WO2022114461A1 - Composition for skin lightening - Google Patents

Composition for skin lightening Download PDF

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Publication number
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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French (fr)
Korean (ko)
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박순형
한은수
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주식회사 에이엠메딕스
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Publication of WO2022114461A1 publication Critical patent/WO2022114461A1/en

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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.

Abstract

The present invention relates to a novel polypeptide fragment and a composition for skin lightening comprising same. The polypeptide according to the present invention is superbly effective in inhibiting melanin biosynthesis and thus can be usefully employed in the composition for the described use.

Description

피부미백용 조성물Composition for skin whitening
본 발명은 신규한 폴리펩타이드 단편 및 이를 포함하는 피부미백용 조성물에 관한 것이다.The present invention relates to a novel polypeptide fragment and a composition for skin whitening comprising the same.
사람의 피부 표피 기저층에 존재하는 멜라닌 생성세포(melanocytes)는 자외선, 방사선 등의 자극을 받게 되면서 티로시나아제(tyrosinase)가 활성화가 되어 티로신(tyrosine)을 도파(dopa), 도파퀴논(dopaquinone)으로 변환시키고 도파크롬(dopachrome)을 거쳐 최종적으로 검은색 및 갈색을 띄는 멜라닌을 생성하게 된다. 멜라닌 생합성 과정은 최근 집중적으로 연구되고 있고, 이 연구 결과는 다양한 멜라닌 생성 저해 물질을 이용한 치료제 및 미백용 제품에 적용 되고 있다.When 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.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
대한민국 공개특허문헌 제10-2013-0099694호 (2013.09.06. 공개)Korean Patent Publication No. 10-2013-0099694 (published on Sep. 6, 2013)
본 발명자들은 멜라닌 생성을 저해하여 피부미백 효과가 있는 제제를 개발하고자 예의 연구 노력하였다. 그 결과 본 발명의 서열번호 1의 아미노산 서열로 이루어진 8mer의 짧은 신규한 폴리펩타이드를 개발하고, 이 폴리펩타이드가 세포독성을 나타내지 않으면서 멜라닌의 생성을 저해하는 효과가 우수함을 규명함으로써, 본 발명을 완성하게 되었다. The present inventors made intensive research efforts to develop a formulation having a skin whitening effect by inhibiting melanin production. As a result, 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
따라서, 본 발명의 목적은 서열번호 1의 아미노산 서열로 이루어진 폴리펩타이드를 제공하는 것이다. Accordingly, an object of the present invention is to provide a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
본 발명의 다른 목적은 상기 폴리펩타이드를 유효성분으로 포함하는 피부미백용 화장료 조성물, 외용제 조성물, 또는 색소침착의 예방, 개선, 치료 또는 저해용 약제학적 조성물을 제공하는 것이다.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.
본 발명의 또 다른 목적은 상기 폴리펩타이드를 인코딩하는 핵산 분자, 상기 핵산 분자를 포함하는 재조합 벡터, 상기 재조합 벡터를 포함하는 숙주 세포를 제공하는 것이다. 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.
본 발명의 일 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 이루어진 폴리펩타이드를 제공한다. According to one aspect of the present invention, the present invention provides a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
본 발명의 일 구현예에 있어서, 본 발명의 서열번호 1의 아미노산 서열은 "RLPRPSVR"이다. In one embodiment of the present invention, the amino acid sequence of SEQ ID NO: 1 of the present invention is "RLPRPSVR".
본 발명의 일 구현예에 있어서, 본 발명의 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드는 세포독성이 없고, 멜라닌의 생합성을 저해하여 피부미백 효과, 또는 색소침착 저해효과를 나타낸다. In one embodiment of the present invention, 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.
본 발명의 다른 일 구현예에 있어서, 본 발명의 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드는 DOPA 산화반응 저해효과를 나타낸다. In another embodiment of the present invention, the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention exhibits an inhibitory effect on the DOPA oxidation reaction.
본 발명의 다른 일 구현예에 있어서, 본 발명의 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드는 티로시나아제의 전사를 촉진하는 유전자인 MITF 및 TRP-1의 발현을 억제한다. In another embodiment of the present invention, 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.
본 명세서에서 사용되는 용어 “폴리 펩타이드”는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미한다. 본 발명의 폴리 펩타이드는 당업계에 공지된 화학적 합성 방법, 특히 고상 합성 기술 (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)) 또는 액상 합성 기술(US 등록특허 제5,516,891호)에 따라 제조될 수 있다.As used herein, the term “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).
본 발명의 폴리 펩타이드는 아미노산 서열의 일부 부위를 선정하고 그 활성을 증가시키기 위해 N-말단 또는 C-말단에 변형을 유도할 수 있다. 이러한 변형을 통해 본 발명의 폴리 펩타이드는 생체내 투여시의 반감기를 증가시킨 높은 반감기를 가질 수 있다.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.
본 발명의 다른 구현예에 있어서, 본 발명의 폴리 펩타이드의 C-말단은 히드록시기(-OH), 아미노기(-NH2), 아자이드기(-NHNH2) 등으로 변형될 수 있으며, 폴리 펩타이드의 N-말단은 아세틸기, 플루오레닐 메톡시 카르보닐기, 포르밀기, 팔미토일기, 미리스틸기, 스테아릴기 및 폴리에틸렌글리콜(PEG)로 구성된 군으로부터 선택되는 보호기가 결합될 수 있다.In another embodiment of the present invention, 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.
상술한 아미노산의 변형은 본 발명의 폴리 펩타이드의 안정성을 크게 개선하는 작용을 한다. 본 명세서에서 용어 “안정성”은 인 비보 안정성뿐만 아니라, 저장 안정성(예컨대, 상온 저장 안정성)도 의미한다. 상술한 보호기는 생체 내의 단백질 절단효소의 공격으로부터 본 발명의 폴리 펩타이드를 보호하는 작용을 한다.Modification of the above-described amino acids acts to greatly improve the stability of the polypeptide of the present invention. As used herein, the term “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.
본 발명의 다른 일 양태에 따르면, 본 발명은 상기 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드를 유효성분으로 포함하는 피부미백용 화장료 조성물을 제공한다. According to another aspect of the present invention, 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.
본 발명의 다른 일 양태에 따르면, 본 발명은 상기 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드를 유효성분으로 포함하는 피부미백용 외용제 조성물을 제공한다. According to another aspect of the present invention, 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.
본 발명의 또 다른 일 양태에 따르면, 본 발명은 상기 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드를 유효성분으로 포함하는 색소침착의 예방, 개선, 치료 또는 저해용 약제학적 조성물을 제공한다.According to another aspect of the present invention, 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.
본 발명의 일 구현예에 있어서, 상기 화장료 조성물, 외용제 조성물, 및 약제학적 조성물은 상기한 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드 외에 의도된 투여 경로 및 표준 약제학적 또는 미용적 적용에 관련하여 선택되는, 적어도 하나의 약제학적, 및/또는 미용적으로 허용가능한 부형제, 담체, 또는 희석제를 포함한다. (예를 들어, 본 명세서 참조로 포함되는, Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA 참조).In one embodiment of the present invention, 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. (See, eg, Remington: The Science and Practice of Pharmacy, 19 th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA, incorporated herein by reference).
본 발명의 상기 폴리펩타이드는 보관을 위해 동결건조(lyophilise)될 수 있고 사용 전 적절한 담체로 재구성될(reconstituted) 수 있다. The polypeptides of the invention may be lyophilized for storage and reconstituted with an appropriate carrier prior to use.
본 발명의 용어, "약제학적으로 허용가능한(pharmaceutically acceptable)"은 제제가 멸균이고 발열원(pyrogen)이 없는 것이다. 적절한 약제학적 담체는 약제학 분야에서 잘 알려져 있다. 상기 담체(들)은 본 발명의 유효성분의 효능에 악영향을 미치지 않고 그의 수용자(recipient)에게 해롭지 않다는 의미에서 "허용가능(acceptable)"해야 한다. 전형적으로, 상기 담체는 멸균되고 발열원이 없는 물 또는 식염수일 것이다; 그러나, 다른 허용가능한 담체가 사용될 수 있다. 따라서, "약제학적으로 허용가능한 담체(pharmaceutically acceptable carrier)" 및 "약제학적으로 허용가능한 부형제(pharmaceutically acceptable excipient)"는 단순히 담체로서 작용하도록 의도된, 즉, 그 자체의 생물학적 활성을 갖는 것으로 의도되지 않는, 제제의 일부를 형성하는데 사용된 화합물(들)을 포함한다. 약제학적으로 허용가능한 담체 또는 부형제는 일반적으로 안전하고, 무독성이다. 본 명세서에 사용된 것과 같은 약제학적으로 허용가능한 담체 및/또는 부형제는 1 이상의 담체 및/또는 부형제를 모두 포함한다.As used herein, "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. Typically, the carrier will be sterile, pyrogen-free water or saline; However, other acceptable carriers may be used. Thus, "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.
마찬가지로, 용어 "미용적으로 허용가능한(cosmetically acceptable)"은 화장품으로서 사용하기에 적절한 제제를 나타내는데 사용된다. 적절한 미용적 담체는 샴푸, 로션, 크림, 스프레이 및 다른 이러한 제품에 일반적으로 사용되는 것과 같이, 당해 기술 분야에서 잘 알려져 있다.Likewise, the term “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.
부형제는 하나 이상의 탄수화물, 폴리머, 지질 및 미네랄일 수 있다. 탄수화물의 예는 락토스, 수크로스, 만니톨, 및 시클로덱스트린을 포함하고, 이것은 예를 들여, 동결건조를 가능하게 하기 위해 조성물에 첨가된다. 폴리머의 예는 전분, 셀룰로스 에테르, 셀룰로스 카르복시메틸셀룰로스, 히드록시프로필메틸 셀룰로스, 히드록시에틸 셀룰로스, 에틸히드록시에틸 셀룰로스, 알기네이트, 카라기난, 히알루론산 및 그의 유도체, 폴리아크릴산, 폴리술포네이트, 폴리에틸렌글리콜/폴리에틸렌 옥시드, 폴리에틸렌옥시드/폴리프로필렌 옥시드 코폴리머, 상이한 가수분해도(degree of hydrolysis)의 폴리비닐알콜/폴리비닐아세테이트, 및 모든 상이한 분자량의 폴리비닐피롤리돈이고, 이들은 예를 들어, 점도 조절을 위해, 접착을 이루기 위해, 또는 화학적 및 단백질 분해성 분해로부터 지질을 보호하기 위해 상기 조성물에 첨가된다. 지질의 예는 지방산, 인지질, 인산, 모노-, 다이-, 및 트리글리세리드, 세라마이드, 모든 상이한 아실 사슬 길이 및 포화의 스핑고지질 및 당지질, 달걀 레시틴, 콩 레시틴, 수소화된 달걀 및 콩 레시틴이고, 이들은 폴리머에 대한 이유와 유사한 이유로 조성물에 첨가된다. 미네랄의 예는 탈크, 산화마그네슘, 산화아연 및 산화티타늄이고, 이들은 액체 축적의 감소 또는 유리한 색소 특성과 같은 이점을 수득하기 위해 상기 조성물에 첨가된다.An excipient may be one or more carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition to facilitate lyophilization, for example. Examples of 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. Examples of 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. Examples of 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.
용어 "희석제(diluent)"는 약제학적 제조에서 펩타이드를 희석할 목적을 갖는 수성 또는 비-수성 용액을 의미하는 것으로 의도된다. 희석제는 하나 이상의 식염수(saline), 물, 폴리에틸렌 글리콜, 프로필렌 글리콜, 에탄올 또는 (홍화유, 옥수수유, 땅콩 기름, 면실유 또는 참기름과 같은) 오일일 수 있다.The term "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).
희석제는 또한 완충액으로서 기능할 수 있다. 용어 "완충액(buffer)"은 pH를 안정화하는 목적을 갖는 산-염기 혼합물을 함유하는 수성 용액을 의미하는 것으로 의도된다. 완충액의 예는 Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, 포스페이트, 카르보네이트, 아세테이트, 시트레이트, 글리콜레이트, 락테이트, 보레이트, ACES, ADA, 타르타레이트, AMP, AMPD, AMPSO, BES, CABS, 카코딜레이트, CHES, DIPSO, EPPS, 에탄올아민, 글리신, HEPPSO, 이미다졸, 이미다졸락트산, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO 및 TES이다.The diluent may also function as a buffer. The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilizing the pH. Examples of 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.
본 발명의 약제학적 조성물, 화장료 조성물, 또는 외용제 조성물은 또한 폴리머 겔, 또는 폴리머로 이루어진 마이크로니들의 형태일 수 있고, 상기 폴리머는 전분, 셀룰로스 에테르, 셀룰로스 카르복시메틸셀룰로스, 히드록시프로필메틸 셀룰로스, 히드록시에틸 셀룰로스, 에틸히드록시에틸 셀룰로스, 알기네이트, 카라기난, 히알루론산 및 그의 유도체, 폴리아크릴산, 폴리술포네이트 등이다.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.
상기 본 발명의 폴리펩타이드는 사용되는 유효성분의 효능 또는 독성에 따라, 다양한 농도로 제제화 될 수 있다. 바람직하게는, 상기 조성물은 1 nM 내지 1 M, 예를 들어 0.1 μM (micromole) 내지 1 mM, 1 μM 내지 100 μM, 5 μM 내지 50 μM, 10 μM 내지 50 μM, 20 μM 내지 40 μM 및 선택적으로 약 30 μM의 농도로 상기 폴리펩타이드를 포함한다. 생체 외(ex vivo) 및 시험관 내 적용을 위해, 조성물은 더 낮은 농도, 예를 들어, 0.0025 μM 내지 1 μM 의 폴리펩타이드를 포함할 수 있다.The polypeptide of the present invention may be formulated at various concentrations depending on the efficacy or toxicity of the active ingredient used. Preferably, the composition comprises 1 nM to 1 M, for example 0.1 μM (micromole) to 1 mM, 1 μM to 100 μM, 5 μM to 50 μM, 10 μM to 50 μM, 20 μM to 40 μM and optionally and the polypeptide at a concentration of about 30 μM. For ex vivo and in vitro applications, the composition may comprise a lower concentration of the polypeptide, for example, between 0.0025 μM and 1 μM.
본 발명의 조성물은 다양한 경로 예를 들어, 국부, 피하, 비경구 또는 경구 투여로 투여될 수 있다는 것이 당해 기술 분야의 통상의 기술자에게 인식되어 있다.It will be appreciated by those skilled in the art that the compositions of the present invention may be administered by a variety of routes, for example, topical, subcutaneous, parenteral or oral administration.
바람직하게는, 본 발명의 조성물은 국부 투여 또는 피내(intracutaneous) 투여에 적절하다. 따라서, 본 발명의 조성물은 피부(예를 들어, 두피)에 국부적으로 투여될 수 있다. 예를 들어, 상기 조성물은 예를 들어, 하기 중 하나 이상을 갖는 혼합물에 현탁 또는 용해된 활성 폴리펩타이드를 함유하는 연고의 형태로 제공될 수 있다: 미네랄 오일, 액체 페트롤라툼, 백색 페트롤라툼, 프로필렌 글리콜, 폴리옥시에틸렌 폴리옥시프로필렌 화합물, 유화(emulsifying) 왁스 및 물. 대안적으로, 상기 폴리펩타이드는 예를 들어 하기 중 하나 이상의 혼합물에 현탁 또는 용해된, 적절한 로션 또는 크림, 스프레이, 겔, 에멀젼, 페이스트, 비누, 분말, 또는 이들의 조합 등으로 제제화될 수 있다: 미네랄 오일, 소르비탄 모노스테아레이트, 폴리에틸렌 글리콜, 액체 파라핀, 폴리소르베이트 60, 세틸 에스테르 왁스, 세테아릴 알콜, 2-옥틸도데카놀, 벤질 알콜 및 물.Preferably, the composition of the present invention is suitable for topical or intracutaneous administration. Accordingly, the compositions of the present invention may be administered topically to the skin (eg, the scalp). For example, 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. Alternatively, 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.
선택적으로, 국부 투여용 조성물은 투과 증진제(penetration enhancer)를 포함할 수 있다(예를 들어, 그 개시가 본 명세서에 참조로서 포함된, Osborne & Henke, 1997, Pharmaceutical Technology, November: 58-82 and Pathan & Setty, 2009, Tropical Journal of Pharmaceutical Research 8(2): 173-179에 기재된 바와 같음).Optionally, 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). As described in Patan & Setty, 2009, Tropical Journal of Pharmaceutical Research 8(2): 173-179).
대안적으로, 본 발명의 조성물은 비경구적으로, 예를 들어 피내로 투여될 수 있다. 이러한 조성물은 다른 물질, 예를 들어 혈액과 등장성인 용액을 만드는데 충분한 염 또는 글루코스를 함유할 수 있는 멸균 수성 용액의 형태로 가장 잘 사용된다. 상기 수성 용액은 필요하면, 적절하게 (바람직하게는 pH 3 내지 9로) 완충되어야 한다. 멸균 상태 하의 적절한 비경구 제제의 제조는 당해 기술 분야의 통상의 기술자에게 잘 알려진 표준 약제학적 기법에 의해 손쉽게 달성된다. Alternatively, the 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.
본 발명의 폴리펩타이드를 포함하는 조성물은 유효량으로 대상체(subject)투여된다. 본 명세서에 사용된, '치료적 유효량(therapeutically effective amount)', 또는 '유효량(effective amount)', 또는 '치료적으로 유효한(therapeutically effective)'은 피부미백 효과 또는 색소침착의 예방, 경감, 치료 또는 억제 효과를 제공하는 양을 말한다. 이것은 원하는 치료 효과를 생성하도록 산출된 활성 물질의 미리 결정된 양이다. 당해 기술 분야의 통상의 기술자에게 인식되는 바와 같이, 유효성분의 양은 그의 특이적 활성에 따라 달라질 수 있다. 적절한 용량은 요구되는 희석제와 함께 원하는 치료적 효과를 생성하도록 산출된 활성 물질의 미리 결정된 양을 함유할 수 있다. 본 발명의 조성물의 제조를 위한 방법 및 용도에서, 활성 성분의 치료적 유효량이 제공된다. 치료적 유효량은 당해 기술 분야에 잘 알려진 바와 같이, 연령, 체중, 성별, 상태, 합병증, 기타 질병 등과 같은, 환자 특성에 기초하여 통상의 의료계 또는 수의계 종사자에 의해 결정될 수 있다.A composition comprising the polypeptide of the present invention is administered to a subject in an effective amount. As used herein, '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. As will be appreciated by those skilled in the art, 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. In the methods and uses for the preparation of the compositions of the present invention, a therapeutically effective amount of an active ingredient is provided. 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.
본 발명의 일 구현예에 있어서, "색소침착"은 멜라닌 합성, 멜라닌 세포의 증식, 멜라닌 세포의 아폽토시스에 의한 결과이다. 상기 멜라닌 합성, 멜라닌 세포의 증식, 멜라닌 세포의 아폽토시스는 염증후 색소과다침착(post inflammatory hyperpigmentation, PIH)의 결과일 수 있다. 염증후 색소과다침착은 여드름, 아토피성 피부염, 접촉성 알러지 피부염, 색소 실조증, 편평태선, 만성 홍반성 낭창, 반상경피증, 기계적 트라우마, 이온화 또는 비이온화 방사선, 화상, 레이저 또는 약물 요법, 피부 감염 또는 이들의 조합에서 유도된다. 예를 들어, 염증후 색소과다침착(PIH)은 여드름에서 유도된다.In one embodiment of the present invention, "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. For example, post-inflammatory hyperpigmentation (PIH) is induced in acne.
본 발명의 다른 일 양태에 따르면, 본 발명은 상술한 서열번호 1의 아미노산 서열을 포함하는 폴리펩타이드를 인코딩하는 핵산 분자를 제공한다. According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 described above.
본 명세서에서 용어 "핵산 분자”는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 갖으며, 핵산 분자에서 기본 구성 단위인 뉴클레오타이드는 자연의 뉴클레오타이드뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다(Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman 및 Peyman, Chemical Reviews, 90:543-584(1990)). As used herein, the term "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)).
본 발명의 상기 폴리 펩타이드를 인코딩하는 뉴클레오타이드 서열은 상기 폴리 펩타이드를 구성하는 아미노산 서열을 인코딩하는 뉴클레오타이드 서열인 것으로 족하며, 어느 특정 뉴클레오타이드 서열에 한정되지 않는다는 것은 당업자에게 자명하다. 이는 뉴클레오티드 서열의 변이가 발생하더라도 변이된 뉴클레오타이드 서열을 단백질로 발현하면 단백질 서열에서 변화를 가져오지 않는 경우도 있기 때문이다. 이를 코돈의 축퇴성이라고 한다. 따라서 상기 뉴클레오타이드 서열은 기능적으로 균등한 코돈 또는 동일한 아미노산을 코딩하는 코돈 (예를 들어, 코돈의 축퇴성에 의해, 아르기닌 또는 세린에 대한 코돈은 여섯 개이다), 또는 생물학적으로 균등한 아미노산을 코딩하는 코돈을 포함하는 뉴클레오타이드 서열을 포함한다.The 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. Thus, 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.
상기 폴리 펩타이드를 인코딩하는 본 발명의 핵산 분자는 상기한 뉴클레오타이드 서열에 대하여 실질적인 동일성을 나타내는 뉴클레오타이드 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 뉴클레오타이드 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80%의 상동성, 보다 바람직하게는 최소 90%의 상동성, 가장 바람직하게는 최소 95%, 97%, 98%, 또는 99%의 상동성을 나타내는 뉴클레오타이드 서열을 의미한다.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.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 폴리 펩타이드를 인코딩하는 핵산 분자는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 61%의 상동성, 보다 바람직하게는 70%의 상동성, 보다 더 바람직하게는 80%의 상동성, 가장 바람직하게는 90%의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. 얼라인먼트에 대한 다양한 방법 및 알고리즘은 Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5:151-3(1989); Corpet et al., Nuc. Acids Res. 16:10881-90(1988); Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994)에 개시되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al., J. Mol. Biol. 215:403-10(1990))은 NBCI(National Center for Biological Information) 등에서 접근 가능하며, 인터넷 상에서 blastp, blastn, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLAST는 ncbi 웹사이트의 BLAST 페이지를 통하여 접속 가능하다. 이 프로그램을 이용한 서열 상동성 비교 방법은 ncbi 웹사이트의 BLAST help 페이지에서 확인할 수 있다.Considering the above-described variation having biological equivalent activity, it is construed that 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. 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. 2:482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10(1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. 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.
본 발명의 또 다른 일 양태에 따르면, 본 발명은 상술한 폴리 펩타이드를 인코딩하는 핵산 분자를 포함하는 재조합 벡터를 제공한다. According to another aspect of the present invention, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the above-described polypeptide.
본 발명의 일 구현예에 따르면, 상기 발현 벡터는 상기 폴리 펩타이드를 인코딩하는 핵산 분자가 삽입된 벡터로서, 상기 핵산 분자의 뉴클레오티드 서열에 작동적으로 결합(operatively linked)되어 있고 숙주세포에서 RNA 분자를 형성시키는 프로모터 및 숙주세포에서 작용하여 RNA 분자의 3'-말단의 폴리아데닐화를 야기시키는 폴리 A 시그널 서열을 포함하는 숙주세포 발현용 재조합 벡터이다.According to one embodiment of the present invention, 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.
본 명세서에서 용어 "작동적으로 결합(operatively linked)"은 핵산 발현 조절 서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 해독(translation)을 조절하게 된다.As used herein, the term "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.
본 발명의 벡터 시스템은 당업계에 공지된 다양한 방법을 통해 구축될 수 있으며, 이에 대한 구체적인 방법은 Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001)에 개시되어 있으며, 이 문헌은 본 명세서에 참조로서 삽입된다.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.
본 발명의 벡터는 전형적으로 클로닝을 위한 벡터 또는 발현을 위한 벡터로서 구축될 수 있다. 또한, 본 발명의 벡터는 원핵 세포 또는 진핵 세포를 숙주로 하여 구축될 수 있다.The vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
예를 들어, 본 발명의 벡터가 발현 벡터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예컨대, pLλ 프로모터, trp 프로모터, lac 프로모터, T7 프로모터, tac 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 숙주 세포로서 E. coli가 이용되는 경우, E. coli 트립토판 생합성 경로의 프로모터 및 오퍼레이터 부위(Yanofsky, C., J. Bacteriol., 158:1018-1024(1984)) 그리고 파아지 λ의 좌향 프로모터(pLλ 프로모터, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445(1980))가 조절 부위로서 이용될 수 있다.For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of propagating transcription (eg, pLλ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , a ribosome binding site for initiation of translation and a transcription/translation termination sequence. When 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 λ (pL) The λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as a regulatory region.
한편, 본 발명에 이용될 수 있는 벡터는 당업계에서 종종 사용되는 플라스미드(예: pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX 시리즈, pET 시리즈 및 pUC19 등), 파지(예: λgt·λ4B, λ-Charon, λΔz1 및 M13 등) 또는 바이러스(예: SV40 등)를 조작하여 제작될 수 있다.On the other hand, 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, λgt ·λ4B, λ-Charon, λΔz1 and M13, etc.) or viruses (eg, SV40, etc.) can be manufactured.
본 발명의 벡터는 그로부터 발현되는 폴리펩티드의 정제를 용이하게 하기 위하여, 다른 서열과 융합될 수도 있다. 융합되는 서열은 예컨대, 글루타티온 S-트랜스퍼라제(Pharmacia, USA), 말토스 결합 단백질(NEB, USA), FLAG(IBI, USA) 및 6x His(hexahistidine; Quiagen, USA) 등이 있다. 상기 정제를 위한 추가적인 서열 때문에, 숙주에서 발현된 단백질은 친화성 크로마토그래피를 통하여 신속하고, 용이하게 정제된다.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.
본 발명의 벡터는 선택표지로서, 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함할 수 있으며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.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.
한편, 본 발명의 벡터가 발현 벡터이고, 진핵 세포를 숙주로 하는 경우에는, 포유동물 세포의 지놈으로부터 유래된 프로모터(예: 메탈로티오닌 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예: 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, 사이토메갈로바이러스 프로모터 및 HSV의 tk 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다.On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter) or 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) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.
선택적으로, 상기 벡터는 리포터 분자(예: 루시퍼라아제 및 -글루쿠로니다아제)를 코딩하는 유전자를 추가적으로 운반할 수 있다. Optionally, the vector may additionally carry genes encoding reporter molecules (eg, luciferase and -glucuronidase).
본 발명의 또 다른 일 양태에 따르면, 본 발명은 상기 재조합 벡터로 형질전환된 숙주세포를 제공한다.According to another aspect of the present invention, the present invention provides a host cell transformed with the recombinant vector.
본 발명의 벡터를 안정하게 연속적으로 클로닝 및 발현시킬 수 있는 숙주 세포는 당업계에 공지되어 있는 어떠한 숙주 세포도 이용할 수 있으며, 예컨대, E. coli Origami2, E. coli JM109, E. coli BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110와 같은 E.coli 균주, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있다.As a host cell capable of stably and continuously cloning and expressing the vector of the present invention, 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.
또한, 본 발명의 벡터를 진핵 세포에 형질전환시키는 경우에는 숙주 세포로서, 이스트(Saccharomyce cerevisiae), 곤충 세포 및 동물 세포(예컨대, CHO 세포주(Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주) 등이 이용될 수 있다. In addition, when the vector of the present invention is transformed into eukaryotic cells, as host cells, yeast (Saccharomyce 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) can be used.
본 발명의 벡터를 숙주 세포 내로 운반하는 방법은, 숙주 세포가 원핵 세포인 경우, CaCl2 방법(Cohen, S.N. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973)), 하나한 방법(Cohen, S.N. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973); 및 Hanahan, D., J. Mol. Biol., 166:557-580(1983)) 및 전기 천공 방법(Dower, W.J. et al., Nucleic. Acids Res., 16:6127-6145(1988)) 등에 의해 실시될 수 있다. 또한, 숙주세포가 진핵 세포인 경우에는, 미세 주입법(Capecchi, M.R., Cell, 22:479(1980)), 칼슘 포스페이트 침전법(Graham, F.L. et al., Virology, 52:456(1973)), 전기 천공법(Neumann, E. et al., EMBO J., 1:841(1982)), 리포좀-매개 형질감염법(Wong, T.K. et al., Gene, 10:87(1980)), DEAE-덱스트란 처리법(Gopal, Mol. Cell Biol., 5:1188-1190(1985)), 및 유전자 밤바드먼트(Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572(1990)) 등에 의해 벡터를 숙주 세포 내로 주입할 수 있다.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)). In addition, when the host cell is a eukaryotic cell, 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.
본 발명에서 숙주세포 내로 주입된 재조합 벡터는 숙주 세포 내에서 재조합된 상기의 폴리 펩타이드를 발현할 수 있으며, 이러한 경우에는 다량의 폴리 펩타이드를 얻게 된다. 예를 들어, 상기 발현 벡터가 lac 프로모터를 포함하는 경우에는 숙주 세포에 IPTG를 처리하여 유전자 발현을 유도할 수 있다.In the present invention, 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. For example, when the expression vector includes the lac promoter, the host cell may be treated with IPTG to induce gene expression.
형질전환된 숙주세포의 배양은 공지된 숙주세포 배양 방법 또는 이를 변형한 방법으로 행할 수 있다. 예를 들어, 숙주세포가 대장균(E.coli)인 경우 형질전환 숙주세포의 배양을 위한 배지는 대장균이 효율적으로 이용할 수 있는 탄소원, 질소원, 무기염 등을 포함한다면 천연 배지 또는 합성 배지를 사용할 수 있다. 사용될 수 있는 탄소원은 글루코오스, 프럭토오스, 수크로오스와 같은 탄수화물; 녹말, 녹말의 가수분해물; 아세트산 및 프로피온산과 같은 유기산; 에탄올, 프로판올, 글리세롤과 같은 알코올 등을 포함한다. 질소원은 암모니아; 염화암모늄, 암모늄설페이트, 암모늄아세테이트 및 암모늄포스페이트와 같은 무기산 또는 유기산의 암모늄염; 펩톤, 고기추출물(meat extract), 이스트추출물, 옥수수 침지액, 카제인 가수분해물, 대두추출물, 대두가수분해물; 다양한 발효된 세포 및 이들의 분해물 등을 포함한다. 무기염은 포타슘디하이드로젠 포스페이트, 다이포타슘하이드로젠 포스페이트, 마그네슘 포스페이트, 마그네슘 설페이트, 소디엄 클로라이드, 망간 설페이트, 구리 설페이트, 칼슘 카보네이트 등을 포함한다.The transformed host cell can be cultured by a known host cell culture method or a modified method thereof. For example, when the host cell is E. coli , 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.
상기 배양은 통상적으로 진탕배양 또는 회전기에 의한 회전에 의한 것과 같은 호기성 조건하에서 행한다. 배양 온도는 바람직하게는 10 내지 40℃의 범위에서 행하고, 배양시간은 일반적으로 5 시간 내지 7 일간 행한다. 배지의 pH는 배양 중에서 바람직하게는 3.0 내지 9.0의 범위를 유지한다. 배지의 pH는 무기 또는 유기산, 알칼리 용액, 우레아, 칼슘 카보네이트, 암모니아 등으로 조절할 수 있다. 배양 중에는 필요한 경우 재조합 벡터의 유지 및 발현을 위해 암피실린, 스트렙토마이신, 클로람페니콜, 카나마이신 및 테트라사이클린과 같은 항생제를 첨가할 수 있다. 유도(induction) 가능한 프로모터를 갖는 재조합 발현 벡터로 형질전환된 숙주세포를 배양하는 경우 필요하다면 배지에 적합한 유도제(inducer)를 첨가할 수 있다. 예를 들어, 발현 벡터가 lac 프로모터를 함유하는 경우 IPTG (isopropyl-beta-D-thiogalactopyranoside)를 첨가하고, trp 프로모터를 포함하는 경우 인돌아크릴산(indoleacrylic acid)을 배지에 첨가할 수 있다.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. During culture, if necessary, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin and tetracycline may be added for maintenance and expression of the recombinant vector. When culturing a host cell transformed with a recombinant expression vector having an inducible promoter, if necessary, a suitable inducer may be added to the medium. For example, if the expression vector contains the lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) may be added, and if the expression vector contains a trp promoter, indoleacrylic acid may be added to the medium.
본 발명은 서열번호 1의 아미노산 서열로 이루어진 폴리펩타이드, 및 이를 포함하는 피부미백용 화장료 조성물, 외용제 조성물, 및 색소침착의 예방, 개선, 치료 또는 저해용 약제학적 조성물을 제공한다. 본 발명의 폴리펩타이드는 멜라닌의 생합성을 저해하는 효과가 우수한 바, 상술한 용도의 조성물로 유용하게 사용될 수 있다.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.
도 1은 실시예 1에서 확인한 본 발명의 시험물질 (AMPEP-AP-1)의 세포독성 시험결과를 나타낸 도이다.1 is a view showing the cytotoxicity test result of the test substance (AMPEP-AP-1) of the present invention confirmed in Example 1.
도 2는 본 발명의 실시예 2 및 3의 시험의 원리를 나타낸 도이다. 2 is a diagram showing the principle of testing in Examples 2 and 3 of the present invention.
도 3은 실시예 2에서 확인한 본 발명의 시험물질 (AMPEP-AP-1)이 멜라닌생성에 미치는 효과를 나타낸 도이다.3 is a diagram showing the effect of the test substance (AMPEP-AP-1) of the present invention confirmed in Example 2 on melanogenesis.
도 4는 실시예 3에서 확인한 본 발명의 시험물질 (AMPEP-AP-1)이 tyrosinase 활성에 미치는 효과를 나타낸 도이다.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.
도 5는 실시예 4에서 확인한 본 발명의 시험물질 (AMPEP-AP-1)이 DOPA 산화반응 저해에 미치는 효과를 나타낸 도이다.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.
도 6 및 도 7은 실시예 5에서 확인한 본 발명의 시험물질 (AMPEP-AP-1)이 tyrosinase 전사를 촉진시키는 MITF 및 TRP-1의 mRNA 발현에 미치는 효과를 나타낸 도이다.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.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout this specification, "%" 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) %.
실험방법 및 실험재료Experimental methods and materials
본 발명의 시험물질은 RLPRPSVR의 아미노산 서열(서열번호 1)로 이루어진 폴리펩타이드로, 본 발명자들에 의해 "AMPEP-AP-1"으로 명명되었다. The 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.
본 발명의 시험물질(AMPEP-AP-1)은 정제수 1 ml에 용해하여 사용하였으며, 하기 시험예에서는 sample A로 표시되었다. The 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.
본 발명의 대조군으로는 정제수에 용해시킨 알부틴(arbutin) 1 mM을 사용하였다.As a control of the present invention, 1 mM of arbutin dissolved in purified water was used.
본 발명의 실시예에서 멜라닌 형성을 촉진하기 위한 시료로는 L-tyrosine 4 mM을 사용하였다.4 mM of L-tyrosine was used as a sample for promoting melanin formation in an embodiment of the present invention.
본 발명의 세포주는 B16F1(mouse melanoma cell)을 사용하였으며 5±1% CO2, 37±1℃의 온도 및 습도 조건에서 2~3일 마다 계대 배양을 하였고, 배양 배지로는 10% FBS, Penicillin (100 Units/ml)/Streptomycin (100μg/ml)을 포함하는 Dulbecco's modified Eagle's medium (DMEM)을 사용하였다.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 μg/ml) was used.
실시예 1. 세포 독성 시험Example 1. Cytotoxicity test
본 발명의 시험물질의 세포 독성 유무를 확인하기 위하여, 다음과 같은 실험을 통하여 B16F1 세포에 5가지 농도(0.1, 1, 5, 10, 및 50 μg/ml)의 시료에 대해 독성 여부를 확인하였다.In order to confirm the presence or absence of cytotoxicity of the test substance of the present invention, it was confirmed whether the samples at 5 concentrations (0.1, 1, 5, 10, and 50 μg/ml) were toxic to B16F1 cells through the following experiment. .
먼저 96 웰 세포 배양 플레이트에 B16F1 세포를 1x104/well (100㎕)로 분주하였다. CO2 배양기에서 24시간 동안 배양한 후, 96-웰 플레이트를 현미경을 이용하여 세포가 바닥에 골고루 분포되어 자라는지(confluency) 확인하였다. 각 웰의 배지를 제거하고, 본 발명의 시험물질을 각각 0, 0.1, 1, 5, 10, 및 50 μg/ml의 농도로 포함하는 배지를 100 ㎕/well씩 분주하고, CO2 배양기에서 24시간 동안 배양하였다. 각 웰의 배지를 제거한 후, MTT 시약 (1㎎/㎖)를 50 ㎕/well씩 분주하고, 플레이트를 차광한 후 37℃에서 2시간 동안 배양하였다. 2시간 후, 세포에 보라색 침전물이 생겼는지 확인하였다. 각 웰의 MTT 시약을 제거한 후, 100% 이소프로판올을 100 ㎕/well씩 넣고 침전물을 용해하였다. 마지막으로 Spectrophotometer를 이용하여 검출 파장(detection wavelength, 570 nm) 및 참조 파장(reference wavelength, 650 nm)에서 플레이트의 흡광도를 측정하였다. 살아있는 세포의 수가 감소하면 MTT 시약이 미토콘드리아의 탈수소효소에 의해 포르마잔(formazan)으로 변형되는 양이 감소한다. 이에 따라, 생존율은 다음과 같이 계산하였다.First, B16F1 cells were seeded at 1x10 4 /well (100 μl) 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 μl/well of the medium containing the test substance of the present invention at a concentration of 0, 0.1, 1, 5, 10, and 50 μg/ml is dispensed, and in a CO 2 incubator 24 incubated for hours. After removing the medium from each well, 50 μl/well of 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 μl/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.
Figure PCTKR2021011497-appb-img-000001
Figure PCTKR2021011497-appb-img-000001
결과는 도 1에 나타내었다. 도 1의 결과에서, 살아있는 세포의 수가 감소하면 대사 활동이 감소하게 되므로, 생존율(%)이 낮을수록 시료의 잠재적인 세포 독성은 더 높아지며, 생존율이 공 시험액의 70% 미만(<70%)으로 감소되는 경우, 이를 세포 독성이 있는 것으로 간주한다. The results are shown in FIG. 1 . In the results of Figure 1, since the decrease in the number of living cells leads to a decrease in metabolic activity, the lower the viability (%), the higher the potential cytotoxicity of the sample, and the viability is less than 70% (<70%) of the blank test solution. If reduced, it is considered cytotoxic.
도 1에 나타낸 바와 같이, 본 발명의 시험물질은 모든 농도(0.1, 1, 5, 10, 및 50 μg/ml)에서 생존율이 70% 이상 되어, 독성이 없음을 확인하였다.As shown in FIG. 1 , 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 μg/ml), confirming that there was no toxicity.
실시예 2. 멜라닌 정량 시험Example 2. Melanin quantitative test
본 발명의 시험물질(sample A)이 멜라닌 색소의 생성에 미치는 영향을 확인하기 위하여, 본 발명의 시험물질을 B16F1 세포에 10, 25, 50μg/mL 농도로 각각 처리한 경우의 멜라닌의 정량 시험을 수행하였다. 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 μg/mL in B16F1 cells, respectively carried out.
먼저 6-웰 세포배양 플레이트에 B16F1 세포를 1x105 cells/well (2 mL)로 분주하고, CO2 배양기에서 24시간 동안 배양하였다. 24시간 동안 배양한 6-웰 플레이트를 현미경을 이용하여 세포가 바닥에 골고루 분포되었는지(confluency) 확인하였다. 각 웰의 배지를 제거하고 L-tyrosine 4 mM이 포함된 페놀 레드(phenol red)가 포함되지 않는 배지를 2 mL/well씩 분주하였다. CO2 배양기에서 24시간 배양 후, 시험물질과 대조군(Arbutin, 1 mM)을 처리하여 CO2 배양기에서 24시간 배양하였다. 각 웰의 배지를 제거한 후, PBS로 세척하고, 트립신을 처리하였다. 수집된 세포 펠렛에 1N NaOH 용액 100㎕를 넣고 60℃에서 1시간 동안 배양하였다. 1시간 배양하는 동안 합성 멜라닌을 이용한 멜라닌 standard curve와 BSA를 이용한 protein standard curve를 측정하였다. 다음으로 상기 Cell lysate의 멜라닌 색소의 양과 단백질 양을 측정하였고, 샘플 내의 멜라닌 색소의 양을 측정된 단백질의 양으로 보정하였다. First, 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. After removing the medium from each well, it was washed with PBS and treated with trypsin. 100 μl of 1N NaOH solution was added to the collected cell pellet and incubated at 60° C. for 1 hour. Melanin standard curve using synthetic melanin and protein standard curve using BSA were measured during 1 hour incubation. Next, the amount of melanin pigment and the amount of protein of the cell lysate were measured, and the amount of melanin pigment in the sample was corrected with the amount of the measured protein.
본 실시예 2의 멜라닌 정량시험은 도 2에 나타낸 멜라닌 색소 생성 메커니즘을 이용하여 수행된 것이다. 도 2에 나타낸 바와 같이, L-tyrosine을 첨가하면, 멜라닌 생성이 촉진되어 측정되는 멜라닌의 양이 증가한다. 그러나 L-tyrosine과 함께 tyrosinase inhibitor인 Arbutin을 처리하면 측정되는 멜라닌의 양은 감소한다.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.
결과는 도 3에 나타내었다. The results are shown in FIG. 3 .
도 3에 나타낸 바와 같이, 멜라닌 생성을 촉진하는 L-tyrosine 4 mM만을 처리하였을 때(C, control) 아무것도 처리하지 않은 음성대조군(-, Blank)에 대하여 멜라닌의 양이 약 6.6배 증가하였고, tyrosinase inhibitor인 arbutin 1 mM을 처리하였을 때(A, Arbutin 1 mM)에는 음성대조군(-, Blank)에 대하여 멜라닌의 양이 약 5.5배 증가되어 알부틴에 의해 멜라닌의 생성량이 감소됨을 확인하였다. 또한, 본 발명의 시험물질을 처리하는 경우 L-tyrosine만 처리한 군(C, Control)에 비해 시험물질의 농도에 의존적으로 멜라닌 생성량이 감소되었으며, 특히 시험물질 50 μg/mL에서 p<0.005 유의 수준으로 멜라닌 생성량이 감소하였다. 따라서, 본 발명의 시험물질은 멜라닌 생합성을 저해하여 미백에 효과적인 화장료 조성물로 사용될 수 있음을 확인하였다.As shown in Figure 3, when only 4 mM of L-tyrosine, which promotes melanin production, was treated (C, control), the amount of melanin was increased about 6.6 times with respect to the negative control group (-, Blank) that was not treated with anything, tyrosinase When the inhibitor, arbutin 1 mM, was treated (A, Arbutin 1 mM), the amount of melanin was increased about 5.5 times compared to the negative control group (-, Blank), and it was confirmed that the amount of melanin produced by arbutin was decreased. In addition, 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 μg/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.
실시예 3. 세포 내 Tysosinase activity 측정Example 3. Measurement of Intracellular Tysosinase Activity
본 발명자들은 본 발명의 시험물질(sample A)이 멜라닌 생성을 촉진하는 tyrosinase의 활성에 미치는 영향을 확인하고자, 다음과 같은 시험을 수행하였다. The present inventors performed the following test in order to confirm the effect of the test substance (sample A) of the present invention on the activity of tyrosinase that promotes melanin production.
먼저, B16F10 세포를 6 웰 플레이트에 5×104 cells/well이 되도록 24시간 배양하였다. L-tyrosine 4 mM을 음성대조군(C, Control)을 제외한 나머지 시료군에 모두 적용하였다. 1시간 후 각 웰에 본 발명의 시험물질을 10, 25, 50μg/ml 농도를 48시간 동안 처리하였다. PBS로 2회 세척한 후, 각 웰에 lysis buffer (1% triton X-100, 0.1 M sodium phosphate buffer, 50mM phenylmethylsulfonyl fluoride, pH 6.8)를 추가하였다. 얼음 위에서 세포를 용출시키고 13,500× g, 4℃, 30 분간 원심 분리한 후 상층액만 따로 모아 효소 용액으로 사용하였다. L-DOPA를 2 mg/ml 농도로 0.1 M sodium phosphate buffer (pH 6.8)에 녹여 기질을 준비하고, 상기 기질 160 μl에 상기에서 준비한 효소 용액 40μl를 가하고 37℃에서 1 시간 동안 가온하여 생성된 DOPA chrome의 양을 490 nm에서 흡광도를 측정한 후 억제율을 계산하였다. First, 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 μg/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. 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 μl of the enzyme solution prepared above was added to 160 μl 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.
결과는 도 4에 나타내었다. The results are shown in FIG. 4 .
도 4에 나타낸 바와 같이, B16F1 세포를 이용한 시료의 tyrosinase 활성을 분석한 결과, L-tyrosine 자극에 의해 증가된 tyrosinase 대비 양성대조군으로 사용한 arbutin에서 tyrosinase 활성이 68.69%로 유의적으로 감소하였다. 반면, 시료에 대한 tyrosinase 활성은 모든 농도에서 감소하는 경향을 보이지는 않았다. 상기 결과로부터 본 발명의 시험물질은 tyrosinase 활성의 저해보다는, 멜라닌의 생합성량을 저해하는 것을 알 수 있었다. 따라서, 본 발명의 시험물질은 멜라닌 생합성을 저해하여 미백에 효과적인 화장료 조성물로 사용될 수 있음을 확인하였다. As shown in FIG. 4 , as a result of analyzing the tyrosinase activity of the sample using B16F1 cells, the tyrosinase activity was significantly reduced to 68.69% in arbutin used as a positive control compared to tyrosinase increased by L-tyrosine stimulation. On the other hand, 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.
실시예 4. DOPA 산화반응 저해 효과 확인Example 4. Confirmation of DOPA oxidation inhibitory effect
본 발명자들은 본 발명의 시험물질(AMPEP-AP-1)이 멜라닌 색소의 합성과정의 속도 결정 단계에 관여하는 tyrosinase의 DOPA 산화 반응에 대한 활성 저해 효과를 측정함으로써 미백 효과를 평가하였다. Tyrosinase의 기질로는 L-DOPA를 사용하였다. DOPA 산화 반응 활성 저해 어세이(DOPA oxidation inhibition assay)는 tyrosinase의 작용에 의한 도파 크롬의 생성을 흡광도로 측정하여 미백 성분의 효과를 평가하는 시험법이다.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.
본 발명의 시험물질(AMPEP-AP-1) 및 양성대조군인 아스코르브산(ascorbic acid) 시료는 적당한 용매에 0, 10, 25, 50㎍/mL의 농도로 녹인 후 시험에 사용하였다.The 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 μg/mL, and then used for the test.
DOPA 산화 반응에 대한 tyrosinase 활성 저해를 확인할 수 있는 농도 범위를 알아보았다.The concentration range in which inhibition of tyrosinase activity for DOPA oxidation reaction can be confirmed was investigated.
96-well plate에 0.1M PBS (pH6.5)를 185㎕, mushroom tyrosinase (2,000 U/㎖) 5㎕, 각 시료 5㎕와 10mM L-DOPA 5㎕를 넣고 37℃에서 15분간 반응시켰다. 반응 후 spectrometer를 이용하여, 475nm에서 흡광도를 측정하였다. In a 96-well plate, 185 μl of 0.1M PBS (pH 6.5), 5 μl of mushroom tyrosinase (2,000 U/ml), 5 μl of each sample and 5 μl of 10 mM L-DOPA were added, and reacted at 37° C. for 15 minutes. After the reaction, the absorbance was measured at 475 nm using a spectrometer.
결과는 도 5에 나타내었다.The results are shown in FIG. 5 .
도 5에 나타낸 바와 같이, 각 시료의 DOPA 산화 저해 효능을 측정한 결과, 10㎍/mL의 농도에서 26.4%, 25㎍/mL의 농도에서 29.6%와 50㎍/mL에서 26.9%의 DOPA 산화 저해 효능을 나타냄을 확인하였다. 따라서, 본 발명의 시험물질(AMPEP-AP-1)은 DOPA의 산화반응을 억제하여 피부 미백 효과를 나타냄을 알 수 있었다. 5, as a result of measuring the DOPA oxidation inhibitory efficacy of each sample, DOPA oxidation inhibition of 26.4% at a concentration of 10 μg/mL, 29.6% at a concentration of 25 μg/mL, and 26.9% at a concentration of 50 μg/mL It was confirmed that the effect was shown. Therefore, it was found that the test substance (AMPEP-AP-1) of the present invention inhibits the oxidation reaction of DOPA, thereby exhibiting a skin whitening effect.
실시예 5. Real time PCR을 이용한 유전자 발현Example 5. Gene expression using real time PCR
인체 내에서 멜라닌 생성은 주로 산화적 반응이 원인이 되며, 이와 관련된 효소에는 tyrosinase, tyrosinase related protein-1(TRP-1), tyrosinase related protein-2(TRP-2) 등이 있다. 또한, MITF는 microphthalamia transcription factor로서 tyrosinase 및 TRPs의 M-box sequences에 결합하여 전사를 촉진한다. 본 발명자들은 본 발명의 시험물질(AMPEP-AP-1)이 멜라닌 합성에 관여하는 MITF와 TRP-1의 유전자 발현에 미치는 영향을 확인하기 위하여 다음과 같은 시험을 수행하였다. In the human body, 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. In addition, 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.
5-1. RNA 추출5-1. RNA extraction
먼저 B16F1 세포를 6-well plate에 1x107 cells/well로 24시간 배양하였다. 시료 2가지 (AMPEP-AP-1, arbutin)를 농도별 (10, 25, 50μg/mL)로 처리하였다. 배지 상등액을 제거한 후, trizol lysis buffer를 각 well에 500㎕씩 분주하여 세포를 분쇄하였다. 균질화된 용액을 상온 (15-25℃에 5분간 방치하였다. Chloroform 200㎕을 분주하여 15-20초 흔들어주며, 3분간 방치하였다. 그 후, 12,000 x g에서 15분간 원심분리하였다. 분리된 맑은 층인 aqueous phase를 새 tube에 옮겼다. 옮긴 tube에 100% isopropanol 500㎕ 첨가 후 실온에서 15분간 방치하였다. 10분간 12,000 x g에서 원심분리하였다. Pellet을 제외한 나머지 상등액을 모두 제거하였다. DEPC. D.W로 만든 70% ethanol을 pellet이 담긴 EP-tube에 1㎖ 첨가한 후 혼합하였다. 5분간 7,500 x g에서 원심분리 후, pellet을 제외한 상등액 모두 제거하는 과정을 2회 반복하였다. pellet이 담긴 tube를 실온에서 5-10분간 건조하였다. pellet만 남은 tube에 DEPC. DW를 20-30㎕ 첨가 후 혼합하였다. Nano-drop으로 1-2 ㎕ RNA를 취하여 정량하였다.First, 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 μg/mL). After removing the medium supernatant, 500 μl 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 μl 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. 500 μl of 100% isopropanol was added to the transferred tube, and then left at room temperature for 15 minutes. Centrifuged for 10 minutes at 12,000 x g. All supernatant except the pellet was removed. DEPC 70 made with DW After adding 1 ml of % ethanol to the EP-tube containing the pellet, it was mixed. After centrifugation at 7,500 x g for 5 minutes, the process of removing all the supernatant except the pellet was repeated twice. After drying for 10 minutes, 20-30 μl of DEPC and DW was added to the tube remaining in the pellet, and then mixed. 1-2 μl of RNA was quantified with a nano-drop.
5-2. Reverse transcription PCR5-2. Reverse transcription PCR
MITF와 TRP-1에 대한 mRNA 발현을 알아보기 위해 시험에 사용한 프라이머의 서열은 하기 표 1에 나타낸 것과 같았다.The sequences of the primers used in the test to examine the mRNA expression for MITF and TRP-1 were as shown in Table 1 below.
Figure PCTKR2021011497-appb-img-000002
Figure PCTKR2021011497-appb-img-000002
PCR tube에 green GoTaq Flexi buffer, MgCl2, PCR nucleotide mix (10 mM), primer, GoTaq DNA polymerase, nuclease free water에 상기 5-1에서 추출한 RNA로부터 합성한 cDNA를 첨가하였다. 각 PCR 튜브를 95℃ 30 sec, 56℃ 60 sec, 72℃ 1 min를 1 사이클로, 40 사이클 반응을 진행하였다. MITF, TRP-1 유전자의 발현된 양을 β-actin으로 보정하여 정량하였다.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 β-actin.
결과는 도 6(MIFT) 및 도 7(TRP-1)에 나타내었다.The results are shown in FIGS. 6 (MIFT) and 7 (TRP-1).
도 6에 나타낸 바와 같이, 본 발명의 시험물질(AMPEP-AP-1)은 10 및 25 μg/mL의 농도에서 멜라닌 색소를 생성하는 tyrosinase의 전사를 촉진시키는 MITF 유전자의 발현을 억제하였다.As shown in FIG. 6 , the 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 μg/mL.
또한, 도 7에 나타낸 바와 같이, 본 발명의 시험물질(AMPEP-AP-1)은 10 및 25 μg/mL의 농도에서 멜라닌 색소를 생성하는 tyrosinase의 전사를 촉진시키는 TRP-1 유전자의 발현을 억제하였다.In addition, as shown in FIG. 7 , the 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 μg/mL. did.
따라서, 상기 결과로부터, 본 발명의 시험물질 (AMPEP-AP-1)은 멜라닌 색소의 생성에 영향을 미치는 유전자의 발현을 억제하여 미백 효능이 있음을 알 수 있었다. Therefore, from the above results, it can be seen that the test substance (AMPEP-AP-1) of the present invention has a whitening effect by suppressing the expression of genes affecting the production of melanin.

Claims (6)

  1. 서열번호 1의 아미노산 서열로 이루어진 폴리펩타이드.A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
  2. 제1항의 폴리펩타이드를 유효성분으로 포함하는 피부미백용 화장료 조성물.A cosmetic composition for skin whitening comprising the polypeptide of claim 1 as an active ingredient.
  3. 제1항의 폴리펩타이드를 유효성분으로 포함하는 피부미백용 외용제 조성물. A composition for external application for skin whitening comprising the polypeptide of claim 1 as an active ingredient.
  4. 제1항의 폴리펩타이드를 유효성분으로 포함하는 색소침착의 예방, 개선, 치료 또는 저해용 약제학적 조성물. A pharmaceutical composition for preventing, improving, treating or inhibiting pigmentation comprising the polypeptide of claim 1 as an active ingredient.
  5. 제1항의 폴리펩타이드를 인코딩하는 핵산 분자.A nucleic acid molecule encoding the polypeptide of claim 1 .
  6. 제5항의 핵산 분자를 포함하는 재조합 벡터.A recombinant vector comprising the nucleic acid molecule of claim 5 .
PCT/KR2021/011497 2020-11-24 2021-08-27 Composition for skin lightening WO2022114461A1 (en)

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