WO2013077681A1 - Système d'administration transdermique de molécules bioactives de peau au moyen de peptides d'administration intracellulaire de molécules - Google Patents

Système d'administration transdermique de molécules bioactives de peau au moyen de peptides d'administration intracellulaire de molécules Download PDF

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WO2013077681A1
WO2013077681A1 PCT/KR2012/009998 KR2012009998W WO2013077681A1 WO 2013077681 A1 WO2013077681 A1 WO 2013077681A1 KR 2012009998 W KR2012009998 W KR 2012009998W WO 2013077681 A1 WO2013077681 A1 WO 2013077681A1
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skin
peptide
composition
vitamin
group
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PCT/KR2012/009998
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English (en)
Korean (ko)
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이강진
임선희
신기덕
이병규
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주식회사 프로셀제약
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Priority to KR1020127031205A priority Critical patent/KR101393397B1/ko
Publication of WO2013077681A1 publication Critical patent/WO2013077681A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/671—Vitamin A; Derivatives thereof, e.g. ester of vitamin A acid, ester of retinol, retinol, retinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/676—Ascorbic acid, i.e. vitamin C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/007—Preparations for dry skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists

Definitions

  • the present invention relates to a transdermal delivery composition
  • a transdermal delivery composition comprising a skin bioactive molecule to which intracellular molecular transport peptides are bound, a transdermal delivery system using the skin bioactive molecule, and a method for intracellular skin delivery of the skin bioactive molecule.
  • the skin is a tissue that is always in contact with the outside environment. Its main function is to protect body fluids from leakage and infection, as well as act as a protective barrier against water loss.
  • the stratum corneum of the epidermis is located on the outermost surface of the skin and prevents the loss of moisture and electrolytes outside the skin, preventing the drying of the skin and providing an environment for normal biochemical metabolism of the skin. It protects the human body from humans and plays an important role in preventing bacteria, fungi, viruses, etc. from invading the skin (Bouwstra J. A, Honeywell-Nguyen PL Gooris GS and Ponec M. Prog Lipid Res. 42: 1-36 (2003)).
  • the stratum corneum of the skin is the natural constituent of the keratinocyte (keratinocyte) is a natural death and forms a dense structure in the outermost layer of the skin, not only evaporation of moisture but also inhibits the penetration of foreign substances, sweat and various lipid components Due to the acidity is showing around pH 5.
  • the molecular weight In order to penetrate the stratum corneum barrier, the molecular weight must be as small as 1,000 or less and possess lipophilic properties (Metha R. C. and Fitzpatrick R. E. Dermatol. Ther. 20: 350-359 (2007)).
  • TDD transdermal drug delivery
  • peptides having cell permeability has several advantages, mainly due to the various modifications that can be made to the peptide sequence at all times. It can designate different subcellular domains and allows manipulation of carriers that can carry various forms of cargo molecules.
  • Representative cell membrane permeable peptides are as follows.
  • the transcriptional protein Tat of HIV-1 a virus that causes acquired immune deficiency syndrome (AIDS)
  • AIDS acquired immune deficiency syndrome
  • the cell permeable Tat domain is the 48th to 57th basic amino acid sequence (GRKKRRQRRR) of the Tat protein, which has been found to play an important role in the passage of cell membranes (Vives et al., J. Biol. Chem.
  • RQIKIYFQNRRMKWKK consisting of 16 amino acids derived from the antennapedia homeodomain of Drosophila (Joliot et al., Proc Natl Acad Sci USA 88: 1864-1868 (1991) Derossi et al., J Biol Chem 269, 10444-10450 (1994); Joliot, A. and A. Prochiantz, Nat. Cell Biol. 6 (3): 189-96 (2004).
  • a peptide based on a membrane translocating sequence (MTS) or signal sequence which is a receptor protein that helps to move a newly synthesized protein by RNA to the biofilm of suitable intracellular organelles in vivo.
  • MTS membrane translocating sequence
  • NLS nuclear localization signals
  • peptides bind to a cargo molecule and act as an import signal when in close proximity to the cell, inducing the influx of the cargo molecule into the cell. Accordingly, attempts have been actively made to link cell membrane permeable peptides with proteins originally present in cells to be applied to biological research and disease treatment (F. Milletti, Drug Discovery Today 17: 850-860 (2012)). If cell membrane-penetrating peptides are derived from viruses such as Tat or VP22, there is a possibility of inducing an immune response in vivo, and thus, new membrane-penetrating peptides are mainly identified from secreted proteins found in humans (Eguchi). , A. and Dowdy, SF Trends Pharmacol.Sci.
  • TAT proteins do not actually undergo metastasis, but rather strongly induce electrostatic interactions with the cell surface, causing inclusions and accumulating inside the endosome, and endosomes are fused with lysosomes. It is suggested that peptides are easily degraded by proteolytic enzymes present in lysosomes.
  • a substance transport carrier composed of a basic amino acid such as TAT to deliver a specific active ingredient into the cell, it is difficult to directly access to the target in the cytoplasm and high concentration treatment to deliver the active ingredient to a level that can exhibit the desired activity. There is a problem in that it is not possible to obtain the expected effect when applying it (JS Wadia, et al., Nat. Med.
  • MTD has a higher delivery efficiency of cargo material, which can be selected from compounds, peptides, and proteins, and has the advantage of cell-to-cell delivery, compared to the conventional cell membrane permeable peptide, TAT. Its applicability is highly appreciated in the development of cosmetics.
  • the present inventors apply an improved MTD peptide having better cell permeability by modifying the amino acid sequence of the macromolecular transport domain (MTD) to the intracellular molecular transport peptide of the present invention.
  • MTD macromolecular transport domain
  • the present invention is designed to efficiently introduce skin bioactive molecules into the skin cells, which are difficult to deliver through the skin due to the size of the molecular weight or the intrinsic properties of the stratum corneum as described above, the cell improved cell permeability compared to the existing MTD peptide
  • a transdermal delivery composition comprising a skin bioactive molecule to which an intramolecular transport peptide is bound, a transdermal delivery system using a skin bioactive molecule to which the peptide is bound, and a skin bioactive molecule to which an intracellular molecular transport peptide is bound Or to provide a method for delivery into the stratum corneum of the skin.
  • the present invention provides a composition for transdermal delivery comprising a skin bioactive molecule to which an intracellular molecular transport peptide is bound, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35. .
  • the composition is characterized in that the cosmetic composition.
  • the peptide is characterized in that it is encoded from a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 36 to 56.
  • amino acid sequence of SEQ ID NO: 15 may be encoded by the polynucleotide sequence of SEQ ID NO: 36, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 16 may be encoded by the polynucleotide sequence of SEQ ID NO: 37, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 17 may be encoded by the polynucleotide sequence of SEQ ID NO: 38, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 18 may be encoded by the polynucleotide sequence of SEQ ID NO: 39, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 19 may be encoded by a polynucleotide sequence of SEQ ID NO: 40, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 20 may be encoded by a polynucleotide sequence of SEQ ID NO: 41, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 21 may be encoded by the polynucleotide sequence of SEQ ID NO: 42, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 22 may be encoded by the polynucleotide sequence of SEQ ID NO: 43, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 23 may be encoded by the polynucleotide sequence of SEQ ID NO: 44, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 24 may be encoded by the polynucleotide sequence of SEQ ID NO: 45, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 25 may be encoded by a polynucleotide sequence of SEQ ID NO: 46, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 26 may be encoded by a polynucleotide sequence of SEQ ID NO: 47, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 27 may be encoded by a polynucleotide sequence of SEQ ID NO: 48, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 28 may be encoded by a polynucleotide sequence of SEQ ID NO: 49, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 29 may be encoded by a polynucleotide sequence of SEQ ID NO: 50, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 30 may be encoded by the polynucleotide sequence of SEQ ID NO: 51, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 31 may be encoded by the polynucleotide sequence of SEQ ID NO: 52, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 32 may be encoded by the polynucleotide sequence of SEQ ID NO: 53, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 33 may be encoded by the polynucleotide sequence of SEQ ID NO: 54, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 34 may be encoded by the polynucleotide sequence of SEQ ID NO: 55, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 35 may be encoded by a polynucleotide sequence of SEQ ID NO: 56, but is not limited thereto.
  • the composition is characterized in that it penetrates the stratum corneum.
  • the skin bioactive molecule is characterized by binding to the N-terminus, C-terminus or sock end of the intracellular molecular transport peptide.
  • the skin bioactive molecule is characterized in that the binding of the amino acid of the intracellular molecule transfer peptide in the form arranged in reverse order.
  • the linkage is characterized by a peptide bond or a chemical bond.
  • the chemical bonds are disulfide bonds, diamine bonds, sulfide-amine bonds, carboxyl-amine bonds, ester bonds and covalent bonds. Characterized in that selected from the group consisting of.
  • the vitamin is vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D1, vitamin D2, vitamin D3 , Vitamin D4, vitamin D5, vitamin E and vitamin K is selected from the group consisting of.
  • the retinoid is characterized in that it is selected from the group consisting of retinol, retinol mutated and synthetic analogues, retinal, trans, 9-cis, and 13-cis retinoic acid and etretinate.
  • the fatty acid is characterized in that selected from the group consisting of lauric acid, stearic acid, palmitic acid, undecylenic acid, paritoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid and erucin acid .
  • the skin bioactive molecule is characterized in that it is selected from the group consisting of anti-wrinkle agent, whitening agent, antioxidant and moisturizer.
  • the anti-wrinkle agent is selected from the group consisting of a skin functioning growth factor peptide or protein, retinol, retinyl palmitate, adenosine and polyethoxylateddretinamide.
  • the whitening agent is characterized in that it is selected from the group consisting of a whitening peptide, niacinamide, turmeric extract, arbutin, ethylascomilletel, licorice extract, ascorbyl glucoside, and magnesium ascorbyl phosphate It is done.
  • the skin bioactive molecule is characterized in that it is selected from the group consisting of coumaric acid (coumaric acid) or acetyl pentapeptide and acetyl hexapeptide (Acetyl Hexapeptide).
  • the composition is characterized in that it is prepared in a formulation selected from the group consisting of emulsions, creams, essences, skins, liposomes, microcapsules, composite particles, shampoos and rinses.
  • the present invention also provides transdermal delivery of skin bioactive molecules into skin cells, wherein the intracellular molecular transport peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35 are delivered in combination with a skin bioactive molecule.
  • the intracellular molecular transport peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35 are delivered in combination with a skin bioactive molecule.
  • the peptide is characterized in that encoded from a polynucleotide selected from the group consisting of SEQ ID NO: 36 to 56.
  • the present invention provides a method for delivering the skin bioactive molecules into the skin cells comprising the step of binding and delivering the intracellular molecular transport peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to 35 with the skin bioactive molecules Provide a method.
  • the present invention is to deliver the skin bioactive molecules into the stratum corneum of the skin comprising the step of delivering the intracellular molecular transport peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to 35 in combination with the skin bioactive molecules.
  • the intracellular molecular transport peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to 35 in combination with the skin bioactive molecules.
  • composition for transdermal delivery of the present invention binds a peptide having excellent molecular transport ability in vivo to the skin bioactive molecules that are not easily permeable to the stratum corneum due to the size and physical properties of the skin stratum corneum, thereby allowing the skin bioactive molecules to be transferred to skin cells or skin Up to the stratum corneum.
  • the compositions of the present invention have the advantages of antioxidant efficacy, increased angiogenesis, reduced acne symptoms, decreased line secretion, decreased aging effects, reduced wrinkles, reduced melanogenesis, lessened skin inflammation or improved skin dryness. Not only can the compound be effectively delivered to the skin tissue depth, but also can be formulated in various forms, it can be usefully used as a functional cosmetic raw material.
  • 1 to 4 show the results of analyzing the secondary structure of the intracellular molecular transport peptide of the present invention using the PEP FOLD server program.
  • 5 is a flow cytometry analysis result for measuring the skin cell permeability of the intracellular molecular transport peptide of the present invention.
  • 6 to 8 are the results of observation with confocal microscopy to measure the skin cell permeability of the intracellular molecular transport peptide of the present invention:
  • MR-form improved MTD in the form A1-A2-MTD wherein A1 is methionine and A2 is arginine;
  • MH-form improved MTD in the form A1-A2-MTD, wherein A1 is methionine and A2 is histidine;
  • MK-form Enhanced MTD of the form wherein A1 is methionine and A2 is lysine in the formula A1-A2-MTD.
  • Figure 9 is a photograph of the intracellular molecular transmission peptide of the intracellular molecular transmission peptide in the EpiOral artificial tissue model visualized by confocal microscopy.
  • FIG. 10 is a confocal microscope picture of the permeability in the stratum corneum and skin tissue of intracellular molecular transport peptides in an EpiDerm artificial skin model.
  • Figure 13 is the result confirming the inhibitory effect of the intracellular melanin production of the coumalic acid derivative coupled to the intracellular molecular transport peptides.
  • 15 is a flow cytometry analysis for measuring skin cell permeability of intracellular molecular transport peptides and acetylhexapeptide binding derivatives.
  • Figure 16 shows the results of confirming the skin cell permeation position of intracellular molecular transport peptides and acetylhexapeptide binding derivatives by confocal microscopy.
  • the stratum corneum of the skin is keratinocyte (the major constituent cell of the skin) is a natural death and forms a dense structure in the outermost layer of the skin, inhibits the evaporation of moisture as well as the penetration of foreign substances, sweat and various lipid components Due to its acidic region near pH 5.
  • the molecular weight In order to penetrate the stratum corneum, the molecular weight must be less than 1,000 and possess lipophilic properties (Metha R. C. and Fitzpatrick R. E. Dermatol. Ther. 20: 350-359 (2007)).
  • Macromolecules such as proteins, peptides and nucleic acids are difficult to penetrate into cell membranes having a double lipid membrane structure due to their molecular weight.
  • stratum corneum which constitutes the skin barrier, they are known to have extremely low permeation efficiency of low molecular weight materials and lower permeation efficiency of high molecular weight materials.
  • Macromolecule Intracellular Transduction Technologiy can be used as a method for amplifying the efficiency of the small molecules and macromolecules through the plasma membrane of the cell.
  • a negative charge is applied to the hydrophobic domain by applying one or two hydrophilic (polar) amino acids having a positive charge to the hydrophobic macromolecular transfer domain (MTD) that has been invented (Korean Patent Publication No. 10-2009-0103957).
  • MTD macromolecular transfer domain
  • the improved MTD peptide is a peptide described by the following Formula 1,
  • a 1 is methionine (M, Met);
  • A2 is an amino acid selected from the group consisting of arginine (R, Arg), histidine (H, His) and lysine (K, Lys);
  • MTD characterized in that having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7;
  • the peptide is a peptide capable of mediating the delivery of a biologically active molecule into cells, and is a peptide that exhibits superior cell permeability compared to the MTD peptide described in Korean Patent Application Laid-Open No. 10-2009-0103957. My molecular transport peptide ".
  • the peptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35, but is not limited thereto.
  • the peptide is preferably a sequence satisfying the following conditions among quantified and comparatively comparable sequences. It is not limited to:
  • the instability index assessed using the Protparam program is evaluated to a value between 30 and 60;
  • amino acid sequence of SEQ ID NO: 1 may be encoded by the polynucleotide sequence of SEQ ID NO: 8, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 2 may be encoded by the polynucleotide sequence of SEQ ID NO: 9, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 3 may be encoded by the polynucleotide sequence of SEQ ID NO: 10, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 4 may be encoded by the polynucleotide sequence of SEQ ID NO: 11, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 5 may be encoded by the polynucleotide sequence of SEQ ID NO: 12, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 6 may be encoded by the polynucleotide sequence of SEQ ID NO: 13, but is not limited thereto.
  • amino acid sequence of SEQ ID NO: 7 may be encoded by the polynucleotide sequence of SEQ ID NO: 14, but is not limited thereto.
  • Synthesis of the peptides of the present invention and the synthesis of peptides containing cargo, which is a skin bioactive molecule, can be carried out, for example, using a device or using genetic engineering techniques.
  • topical application of compositions comprising them can provide a beneficial effect on the skin.
  • the beneficial effect is to reduce or prevent damage caused by sunlight, to provide antioxidant activity, to reduce the appearance of wrinkles, including fine wrinkles on the skin, to reduce gland secretion, the aging effect It includes, but is not limited to, reducing acne, treating acne, inducing angiogenesis in hair follicles, hair growth, conducive to skin moisturizing, and inhibiting melanin production to brighten skin color.
  • the active ingredient deliverable into the skin cells by the intracellular molecular transfer peptide may be a compound or a protein or fragments thereof, further providing additional benefits for the skin. They are non-toxic, non-allergic and non-irritating and therefore friendly with skin tissue.
  • suitable active cars may include, but are not limited to, vitamins and derivatives thereof, retinoids and fatty acids.
  • vitamin C ascorbic acid
  • vitamin E alpha-tocopherol
  • vitamin A retinoid
  • 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 other types of cellular damage due to fatty oxidation and UV exposure (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 to inhibit melanin formation and histamine secretion of 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 deleterious effects of phospholipids and free radicals on cell membranes (J.B Chazan et al. Free Radicals and Vitamin E. Cah. Nutr. Diet. 1987 22 (l): 66-76).
  • Retinoids block the mediators of inflammation in the skin and increase the production of procollagen, allowing for more production of type I and type III collagen.
  • the cargo compound of the present invention is vitamin A, vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin Dl, vitamin D2, vitamin D3, vitamin D4, Vitamins such as vitamin D5, vitamin E, and vitamin K.
  • Yet another cargo compound of the present invention is a retinoid.
  • Retinoids are retinol, natural and synthetic analogues of vitamin A (retinol), vitamin A aldehydes (retinal), all trans, 9-cis, and 13-cis retinoic acid, etretinate and vitamins described in several prior documents.
  • the cargo compounds of the invention comprise fatty acids.
  • Fatty acids are monocarboxylic acids with saturated or unsaturated fatty tails. As defined in the International Cosmetic Ingredient Dictionary and Handbook, 7th Ed. (1997) volume 2, page 1567 (the disclosure is incorporated herein by reference), Having at least about 7 carbon atoms.
  • 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 of the skin produced by the sebaceous gland and is used in skin care and cosmetic preparations as a moisturizer. It stabilizes the oil balance to keep the skin normal and healthy, softens the skin and reacts like an anti-keratinizing agent.
  • Esters of palmitic acid are used to provide silky silkiness to the skin and hair.
  • the palmitic acid acts as a carrier that can penetrate the pentapeptide into the skin, is widely used as a lubricant, and is used as an emulsifier, surfactant, and formula texturizer.
  • suitable fatty acids of 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.
  • the intracellular molecular transport peptide and the cargo compound, ie, the skin bioactive molecule, are bound by covalent bonds, and include, but are not limited to, ester, amide, ether and carbamide bonds.
  • Embodiments of the invention include, but are not limited to, coumalic acid, acetylpentapeptide or acetylhexapeptide and selected intracellular molecular transport peptides.
  • the compounds of the present invention can be used in many cosmetic or dermatological compositions intended for topical application. Since intracellular molecular transport peptides and cargo compounds can penetrate the skin and stimulate angiogenesis in hair follicles, these compositions can be used to prevent hair loss or to improve hair growth. If a compound is used that affects hair growth, it is applied to the scalp, eyebrows or eyelashes. In addition, when applied to the skin, the intracellular molecular transport peptides and cargo compounds may reduce skin texture to reduce the appearance of fine lines and wrinkles, to improve skin elasticity, and to reduce puffiness.
  • To soften to provide a silky texture to the skin, to provide a moisturizing effect, to provide lubricity, to provide a bright complexion through inhibition of melanin production, to reduce the aging effect, or to other cosmetics It can be used to provide benefits.
  • skin bioactive molecules are defined as “cosmetically or dermatologically acceptable molecules” and contact physiological tissue without excessive toxicity, incompatibility, instability, etc. And suitable compositions or compounds for use.
  • composition of the present invention may further comprise a pharmacologically or physiologically acceptable carrier, excipient, diluent.
  • compositions examples include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
  • the composition may further include conventional fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like.
  • compositions of the invention may be solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solids used for application to the skin and other tissues to which the compositions can be applied.
  • emulsions including microemulsions
  • suspensions creams, lotions, gels, powders, or other typical solids used for application to the skin and other tissues to which the compositions can be applied.
  • compositions may comprise additional antimicrobial, moisturizing and hydrating agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents ( may include gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyestuffs, colorants, powders, viscosity-controlling agents and water And optionally, anesthetics, anti-itch actives, botanical extracts, conditioning agents, darkening or lightening agents, glitters, wetting agents ( humectant, mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins, and phytomedicinal.
  • the compositions of the present invention are formulated with the aforementioned ingredients to be stable for long periods of time, which may be useful if continuous or long term use is intended.
  • the composition is used to treat, ameliorate or prevent skin aging.
  • Skin damage from aging can include fine and deep wrinkles, skin lines, crevices, bumps, large holes, scalyness, loss of skin elasticity, sagging, loss of skin firmness or tautness, and discoloration.
  • Hyperpigmented areas such as age spots and freckles, keratosis, abnormal differentiation, hyperkeratinization, elastic fibrosis, collagen breakdown, stratum corneum, dermis, epidermis, skin vascular system, And other histological changes in underlying tissues particularly in proximity to the skin.
  • skin aging is aging due to external factors such as chronological aging or continuous exposure to sunlight, the compositions of the present invention disclose methods for treating, ameliorating or preventing such skin aging and other types of skin damage.
  • the present inventors treated the skin cells at a constant concentration using a complex in which the FITC fluorescent substance was linked to the intracellular molecular transport peptide to quantitatively determine the cell permeability of the intracellular molecular transport peptide. Afterwards, fluorescence intensity was measured using flow cytometry, and as a result, several sequences were confirmed by intracellular molecular transport peptides that delivered FITC into cells more efficiently than known protein transduction domains (PTDs). The invasion effect of the intracellular molecular transport peptides in skin cells was demonstrated.
  • PTDs protein transduction domains
  • the inventors have performed cell permeability and intracellular localization through confocal microscope analysis using a complex linking FITC fluorescent material to intracellular molecular transport peptides. The results can be visually confirmed. As a result, as shown in the flow cytometry results, it was confirmed that exhibited high skin cell permeability in the improved intracellular molecular transport peptide than the existing MTD peptide. In addition, it was confirmed that the intracellular molecular transport peptide has a higher cell permeability than Tat, which is known to have a relatively excellent intracellular transport efficiency among known protein transduction domains (PTDs).
  • PTDs protein transduction domains
  • the peptide having a cargo having a skin physiological activity was confirmed to increase the physiological activity through skin permeation, thereby demonstrating that it can be used as a cosmetic raw material having a whitening or wrinkle improvement effect.
  • the inventors of the present invention among the 193 MTD peptides developed in Korean Patent Application Publication No. 110-2009-0103957), which have been developed by the present inventors, are quantified and transmitted in a sequence that satisfies the following conditions among relative comparable sequences. Subject MTD peptide sequences were selected for ability improvement.
  • sequences having a high possibility of inducing extracellular secretion are selected.
  • sequences of the macromolecular delivery domain a sequence satisfying a specific level of the aliphatic index, which is an element that determines the physicochemical properties of the macromolecular delivery domain, is selected.
  • sequences of the macromolecular delivery domain a sequence that satisfies a certain level of flexibility, which is a factor that determines the physicochemical properties of the macromolecular delivery domain, is selected.
  • sequences of the macromolecular delivery domain a sequence that satisfies a certain level of hydropathicity, which is a factor that determines the physicochemical properties of the macromolecular delivery domain, is selected.
  • a sequence that satisfies a specific level is selected for the instability index, which is a factor that determines the physicochemical properties of the macromolecular delivery domain.
  • sequences of the macromolecular delivery domain a sequence is selected in which polarity, an element that determines the physicochemical properties of the macromolecular delivery domain, satisfies a certain level.
  • representative sequences were determined based on the presence and location of proline, as described in step 1) above.
  • the amino acids alanine, valine, proline, leucine, and isoleucine that make up the sequence of the macromolecular delivery domain.
  • the shorter and smaller size of the proximal side affects the degree of freedom of secondary structure formation of the amino acid sequence, which is the macromolecular delivery domain. Contributes to cell membrane permeation.
  • MTDs macromolecular delivery domains known in the Republic of Korea Publication No.
  • 10-2009-0103957 Novel macromolecular delivery domains and methods and uses thereof, proline is present on the sequences constituting it and the Forty-nine macromolecular delivery domains were determined, whose positions were located in the middle of the amino acid sequence of the macromolecular delivery domain and were classified as having high degree of freedom of secondary structure formation of the amino acid sequence.
  • sequences with high possibility of extracellular secretion were selected from the macromolecular delivery domains selected in step 1).
  • Some water-soluble proteins have signals that can interact with receptors that mediate transport.
  • signal mediated transport a protein has one or more signal sequences that specify the target to which it is delivered. Therefore, under the assumption that the similarity with the extracellular secretion inducing sequence can improve the transmission ability, the possibility of inducing extracellular secretion was evaluated using the Signal P program. Probability between 10% and 90% is assessed for each domain, with sequences rated at least 60% of the macromolecular delivery domains determined in step 1) selected as improved subject sequences.
  • step 3 sequences satisfying a specific level of the aliphatic index, which is an element that determines the physicochemical properties of the macromolecular delivery domain, were selected.
  • Aliphatic index is a physical feature that determines the volume of the entire molecule, which is determined by the carbon chain of the side chain of amino acids, and is evaluated as a feature that modifies the structure of the cell membrane as the macromolecular delivery domain penetrates the cell membrane.
  • Aliphatic index is determined by the unique value of each amino acid sequence of the macromolecular delivery domain and the average of the entire sequence, and was determined using the Protparam program (see http://web.expasy.org/protparam/).
  • the Protparam program is a useful tool that quantifies the physical properties of proteins or peptides consisting of amino acids.
  • the aliphatic index was selected from 100 to 300 as the representative sequence for improving the transfer capacity.
  • step 4 among the macromolecular delivery domains selected in step 3), a sequence was selected in which flexibility, an element that determines the physicochemical properties of the macromolecular delivery domain, satisfies a certain level.
  • Flexibility is a physicochemical feature that represents the degree of freedom and degrees of freedom between the N- and C-terminal amino acids of the macromolecular delivery domain, and provides structural flexibility and is associated with affinity for cell membranes. Flexibility was evaluated according to the length of the amino acid sequence and the composition of the side chain sequence of the amino acid, and evaluated using the Protscale (Average flexibility) program (see http://web.expasy.org/protscale/).
  • the Protscale program was used as a tool to quantify the physical properties of proteins or peptides consisting of amino acids.
  • the evaluation result of flexibility using the Protscale (Average flexibility) program was 0.36 or higher. Selected sequences were selected as representative sequences for improving transmission capacity.
  • hydropathicity which is a factor determining the physicochemical properties of the macromolecular delivery domain, satisfies a certain level.
  • Hydropathicity is a physical property determined by the unique properties of amino acids and is considered to be a property that determines physical properties. It is known to cause severe entanglement at 3.0 or higher. Hydropathicity was also determined by the unique value of each of the amino acid sequences of the macromolecular delivery domain and the average of the entire sequence, and evaluated using the Protparam program. Therefore, among the hydropathicity results of 1.3 to 3.8, a sequence whose hydropathicity was evaluated to 3.0 or less in the macromolecular delivery domain selected in step 4) was determined as a representative sequence for improvement.
  • step 6 a sequence was determined in which the instability index of the macromolecular delivery domain satisfies a certain level.
  • Instability index is a property that indicates the stability of the amino acid sequence is determined by the sequence of amino acids on the sequence, the higher the value has instability. This is a factor that determines the physicochemical properties of the macromolecular delivery domain and is considered to be a characteristic that affects the intracellular stability of the domain and was evaluated using the Protparam program.
  • a sequence having an instability index of 30 to 60 within the macromolecular delivery domain determined in step 5) among the macromolecular delivery domains represented by instability index 0 to 130 was selected as a representative sequence for improvement.
  • a sequence was determined in which Polarity, an element that determines the physicochemical properties of the macromolecular delivery domain, satisfies a certain level.
  • Polarity is a measure of the affinity for water as judged by the length of the carbon chain and the presence or absence of hydroxyl groups among the components of amino acids. This, together with hydropathicity, determines the properties of the macromolecular transport domain and is thought to affect affinity to cell membranes.
  • Polarity is determined by the unique value of each of the amino acid sequences of the macromolecular delivery domain and the average of the entire sequence, and was evaluated using the Protscale (polarity) program. Accordingly, among the macromolecular transfer domains determined in step 6), sequences whose evaluation results were greater than 0.1 using the Protscale (polarity) program were selected as representative sequences for improvement.
  • JO-103 SEQ ID NO: 3
  • JO-103 SEQ ID NO: 3
  • JO-103 consists of the sequence of LALPVLLLA;
  • JO-103 determined as a target sequence for improving transmission capacity is a target domain that satisfies all seven steps.
  • the selected target MTD peptides were MTD JO-18 (SEQ ID NO: 1), MTD JO-067 (SEQ ID NO: 2), MTD JO-103 (SEQ ID NO: 3), MTD JO-159 (SEQ ID NO: 4), and MTD JO-173 (SEQ ID NO: 5).
  • the MTD 173A peptide (SEQ ID NO: 7), which is an intermediate peptide, was prepared.
  • the MTD 173A peptide thus prepared also satisfies the selection conditions of the above seven steps, and was included in the target MTD sequence for designing an improved MTD peptide.
  • intermediate peptide MTD 18m (SEQ ID NO: 6) was derived to improve the physical properties by substituting the amino acid with low hydropathicity for the JO-18 No. among the selected MTD. Satisfaction was included in the subject MTD sequence for the design of the improved MTD peptide.
  • a 1 is methionine (M, Met);
  • A2 is an amino acid selected from the group consisting of positively charged arginine (R, Arg), histidine (H, His) and lysine (K, Lys);
  • MTD is 7 amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 7 selected in Example 1.
  • the sequence of the improved MTD peptide designed through Equation 1 is the same as the amino acid sequence shown in SEQ ID NOs: 15 to 35, and the peptide was synthesized based on the designed amino acid sequence to confirm its cell permeability.
  • the improved MTD peptide designed in Example 2 was applied to intracellular molecular transport peptides fused to the skin bioactive molecules of the present invention, and for the synthesis thereof, C-term using a general Fmoc solid phase peptide synthesis (SPPS) method. Coupling one by one from (coupling).
  • SPPS general Fmoc solid phase peptide synthesis
  • the C-terminal first amino acid of the peptide was attached to the resin.
  • Resin that can be used is NH 2 -Lys (Dde) -2-chloro-Trityl resin, NH 2 -Met-2-chloro-Trityl resin, or NH 2 -Ser (tBu) -2-chloro-Trityl resin as needed. Appropriate resins were selected and used for peptide synthesis.
  • Fmoc was removed by reacting twice at room temperature for 5 minutes using 20% piperidine in DMF.
  • TFA trifluoroacetic acid
  • EDT 1,2-ethanedithiol
  • TIS triisopropylsilane.
  • the molecular weight was confirmed by a mass spectrometer (mass spectrometer) and lyophilized to obtain an intracellular molecule transfer peptide synthesis product.
  • intracellular molecular transport peptides were synthesized by the above synthesis method, and finally, lysine (K, Lysine) was added to proceed with peptide synthesis. FITC was then bound to the free amine residue of lysine.
  • the synthesized intracellular molecular transfer peptide-FITC peptide was separated from the resin and purified by HPLC, and then the molecular weight of the peptide was confirmed by mass spectrometer and lyophilized to obtain intracellular molecular transfer peptide fluorescent substance.
  • the synthesized intracellular molecular transport peptide phosphor was dissolved in DMSO so as to have a concentration of 1 mM in a light shielding state, and then aliquoted in a small amount in a 1.5 mL centrifuge container and stored frozen until just before use.
  • MTD-FITC intracellular molecular transport peptide phosphor
  • MTD-FITC peptide was applied to a flow cytometer (FACS Calibur, Beckton-Dickinson, San Diego CA, USA). For each sample, cells (1 ⁇ 10 4 ) were analyzed using CellQuest Pro cytometric analysis software, and each experiment was performed three or more times. Cell permeability of each of the intracellular molecular transport peptides applied to the present invention was quantitatively analyzed for cell permeation efficiency using the existing MTD-FITC as a control.
  • FIG. 5 shows the results of flow cytometry analysis.
  • the cell influx of each intracellular molecular transport peptide treated in the cell was measured using the CellQues Pro cytometric analysis software.
  • Cell influx efficiency was compared by relatively evaluating the change in fluorescence.
  • the intracellular molecular transport peptide shows the excellent cell inflow efficiency of the existing MTD peptide, and also shows the cell inflow efficiency higher by at least 140% to up to 400% than the known PTD.
  • Intracellular Molecular Transport Peptides and PTD (Tat) Proteins Known to Be Permeable to Cells were Treated at a Concentration of 3 ⁇ M in HaCaT cells, immortalized human keratinocytes, Cat No. 300493, CLS, Germany 37 After incubation for 1 hour at °C, it was visually observed using a confocal microscope (confocal microscopy, Nikon, Germany). The day before the experiment, HaCaT cells were incubated for 24 hours in a 12-well culture plate containing glass coverslips.
  • HaCaT cells were maintained in DMEM medium containing 10% Fetal Bovine Serum (FBS), and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 ⁇ g / mL streptomycin, Invitrogen, USA), and 5% CO Incubated at 37 ° C. under a humidified atmosphere of 2 .
  • HaCaT cells were treated with scrambled peptide, MTD-FITC and PTD-FITC peptide for 1 hour at a concentration of 3 ⁇ M.
  • One hour after the treatment cells were fixed for 20 minutes with 4% paraformaldehyde solution (paraformaldehyde, PFA) at room temperature for observation.
  • the cells were washed three times with PBS for direct detection of internalized FITC-peptide and counterstained with 5 mM concentration of DAPI (4 ', 6-diamidino-2-phenylindole), a nuclear fluorescence staining solution. Was performed. After 10 minutes of DAPI staining, the cells were washed three times with PBS and 20 ⁇ l of mounting media were dropped onto the slides and observed to preserve the fluorescent label of the protein. Cells treated with the intracellular molecular transport peptides were transferred to the nucleus through DAPI staining for easy identification of intracellular delivery sites of FITC-peptides and confirmed cell permeability.
  • the confocal microscope is a Normaski filter.
  • the prototype of the cells was observed using FITC fluorescence and DAPI fluorescence with a filter suitable for each fluorochrome. As shown in Figures 6 to 8, it was confirmed that all of the intracellular molecular transport peptides to be applied to the present invention was clearly transmitted into the cell compared to the PTD (Tat) attached to the outside of the cell membrane, and improved intracellular molecular transport It was demonstrated that the peptide has good cell permeability for skin cells.
  • MTD M18m, M173
  • M1018m, M1173A intracellular molecular transport peptide
  • FITC FITC
  • EpiOral skin model MatTek, MA, USA
  • confocal microscopy confocal microscopy, Carl Zeisse, Germany
  • EpiOral artificial tissue model was incubated at 37 ° C. for 15 hours under a humidified atmosphere of 5% CO 2 in a 12-well plate containing 0.5 mL of the test medium provided by MatTek.
  • an epiderm skin model (MatTek, MA, USA) was used to select and process intracellular molecular transport peptides (M1067) and Tat peptides conjugated to FITC. confocal microscopy, Carl Zeiss, Germany).
  • EpiDerm skin models were incubated at 37 ° C. for 15 hours under a humidified atmosphere of 5% CO 2 in a 12-well plate containing 0.5 ml of the test medium provided by MatTek. The next day it was exchanged with fresh medium and 40 ⁇ l of 100 ⁇ M peptide was treated over EpiDerm skin and incubated at 37 ° C. under a humidified atmosphere of 5% CO 2 for 24 hours.
  • cryosections (6 ⁇ m) were prepared using a Microm HM520 cryostat, Thermo. This was put on a glass slide. The prepared slides were stained with PBS buffer for 10 minutes and stained with cell nuclei in tissues by exposure to 0.5 mM DAPI solution for 5 minutes. The stained tissues were washed with PBS buffer three times for 10 minutes, and then fixed with a mounting medium, and observed with a confocal microscope. The results are shown in FIG. 10.
  • Sections were obtained to produce slides.
  • the prepared slides were washed with PBS buffer for 10 minutes, and then exposed to 0.5 mM DAPI solution for 5 minutes to stain the cell nuclei in tissues.
  • the stained tissues were washed again with PBS buffer three times for 10 minutes, and then fixed by using a mounting medium, followed by confocal microscopy, shown in FIG. 11.
  • a 20% piperidine / N-methylpyrrolidone solution was added to the intracellular molecular transfer peptides M1067 and M2067 synthesized in Example 4 coupled to the N-terminal amino acid to remove the Fmoc group. After washing with N-methylpyrrolidone and dichloromethane (coumaric acid, Sigma, USA) commercially available was coupled. After coupling, the mixture was washed several times with N-methylpyrrolidone and dichloromethane and dried with nitrogen gas.
  • N-methylpyrrolidone and dichloromethane coumaric acid, Sigma, USA
  • Trifluoroacetic acid phenol: thioanisole: water: trifluoroacetic acid (phenol: thioanisole: water: triisopropylsilane) 90: 2.5: 2.5: 2.5: 2.5 (v / v) solution of 2 to The reaction was carried out for 3 hours to remove the peptide protecting group, and the peptide-bound coumalic acid was separated from the resin, and the peptide was precipitated with diethyl ether. 10% Pd / C was added to methanol to remove the benzyl group protecting the alcohol group bonded to carbon 9 of the coumalic acid, stirred at room temperature for about 1 hour under hydrogen, and then Pd using Celite. / C was removed and the filtrate was concentrated under reduced pressure.
  • the intracellular molecular transport peptide-coumaric acid derivative thus obtained was purified with acetonitrile containing 0.1% trifluoroacetic acid as a gradient, followed by a purified reverse phase high performance liquid chromatography column (Zobax, C8 300 ⁇ , 21.1). mm ⁇ 25 cm) to obtain intracellular molecular transport peptide-coumaric acid derivatives in which coumaric acid is bound to M1067 or M2067 intracellular molecular transport peptides having the amino acid sequence of SEQ ID NO: 16 or 23, as shown in Equation 2 below. Synthesized.
  • the inhibitory effect of tyrosinase activity was measured using the intracellular molecule transfer peptide-coumaric acid, a compound synthesized in Example 10-1.
  • Tyrosinase was isolated and purified from mushrooms and purchased from Sigma, USA.
  • Tyrosine, a substrate was dissolved in 0.05 M potassium phosphate buffer (pH6.8) and used as a 0.3 mg / mL solution.
  • the compound was dissolved in distilled water at a concentration of 100 mg / mL, and the coumalic acid was dissolved in ethyl alcohol at a concentration of 100 mg / mL, and diluted again to an appropriate concentration.
  • A is the absorbance at 450 nm of no inhibitor added
  • B is the absorbance at 450 nm of the inhibitor added.
  • Intracellular melanocyte production was compared using the intracellular molecular transfer peptide-coumaric acid compound synthesized in Example 10-1.
  • Mouse-derived melanoma cells murine melanoma, B16F1, Korea Cell Line Bank KCLB No.80007
  • DMEM Dubelcco's modified eagle medium
  • FBS fetal bovine serum
  • the medium is removed, the sample is replaced with medium diluted to an appropriate concentration (1, 10, 100 ⁇ g / mL), and then incubated for 3 days with changing medium every day under 5% CO 2 , 37 ° C.
  • the day after the 3 day culture the cells from which the medium was removed are washed with PBS (phosphated buffer saline), and the cells are recovered by treating with trypsin.
  • the recovered cells were centrifuged at 10,000 rpm for 10 minutes, and then the supernatant was removed to obtain cell pellets.
  • the cell pellets were dried at 60 ° C., and 200 ⁇ L of 1M sodium hydroxide solution containing 10% DMSO was dissolved at 60 ° C ..
  • the intracellular molecular transport peptide and intracellular molecular transport peptide-coumaric acid were identified as substances in the hypoallergenic category in terms of human skin primary stimulation. As a result, it was proved that the intracellular molecular transport peptides can be safely used in the human body while maintaining the functions of the skin bioactive molecules through clinical trials of specialized test institutes. Giving.
  • compositions containing intracellular molecular transfer peptide-coumaric acid or intracellular molecular transfer peptide were formulated as follows with the composition of Table 3 above.
  • Comparative Example 1 1 to 6 in the water melting tank and completely dissolved while warming up to 70 °C and put into the emulsion tank. 7 to 11 is added to the oil-dissolving tank and dissolved completely while warming up to 70 ° C. After the contents were cooled to 40 ° C., raw materials 12 to 14 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition containing an intracellular molecular transfer peptide.
  • Comparative Example 2 1 to 6 in the water melting tank and completely dissolved while warming up to 70 °C and put into the emulsion tank. 7 to 11 and 16 are added to the oil-dissolving tank, dissolved completely while warming up to 70 ° C, and then mixed in an emulsifying tank. After the contents were cooled to 40 ° C., raw materials 12 and 13 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition containing kumaric acid.
  • Trifluoroacetic acid phenol: phenol (phenol): thioanisole (thioanisole): water (water): triisopropylsilane (triisopropylsilane) 90: 2.5: 2.5: 2.5: 2.5 (v / v)
  • the mixture was reacted for 2 hours to 3 hours to remove the peptide protecting group, the peptide was separated from the resin, and the peptide was precipitated with diethyl ether.
  • the MTD peptide-acetylpentapeptide derivatives thus obtained were purified by reverse phase high performance liquid chromatography column (Zobax, C8 300 ⁇ , 21.1 mm X) with acetonitrile containing 0.1% trifluoroacetic acid as a gradient. 25 cm) was used to synthesize an acetyl pentapeptide derivative in which an acetyl pentapeptide was bound to an M1067 intracellular molecular transport peptide having the amino acid sequence of SEQ ID
  • the inhibitory effect of collagenase activity in human normal skin fibroblasts was tested using the intracellular molecule transfer peptide-acetylpentapeptide, a compound synthesized in Example 11-1. Intracellular collagenase activity inhibition was measured by the method of Baeer EA et al, J Invest Dermatol, 82 (2): 162-9, 1983, and 100% of the sample was not added. .
  • Detailed experimental method is as follows. Human normal dermal fibroblasts were dispensed into 6-well plates for cell culture and inoculated with a certain number of cells, treated with intracellular molecule transfer peptide-acetylpentapeptides at a constant concentration and incubated for 24 hours, followed by 100 ⁇ l of culture solution.
  • the amount of collagenase in the culture was measured using the method described in Matrix metalloproteinase-1 (MMP-1) human biotrak ELISA system (GEHealthcare, USA).
  • MMP-1 Matrix metalloproteinase-1
  • GEHealthcare USA
  • 100 ⁇ l of the quantitative buffer solution 2 was placed in a 96-well plate, and 100 ⁇ l of the culture solution and the standard solution diluted to 1/10 were added thereto, and the cells were incubated at room temperature for 2 hours. Remove the culture medium from the well plate, wash three times with 400 ⁇ l of wash buffer, add 100 ⁇ l of antibody solution, react at room temperature for 2 hours, and wash three times with 400 ⁇ l of wash buffer.
  • A is the absorbance at 450 nm of no sample added
  • B is the absorbance at 450 nm of the sample added.
  • the intracellular molecular transport peptide and the intracellular molecular transport peptide-acetylpentapeptide were shown to be substances in the hypoallergenic category in terms of human skin primary stimulation.
  • clinical trials of specialized test institutes demonstrated that intracellular molecular transport peptides can be safely used in the human body while maintaining the function of skin bioactive molecules. It can be said that the results indicate that the value of use is remarkably superior.
  • Comparative Example 1 1 to 5 in the water melting tank and completely dissolved while warming up to 70 °C and put into the emulsion tank. 6 to 10 is added to the oil-dissolving tank, dissolved completely while warming up to 70 ° C, and then mixed in an emulsifying tank. After the contents were cooled to 40 ° C., raw materials 11 to 13 were added to an emulsifying tank, mixed, and the contents were cooled to room temperature to prepare a composition containing intracellular molecular transfer peptides.
  • Comparative Example 2 1 to 5 in the water melting tank and completely dissolved while warming up to 70 °C and put into the emulsion tank. 6 to 10 is added to the oil-dissolving tank, dissolved completely while warming up to 70 ° C, and then mixed in an emulsifying tank. After the contents were cooled to 40 ° C., raw materials 11, 12, and 15 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition containing acetylpentapeptide.
  • Percutaneous absorption experiments were performed in accordance with published guidelines to confirm penetration of the material obtained in the present invention into skin tissue (commonly referred to as transdermal absorption) (Test Guideline 428: Skin absroption: in vitro Method, OECD, Paris, (2004), In Vitro Skin Absorption Test Guidelines, Korea Food and Drug Administration (2010)).
  • Percutaneous absorption experiments were used by thawing frozen stored body skin (cadaver skin, Cat No. SK11122, Hans Biomed) in PBS warmed to 32 °C.
  • the thawed cadaveric skin has the epidermis upward (in the direction of the donor) between the donor and the receptor of a vertical diffusion cell (Logan FDC-6, Logan instrument Corp. Somerset, NJ, USA).
  • the receptor was then filled with PBS solution (phosphate-buffered saline, pH 7.4, 32 ° C.) and left for 1 hour to allow the cadaveric skin to equilibrate with the PBS solution. Thereafter, 1 mg of 1% DMSO was added to the intracellular molecular transfer peptide, intracellular molecular transfer peptide-coumaric acid, and intracellular molecular transfer peptide-acetylpentapeptipeptides prepared in Examples 4, 10-1, and 11-1, respectively.
  • PBS solution phosphate-buffered saline, pH 7.4, 32 ° C.
  • the solution was sufficiently dissolved in 1 mL of the contained PBS solution and applied to the epidermis (1.7 cm 3 coated area), and the donor was sealed with parafilm to prevent the sample from evaporating. After 24 hours, 0.2 mL of sample was taken from the receptor and mass spectrometer was used to determine the amount of intracellular molecular transport peptide, intracellular molecular transport peptide-coumaric acid, and intracellular molecular transport peptide-acetylpentapeptipeptide. Analyze and quantify the molecular weight as shown in Table 7 below.
  • the intracellular molecular transport peptide alone had a relatively high skin permeability of 11.8%.
  • the intracellular molecular transport peptide combined with a skin physiologically active substance, it was able to penetrate the stratum corneum of its own skin.
  • the intracellular molecular transport peptide-bound coumalic acid was confirmed to have doubled the permeation rate.
  • the skin permeability increased by 28 times when binding to the intracellular molecular transport peptide.
  • acetyl hexapeptide acetyl-Glu Glu Met Gln Arg Arg
  • peptides were synthesized by adding lysine (K, lysine) and then free of lysine.
  • FITC was bound to the amine residue.
  • the synthesized peptide-FITC was removed by adding 20% piperidine / N-methylpyrrolidone solution to remove the Fmoc group and washed several times with N-methylpyrrolidone and dichloromethane. Dry with nitrogen gas.
  • Trifluoroacetic acid phenol: phenol (phenol): thioanisole (thioanisole): water (water): triisopropylsilane (triisopropylsilane) 90: 2.5: 2.5: 2.5: 2.5: 2.5 (v / v)
  • the mixture was reacted for 2 hours to 3 hours to remove the peptide protecting group, the peptide was separated from the resin, and the peptide was precipitated with diethyl ether.
  • the intracellular molecular transport peptide-acetylhexapeptide fluorescent derivative thus obtained was purified with acetonitrile containing 0.1% trifluoroacetic acid as a gradient, followed by purified reverse phase high performance liquid chromatography column (Zobax, C8 300 ⁇ ). , 21.1 mm X 25 cm) to the intracellular molecular transport peptide having an amino acid sequence of SEQ ID NO: 16 or 48 as shown in the following formula 4 acetyl hexapeptide and FITC combined intracellular molecular transport peptide-acetylhexa Peptide fluorescent derivatives were synthesized.
  • the synthesized peptide-FITC was dissolved in DMSO so as to have a concentration of 1 mM in a light-shielded state, and then aliquoted in small amounts in a 1.5 ml centrifuge container and stored frozen until just before use.
  • intracellular molecule transfer peptide-acetylhexapeptide phosphor (MTD-AH-FTIC) at a concentration of 3 ⁇ M was applied to human keratinocyte cells (Human Keratinocyte cell line, HaCaT cell, Order No. 300493, CLS cell line). service, Germany) and incubated for 1 hour.
  • human keratinocyte cells Human Keratinocyte cell line, HaCaT cell, Order No. 300493, CLS cell line). service, Germany
  • the HaCaT cells were maintained in DMED (Dubelcco's modified eagle medium) medium containing 10% Fetal bovine serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 ⁇ g / mL streptomycin, invitrogen) , Incubated at 37 ° C.
  • DMED Dubelcco's modified eagle medium
  • FBS Fetal bovine serum
  • penicillin / streptomycin 10,000 units penicillin and 10,000 ⁇
  • the prepared MTD-AH-FITC peptide was applied to a flow cytometer (FACS Calibur, Beckton-Dickinson, San Diego CA, USA), and for each sample, cells (1 ⁇ 10 4 ) were analyzed for CellQuest Pro cytometric analysis (CellQues Pro cytometric). analysis) The software was analyzed and each experiment was performed three or more times.
  • Cell permeability of each intracellular molecule transfer peptide applied to the present invention was quantitatively analyzed for cell permeability efficiency using acetylhexapeptide itself, a skin bioactive substance as a control.
  • the results were compared with the fluorescence change of each experimental group by the geometric mean in the CellQues Pro cytometric analysis software as shown in FIG. 15 to show the cellular influx of the intracellular molecular transport peptides.
  • the cell inflow efficiency of the acetylhexapeptide to which the intracellular molecular transport peptide is bound shows a high cell inflow efficiency from 200% to up to 400% compared to the efficiency of the acetylpexapeptide itself.
  • the cells were treated at 3 ⁇ M concentrations in HaCaT cells, immortalized human keratinocytes, Cat No. 300493, CLS, Germany. After incubation for 1 hour at 37 °C, it was visually observed using a confocal microscope (confocal microscopy, Nikon, Germany). The day before the experiment, HaCaT cells were incubated for 24 hours in a 12-well culture plate containing glass coverslips.
  • HaCaT cells were maintained in DMEM medium containing 10% Fetal Bovine Serum (FBS), and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 ⁇ g / mL streptomycin, Invitrogen, USA), and 5% CO Incubated at 37 ° C. under a humidified atmosphere of 2 .
  • HaCaT cells were treated with scrambled peptides, MTD-FITC and PTD-FITC peptides for 1 hour at a concentration of 5 ⁇ M.
  • the cells were fixed for 20 minutes with 4% paraformaldehyde solution (paraformaldehyde, PFA) at room temperature for observation, and washed three times with PBS, followed by DAPI (4 ', 6-diamidino-) at 5 mM concentration. Counterstain was performed with 2-phenylindole). After 10 minutes of DAPI staining, it was washed three times again with PBS, and 20 ⁇ l of mounting medium was dropped and observed on the slide to preserve the fluorescent label of the protein.
  • paraformaldehyde paraformaldehyde

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  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cosmetics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une composition d'administration transdermique contenant des molécules bioactives de peau, dans laquelle des peptides d'administration intracellulaire de molécules sont fusionnés. Plus particulièrement, la composition est une de celles dans laquelle des peptides possédant une capacité supérieure d'administration moléculaire in vivo sont fusionnés à des molécules bioactives de peau qui ne peuvent pas traverser facilement la couche cornée de l'épiderme de la peau en raison de leur taille moléculaire et de leurs propriétés physiques. La composition peut présenter une excellente perméabilité cutanée in vitro et in vivo, et elle peut donc être utilisée de manière appropriée pour administrer dans des cellules de la peau des composés possédant divers avantages et être préparée sous diverses formes. De ce fait, la composition peut être utilisée de manière appropriée en tant que matière pour des cosmétiques fonctionnels.
PCT/KR2012/009998 2011-11-23 2012-11-23 Système d'administration transdermique de molécules bioactives de peau au moyen de peptides d'administration intracellulaire de molécules WO2013077681A1 (fr)

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WO2022090413A1 (fr) * 2020-10-28 2022-05-05 Follicum Ab Peptides destinés à être utilisés dans la pigmentation de la peau et des cheveux
CN114432176A (zh) * 2021-12-28 2022-05-06 云南云科特色植物提取实验室有限公司 一种靶向肽修饰的美容肽组合物

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KR101746787B1 (ko) 2015-11-12 2017-06-13 주식회사 펩트론 미백, 피부 탄력, 주름 개선 및 상처치유 활성을 갖는 다기능성 피부투과 펩타이드
KR101944388B1 (ko) 2016-09-09 2019-04-17 한양대학교 산학협력단 피부 투과성 펩티드 및 그 이용방법
US20200165312A1 (en) * 2017-06-28 2020-05-28 Lg Household & Health Care Ltd. Cosmetic composition for improving skin conditions comprising fusion protein including skin penetration enhancing peptide
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KR102459219B1 (ko) * 2022-03-31 2022-10-26 주식회사 셀아이콘랩 신규한 피부투과성 펩타이드, 이를 포함하는 피부 및 점막 조성물과 이의 용도
KR102598095B1 (ko) * 2022-08-31 2023-11-07 주식회사 레메디 피부 미백 활성을 가지는 펩타이드를 포함하는 조성물

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022090413A1 (fr) * 2020-10-28 2022-05-05 Follicum Ab Peptides destinés à être utilisés dans la pigmentation de la peau et des cheveux
CN114432176A (zh) * 2021-12-28 2022-05-06 云南云科特色植物提取实验室有限公司 一种靶向肽修饰的美容肽组合物

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KR20130070607A (ko) 2013-06-27

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