WO2021125346A1 - Procédé d'activation de cellules souches cutanées par suppression de mpc1, et agent d'activation de cellules souches cutanées - Google Patents

Procédé d'activation de cellules souches cutanées par suppression de mpc1, et agent d'activation de cellules souches cutanées Download PDF

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WO2021125346A1
WO2021125346A1 PCT/JP2020/047530 JP2020047530W WO2021125346A1 WO 2021125346 A1 WO2021125346 A1 WO 2021125346A1 JP 2020047530 W JP2020047530 W JP 2020047530W WO 2021125346 A1 WO2021125346 A1 WO 2021125346A1
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extract
mpc1
skin stem
skin
stem cell
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俊介 入山
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株式会社 資生堂
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • 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
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • 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
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a method of activating skin stem cells by suppressing MPC1.
  • Patent Document 1 discloses a cell senescence inhibitor for stem cells containing astaxanthin.
  • Patent Document 2 discloses a stem cell-maintaining and activating agent for mesenchymal stem cells, which contains hydroxyproline or a pharmacologically acceptable salt thereof as an active ingredient.
  • Non-Patent Document 1 stem cells in the S phase increase at night when the NADH / NAD + ratio is high and glycolysis is predominant, but decrease during the daytime when the NADH / NAD + ratio is low and the electron transport chain is predominant. It has been reported (Non-Patent Document 2).
  • Non-Patent Document 3 In addition, in hair follicle cells genetically engineered to block the pathway to the electron transport chain by deleting the function of the mitochondrial pyruvate carrier (MPC1), stem cell proliferation is activated and the hair cycle is promoted. Was shown (Non-Patent Document 3).
  • Patent Document 3 discloses a beauty composition containing a mitochondrial transfer promoter, and its use in skin fibroblasts, epidermal keratinocytes, adipose-derived mesenchymal stem cells, epidermal stem cells and dermal stem cells can be used. Are listed.
  • Patent Document 4 includes exposing keratinocytes to a stressor, detecting metabolic indicators associated with glycolysis and oxidative phosphorylation, respectively, and providing a response to the stressor. Disclose a method for identifying a test substance as a skin care activator that improves metabolism.
  • Patent Document 5 non-lethally detects metabolic indicators associated with contacting cells with the first and second test substances, glycolysis and oxidative phosphorylation, respectively, and responds to the test substance with respect to the metabolic pathway. Disclosed are methods of identifying or evaluating synergistic combinations of active ingredients for use in cosmetic compositions, including providing.
  • An object of the present invention is to provide a method for activating skin stem cells, a skin stem cell activator, and a screening method for a skin stem cell activator.
  • the present application includes the following inventions: (1) A cosmetic method that activates skin stem cells by applying an MPC1 inhibitor. (2) The beauty method according to (1), wherein the MPC1 inhibitor contains at least one of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. (3) An MPC1 inhibitor containing at least one of akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. (4) A skin stem cell activator containing an MPC1 inhibitor.
  • Agent. (6) A method for screening a skin stem cell activator using the MPC1 inhibitory effect as an index. (7) Step of bringing the skin sample into contact with the candidate drug, A step of measuring the activity, amount, and / or expression level of MPC1 in a skin sample contacted with a candidate drug.
  • a step of determining the MPC1 inhibitory effect of a candidate drug from the measured MPC1 activity, amount, and / or expression level The step of selecting a skin stem cell activator based on the determined MPC1 inhibitory effect, The method according to (6).
  • an agent and a method effective for activating skin stem cells by suppressing MPC1 are provided. If skin stem cells are activated, it is effective in suppressing skin aging.
  • FIG. 1 shows the results of Experiment 1-1, in which the MCSP / B2M value when control (Cont), UK5099 (10 ⁇ M UK5099, 20 ⁇ M UK5099), and echinomycin (10nM Echinomycin, 20nM Echinomycin) were added was controlled (Cont). It is a graph showing a relative value with the result of) as 1.
  • FIG. 2 shows the results of Experiment 1-2 and is a photograph showing the expression of MCSP and DAPI when Control (Cont) and UK5099 (20 ⁇ M UK5099) were added, respectively.
  • FIG. 3 shows the results of Experiment 1-2 and is a photograph showing the expression of Integrin ⁇ 1 and DAPI when Control (Cont) and UK5099 (20 ⁇ M UK5099) were added, respectively.
  • FIG. 4 shows the results of Experiment 1-3, showing the number of Integrin ⁇ 1-positive cells when no antibody was added (Negative cont), when control was added (Cont), and when UK5099 was added (20 ⁇ M UK5099).
  • FIG. 5 shows the results of Experiment 1-4. Integrin ⁇ 6, MCSP, and DAPI in the tissue section of a three-dimensional cultured skin model in which control (Cont) and UK5099 (1 ⁇ M UK5099, 10 ⁇ M UK5099) were added and cultured for 4 days, respectively.
  • FIG. 6 shows the results of Experiments 1-5, showing the expression of Integrin ⁇ 6, MCSP, and DAPI in human skin organ culture model tissue sections cultured for 5 days with the addition of control (Cont) and UK5099 (10 ⁇ M UK5099), respectively. It is a photograph.
  • FIG. 7 shows the results of the primary screening of Experiment 2-2, and the MPC1 / B2M values when control (DMSO; Control) and each evaluation target sample (drug Nos. 1 to 124) were added, and the control results were shown. Shown as a relative value of 1.0.
  • FIG. 6 shows the results of Experiments 1-5, showing the expression of Integrin ⁇ 6, MCSP, and DAPI in human skin organ culture model tissue sections cultured for 5 days with the addition of control (Cont) and UK5099 (10 ⁇ M UK5099), respectively. It is a photograph.
  • FIG. 7 shows the results of the primary screening of Experiment 2-2, and the MPC1 / B2M values when control (DM
  • FIG. 8 shows the results of the secondary screening of Experiment 2-2, and the MPC1 / B2M values when each evaluation target sample (37 out of drug Nos. 1 to 124) selected in the primary screening was added.
  • the result of control (DMSO) is shown as a ratio (% of control) of 100.0.
  • FIG. 9 shows the results of the tertiary screening in Experiment 2-2, when the control (DMSO; Cont) and each evaluation target sample (15 out of drug Nos. 1 to 124) selected in the secondary screening were added.
  • the MPC1 / B2M value of is shown as a ratio (% of control) with the control result as 100.0.
  • FIG. 10 shows a graph in which the results of FIG. 9 were statistically processed by Dunnett's test for each of the 6 products selected in the tertiary screening of Experiment 2-2 (* P ⁇ 0.05, ** P ⁇ 0.01).
  • the present inventors have come up with the idea that skin stem cells are activated by MPC1 inhibition, and have discovered a novel substance having an MPC1 inhibitory effect.
  • akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine have a high MPC1 inhibitory effect.
  • the present invention describes MPC1 inhibitors and skin stem cell activators (hereinafter, these may be collectively referred to as "agents of the present invention"), screening methods thereof, and skin stem cells by applying them.
  • agents of the present invention provide a method of activating.
  • the method of the present invention may be for the purpose of cosmetology and may not be treated by a doctor or healthcare professional.
  • MPC1 is a protein that forms a heterodimer with MPC2 and forms a mitochondrial pyruvate carrier, which is a transporter that delivers pyruvate into the mitochondria.
  • MPC1 inhibition refers to reducing and / or inhibiting the action, production, and / or translation of MPC1 and / or reducing the activity, amount, and / or expression of MPC1.
  • the function of MPC1 is, for example, the function of MPC1 to deliver pyruvic acid into mitochondria.
  • MPC1 suppression results in, for example, the activity, amount, and / or expression level of MPC1 when the agent of the present invention is applied, as compared to the state (control) where the agent of the present invention is not applied.
  • Decreasing with a statistically significant difference eg Dunnett's test with a significance level of 5%, or, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% Above, it can mean that it is reduced by 60% or more, 70% or more, 80% or more, 90% or more, or 100%.
  • the gene expression level of MPC1 may be measured by using a quantitative PCR method as in the examples, or the protein expression level may be measured by staining with an antibody, and a commercially available kit may be used. , Or can be obtained by the method described in documents such as Patent Document 3, but the present invention is not limited to these, and any known technique can be used.
  • activation of skin stem cells refers to promotion of stem cell proliferation in skin cells such as keratinocytes and fibroblasts.
  • Stem cell proliferation can be determined by counting the number of stem cells, measuring the expression level of stem cell markers such as integrin ⁇ 1, and the like. If skin stem cells are activated, it can be expected to promote turnover, rejuvenate the skin, improve the texture of the skin, and recover from unfavorable skin conditions such as acne and pigmentation at an early stage.
  • the MPC1 inhibitor of the present invention may consist of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine, or may be contained as an active ingredient.
  • the substance is not limited to these as long as it can suppress MPC1.
  • the agent of the present invention may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.
  • the agent of the present invention may combine the above active ingredient with one or more other ingredients such as excipients, carriers and / or diluents.
  • Akebia quinata is a deciduous shrub belonging to the genus Akebia in the family Lardizabalaceae.
  • Akevi has a hyaluronic acid production promoting effect, a collagen production promoting effect, and an MMP inhibiting effect, and has a wrinkle formation preventing / improving effect, a lipase inhibiting activity, a blood triglyceride concentration increasing inhibitory activity, or an anti-obesity activity.
  • Japanese Patent Laid-Open No. 2015-17048, Japanese Patent Application Laid-Open No. 2010-265182, etc. has been reported (Japanese Patent Laid-Open No. 2015-17048, Japanese Patent Application Laid-Open No. 2010-265182, etc.).
  • an extract of akebi stem is preferable, but since the active ingredient is also contained in the fruit, peel, seed, leaf, flower, root and the like of akebi, any of these Or one or more extracts can also be used.
  • Kuromame (Glycine max'Kuromame') is a soybean variety that is an annual plant of the legume family, and each variety such as “Tamba black” and “Wachi black” can be used. It has been reported that it is rich in polyphenols such as anthocyanins, and that black soybean-derived components have anti-obesity, anti-inflammatory, antioxidant, glucose intolerant, and other effects (Japanese Patent Laid-Open No. 2015-140298). etc).
  • the black soybean extract is preferably an extract of black soybean legume, but since the active ingredient is also contained in the pod, leaves, stems, flowers, roots, etc. of black soybean, one or more of these are extracted. You can also use things.
  • Peony (Paeonia lactiflora) is a perennial plant of the Peony family. Peony has been reported to have an epidermal thickening inhibitory effect, a VEGF production promoting effect, an IGF-1 production promoting effect, an HGF production promoting effect, a BMP-2 production promoting effect, and the like (Japanese Patent Laid-Open No. 2009-11252, JP2012). -121856 Gazette, etc.).
  • the syrup extract the extract of syrup root is preferable, but since the active ingredient is also contained in the leaves, stems, flowers, fruits, pericarps, seeds, etc. of syrup, one or more of these are one or more. Extracts can also be used.
  • Tea is an evergreen tree belonging to the genus Camellia of the family Camellia, and various varieties such as tea plant (Camellia sinensis var. Sinnsis) and assamcha (Camellia sinensis var. Assamica) can be used.
  • tea plant Camellia sinensis var. Sinnsis
  • assamcha Camellia sinensis var. Assamica
  • Uji tea or the like can be used. It is known that tea contains abundant polyphenols such as catechin, and has lipid metabolism improving action, antioxidant action, anticancer action, blood pressure increase suppressing action and the like.
  • the tea extract is preferably an extract of tea leaves or stems, but since the active ingredient is also contained in tea nuts, flowers, roots, seeds, etc., one or more of these extracts. Can also be used.
  • Jojoba (Simmondsia chinansis) is an evergreen shrub belonging to the family Caryophyllales jojoba. Jojoba is known to have a moisturizing effect, an emollient effect, an anti-inflammatory effect, a collagenase inhibitory effect, an elastase inhibitory effect, a hyaluronidase inhibitory effect, and the like (Japanese Patent Laid-Open No. 2003-48846, JP-A-2003-048812, JP-A-2003-048812). 2003-03464 No. 4). Jojoba leaf extract is an extract of jojoba leaves, but since jojoba seeds, fruits, flowers, roots, etc. also contain active ingredients, one or more of these extracts should be used. It can also be used.
  • Ergothioneine is an amino acid having the following structure and a molecular weight of 229.3, and is known to have antioxidant, anti-inflammatory, elastase-inhibiting, tyrosinase-inhibiting, etc. (Japanese Patent Laid-Open No. 2019-149972). etc). Ergothioneine may be chemically synthesized, or it may be used in the form of an extract of such a natural product because it is also contained in microorganisms such as basidiomycetes, animals and plants.
  • the above-mentioned various extracts may be commercially available as cosmetic raw materials or health food materials, or may be obtained by a conventional method.
  • the extraction method is not particularly limited, and examples thereof include an extraction method using a solvent and an extraction method by hydrolysis.
  • the raw material When performing extraction using a solvent, the raw material is immersed or heated at room temperature or heated with an extraction solvent at room temperature or heated and refluxed, and then when extraction by hydrolysis is performed, for example, chemical treatment with alkali, acid, etc., heat. It can be obtained by performing arbitrary hydrolysis treatment such as physical treatment with pressure or the like, biological treatment with an enzyme or the like, and then filtering and concentrating.
  • the raw material can be used as it is, but if it is crushed into granules or powder and used for extraction, the active ingredient can be extracted in a short time with high extraction efficiency under mild conditions.
  • the extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, and the like. Usually, it is set in the range from room temperature to the boiling point of the solvent.
  • the extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, the extraction temperature, and the like. Further, at the time of extraction, stirring may be performed, the mixture may be allowed to stand without stirring, or ultrasonic waves may be applied.
  • any solvent can be used as long as it is usually used for extraction.
  • an aqueous solvent such as water, physiological saline, a phosphate buffer, a borate buffer, or an organic solvent such as ethanol can be used.
  • Alcohols such as propylene glycol, 1,3-butylene glycol and glycerin, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane and the like can be used alone or in combination.
  • the active ingredient is extracted and dissolved in a solvent or hydrolyzed.
  • the solvent or hydrolyzate containing the extract may be used as it is, or may be used after undergoing conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. If necessary, it may be concentrated by freeze-drying or diluted with an arbitrary solvent before use. Further, the solvent or the hydrolyzate may be completely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.
  • the squeezed liquid obtained by squeezing the raw material also contains the same active ingredient as the extract, the squeezed liquid can be used instead of the extract.
  • the present application also provides a composition containing the agent of the present invention.
  • the composition of the present invention may be a cosmetic composition or a food composition.
  • the composition of the present invention may be, for example, a composition that activates skin stem cells through an MPC1 inhibitory action.
  • the subject to which the agent or composition of the present invention is applied may be a subject that requires activation of skin stem cells from an objective or subjective viewpoint such as delayed skin turnover, skin aging, and pigmentation. , It may be a subject who desires prophylactic activation of skin stem cells.
  • the agent or composition of the present invention can be applied by any route such as external administration or oral administration, but it is preferably blended with an external preparation for skin that can be directly applied to the skin.
  • an external preparation for skin for example, liquid, emulsion, cream, solid, sheet, spray, gel, foam, powder and the like can be arbitrarily selected.
  • It may be a cosmetic composition such as a milky lotion, a cream, a beauty essence, a lotion, a facial mask, or a facial cleanser.
  • oral administration for example, tablets, supplements, beverages, powders and the like can be arbitrarily selected.
  • any compounding ingredients used in compositions such as cosmetics and pharmaceuticals can be appropriately blended as necessary, as long as the effects are not impaired.
  • optional compounding ingredients include excipients, carriers, diluents, oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, and ultraviolet absorbers.
  • examples include agents, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers and the like. For example, it may contain other medicinal ingredients that promote the activation of skin stem cells.
  • the frequency of administration is once every four weeks, once every two weeks, once a week, once every three days, once every two days, once a day, twice a day, three times a day. It can be arbitrarily selected, such as 4 times a day, 5 times a day, and each administration, but the present invention is not limited to these.
  • the possible forms of the agent or composition of the present invention are not limited to the above-mentioned dosage forms and forms.
  • the amount of the active ingredient such as akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine in the agent or composition of the present invention depends on the type, purpose, form, usage, etc. It can be decided as appropriate.
  • the blending amounts of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine are 0.0001 to 100% by weight, 0.0001 to 0.0001 to the total weight of the agent or composition of the present invention. It can be 90% by weight, 0.001 to 50% by weight, 0.01 to 5% by weight, 0.01 to 1% by weight, 0.1 to 0.5% by weight, etc., but is not limited as long as the effect of the present invention is exhibited.
  • the present application also provides a screening method for a skin stem cell activator using the MPC1 inhibitory action as an index.
  • the screening method of the present invention is a step of contacting a skin sample with a candidate drug; a step of measuring the activity, amount, and / or expression level of MPC1 in the skin sample contacted with the candidate drug; And / or a step of determining the MPC1 inhibitory action of the candidate drug from the expression level; a step of selecting a skin stem cell activator based on the determined MPC1 inhibitory action; may be included.
  • it becomes possible to screen whether or not a candidate drug has a skin stem cell activating effect and it becomes possible to develop products and propose new skin care.
  • the skin sample may be a skin sample after collection, for example, a skin sample in an ex vivo state after being collected from an animal such as a human, or a cultured skin cell, for example, a cultured keratinocyte or a cultured fibroblast. It may be in vitro, such as a cell. Alternatively, it may be an artificial skin sample such as a three-dimensional skin model. Skin samples are not limited as long as the MPC1 inhibitory effect can be measured.
  • the present application also provides akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine that activate skin stem cells through MPC1 inhibition.
  • Experiment 1 Effect of MPC1 inhibition on stem cells
  • Experiment 1-1 Analysis of MCSP gene expression by PCR method
  • the epidermis was used using UK5099, which is known as an MPC1 inhibitor.
  • the expression of the stem cell marker Melanoma-associated chondroitin sulfate proteoglycan (MCSP) was analyzed.
  • MCSP Melanoma-associated chondroitin sulfate proteoglycan
  • Equinomycin is an inhibitor of HIF-1 ⁇ that regulates mitochondrial activity like MPC1, but differs in that it is a substance that inhibits the conversion of pyruvate to acetyl-CoA in mitochondria.
  • Echinomycin (Abcam Echinomycin: product number ab144247) is prepared by preparing a stock solution diluted with DMSO to a concentration of 10 ⁇ M and 20 ⁇ M in advance, and added to the medium at a dilution of 1000 times to obtain a final concentration of 10 nM and 20 nM. did. The mRNA was recovered 24 hours after the addition of these substances.
  • the gene expression levels of MCSP and B2M were measured by the Cyber Green method using Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan). The primers used are as follows.
  • B2M forward 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1)
  • Experiment 1-2 Protein expression analysis of MCSP and Integrin ⁇ 1 by cell staining Furthermore, not only MCSP but also another stem cell marker, Integrin ⁇ 1, was stained and analyzed. Subcultured epidermal keratinized cells were added to 20 ⁇ M in the same manner as in Experiment 1-1, and the same amount of DMSO was added to the control without treatment with UK5099 to obtain 1.0 ⁇ 10 4 cells / well / 0.5. The cells were seeded in mL in a 4-well chamber slide (Falcon, 354114) and cultured for 48 hours. 48 hours after the addition, the cells were reacted with 4% PFA for 10 minutes and fixed.
  • the reaction was carried out with 0.01% Triton-X100 / PBS for 15 minutes to partially lyse the cell membrane. After blocking with 12% BSA / PBS, it was reacted overnight at 4 ° C with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti- ⁇ 1 integrin antibody (Santa Cruz, sc-13590, mouse mAb) as primary antibodies. .. The next day, the cells were washed 3 times with PBS for 5 minutes and then reacted with Alexa 488-anti-mouse IgG antibody as a secondary antibody at room temperature for 1 hour.
  • anti-MCSP antibody Micropore, MAB2029, mouse mAb
  • anti- ⁇ 1 integrin antibody Santa Cruz, sc-13590, mouse mAb
  • Experiment 1-3 FACS analysis of Integrin ⁇ 1-positive cells
  • FACS analysis of the number of Integrin ⁇ 1-positive cells was performed.
  • Subcultured epidermal keratinized cells were added to 20 ⁇ M in the same manner as in Experiment 1-1, and the same amount of DMSO was added to the control without treatment with UK5099 and cultured for 48 hours.
  • These cells were stripped with Trypsin and a cell suspension was prepared at a concentration of 1.0 ⁇ 10 6 cells / 500 uL. After removing the supernatant by centrifugation, blocking was performed with 0.5% BSA / PBS on ice for 10 minutes.
  • IgG antibody and Pacific blue-labeled anti- ⁇ 1 integrin antibody (BioLegend, 313620) were added to the cell suspension, reacted on ice for 1 hour, centrifuged 3 times with 0.1% BSA / PBS, and cell sorter. FACS analysis was performed on SH800. In addition, no antibody was added to the negative control (Negative cont), and the same treatment was performed.
  • Sections were prepared with a thickness of 3 ⁇ m using a microtome. After deparaffinization with xylene and blocking with 12% BSA / PBS, anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti- ⁇ 6 integrin antibody (Santa Cruz, sc-19622, rat mAb) were used as primary antibodies. The reaction was carried out at 4 ° C. overnight. The next day, after washing with PBS three times for 5 minutes, the cells were reacted with Alexa 488-anti-mouse IgG antibody and Alexa 594-anti-rat IgG antibody as secondary antibodies at room temperature for 1 hour. After washing with PBS for 5 minutes and 3 times, the cells were encapsulated with a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.
  • DAPI Vectashield, H-1200
  • Figure 5 shows the results of adding 1 ⁇ M and 5 ⁇ M UK5099 and controls, respectively.
  • the addition of UK5099 increased MCSP and Integrin ⁇ 1-expressing cells, which was consistent with the results of Experiments 1-1, 1-2, and 1-3 using the three-dimensional skin model.
  • Sections were prepared with a thickness of 3 ⁇ m using a microtome. After deparaffinization with xylene and blocking with 12% BSA / PBS, anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti- ⁇ 6 integrin antibody (Santa Cruz, sc-19622, rat mAb) were used as primary antibodies. The reaction was carried out at 4 ° C. overnight. The next day, after washing with PBS three times for 5 minutes, the cells were reacted with Alexa 488-anti-mouse IgG antibody and Alexa 594-anti-rat IgG antibody as secondary antibodies at room temperature for 1 hour. After washing with PBS for 5 minutes and 3 times, the cells were encapsulated with a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.
  • DAPI Vectashield, H-1200
  • Figure 6 shows the results of adding 10 ⁇ M UK5099 and control respectively.
  • the addition of UK5099 increased MCSP and Integrin ⁇ 1-expressing cells, which is consistent with the results of Experiments 1-1, 1-2, 1-3, and 1-4 using ex vivo human skin samples. It was.
  • Experiment 2 Screening of substances with MCP1 inhibitory effect
  • Experiment 1 suggested that inhibition of MCP1 activates stem cells. Therefore, we screened substances that have an MCP1 inhibitory effect.
  • Experiment 2-1 Sample preparation The following samples were used for evaluation of the MPC1 inhibitory effect.
  • a total of 124 types of candidate samples were prepared, including naturally derived components such as extracts of animals and plants and synthetic components.
  • Experiment 2-2 Evaluation of MCP1 inhibitory effect
  • normal human fetal-derived epidermal keratinocytes were cultured, and each drug was added 24 hours after seeding.
  • Candidate samples are prepared by preliminarily making 10% in DMSO and added to the medium at 1000-fold dilution in the primary and secondary screenings to a final concentration of 0.01%. The concentrations were adjusted to 0.001%, 0.01%, and 0.1%.
  • As a positive control UK5099 was added to 20 ⁇ M in the same manner as in Experiment 1.
  • DMSO was added as a negative control at a 1000-fold dilution. The mRNA was recovered 24 hours after the addition of each drug.
  • the gene expression levels of MPC1 and B2M were measured by the Cyber Green method using Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan).
  • the primers used are as follows.
  • B2M forward 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1)
  • the result of the primary screening is shown in FIG. Thirty-seven products (substances below the line shown in FIG. 7) were selected from 124 products as candidates for substances having an MPC1 inhibitory effect.
  • the result of the secondary screening is shown in FIG. Thirty-seven to fifteen substances (dark gray substances shown in FIG. 8) were selected as candidates for substances having an MPC1 inhibitory effect in a concentration-dependent manner.
  • the result of the tertiary screening is shown in FIG. From 15 products as candidates for substances that have a reproducible and concentration-dependent MPC1 inhibitory effect, 6 products: akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine (Fig. 9). (Dark gray substance shown in) was selected.
  • Fig. 10 shows a graph in which the results of Fig. 9 were statistically processed by Dunnett's test for each of the 6 selected products. As shown in FIG. 10, all the substances exerted an MPC1 inhibitory effect with a statistically significant difference in a concentration-dependent manner.
  • skin stem cells are activated by MCP1 inhibition, and akebi extract, black bean extract, sycamore extract, tea extract, jojoba leaf extract, and ergothioneine have an MCP1 inhibitory effect. I understood it. If skin stem cells are activated by the MCP1 inhibitor of the present invention, it is effective in suppressing skin aging.

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Abstract

L'invention concerne un procédé d'activation de cellules souches cutanées, un agent d'activation de cellules souches cutanées, et un procédé de criblage pour des agents d'activation de cellules souches cutanées.　L'application d'un suppresseur de MPC1 est efficace pour activer des cellules souches cutanées. Des exemples de tels suppresseurs de MPC1 comprennent des agents qui contiennent au moins un extrait d'akebia, un extrait de haricot noir, un extrait de pivoine, un extrait de thé, un extrait de feuilles de jojoba ou de l'ergothionéine en tant que principe actif. De plus, des agents d'activation de cellules souches cutanées peuvent être criblés par une action de suppression de MPC1 en tant qu'indice.
PCT/JP2020/047530 2019-12-20 2020-12-18 Procédé d'activation de cellules souches cutanées par suppression de mpc1, et agent d'activation de cellules souches cutanées WO2021125346A1 (fr)

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Citations (4)

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US20150099790A1 (en) * 2012-05-10 2015-04-09 University Of Utah Research Foundation Compositions and methods for modulating mitochondrial pyruvate carrier activity
KR20170025365A (ko) * 2015-08-28 2017-03-08 주식회사 엘지생활건강 피부 개선용 조성물
JP2018123130A (ja) * 2017-02-01 2018-08-09 ロート製薬株式会社 美容組成物及びミトコンドリアトランスファー促進剤
CN108392446A (zh) * 2018-04-16 2018-08-14 广州赛莱拉干细胞科技股份有限公司 基于泡叶藻提取物的护肤组合物及其应用

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KR20140012456A (ko) * 2012-07-20 2014-02-03 바이오스펙트럼 주식회사 아케비아 사포닌-디를 포함하는 피부 상태 개선용 조성물
WO2018039612A1 (fr) 2016-08-26 2018-03-01 The Regents Of The University Of California Compositions et méthodes favorisant la croissance des cheveux au moyen d'inhibiteurs de mpc1

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150099790A1 (en) * 2012-05-10 2015-04-09 University Of Utah Research Foundation Compositions and methods for modulating mitochondrial pyruvate carrier activity
KR20170025365A (ko) * 2015-08-28 2017-03-08 주식회사 엘지생활건강 피부 개선용 조성물
JP2018123130A (ja) * 2017-02-01 2018-08-09 ロート製薬株式会社 美容組成物及びミトコンドリアトランスファー促進剤
CN108392446A (zh) * 2018-04-16 2018-08-14 广州赛莱拉干细胞科技股份有限公司 基于泡叶藻提取物的护肤组合物及其应用

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