WO2022114461A1 - Composition destinée à éclaircir la peau - Google Patents

Composition destinée à éclaircir la peau Download PDF

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WO2022114461A1
WO2022114461A1 PCT/KR2021/011497 KR2021011497W WO2022114461A1 WO 2022114461 A1 WO2022114461 A1 WO 2022114461A1 KR 2021011497 W KR2021011497 W KR 2021011497W WO 2022114461 A1 WO2022114461 A1 WO 2022114461A1
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present
polypeptide
composition
melanin
sequence
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PCT/KR2021/011497
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English (en)
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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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

Definitions

  • the present invention relates to a novel polypeptide fragment and a composition for skin whitening comprising the same.
  • melanocytes present in the basal layer of the human skin epidermis are stimulated by ultraviolet rays or radiation, tyrosinase is activated and tyrosine is converted into dopa and dopaquinone. It is converted and undergoes dopachrome to finally produce black and brown melanin.
  • the process of melanin biosynthesis has been intensively studied recently, and the results of this study are being applied to therapeutics and whitening products using various melanin production inhibitors.
  • the present inventors made intensive research efforts to develop a formulation having a skin whitening effect by inhibiting melanin production.
  • the present invention was developed by developing a novel 8mer short polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention, and identifying that this polypeptide has an excellent effect of inhibiting the production of melanin without exhibiting cytotoxicity. 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 skin whitening, a composition for external application, or a pharmaceutical composition for preventing, improving, treating or inhibiting pigmentation 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.
  • amino acid sequence of SEQ ID NO: 1 of the present invention is "RLPRPSVR".
  • the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention has no cytotoxicity, and inhibits the biosynthesis of melanin to exhibit a skin whitening effect or a pigmentation inhibitory effect.
  • polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention exhibits an inhibitory effect on the DOPA oxidation reaction.
  • the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention suppresses the expression of MITF and TRP-1, which are genes promoting transcription of tyrosinase.
  • 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 skin whitening 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 skin whitening comprising the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing, improving, treating or inhibiting pigmentation comprising the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
  • the cosmetic composition, the composition for topical application, and the pharmaceutical composition are the intended administration route 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 of choice.
  • the cosmetic composition, the composition for topical application, and the pharmaceutical composition are the intended administration route 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 of choice.
  • 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 reduction of 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 to be.
  • the pharmaceutical composition, cosmetic composition, or composition for external 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, hydroxy 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 a skin lightening effect or prevention, alleviation, or treatment of pigmentation. or an amount that provides an inhibitory effect.
  • This is a predetermined amount of active substance calculated to produce the desired 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 therapeutic effect in association with the required diluent.
  • a therapeutically effective amount of an active ingredient is provided in the methods and uses for the preparation of the compositions of the present invention.
  • a therapeutically effective amount can be determined by one of ordinary skill in the medical or veterinary arts based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, and the like, as is well known in the art.
  • pigmentation is a result of melanin synthesis, proliferation of melanocytes, and apoptosis of melanocytes.
  • the melanin synthesis, proliferation of melanocytes, and apoptosis of melanocytes may be a result of post inflammatory hyperpigmentation (PIH).
  • Post-inflammatory hyperpigmentation can be caused by acne, atopic dermatitis, allergic contact dermatitis, ataxia, lichen planus, chronic lupus erythematosus, ecchymosis, mechanical trauma, ionizing or non-ionizing radiation, burns, laser or drug therapy, skin infections, or derived from a combination of these.
  • post-inflammatory hyperpigmentation is induced in acne.
  • 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 above-described 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 described in the sequence listing.
  • the substantial identity is at least 61% when the above-described 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.
  • an algorithm commonly used in the art means a sequence that exhibits homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.
  • 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.
  • 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 thereof 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. have.
  • 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 skin whitening, a composition for topical application, and a pharmaceutical composition for preventing, improving, treating or inhibiting pigmentation comprising the same. Since the polypeptide of the present invention has an excellent effect of inhibiting the biosynthesis of melanin, it can be usefully used as a composition for the above-mentioned use.
  • Example 1 is a view showing the cytotoxicity test result of the test substance (AMPEP-AP-1) of the present invention confirmed in Example 1.
  • FIG. 2 is a diagram showing the principle of testing in Examples 2 and 3 of the present invention.
  • FIG. 3 is a diagram showing the effect of the test substance (AMPEP-AP-1) of the present invention confirmed in Example 2 on melanogenesis.
  • Example 4 is a diagram showing the effect of the test substance (AMPEP-AP-1) of the present invention confirmed in Example 3 on tyrosinase activity.
  • Example 5 is a diagram showing the effect of the test substance (AMPEP-AP-1) of the present invention confirmed in Example 4 on the inhibition of DOPA oxidation reaction.
  • FIGS. 6 and 7 are diagrams showing the effect of the test substance (AMPEP-AP-1) of the present invention confirmed in Example 5 on the mRNA expression of MITF and TRP-1, which promote tyrosinase transcription.
  • % 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) %.
  • test substance of the present invention is a polypeptide consisting of the amino acid sequence of RLPRPSVR (SEQ ID NO: 1), and was named "AMPEP-AP-1" by the present inventors.
  • test substance (AMPEP-AP-1) of the present invention was used by dissolving it in 1 ml of purified water, and was denoted as sample A in the following test example.
  • the cell line of the present invention used B16F1 (mouse melanoma cell) and subcultured every 2-3 days at 5 ⁇ 1% CO 2 , 37 ⁇ 1° C. temperature and humidity conditions, and the culture medium was 10% FBS, Penicillin. Dulbecco's modified Eagle's medium (DMEM) containing (100 Units/ml)/Streptomycin (100 ⁇ g/ml) was used.
  • B16F1 mouse melanoma cell
  • DMEM Dulbecco's modified Eagle's medium
  • B16F1 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.
  • test substance (sample A) of the present invention In order to confirm the effect of the test substance (sample A) of the present invention on the production of melanin pigment, a quantitative test of melanin when the test substance of the present invention is treated at a concentration of 10, 25, and 50 ⁇ g/mL in B16F1 cells, respectively carried out.
  • B16F1 cells were seeded at 1x10 5 cells/well (2 mL) in a 6-well cell culture plate, and cultured in a CO 2 incubator for 24 hours. Using a microscope in a 6-well plate cultured for 24 hours, it was confirmed whether the cells were evenly distributed on the bottom (confluency). The medium from each well was removed, and 2 mL/well of a medium not containing phenol red containing 4 mM of L-tyrosine was dispensed. CO 2 After culturing for 24 hours in an incubator, a test substance and a control (Arbutin, 1 mM) were treated and cultured for 24 hours in a CO 2 incubator.
  • Example 2 The melanin quantitative test of Example 2 was performed using the melanin pigment generation mechanism shown in FIG. 2 . As shown in Figure 2, when L-tyrosine is added, melanin production is promoted and the amount of melanin measured increases. However, when the tyrosinase inhibitor Arbutin is treated together with L-tyrosine, the amount of melanin measured decreases.
  • test substance of the present invention when the test substance of the present invention was treated, the amount of melanin production was reduced depending on the concentration of the test substance compared to the group treated only with L-tyrosine (C, Control), and p ⁇ 0.005 was particularly significant at 50 ⁇ g/mL of the test substance The level of melanin production was reduced. Therefore, it was confirmed that the test substance of the present invention can be used as an effective cosmetic composition for whitening by inhibiting melanin biosynthesis.
  • test substance sample A
  • tyrosinase that promotes melanin production.
  • B16F10 cells were cultured for 24 hours in a 6-well plate to 5 ⁇ 10 4 cells/well. 4 mM L-tyrosine was applied to all samples except for the negative control group (C, Control). After 1 hour, each well was treated with 10, 25, and 50 ⁇ g/ml concentrations of the test substance of the present invention for 48 hours. After washing twice with PBS, lysis buffer (1% triton X-100, 0.1 M sodium phosphate buffer, 50 mM phenylmethylsulfonyl fluoride, pH 6.8) was added to each well. Cells were eluted on ice and centrifuged at 13,500 ⁇ g, 4°C, for 30 minutes. Then, only the supernatant was collected and used as an enzyme solution.
  • lysis buffer 1% triton X-100, 0.1 M sodium phosphate buffer, 50 mM phenylmethylsulfonyl fluoride, pH 6.8
  • the substrate was prepared by dissolving L-DOPA in 0.1 M sodium phosphate buffer (pH 6.8) at a concentration of 2 mg/ml, and 40 ⁇ l of the enzyme solution prepared above was added to 160 ⁇ l of the substrate, followed by heating at 37° C. for 1 hour. After measuring the absorbance at 490 nm for the amount of chrome, the inhibition rate was calculated.
  • the tyrosinase activity was significantly reduced to 68.69% in arbutin used as a positive control compared to tyrosinase increased by L-tyrosine stimulation.
  • the tyrosinase activity for the sample did not show a tendency to decrease at all concentrations. From the above results, it was found that the test substance of the present invention inhibited the amount of melanin biosynthesis rather than inhibition of tyrosinase activity. Therefore, it was confirmed that the test substance of the present invention can be used as an effective cosmetic composition for whitening by inhibiting melanin biosynthesis.
  • the present inventors evaluated the whitening effect by measuring the activity inhibitory effect of the test substance (AMPEP-AP-1) of the present invention on the DOPA oxidation reaction of tyrosinase, which is involved in the rate determining step of the melanin synthesis process.
  • L-DOPA was used as a substrate for tyrosinase.
  • the DOPA oxidation inhibition assay is a test method to evaluate the effect of a whitening component by measuring the absorbance of dopa chromium by the action of tyrosinase.
  • test substance (AMPEP-AP-1) of the present invention and a sample of ascorbic acid as a positive control were dissolved in an appropriate solvent at concentrations of 0, 10, 25, and 50 ⁇ g/mL, and then used for the test.
  • melanin production is mainly caused by an oxidative reaction, and related enzymes include tyrosinase, tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2), and the like.
  • MITF promotes transcription by binding to M-box sequences of tyrosinase and TRPs as a microphthalamia transcription factor.
  • the present inventors performed the following test to confirm the effect of the test substance (AMPEP-AP-1) of the present invention on the gene expression of MITF and TRP-1 involved in melanin synthesis.
  • B16F1 cells were cultured in a 6-well plate at 1x10 7 cells/well for 24 hours. Two samples (AMPEP-AP-1, arbutin) were treated by concentration (10, 25, 50 ⁇ g/mL). After removing the medium supernatant, 500 ⁇ l of trizol lysis buffer was dispensed into each well to disrupt the cells. The homogenized solution was left at room temperature (15-25° C. for 5 minutes. 200 ⁇ l of Chloroform was dispensed, shaken for 15-20 seconds, and left for 3 minutes. Then, centrifuged at 12,000 x g for 15 minutes. The separated clear layer was The aqueous phase was transferred to a new tube.
  • cDNA synthesized from the RNA extracted in 5-1 was added to green GoTaq Flexi buffer, MgCl 2 , PCR nucleotide mix (10 mM), primer, GoTaq DNA polymerase, and nuclease free water in a PCR tube. Each PCR tube was subjected to 1 cycle of 95°C 30 sec, 56°C 60 sec, 72°C 1 min, and 40 cycles of reaction. The amount of expression of MITF and TRP-1 genes was quantified by correcting with ⁇ -actin.
  • test substance (AMPEP-AP-1) of the present invention inhibited the expression of the MITF gene, which promotes the transcription of tyrosinase that produces melanin at concentrations of 10 and 25 ⁇ g/mL.
  • test substance (AMPEP-AP-1) of the present invention inhibits the expression of the TRP-1 gene, which promotes the transcription of tyrosinase that produces melanin at concentrations of 10 and 25 ⁇ g/mL. did.
  • test substance (AMPEP-AP-1) of the present invention has a whitening effect by suppressing the expression of genes affecting the production of melanin.

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Abstract

L'invention se rapporte à un nouveau fragment polypeptidique et à une composition destinée à éclaircir la peau le comprenant. Le polypeptide selon la présente invention est très efficace dans l'inhibition de la biosynthèse de la mélanine et peut donc être utilisé utilement dans la composition destinée à l'utilisation décrite.
PCT/KR2021/011497 2020-11-24 2021-08-27 Composition destinée à éclaircir la peau WO2022114461A1 (fr)

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Citations (3)

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KR20060022208A (ko) * 2004-09-06 2006-03-09 재단법인서울대학교산학협력재단 펩타이드가 결합된 아스코르빈산 유도체
KR102055175B1 (ko) * 2018-03-09 2019-12-12 동신대학교산학협력단 여드름 개선용 화장료 조성물
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KR101436199B1 (ko) 2012-02-29 2014-09-01 바이오스펙트럼 주식회사 호데닌을 포함하는 피부 상태 개선용 조성물
WO2015081306A2 (fr) 2013-11-29 2015-06-04 Escape Therapeutics, Inc. Activateurs d'un peptide tyrosinase
AU2016366220B2 (en) 2015-12-10 2021-03-11 Lubrizol Advanced Materials, Inc. Compounds useful in the treatment and/or care of the skin, hair, nails, and/or mucous membranes
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KR102055175B1 (ko) * 2018-03-09 2019-12-12 동신대학교산학협력단 여드름 개선용 화장료 조성물
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