WO2023282145A1 - Composition for preventing or treating tissue aging - Google Patents

Composition for preventing or treating tissue aging Download PDF

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WO2023282145A1
WO2023282145A1 PCT/JP2022/025977 JP2022025977W WO2023282145A1 WO 2023282145 A1 WO2023282145 A1 WO 2023282145A1 JP 2022025977 W JP2022025977 W JP 2022025977W WO 2023282145 A1 WO2023282145 A1 WO 2023282145A1
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tissue
sam
aging
composition
drosophila
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French (fr)
Japanese (ja)
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昌一 波平
良樹 林
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国立研究開発法人産業技術総合研究所
国立大学法人 筑波大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • 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
    • G01N33/02—Food
    • 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 compositions for preventing or treating aging of various tissues such as skin tissue, reproductive tissue and nerve tissue, and screening methods for anti-aging substances using S-adenosylmethionine as an indicator.
  • SAM S-adenosylmethionine
  • the purpose of the present invention is to provide a composition for preventing or treating age-related functional deterioration of various tissues and a screening method thereof.
  • the inventors found that an increase in the amount of SAM accelerates the aging of reproductive tissues in Drosophila, that the amount of SAM in reproductive tissues and brain tissues also increases with aging in mice, and that SAM is associated with human fibers. It was confirmed for the first time that it affects the proliferation ability of blast cells and the proliferation and differentiation ability of human neural stem cells. Based on this novel discovery, the present inventors found that substances that reduce SAM biosynthesis are useful for preventing or treating aging of various tissues, including skin tissue, reproductive tissue and nerve tissue. .
  • the present invention provides a composition for preventing or treating tissue aging, comprising an S-adenosylmethionine synthase inhibitor.
  • the tissue is preferably selected from the group consisting of skin tissue, reproductive tissue and nerve tissue.
  • the S-adenosylmethionine synthase is preferably methionine adenosyltransferase 2.
  • the S-adenosylmethionine synthase inhibitor is 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a ]pyrimidin-7(4H)-one, (E)-4-(2-chloro-6-fluorostyryl)-N-methylaniline, and 3-(cyclohex-1-en-1-yl)-6- It is preferably selected from the group consisting of (4-methoxyphenyl)-2-phenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-ones.
  • the S-adenosylmethionine synthase inhibitor is preferably an expression vector containing an RNA that inhibits expression of the methionine adenosyltransferase 2 gene or a nucleic acid encoding the same.
  • composition is preferably a pharmaceutical.
  • the composition is preferably a food or drink.
  • fibroblasts (1) culturing fibroblasts in the presence of a candidate compound, (2) evaluating the proliferation ability of the fibroblasts, and (3) and quantifying S-adenosylmethionine in said fibroblasts.
  • the above method preferably further comprises the steps of (7) administering the candidate compound to Drosophila, and (8) analyzing the reproductive tissue of the Drosophila.
  • composition according to the present invention is useful for preventing or treating age-related functional decline in various tissues.
  • screening method according to the present invention can obtain compounds capable of suppressing aging of various tissues including skin tissue, reproductive tissue and nerve tissue by reducing SAM biosynthesis in cells or tissues. , is useful.
  • FIG. 1 shows immunohistochemical staining images of ovarian canaliculi of young and old control Drosophila.
  • FIG. 2 is a graph showing the expression levels of SAM-S in young and old control Drosophila.
  • FIG. 3 is a graph showing the expression levels of SAM-S in young and aged control and Sam-S( ⁇ ) Drosophila ovaries.
  • FIG. 4 is a graph showing age-related changes in the number of germinal stem cells per ovarian canaliculus in each Drosophila strain.
  • FIG. 5 is a graph showing the frequency of abnormal ovaries in control and Sam-S(++) Drosophila.
  • FIG. 6 is a graph showing the frequency of abnormal ovaries in control and Sam-S( ⁇ ) Drosophila.
  • FIG. 1 shows immunohistochemical staining images of ovarian canaliculi of young and old control Drosophila.
  • FIG. 2 is a graph showing the expression levels of SAM-S in young and old control Drosophila.
  • FIG. 7 is a plot showing SAM abundance in young and aged mouse reproductive and brain tissue.
  • FIG. 8 is a graph showing the proportion of cell proliferation marker Ki-67-positive human neural stem cells in the presence of a SAM synthase inhibitor.
  • FIG. 9 is a graph showing the proportion of neural differentiation marker DCX-positive human neural stem cells in the presence of a SAM synthase inhibitor.
  • FIG. 10 is a graph showing the proportion of cell proliferation marker Ki-67-positive human dermal fibroblasts in the presence of SAM synthase inhibitors.
  • FIG. 11 is a graph showing the percentage of cell proliferation marker Ki-67-positive human dermal fibroblasts in the presence of methotrexate.
  • the first embodiment of the present invention is a composition for preventing or treating tissue aging, comprising an S-adenosylmethionine synthase inhibitor.
  • preventing means not only preventing aging of reproductive tissue or nervous tissue in a subject, but also reducing the risk of aging, or preventing reproductive It also includes slowing or reducing aging of tissue or nerve tissue.
  • treating includes not only complete cure of aging of reproductive tissue or nervous tissue, but also remission or alleviation thereof, and delaying or stopping its progression.
  • tissue in this embodiment may be in any vertebrate, preferably mammalian tissue such as mouse, rat, rabbit, sheep, pig, cow, goat, monkey, human, etc. Human tissue is preferred.
  • the type of tissue is also not particularly limited, and may be, for example, skin tissue, reproductive tissue, nerve tissue, muscle tissue, bone and cartilage tissue, immune system tissue, or the like.
  • the tissue in this embodiment is preferably skin tissue, reproductive tissue or nerve tissue.
  • Skin tissue in this embodiment may include all tissues that constitute the skin, ie, epidermis, dermis and subcutaneous tissue.
  • Reproductive tissue in this embodiment may include ovaries and testes.
  • Neve tissue in this embodiment may include central nerve tissue and peripheral nerve tissue.
  • aging of tissue refers to the decrease or deterioration of cells that make up the tissue due to aging.
  • degradation of cells means reduction or loss of cell differentiation ability, proliferation ability and/or function.
  • Cells that make up skin tissue include, but are not limited to, fibroblasts, epidermal stem cells, dermal stem cells, hair follicle stem cells, keratinocytes, pigment cells, and Langerhans cells.
  • Cells constituting reproductive tissue may include general cells determined to differentiate into germ cells, such as egg stem cells (oogonia), primary oocytes, secondary oocytes, egg cells, and sperm. They include, but are not limited to, stem cells (spermatogonia), primary spermatocytes, secondary spermatocytes and spermatids.
  • Cells constituting neural tissue may include all cells determined to differentiate into neural cells, such as, but not limited to, neural stem cells, neural progenitor cells, and neural cells.
  • S-adenosylmethionine (hereinafter also referred to as "SAM") is produced by methionine adenosyltransferase (hereinafter also referred to as "MAT") using methionine and ATP as substrates. Therefore, "S-adenosylmethionine (SAM) synthase” in the present embodiment is synonymous with methionine adenosyltransferase (MAT).
  • MAT has multiple isozymes, and the SAM synthase in this embodiment may be any isozyme.
  • mammals have two types of isozymes, MAT1 and MAT2, both of which may be included in the SAM synthase in this embodiment.
  • the SAM synthase in this embodiment is preferably MAT2.
  • the "S-adenosylmethionine synthase inhibitor” in this embodiment may be any substance that can directly or indirectly decrease the expression and/or activity of MAT. Therefore, as the SAM synthetase inhibitor in this embodiment, for example, proteins, peptides, nucleic acids, lipids, low-molecular-weight compounds and the like can be used, but are not limited to these. Moreover, such substances may be known substances or novel substances.
  • Preferred SAM synthase inhibitors in this embodiment include, for example, 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1, 5-a]pyrimidin-7(4H)-one, (E)-4-(2-chloro-6-fluorostyryl)-N-methylaniline, and 3-(cyclohex-1-en-1-yl) -6-(4-methoxyphenyl)-2-phenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-one and the like, but are not limited thereto.
  • the SAM synthase inhibitor in the present embodiment is preferably 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1,5 -a]pyrimidin-7(4H)-ones can be used.
  • SAM synthetase inhibitors that can be used in this embodiment are commercially available, and commercial products can also be used.
  • Preferred commercial products include, but are not limited to, MAT2A inhibitor 1 (MedChem Express), MAT2A inhibitor 2 (FIDAS-5) (MedChemExpress), MAT2A inhibitor (AG-270) (MedChemExpress), and the like.
  • RNA that inhibits the expression of the methionine adenosyltransferase 2 gene may be used as a preferred SAM synthetase inhibitor in this embodiment.
  • RNA that inhibits gene expression includes RNA molecules with RNA interference effects such as siRNA, shRNA, and dsRNA, as well as target gene (methionine adenosyltransferase 2 gene)-derived mRNA to control its expression. Examples include, but are not limited to, miRNAs that are thought to Preferred RNAs in this embodiment are siRNAs or shRNAs.
  • the RNA in this embodiment can be designed according to a well-established siRNA or shRNA design method in the art. Nucleic acid sequence information of the methionine adenosyltransferase 2 gene can be obtained from predetermined databases. For example, for human MAT2A, NCBI Reference Sequence (RefSeq) ID: NM_005911.6 is available.
  • the RNA in this embodiment may contain a sequence consisting of 17 to 30 nucleotides complementary to any region selected from the coding sequence of the MAT2A gene, preferably the nucleotides of the coding sequence of the human MAT2A gene.
  • 121-1308 more preferably comprising a sequence consisting of 17-30 nucleotides complementary to a region selected from 121-1308, more preferably 19-25 nucleotides, for example AAGGAGAAAGUCAUCAAAGCA (SEQ ID NO: 8). preferable.
  • RNA in this embodiment may be entirely composed of RNA, or part thereof may contain modified RNA.
  • modified RNA include phosphorothioated RNA, boranophosphated RNA, 2'-O-methylated RNA, 2'-F-RNA, 2',4'-BNA (also known as LNA (Locked Nucleic Acid)), etc.
  • the RNA in this embodiment may be expressed intracellularly by using an expression vector containing a nucleic acid encoding the above RNA. Therefore, the composition of this embodiment may contain an expression vector containing a nucleic acid encoding the above RNA as a SAM synthase inhibitor.
  • an appropriate viral vector or non-viral vector can be selected and used depending on the type of cell into which the RNA aptamer is introduced.
  • the composition of the present embodiment can contain one or more SAM synthase inhibitors selected from the above as active ingredients.
  • the SAM synthase inhibitor should be contained so as to be an appropriate intake amount for the subject.
  • 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidine-7 as a SAM synthase inhibitor.
  • the intake of the SAM synthase inhibitor is, for example, 10 to 200 mg/kg (body weight), preferably 100 to 200 mg/kg (body weight) per day for adults. However, it is not limited to such a range, and can be appropriately adjusted depending on the form of the composition, subject's condition, age, sex, and the like.
  • composition of the present embodiment may be composed only of active ingredients, but generally may further contain known pharmaceutically acceptable carriers and additives as optional ingredients.
  • composition of this embodiment can be produced, for example, as a pharmaceutical.
  • the composition of this embodiment can be formulated into various dosage forms such as tablets, granules, powders, capsules, jellies, syrups and injections. Therefore, the composition of this embodiment can be administered by various methods such as oral administration, intraperitoneal administration, intravenous administration, transdermal administration, and direct injection into the target tissue.
  • the composition of the present embodiment is preferably an oral formulation, and therefore is preferably administered orally.
  • the drug of the present embodiment when it is an oral formulation, it can be, for example, a solid formulation such as tablets, granules, and powders.
  • suitable additives such as starch, mannitol, carboxymethyl cellulose, corn starch, inorganic salts, etc., and if desired, binders, disintegrants, lubricants and the like can be added.
  • the drug of this embodiment is made into a tablet, the composition may be coated with sucrose, gelatin, hydroxypropylcellulose, or the like, if desired.
  • a liquid formulation such as a syrup, sterilized water, physiological saline, ethanol and the like can be used as carriers, and if desired, an adjuvant such as a suspending agent may be added.
  • the drug of this embodiment when it is a parenteral formulation, it can be a liquid formulation such as an injection.
  • the active ingredient is dissolved or suspended in a diluent such as distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, polyethylene glycol, etc. It can be prepared by adding an agent or the like.
  • compositions of the present embodiment can be added to the pharmaceuticals of the present embodiment, if desired.
  • the composition of this embodiment can be produced as a food or drink.
  • the composition of this embodiment can be, for example, processed food for general use.
  • processed foods for general use include, but are not limited to, breads, noodles, confectionery, edible fats and oils, seasonings, and beverages.
  • composition of the present embodiment can be used as, for example, a nutritional supplement.
  • Dietary supplements may include, for example, supplements, nutritional supplements, foods for specified health uses, foods with nutrient function claims, and the like.
  • the nutritional supplement of the present embodiment may be prepared, for example, as powders, granules, tablets, capsules, jelly preparations, liquid preparations, and if desired, additives such as flavorings, coloring agents, sweeteners, preservatives, etc. , can be combined with other nutritional ingredients.
  • composition of the present embodiment is useful for preventing or treating age-related decline in reproductive function and/or nerve function.
  • step (2) (1) culturing fibroblasts in the presence of a candidate compound, (2) evaluating the proliferation ability of said fibroblasts, and (3) and quantifying S-adenosylmethionine in said fibroblasts.
  • step numbers do not limit the order in which each step is performed. For example, step (2) may be performed after step (3) is performed.
  • Candidate compounds may be small compounds, nucleic acids, proteins, peptides, antibodies, lipids, animal tissue or cell extracts, plant extracts, etc. These candidate compounds may be novel, It may be a known one.
  • fibroblasts collected from any vertebrate can be used as fibroblasts in this embodiment.
  • the fibroblasts in this embodiment are preferably derived from mammals such as mice, rats, rabbits, sheep, pigs, cows, goats, monkeys and humans, and particularly preferably derived from humans.
  • the method of this embodiment may use an already established fibroblast cell line.
  • Such cell lines can be obtained, for example, from RIKEN BioResource Research Center (RIKEN BRC), American Type Culture Collection (ATCC), and the like.
  • fibroblasts should be cultured for a certain period of time in a medium with or without the addition of candidate compounds.
  • concentration of the candidate compound to be added varies depending on the type of compound, but can be appropriately selected, for example, within the range of 1 nM to 1 mM.
  • the culture period may be, for example, 8 hours to 4 days.
  • the medium one commonly used for culturing fibroblasts can be used.
  • it may be a basal medium such as DMEM or RPMI1640, and a mixture of two or more selected from these may be used. be able to.
  • media for fibroblasts are commercially available, and these commercial products may be used.
  • the proliferative ability of fibroblasts can be evaluated by measuring the doubling time, passage number and/or specific growth rate by methods well known in the art.
  • Quantification of S-adenosylmethionine in fibroblasts may be performed by methods well known in the art, for example, mass spectrometry by liquid chromatography-mass spectrometry (LC-MS/MS), mass microscopy, and ELISA. etc. can be implemented.
  • cultures in the presence or absence of the candidate compound may be performed in parallel and compared. You may compare with the analysis result about culture
  • the candidate compound when fibroblasts cultured in the presence of a candidate compound show significantly improved proliferation ability compared to fibroblasts cultured in the absence of the candidate compound In the future, the candidate compound can be evaluated as promising as an anti-aging substance.
  • the candidate compound can be evaluated as not promising as an anti-aging substance.
  • the screening method of this embodiment includes the steps of (4) culturing neural stem cells in the presence of the candidate compound, (5) evaluating the proliferation and/or differentiation potential of the neural stem cells, and (6) quantifying S-adenosylmethionine in neural stem cells and/or (7) administering said candidate compound to Drosophila; (8) analyzing reproductive tissue of said Drosophila; may further include Steps (4)-(8) are preferably carried out on candidate compounds evaluated as promising anti-aging substances based on the results of steps (1)-(3). Note that the numbers of steps (4) to (8) do not limit the order of execution of each step. For example, after steps (7) and (8) are performed, steps (4) to (6) may be implemented.
  • the neural stem cells used in steps (4) to (6) may be any stem cells that can acquire the ability to differentiate into neural lineage cells by the differentiation induction procedure described later.
  • Neural stem cells that can be used in the method of this embodiment may be derived from any vertebrate, but are preferably derived from mammals such as mice, rats, rabbits, sheep, pigs, cows, goats, monkeys, and humans. It is of animal origin, and particularly preferably of human origin.
  • Neural stem cells that can be used in the method of the present embodiment may be prepared from fetal or adult neural tissue, or may be prepared from ES cells or iPS cells. Alternatively, the method of this embodiment may use an already established neural stem cell line. Such cell lines can be obtained, for example, from RIKEN BioResource Research Center (RIKEN BRC), American Type Culture Collection (ATCC), and the like.
  • neural stem cells may be cultured for a certain period of time in a maintenance medium with or without the addition of candidate compounds.
  • concentration of the candidate compound to be added varies depending on the type of compound, but can be appropriately selected, for example, within the range of 1 nM to 1 mM.
  • the culture period may be, for example, 8 hours to 7 days.
  • a maintenance medium for neural stem cells is known (Hirano K and Namihira M, FEBS Open bio., 7(12): 1932-1942, 2017), for example, DMEM/F12 as a basal medium, N2 supplement, B27 It can be prepared by adding supplements, basic fibroblast growth factor, epidermal growth factor and the like.
  • maintenance media for neural stem cells are commercially available, and these commercial products may be used.
  • the proliferative ability of neural stem cells can be evaluated by measuring the doubling time, passage number and/or specific growth rate by methods well known in the art.
  • neural stem cells may be cultured for a certain period of time in a differentiation-inducing medium with or without the addition of candidate compounds.
  • concentration of the candidate compound to be added varies depending on the type of compound, but can be appropriately selected, for example, within the range of 1 nM to 1 mM.
  • the culture period may be, for example, 8 hours to 7 days.
  • Differentiation induction medium for neural stem cells is known (Hirano K and Namihira M, FEBS Open bio., 7(12): 1932-1942, 2017). It can be prepared by adding glutamine or the like. In addition, differentiation-inducing media for neural stem cells are commercially available, and these commercial products may be used.
  • the differentiation potential of neural stem cells can be evaluated by culturing neural stem cells under differentiation-inducing conditions already established in the field and detecting known differentiation markers.
  • Neuronal differentiation markers include, but are not limited to, doublecortin (DCX), ⁇ III tubulin, and the like.
  • detection of neuronal differentiation markers can be performed by immune cell staining, flow cytometry, or the like.
  • culture in the presence or absence of the candidate compound may be performed in parallel and compared. Analysis results for cultures in the absence of the candidate compound may be compared.
  • the candidate The compounds can be evaluated as promising antiaging agents, especially for neural tissue.
  • the candidate compound can be evaluated as not promising as an anti-aging agent for neural tissue.
  • the fruit fly used in steps (7) and (8) may be any fly of the genus Drosophila, but is preferably Drosophila melanogaster.
  • concentrations of the candidate compound may be added to food and ingested.
  • concentration of the candidate compound added to the feed varies depending on the type of compound, but can be appropriately selected, for example, within the range of 0.1 mM to 100 mM.
  • Administration of the candidate compound can be carried out, for example, over a period of 1 day to 2 months.
  • Drosophila reproductive tissue analysis can be performed by counting the number of germline cells and/or observing the formation of reproductive tissue by methods well known in the art, such as immunohistochemical staining.
  • reproductive tissue from Drosophila not administered with the candidate compound may be analyzed in parallel and compared. and may be compared with the results of past analyzes of reproductive tissues of Drosophila to which no candidate compound was administered.
  • a candidate compound is particularly preferred if administration of the candidate compound results in an increase in the number of germline cells and/or a decrease in reproductive tissue dysplasia compared to the reproductive tissue of Drosophila to which the candidate compound has not been administered. It can be evaluated as a promising anti-aging substance for reproductive tissues.
  • the candidate compound results in no change or a decrease in the number of germline cells and/or changes in germ tissue dysplasia compared to Drosophila that have not been administered the candidate compound. If it is absent or increased, the candidate compound can be evaluated as not promising as an anti-aging substance for reproductive tissue.
  • the method of the present embodiment it is possible to screen for anti-aging substances capable of preventing or treating age-related functional decline in various tissues, including nervous tissues and reproductive tissues.
  • Control Drosophila ZH-86Fa strain (#24486, Bloomington Drosophila Stock Center) or p(CaryP)attP2 strain (#36303, Bloomington Drosophila Stock Center) not modified for expression of the Sam-S gene; hereinafter "control Drosophila” or simply
  • control Drosophila As shown by the immunohistochemical staining image of the ovarian canaliculus of the "control"), the old (8-week-old) individual has a normal egg chamber (Fig. 1, left) and the young (2-3 days old) individual. Different abnormal egg chambers containing more than 16 germ cell cysts were observed (Fig. 1, right), so we tested whether there is a correlation between such abnormal egg chamber formation and the amount of SAM.
  • the pUASp-K10attB vector into which a DNA fragment encoding the Sam-S gene was inserted was introduced into a Drosophila strain having an attP site (#24486 strain, Bloomington Drosophila Stock Center), and the resulting Drosophila strain was further transfected with nanos-Gal4.
  • a strain that overexpresses the Sam-S gene in a germline-specific manner hereinafter referred to as “Sam-S(++)
  • a DNA fragment encoding the Sam-S gene was prepared by PCR using GH08738 (Drosophila Genomics Resource Center) as a template and the following primers.
  • Forward primer 5'-CGGGGTACCTTCAAAACTTCGAGTTACATATTAC-3' (SEQ ID NO: 1)
  • reverse primer 5'-CGGTCTAGATCAGTTGTCAATCTCCAGAGGCTTG-3' (SEQ ID NO: 2).
  • the primers used were as follows: 5'-ACAAATGTGCGACCAAATCAGC-3' (SEQ ID NO: 3) and 5'-CAATCTTTTCGTTTAGTTTGTGAGC-3' (SEQ ID NO: 4). Also, for detection of the internal standard (rp49), the following primers were used: 5'-CACGATAGCATACAGGCCCAAGATCGG-3' (SEQ ID NO: 5) and 5'-GCCATTTGTGCGACAGCTTAG-3' (SEQ ID NO: 6).
  • the eluate was filtered through a 0.22 ⁇ m PVDF filter, and the filtrate was mixed with an equal amount of 100 ⁇ M dithiothreitol (DTT)/50 mM Tris-HCl (pH 8.8) to prepare an analytical sample.
  • An analytical sample was subjected to a UPLC-MS/MS analyzer (Waters) to detect SAM.
  • the protein removed by the above process was quantified by the BCA method, and the amount of SAM detected was corrected with the amount of protein.
  • VASA-positive spherical cells in contact with Cap cells at the anterior end of the cambium layer those having spectrosomes (stained with Hts) were observed as germinal stem cells.
  • egg chambers usually having germ cell cysts consisting of 16 cells
  • egg chambers containing germ cell cysts of 16 cells or more were observed as abnormal egg chambers.
  • SAM levels in mouse reproductive tissues and brain tissues were quantified by the following procedure. Young (2 months old) and old (1 year and 6 months old) C57BL/6 mice (3 mice each) were euthanized by cervical dislocation, and the cerebellum, cerebrum, hippocampus, testes and ovaries were collected and dried. After quick freezing on ice, it was stored at -80°C. After crushing the frozen tissue with a bead crusher, metabolites were extracted and proteins were removed with a 50% methanol solution containing 10 mM acetic acid.
  • the metabolite fraction was concentrated with a centrifugal concentrator (TOMY Seiko) and eluted with ultrapure water.
  • the eluate was filtered through a 0.22 ⁇ m PVDF filter, and the filtrate was mixed with an equal amount of ultrapure water to obtain an analytical sample.
  • An analytical sample was subjected to a UPLC-MS/MS analyzer (Waters) to measure metabolites.
  • the protein removed by the above process was quantified by the BCA method, and the amount of SAM detected was corrected with the amount of protein. Statistics were performed using analysis of variance and T-test.
  • Human neural stem cells derived from embryonic 14-week human fetal cerebral cortex, purchased from PhoenixSongs Biologicals
  • fetal cerebral cortex purchased from PhoenixSongs Biologicals
  • neural stem cell maintenance medium (1 ⁇ N2 supplement and 0.1% B27
  • the cells were maintained and cultured in DMEM/F12 containing supplements (medium supplemented with 10 ng/ml human basic fibroblast growth factor (hbFGF) and 20 ng/ml human epidermal growth factor (hEGF)).
  • hbFGF human basic fibroblast growth factor
  • hEGF human epidermal growth factor
  • Human neural stem cells were seeded at 2 ⁇ 10 5 cells/well in a laminin-coated 12-well plate, cultured in the above medium for 2 days until 80-90% confluent, and transferred to Neurobasal medium (Thermo Fisher Scientific) for 2 days. Differentiation was induced by substituting a differentiation-inducing medium supplemented with % B27 supplement and 0.5 mM L-glutamine. Here, 0.1% DMSO was added to the control group, and MAT2A inhibitor 1 (MedChemExpress) dissolved in DMSO was added to the medium at each concentration for the SAM synthase inhibition group.
  • MAT2A inhibitor 1 MedChemExpress
  • mice Seven days after the start of differentiation induction, the cells were fixed with 4% paraformaldehyde/PBS, and fluorescence immunostaining was performed according to a normal procedure.
  • Mouse Anti-Ki-67 (Nippon Becton Dickinson, 550609) (1:500 dilution) and Rabbit Anti-Doublecortin antibody (Ab18723, Abcam) (1:500 dilution) were used as primary antibodies.
  • Secondary antibodies included CF488 donkey anti-mouse IgG (HCL), highly cross-adsorbed (Biotium, 20014) (1:500 dilution) and CF568 donkey anti-rat IgG (HCL), highly cross-adsorbed (B2002, B2009).
  • Ki-67 cell proliferation marker
  • DCX neural differentiation marker
  • Mouse Anti-Ki-67 (Nippon Becton Dickinson, 550609) (1:500 dilution) was used as the primary antibody.
  • Hoechst33342 (1/2000 dilution) was added together with the secondary antibody, and cell nuclei were also stained at the same time. The ratio of Ki-67 positive cells to the total number of Hoechst-stained cells was calculated. Statistics were performed using analysis of variance and T-test.
  • Methotrexate is a well-known antifolate drug that inhibits dihydrofolate reductase (DHFR), thereby decreasing the production of tetrahydrofolate (THF) and, consequently, the downstream production of SAM.
  • DHFR dihydrofolate reductase
  • MTX is also known to directly inhibit MAT expression and activity. Therefore, in this example, it was tested whether MTX affects the proliferative ability of human dermal fibroblasts.
  • Human skin fibroblasts were prepared by the same procedure as in 4 above, and each concentration of methotrexate (Fujifilm Wako Pure Chemical Industries) was added (no addition to the control). After culturing for 2 days, fluorescence immunostaining for the cell proliferation marker Ki-67 and Hoechst staining were performed in the same manner as in 4 above, and the ratio of Ki-67 positive cells to the total number of cells was calculated. Statistics were performed using analysis of variance and T-test.

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Abstract

Provided is a composition for preventing or treating tissue aging which contains an S-adenosylmethionine synthase inhibitor. Also provided is a method for screening an anti-aging substance, the method comprising (1) a step for culturing fibroblasts in the presence of a candidate compound, (2) a step for evaluating the propagating capability of the fibroblasts, and (3) a step for quantitatively determining the S-adenosylmethionine in the fibroblasts.

Description

組織の老化を予防または治療するための組成物Composition for preventing or treating tissue aging
　本発明は、皮膚組織、生殖組織および神経組織などの種々の組織の老化を予防または治療するための組成物、およびＳ－アデノシルメチオニンを指標とした抗老化物質のスクリーニング方法に関する。 The present invention relates to compositions for preventing or treating aging of various tissues such as skin tissue, reproductive tissue and nerve tissue, and screening methods for anti-aging substances using S-adenosylmethionine as an indicator.
　少子高齢化が進む社会において、加齢に伴う組織の機能低下（老化）の克服は喫緊の課題である。とりわけ、晩婚化による出産年齢の上昇や、高齢化に伴う認知症患者の増加は大きな問題となっており、生殖機能および脳機能の老化を抑制することが、少子化に歯止めをかけ、健康長寿を達成する上で極めて重要である。しかし、特定の疾患を対象とした従来の創薬アプローチによっては抗老化に十分に対処することは困難であり、異なった視点からの抗老化アプローチが求められている。 In a society where the birthrate is declining and the population is aging, overcoming the decline in tissue function (aging) that accompanies aging is an urgent issue. In particular, the rise in childbearing age due to later marriage and the increase in dementia patients due to aging are major problems. is extremely important in achieving However, it is difficult to sufficiently deal with anti-aging by conventional drug discovery approaches targeting specific diseases, and an anti-aging approach from a different viewpoint is required.
　近年、メチオニンの代謝産物であるＳ－アデノシルメチオニン（ＳＡＭ）がショウジョウバエ個体の寿命に影響を及ぼすことが報告された（非特許文献１）。この報告によれば、ショウジョウバエ体内のＳＡＭ量が加齢に伴って増加する一方、ＳＡＭ量の増加を抑制するとショウジョウバエの寿命が延長される。しかし、ショウジョウバエの各組織の老化に対するＳＡＭの影響は不明である。さらに、ショウジョウバエ以外の動物における老化とＳＡＭの関連性は、一切明らかにされていない。 In recent years, it has been reported that S-adenosylmethionine (SAM), a metabolite of methionine, affects the lifespan of Drosophila individuals (Non-Patent Document 1). According to this report, the amount of SAM in the body of Drosophila increases with age, and suppressing the increase in the amount of SAM extends the lifespan of Drosophila. However, the effect of SAMs on aging of Drosophila tissues is unclear. Furthermore, no link between aging and SAM in animals other than Drosophila has been demonstrated.
　本発明は、加齢に伴う種々の組織の機能低下を予防または治療するための組成物ならびにそのスクリーニング方法を提供することを目的としてなされたものである。 The purpose of the present invention is to provide a composition for preventing or treating age-related functional deterioration of various tissues and a screening method thereof.
　発明者らは、鋭意研究の結果、ショウジョウバエにおいてＳＡＭ量の増加が生殖組織の老化を促進すること、マウスにおいても加齢に伴い生殖組織および脳組織におけるＳＡＭ量が増加すること、ＳＡＭがヒト線維芽細胞の増殖能やヒト神経幹細胞の増殖能および分化能に影響することを初めて確認した。本発明者らは、この新規な発見に基づき、ＳＡＭの生合成を低減する物質が、皮膚組織、生殖組織および神経組織を含む種々の組織の老化の予防または治療に有用であることを見出した。 As a result of intensive research, the inventors found that an increase in the amount of SAM accelerates the aging of reproductive tissues in Drosophila, that the amount of SAM in reproductive tissues and brain tissues also increases with aging in mice, and that SAM is associated with human fibers. It was confirmed for the first time that it affects the proliferation ability of blast cells and the proliferation and differentiation ability of human neural stem cells. Based on this novel discovery, the present inventors found that substances that reduce SAM biosynthesis are useful for preventing or treating aging of various tissues, including skin tissue, reproductive tissue and nerve tissue. .
　すなわち、本発明は、一実施形態によれば、Ｓ－アデノシルメチオニン合成酵素阻害剤を含んでなる、組織の老化を予防または治療するための組成物を提供するものである。 That is, according to one embodiment, the present invention provides a composition for preventing or treating tissue aging, comprising an S-adenosylmethionine synthase inhibitor.
　前記組織は、皮膚組織、生殖組織および神経組織からなる群から選択されることが好ましい。 The tissue is preferably selected from the group consisting of skin tissue, reproductive tissue and nerve tissue.
　前記Ｓ－アデノシルメチオニン合成酵素は、メチオニンアデノシルトランスフェラーゼ２であることが好ましい。 The S-adenosylmethionine synthase is preferably methionine adenosyltransferase 2.
　前記Ｓ－アデノシルメチオニン合成酵素阻害剤は、６－（２－メチルベンゾ［ｄ］チアゾール－６－イル）－２，３－ジフェニル－５－（ピリジン－２－イルアミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オン、（Ｅ）－４－（２－クロロ－６－フルオロスチリル）－Ｎ－メチルアニリン、および３－（サイクロヘクス－１－エン－１－イル）－６－（４－メトキシフェニル）－２－フェニル－５－（ピリジン－２－イラミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オンからなる群から選択されることが好ましい。 The S-adenosylmethionine synthase inhibitor is 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a ]pyrimidin-7(4H)-one, (E)-4-(2-chloro-6-fluorostyryl)-N-methylaniline, and 3-(cyclohex-1-en-1-yl)-6- It is preferably selected from the group consisting of (4-methoxyphenyl)-2-phenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-ones.
　あるいは、前記Ｓ－アデノシルメチオニン合成酵素阻害剤は、メチオニンアデノシルトランスフェラーゼ２遺伝子の発現を阻害するＲＮＡまたはそれをコードする核酸を含む発現ベクターであることが好ましい。 Alternatively, the S-adenosylmethionine synthase inhibitor is preferably an expression vector containing an RNA that inhibits expression of the methionine adenosyltransferase 2 gene or a nucleic acid encoding the same.
　前記組成物は、医薬品であることが好ましい。 The composition is preferably a pharmaceutical.
　あるいは、前記組成物は、飲食品であることが好ましい。 Alternatively, the composition is preferably a food or drink.
　また、本発明は、一実施形態によれば、（１）候補化合物の存在下で線維芽細胞を培養するステップと、（２）前記線維芽細胞の増殖能を評価するステップと、（３）前記線維芽細胞におけるＳ－アデノシルメチオニンを定量するステップとを含む、抗老化物質のスクリーニング方法を提供するものである。 Further, according to one embodiment of the present invention, (1) culturing fibroblasts in the presence of a candidate compound, (2) evaluating the proliferation ability of the fibroblasts, and (3) and quantifying S-adenosylmethionine in said fibroblasts.
　上記方法は、（４）前記候補化合物の存在下で神経幹細胞を培養するステップと、（５）前記神経幹細胞の増殖能および／または分化能を評価するステップと、（６）前記神経幹細胞におけるＳ－アデノシルメチオニンを定量するステップとをさらに含むことが好ましい。 (4) culturing neural stem cells in the presence of the candidate compound; (5) evaluating the proliferation and/or differentiation potential of the neural stem cells; - quantifying adenosylmethionine.
　上記方法は、（７）ショウジョウバエに前記候補化合物を投与するステップと、（８）前記ショウジョウバエの生殖組織を解析するステップとをさらに含むことが好ましい。 The above method preferably further comprises the steps of (7) administering the candidate compound to Drosophila, and (8) analyzing the reproductive tissue of the Drosophila.
　本発明に係る組成物は、加齢に伴う種々の組織の機能低下を予防または治療するために有用である。また、本発明に係るスクリーニング方法は、細胞または組織におけるＳＡＭの生合成を低減することにより、皮膚組織、生殖組織および神経組織を含む種々の組織の老化を抑制し得る化合物を取得することができ、有用である。 The composition according to the present invention is useful for preventing or treating age-related functional decline in various tissues. In addition, the screening method according to the present invention can obtain compounds capable of suppressing aging of various tissues including skin tissue, reproductive tissue and nerve tissue by reducing SAM biosynthesis in cells or tissues. , is useful.
図１は、若齢および老齢の対照ショウジョウバエの卵巣小管の免疫組織染色像である。FIG. 1 shows immunohistochemical staining images of ovarian canaliculi of young and old control Drosophila. 図２は、若齢および老齢の対照ショウジョウバエにおけるＳＡＭ－Ｓの発現量を示すグラフである。FIG. 2 is a graph showing the expression levels of SAM-S in young and old control Drosophila. 図３は、若齢ならびに老齢の対照およびＳａｍ－Ｓ（－）ショウジョウバエ卵巣におけるＳＡＭ－Ｓの発現量を示すグラフである。FIG. 3 is a graph showing the expression levels of SAM-S in young and aged control and Sam-S(−) Drosophila ovaries. 図４は、各ショウジョウバエ系統における卵巣小管あたりの生殖幹細胞の数の加齢に伴う変化を示すグラフである。FIG. 4 is a graph showing age-related changes in the number of germinal stem cells per ovarian canaliculus in each Drosophila strain. 図５は、対照およびＳａｍ－Ｓ（＋＋）ショウジョウバエにおける異常卵巣の出現頻度を示すグラフである。FIG. 5 is a graph showing the frequency of abnormal ovaries in control and Sam-S(++) Drosophila. 図６は、対照およびＳａｍ－Ｓ（－）ショウジョウバエにおける異常卵巣の出現頻度を示すグラフである。FIG. 6 is a graph showing the frequency of abnormal ovaries in control and Sam-S(−) Drosophila. 図７は、若齢ならびに老齢のマウス生殖組織および脳組織におけるＳＡＭ量を示すプロットである。FIG. 7 is a plot showing SAM abundance in young and aged mouse reproductive and brain tissue. 図８は、ＳＡＭ合成酵素阻害剤の存在下における、細胞増殖マーカーＫｉ－６７陽性のヒト神経幹細胞の割合を示すグラフである。FIG. 8 is a graph showing the proportion of cell proliferation marker Ki-67-positive human neural stem cells in the presence of a SAM synthase inhibitor. 図９は、ＳＡＭ合成酵素阻害剤の存在下における、神経分化マーカーＤＣＸ陽性のヒト神経幹細胞の割合を示すグラフである。FIG. 9 is a graph showing the proportion of neural differentiation marker DCX-positive human neural stem cells in the presence of a SAM synthase inhibitor. 図１０は、ＳＡＭ合成酵素阻害剤の存在下における、細胞増殖マーカーＫｉ－６７陽性のヒト皮膚線維芽細胞の割合を示すグラフである。FIG. 10 is a graph showing the proportion of cell proliferation marker Ki-67-positive human dermal fibroblasts in the presence of SAM synthase inhibitors. 図１１は、メトトレキサートの存在下における、細胞増殖マーカーＫｉ－６７陽性のヒト皮膚線維芽細胞の割合を示すグラフである。FIG. 11 is a graph showing the percentage of cell proliferation marker Ki-67-positive human dermal fibroblasts in the presence of methotrexate.
　以下、本発明を詳細に説明するが、本発明は本明細書中に説明した実施形態に限定されるものではない。 Although the present invention will be described in detail below, the present invention is not limited to the embodiments described herein.
　本発明は、第一の実施形態によれば、Ｓ－アデノシルメチオニン合成酵素阻害剤を含んでなる、組織の老化を予防または治療するための組成物である。 The first embodiment of the present invention is a composition for preventing or treating tissue aging, comprising an S-adenosylmethionine synthase inhibitor.
　本実施形態において、「予防する」とは、生殖組織または神経組織が老化するおそれのある対象において、それを未然に防ぐことのみならず、そのリスクを低減することや、事前の処置により、生殖組織または神経組織の老化を遅延または軽減することをも含む。 In the present embodiment, "preventing" means not only preventing aging of reproductive tissue or nervous tissue in a subject, but also reducing the risk of aging, or preventing reproductive It also includes slowing or reducing aging of tissue or nerve tissue.
　本実施形態において、「治療する」とは、生殖組織または神経組織の老化を完全に治癒することのみならず、それを寛解または緩和することや、その進行を遅延または停止させることをも含む。 In the present embodiment, "treating" includes not only complete cure of aging of reproductive tissue or nervous tissue, but also remission or alleviation thereof, and delaying or stopping its progression.
　本実施形態における「組織」は、任意の脊椎動物におけるものであってよく、好ましくは、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ヤギ、サル、ヒトなどの哺乳動物の組織であり、特に好ましくはヒトの組織である。また、組織の種類も特に限定されず、例えば、皮膚組織、生殖組織、神経組織、筋組織、骨および軟骨組織、免疫系組織などであってよい。 The "tissue" in this embodiment may be in any vertebrate, preferably mammalian tissue such as mouse, rat, rabbit, sheep, pig, cow, goat, monkey, human, etc. Human tissue is preferred. The type of tissue is also not particularly limited, and may be, for example, skin tissue, reproductive tissue, nerve tissue, muscle tissue, bone and cartilage tissue, immune system tissue, or the like.
　本実施形態における組織は、好ましくは、皮膚組織、生殖組織または神経組織である。本実施形態における「皮膚組織」には、皮膚を構成するすべての組織、すなわち、表皮、真皮および皮下組織が含まれてよい。本実施形態における「生殖組織」には、卵巣および精巣が含まれてよい。本実施形態における「神経組織」には、中枢神経組織および末梢神経組織が含まれてよい。 The tissue in this embodiment is preferably skin tissue, reproductive tissue or nerve tissue. "Skin tissue" in this embodiment may include all tissues that constitute the skin, ie, epidermis, dermis and subcutaneous tissue. "Reproductive tissue" in this embodiment may include ovaries and testes. "Nerve tissue" in this embodiment may include central nerve tissue and peripheral nerve tissue.
　本実施形態において、組織の「老化」とは、その組織を構成する細胞が加齢により減少または劣化することをいう。ここで、細胞の「劣化」とは、細胞の分化能、増殖能および／または機能が低下または消失することを意味する。 In the present embodiment, "aging" of tissue refers to the decrease or deterioration of cells that make up the tissue due to aging. Here, "degradation" of cells means reduction or loss of cell differentiation ability, proliferation ability and/or function.
　皮膚組織を構成する細胞には、例えば、線維芽細胞、表皮幹細胞、真皮幹細胞、毛包幹細胞、角化細胞、色素細胞、ランゲルハンス細胞などが挙げられるが、これらに限定されない。生殖組織を構成する細胞には、生殖細胞への分化が決定づけられた細胞全般が含まれてよく、例えば、卵子幹細胞（卵原細胞）、一次卵母細胞、二次卵母細胞、卵細胞、精子幹細胞（精原細胞）、一次精母細胞、二次精母細胞および精細胞などが挙げられるが、これらに限定されない。神経組織を構成する細胞には、神経細胞への分化が決定づけられた細胞全般が含まれてよく、例えば、神経幹細胞、神経前駆細胞、神経細胞などが挙げられるが、これらに限定されない。 Cells that make up skin tissue include, but are not limited to, fibroblasts, epidermal stem cells, dermal stem cells, hair follicle stem cells, keratinocytes, pigment cells, and Langerhans cells. Cells constituting reproductive tissue may include general cells determined to differentiate into germ cells, such as egg stem cells (oogonia), primary oocytes, secondary oocytes, egg cells, and sperm. They include, but are not limited to, stem cells (spermatogonia), primary spermatocytes, secondary spermatocytes and spermatids. Cells constituting neural tissue may include all cells determined to differentiate into neural cells, such as, but not limited to, neural stem cells, neural progenitor cells, and neural cells.
　Ｓ－アデノシルメチオニン（以下、「ＳＡＭ」とも記載する）は、メチオニンとＡＴＰを基質として、メチオニンアデノシルトランスフェラーゼ（以下、「ＭＡＴ」とも記載する）により生成される。したがって、本実施形態における「Ｓ－アデノシルメチオニン（ＳＡＭ）合成酵素」は、メチオニンアデノシルトランスフェラーゼ（ＭＡＴ）と同義である。ＭＡＴには複数のアイソザイムが存在し、本実施形態におけるＳＡＭ合成酵素は、いずれのアイソザイムであってもよい。例えば、哺乳動物にはＭＡＴ１およびＭＡＴ２の２種類のアイソザイムが存在し、いずれも本実施形態におけるＳＡＭ合成酵素に含まれてよい。本実施形態におけるＳＡＭ合成酵素は、好ましくはＭＡＴ２である。 S-adenosylmethionine (hereinafter also referred to as "SAM") is produced by methionine adenosyltransferase (hereinafter also referred to as "MAT") using methionine and ATP as substrates. Therefore, "S-adenosylmethionine (SAM) synthase" in the present embodiment is synonymous with methionine adenosyltransferase (MAT). MAT has multiple isozymes, and the SAM synthase in this embodiment may be any isozyme. For example, mammals have two types of isozymes, MAT1 and MAT2, both of which may be included in the SAM synthase in this embodiment. The SAM synthase in this embodiment is preferably MAT2.
　本実施形態における「Ｓ－アデノシルメチオニン合成酵素阻害剤」は、ＭＡＴの発現および／または活性を直接的または間接的に減少させることができる任意の物質であってよい。したがって、本実施形態におけるＳＡＭ合成酵素阻害剤としては、例えば、タンパク質、ペプチド、核酸、脂質、低分子化合物などを用いることができるが、これらに限定されない。また、そのような物質は、公知のものであってもよいし、新規のものであってもよい。 The "S-adenosylmethionine synthase inhibitor" in this embodiment may be any substance that can directly or indirectly decrease the expression and/or activity of MAT. Therefore, as the SAM synthetase inhibitor in this embodiment, for example, proteins, peptides, nucleic acids, lipids, low-molecular-weight compounds and the like can be used, but are not limited to these. Moreover, such substances may be known substances or novel substances.
　本実施形態における好ましいＳＡＭ合成酵素阻害剤としては、例えば、６－（２－メチルベンゾ［ｄ］チアゾール－６－イル）－２，３－ジフェニル－５－（ピリジン－２－イルアミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オン、（Ｅ）－４－（２－クロロ－６－フルオロスチリル）－Ｎ－メチルアニリン、および３－（サイクロヘクス－１－エン－１－イル）－６－（４－メトキシフェニル）－２－フェニル－５－（ピリジン－２－イラミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オンなどが挙げられるが、これらに限定されない。本実施形態におけるＳＡＭ合成酵素阻害剤としては、好ましくは６－（２－メチルベンゾ［ｄ］チアゾール－６－イル）－２，３－ジフェニル－５－（ピリジン－２－イルアミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オンを用いることができる。 Preferred SAM synthase inhibitors in this embodiment include, for example, 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1, 5-a]pyrimidin-7(4H)-one, (E)-4-(2-chloro-6-fluorostyryl)-N-methylaniline, and 3-(cyclohex-1-en-1-yl) -6-(4-methoxyphenyl)-2-phenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-one and the like, but are not limited thereto. The SAM synthase inhibitor in the present embodiment is preferably 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1,5 -a]pyrimidin-7(4H)-ones can be used.
　本実施形態において使用できるＳＡＭ合成酵素阻害剤は市販されており、市販品を使用することもできる。好ましい市販品としては、例えば、ＭＡＴ２Ａ　ｉｎｈｉｂｉｔｏｒ　１（ＭｅｄＣｈｅｍＥｘｐｒｅｓｓ）、ＭＡＴ２Ａ　Ｉｎｈｉｂｉｔｏｒ　２（ＦＩＤＡＳ－５）（ＭｅｄＣｈｅｍＥｘｐｒｅｓｓ）、ＭＡＴ２Ａ　Ｉｎｈｉｂｉｔｏｒ（ＡＧ－２７０）（ＭｅｄＣｈｅｍＥｘｐｒｅｓｓ）などが挙げられるが、これらに限定されない。 SAM synthetase inhibitors that can be used in this embodiment are commercially available, and commercial products can also be used. Preferred commercial products include, but are not limited to, MAT2A inhibitor 1 (MedChem Express), MAT2A inhibitor 2 (FIDAS-5) (MedChemExpress), MAT2A inhibitor (AG-270) (MedChemExpress), and the like.
　あるいは、本実施形態における好ましいＳＡＭ合成酵素阻害剤として、メチオニンアデノシルトランスフェラーゼ２遺伝子の発現を阻害するＲＮＡを用いてもよい。「遺伝子の発現を阻害するＲＮＡ」には、ｓｉＲＮＡ、ｓｈＲＮＡ、ｄｓＲＮＡなどのＲＮＡ干渉作用を有するＲＮＡ分子の他、標的遺伝子（メチオニンアデノシルトランスフェラーゼ２遺伝子）由来のｍＲＮＡに作用してその発現を制御すると考えられるｍｉＲＮＡなどが挙げられるが、これらに限定されない。本実施形態における好ましいＲＮＡは、ｓｉＲＮＡまたはｓｈＲＮＡである。 Alternatively, an RNA that inhibits the expression of the methionine adenosyltransferase 2 gene may be used as a preferred SAM synthetase inhibitor in this embodiment. "RNA that inhibits gene expression" includes RNA molecules with RNA interference effects such as siRNA, shRNA, and dsRNA, as well as target gene (methionine adenosyltransferase 2 gene)-derived mRNA to control its expression. Examples include, but are not limited to, miRNAs that are thought to Preferred RNAs in this embodiment are siRNAs or shRNAs.
　本実施形態におけるＲＮＡは、当分野において十分に確立されたｓｉＲＮＡまたはｓｈＲＮＡの設計方法にしたがって設計することができる。メチオニンアデノシルトランスフェラーゼ２遺伝子の核酸配列情報は、所定のデータベースから入手することができる。例えば、ヒトＭＡＴ２Ａであれば、ＮＣＢＩ　Ｒｅｆｅｒｅｎｃｅ　Ｓｅｑｕｅｎｃｅ（ＲｅｆＳｅｑ）ＩＤ：ＮＭ＿００５９１１．６が利用可能である。本実施形態におけるＲＮＡは、ＭＡＴ２Ａ遺伝子のコード配列から選択される任意の領域に対して相補的な１７～３０ヌクレオチドからなる配列を含むものであってよく、好ましくはヒトＭＡＴ２Ａ遺伝子のコード配列のヌクレオチド１２１～１３０８から選択される領域に対して相補的な１７～３０ヌクレオチド、より好ましくは１９～２５ヌクレオチドからなる配列を含むものであってよく、例えば、ＡＡＧＧＡＧＡＡＡＧＵＣＡＵＣＡＡＡＧＣＡ（配列番号８）を含むことが特に好ましい。 The RNA in this embodiment can be designed according to a well-established siRNA or shRNA design method in the art. Nucleic acid sequence information of the methionine adenosyltransferase 2 gene can be obtained from predetermined databases. For example, for human MAT2A, NCBI Reference Sequence (RefSeq) ID: NM_005911.6 is available. The RNA in this embodiment may contain a sequence consisting of 17 to 30 nucleotides complementary to any region selected from the coding sequence of the MAT2A gene, preferably the nucleotides of the coding sequence of the human MAT2A gene. 121-1308, more preferably comprising a sequence consisting of 17-30 nucleotides complementary to a region selected from 121-1308, more preferably 19-25 nucleotides, for example AAGGAGAAAGUCAUCAAAGCA (SEQ ID NO: 8). preferable.
　なお、本実施形態におけるＲＮＡは、すべてＲＮＡから構成されてもよいし、その一部に修飾ＲＮＡが含まれてもよい。修飾ＲＮＡとしては、例えば、ホスホロチオエート化ＲＮＡ、ボラノホスフェート化ＲＮＡ、２’－Ｏ－メチル化ＲＮＡ、２’－Ｆ化ＲＮＡ、２’，４’－ＢＮＡ（別名ＬＮＡ（Ｌｏｃｋｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ））、などが挙げられる。 It should be noted that the RNA in this embodiment may be entirely composed of RNA, or part thereof may contain modified RNA. Examples of modified RNA include phosphorothioated RNA, boranophosphated RNA, 2'-O-methylated RNA, 2'-F-RNA, 2',4'-BNA (also known as LNA (Locked Nucleic Acid)), etc.
　本実施形態におけるＲＮＡは、上記ＲＮＡをコードする核酸を含む発現ベクターを用いることにより、細胞内で発現させてもよい。したがって、本実施形態の組成物は、ＳＡＭ合成酵素阻害剤として、上記ＲＮＡをコードする核酸を含む発現ベクターを含むものであってもよい。発現ベクターは、ＲＮＡアプタマーを導入する細胞の種類に応じて、適切なウイルスベクターまたは非ウイルスベクターを選択して用いることができる。 The RNA in this embodiment may be expressed intracellularly by using an expression vector containing a nucleic acid encoding the above RNA. Therefore, the composition of this embodiment may contain an expression vector containing a nucleic acid encoding the above RNA as a SAM synthase inhibitor. As the expression vector, an appropriate viral vector or non-viral vector can be selected and used depending on the type of cell into which the RNA aptamer is introduced.
　本実施形態の組成物は、上記から選択される１または複数のＳＡＭ合成酵素阻害剤を有効成分として含むことができる。本実施形態の組成物において、ＳＡＭ合成酵素阻害剤は、対象において適切な摂取量となるように含有されればよい。例えば、ＳＡＭ合成酵素阻害剤として６－（２－メチルベンゾ［ｄ］チアゾール－６－イル）－２，３－ジフェニル－５－（ピリジン－２－イルアミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オンを用いる場合であれば、ＳＡＭ合成酵素阻害剤は、その摂取量が、成人１日当たり、例えば１０～２００ｍｇ／ｋｇ（体重）、好ましくは　１００～２００ｍｇ／ｋｇ（体重）となるように組成物中に含有されてよいが、かかる範囲には限定されず、組成物の形態、対象の状態、年齢、性別などにより適宜調整され得る。 The composition of the present embodiment can contain one or more SAM synthase inhibitors selected from the above as active ingredients. In the composition of the present embodiment, the SAM synthase inhibitor should be contained so as to be an appropriate intake amount for the subject. For example, 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidine-7 as a SAM synthase inhibitor. When (4H)-one is used, the intake of the SAM synthase inhibitor is, for example, 10 to 200 mg/kg (body weight), preferably 100 to 200 mg/kg (body weight) per day for adults. However, it is not limited to such a range, and can be appropriately adjusted depending on the form of the composition, subject's condition, age, sex, and the like.
　本実施形態の組成物は、有効成分のみから構成されてもよいが、一般的には、さらに任意の成分として、薬学的に許容される公知の担体および添加物を含んでもよい。 The composition of the present embodiment may be composed only of active ingredients, but generally may further contain known pharmaceutically acceptable carriers and additives as optional ingredients.
　本実施形態の組成物は、例えば医薬品として製造することができる。この場合、本実施形態の組成物は、例えば、錠剤、顆粒剤、散剤、カプセル剤、ゼリー剤、シロップ剤、注射剤などの、種々の剤型に製剤化することができる。したがって、本実施形態の組成物は、経口投与、腹腔内投与、静脈内投与、経皮投与、標的組織への直接注射など、種々の方法により投与することができる。本実施形態の組成物は、経口用製剤とすることが好ましく、したがって、経口投与されることが好ましい。 The composition of this embodiment can be produced, for example, as a pharmaceutical. In this case, the composition of this embodiment can be formulated into various dosage forms such as tablets, granules, powders, capsules, jellies, syrups and injections. Therefore, the composition of this embodiment can be administered by various methods such as oral administration, intraperitoneal administration, intravenous administration, transdermal administration, and direct injection into the target tissue. The composition of the present embodiment is preferably an oral formulation, and therefore is preferably administered orally.
　本実施形態の医薬品を経口用製剤とする場合には、例えば、錠剤、顆粒剤、散剤などの固形剤とすることができる。この場合には、適切な添加物、例えば、デンプン、マンニトール、カルボキシメチルセルロース、コーンスターチ、無機塩などの添加剤や、さらに所望により結合剤、崩壊剤、滑沢剤などを配合することができる。本実施形態の医薬品を錠剤とする場合には、所望によりショ糖、ゼラチン、ヒドロキシプロピルセルロースなどにより組成物を被覆してもよい。本実施形態の医薬品をシロップ剤などの液剤とする場合には、滅菌水、生理食塩水、エタノールなどを担体として使用でき、さらに所望により、懸濁剤などの補助剤を添加してもよい。 When the drug of the present embodiment is an oral formulation, it can be, for example, a solid formulation such as tablets, granules, and powders. In this case, suitable additives such as starch, mannitol, carboxymethyl cellulose, corn starch, inorganic salts, etc., and if desired, binders, disintegrants, lubricants and the like can be added. When the drug of this embodiment is made into a tablet, the composition may be coated with sucrose, gelatin, hydroxypropylcellulose, or the like, if desired. When the drug of this embodiment is made into a liquid formulation such as a syrup, sterilized water, physiological saline, ethanol and the like can be used as carriers, and if desired, an adjuvant such as a suspending agent may be added.
　本実施形態の医薬品を非経口用製剤とする場合には、例えば注射剤などの液剤とすることができる。この場合には、有効成分を注射用蒸留水、生理食塩水、ブドウ糖水溶液、注射用植物油、ポリエチレングリコールなどの希釈剤に溶解または懸濁させ、必要に応じ、殺菌剤、等張化剤、無痛化剤などを加えることにより調製することができる。 When the drug of this embodiment is a parenteral formulation, it can be a liquid formulation such as an injection. In this case, the active ingredient is dissolved or suspended in a diluent such as distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, polyethylene glycol, etc. It can be prepared by adding an agent or the like.
　本実施形態の医薬品には、さらに所望により、保存料や安定化剤などの薬学的に許容される添加物や、他の治療薬を配合することができる。 Pharmaceutically acceptable additives such as preservatives and stabilizers, and other therapeutic agents can be added to the pharmaceuticals of the present embodiment, if desired.
　あるいは、本実施形態の組成物は、飲食品として製造することができる。この場合、本実施形態の組成物は、例えば、一般用加工食品とすることができる。一般用加工食品には、例えば、パン類、麺類、菓子類、食用油脂、調味料、飲料類などが挙げられるが、これらに限定されない。 Alternatively, the composition of this embodiment can be produced as a food or drink. In this case, the composition of this embodiment can be, for example, processed food for general use. Examples of processed foods for general use include, but are not limited to, breads, noodles, confectionery, edible fats and oils, seasonings, and beverages.
　また、本実施形態の組成物は、例えば、栄養補助食品とすることができる。栄養補助食品には、例えば、サプリメント、栄養補助飲料、特定保健用食品、栄養機能食品などが含まれてよい。本実施形態の栄養補助食品は、例えば、散剤、顆粒剤、錠剤、カプセル剤、ゼリー剤、液剤として調製されてよく、さらに所望により、香料、着色料、甘味料、保存料などの添加物や、他の栄養成分を配合することができる。 Also, the composition of the present embodiment can be used as, for example, a nutritional supplement. Dietary supplements may include, for example, supplements, nutritional supplements, foods for specified health uses, foods with nutrient function claims, and the like. The nutritional supplement of the present embodiment may be prepared, for example, as powders, granules, tablets, capsules, jelly preparations, liquid preparations, and if desired, additives such as flavorings, coloring agents, sweeteners, preservatives, etc. , can be combined with other nutritional ingredients.
　本実施形態の組成物は、加齢に伴う生殖機能および／もしくは神経機能の低下を予防または治療するために有用である。 The composition of the present embodiment is useful for preventing or treating age-related decline in reproductive function and/or nerve function.
　本発明は、第二の実施形態によれば、（１）候補化合物の存在下で線維芽細胞を培養するステップと、（２）前記線維芽細胞の増殖能を評価するステップと、（３）前記線維芽細胞におけるＳ－アデノシルメチオニンを定量するステップとを含む、抗老化物質のスクリーニング方法である。なお、ステップの番号は、各ステップの実施の順番を限定するものではなく、例えば、ステップ（３）を実施した後に、ステップ（２）を実施してもよい。 According to a second embodiment of the present invention, (1) culturing fibroblasts in the presence of a candidate compound, (2) evaluating the proliferation ability of said fibroblasts, and (3) and quantifying S-adenosylmethionine in said fibroblasts. Note that the step numbers do not limit the order in which each step is performed. For example, step (2) may be performed after step (3) is performed.
　候補化合物は、低分子化合物、核酸、タンパク質、ペプチド、抗体、脂質、動物組織または細胞抽出物、植物抽出物などであってよく、これらの候補化合物は、新規なものであってもよいし、公知のものであってもよい。 Candidate compounds may be small compounds, nucleic acids, proteins, peptides, antibodies, lipids, animal tissue or cell extracts, plant extracts, etc. These candidate compounds may be novel, It may be a known one.
　本実施形態における線維芽細胞は、任意の脊椎動物から採取された細胞を用いることができる。本実施形態における線維芽細胞は、好ましくは、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ヤギ、サル、ヒトなどの哺乳動物由来であり、特に好ましくはヒト由来である。あるいは、本実施形態の方法では、すでに確立された線維芽細胞株を用いてもよい。そのような細胞株は、例えば理化学研究所バイオリソース研究センター（ＲＩＫＥＮ　ＢＲＣ）、Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）などから入手することができる。 Cells collected from any vertebrate can be used as fibroblasts in this embodiment. The fibroblasts in this embodiment are preferably derived from mammals such as mice, rats, rabbits, sheep, pigs, cows, goats, monkeys and humans, and particularly preferably derived from humans. Alternatively, the method of this embodiment may use an already established fibroblast cell line. Such cell lines can be obtained, for example, from RIKEN BioResource Research Center (RIKEN BRC), American Type Culture Collection (ATCC), and the like.
　線維芽細胞の増殖能を評価するためには、線維芽細胞を、候補化合物を添加したまたは添加していない培地中で一定期間培養すればよい。添加される候補化合物の濃度は、化合物の種類により異なるが、例えば、１ｎＭ～１ｍＭの範囲で適宜選択することができる。培養期間は、例えば、８時間～４日であってよい。培地は、線維芽細胞を培養するために通常用いられているものを使用でき、例えば、ＤＭＥＭやＲＰＭＩ１６４０などの基礎培地であってよく、これらから選択される単独または２種類以上を混合して用いることができる。また、線維芽細胞用の培地が市販されており、それら市販品を用いてもよい。 In order to evaluate the proliferative ability of fibroblasts, fibroblasts should be cultured for a certain period of time in a medium with or without the addition of candidate compounds. The concentration of the candidate compound to be added varies depending on the type of compound, but can be appropriately selected, for example, within the range of 1 nM to 1 mM. The culture period may be, for example, 8 hours to 4 days. As the medium, one commonly used for culturing fibroblasts can be used. For example, it may be a basal medium such as DMEM or RPMI1640, and a mixture of two or more selected from these may be used. be able to. In addition, media for fibroblasts are commercially available, and these commercial products may be used.
　線維芽細胞の増殖能は、当分野において周知の方法により、倍加時間、継代数および／または比増殖速度を計測することにより評価することができる。 The proliferative ability of fibroblasts can be evaluated by measuring the doubling time, passage number and/or specific growth rate by methods well known in the art.
　線維芽細胞におけるＳ－アデノシルメチオニンの定量は、当分野において周知の方法により実施すればよく、例えば、液体クロマトグラフィー質量分析（ＬＣ－ＭＳ／ＭＳ）や質量顕微鏡法などによる質量分析や、ＥＬＩＳＡなどにより実施することができる。 Quantification of S-adenosylmethionine in fibroblasts may be performed by methods well known in the art, for example, mass spectrometry by liquid chromatography-mass spectrometry (LC-MS/MS), mass microscopy, and ELISA. etc. can be implemented.
　候補化合物により線維芽細胞の増殖能が変化したかどうかを判定するためには、候補化合物の存在下または非存在下での培養を並行して実施して比較してもよいし、過去に実施した候補化合物の非存在下での培養についての解析結果と比較してもよい。本実施形態のスクリーニング方法において、候補化合物の存在下で培養された線維芽細胞が、候補化合物の非存在下で培養された線維芽細胞と比較して、有意に改善した増殖能を示した場合には、当該候補化合物は、抗老化物質として有望であると評価することができる。一方、候補化合物の存在下で培養された線維芽細胞が、候補化合物の非存在下で培養された線維芽細胞と比較して、低下した増殖能を示した場合、または両者の増殖能に有意な差が見られなかった場合には、当該候補化合物は、抗老化物質として有望ではないと評価することができる。 In order to determine whether the candidate compound has altered the proliferative ability of fibroblasts, cultures in the presence or absence of the candidate compound may be performed in parallel and compared. You may compare with the analysis result about culture|cultivation by the absence of the candidate compound which carried out. In the screening method of the present embodiment, when fibroblasts cultured in the presence of a candidate compound show significantly improved proliferation ability compared to fibroblasts cultured in the absence of the candidate compound In the future, the candidate compound can be evaluated as promising as an anti-aging substance. On the other hand, if fibroblasts cultured in the presence of the candidate compound exhibited a reduced proliferative capacity compared to fibroblasts cultured in the absence of the candidate compound, or both If no significant difference is observed, the candidate compound can be evaluated as not promising as an anti-aging substance.
　本実施形態のスクリーニング方法は、（４）前記候補化合物の存在下で神経幹細胞を培養するステップと、（５）前記神経幹細胞の増殖能および／または分化能を評価するステップと、（６）前記神経幹細胞におけるＳ－アデノシルメチオニンを定量するステップとをさらに含んでもよく、かつ／または、（７）ショウジョウバエに前記候補化合物を投与するステップと、（８）前記ショウジョウバエの生殖組織を解析するステップとをさらに含んでもよい。ステップ（４）～（８）は、ステップ（１）～（３）の結果から抗老化物質として有望であると評価された候補化合物について実施することが好ましい。なお、ステップ（４）～（８）の番号は、各ステップの実施の順番を限定するものではなく、例えば、ステップ（７）および（８）を実施した後に、ステップ（４）～（６）を実施してもよい。 The screening method of this embodiment includes the steps of (4) culturing neural stem cells in the presence of the candidate compound, (5) evaluating the proliferation and/or differentiation potential of the neural stem cells, and (6) quantifying S-adenosylmethionine in neural stem cells and/or (7) administering said candidate compound to Drosophila; (8) analyzing reproductive tissue of said Drosophila; may further include Steps (4)-(8) are preferably carried out on candidate compounds evaluated as promising anti-aging substances based on the results of steps (1)-(3). Note that the numbers of steps (4) to (8) do not limit the order of execution of each step. For example, after steps (7) and (8) are performed, steps (4) to (6) may be implemented.
　ステップ（４）～（６）で用いられる神経幹細胞は、後述する分化誘導手順により神経系列細胞に分化する能力を獲得し得る幹細胞であれば、任意のものであってよい。本実施形態の方法において用いることができる神経幹細胞は、任意の脊椎動物由来のものであってよいが、好ましくは、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ヤギ、サル、ヒトなどの哺乳動物由来であり、特に好ましくはヒト由来である。本実施形態の方法において用いることができる神経幹細胞は、胎児または成体の神経組織から調製されたものであってもよいし、ＥＳ細胞やｉＰＳ細胞から調製されたものであってもよい。あるいは、本実施形態の方法では、すでに確立された神経幹細胞株を用いてもよい。そのような細胞株は、例えば理化学研究所バイオリソース研究センター（ＲＩＫＥＮ　ＢＲＣ）、Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）などから入手することができる。 The neural stem cells used in steps (4) to (6) may be any stem cells that can acquire the ability to differentiate into neural lineage cells by the differentiation induction procedure described later. Neural stem cells that can be used in the method of this embodiment may be derived from any vertebrate, but are preferably derived from mammals such as mice, rats, rabbits, sheep, pigs, cows, goats, monkeys, and humans. It is of animal origin, and particularly preferably of human origin. Neural stem cells that can be used in the method of the present embodiment may be prepared from fetal or adult neural tissue, or may be prepared from ES cells or iPS cells. Alternatively, the method of this embodiment may use an already established neural stem cell line. Such cell lines can be obtained, for example, from RIKEN BioResource Research Center (RIKEN BRC), American Type Culture Collection (ATCC), and the like.
　神経幹細胞の増殖能を評価するためには、神経幹細胞を、候補化合物を添加したまたは添加していない維持培地中で一定期間培養すればよい。添加される候補化合物の濃度は、化合物の種類により異なるが、例えば、１ｎＭ～１ｍＭの範囲で適宜選択することができる。培養期間は、例えば、８時間～７日であってよい。神経幹細胞のための維持培地は公知であり（Ｈｉｒａｎｏ　Ｋ　ａｎｄ　Ｎａｍｉｈｉｒａ　Ｍ、ＦＥＢＳ　Ｏｐｅｎ　ｂｉｏ．，　７（１２）：１９３２－１９４２，　２０１７）、例えば、ＤＭＥＭ／Ｆ１２などを基本培地として、Ｎ２サプリメント、Ｂ２７サプリメント、塩基性線維芽細胞増殖因子、上皮細胞増殖因子などを添加することにより調製することができる。また、神経幹細胞のための維持培地が市販されており、それら市販品を用いてもよい。 In order to evaluate the proliferative ability of neural stem cells, neural stem cells may be cultured for a certain period of time in a maintenance medium with or without the addition of candidate compounds. The concentration of the candidate compound to be added varies depending on the type of compound, but can be appropriately selected, for example, within the range of 1 nM to 1 mM. The culture period may be, for example, 8 hours to 7 days. A maintenance medium for neural stem cells is known (Hirano K and Namihira M, FEBS Open bio., 7(12): 1932-1942, 2017), for example, DMEM/F12 as a basal medium, N2 supplement, B27 It can be prepared by adding supplements, basic fibroblast growth factor, epidermal growth factor and the like. In addition, maintenance media for neural stem cells are commercially available, and these commercial products may be used.
　神経幹細胞の増殖能は、当分野において周知の方法により、倍加時間、継代数および／または比増殖速度を計測することにより評価することができる。 The proliferative ability of neural stem cells can be evaluated by measuring the doubling time, passage number and/or specific growth rate by methods well known in the art.
　神経幹細胞の分化能を評価するためには、神経幹細胞を、候補化合物を添加したまたは添加していない分化誘導培地中で一定期間培養すればよい。添加される候補化合物の濃度は、化合物の種類により異なるが、例えば、１ｎＭ～１ｍＭの範囲で適宜選択することができる。培養期間は、例えば、８時間～７日であってよい。 In order to evaluate the differentiation potential of neural stem cells, neural stem cells may be cultured for a certain period of time in a differentiation-inducing medium with or without the addition of candidate compounds. The concentration of the candidate compound to be added varies depending on the type of compound, but can be appropriately selected, for example, within the range of 1 nM to 1 mM. The culture period may be, for example, 8 hours to 7 days.
　神経幹細胞のための分化誘導培地は公知であり（Ｈｉｒａｎｏ　Ｋ　ａｎｄ　Ｎａｍｉｈｉｒａ　Ｍ、ＦＥＢＳ　Ｏｐｅｎ　ｂｉｏ．，　７（１２）：１９３２－１９４２，　２０１７）例えば、Ｎｅｕｒｏｂａｓａｌ培地などを基本培地として、Ｂ２７サプリメント、Ｌ－グルタミンなどを添加することにより調製することができる。また、神経幹細胞のための分化誘導培地が市販されており、それら市販品を用いてもよい。 Differentiation induction medium for neural stem cells is known (Hirano K and Namihira M, FEBS Open bio., 7(12): 1932-1942, 2017). It can be prepared by adding glutamine or the like. In addition, differentiation-inducing media for neural stem cells are commercially available, and these commercial products may be used.
　神経幹細胞の分化能は、当分野においてすでに確立された分化誘導条件下で神経幹細胞を培養し、公知の分化マーカーを検出することにより評価することができる。神経分化マーカーには、例えば、ダブルコルチン（ＤＣＸ）、βＩＩＩチューブリンなどが挙げられるが、これらに限定されない。また、神経分化マーカーの検出は、免疫細胞染色やフローサイトメトリーなどにより行うことができる。 The differentiation potential of neural stem cells can be evaluated by culturing neural stem cells under differentiation-inducing conditions already established in the field and detecting known differentiation markers. Neuronal differentiation markers include, but are not limited to, doublecortin (DCX), βIII tubulin, and the like. In addition, detection of neuronal differentiation markers can be performed by immune cell staining, flow cytometry, or the like.
　候補化合物により神経幹細胞の分化能が変化したかどうかを判定するためには、候補化合物の存在下または非存在下での培養を並行して実施して比較してもよいし、過去に実施した候補化合物の非存在下での培養についての解析結果と比較してもよい。候補化合物の存在下で培養された神経幹細胞が、候補化合物の非存在下で培養された神経幹細胞と比較して、有意に改善した増殖能および／または分化能を示した場合には、当該候補化合物は、特に神経組織に対する抗老化物質として有望であると評価することができる。一方、候補化合物の存在下で培養された神経幹細胞が、候補化合物の非存在下で培養された神経幹細胞と比較して、低下した増殖能および／または分化能を示した場合、または両者の増殖能および／または分化能に有意な差が見られなかった場合には、当該候補化合物は、神経組織に対する抗老化物質としては有望ではないと評価することができる。 In order to determine whether the candidate compound has changed the differentiation potential of neural stem cells, culture in the presence or absence of the candidate compound may be performed in parallel and compared. Analysis results for cultures in the absence of the candidate compound may be compared. When neural stem cells cultured in the presence of a candidate compound exhibit significantly improved proliferation and/or differentiation potential compared to neural stem cells cultured in the absence of the candidate compound, the candidate The compounds can be evaluated as promising antiaging agents, especially for neural tissue. On the other hand, if the neural stem cells cultured in the presence of the candidate compound show reduced proliferation and/or differentiation potential compared to the neural stem cells cultured in the absence of the candidate compound, or the proliferation of both If no significant difference in potency and/or differentiation potential is observed, the candidate compound can be evaluated as not promising as an anti-aging agent for neural tissue.
　ステップ（７）および（８）で用いられるショウジョウバエは、ショウジョウバエ属（Ｄｒｏｓｏｐｈｉｌａ）の任意のハエであってよいが、好ましくはキイロショウジョウバエ（Ｄｒｏｓｏｐｈｉｌａ　ｍｅｌａｎｏｇａｓｔｅｒ）である。 The fruit fly used in steps (7) and (8) may be any fly of the genus Drosophila, but is preferably Drosophila melanogaster.
　候補化合物をショウジョウバエに投与するには、種々の濃度の候補化合物を、餌に添加して摂取させればよい。餌に添加される候補化合物の濃度は、化合物の種類により異なるが、例えば、０．１ｍＭ～１００ｍＭの範囲で適宜選択することができる。候補化合物の投与は、例えば、１日～２ヶ月間にわたって行うことができる。 In order to administer a candidate compound to Drosophila, various concentrations of the candidate compound may be added to food and ingested. The concentration of the candidate compound added to the feed varies depending on the type of compound, but can be appropriately selected, for example, within the range of 0.1 mM to 100 mM. Administration of the candidate compound can be carried out, for example, over a period of 1 day to 2 months.
　ショウジョウバエの生殖組織の解析は、当分野において周知の方法により、例えば免疫組織染色などにより、生殖系列細胞の数を計測および／または生殖組織の形成を観察することにより行うことができる。 Drosophila reproductive tissue analysis can be performed by counting the number of germline cells and/or observing the formation of reproductive tissue by methods well known in the art, such as immunohistochemical staining.
　候補化合物によりショウジョウバエの生殖系列細胞の数または生殖組織の形成が変化したかどうかを判定するためには、候補化合物を投与しなかったショウジョウバエの生殖組織を並行して解析して比較してもよいし、過去に実施した候補化合物を投与しなかったショウジョウバエの生殖組織についての解析結果と比較してもよい。候補化合物の投与により、候補化合物を投与しなかったショウジョウバエの生殖組織と比較して、生殖系列細胞の数が増加および／または生殖組織の形成異常が減少した場合には、当該候補化合物は、特に生殖組織に対する抗老化物質として有望であると評価することができる。一方、候補化合物の投与により、候補化合物を投与しなかったショウジョウバエと比較して、生殖系列細胞の数に変化が見られないもしくは減少した場合、かつ／または生殖組織の形成異常に変化が見られないもしくは増加した場合には、当該候補化合物は、生殖組織に対する抗老化物質として有望ではないと評価することができる。 To determine whether the candidate compound alters the number of germline cells or the formation of reproductive tissue in Drosophila, reproductive tissue from Drosophila not administered with the candidate compound may be analyzed in parallel and compared. and may be compared with the results of past analyzes of reproductive tissues of Drosophila to which no candidate compound was administered. A candidate compound is particularly preferred if administration of the candidate compound results in an increase in the number of germline cells and/or a decrease in reproductive tissue dysplasia compared to the reproductive tissue of Drosophila to which the candidate compound has not been administered. It can be evaluated as a promising anti-aging substance for reproductive tissues. On the other hand, if administration of the candidate compound results in no change or a decrease in the number of germline cells and/or changes in germ tissue dysplasia compared to Drosophila that have not been administered the candidate compound. If it is absent or increased, the candidate compound can be evaluated as not promising as an anti-aging substance for reproductive tissue.
　本実施形態の方法によれば、神経組織や生殖組織を含む種々の組織の加齢に伴う機能低下を予防または治療し得る抗老化物質をスクリーニングすることが可能である。 According to the method of the present embodiment, it is possible to screen for anti-aging substances capable of preventing or treating age-related functional decline in various tissues, including nervous tissues and reproductive tissues.
　以下に実施例を挙げ、本発明についてさらに説明する。なお、これらは本発明を何ら限定するものではない。 The present invention will be further described with reference to examples below. In addition, these do not limit this invention at all.
＜１．ショウジョウバエ卵巣の老化とＳＡＭの生合成との相関＞
　発明者らはこれまで、ショウジョウバエの生殖細胞の老化について研究を行い、ショウジョウバエの卵巣において、老化に伴って生殖幹細胞の数が減少するとともに、異常卵室の形成が増加することを明らかにしている。Ｓａｍ－Ｓ遺伝子の発現に関して改変されていない対照ショウジョウバエ（ＺＨ－８６Ｆａ系統（＃２４４８６、Ｂｌｏｏｍｉｎｇｔｏｎ　Ｄｒｏｓｏｐｈｉｌａ　Ｓｔｏｃｋ　Ｃｅｎｔｅｒ）またはｐ（ＣａｒｙＰ）ａｔｔＰ２系統（＃３６３０３、Ｂｌｏｏｍｉｎｇｔｏｎ　Ｄｒｏｓｏｐｈｉｌａ　Ｓｔｏｃｋ　Ｃｅｎｔｅｒ；以下「対照ショウジョウバエ」または単に「対照」と記載する）の卵巣小管の免疫組織染色像が示すように、老齢（８週齢）個体では、若齢（２～３日齢）個体における正常な卵室（図１左）と異なる、１６個を超える生殖細胞シストを含む異常卵室が観察される（図１右）。そこで、このような異常卵室形成とＳＡＭ量との間に相関が見られるかどうかについて試験した。 <1. Correlation between Drosophila ovary aging and SAM biosynthesis>
The inventors have conducted research on the senescence of Drosophila germ cells, and have clarified that the number of germ stem cells decreases and the formation of abnormal egg chambers increases with aging in the Drosophila ovary. . Control Drosophila (ZH-86Fa strain (#24486, Bloomington Drosophila Stock Center) or p(CaryP)attP2 strain (#36303, Bloomington Drosophila Stock Center) not modified for expression of the Sam-S gene; hereinafter "control Drosophila" or simply As shown by the immunohistochemical staining image of the ovarian canaliculus of the "control"), the old (8-week-old) individual has a normal egg chamber (Fig. 1, left) and the young (2-3 days old) individual. Different abnormal egg chambers containing more than 16 germ cell cysts were observed (Fig. 1, right), so we tested whether there is a correlation between such abnormal egg chamber formation and the amount of SAM.
（１－１）ショウジョウバエ系統の作製および飼育
　ショウジョウバエは、通常の餌を用いて２５℃で飼育した。生殖系列特異的に発現するＧａｌ４系統であるｎａｎｏｓ－Ｇａｌ４系統と、ＵＡＳ－Ｓａｍ－Ｓ　ＲＮＡｉ系統（＃３６３０６、Ｂｌｏｏｍｉｎｇｔｏｎ　Ｄｒｏｓｏｐｈｉｌａ　Ｓｔｏｃｋ　Ｃｅｎｔｅｒ）を交配することにより、生殖系列特異的にＳＡＭ合成酵素（Ｓａｍ－Ｓ）遺伝子の発現をノックダウンした系統（以下、「Ｓａｍ－Ｓ（－）」と記載する）を得た。Ｓａｍ－Ｓ（－）ショウジョウバエ系統は、Ｓａｍ－Ｓ遺伝子中の配列：５’－ＡＴＧＧＡＧＡＡＡＧＴＴＧＴＴＡＡＡＧＴＡ－３’（配列番号７）を標的とするｓｈＲＮＡを生殖系列特異的に発現する。 (1-1) Preparation and Breeding of Drosophila Lines Drosophila were bred at 25° C. using normal feed. Germline-specific SAM synthase (Sam- S) A strain in which gene expression was knocked down (hereinafter referred to as "Sam-S(-)") was obtained. The Sam-S(-) Drosophila strain expresses germline-specifically an shRNA targeting the sequence: 5'-ATGGAGAAAGTTGTTAAAGTA-3' (SEQ ID NO: 7) in the Sam-S gene.
　一方、Ｓａｍ－Ｓ遺伝子をコードするＤＮＡ断片を挿入したｐＵＡＳｐ－Ｋ１０ａｔｔＢベクターを、ａｔｔＰサイトをもつショウジョウバエ系統（＃２４４８６系統、Ｂｌｏｏｍｉｎｇｔｏｎ　Ｄｒｏｓｏｐｈｉｌａ　Ｓｔｏｃｋ　Ｃｅｎｔｅｒ）に導入し、得られたショウジョウバエ系統をさらにｎａｎｏｓ－Ｇａｌ４系統と交配することにより、生殖系列特異的にＳａｍ－Ｓ遺伝子を過剰発現する系統（以下、「Ｓａｍ－Ｓ（＋＋）」と記載する）を得た。Ｓａｍ－Ｓ遺伝子をコードするＤＮＡ断片は、ＧＨ０８７３８（Ｄｒｏｓｏｐｈｉｌａ　Ｇｅｎｏｍｉｃｓ　Ｒｅｓｏｕｒｃｅ　Ｃｅｎｔｅｒ）を鋳型とし、以下のプライマーを用いたＰＣＲにより調製した。フォワードプライマー：５’－ＣＧＧＧＧＴＡＣＣＴＴＣＡＡＡＣＴＴＣＧＡＧＴＴＡＣＡＴＡＴＴＡＣ－３’（配列番号１）、リバースプライマー：５’－ＣＧＧＴＣＴＡＧＡＴＣＡＧＴＴＧＴＣＡＡＴＣＴＣＣＡＧＡＧＧＣＴＴＧ－３’（配列番号２）。 On the other hand, the pUASp-K10attB vector into which a DNA fragment encoding the Sam-S gene was inserted was introduced into a Drosophila strain having an attP site (#24486 strain, Bloomington Drosophila Stock Center), and the resulting Drosophila strain was further transfected with nanos-Gal4. By mating with the strain, a strain that overexpresses the Sam-S gene in a germline-specific manner (hereinafter referred to as “Sam-S(++)”) was obtained. A DNA fragment encoding the Sam-S gene was prepared by PCR using GH08738 (Drosophila Genomics Resource Center) as a template and the following primers. Forward primer: 5'-CGGGGTACCTTCAAAACTTCGAGTTACATATTAC-3' (SEQ ID NO: 1), reverse primer: 5'-CGGTCTAGATCAGTTGTCAATCTCCAGAGGCTTG-3' (SEQ ID NO: 2).
（１－２）ショウジョウバエ卵巣におけるＳＡＭ－Ｓの発現の定量
　羽化後２～３日齢および４週齢の雌の対照ショウジョウバエ（２０匹）より卵巣を摘出し、液体窒素にて凍結後、ＴＲＩｚｏｌ（商標）Ｒｅａｇｅｎｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）中でビーズ破砕機により破砕した。ＴＲＩｚｏｌ（商標）ＲｅａｇｅｎｔおよびＳｕｐｅｒＳｃｒｉｐｔ（商標）ＩＩＩ　Ｆｉｒｓｔ－Ｓｔｒａｎｄ　Ｓｙｎｔｈｅｓｉｓ　Ｓｙｓｔｅｍ　ｆｏｒ　ＲＴ－ＰＣＲ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用い、付属のプロトコールに従って、ＳＡＭ合成酵素（Ｓａｍ－Ｓ）についてｑＰＣＲを実施した。使用したプライマーは以下の通りである：５’－ＡＣＡＡＡＡＴＧＴＧＣＧＡＣＣＡＡＡＴＣＡＧＣ－３’（配列番号３）および５’－ＣＡＡＴＣＴＴＴＴＣＧＴＴＴＡＧＴＴＴＧＴＧＡＧＣ－３’（配列番号４）。また、内部標準（ｒｐ４９）の検出のために、以下のプライマーを用いた：５’－ＣＡＣＧＡＴＡＧＣＡＴＡＣＡＧＧＣＣＣＡＡＧＡＴＣＧＧ－３’（配列番号５）および５’－ＧＣＣＡＴＴＴＧＴＧＣＧＡＣＡＧＣＴＴＡＧ－３’（配列番号６）。 (1-2) Quantification of expression of SAM-S in Drosophila ovaries Ovaries were removed from female control Drosophila (20 mice) aged 2 to 3 days and 4 weeks after eclosion, frozen in liquid nitrogen, and treated with TRIzol ( TM Reagent (Thermo Fisher Scientific) with a bead crusher. qPCR was performed on SAM synthase (Sam-S) using TRIzol™ Reagent and SuperScript™ III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific) according to the attached protocol. The primers used were as follows: 5'-ACAAATGTGCGACCAAATCAGC-3' (SEQ ID NO: 3) and 5'-CAATCTTTTCGTTTAGTTTGTGAGC-3' (SEQ ID NO: 4). Also, for detection of the internal standard (rp49), the following primers were used: 5'-CACGATAGCATACAGGCCCAAGATCGG-3' (SEQ ID NO: 5) and 5'-GCCATTTGTGCGACAGCTTAG-3' (SEQ ID NO: 6).
　結果を図２に示す。対照ショウジョウバエでは、加齢に伴ってＳａｍ－Ｓの発現量が増加していることが確認された。 The results are shown in Figure 2. In the control Drosophila, it was confirmed that the expression level of Sam-S increased with aging.
（１－３）ショウジョウバエ卵巣におけるＳＡＭ量の計測
　羽化後２～３日齢および４週齢の雌（対照およびＳａｍ－Ｓ（－）、各２０匹）より卵巣を摘出し、液体窒素にて凍結後、５０％メタノール溶液中でビーズ破砕機により破砕した。５０％アセトニトリル溶液によりタンパク質を除去して得られた溶液を遠心濃縮機（ＴＯＭＹ精工）により濃縮し、１０ｍＭ　ＨＣｌ溶液により溶出した。溶出液を０．２２μｍＰＶＤＦフィルターにより濾過し、濾液と等量の１００μＭジチオスレイトール（ＤＴＴ）／５０ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ８．８）と混合し、分析試料とした。分析試料をＵＰＬＣ－ＭＳ／ＭＳ分析計（Ｗａｔｅｒｓ）に供し、ＳＡＭを検出した。上記過程により除去されたタンパク質をＢＣＡ法により定量し、検出されたＳＡＭ量をタンパク質量で補正した。 (1-3) Measurement of SAM content in Drosophila ovaries Ovaries were removed from females (control and Sam-S(-), 20 each) at 2-3 days and 4 weeks after eclosion, and frozen in liquid nitrogen. After that, it was crushed with a bead crusher in a 50% methanol solution. The solution obtained by removing proteins with a 50% acetonitrile solution was concentrated with a centrifugal concentrator (TOMY Seiko) and eluted with a 10 mM HCl solution. The eluate was filtered through a 0.22 μm PVDF filter, and the filtrate was mixed with an equal amount of 100 μM dithiothreitol (DTT)/50 mM Tris-HCl (pH 8.8) to prepare an analytical sample. An analytical sample was subjected to a UPLC-MS/MS analyzer (Waters) to detect SAM. The protein removed by the above process was quantified by the BCA method, and the amount of SAM detected was corrected with the amount of protein.
　結果を図３に示す。対照ショウジョウバエの卵巣では、加齢に伴ってＳＡＭ量が増加しており（対照、図３左）、この結果は、上記（１－１）の結果と整合するものであった。これに対し、Ｓａｍ－Ｓ（－）ショウジョウバエの卵巣では、加齢に伴うＳＡＭ量の有意な増加はみられなかった（図３右）。これらの結果から、Ｓａｍ－Ｓの発現量を低下させることにより、加齢に伴うＳＡＭ量の増加を緩和できることが明らかになった。 The results are shown in Figure 3. In the control Drosophila ovary, the amount of SAM increased with aging (control, left of FIG. 3), and this result was consistent with the above (1-1). In contrast, in Sam-S(-) Drosophila ovaries, no significant increase in SAM levels was observed with aging (Fig. 3, right). From these results, it was clarified that an increase in the amount of SAM associated with aging can be mitigated by reducing the expression level of Sam-S.
（１－４）ショウジョウバエ卵巣の免疫組織染色
　羽化後２～３日齢、２週齢、４週齢および８週齢の雌（対照、Ｓａｍ－Ｓ（＋＋）およびＳａｍ－Ｓ（－））より卵巣を摘出し、４％パラホルムアルデヒド／ＰＢＳにて１５分間固定した後、通常の蛍光免疫化学染色を行った。一次抗体には、ｃｈｉｃｋ　ａｎｔｉ－ＶＡＳＡ抗体（１：２０００）およびｍｏｕｓｅ　ａｎｔｉ－Ｈｔｓ抗体（１：５）（いずれもＤｅｖｅｌｏｐｍｅｎｔａｌ　Ｓｔｕｄｉｅｓ　Ｈｙｂｒｉｄｏｍａ　Ｂａｎｋ（ＤＳＨＢ））、二次抗体には、ｇｏａｔ　ａｎｔｉ－Ｃｈｉｃｋ　ＩｇＹ　Ａｌｅｘａ　Ｆｌｕｏｒ　４８８抗体およびｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ　Ａｌｅｘａ　Ｆｌｕｏｒ　５４６抗体（いずれもＴｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いた。形成細胞層前端のＣａｐ細胞に接するＶＡＳＡ陽性の球形細胞のうち、スペクトロゾーム（Ｈｔｓにより染色される）を有するものを、生殖幹細胞として観察した。また、形成細胞層の領域３以降に存在する卵室（通常は１６細胞からなる生殖細胞シストを有する）において、１６細胞以上の生殖細胞シストを含む卵室を異常卵室として観察した。 (1-4) Immunohistochemical staining of Drosophila ovaries From females (control, Sam-S (++) and Sam-S (-)) aged 2 to 3 days, 2 weeks, 4 weeks and 8 weeks after eclosion Ovaries were removed and fixed with 4% paraformaldehyde/PBS for 15 minutes, followed by routine fluorescent immunochemical staining. Primary antibody: chick anti-VASA antibody (1:2000) and mouse anti-Hts antibody (1:5) (both from Development Studies Hybridoma Bank (DSHB)), secondary antibody: goat anti-Chick IgY Alexa Fluor 488 antibody and goat anti-mouse IgG Alexa Fluor 546 antibody (both from Thermo Fisher Scientific) were used. Among VASA-positive spherical cells in contact with Cap cells at the anterior end of the cambium layer, those having spectrosomes (stained with Hts) were observed as germinal stem cells. In addition, among the egg chambers (usually having germ cell cysts consisting of 16 cells) present in region 3 and beyond of the cambium layer, egg chambers containing germ cell cysts of 16 cells or more were observed as abnormal egg chambers.
　結果を図４～６に示す。図５および６中、バーの黒い領域が正常な卵室を含む卵巣小管を、白い領域が異常卵室を含む卵巣小管の出現頻度を示す。Ｓａｍ－Ｓ（＋＋）では、加齢に伴う生殖幹細胞の減少が促進されたのに対し、Ｓａｍ－Ｓ（－）では、加齢に伴う生殖幹細胞の減少が緩和された（図４）。また、Ｓａｍ－Ｓ（＋＋）では、加齢に伴う異常卵室の増加が促進されたのに対し（図５）、Ｓａｍ－Ｓ（－）では、加齢に伴う異常卵室の増加が大幅に緩和された（図６）。これらの結果から、加齢に伴いＳＡＭ合成酵素の発現が上昇し、ＳＡＭ量が増加することが、卵巣の老化の主要因であることが示された。さらに、Ｓａｍ－Ｓ（－）の結果から、ＳＡＭ合成酵素を阻害することにより、卵巣の老化を緩和することが可能であることが示された。 The results are shown in Figures 4-6. In FIGS. 5 and 6, the black area of the bar indicates the appearance frequency of ovarian canaliculi containing normal egg chambers, and the white area indicates the appearance frequency of ovarian canaliculi containing abnormal egg chambers. Sam-S(++) accelerated the age-related decrease in germ stem cells, whereas Sam-S(−) moderated the age-related decrease in germ stem cells (FIG. 4). In Sam-S(++), the number of abnormal egg chambers increased with age (Fig. 5). (Fig. 6). These results indicated that the increase in the expression of SAM synthase and the increase in the amount of SAM with aging are the main factors of ovarian aging. Furthermore, the results of Sam-S(-) indicated that ovarian aging could be alleviated by inhibiting SAM synthase.
＜２．マウス生殖組織および脳組織におけるＳＡＭ量の計測＞
　哺乳動物の組織の老化とＳＡＭ量との関連を調べるために、マウス生殖組織および脳組織におけるＳＡＭ量を以下の手順により定量した。若齢（生後２ヶ月齢）と老齢（生後１年６ヶ月齢）のＣ５７ＢＬ／６マウス（各３匹）を頸椎脱臼により安楽死させ、小脳、大脳、海馬、精巣および卵巣を採取し、ドライアイス上で急速凍結後、－８０℃で保存した。凍結された組織をビーズ破砕機により破砕した後、１０ｍＭの酢酸が入った５０％メタノール溶液により代謝物質の抽出およびタンパク質の除去を行った。代謝物質分画を遠心濃縮機（ＴＯＭＹ精工）により濃縮し、超純水により溶出した。溶出液を０．２２μｍＰＶＤＦフィルターにより濾過し、濾液と等量の超純水と混合し、分析試料とした。分析試料をＵＰＬＣ－ＭＳ／ＭＳ分析計（Ｗａｔｅｒｓ）に供し、代謝物質を計測した。上記過程により除去されたタンパク質をＢＣＡ法により定量し、検出されたＳＡＭ量をタンパク質量で補正した。分散分析およびＴ検定を用いて統計処理を行った。 <2. Measurement of SAM amount in mouse reproductive tissue and brain tissue>
In order to examine the relationship between mammalian tissue aging and SAM levels, SAM levels in mouse reproductive tissues and brain tissues were quantified by the following procedure. Young (2 months old) and old (1 year and 6 months old) C57BL/6 mice (3 mice each) were euthanized by cervical dislocation, and the cerebellum, cerebrum, hippocampus, testes and ovaries were collected and dried. After quick freezing on ice, it was stored at -80°C. After crushing the frozen tissue with a bead crusher, metabolites were extracted and proteins were removed with a 50% methanol solution containing 10 mM acetic acid. The metabolite fraction was concentrated with a centrifugal concentrator (TOMY Seiko) and eluted with ultrapure water. The eluate was filtered through a 0.22 μm PVDF filter, and the filtrate was mixed with an equal amount of ultrapure water to obtain an analytical sample. An analytical sample was subjected to a UPLC-MS/MS analyzer (Waters) to measure metabolites. The protein removed by the above process was quantified by the BCA method, and the amount of SAM detected was corrected with the amount of protein. Statistics were performed using analysis of variance and T-test.
　結果を図７に示す（＊Ｐ＜０．０５，＊＊Ｐ＜０．０１，ＮＳ：有意差なし）。エラーバーは標準偏差を示す。ショウジョウバエと同様、加齢に伴って生殖組織におけるＳＡＭ量が増加する傾向がみられた。脳組織においても、加齢に伴ってＳＡＭ量が増加する傾向にある。これらの結果から、哺乳動物においてもＳＡＭ量の増加が組織の老化に関連する可能性が示唆された。 The results are shown in FIG. 7 (*P<0.05, **P<0.01, NS: no significant difference). Error bars indicate standard deviation. Similar to Drosophila, the amount of SAM in reproductive tissues tended to increase with aging. Also in brain tissue, the amount of SAM tends to increase with aging. These results suggested the possibility that an increase in the amount of SAM is related to tissue aging in mammals as well.
＜３．ヒト神経幹細胞の増殖能および分化能に対するＳＡＭの影響＞
　ヒトの神経組織の老化とＳＡＭとの関連を調べるために、ＳＡＭ合成酵素阻害剤の存在下および非存在下におけるヒト神経幹細胞の増殖能および分化能を比較した。ヒト神経幹細胞（胎生１４週のヒト胎児大脳皮質由来、ＰｈｏｅｎｉｘＳｏｎｇｓ　Ｂｉｏｌｏｇｉｃａｌｓから購入）を、ポリ－Ｌ－オルニチン／ラミニンコーティングディッシュに接着させ、神経幹細胞維持用培地（１×Ｎ２サプリメントおよび０．１％Ｂ２７サプリメントを含むＤＭＥＭ／Ｆ１２に、１０ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子（ｈｂＦＧＦ）および２０ｎｇ／ｍｌヒト上皮成長因子（ｈＥＧＦ）を加えた培地）中で維持培養した。ラミニンコートした１２ウェルプレートに２×１０５細胞／ウェルのヒト神経幹細胞を播種し、上記培地中で２日間、８０～９０％コンフルエントになるまで培養した後、Ｎｅｕｒｏｂａｓａｌ培地（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）に２％　Ｂ２７サプリメントおよび０．５ｍＭ　Ｌ－グルタミンを加えた分化誘導培地に置換することにより分化誘導を行った。ここで、対照群については０．１％ＤＭＳＯを、ＳＡＭ合成酵素阻害群についてはＤＭＳＯに溶解させたＭＡＴ２Ａ　ｉｎｈｉｂｉｔｏｒ　１（ＭｅｄＣｈｅｍＥｘｐｒｅｓｓ）を各濃度で培地に添加した。分化誘導開始から７日後、４％パラホルムアルデヒド／ＰＢＳで細胞を固定し、通常の手順により蛍光免疫染色を行った。一次抗体には、Ｍｏｕｓｅ　Ａｎｔｉ－Ｋｉ－６７（日本ベクトン・ディッキンソン、５５０６０９）（１：５００希釈）およびＲａｂｂｉｔ　Ａｎｔｉ－Ｄｏｕｂｌｅｃｏｒｔｉｎ抗体（アブカム、Ａｂ１８７２３）（１：５００希釈）を用いた。二次抗体には、ＣＦ４８８　ｄｏｎｋｅｙ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ（ＨＣＬ），　ｈｉｇｈｌｙ　ｃｒｏｓｓ－ａｄｓｏｒｂｅｄ（Ｂｉｏｔｉｕｍ、２００１４）（１：５００希釈）およびＣＦ５６８　ｄｏｎｋｅｙ　ａｎｔｉ－ｒａｔ　ＩｇＧ（ＨＣＬ），　ｈｉｇｈｌｙ　ｃｒｏｓｓ－ａｄｓｏｒｂｅｄ（Ｂｉｏｔｉｕｍ、２００９２）（１：５００希釈）を用いた。さらに、二次抗体とともにＨｏｅｃｈｓｔ３３３４２（１／２０００希釈）を添加し、細胞核も同時に染色した。Ｈｏｅｃｈｓｔで染色された全細胞数に対するＫｉ－６７陽性細胞またはダブルコルチン（ＤＣＸ）陽性細胞の割合を算出した。分散分析およびＴ検定を用いて統計処理を行った。 <3. Effect of SAM on Proliferative and Differentiative Potential of Human Neural Stem Cells>
In order to investigate the relationship between aging of human neural tissue and SAM, we compared the proliferative and differentiation abilities of human neural stem cells in the presence and absence of SAM synthase inhibitors. Human neural stem cells (derived from embryonic 14-week human fetal cerebral cortex, purchased from PhoenixSongs Biologicals) were allowed to adhere to poly-L-ornithine/laminin-coated dishes and supplemented with neural stem cell maintenance medium (1×N2 supplement and 0.1% B27 The cells were maintained and cultured in DMEM/F12 containing supplements (medium supplemented with 10 ng/ml human basic fibroblast growth factor (hbFGF) and 20 ng/ml human epidermal growth factor (hEGF)). Human neural stem cells were seeded at 2×10 5 cells/well in a laminin-coated 12-well plate, cultured in the above medium for 2 days until 80-90% confluent, and transferred to Neurobasal medium (Thermo Fisher Scientific) for 2 days. Differentiation was induced by substituting a differentiation-inducing medium supplemented with % B27 supplement and 0.5 mM L-glutamine. Here, 0.1% DMSO was added to the control group, and MAT2A inhibitor 1 (MedChemExpress) dissolved in DMSO was added to the medium at each concentration for the SAM synthase inhibition group. Seven days after the start of differentiation induction, the cells were fixed with 4% paraformaldehyde/PBS, and fluorescence immunostaining was performed according to a normal procedure. Mouse Anti-Ki-67 (Nippon Becton Dickinson, 550609) (1:500 dilution) and Rabbit Anti-Doublecortin antibody (Ab18723, Abcam) (1:500 dilution) were used as primary antibodies. Secondary antibodies included CF488 donkey anti-mouse IgG (HCL), highly cross-adsorbed (Biotium, 20014) (1:500 dilution) and CF568 donkey anti-rat IgG (HCL), highly cross-adsorbed (B2002, B2009). ) (1:500 dilution) was used. Furthermore, Hoechst33342 (1/2000 dilution) was added together with the secondary antibody, and cell nuclei were also stained at the same time. The ratio of Ki-67-positive cells or doublecortin (DCX)-positive cells to the total number of Hoechst-stained cells was calculated. Statistics were performed using analysis of variance and T-test.
　Ｋｉ－６７（細胞増殖マーカー）陽性細胞の割合を図８に、ＤＣＸ（神経分化マーカー）陽性細胞の割合を図９に示す（ｎ＝４，＊Ｐ＜０．０５）。ＳＡＭ合成酵素を阻害されたヒト神経幹細胞は、分化能を維持したまま（図８）増殖能の上昇を獲得した（図７）。 The percentage of Ki-67 (cell proliferation marker)-positive cells is shown in FIG. 8, and the percentage of DCX (neural differentiation marker)-positive cells is shown in FIG. 9 (n=4, *P<0.05). SAM synthase-inhibited human neural stem cells acquired increased proliferative potential (FIG. 7) while maintaining differentiation potential (FIG. 8).
＜４．ヒト皮膚線維芽細胞の増殖能に対するＳＡＭの影響＞
　ヒト健常者由来の皮膚線維芽細胞（ＳＦ８４０５、ＲＩＫＥＮ　ＢＲＣより入手）を、最終濃度１５％ウシ胎児血清（ＦＢＳ）を含むＭＥＭ　ＡＬＰＨＡ中で維持培養した。２４ウェルプレートに１×１０４細胞／ウェルのヒト線維芽細胞を播種し、翌日に、各濃度のＭＡＴ２Ａ　ｉｎｈｉｂｉｔｏｒ　１（対照には０．１％ＤＭＳＯ）を添加した。２日間の培養後、４％パラホルムアルデヒド／ＰＢＳで細胞を固定し、通常の手順により蛍光免疫染色を行った。一次抗体には、Ｍｏｕｓｅ　Ａｎｔｉ－Ｋｉ－６７（日本ベクトン・ディッキンソン、５５０６０９）（１：５００希釈）を用いた。二次抗体には、ＣＦ４８８　ｄｏｎｋｅｙ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ（ＨＣＬ），　ｈｉｇｈｌｙ　ｃｒｏｓｓ－ａｄｓｏｒｂｅｄ（Ｂｉｏｔｉｕｍ、２００１４）（１：５００希釈）を用いた。さらに、二次抗体とともにＨｏｅｃｈｓｔ３３３４２（１／２０００希釈）を添加し、細胞核も同時に染色した。Ｈｏｅｃｈｓｔで染色された全細胞数に対するＫｉ－６７陽性細胞の割合を算出した。分散分析およびＴ検定を用いて統計処理を行った。 <4. Effect of SAM on proliferation ability of human skin fibroblasts>
Skin fibroblasts derived from healthy human subjects (SF8405, obtained from RIKEN BRC) were maintained and cultured in MEM ALPHA containing fetal bovine serum (FBS) at a final concentration of 15%. 1×10 4 cells/well of human fibroblasts were seeded in a 24-well plate, and the next day, each concentration of MAT2A inhibitor 1 (0.1% DMSO for control) was added. After culturing for 2 days, the cells were fixed with 4% paraformaldehyde/PBS, and fluorescence immunostaining was performed according to a normal procedure. Mouse Anti-Ki-67 (Nippon Becton Dickinson, 550609) (1:500 dilution) was used as the primary antibody. CF488 donkey anti-mouse IgG (HCL), highly cross-adsorbed (Biotium, 20014) (1:500 dilution) was used as the secondary antibody. Furthermore, Hoechst33342 (1/2000 dilution) was added together with the secondary antibody, and cell nuclei were also stained at the same time. The ratio of Ki-67 positive cells to the total number of Hoechst-stained cells was calculated. Statistics were performed using analysis of variance and T-test.
　結果を図１０に示す（ｎ＝３，＊Ｐ＜０．０５）。エラーバーは標準偏差を示す。ＳＡＭの生合成を阻害することにより、ヒト皮膚線維芽細胞の増殖能が顕著に上昇した。以上の結果から、ヒト皮膚線維芽細胞の増殖能およびＳＡＭの生合成を指標として、神経組織および生殖組織を含む種々の組織の老化を評価できる可能性が示された。また、ＳＡＭの生合成を阻害する物質が、皮膚組織、神経組織および生殖組織を含む種々の組織の老化を予防または治療し得ることが示唆された。 The results are shown in FIG. 10 (n=3, *P<0.05). Error bars indicate standard deviation. Inhibition of SAM biosynthesis markedly increased the proliferative capacity of human skin fibroblasts. The above results indicated the possibility of evaluating the aging of various tissues, including neural tissue and reproductive tissue, using the proliferative ability of human skin fibroblasts and the biosynthesis of SAM as indices. It has also been suggested that substances that inhibit SAM biosynthesis can prevent or treat aging of various tissues, including skin tissue, nerve tissue and reproductive tissue.
＜５．ヒト皮膚線維芽細胞の増殖能に対するメトトレキサートの影響＞
　メトトレキサート（ＭＴＸ）は周知の葉酸代謝拮抗薬であり、ジヒドロ葉酸レダクターゼ（ＤＨＦＲ）を阻害することによりテトラヒドロ葉酸（ＴＨＦ）の産生を減少させ、その結果、その下流のＳＡＭの産生を減少させる。また、ＭＴＸは、ＭＡＴの発現および活性を直接的にも阻害することが知られている。そこで、本実施例では、ＭＴＸがヒト皮膚線維芽細胞の増殖能に影響するかどうかを試験した。 <5. Effect of methotrexate on the proliferative ability of human skin fibroblasts>
Methotrexate (MTX) is a well-known antifolate drug that inhibits dihydrofolate reductase (DHFR), thereby decreasing the production of tetrahydrofolate (THF) and, consequently, the downstream production of SAM. MTX is also known to directly inhibit MAT expression and activity. Therefore, in this example, it was tested whether MTX affects the proliferative ability of human dermal fibroblasts.
　上記４と同様の手順により、ヒト皮膚線維芽細胞を準備し、各濃度のメトトレキサート（富士フイルム和光純薬）を添加した（対照には無添加）。２日間の培養後、上記４と同様の手順により、細胞増殖マーカーＫｉ－６７の蛍光免疫染色およびＨｏｅｃｈｓｔ染色を行い、全細胞数に対するＫｉ－６７陽性細胞の割合を算出した。分散分析およびＴ検定を用いて統計処理を行った。 Human skin fibroblasts were prepared by the same procedure as in 4 above, and each concentration of methotrexate (Fujifilm Wako Pure Chemical Industries) was added (no addition to the control). After culturing for 2 days, fluorescence immunostaining for the cell proliferation marker Ki-67 and Hoechst staining were performed in the same manner as in 4 above, and the ratio of Ki-67 positive cells to the total number of cells was calculated. Statistics were performed using analysis of variance and T-test.
　結果を図１１に示す（ｎ＝４，＊Ｐ＜０．０５）。エラーバーは標準偏差を示す。ＭＴＸの添加量依存的にヒト皮膚線維芽細胞の増殖能が上昇したことが確認された。この結果から、ＭＴＸはＳＡＭを低減させることにより皮膚組織の老化を抑制し得ることが示唆された。 The results are shown in FIG. 11 (n=4, *P<0.05). Error bars indicate standard deviation. It was confirmed that the proliferative ability of human skin fibroblasts increased in a manner dependent on the amount of MTX added. This result suggested that MTX could suppress skin tissue aging by reducing SAM.

Claims (10)

  1. 　Ｓ－アデノシルメチオニン合成酵素阻害剤を含んでなる、組織の老化を予防または治療するための組成物。 A composition for preventing or treating tissue aging, comprising an S-adenosylmethionine synthase inhibitor.
  2. 　前記組織が、皮膚組織、生殖組織および神経組織からなる群から選択される、請求項１に記載の組成物。 The composition according to claim 1, wherein said tissue is selected from the group consisting of skin tissue, reproductive tissue and nerve tissue.
  3. 　前記Ｓ－アデノシルメチオニン合成酵素がメチオニンアデノシルトランスフェラーゼ２である、請求項１または２に記載の組成物。 The composition according to claim 1 or 2, wherein the S-adenosylmethionine synthase is methionine adenosyltransferase-2.
  4. 　前記Ｓ－アデノシルメチオニン合成酵素阻害剤が、６－（２－メチルベンゾ［ｄ］チアゾール－６－イル）－２，３－ジフェニル－５－（ピリジン－２－イルアミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オン、（Ｅ）－４－（２－クロロ－６－フルオロスチリル）－Ｎ－メチルアニリン、および３－（サイクロヘクス－１－エン－１－イル）－６－（４－メトキシフェニル）－２－フェニル－５－（ピリジン－２－イラミノ）ピラゾロ［１，５－ａ］ピリミジン－７（４Ｈ）－オンからなる群から選択される、請求項１～３のいずれか１項に記載の組成物。 The S-adenosylmethionine synthase inhibitor is 6-(2-methylbenzo[d]thiazol-6-yl)-2,3-diphenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a ]pyrimidin-7(4H)-one, (E)-4-(2-chloro-6-fluorostyryl)-N-methylaniline, and 3-(cyclohex-1-en-1-yl)-6- (4-Methoxyphenyl)-2-phenyl-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-ones. A composition according to any one of the preceding claims.
  5. 　前記Ｓ－アデノシルメチオニン合成酵素阻害剤が、メチオニンアデノシルトランスフェラーゼ２遺伝子の発現を阻害するＲＮＡまたはそれをコードする核酸を含む発現ベクターである、請求項１～３のいずれか１項に記載の組成物。 The S-adenosylmethionine synthase inhibitor is an expression vector comprising an RNA that inhibits expression of the methionine adenosyltransferase 2 gene or a nucleic acid encoding the same, according to any one of claims 1 to 3. Composition.
  6. 　前記組成物が医薬品である、請求項１～５のいずれか１項に記載の組成物。 The composition according to any one of claims 1 to 5, wherein the composition is a pharmaceutical.
  7. 　前記組成物が飲食品である、請求項１～５のいずれか１項に記載の組成物。 The composition according to any one of claims 1 to 5, wherein the composition is a food or drink.
  8. 　（１）候補化合物の存在下で線維芽細胞を培養するステップと、
    　（２）前記線維芽細胞の増殖能を評価するステップと、
    　（３）前記線維芽細胞におけるＳ－アデノシルメチオニンを定量するステップと
    を含む、抗老化物質のスクリーニング方法。     (1) culturing fibroblasts in the presence of a candidate compound;
    (2) assessing the proliferative capacity of said fibroblasts;
    (3) A method of screening for an anti-aging substance, comprising the step of quantifying S-adenosylmethionine in said fibroblasts.
  9. 　（４）前記候補化合物の存在下で神経幹細胞を培養するステップと、
    　（５）前記神経幹細胞の増殖能および／または分化能を評価するステップと、
    　（６）前記神経幹細胞におけるＳ－アデノシルメチオニンを定量するステップと
    をさらに含む、請求項８に記載の方法。     (4) culturing neural stem cells in the presence of said candidate compound;
    (5) assessing the proliferation and/or differentiation potential of said neural stem cells;
    (6) quantifying S-adenosylmethionine in said neural stem cells.
  10. 　（７）ショウジョウバエに前記候補化合物を投与するステップと、
    　（８）前記ショウジョウバエの生殖組織を解析するステップと
    をさらに含む、請求項８または９に記載の方法。
          (7) administering the candidate compound to Drosophila;
    (8) analyzing the reproductive tissue of the Drosophila.
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JP2014512337A (en) * 2011-01-28 2014-05-22 ユニバーシティ オブ ケンタッキー リサーチ ファウンデーション Stilbene analogs and methods of treating cancer
JP2019529529A (en) * 2016-08-31 2019-10-17 アジオス ファーマシューティカルズ， インコーポレイテッド Inhibitors of cellular metabolic processes

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JP2019529529A (en) * 2016-08-31 2019-10-17 アジオス ファーマシューティカルズ， インコーポレイテッド Inhibitors of cellular metabolic processes

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