WO2022244460A1 - Netrin-1 INHIBITOR - Google Patents

Netrin-1 INHIBITOR Download PDF

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WO2022244460A1
WO2022244460A1 PCT/JP2022/013694 JP2022013694W WO2022244460A1 WO 2022244460 A1 WO2022244460 A1 WO 2022244460A1 JP 2022013694 W JP2022013694 W JP 2022013694W WO 2022244460 A1 WO2022244460 A1 WO 2022244460A1
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Prior art keywords
netrin
epidermal
epidermis
skin
extract
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PCT/JP2022/013694
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French (fr)
Japanese (ja)
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美加 加治屋
亮太 上
大気 筒井
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株式会社 資生堂
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Priority to JP2023522287A priority Critical patent/JPWO2022244460A1/ja
Publication of WO2022244460A1 publication Critical patent/WO2022244460A1/en

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    • 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)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • 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
    • 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
    • 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
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • 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 Netrin-1 inhibitor.
  • the skin epidermis has a great influence on the apparent age and appearance, and maintaining the epidermis in a healthy state and improving it to a better state is a major cosmetic issue.
  • stem cells in the basal layer repeatedly divide and differentiate, migrate to the surface of the skin, and the old epidermal cells are sloughed off from the skin.
  • Patent Literature 1 discloses a laminin 511 expression promoter that suppresses the decrease or increases the number of epidermal stem cells.
  • Patent Document 2 discloses an agent for promoting the differentiation of epidermal stem cells into epidermal keratinocytes, containing a supercritical extract of Ganoderma lucidum spores as an active ingredient.
  • Patent Document 3 discloses a composition for improving the condition of the skin by strengthening the dermal-epidermal junction.
  • An object of the present invention is to provide a substance that promotes epidermal normalization and a method of screening for a substance that promotes epidermal normalization.
  • the present inventors have found that blood vessels in the papillary dermis are closely related to the production of epidermal stem cells, and that disturbance of the dermal vasculature and suppression of epidermal stem cell production are one of the factors that suppress epidermal normalization. I have learned that there is. Furthermore, the present inventors discovered that such disruption of vascular structures is caused by Netrin-1, and also discovered a substance that inhibits Netrin-1.
  • the present invention relates to the following inventions: (1) Netrin-1 inhibitors for epidermal normalization. (2) a Netrin-1 inhibitor consisting of one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil; (3) An agent for normalizing epidermis, comprising the Netrin-1 inhibitor according to (1) or (2). (4) The agent for normalizing the epidermis according to (3), wherein the normalization of the epidermis is achieved through inhibition of disturbance of vascular structures by inhibition of Netrin-1. (5) Screening method for an epidermal normalizing agent using Netrin-1 as an index.
  • the screening method according to (5) comprising (7) A cosmetic method for normalizing the epidermis of a subject comprising suppressing Netrin-1 in the subject. (8) The method of (7), wherein suppression of Netrin-1 in the subject is achieved by administering the epidermal normalizing agent of (3) or (4) to the subject.
  • the Netrin-1 inhibitor of the present invention can suppress Netrin-1. Inhibition of Netrin-1 inhibits disturbance of vasculature. Suppression of disturbance of vascular structures promotes the production of epidermal stem cells, which in turn contributes to normalization of the epidermis.
  • a Netrin-1 inhibitor containing one or more herbal medicines selected from the group consisting of yuzu extract, chimp extract, and lactobiotil is effective in epidermal normalization through inhibition of disturbance of vascular structures.
  • the screening method of the present invention enables selection of a substance having an epidermal normalizing effect. Epidermal normalization agents selected by the screening method of the present invention contribute to epidermal normalization by suppressing Netrin-1.
  • FIG. 1 shows the results of Experiment 1.
  • FIG. 1 top, shows the epidermal-dermal junction in young (33 years old) and old (69 years old) skin. Green indicates CD31-stained portions (vascular endothelial cells) and white indicates DAPI-stained portions (nuclei).
  • FIG. 2 shows the results of Experiment 2.
  • FIG. 2 shows the results of Experiment 2.
  • FIG. 2 shows the epidermal-dermal junction in young (27 years old) (left and top center) and aged (60 years) (bottom center) skin.
  • Green indicates CD31-stained portions (vascular endothelial cells)
  • white indicates DAPI-stained portions (nuclei)
  • red indicates MCSP-stained portions (epidermal stem cells and pericytes).
  • * indicates p ⁇ 0.05 (t-test).
  • FIG. 3 shows the results of Experiment 3.
  • FIG. 3 shows the epidermal-dermal junction in the skin of a young person (27 years old). Green indicates areas stained with CD31 (vascular endothelial cells), white indicates areas stained with DAPI (nuclei), red indicates areas stained with MCSP (epidermal stem cells and pericytes), and blue indicates areas stained with anti-integrin alpha 6 antibody (basement membrane). The left shows all stained sections, the right shows only the basement membrane (blue).
  • FIG. 4 shows the results of Experiment 3, and shows images taken while changing the depth of the epidermal-dermal junction in FIG. 3 (moving in the Z-axis direction). Staining is the same as in FIG. FIG.
  • FIG. 5 shows the results of Experiment 4, showing the dermal vascular structure of a young (18 year old) subject on the left and an elderly (50 year old) subject on the right.
  • FIG. 6 shows the relationship between the Netrin-1 expression level obtained from Experiment 5 and age.
  • FIG. 7 shows the epidermal-dermal junction in the skin of a young person (27 years old), the result of Experiment 6.
  • Green indicates CD31-stained portions (vascular endothelial cells), white indicates DAPI-stained portions (nuclei), red indicates MCSP-stained portions (epidermal stem cells and pericytes), and blue indicates UNC5B-stained portions (Netrin-1 receptor).
  • FIG. 8 shows the method (left) and results (right) of Experiment 7.
  • the vertical axis of the central graph indicates human recombinant Netrin-1 at a final concentration of 0.1 ⁇ g/ml or 1 ⁇ g/ml, and/or vascular endothelial growth factor VEGFA165 (450-32, Peprotech, NJ) at a final concentration of 50 ng/ml. Shows the number of cells that migrated when As a control, a medium with the same composition was used except that the above substances were not added (Ctrl).
  • FIG. 9 shows a 3D blood vessel model constructed by the method of Experiment 8 with (right) or without (left) Netrin-1. Green indicates areas stained with rabbit anti-VE-Cadherin antibody (vascular endothelial cells), blue indicates areas stained with DAPI (nuclei), and red indicates areas stained with Phalloidin (cytoskeletal f-actin).
  • FIG. 10 is a schematic diagram showing the repelling effect of the Netrin-1 receptor UNC5B on vascular cells and the disorder of the vascular structure by epidermal Netrin-1, which increases with aging, as a concept derived from the present application.
  • FIG. 11 shows the expression of Netrin-1 when yuzu extract, chimp extract, and lactobiotil were used as relative values (%) when the control was set to 100. In the figure, * indicates p ⁇ 0.05 (t-test).
  • the present inventors surprisingly discovered that capillaries in the papillary dermis have a great influence on epidermal normalization.
  • epidermal stem cells are supplied by pericytes present around papillary capillaries, and that with aging, the number of papillary capillaries decreases and their structure is disturbed, resulting in flattening of the dermal-epidermal junction.
  • Netrin-1 as a substance that induces such disorder of vascular structure, and obtained the knowledge that epidermal Netrin-1 exerts an adverse effect on the epidermis through its action on blood vessels in the dermis.
  • epidermal Netrin-1 increases with aging, and epidermal Netrin-1 repels papillary capillaries from the dermal-epidermal junction, resulting in disturbance of the papillary layer vasculature.
  • capillaries in the papillary layer are repelled from the dermal-epidermal junction, the vascular structure of the papillary layer is disturbed, the dermal-epidermal junction becomes flattened, the number of epidermal stem cells supplied through the epidermal basement membrane decreases, and the epidermis normalizes. is thought to be suppressed.
  • the present invention is based on these findings of the inventors, and relates to a Netrin-1 inhibitor and an epidermal normalizing agent containing a Netrin-1 inhibitor. Suppression of Netrin-1 suppresses disturbance of the vascular structure and promotes the supply of epidermal stem cells by making the relationship between the capillaries of the papillary layer and the epidermal basement membrane closer. Accelerated supply of epidermal stem cells promotes turnover and contributes to normalization of the epidermis.
  • Netrin-1 (Laminin-related secret protein; NTN1) is a 50-75 kDa laminin-like polypeptide. Netrin-1 functions as an axonal guidance factor and is known to be expressed in the skin and involved in photoaging. It has been reported that it has a regulating effect on the proliferation and migration of vascular cells and endothelial cells and that it is involved in the onset of pain (Patent Documents 7, 8 and 9). However, it is not known that Netrin-1 adversely affects epidermis by acting on dermal blood vessels.
  • Pericytes are cells that surround microvessels. It is thought to be involved in maintenance of blood vessels, regulation of blood flow, regulation of differentiation/proliferation of vascular endothelial cells, angiogenesis, etc., but the details are often unknown. It has also been suggested that pericytes are mesenchymal cells with pluripotency. In the skin, it has been reported or suggested that it participates in the wound healing process, regulates the microenvironment of the skin, and induces the polarity and direction of division of epidermal cells to promote skin regeneration. However, it is completely unknown that pericytes present around dermal blood vessels directly supply epidermal stem cells.
  • the Netrin-1 inhibitor of the present invention consists of or contains as an active ingredient one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil.
  • the substance is not limited to these as long as it can suppress Netrin-1.
  • Citrus junos is a citrus fruit belonging to the genus Citrus of the family Rutaceae. Also known as honyuzu, it is widely distributed throughout Japan, China, and other Asian regions such as Vietnam.
  • the fruit and pericarp are known to have blood circulation promoting action, active oxygen scavenging action, antibacterial action, and the like.
  • Yuzu extract refers to an extract obtained by extracting yuzu fruit and pericarp.
  • the fruit (flesh and peel) of yuzu is preferable, but since active ingredients are also contained in seeds, leaves, stems, flowers, roots, etc., extracts of one or more of these may also be used. can.
  • Mandarin orange (Citrus unshiu) is an evergreen shrub belonging to the genus Citrus of the family Rutaceae, order Sapindaceae, and has been reported to maintain an undifferentiated state of stem cells and promote proliferation (Patent Document 10).
  • Chimpi extract refers to an extract of tangerine peel. Extracts of pericarp are preferred, but since active ingredients are also contained in fruits, seeds, leaves, stems, flowers, roots, etc., extracts from any one or more of these may be used.
  • Lactobiotyl is a product of Silab (France) and is a mixture of maltodextrin and water of the filtrate obtained by culturing lactic acid bacteria (Lactobacillus arizonensis) with jojoba. .
  • Yuzu extract and chimp extract can be easily dried, purified, and extracted from the above plants by known methods, and are readily available commercially. It can be used raw or dried, but it can also be used as an extract, dried product, dried powder, raw material powder, squeezed liquid, etc., and it is possible to appropriately select which form to use. and may be subjected to treatment such as sterilization as necessary.
  • the extraction method of the above extract can be performed, for example, by solvent extraction.
  • solvent extraction the whole or part of the plant is optionally dried and optionally chopped or ground before being treated with an aqueous extractant, water, such as cold water, hot water, or boiling point or below.
  • Hot water, anhydrous or water-containing organic solvents, organic solvents such as ethanol, methanol, ether, 1,3-butylene glycol, propylene glycol, etc. are appropriately selected according to the properties of the raw material and the application of the composition, and the solvent is heated at room temperature. Or it is extracted by heating and using.
  • the extraction method is not limited to solvent extraction, and may be a conventional method known in the art.
  • the form of the above-mentioned extract is not limited to the liquid extract itself, but may be one obtained by diluting or concentrating it by a conventional technique. good too.
  • the extraction solvent used in the present invention is preferably a polar solvent such as water, a lower alcohol, or a liquid polyhydric alcohol. , particularly water, or lower alcohols such as methanol, ethanol or 1,3-butylene glycol are preferred.
  • the lower alcohol may be, for example, a hydrous lower alcohol, in which case the water content is, for example, 0-10 v/v%, 10-40 v/v%, 20-30 v/v%, 30-50 v/v%. , 50 to 80 v/v%, 80 to 99.5 v/v%, and the like.
  • the lower alcohol may be, for example, a C1-C5 lower alcohol.
  • the solvent containing the extract may be used as it is, or may be used after being subjected to conventional purification treatments such as sterilization, washing, filtration, decolorization and deodorization. In addition, if necessary, it may be used after being concentrated by freeze-drying or the like or diluted with an arbitrary solvent. Further, the solvent may be entirely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.
  • the pressed liquid obtained by pressing the raw material plant also contains the same active ingredients as the extract, so the pressed liquid can be used instead of the extract.
  • the present invention also provides an epidermal normalizing agent containing the Netrin-1 inhibitor of the present invention.
  • the epidermal normalizing agent of the present invention can improve disturbance of the vascular structure, promote the supply of epidermal stem cells, and contribute to epidermal normalization.
  • Epidermal normalization refers to restoring the epidermis to a normal state by suppressing disturbance of the vascular structure.
  • inhibition of vasculature disruption achieves epidermal normalization via inhibition of structural flattening of the dermoepidermal junction and/or enhancement of epidermal stem cell supply.
  • epidermal normalization refers to improvement of skin condition by supplying epidermal stem cells. The epidermis is maintained in a normal state by repeating turnover at regular intervals. In other words, due to turnover, epidermal cells repeatedly divide and differentiate from stem cells in the basal layer in a normal cycle and migrate to the skin surface to regenerate new skin, while old epidermal cells are peeled off from the skin surface.
  • epidermal stem cells decrease with aging and turnover is delayed.
  • Disturbance of the vascular structure refers to a decrease in the amount of capillaries in the papillary layer and/or a disturbance in the direction of the capillaries and/or a movement of the capillaries away from the basement membrane.
  • the number of capillaries in the papillary layer is large and extends toward the basement membrane. move away from
  • such perturbation of vasculature is due to the action of epidermal Netrin-1.
  • Suppression of Netrin-1 means that the expression or activity of Netrin-1, for example, the amount of Netrin-1 mRNA or protein, is reduced when the Netrin-1 inhibitor of the present invention is added compared to when it is not added.
  • the reduction may be, for example, a reduction having statistical significance (e.g., Student's t-test) with a significance level of 5%, and/or, for example, 10% or more, 20% or more, 30% or more, The reduction may be 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, or 100% reduction.
  • the Netrin-1 inhibitor and epidermis normalizing agent of the present invention may contain any one of the above active ingredients alone. Alternatively, two or more types may be contained in any combination and ratio.
  • the agent of the present invention can also be a composition in which the above active ingredients are combined with one or more other ingredients such as excipients, carriers and/or diluents.
  • the composition and form of the composition are arbitrary, and may be appropriately selected according to conditions such as the active ingredient and application.
  • the composition can be produced by a conventional method in a formulation in which an excipient, carrier and/or diluent and other ingredients are appropriately combined according to the dosage form.
  • the agent of the present invention may be appropriately used orally or parenterally (percutaneous administration, intravenous administration, intraperitoneal administration, etc.) and may be administered by any route.
  • Transdermal administration is preferred.
  • the agent of the present invention can be used for humans and animals by being incorporated into cosmetics, pharmaceuticals, quasi-drugs, and the like.
  • the form of transdermal administration can be arbitrarily selected, for example, from creams, milky lotions, liquids, sheets, sprays, gels, and the like.
  • the cosmetic composition of the present invention may be various cosmetics such as milky lotion, cream, serum, lotion, pack, facial cleanser, soap, body soap, shampoo, etc., and may be liquid, milky, creamy or solid. , sheet-like, spray-like, gel-like, foam-like, and powder-like.
  • the present application also provides a cosmetic method for normalizing the epidermis, for example, for normalizing the epidermis through suppression of vascular structure disturbance, by administering the agent or composition of the present invention to a subject.
  • the method of the present invention may be a cosmetic method and not a treatment by a doctor or medical practitioner.
  • the administration frequency is once every 4 weeks, once every 2 weeks, once a week, once every 3 days, once every 2 days, once a day, twice a day, 3 times a day, 1 4 times a day, 5 times a day, each time administration, etc. can be arbitrarily selected, but are not limited to these.
  • the amount of yuzu extract, chimp extract, and/or lactobiotil in the agent or composition of the present invention can be appropriately determined according to the type, purpose, form, method of use, and the like.
  • the amount of these active ingredients is 0.0001 to 100% by weight, 0.0001 to 90% by weight, 0.001 to 50% by weight, 0.01 to 10% by weight, 0.1 to 10% by weight, based on the total weight of the agent or composition of the present invention. 1-10% by weight, 5-10% by weight, 6-7% by weight, 0.1-0.7% by weight, 0.7-6% by weight, 0.05-0.2% by weight, 0.1% by weight, etc. is not limited as long as the effect of is exhibited.
  • the agent and composition of the present invention can be produced using a conventional method in a formulation that appropriately combines excipients, carriers and/or diluents and other ingredients according to the dosage form.
  • Additives can be arbitrarily selected and used together as needed. Additives include excipients, colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, etc. As for, known ones can be appropriately selected and used.
  • the present invention also relates to a screening method for an epidermis-normalizing agent using Netrin-1 as an index in biological samples.
  • Screening methods of the present invention include, for example, the following steps: culturing a biological sample in medium containing a drug candidate; measuring Netrin-1 expression or activity in the biological sample; determining the candidate agent as having an epidermal normalizing effect if the expression or activity of is decreased.
  • the control is the expression or activity of Netrin-1 when cultured in media without the drug candidate.
  • a control experiment may be performed in parallel with the screening method of the present invention, or may be performed in advance.
  • epidermal cells are preferably used.
  • epidermal cells collected from animals such as humans, or their passaged cells may be cell lines, epidermal cells in skin models, Epidermal cells in the living body of animals such as humans may also be used.
  • a pre-culturing step of culturing the biological sample as described above may be included before the step of culturing the biological sample in a medium containing the candidate drug.
  • a post-culturing step of further culturing in a medium not containing the drug candidate may be included.
  • the culture obtained in the pre-culturing step may be cultured by directly adding the candidate drug or a diluted solution thereof, or by substituting the medium containing the candidate drug. Cultivation may be performed as follows.
  • the measurement of Netrin-1 expression or activity can be determined by measuring the amount of Netrin-1 mRNA or protein in a biological sample.
  • the amount of mRNA can be measured using techniques known in this technical field, such as quantitative PCR and northern blotting.
  • a probe for Netrin-1 mRNA may be used, as described in the Examples.
  • Protein amounts can be determined using any technique known in the art, such as Western blotting, immunostaining, FACS.
  • antibodies that specifically bind to Netrin-1 may be used.
  • a screening system using Netrin-1-expressing cells may be established and used.
  • the present invention also provides kits for carrying out the screening methods of the present invention, which contain reagents for determining Netrin-1 expression or activity.
  • a substance having an inhibitory effect on Netrin-1 screened by the screening method of the present invention may be any substance as long as it can suppress the expression or activity of Netrin-1.
  • One embodiment of the screening method of the present invention is to perform screening using an arbitrary library of cosmetic materials, food materials, pharmaceutical materials, etc., using Netrin-1 expression in biological samples such as epidermal cells as an indicator.
  • Substances having an inhibitory effect on Netrin-1 determined in this manner include one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil.
  • Netrin-1 inhibitors, including one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil are effective in epidermal normalization through inhibition of vascular structural disturbance.
  • the screening method of the present invention enables selection of a substance having an epidermal normalizing effect.
  • CA Obio LLC.
  • Sheep anti-Pecam-1/CD31 antibody against vascular endothelial cells was used as the primary antibody, and Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR) was used.
  • DAPI D9542, Merck, Germany
  • 3D skin vessels and cell nuclei were detected and imaged using a confocal microscope LSM880 (CarlZeiss, Germany).
  • the blood vessels in the dermis region extend in the direction toward the epidermis, and the epidermal-dermal junction has a large wavy structure.
  • Experiment 2 Structural Visualization of Skin Capillaries and Epidermal Stem Cells
  • skin tissue was immunostained.
  • Sheep anti-Pecam-1/CD31 antibody AF806, R&D systems, MN
  • mouse anti-MCSP antibody MAB2029, Merck, Germany
  • Secondary antibodies used were Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR) and Alexa Fluor568 donkey anti-mouse antibody (A10037, Molecular Probes, Eugene, OR).
  • DAPI D9542, Merck, Germany
  • epidermal stem cells white arrows
  • capillaries in the papillary layer
  • pericytes yellow arrows
  • Non-Patent Documents 1 to 4 mesenchymal stem cells gather in the skin wound and differentiate to promote repair. Therefore, the present inventors made more detailed observations on the relationship between pericytes present in papillary capillaries and epidermal stem cells.
  • Experiment 3 Structure Visualization of Skin Capillaries, Epidermal Stem Cells, and Basement Membrane
  • Primary antibodies include sheep anti-Pecam-1/CD31 antibody (AF806, R&D systems, MN) against vascular endothelial cells, mouse anti-MCSP antibody (MAB2029, Merck, Germany) against epidermal stem cells and pericytes, basement membrane A rat anti-Integrin alpha 6 antibody (sc-19622, Santa Cruz Biotechnology, Calif.) was used as a secondary antibody against Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR), Alexa Fluor568 donkey anti- Mouse antibody (A10037, Molecular Probes, Eugene, OR), Alexa Fluor647 donkey anti-rat antibody (ab150155, Abcam, UK) were used.
  • DAPI D9542, Merck, Germany
  • LSM880 CarlZeiss, Germany
  • the fixed skin tissue was washed with PBS, permeabilized with 0.5% Triton X-100, soaked in 1% Triton X-100/0.5% Tween-20 solution and incubated, then added with Perm block solution (0.5g BSA, 50ul Blocked with Tween20, 300ul 5% sodium azide in PBS) for 3 days. After that, it was incubated with sheep anti-Pecam-1/CD31 antibody (AF806, R&D systems, Minn.) as a primary antibody at 37°C for 3 days.
  • Perm block solution 0.5g BSA, 50ul Blocked with Tween20, 300ul 5% sodium azide in PBS
  • Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, Oreg.) was used as a secondary antibody and incubated at 37° C. for 3 days. After washing the antibody, it was dehydrated in 50% methanol for 3 hours, 70% methanol for 3 hours, and 100% methanol overnight. After that, it was shaken twice with dichloromethane (Sigma) for 15 minutes, and then allowed to stand overnight with dibenzyl ether (Sigma, MO) for clarifying treatment. The cleared skin tissue was observed with a light sheet fluorescence microscope Lightsheet Microscopy Z.1 (CarlZeiss, Germany) and images were obtained. From the resulting sheet images, 3D images were constructed using Imaris (Bitplane, Concord, Mass.).
  • FIG. 6 shows the results of the relationship between Netrin-1 expression and age obtained from Experiment 5. Therefore, we analyzed how Netrin-1 actually participates in skin vasculature and epidermis by the following experiments.
  • Experiment 7 Migration Test Using Vascular Endothelial Cells
  • human umbilical cord blood vein endothelial cells (HUVEC) were seeded on Fluoroblock inserts (BD Falcon), and human recombinant Netrin-1 (6419-N1, 6419-N1, R&D systems, MN) at a final concentration of 0.1 ⁇ g/ml or 1 ⁇ g/ml and/or vascular endothelial growth factor VEGFA165 (450-32, Peprotech, NJ) at a final concentration of 50 ng/ml. did.
  • EBM-2 medium with the same composition was used, except that these substances were not added.
  • the cells were fixed with 10% buffered formalin, and the cell nuclei were stained with Hoechst33432 (H3570, Invitrogen, Calif.). The number of cells that migrated to the membrane underneath the Fluoroblock insert was then counted.
  • Experiment 8 Preparation of 3D model of blood vessels and analysis of action of Netrin-1 A 3D model of blood vessels was prepared in a collagen gel, and the effect of adding Netrin-1 was observed. A 4 mg/ml collagen solution was prepared on ice using 5 mg/ml rat-derived collagen type 1 (3447-020-01, R&D systems, Minn.), 1M HEPES, 37 g/l NaHCO3. Using OrganoPlate (registered trademark) 3-lane 40 (4004-400-B, MIMETAS BV, Netherlands), according to the attached instructions and the method described in Non-Patent Document 7, the collagen solution described above was added to the gel lane (lane 2). was added and incubated at 37°C for 15 minutes to allow gelation.
  • OrganoPlate registered trademark 3-lane 40
  • 20,000 human umbilical vein endothelial cells were suspended in 2 ⁇ l of endothelial cell growth medium MV2 kit (C-22121, PromoCell, Germany) and seeded into the perfusion lane (lane 1) inlet of the OrganoPlate. After that, 50 ⁇ l of MV2 medium was added from the same inlet, and incubated on the OrganoPlate Stand at 37° C. for 1.5 hours. After confirming that the cells adhered to the gel, 50 ⁇ l each of MV2 medium was added to the outlet of the perfusion lane (lane 1) where the cells were seeded, and the inlet and outlet of the perfusion lane (lane 3) on the opposite side.
  • MV2 kit C-22121, PromoCell, Germany
  • Program 1 (Interval 8 min, 7 ° C.) was set on MIMETAS Rocker Mini (MIMETAS BV, Netherland) and cultured at 37° C. for 3 days to fabricate a blood vessel having a 3D structure.
  • VEGF165 450-32, Peprotech, NJ
  • bFGF 100 -18B, Peprotech, NJ
  • PMA P1585, Sigma, MO
  • Sphingosin-1-Phosphate S9666, Sigma, MO
  • Human recombinant Netrin at a final concentration of 1ug/ml- 1 (6419-N1, R&D systems, MN) was added, cultured on MIMETAS Rocker Mini at 37°C for 3 days, fixed by incubating with 3.7% paraformaldehyde at RT for 15 minutes, and immunostained.
  • Primary antibodies were rabbit anti-VE-Cadherin antibody (#2158, Cell Signaling Technology, MA) against vascular endothelial cells and CF568 conjugated Phalloidin (00040, Biotium, Inc., CA) against cytoskeletal f-actin.
  • the secondary antibody used was Alexa Fluor488 donkey anti-rabbit antibody (A21206, Molecular Probes, Eugene, Oreg.).
  • Hoechst33432 H3570, Invitrogen, Calif. was used for nuclear staining. After staining, a confocal microscope LSM880 (CarlZeiss, Germany) was used to detect 3D vascular structures and angiogenesis to obtain image information.
  • a 3D image was constructed from the obtained information using Imaris (Bitplane, Concord, Mass.) (Fig. 9, right).
  • a 3D model was prepared under the same conditions except that Netrin-1 was not added, and staining and image construction were performed (FIG. 9, left).
  • the constructed 3D image is shown in Fig. 9.
  • Fig. 9 When Netrin-1 was added (added to the upper right part of Fig. 9), a decrease in the amount of blood vessels formed in the collagen gel and disturbance of the vascular structure were observed (Fig. 9 right center part). According to 1, the result was consistent with the knowledge obtained from experiments 7 and 8 that the capillaries acted in the direction of repelling.
  • Fig. 10 As shown in Fig. 10 as a schematic diagram, epidermal Netrin-1, which increases with age, binds to the Netrin-1 receptor UNC5B on capillaries in the papillary layer.
  • blood vessels repel the epidermis, disturbing the vascular structure and suppressing the supply of epidermal stem cells from pericytes, leading to suppression of normalization of the epidermis.
  • Real-time PCR was performed using a reaction kit (ThermoFishser scientific, TaqMan RNA-to-CT 1-Step Kit, 4392938) and Netrin-1 and ⁇ -actin specific probes (ThermoFishser scientific, Netrin-1: Hs00924151_m1, ⁇ -actin: Hs01060665_g1). was used.
  • ⁇ -actin was used as an endogenous control. We searched for those in which the Netrin-1 expression level was significantly reduced compared to the control (cont).

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Abstract

The present invention addresses the problem of providing: a substance for making skin normal; and a method for screening for a substance having an activity to make skin normal.　Provided are: a Netrin-1 inhibitor; and an agent which comprises the Netrin-1 inhibitor and is effective for making skin normal through the inhibition of the disarrangement of a vascular structure by the inhibition of Netrin-1. A Netrin-1 inhibitor comprising one or a plurality selected from the group consisting of Yuzu (Citrus junos) extract, a citrus unshiu peel extract and lactobiotyl is effective for making skin normal through the inhibition of the disarrangement of a vascular structure. The screening method according to the present invention makes it possible to select a substance having an activity to make skin normal.

Description

Ｎｅｔｒｉｎ－１抑制剤Netrin-1 inhibitor
　本発明は、Netrin-1抑制剤に関する。 The present invention relates to a Netrin-1 inhibitor.
　皮膚表皮は、見た目の年齢や外観に影響が大きく、表皮を健全な状態に維持することおよびより良い状態に改善することは、美容上の大きな課題である。表皮では、基底層にある幹細胞から分裂を繰り返し分化するとともに皮膚表面に移動し、古い表皮細胞が皮膚から剥がれ落ちるターンオーバーを繰り返すことで正常な状態に保たれている。 The skin epidermis has a great influence on the apparent age and appearance, and maintaining the epidermis in a healthy state and improving it to a better state is a major cosmetic issue. In the epidermis, stem cells in the basal layer repeatedly divide and differentiate, migrate to the surface of the skin, and the old epidermal cells are sloughed off from the skin.
　しかしながら、表皮幹細胞がうまく供給されないと表皮が正常な状態に保つことができない。例えば、加齢などの要因により表皮幹細胞が減少することが知られており、皮膚が老化する一因となっている。表皮幹細胞の産生を促進するための各種方策が知られている。例えば、特許文献１では、表皮幹細胞の減少抑制又は増加促進剤であるラミニン５１１発現促進剤を開示する。特許文献２では、マンネンタケ胞子の超臨界抽出物を有効成分として含有する、表皮幹細胞の表皮角化細胞への分化促進剤を開示する。 However, if epidermal stem cells are not well supplied, the epidermis cannot be maintained in a normal state. For example, it is known that epidermal stem cells decrease due to factors such as aging, which contributes to skin aging. Various strategies are known for promoting the production of epidermal stem cells. For example, Patent Literature 1 discloses a laminin 511 expression promoter that suppresses the decrease or increases the number of epidermal stem cells. Patent Document 2 discloses an agent for promoting the differentiation of epidermal stem cells into epidermal keratinocytes, containing a supercritical extract of Ganoderma lucidum spores as an active ingredient.
　また、加齢により真皮表皮接合部の構造が平坦化し、皮膚の弾力を低下させる一因となっていることも知られている。真皮表皮接合部の構造が平坦化を抑制するための方策として、例えば、特許文献３では、真皮表皮接合部を強化することにより皮膚の状態を改善するための組成物を開示する。 It is also known that aging causes the structure of the dermal-epidermal junction to flatten, which is one of the factors that reduces skin elasticity. As a measure for suppressing flattening of the structure of the dermal-epidermal junction, for example, Patent Document 3 discloses a composition for improving the condition of the skin by strengthening the dermal-epidermal junction.
国際公開第2018/074606号WO2018/074606 特開2020-174543号公報JP 2020-174543 A 国際公開第2018/125237号WO2018/125237 国際公開第2019/098302号WO2019/098302 国際公開第2019/097958号WO 2019/097958 国際公開第2020/230331号WO2020/230331 特許第6042904号公報Patent No. 6042904 国際公開第2007/521634号WO2007/521634 特許第6306768号公報Patent No. 6306768 特開2018-57304号公報Japanese Patent Application Laid-Open No. 2018-57304
　本発明は、表皮正常化を促進する物質、並びに、表皮正常化促進作用を有する物質をスクリーニングする方法を提供することを課題とする。 An object of the present invention is to provide a substance that promotes epidermal normalization and a method of screening for a substance that promotes epidermal normalization.
　本発明者らは鋭意研究の結果、真皮乳頭層における血管と表皮幹細胞の産生が密接に関連すること、および真皮血管構造の乱れと表皮幹細胞の産生の抑制が表皮正常化を抑制する一因であるという知見を得た。更に本発明者らはかかる血管構造の乱れがNetrin-1により引き起こされることを発見し、Netrin-1を抑制する物質も発見した。 As a result of intensive research, the present inventors have found that blood vessels in the papillary dermis are closely related to the production of epidermal stem cells, and that disturbance of the dermal vasculature and suppression of epidermal stem cell production are one of the factors that suppress epidermal normalization. I have learned that there is. Furthermore, the present inventors discovered that such disruption of vascular structures is caused by Netrin-1, and also discovered a substance that inhibits Netrin-1.
　本発明は、下記の発明に関する：
（１）表皮正常化のための、Netrin-1抑制剤。
（２）ユズエキス、チンピエキス、およびラクトビオチルからなる群から選択される1又は複数からなる、Netrin-1抑制剤。
（３）（１）又は（２）に記載のNetrin-1抑制剤を含む、表皮正常化剤。
（４）前記表皮正常化は、Netrin-1の抑制により血管構造の乱れを抑制することを介して達成される、（３）に記載の表皮正常化剤。
（５）Netrin-1を指標とした表皮正常化剤のスクリーニング方法。
（６）候補薬剤を含む培地で生体試料を培養すること；
　生体試料におけるNetrin-1の発現又は活性を測定すること；および
　対照に比較してNetrin-1の発現又は活性が減少した場合に、候補薬剤を表皮正常化作用を有する物質として決定すること；
　を含む、（５）に記載のスクリーニング方法。
（７）対象のNetrin-1を抑制させることを含む、対象の表皮を正常化するための美容目的の方法。
（８）対象のNetrin-1の抑制は、（３）又は（４）に記載の表皮正常化剤を対象に投与することにより達成される、（７）に記載の方法。 The present invention relates to the following inventions:
(1) Netrin-1 inhibitors for epidermal normalization.
(2) a Netrin-1 inhibitor consisting of one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil;
(3) An agent for normalizing epidermis, comprising the Netrin-1 inhibitor according to (1) or (2).
(4) The agent for normalizing the epidermis according to (3), wherein the normalization of the epidermis is achieved through inhibition of disturbance of vascular structures by inhibition of Netrin-1.
(5) Screening method for an epidermal normalizing agent using Netrin-1 as an index.
(6) culturing the biological sample in a medium containing the candidate agent;
measuring Netrin-1 expression or activity in a biological sample; and determining a candidate agent as having epidermal normalizing activity if Netrin-1 expression or activity is reduced compared to a control;
The screening method according to (5), comprising
(7) A cosmetic method for normalizing the epidermis of a subject comprising suppressing Netrin-1 in the subject.
(8) The method of (7), wherein suppression of Netrin-1 in the subject is achieved by administering the epidermal normalizing agent of (3) or (4) to the subject.
　本発明のNetrin-1抑制剤は、Netrin-1を抑制することができる。Netrin-1が抑制されると、血管構造の乱れが抑制される。血管構造の乱れの抑制により表皮幹細胞の産生が促進され、ひいては表皮正常化に寄与する。ユズエキス、チンピエキス、およびラクトビオチルからなる群から選択される1又は複数の生薬を含むNetrin-1抑制剤は、血管構造の乱れの抑制を介し表皮正常化に有効である。また、本発明のスクリーニング方法により、表皮正常化作用を有する物質を選択することができる。本発明のスクリーニング方法により選択された表皮正常化剤は、Netrin-1を抑制することにより表皮正常化に寄与する。 The Netrin-1 inhibitor of the present invention can suppress Netrin-1. Inhibition of Netrin-1 inhibits disturbance of vasculature. Suppression of disturbance of vascular structures promotes the production of epidermal stem cells, which in turn contributes to normalization of the epidermis. A Netrin-1 inhibitor containing one or more herbal medicines selected from the group consisting of yuzu extract, chimp extract, and lactobiotil is effective in epidermal normalization through inhibition of disturbance of vascular structures. In addition, the screening method of the present invention enables selection of a substance having an epidermal normalizing effect. Epidermal normalization agents selected by the screening method of the present invention contribute to epidermal normalization by suppressing Netrin-1.
図1は、実験1の結果を示す。図1上は、若齢者（33歳）及び高齢者（69歳）の皮膚における表皮真皮接合部を示す。緑はCD31染色部分（血管内皮細胞）、白はDAPI染色部分（核）を示す。図1下は、若齢群（20-30代：N=3）及び高齢群（60-90代：N=3)の表皮真皮接合部の長さ（μｍ）（下左）と真皮領域における血管部分の面積の割合（％）(下右)を示す。図中、＊はp<0.05、＊＊はp<0.01を示す（t検定）。FIG. 1 shows the results of Experiment 1. FIG. 1, top, shows the epidermal-dermal junction in young (33 years old) and old (69 years old) skin. Green indicates CD31-stained portions (vascular endothelial cells) and white indicates DAPI-stained portions (nuclei). The lower part of Fig. 1 shows the length of the epidermal-dermal junction (μm) (lower left) and the dermal area of the young group (20s-30s: N = 3) and the old group (60s-90s: N = 3). The ratio (%) of the area of the blood vessel part (lower right) is shown. In the figure, * indicates p<0.05 and ** indicates p<0.01 (t-test). 図2は、実験2の結果を示す。図2左と中央は、若齢者（27歳）（左及び中央上）及び高齢者（60歳）（中央下）の皮膚における表皮真皮接合部を示す。緑はCD31染色部分（血管内皮細胞）、白はDAPI染色部分（核）、赤はMCSP染色部分（表皮幹細胞及びペリサイト）を示す。図2右は、若齢群（20-30代：N=3）及び高齢群（60-90代：N=3)の表皮領域におけるMCSP染色部分の面積の割合（％）を示す。図中、＊はp<0.05を示す（t検定）。FIG. 2 shows the results of Experiment 2. FIG. 2, left and middle, shows the epidermal-dermal junction in young (27 years old) (left and top center) and aged (60 years) (bottom center) skin. Green indicates CD31-stained portions (vascular endothelial cells), white indicates DAPI-stained portions (nuclei), and red indicates MCSP-stained portions (epidermal stem cells and pericytes). The right side of FIG. 2 shows the ratio (%) of the area of the MCSP-stained part in the epidermal region of the young group (20s to 30s: N=3) and the old group (60s to 90s: N=3). In the figure, * indicates p<0.05 (t-test). 図3は、実験3の結果を示す。図3左は、若齢者（27歳）の皮膚における表皮真皮接合部を示す。緑はCD31染色部分（血管内皮細胞）、白はDAPI染色部分（核）、赤はMCSP染色部分（表皮幹細胞及びペリサイト）、青は抗Integrin alpha 6抗体による染色部分(基底膜）を示す。左は全ての染色部分を示し、右は基底膜（青）のみを示す。FIG. 3 shows the results of Experiment 3. Fig. 3 (left) shows the epidermal-dermal junction in the skin of a young person (27 years old). Green indicates areas stained with CD31 (vascular endothelial cells), white indicates areas stained with DAPI (nuclei), red indicates areas stained with MCSP (epidermal stem cells and pericytes), and blue indicates areas stained with anti-integrin alpha 6 antibody (basement membrane). The left shows all stained sections, the right shows only the basement membrane (blue). 図4は、実験3の結果を示し、図3の表皮真皮接合部の深度を変えて（Z軸方向に移動）撮影した図を示す。染色は図3と同じである。FIG. 4 shows the results of Experiment 3, and shows images taken while changing the depth of the epidermal-dermal junction in FIG. 3 (moving in the Z-axis direction). Staining is the same as in FIG. 図5は、実験4の結果を示し、左は若齢者（18歳）、右は高齢者（50歳）の被験者の真皮の血管構造を示す。FIG. 5 shows the results of Experiment 4, showing the dermal vascular structure of a young (18 year old) subject on the left and an elderly (50 year old) subject on the right. 図6は、実験5より得られたNetrin-1発現量と年齢の関係を示す。FIG. 6 shows the relationship between the Netrin-1 expression level obtained from Experiment 5 and age. 図7は、実験6の結果である若齢者（27歳）の皮膚における表皮真皮接合部を示す。緑はCD31染色部分（血管内皮細胞）、白はDAPI染色部分（核）、赤はMCSP染色部分（表皮幹細胞及びペリサイト）、青はUNC5B染色部分(Netrin-1受容体）を示す。左は全ての染色部分、中央は、血管（緑）、幹細胞・ペリサイト（赤）及びNetrin-1受容体（青）、右はNetrin-1受容体（青）のみを示す。FIG. 7 shows the epidermal-dermal junction in the skin of a young person (27 years old), the result of Experiment 6. Green indicates CD31-stained portions (vascular endothelial cells), white indicates DAPI-stained portions (nuclei), red indicates MCSP-stained portions (epidermal stem cells and pericytes), and blue indicates UNC5B-stained portions (Netrin-1 receptor). Left shows all stained parts, middle shows blood vessels (green), stem cells/pericytes (red) and Netrin-1 receptor (blue), right shows only Netrin-1 receptor (blue). 図8は、実験7の方法（左）および結果（右）を示す。中央グラフの縦軸は、Human recombinant Netrin-1を最終濃度0.1μg/ml又は1μg/ml、及び／又は血管内皮増殖因子VEGFA165(450-32, Peprotech, NJ)を50ng/mlの最終濃度で添加した場合に遊走した細胞数を示す。対照には、上記物質を添加しない以外は同じ組成の培地を使用した（Ctrl）。図中、＊＊＊＊はp<0.0001、＊＊＊はp<0.001を示す（t検定）。FIG. 8 shows the method (left) and results (right) of Experiment 7. The vertical axis of the central graph indicates human recombinant Netrin-1 at a final concentration of 0.1 μg/ml or 1 μg/ml, and/or vascular endothelial growth factor VEGFA165 (450-32, Peprotech, NJ) at a final concentration of 50 ng/ml. Shows the number of cells that migrated when As a control, a medium with the same composition was used except that the above substances were not added (Ctrl). In the figure, *** indicates p<0.0001, *** indicates p<0.001 (t-test). 図9は、実験8の方法によりNetrin-1を添加した（右）又は添加しない（左）条件で構築した３Ｄ血管モデルを示す。緑はラビット抗VE-Cadherin抗体による染色部分（血管内皮細胞）、青はDAPI染色部分（核）、赤はPhalloidin染色部分（細胞骨格f-アクチン）を示す。FIG. 9 shows a 3D blood vessel model constructed by the method of Experiment 8 with (right) or without (left) Netrin-1. Green indicates areas stained with rabbit anti-VE-Cadherin antibody (vascular endothelial cells), blue indicates areas stained with DAPI (nuclei), and red indicates areas stained with Phalloidin (cytoskeletal f-actin). 図10は、老化により増加する表皮Netrin-1による血管細胞上にあるNetrin-1受容体UNC5Bの忌避作用と血管構造の乱れを本願から導かれる概念として示す模式図である。FIG. 10 is a schematic diagram showing the repelling effect of the Netrin-1 receptor UNC5B on vascular cells and the disorder of the vascular structure by epidermal Netrin-1, which increases with aging, as a concept derived from the present application. 図11は、ユズエキス、チンピエキス、およびラクトビオチルを用いた場合のNetrin-1の発現を、対照（control）を100とした場合の相対値（％）として示す。図中、＊はp<0.05を示す（t検定）。FIG. 11 shows the expression of Netrin-1 when yuzu extract, chimp extract, and lactobiotil were used as relative values (%) when the control was set to 100. In the figure, * indicates p<0.05 (t-test).
　血管と皮膚の状態には何らかの関連性があることは知られており、例えば、毛細血管の保護、改善、増加、機能向上を介してむくみ、くすみ、アトピー性皮膚炎などの改善に機能すること、VEカドヘリン及びインテグリンα５により皮膚のハリを改善すること等が報告されている（特許文献４、５）。また、本発明者らは、血管の安定化や機能向上により血管周囲のコラーゲン合成を促進し皮膚弾力を改善することを報告している（特許文献６）。しかしながら、真皮内に存在する血管と表皮が実際にどのように関係するかについては不明な点が多くあまり解明されていなかった。 It is known that there is some relationship between blood vessels and skin conditions. For example, it functions to improve swelling, dullness, atopic dermatitis, etc. , VE-cadherin and integrin α5 have been reported to improve skin elasticity (Patent Documents 4 and 5). In addition, the present inventors have reported that by stabilizing and improving the functions of blood vessels, collagen synthesis around blood vessels is promoted and skin elasticity is improved (Patent Document 6). However, the actual relationship between the blood vessels in the dermis and the epidermis has not been elucidated due to many unclear points.
　しかしながら、本発明者らは、更なる鋭意研究の結果、驚くことに真皮乳頭層の毛細血管が表皮正常化に大きな影響を与えていることを発見した。とりわけ、本発明者らは、乳頭層毛細血管周囲に存在するペリサイトにより表皮幹細胞が供給されること、加齢により乳頭層毛細血管が減少しその構造が乱れ真皮表皮接合部の平坦化が起こること、更に、このような血管構造の乱れを引き起こす物質としてNetrin-1を発見し、表皮Netrin-1が真皮の血管に作用することを介して表皮に悪影響を及ぼすという知見を得た。より詳細には、加齢に伴い表皮Netrin-1が増加すること、並びに、表皮Netrin-1により乳頭層毛細血管が真皮表皮接合部から忌避され、その結果、乳頭層血管構造が乱れる要因となっているという知見を得た。乳頭層の毛細血管が真皮表皮接合部から忌避されると、乳頭層の血管構造が乱れ真皮表皮接合部が平坦化し、表皮基底膜を介して供給される表皮幹細胞数が減少し、表皮正常化が抑制されると考えられる。 However, as a result of further intensive research, the present inventors surprisingly discovered that capillaries in the papillary dermis have a great influence on epidermal normalization. In particular, the present inventors have found that epidermal stem cells are supplied by pericytes present around papillary capillaries, and that with aging, the number of papillary capillaries decreases and their structure is disturbed, resulting in flattening of the dermal-epidermal junction. Furthermore, we discovered Netrin-1 as a substance that induces such disorder of vascular structure, and obtained the knowledge that epidermal Netrin-1 exerts an adverse effect on the epidermis through its action on blood vessels in the dermis. More specifically, epidermal Netrin-1 increases with aging, and epidermal Netrin-1 repels papillary capillaries from the dermal-epidermal junction, resulting in disturbance of the papillary layer vasculature. I got the knowledge that When capillaries in the papillary layer are repelled from the dermal-epidermal junction, the vascular structure of the papillary layer is disturbed, the dermal-epidermal junction becomes flattened, the number of epidermal stem cells supplied through the epidermal basement membrane decreases, and the epidermis normalizes. is thought to be suppressed.
　本発明は、このような発明者らの知見に基づくものであり、Netrin-1抑制剤およびNetrin-1抑制剤を含む表皮正常化剤に関する。Netrin-1が抑制されると、血管構造の乱れが抑制され、乳頭層毛細血管と表皮基底膜との関連が密接になり表皮幹細胞の供給が促進される。表皮幹細胞の供給が促進されるとターンオーバーが促進され表皮正常化に寄与する。 The present invention is based on these findings of the inventors, and relates to a Netrin-1 inhibitor and an epidermal normalizing agent containing a Netrin-1 inhibitor. Suppression of Netrin-1 suppresses disturbance of the vascular structure and promotes the supply of epidermal stem cells by making the relationship between the capillaries of the papillary layer and the epidermal basement membrane closer. Accelerated supply of epidermal stem cells promotes turnover and contributes to normalization of the epidermis.
　Netrin-1（Laminin-related secreted protein；NTN1）は、50～75 kDaのラミニン様ポリペプチドである。Netrin-1は、軸索ガイダンス因子として機能し、皮膚にもその発現が見られ光老化に関与すること等知られている。血管細胞及び内皮細胞の増殖及び遊走を調節作用があること、疼痛発症に関与することなどが報告されている(特許文献７，８，９）。しかしながら、Netrin-1が真皮の血管に作用することにより表皮に悪影響を及ぼすことは知られていない。 　Netrin-1 (Laminin-related secret protein; NTN1) is a 50-75 kDa laminin-like polypeptide. Netrin-1 functions as an axonal guidance factor and is known to be expressed in the skin and involved in photoaging. It has been reported that it has a regulating effect on the proliferation and migration of vascular cells and endothelial cells and that it is involved in the onset of pain (Patent Documents 7, 8 and 9). However, it is not known that Netrin-1 adversely affects epidermis by acting on dermal blood vessels.
　ペリサイト（周皮細胞）とは、微小血管周囲を取り角むように存在する細胞である。血管の維持、血流調節、血管内皮細胞の分化・増殖制御、血管形成等に関与すると考えられているが、その詳細は不明なことが多い。ペリサイトが多分化能を有する間葉系細胞であるという示唆もされている。皮膚においては、創傷治癒過程に関与すること、皮膚の微小環境を調節していること、表皮細胞の分裂の極性や方向性を誘導して皮膚再生を促していること等が報告又は示唆されている（非特許文献１～４）ものの、真皮血管周囲に存在するペリサイトが直接表皮幹細胞を供給することは全く知られていない。 Pericytes (pericytes) are cells that surround microvessels. It is thought to be involved in maintenance of blood vessels, regulation of blood flow, regulation of differentiation/proliferation of vascular endothelial cells, angiogenesis, etc., but the details are often unknown. It has also been suggested that pericytes are mesenchymal cells with pluripotency. In the skin, it has been reported or suggested that it participates in the wound healing process, regulates the microenvironment of the skin, and induces the polarity and direction of division of epidermal cells to promote skin regeneration. However, it is completely unknown that pericytes present around dermal blood vessels directly supply epidermal stem cells.
　1実施形態では、本発明のNetrin-1抑制剤は、ユズエキス、チンピエキス、およびラクトビオチルからなる群から選択される1又は複数からなる又は有効成分として含有する。しかしながら、Netrin-1を抑制することができる物質であればこれらに限定されない。 In one embodiment, the Netrin-1 inhibitor of the present invention consists of or contains as an active ingredient one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil. However, the substance is not limited to these as long as it can suppress Netrin-1.
　ユズ（Citrus junos）はミカン科ミカン属ユズ種の柑橘類である。別名ホンユズとも呼ばれ、日本各地、中国、ベトナムなどのアジア地域に広く分布する。果実や果皮に、血行促進作用、活性酸素消去作用、抗菌作用等が知られている。ユズエキスは、ユズの果実や果皮を抽出した抽出物のことをいう。ユズは果実（果肉および果皮）が好ましいが、種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。 Citrus junos is a citrus fruit belonging to the genus Citrus of the family Rutaceae. Also known as honyuzu, it is widely distributed throughout Japan, China, and other Asian regions such as Vietnam. The fruit and pericarp are known to have blood circulation promoting action, active oxygen scavenging action, antibacterial action, and the like. Yuzu extract refers to an extract obtained by extracting yuzu fruit and pericarp. The fruit (flesh and peel) of yuzu is preferable, but since active ingredients are also contained in seeds, leaves, stems, flowers, roots, etc., extracts of one or more of these may also be used. can.
　ミカン（Citrus unshiu）は、ムクロジ目ミカン科ミカン属の常緑低木であり、幹細胞未分化状態維持及び増殖促進作用等が報告されている（特許文献１０）。チンピエキスは、ミカンの果皮の抽出物を指す。果皮の抽出物が好ましいが、果実、種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。 Mandarin orange (Citrus unshiu) is an evergreen shrub belonging to the genus Citrus of the family Rutaceae, order Sapindaceae, and has been reported to maintain an undifferentiated state of stem cells and promote proliferation (Patent Document 10). Chimpi extract refers to an extract of tangerine peel. Extracts of pericarp are preferred, but since active ingredients are also contained in fruits, seeds, leaves, stems, flowers, roots, etc., extracts from any one or more of these may be used.
　ラクトビオチル(Lactobiotyl）は、シラブ（Silab）社（フランス）の製品であり、乳酸菌（ラクトバチルス アリゾネンシス）をホホバとともに培養することにより得られる培養液の濾過物をマルトデキストリンおよび水と混合した混合物である。 Lactobiotyl is a product of Silab (France) and is a mixture of maltodextrin and water of the filtrate obtained by culturing lactic acid bacteria (Lactobacillus arizonensis) with jojoba. .
　ユズエキスおよびチンピエキスは、上記植物から公知の方法により容易に乾燥、精製、抽出ができ、また市販品を容易に入手可能である。生のままでも乾燥したものでも使用することができるが、抽出物、乾燥物、乾燥粉末、原料の粉末物、搾汁液等として用いることもでき、いずれの形態を用いるかは適宜選択することができ、必要に応じて殺菌等の処理を施してもよい。 Yuzu extract and chimp extract can be easily dried, purified, and extracted from the above plants by known methods, and are readily available commercially. It can be used raw or dried, but it can also be used as an extract, dried product, dried powder, raw material powder, squeezed liquid, etc., and it is possible to appropriately select which form to use. and may be subjected to treatment such as sterilization as necessary.
　上記エキスの抽出方法は例えば溶媒抽出により行うことができる。溶媒抽出の場合には、植物の全体又は部分を必要に応じて乾燥させ、更に必要に応じて細断又は粉砕した後、水性抽出剤、水、例えば冷水、温水、又は沸点若しくはそれより低温の熱水、あるいは無水又は含水有機溶媒、有機溶媒、例えばエタノール、メタノール、エーテル、１，３－ブチレングリコール、プロピレングリコール等を原料の性質や組成物の用途等により好ましい溶媒を適宜選択して常温で又は加熱して用いることにより抽出される。しかしながら、抽出方法は溶媒抽出に限定されず、当業界で知られている常用の手法によってもよく、本発明で用いる抽出物の抽出方法や抽出物の形態は、本発明の効果を損なわない限り任意である。上記抽出物の形態は、抽出液自体だけでなく、常用の手法により適宜希釈又は濃縮したものであってもよく、更に、抽出液を乾燥することによって得られる粉状あるいは塊状の固体であってもよい。 The extraction method of the above extract can be performed, for example, by solvent extraction. In the case of solvent extraction, the whole or part of the plant is optionally dried and optionally chopped or ground before being treated with an aqueous extractant, water, such as cold water, hot water, or boiling point or below. Hot water, anhydrous or water-containing organic solvents, organic solvents such as ethanol, methanol, ether, 1,3-butylene glycol, propylene glycol, etc. are appropriately selected according to the properties of the raw material and the application of the composition, and the solvent is heated at room temperature. Or it is extracted by heating and using. However, the extraction method is not limited to solvent extraction, and may be a conventional method known in the art. Optional. The form of the above-mentioned extract is not limited to the liquid extract itself, but may be one obtained by diluting or concentrating it by a conventional technique. good too.
　本発明で用いる抽出物の抽出方法や形態は本発明の効果を損なわない限り、任意であるが、本発明に用いられる抽出溶媒は、水、低級アルコール及び液状多価アルコール等の極性溶媒が好ましく、特に水、あるいはメタノール、エタノール又は１，３－ブチレングリコール等の低級アルコールが好ましい。低級アルコールは、例えば、含水低級アルコールであってもよく、その場合の含水率は、例えば0～10v/v％、10～40v/v％、20～30v/v％、30～50v/v％、50～80v/v％、80～99.5v/v％等であってもよい。低級アルコールは、例えば、Ｃ１～Ｃ５の低級アルコールであってもよい。これらの溶媒は一種でも二種以上を混合して用いても良い。 The extraction method and form of the extract used in the present invention are arbitrary as long as the effects of the present invention are not impaired, but the extraction solvent used in the present invention is preferably a polar solvent such as water, a lower alcohol, or a liquid polyhydric alcohol. , particularly water, or lower alcohols such as methanol, ethanol or 1,3-butylene glycol are preferred. The lower alcohol may be, for example, a hydrous lower alcohol, in which case the water content is, for example, 0-10 v/v%, 10-40 v/v%, 20-30 v/v%, 30-50 v/v%. , 50 to 80 v/v%, 80 to 99.5 v/v%, and the like. The lower alcohol may be, for example, a C1-C5 lower alcohol. These solvents may be used singly or in combination of two or more.
　このような抽出操作により、有効成分が抽出され、溶媒に溶け込む。抽出物を含む溶媒は、そのまま使用してもよいが、滅菌、洗浄、濾過、脱色、脱臭等の慣用の精製処理を加えてから使用してもよい。また、必要により凍結乾燥などにより濃縮あるいは任意の溶媒で希釈してから使用してもよい。さらに、溶媒を全て揮発させて固体状（乾燥物）としてから使用してもよいし、該乾燥物を任意の溶媒に再溶解してから使用してもよい。 Through such an extraction operation, the active ingredients are extracted and dissolved in the solvent. The solvent containing the extract may be used as it is, or may be used after being subjected to conventional purification treatments such as sterilization, washing, filtration, decolorization and deodorization. In addition, if necessary, it may be used after being concentrated by freeze-drying or the like or diluted with an arbitrary solvent. Further, the solvent may be entirely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.
　また、原料の植物を圧搾することにより得られる圧搾液にも抽出物と同様の有効成分が含まれているので、抽出物の代わりに圧搾液を使用することもできる。 In addition, the pressed liquid obtained by pressing the raw material plant also contains the same active ingredients as the extract, so the pressed liquid can be used instead of the extract.
　また、本発明は、本発明のNetrin-1抑制剤を含有する表皮正常化剤も提供する。本発明の表皮正常化剤は、表皮におけるNetrin-1を抑制することにより、血管構造の乱れが改善され、表皮幹細胞の供給が促進され、表皮正常化に寄与することができる。 The present invention also provides an epidermal normalizing agent containing the Netrin-1 inhibitor of the present invention. By suppressing Netrin-1 in the epidermis, the epidermal normalizing agent of the present invention can improve disturbance of the vascular structure, promote the supply of epidermal stem cells, and contribute to epidermal normalization.
　表皮正常化とは、血管構造の乱れを抑制することにより表皮を正常な状態にすることを指す。１実施形態では、血管構造の乱れの抑制により、真皮表皮接合部の構造の平坦化の抑制及び／又は表皮幹細胞の供給の促進を介して表皮正常化が達成される。１実施形態では、表皮正常化とは、表皮幹細胞が供給されることによる皮膚状態の改善を指す。表皮は一定の周期でターンオーバーを繰り返すことで正常な状態に保たれている。つまり、ターンオーバーにより、表皮細胞が正常な周期で基底層にある幹細胞から分裂を繰り返し分化するとともに皮膚表面に移動し新しい皮膚が再生されるとともに、古い表皮細胞が皮膚表面から剥がれ落ちる。しかしながら、加齢といった要因によるターンオーバーの乱れや遅延により、シミ、しわ、たるみといった老化現象を引き起こす。表皮幹細胞の減少は、このようなターンオーバーの乱れや遅延の要因となる。とりわけ、加齢に伴い表皮幹細胞が減少し、ターンオーバーが遅くなることが知られている。 Epidermal normalization refers to restoring the epidermis to a normal state by suppressing disturbance of the vascular structure. In one embodiment, inhibition of vasculature disruption achieves epidermal normalization via inhibition of structural flattening of the dermoepidermal junction and/or enhancement of epidermal stem cell supply. In one embodiment, epidermal normalization refers to improvement of skin condition by supplying epidermal stem cells. The epidermis is maintained in a normal state by repeating turnover at regular intervals. In other words, due to turnover, epidermal cells repeatedly divide and differentiate from stem cells in the basal layer in a normal cycle and migrate to the skin surface to regenerate new skin, while old epidermal cells are peeled off from the skin surface. However, disturbance or delay in turnover due to factors such as aging causes aging phenomena such as spots, wrinkles, and sagging. Decrease in epidermal stem cells is one of the factors that disrupts and delays such turnover. In particular, it is known that epidermal stem cells decrease with aging and turnover is delayed.
　血管構造の乱れとは、乳頭層における毛細血管の量が減少すること及び／又は毛細血管の方向が乱れること及び／又は毛細血管が基底膜から遠ざかることを指す。図５に示すように若齢者の皮膚では乳頭層における毛細血管はその量が多く基底膜に向かって伸びているが、加齢に伴い毛細血管の量が減少し、方向が乱れ、基底膜から遠ざかる。1実施形態では、このような血管構造の乱れは表皮Netrin-1の作用によるものである。 Disturbance of the vascular structure refers to a decrease in the amount of capillaries in the papillary layer and/or a disturbance in the direction of the capillaries and/or a movement of the capillaries away from the basement membrane. As shown in Fig. 5, in young skin, the number of capillaries in the papillary layer is large and extends toward the basement membrane. move away from In one embodiment, such perturbation of vasculature is due to the action of epidermal Netrin-1.
　Netrin-1の抑制は、Netrin-1の発現又は活性、例えば、Netrin-1のmRNA量又はタンパク質量が、本発明のNetrin-1抑制剤を添加すると、添加しない場合と比べて減少することを意味しうる。減少は、例えば、有意水準を5％とした統計学的有意差（例えばスチューデントのt検定）を有する減少であってもよく、及び／又は、例えば10％以上、20％以上、30％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、又は100％の減少であってもよい。 Suppression of Netrin-1 means that the expression or activity of Netrin-1, for example, the amount of Netrin-1 mRNA or protein, is reduced when the Netrin-1 inhibitor of the present invention is added compared to when it is not added. can mean The reduction may be, for example, a reduction having statistical significance (e.g., Student's t-test) with a significance level of 5%, and/or, for example, 10% or more, 20% or more, 30% or more, The reduction may be 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, or 100% reduction.
　本発明のNetrin-1抑制剤及び表皮正常化剤（以降これらを総称して「本発明の剤」という場合がある。）は、上記の有効成分の何れか1種を単独で含有してもよく、2種類以上を任意の組み合わせ及び比率で含有してもよい。 The Netrin-1 inhibitor and epidermis normalizing agent of the present invention (hereinafter collectively referred to as "the agent of the present invention") may contain any one of the above active ingredients alone. Alternatively, two or more types may be contained in any combination and ratio.
　本発明の剤は、上記の有効成分を、1種又は2種以上の他の成分、例えば賦形剤、担体及び/又は希釈剤等と組み合わせた組成物とすることもできる。組成物の組成や形態は任意であり、有効成分や用途等の条件に応じて適切に選択すればよい。当該組成物は、その剤形に応じ、賦形剤、担体及び/又は希釈剤等及び他の成分と適宜組み合わせた処方で、常法を用いて製造することができる。 The agent of the present invention can also be a composition in which the above active ingredients are combined with one or more other ingredients such as excipients, carriers and/or diluents. The composition and form of the composition are arbitrary, and may be appropriately selected according to conditions such as the active ingredient and application. The composition can be produced by a conventional method in a formulation in which an excipient, carrier and/or diluent and other ingredients are appropriately combined according to the dosage form.
　本発明の剤は経口的にあるいは非経口的（経皮投与、静脈投与、腹腔内投与、等）に適宜に使用され、任意の経路で投与されてもよいが、皮膚の血管に作用するよう経皮投与が好ましい。例えば、本発明の剤は、化粧品、医薬品、医薬部外品等に配合してヒト及び動物に使用することができる。経皮投与の形態としては、例えば、クリーム、乳液、液体、シート、スプレー、ゲルなど任意に選択することができる。 The agent of the present invention may be appropriately used orally or parenterally (percutaneous administration, intravenous administration, intraperitoneal administration, etc.) and may be administered by any route. Transdermal administration is preferred. For example, the agent of the present invention can be used for humans and animals by being incorporated into cosmetics, pharmaceuticals, quasi-drugs, and the like. The form of transdermal administration can be arbitrarily selected, for example, from creams, milky lotions, liquids, sheets, sprays, gels, and the like.
　例えば、本発明の化粧品組成物は、乳液、クリーム、美容液、ローション、パック、洗顔料、石鹸、ボディソープ、シャンプー等の各種化粧品であってもよく、液状、乳液状、クリーム状、固形状、シート状、スプレー状、ゲル状、泡状、パウダー状等の様々な形態であり得る。 For example, the cosmetic composition of the present invention may be various cosmetics such as milky lotion, cream, serum, lotion, pack, facial cleanser, soap, body soap, shampoo, etc., and may be liquid, milky, creamy or solid. , sheet-like, spray-like, gel-like, foam-like, and powder-like.
　また、本願は、本発明の剤又は組成物を対象に投与することにより、表皮を正常化するため、例えば血管構造の乱れの抑制を介した表皮正常化のための美容方法も提供する。本発明の方法は、美容を目的とする方法であり、医師や医療従事者による治療ではないことがある。 The present application also provides a cosmetic method for normalizing the epidermis, for example, for normalizing the epidermis through suppression of vascular structure disturbance, by administering the agent or composition of the present invention to a subject. The method of the present invention may be a cosmetic method and not a treatment by a doctor or medical practitioner.
　投与頻度は、４週間に１回、２週間に１回、１週間に１回、３日に１回、２日に１回、１日１回、１日２回、１日３回、１日４回、１日５回、都度投与等任意に選択できるがこれらに限定されない。 The administration frequency is once every 4 weeks, once every 2 weeks, once a week, once every 3 days, once every 2 days, once a day, twice a day, 3 times a day, 1 4 times a day, 5 times a day, each time administration, etc. can be arbitrarily selected, but are not limited to these.
　本発明の剤又は組成物におけるユズエキス、チンピエキス、及び／又はラクトビオチルの配合量は、種類、目的、形態、利用方法などに応じて、適宜決めることができる。例えば、これら有効成分の配合量は、本発明剤又は組成物の総重量当たり0.0001～100重量％、0.0001～90重量％、0.001～50重量％、0.01～10重量％、0.1～10重量％、1～10重量％、5～10重量％、6～7重量％、0.1～0.7重量％、0.7～6重量％、0.05～0.2重量％、0.1重量％、等とすることができるが、本発明の効果が発揮されれば限定されない。 The amount of yuzu extract, chimp extract, and/or lactobiotil in the agent or composition of the present invention can be appropriately determined according to the type, purpose, form, method of use, and the like. For example, the amount of these active ingredients is 0.0001 to 100% by weight, 0.0001 to 90% by weight, 0.001 to 50% by weight, 0.01 to 10% by weight, 0.1 to 10% by weight, based on the total weight of the agent or composition of the present invention. 1-10% by weight, 5-10% by weight, 6-7% by weight, 0.1-0.7% by weight, 0.7-6% by weight, 0.05-0.2% by weight, 0.1% by weight, etc. is not limited as long as the effect of is exhibited.
　本発明の剤及び組成物は、その剤形に応じ、賦形剤、担体及び/又は希釈剤等及び他の成分と適宜組み合わせた処方で、常法を用いて製造することができる。必要に応じて添加剤を任意に選択し併用することができる。添加剤としては賦形剤、着色剤、保存剤、増粘剤、結合剤、崩壊剤、分散剤、安定化剤、ゲル化剤、酸化防止剤、界面活性剤、保存剤、ｐＨ調整剤等については、公知のものを適宜選択して使用できる。 The agent and composition of the present invention can be produced using a conventional method in a formulation that appropriately combines excipients, carriers and/or diluents and other ingredients according to the dosage form. Additives can be arbitrarily selected and used together as needed. Additives include excipients, colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, etc. As for, known ones can be appropriately selected and used.
　しかしながら、本発明の剤の採り得る形態は、上述のものに限定されるものではない。 However, the forms that the agent of the present invention can take are not limited to those described above.
　また、本発明は、生体試料におけるNetrin-1を指標とした表皮正常化剤のスクリーニング方法に関する。本発明のスクリーニング方法は、例えば以下の工程：候補薬剤を含む培地で生体試料を培養すること；生体試料におけるNetrin-1の発現又は活性を測定すること；および、対照に比較してNetrin-1の発現又は活性が減少した場合に、候補薬剤を表皮正常化作用を有する物質として決定すること；を含む。ここで、対照は、候補薬剤が含まれない培地で培養している点で異なる場合のNetrin-1の発現又は活性である。対照についての実験は、本発明のスクリーニング方法と並行して行われていてもよいし、予め行われていてもよい。 The present invention also relates to a screening method for an epidermis-normalizing agent using Netrin-1 as an index in biological samples. Screening methods of the present invention include, for example, the following steps: culturing a biological sample in medium containing a drug candidate; measuring Netrin-1 expression or activity in the biological sample; determining the candidate agent as having an epidermal normalizing effect if the expression or activity of is decreased. Here, the control is the expression or activity of Netrin-1 when cultured in media without the drug candidate. A control experiment may be performed in parallel with the screening method of the present invention, or may be performed in advance.
　本発明で使用する生体試料としては、Netrin-1の発現又は活性が測定できれば限定されないが、表皮細胞を用いることが好ましい。例えば、実施例に記載のようにヒト等の動物から採取した表皮細胞、又はそれらの継代された細胞であってもよく、株化された細胞であってもよく、皮膚モデルにおける表皮細胞、ヒト等の動物の生体における表皮細胞であってもよい。 The biological sample used in the present invention is not limited as long as the expression or activity of Netrin-1 can be measured, but epidermal cells are preferably used. For example, as described in Examples, epidermal cells collected from animals such as humans, or their passaged cells, may be cell lines, epidermal cells in skin models, Epidermal cells in the living body of animals such as humans may also be used.
　また、候補薬剤を含む培地で生体試料を培養する工程の前に、上述のような生体試料を培養する前培養工程を含んでもよい。また、候補薬剤を含む培地で生体試料を培養する工程の後に、候補薬剤を含まない培地でさらに培養する後培養工程を含んでもよい。候補薬剤を含む培地で生体試料を培養する工程は、前培養工程で得られた培養物に、候補薬剤又はその希釈液を直接添加して培養してもよいし、候補薬剤を含む培地に置換して培養が行われてもよい。 In addition, a pre-culturing step of culturing the biological sample as described above may be included before the step of culturing the biological sample in a medium containing the candidate drug. Moreover, after the step of culturing the biological sample in a medium containing the candidate drug, a post-culturing step of further culturing in a medium not containing the drug candidate may be included. In the step of culturing the biological sample in a medium containing the candidate drug, the culture obtained in the pre-culturing step may be cultured by directly adding the candidate drug or a diluted solution thereof, or by substituting the medium containing the candidate drug. Cultivation may be performed as follows.
　Netrin-1の発現又は活性の測定は、生体試料におけるNetrin-1のmRNA量又はタンパク質量を測定することにより決定されうる。mRNA量の測定としては、定量的PCRやノーザンブロティングなど本技術分野に既知の手法を用いて行うことができる。例えば、実施例に記載のように、Netrin-1 mRNAに対するプローブを用いてもよい。タンパク質量についは、ウエスタンブロッティング、免疫染色、FACSなどの本技術分野に既知の任意の手法を用いて行うことができる。例えば、Netrin-1に特異的に結合する抗体を使用してもよい。あるいは、例えばNetrin-1発現細胞を用いたスクリーニング系を確立して使用してもよい。また、本発明は、Netrin-1の発現又は活性を決定するための試薬を含む、本発明のスクリーニング方法を実施するためのキットも提供する。 The measurement of Netrin-1 expression or activity can be determined by measuring the amount of Netrin-1 mRNA or protein in a biological sample. The amount of mRNA can be measured using techniques known in this technical field, such as quantitative PCR and northern blotting. For example, a probe for Netrin-1 mRNA may be used, as described in the Examples. Protein amounts can be determined using any technique known in the art, such as Western blotting, immunostaining, FACS. For example, antibodies that specifically bind to Netrin-1 may be used. Alternatively, for example, a screening system using Netrin-1-expressing cells may be established and used. The present invention also provides kits for carrying out the screening methods of the present invention, which contain reagents for determining Netrin-1 expression or activity.
　本発明のスクリーニング方法によりスクリーニングされた、Netrin-1抑制作用を有する物質は、Netrin-1の発現又は活性を抑制できれば任意の物質であってよい。本発明のスクリーニング方法の1の態様は、表皮細胞などの生体試料におけるNetrin-1発現を指標として、化粧品素材、食品素材、医薬品素材などの任意のライブラリーを用いてスクリーニングを行うことである。このようにして決定されたNetrin-1抑制作用を有する物質として、ユズエキス、チンピエキス、およびラクトビオチルからなる群から選択される1又は複数が挙げられる。ユズエキス、チンピエキス、およびラクトビオチルからなる群から選択される1又は複数を含むNetrin-1抑制剤は、血管構造の乱れの抑制を介し表皮正常化に有効である。また、本発明のスクリーニング方法により、表皮正常化作用を有する物質を選択することができる。 A substance having an inhibitory effect on Netrin-1 screened by the screening method of the present invention may be any substance as long as it can suppress the expression or activity of Netrin-1. One embodiment of the screening method of the present invention is to perform screening using an arbitrary library of cosmetic materials, food materials, pharmaceutical materials, etc., using Netrin-1 expression in biological samples such as epidermal cells as an indicator. Substances having an inhibitory effect on Netrin-1 determined in this manner include one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil. Netrin-1 inhibitors, including one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil, are effective in epidermal normalization through inhibition of vascular structural disturbance. In addition, the screening method of the present invention enables selection of a substance having an epidermal normalizing effect.
　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 in this specification are hereby incorporated 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 illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the claims. Modifications of the present invention, such as additions, deletions and replacements of constituent elements of the present invention, can be made without departing from the gist of the present invention.
　実験1：血管と表皮の加齢構造変化の可視化
　インフォームドコンセントを使用し、ヒト臀部皮膚組織をObio LLC. (CA）より提供を受けた。インフォームドコンセントはＩＲＢによって承認されている。20-30代（N=3）を若齢群、60-90代(N=3)を高齢群として、Tissue-Tech OCTコンパウンド(Sakura Finetechnical, Tokyo, Japan)に包埋した。その後、包埋ブロックをクリオスタットにより100μmに薄切し、凍結切片を作製した。作製した組織標本にメタノール固定を施し、免疫染色を実施した。一次抗体には、血管内皮細胞に対してシープ抗Pecam-1/CD31抗体（AF806, R＆D systems, MN）を用い、二次抗体には、Alexa Fluor488ドンキー抗シープ抗体（A11015, Molecular Probes, Eugene, OR）を用いた。また、核染色には、DAPI(D9542, Merck, Germany)を用いた。染色後、共焦点顕微鏡LSM880 (CarlZeiss, Germany)を用いて3Dの皮膚血管と細胞核を検出し、画像化した。当該画像から血管密度、及び表皮真皮の境界をImage Jを用いて定量化し、真皮領域における血管部分の面積の割合（＝血管部分の面積／真皮領域の面積×100（％））を求めた。 Experiment 1: Visualization of age-related structural changes in blood vessels and epidermis Using informed consent, human buttock skin tissue was provided by Obio LLC. (CA). Informed consent is approved by the IRB. The 20s to 30s (N=3) were the young group and the 60s to 90s (N=3) were the old group, and were embedded in a Tissue-Tech OCT compound (Sakura Finetechnical, Tokyo, Japan). Then, the embedded block was sliced to 100 μm by a cryostat to prepare frozen sections. The prepared tissue specimen was fixed with methanol and immunostained. Sheep anti-Pecam-1/CD31 antibody against vascular endothelial cells (AF806, R&D systems, MN) was used as the primary antibody, and Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR) was used. DAPI (D9542, Merck, Germany) was used for nuclear staining. After staining, 3D skin vessels and cell nuclei were detected and imaged using a confocal microscope LSM880 (CarlZeiss, Germany). From the images, the blood vessel density and the epidermis-dermis boundary were quantified using Image J, and the ratio of the area of the blood vessel portion in the dermis region (=area of the blood vessel portion/area of the dermis region×100 (%)) was determined.
　図1は、実験1の結果を示し、若齢者（33歳）及び高齢者（69歳）の皮膚における表皮真皮接合部分（上図）、並びに、若齢群（20-30代：N=3）及び高齢群（60-90代：N=3)の表皮真皮接合部分の長さ（μｍ）（下左）と真皮領域における血管部分の面積の割合（％）(下右)を示す。図1上に見られるように、若齢群では真皮領域における血管は表皮に向かう方向に延びており、表皮真皮接合部分が大きく波うち構造が顕著であることがわかるが、高齢群では血管の量が減り、血管の方向も揃わず血管構造が乱れており、波うち構造も平坦化していることがわかる。実験１より、真皮血管構造と表皮が何らかの関連性を有することが示唆された。そのため、どのように関与しているかを詳細に調べるため実験２により更なる可視化を行った。 Figure 1 shows the results of experiment 1, the epidermal-dermal junction (upper figure) in the skin of young (33 years old) and old (69 years old), and the young group (20s-30s: N = 3) and the aged group (60s-90s: N=3) show the length (μm) of the epidermal-dermal junction (lower left) and the ratio (%) of the vascular portion in the dermal region (lower right). As can be seen in Fig. 1, in the young group, the blood vessels in the dermis region extend in the direction toward the epidermis, and the epidermal-dermal junction has a large wavy structure. It can be seen that the amount is reduced, the direction of the blood vessels is not aligned, the vascular structure is disturbed, and the wavy structure is flattened. Experiment 1 suggested that the dermal vasculature and the epidermis have some kind of relationship. Therefore, in order to investigate in detail how they are involved, Experiment 2 was used for further visualization.
　実験２：皮膚毛細血管と表皮幹細胞の構造可視化
　実験１と同様の方法で、皮膚組織に免疫染色を実施した。一次抗体には、血管内皮細胞に対してシープ抗Pecam-1/CD31抗体（AF806, R＆D systems, MN）、表皮幹細胞・ペリサイトに対してマウス抗MCSP抗体(MAB2029, Merck, Germany)を用い、二次抗体には、Alexa Fluor488ドンキー抗シープ抗体（A11015, Molecular Probes, Eugene, OR）及びAlexa Fluor568 ドンキー抗マウス抗体(A10037, Molecular Probes, Eugene, OR)を用いた。また、核染色には、DAPI(D9542, Merck, Germany)を用いた。染色後、共焦点顕微鏡LSM880 (CarlZeiss, Germany)を用いて3Dの皮膚血管と表皮幹細胞・ペリサイト、及び細胞核を検出し、画像化した。当該画像から表皮あたりの表皮幹細胞の占めるエリアをImage Jを用いて定量化し、表皮領域におけるMCSP染色部分の面積の割合（＝表皮領域におけるMCSP染色部分の面積／表皮領域の面積×100（％））を求めた。 Experiment 2: Structural Visualization of Skin Capillaries and Epidermal Stem Cells In the same manner as in Experiment 1, skin tissue was immunostained. Sheep anti-Pecam-1/CD31 antibody (AF806, R&D systems, MN) for vascular endothelial cells and mouse anti-MCSP antibody (MAB2029, Merck, Germany) for epidermal stem cells and pericytes were used as primary antibodies. Secondary antibodies used were Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR) and Alexa Fluor568 donkey anti-mouse antibody (A10037, Molecular Probes, Eugene, OR). DAPI (D9542, Merck, Germany) was used for nuclear staining. After staining, 3D skin blood vessels, epidermal stem cells/pericytes, and cell nuclei were detected and imaged using a confocal microscope LSM880 (CarlZeiss, Germany). From the image, the area occupied by epidermal stem cells per epidermis was quantified using Image J, and the ratio of the area of the MCSP-stained part in the epidermal region (= area of MCSP-stained part in the epidermal region / area of the epidermal region × 100 (%) ).
　図2は、実験2の結果を示し、若齢者（27歳）及び高齢者（60歳）の皮膚における表皮真皮接合部（左及び中央）、並びに若齢群（20-30代：N=3）及び高齢群（60-90代：N=3)の表皮領域におけるMCSP染色部分の面積の割合（％）を示す。図2左では、基底膜に存在する表皮幹細胞（白矢印部分）と乳頭層に存在する毛細血管さらにその上のペリサイト（黄色矢印部分）が非常に近くに存在しているのが分かる。図2中央に示すように若年者ではこの関連が強くみられたが、高齢者では血管の量が減少するとともに血管の構造も脆弱になり、基底膜及び乳頭層が平坦化していることがわかる。また、表皮領域におけるMCSP染色部分は高齢群で有意に減少していた。これらの結果より、毛細血管とペリサイトが表皮幹細胞および基底膜の構造に何らかの関与をしているが加齢によりその関連性が減少している可能性が考えられる。ここで、毛細血管周囲のペリサイトは万能間葉系幹細胞に類似する働きがある可能性、ぺリサイトとは皮膚微小環境における皮膚組織再生の調節因子である可能性、ペリサイトは皮膚再生を促している可能性、皮膚の創傷部に間葉系幹細胞が集まり分化することにより修復を促している可能性が示唆されている（非特許文献１～４)。そこで、本発明者らは乳頭層毛細血管に存在するペリサイトと表皮幹細胞の関連性について更に詳細な観察を行った。 Figure 2 shows the results of Experiment 2, showing the epidermal-dermal junction (left and middle) in young (27 years old) and old (60 years old) skin, and the young group (20-30s: N = 3) and the ratio (%) of the area of the MCSP-stained part in the epidermal region of the aged group (60s to 90s: N=3). In the left side of Fig. 2, epidermal stem cells (white arrows) in the basement membrane, capillaries in the papillary layer, and pericytes (yellow arrows) are located very close to each other. As shown in the center of Fig. 2, this relationship was strong in young people, but in elderly people, the volume of blood vessels decreased and the structure of blood vessels became fragile, and the basement membrane and papillary layer became flat. . In addition, the MCSP-stained area in the epidermis area was significantly decreased in the aged group. These results suggest that capillaries and pericytes are involved in the structure of epidermal stem cells and basement membranes, but their relevance decreases with age. Here, it is possible that pericytes around capillaries have a function similar to that of pluripotent mesenchymal stem cells, that pericytes are regulators of skin tissue regeneration in the skin microenvironment, and that pericytes promote skin regeneration. It has been suggested that mesenchymal stem cells gather in the skin wound and differentiate to promote repair (Non-Patent Documents 1 to 4). Therefore, the present inventors made more detailed observations on the relationship between pericytes present in papillary capillaries and epidermal stem cells.
　実験３：皮膚毛細血管と表皮幹細胞、及び基底膜の構造可視化
　実験１、２と同様の方法で、若齢群の皮膚組織に免疫染色を実施した。一次抗体には、血管内皮細胞に対してシープ抗Pecam-1/CD31抗体（AF806, R＆D systems, MN）、表皮幹細胞・ペリサイトに対してマウス抗MCSP抗体(MAB2029, Merck, Germany)、基底膜に対してラット抗Integrin alpha 6抗体(sc-19622, Santa Cruz Biotechnology, CA)を用い、二次抗体には、Alexa Fluor488ドンキー抗シープ抗体（A11015, Molecular Probes, Eugene, OR）、Alexa Fluor568 ドンキー抗マウス抗体(A10037, Molecular Probes, Eugene, OR)、Alexa Fluor647 ドンキー抗ラット抗体(ab150155, Abcam, UK)を用いた。また、核染色には、DAPI(D9542, Merck, Germany)を用いた。染色後、共焦点顕微鏡LSM880 (CarlZeiss, Germany)を用いて３Ｄの皮膚血管と表皮幹細胞・ペリサイト、及び基底膜を検出し、画像化した。得られた３Ｄ画像から一平面の情報を抽出し、Z軸の深さ方向に画像を得た。 Experiment 3: Structure Visualization of Skin Capillaries, Epidermal Stem Cells, and Basement Membrane In the same manner as in Experiments 1 and 2, immunostaining was performed on the skin tissue of the young group. Primary antibodies include sheep anti-Pecam-1/CD31 antibody (AF806, R&D systems, MN) against vascular endothelial cells, mouse anti-MCSP antibody (MAB2029, Merck, Germany) against epidermal stem cells and pericytes, basement membrane A rat anti-Integrin alpha 6 antibody (sc-19622, Santa Cruz Biotechnology, Calif.) was used as a secondary antibody against Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR), Alexa Fluor568 donkey anti- Mouse antibody (A10037, Molecular Probes, Eugene, OR), Alexa Fluor647 donkey anti-rat antibody (ab150155, Abcam, UK) were used. DAPI (D9542, Merck, Germany) was used for nuclear staining. After staining, 3D cutaneous blood vessels and epidermal stem cells/pericytes, and basement membrane were detected and imaged using a confocal microscope LSM880 (CarlZeiss, Germany). One-plane information was extracted from the obtained 3D image, and an image was obtained in the depth direction of the Z axis.
　実験3の結果を図3、4に示す。驚くことに血管周囲のペリサイトに近い部分の基底膜が抜け、ペリサイトが基底膜を超えて表皮側に近づく様子が観察され（矢印部）、乳頭層毛細血管に存在するペリサイトが基底膜を超えて表皮幹細胞を供給していることが確認された。更に、本発明者らは、加齢により毛細血管の構造が変化することによりペリサイトからの表皮幹細胞の供給が抑制されている可能性に想到し、その要因を検討した。 The results of Experiment 3 are shown in Figures 3 and 4. Surprisingly, it was observed that the basement membrane near the pericytes around the blood vessel was removed, and the pericytes crossed over the basement membrane and approached the epidermis (arrows). It was confirmed that epidermal stem cells were supplied beyond Furthermore, the present inventors considered the possibility that the supply of epidermal stem cells from pericytes may be suppressed due to changes in the structure of capillaries due to aging, and investigated the factors.
　実験４：皮膚組織透明化と皮膚血管網の加齢変化
　18歳、50歳のヒト皮膚組織をILSBIO LLC (Chestertown, MD)から取得した。ILSBIOから取得されたアジア人の背中の皮膚試料は全て、米国および国際倫理ガイドラインに基づいて取得した。ILSBIOプロトコールは、ヘルスアンドヒューマンサービス認証知見審査委員会により承認されている。収集の前にインフォームドコンセントを得た。得られた皮膚組織をDispase I(Roche, Switzerland)で酵素処理を施し、表皮を剥した。表皮を剥した皮膚組織を4%パラホルムアルデヒドに浸漬し固定した。固定された皮膚組織をPBSで洗浄し、0.5% Triton X-100で透過処理し、1% Triton X-100/ 0.5% Tween-20溶液に浸けてインキュベートし、Perm block solution (0.5g BSA、50ul Tween20、300ul 5%アジ化ナトリウムをPBSに溶解したもの)で3日間ブロッキングした。その後、一次抗体として、シープ抗Pecam-1/CD31抗体（AF806, R＆D systems, MN）で37℃3日間インキュベートした。2日間PBSTで洗浄後、二次抗体としてAlexa Fluor488ドンキー抗シープ抗体（A11015, Molecular Probes, Eugene, OR）を用いて、37℃3日間インキュベートした。抗体を洗浄後、50%メタノールで３時間、70%メタノールで３時間、100%メタノールで一晩脱水した。その後、ジクロロメタン（Sigma）で15分２回振とうし、ジベンジルエーテル(Sigma, MO)で一晩静置し透明化処理を行った。透明化した皮膚組織を、光シート蛍光顕微鏡Lightsheet Microscopy Z.1 (CarlZeiss, Germany)で観察し、画像を得た。得られたシート画像から、Imaris(Bitplane, Concord, MA)を用いて、３Ｄ画像を構築した。 Experiment 4: Clearing of Skin Tissue and Age-related Changes in Skin Vascular Network Human skin tissues of 18 and 50 years old were obtained from ILSBIO LLC (Chestertown, Md.). All Asian back skin samples obtained from ILSBIO were obtained in accordance with US and international ethical guidelines. The ILSBIO protocol has been approved by the Health and Human Services Accreditation Review Board. Informed consent was obtained prior to collection. The obtained skin tissue was enzymatically treated with Dispase I (Roche, Switzerland) and the epidermis was peeled off. The skin tissue from which the epidermis was removed was immersed in 4% paraformaldehyde and fixed. The fixed skin tissue was washed with PBS, permeabilized with 0.5% Triton X-100, soaked in 1% Triton X-100/0.5% Tween-20 solution and incubated, then added with Perm block solution (0.5g BSA, 50ul Blocked with Tween20, 300ul 5% sodium azide in PBS) for 3 days. After that, it was incubated with sheep anti-Pecam-1/CD31 antibody (AF806, R&D systems, Minn.) as a primary antibody at 37°C for 3 days. After washing with PBST for 2 days, Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, Oreg.) was used as a secondary antibody and incubated at 37° C. for 3 days. After washing the antibody, it was dehydrated in 50% methanol for 3 hours, 70% methanol for 3 hours, and 100% methanol overnight. After that, it was shaken twice with dichloromethane (Sigma) for 15 minutes, and then allowed to stand overnight with dibenzyl ether (Sigma, MO) for clarifying treatment. The cleared skin tissue was observed with a light sheet fluorescence microscope Lightsheet Microscopy Z.1 (CarlZeiss, Germany) and images were obtained. From the resulting sheet images, 3D images were constructed using Imaris (Bitplane, Concord, Mass.).
　結果を図5に示す。18歳の試料では毛細血管が表皮方向に向いているが一方、50歳の試料では血管の方向と構造に乱れが生じていることが分かる。つまり、加齢により血管の量及び血管の長さが減少し、構造や方向が乱れ表皮から離れる方向に作用することが分かった。そこで、以下の実験により血管構造を変化させている要因について解析を行った。 The results are shown in Fig. 5. In the 18-year-old sample, the capillaries are oriented toward the epidermis, while in the 50-year-old sample, the direction and structure of the blood vessels are disturbed. In other words, it was found that the amount and length of blood vessels decreased with aging, and the structure and direction were disturbed, acting in the direction away from the epidermis. Therefore, the following experiment was conducted to analyze the factors that change the vascular structure.
　実験５：表皮加齢に伴って発現変化する分泌因子の検出
　公開されている表皮の遺伝子発現量情報をもとに解析を行った(E-MTAB-4382)（非特許文献５）。発現情報の解析はR(R Core Team, 2019)のlimma package（非特許文献６）を用いた。発現データはbackgroundCorrectによってバックグラウンドシグナルの補正を行い、その後normalizeBetweenArrayを用いたQuantile normalizationで標準化した。そして代表的な加齢マーカー遺伝子であるCDKN2A（p16）の発現量と年齢間のSpearman相関係数（rho）を計算した。この結果CDKN2Aと年齢間の相関係数（rho）がrho=0.533であったことから、相関係数（rho）0.533以上の遺伝子を加齢とともに発現上昇する遺伝子、相関係数（rho）-0.533以下の遺伝子を加齢とともに発現低下する遺伝子とした。また分泌因子の定義はGo termを用いた（GO:0005615, GO:0005576）。 Experiment 5: Detection of secreted factors whose expression changes with aging of the epidermis An analysis was performed based on published epidermal gene expression level information (E-MTAB-4382) (Non-Patent Document 5). Expression information was analyzed using R (R Core Team, 2019) limma package (Non-Patent Document 6). The expression data were corrected for background signals by backgroundCorrect, and then normalized by Quantile normalization using normalizeBetweenArray. We then calculated the Spearman correlation coefficient (rho) between the expression level of CDKN2A (p16), a representative aging marker gene, and age. As a result, the correlation coefficient (rho) between CDKN2A and age was rho = 0.533. The following genes were defined as genes whose expression decreases with aging. Go term was used for the definition of secretory factors (GO:0005615, GO:0005576).
　表皮におけるリガンド候補のうち、加齢により上方制御される遺伝子は6個、下方制御される遺伝子は13個発見され、そのうち血管機能に関与する遺伝子としてNetrin-1が発見された。実験5より得られたNetrin-1発現と年齢の関係についての結果を図6に示す。そこで、次の実験によりNetrin-1が実際に皮膚血管構造および表皮にどのように関与するかについて解析を行った。 Among the candidate ligands in the epidermis, 6 genes were found to be up-regulated and 13 genes were down-regulated with aging, among which Netrin-1 was discovered as a gene involved in vascular function. FIG. 6 shows the results of the relationship between Netrin-1 expression and age obtained from Experiment 5. Therefore, we analyzed how Netrin-1 actually participates in skin vasculature and epidermis by the following experiments.
　実験６：皮膚血管におけるNetrin-1受容体の可視化
　上記と同様の方法で、免疫染色を実施した。一次抗体には、血管内皮細胞に対してシープ抗Pecam-1/CD31抗体（AF806, R＆D systems, MN）、表皮幹細胞・ペリサイトに対してマウス抗MCSP抗体(MAB2029, Merck, Germany)、Netrin-1受容体に対してラビット抗UNC5B抗体(PA5-63671, Invitrogen, CA)を用い、二次抗体には、Alexa Fluor488ドンキー抗シープ抗体（A11015, Molecular Probes, Eugene, OR）、Alexa Fluor568 ドンキー抗マウス抗体(A10037, Molecular Probes, Eugene, OR)、Alexa Fluor647 ドンキー抗ラビット抗体(A31573, Molecular Probes, Eugene, OR)を用いた。また、核染色には、DAPI(D9542, Merck, Germany)を用いた。染色後、共焦点顕微鏡LSM880 (CarlZeiss, Germany)を用いて3Dの皮膚血管と表皮幹細胞・ペリサイト、及びNetrin-1受容体UNC5Bを検出し、画像化した。 Experiment 6: Visualization of Netrin-1 Receptor in Skin Blood Vessels Immunostaining was performed in the same manner as above. Primary antibodies included sheep anti-Pecam-1/CD31 antibody (AF806, R&D systems, MN) against vascular endothelial cells, mouse anti-MCSP antibody (MAB2029, Merck, Germany) against epidermal stem cells and pericytes, Netrin- Rabbit anti-UNC5B antibody (PA5-63671, Invitrogen, Calif.) was used against 1 receptor, and secondary antibodies were Alexa Fluor488 donkey anti-sheep antibody (A11015, Molecular Probes, Eugene, OR), Alexa Fluor568 donkey anti-mouse. Antibody (A10037, Molecular Probes, Eugene, OR), Alexa Fluor647 donkey anti-rabbit antibody (A31573, Molecular Probes, Eugene, OR) was used. DAPI (D9542, Merck, Germany) was used for nuclear staining. After staining, 3D cutaneous blood vessels and epidermal stem cells/pericytes and Netrin-1 receptor UNC5B were detected and imaged using a confocal microscope LSM880 (CarlZeiss, Germany).
　結果を図7に示す。毛細血管ループの先端の表皮に近い部分でNetrin-1受容体であるUNC5Bが高度に発現しているのが見られた（矢印部分）。表皮に存在するNetrin-1と真皮内の血管にあるNetrin-1受容体が関与しており、これらの作用により血管構造が変化している可能性が示唆される。そこで、以下の実験によりNetrin-1と血管構造の関連性について解析を行った。 The results are shown in Fig. 7. UNC5B, a Netrin-1 receptor, was found to be highly expressed in the tip of the capillary loop near the epidermis (arrow). Netrin-1 present in the epidermis and Netrin-1 receptors in blood vessels in the dermis are involved, suggesting the possibility that these actions alter the vascular structure. Therefore, we analyzed the relationship between Netrin-1 and vasculature by the following experiments.
　実験７：血管内皮細胞を用いた遊走試験
　図８左に示すように、ヒト臍帯血静脈内皮細胞(HUVEC)をフルオロブロックインサート（ＢＤファルコン）に播種し、Human recombinant Netrin-1(6419-N1, R＆D systems, MN)を最終濃度0.1μg/ml又は1μg/ml、及び／又は血管内皮増殖因子VEGFA165(450-32, Peprotech, NJ)を50ng/mlの最終濃度で添加したEBM-2中で培養した。対照には、これらの物質を添加しない以外は同じ組成のEBM-2培地を使用した。添加4時間半後、10%緩衝ホルマリンにて細胞を固定の後、Hoechst33432(H3570, Invitrogen, CA)にて細胞核を染色した。その後、フルオロブロックインサートの下側のメンブレンに遊走した細胞数を計測した。 Experiment 7: Migration Test Using Vascular Endothelial Cells As shown on the left side of Fig. 8, human umbilical cord blood vein endothelial cells (HUVEC) were seeded on Fluoroblock inserts (BD Falcon), and human recombinant Netrin-1 (6419-N1, 6419-N1, R&D systems, MN) at a final concentration of 0.1 μg/ml or 1 μg/ml and/or vascular endothelial growth factor VEGFA165 (450-32, Peprotech, NJ) at a final concentration of 50 ng/ml. did. As a control, EBM-2 medium with the same composition was used, except that these substances were not added. Four and a half hours after the addition, the cells were fixed with 10% buffered formalin, and the cell nuclei were stained with Hoechst33432 (H3570, Invitrogen, Calif.). The number of cells that migrated to the membrane underneath the Fluoroblock insert was then counted.
　結果を図８右に示す。対照に比べてVEGFA入りの培地に対して遊走した細胞数は多かったものの、Netrin-1を同時に添加するとその数は減少した。このことより血管内皮細胞はNetrin-1を忌避する方向に作用し、このことが毛細血管を表皮から遠ざける要因となっている可能性が考えられる。そのため、次の実験により３Ｄ血管新生モデルに対しNetrin-1を添加し血管構造にどのように影響するかについての解析を行った。 The results are shown on the right of Fig. 8. Although more cells migrated to media containing VEGFA compared to controls, the number decreased when Netrin-1 was added simultaneously. This suggests that vascular endothelial cells act in the direction of repelling Netrin-1, which may be a factor in keeping capillaries away from the epidermis. Therefore, the following experiment was conducted to analyze how Netrin-1 was added to the 3D angiogenesis model and how it affected the vascular structure.
　実験８：血管３Ｄモデルの作製とNetrin-1の作用解析
　コラーゲンゲル内に血管3Dモデルを作製し、Netrin-1を添加することによる影響を観察した。5mg/mlのラット由来一型コラーゲン(3447-020-01, R＆D systems, MN)と1M HEPES、37g/l NaHCO3を用いて4mg/ml コラーゲン溶液を氷上で作製した。OrganoPlate（登録商標）3-lane 40 (4004-400-B, MIMETAS B.V., Netherland)を用い、添付の説明書および非特許文献7に記載の方法に従い、gel lane（レーン２）に前述のコラーゲン溶液を添加し、37℃15分インキュベートして、ゲル化させた。ヒト臍帯血静脈内皮細胞(HUVEC)20,000細胞を内皮細胞増殖培地MV2キット(C-22121, PromoCell, Germany) 2μlで懸濁し、OrganoPlateのperfusion lane （レーン１）のinletに播種した。その後、MV2培地を50μl同inletから加え、OrganoPlate Stand上で、37℃1時間半インキュベートした。細胞がゲルに接着したことを確認した後、MV2培地を各50μlずつ細胞が播種されているperfusion lane（レーン１）のoutlet、逆側のperfusion lane（レーン３）のinletとoutletに加えた。その後、MIMETAS Rocker Mini (MIMETAS B.V., Netherland)上でProgram1 (Interval 8min, 7o)を設定し、37℃3日間培養して、３Ｄ構造をもつ血管を作製した。培養3日目に、細胞が播種されている逆側のperfusion lane（レーン３）の培地に最終濃度50ng/mlのVEGF165(450-32, Peprotech, NJ)、最終濃度50ng/mlのbFGF(100-18B, Peprotech, NJ)、最終濃度2ng/mlのPMA(P1585, Sigma, MO)、最終濃度500nMのSphingosin-1-Phosphate(S9666, Sigma, MO)、最終濃度1ug/mlのHuman recombinant Netrin-1(6419-N1, R＆D systems, MN)を加え、MIMETAS Rocker Mini上で37℃3日間培養し、3.7%パラホルムアルデヒドでRT 15分インキュベートすることで固定し、免疫染色を実施した。一次抗体には、血管内皮細胞に対してラビット抗VE-Cadherin抗体（#2158, Cell Signaling Technology, MA）、細胞骨格f-アクチンに対してCF568 conjugated Phalloidin (00040, Biotium, Inc., CA)を用い、二次抗体に、Alexa Fluor488ドンキー抗ラビット抗体（A21206, Molecular Probes, Eugene, OR）を用いた。また、核染色には、Hoechst33432(H3570, Invitrogen, CA)を用いた。染色後、共焦点顕微鏡LSM880 (CarlZeiss, Germany)を用いて3Dの血管構造と血管形成を検出し、画像情報を得た。得られた情報からImaris(Bitplane, Concord, MA)を用いて、３Ｄ画像を構築した（図9右）。対照はNetrin-1を添加しない以外は同じ条件で３Ｄモデルを作製し、染色および画像構築を行った（図9左）。 Experiment 8: Preparation of 3D model of blood vessels and analysis of action of Netrin-1 A 3D model of blood vessels was prepared in a collagen gel, and the effect of adding Netrin-1 was observed. A 4 mg/ml collagen solution was prepared on ice using 5 mg/ml rat-derived collagen type 1 (3447-020-01, R&D systems, Minn.), 1M HEPES, 37 g/l NaHCO3. Using OrganoPlate (registered trademark) 3-lane 40 (4004-400-B, MIMETAS BV, Netherlands), according to the attached instructions and the method described in Non-Patent Document 7, the collagen solution described above was added to the gel lane (lane 2). was added and incubated at 37°C for 15 minutes to allow gelation. 20,000 human umbilical vein endothelial cells (HUVEC) were suspended in 2 μl of endothelial cell growth medium MV2 kit (C-22121, PromoCell, Germany) and seeded into the perfusion lane (lane 1) inlet of the OrganoPlate. After that, 50 μl of MV2 medium was added from the same inlet, and incubated on the OrganoPlate Stand at 37° C. for 1.5 hours. After confirming that the cells adhered to the gel, 50 μl each of MV2 medium was added to the outlet of the perfusion lane (lane 1) where the cells were seeded, and the inlet and outlet of the perfusion lane (lane 3) on the opposite side. Thereafter, Program 1 (Interval 8 min, 7 ° C.) was set on MIMETAS Rocker Mini (MIMETAS BV, Netherland) and cultured at 37° C. for 3 days to fabricate a blood vessel having a 3D structure. On day 3 of culture, VEGF165 (450-32, Peprotech, NJ) at a final concentration of 50 ng/ml and bFGF (100 -18B, Peprotech, NJ), PMA (P1585, Sigma, MO) at a final concentration of 2ng/ml, Sphingosin-1-Phosphate (S9666, Sigma, MO) at a final concentration of 500nM, Human recombinant Netrin at a final concentration of 1ug/ml- 1 (6419-N1, R&D systems, MN) was added, cultured on MIMETAS Rocker Mini at 37°C for 3 days, fixed by incubating with 3.7% paraformaldehyde at RT for 15 minutes, and immunostained. Primary antibodies were rabbit anti-VE-Cadherin antibody (#2158, Cell Signaling Technology, MA) against vascular endothelial cells and CF568 conjugated Phalloidin (00040, Biotium, Inc., CA) against cytoskeletal f-actin. The secondary antibody used was Alexa Fluor488 donkey anti-rabbit antibody (A21206, Molecular Probes, Eugene, Oreg.). Hoechst33432 (H3570, Invitrogen, Calif.) was used for nuclear staining. After staining, a confocal microscope LSM880 (CarlZeiss, Germany) was used to detect 3D vascular structures and angiogenesis to obtain image information. A 3D image was constructed from the obtained information using Imaris (Bitplane, Concord, Mass.) (Fig. 9, right). As a control, a 3D model was prepared under the same conditions except that Netrin-1 was not added, and staining and image construction were performed (FIG. 9, left).
　構築した３Ｄ画像を図9に示す。図9より、Netrin-1を添加（図9右の上部に添加）すると、コラーゲンゲル内に形成される血管の量の減少と血管構造の乱れが観察され（図9右中央部）、Netrin-1により毛細血管が忌避する方向に作用しているという実験7,8から得た知見と一致する結果となった。更に、実験1～6の結果を併せると、例えば模式的な図として図10に示すように、加齢により増加する表皮Netrin-1が乳頭層内の毛細血管上のNetrin-1受容体UNC5Bに働いて、血管が表皮から忌避する方向に作用し血管構造が乱され、ペリサイトから表皮幹細胞の供給が抑制されることにより表皮正常化が抑制されることが導かれる。 The constructed 3D image is shown in Fig. 9. From Fig. 9, when Netrin-1 was added (added to the upper right part of Fig. 9), a decrease in the amount of blood vessels formed in the collagen gel and disturbance of the vascular structure were observed (Fig. 9 right center part). According to 1, the result was consistent with the knowledge obtained from experiments 7 and 8 that the capillaries acted in the direction of repelling. Furthermore, when the results of Experiments 1 to 6 are combined, for example, as shown in Fig. 10 as a schematic diagram, epidermal Netrin-1, which increases with age, binds to the Netrin-1 receptor UNC5B on capillaries in the papillary layer. As a result, blood vessels repel the epidermis, disturbing the vascular structure and suppressing the supply of epidermal stem cells from pericytes, leading to suppression of normalization of the epidermis.
　よって、老化等により表皮幹細胞が減少した皮膚であっても、Netrin-1を抑制することにより表皮を正常化させることが可能であると考えられる。したがって、以下の実験にて、Netrin-1抑制作用を有する物質のスクリーニングを行った。 Therefore, it is considered possible to normalize the epidermis by suppressing Netrin-1, even in skin where epidermal stem cells have decreased due to aging. Therefore, in the following experiments, screening for substances having Netrin-1 inhibitory activity was performed.
　実験９：表皮細胞におけるNetrin-1抑制薬剤スクリーニング
　クラボウ社より得た表皮細胞を、増殖添加剤（クラボウ、HuMedia-KＧ増殖添加剤セット、KK-6150）を加えた培地（ThermoFishser scientific、EpiLife Medium、MEPI500CA）にて37℃で24時間培養した。ユズエキス（丸善製薬株式会社製、ユズ抽出液―Ｊ)、チンピキス（丸善製薬株式会社製、チンピ抽出液ＢＧ）、およびラクトビオチル（シラブ社）を含む16種類の候補薬剤を培地に対し0.1重量％となるようにそれぞれ添加し（ラクトビオチルについては0.1重量％、0.77重量%、及び6.0重量％），37℃でさらに24時間培養した。細胞を回収してRNAを抽出し（RNeasy Plus Mini Kit、QIAGEN、74136），Netrin-1の発現量をreal time PCRで定量した。real time PCRは反応キット（ThermoFishser scientific、TaqMan RNA-to-CT 1-Step Kit、4392938）及びNetrin-1とβ-actin特異的プローブ（ThermoFishser scientific、 Netrin-1：Hs00924151_m1、 β-actin：Hs01060665_g1）を用いて行った。なおβ-actinは内在性コントロールとして用いた。対照(cont)よりも有意にNetrin-1発現量が減少するものを探索した。 Experiment 9: Netrin-1 inhibitory drug screening in epidermal cells Epidermal cells obtained from Kurabo Industries, Inc. were added with growth additives (Kurabo, HuMedia-KG growth additive set, KK-6150) in a medium (ThermoFishser scientific, EpiLife Medium, MEPI500CA) and cultured at 37°C for 24 hours. Yuzu extract (manufactured by Maruzen Pharmaceutical Co., Ltd., yuzu extract-J), chimpikis (manufactured by Maruzen Pharmaceutical Co., Ltd., chimp extract BG), and 16 drug candidates, including lactobiotil (Shilab), were added at 0.1% by weight to the medium. (0.1% by weight, 0.77% by weight, and 6.0% by weight for lactobiotil) and cultured at 37° C. for an additional 24 hours. Cells were collected, RNA was extracted (RNeasy Plus Mini Kit, QIAGEN, 74136), and the expression level of Netrin-1 was quantified by real time PCR. Real-time PCR was performed using a reaction kit (ThermoFishser scientific, TaqMan RNA-to-CT 1-Step Kit, 4392938) and Netrin-1 and β-actin specific probes (ThermoFishser scientific, Netrin-1: Hs00924151_m1, β-actin: Hs01060665_g1). was used. β-actin was used as an endogenous control. We searched for those in which the Netrin-1 expression level was significantly reduced compared to the control (cont).
　結果を図11に示す。図11より、候補薬剤として、ユズエキス、チンピエキス、およびラクトビオチルを用いた場合に、対照に比較してNetrin-1の発現が有意に減少した。これらのエキスを、Netrin-1抑制作用を有する物質として選択した。 The results are shown in Fig. 11. As shown in FIG. 11, when yuzu extract, chimp extract, and lactobiotil were used as candidate drugs, the expression of Netrin-1 was significantly decreased compared to the control. These extracts were selected as substances with Netrin-1 inhibitory activity.

Claims (8)

  1. 　表皮正常化のための、Netrin-1抑制剤。 A Netrin-1 inhibitor for epidermal normalization.
  2. 　ユズエキス、チンピエキス、およびラクトビオチルからなる群から選択される1又は複数からなる、Netrin-1抑制剤。 A Netrin-1 inhibitor consisting of one or more selected from the group consisting of yuzu extract, chimp extract, and lactobiotil.
  3. 　請求項１又は２に記載のNetrin-1抑制剤を含む、表皮正常化剤。 An epidermal normalizing agent comprising the Netrin-1 inhibitor according to claim 1 or 2.
  4. 　前記表皮正常化は、Netrin-1の抑制により血管構造の乱れを抑制することを介して達成される、請求項３に記載の表皮正常化剤。 The agent for normalizing the epidermis according to claim 3, wherein the normalization of the epidermis is achieved through inhibition of disturbance of vascular structures by inhibition of Netrin-1.
  5. 　Netrin-1を指標とした表皮正常化剤のスクリーニング方法。 A screening method for an epidermal normalizing agent using Netrin-1 as an index.
  6. 　候補薬剤を含む培地で生体試料を培養すること；
    　生体試料におけるNetrin-1の発現又は活性を測定すること；および
    　対照に比較してNetrin-1の発現又は活性が減少した場合に、候補薬剤を表皮正常化作用を有する物質として決定すること；
    　を含む、請求項５に記載のスクリーニング方法。     culturing a biological sample in a medium containing the candidate agent;
    measuring Netrin-1 expression or activity in a biological sample; and determining a candidate agent as having epidermal normalizing activity if Netrin-1 expression or activity is reduced compared to a control;
    The screening method of claim 5, comprising:
  7. 　対象のNetrin-1を抑制させることを含む、対象の表皮を正常化するための美容目的の方法。 A cosmetic method for normalizing a subject's epidermis, including inhibiting the subject's Netrin-1.
  8. 　対象のNetrin-1の抑制は、請求項３又は４に記載の表皮正常化剤を対象に投与することにより達成される、請求項７に記載の方法。 The method according to claim 7, wherein the subject's suppression of Netrin-1 is achieved by administering the epidermal normalizing agent according to claim 3 or 4 to the subject.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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