WO2022092513A1 - Composition destinée à la prévention ou à l'atténuation du vieillissement ou des rides de la peau - Google Patents

Composition destinée à la prévention ou à l'atténuation du vieillissement ou des rides de la peau Download PDF

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WO2022092513A1
WO2022092513A1 PCT/KR2021/011278 KR2021011278W WO2022092513A1 WO 2022092513 A1 WO2022092513 A1 WO 2022092513A1 KR 2021011278 W KR2021011278 W KR 2021011278W WO 2022092513 A1 WO2022092513 A1 WO 2022092513A1
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present
polypeptide
skin
expression
composition
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PCT/KR2021/011278
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Korean (ko)
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박순형
한은수
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주식회사 에이엠메딕스
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention relates to a novel polypeptide fragment and a composition for preventing or improving skin aging or wrinkles comprising the same.
  • the skin is a membrane that covers the outside of the body, and at the same time protects the body from various environmental factors and is responsible for the aesthetic function reflected from the outside.
  • This skin aging process can be classified into intrinsic aging and extrinsic aging. Intrinsic aging is a natural aging process that occurs naturally with aging, and it is difficult to artificially control it because it is mainly influenced by genetic factors, whereas in the case of exogenous aging, it is influenced by environmental factors such as ultraviolet rays, so it is artificially controlled.
  • the present inventors made intensive research efforts to develop a formulation for wrinkle generation and improvement.
  • a novel 7mer short polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention was developed, and this polypeptide has excellent inhibitory effect on MMP-1 without showing cytotoxicity, and By finding out that it promotes the production, the present invention has been completed.
  • an object of the present invention is to provide a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • Another object of the present invention is to provide a cosmetic composition for preventing or improving skin aging or wrinkles, or an external composition comprising the polypeptide as an active ingredient.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the polypeptide, a recombinant vector comprising the nucleic acid molecule, and a host cell comprising the recombinant vector.
  • the present invention provides a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
  • the amino acid sequence of SEQ ID NO: 1 of the present invention is "KAIGELD".
  • the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention has no cytotoxicity, inhibits the increase in the expression of collagenase MMP-1 by UVB ultraviolet irradiation, and type 1 collagen increase the expression of Accordingly, the polypeptide can be usefully used for preventing or improving skin aging or wrinkles.
  • the aging includes photoaging.
  • Photoaging reduces the synthesis of collagen I and collagen III, the main components of the dermis, by inhibiting TGF- ⁇ and TGF- ⁇ by activating AP-1 and by activating AP-1.
  • Activation of -1 and NF- ⁇ proceeds by activating MMPs, particularly MMP-1, MMP-3, and MMP-9, to promote the breakdown of connective tissue in the dermis.
  • MMPs particularly MMP-1, MMP-3, and MMP-9
  • substances including amorphous elastin are deposited on the epidermis and dermis connecting layer, and in the dermal layer, the fibrous structure of collagen and elastin is deformed, and solar elastic fibrosis (elastosis) occurs due to the increase of elastic fibers.
  • Wrinkles of the skin are caused by activation of MMPs and degradation of collagen. Skin wrinkles are mainly caused by aging and UV rays. Skin exposure due to aging and UV light activates mitogen-activated protein kinase (MAPK).
  • MAPK mitogen-activated protein kinase
  • the factor most affected by MAPK is AP-1.
  • AP-1 is a transcription factor composed of Jun and Fos family proteins, and its transcriptional activity is the highest when it exists as a heterodimer of c-Jun and c-Fos. When human skin is not exposed to UV light, it exists as a dimer of c-Fos and JunD.
  • MMP is an endoprotease that requires zinc ions and is an enzyme that breaks down extracellular matrix proteins.
  • MMP-1 is known as collagenase 1, and uses type I and III collagen as substrates.
  • MMP-3 also called stromelysin 1, decomposes type IV collagen in the basement membrane and activates proMMP-1, a zymogen.
  • MMP-9 is gelatinase B, which hydrolyzes the products degraded by collagenase to a smaller extent. UV light breaks down MMP-1 into fibillar collagen (types I and III), decomposing collagen, and subsequently increasing MMP-3 and MMP-9.
  • collagen is also called collagen fiber and occupies 85-90% of the dermis.
  • the elasticity or moisture of the skin is maintained by the moisture present in the stratum corneum of the epidermis, which is the outermost layer of the skin, and the collagen present in the dermis.
  • Most of the skin collagen is type-1 collagen.
  • the main functions are the mechanical firmness of the skin, the resistance of connective tissue and the binding force of tissues, the support of cell adhesion, and the induction of cell division and differentiation.
  • Collagen exists in high concentrations in skin, bone, ligaments, cartilage and teeth, etc., and is not affected by proteolytic enzymes such as trypsin, but is degraded by collagenase.
  • polypeptide refers to a linear molecule formed by bonding amino acid residues to each other by peptide bonds.
  • Polypeptides of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques; Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al. , Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid phase synthesis technology (US Patent No. 5,516,891).
  • the polypeptide of the present invention may select a portion of the amino acid sequence and induce modifications at the N-terminus or C-terminus to increase its activity. Through this modification, the polypeptide of the present invention can have a high half-life with increased half-life upon in vivo administration.
  • the C-terminus of the polypeptide of the present invention may be modified with a hydroxyl group (-OH), an amino group (-NH 2 ), an azide group (-NHNH 2 ), etc., of the polypeptide
  • a protecting group selected from the group consisting of an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) may be bonded to the N-terminus.
  • Stability refers to storage stability (eg, room temperature storage stability) as well as in vivo stability.
  • the above-mentioned protecting group functions to protect the polypeptide of the present invention from attack by proteolytic enzymes in vivo.
  • the present invention provides a cosmetic composition for preventing or improving skin aging or wrinkles, comprising the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
  • the present invention provides an external composition for preventing or improving skin aging or wrinkles, comprising the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
  • the cosmetic composition, and the composition for topical application are selected in relation to the intended route of administration and standard pharmaceutical or cosmetic application in addition to the polypeptide comprising the amino acid sequence of SEQ ID NO: 1, at least one pharmaceutically and/or cosmetically acceptable excipient, carrier, or diluent.
  • the polypeptide comprising the amino acid sequence of SEQ ID NO: 1, at least one pharmaceutically and/or cosmetically acceptable excipient, carrier, or diluent.
  • polypeptides of the invention may be lyophilized for storage and reconstituted with an appropriate carrier prior to use.
  • pharmaceutically acceptable means that the preparation is sterile and pyrogen-free.
  • Suitable pharmaceutical carriers are well known in the pharmaceutical art.
  • the carrier(s) must be “acceptable” in the sense of not adversely affecting the efficacy of the active ingredient of the present invention and not harmful to its recipient.
  • the carrier will be sterile, pyrogen-free water or saline; However, other acceptable carriers may be used.
  • pharmaceutically acceptable carrier and “pharmaceutically acceptable excipient” are merely intended to act as carriers, i.e., not intended to have their own biological activity. compound(s) used to form part of the formulation.
  • Pharmaceutically acceptable carriers or excipients are generally safe and nontoxic.
  • a pharmaceutically acceptable carrier and/or excipient as used herein includes both one or more carriers and/or excipients.
  • cosmetically acceptable is used to denote an agent suitable for use as a cosmetic.
  • Suitable cosmetic carriers are well known in the art, as are commonly used in shampoos, lotions, creams, sprays and other such products.
  • An excipient may be one or more carbohydrates, polymers, lipids and minerals.
  • carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition to facilitate lyophilization, for example.
  • polymers include starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenan, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulfonates, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degrees of hydrolysis, and polyvinylpyrrolidone of all different molecular weights, which are for example , to control viscosity, to achieve adhesion, or to protect lipids from chemical and proteolytic degradation.
  • lipids are fatty acids, phospholipids, phosphoric acid, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids of all different acyl chain lengths and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are It is added to the composition for reasons similar to those for polymers.
  • minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain advantages such as reduced liquid build-up or advantageous pigment properties.
  • diluent is intended to mean an aqueous or non-aqueous solution with the purpose of diluting a peptide in pharmaceutical preparation.
  • the diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or an oil (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).
  • the diluent may also function as a buffer.
  • buffer is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilizing the pH.
  • buffers include Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate , AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolamic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES am.
  • the cosmetic composition or composition for topical application of the present invention may also be in the form of a polymer gel or microneedle made of a polymer, wherein the polymer is starch, cellulose ether, cellulose carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonate, and the like.
  • the polypeptide of the present invention may be formulated at various concentrations depending on the efficacy or toxicity of the active ingredient used.
  • the composition comprises 1 nM to 1 M, for example 0.1 ⁇ M (micromole) to 1 mM, 1 ⁇ M to 100 ⁇ M, 5 ⁇ M to 50 ⁇ M, 10 ⁇ M to 50 ⁇ M, 20 ⁇ M to 40 ⁇ M and optionally and the polypeptide at a concentration of about 30 ⁇ M.
  • the composition may comprise a lower concentration of the polypeptide, for example, between 0.0025 ⁇ M and 1 ⁇ M.
  • compositions of the present invention may be administered by a variety of routes, for example, topical, subcutaneous, parenteral or oral administration.
  • the composition of the present invention is suitable for topical or intracutaneous administration.
  • the compositions of the present invention may be administered topically to the skin (eg, the scalp).
  • the composition may be provided in the form of an ointment containing the active polypeptide suspended or dissolved in a mixture having, for example, one or more of: mineral oil, liquid petrolatum, white petrolatum, propylene glycols, polyoxyethylene polyoxypropylene compounds, emulsifying waxes and water.
  • the polypeptide may be formulated as a suitable lotion or cream, spray, gel, emulsion, paste, soap, powder, or combination thereof, etc., suspended or dissolved, for example, in a mixture of one or more of the following: Mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • compositions for topical administration may include a penetration enhancer (eg, Osborne & Henke, 1997, Pharmaceutical Technology, November: 58-82 and the disclosure of which is incorporated herein by reference).
  • a penetration enhancer eg, Osborne & Henke, 1997, Pharmaceutical Technology, November: 58-82 and the disclosure of which is incorporated herein by reference.
  • compositions of the present invention may be administered parenterally, for example intradermally.
  • Such compositions are best used in the form of a sterile aqueous solution which may contain sufficient salt or glucose to make the solution isotonic with other substances, for example, blood.
  • the aqueous solution should be suitably buffered (preferably to pH 3-9), if necessary.
  • the preparation of suitable parenteral preparations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • a composition comprising the polypeptide of the present invention is administered to a subject in an effective amount.
  • 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective' refers to an effect of preventing or improving (reducing) skin aging and wrinkles.
  • amount to provide is a predetermined amount of active substance calculated to produce the desired prophylactic or ameliorating (therapeutic) effect.
  • the amount of an active ingredient may vary depending on its specific activity. Appropriate dosages may contain a predetermined amount of active substance calculated to produce the desired prophylactic or ameliorating (therapeutic) effect in association with the required diluent.
  • a prophylactically or ameliorating (therapeutically) effective amount of an active ingredient is provided.
  • a prophylactically or ameliorated (therapeutically) effective amount can be determined by a person skilled in the art of ordinary medical or veterinary medicine based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art. .
  • the present invention provides a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 described above.
  • nucleic acid molecule has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).
  • nucleotide sequence encoding the polypeptide of the present invention suffices to be a nucleotide sequence encoding an amino acid sequence constituting the polypeptide, and it is apparent to those skilled in the art that it is not limited to any specific nucleotide sequence. This is because, even if a nucleotide sequence mutation occurs, the protein sequence does not change when the mutated nucleotide sequence is expressed as a protein in some cases. This is called codon degeneracy.
  • the nucleotide sequence is a functionally equivalent codon or codon encoding the same amino acid (eg, due to codon degeneracy, there are six codons for arginine or serine), or a codon encoding a biologically equivalent amino acid It contains a nucleotide sequence comprising a.
  • the nucleic acid molecule of the present invention encoding the polypeptide is construed to include a nucleotide sequence exhibiting substantial identity to the nucleotide sequence described above.
  • the substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. , more preferably at least 90% homology, and most preferably at least 95%, 97%, 98%, or 99% homology.
  • the nucleic acid molecule encoding the polypeptide of the present invention includes a sequence exhibiting substantial identity to the sequence shown in the sequence listing.
  • the substantial identity is at least 61% when the sequence of the present invention and any other sequence are aligned to the maximum correspondence, and the aligned sequence is analyzed using an algorithm commonly used in the art.
  • Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) ; Needleman and Wunsch, J. Mol.
  • BLAST can be accessed through the BLAST page of the ncbi website. A method for comparing sequence homology using this program can be found on the BLAST help page of the ncbi website.
  • the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the above-described polypeptide.
  • the expression vector is a vector into which a nucleic acid molecule encoding the polypeptide is inserted, is operatively linked to a nucleotide sequence of the nucleic acid molecule, and is an RNA molecule in a host cell. It is a recombinant vector for host cell expression comprising a promoter that forms it and a poly A signal sequence that acts in the host cell to cause polyadenylation of the 3'-end of the RNA molecule.
  • operatively linked refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, and By this, the regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.
  • a nucleic acid expression control sequence eg, a promoter, signal sequence, or an array of transcriptional regulator binding sites
  • the vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). , this document is incorporated herein by reference.
  • the vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
  • a strong promoter capable of propagating transcription eg, pL ⁇ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.
  • a ribosome binding site for initiation of translation and a transcription/translation termination sequence e.g., pL ⁇ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.
  • E. coli E. coli is used as a host cell
  • the promoter and operator site of the E. coli tryptophan biosynthesis pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)) and the left-handed promoter of phage ⁇ (pL)
  • the ⁇ promoter Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)
  • Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980) can
  • vectors that can be used in the present invention include plasmids (eg, pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (eg, ⁇ gt ⁇ 4B, ⁇ -Charon, ⁇ z1 and M13, etc.) or viruses (eg, SV40, etc.) can be manufactured.
  • plasmids eg, pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.
  • phage eg, ⁇ gt ⁇ 4B, ⁇ -Charon, ⁇ z1 and M13, etc.
  • viruses eg, SV40, etc.
  • the vector of the present invention may be fused with other sequences to facilitate purification of the polypeptide expressed therefrom.
  • the sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). Because of the additional sequences for purification, the protein expressed in the host is rapidly and easily purified via affinity chromatography.
  • the vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a gene for resistance to tetracycline.
  • an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a gene for resistance to tetracycline.
  • a promoter derived from the genome of a mammalian cell eg, a metallotionine promoter
  • a promoter derived from a mammalian virus eg, adeno viral late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV
  • a promoter derived from the genome of a mammalian cell eg, a metallotionine promoter
  • a promoter derived from a mammalian virus eg, adeno viral late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV
  • the vector may additionally carry genes encoding reporter molecules (eg, luciferase and -glucuronidase).
  • reporter molecules eg, luciferase and -glucuronidase
  • the present invention provides a host cell transformed with the recombinant vector.
  • any host cell known in the art may be used, for example, E. coli Origami2, E. coli JM109, E. coli BL21 (DE3). ), E. coli strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus genus strains such as Bacillus subtilis, Bacillus thuringiensis, and Enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcesens, and various Pseudomonas species.
  • yeast Sacharomyce cerevisiae
  • insect cells and animal cells eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines
  • yeast Sacharomyce cerevisiae
  • insect cells and animal cells eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines
  • the method of delivering the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl 2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)) ), Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580). (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)).
  • the microinjection method (Capecchi, MR, Cell, 22:479 (1980)), the calcium phosphate precipitation method (Graham, FL et al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bambadment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) ), and the like, to inject the vector into the host cell.
  • the microinjection method Capecchi, MR, Cell, 22:479 (1980)
  • the calcium phosphate precipitation method Graham, FL et al., Virology, 52:456 (1973)
  • the recombinant vector injected into the host cell can express the above-recombined polypeptide in the host cell, and in this case, a large amount of the polypeptide is obtained.
  • the expression vector includes the lac promoter
  • the host cell may be treated with IPTG to induce gene expression.
  • the transformed host cell can be cultured by a known host cell culture method or a modified method thereof.
  • a natural medium or synthetic medium can be used as the medium for culturing the transformed host cell if it contains a carbon source, nitrogen source, inorganic salt, etc. that can be efficiently used by E. coli.
  • Carbon sources that can be used include carbohydrates such as glucose, fructose, sucrose; starch, a hydrolyzate of starch; organic acids such as acetic acid and propionic acid; alcohols such as ethanol, propanol, glycerol, and the like.
  • the nitrogen source is ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean extract, soybean hydrolyzate; various fermented cells and their lysates; and the like.
  • Inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, calcium carbonate, and the like.
  • the culture is usually carried out under aerobic conditions, such as by shaking culture or rotation by a rotary machine.
  • the culture temperature is preferably in the range of 10 to 40° C., and the culture time is generally 5 hours to 7 days.
  • the pH of the medium is preferably maintained in the range of 3.0 to 9.0 during culture.
  • the pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.
  • antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin and tetracycline may be added for maintenance and expression of the recombinant vector.
  • a suitable inducer may be added to the medium.
  • a suitable inducer may be added to the medium.
  • IPTG isopropyl-beta-D-thiogalactopyranoside
  • indoleacrylic acid may be added to the medium.
  • the present invention provides a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and a cosmetic composition for preventing or improving skin aging or wrinkles comprising the same, and an external composition.
  • the polypeptide of the present invention inhibits the expression of the MMP-1 enzyme that decomposes collagen and has an excellent effect of promoting the expression of type 1 collagen, and thus can be usefully used as a composition for the above-mentioned use.
  • Example 1 is a diagram showing the cytotoxicity test results of the test substance (AMPEP-AA-1) of the present invention confirmed in Example 1.
  • Example 2 is a diagram showing the effect of the test substance (AMPEP-AA-1) of the present invention confirmed in Example 2 on the expression of the collagen-degrading enzyme MMP-1.
  • FIG. 3 is a diagram showing the effect of the test substance (AMPEP-AA-1) of the present invention confirmed in Example 3 on the expression of type 1 collagen.
  • % used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.
  • the test substance of the present invention is a polypeptide consisting of the amino acid sequence of KAIGELD (SEQ ID NO: 1), and was named "AMPEP-AA-1" by the present inventors.
  • the test substance of the present invention (AMPEP-AA-1) was dissolved in 1 ml of purified water and used, and was denoted as sample A in the following test example.
  • CCD-986SK cells were seeded at 1x10 4 /well (100 ⁇ l) in a 96-well cell culture plate. After culturing for 24 hours in a CO 2 incubator, it was confirmed whether the cells were evenly distributed on the bottom of the 96-well plate using a microscope to grow (confluency). Remove the medium from each well, and each 100 ⁇ l/well of the medium containing the test substance of the present invention at a concentration of 0, 0.1, 1, 5, 10, and 50 ⁇ g/ml is dispensed, and in a CO 2 incubator 24 incubated for hours.
  • MTT reagent (1 mg/ml) was dispensed, and the plate was incubated at 37° C. for 2 hours after blocking light. After 2 hours, it was confirmed whether a purple precipitate was formed in the cells. After removing the MTT reagent from each well, 100% isopropanol was added at a time of 100 ⁇ l/well to dissolve the precipitate. Finally, the absorbance of the plate was measured at the detection wavelength (570 nm) and the reference wavelength (650 nm) using a spectrophotometer. When the number of living cells decreases, the amount of MTT reagent converted into formazan by mitochondrial dehydrogenase decreases. Accordingly, the survival rate was calculated as follows.
  • the test substance of the present invention had a survival rate of 70% or more at all concentrations (0.1, 1, 5, 10, and 50 ⁇ g/ml), confirming that there was no toxicity.
  • CCD-986SK cells were treated with the test substance of the present invention by concentration with UVB ultraviolet rays, and the expression levels of MMP-1 and Collagen I were quantitatively measured.
  • CCD-986SK cells were treated with a test substance and RNA was extracted as follows.
  • CCD-986SK cells were seeded in a 6-well plate at 5 ⁇ 10 4 cells/well and cultured for 24 hours.
  • the test substance AMPEP-AA-1 (Sample A) of the present invention was treated with each concentration (0, 1, 10, 50 ⁇ g/mL).
  • 500 ⁇ l of trizol lysis buffer was dispensed into each well and the cells were homogenized. The homogenized solution was left at room temperature (15-25 ° C. for 5 minutes. 200 ⁇ L of chloroform was dispensed into each well, shaken every 15-20 seconds, and left for 3 minutes.
  • mRNA was quantified using the primer sequences shown in Table 1 below.
  • RT PCR reverse transcription PCR
  • Green GoTaq Flexi buffer, MgCl 2 , PCR nucleotide mix (10 mM), primer, GoTaq DNA polymerase, and nuclease free water were added to the synthesized cDNA in a PCR tube.
  • the expression level was measured by performing PCR on the samples of each experimental group under the conditions of 95°C 30 sec, 56°C, 60 sec, 72°C 1 min (40 cycles) for each gene (MMP-1, Collagen I, GAPDH).
  • the expression levels of MMP-1 and Collagen I were quantitatively analyzed by correcting with GAPDH.
  • Example 2 expression level of MMP-1 is shown in FIG. As shown in Figure 2, compared with the group irradiated with UVB 20mJ / cm 2 MMP-1 of the test group treated with the test substance of the present invention at a significant level at all concentrations (1, 5, 10 ⁇ g / mL) expression decreased.
  • Example 3 The results of Example 3 (collagen I expression level) are shown in FIG. 3 .
  • the test substance of the present invention reduces the expression of MMP-1 that decomposes collagen, and has an excellent effect of suppressing skin wrinkles and increasing the expression of type 1 collagen related to skin elasticity and skin moisture. Or it was found that it can be usefully used as a composition for preventing or improving wrinkles.

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Abstract

La présente invention concerne un nouveau fragment polypeptidique et une composition la comprenant, pour la prévention ou l'atténuation du vieillissement ou des rides de la peau. Le polypeptide de la présente invention a d'excellents effets de régulation négative de l'expression de MMP-1, ce qui dégrade le collagène, réduit les rides de la peau et régule positivement l'expression du collagène de type 1, qui est associé à l'élasticité de la peau et à l'humidification de la peau, trouvant ainsi des applications avantageuses en tant que composition destinée à la prévention et à l'atténuation du vieillissement ou des rides de la peau.
PCT/KR2021/011278 2020-10-28 2021-08-24 Composition destinée à la prévention ou à l'atténuation du vieillissement ou des rides de la peau WO2022092513A1 (fr)

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KR10-2020-0140862 2020-10-28
KR1020200140862A KR102462330B1 (ko) 2020-10-28 2020-10-28 피부노화 또는 주름의 예방 또는 개선용 조성물

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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
KR101886123B1 (ko) * 2016-08-17 2018-08-07 (주)진셀팜 주름 개선용 펩타이드, 및 이의 용도
KR101889322B1 (ko) * 2016-08-17 2018-08-20 (주)진셀팜 주름 개선 및 항노화 효과를 가지는 펩타이드, 및 이의 용도
KR101943081B1 (ko) * 2017-08-31 2019-01-29 (주)케어젠 주름 개선 활성을 나타내는 펩타이드 및 이의 용도

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KR20110099730A (ko) * 2008-12-15 2011-09-08 칼피스가부시키가이샤 피부 노화 억제 펩타이드
KR101617197B1 (ko) 2014-07-21 2016-05-03 경북대학교 산학협력단 피부노화 또는 피부주름 예방, 개선 또는 치료용 조성물
KR101613302B1 (ko) * 2015-12-30 2016-04-18 (주)넥스젠바이오텍 Sv82 폴리펩타이드 및 이를 유효성분으로 함유하는 피부 주름 개선 및 탄력 유지용 화장료 조성물

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140089295A (ko) * 2012-12-13 2014-07-14 주식회사 카엘젬백스 피부 노화 개선용 조성물
KR101746787B1 (ko) * 2015-11-12 2017-06-13 주식회사 펩트론 미백, 피부 탄력, 주름 개선 및 상처치유 활성을 갖는 다기능성 피부투과 펩타이드
KR101886123B1 (ko) * 2016-08-17 2018-08-07 (주)진셀팜 주름 개선용 펩타이드, 및 이의 용도
KR101889322B1 (ko) * 2016-08-17 2018-08-20 (주)진셀팜 주름 개선 및 항노화 효과를 가지는 펩타이드, 및 이의 용도
KR101943081B1 (ko) * 2017-08-31 2019-01-29 (주)케어젠 주름 개선 활성을 나타내는 펩타이드 및 이의 용도

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