WO2022045261A1 - Inhibitor of downregulation of cd39 gene - Google Patents

Inhibitor of downregulation of cd39 gene Download PDF

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Publication number
WO2022045261A1
WO2022045261A1 PCT/JP2021/031403 JP2021031403W WO2022045261A1 WO 2022045261 A1 WO2022045261 A1 WO 2022045261A1 JP 2021031403 W JP2021031403 W JP 2021031403W WO 2022045261 A1 WO2022045261 A1 WO 2022045261A1
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expression
skin
gene
glucan
poe
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PCT/JP2021/031403
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French (fr)
Japanese (ja)
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純一 細井
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株式会社 資生堂
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Priority to JP2022540597A priority Critical patent/JPWO2022045261A1/ja
Priority to CN202180013807.7A priority patent/CN115066251A/en
Publication of WO2022045261A1 publication Critical patent/WO2022045261A1/en

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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/717—Celluloses
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A61K31/77—Polymers containing oxygen of oxiranes
    • 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)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
    • A61K8/86—Polyethers
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • 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]

Definitions

  • the present invention relates to a drug that suppresses a decrease in the expression of the CD39 gene.
  • a drug that suppresses a decrease in the expression of the CD39 gene By suppressing the decrease in the expression of the CD39 gene, the immune response of the skin can be regulated.
  • the skin is an organ located in the outermost layer of the living body and is constantly exposed to external stimuli such as ultraviolet rays, physical stimuli, chemical stimuli and biological invasion.
  • the skin has a three-layer structure of epidermis, dermis, and subcutaneous tissue.
  • the epidermis of the outermost layer is composed of keratinocytes (keratinocytes), Langerhans cells, melanocytes and the like.
  • the outermost epidermis has a function of protecting the human body from the external environment such as evaporation of water, invasion of foreign substances, and ultraviolet rays.
  • Externally stimulated keratinocytes release stimulatory response factors.
  • the surrounding keratinocytes that receive the signal of the released stimulus response factor secrete inflammatory cytokines.
  • Non-Patent Document 1 Immune cells are attracted by the secreted cytokines, causing inflammation at the stimulation site.
  • Non-Patent Document 1 Recent studies have revealed that Langerhans cells play an important role in skin immune function by antigen processing and antigen presenting ability. When an antigen as a foreign substance enters from the outside, the Langerhans cells promptly contact and process the antigen, move to the lymph node, present the antigen to the T cell, and elicit a series of subsequent immune response reactions. It has been shown that Langerhans cells contribute to the function of countering chemical stimuli and biological invasion.
  • Non-Patent Document 2 Langerhans cells also have the ability to express CD39, which acts as an ATPase, thereby reducing the amount of ATP, which is an extracellular stimulus response factor.
  • Extracellular ATP is recognized as one of the signals of the inflammatory process. Therefore, it is believed that Langerhans cells contribute to the sedation of inflammation through the degradation of extracellular ATP and reduce skin damage caused by ultraviolet rays and physical stimuli. It has been reported that when the number of Langerhans cells decreases due to zinc deficiency, the sedative function of the skin's response to external stimuli by CD39 molecules decreases, and the stimulus response becomes excessive, causing excessive inflammation (Non-Patent Documents). 3).
  • Patent Document 1 a drug screening method using the number of Langerhans cells as an index (Patent Document 1). ), And a drug or a Langerhans cell depletion inhibitor consisting of a specific plant extract has been developed (Patent Document 2). It is also disclosed that carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and rose water promote the gene expression of CD39 in Langerhans cells. (Patent Document 4).
  • An object of the present invention is to develop a drug capable of suppressing the decrease in the expression of the CD39 gene involved in the activation of Langerhans cells and regulating the immune response of the skin.
  • the present inventors conducted screening using the gene expression of CD39, which can be used as an index of activation of Langerhans cells, as an index.
  • the present invention relates to: [1-1] An agent for suppressing the decrease in the expression of the CD39 gene, which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract. [1-2] Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract for the production of an agent for suppressing the expression reduction of the CD39 gene. Use of 3 components consisting of.
  • a method for suppressing a decrease in the expression of the CD39 gene which comprises administering a composition containing the three components comprising the same.
  • Three components consisting of combined dimethyl ether and camellia extract.
  • a sedative agent for an external skin stimulus response which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
  • Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract for the production of a sedative for an external skin stimulus response.
  • Use of 3 ingredients consisting of.
  • a method for quelling an external skin stimulus response which comprises administering a composition comprising three components comprising.
  • Three components consisting of random copolymer dimethyl ether and camellia extract.
  • a sedative agent for a skin immune response which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
  • POP polyoxypropylene
  • a method for quelling a skin immune response which comprises administering a composition containing the three components.
  • Three components consisting of copolymer dimethyl ether and camellia extract.
  • a step of culturing Langerhans cells in the presence of testosterone and a candidate substance A method for screening an agent for suppressing the decrease in CD39 expression, which comprises a step of measuring the expression of the CD39 gene and a step of determining the CD39 expression promoting action of the candidate substance based on the expression level of the CD39 gene.
  • the inhibitor for reducing the expression of CD39 is a sedative for an external skin stimulus response.
  • the inhibitor for reducing the expression of CD39 is a skin immune response sedative.
  • the present invention can exert at least one of the effects of suppressing the decrease in the expression of the CD39 gene, increasing the expression of the CD39 gene, sedating the skin response by external stimuli, and sedating the skin immune response.
  • FIG. 1 when testosterone was applied to cultured monocytic cells (T), the expression of the CD39 gene was decreased as compared with the control (no T), while CM-glucan (G) and polyoxy were previously shown.
  • FIG. 5 is a graph showing that when ethylene (POE) 14 / polyoxypropylene (POP) 7 random copolymer dimethyl ether (A) was applied (+ GA), the decrease in the expression of the CD39 gene was suppressed.
  • the graph in FIG. 1 further shows the camellia extract (flowers: F, leaves:) in addition to CM-glucan (G) and polyoxyethylene (POE) 14 / polyoxypropylene (POP) 7 random copolymer dimethyl ether (A).
  • L was applied (+ GA + FL
  • the present invention is an inhibitor of CD39 gene expression reduction, which comprises three components consisting of carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
  • the present invention relates to a skin external stimulus response sedative agent and a skin immune response sedative agent.
  • CD39 refers to a protein also called ectnucleoside triphosphate diphosphohydrolase-1 (ENTPDl), which is an enzyme that hydrolyzes ATP / UTP and ADP / UDP to nucleosides such as AMP, respectively.
  • ENTPDl ectnucleoside triphosphate diphosphohydrolase-1
  • CD39 is a membrane protein having two transmembrane domains, and its expression is observed in Langerhans cells, T cell subsets, B cells, dendritic cells and the like. It is known that CD39 has an action of degrading extracellular ATP, and that the cell expressing CD39 has a role of calming inflammation by such action.
  • ATP which is degraded by the enzymatic activity of CD39, is used as a medium of energy inside the cell, but is a substance that plays a role as a signal transducing substance outside the cell.
  • ATP is known to act as a neurotransmitter in the nervous system.
  • ATP is released extracellularly in response to mechanical, chemical or ultraviolet stimuli, or when cells are destroyed, and acts on nearby immune cells to cause cytokines and chemokines. Is stimulated to produce.
  • immune cells are attracted, and extracellular ATP is considered to contribute to immune, allergic, and inflammatory reactions in the skin (Non-Patent Document 1). Therefore, by degrading extracellular ATP, cells expressing CD39 can sedate excessive responses to external stimuli of the skin, especially the epidermis, and excessive immune responses.
  • examples of cells expressing CD39 include melanocytes and Langerhans cells.
  • Langerhans cells are dendritic cells derived from bone marrow and are migratory cells existing in the upper layer from the spinous layer.
  • Langerhans cells are cells that express markers such as CD1a in addition to CD39.
  • the functions of Langerhans cells include the function of capturing and recognizing foreign substances invading from the outside world and presenting them to T cells, the function of recognizing cancer cells, the function of inducing immune tolerance, and the function of degrading extracellular ATP. , Langerhans cells are primarily involved in the immune response response in the skin.
  • the function of degrading extracellular ATP via CD39 enables the suppression of excessive response to external stimuli and excessive immune response in the skin, especially the epidermis (FRAGRANCE JOURNAL 2018). -1, 16-20).
  • the androgen testosterone is known to be produced in the skin in response to external stimuli (Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 107-123). It has been reported that the number of Langerhans cells in the skin is reduced by the action of testosterone produced in the skin (The Journal of Investigative Dermatology (1989) vol. 92, no. 1, 86-89). In addition, testosterone generally acts suppressively on the immune system (Cellular Immunology 294 (2015) 87-94).
  • the agent for suppressing the decrease in the expression of the CD39 gene of the present invention refers to an agent that suppresses the decrease in the expression of the CD39 gene in cells expressing the CD39 gene, particularly Langerhans cells.
  • the decreased expression of the CD39 gene in the present invention may be any cause, but as an example, it may be caused by testosterone generated in the skin in response to an external stimulus.
  • the agent for suppressing the decrease in the expression of the CD39 gene of the present invention can also be said to be the agent for promoting the expression of the CD39 gene.
  • the agent for suppressing the decrease in the expression of the CD39 gene can also be used as an external stimulus response sedative.
  • the external stimulus response sedative of the present invention sedates the response caused by external stimuli on the skin, especially the epidermis.
  • External skin stimuli include, for example, UV light, mechanical stimuli, thermal stimuli, cold stimuli, chemical stimuli and biological invasion.
  • keratinocytes in the epidermis release stimulatory response factors, especially ATP, extracellularly, and as explained above, extracellular ATP acts as a signaling substance, resulting in immune, allergic and inflammatory reactions. Occurs.
  • Mechanical stimuli include scratches, scrapes, insect bites, etc., such as scratches, lacerations, wounds, redness, swelling, etc.
  • Burns, chilblains, chilblains, frostbite, and frostbite occur as a result of heat and cold stimuli. Sunburn, burns, etc. occur as a result of UV stimulation.
  • Chemical irritation is triggered by chemicals that are irritating to the skin, such as acids, alkalis, organic solvents, etc., resulting in eczema, rashes, dermatitis and the like.
  • Biological invasion includes bacterial infections and the like.
  • Decreased CD39 gene expression by external stimuli results in an excessive immune response.
  • Excessive immune response is also involved in skin aging. Therefore, an agent that suppresses the decrease in the expression of the CD39 gene can also be said to be an agent that suppresses skin aging.
  • the skin external stimulus response sedative of the present invention may alleviate or alleviate the response to these external stimuli on the skin, especially the epidermis, such as inflammation, or itching, redness, pain, warmth, swelling, etc. caused by inflammation.
  • the external skin stimulus response sedative of the present invention may be an agent for alleviating, treating, or preventing the above-mentioned symptoms.
  • the skin external stimulus response sedative agent of the present invention may be a skin immune response sedative agent.
  • the external skin stimulus response sedative of the present invention does not directly suppress the stimulus response cascade in the skin, but indirectly stimulates the stimulus response by promoting the expression of the CD39 gene having an ATPase function.
  • the external stimulus response sedative agent enhances the function normally possessed by the skin of quelling an excessive response to an external stimulus, and can also be referred to as a stimulus response sedative function enhancer.
  • External stimulus response The sedative function enhancer is used in normal times when there is no inflammation, rather than being administered directly to the site of inflammation, and the inflammatory response when there is an external stimulus, such as itching, redness, and pain. It can function to control the feeling of heat, swelling, and rashes.
  • the external stimulus response sedative may be added to a pharmaceutical product, but is preferably added to a cosmetic product intended for daily use and used prophylactically against external stimuli. If the sedative agent for external skin stimulus response of the present invention is to be added to a pharmaceutical product, it may be used together with other anti-inflammatory agents to supplement the anti-inflammatory effect of such agents. Can act or enhance.
  • the skin immune response sedative agent of the present invention is a drug that calms the immune response in the skin, and particularly contributes to the suppression of the immune response caused by the decrease of Langerhans cells and the increase of extracellular ATP, particularly the suppression of excessive inflammation. can do.
  • Excessive inflammation includes chronic dermatitis, such as atopic dermatitis, psoriasis, erythema, and acute dermatitis, such as contact dermatitis such as rash, seborrheic dermatitis, and sunlight dermatitis.
  • an excessive reaction such as redness and itch that occurs when the skin receives various external stimuli occurs. It becomes difficult and leads to the treatment or prevention of the onset of inflammatory skin diseases.
  • skin diseases such as atopic dermatitis
  • the symptoms are exacerbated by scratching the affected area accompanied by itching, so it is useful for treatment to make it difficult for an excessive inflammatory reaction to occur.
  • the carboxymethyl (CM) beta-glucan used in the present invention is usually a beta-glucan modified to be water-soluble by carboxymethylating an insoluble beta-glucan.
  • Carboxymethyl beta-glucans may be produced on the basis of or synthetically from beta-glucans derived from any plant, fungus or bacterium. Natural beta-glucans may be produced from mushrooms such as Agaricus, Reishi, Schizophyllum communis, and yeast.
  • the weight average molecular weight of carboxymethyl beta-glucan is, for example, 1,000 to 5,000,000 daltons depending on its origin, and in the case of yeast-based carboxymethyl beta-glucan, the upper limit is 5,000.
  • the carboxymethyl beta glucan may be in the form of any salt, for example a sodium salt, a potassium salt, an ammonium salt, a triethanolamine salt and the like.
  • CM-glucan Mobelle Biochemistry which is a carboxymethyl beta-glucan sodium can be used, but the present invention is not limited thereto.
  • the blending amount may vary depending on the dosage form of the cosmetic or pharmaceutical product to be blended, but from the viewpoint of exerting sufficient efficacy, for example, 0.0001% by mass or more, preferably 0.0002% by mass or more, more preferably 0.002. It is blended in an amount of% by mass or more. On the other hand, from the viewpoint of solubility, it is used in an amount of 0.5% by mass or less, more preferably 0.1% by mass or less, still more preferably 0.01% by mass or less.
  • the average number of moles of polyoxypropylene (POP) used in the polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether used in the present invention is 2 to 50, and the average number of polyoxyethylene (POE) is 2.
  • POE and POP are abbreviations for polyoxyethylene and polyoxypropylene, respectively, and may be abbreviated as described below.
  • the molar ratio of polyoxyethylene to polyoxypropylene used in the polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether is set to 1: from the viewpoint of obtaining a polymer exhibiting a desired action.
  • the range is 5 to 5: 1, preferably 1: 3 to 3: 1, and more preferably 1: 2 to 2: 1.
  • a POE: POP ratio of 2: 1 can be used.
  • the polyoxypropylene / polyoxyethylene random copolymer dialkyl ether can be produced by a known method (Patent Document 3).
  • the blending amount may vary depending on the dosage form of the cosmetic or pharmaceutical product to be blended, but from the viewpoint of exerting sufficient efficacy, for example, 0.01% by mass or more, preferably 0.05% by mass or more, more preferably 0. It is blended in an amount of 1% by mass or more. On the other hand, from the viewpoint of solubility, it is used in an amount of 3% by mass or less, more preferably 1% by mass or less, still more preferably 0.5% by mass or less.
  • the polyoxyethylene / polyoxypropylene random copolymer dialkyl ether used in the present invention is known to have a moisturizing effect alone or in combination with other components, but the polyoxyethylene / polyoxypropylene random
  • the effect of the copolymer dialkyl ether on the expression of the CD39 gene is unknown.
  • the camellia extract used in the present invention refers to an extract obtained from a plant belonging to the genus Camellia of the Theaceae family.
  • Camellia family Camellia plant include Camellia japonica, and related species such as Camellia rusticana, Camellia sasanqua, and hybrid species.
  • Camellia japonica is native to Japan, and extracts can be extracted from its plants such as flowers, fruits, leaves, branches, trees and seeds.
  • an organic solvent or an aqueous solvent can be used, for example, alcohols such as butylene glycol, propylene glycol, ethanol and glycerin, ethers such as diethylene glycol monoethyl ether, esters such as ethyl acetate and butyl acetate, or Water or any mixture thereof can be used.
  • camellia flower extract can be obtained by extracting camellia flowers with a 50% aqueous solution of butylene glycol.
  • the camellia leaf extract can be obtained by extracting camellia leaves with a 50% aqueous solution of butylene glycol.
  • the blending amount is, for example, 0.0001% (v / v) or more, preferably 0.0002% (v / v) or more, and more preferably 0.0005% (v / v) from the viewpoint of exhibiting sufficient efficacy. ) It is mixed with the above. On the other hand, from the viewpoint of stability and safety, it is used at 5% (v / v) or less, more preferably 3% (v / v) or less, still more preferably 1% (v / v) or less.
  • the compounding ratio of carboxymethyl beta-glucan or a salt thereof used in the present invention, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract is 2 of carboxymethyl beta-glucan or a salt thereof.
  • the blending amount of the% aqueous solution is 1, it is 1: 1 to 100: 1 to 100, preferably 1: 1 to 20: 1 to 20, and more preferably 1:10:10.
  • the three components of carboxymethyl beta glucan or a salt thereof used in the present invention, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract may be blended into one pharmaceutical product. However, it may be compounded in a plurality of formulations and administered sequentially.
  • the inhibitor of the expression decrease of the CD39 gene of the present invention, the external stimulus response sedative agent, and the skin immune response sedative agent may be incorporated into cosmetics, pharmaceuticals or quasi-drugs, respectively. These agents may be administered orally or parenterally, eg, transdermally. When administered transdermally, it can be formulated into a skin external preparation.
  • the external skin preparation is not particularly limited as long as it is applicable to the skin, and is, for example, solution-like, emulsified-like, solid-like, semi-solid-like, powder-like, powder-dispersed, water-oil two-layer separated form, water-oil. -Any dosage form such as powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc.
  • a base usually used for the skin external preparation and an excipient such as a preservative, an emulsifier, a pH adjuster and the like may be used, and further, an anti-inflammatory component.
  • steroids, antihistamines and the like may be further added.
  • facial or body cosmetics such as lotions, milky lotions, serums, creams, lotions, packs, essences, gels, makeup cosmetics such as foundations, makeup bases, and concealers, and even It can be added to bathing agents and the like.
  • the cosmetics containing the ingredients of the present invention the inflammatory reaction can be alleviated or prevented, so that it is possible to maintain healthy skin that is less likely to cause redness, itchiness and rashes.
  • the present invention also presents an inhibitor of the expression reduction of the CD39 gene of the present invention, an external stimulus response sedative, and a skin immune response sedative, that is, carboxymethyl beta glucan or a salt thereof, polyoxyethylene (POE). / Polyoxypropylene (POP) Random copolymer dimethyl ether, including administration of three components consisting of camellia extract, cosmetic method, external stimulus response sedation method, healthy skin maintenance method, inflammation treatment method good.
  • the administration of the three components of the present invention may be carried out by using a skin external preparation or cosmetic containing all the components, or a skin external preparation or a cosmetic containing individual components. It may be done by using the cosmetics continuously.
  • the invention also relates to a method of screening for an agent that suppresses decreased expression of the CD39 gene.
  • the screening method is as follows: The step of culturing CD39-positive cells in the presence of testosterone and candidate substances, It includes a step of measuring the expression of the CD39 gene and a step of determining the inhibitory effect on the decrease in the expression of the CD39 gene of the candidate substance based on the expression level of the CD39 gene. Testosterone may be added to the culture of CD39-positive cells cultured in the presence of the candidate substance or by medium replacement.
  • the inhibitory effect on the decrease in the expression of the CD39 gene of the candidate substance can be determined by measuring the expression level of the CD39 gene.
  • the amount of mRNA may be measured by a known method, or the amount of protein expressed by a known method may be measured.
  • the inhibitory effect on the decrease in the expression of the CD39 gene can also be said to be the effect of promoting the expression of the CD39 gene.
  • the agent for suppressing the decrease in the expression of the CD39 gene screened in this way can also be referred to as a sedative for an external skin stimulus response or a sedative for a skin immune response.
  • the CD39-positive cells Langerhans cells or alternative cells of Langerhans cells, for example, THP-1 cells can be used.
  • THP-1 Human Monosphere Cell Lines, Virginia USA
  • FCS fetal bovine serum
  • FCS Santa Ana, CA
  • non-essential amino acids and essential amino acids Nacalai Tesque, Japan
  • RPMI 1640 medium Nacalai Tesque, Japan
  • each test substance was as follows: Group A (noT): PBS, EtOH only Group B (T): 1 ng / ml testosterone (TST) Group C (+ GA): 0.01% carboxymethyl beta glucan (CM-Gulucan, Mibelle Biochemistry), 1 ng / mL TST, and 0.1% polyoxyethylene (POE) 14 / polyoxypropylene (POP) 7 random. Copolymer dimethyl ether group D (+ GA + FL0.001): 0.01% carboxymethyl beta glucan (CM-Gulucan, Mibelle Biochemistry) and 1 ng / mL TST, 0.1% polyoxyethylene (POE) 14 / polyoxypropylene.
  • CD39 The expression level of CD39 was normalized by ribosomal protein S9 (rpS9), and a statistically significant difference was determined by ANOVA and post-hook Tukey's test. The results are shown in FIG.

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Abstract

The present invention addresses the problem of developing of a drug capable of inhibiting downregulation of a CD39 gene involved in activation of Langerhans cells and modulating an immune response of the skin. The invention is based on the discovery that a composition that includes carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE)-polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract inhibits downregulation of a CD39 gene by testosterone.

Description

ＣＤ３９遺伝子の発現低下の抑制剤Suppressor of decreased expression of CD39 gene
　本発明は、ＣＤ３９遺伝子の発現低下を抑制する薬剤に関する。ＣＤ３９遺伝子の発現低下の抑制により、皮膚の免疫応答を調節することができる。 The present invention relates to a drug that suppresses a decrease in the expression of the CD39 gene. By suppressing the decrease in the expression of the CD39 gene, the immune response of the skin can be regulated.
　皮膚は生体の最外層に位置する臓器であり、外部からの刺激、例えば紫外線、物理的刺激、化学的刺激及び生物学的侵襲に常に晒されている。皮膚は、表皮、真皮、皮下組織の三層構造をとっている。このうち、最外層の表皮は、角化細胞（ケラチノサイト）、ランゲルハンス細胞、メラノサイトなどにより構成されている。最外層の表皮は、水分の蒸発や異物の侵入、紫外線などの外的環境から人体を防御する機能を有している。外部刺激を受けたケラチノサイトは、刺激応答因子を放出する。放出された刺激応答因子の信号を受けた周囲のケラチノサイトが、炎症性のサイトカインを分泌する。分泌されたサイトカインにより免疫細胞が誘引されて、刺激部位に炎症が引き起こされる（非特許文献１）。また、近年の研究により、ランゲルハンス細胞が、抗原処理、抗原提示能力によって皮膚免疫機能における重要な役割を果たすことが分かってきている。外部からの異物としての抗原が進入すると、ランゲルハンス細胞は、すみやかに抗原と接触して処理し、リンパ節へ移動してＴ細胞に抗原を提示し、以後の一連の免疫応答反応を惹起する。それにより、ランゲルハンス細胞は、化学的刺激や生物学的侵襲に対抗する機能に寄与することが分かってきている。一方で、ランゲルハンス細胞は、ＡＴＰａｓｅとして働くＣＤ３９を発現し、それにより細胞外の刺激応答因子であるＡＴＰ量を低下させる能力も有している（非特許文献２）。細胞外ＡＴＰは、炎症過程のシグナルの一つとして認知されている。したがって、ランゲルハンス細胞が細胞外ＡＴＰの分解を介して、炎症の鎮静化に寄与し、紫外線や物理的刺激による皮膚障害を低減すると考えられている。亜鉛欠乏によりランゲルハンス細胞の数が減少すると、ＣＤ３９分子による外部刺激に対する皮膚の応答の鎮静化機能が低下し、刺激応答が過剰となって過剰な炎症を引き起こすことが報告されている（非特許文献３）。 The skin is an organ located in the outermost layer of the living body and is constantly exposed to external stimuli such as ultraviolet rays, physical stimuli, chemical stimuli and biological invasion. The skin has a three-layer structure of epidermis, dermis, and subcutaneous tissue. Of these, the epidermis of the outermost layer is composed of keratinocytes (keratinocytes), Langerhans cells, melanocytes and the like. The outermost epidermis has a function of protecting the human body from the external environment such as evaporation of water, invasion of foreign substances, and ultraviolet rays. Externally stimulated keratinocytes release stimulatory response factors. The surrounding keratinocytes that receive the signal of the released stimulus response factor secrete inflammatory cytokines. Immune cells are attracted by the secreted cytokines, causing inflammation at the stimulation site (Non-Patent Document 1). In addition, recent studies have revealed that Langerhans cells play an important role in skin immune function by antigen processing and antigen presenting ability. When an antigen as a foreign substance enters from the outside, the Langerhans cells promptly contact and process the antigen, move to the lymph node, present the antigen to the T cell, and elicit a series of subsequent immune response reactions. It has been shown that Langerhans cells contribute to the function of countering chemical stimuli and biological invasion. On the other hand, Langerhans cells also have the ability to express CD39, which acts as an ATPase, thereby reducing the amount of ATP, which is an extracellular stimulus response factor (Non-Patent Document 2). Extracellular ATP is recognized as one of the signals of the inflammatory process. Therefore, it is believed that Langerhans cells contribute to the sedation of inflammation through the degradation of extracellular ATP and reduce skin damage caused by ultraviolet rays and physical stimuli. It has been reported that when the number of Langerhans cells decreases due to zinc deficiency, the sedative function of the skin's response to external stimuli by CD39 molecules decreases, and the stimulus response becomes excessive, causing excessive inflammation (Non-Patent Documents). 3).
　したがって、ランゲルハンス細胞を増加又は活性化させることにより、皮膚免疫機能の亢進、炎症の低下や日焼けなどの皮膚障害の低減が期待できることから、ランゲルハンス細胞数を指標とした薬剤のスクリーニング方法（特許文献１）、及び特定の植物抽出物からなる医薬又はランゲルハンス細胞減少抑制剤の開発が行われている（特許文献２）。また、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びローズ水によって、ランゲルハンス細胞におけるＣＤ３９の遺伝子発現が促進されることが開示されている（特許文献４）。 Therefore, by increasing or activating Langerhans cells, it can be expected to enhance skin immune function, reduce inflammation, and reduce skin disorders such as sunburn. Therefore, a drug screening method using the number of Langerhans cells as an index (Patent Document 1). ), And a drug or a Langerhans cell depletion inhibitor consisting of a specific plant extract has been developed (Patent Document 2). It is also disclosed that carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and rose water promote the gene expression of CD39 in Langerhans cells. (Patent Document 4).
特開２０００－２３６７７５号公報Japanese Unexamined Patent Publication No. 2000-236775 特開２０００－２３９１４４号公報Japanese Unexamined Patent Publication No. 2000-239144 特開２００４－８３５４１号公報Japanese Unexamined Patent Publication No. 2004-83541 特表２０１７－５１１７９０号公報Special Table 2017-511790
　本発明の課題は、ランゲルハンス細胞の活性化に関与するＣＤ３９遺伝子の発現低下を抑制し、皮膚の免疫応答を調節することができる薬剤を開発することにある。 An object of the present invention is to develop a drug capable of suppressing the decrease in the expression of the CD39 gene involved in the activation of Langerhans cells and regulating the immune response of the skin.
　本発明者らは、ランゲルハンス細胞を活性化の指標として使用可能なＣＤ３９の遺伝子発現を指標として、スクリーニングを行ったところ、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル及びカルボキシメチル・ベータグルカン・ナトリウムが、テストステロン適用によるランゲルハンス細胞におけるＣＤ３９の遺伝子発現の低下を抑制することができることを見いだした。さらに、驚くべきことに、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、カルボキシメチル・ベータグルカン・ナトリウム、及びツバキエキスを組み合わせて用いた場合に、テストステロン適用によるＣＤ３９遺伝子の発現低下の影響を完全に抑制し、むしろＣＤ３９遺伝子発現を増大させることができることを見出し、下記の本発明に至った。 The present inventors conducted screening using the gene expression of CD39, which can be used as an index of activation of Langerhans cells, as an index. As a result, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether and It was found that carboxymethyl betaglucan sodium can suppress the decrease in the gene expression of CD39 in Langerhans cells due to the application of testosterone. Furthermore, surprisingly, the CD39 gene by testosterone application when used in combination with polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, carboxymethyl beta-glucan sodium, and camellia extract. It was found that the influence of the decrease in the expression of glucan can be completely suppressed, and rather the expression of the CD39 gene can be increased, leading to the following invention.
　したがって、本発明は以下のものに関する：
［１－１］　カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスを含む、ＣＤ３９遺伝子の発現低下の抑制剤。
［１－２］　ＣＤ３９遺伝子の発現低下の抑制剤の製造のための、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分の使用。
［１－３］　ＣＤ３９遺伝子の発現低下の抑制を必要とする対象において、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分を含む組成物を投与することを含むＣＤ３９遺伝子の発現低下の抑制方法。
［１－４］　ＣＤ３９遺伝子の発現低下を介して、皮膚炎症の治療又は予防において使用するための、ルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分。
［２］　ＣＤ３９遺伝子の発現低下が、外部刺激に起因する、項目１－１に記載の抑制剤、項目１－２に記載の使用、項目１－３に記載の方法、項目１－４に記載の３成分。
［３］　ＣＤ３９遺伝子の発現低下が、テストステロンの増加に起因する、項目１－１に記載の抑制剤、項目１－２に記載の使用、項目１－３に記載の方法、項目１－４に記載の３成分。
［４］　皮膚のランゲルハンス細胞においてＣＤ３９遺伝子発現低下を抑制する、項目１－１、１－２、１－３、１－４、２及び３のいずれか一項に記載の抑制剤、使用、方法、又は３成分。
［５－１］　カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスを含む、皮膚外部刺激応答の鎮静化剤。
［５－２］　皮膚外部刺激応答の鎮静化剤の製造のための、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分の使用。
［５－３］　皮膚外部刺激応答の鎮静化を必要とする対象において、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分を含む組成物を投与することを含む、皮膚外部刺激応答の鎮静化方法。
［５－４］　皮膚外部刺激応答の鎮静化を介して、皮膚炎症の治療又は予防において使用するための、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分。
［６－１］　カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスを含む、皮膚免疫応答の鎮静化剤。
［６－２］　皮膚免疫応答の鎮静化剤の製造のための、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分の使用。
［６－３］　皮膚免疫応答の鎮静化を必要とする対象において、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分を含む組成物を投与することを含む皮膚免疫応答の鎮静化方法。
［６－４］　皮膚免疫応答の鎮静化を介して、皮膚炎症の治療又は予防において使用するための、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分。
［７］　テストステロン及び候補物質の存在下でランゲルハンス細胞を培養する工程、
　ＣＤ３９遺伝子の発現を測定する工程、及び
　ＣＤ３９遺伝子の発現量に基づき、候補物質のＣＤ３９発現促進作用を決定する工程
を含む、ＣＤ３９発現低下の抑制剤のスクリーニング方法。
［８］　ＣＤ３９発現低下の抑制剤が、皮膚外部刺激応答の鎮静化剤である、項目７に記載の方法。
［９］　ＣＤ３９発現低下の抑制剤が、皮膚免疫応答鎮静化剤である、項目７に記載の方法。 Therefore, the present invention relates to:
[1-1] An agent for suppressing the decrease in the expression of the CD39 gene, which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
[1-2] Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract for the production of an agent for suppressing the expression reduction of the CD39 gene. Use of 3 components consisting of.
[1-3] Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract in subjects requiring suppression of decreased expression of the CD39 gene. A method for suppressing a decrease in the expression of the CD39 gene, which comprises administering a composition containing the three components comprising the same.
[1-4] Luboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random co-weight for use in the treatment or prevention of skin inflammation through decreased expression of the CD39 gene. Three components consisting of combined dimethyl ether and camellia extract.
[2] The inhibitor according to item 1-1, the use according to item 1-2, the method according to item 1-3, and item 1-4, wherein the decreased expression of the CD39 gene is caused by an external stimulus. 3 ingredients.
[3] The inhibitor according to item 1-1, the use according to item 1-2, the method according to item 1-3, and item 1-4, in which the decrease in the expression of the CD39 gene is caused by the increase in testosterone. The three components described.
[4] The inhibitor, use, and method according to any one of items 1-1, 1-2, 1-3, 1-4, 2 and 3, which suppresses the decrease in CD39 gene expression in Langerhans cells of the skin. , Or 3 components.
[5-1] A sedative agent for an external skin stimulus response, which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
[5-2] Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract for the production of a sedative for an external skin stimulus response. Use of 3 ingredients consisting of.
[5-3] Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract in subjects requiring sedation of external skin stimulus response. A method for quelling an external skin stimulus response, which comprises administering a composition comprising three components comprising.
[5-4] Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP), for use in the treatment or prevention of skin inflammation through sedation of the external skin stimulus response. Three components consisting of random copolymer dimethyl ether and camellia extract.
[6-1] A sedative agent for a skin immune response, which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
[6-2] From carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract for the production of a sedative agent for skin immune response. Use of 3 ingredients.
[6-3] From carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract in subjects requiring sedation of the skin immune response. A method for quelling a skin immune response, which comprises administering a composition containing the three components.
[6-4] Carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random for use in the treatment or prevention of skin inflammation through sedation of the skin immune response. Three components consisting of copolymer dimethyl ether and camellia extract.
[7] A step of culturing Langerhans cells in the presence of testosterone and a candidate substance,
A method for screening an agent for suppressing the decrease in CD39 expression, which comprises a step of measuring the expression of the CD39 gene and a step of determining the CD39 expression promoting action of the candidate substance based on the expression level of the CD39 gene.
[8] The method according to item 7, wherein the inhibitor for reducing the expression of CD39 is a sedative for an external skin stimulus response.
[9] The method according to item 7, wherein the inhibitor for reducing the expression of CD39 is a skin immune response sedative.
　本発明は、ＣＤ３９遺伝子の発現低下の抑制、ＣＤ３９遺伝子の発現増加、外部刺激による皮膚応答鎮静化及び皮膚免疫応答鎮静化のうちの少なくとも１以上の効果を発揮することができる。 The present invention can exert at least one of the effects of suppressing the decrease in the expression of the CD39 gene, increasing the expression of the CD39 gene, sedating the skin response by external stimuli, and sedating the skin immune response.
図１は、培養単球細胞に対してテストステロンを適用した場合（Ｔ）に、対照（ｎｏ　Ｔ）と比較してＣＤ３９遺伝子の発現低下がみられる一方、予めＣＭ－グルカン（Ｇ）及びポリオキシエチレン（ＰＯＥ）１４／ポリオキシプロピレン（ＰＯＰ）７ランダム共重合体ジメチルエーテル（Ａ）を適用していた場合には（＋ＧＡ）、ＣＤ３９遺伝子の発現低下が抑制されることを示すグラフである。図１のグラフは、さらに、ＣＭ－グルカン（Ｇ）及びポリオキシエチレン（ＰＯＥ）１４／ポリオキシプロピレン（ＰＯＰ）７ランダム共重合体ジメチルエーテル（Ａ）に加えてツバキエキス（花：Ｆ、葉：Ｌ）を適用していた場合には（＋ＧＡ＋ＦＬ）、ＣＤ３９遺伝子の発現が増加することを示す。In FIG. 1, when testosterone was applied to cultured monocytic cells (T), the expression of the CD39 gene was decreased as compared with the control (no T), while CM-glucan (G) and polyoxy were previously shown. FIG. 5 is a graph showing that when ethylene (POE) 14 / polyoxypropylene (POP) 7 random copolymer dimethyl ether (A) was applied (+ GA), the decrease in the expression of the CD39 gene was suppressed. The graph in FIG. 1 further shows the camellia extract (flowers: F, leaves:) in addition to CM-glucan (G) and polyoxyethylene (POE) 14 / polyoxypropylene (POP) 7 random copolymer dimethyl ether (A). When L) was applied (+ GA + FL), it is shown that the expression of the CD39 gene is increased.
　本発明は、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分を含む、ＣＤ３９遺伝子発現低下の抑制剤、皮膚外部刺激応答鎮静化剤、及び皮膚免疫応答鎮静化剤に関する。 The present invention is an inhibitor of CD39 gene expression reduction, which comprises three components consisting of carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract. The present invention relates to a skin external stimulus response sedative agent and a skin immune response sedative agent.
　ＣＤ３９は、エクトヌクレオシド三リン酸塩ジホスホヒドロラーゼ－１（ＥＮＴＰＤｌ）とも呼ばれるタンパク質を指し、ＡＴＰ／ＵＴＰおよびＡＤＰ／ＵＤＰを、それぞれ、ＡＭＰなどのヌクレオシドに加水分解する酵素である。ＣＤ３９は、２つの膜貫通ドメインを有する膜タンパク質であり、ランゲルハンス細胞の他、Ｔ細胞サブセット、Ｂ細胞、樹状細胞などで発現が認められる。ＣＤ３９は細胞外のＡＴＰを分解する作用を有し、かかる作用により、ＣＤ３９を発現する細胞が、炎症を鎮静化する役割を有することが分かっている。 CD39 refers to a protein also called ectnucleoside triphosphate diphosphohydrolase-1 (ENTPDl), which is an enzyme that hydrolyzes ATP / UTP and ADP / UDP to nucleosides such as AMP, respectively. CD39 is a membrane protein having two transmembrane domains, and its expression is observed in Langerhans cells, T cell subsets, B cells, dendritic cells and the like. It is known that CD39 has an action of degrading extracellular ATP, and that the cell expressing CD39 has a role of calming inflammation by such action.
　ＣＤ３９の酵素活性により分解されるＡＴＰは、細胞内ではエネルギーの媒体として使用されているが、細胞外では情報伝達物質としての役割を果たす物質である。例えば、ＡＴＰは、神経系において神経伝達物質として作用することが知られている。一方で皮膚においては、機械的刺激、化学的刺激や紫外線刺激に応答して、又は細胞が破壊されることによりＡＴＰが細胞外に放出され、近傍の免疫細胞に作用することによりサイトカインやケモカイン類の産生を促される。それにより、免疫細胞が誘引されることから、細胞外ＡＴＰは、皮膚における免疫、アレルギー、や炎症反応に寄与すると考えられている（非特許文献１）。したがって、ＣＤ３９を発現する細胞が、細胞外のＡＴＰを分解することで、皮膚、特に表皮の外部刺激に対する過剰な応答や、過剰な免疫応答を鎮静化することができる ATP, which is degraded by the enzymatic activity of CD39, is used as a medium of energy inside the cell, but is a substance that plays a role as a signal transducing substance outside the cell. For example, ATP is known to act as a neurotransmitter in the nervous system. On the other hand, in the skin, ATP is released extracellularly in response to mechanical, chemical or ultraviolet stimuli, or when cells are destroyed, and acts on nearby immune cells to cause cytokines and chemokines. Is stimulated to produce. As a result, immune cells are attracted, and extracellular ATP is considered to contribute to immune, allergic, and inflammatory reactions in the skin (Non-Patent Document 1). Therefore, by degrading extracellular ATP, cells expressing CD39 can sedate excessive responses to external stimuli of the skin, especially the epidermis, and excessive immune responses.
　皮膚においては、ＣＤ３９を発現する細胞（以下、ＣＤ３９陽性細胞ともいう）としては、メラノサイトやランゲルハンス細胞が挙げられる。ランゲルハンス細胞は、骨髄由来の樹状細胞であり、有棘層の中から上層に存在する遊走性を有する細胞である。ランゲルハンス細胞は、ＣＤ３９の他に、ＣＤ１ａ等のマーカーを発現する細胞である。ランゲルハンス細胞の機能として、外界から侵入した異物を捕捉・認識してＴ細胞へ提示する機能、癌細胞を認識する機能、免疫寛容を誘導する機能、細胞外のＡＴＰを分解する機能などが挙げられ、ランゲルハンス細胞は主に皮膚における免疫応答反応に関与する。ランゲルハンス細胞の有する多くの機能のうち、ＣＤ３９を介して細胞外ＡＴＰを分解する機能が、皮膚、特に表皮における外部刺激に対する過剰な応答や過剰な免疫応答の鎮静化を可能にする(FRAGRANCE JOURNAL 2018-1, 16-20)。男性ホルモンであるテストステロンは、外部刺激に応じて皮膚において産生されることが知られている(Journal of Steroid Biochemistry & Molecular Biology 137（2013）107-123)。皮膚において産生されたテストステロンの作用により、皮膚中のランゲルハンス細胞数が低減することが報告されている（The Journal of Investigative Dermatology (1989) vol. 92, no.1, 86-89)。また、一般的にテストステロンは免疫系に対し抑制的に作用する（Cellular Immunology 294 (2015)87-94）。その一方で、本発明者らにより、テストステロンはランゲルハンス細胞においてＣＤ３９の発現を低下させることが示されている。それにより、テストステロンは細胞外ＡＴＰ増大を介して過剰免疫を引き起こすことが示唆される。そうするとテストステロンは皮膚において免疫の抑制と過剰免疫の誘導という相反する作用を引き起こし、皮膚免疫を攪乱する。本発明のＣＤ３９遺伝子の発現低下の抑制剤は、ＣＤ３９遺伝子を発現する細胞、特にランゲルハンス細胞において、ＣＤ３９遺伝子の発現低下を抑制させる薬剤のことをいう。本発明におけるＣＤ３９遺伝子の発現低下は、任意の原因であってもよいが、一例として外部刺激に応じて皮膚で生じたテストステロンに起因して生じうる。 In the skin, examples of cells expressing CD39 (hereinafter, also referred to as CD39-positive cells) include melanocytes and Langerhans cells. Langerhans cells are dendritic cells derived from bone marrow and are migratory cells existing in the upper layer from the spinous layer. Langerhans cells are cells that express markers such as CD1a in addition to CD39. The functions of Langerhans cells include the function of capturing and recognizing foreign substances invading from the outside world and presenting them to T cells, the function of recognizing cancer cells, the function of inducing immune tolerance, and the function of degrading extracellular ATP. , Langerhans cells are primarily involved in the immune response response in the skin. Among the many functions of Langerhans cells, the function of degrading extracellular ATP via CD39 enables the suppression of excessive response to external stimuli and excessive immune response in the skin, especially the epidermis (FRAGRANCE JOURNAL 2018). -1, 16-20). The androgen testosterone is known to be produced in the skin in response to external stimuli (Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 107-123). It has been reported that the number of Langerhans cells in the skin is reduced by the action of testosterone produced in the skin (The Journal of Investigative Dermatology (1989) vol. 92, no. 1, 86-89). In addition, testosterone generally acts suppressively on the immune system (Cellular Immunology 294 (2015) 87-94). On the other hand, we have shown that testosterone reduces the expression of CD39 in Langerhans cells. This suggests that testosterone causes hyperimmunity through extracellular ATP increase. Testosterone then causes the contradictory effects of suppressing immunity and inducing hyperimmunity in the skin, disrupting skin immunity. The agent for suppressing the decrease in the expression of the CD39 gene of the present invention refers to an agent that suppresses the decrease in the expression of the CD39 gene in cells expressing the CD39 gene, particularly Langerhans cells. The decreased expression of the CD39 gene in the present invention may be any cause, but as an example, it may be caused by testosterone generated in the skin in response to an external stimulus.
　したがって、本発明のＣＤ３９遺伝子の発現低下の抑制剤は、ＣＤ３９遺伝子発現促進剤ということもできる。ＣＤ３９遺伝子の発現低下の抑制剤は、外部刺激応答鎮静化剤として使用することもできる。本発明の外部刺激応答鎮静化剤によって、皮膚、特に表皮における外部刺激により生じる応答が鎮静化される。皮膚の外部刺激としては、例えば紫外線、機械的刺激、熱刺激、冷刺激、化学的刺激及び生物学的侵襲が挙げられる。これらの外部刺激を皮膚が受けると、表皮のケラチノサイトが細胞外に刺激応答因子、特にＡＴＰを放出し、上で説明したとおり、細胞外ＡＴＰが情報伝達物質として作用し、免疫、アレルギーや炎症反応が生じる。機械的刺激として、ひっかき、こすり、虫刺されなどが挙げられ、例えば掻傷、裂傷、創傷、発赤、腫れなどが生じる。熱刺激や冷刺激の結果として、やけど、しもやけ、あかぎれ、凍傷、凍瘡などが生じる。紫外線刺激の結果として日焼け、熱傷などが生じる。化学的刺激は、皮膚に対して刺激性を有する化学物質により誘発され、例えば酸やアルカリ、有機溶媒などにより誘発され、結果として湿疹、かぶれ、皮膚炎などが生じる。生物学的侵襲としては、細菌感染などが挙げられる。外部刺激によるＣＤ３９遺伝子発現が低下することで、過剰な免疫応答が生じる。過剰な免疫応答は、皮膚老化にも関与する。したがって、ＣＤ３９遺伝子の発現低下の抑制剤は、皮膚老化の抑制剤ともいうことができる。 Therefore, the agent for suppressing the decrease in the expression of the CD39 gene of the present invention can also be said to be the agent for promoting the expression of the CD39 gene. The agent for suppressing the decrease in the expression of the CD39 gene can also be used as an external stimulus response sedative. The external stimulus response sedative of the present invention sedates the response caused by external stimuli on the skin, especially the epidermis. External skin stimuli include, for example, UV light, mechanical stimuli, thermal stimuli, cold stimuli, chemical stimuli and biological invasion. When the skin receives these external stimuli, keratinocytes in the epidermis release stimulatory response factors, especially ATP, extracellularly, and as explained above, extracellular ATP acts as a signaling substance, resulting in immune, allergic and inflammatory reactions. Occurs. Mechanical stimuli include scratches, scrapes, insect bites, etc., such as scratches, lacerations, wounds, redness, swelling, etc. Burns, chilblains, chilblains, frostbite, and frostbite occur as a result of heat and cold stimuli. Sunburn, burns, etc. occur as a result of UV stimulation. Chemical irritation is triggered by chemicals that are irritating to the skin, such as acids, alkalis, organic solvents, etc., resulting in eczema, rashes, dermatitis and the like. Biological invasion includes bacterial infections and the like. Decreased CD39 gene expression by external stimuli results in an excessive immune response. Excessive immune response is also involved in skin aging. Therefore, an agent that suppresses the decrease in the expression of the CD39 gene can also be said to be an agent that suppresses skin aging.
　本発明の皮膚外部刺激応答鎮静化剤は、これらの外部刺激による皮膚、特に表皮における応答、例えば炎症、又は炎症に起因するかゆみ、発赤、疼痛、熱感、腫脹などを緩和又は軽減することができ、結果として上に挙げられた症状を緩和、軽減又は治療することが可能になる。したがって、本発明の皮膚外部刺激応答鎮静化剤は、上記の症状の緩和、治療、又は予防剤であってもよい。また、皮膚外部刺激応答を鎮静化する結果として、皮膚における免疫応答を鎮静化できることから、本発明の皮膚外部刺激応答鎮静化剤は、皮膚免疫応答鎮静化剤であってもよい。 The skin external stimulus response sedative of the present invention may alleviate or alleviate the response to these external stimuli on the skin, especially the epidermis, such as inflammation, or itching, redness, pain, warmth, swelling, etc. caused by inflammation. As a result, it becomes possible to alleviate, alleviate or treat the symptoms listed above. Therefore, the external skin stimulus response sedative of the present invention may be an agent for alleviating, treating, or preventing the above-mentioned symptoms. Further, since the immune response in the skin can be calmed as a result of calming the external skin stimulus response, the skin external stimulus response sedative agent of the present invention may be a skin immune response sedative agent.
　その一方で、本発明の皮膚外部刺激応答鎮静化剤は、皮膚における刺激応答のカスケードを直接的に抑制する訳ではなく、ＡＴＰａｓｅ機能を有するＣＤ３９遺伝子発現の促進を介して、間接的に刺激応答を緩和すると考えられている。したがって、外部刺激応答鎮静化剤は、外部刺激に対する過剰な応答を鎮静化するという、皮膚が通常有している機能を増強するものであり、刺激応答鎮静化機能増強剤ということもできる。外部刺激応答鎮静化機能増強剤は、炎症が起きている部位に直接投与するよりも、炎症が生じていない平常時に用いることにより、外部刺激があった際の炎症応答、例えばかゆみ、発赤、疼痛、熱感、腫脹、かぶれを抑制するように機能することができる。したがって、外部刺激応答鎮静化剤は、医薬品に配合されてもよいが、日常的に使用することが意図される化粧料に配合され、外部刺激に対して予防的に使用されることが好ましい。本発明の皮膚外部刺激応答鎮静化剤が、医薬品に配合される場合であれば、抗炎症作用を有する他の薬剤と共に配合されることで、そのような薬剤の抗炎症作用に対し補助的に作用又は増強することができる。 On the other hand, the external skin stimulus response sedative of the present invention does not directly suppress the stimulus response cascade in the skin, but indirectly stimulates the stimulus response by promoting the expression of the CD39 gene having an ATPase function. Is believed to ease. Therefore, the external stimulus response sedative agent enhances the function normally possessed by the skin of quelling an excessive response to an external stimulus, and can also be referred to as a stimulus response sedative function enhancer. External stimulus response The sedative function enhancer is used in normal times when there is no inflammation, rather than being administered directly to the site of inflammation, and the inflammatory response when there is an external stimulus, such as itching, redness, and pain. It can function to control the feeling of heat, swelling, and rashes. Therefore, the external stimulus response sedative may be added to a pharmaceutical product, but is preferably added to a cosmetic product intended for daily use and used prophylactically against external stimuli. If the sedative agent for external skin stimulus response of the present invention is to be added to a pharmaceutical product, it may be used together with other anti-inflammatory agents to supplement the anti-inflammatory effect of such agents. Can act or enhance.
　本発明の皮膚免疫応答鎮静化剤は、皮膚における免疫応答を鎮静化させる薬剤であり、特にランゲルハンス細胞の減少や、細胞外ＡＴＰの増加に起因する免疫応答、特に過剰な炎症の鎮静化に寄与することができる。過剰な炎症としては、慢性皮膚炎、例えばアトピー性皮膚炎、乾癬、紅皮症など、急性皮膚炎、例えばかぶれなどの接触性皮膚炎や脂漏性皮膚炎、日光皮膚炎などが挙げられる。本発明の皮膚免疫応答鎮静化剤を配合した医薬品や、医薬部外品、又は化粧料を使用することにより、皮膚が各種の外部刺激を受けたときに生じる発赤やかゆみといった過剰な反応がおこりにくくなり、ひいては炎症性皮膚疾患の発症の治療又は予防につながる。特にアトピー性皮膚炎などの皮膚疾患では、かゆみを伴う罹患部位を掻くことにより症状が悪化することから、過剰な炎症反応を生じにくくさせることは、治療に有用である。 The skin immune response sedative agent of the present invention is a drug that calms the immune response in the skin, and particularly contributes to the suppression of the immune response caused by the decrease of Langerhans cells and the increase of extracellular ATP, particularly the suppression of excessive inflammation. can do. Excessive inflammation includes chronic dermatitis, such as atopic dermatitis, psoriasis, erythema, and acute dermatitis, such as contact dermatitis such as rash, seborrheic dermatitis, and sunlight dermatitis. By using a drug containing the skin immune response sedative of the present invention, a non-pharmaceutical product, or a cosmetic, an excessive reaction such as redness and itch that occurs when the skin receives various external stimuli occurs. It becomes difficult and leads to the treatment or prevention of the onset of inflammatory skin diseases. In particular, in skin diseases such as atopic dermatitis, the symptoms are exacerbated by scratching the affected area accompanied by itching, so it is useful for treatment to make it difficult for an excessive inflammatory reaction to occur.
　本発明で用いられるカルボキシメチル（ＣＭ）・ベータグルカンは、通常、不溶性のベータグルカンをカルボキシメチル化することにより、水溶性に改変したベータグルカンである。カルボキシメチル・ベータグルカンは、任意の植物、菌類、細菌に由来のベータグルカンを元に又は合成により製造されてもよい。天然のベータグルカンは、例えばアガリクス、霊芝、スエヒロタケなどのキノコ類、や酵母から生成されてもよい。カルボキシメチル・ベータグルカンの重量平均分子量は、その由来に応じて例えば、１，０００～５，０００，０００ダルトンであり、酵母を原料としたカルボキシメチル・ベータグルカンの場合、上限は５，０００，０００ダルトン、より好ましくは３,０００，０００ダルトン、さらに好ましくは２，０００，０００ダルトンであり、下限は、１０，０００ダルトン、より好ましくは１００，０００ダルトン、さらに好ましくは５００，０００ダルトンである。カルボキシメチル・ベータグルカンは、任意の塩の形態であってよく、例えばナトリウム塩、カリウム塩、アンモニウム塩、トリエタノールアミン塩などであってよい。市販されているカルボキシメチル・ベータグルカンとして、カルボキシメチル・ベータグルカン・ナトリウムであるＣＭ－グルカン（Mibelle Biochemistry）等を用いることができるが、それに限定されるものではない。その配合量は、配合する化粧料や医薬品の剤形によって異なりうるが、十分な効力を発揮する観点から例えば０．０００１質量％以上、好ましくは０．０００２質量％以上、より好ましくは０．００２質量％以上で配合される。一方で、溶解性の観点から、０．５質量％以下、より好ましくは０．１質量％以下、さらに好ましくは０．０１質量％以下で用いられる。 The carboxymethyl (CM) beta-glucan used in the present invention is usually a beta-glucan modified to be water-soluble by carboxymethylating an insoluble beta-glucan. Carboxymethyl beta-glucans may be produced on the basis of or synthetically from beta-glucans derived from any plant, fungus or bacterium. Natural beta-glucans may be produced from mushrooms such as Agaricus, Reishi, Schizophyllum communis, and yeast. The weight average molecular weight of carboxymethyl beta-glucan is, for example, 1,000 to 5,000,000 daltons depending on its origin, and in the case of yeast-based carboxymethyl beta-glucan, the upper limit is 5,000. It is 000 daltons, more preferably 3,000,000 daltons, even more preferably 2,000,000 daltons, with a lower limit of 10,000 daltons, more preferably 100,000 daltons, even more preferably 500,000 daltons. .. The carboxymethyl beta glucan may be in the form of any salt, for example a sodium salt, a potassium salt, an ammonium salt, a triethanolamine salt and the like. As a commercially available carboxymethyl beta-glucan, CM-glucan (Mibelle Biochemistry) which is a carboxymethyl beta-glucan sodium can be used, but the present invention is not limited thereto. The blending amount may vary depending on the dosage form of the cosmetic or pharmaceutical product to be blended, but from the viewpoint of exerting sufficient efficacy, for example, 0.0001% by mass or more, preferably 0.0002% by mass or more, more preferably 0.002. It is blended in an amount of% by mass or more. On the other hand, from the viewpoint of solubility, it is used in an amount of 0.5% by mass or less, more preferably 0.1% by mass or less, still more preferably 0.01% by mass or less.
　本発明で用いられるポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテルに用いられるポリオキシプロピレン（ＰＯＰ）の平均付加モル数は２～５０、ポリオキシエチレン（ＰＯＥ）の平均付加モル数は８～１００であり、ポリオキシエチレン（ＰＯＥ）とポリオキシプロピレン（ＰＯＰ）の平均付加モル数の合計に対するポリオキシエチレン（ＰＯＥ）の平均付加モル数の割合 [ＰＯＥ／（ＰＯＥ＋ＰＯＰ）］は０．５以上であることが好ましい。ここで、ＰＯＥ及びＰＯＰは、それぞれポリオキシエチレン、ポリオキシプロピレンの略であり、以下、このように略して記載することがある。また、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテルにおいて用いられるポリオキシエチレンとポリオキシプロピレンのモル比は、所望の作用を発揮するポリマーを取得する観点から、１：５～５：１、好ましくは１：３～３：１、さらに好ましくは１：２～２：１の範囲である。例えばＰＯＥ：ＰＯＰの比が２：１のものを用いることができる。ポリオキシプロピレン・ポリオキシエチレンランダム共重合体ジアルキルエーテルは公知の方法で製造することができる（特許文献３）。例えば、水酸基を有している化合物にエチレンオキシドおよびプロピレンオキシドを付加重合した後、ハロゲン化アルキルをアルカリ触媒の存在下にエーテル反応させることによって得られる。その配合量は、配合する化粧料や医薬品の剤形によって異なりうるが、十分な効力を発揮する観点から、例えば０．０１質量％以上、好ましくは０．０５質量％以上、より好ましくは０．１質量％以上で配合される。一方で、溶解性の観点から、３質量％以下、より好ましくは１質量％以下、さらに好ましくは０．５質量％以下で用いられる。なお、本発明で用いられる、ポリオキシエチレン・ポリオキシプロピレンランダム共重合体ジアルキルエーテルは、単独またはその他の成分との組み合わせにより保湿効果などが知られているが、ポリオキシエチレン・ポリオキシプロピレンランダム共重合体ジアルキルエーテルのＣＤ３９遺伝子発現への効果は知られていない。 The average number of moles of polyoxypropylene (POP) used in the polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether used in the present invention is 2 to 50, and the average number of polyoxyethylene (POE) is 2. The number of added moles is 8 to 100, and the ratio of the average number of added moles of polyoxyethylene (POE) to the total number of average added moles of polyoxyethylene (POE) and polyoxypropylene (POP) [POE / (POE + POP)). ] Is preferably 0.5 or more. Here, POE and POP are abbreviations for polyoxyethylene and polyoxypropylene, respectively, and may be abbreviated as described below. Further, the molar ratio of polyoxyethylene to polyoxypropylene used in the polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether is set to 1: from the viewpoint of obtaining a polymer exhibiting a desired action. The range is 5 to 5: 1, preferably 1: 3 to 3: 1, and more preferably 1: 2 to 2: 1. For example, a POE: POP ratio of 2: 1 can be used. The polyoxypropylene / polyoxyethylene random copolymer dialkyl ether can be produced by a known method (Patent Document 3). For example, it is obtained by addition-polymerizing ethylene oxide and propylene oxide to a compound having a hydroxyl group, and then subjecting an alkyl halide to an ether reaction in the presence of an alkaline catalyst. The blending amount may vary depending on the dosage form of the cosmetic or pharmaceutical product to be blended, but from the viewpoint of exerting sufficient efficacy, for example, 0.01% by mass or more, preferably 0.05% by mass or more, more preferably 0. It is blended in an amount of 1% by mass or more. On the other hand, from the viewpoint of solubility, it is used in an amount of 3% by mass or less, more preferably 1% by mass or less, still more preferably 0.5% by mass or less. The polyoxyethylene / polyoxypropylene random copolymer dialkyl ether used in the present invention is known to have a moisturizing effect alone or in combination with other components, but the polyoxyethylene / polyoxypropylene random The effect of the copolymer dialkyl ether on the expression of the CD39 gene is unknown.
　本発明で用いられるツバキエキスは、ツバキ科ツバキ属の植物体から得られるエキスをいう。ツバキ科ツバキ属の植物として、ヤブツバキ（Camellia japonica)が挙げられ、またその近縁種、例えばユキツバキ（Camellia rusticana）、サザンカ（Camellia sasanqua）等や交配種が挙げられる。ヤブツバキは日本原産であり、その植物体、例えば花、果実、葉、枝、樹木、種子からエキスを抽出することができる。抽出溶媒としては、有機溶媒又は水性溶媒を使用することができ、一例としてブチレングリコール、プロピレングリコール、エタノール、グリセリンなどのアルコール、ジエチレングリコールモノエチルエーテルなどのエーテル、酢酸エチル、酢酸ブチルなどのエステル、又は水、或いはそれらの任意の混合液を使用することができる。一例として、ツバキ花エキスは、ツバキの花を、ブチレングリコールの５０％水溶液で抽出して得ることができる。ツバキ葉エキスは、ツバキの葉を、ブチレングリコールの５０％水溶液で抽出して得ることができる。配合量としては、十分な効力を発揮する観点から、例えば０．０００１％（ｖ/ｖ）以上、好ましくは０．０００２％（ｖ/ｖ）以上、より好ましくは０．０００５％（ｖ/ｖ）以上で配合される。一方で、安定性・安全性の観点から、５％（ｖ/ｖ）以下、より好ましくは３％（ｖ/ｖ）以下、さらに好ましくは１％（ｖ/ｖ）以下で用いられる。 The camellia extract used in the present invention refers to an extract obtained from a plant belonging to the genus Camellia of the Theaceae family. Examples of the Camellia family Camellia plant include Camellia japonica, and related species such as Camellia rusticana, Camellia sasanqua, and hybrid species. Camellia japonica is native to Japan, and extracts can be extracted from its plants such as flowers, fruits, leaves, branches, trees and seeds. As the extraction solvent, an organic solvent or an aqueous solvent can be used, for example, alcohols such as butylene glycol, propylene glycol, ethanol and glycerin, ethers such as diethylene glycol monoethyl ether, esters such as ethyl acetate and butyl acetate, or Water or any mixture thereof can be used. As an example, camellia flower extract can be obtained by extracting camellia flowers with a 50% aqueous solution of butylene glycol. The camellia leaf extract can be obtained by extracting camellia leaves with a 50% aqueous solution of butylene glycol. The blending amount is, for example, 0.0001% (v / v) or more, preferably 0.0002% (v / v) or more, and more preferably 0.0005% (v / v) from the viewpoint of exhibiting sufficient efficacy. ) It is mixed with the above. On the other hand, from the viewpoint of stability and safety, it is used at 5% (v / v) or less, more preferably 3% (v / v) or less, still more preferably 1% (v / v) or less.
　本発明で用いられるカルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、ツバキエキスの配合比は、カルボキシメチル・ベータグルカン又はその塩の２％水溶液の配合量を１とした場合に、１：１～１００：１～１００であり、好ましくは１：１～２０：１～２０、さらに好ましくは１：１０：１０である。本発明で用いられるカルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスの３成分は、１つの製剤に配合されてもよいし、複数の製剤に配合されて、逐次投与されてもよい。 The compounding ratio of carboxymethyl beta-glucan or a salt thereof used in the present invention, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract is 2 of carboxymethyl beta-glucan or a salt thereof. When the blending amount of the% aqueous solution is 1, it is 1: 1 to 100: 1 to 100, preferably 1: 1 to 20: 1 to 20, and more preferably 1:10:10. The three components of carboxymethyl beta glucan or a salt thereof used in the present invention, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract may be blended into one pharmaceutical product. However, it may be compounded in a plurality of formulations and administered sequentially.
　本発明のＣＤ３９遺伝子の発現低下の抑制剤、外部刺激応答鎮静化剤、及び皮膚免疫応答鎮静化剤は、それぞれ化粧料、医薬品又は医薬部外品に配合されてもよい。これらの薬剤は、経口、又は非経口、例えば経皮投与されてもよい。経皮投与される場合、皮膚外用剤に剤形することができる。皮膚外用剤は、皮膚に適用可能であれば特に限定されず、例えば、溶液状、乳化状、固形状、半固形状、粉末状、粉末分散状、水－油二層分離状、水－油－粉末三層分離状、軟膏状、ゲル状、エアゾール状、ムース状、スティック状等、任意の剤形が適用できる。皮膚外用剤に剤形される場合には、皮膚外用剤に通常用いられる基剤、及び賦形剤、例えば保存剤、乳化剤、ｐＨ調整剤などが用いられてもよく、さらに抗炎症性の成分、例えばステロイド、抗ヒスタミン剤などがさらに添加されてもよい。化粧料に配合する場合、化粧水、乳液、美容液、クリーム、ローション、パック、エッセンス、ジェル等の顔用又は体用の化粧料や、ファンデーション、化粧下地、コンシーラー等のメーキャップ化粧料、さらには浴用剤などに配合することができる。本発明の成分を含む化粧品を用いることにより、炎症反応を緩和又は予防することができることから、発赤、かゆみ、かぶれが生じにくい健康な肌の維持を可能にする。 The inhibitor of the expression decrease of the CD39 gene of the present invention, the external stimulus response sedative agent, and the skin immune response sedative agent may be incorporated into cosmetics, pharmaceuticals or quasi-drugs, respectively. These agents may be administered orally or parenterally, eg, transdermally. When administered transdermally, it can be formulated into a skin external preparation. The external skin preparation is not particularly limited as long as it is applicable to the skin, and is, for example, solution-like, emulsified-like, solid-like, semi-solid-like, powder-like, powder-dispersed, water-oil two-layer separated form, water-oil. -Any dosage form such as powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. can be applied. When formulated into a skin external preparation, a base usually used for the skin external preparation and an excipient such as a preservative, an emulsifier, a pH adjuster and the like may be used, and further, an anti-inflammatory component. , For example, steroids, antihistamines and the like may be further added. When blended in cosmetics, facial or body cosmetics such as lotions, milky lotions, serums, creams, lotions, packs, essences, gels, makeup cosmetics such as foundations, makeup bases, and concealers, and even It can be added to bathing agents and the like. By using the cosmetics containing the ingredients of the present invention, the inflammatory reaction can be alleviated or prevented, so that it is possible to maintain healthy skin that is less likely to cause redness, itchiness and rashes.
　本発明は、また、本発明のＣＤ３９遺伝子の発現低下の抑制剤、外部刺激応答鎮静化剤、及び皮膚免疫応答鎮静化剤、すなわち、カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）／ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスからなる３成分を投与することを含む、化粧方法、外部刺激応答鎮静化方法、健常肌維持方法、炎症治療方法にも関してもよい。これらの方法において、本発明の３成分の投与は、全ての成分を配合された皮膚外用剤又は化粧料を使用することにより行われてもよいし、個別の成分を配合された皮膚外用剤又は化粧料を連続して使用することにより行われてもよい。 The present invention also presents an inhibitor of the expression reduction of the CD39 gene of the present invention, an external stimulus response sedative, and a skin immune response sedative, that is, carboxymethyl beta glucan or a salt thereof, polyoxyethylene (POE). / Polyoxypropylene (POP) Random copolymer dimethyl ether, including administration of three components consisting of camellia extract, cosmetic method, external stimulus response sedation method, healthy skin maintenance method, inflammation treatment method good. In these methods, the administration of the three components of the present invention may be carried out by using a skin external preparation or cosmetic containing all the components, or a skin external preparation or a cosmetic containing individual components. It may be done by using the cosmetics continuously.
　本発明の別の態様では、本発明は、ＣＤ３９遺伝子の発現低下の抑制剤のスクリーニング方法にも関する。当該スクリーニング方法は、以下の：
テストステロン及び候補物質の存在下でＣＤ３９陽性細胞を培養する工程、
　ＣＤ３９遺伝子の発現を測定する工程、及び
　ＣＤ３９遺伝子の発現量に基づき、候補物質のＣＤ３９遺伝子の発現低下の抑制作用を決定する工程
を含む。テストステロンは、候補物質の存在下で培養されたＣＤ３９陽性細胞の培養物中に添加又は培地置換により加えられてもよい。テストステロンは、ＣＤ３９陽性細胞においてＣＤ３９遺伝子の発現低下を誘導することから、ＣＤ３９遺伝子発現量を測定することで、候補物質のＣＤ３９遺伝子の発現低下に対する抑制作用を決定することができる。ＣＤ３９遺伝子発現量は、既知の手法によりｍＲＮＡ量を測定してもよいし、既知の手法により発現したタンパク質量を測定してもよい。ＣＤ３９遺伝子の発現低下に対する抑制作用は、ＣＤ３９遺伝子の発現促進作用ということもできる。こうしてスクリーニングされたＣＤ３９遺伝子の発現低下の抑制剤は、皮膚外部刺激応答の鎮静化剤又は皮膚免疫応答鎮静化剤ともいうことができる。ＣＤ３９陽性細胞としては、ランゲルハンス細胞又はランゲルハンス細胞の代替細胞、例えばＴＨＰ－１細胞を使用することができる。 In another aspect of the invention, the invention also relates to a method of screening for an agent that suppresses decreased expression of the CD39 gene. The screening method is as follows:
The step of culturing CD39-positive cells in the presence of testosterone and candidate substances,
It includes a step of measuring the expression of the CD39 gene and a step of determining the inhibitory effect on the decrease in the expression of the CD39 gene of the candidate substance based on the expression level of the CD39 gene. Testosterone may be added to the culture of CD39-positive cells cultured in the presence of the candidate substance or by medium replacement. Since testosterone induces a decrease in the expression of the CD39 gene in CD39-positive cells, the inhibitory effect on the decrease in the expression of the CD39 gene of the candidate substance can be determined by measuring the expression level of the CD39 gene. As for the expression level of the CD39 gene, the amount of mRNA may be measured by a known method, or the amount of protein expressed by a known method may be measured. The inhibitory effect on the decrease in the expression of the CD39 gene can also be said to be the effect of promoting the expression of the CD39 gene. The agent for suppressing the decrease in the expression of the CD39 gene screened in this way can also be referred to as a sedative for an external skin stimulus response or a sedative for a skin immune response. As the CD39-positive cells, Langerhans cells or alternative cells of Langerhans cells, for example, THP-1 cells can be used.
　本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。 All documents referred to herein are incorporated herein by reference in their entirety.
　以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。 The examples of the present invention described below are for illustration purposes only and do not limit the technical scope of the present invention. The technical scope of the invention is limited only by the description of the claims. Modifications of the present invention, for example, addition, deletion and replacement of the constituent elements of the present invention may be made on condition that the gist of the present invention is not deviated.
　１０5個のＴＨＰ－１（ヒト単球細胞株、Virginia USA）細胞を、１０％　胎児ウシ血清 (FCS; Santa Ana, CA)、非必須アミノ酸及び必須アミノ酸 (Nacalai Tesque, Japan)を添加した２ｍｌのＲＰＭＩ１６４０培地（Nacalai Tesque, Japan)中で１２ウェルプレートの各ウェルに播種し、そして２４時間培養した。１０μＬのリン酸緩衝生理食塩水（ＰＢＳ）又はＰＢＳで希釈された１０μＬの試験物質を培養培地に添加した。３７℃で１時間インキュベート後に、１０μＬのＥｔＯＨ又は１０μＬのエタノールに溶解したテストステロン（ＴＳＴ：SIGMA, St. Louise, MO）を加えた。各試験物質の最終濃度は、下記のとおりであった：
群Ａ（ｎｏＴ）：ＰＢＳ、ＥｔＯＨのみ
群Ｂ（Ｔ）：１ｎｇ／ｍｌテストステロン（ＴＳＴ）
群Ｃ（＋ＧＡ）：　０．０１％　カルボキシメチルベータグルカン（CM-Gulucan、Mibelle Biochemistry社）、１ｎｇ／ｍＬ　ＴＳＴ、及び０．１％ポリオキシエチレン（ＰＯＥ）１４／ポリオキシプロピレン（ＰＯＰ）７ランダム共重合体ジメチルエーテル
群Ｄ（＋ＧＡ＋ＦＬ０．００１）：０．０１％　カルボキシメチルベータグルカン（CM-Gulucan、Mibelle Biochemistry社）及び１ｎｇ／ｍＬ　ＴＳＴ、０．１％ポリオキシエチレン（ＰＯＥ）１４／ポリオキシプロピレン（ＰＯＰ）７ランダム共重合体ジメチルエーテル、０．００１％ツバキ花エキス（東亜化成（株））及び０．００１％ツバキ葉エキス（東亜化成（株））
群Ｅ（＋ＧＡ＋ＦＬ０．０１）：０．０１％　カルボキシメチルベータグルカン及び１ｎｇ／ｍＬ　ＴＳＴ、０．１％ポリオキシエチレン（ＰＯＥ）１４／ポリオキシプロピレン（ＰＯＰ）７ランダム共重合体ジメチルエーテル、０．０１％ツバキ花エキス及び０．０１％ツバキ葉エキス 10 5 THP-1 (Human Monosphere Cell Lines, Virginia USA) cells 2 ml supplemented with 10% fetal bovine serum (FCS; Santa Ana, CA), non-essential amino acids and essential amino acids (Nacalai Tesque, Japan) In RPMI 1640 medium (Nacalai Tesque, Japan), each well of a 12-well plate was seeded and cultured for 24 hours. 10 μL of phosphate buffered saline (PBS) or 10 μL of test material diluted with PBS was added to the culture medium. After incubation at 37 ° C. for 1 hour, testosterone (TST: SIGMA, St. Louise, MO) dissolved in 10 μL EtOH or 10 μL ethanol was added. The final concentration of each test substance was as follows:
Group A (noT): PBS, EtOH only Group B (T): 1 ng / ml testosterone (TST)
Group C (+ GA): 0.01% carboxymethyl beta glucan (CM-Gulucan, Mibelle Biochemistry), 1 ng / mL TST, and 0.1% polyoxyethylene (POE) 14 / polyoxypropylene (POP) 7 random. Copolymer dimethyl ether group D (+ GA + FL0.001): 0.01% carboxymethyl beta glucan (CM-Gulucan, Mibelle Biochemistry) and 1 ng / mL TST, 0.1% polyoxyethylene (POE) 14 / polyoxypropylene. (POP) 7 Random Copolymer Dimethyl Ether, 0.001% Tsubaki Flower Extract (Toa Kasei Co., Ltd.) and 0.001% Tsubaki Leaf Extract (Toa Kasei Co., Ltd.)
Group E (+ GA + FL0.01): 0.01% carboxymethyl beta-glucan and 1 ng / mL TST, 0.1% polyoxyethylene (POE) 14 / polyoxypropylene (POP) 7 random copolymer dimethyl ether, 0.01 % Camellia flower extract and 0.01% camellia leaf extract
　さらに２４時間の培養後に、RNeasy micro plus kit (Qiagen, Netherlands)を用いてRNAを抽出した。GoScript reverse transcriptase kit (Promega, Japan)でｃＤＮＡを合成した。GoTaq qPCR master mix (Promega, Japan)及び LyteCycler 480II (Roche, Switzerland)を用いてRT-PCRを行った。使用したプライマーは下記の通りであった：
Figure JPOXMLDOC01-appb-T000001
 After an additional 24 hours of culture, RNA was extracted using the RNeasy micro plus kit (Qiagen, Netherlands). CDNA was synthesized with the GoScript reverse transcriptase kit (Promega, Japan). RT-PCR was performed using GoTaq qPCR master mix (Promega, Japan) and LyteCycler 480II (Roche, Switzerland). The primers used were:
Figure JPOXMLDOC01-appb-T000001
　ＣＤ３９の発現値をリボソームタンパク質Ｓ９（ｒｐＳ９）で正規化し、ＡＮＯＶＡ及びポストホックＴｕｋｅｙ検定で統計的有意差を決定した。結果を図１に示す。 The expression level of CD39 was normalized by ribosomal protein S9 (rpS9), and a statistically significant difference was determined by ANOVA and post-hook Tukey's test. The results are shown in FIG.

Claims (9)

  1. 　カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスを含む、ＣＤ３９遺伝子の発現低下の抑制剤。 An inhibitor of decreased expression of the CD39 gene, which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
  2. 　ＣＤ３９遺伝子の発現低下が、外部刺激に起因する、請求項１に記載の抑制剤。 The inhibitor according to claim 1, wherein the decreased expression of the CD39 gene is caused by an external stimulus.
  3. 　ＣＤ３９遺伝子の発現低下が、テストステロンの増加に起因する、請求項１に記載の抑制剤。 The inhibitor according to claim 1, wherein the decrease in the expression of the CD39 gene is caused by the increase in testosterone.
  4. 　皮膚のランゲルハンス細胞においてＣＤ３９遺伝子発現低下を抑制する、請求項１～３のいずれか一項に記載の抑制剤。 The inhibitor according to any one of claims 1 to 3, which suppresses the decrease in CD39 gene expression in Langerhans cells of the skin.
  5. 　カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスを含む、皮膚外部刺激応答の鎮静化剤。 A sedative for external skin irritation, including carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
  6. 　カルボキシメチル・ベータグルカン又はその塩、ポリオキシエチレン（ＰＯＥ）・ポリオキシプロピレン（ＰＯＰ）ランダム共重合体ジメチルエーテル、及びツバキエキスを含む、皮膚免疫応答の鎮静化剤。 A sedative agent for the skin immune response, which comprises carboxymethyl beta-glucan or a salt thereof, polyoxyethylene (POE) / polyoxypropylene (POP) random copolymer dimethyl ether, and camellia extract.
  7. 　テストステロン及び候補物質の存在下でランゲルハンス細胞を培養する工程、
    　ＣＤ３９遺伝子の発現を測定する工程、及び
    　ＣＤ３９遺伝子の発現量に基づき、候補物質のＣＤ３９発現促進作用を決定する工程
    を含む、ＣＤ３９発現低下の抑制剤のスクリーニング方法。     The step of culturing Langerhans cells in the presence of testosterone and candidate substances,
    A method for screening an agent for suppressing the decrease in CD39 expression, which comprises a step of measuring the expression of the CD39 gene and a step of determining the CD39 expression promoting action of the candidate substance based on the expression level of the CD39 gene.
  8. 　ＣＤ３９発現低下の抑制剤が、皮膚外部刺激応答の鎮静化剤である、請求項７に記載の方法。 The method according to claim 7, wherein the inhibitor for reducing the expression of CD39 is a sedative for an external skin stimulus response.
  9. 　ＣＤ３９発現低下の抑制剤が、皮膚免疫応答鎮静化剤である、請求項７に記載の方法。 The method according to claim 7, wherein the inhibitor for reducing the expression of CD39 is a skin immune response sedative.
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Citations (4)

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JP2000236775A (en) * 1999-02-23 2000-09-05 Shiseido Co Ltd Control of immune response in sensitized animal
JP2009155317A (en) * 2007-12-27 2009-07-16 Bhn Kk Anti-inflammatory agent and skin external medicine
JP2015134737A (en) * 2014-01-18 2015-07-27 共栄化学工業株式会社 External preparation for skin
JP2017511790A (en) * 2014-03-12 2017-04-27 株式会社 資生堂 Sedative or method of sedation of external stimulus response in skin

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Publication number Priority date Publication date Assignee Title
JP2000236775A (en) * 1999-02-23 2000-09-05 Shiseido Co Ltd Control of immune response in sensitized animal
JP2009155317A (en) * 2007-12-27 2009-07-16 Bhn Kk Anti-inflammatory agent and skin external medicine
JP2015134737A (en) * 2014-01-18 2015-07-27 共栄化学工業株式会社 External preparation for skin
JP2017511790A (en) * 2014-03-12 2017-04-27 株式会社 資生堂 Sedative or method of sedation of external stimulus response in skin

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KOYAMA YOH-ICHI, NAGAO SHIZUKO, OHASHI KOJI, TAKAHASHI HISAHIDE, MARUNOUCHI TOHRU: "Effect of Systemic and Topical Application of Testosterone Propionate on the Density of Epidermal Langerhans Cells in the Mouse", JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 92, no. 1, 1 January 1989 (1989-01-01), NL , pages 86 - 90, XP055903280, ISSN: 0022-202X, DOI: 10.1111/1523-1747.ep13071282 *

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