Fusion peptide comprising cell-penetrating repeating peptide and use thereof

Abstract

The present invention relates to a cell-penetrating repeating peptide in which a plurality of cell-penetrating peptides are repeatedly linked, and has been completed by confirming that the cell-penetrating repeating peptide has significantly higher cell permeability than a single peptide, and thus can enhance the efficiency of intracellular transport of a conjugated protein. Thus, a neurotransmitter secretion-blocking peptide conjugated to the cell-penetrating repeating peptide, of the present invention, can move into cells with high efficiency, and thus can more effectively block the secretion of neurotransmitters by cells. Therefore, the neurotransmitter secretion-blocking peptide can be used not only for cosmetic purposes such as improving skin wrinkles, but also for ameliorating or treating neuromuscular diseases caused by excessive neuronal signal transduction, muscle tension, and the like.

Description

Fusion peptide comprising cell-penetrating repeat peptide and use thereof

[0001] The present invention relates to a cell-penetrating repeat peptide in which cell-penetrating peptides are repeatedly linked; a fusion peptide in which the cell-penetrating repeat peptide and a neurotransmitter-blocking peptide are conjugated; and uses thereof.

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2023-0091867, filed July 14, 2023, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Neurons store neurotransmitters such as acetylcholine in small sacs called synapse vesicles, and when they receive a specific stimulus, these vesicles fuse with the cell membrane and release the neurotransmitters inside, thereby causing neurotransmission to the next neuron. At this time, the SNARE membrane fusion complex present on the surface of the vesicle is involved in the release of neurotransmitters. The SNARE complex is composed of three proteins: synaptobrevin (vesicle-associate membrane protein, VAMP), SNAP25 present in the pre-synaptic plasma membrane, and syntaxin. When stimulated, intracellular Ca 2+ As the concentration increases, vesicles bind to nerve terminals and fuse with them through the SNARE complex.

[0005] Botulinum toxin is a toxic substance that inhibits the release of acetylcholine from peripheral nerves. Because it blocks acetylcholine release from all parasympathetic and cholinergic postganglionic sympathetic neurons, it can be used for excessively hypertonic smooth muscle and glandular tissue. Depending on its subtype, botulinum toxin cleaves specific sites on VAMP, SNAP25, and syntaxin, respectively, and interfere with the formation of SNARE complexes, thereby blocking vesicle membrane fusion and acetylcholine release.

[0006] However, botulinum toxin has high toxicity, which limits its use. While peptide fragments, which are truncated botulinum toxin fragments, are sometimes used, their activity is reduced compared to the botulinum toxin itself. Furthermore, as the peptide size increases, the efficiency of intracellular delivery decreases, resulting in lower cell penetration efficiency compared to botulinum toxin, which enters cells through membrane receptors. Peptides derived from the human SNAP-25 protein are being used as alternatives to botulinum toxin, but their large molecular weight and hydrophilic nature limit their low transdermal absorption. Therefore, a general method is needed to effectively deliver biologically active molecules into cells without damaging cells both inside and outside the body.

[0007] Thus, the cell membrane, due to its inherent impermeability, acts as a barrier to the intracellular penetration of proteins, nucleic acids, peptides, and poorly soluble compounds. Intracellular drug delivery is a critical issue that must be addressed in fields such as disease treatment, prevention, and diagnosis. Common methods for delivering biologically active macromolecules into the cell membrane include electroporation, membrane fusion using liposomes, transfection using cationic polymers such as polyethyleneimine (PEI) and DEAE-dextran, viral nucleic acid infection, and single-cell microinjection. Recently, technologies that utilize the endocytosis of drug carriers such as nanoparticles have been utilized for effective intracellular drug delivery. However, further research is required due to issues such as toxicity, rapid clearance from the immune system, decreased delivery efficiency, and steric hindrance caused by interactions with cells. To solve this problem, many attempts have been made recently to use cell penetrating peptides (CPPs).

[0008] Cell-penetrating peptides have the ability to transport biologically active molecules such as liposomes, nucleic acids, proteins, and compounds into cells without a specific receptor. Cell-penetrating peptides were first discovered in 1988 when TAT, a transcription factor of HIV (human immunodeficiency virus), was confirmed to be transported into cells. Since then, various cell-penetrating peptides have been developed, including penetratin, VP22, and MTS. However, when existing cell-penetrating peptides are conjugated to biologically active macromolecules (cargo), the cell permeability, pattern, solubility, and structure of the peptide change, preventing the peptide from passing through the cell membrane or preventing the activity of the biologically active molecule from being maintained. Therefore, the number of cell-penetrating peptides that can be practically industrialized and utilized is insufficient. Therefore, if a novel cell-penetrating peptide is developed that can effectively deliver a delivery substance into the cell by passing through the cell membrane even when bound to a biologically active macromolecule, it is expected to be effectively applied in various fields such as cosmetics, diagnosis, and medicine.

[0009]

[0010] The present invention was conceived to solve the above problems, and was completed by confirming that a cell-penetrating repeat peptide in which 2 to 3 cell-penetrating peptides are repeatedly linked exhibits significantly superior cell-penetrating ability compared to a single cell-penetrating peptide, and thus can enhance the efficiency of movement into cells of other proteins conjugated thereto.

[0011] Accordingly, the present invention aims to provide a cell-penetrating repeat peptide in which two to three cell-penetrating peptides are repeatedly linked.

[0012] Another object of the present invention is to provide a cell-penetrating repeat peptide in which two to three cell-penetrating peptides are repeatedly linked; and a fusion peptide conjugated with a SNAP-25-derived peptide.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neuromuscular diseases or skin diseases, comprising the above fusion peptide as an active ingredient.

[0014] Another object of the present invention is to provide a cosmetic composition for improving skin condition, comprising the above fusion peptide as an active ingredient.

[0015]

[0016] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0017]

[0018] The present invention is a fusion peptide comprising a cell-penetrating repeat peptide in which two to three cell-penetrating peptides are repeatedly linked; and a SNAP-25-derived peptide,

[0019] The present invention provides a fusion peptide, wherein each of the above cell-penetrating peptides is composed of Tyr, Ala, Arg, Val, Gly, and Pro, and each is 6 to 15 amino acids long.

[0020] More specifically, the SNAP-25-derived peptide provides a fusion peptide comprising amino acids 181 to 206 of the human SNAP-25 protein.

[0021] More specifically, the cell-penetrating repeat peptide provides a fusion peptide in which two to three cell-penetrating peptides are linked to each other via a linker.

[0022] More specifically, the linker provides a fusion peptide comprising 1 to 5 Gly groups.

[0023] More specifically, the cell-penetrating repeat peptide provides a fusion peptide conjugated to the SNAP-25 derived peptide via a linker.

[0024] More specifically, the linker provides a fusion peptide comprising 1 to 5 Gly groups.

[0025] More specifically, the cell-penetrating repeat peptide provides a fusion peptide characterized by having a cell-penetrating ability that is at least 5 times higher than that of a single cell-penetrating peptide.

[0026] In another aspect, the present invention provides a nucleic acid molecule encoding the fusion peptide described above.

[0027] In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule described above.

[0028] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating neuromuscular diseases or skin diseases, comprising the above-described fusion peptide as an active ingredient.

[0029] More specifically, the neuromuscular disease is at least one selected from the group consisting of hyperhidrosis, urinary incontinence, overactive bladder, myoclonus, blepharospasm, hemifacial spasm, spasmodic torticollis, anal fissure, blepharospasm, facial spasm, cerebral palsy, medication overuse headache, chronic migraine, myalgia, strabismus, temporomandibular disorder, neuralgia, rhinitis, dystonia, and vocal fold granuloma. Provides.

[0030] More specifically, the pharmaceutical composition provides a skin disease, wherein the skin disease is at least one selected from the group consisting of skin wrinkles and acne.

[0031] In another aspect, the present invention provides a cosmetic composition for improving skin condition, comprising the above-described fusion peptide as an active ingredient.

[0032] More specifically, the cosmetic composition provides a cosmetic composition for at least one selected from the group consisting of reducing the appearance of fine lines, reducing the appearance of wrinkles, widening the eyes, lifting the corner of the mouth, reducing muscle mass, flattening lines extending from the upper lip, and improving acne.

[0033] In another aspect, the present invention provides a cosmetic method for improving skin condition, comprising the step of administering an effective amount of the above-described fusion peptide to a subject in need thereof.

[0034] In another aspect, the present invention provides a topical application preparation for preventing or improving neuromuscular diseases or skin diseases, comprising the above-described fusion peptide as an active ingredient.

[0035] In addition, the present invention provides a method for preventing, improving, or treating a neuromuscular disease or skin disease, comprising administering a pharmaceutically effective amount of the above-described fusion peptide or a composition containing the same as an active ingredient to a subject in need thereof.

[0036] In addition, a pharmaceutical composition comprising the above-described fusion peptide as an active ingredient is provided.

[0037] In addition, the present invention provides a use of the above-described fusion peptide; or a composition comprising the same as an active ingredient, for preventing, improving, or treating neuromuscular diseases or skin diseases.

[0038] In addition, the present invention provides a use for the above-described fusion peptide; or a composition comprising the same as an active ingredient, in the manufacture of a drug for preventing, improving, or treating neuromuscular diseases or skin diseases.

[0039] In addition, a kit for preventing, improving, or treating a neuromuscular disease or skin disease is provided, including the above-described fusion peptide; or a composition comprising the same as an active ingredient; and an instruction manual.

[0040]

[0041] The present invention relates to a cell-penetrating repeat peptide in which a plurality of cell-penetrating peptides are repeatedly linked, and has been completed by confirming that the cell-penetrating repeat peptide has a significantly higher cell-penetrating ability than a single peptide, and thus can increase the efficiency of movement of a conjugated protein into a cell. Accordingly, the neurotransmitter secretion blocking peptide conjugated to the cell-penetrating repeat peptide of the present invention can move into a cell with high efficiency, and thus can more effectively block the secretion of neurotransmitters in the cell, and thus can be utilized for cosmetic purposes such as improving skin wrinkles, as well as for improving or treating neuromuscular diseases caused by excessive nerve signal transmission and muscle tension.

[0042]

[0043] FIG. 1 is a diagram showing the sequence of a fusion peptide in which a cell-penetrating peptide (CPP) is linked three times and a SNAP-25-derived peptide is conjugated according to one embodiment of the present invention.

[0044] Figures 2a and 2b show the results of confirming the change in cell penetration ability according to the repeated binding of the CPP of the present invention using a fluorescence microscope. FITC was conjugated to a peptide in which one, two, or three CPPs were repeatedly bound (1X-CPP, 2X-CPP, and 3X-CPP, respectively), and the human skin cell lines HaCAT (Figure 2a) and HT1080 (Figure 2b) were treated to observe the fluorescence signal.

[0045] Figures 3a to 3d show the results of confirming the change in cell penetration ability according to the repeated binding of the CPP of the present invention by FACS. FITC was conjugated to 1X-CPP, 2X-CPP, and 3X-CPP, and treated on human neuroblastoma SH-SY5Y (Figure 3a) and HaCAT (Figure 3b), and the fluorescent signal was detected through FACS, and the cell penetration ability of 1X-CPP, 2X-CPP, and 3X-CPP, respectively, compared to TAT (Figure 3c) and the cell penetration ability of 2X-CPP and 3X-CPP, respectively, compared to 1X-CPP (Figure 3d) were calculated.

[0046] Figures 4a and 4b show the results of confirming the intracellular delivery efficiency of a SNAP25-derived peptide (SNAP25) according to the repeated binding of the CPP of the present invention using a fluorescence microscope. SNAP25 was conjugated to each of 1X-CPP, 2X-CPP, and 3X-CPP, and the human skin cell lines HaCAT (Figure 4a) and HT1080 (Figure 4b) were treated to observe the fluorescence signals.

[0047] Figure 5 shows the results of confirming the binding of SNAP25 and VAMP2 conjugated to the CPP (2XCPP or 3XCPP) of the present invention by precipitating the protein with a VAMP2 targeting antibody and then confirming the presence of VAMP2 and CPP-SNAP2 proteins by Western blot.

[0048] Figures 6a and 6b show the results of measuring the number of sweat glands after administering pilocarpine to ICR mice to establish an animal model of hyperhidrosis.

[0049] Figures 7a and 7b show the results of observing sweat glands stained with iodine after applying Sweatrol or 3XCPP SNAP25, a positive control, to an animal model of hyperhidrosis and inducing sweating on the 7th day (Figure 7a) and calculating the number of sweat glands (Figure 7b).

[0050]

[0051] The present invention relates to a cell-penetrating repeat peptide in which two to three cell-penetrating peptides are repeatedly linked, and the cell-penetrating repeat peptide has a significantly higher cell-penetrating ability than a single peptide, and thus can enhance the efficiency of movement into cells of other proteins (e.g., neurotransmitter blocking peptides) conjugated thereto, and thus has been completed.

[0052] Specifically, in one embodiment of the present invention, 1 to 3 cell penetrating peptides (CPPs) according to the present invention were repeatedly linked, and a SNAP25-derived peptide corresponding to amino acids 181 to 206 of the SNAP25 protein, which is a peptide having a neurotransmitter blocking effect, was conjugated to prepare fusion peptides (1XCPP-SNAP25, 2XCPP-SNAP25, and 3XCPP-SNAP25) (Example 1).

[0053] In another embodiment of the present invention, in order to confirm the cell penetrability according to the repeated binding of the cell penetrable peptide according to the present invention, cell penetrable peptides to which 1, 2, or 3 CPPs were bound were prepared, and a cell penetrability comparison experiment using human skin cells or neuroblastoma cells was performed (Examples 2 and 3). As a result, 2X-CPP, in which 2 CPPs were repeatedly bound, showed a cell penetrability about 8 times or more higher than that of a single CPP (1X-CPP), and 3X-CPP, in which 3 CPPs were repeatedly bound, showed a cell penetrability about 20 times or more higher than that of 1X-CPP. Therefore, it was confirmed that the cell membrane penetration efficiency was significantly increased as the CPP according to the present invention was repeatedly bound.

[0054] In another embodiment of the present invention, the cell penetrability of the fusion peptides conjugated with the cell-penetrating repeat peptide and SNAP25 was confirmed for human skin cells, and it was confirmed that the intracellular movement efficiency of 3XCPP-SNAP25 was significantly higher than that of 1XCPP-SNAP25 (Example 4).

[0055] In another embodiment of the present invention, it was confirmed that the fusion peptide conjugated with the cell-penetrating repeat peptide and SNAP25 normally binds to VAMP2, and it was confirmed that SNAP25 can normally bind to VAMP2 even when bound to the cell-penetrating repeat peptide of the present invention, and thus can inhibit the formation of the SNARE complex (Example 5).

[0056] In another embodiment of the present invention, in order to confirm the biological efficacy of the fusion peptide according to the present invention, the peptide was applied to a hyperhidrosis model, and as a result, the number of sweat glands was further reduced compared to the positive control group, confirming the hyperhidrosis treatment effect of the fusion peptide of the present invention (Example 6).

[0057] That is, the cell-permeable repeat peptide according to the present invention can significantly increase the intracellular delivery efficiency of a neurotransmitter blocking protein conjugated thereto, and can effectively block acetylcholine secretion from nerve cells, etc., and is therefore expected to be used for cosmetic purposes such as improving skin wrinkles, as well as for improving or treating various neuromuscular diseases caused by excessive nerve signal transmission and muscle tension.

[0058] Accordingly, the main purpose of the present invention is to provide a fusion peptide characterized by a cell-penetrating repeat peptide in which cell-penetrating peptides are repeatedly linked, wherein the cell-penetrating peptides are each composed of Tyr, Ala, Arg, Val, Gly, and Pro, and each has a length of 6 to 15 amino acids.

[0059] Another object of the present invention is to provide a fusion peptide conjugated with the cell-permeable repeat peptide and a neurotransmitter blocking protein. Preferably, the neurotransmitter blocking protein is an acetylcholine blocking protein, and most preferably, a SNAP-25-derived peptide.

[0060] In the present invention, “cell penetrating peptide (CPP)” refers to a peptide having cell penetrability and having the ability to deliver a cargo into a cell in vitro and / or in vivo. In the present invention, “cell penetrating repeated peptide” refers to a fusion peptide in which the cell penetrating peptides are repeatedly linked. In other words, the cell penetrating repeated peptide comprises two or more CPPs, wherein the cell penetrating peptides are linked to each other directly or through a linker or the like. In this case, the CPPs may have the same or different sequences.

[0061] In one embodiment of the present invention, the cell-penetrating repeat peptide may be a repeat-linked 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 CPPs. In a preferred embodiment, the cell-penetrating repeat peptide may be a repeat-linked 2 to 3 CPPs. That is, the cell-penetrating repeat peptide according to the present invention has a higher cell-penetrating ability than a single CPP when it includes 2 to 3 CPPs, whereas the cell-penetrating ability may be reduced when it includes 4 or more CPPs. The cell-penetrating repeat peptide according to the present invention is characterized in that it exhibits a significantly higher cell-penetrating ability (cell membrane penetration efficiency) than any single CPP constituting the repeat peptide, by repeatedly linking the CPPs. For example, the cell-penetrating repeat peptide of the present invention may have a cell-penetrating ability that is at least 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 25 times, or 30 times or more higher than a single CPP, but is not limited thereto. For example, the cell-penetrating repeat peptide of the present invention may have a cell-penetrating ability that is at least 2 to 30 times, 4 to 30 times, 6 to 30 times, 8 to 30 times, 10 to 30 times, 2 to 25 times, 4 to 25 times, 6 to 25 times, 8 to 25 times, 2 to 20 times, 4 to 20 times, 6 to 20 times, or 8 to 20 times or more higher than a single CPP, but is not limited thereto.

[0062] Throughout this specification, the term “connection” may be used interchangeably with fusion, joining, bonding, conjugation, and the like.

[0063] In the present invention, the term "cargo" includes any substance that can bind to a cell-penetrating peptide and move into cells, and may include all types of biologically active molecules. The cargo includes, for example, any substance whose cell penetration efficiency is desired to be increased, specifically, an effective substance of a drug, cosmetic, or health food, and more specifically, a substance that does not easily move into cells through a general route, and more specifically, proteins, nucleic acids, peptides, minerals, sugars such as glucose, nanoparticles, biological agents, viruses, contrast agents, or other chemical substances, but is not limited thereto.

[0064] The cell-penetrating peptide according to the present invention may be composed of Tyr, Ala, Arg, Val, Gly, and Pro.

[0065] In one embodiment of the present invention, the cell-penetrating peptide may be cationic. Accordingly, the proportion of Arg relative to the total amino acids constituting the cell-penetrating peptide may be, but is not limited to, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, or 45% or more. Alternatively, the proportion of Arg relative to the total amino acids constituting the cell-penetrating peptide may be, but is not limited to, 40 to 95%, 40 to 90%, 40 to 80%, 40 to 70%, 40 to 60%, 40 to 50%, 40 to 48%, 40 to 46%, 42 to 46%, 43 to 46%, 44 to 46%, or 45 to 46%.

[0066] In one embodiment of the present invention, the cell-penetrating peptide may be composed of 6 to 15 amino acids. That is, the cell-penetrating peptide may be 6 to 15 amino acids in length. Specifically, the cell-penetrating peptide may be 6 to 15 amino acids, 8 to 15 amino acids, 9 to 15 amino acids, 10 to 15 amino acids, 6 to 14 amino acids, 6 to 13 amino acids, 6 to 12 amino acids, 7 to 12 amino acids, 9 to 12 amino acids, 10 to 12 amino acids, or 11 amino acids in length, but is not limited thereto.

[0067] In one embodiment of the present invention, the cell-penetrating peptide is 11 amino acids long, and the ratio of Arg to the total amino acids constituting the cell-penetrating peptide is 40% or more, and may include at least one of Tyr, Ala, Val, Gly, and Pro, but is not limited thereto.

[0068] In another embodiment of the present invention, the cell-penetrating peptide may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 1.

[0069] The cell-penetrating peptide according to the present invention includes derivatives and analogs (mimetics, or peptidomimetics) of the peptide. As used herein, “derivative” refers to a general term for a similar peptide obtained by changing a portion of the cell-penetrating peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention, and preferably, it may be a peptide in which one or more amino acids are substituted with other amino acids, one or more amino acids are added, one or more amino acids are deleted, or a peptide is fused with a compound that increases the half-life of the peptide (e.g., polyethylene glycol, etc.). In the present invention, the derivative may maintain, increase, or decrease the function or characteristic (e.g., cell permeability) of the cell-penetrating peptide according to the present invention.

[0070] More specifically, the derivative may have one or more amino acid substitutions while maintaining the properties of the cell-penetrating peptide according to the present invention. The amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, as conservative or semi-conservative substitutions, glycine, alanine, valine, leucine, and isoleucine, which have aliphatic side chains, may be substituted for each other; glycine and alanine, which have relatively short side chains, may be substituted for each other; valine, leucine, and isoleucine, which have larger hydrophobic aliphatic side chains, may be substituted for each other; phenylalanine, tyrosine, and tryptophan, which have aromatic side chains, may be substituted for each other; lysine, arginine, and histidine, which have basic side chains, may be substituted for each other; Aspartate and glutamate, which have acidic side chains, can be substituted for each other; cysteine ​​and methionine, which have sulfur-containing side chains, can be substituted for each other.

[0071] In one embodiment of the present invention, each cell-penetrating peptide constituting the cell-penetrating repeat peptide may be connected to each other by a physical bond, a chemical bond, a covalent bond, a non-covalent bond, a peptide bond, or self-assembly, or may be connected in an integrated or fused form using a mediator (e.g., a linker).

[0072] The linker can link each cell-penetrating peptide to each other and improve the fluidity of the fusion peptide, thereby increasing the binding efficiency between the fusion peptide and VAMP. The linker according to the present invention may be applied without limitation to any linker known in the art, but preferably, it may be a linker composed of 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 6, 2 to 5, 2 to 4, or 3 amino acids. More preferably, the linker may be composed of Gly. Each of the plurality of linkers connecting the plurality of cell-penetrating peptides may have the same or different sequences. Preferably, each of the linkers connecting the cell-penetrating peptides has the same sequence.

[0073] In the present invention, the SNAP-25-derived peptide is derived from human SNAP-25 protein and can be included without limitation as long as it can inhibit the secretion of neurotransmitters that induce muscle contraction, such as acetylcholine and catecholamine, by inhibiting SNARE formation. In particular, the SNAP-25-derived peptide according to the present invention is characterized by inhibiting SNARE formation by binding to VAMP2. The SNAP-25-derived peptide may be a part (fragment) of the human SNAP-25 protein, and may be obtained by deleting or substituting some of the amino acids of the protein, or adding amino acids to the protein or a fragment thereof.

[0074] In one embodiment of the present invention, the SNAP-25-derived peptide is a peptide derived from the C-terminus of human SNAP-2 protein, and preferably may be a C-terminal peptide fragment of human SNAP-25 cleaved by botulinum toxin. Full-length human SNAP-2 protein forms a SNARE complex with VAMP and the like to promote the secretion of neurotransmitters, but the C-terminal fragment of SNAP-25 cleaved by botulinum toxin and the like can block the secretion of neurotransmitters by binding to VAMP and interfering with the formation of a normal SNARE complex. Preferably, the SNAP-25-derived peptide according to the present invention may be a peptide comprising amino acids 181 to 206 of human SNAP-25 protein, or consisting of amino acids 181 to 206. The sequence information of the full-length human SNAP-25 protein can be found in NCBI Sequence Reference Number NP_001309832.1 or NP_001309831.1; GenBank Accession Number BAD97337.1, etc. In one embodiment of the present invention, the SNAP-25-derived peptide may comprise the amino acid sequence of SEQ ID NO: 3 or may consist of the amino acid sequence of SEQ ID NO: 3. Throughout this specification, the SNAP-25-derived peptide may be conveniently referred to as “SNAP-25.” In addition, throughout this specification, SNAP-25 may also be expressed as SNAP25.

[0075] In the present invention, the cell-penetrating repeat peptide and the SNAP-25-derived peptide may be conjugated to each other through chemical bonding, bonding via a linker, or peptide bonding, but are not limited thereto.

[0076] Preferably, the cell-penetrating repeat peptide and the SNAP-25-derived peptide may be linked via a linker. The linker may link the cell-penetrating repeat peptide and the SNAP-25-derived peptide to each other and may increase the efficiency of the fusion peptide's uptake into cells by improving the fluidity of the fusion peptide, and may also increase the binding efficiency between the fusion peptide and VAMP. The linker according to the present invention may be applied without limitation to any linker known in the art, but preferably, it may be a linker composed of 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 6, 2 to 5, 2 to 4, or 3 amino acids. More preferably, the linker may be composed of Gly. A linker that links each CPP constituting the cell-penetrating repeat peptide to each other; And the linker connecting the cell-penetrating repeat peptide and the SNAP-25-derived peptide may be the same or different.

[0077] The fusion peptide according to the present invention may comprise the amino acid sequence of SEQ ID NO: 3 or may consist of the amino acid sequence of SEQ ID NO: 3.

[0078] In this specification, a polypeptide (or nucleic acid molecule) indicated by a specific sequence may include not only the sequence but also biological equivalents thereof. That is, considering a variant having a biological equivalent activity of the polypeptide (nucleic acid molecule), it is interpreted that one aspect of the polypeptide (or nucleic acid molecule) also includes a sequence that shows substantial identity with the sequence set forth in the sequence number. Specifically, a polypeptide (nucleic acid molecule) comprising an amino acid sequence (nucleotide sequence) indicated by a specific sequence number is not limited to the amino acid sequence (nucleotide sequence), and variants of the amino acid sequence (nucleotide sequence) are included within the scope of the present invention. The polypeptide molecule (nucleic acid molecule) composed of an amino acid sequence (nucleotide sequence) represented by a specific sequence number of the present invention is a concept that includes functional equivalents of the polypeptide molecule (nucleic acid molecule) constituting it, for example, variants in which a part of the amino acid sequence (nucleotide sequence) of the polypeptide molecule (nucleic acid molecule) is modified by deletion, substitution, or insertion, but which can perform the same function functionally as the polypeptide (nucleic acid molecule). Specifically, the polypeptide (nucleic acid molecule) disclosed in the present invention may include an amino acid sequence (nucleotide sequence) having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the amino acid sequence represented by the specific sequence number.For example, it includes polypeptides (nucleic acid molecules) having sequence homology of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The "% sequence identity" for a polypeptide (nucleic acid molecule) is determined by comparing two optimally aligned sequences over a comparison region, where portions of the polypeptide sequence (nucleotide sequence) in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0079] Meanwhile, the method for producing a fusion peptide according to the present invention is not particularly limited to any method, and various fusion peptide production methods known in the art can be applied to the present invention, as long as the fusion peptide as the final product produced can exhibit improved effects (compared to a SNAP-25-derived peptide conjugated to a single cell-penetrating peptide) in terms of cell penetration ability, skin absorption efficiency, and prevention, improvement, and / or treatment effects of neuromuscular diseases or skin diseases. For example, a method known in the art, such as a solid-phase synthesis method or a synthesis method using a recombinant expression system, can be used to produce the fusion peptide according to the present invention.

[0080] As a non-limiting example, the fusion peptide according to the present invention can be synthesized as a pure fusion peptide using an organic synthesis device for peptide synthesis. This method is Merrifield's solid phase peptide synthesis method (J. Am. Chem. Soc. 85, 2149-21, 54 (1963)), and a peptide semi-automatic synthesizer (Peti-Syzer Model PSS-510) is used to synthesize a fusion peptide by condensation reaction of a monomer having reactivity between the C-terminus and the N-terminus of an amino acid.

[0081] Solid-phase synthesis begins with coupling an amino acid whose α-amino group is protected to a suitable resin, starting with the carboxyl-terminal amino acid. The α-amino-protected amino acid is then attached to a hydroxymethyl resin or a chloromethylated resin via an ester bond. Fmoc (9-fluorenyl methoxycarbonyl) is used as the protecting group for the α-amino group (Fmoc-protected amino acids can be purchased from Bidtech Co., Ltd.). For example, the amino group of Arg is reactive. Reactive residues of Gly, Lys, and Gln utilize Fmoc-amino acids protected with suitable groups such as tert-butyl (t-Bu) and Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl). Peptide synthesis proceeds by sequentially removing protecting groups from the amino terminus of a peptide chain attached to a solid support resin using reagents such as trifluoroacetic acid (TFA) and phenol. Peptides are isolated by extraction from the TFA solution with diethyl ether and can be purified by high-performance liquid chromatography (HPLC).

[0082] The method for producing a fusion peptide using a biological method includes the steps of (a) cloning a nucleic acid molecule encoding the fusion peptide according to the present invention into an expression vector capable of expressing a specific peptide to produce a recombinant vector capable of expressing the fusion peptide in the form of a fusion peptide, and (b) expressing the fusion peptide in large quantities in E. coli using the recombinant vector, and then isolating and purifying the fusion peptide. At this time, in order to produce the recombinant vector, a person skilled in the art can easily infer the base sequence encoding the fusion peptide from the amino acid sequence disclosed herein, and selection and overall technical details regarding a cloning vector for inserting such a base sequence, a recombinant expression vector produced using cloning technology, a host cell for transforming the recombinant vector to express the target fusion peptide, a method for transforming the host with the recombinant vector, a method for expressing the target fusion peptide from the transformed host cell, and a method for obtaining the target fusion peptide from the final product, etc., can be easily recognized by a person skilled in the art.

[0083] The present invention also provides a nucleic acid molecule encoding a fusion peptide according to the present invention and / or an expression vector comprising the nucleic acid molecule.

[0084] In addition, the present invention provides a pharmaceutical composition for preventing or treating neuromuscular diseases or skin diseases, comprising the fusion peptide according to the present invention as an active ingredient.

[0085] In the present invention, the neuromuscular disease or skin disease may be included without limitation as long as it is a disease that can be caused by excessive secretion or continuous secretion of a neurotransmitter (preferably, catecholamine).

[0086] In the present invention, the “neuromuscular disease” may be included without limitation as a disease caused by a direct disease of a muscle, an indirect abnormality of muscle function due to an abnormality of a nerve or neuromuscular junction, an abnormality of a muscle or the nervous system that controls muscle movement, etc., as long as the disease can be prevented, improved, and / or treated by inhibiting the formation of a SNARE complex. The above neuromuscular disorders are not limited to a specific type, but may be selected from the group consisting of hyperhidrosis, urinary incontinence, overactive bladder, myoclonus, blepharospasm, hemifacial spasm, spasmodic torticollis, anal fissure, blepharospasm, facial spasm, cerebral palsy, medication overuse headache, chronic migraine, myalgia, strabismus, temporomandibular disorder, neuralgia, rhinitis, dystonia, and vocal fold granuloma.

[0087] In the present invention, the skin disease may be any disease that can be caused by excessive neurotransmitter secretion or excessive muscle tension, and can be prevented, improved, and / or treated by inhibiting the formation of SNARE complexes, without limitation. The skin disease is not limited to a specific type, but may be selected from the group consisting of skin wrinkles and acne.

[0088] The content of the fusion peptide in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt% or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0089] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0090] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0091] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0092] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0093] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0094] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0095] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity increasing agent, and the like, as needed.

[0096] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0097] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0098] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0099] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0100] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0101] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0102] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0103] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0104] The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration are conceivable, and include, for example, oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and skin application.

[0105] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0106] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0107] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0108] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0109] In addition, the present invention provides a pharmaceutical composition comprising a fusion peptide according to the present invention as an active ingredient.

[0110] In addition, the present invention provides a method for preventing, improving, or treating a neuromuscular disease or skin disease, comprising administering a pharmaceutically effective amount of a fusion peptide according to the present invention or a composition containing the same as an active ingredient to a subject in need thereof.

[0111] In addition, the present invention provides a use of a fusion peptide according to the present invention; or a composition comprising the same as an active ingredient; for use in the prevention, improvement, or treatment of neuromuscular diseases or skin diseases.

[0112] In addition, the present invention provides a use of a fusion peptide according to the present invention; or a composition comprising the same as an active ingredient; for use in the manufacture of a medicament for preventing, improving, or treating neuromuscular diseases or skin diseases.

[0113] The present invention also provides a cosmetic composition comprising the fusion peptide according to the present invention as an active ingredient. Specifically, the cosmetic composition may be a cosmetic composition for improving skin condition. The present invention also provides a cosmetic composition for preventing or improving skin diseases, comprising the fusion peptide as an active ingredient. The cosmetic composition may be administered to a subject via the various administration routes mentioned above.

[0114] In the present invention, the cosmetic composition or cosmetic composition may be for one or more selected from the group consisting of, but not limited to, reducing the appearance of fine lines, reducing the appearance of wrinkles, widening the eyes, lifting the corner of the mouth, reducing muscle mass, flattening lines extending from the upper lip, and improving acne.

[0115] The formulation of the cosmetic composition according to the present invention may be in the form of a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, mist, moisture cream, hand cream, hand lotion, foundation, essence, nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, cleansing oil, cleansing balm, body lotion, or body cleanser.

[0116] The cosmetic composition of the present invention may further include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, and sphingolipids.

[0117] In addition to the above essential ingredients, the cosmetic composition of the present invention may contain other ingredients commonly incorporated into cosmetics as needed.

[0118] In addition, other compounding ingredients that may be added include oil components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

[0119] In addition, the mixing components that may be added are not limited to these, and any of the above components may be mixed within a range that does not impair the purpose and effect of the present invention, but may be mixed at a weight percentage of 0.01-5% or a weight percentage of 0.01-3% with respect to the total weight.

[0120] The present invention also provides a cosmetic method for improving skin condition, comprising administering an effective amount of the fusion peptide to a subject in need thereof. The administration may be accomplished via any of the various routes of administration mentioned above.

[0121] The present invention also provides a topical formulation for the prevention or improvement of neuromuscular or skin diseases, comprising the fusion peptide as an active ingredient. The topical formulation may be in the form of a cream, gel, lotion, powder, powder spray, oil, roll-on formulation, ointment, foam, spray, stick, or tincture. Furthermore, the formulation may be an injectable formulation for administration by injection.

[0122]

[0123] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0124]

[0125] [Example]

[0126] Example 1. Production of cell-permeable repeat peptides

[0127] The cell-penetrating repeat peptide 3XCPP-SNAP25 according to the present invention was prepared by repeatedly binding cationic cell-penetrating peptides (CPPs) and then further conjugating a SNAP25-derived peptide corresponding to amino acids 181 to 206 of the human SNAP25 protein (hereinafter, referred to simply as “SNAP25” in the examples) (Fig. 1). At this time, each CPP was linked to each other with a linker peptide, GGG. In addition, as comparative examples, 1XCPP-SNAP25 and 2XCPP-SNAP25, in which CPPs were repeatedly bound once or twice and SANP25 was conjugated, were additionally prepared. The amino acid sequences of each peptide related to 3XCPP-SNAP25 according to the present invention are summarized in Table 1 below. Hereinafter, a peptide in which CPP is repeatedly bonded may be referred to as a “cell-permeable repeat peptide,” “repeated CPP,” “repeated peptide,” etc.

[0128]

[0129]

[0130] Example 2. Evaluation of cell penetration efficacy in human epidermal cells according to repeated binding of cell-penetrating peptides (fluorescence microscopy analysis)

[0131] First, in order to confirm whether the cell penetration ability is enhanced by repeated combination of CPPs according to the present invention, cell-penetrating peptides in which 1, 2, or 3 CPPs are combined were prepared (referred to as 1X-CPP, 2X-CPP, and 3X-CPP, respectively), and a cell penetration ability comparison experiment was performed. FITC was conjugated to each peptide to detect the intracellular location of the CPPs using a fluorescence microscope. Human epidermal cell lines HaCAT cell line and HT1080 cell line were used as target cells.

[0132] Specifically, 5 × 10 HaCAT cells or SH-SY5Y cells were seeded on 24-well plates with circular cover glasses for microscope (Microscope Cover Glasses, 12 mm Φ). 4 Cells were seeded at 10 cells / well and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 18 hours to allow the cells to attach to the cover glass. After the culture was completed, the medium was removed, and the cells were treated with 500 μL of D-PBS and 300 μL of DMEM containing 4 μ of CPP, and cultured for 1 hour. After the culture was completed, the cells were washed three times with 1 mL of D-PBS to remove extracellular proteins. The washed cells were fixed with 4% paraformaldehyde and washed once again with 1 mL of D-PBS. The fixed cells were mounted using Mounting medium with DAPI Aqueous, Fluoroshield (abcam) and observed using a fluorescence microscope.

[0133] The experimental results using HaCAT and HT1080 are shown in Figs. 2a and 2b, respectively. In the group treated with 1X-CPP, almost no fluorescence signal was detected, whereas in the group treated with 2X-CPP, fluorescence signals were detected in a large number of cells, and in the group treated with 3X-CPP, stronger fluorescence signals were detected in a larger number of cells. The above results show that the cell-penetrating peptide according to the present invention can not only effectively pass through the cell membrane of human skin cells, but also that when the peptide is repeatedly bound, the cell-penetrating ability for human skin cells increases to a significant level.

[0134]

[0135] Example 3. Evaluation of cell penetration efficacy in human epidermal cells according to repeated binding of cell-penetrating peptides (FACS analysis)

[0136] In this example, the degree of cell penetration according to repeated binding of CPPs according to the present invention was verified by FACS. In this experiment, HaCAT cells, a human epidermal cell line, and SH-SY5Y cells, a human neuroblastoma cell line, were used.

[0137] Specifically, 1 × 10 SH-SY5Y and HaCAT cells were seeded in 24-well plates. 7 Cells were seeded at 10 cells / well and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 24 hours. Subsequently, 4 μM of FITC-conjugated CPP or FITC-conjugated TAT were treated and cultured for 1 hour. After completion of the culture, the cells were transferred to a sterile tube using 0.25% trypsin and centrifuged to remove the medium. After that, the cells were washed twice with 500 μL of D-PBS to completely remove the medium, and finally, the cells were resuspended in 400 μL of D-PBS and intracellular fluorescence was measured using a flow cytometer (FACSCanto II).

[0138] The results are shown in Figs. 3a to 3d. In both cell lines, the CPP according to the present invention (1X-CPP) showed higher cell penetrability than TAT, a cell-penetrating peptide well known in the art, and it was confirmed that the cell penetrability was significantly increased as the number of repetitions of CPP increased (Figs. 3a and 3b). As a result of calculating the cell penetrability of each cell-penetrating peptide compared to TAT, 1X-CPP showed slightly higher cell penetrability than TAT, whereas 2X-CPP showed cell penetrability 10 to 20 times higher than that of TAT, and 3X-CPP showed cell penetrability about 30 times higher than that of TAT (Fig. 3c). In addition, as a result of comparing the cell penetration ability between 1X-CPP, 2X-CPP, and 3X-CPP, 2X-CPP showed about 8 to 16 times higher cell penetration ability than 1X-CPP, and 3X-CPP showed about 20 times higher cell penetration ability than 1X-CPP (Fig. 3d). The above results show that the cell-penetrating peptide of the present invention shows higher cell penetration ability than TAT, and particularly, has significantly superior cell penetration ability when repeatedly bound.

[0139]

[0140] Example 4. Evaluation of cell penetration efficacy of cell-penetrating repeat peptides conjugated with SNAP-25 (fluorescence microscopy analysis)

[0141] In this example, the cell penetration ability of the cell-penetrating peptide conjugated with SNAP25 according to Example 1 was confirmed, and in particular, the cell penetration ability according to the number of repetitions of CPP was compared (1XCPP-SNAP25, 2XCPP-SNAP25, and 3XCPP-SNAP25). Human epidermal cell lines HaCAT and HT1080 were used as target cells.

[0142] Specifically, 5 × 10 HaCAT cells or SH-SY5Y cells were seeded on 24-well plates with circular cover glasses for microscope (Microscope Cover Glasses, 12 mm Φ). 4 Cells were seeded at 10 cells / well and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 18 hours to allow the cells to attach to the cover glass. After the culture was completed, the medium was removed, and 500 μL of D-PBS and 300 μL of DMEM containing 1 μ CPP were added to the cells, and the cells were cultured for 1 hour. After the culture was completed, the cells were washed three times with 1 mL of D-PBS to remove extracellular proteins. The washed cells were fixed with 4% paraformaldehyde and washed once again with 1 mL of D-PBS. The fixed cells were mounted using Mounting medium with DAPI Aqueous, Fluoroshield (abcam) and observed using a fluorescence microscope.

[0143] The results are shown in Figs. 4a and 4b. First, in the case of the HaCAT cell line, only a very weak fluorescent signal was detected in the 1XCPP-SNAP25 treatment group, whereas a relatively strong fluorescent signal was detected within the cells in the 2XCPP-SNAP25 treatment group, and a very strong fluorescent signal was confirmed in cells treated with 3XCPP-SNAP25 (Fig. 4a). In the HT1080 cell line, a strong fluorescent signal was detected in the 2XCPP-SNAP25 treatment group compared to 1XCPP-SNAP25, and a much stronger fluorescent signal was detected in the 3XCPP-SNAP25 treatment group (Fig. 4b). The above results show that the cell-penetrating peptide according to the present invention can effectively pass through the cell membrane and deliver SNAP25 into cells even when bound to SNAP25, and that the fusion peptide in which the peptide is repeatedly bound significantly increases the cell-penetrating ability and the delivery rate of SNAP25.

[0144]

[0145] Example 5. Binding analysis of SNAP25 and VAMP2 conjugated to cell-permeable repeat peptides.

[0146] The SNARE membrane fusion complex is a protein complex composed of VAMP2, SNAP25 (full-length SNAP25 protein), and syntaxin, and is responsible for neurotransmission in neurons. The SNAP25-derived peptide Faux-Tox according to the present invention binds to VAMP2 to inhibit SNARE formation, thereby reducing the secretion of catecholamines, neurotransmitters that induce muscle contraction, and consequently improving skin wrinkles as well as neuromuscular diseases such as hyperhidrosis, urinary incontinence, and convulsions. Therefore, in this example, the degree of binding between a repeat CPP conjugated with SNAP25 (a SNAP25-derived peptide according to the present invention, i.e., Faux-Tox) and VAMP2 was observed to confirm whether SNAP25 bound to the CPP according to the present invention can exert a wrinkle-inhibiting effect through inhibition of SNARE formation.

[0147] The degree of protein binding was analyzed by immunoprecipitation. After adding an antibody specific for the His-tag to a sample containing both His-tagged VAMP2 and SNAP25 bound to the CPP of the present invention (2XCPP or 3XCPP), immunoprecipitation was performed, and it was confirmed that CPP-SNAP25 was precipitated together with VAMP2. The presence of each protein was confirmed by Western blotting.

[0148] As a result, as shown in Fig. 5, it was confirmed that 2XCPP-SNAP25 and 3XCPP-SNAP25 proteins were also detected in the sample in which VAMP2 was precipitated by the antibody. The above results show that SNAP25 conjugated to the CPP according to the present invention binds to VAMP2, thereby inhibiting the formation of the SNARE complex and exerting a therapeutic effect on wrinkles, hyperhidrosis, urinary incontinence, etc.

[0149]

[0150] Example 6. Confirmation of the therapeutic effect of SNAP25 conjugated to a cell-penetrating repeat peptide on hyperhidrosis.

[0151] To confirm the biological efficacy of SNAP25 conjugated to a cell-penetrating repeat peptide according to the present invention, a hyperhidrosis treatment efficacy test was performed using a hyperhidrosis animal model. An animal model in which hyperhidrosis (sweat secretion) was induced was established by applying pilocarpine hydrochloride (2 mg / kg), a sweat inducer, to ICR mice (Figs. 6a and 6b). 10 μl of 3XCPP-SNAP25 according to the present invention at a concentration of 5 mg / ml was applied to the animal model on days 1, 3, and 5. On day 7, sweating was induced and iodine was treated, and the degree of iodine infiltration into the sweat glands was counted. In addition, a sweatol solution was used as a positive control to compare the efficacy of 3XCPP-SNAP25.

[0152] The results are shown in Figs. 7a and 7b. In the feet of the negative control group mice, a large number of sweat glands stained with iodine were observed, indicating that the sweat secretion animal model was normally established. The number of sweat glands stained with iodine was slightly reduced in the mice treated with the sweatol solution, but it was confirmed that the number of sweat glands was significantly reduced in the mice treated with the 3XCPP-SNAP25 of the present invention compared to the positive control group. The above results show that SNAP25 conjugated to the repetitive CPP of the present invention effectively penetrates into skin cells, etc., induces a reduction in the number of sweat glands, and exhibits a therapeutic effect on hyperhidrosis.

[0153]

[0154] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0155]

[0156] The present invention relates to a cell-penetrating repeat peptide in which a plurality of cell-penetrating peptides are repeatedly linked, and has been completed by confirming that the cell-penetrating repeat peptide has a significantly higher cell-penetrating ability than a single peptide, and thus can increase the efficiency of movement of a conjugated protein into a cell. Accordingly, the neurotransmitter secretion blocking peptide conjugated to the cell-penetrating repeat peptide of the present invention can move into a cell with high efficiency, and thus can more effectively block the secretion of neurotransmitters in the cell, and thus can be utilized for cosmetic purposes such as improving skin wrinkles, as well as for improving or treating neuromuscular diseases caused by excessive nerve signal transmission and muscle tension, and thus has industrial applicability.

Claims

A cell-penetrating repeat peptide comprising 1.2 to 3 cell-penetrating peptides repeatedly linked together; and a fusion peptide conjugated with a SNAP-25-derived peptide, A fusion peptide characterized in that each of the above cell-penetrating peptides is composed of Tyr, Ala, Arg, Val, Gly, and Pro, and each is 6 to 15 amino acids long.

2. In paragraph 1, The above SNAP-25-derived peptide is a fusion peptide comprising amino acids 181 to 206 of the human SNAP-25 protein.

3. In any one of paragraphs 1 and 2, The above cell-penetrating repeat peptide is a fusion peptide in which two to three cell-penetrating peptides are linked to each other via a linker.

4. In any one of paragraphs 1 to 3, A fusion peptide wherein the linker is composed of 1 to 5 Gly.

5. In any one of paragraphs 1 to 4, A fusion peptide wherein the cell-penetrating repeat peptide is conjugated to the SNAP-25-derived peptide via a linker.

6. In any one of paragraphs 1 to 5, A fusion peptide wherein the linker is composed of 1 to 5 Gly.

7. In any one of paragraphs 1 to 6, A fusion peptide characterized in that the cell-penetrating repeat peptide has a cell-penetrating ability that is at least 5 times higher than that of a single cell-penetrating peptide.

8. A nucleic acid molecule encoding a fusion peptide of any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid molecule of paragraph 8.

10. A pharmaceutical composition for the prevention or treatment of neuromuscular disease or skin disease, comprising the fusion peptide of any one of claims 1 to 7 as an active ingredient.

11. In paragraph 10, A pharmaceutical composition according to claim 1, wherein the neuromuscular disease is at least one selected from the group consisting of hyperhidrosis, urinary incontinence, overactive bladder, myoclonus, blepharospasm, hemifacial spasm, spasmodic torticollis, anal fissure, blepharospasm, facial spasm, cerebral palsy, medication overuse headache, chronic migraine, myalgia, strabismus, temporomandibular disorder, neuralgia, rhinitis, dystonia, and vocal fold granuloma.

12. In any one of paragraphs 10 to 11, A pharmaceutical composition, wherein the above skin disease is at least one selected from the group consisting of skin wrinkles and acne.

13. A cosmetic composition for improving skin condition, comprising the fusion peptide of any one of claims 1 to 7 as an active ingredient.

14. In paragraph 13, A cosmetic composition for at least one selected from the group consisting of reducing the appearance of fine lines, reducing the appearance of wrinkles, widening the eyes, lifting the corner of the mouth, reducing muscle mass, flattening lines extending from the upper lip, and improving acne.

15. A cosmetic method for improving skin condition, comprising a step of administering an effective amount of a fusion peptide of any one of claims 1 to 7 to an individual in need thereof.

16. A topical preparation for preventing or improving neuromuscular or skin diseases, comprising the fusion peptide of any one of claims 1 to 7 as an active ingredient.

17. A pharmaceutical composition comprising the fusion peptide of any one of claims 1 to 7 as an active ingredient.

18. Use of a fusion peptide according to any one of claims 1 to 7 for the prevention, improvement, or treatment of neuromuscular diseases or skin diseases; or a composition comprising the same as an active ingredient.

19. Use of a fusion peptide according to any one of claims 1 to 7 for use in the manufacture of a medicament for preventing, improving, or treating neuromuscular diseases or skin diseases; or a composition comprising the same as an active ingredient.

20. A method for preventing, improving, or treating a neuromuscular disease or skin disease, comprising administering a pharmaceutically effective amount of a fusion peptide according to any one of claims 1 to 7; or a composition containing the fusion peptide as an active ingredient, to a subject in need thereof.

21. A kit for preventing, improving, or treating neuromuscular or skin diseases, comprising a fusion peptide according to any one of claims 1 to 7; or a composition comprising the fusion peptide as an active ingredient; and an instruction manual.

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