragacanth gum, cellulose derivatives, polyethylene glycol, silicones, bentonite, silica, talc, zinc oxide, etc., may be used as carrier components, but are not limited thereto.

[0143] When the pharmaceutical compositions of this disclosure are formulated for external application to the skin, they may more preferably have a formulation selected from gels, patches, sprays, ointments, plasters, lotions, liniments, pastes and poultices, but are not limited thereto.

[0144] The pharmaceutically effective amount and topical effective dose of the pharmaceutical compositions disclosed herein may vary depending on the formulation, method of administration, time of administration, and / or route of administration of the pharmaceutical composition, and may vary depending on various factors, including the type and extent of the response to be achieved by administering the pharmaceutical composition, the type of subject, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, drugs used concurrently or alone in the subject, and other components of the composition, as well as similar factors well known in the medical field, and those skilled in the art can readily determine and prescribe an effective dose for the desired treatment. The pharmaceutical compositions of this disclosure may be administered once daily or divided into several doses. Therefore, the dosage is not intended to limit the scope of this disclosure in any way.

[0145] In addition, the application in this disclosure can be for external use, and the preferred dosage of the pharmaceutical composition of this disclosure can be from 1 mg / kg to 1000 mg / kg per day.

[0146] The route of administration and mode of administration of the pharmaceutical compositions disclosed herein can be independent and are not particularly limited to such a mode, and the pharmaceutical compositions can follow any route of administration or mode of administration as long as they can reach the target site. The pharmaceutical compositions can be administered as a topical preparation. Specifically, methods such as applying or spraying the composition to the disease site can be used, but are not limited thereto.

[0147] The pharmaceutical compositions disclosed herein can preferably be applied by smearing or spraying, and specifically, they are applied by topically applying to areas where skin condition needs to be improved or where skin diseases need to be prevented, improved or treated.

[0148] Another aspect of this disclosure provides a kit comprising the composition of this disclosure for transdermal delivery; and a bioactive substance.

[0149] The kit can be used for transdermal delivery, skin penetration, skin improvement, prevention or treatment of skin diseases, but is not limited thereto. Kits may be provided that allow the compositions and bioactive substances for transdermal delivery to be stored in separate containers and mixed upon use, but are not limited thereto.

[0150] Additionally, the kits disclosed herein may include, but are not limited to, compositions, solutions, or devices comprising one or more other components for percutaneous delivery, skin penetration, skin improvement, prevention, or treatment of skin diseases. Examples of other components may include, but are not limited to, suitable carriers, solubilizers, buffers, stabilizers, etc. Carriers may include soluble or insoluble carriers, and examples of soluble carriers may include physiologically acceptable buffers known in the art (e.g., PBS), and examples of insoluble carriers may include polystyrene, polyethylene, polypropylene and polyesters, polyacrylonitrile, fluoropolymers, cross-linked dextran, polysaccharides, polymers (such as magnetic microparticles coated with metal on latex, other paper, glass, metal, agarose), and combinations thereof.

[0151] Additionally, the kit disclosed herein may include a user guide describing the conditions for conducting the optimal reaction. This guide is a printed document explaining how to use the kit, such as reaction conditions. The guide may include a booklet or leaflet, a label affixed to the kit, and instructions on the surface of the packaging containing the kit. Furthermore, the guide may include information disclosed or provided through electronic media such as the Internet.

[0152] Another aspect of this disclosure provides a method for preparing the compositions of this disclosure for transdermal delivery.

[0153] Another objective of this disclosure is to provide a method for the transdermal delivery of a physiologically active substance, the method comprising applying to the skin or mucous membrane the composition of this disclosure for transdermal delivery; and the bioactive substance.

[0154] Another objective of this disclosure is to provide a method for improving skin, the method comprising applying the disclosed composition for transdermal delivery to the skin or mucous membrane; and a bioactive substance.

[0155] Another objective of this disclosure is to provide methods for preventing or treating skin or mucous membrane diseases, the methods comprising applying the pharmaceutical compositions of this disclosure to the skin or mucous membranes of a subject excluding humans.

[0156] Another objective of this disclosure is to provide methods for preventing or improving skin or mucous membrane diseases, the methods comprising applying a cosmetic composition or quasi-pharmaceutical composition of this disclosure to the skin.

[0157] Another object of this disclosure is to provide a method for preparing a cosmetic composition, a quasi-pharmaceutical composition, or a pharmaceutical composition, said method comprising mixing a composition of the present disclosure for transdermal delivery with a bioactive substance.

[0158] Another objective of this disclosure is to provide the use of peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 for the skin penetration of bioactive substances.

[0159] Another objective of this disclosure is to provide the use of peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences in SEQ ID NO:5 to SEQ ID NO:8 for the transdermal delivery of bioactive substances.

[0160] Another objective of this disclosure is to provide the use of peptides, derivatives thereof, or fragments thereof and bioactive substances comprising any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8; cosmetic compositions thereof; or quasi-pharmaceutical compositions thereof for the improvement or prevention or improvement of skin diseases.

[0161] Another objective of this disclosure is to provide the use of pharmaceutical compositions comprising peptides, derivatives thereof, or fragments thereof consisting of any of the amino acid sequences of SEQ ID NO:5 to SEQ ID NO:8, and bioactive substances of this disclosure for the prevention or treatment of skin diseases. Compositions for transdermal delivery, bioactive substances, skin improvement, pharmaceutical compositions, cosmetic compositions, quasi-pharmaceutical compositions, skin diseases, prevention, improvement, and treatment are as described above.

[0162] As used herein, the term “object” refers to any animal, including, but not limited to, mice, livestock, and mammals (including humans).

[0163] As used herein, the term “application” means, by any suitable method, bringing the compositions and bioactive substances of this disclosure for transdermal delivery into contact with the skin of a subject, and includes, but is not limited to, all actions intended to absorb the compositions and bioactive substances of this disclosure for transdermal delivery into the skin.

[0164] Forms for implementing the present invention

[0165] The present disclosure will be described in more detail below with reference to embodiments. However, these embodiments and experimental examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these embodiments and experimental examples.

[0166] Preparation Example 1: Preparation of Skin-Permeable Peptides

[0167] To examine the function of skin-permeable peptides, peptides and their derivative peptides of SEQ ID NO:5 to SEQ ID NO:8 were prepared.

[0168] 1-1: Synthesis of the peptide of SEQ ID NO:1

[0169] Weigh 71.4 mg (0.10 mmol) of 2-chlorotriphenylmethyl chloride resin (1.4 mmol / g loaded resin) purchased from Novabiochem, solubilize the resin with 7.5 mL of methyl chloride and react for 5 minutes, then remove the methyl chloride.

[0170] Subsequently, Fmoc–Lys(Boc)–OH (281.1 mg, 0.60 mmol) was completely dissolved in 7.5 mL of MC solvent, and 0.21 mL of 1.2 mmol of N,N′-diisopropylethylamine (DIPEA) was added. The solution was then 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.

[0171] Then, 7.5 mL of 20% (w / v) pipeidine DMF solution was added, followed by shaking for 15 minutes. The protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was completely removed from the substance added to the resin, and the substance was washed four times with 10 mL of DMF solvent (10 mL each time, four washes). At this stage, the deprotection reaction of the Fmoc protecting group was confirmed by Kaiser tests (E. Kaiser et al., Anal. Biochem., 1970, 34(2), 595–598).

[0172] Simultaneously, 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. Then, DIPEA (0.21 mL, 1.2 mmol) was added, and the mixture was added to the resin. After shaking at room temperature for 3 hours, the reaction solution was washed four times with 10 mL of DMF solvent each time. At this stage, a Kaiser test was performed to confirm the completion of the reaction.

[0173] Next, the peptides are continuously condensed (coupled) according to the same synthetic cycle as below.

[0174] (1) Wash 4 times with DMF solvent (10 mL);

[0175] (2) Deprotection was performed for 15 minutes using 7.5 mL of 20% (w / v) piperidine DMF solution;

[0176] (3) Wash 4 times with DMF solvent (10 mL);

[0177] (4) Add Fmoc-amino acids, HBTU, HOBt, and DIPEA;

[0178] (5) The amino acids were activated by adding HBTU, HOBt and DIPEA condensation reagents and condensed for 3 hours;

[0179] (6) Wash 4 times with DMF solvent (10 mL).

[0180] Repeat steps (1)-(6) above, and at this time, add the Fmoc-protected amino acid (0.60 mmol) after Fmoc–Leu–OH into the resin reaction vessel in the following order and condense.

[0181] (i)Fmoc–Tyr(tBu)–OH;

[0182] (ii)Fmoc–Lys(Boc)–OH;

[0183] (iii)Fmoc–Leu–OH;

[0184] (iv)Fmoc–Ile–OH;

[0185] (v)Fmoc–Lys(Boc)–OH;

[0186] (vi)Fmoc–Lys(Boc)–OH;

[0187] (vii)Fmoc–Phe–OH;

[0188] (viii)Fmoc–Leu–OH;

[0189] (ix)Fmoc–Lys(Boc)–OH;

[0190] (x)Fmoc–Lys(Boc)–OH.

[0191] After the condensation of (6)Fmoc–Lys(Boc)–OH, the final treatment was a 20% piperidine DMF solution (7.5 mL), followed by washing three times with 10 mL of DMF solvent each time, three times with MC solvent, and three times with diethyl ether solvent.

[0192] Immediately after synthesis, the peptide was cleaved from the peptide coupling resin for 3 hours using a mixture of trifluoroacetic acid / triisopropylsilane / water (95:2.5:2.5) (10 mL). The resulting mixture was treated with 100 mL of diethyl ether, stored in a refrigerator, to produce a precipitate. The precipitate was completely precipitated by centrifugation, primarily removing trifluoroacetic acid. The above procedure (washing the precipitate with 100 mL of diethyl ether and centrifuging; the procedure for removing trifluoroacetic acid, attempting its primary removal) was repeated twice to obtain a solidified precipitate.

[0193] The precipitate (peptide) was purified by HPLC within 50 minutes using a C-18 column and a 5%-100% acetonitrile / water gradient solvent system containing 0.001% trifluoroacetic acid. The purified fraction was lyophilized to obtain 155 mg of the skin-permeable peptide of SEQ ID NO:1, which is a white powder of trifluoroacetic acid salt.

[0194] SEQ ID NO: 1: H2N–[Lys–Lys–Leu–Phe–Lys–Lys–Ile–Leu–Lys–Tyr–Leu–Lys]–CO2H

[0195] MS(ESI)m / e, [M+H] + =1550.17; (155mg).

[0196] 1-2: Synthesis of peptides of SEQ ID NO:2 to SEQ ID NO:8

[0197] The peptides SEQ ID NO:2 to SEQ ID NO:8 were prepared in the same preparation process as in Preparation Examples 1-1, except that the order of the Fmoc-protected amino acids was changed.

[0198] SEQ ID NO:2: H2N–[Arg–Arg–Leu–Phe–Arg–Arg–Ile–Leu–Arg–Tyr–Leu–Arg]–CO2H

[0199] MS(ESI)m / e, [M+H] + =1718.23; (171mg)

[0200] SEQ ID NO:3: H2N–[Arg–Phe–Leu–Phe–Arg–Leu–Ile–Leu–Arg–Tyr–Leu–Arg]–CO2H

[0201] MS(ESI)m / e, [M+H] + =1666.18; (166mg)

[0202] SEQ ID NO:4: H2N–[Arg–Arg–Leu–Phe–Arg–Leu–Ile–Leu–Arg–Tyr–Leu–Phe]–CO2H

[0203] MS(ESI)m / e, [M+H] + =1666.18; (166mg)

[0204] SEQ ID NO:5: H2N–[Arg–Lys–Leu–Phe–Lys–Arg–Ile–Leu–Arg–Tyr–Leu–Lys]–CO2H

[0205] MS(ESI)m / e, [M+H] + =1634.2; (153mg)

[0206] SEQ ID NO:6: H2N–[Lys–Arg–Leu–Phe–Arg–Lys–Ile–Leu–Lys–Tyr–Leu–Arg]–CO2H

[0207] MS(ESI)m / e, [M+H] + =1634.2; (143mg)

[0208] SEQ ID NO:7: H2N–[Lys–Arg–Leu–Phe–Lys–Arg–Ile–Leu–Arg–Tyr–Leu–Lys]–CO2H

[0209] MS(ESI)m / e, [M+H] + =1634.2; (132mg)

[0210] SEQ ID NO:8: H2N–[Arg–Lys–Leu–Phe–Arg–Lys–Ile–Leu–Lys–Tyr–Leu–Arg]–CO2H

[0211] MS(ESI)m / e, [M+H] + =1634.2; (172mg).

[0212] Example 1: Skin permeability of SEQ ID NO:5 to SEQ ID NO:8 and their derivatives to bioactive substances Assessment

[0213] To confirm its function as a skin-permeable peptide, the skin permeability of lidocaine was evaluated by comparison with known peptides. Mixtures of skin-permeable peptides consisting of any amino acid sequence selected from SEQ ID NO:5 to SEQ ID NO:8 and their derivatives thereof SEQ ID NO:1 to SEQ ID NO:4, or known peptides and lidocaine, or lidocaine alone, were treated to evaluate skin permeability.

[0214] In detail, to assess skin permeability, real human skin was used, and skin permeability was measured by Franz diffusion cell assay; the permeation of lidocaine was measured by HPLC, and then compared. The known peptide sequences are as follows.

[0215] - Known peptide sequence (TAT): GRKKRRQRRRPQ

[0216] Franz diffusion cell measurement

[0217] The human skin used in this experiment was purchased from UMC Science Ltd., South Korea, and the PBS (phosphate-buffered saline, Gibco, USA) was purchased from Sigma-Aldrich.

[0218] To measure the skin permeability of skin-permeable peptides, Franz diffusion cell determination was performed in accordance with the OECD (2004, Paris) guidance document for skin absorption studies and the Ministry of Food and Drug Safety (2009) guidelines on alternative methods for animal testing of cosmetics (in vitro skin absorption test methods).

[0219] First, set the skin area to 3cm. 2 Place the skin, with the stratum corneum facing upwards, onto the receiving chamber. Then, place the supply chamber onto the stratum corneum and clamp it in place. Insert the magnetic strip into the receiver at the bottom of the Franz diffusion cell and fill the buffer via the sampling arm. When the test substance is water-soluble, the buffer is PBS at pH 7.4, or when the test substance is oil-soluble, ethanol:water = 1:1.

[0220] The temperature of the receptor fluid was kept constant at 32℃ ± 1℃, and the humidity was maintained at 30%–70%, then stabilized for 30 minutes. Afterward, the stirrer was set to 600 rpm. Each group's skin was treated with 1 mL of a 2 mM sample. The aqueous solution was then thoroughly mixed by maintaining the speed of the magnetic stir bar inserted into the receptor at 600 rpm. After 6 hours, samples were taken through the receptor's sampling arm, and for quantification, the STD (100 μM standard sample) and the sample were analyzed by HPLC, and permeability was expressed as a percentage (%) relative to the STD.

[0221] The results confirmed that when using SEQ ID NOS:5-8 and its derivatives, the skin penetration of lidocaine was significantly better than that when using known peptides and when not using peptides. Figure 1 ).

[0222] Example 2: Evaluation of the skin permeability of skin-permeable peptides to bioactive substances

[0223] Among the peptides listed in SEQ ID NO:1 to SEQ ID NO:8, the skin permeability of the skin-permeable peptide SEQ ID NO:8, which exhibits the highest effect on caffeine, was measured over time. The skin permeability was assessed by treating a mixture of the skin-permeable peptide SEQ ID NO:8 and caffeine, or by treating caffeine alone.

[0224] In detail, the Franz diffusion cell assay was performed using real human skin as described in Example 1, and the permeation of caffeine was measured by HPLC, and then compared. Figure 2 ).

[0225] The results confirmed that when using SEQ ID NO:8, the skin penetration of caffeine was significantly better than when using known peptides and when not using peptides.

[0226] Example 3: Evaluation of the skin permeability of skin-permeable peptides to bioactive substances with different molecular weights

[0227] To assess whether skin-permeable peptides possess skin permeability even for bioactive substances with different molecular weights, aqueous solutions of collagen (COLA) and hyaluronic acid (HA) conjugated with 3 kDa, 30 kDa, and 300 kDa FITC, or HA, and treated alone with the peptide of SEQ ID NO:8, were obtained by measuring the FITC fluorescence values ​​using a Franz diffusion cell.

[0228] According to the manufacturer's instructions, the FITC conjugation kit was purchased from Abcam (Cambridge, MA, USA). (ab188285)) is used to perform FITC concatenation.

[0229] The results confirmed that when a mixture of peptides with SEQ ID NO:8 was used, a strong skin penetration effect was observed in collagen and hyaluronic acid with different molecular weights (3kDa, 30kDa, and 300kDa). Figure 3 ).

[0230] Example 4: Evaluation of the skin permeability of skin-permeable peptides to liposomes

[0231] To carefully examine the effect of skin-permeable peptides on the skin penetration of liposomes, a physiologically and bioactive substance, human skin was treated with a mixture of the peptide and liposomes of SEQ ID NO:8 or liposomes alone for 3 hours. The resulting aqueous solutions were then measured using a Franz diffusion cell, and FITC fluorescence values ​​were analyzed using a fluorescence microplate reader (SpectraMax i3, Molecular Devices, USA).

[0232] Luciferin was liposomalized using a liposome kit purchased from Sigma-Aldrich (MO, USA), and the liposomalized luciferin was used as liposomes for skin permeability testing. The size of the liposomes was measured to be 180 nm by SEM.

[0233] The results confirmed that when the mixture of peptide and liposome of SEQ ID NO:8 was treated, the skin penetration effect of the liposomes was significantly increased. Figure 4 ).

[0234] Example 5: Bioactive substances caused by the skin penetration effect of skin-permeable peptides on bioactive substances Evaluation of quality and efficacy improvement

[0235] To examine the efficacy improvement of the bioactive substance due to the skin penetration effect of the active ingredient via the peptide of SEQ ID NO:8, HDF cells were treated with a mixture of the KTTKS peptide and the peptide of SEQ ID NO:8 as bioactive substances, or with KTTKS peptide alone, and the amount of collagen produced was analyzed using an ELISA assay.

[0236] The HDF cells used in this experiment were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The HDF cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, Life Technologies, Grand Island, NY, USA) and incubated at 37°C in a 5% CO2 incubator.

[0237] With a density of 2×10 5 HDF cells per cell / mL were dispensed into 48 wells at a volume of 250 μL and allowed to stabilize for 24 hours. Samples were treated with different concentrations and cultured in a CO2 incubator for 48 hours to obtain each culture. Antibodies were diluted in coating buffer and coated onto microwells according to the manufacturer's instructions (Collagen Type I ELISA Kit, R&D Biosystems), and incubated overnight at 4°C. After washing each well three times with washing buffer, 100 μL of culture was dispensed. The wells were incubated at room temperature for 1 hour, washed three times with washing buffer, treated with 100 μL of avidin-HRP conjugated antibody, incubated at room temperature for 1 hour, and then washed three times again. 100 μL of TMB substrate was dispensed, incubated in the dark for 30 minutes, treated with 50 μL of stop solution, and the absorbance was measured at 450 nm using a microspectrophotometer (Molecular Device, Sunnyvale, CA, USA).

[0238] The results confirmed that when the mixture of the peptide of SEQ ID NO:8 and KTTKS was treated, the amount of collagen produced increased significantly due to the increased skin penetration effect of the KTTKS peptide. Figure 5 ).

[0239] Example 6: Improved efficacy due to the skin penetration effect of skin-permeable peptides on bioactive substances Evaluation of human application testing

[0240] To verify the improved efficacy resulting from the skin penetration effect of the active ingredient, the peptide of SEQ ID NO:8, through human application testing, a human application test was conducted on 20 subjects at the SKNMED Clinical Trial Center. The test group was prescribed a mixture of the peptide of SEQ ID NO:8 and 1% ultra-low molecular weight hyaluronic acid, while the control group was prescribed 1% ultra-low molecular weight hyaluronic acid without the peptide of SEQ ID NO:8.

[0241] For the test subjects, the product was applied to the forearm, with an application amount of approximately 0.5g ± 0.1g per application, and the application cycle and method were twice daily (other lifestyles remained the same). The 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 airflow or direct sunlight. To accurately determine the effects of this disclosure, the application area was prevented from contact with water or sweat during the test, thus preserving as much of the product application area as possible. Furthermore, to minimize measurement error, each measurement was repeated three times using the same testing instrument, and the average value was evaluated.

[0242] To examine whether skin texture had improved, Visioscan was used to uniformly distribute light onto a reflector, and images were captured using a video sensor chip and objective lens. A non-glossy image of the skin surface (approximately 10mm × 8mm) representing 255 gray levels was exported, and the exported measurements reflected the SEr (Roughness) value, which indicates skin roughness. Predetermined areas were measured, and the same areas were measured again 24 hours after product use, comparing the area before product application to the measured area afterward, to examine skin roughness. As SEr increased, skin roughness decreased, indicating improved skin texture.

[0243] The Cutometer Dual MPA 580 was used to measure whether skin elasticity had improved. The Cutometer's measurement principle is based on the suction method, where negative pressure mechanically deforms the skin. Pressure is generated in the device, drawing skin into the orifice of a probe after a defined time. Inside the probe, the penetration depth is determined via a non-contact optical measurement system. This system consists of a light source, a light receiver, and two prisms facing each other, which project light from the emitter to the receiver. The light intensity varies depending on the penetration depth of the skin. During the measurement, the skin's resistance to negative pressure (hardness) and its ability to return to its original position (elasticity) are displayed in real time as curves (expressed in mm / time). From these curves, the R² value, as a measurement parameter, is calculated to correspond to the elastic and viscoelastic properties of the skin surface and skin aging. The closer this value is to 1 (100%), the greater the elasticity. In this human application test, the R² value (R² = U / 2) representing the skin elasticity of the test area was measured 24 hours after product use. a / U f (The proportion of the maximum amplitude).

[0244] Glossiness of the forearm area was measured using a SkinGlossMeter (SGM, Delfin). The amount of reflection was measured when light from a 635nm red diode laser was reflected from the skin surface. Light passing through the diffraction microstructure within the device was used to calculate the glossiness value, which was then used as the SGU (Skin Gloss Unit) parameter. The higher the skin glossiness, the higher the measured value. In this human application test, SGU values ​​representing the skin glossiness of the test area were measured before product use and 24 hours after product use.

[0245] In addition, the improvement rate is calculated using the following formula.

[0246] - Improvement rate (%) formula = {(before use - after use) / before use} × 100

[0247] The results showed that, after 24 hours, compared with the control group, the test group showed an average improvement rate of approximately 40-fold, 4-fold, and 5-fold in skin texture, skin elasticity, and radiance, respectively (Table 1).

[0248] [Table 1]

[0249]

[0250] The above results confirm that the skin-permeable peptides, their derivatives or fragments disclosed herein have excellent skin permeability, and therefore have significant effects as skin-permeable peptides.

[0251] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims and not by its preceding description, and therefore all variations and modifications falling within the boundaries and limits of the claims, or equivalents of such boundaries and limits, are thus intended to be included in the claims. sequence list <110> Cellicon Lab Inc. <120> Novel skin-permeable peptides for skin and mucous membranes, compositions thereof, and their uses <130> OPA22040 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 1 <400> 1 Lys Lys Leu Phe Lys Lys Ile Leu Lys Tyr Leu Lys 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 2 <400> 2 Arg Arg Leu Phe Arg Arg Ile Leu Arg Tyr Leu Arg 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 3 <400> 3 Arg Phe Leu Phe Arg Leu Ile Leu Arg Tyr Leu Arg 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 4 <400> 4 Arg Arg Leu Phe Arg Leu Ile Leu Arg Tyr Leu Phe 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 5 <400> 5 Arg Lys Leu Phe Lys Arg Ile Leu Arg Tyr Leu Lys 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 6 <400> 6 Lys Arg Leu Phe Arg Lys Ile Leu Lys Tyr Leu Arg 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 7 <400> 7 Lys Arg Leu Phe Lys Arg Ile Leu Arg Tyr Leu Lys 1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin Penetrating Peptide 8 <400> 8 Arg Lys Leu Phe Arg Lys Ile Leu Lys Tyr Leu Arg 1 5 10

Claims

1. A composition for transdermal delivery of a bioactive substance, said composition comprising a skin-permeable peptide or a derivative thereof comprising the amino acid sequence of SEQ ID NO:

1. The derivative therein, compared to the skin-permeable peptide shown in SEQ ID NO: 1, has one or more amino acids substituted with another amino acid at the following positions: positions 1, 2, 5, 6, 9, and 12 from the N-terminus of the skin-permeable peptide shown in SEQ ID NO:

1. The amino acids corresponding to positions 1, 5, and 9 are replaced by arginine; The amino acid corresponding to position 6 is replaced by arginine or leucine; and The amino acids corresponding to positions 2 and 12 are replaced by arginine or phenylalanine.

2. The composition for transdermal delivery of a bioactive substance as claimed in claim 1, wherein the derivative is one or more selected from SEQ ID NO: 2 to SEQ ID NO:

8.

3. The composition for transdermal delivery of a bioactive substance as claimed in claim 1, wherein the composition for transdermal delivery increases the permeability of the bioactive substance to any one or more selected from skin and mucous membranes.

4. The composition for transdermal delivery of bioactive substances as claimed in claim 1, further comprising a carrier.

5. The composition for transdermal delivery of bioactive substances as claimed in claim 4, wherein the carrier is a liposome.

6. The composition for transdermal delivery of a bioactive substance as claimed in claim 1, wherein the bioactive substance is a substance that has physiological effects in vivo.

7. A composition for skin penetration of a bioactive substance, said composition comprising a skin-permeable peptide or a derivative thereof consisting of the amino acid sequence of SEQ ID NO:

1. The derivative therein, compared to the skin-permeable peptide shown in SEQ ID NO: 1, has one or more amino acids substituted with another amino acid at the following positions: positions 1, 2, 5, 6, 9, and 12 from the N-terminus of the skin-permeable peptide shown in SEQ ID NO:

1. The amino acids corresponding to positions 1, 5, and 9 are replaced by arginine; The amino acid corresponding to position 6 is replaced by arginine or leucine; and The amino acids corresponding to positions 2 and 12 are replaced by arginine or phenylalanine.

8. A carrier for the transdermal delivery of a bioactive substance, said carrier comprising a skin-permeable peptide or a derivative thereof consisting of the amino acid sequence of SEQ ID NO:

1. The derivative therein, compared to the skin-permeable peptide shown in SEQ ID NO: 1, has one or more amino acids substituted with another amino acid at the following positions: positions 1, 2, 5, 6, 9, and 12 from the N-terminus of the skin-permeable peptide shown in SEQ ID NO:

1. The amino acids corresponding to positions 1, 5, and 9 are replaced by arginine; The amino acid corresponding to position 6 is replaced by arginine or leucine; and The amino acids corresponding to positions 2 and 12 are replaced by arginine or phenylalanine.

9. A skin-permeable carrier for a bioactive substance, said carrier comprising a skin-permeable peptide or a derivative thereof consisting of the amino acid sequence of SEQ ID NO:

1. The derivative therein, compared to the skin-permeable peptide shown in SEQ ID NO: 1, has one or more amino acids substituted with another amino acid at the following positions: positions 1, 2, 5, 6, 9, and 12 from the N-terminus of the skin-permeable peptide shown in SEQ ID NO:

1. The amino acids corresponding to positions 1, 5, and 9 are replaced by arginine; The amino acid corresponding to position 6 is replaced by arginine or leucine; and The amino acids corresponding to positions 2 and 12 are replaced by arginine or phenylalanine.

10. A cosmetic composition comprising the composition for transdermal delivery as claimed in claim 1.

11. A quasi-pharmaceutical composition comprising the composition for transdermal delivery as claimed in claim 1.

12. A pharmaceutical composition for external application to the skin or mucous membranes, said pharmaceutical composition comprising the composition for transdermal delivery as claimed in claim 1.

13. A kit comprising the composition for transdermal delivery as claimed in claim 1; and a bioactive substance.

14. A method for preparing the composition for transdermal delivery as claimed in claim 1.

15. A method for preparing a cosmetic composition, a quasi-pharmaceutical composition, or a pharmaceutical composition, said method comprising mixing the composition of claim 1 for transdermal delivery with a bioactive substance.

16. Use of the peptide or a derivative thereof in the composition of claim 1 in the preparation of a composition for the skin penetration of a bioactive substance.

17. Use of the peptide or its derivative in the composition of claim 1 in the preparation of a composition for transdermal delivery of a bioactive substance.

Images