KR20170031068A - Skin penetrating peptide and method of use thereof - Google Patents

Abstract

The present invention relates to a novel skin permeable peptide, a biologically active substance-binding fusion, a cosmetic composition comprising the same, and a pharmaceutical composition for external application to the skin. The skin permeable peptide of the present invention has low possibility of causing an immune reaction with respect to a conventional skin permeable peptide and exhibits remarkably improved skin permeability, thereby being able to effectively transfer a biologically active substance through the skin, and especially stratum corneum.

Description

Skin penetrating peptide and method of use thereof

The present invention relates to a skin-penetrating peptide or a derivative thereof capable of transmitting a biologically active substance through the skin, a pharmaceutical composition for external application for skin comprising the peptide or a derivative thereof, and a cosmetic composition comprising the peptide or a derivative thereof.

The skin is a tissue that is always in contact with the external environment, and acts as a protective barrier to prevent body fluid leakage and infection, and to prevent moisture loss. In particular, the stratum corneum of the epidermis exists on the outermost side of the skin and prevents drying of the skin by suppressing the loss of moisture and electrolytes outside the skin, and provides an environment for normal biochemical metabolism of the skin. It protects the human body from substances and plays an important role in preventing bacteria, fungi, and viruses from invading the skin (Bouwstra J. A, Honeywell-Nguyen PL Gooris GS and Ponec M. Prog Lipid Res. 42:1-36 (2003)).

There are three pathways for absorption through the skin: absorption through the stratum corneum, absorption through hair follicles and sebaceous glands, and absorption through sweat glands (Prausnitz M. R. and Langer R. Nat Biotech. 26:1261-1268 (2008)). The delivery of physiologically active molecules through the skin is subject to various limitations due to the structural and physical properties of the skin, and absorption through the stratum corneum is known to be the most important absorption pathway. In particular, the stratum corneum of the skin has a dense structure in the outermost layer of the skin due to the natural death of keratinocytes, which are the major constituent cells of the skin. Because of this, it shows acidity around pH 5. In order to penetrate the stratum corneum barrier, the molecular weight must be as small as 1,000 or less, and it must have lipophilic properties (Metha R. C. and Fitzpatrick R.E. Dermatol. Ther. 20:350-359 (2007)).

It is known that low molecular weight synthetic compounds or natural compounds with a molecular weight of 1,000 kDa or less, which are frequently used as raw materials for cosmetics, can be easily delivered into cells, but the penetration efficiency of low molecular weight substances is also low due to the inherent characteristics of the stratum corneum constituting the skin barrier. Macromolecules such as proteins, peptides and nucleic acids having a molecular weight of 1,000 kDa or more are more difficult to penetrate into cell membranes having a double lipid membrane structure due to their large molecular weight. As a method for amplifying the efficiency of these small and macromolecules passing through the plasma membrane of cells, a method of administration through the skin (Transdermal drug delivery, TDD), which has recently gained increasing interest (Prausnitz MR and Langer R. Nat Biotech. 26:1261-1268 (2008)). However, the biggest barrier to the method of administration through the skin is the keratinocytes in the stratum corneum and the lipids between keratinocytes between them. Molecules that are physiologically active in most of the skin (hereinafter, skin physiologically active molecules) have a lower transdermal transmittance due to the resistance of the stratum corneum of the skin.

Various methods for promoting the transdermal permeability of such skin physiologically active molecules have been studied. Recently, a lot of interest has been focused on delivery systems using cell-penetrating peptides. The use of peptides with cell permeability has several advantages, mainly due to the various modifications that can be made to the constant peptide sequence. This allows the manipulation of carriers that can designate different subcellular domains and carry various types of cargo molecules.

For example, a representative cell membrane-penetrating peptide is the first protein that has been confirmed to penetrate the cell membrane of a substance called TAT as one of the mechanisms of infection of HIV-1 (Human immunodeficiency virus-1). Peptides are the most widely applied and active research is being conducted (Mann, DA et al., Embo J 10: 1733-1739, 1991). TAT peptides were used to deliver β-galactosidase, horseradish peroxidase, RNase A, and domain of Pseudomonas exotoxin A (PE) into cells, and studies on their function and intracellular localization have been conducted (Fawell, S. et al. , PNAS 91:664-668, 1994), TAT peptide was found to be due to endocytosis involving Lipid Raft, which occurs after interaction with Heparan sulfate present in the cell membrane (Jehangir SW et al., Nature). Med 10:310-315, 2004).

In addition, a cell-penetrating peptide called Penetratin (Antp) consisting of 16 amino acid sequences derived from Antennapedia homeoprotein, a transcription factor essential for the development of Drosophila, is derived from VP22, a protein expressed by HSV-1 (Herpes simplex virus type 1). Cell-penetrating peptide VP22 of the same name, Transportan composed of an artificially synthesized 27 amino acid sequence, and poly-Arginine artificially repeated arginine, which is expected to play the most important function in cell-penetrating peptides, are cell permeable. It is well known as a peptide.

These conventional cell-penetrating peptides are sequences derived from viral proteins such as HIV-1, derived from proteins expressing other species such as Drosophila, or characteristic amino acid sequences through amino acid sequence analysis constituting conventional cell-penetrating peptides. Since it is a selected and artificially synthesized amino acid sequence, it may cause side effects such as an immune response when applied to the human body.

In addition, since they are composed of relatively long amino acid chains, they are more likely to cause unwanted immune reactions, and because they can affect the structure and function of the protein to be delivered, their efficiency decreases when linking with biologically active substances to be delivered into cells. There was a problem of becoming.

The inventors of the present invention believe that the peptide sequence derived from the human ASTN1 (Astroractin 1) protein, which is one of the neuroproteins involved in the development of the cerebellum and nerve movement, is compared to the cell-penetrating peptide TAT or the conventionally known skin-penetrating peptide. The present invention was completed by proving that the skin tissue penetration efficiency was remarkably excellent.

Korean Patent Publication No. 10-2015-0056022 Korean Patent Publication No. 10-2013-0070607

The present invention is a skin-penetrating peptide for efficiently introducing into skin cells a skin physiologically active molecule that is difficult to pass through the skin due to the size of the molecular weight or the intrinsic characteristics of the stratum corneum. It is intended to provide a cosmetic composition or a pharmaceutical composition for external application for skin that is capable of efficiently delivering a biologically active substance through the skin, in particular, the stratum corneum, because it is low and exhibits remarkably improved skin permeability.

The present invention provides a skin-penetrating peptide having the sequence _{of (X1) n} -X2-(cysteine)-(X3) _m. Wherein n is an integer from 3 to 14, m is an integer from 4 to 14, X1 and X3 are each independently arginine, lysine or histidine, and X2 is alanine, glycine, proline, Tryptophan, phenylalanine, leucine, isoleucine, methionine, valine, arginine, lysine or histidine.

The present invention provides a fusion body in which the skin-penetrating peptide and a biologically active substance are combined.

The present invention provides a cosmetic composition comprising the fusion body as an active ingredient.

The present invention provides a pharmaceutical composition for external application for skin comprising the fusion as an active ingredient.

The skin-penetrating peptide of the present invention can effectively deliver proteins into epidermal cell lines, dermal cell lines, and skin tissues, and can deliver proteins with higher efficiency compared to conventional TAT peptides or skin-penetrating peptides, and are delivered through the skin for treatment. It can be usefully used for the delivery of biologically active substances such as proteins, genetic materials, and chemical compounds that can be used for purposes.

FIG. 1 is a photograph of a 789 bp double-stranded DNA fragment, which is a PCR product encoding AP of Preparation Example 2 and EGFP linked thereto (AP-EGFP), confirmed by 1% agarose gel electrophoresis.
FIG. 2 is an enzyme reaction of an AP-EGFP DNA fragment (789 bp) and a pRSET-b vector (2.9 Kb) using NheI and HindIII restriction enzymes, respectively, in order to insert the AP-EGFP DNA fragment of Preparation Example 3, and then 1% This is a picture confirming the amount of DNA through agarose gel electrophoresis.
3 is a plasmid after transforming the pRSET-b vector into which the DNA fragment encoding AP-EGFP of Preparation Example 3 is inserted into the DH5α Escherichia coli strain, culturing it in a plate LB medium, and inoculating the selected colony in a liquid LB medium. This is a 1% agarose gel electrophoresis picture of the DNA separated through the mini preparation process.
FIG. 4 shows that the isolated plasmid DNA of Preparation Example 3 was composed of a DNA fragment encoding AP-EGFP (789 bp) and a pRSET-b vector (2.9 Kb) after enzymatic reaction using NheI and HindIII restriction enzymes, followed by 1% agar. This is a picture confirmed through Ross Gel electrophoresis.
5 is a schematic diagram showing the structure of a pRSET-b vector into which AP-EGFP of Preparation Example 3 is inserted.
6 shows the purified AP-EGFP protein of Preparation Example 4 and the EGFP protein to which the cell penetrating peptide corresponding to the negative control is not connected, and the TAT-EGFP protein, which is the most widely known cell penetrating peptide corresponding to the positive control group. % SDS This is a picture confirmed through gel electrophoresis.
7 is a graph showing the concentration-dependent and time-dependent delivery of AP-EGFP protein into Jurkat cells in Experimental Example 1 through intracellular fluorescence intensity analysis using flow cytometry.
FIG. 8 is a graph of intracellular fluorescence intensity analysis in Experimental Example 2 by comparing the efficiency of AP to Jurkat cells to deliver proteins into Jurkat cells by setting conventional cell-penetrating peptides as positive controls and analyzing them by flow cytometry.
FIG. 9 is a graph of intracellular fluorescence intensity analysis by comparing and analyzing the efficiency of AP to Jurkat cells in Experimental Example 3 by using conventional skin-penetrating peptides as positive controls.
FIG. 10 is a graph of intracellular fluorescence intensity analysis by comparing and analyzing the efficiency of AP delivering proteins into HaCaT cells, which is a skin epidermal cell line, in Experimental Example 4 using conventional cell-penetrating peptides as a positive control group and analyzed by flow cytometry. to be.
FIG. 11 is a graph of intracellular fluorescence intensity analysis obtained by comparing the efficiency of AP delivering proteins into NIH3T3 cells, which is a skin dermal cell line, in Experimental Example 4 by comparing conventional skin-permeable peptides as a positive control group and analyzed by flow cytometry. to be.
FIG. 12 is a graph of intracellular fluorescence intensity analysis obtained by comparing the efficiency of AP delivering proteins into HaCaT cells, which is a skin epidermal cell line, into HaCaT cells, which is a skin epidermal cell line, in Experimental Example 5 by comparing conventional skin-permeable peptides as positive controls and analyzed by flow cytometry. to be.
FIG. 13 is a graph of intracellular fluorescence intensity analysis obtained by comparing the efficiency of AP delivering proteins into NIH3T3 cells, which is a skin dermal cell line, in Experimental Example 5 by comparing conventional skin-permeable peptides as a positive control group and analyzed by flow cytometry. to be.
14 is a comparison of the effect of arginine, which is the amino acid sequence occupying the most part of the AP in Experimental Example 6, on the delivery of proteins into the cells of the AP by setting a control group removing arginine one by one and using a flow cytometry This is a graph of the analysis of the fluorescence intensity in the analyzed cells.
15 is a substitution with alanine to confirm the effect of tryptophan (X2), cysteine, and lysine (the first amino acid of X3), which are amino acid sequences constituting AP in Experimental Example 6, on the delivery of proteins into the cells of AP. This is a graph of intracellular fluorescence intensity analysis compared and analyzed through flow cytometry.
16 is a substitution of arginine, respectively, to confirm the effect of tryptophan (X2), cysteine, and lysine (the first amino acid of X3), which are amino acid sequences constituting AP in Experimental Example 6, on the delivery of proteins into the cells of AP. This is a graph of intracellular fluorescence intensity analysis compared and analyzed through flow cytometry.
FIG. 17 is a graph of intracellular fluorescence intensity analysis comparing and analyzing changes in intracellular delivery efficiency of AP according to changes in temperature and serum concentration in the medium in Experimental Example 7 using conventional cell penetrating peptides as positive controls. .
FIG. 18 is a graph comparing and analyzing changes in intracellular delivery efficiency of AP-EGFP according to changes in concentrations of Heparin and MβCD (Methyl-beta-cyclodextrin) in Experimental Example 8 using conventional cell penetrating peptides as positive controls. .
19 is a fluorescence micrograph showing that AP-EGFP was delivered into HeLa cells in Experimental Example 9. FIG.
Figure 20 Comparison of the intracellular fluorescence intensity analysis and cellular location via the 0 seconds point microscope AP sets a skin epidermal cell line, HaCaT cells, efficiency of the conventional cell permeable peptide to deliver the protein within a positive control in Experimental Example 10 It is an image.
FIG. 21 is an analysis of intracellular fluorescence intensity and intracellular location compared with a confocal microscope with conventional cell penetrating peptides set as positive controls for the efficiency of AP delivering proteins into NIH3T3 cells, which is a skin dermal cell line, in Experimental Example 10. It is an image.
22 is a fluorescence micrograph showing that AP-EGFP was delivered into each organ cell of a mouse in Experimental Example 11. FIG.
FIG. 23 is a fluorescence micrograph showing a change in delivery efficiency of AP-EGFP to mouse skin tissue over time in Experimental Example 12 and a comparative analysis of a conventional cell penetrating peptide as a positive control.
FIG. 24 is a fluorescence micrograph showing a comparative analysis of protein delivery efficiency between AP-dTomato and conventional skin-penetrating peptides to the skin of a mouse in Experimental Example 13. FIG.
FIG. 25 is a confocal micrograph for confirming that AP-dTomato was delivered to the skin of mice hit with green fluorescent protein in Experimental Example 14. FIG.
26 is an enlarged photograph of FIG. 25.
FIG. 27 is a photograph confirming the AP-EGFP protein and AP-PTPN2 protein purified in Experimental Example 15 through 12% SDS gel electrophoresis.
28 is a diagram showing a plan of an animal model of Oxazolone-induced contact dermatitis of Experimental Example 16.
29 is a photograph showing that the morphology of the ear induced contact dermatitis after 6 days of sensitization in Experimental Example 16 was observed, and the inflammation of the rat ear of the AP-PTPN2 treatment group was inhibited.
FIG. 30 is a graph showing that the ear thickness of the AP-PTPN2-treated rat group was decreased compared to the PBS-treated rat group as a control group in Experimental Example 16. FIG.
31 is a graph showing that the ear weight of the AP-PTPN2-treated rat group in Experimental Example 16 decreased compared to the PBS-treated rat group as a control group.
32 is an image obtained by observing the difference in ear thickness with an optical microscope by staining the ears of the rat group treated with AP-PTPN2 in Experimental Example 16 and the ears of the rat group treated with PBS as a control by H&E staining.

The present invention provides a skin-penetrating peptide having the sequence _{of (X1) n} -X2-(cysteine)-(X3) _m.

N is an integer of 3 to 14, preferably an integer of 3 to 6, and m is an integer of 4 to 14, preferably an integer of 4 to 7 to be.

The skin-penetrating peptide is preferably composed of 9 to 14, more preferably 9 to 12, and most preferably 9 to 10 amino acids. If it is less than the lower limit, the cell permeation efficiency decreases rapidly, and if it exceeds the upper limit, the possibility of generating an immune response increases.

In the skin-penetrating peptide, X1 and X3 are each independently one of positively charged amino acids, arginine (Arg, R), lysine (Lys, K), or histidine (His, H), preferably arginine or lysine. The (X1) _n is a combination of n positively charged amino acids corresponding to X1, and n amino acids include not only n identical amino acids but also n different positively charged amino acids. Similarly, the (X3) _m includes those in which m different positively charged amino acids are present.

In the skin-penetrating peptide, X2 is any one of non-polar or positively charged amino acids, such as alanine (Ala, A), glycine (Gly, G), proline (Pro, P), tryptophan (Trp, W), phenylalanine (Phe, F). , Leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), valine (Val, V), arginine (Arg, R), lysine (Lys, K) or histidine (His, H) And, preferably, alanine, tryptophan or arginine.

The skin-penetrating peptide more preferably consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 12.

The present invention provides a fusion body in which a skin-penetrating peptide and a biologically active substance are combined.

The biological activity is an activity related to a physiological phenomenon or an activity related to a therapeutic purpose by being delivered into cells or in vivo through the skin. The biologically active substance is also referred to as a cargo because it is delivered through the skin-penetrating peptide of the present invention, and may be a protein, a genetic material, a fat, a carbohydrate, and a chemical compound.

Proteins that bind to the skin permeable peptide include, for example, cytokines and their receptors, as well as chimeric proteins including cytokines or their receptors, such as tumor necrosis factors alpha and beta, their receptors and their derivatives; Lenin; Growth hormones such as human growth hormone, bovine growth hormone, methion human growth hormone, des-phenylalanine human growth hormone, and pig growth hormone; Growth hormone releasing factor (GRF); Parathyroid and pituitary hormones; Thyroid stimulating hormone; Human pancreatic hormone releasing factor; Lipoprotein; Colchicine; Prolactin; Corticotropin; Thyroid stimulating hormone; Oxytocin; Vasopressin; Somatostatin; Repressin; Pancreatin; Leuprolide; Alpha-1-antitrypsin; Insulin A-chain; Insulin B-chain; Proinsulin; Follicle stimulating hormone; Calcitonin; Luteinizing hormone; Luteinizing hormone releasing hormone (LHRH); LHRH agonists and antagonists; Glucagon; Coagulation factors such as factor VIIIC, factor IX, tissue factor and von Willebrand factor; Anticoagulant factors such as protein C; Atrial natriuretic factor; Lung surfactant; Plasminogen activators other than tissue-type plasminogen activators (t-PA), such as urokinase; Bombesin; Thrombin; Hematopoietic growth factor; Enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); Human macrophage inflammatory protein (MIP-1-alpha); Serum albumin such as human serum albumin; Mueller's duct inhibitory substances; Relaxin A-chain; Relaxin B-chain; Prorelaxin; Mouse gonadotropin-associated peptide; Chorionic gonadotropin; Gonadotropin-releasing hormone; Bovine somatotropin; Porcine somatotropin; Microbial proteins such as beta-lactamase; DNase; Inhibin; Activin; Vascular endothelial growth factor (VEGF); Receptors for hormones or growth factors; Integrin; Protein A or D; Rheumatic factor; Neurotrophic factors such as bone-derived neurotrophic tension (BDNF), Neutropin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor , Such as NGF-β; Platelet derived growth factor (PDGF); Fibroblast growth factors such as acidic FGF and basic FGF; Epidermal growth factor (EGF); Transforming growth factors (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; Insulin-like growth factor-I and -II (IGF-I and IGF-II); Des(1-3)-IGF-I (bone IGF-I), insulin-like growth factor binding protein; CD proteins such as CD-3, CD-4, CD-8 and CD-19; Erythropoietin; Bone inducing factor; Immunotoxin; Bone morphogenetic protein (BMP); Interferons such as interferon-alpha (eg interferon α2A), -beta, -gamma, -lambda and consensus interferon; Colony stimulating factors (CSF) such as M-CSF, GM-CSF and G-CSF; Interleukin (IL) such as IL-1 to IL-10; Superoxide dismutase; T-cell receptor; Surface membrane protein; Decay acceleration factor; Viral antigens, such as portions of HIV-1 envelope glycoproteins, gp120, gp160 or fragments thereof; Transport protein; Homing receptor; Addressin; Reproductive inhibitors such as prostaglandins; Reproductive stimulants; Regulatory protein; Antibodies (including fragments thereof) and chimeric proteins such as immunoconjugates; Precursors, derivatives, prodrugs and analogs of these compounds, and pharmaceutically acceptable salts of these compounds, or precursors, derivatives, prodrugs and analogs thereof.

Preferably, the protein is a growth hormone such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methione human growth hormone, des-phenylalanine human growth hormone and pig growth hormone; Insulin, insulin A-chain, insulin B-chain and proinsulin; Or growth factors such as vascular endothelial growth factor (VEGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF) , And insulin-like growth factors-I and -II (IGF-I and IGF-II).

In addition, the PTPN2 protein may be, and the PTPN2 protein combined with the skin-permeable peptide of the present invention is topically applied to the skin to improve or treat dermatitis symptoms.

The genetic material associated with the skin-penetrating peptide may include nucleic acids as well as precursors, derivatives, prodrugs and analogs thereof such as therapeutic nucleotides, nucleosides and analogs thereof; Therapeutic oligonucleotides; And therapeutic polynucleotides.

The genetic material can find specific uses as anticancer agents and antiviral agents. Examples of the genetic material include ribozyme, antisense oligodeoxynucleotide, aptamer, and siRNA. Examples of suitable nucleoside analogs include cytarabine (araCTP), gemcitabine (dFdCTP) and floxuridine (FdUTP). Also, the genetic material is, for example, an interfering RNA such as shRNA, miRNA or siRNA. Suitable siRNAs include, for example, IL-7 (interleukin-7) siRNA, IL-10 (interleukin-10) siRNA, IL-22 (interleukin-22) siRNA, IL-23 (interleukin 23) siRNA, CD86 siRNA, KRT6a (keratin 6A) siRNA, K6a N171K (keratin 6a N171K) siRNA, TNFα (tumor necrosis factor α) siRNA, TNFR1 (tumor necrosis factor receptor-1) siRNA, TACE (tumor necrosis factor (TNF)-α converting enzyme) siRNA , RRM2 (ribonucleotide reductase subunit) siRNA, and VEGF (vascular endothelial growth factor) siRNA. The mRNA sequences of human gene targets of these siRNAs are known in the art. In addition, various methods and techniques for selecting specific mRNA target sequences during siRNA design are known in the art.

Fat bound to the skin-penetrating peptide contains fatty acids. Fatty acids are monocarboxylic acids with saturated or unsaturated fatty tails. Fatty acids have about 7 or more carbon atoms, as defined in the International Cosmetic Ingredient Dictionary and Handbook, 7th Ed. (1997) volume 2, page 1567. For example, palmitic acid is the most abundant natural fatty acid, a saturated fatty acid found in palm oil and other fats. Palmitic acid is also one of the major fatty acids in the skin produced by sebaceous gland, and is used in skin care and cosmetic formulations as a moisturizer. It stabilizes the oil balance, allowing the skin to remain in a normal, healthy condition, softens the skin and reacts like an anti-keratin agent. Esters of palmitic acid are used to provide silkyness to the skin and hair. The palmitic acid acts as a carrier capable of permeating pentapeptide into the skin, is widely used as a lubricant, and is used as an emulsifier, surfactant, and formula texturizer.

Suitable fatty acids in the present invention include, but are not limited to, lauric acid, stearic acid, palmitic acid, undecylenic acid, palmitoleic acid, oleic acid, linoleic acid, linoleic acid, arachidonic acid and erucic acid. Additional suitable fatty acids are disclosed in the International Cosmetic Ingredient Dictionary and Handbook, 7th Ed. (1997) volume 2, page 1567.

Chemical compounds bound to the skin-penetrating peptide may include vitamins, derivatives thereof, and retinoids, but are not limited thereto. According to the present invention, ascorbic acid (vitamin C), alpha-tocopherol (vitamin E) and retinoid (vitamin A) can provide beneficial properties to the skin. Ascorbic acid stimulates the synthesis of connective tissue, and is particularly involved in the stimulation and regulation of collagen production. It helps to prevent or minimize fat oxidation and other types of cell damage due to continuous exposure to UV rays (Varani, J. et al., J. Invest. Dermatol. 114: 480-486 (2000), Offord, EA et al, Free, Radical Biol. & Med. 32:1293-1303, (2002)). Ascorbic acid helps inhibit melanin formation and histamine secretion in cell membranes, compensates for vitamin E deficiency in the skin, is involved in preventing depigmentation of the skin, and has anti-free radical activity. Alpha-tocopherol is an antioxidant that prevents the harmful effects of cell membrane phospholipids and free radicals (JB Chazan et al. Free Radicals and Vitamin E. Cah. Nutr. Diet. 1987 22(l):66-76). Retinoids block mediators of inflammation in the skin and increase the production of procollagen, resulting in greater production of type I and type III collagen.

In the cosmetic composition of the present invention, the biologically active substance bound to the skin-penetrating peptide is an antioxidant, increases angiogenesis, reduces acne symptoms, reduces line secretion, reduces aging effects, reduces wrinkles, reduces melanin production, relieves skin inflammation, or It may be a material having an activity such as improving skin dryness.

In the pharmaceutical composition for external application for skin of the present invention, biologically active substances bound to skin-penetrating peptides are peripheral nerves, adrenaline receptors, choline receptors, skeletal muscle, cardiovascular system, smooth muscle, blood circulation system, synapse site, nerve effector junction site, endocrine and hormonal system, It may be a chemical compound acting on the immune system, reproductive system, skeletal system, otacoid system, digestive and excretory system, histamine system and central nervous system.

The chemical compound may include preparations for psoriasis such as local anesthetics, anti-inflammatory agents, anti-infectives, acne treatments, antivirals, antifungal agents, and topical corticosteroids.

For example, the chemical compounds are listed below: 16-17A-epoxyprogesterone (CAS registration number: 1097-51-4), P-methoxycinnamic acid/4-methoxycinnamic acid (CAS registration number: 830-09-1 ), octyl methoxycinnamate (CAS registration number:5466-77-3), octyl methoxycinnamate (CAS registration number: 5466-77-3), methyl p-methoxy cinnamate (CAS registration number:832- 01-9), 4-estrene-17β-OL-3-one (CAS registration number: 62-90-8), ethyl-p-anisoyl acetate (CAS registration number: 2881-83-6), dihydro Eurasil (CAS registration number: 1904-98-9), Lopinavir (CAS registration number: 192725-17-0), RITANSERIN (CAS registration number: 87051-43-2), Neil Nilotinib (CAS registration number: 641571-10-0); Rocuronium bromide (CAS registration number: 119302-91-9), p-nitrobenzyl-6-(1-hydroxyethyl)-1-azabicyclo(3.2.0)heptane-3, 7-dione-2-carboxylate (CAS registration number: 74288-40-7), Abamectin (CAS registration number: 71751-41-2), Paliperidone (CAS registration number: 144598- 75-4), Gemifioxacin (CAS registration number: 175463-14-6), Valrubicin (CAS registration number: 56124-62-0), Mizoribine (CAS registration number) : 50924-49-7), Solifenacin succinate (CAS registration number:242478-38-2), Lapatinib (CAS registration number: 231277-92-2), Didrogesterone ) (CAS registration number: 152-62-5), 2,2-dichloro-N-[(1R,2S)-3-fluoro-1-hydroxy-1-(4-methylsulfonylphenyl)propane -2-yl]acetamide (CAS registration number: 73231-34-2), Tilmicosin (CAS registration number: 108050-54-0), Efavirenz (CAS registration number: 154598- 52-4), Pirarubicin (CAS registration number: 72496-41-4), Nateglinide (CAS registration number: 105816-04-4), Epirubicin (CAS registration) Number: 56420-45-2), Entecavir (CAS registration number: 142217-69-4), Etoricoxib (CAS registration number: 202409-33-4), Silnidipine (CAS Registration number: 132203-70-4), doxorubicin hydrochloride (CAS registration number: 25316-40-9), Escitalopram (CAS registration number) No.: 128196-01-0), Sitagliptin phosphate monohydrate (CAS registration number: 654671-77-9), Acitretin (CAS registration number: 55079-83-9), Lizatrip Rizatriptan benzoate (CAS registration number: 145202-66-0), Doripenem (CAS registration number: 148016-81-3), Atracurium besylate (CAS registration number: 64228-81-5), Nilutamide (CAS registration number: 63612-50-0), 3,4-dihydroxyphenylethanol (CAS registration number: 10597-60-1), ketanserine tartrate (KETANSERIN TARTRATE) (CAS registration number: 83846-83-7), Ozagrel (CAS registration number: 8251-53-7), Eprosartan mesylate (CAS registration number: 144143-96) -4), Ranitidine hydrochloride (CAS registration number: 66357-35-5), 6,7-dihydro-6-mercapto-5H-pyrazolo[1,2-a][,2, 4] Triazolium chloride (CAS registration number: 153851-71-9), Sulfapyridine (CAS registration number: 144-83-2), Teicoplanin (CAS registration number: 61036-62) -2), Tacrolimus (CAS registration number: 104987-11-3), LUMIRACOXIB (CAS registration number: 220991-20-8), allyl alcohol (CAS registration number: 107-18 -6), Protected meropenem (CAS registration number: 96036-02-1), Nelarabine (CAS registration number: 121032-29-9), Pimecrolimus (CAS Registration Number : 137071-32-0), 4-[-methoxy-7-(3-piperidin-1-ylpropoxy)quinazolin-4-yl]-N-(4-propan-2-yloxyphenyl ) Piperazine-1-carboxamide (CAS registration number: 387867-13-2), Ritonavir (CAS registration number: 155213-67-5), Adapalene (CAS registration number: 106685) -40-9), Aprepitant (CAS registration number: 170729-80-3), Eplerenone (CAS registration number: 107724-20-9), Rasagiline mesylate (CAS registration number: 161735-79-1), Miltefosine (CAS registration number: 58066-85-6), Raltegravir potassium (CAS registration number: 871038-72-1), Nasatinib Dasatinib monohydrate (CAS registration number: 863127-77-9), OXOMEMAZINE (CAS registration number: 3689-50-7), Pramipexole (CAS registration number: 104632-26) -0), PARECOXIB SODIUM (CAS registration number: 198470-85-8), Tigecycline (CAS registration number: 220620-09-7), Toltrazuril (CAS registration) Number: 69004-03-1), Vinflunine (CAS registration number: 162652-95-1), Drospirenone (CAS registration number: 67392-87-4), Daptomycin (CAS registration number: 103060-53-3), Montelukast sodium (CAS registration number: 151767-02-1), Brnzolamide (CAS registration number: 138890-62-7), Maraviroc (CAS registration number: 376348-65-1), doxecalciferol (D oxercalciferol) (CAS registration number: 54573-75-0), Oxolinic acid (CAS registration number: 14698-29-4), Daunorubicin hydrochloride (CAS registration number: 23541-50- 6), Nizatidine (CAS registration number: 76963-41-2), Idarubicin (CAS registration number: 58957-92-9), fluoxetine hydrochloride (FLUOXETINE HYDROCHLORIDE) (CAS registration number : 59333-67-4), Ascomycin (CAS registration number: 11011-38-4), beta-methyl vinyl phosphate (MAP) (CAS registration number: 90776-59-3), amorolfine (CAS registration number: 67467-83-8), Fexofenadine HCl (CAS registration number: 83799-24-0), Ketoconazole (CAS registration number: 65277-42-1), 9,10-Die Fluoro-2,3-dihydro-3-me-7-oxo-7H-pyrido-1 (CAS registration number: 82419-35-0), ketoconazole (CAS registration number: 65277-42-1) ), Terbinafine HCl (CAS registration number: 78628-80-5), Amorolfine (CAS registration number: 78613-35-1), Methoxsalen (CAS registration number: 298- 81-7), olopatadine HCl (CAS registration number: 113806-05-6), zinc pyrithione (CAS registration number: 13463-41-7), olopatadine HCl (CAS registration number: 140462- 76-6), cyclosporine (CAS registration number: 59865-13-3), and botulinum toxin and its analogs and vaccine components.

The skin-penetrating peptide and the biologically active substance are bonded by covalent bonds, and include, but are not limited to, ester, amide, ether, and carbamide bonds.

Since the skin-penetrating peptide is a very small peptide, biological interference with an active substance that may occur may be minimized. The fusion body of the skin-penetrating peptide and the biologically active substance may be delivered in vivo through the skin.

In the cosmetic composition of the present invention or a pharmaceutical composition for external application for skin, the fusion is contained in an amount of 0.0001 to 50% by weight based on the total weight of the composition. The composition may further contain one or more active ingredients exhibiting the same or similar function to the fusion body.

The composition may further include a pharmaceutically or physiologically acceptable carrier, excipient, and diluent in addition to the above-described fusion for administration. Examples of suitable carriers, excipients and diluents that may be included in such compositions include lactose. , Dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydride Oxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The composition may further contain conventional fillers, extenders, binders, disintegrants, surfactants, anti-aggregating agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

Formulations of the composition may include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solid or liquid compositions used for application to the skin and other tissues to which the composition may be applied. I can. Such compositions include additional antimicrobial, moisturizing and hydration agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents ( gelling agents), emollients, antioxidants, flavors, fillers, thickeners, waxes, odor absorbers, dyestuffs, colorants, powders, viscosity-controlling agents, and water. And, optionally, an anesthetic, an anti-itch active, a botanical extract, a conditioning agent, a darkening or lightening agent, a glitter, a humectant ( humectant), mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins, and phytomedicinals.

The dosage of the composition varies according to the subject's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the compound is about 0.01 to 100 mg/kg, preferably 0.5 to 10 mg/kg, and it is more preferable to administer the compound once to several times a day.

The present invention provides a recombinant expression vector expressing a recombinant protein in which the skin-penetrating peptide and a biologically active protein are fused, and a recombinant expression vector comprising a DNA encoding a cell-penetrating peptide and a DNA encoding a biologically active protein.

The biologically active protein may be delivered into a cell or in a living body to exhibit an activity related to a physiological phenomenon or an activity related to a therapeutic purpose.

The recombinant expression vector includes a sequence of the cell-penetrating peptide and a biologically active protein, and a tag sequence that facilitates purification of the fusion protein, for example, a contiguous histidine codon, a maltose binding protein codon, and a Myc codon. It may be possible, and may further include a fusion partner for increasing the solubility of the fusion. In addition, a spacer amino acid or nucleotide sequence may be additionally included for stability of the overall structure and function of the recombinant protein or for flexibility of the protein encoded by each gene. Examples of the spacer include AAY (PM Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (RPM Sutmuller et al., J Immunol. 2000, 165: 7308-7315) or, one to a plurality of spacers. Lysine residues (S. Ota et al., Can Res. 62, 1471-1476, KS Kawamura et al., J Immunol. 2002, 168: 5709-5715). In addition, it may include a sequence specifically cleaved by an enzyme in order to remove an unnecessary portion of the recombinant protein, an expression control sequence, and a marker or reporter gene sequence for confirming intracellular delivery, but are limited thereto. no.

The expression control sequence used in the recombinant expression vector may be composed of a regulatory domain including a promoter specific to cells, tissues, and organs in which the target DNA and/or RNA is selectively delivered or expressed.

The present invention comprises the steps of preparing a delivery complex by combining the skin-penetrating peptide with a biologically active substance; And it provides a transdermal delivery method of a biologically active material comprising the step of injecting the delivery complex into a living body or cells through the skin.

The binding of the skin-penetrating peptide and the biologically active substance may be by indirect linkage by cloning technique using an expression vector at the nucleotide level, or by direct linkage by chemical or physical covalent or non-covalent bonding between the peptide and the biologically active substance. have.

The present invention comprises the steps of preparing a delivery complex by combining the skin-penetrating peptide with a genetic material; And injecting the delivery complex into skin cells.

The skin-penetrating peptide and the genetic material may be directly linked by a chemical or physical covalent bond or a non-covalent bond. Injecting the genetic material delivery complex into a living body or cells through the skin may be administered by the same route as described above.

The delivery complex of the genetic material is non-immunogenic and non-infectious, and is not limited by the size of the plasmid because the DNA is not packaged in a vector organism such as a retrovirus or adenovirus. Therefore, it can be used for any practically sized recombinant gene expression construct.

Hereinafter, the present invention will be described in more detail with reference to preferred examples. However, these examples are intended to illustrate the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Preparation Example 1: Peptide Synthesis and Separation and Purification

Peptides having amino acid sequences of SEQ ID NOs: 1 to 12, 15 and 17 were synthesized.

After synthesizing sense and antisense oligodeoxy nucleotides suitable for the amino acid sequence, respectively, they were left at 95° C. for 3 minutes to remove secondary or tertiary structures formed (denaturation) at 50° C. and By changing the temperature to 72℃, DNA double strands were made. Restriction enzyme specific sequences other than sense and antisense oligodeoxynucleotides were put into 5'and 3'to insert into the pRSET-b vector. Then, it was put into E. coli (transformaiton) and mass amplified. After confirming the integrity of the sequence, it was put into E. coli to induce expression.

In order to fuse a peptide having the amino acid sequence of SEQ ID NO: 1 (hereinafter, referred to as'AP') with a green fluorescent protein (EGFP), a primer was prepared to allow EGFP to be ligated to the N-terminus of the AP. It was produced through a PCR reaction and inserted into a vector (pRSET-b) to express and purify the protein in the E. coli strain, and then an experiment was conducted to confirm the delivery efficiency in the cell.

Preparation Example 2: Preparation of double-stranded DNA encoding EGFP where AP is linked to the N-terminus

A forward primer was prepared by adding a DNA nucleotide sequence encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to a DNA nucleotide sequence encoding a part of the N-terminus of the green fluorescent protein (hereinafter, also referred to as'EGFP').

The forward primer of SEQ ID NO: 13 includes a NheI restriction enzyme recognition site for DNA cloning at the 5′ end, and a BamHI restriction enzyme recognition site between the base sequences of AP and EGFP. Meanwhile, in order to amplify AP-EGFP by PCR reaction, a reverse primer of SEQ ID NO: 14 was prepared. The reverse primer contains a DNA sequence encoding a part of the C-terminus of EGFP, and a HindIII restriction enzyme recognition site was inserted for DNA cloning at the 5'most end of the primer.

The PCR reaction was performed using the primers of SEQ ID NO: 13 and SEQ ID NO: 14 using the pRSETb vector containing the EGFP gene as a template. After initial thermal denaturation reaction at 95°C for 3 minutes, thermal denaturation reaction of the template at 95°C for 20 seconds, polymerization reaction in which primer and template are combined at 50°C for 20 seconds, and extension reaction at 72°C for 30 seconds. 1 cycle was set to be, and 30 cycles were performed using a PCR reactor (Biorad).

The amplified product of AP-EGFP amplified from the reaction was confirmed that a 789 bp DNA fragment was amplified through 1% agarose gel electrophoresis (FIG. 1).

Preparation Example 3: Preparation of pRSETb vector into which AP-EGFP is inserted

In order to express the AP-EGFP protein, the 789 bp DNA fragment prepared in Preparation Example 2 was cut into pRSETb, a protein expression vector, with a restriction enzyme, and then inserted into the vector using a ligase.

The DNA fragment amplified in Preparation Example 2 was subjected to enzymatic reaction using NheI and HindIII ( NEB ) so that the 5′/3′ end of the DNA became a sticky end. Meanwhile, by enzymatic reaction of pRSETb using the same two restriction enzymes, a linear pRSETb vector having NheI and HindIII insertion sites was made. After each enzymatic reaction, it was isolated using a PCR purification kit (Cosmo Genetech).

The separated AP-EGFP double-stranded DNA fragment and the pRSET-b vector were enzymatically reacted at 25° C. for two hours using T4 Ligase (NEB). Each concentration of the AP-EGFP double-stranded DNA fragment and the pRSET-b vector was confirmed by 1% agarose gel electrophoresis (FIG. 2).

The circular pRSETb vector into which the AP-EGFP was inserted was transformed into a DH5α E. coli strain and cultured in an LB plate medium containing 50 μg/ml of the antibiotic ampicillin to select transformed E. coli to form a colony. The selected E. coli colonies were again inoculated and cultured in a liquid LB medium containing 50 μg/ml of ampicillin, and then the plasmid vector was isolated using a plasmid Mini Preparation kit (Cosmo Genetec) (FIG. 3).

In order to confirm that the plasmid vector isolated through the above process was a pRSETb vector into which AP-EGFP was inserted, it was first subjected to enzymatic reaction using NheI and HindIII restriction enzymes, and then confirmed by 1% agarose gel electrophoresis. As a result, it was confirmed that the AP-EGFP DNA fragment corresponding to 789 bp was inserted into the pRSET-b vector corresponding to 2.9 kbp (FIG. 4). And this was finally confirmed through DNA sequencing ( Bionics). The structure of the pRSETb vector into which the thus prepared AP-EGFP is inserted is shown in FIG. 5.

Preparation Example 4: Expression and purification of AP-EGFP protein in E. coli

After transforming the pRSETb vector into which the AP-EGFP of Preparation 3 was inserted into the E. coli BL21 (DE3) star pLysS strain, colonies formed in LB plate medium containing 34 μg/ml of chloramphenicol and 50 μg/ml of ampicillin, which are antibiotics, were transformed. It was inoculated into 50 ml of liquid LB medium, cultured at 37°C for 10 hours, and inoculated into 500 ml of new liquid LB medium. When the amount of E. coli was measured with a spectrophotometer at the same temperature, the culture was incubated until the OD value became 0.5, and then added so that the concentration of IPTG (Isopropyl β-D-1-thiogalactopyranoside) was 1 mM, and the temperature was 20. Incubation was carried out for 14 hours in a shaking incubator set at ℃ and rotation speed of 150 rpm. The protein expressed by the E. coli strain contains a 6X-His tag encoded in the pRSET-b vector in front of AP-EGFP. Using this, the protein was purified by the following experimental method.

After the culture was collected by centrifugation, it was resuspended in a native condition solution (0.5M NaCl, 5mM imidazole, 20mM Tris-HCl, pH 8.0). In order to break the cell walls and membranes of E. coli, they were left suspended in the lysis solution for 10 minutes. Then, the cells were crushed using an ultrasonic cell disruptor, VCX-130 ( Sonics & Materials ), and the supernatant was separated by centrifugation. The separated supernatant was filtered once using a 0.45 µm filter (Advantec ) and allowed to combine with Ni-NTA agarose ( Qiagen ) for 1 hour at room temperature. After that, a histidine column (His-column, Biorad ) was used to allow only the protein product bound to Ni-NTA agarose to be bound to the column. After washing with 20 mM imidazole solution, it was finally eluted with 250 mM imidazole solution. The protein product thus eluted was applied to a PD-10 desalting column ( Amersham Bioscience ) to finally separate and purify AP-EGFP (FIG. 6).

Experimental Example 1: Comparison of the delivery efficiency of AP-EGFP protein into the Jurkat cell line, which is a cancerized human T cell

The AP-EGFP protein purified in the process of Preparation Example 4 was transferred into the Jurkat cell line, which is a cancerized human T cell line, and its efficiency was confirmed. Jurkat cells were cultured using RPMI medium (HyClone ), and 350 µl of RPMI medium was dispensed into a 24-well plate ( SPL). lifescience ) cells were dispensed by adding 100 µl of RPMI medium containing ^{1×10 6} cells to each well. In 50 µl, D-PBS (Welgene) and a protein adjusted to an appropriate concentration were mixed, the protein was prepared so that the total volume was 500 µl, and then the protein was treated according to various conditions as specified. In the following experimental examples, unless otherwise specified, the protein treatment conditions were treated with 5 μM of each protein in the culture medium and incubated for 1 hour at 37° C. in _{a 5% CO 2 cell incubator.}

First, the concentration of AP-EGFP protein is 1 μM and 5 μM, respectively, and then 5% CO₂Incubated for 1 hour at 37 ℃ in a cell incubator. As a control, an EGFP protein to which AP is not linked and a TAT-EGFP protein, one of the conventional cell-penetrating peptides, were used. After 1 hour, collect all the cells, transfer them to a tube, and centrifuge the supernatant. I removed it. In addition, the process of washing with 1 ml of D-PBS, resuspending, and centrifugation was repeated twice so that the cell surface was not affected or the medium remained and was not affected to measure the efficiency. The cells obtained after two washing procedures were finally resuspended with 500 µl of D-PBS, andBD science FACScantoII) Was used to measure the intracellular fluorescence to measure the intracellular delivery efficiency of the protein. As a result, it was confirmed that AP-EGFP was delivered into the Jurkat cell line depending on the treatment concentration of the protein.

Subsequently, the concentration of the AP-EGFP protein was maintained at 5 μM and incubated for 30 minutes to 4 hours at 37° C. in _{a 5% CO 2 cell incubator.} After the corresponding treatment time had elapsed, the cells were washed in the same manner as described above, and the intracellular fluorescence was measured with a flow cytometer. As a result, it was confirmed that AP-EGFP was delivered into the Jurkat cell line depending on the processing time of the protein (FIG. 7).

Experimental Example 2: Comparison of protein delivery efficiency with conventional cell-penetrating peptides into Jurkat cell line

In addition, in order to compare the efficiency of the conventional cell-permeable peptides with the same concentration as in Experimental Example 1, an experiment was carried out in which each protein was delivered into the Jurkat cell line in the same manner as above for the same time.

EGFP protein to which the cell penetrating peptide is not linked was used as a negative control, and TAT-EGFP, R9-EGFP, and Hph-1 were each bound to the cell penetrating peptide and EGFP (Enhanced Green Fluorescence Protein) as a positive control. It was compared using EGFP. As a result, it was confirmed that the AP sequence of the present invention transfers proteins into Jurkat cells with higher efficiency than TAT (FIG. 8).

Experimental Example 3: Comparison of protein delivery efficiency with conventional skin-penetrating peptides into Jurkat cell line

In addition, in order to compare the efficiency of the conventional skin-penetrating peptides with the same concentration as in Experimental Example 1, an experiment was conducted in which each protein was delivered into the Jurkat cell line in the same manner as above for the same time period.

As a negative control, dTomato fluorescent protein to which the cell-penetrating peptide was not linked was used, and as a positive control, TDP1-dTomato and TDP2-dTomato in which the dTomato fluorescent protein was bound to the skin-permeable peptide were used for comparison.

The TDP1-dTomato is a skin-penetrating peptide disclosed in Korean Laid-Open Patent Publication No. 10-2013-0135207 (amino acid sequence'NGSLNTHLAPIL', hereinafter referred to as'a peptide having an amino acid sequence of SEQ ID NO: 15' or'TDP1'), and TDP1 It is a fusion having an amino acid sequence of SEQ ID NO: 16 in which a linker (amino acid sequence'GS') that can be recognized by a restriction enzyme is bonded to the C-terminus of the linker, and dTomato fluorescent protein is bonded to the C-terminus of the linker.

The TDP2-dTomato is a skin-penetrating peptide disclosed in Korean Laid-Open Patent Publication No. 10-2013-0070607 (amino acid sequence'MRAAAPAVAA', hereinafter referred to as'peptide having an amino acid sequence of SEQ ID NO: 17' or'TDP2'), and TDP2 It is a fusion having an amino acid sequence of SEQ ID NO: 18 in which a linker (amino acid sequence'GS') that can be recognized by a restriction enzyme is bonded to the C-terminus of the linker, and dTomato fluorescent protein is bonded to the C-terminus of the linker.

In the same way that the fusion of AP and EGFP was efficiently delivered to Jurkat cells in Experimental Example 2, the fusion of dTomato to AP also significantly improved the delivery efficiency compared to the negative control (dTomato alone). In addition, it was confirmed that TDP1 or TDP2, which are conventionally known as skin-penetrating peptides, have significantly lower delivery efficiency compared to AP-dTomato when combined with dTomato, a fluorescent protein having a large molecular weight (FIG. 9).

Experimental Example 4: Comparison of protein delivery efficiency with conventional cell-penetrating peptides into skin cells

The HaCaT cell line is a human epidermal cell line that has been used in transdermal delivery experiments and skin disease papers for in vitro delivery efficiency and mechanism confirmation [J Invest Dermatol. 2011 Jul;131(7):1477-85; Biochem Pharmacol. 2008 Mar 15;75(6):1348-57.]. In addition, the NIH3T3 cell line is a mouse dermal cell line, and is a cell line frequently used in in vitro experiments to confirm the mechanism of transdermal delivery and skin disease like HaCaT [J Pharm Sci. 2013 Nov;102(11):4109-20; J Immunol. 2003 Jan 1;170(1):548-55.].

Whether the AP of the present invention can shear EGFP fluorescent protein to skin cells using the HaCaT cell line and the NIH3T3 cell line is shown in FIGS. 10 and 11 in comparison with the conventional cell-penetrating peptide.

In the same manner as in Experimental Example 2, an EGFP protein to which a cell-penetrating peptide was not linked was used as a negative control, and TAT-EGFP and R9-EGFP were used as a positive control, respectively, in which EGFP was bound to a cell-penetrating peptide. In addition, AP C→A EGFP in which the cysteine of the AP sequence was substituted with alanine was added to the experimental group (SEQ ID NO: 6).

In addition to immune cells (Jurkat cells), it was confirmed that the AP of the present invention efficiently transfers proteins with high molecular weight compared to TAT or R9 in HaCaT cell lines and NIH3T3 cell lines, and the transfer efficiency of these APs is cysteine contained in the amino acid sequence. Was significantly reduced by substituting for alanine.

Experimental Example 5: Comparison of delivery efficiency of proteins with conventional skin-penetrating peptides into skin cells

It is shown in FIGS. 12 and 13 whether the AP of the present invention can shear dTomato fluorescent protein to skin cells using the HaCaT cell line of Experimental Example 3 and the NIH3T3 cell line compared with the conventional cell-penetrating peptide.

In the same manner as in Experimental Example 3, dTomato fluorescent protein to which the cell-penetrating peptide was not linked was used as a negative control, and TDP1-dTomato and TDP2-dTomato were used as a positive control, in which the dTomato fluorescent protein was bound to the skin-permeable peptide. saw.

In addition to immune cells (Jurkat cells), the AP of the present invention delivers high molecular weight proteins with high efficiency in HaCaT cell lines and NIH3T3 cell lines, whereas conventional TDP1 and TDP2 skin-penetrating peptides have increased permeation efficiency when proteins with high molecular weight are combined. Was insignificant.

Experimental example 6: Comparison of protein transfer efficiency according to substitution, removal, and addition of amino acids constituting AP

To find out the role of each amino acid constituting AP, various variants were made and compared and analyzed.

(1) Comparison of protein delivery efficiency according to the removal of terminal amino acids

First, one arginine constituting both ends of the AP is excluded at the N-terminus (AP_D1, SEQ ID NO: 2), one at the C-terminus (AP_D2, SEQ ID NO: 3), and one at the N and C-terminus (AP_D3, sequence Number 4) After making one variant, EGFP, AP-EGFP, TAT-EGFP, and R9-EGFP were compared as controls.

As a result, when there is no arginine, that is, when X1 is less than 3 or X2 is less than 4, the efficiency decreases significantly compared to the existing AP, so the lower limit of the number of amino acids of X1 and X2 in the role of intracellular protein delivery of AP. The critical significance of was confirmed (FIG. 14).

(2) Comparison of protein delivery efficiency according to substitution of amino acids X2, cysteine, and X3

In order to investigate the role of tryptophan (X2), cysteine, and lysine (the first amino acid of X3), a variant in which each amino acid was substituted with alanine or arginine was prepared and compared. Alanine is suitable as a control because it has no charge and is the simplest and smallest structure.

The positively charged arginine substituent was used as a control to compare the efficiency with R9 with 9 arginine attached. As a result, the alanine substituent and the arginine substituent showed similar patterns. It was confirmed that tryptophan did not significantly contribute to the functional role of AP as it did not show a significant difference in efficiency from the existing AP sequence even if it was changed to another amino acid. When arginine is substituted with another amino acid, it can be confirmed that the efficiency is greatly reduced. It is believed that arginine's positively charged property had a positive effect. When cysteine was substituted with another amino acid, the most rapid decrease in efficiency was shown, which suggests that cysteine plays a very important function and role in AP (Figs. 15 and 16) (SEQ ID NOs: 7 to 12).

Experimental Example 7: Confirmation of AP cell permeation mechanism

Meanwhile, in order to confirm whether AP is delivered into cells through endocytosis, like TAT, which is the most well-known cell-penetrating peptide in the past, changes in intracellular delivery depending on temperature were measured, and according to the serum concentration in the medium. We checked whether it was affected by other proteins. EGFP to which the cell-penetrating peptide was not linked was used as a negative control, and TAT-EGFP was used as a positive control.

Each protein was treated with Jurkat cells to a concentration of 5 μM in the same manner as described above, and independently 1 hour at 4° C., 25° C., and 37° C. temperature. As a result, it was confirmed that unlike the existing TAT, it was not affected by temperature. This is indirect evidence that AP is delivered in a different way than TAT, which is delivered through energy-dependent endocytosis. It was confirmed that it was a permeable peptide. In addition, it was confirmed that the transfer efficiency was higher than that of TAT at each temperature (top of FIG. 17).

In addition, when the concentration of serum added to the RPMI medium was changed to 0% and 10%, respectively, the delivery efficiency of each protein treated to be 5 μM was analyzed. Cells were cultured at 37° C. for 1 hour after protein treatment, and EGFP to which the cell-penetrating peptide was not linked as a negative control group and TAT-EGFP were used as a positive control group.

As a result, it was confirmed that as the serum concentration of the medium increased, the efficiency of the AP-EGFP protein passing through the cell membrane and transferred into the cells decreased. This is also a result of indirectly showing that the cell-penetrating peptide is affected by competition or interaction with other proteins, and is a result of reconfirming the function of the AP developed in the present invention as a cell-penetrating peptide. On the other hand, it was confirmed that AP at each serum concentration still showed higher delivery efficiency compared to other positive controls (bottom of FIG. 17).

Experimental example 8: AP- according to the change in the concentration of Heparin and MβCD EGFP Intracellular protein delivery efficiency

(1) Intracellular protein delivery efficiency of AP-EGFP according to the change of Heparin concentration

As the mechanism of the transfer efficiency of AP-EGFP was confirmed through Experimental Example 4, heparin of a similar structure that may interfere with the binding of the cell-penetrating peptide to the heparan sulfate on the cell surface was previously treated to mutually Heparin (Heparin sodium salt from porcine intestinal mucosa, Sigma ) in 4 concentrations of 0 μm/ml, 10 μg/ml, 20 μg/ml, and 50 μg/ml with the expectation that it will be directly or indirectly affected by the action. The Jurkat cell line was treated for 30 minutes first, and then 10 μM of AP-EGFP protein was treated with D-PBS so that the final volume was 100 μl. As a positive control, TAT-EGFP was used for comparison. And incubated in a 5% CO ₂ cell incubator at 37°C for 1 hour. After 1 hour, collect all the cells, transfer them to a tube, and centrifuge the supernatant. I removed it. In addition, the process of washing, resuspending and centrifuging with 1 ml of D-PBS was repeated twice so that the cell surface was not affected by the measurement of the efficiency due to the presence of the medium or the remaining medium. Cells obtained after two washing procedures are finally resuspended with 500 µl of D-PBS, and the intracellular delivery efficiency of proteins is measured by measuring intracellular fluorescence using a flow cytometry (FACS machine, BD science FACS Canto). It was measured.

As a result, it was confirmed that the intracellular delivery efficiency of AP-EGFP significantly decreased as the concentration of Heparin treatment increased (top of FIG. 18).

(2) Intracellular protein delivery efficiency of AP-EGFP according to the change of MβCD concentration

In addition, it is predicted that AP-EGFP will be affected by endocytosis, which is associated with the lipid raft constituting the phospholipid bilayer cell membrane, in the process of delivery into the cell, thereby removing cholesterol from the cell membrane. After inhibiting endocytosis due to lipids by treatment with known methyl-beta-cyclodextrin (MβCD), MβCD was added to Jurkat cell lines at three concentrations of 0 mM, 3 mM, and 5 mM on ice for 20 minutes first. After treatment, 10 μM of AP-EGFP protein was treated for one hour. As a positive control, TAT-EGFP was used for comparison. As a result, it was confirmed that the intracellular delivery efficiency of AP-EGFP significantly decreased as the MβCD treatment concentration increased (bottom of FIG. 18).

This is also a result of directly or indirectly showing that the cell-penetrating peptide is affected by competition or interaction with other proteins, and is a result of reconfirming the function of the AP developed in the present invention as a cell-penetrating peptide.

Experimental Example 9: Delivery of AP-EGFP into HeLa cells, a cancer cell line

In order to directly confirm whether AP is actually delivered to the cell along with the protein, and where it exists in the cell, AP-EGFP was delivered into HeLa cells, a cervical cancer cell line, and analyzed using a confocal microscope.

12-well plate ( SPL lifescience ) in advance on a microscopic circular cover glass ( Marin field ), and the number of HeLa cells ^{was divided into 1×10 5} , and then cultured in DMEM medium (HyClone ) for 18 hours to allow the cells to be placed on the cover glass. So that it can be attached. Thereafter, after all the medium was sucked away, 450 µl of new DMEM was added, and the AP-EGFP protein purified through Example 3 was mixed with D-PBS to a concentration of 5 µM and treated to a volume of 50 µl. I did. Then, it was incubated in a 5% CO ₂ cell incubator at 37° C. for 30 minutes. After the 30-minute treatment time had elapsed, all the medium was sucked and discarded in order to remove the protein outside the cells, and the process of washing with 1 ml of D-PBS was repeated 5 times. Then, 1 ml of formaldehyde (formaldehyde 37% solution, formalin, SIGMA ) was used to fix the cells. After fixation, it was washed 5 times with 1 ml of D-PBS. After that, in order to identify the cells and confirm the location of the nucleus , 500 μl of Hoechst (Hoechst AG ) diluted 1:4000 was used to stain the nuclei to be fluorescent. After a reaction time of 10 minutes, 5 times with 1 ml of D-PBS. Washed. The prepared cover glass is mounted on the slide glass using a mounting medium (SIGMA ), and a fluorescence microscope AX-10 (Fluorescence microscope, Carl zeiss ) As a result, it was confirmed that the green fluorescent protein was located and transferred into the cell. As a result, it was confirmed that the green fluorescent protein to which AP was bound penetrated the cell membrane and passed into the cell, and the intracellular location remained in the cytoplasm (FIG. 19).

Experimental Example 10: Delivery of AP-EGFP into skin cell lines HaCaT and NIH3T3 cells

The same method as in Experimental Example 9 using a confocal microscope by delivering AP-EGFP into the skin cell lines HaCaT and NIH3T3 cells to directly confirm whether AP is actually delivered to the skin cells along with the protein and where it exists in the skin cells. to Analyzed. As positive controls, TAT-EGFP and R9-EGFP were used for comparison. In addition, AP C→A EGFP in which the cysteine of the AP sequence was substituted with alanine was added as an experimental group.

As a result, the green fluorescent protein to which AP is bound penetrates the cell membrane with higher efficiency than the positive control or C→A EGFP, and passes into the skin cells and enters the intracellular nucleus. And it was confirmed that it stays in the cytoplasm (FIGS. 20 and 21).

Experimental Example 11: Delivery of AP-EGFP to the inside of each organ of a mouse

In order to directly check whether AP is delivered under actual in vivo conditions and to which organ it can be delivered, 5 mg of AP-EGFP protein was intraperitoneally injected into 6-week-old female mice of C57BL/6 for 2 hours. Afterwards, organs such as brain, heart, kidney, liver, lungs, spleen, and intestine were obtained, fixed with 4% paraformaldehyde, washed 2 to 3 times with D-PBS, and frozen blocks using OCT compound. Subsequently, a slide sample was prepared by making a section with a thickness of 6 μm using a cryostat, and the delivery of AP-EGFP in several organ cells was confirmed through a fluorescence microscope. After making the slide, it was stained with Hoechst for 10 minutes, and then it was checked whether it entered the cell by overlapping and comparing it with the fluorescent protein. As a control, EGFP to which the cell-penetrating peptide was not linked was used. As a result, compared to EGFP, it was confirmed that the green fluorescent protein to which AP is linked clearly enters cells constituting organs such as brain, heart, kidney, liver, lung, spleen, and intestine (FIG. 22).

Experimental Example 12: Delivery of AP-EGFP to the skin of a mouse over time

After removing the hair of a 7-week-old female rat of C57BL/6 species to confirm whether the AP of the present invention is delivered to the skin tissue or to what depth it can be delivered, the stratum corneum was removed by tape bonding 10 times. 100 μg of AP-EGFP was attached to the skin using a paper patch. After 2, 4, 6 and 8 hours, respectively, skin tissue was obtained, fixed with 4% paraformaldehyde, and then frozen blocks were prepared using an O.C.T compound. Subsequently, a slide sample was prepared by making a section with a thickness of 7 μm using a cryostat, and the delivery of AP-EGFP in the skin tissue was confirmed through a fluorescence microscope. After making the slide, it was stained with Hoechst for 15 minutes, and then it was checked whether it entered the cell through the process of overlapping and comparing with the fluorescent protein. TAT-EGFP to which a conventional cell penetrating peptide was bound was used as a positive control.

TAT-EGFP did not deliver the green fluorescent protein into the skin tissue until 8 hours, but it was confirmed that the green fluorescent protein (AP-EGFP) to which AP is linked enters the cells constituting the skin tissue clearly from the 2nd hour ( Fig. 23).

Experimental Example 13: Comparison of protein delivery efficiency between AP-dTomato and conventional skin-penetrating peptides to mouse skin

Even when the AP of the present invention is combined with a dTomato fluorescent protein having a large molecular weight, it was tested whether it delivered the fluorescent protein to the skin tissue by comparing with conventional skin permeable peptides. In the same manner as in Experimental Example 3, dTomato fluorescent protein to which the cell-penetrating peptide was not linked was used as a negative control, and TAT-dTomato, TDP1-dTomato, TDP2-dTomato in which the dTomato fluorescent protein was bound to the skin-permeable peptide was used as a positive control. I used to compare. The experiment was the same as in Experimental Example 12, but skin tissue was obtained at 4 hours and used in the experiment.

It was confirmed that only the AP-dTomato of the present invention delivered the dTomato fluorescent protein into the skin tissue (FIG. 24).

Experimental Example 14: Delivery of AP-dTomato to the skin of mice hit with green fluorescent protein

In order to clearly confirm whether the AP of the present invention is delivered to skin cells in the skin tissue, the GFP gene expressing green fluorescent protein (GFP) was hit in all cells. dTomato fluorescent protein was attached. After 4 hours, a skin tissue was obtained, fixed with 4% paraformaldehyde, and then a frozen block was prepared using an O.C.T compound. Subsequently, a slide sample was prepared by making a section with a thickness of 20 μm using a cryostat, and the delivery of AP-EGFP in the skin tissue was confirmed through a confocal microscope.

After making the slide, it was stained with Hoechst for 15 minutes, and then it was confirmed whether or not it entered the cell through the process of overlapping and comparing with the fluorescent protein, and shown in FIGS. 25 and 26. As a control, dTomato to which the skin penetrating peptide was not linked was used as a negative control.

As a result, compared to dTomato, it was confirmed that the red fluorescent protein to which AP is connected clearly enters the cells constituting the skin tissue (FIG. 25), and green cells in which the AP-dTomato fluorescent protein expresses GFP through a high magnification image. It was confirmed that it was well delivered within (FIG. 26).

Experimental Example 15: Expression and purification of AP-PTPN2 protein in E. coli

The AP-PTPN2 protein having the amino acid sequence of SEQ ID NO: 19 in which the AP of the present invention was bound to the PTPN2 protein was expressed in E. coli and purified.

First, the PET28a vector inserted with AP-PTPN2 was transformed into E. coli Rosetta strain, and then colonies formed in the LB plate medium containing antibiotics chloramphenicol (34 ㎍/ml) and kanamycin (50 ㎍/ml) were transferred to liquid LB medium 50. It was inoculated and cultured at 37 for 10 hours and inoculated into a new liquid LB medium 500. When the amount of E. coli was measured with a spectrophotometer at the same temperature, the culture was incubated until the OD value became 0.5, and then added so that the concentration of IPTG (Isopropyl β-D-1-thiogalactopyranoside) was 1 mM, and the temperature was 20. Incubation was carried out for 14 hours in a shaking incubator set at ℃ and rotation speed of 150 rpm. The protein expressed by E. coli in this way contains the 6X-His tag encoded in the PET28a vector in front of AP-PTPN2. Using this, the protein was purified by the following experimental method.

After collecting the culture medium by centrifugation, it was resuspended in a native condition dissolution solution (0.5M NaCl, 5mM imidazole, 20mM Tris-HCl, pH 8.0). In order to break the cell walls and membranes of E. coli, they were left suspended in the lysis solution for 10 minutes. Then, the cells were crushed using an ultrasonic cell disruptor, VCX-130 ( Sonics & Materials ), and the supernatant was separated by centrifugation. The separated supernatant was filtered once using a 0.45 µm filter (Advantec ) and allowed to combine with Ni-NTA agarose ( Qiagen ) for 1 hour at room temperature. After that, a histidine column (His-column, Biorad ) was used to allow only the protein product bound to Ni-NTA agarose to be bound to the column. After washing with 20 mM imidazole solution, final elution was performed using 250 mM imidazole solution. The protein product thus eluted was applied to a PD-10 desalting column ( Amersham Bioscience ) to finally separate and purify AP-EGFP (FIG. 27).

Experimental Example 16: Confirmation of the therapeutic effect of AP-PTPN2 protein in an Oxazolone-induced contact dermatitis animal model

The next day after removing the hairs of female 7-week-old C57BL/6 mice with a depilatory agent, sensitize by spraying 10 µl of 2% oxazolone mixed with acetone: olive oil (4:1) solution per side of the ear of the mouse. 5 days after sensitization, 1% oxazolone solution is sprayed equally to induce dermatitis. AP-PTPN2 protein is applied by 100 μg each after attaching it to both sides of the ear using a paper patch 6 times between sensitization and induction. The analysis proceeds the day after induction of 1% oxazolone (Fig. 28).

As a result, it was confirmed that the ear of the rat group to which AP-PTPN2 was applied had inhibition of inflammation compared to the PBS-treated ear, which is a negative control group (Fig. 29), and as a result of measuring the ear thickness using a micrometer (mitutoyo), AP-PTPN2 was applied. It was observed that the ear thickness of one group became thinner compared to the negative control group (FIG. 30), and the ear weight was also decreased (FIG. 31).

In order to confirm more specifically, the ear was fixed with 4% formaldehyde for one day, and then the tissue was dehydrated by immersing it in 30% sucrose for one day, and a frozen section was made using an O.C.T compound. After making a section with 10 μm, the section was stained with H&E staining technique and observed with an optical microscope. As a result, it was confirmed that the ear thickness decreased compared to the negative control group (FIG. 32).

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Skin penetrating peptide and method of use thereof <130> HPC6909 <160> 19 <170> KopatentIn 2.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 1 Arg Arg Arg Trp Cys Lys Arg Arg Arg 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 2 Arg Arg Trp Cys Lys Arg Arg Arg 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 3 Arg Arg Arg Trp Cys Lys Arg Arg 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 4 Arg Arg Trp Cys Lys Arg Arg 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 5 Arg Arg Arg Ala Cys Lys Arg Arg Arg 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 6 Arg Arg Arg Trp Ala Lys Arg Arg Arg 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 7 Arg Arg Arg Trp Cys Ala Arg Arg Arg 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 8 Arg Arg Arg Arg Cys Lys Arg Arg Arg 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 9 Arg Arg Arg Trp Arg Lys Arg Arg Arg 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 10 Arg Arg Arg Trp Cys Arg Arg Arg Arg 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 11 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 12 Arg Arg Arg Arg Cys Arg Arg Arg Arg 1 5 <210> 13 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> EGFP forward primer <400> 13 ctagctagcc gccggcgctg gtgcaaacgc cgccggggat ccgtgagcaa gggcgaggag 60 ctgttcac 68 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> EGFP reverse primer <400> 14 caagctttta cttgtatagc tcgtc 25 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 15 Asn Gly Ser Leu Asn Thr His Leu Ala Pro Ile Leu 1 5 10 <210> 16 <211> 262 <212> PRT <213> Artificial Sequence <220> <223> TDP1-dTomato <400> 16 Met Arg Gly Ser His His His His His His His Gly Met Ala Ser Asn Gly 1 5 10 15 Ser Leu Asn Thr His Leu Ala Pro Ile Leu Gly Ser Met Val Ser Lys 20 25 30 Gly Glu Glu Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu 35 40 45 Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly 50 55 60 Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly 65 70 75 80 Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr 85 90 95 Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys 100 105 110 Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe 115 120 125 Glu Asp Gly Gly Leu Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp 130 135 140 Gly Thr Leu Ile Tyr Lys Val Lys Met Arg Gly Thr Asn Phe Pro Pro 145 150 155 160 Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Thr 165 170 175 Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu Ile His Gln 180 185 190 Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu Phe Lys Thr 195 200 205 Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr Tyr Tyr Val 210 215 220 Asp Thr Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val 225 230 235 240 Glu Gln Tyr Glu Arg Ser Glu Gly Arg His His Leu Phe Leu Tyr Gly 245 250 255 Met Asp Glu Leu Tyr Lys 260 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Skin penetrating peptide <400> 17 Met Arg Ala Ala Ala Pro Ala Val Ala Ala 1 5 10 <210> 18 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> TDP2-dTomato <400> 18 Met Arg Gly Ser His His His His His His His Gly Met Ala Ser Met Arg 1 5 10 15 Ala Ala Ala Pro Ala Val Ala Ala Gly Ser Met Val Ser Lys Gly Glu 20 25 30 Glu Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly Ser 35 40 45 Met Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro 50 55 60 Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro 65 70 75 80 Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser 85 90 95 Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys Lys Leu 100 105 110 Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp 115 120 125 Gly Gly Leu Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Thr 130 135 140 Leu Ile Tyr Lys Val Lys Met Arg Gly Thr Asn Phe Pro Pro Asp Gly 145 150 155 160 Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Thr Glu Arg 165 170 175 Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu Ile His Gln Ala Leu 180 185 190 Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu Phe Lys Thr Ile Tyr 195 200 205 Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr Tyr Tyr Val Asp Thr 210 215 220 Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln 225 230 235 240 Tyr Glu Arg Ser Glu Gly Arg His His Leu Phe Leu Tyr Gly Met Asp 245 250 255 Glu Leu Tyr Lys 260 <210> 19 <211> 313 <212> PRT <213> Artificial Sequence <220> <223> AP-PTPN2 <400> 19 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Arg Arg Arg Trp Cys Lys Arg Arg Arg 20 25 30 Glu Phe Ile Glu Arg Glu Phe Glu Glu Leu Asp Ala Gln Cys Arg Trp 35 40 45 Gln Pro Leu Tyr Leu Glu Ile Arg Asn Glu Ser His Asp Tyr Pro His 50 55 60 Arg Val Ala Lys Phe Pro Glu Asn Arg Asn Arg Asn Arg Tyr Arg Asp 65 70 75 80 Val Ser Pro Tyr Asp His Ser Arg Val Lys Leu Gln Ser Thr Glu Asn 85 90 95 Asp Tyr Ile Asn Ala Ser Leu Val Asp Ile Glu Glu Glu Ala Gln Arg Ser 100 105 110 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Cys His Phe Trp 115 120 125 Leu Met Val Trp Gln Gln Lys Thr Lys Ala Val Val Met Leu Asn Arg 130 135 140 Thr Val Glu Lys Glu Ser Val Lys Cys Ala Gln Tyr Trp Pro Thr Asp 145 150 155 160 Asp Arg Glu Met Val Phe Lys Glu Thr Gly Phe Ser Val Lys Leu Leu 165 170 175 Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val His Leu Leu Gln Leu Glu 180 185 190 Asn Ile Asn Thr Gly Glu Thr Arg Thr Ile Ser His Phe His Tyr Thr 195 200 205 Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu Asn 210 215 220 Phe Leu Phe Lys Val Arg Glu Ser Gly Cys Leu Thr Pro Asp His Gly 225 230 235 240 Pro Ala Val Ile His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr Phe 245 250 255 Ser Leu Val Asp Thr Cys Leu Val Leu Met Glu Lys Gly Glu Asp Val 260 265 270 Asn Val Lys Gln Leu Leu Leu Asn Met Arg Lys Tyr Arg Met Gly Leu 275 280 285 Ile Gln Thr Pro Asp Gln Leu Arg Phe Ser Tyr Met Ala Ile Ile Glu 290 295 300 Gly Leu Glu His His His His His His 305 310

Claims (13)

(X1) _n- X2- (cysteine) - (X3) _m :
N is an integer of 3 to 14,
M is an integer of any one of 4 to 14,
X1 and X3 are each independently arginine, lysine or histidine,
X2 is alanine, glycine, proline, tryptophan, phenylalanine, leucine, isoleucine, methionine, valine, arginine, lysine or histidine. The skin permeable peptide of claim 1, wherein the skin permeable peptide is comprised of 9 to 14 amino acids. The skin permeable peptide of claim 2, wherein X1 and X3 are each independently arginine or lysine. 3. The cell permeable peptide according to claim 2, wherein X2 is alanine, tryptophan or arginine. 4. The skin permeable peptide of claim 3, wherein the peptide is an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: A fusion substance comprising a skin permeable peptide according to any one of claims 1 to 5 combined with a biologically active substance. The fusion substance according to claim 6, wherein the biologically active substance is at least one selected from a protein, a genetic material, a fat, a carbohydrate, and a chemical compound. The fused product of claim 6, wherein the bond is a peptide bond or a chemical bond. 9. The method of claim 8, wherein the chemical bond is selected from the group consisting of a disulfide bond, a diamine bond, sulfide-amine bonds, carboxyl-amine bonds, ester bonds, &Lt; / RTI &gt; A cosmetic composition comprising a fused product of a skin permeable peptide and a biologically active substance of claim 6 as an active ingredient. The cosmetic composition according to claim 10, wherein the composition is prepared from a formulation selected from the group consisting of emulsion, cream, essence, skin, liposome, microcapsule, composite particle, shampoo and rinse. A pharmaceutical composition for an external preparation for skin comprising the fused product of the skin permeable peptide and the biologically active substance of claim 6 as an active ingredient. 13. The pharmaceutical composition for external application for skin according to claim 12, wherein the substance having biological activity to which the skin permeable peptide is bound is permeable to the skin horny layer.

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