WO2006077954A1 - Remedy for neurological disease - Google Patents

Remedy for neurological disease Download PDF

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Publication number
WO2006077954A1
WO2006077954A1 PCT/JP2006/300821 JP2006300821W WO2006077954A1 WO 2006077954 A1 WO2006077954 A1 WO 2006077954A1 JP 2006300821 W JP2006300821 W JP 2006300821W WO 2006077954 A1 WO2006077954 A1 WO 2006077954A1
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Prior art keywords
neural stem
substance
promotes
stem cell
acceptable salt
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PCT/JP2006/300821
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French (fr)
Japanese (ja)
Inventor
Hideyuki Onodera
Michio Ichimura
Kazuhiro Sakurada
Ayako Kawabata
Toshio Ota
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Kyowa Hakko Kogyo Co., Ltd.
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Publication of WO2006077954A1 publication Critical patent/WO2006077954A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • 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
    • A61P25/22—Anxiolytics
    • 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
    • A61P25/24—Antidepressants
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a therapeutic agent for neurological diseases, an agent for promoting neurogenesis or regeneration, a food and drink for preventing or enhancing nerve function and an additive for food and drink, a neural stem cell proliferation promoter, a neural stem cell, a euron and a glial cell.
  • the present invention relates to a method for screening for euron and glial cells, and a method for screening a proliferation promoting agent for neural stem cells.
  • Neurological diseases collectively refer to neurodegenerative diseases and depression, manic depression, schizophrenia, etc., in which brain and peripheral neurons are damaged by genetic factors, environmental factors, aging factors, etc. It is.
  • Specific examples of neurodegenerative diseases include Parkinson's disease, Arno-i-maima disease, triplet repeat disease, amyotrophic lateral sclerosis, multiple-euro bee, spinal cord injury, and cerebrovascular disorder.
  • a common treatment for these neurodegenerative diseases is to replace neurotransmitters that have been lost due to neuronal injury, but the treatment improves symptoms such as Parkinson's disease, Arno-Ima's disease, etc. It is limited to. Neurotransmitter replacement therapy cannot stop the progression of neuronal cell death.
  • neural stem cells obtained from fetal brain and ES cells obtained from human fertilized eggs in vitro are also being investigated.
  • -Techniques for accurately sorting into eurons have not been established, and there are problems due to the use of embryonic neural stem cells and human ES cells, and clinical applications are not progressing.
  • Non-Patent Document 1 neural stem cells were isolated from the adult brain (see Non-Patent Document 1), and it was reported that the human adult brain also undergoes a life-long development of Euron (see Non-Patent Document 2). Also depression It has been suggested that nerve regeneration by proliferation of neural stem cells is also involved in recovery from diseases (see Non-Patent Document 3) and schizophrenia (see Non-Patent Document 4).
  • cytoforce-in such as insulin-like growth factor 1 (see Non-Patent Document 5), fibroblast growth factor 2 (see Non-Patent Document 6), erythropoietin (see Non-Patent Document 7), It has been reported that in the model of diseases such as forebrain ischemia (see Non-patent Document 8) and epilepsy stimulation (see Non-Patent Document 9), the development of -euron in the lateral subcerebral zone, hippocampus and olfactory bulb is promoted. Has been.
  • Non-Patent Document 10 it has been reported that administration of tumor growth factor ⁇ in the brain causes the formation of dopaminergic-euron in the striatum and improves the symptoms of Parkinson's disease.
  • 40% of CA1 pyramidal cells lost due to ischemic injury were administered by fibroblast growth factor 2 and epidermal growth factor into the brain from day 2 to day 5 after ischemia. It has also been reported to recover (see Non-Patent Document 11).
  • Mammalian 'target'of'rapamycin is a signal molecule essential for protein translation initiation, and its phosphate activity is known to be inhibited by ravamycin. It is known that branched chain amino acids, particularly leucine, promote protein synthesis by strongly activating mTOR signals in the liver (see Non-Patent Document 12). It has been reported that the proliferation of embryonic cortex-derived neural stem cells is promoted by high expression of Akt-1, and the involvement of mTOR signal has been estimated as the mechanism (see Non-Patent Document 13). ). However, in mammals, embryonic neural stem cells and adult-derived neural stem cells are said to be essentially different in nature.
  • telomerase is required for growth of adult-derived neural stem cells, whereas embryonic neural stem cells can proliferate independently of telomerase. It is very likely that the structure is completely different (see Non-Patent Document 14). Therefore, the characteristics of embryonic stem cells cannot be extrapolated to adult-derived neural stem cells.
  • Vinigrol and its derivatives have antiplatelet aggregation action, blood pressure lowering action (see Patent Document 1), tumor necrosis factor antagonism (see Patent Document 2), granulocyte macrophage colony stimulating factor-like action, immunostimulatory action and It is known to have an antitumor action (see Patent Document 3) and to be useful as a therapeutic agent for human immunodeficiency virus infection (see Patent Document 4).
  • an anti-inflammatory drug and an immunosuppressive drug-containing drug that suppresses the production of cytoforce-in containing biniglol are effective for autoimmune diseases (see Patent Document 5), a tumor necrosis factor antagonist containing vinylogol, and It has been reported that a drug containing a selective COX-II inhibitor is useful for inflammatory diseases (see Patent Document 6).
  • Patent Document 1 Japanese Patent Laid-Open No. 63-215650
  • Patent Document 2 Japanese Patent Publication No. 5-504130
  • Patent Document 3 Japanese Patent Laid-Open No. 2003-267930
  • Patent Document 4 Japanese Patent Laid-Open No. 7-206668
  • Patent Literature 5 Japanese Patent Publication No. 11-515020
  • Patent Document 6 International Publication No. 01Z00229 Pamphlet
  • Non-Patent Document 1 “Science”, 1992, 255 ⁇ , p. 1707-1710
  • Non-Patent Document 2 “Nature Medicine”, 1998, 4 ⁇ , p. 13
  • Non-Patent Document 3 “Science”, 2003, 301 ⁇ , p. 805-809
  • Non-Patent Document 4 “Cellular and“ Molekiyura ”Life Sciences (Cellular and
  • Non-Patent Document 5 “The Journal of Neuroscience”, 2000, 20 ⁇ , p. 2896—2903
  • Non-Patent Document 6 “Proceedings of the National Academv of science of the United States” (Proceedings of the National Academv of science of the United States) of America ", 2001, 98 ⁇ , p. 5874-5879
  • Non-Patent Document 7 “The Journal of Neuroscience 2001, 21 ⁇ , p. 9733— 9743
  • Non-Patent Document 8 “The Journal of Neuroscience 1998, 18 ⁇ , p. 7768-7778
  • Non-Patent Document 9 “The Journal of Neuroscience 2002, 22 ⁇ , p. 3174— 3188
  • Non-Patent Document 10 “Proceedings of the National Academy of Science of the United States” (Proceedings of the National Academy of Science of the United States) of America), 2000 cattle, 97 ⁇ , p. 14686-14691
  • Non-Patent Document 11 “Cell”, 2002, 110 ⁇ , p. 429 -441
  • Non-Patent Document 12 “Biochemical and Biophysical Research Communications”, 2004, 313 ⁇ , p. 387- 389
  • Non-Patent Document 13 “The Journal of Neuroscience 2004, 24 ⁇ , p. 8531— 8541
  • Non-Patent Document 14 “Development”, 2004, 131 ⁇ , p. 4059 -4070
  • An object of the present invention is to treat a neurological disease, an agent for neurogenesis or regeneration, a food and drink for preventing or promoting nerve function and an additive for food and drink, a neural stem cell proliferation promoter, a neural stem cell, a neuron, or a glial cell. And a screening method for a substance that promotes proliferation of neurons or glial cells or neural stem cells produced by the production method.
  • the present invention relates to the following (1) to (29).
  • a therapeutic agent for neurological diseases containing as an active ingredient a substance that promotes the activity of Manmarian's target obu rapamycin (hereinafter abbreviated as mTOR).
  • Neurological diseases are Parkinson's disease, Alzheimer's disease, Down's syndrome, cerebrovascular disorder, stroke, spinal cord injury, triplet repeat disease, multiple sclerosis, amyotrophic lateral sclerosis, multiple euroves, epilepsy,
  • the therapeutic agent according to (1) above which is a neurological disease selected from the group consisting of anxiety disorders, schizophrenia, depression and manic depression.
  • R 1 represents hydrogen, R 2 represents hydroxy or protected hydroxy, or R 1 and R 2 together represent oxo, R 3 represents hydroxymethyl, protected
  • the therapeutic agent according to (1) or (2) above which is biniglol represented by the formula (1) or a pharmacologically acceptable salt thereof.
  • a substance capable of promoting mTOR activity The therapeutic agent according to (1) or (2) above, which is phosphatidic acid or a pharmacologically acceptable salt thereof.
  • a neurogenesis or regeneration promoter containing a substance that promotes mTOR activity as an active ingredient is included in the central nervous system.
  • a neural stem cell proliferation promoter containing a substance that promotes mTOR activity as an active ingredient (18) The neural stem cell proliferation promoter according to (17) above, wherein the substance that promotes mTOR activity is compound (I) or a pharmacologically acceptable salt thereof.
  • neural stem cells are cultured to proliferate neural stem cells, and neural stem cells are collected from the culture.
  • a method for producing a neural stem cell which is characterized.
  • (24) A method for producing euron, characterized in that neural stem cells produced by the production method of (23) are cultured and differentiated into neurons, and euron is collected from the culture.
  • a method for producing glial cells comprising culturing neural stem cells produced by the production method according to (23) above to differentiate into glial cells, and collecting glial cells from the culture.
  • a substance having the phosphorylation promoting activity is selected as a neural stem cell proliferation promoting substance Process
  • a powerful screening method for neural stem cell proliferation promoting substances is a powerful screening method for neural stem cell proliferation promoting substances.
  • a therapeutic agent for neurological disease a neurogenesis or regeneration promoter, a food or drink for preventing or promoting neurological function and a food additive, a neural stem cell, comprising a substance that promotes mTOR activity as an active ingredient
  • a method for producing a proliferation promoter, neural stem cell, neuron or glial cell, a method for screening a neuron or glial cell produced by the production method, or a neural stem cell proliferation promoting substance can be provided.
  • FIG. 1 is a graph showing the effects of FK506 and rapamycin on the neural stem cell proliferation promoting activity of biniglol.
  • the values on the horizontal axis of the graph represent the final concentrations of FK506, rapamycin, and vinylogol in the medium.
  • the vertical axis of the graph represents the relative value in the drug addition group when the measured value of the well without cell seeding is 0% and the measured value of the negative control is 100%.
  • * Significant difference with respect to DMSO control P ⁇ 0.01, * *: Significant difference with bigol P ⁇ 0.01, * * * *: Significant difference with rapamycin + binigrol P ⁇ 0.001 (Unpaired Student 'st — Test)
  • the therapeutic agent for neurological diseases, the neurogenesis or regeneration promoter of the present invention, the prevention of nerve function or the food or drink additive for enhancement, or the food additive, is! Promotes the activity of mTOR contained in the neural stem cell proliferation promoter Any substance may be used as long as it promotes mTOR activity.
  • Examples thereof include compounds represented by (III) to (VI).
  • biniglol can be produced by culturing a bigol-producing bacterium belonging to the genus Virgaria in a medium and isolating the resulting culture strength biniglol.
  • the compounds represented by the formulas (III) to (V) can be obtained by subjecting vinylol to diionates by a conventional method.
  • the compound represented by the formula (VI) is vinylogol, the compound represented by the formula (IV) or the formula (V)
  • the compound represented by V) can be obtained by introducing a hydroxy protecting group into the hydroxyl group in the hydroxymethyl group at the 3-position and the hydroxyl group at the Z-position or 4-position by a conventional method.
  • the hydroxy protecting group may be introduced into only one of the hydroxyl group of the hydroxyl group at the 3-position and the hydroxyl group at the 4-position.
  • another hydroxy protecting group may be introduced again into the thus obtained compound.
  • Protected hydroxy is a group usually used as a protecting group for a hydroxyl group (eg, THEODORA W. GREEN, PETER GM WUTS, “PROTECTIV E GROUPS in ORGANIC SYNTHESIS”, 3rd edition, WILEY— INTERSCI ENCE, 1999)), but is not particularly limited, but is preferably a lower alkanoyloxy such as acetoxy, propionyloxy, butyryloxy, isobutyryloxy, tert-tyryloxy, t Lower alkylsilyloxy such as ptyldimethylsilyloxy, and allylooxy such as benzoyloxy.
  • a lower alkanoyloxy such as acetoxy, propionyloxy, butyryloxy, isobutyryloxy, tert-tyryloxy, t Lower alkylsilyloxy such as ptyldimethylsilyloxy, and allylooxy such as
  • hydroxymethyl a group usually used as a protective group for hydroxymethyl group (for example, “PROTECTIVE GROUPS in ORGANIC SYNTHESIS”, 3rd edition, written by THEODORA W. GREEN, PETER GM WUTS, , WILEY — INTERSCIENCE, 1999)), but not particularly limited, preferably acetoxymethyl, propio-oxymethyl, butyryloxymethyl, isobutyryloxymethyl, tert-butyryloxymethyl, etc. And lower alkylsilyloxymethyl such as t-butyldimethylsilyloxymethyl, allylooxymethyl such as benzoyloxymethyl, and the like.
  • branched chain amino acid palin, leucine or isoleucine alone, or a mixture of two or three of phosphorus ⁇ phosphorus, leucine and isoleucine is preferred.
  • the weight ratio of each component in the mixture is 0.5 to It is preferable that the ratio is 1.5: 1 to 3: 0.5 to 1.5.
  • leucine and isoleucine may be used in the L-form, D-form, and a mixture of L-form and D-form, respectively, but preferably the L-form.
  • the method for producing a branched chain amino acid is not particularly limited, and can be produced by a fermentation method, a synthesis method, a purification method, or the like. For example, by purchasing from Kyowa Hakko Kogyo Co., Ltd. or Kyowa Wellness Co., Ltd. Monkey.
  • Phosphatidic acid refers to 1,2 diacylglycerol triphosphate.
  • the phosphatidic acid used in the present invention may be any phosphatidic acid that promotes the activity of mTOR, and examples thereof include 1 palmitoyl 2-oleoylphosphatidic acid.
  • Phosphatidic acid can be obtained by purifying egg yolk, soybean, etc., and can also be obtained as a commercial product or by chemical synthesis (for example, US Pat. No. 3,423,440).
  • a non-toxic water-soluble salt is preferable, for example, hydrochloride, sulfate, nitrate, phosphorus
  • examples include inorganic acid salts such as acid salts, organic acid salts such as acetates, maleates, fumarate salts, and citrate salts.
  • examples of pharmacologically acceptable metal salts include sodium salts and potassium salts.
  • Alkali earth metal salts such as alkali metal salts, magnesium salts, and calcium salts, aluminum salts, zinc salts, and the like.
  • Examples of pharmacologically acceptable ammonium salts include ammonium salts, tetra salts. Examples thereof include salts such as methylammonium, and pharmacologically acceptable organic amine addition salts include addition salts such as morpholine and piperidine.
  • Neural disease therapeutic agent neurogenesis or regenerative agent
  • the neurological disease for which the therapeutic agent for neurological diseases of the present invention is not particularly limited, but preferably Parkinson's disease, Alzheimer's disease, Down's syndrome, cerebrovascular disorder, stroke, spinal cord injury, triplet repeat disease, multiple sclerosis , Amyotrophic lateral sclerosis, multiple-europathy, epilepsy, anxiety disorder, schizophrenia, depression, manic depression and the like.
  • the neurogenesis or regenerative agent of the present invention promotes the proliferation of neural stem cells by acting directly on the neural stem cells in the brain of humans or animals, and as a result, has the effect of promoting neurogenesis or regeneration.
  • neurogenesis refers to the formation of neuronal glia cells by proliferation and differentiation of neural stem cells
  • nerve regeneration refers to neural circuits that have fallen in the above-mentioned neurological diseases due to the born-euron glia cells. Is reconstructed and its functions are regenerated.
  • the substance that promotes mTOR activity or a pharmacologically acceptable salt thereof can be administered alone as it is as a therapeutic agent for neurological diseases, or as a neurogenesis or regenerative agent. It is desirable to provide it as a pharmaceutical formulation. These pharmaceutical preparations are used for animals and humans.
  • the therapeutic agent for neurological disease or neurogenesis or regenerative agent of the present invention is a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof as an active ingredient, alone or for any other treatment. It can contain as a mixture with this active ingredient.
  • These pharmaceutical preparations can be prepared by any method well known in the technical field of pharmaceutics by mixing the active ingredient together with one or more pharmaceutically acceptable carriers. Manufactured.
  • the route of administration is preferably oral or the one that is most effective in treatment, or
  • parenteral such as intravenous can be mentioned.
  • Examples of the dosage form include tablets, powders, granules, syrups, injections and the like.
  • Liquid preparations suitable for oral administration, such as syrups, are water, sucrose, sorbit
  • Sugars such as fructose, Daricols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, preservatives such as P-hydroxybenzoate esters, flavors such as strawberry flavor and peppermint Etc.
  • Tablets, powders and granules are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy It can be produced using a binder such as propylcellulose and gelatin, a surfactant such as a fatty acid ester, and a plasticizer such as glycerin.
  • Formulations suitable for parenteral administration preferably comprise a sterile aqueous preparation containing an active compound which is isotonic with the blood of the recipient.
  • a solution for injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.
  • a carrier such as a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.
  • these parenteral agents one kind selected from diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc., exemplified for oral agents. Or more auxiliary ingredients can be added.
  • the dose and frequency of administration of a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof depends on the dosage form, patient age, body weight, nature or severity of the condition to be treated. Although different, in the case of oral administration, usually 0.01 mg to 20 g, preferably 0.05 mg to 10 g per adult is administered once to several times a day. In the case of parenteral administration such as intravenous administration, 0.001 mg to 20 g per adult, preferably 0. Olmg to: LOg is administered once to several times a day.
  • Examples of the food and drink of the present invention include food and drink obtained by adding a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof.
  • the food / beverage product of the present invention is processed by using a general method for producing a food / beverage product, except that a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof is added to the food / beverage product.
  • a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof is added to the food / beverage product.
  • the food and drink of the present invention are, for example, fluidized bed granulation, stirring granulation, extrusion granulation, rolling granulation, gas flow granulation, compression molding granulation, pulverization granulation, spray granulation, spray granulation, etc.
  • Granulation method pan coating, fluidized bed coating, coating method such as dry coating, puff drying, excess steam method, foam mat method, expansion method such as microwave heating method, extrusion granulator, extrusion method such as extruder, etc. It can also be manufactured using.
  • the food and drink of the present invention includes juices, soft drinks, teas, lactic acid bacteria beverages, fermented milk, frozen desserts, butter, cheese, yogurt, processed milk, skim milk and other dairy products, ham, sausage, hamburger Livestock meat products such as fish, fish paste such as bamboo shoots, bamboo rings, and fish cakes, egg products such as soup rolls, egg tofu, cookies, jelly, chewing gum, candy, throat candy, snacks and other confectionery, breads, potatoes , Pickles, salmon products, dried fish, boiled fish, salted products, soups, seasonings, etc.
  • the food and drink of the present invention are in the form of, for example, powdered food, sheet-like food, bottled food, canned food, retort food, capsule food, tablet food, liquid food, and drink. Also good.
  • the food and drink additive of the present invention can be prepared by the same method as the oral preparation described in 2 above.
  • the food / beverage food additive is usually processed and manufactured in the form of powder, granules, pellets, tablets, or various liquids by mixing or dissolving other food / beverage food additives as necessary.
  • the food / beverage products or food / beverage product additives of the present invention include food / beverage product additives generally used for food / beverage products, such as sweeteners, colorants, preservatives, thickening stabilizers, antioxidants, colorants. Bleaching agents, fungicides, gum bases, bittering agents, enzymes, brighteners, acidulants, seasonings, emulsifiers, fortifiers, manufacturing agents, fragrances, spice extracts and the like may be added.
  • the food or drink of the present invention can be used as a food or drink such as a health food for preventing or enhancing neurological function or a functional food.
  • the amount of the substance that promotes mTOR activity to the food or drink of the present invention or a pharmacologically acceptable salt thereof, or the food or drink additive of the present invention depends on the type of the food or drink, although it is appropriately selected depending on the expected effect of ingestion, etc., as a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof, usually 0.1 to: LOO% by weight, preferably It is added so as to contain 1 to 70% by weight, particularly preferably 5 to 50% by weight.
  • the intake of the food and drink of the present invention varies depending on the intake form, the age of the user, the body weight, etc., but as a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof, For adults, 0.01 mg to 20 g, preferably 0.05 mg or more: Take LOg once or several times.
  • the neural stem cell proliferation promoter of the present invention can promote proliferation of neural stem cells when contacted with neural stem cells in vivo or in vitro.
  • Stem cells refer to cells having multipotency to differentiate into a plurality of different types of cells and self-renewal ability to generate new stem cells by symmetrical or asymmetric division.
  • a progenitor cell a cell that has entered a lineage of a certain amount and is destined to undergo differentiation after a limited number of divisions.
  • neural stem cells, neural progenitor cells, and glial progenitor cells are collectively referred to as neural stem cells. .
  • the neural stem cell is not particularly limited as long as it is a neural stem cell, but is preferably an adult neural stem cell of the brain.
  • the brain may be the brain of any animal, preferably a mammal, more preferably a rat, mouse, monkey, human or the like.
  • a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof can be used alone as a neural stem cell proliferation promoter, but it is usually provided as various pharmaceutical products. desirable. These pharmaceutical preparations are used for animals and humans.
  • the neural stem cell proliferation promoter of the present invention is a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof as an active ingredient, or an active ingredient for any other treatment. And a mixture thereof.
  • These pharmaceutical preparations can be produced by the same method as that for the preparation of the therapeutic agent for neurological diseases described above, and can be administered by the same administration method.
  • the neural stem cell proliferation promoter of the present invention is brought into contact with neural stem cells in vitro, By culturing the neural stem cell, the proliferation of the neural stem cell is promoted, and the neural stem cell can be collected from the culture, and can be used for a method for producing a neural stem cell.
  • a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof is dissolved in a solution that can dissolve the substance or the salt. And preferably used.
  • the solution include water and DMSO.
  • neural stem cell proliferation promoter of the present invention By culturing animal neural stem cells in the presence of the neural stem cell proliferation promoter of the present invention, proliferation of the neural stem cells can be actively promoted.
  • the neural stem cells of an animal may be neural stem cells of any animal, preferably mammals, more preferably rat, mouse, monkey, or human-derived neural stem cells. Can include brain-derived neural stem cells.
  • the neural stem cells may be cells derived from animals of any age or age, but preferably adult neural stem cells.
  • the neural stem cell proliferation promoter is applied to an adult neural stem cell of about 6.25 ⁇ 10 4 Zcm 2 at 10 nm. It is preferable to work at a concentration of 0 lZl to lmmolZl.
  • Adult neural stem cells and the present invention Contacted with a neural stem cell growth promoter, at 37 ° C in a 5% CO atmosphere, 1
  • the growth of neural stem cells can be promoted by culturing for 2 to 14 days every two days while changing the whole amount or a partial amount of the medium.
  • the medium contains 1% N-2 supplement (made by Invitrogen Corp.) as long as the medium does not hinder the promotion of neural stem cell growth! It is preferable to use a medium (Invitrogen) or the like.
  • the neural stem cells obtained by the culture described above do not contain the neural stem cell proliferation promoter of the present invention, lnM to lmM all-trans retinoic acid, lnM to lmM forskolin, or 0.lngZml to lmgZml platelet-derived growth.
  • It can be differentiated into -euron by culturing for 2 to 14 days every two days while changing the whole or partial medium.
  • the medium may be a misaligned medium as long as it does not prevent differentiation into neurons! However, it is preferable to use DMEMZF12 medium (Invitrogen) or the like containing 1% N-2 supplement (Invitrogen).
  • the neural stem cells obtained by the above culture do not contain the neural stem cell proliferation promoter of the present invention, and 0. IngZml to: LmgZml of leukemia inhibitory factor (LIF), or 0. lng Zml to lmgZml of osteogenic factor -2 Medium containing (BMP-2), etc., static culture at 37 ° C in 5% CO atmosphere for 1-14 days, changing the whole or partial volume every 2 days
  • any medium may be used! However, it is preferable to use DMEMZF12 medium (Invitrogen) or the like containing 1% N-2 supplement (Invitrogen).
  • Neural stem cells, neurons, or glial cells obtained by the above culture can be collected from a medium and transplanted to a damaged site of a neurological disease patient by a surgical technique to be used for treatment of the neurological disease.
  • the neurological diseases include Parkinson's disease, Alzheimer's disease, Down's syndrome, cerebrovascular disorder, stroke, spinal cord injury, triplet repeat disease, multiple sclerosis, amyotrophic lateral sclerosis, multiple neurobaty, epilepsy, anxiety disorder, integration Examples include ataxia, depression, manic depression. 6. Screening method for neural stem cell proliferation promoter
  • the method for screening a neural stem cell proliferation promoting substance of the present invention includes (i) a step of measuring the amount of a phosphopeptide in the presence of mTOR, a peptide phosphorylated by mTOR, and ATP, (Ii) a step of measuring the amount of phosphorylated peptide in the presence of test substance, mTOR, peptide phosphorylated by mTOR, and ATP; (iii) measurement of (i) and (ii) A step of evaluating the phosphorylation-promoting activity of the test substance against the peptide from the amount; (iv) a step of selecting the substance having the phosphorylation-promoting activity as a neural stem cell proliferation-promoting substance; Can do.
  • the neural stem cell is not particularly limited as long as it is a neural stem cell, but is preferably an adult neural stem cell of the brain.
  • the brain may be the brain of any animal, but is preferably a mammal, more preferably a rat, mouse, monkey, human or the like.
  • the test substance is not particularly limited.
  • peptides, proteins, cell extracts, purified products derived from the extracts, cell culture supernatants, purified products derived from the supernatants, biological samples such as serum, and the biological samples Purified products derived from microorganisms, purified cells derived from microorganisms, purified products derived from these extracts, purified supernatants from microorganisms, purified products derived from these supernatants, compounds, compounds synthesized using combinatorial chemistry, etc. Can do.
  • the mTOR is not particularly limited as long as it has mTOR activity !, but preferably mTOR can be mentioned from mammals, more preferably rat, mouse, monkey or human.
  • mammals more preferably rat, mouse, monkey or human.
  • the protein described in ["Sciences Signal Transduction 'Narrange Environment (Sciences Signal Transduction Knowledge Environment)' 2003, 212 ⁇ , pp. Rel5] can be mentioned.
  • mTOR can be obtained from cells expressing mTOR! /, By immunoprecipitation.
  • the cells are not particularly limited as long as they express mTOR, but preferably include neural stem cells derived from mammals, more preferably rats, mice, monkeys or humans.
  • peptides phosphorylated by mTOR include ribozomal S6 kinase (S6K) and eukaryotic initiation factor—4E binding protein 1 (4E—BP1).
  • S6K ribozomal S6 kinase
  • E—BP1 eukaryotic initiation factor—4E binding protein 1
  • ⁇ Ob Biochemistry (Europ ean Journal of Biochemistry) "2002, 269 ⁇ , p. 5338-5349].
  • ATP which is a donor of phosphate
  • ⁇ 32 P] ATP a test substance the presence or in the subject matter absence phosphorus Sani of p70S6K or 4 E- BP1 or enzyme, by complex containing mTOR or mTOR obtained by immunoprecipitation
  • An example is a method in which a phosphoric acid-containing reaction of a peptide containing a sputum site is performed, and the amount of 32 P incorporated into the enzyme or peptide is measured using a liquid scintillation counter or the like.
  • an antibody that specifically recognizes the phosphorylated substrate enzyme or peptide is used after the phosphorylation reaction and labeled with fluorescence or chemiluminescence. The following antibody can be used to determine “willow”.
  • the therapeutic agent for neurological diseases of the present invention can proliferate neural stem cells at ⁇ to treat neurological diseases.
  • the therapeutic agent for a neurological disease of the present invention described above is administered to a laboratory animal such as a rat or monkey.
  • the experimental animal may be a healthy animal that does not have an injury, but it is possible to effectively observe the neurogenesis or regeneration by giving hippocampal ischemic injury. ), 2002, 110 ⁇ , p. 429-441], preferred is an animal whose brain is damaged by ischemia, 6-hydroxydopamine (6-OHDA) administration or kainic acid administration.
  • 6-OHDA 6-hydroxydopamine
  • the dosage and administration method are, for example, 100 ng to 500 mg, preferably 500 ng to 200 mg per kg body weight.
  • parenteral administration such as intravenous administration
  • 10 ng to 500 mg preferably 100 ng to 200 mg per kg body weight is administered once to several times a day.
  • Proliferated neural stem cells can be detected by the following method.
  • the first administration of the substance is a retroviral vector that can express cell-labelable genes such as 5-promo 2'-deoxyuridine (BrdU) or Green Fluorescent Protein (GFP) or beta-galactosidase that can label proliferating cells.
  • cell-labelable genes such as 5-promo 2'-deoxyuridine (BrdU) or Green Fluorescent Protein (GFP) or beta-galactosidase that can label proliferating cells.
  • the substance is administered once to several times a day and kept for 10 to 20 days. Thereafter, the brain of the experimental animal is removed, and a frozen section of the brain is prepared and observed using a fluorescence microscope.
  • BrdU is used as a drug for labeling proliferating cells
  • BrdU positive cells per unit area Compare numbers with negative controls
  • the neural stem cell proliferation promoting action and the therapeutic effect on the neurological disease of the therapeutic agent for the neurological disease of the present invention can be evaluated.
  • the neural stem cell proliferation promoter of the present invention can promote the proliferation of neural stem cells in vitro.
  • the neural stem cells obtained by the above method 5 are suspended in DMEMZF12 medium containing 1% N-2 additive at a concentration of 2.0 X 10 4 ⁇ @ / 0. 1 ml, and polyortin and laminin Seed in a 96-well plate, etc. that has been surface-treated, etc., and incubate at 37 ° C in a 5% CO atmosphere.
  • test substance dissolved in PBS or DMSO is diluted stepwise with the above medium or PBS in a concentration range of 0.01 nmolZl to 100 mmolZl and added.
  • Add the same volume of the medium or PBS as a negative control.
  • FGF2 or EGF it is preferable to use as a positive control. Then, under 5% CO atmosphere
  • the growth of neural stem cells can be promoted by culturing at 37 ° C for 1 to 14 days while changing the whole or part of the medium as necessary.
  • the proliferated neural stem cells can be measured by a method such as a direct counting method, an FDSS measurement method, an XTT method, or a BrdU incorporation method.
  • a method such as a direct counting method, an FDSS measurement method, an XTT method, or a BrdU incorporation method.
  • the FDSS measurement method is explained in detail.
  • the grown neural stem cells are fixed with formaldehyde, alcohol, etc., and the washing is repeated using PBS containing 0.3% Triton X-100 (manufactured by Nacalai Testa) (hereinafter referred to as TBST).
  • PBS containing 0.3% Triton X-100 manufactured by Nacalai Testa
  • TBST PBS containing 0.3% Triton X-100
  • Hoechst 33342 Necalai Testa
  • washing with TBST is repeated, and the fluorescence intensity is measured using FDSS6000 (manufactured by Hamamatsu Photonics), etc., and compared with the control.
  • FDSS6000 manufactured by Hamamatsu Photonics
  • Test Example 1 Neural stem cell proliferation-promoting activity by biniglol (1) Direct counting method
  • Rat adult neural stem cell line ANSC-7 cells obtained by the method described in Reference Example 1 were mixed with N-2 supplement [5 ⁇ g / ml insulin (manufactured by Sigma), 100 ⁇ g Zml Usiapotransferrin (Sigma) ), 6.3 ngZml progesterone (manufactured by Sigma), 1.6 gZml putrescine (manufactured by Sigma), 5.2 ngZml sodium selenate (manufactured by Sigma)] and antibiotic mixture [0.
  • hex 33342 (manufactured by Nacalai Testa Co., Ltd.) prepared at 3.3 ⁇ mol / 1 was added with 50 1Z well, and the nucleus was labeled overnight at 4 ° C in the dark. 100 17 wells 3 times twice, followed by 3 83 times, then washed once with 100 1 Zwell PBS, and 0.14 445 mm 2 of nuclear stained cells per fluorescence microscope (Nikon) ).
  • Vinigrol was shown to promote proliferation of neural stem cells in the concentration range of 0.3 to: L0. ⁇ / z molZl.
  • Test Example 2 Activity of promoting proliferation of neural stem cells by biniglol (2) FDSS measurement method
  • the nuclei of ANSC-7 cells seeded in a 96-well plate and cultured were labeled with Hoechst 33342. After washing twice with 100 1Z-well TBST and then once with PBS, the cells were immersed in 100 / zl-well PBS and the fluorescence intensity of the nuclear-stained cells was measured with FDSS6000 (Hamamatsu Photonics). The relative value in the binigrol-added group was calculated assuming that the measured value of the well without cell seeding was 0% and the measured value of the negative control was 100%.
  • Vinigrol was shown to promote neural stem cell proliferation in the concentration range of 0.3-: L0.
  • Test Example 3 Vinigrol promotes proliferation of neural stem cells (3) Acupuncture
  • ANSC—7 cells were suspended in Atsy medium at a concentration of 2.0 ⁇ 10 5 Zml and seeded in 0.1 ml each in a 96-well plate (manufactured by NUNK) that was surface-coated with polyortin and laminin. The cells were cultured at 37 ° C in a 5% CO atmosphere. Then remove 50 1 culture supernatant,
  • XTT sodium 3 '-[1-(phenylamin ocarbonyl) -d, 4-tetrazolium) -bis (4-methoxy-6-nitro) benzene su lfonic acid hydrate
  • a reagent (Roche Diagnostics) was added to the culture medium. Cultivate in a 5% CO incubator for 5.5 hours at 37 ° C, then stir for 1 minute
  • the absorbance at 490 nm (control wavelength: 655 nm) was measured using a microplate spectrophotometer Emax (manufactured by Molecular Devices Corporation). The cells were not seeded! The relative value in the biniglol-added group was calculated with the measured value of the well being 0% and the measured value of the negative control being 100%.
  • Vinigrol is 0.3 ⁇ : Nerve trunk in the concentration range of L0. It has been shown to promote follicle growth.
  • Test Example 4 Growth promotion activity of neural stem cells by biniglol (4) BrdU incorporation method
  • ANSC—7 cells were suspended in Atsy's medium at a concentration of 2.0 ⁇ 10 5 Zml, and 0.1 ml each was seeded in a 96-well plate (manufactured by NUNK) that was surface-coated with polyortin and laminin. The cells were cultured at 37 ° C in a 5% CO atmosphere. Then remove 50 1 culture supernatant,
  • Vinignol, FGF2 (positive control), or DMSO (negative control) diluted to 2 ⁇ molZl with Atsey medium was added at a rate of 50 per well. Two days later, remove 50 ⁇ l of culture supernatant per well, and add a new Atsey medium containing 40 ⁇ molZl of 5-bromo-2, -deoxyuridine (Brd U, Roche Diagnostics). 50 ⁇ l per well was added and cultured for another 2 days. Thereafter, 50 1 of the culture supernatant was removed, and 50 ⁇ l of 15% neutral buffered formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) cooled to 4 ° C. was added and allowed to stand.
  • neutral buffered formalin solution manufactured by Wako Pure Chemical Industries, Ltd.
  • nestin a marker for neural stem cells
  • Alexa Fluor 488 Molecular ⁇ ⁇ ⁇ Probes, diluted 1 to 2000
  • Hoechst 33342 3.3 ⁇ mol / 1
  • the It was dissolved in a 5% skim milk solution diluted with PBS, and added with 50 1Z well, and allowed to stand at 4 ° C for 2 minutes in the dark. After removing the solution, the plate was washed with 100 1Z well TBST and then PBS once, and then 100 1Z well PBS was added.
  • Neural stem cells that express nestin increased as the total number of cells increased due to the treatment with biniglol. That is, it was shown that the cells increased by the treatment with vinylogol were neural stem cells.
  • Test Example 6 Inhibition of neural stem cell proliferation-promoting activity of Vinigrol by ravamycin (1) Rapamycin, a specific inhibitor of mTOR ["Oncogene” 2000, 19 ⁇ , p. 6680-6686] The inhibitory effect of biniglol on the activity of promoting proliferation of neural stem cells was examined.
  • ANSC—7 cells are suspended in Atsy medium at a concentration of 2.0 ⁇ 10 4 ZO. 1 ml, 0.1 ml each in a 96-well plate (manufactured by NUNK) that is surface-coated with poliotin and laminin. The seeds were seeded and cultured at 37 ° C in a 5% CO atmosphere. Then remove 50 1 culture supernatant.
  • rabamycin Sigma
  • DMSO negative control
  • Atsey medium diluted to twice the final concentration with Atsey medium and DMSO (negative control) were added at 50 1 per well.
  • 50 1 of the culture supernatant was removed, and vinigrol diluted to 1 / z molZl in an Atsey medium containing rapamycin or DMSO at the same concentration as above was added to 50 1 per well.
  • the cells were soaked in 100 ⁇ lZ well BS and the fluorescence intensity of the nuclear-stained cells was measured with FDSS6000.
  • the relative value in the drug-added group was calculated assuming that the measured value of the wells without cell seeding was 0% and the measured value of the negative control was 100%.
  • Test Example 7 Inhibition of neural stem cell proliferation-promoting activity of biniglol by ravamycin (2) Rapamycin forms a complex with FKBP-12 immunophilin protein and inhibits mTOR phosphorylation activity. Similarly, FK506 also binds to FKBP-12 immunophilin protein, thus antagonizing rapamycin-FKBP-12 immunophilin protein complex formation, but the FK506-FKBP 12 immunophilin protein complex does not inhibit mTOR. ! ⁇ ["FEBS Letters" 2003, 550 ⁇ , p. 94-100]. Therefore, we analyzed whether the ability of Fava 506 to cancel the inhibitory effect of rabamicin on the proliferation-promoting activity of Vinigrol on neural stem cells was analyzed.
  • ANSC—7 cells were suspended in Atsey medium at a concentration of 2.2 ⁇ 10 4 ZO. 1 ml and surface-coated with poliotin and laminin in a 96-well plate (Nunk). 1 ml each was inoculated and cultured at 37 ° C in 5% CO atmosphere. Then remove 50 1 culture supernatant.
  • the FK506 was diluted serially to twice the final concentration in Atsy's medium, and 50 ⁇ l per well was added. After 30 minutes, rapamycin diluted to 2 nmolZl with Atsy medium containing FK506 was added by exchanging half the culture supernatant. After an additional 30 minutes, biniglol diluted to 1 ⁇ molZl in Atsy medium containing FK506 and lavamycin was added by exchanging half the culture supernatant.
  • Rapamycin a specific inhibitor of mTOR
  • 500 nmolZl of vinylogol completely suppressed the proliferation promoting effect of neural stem cells.
  • the drug FK506, which antagonistically inhibits the complex formation between rapamycin and FKBP-12 immunophilin protein canceled the above-mentioned inhibitory effect of rapamycin in a concentration-dependent manner at lOnmolZl or higher.
  • Test Example 8 Cellular phosphorylation regulator 4E— BP1 and p70 S6 kinase
  • a 6 cm culture dish made of Iwakine soil surface-treated with polyortin and laminin was seeded with 6. 7 1 X 10 6 ANSC—7 cells in Atsy medium at 37 ° C, 5% CO atmosphere
  • the cells were lysed at 4 ° C for 1 hour and then centrifuged. Measure the protein concentration in the supernatant and prepare a sample so that the amount of protein in each lane is the same. Then, separate the protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). I did it. After the separated protein sample is transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), anti-serine 65 phosphorylated 4E-BP1 antibody (Celcidnering) or anti-serine is used as the primary antibody.
  • PVDF polyvinylidene difluoride
  • Threonine 389 phosphorylated p70S6 kinase antibody (manufactured by Cel Signaling) was added and reacted with the protein on the membrane. Thereafter, as a secondary antibody, an anti-rabbit Ig antibody (manufactured by Celsidanaring) labeled with horseradish peroxidase was reacted. Then, color was developed with ECL reagent (Pierce) and detected with LAS-1000 Lumino 'image analyzer (Fuji Film).
  • This activity was completely suppressed by adding rapamycin, a specific mTOR inhibitor, at a final concentration of 20 nmolZl.
  • Test Example 9 Direct phosphorylation promoting action of 4E—BP1 in vitro by vinylogol
  • the ANTOR-7 cell force was also purified by immunoprecipitation, and the 4E-BP1 phosphate was analyzed in vitro.
  • the cells were cultured at 37 ° C for 3 days. 1. After washing twice with 5 ml of PBS, 2.51 ⁇ 10 7 cells were recovered with a scraper (made by Iwakine Earth). 2 ml of cell lysis buffer in which the recovered cells are cooled
  • 50 ⁇ l mTOR complex immunoprecipitation beads were diluted with Atsy buffer to a final concentration of 2.5 ⁇ molZl (10 ⁇ 1) and 50 ⁇ l phosphorylation buffer [ 10 mmol / 1 hepes-sodium hydroxide (adjusted to pH 7.4), 50 mmol Zl sodium chloride, 50 mmol / l ⁇ -glycephosphate, lOmmolZl manganese chloride, 100 / z molZl adenosine triphosphate] Stir at 30 ° C for 30 minutes.
  • 4E-BP1 recombinant peptide (rat PHAS-1, manufactured by Calbiochem) was added as a phosphorylated substrate, and the mixture was further stirred at 30 ° C. for 50 minutes. After adding 50 times 3 times SDS sample buffer and boiling for 5 minutes to stop the reaction, proteins were separated by SDS-PAGE. After the separated protein sample was transferred to a PVDF membrane, an anti-threonine 37 Z46 phosphorylated 4E-BP1 antibody (manufactured by Cell Signaling) was added as a primary antibody to react with the protein on the membrane.
  • anti-rabbit Ig antibody manufactured by Cell Signaling
  • horseradish rust peroxidase horseradish rust peroxidase
  • biniglol increased the phosphorylation of threonine 37Z46 position of the 4E-BP1 substrate peptide by 1.5 times.
  • biniglol exerts a proliferation promoting activity of neural stem cells by directly acting on the mTOR complex.
  • Test Example 10 Growth promotion of neural stem cells by branched chain amino acids Low Insulin N—2-supplemented [0.1 ⁇ g / ml insulin (Sigma), 100 ⁇ gZ ml Ussiapotransferrin (Sigma), 6.3 ngZml progesterone (Sigma), 1.
  • Branched-chain amino acid (L-parin, L-tipped isine, L-isoleucine), FGF2 (positive control), or DMSO (negative) serially diluted to a final concentration of 2 times in low-insulin medium. Control) was added at 50 1 per well. After culturing for 96 hours, remove 50 1 culture supernatant, and add 50% 15% neutral buffered formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) cooled to 4 ° C for 30 minutes. did.
  • Reference Example 1 Isolation and culture of adult neural stem cells from rat brain power
  • the tissue including the periventricular region is segmented to about lmm 3 using ophthalmic scissors and a scalpel, and then 2.5UZml of papa In, 250 U / ml DNase (both manufactured by Worthington, Freehold, NJ), 5 ml HBSS buffer (made by Invitrogen) containing neutral protease (Dispase, manufactured by Boehringer Mannheim) of lU / m 1
  • the digestion reaction was performed at 37 ° C for 30 minutes.
  • the cell and tissue mixture obtained by the reaction was washed 3 times with DMEM (Invitrogen) containing 10% fetal calf serum (Hyclone), and then DMEM containing 10% fetal calf serum.
  • the undigested material was removed using a 10 m nylon mesh.
  • the obtained crude cell extract was cultured on a 10 cm culture dish in a 37 ° C incubator using DMEM ZF 12 medium (Invitrogen) containing 10% fetal calf serum. It was. On the next day, the culture medium was replaced with DMEMZF 12 containing 1% N-2 supplemented product (manufactured by Invitrogen) and 20 ng / ml FGF2 (manufactured by Pebrotech), and culture was started. Once every 3 days, half of the medium was replaced with DMEM / F 12 containing 1% fresh N-2 supplement and 20 ng / ml FGF2, and the culture was continued.
  • DMEM ZF 12 medium Invitrogen
  • N-2 supplemented product manufactured by Invitrogen
  • 20 ng / ml FGF2 manufactured by Pebrotech
  • a tablet having the following composition is prepared by a conventional method.
  • vinylogol was dissolved in DMSO to a concentration of 0.1 mmol and a neural stem cell proliferation promoter containing vinylogol was prepared.
  • a therapeutic agent for neurological disease containing a substance that promotes mTOR activity as an active ingredient, an agent for neurogenesis or regeneration, a food or drink for preventing or enhancing neurological function, and a food or drink additive, It is possible to provide a method for producing a neural stem cell proliferation promoter, a neural stem cell, a neuron or a Daria cell, a neuron or glial cell produced by the production method, or a screening method for a neural stem cell proliferation promoting substance.

Abstract

It is intended to provide a remedy for a neurological disease; a neurogenesis or nerve regeneration promoter; a food or a drink for preventing a decrease in a nerve function or promoting the same and a food additive therefor; a nerve stem cell growth promoter; a method of producing nerve stem cell, neurons or glial cells; neurons or glial cells produced by this method; or a method of screening a nerve stem cell growth promoter. Namely, a remedy for a neurological disease containing a substance, which promotes the activity of mTOR, as the active ingredient; a neurogenesis or nerve regeneration promoter; a food or a drink for preventing a decrease in a nerve function or promoting the same and a food additive therefor; a nerve stem cell growth promoter; a method of producing nerve stem cell, neurons or glial cells; neurons or glial cells produced by this method; or a method of screening a nerve stem cell growth promoter are provided.

Description

明 細 書  Specification
神経疾患治療剤  Neurological treatment
技術分野  Technical field
[0001] 本発明は、神経疾患治療剤、神経新生または再生促進剤、神経機能低下防止ま たは増進用飲食品および飲食品添加剤、神経幹細胞増殖促進剤、神経幹細胞、二 ユーロンおよびグリア細胞の製造方法、該製造方法により製造される-ユーロンおよ びグリア細胞、ならびに神経幹細胞増殖促進物質のスクリーニング方法に関する。 背景技術  [0001] The present invention relates to a therapeutic agent for neurological diseases, an agent for promoting neurogenesis or regeneration, a food and drink for preventing or enhancing nerve function and an additive for food and drink, a neural stem cell proliferation promoter, a neural stem cell, a euron and a glial cell. The present invention relates to a method for screening for euron and glial cells, and a method for screening a proliferation promoting agent for neural stem cells. Background art
[0002] 神経疾患とは、脳や末梢のニューロンが遺伝的要因、環境要因、加齢要因等により 傷害を受ける疾患である神経変性疾患と鬱病、躁鬱病、総合失調症等を総称する疾 患である。神経変性疾患として、具体的には、パーキンソン病、アルッノヽイマ一病、ト リブレットリピート病、筋萎縮性側索硬化症、多発-ユーロバチ一、脊髄損傷、脳血管 障害等があげられる。これら神経変性疾患に対する一般的な治療法は、ニューロン の傷害により失われた神経伝達物質を補充する方法であるが、該治療法で症状が改 善するのはパーキンソン病、アルッノヽイマ一病等に限定される。また神経伝達物質 補充療法では神経細胞死の進行を止めることはできない。  [0002] Neurological diseases collectively refer to neurodegenerative diseases and depression, manic depression, schizophrenia, etc., in which brain and peripheral neurons are damaged by genetic factors, environmental factors, aging factors, etc. It is. Specific examples of neurodegenerative diseases include Parkinson's disease, Arno-i-maima disease, triplet repeat disease, amyotrophic lateral sclerosis, multiple-euro bee, spinal cord injury, and cerebrovascular disorder. A common treatment for these neurodegenerative diseases is to replace neurotransmitters that have been lost due to neuronal injury, but the treatment improves symptoms such as Parkinson's disease, Arno-Ima's disease, etc. It is limited to. Neurotransmitter replacement therapy cannot stop the progression of neuronal cell death.
[0003] 中枢神経系を再生する再生医療は、パーキンソン病で失われたドーパミン作動性 ニューロンの機能を積極的に回復させる治療法として移植という観点力 検討が進め られてきたが、中絶胎児脳を用いることに起因する様々な問題があり、一般的な利用 には至っていない。  [0003] Regenerative medicine that regenerates the central nervous system has been studied for the potential of transplantation as a treatment that actively restores the function of dopaminergic neurons lost in Parkinson's disease. There are various problems caused by its use, and it has not been used in general.
また、胎児脳から取得した神経幹細胞ゃヒト受精卵から取得した ES細胞を生体外 で大量培養した後、目的の-ユーロンへ分化させて移植に用いるという治療法も検 討されている力 目的とする-ユーロンへ正確に分ィ匕させる技術は確立されておらず 、また胎児由来の神経幹細胞ゃヒト ES細胞を用いることに起因する問題もあり、臨床 応用は進んでいない。  In addition, the treatment of neural stem cells obtained from fetal brain and ES cells obtained from human fertilized eggs in vitro, and then differentiated into the desired -euron for use in transplantation is also being investigated. -Techniques for accurately sorting into eurons have not been established, and there are problems due to the use of embryonic neural stem cells and human ES cells, and clinical applications are not progressing.
[0004] 一方、成体脳から神経幹細胞が分離され (非特許文献 1参照）、ヒト成体脳でも生涯 にわたり-ユーロンの新生が起こることが報告された (非特許文献 2参照)。また、鬱 病 (非特許文献 3参照)や統合失調症 (非特許文献 4参照)の回復にも神経幹細胞の 増殖による神経再生が関与することが示唆されて 、る。 [0004] On the other hand, neural stem cells were isolated from the adult brain (see Non-Patent Document 1), and it was reported that the human adult brain also undergoes a life-long development of Euron (see Non-Patent Document 2). Also depression It has been suggested that nerve regeneration by proliferation of neural stem cells is also involved in recovery from diseases (see Non-Patent Document 3) and schizophrenia (see Non-Patent Document 4).
このような背景のもと、患者の脳内に存在する神経幹細胞を薬剤等で刺激して神経 再生を誘導して神経変性疾患を治療する方法が検討されている。すなわち、インスリ ン様増殖因子 1 (非特許文献 5参照)、線維芽細胞増殖因子 2 (非特許文献 6参 照）、エリスロポエチン (非特許文献 7参照)等のサイト力インの脳内投与や、前脳虚 血 (非特許文献 8参照）、てんかん刺激 (非特許文献 9参照)等の疾患モデルにお 、 て側脳質下帯、海馬、嗅球での-ユーロン新生が促進されることが報告されている。 また、腫瘍増殖因子 αの脳内投与により線条体でドーパミン作動性-ユーロンが 新生し、パーキンソン病の症状が改善することも報告されて 、る (非特許文献 10参照 ) οさらに、海馬への虚血傷害により失われた CA1錘体細胞が虚血後 2日目力ら 5日 目にかけて脳内に線維芽細胞増殖因子 2と上皮細胞増殖因子を投与することによ り、その 40%が回復することも報告されている（非特許文献 11参照)。  Against this background, methods for treating neurodegenerative diseases by stimulating neural stem cells present in a patient's brain with drugs or the like to induce nerve regeneration have been studied. In other words, intracerebral administration of cytoforce-in such as insulin-like growth factor 1 (see Non-Patent Document 5), fibroblast growth factor 2 (see Non-Patent Document 6), erythropoietin (see Non-Patent Document 7), It has been reported that in the model of diseases such as forebrain ischemia (see Non-patent Document 8) and epilepsy stimulation (see Non-Patent Document 9), the development of -euron in the lateral subcerebral zone, hippocampus and olfactory bulb is promoted. Has been. In addition, it has been reported that administration of tumor growth factor α in the brain causes the formation of dopaminergic-euron in the striatum and improves the symptoms of Parkinson's disease (see Non-Patent Document 10). 40% of CA1 pyramidal cells lost due to ischemic injury were administered by fibroblast growth factor 2 and epidermal growth factor into the brain from day 2 to day 5 after ischemia. It has also been reported to recover (see Non-Patent Document 11).
[0005] これらの結果は、薬剤等によって成体脳内在性の神経幹細胞の増殖を促進するこ とによって神経細胞数を増加させ、神経再生を促進する方法が、神経疾患の治療に 有効であることを示している。従って、神経幹細胞の増殖を促進させる薬剤は、神経 疾患の治療薬として有用であると考えられる。しかし、上記の方法ではいずれも蛋白 性の因子を脳内に投与することが必要であり、一般的な治療法とすることは難しい。  [0005] These results indicate that the method of increasing the number of nerve cells and promoting nerve regeneration by promoting the proliferation of adult brain endogenous neural stem cells with drugs and the like is effective in treating neurological diseases. Is shown. Therefore, a drug that promotes the proliferation of neural stem cells is considered useful as a therapeutic agent for neurological diseases. However, in any of the above methods, it is necessary to administer a protein factor into the brain, and it is difficult to obtain a general treatment method.
[0006] マンマリアン 'ターゲット'ォブ'ラパマイシン (mTOR)は、蛋白質翻訳開始に必須 のシグナル分子であり、そのリン酸ィ匕活性がラバマイシンにより阻害されることが知ら れている。分岐鎖アミノ酸、特にロイシンが、肝臓における mTORのシグナルを強力 に活性化することにより、蛋白質合成を促進することが知られている（非特許文献 12 参照)。胎生マウス皮質由来の神経幹細胞に対し、 Akt— 1を高発現させることにより 、その増殖が促進されることが報告され、その機構として mTORシグナルの関与が推 測されている（非特許文献 13参照)。しかし、哺乳類では胎生由来の神経幹細胞と 成体由来の神経幹細胞では本質的に性質が異なると言われている。例えば、成体 由来の神経幹細胞の増殖にはテロメラーゼが必要であるのに対し、胎生由来の神経 幹細胞はテロメラーゼ非依存的に増殖可能であるというように、その基本的な増殖機 構が全く異なる可能性が非常に高い (非特許文献 14参照)。従って、胎生由来の神 経幹細胞の特性を成体由来の神経幹細胞に外挿することはできない。 [0006] Mammalian 'target'of'rapamycin (mTOR) is a signal molecule essential for protein translation initiation, and its phosphate activity is known to be inhibited by ravamycin. It is known that branched chain amino acids, particularly leucine, promote protein synthesis by strongly activating mTOR signals in the liver (see Non-Patent Document 12). It has been reported that the proliferation of embryonic cortex-derived neural stem cells is promoted by high expression of Akt-1, and the involvement of mTOR signal has been estimated as the mechanism (see Non-Patent Document 13). ). However, in mammals, embryonic neural stem cells and adult-derived neural stem cells are said to be essentially different in nature. For example, telomerase is required for growth of adult-derived neural stem cells, whereas embryonic neural stem cells can proliferate independently of telomerase. It is very likely that the structure is completely different (see Non-Patent Document 14). Therefore, the characteristics of embryonic stem cells cannot be extrapolated to adult-derived neural stem cells.
ビニグロール (vinigrol)およびその誘導体は、抗血小板凝集作用や血圧降下作用 (特許文献 1参照)、腫瘍壊死因子拮抗作用（特許文献 2参照)、顆粒球マクロファー ジコロニー刺激因子様作用、免疫賦活作用および抗腫瘍作用（特許文献 3参照)を 有すること、またヒト免疫不全ウィルス感染症の治療薬として有用であること (特許文 献 4参照）が知られている。さらに、ビニグロールを含むサイト力インの生産を抑制する 抗炎症性薬剤および免疫抑制剤を含有する薬剤が自己免疫疾患に有効であること（ 特許文献 5参照）、ビニグロールを含む腫瘍壊死因子拮抗剤および選択的 COX— II 阻害剤を含有する薬剤が炎症性疾患に有用であること (特許文献 6参照）が報告され ている。  Vinigrol and its derivatives have antiplatelet aggregation action, blood pressure lowering action (see Patent Document 1), tumor necrosis factor antagonism (see Patent Document 2), granulocyte macrophage colony stimulating factor-like action, immunostimulatory action and It is known to have an antitumor action (see Patent Document 3) and to be useful as a therapeutic agent for human immunodeficiency virus infection (see Patent Document 4). In addition, an anti-inflammatory drug and an immunosuppressive drug-containing drug that suppresses the production of cytoforce-in containing biniglol are effective for autoimmune diseases (see Patent Document 5), a tumor necrosis factor antagonist containing vinylogol, and It has been reported that a drug containing a selective COX-II inhibitor is useful for inflammatory diseases (see Patent Document 6).
特許文献 1：特開昭 63— 215650号公報 Patent Document 1: Japanese Patent Laid-Open No. 63-215650
特許文献 2：特表平 5 - 504130号公報 Patent Document 2: Japanese Patent Publication No. 5-504130
特許文献 3：特開 2003 - 267930号公報 Patent Document 3: Japanese Patent Laid-Open No. 2003-267930
特許文献 4：特開平 7 - 206668号公報 Patent Document 4: Japanese Patent Laid-Open No. 7-206668
特許文献 5：特表平 11— 515020号公報 Patent Literature 5: Japanese Patent Publication No. 11-515020
特許文献 6 :国際公開第 01Z00229号パンフレット Patent Document 6: International Publication No. 01Z00229 Pamphlet
非特許文献 1 :「サイエンス（Science)」、 1992年、 255卷、 p. 1707- 1710 非特許文献 2 :「ネィチヤ一'メディスン（Nature Medicine)」、 1998年、 4卷、 p. 13Non-Patent Document 1: “Science”, 1992, 255 卷, p. 1707-1710 Non-Patent Document 2: “Nature Medicine”, 1998, 4 卷, p. 13
13 - 1317 13-1317
非特許文献 3 :「サイエンス（Science)」、 2003年、 301卷、 p. 805— 809 非特許文献 4：「セルラ一'アンド'モレキユラ一'ライフ ·サイェンシズ (Cellular andNon-Patent Document 3: “Science”, 2003, 301 卷, p. 805-809 Non-Patent Document 4: “Cellular and“ Molekiyura ”Life Sciences (Cellular and
Molecular Life Sciences)」、 2003年、 60卷、 p. 1891— 1902 Molecular Life Sciences), 2003, 60 卷, p. 1891—1902.
非特許文献 5 :「ザ'ジャーナル'ォブ 'ニューロサイエンス（The Journal of Neur oscience)」、 2000年、 20卷、 p. 2896— 2903 Non-Patent Document 5: “The Journal of Neuroscience”, 2000, 20 卷, p. 2896—2903
非特許文献 6：「プロシーディンダス ·ォブ ·ザ ·ナショナル ·アカデミー ·ォブ ·サイェン ス'ォブ ·ザ'ユナイテッド'ステイツ'ォブ 'アメリカ（Proceedings of the National Academv of science of the United States of Americaノ」、 2001年、 98卷、 p. 5874- 5879 Non-Patent Document 6: “Proceedings of the National Academv of science of the United States” (Proceedings of the National Academv of science of the United States) of America ", 2001, 98 卷, p. 5874-5879
非特許文献 7 :「ザ'ジャーナル'ォブ 'ニューロサイエンス（The Journal of Neur oscience 2001年、 21卷、 p. 9733— 9743  Non-Patent Document 7: “The Journal of Neuroscience 2001, 21 卷, p. 9733— 9743
非特許文献 8 :「ザ'ジャーナル'ォブ 'ニューロサイエンス（The Journal of Neur oscience 1998年、 18卷、 p. 7768 - 7778  Non-Patent Document 8: “The Journal of Neuroscience 1998, 18 卷, p. 7768-7778
非特許文献 9 :「ザ'ジャーナル'ォブ 'ニューロサイエンス（The Journal of Neur oscience 2002年、 22卷、 p. 3174— 3188  Non-Patent Document 9: “The Journal of Neuroscience 2002, 22 卷, p. 3174— 3188
非特許文献 10：「プロシーディンダス ·ォブ ·ザ ·ナショナル ·アカデミー ·ォブ ·サイェン ス'ォブ ·ザ'ユナイテッド'ステイツ'ォブ 'アメリカ（Proceedings of the National Academy of Science of the United states of America)」、 2000牛、 97卷、 p. 14686 - 14691  Non-Patent Document 10: “Proceedings of the National Academy of Science of the United States” (Proceedings of the National Academy of Science of the United States) of America), 2000 cattle, 97 卷, p. 14686-14691
非特許文献 11 :「セル（Cell)」、 2002年、 110卷、 p. 429 -441  Non-Patent Document 11: “Cell”, 2002, 110 卷, p. 429 -441
非特許文献 12：「バイオケミカル ·アンド ·バイオフィジカル ·リサーチ ·コミュニケーショ ンズ (Biochemical ana Biophysical Research Communicationsノ」、 2004 年、 313卷、 p. 387- 389  Non-Patent Document 12: “Biochemical and Biophysical Research Communications”, 2004, 313 卷, p. 387- 389
非特許文献 13 :「ザ'ジャーナル'ォブ 'ニューロサイエンス（The Journal of Neur oscience 2004年、 24卷、 p. 8531— 8541  Non-Patent Document 13: “The Journal of Neuroscience 2004, 24 卷, p. 8531— 8541
非特許文献 14 :「ディべロップメント（Development)」、 2004年、 131卷、 p. 4059 -4070  Non-Patent Document 14: “Development”, 2004, 131 卷, p. 4059 -4070
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0008] 本発明の目的は、神経疾患治療剤、神経新生もしくは再生促進剤、神経機能低下 防止もしくは増進用飲食品および飲食品添加剤、神経幹細胞増殖促進剤、神経幹 細胞、ニューロンもしくはグリア細胞の製造方法、該製造方法により製造される-ユー ロンもしくはグリア細胞、または神経幹細胞増殖促進物質のスクリーニング方法を提 供することにある。 [0008] An object of the present invention is to treat a neurological disease, an agent for neurogenesis or regeneration, a food and drink for preventing or promoting nerve function and an additive for food and drink, a neural stem cell proliferation promoter, a neural stem cell, a neuron, or a glial cell. And a screening method for a substance that promotes proliferation of neurons or glial cells or neural stem cells produced by the production method.
課題を解決するための手段  Means for solving the problem
[0009] 本発明は以下の（1)〜（29)に関する。 ( 1)マンマリアン'ターゲット ·ォブ ·ラパマイシン（以下、 mTORと略す）の活性を促進 する物質を有効成分として含有する神経疾患治療剤。 [0009] The present invention relates to the following (1) to (29). (1) A therapeutic agent for neurological diseases containing as an active ingredient a substance that promotes the activity of Manmarian's target obu rapamycin (hereinafter abbreviated as mTOR).
(2)神経疾患が、パーキンソン病、アルツハイマー病、ダウン症、脳血管障害、脳卒 中、脊髄損傷、トリプレットリピート病、多発性硬化症、筋萎縮性側索硬化症、多発二 ユーロバチ一、てんかん、不安障害、統合失調症、鬱病および躁鬱病からなる群より 選ばれる神経疾患である上記（1)の治療剤。  (2) Neurological diseases are Parkinson's disease, Alzheimer's disease, Down's syndrome, cerebrovascular disorder, stroke, spinal cord injury, triplet repeat disease, multiple sclerosis, amyotrophic lateral sclerosis, multiple euroves, epilepsy, The therapeutic agent according to (1) above, which is a neurological disease selected from the group consisting of anxiety disorders, schizophrenia, depression and manic depression.
(3) mTORの活性を促進する物質が、式 (I)  (3) A substance that promotes mTOR activity is represented by the formula (I)
[0010] [化 1] [0010] [Chemical 1]
Figure imgf000006_0001
Figure imgf000006_0001
[0011] (式中、 R1は水素を表し、 R2はヒドロキシまたは保護されたヒドロキシを表す力 または R1と R2が一緒になつてォキソを表わし、 R3はヒドロキシメチル、保護されたヒドロキシメ チルまたはホルミルを表す)で表される化合物 [以下、化合物 (I)という]またはその薬 理学的に許容される塩である上記（1)または（2)の治療剤。 [Wherein R 1 represents hydrogen, R 2 represents hydroxy or protected hydroxy, or R 1 and R 2 together represent oxo, R 3 represents hydroxymethyl, protected The therapeutic agent according to the above (1) or (2), which is a compound represented by hydroxymethyl or formyl) [hereinafter referred to as compound (I)] or a pharmaceutically acceptable salt thereof.
(4) mTORの活性を促進する物質力 式 (II)  (4) Material force that promotes mTOR activity (II)
[0012] [化 2]  [0012] [Chemical 2]
Figure imgf000006_0002
で表されるビニグロールまたはその薬理学的に許容される塩である上記（1)または（ 2)の治療剤。
Figure imgf000006_0002
The therapeutic agent according to (1) or (2) above, which is biniglol represented by the formula (1) or a pharmacologically acceptable salt thereof.
(5) mTORの活性を促進する物質が、分岐鎖アミノ酸またはその薬理学的に許容さ れる塩である上記（1)または（2)の治療剤。  (5) The therapeutic agent according to (1) or (2) above, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
(6) mTORの活性を促進する物質力 ホスファチジン酸またはその薬理学的に許容 される塩である上記（1)または（2)の治療剤。  (6) A substance capable of promoting mTOR activity The therapeutic agent according to (1) or (2) above, which is phosphatidic acid or a pharmacologically acceptable salt thereof.
(7) mTORの活性を促進する物質を有効成分として含有する神経新生または再生 促進剤。  (7) A neurogenesis or regeneration promoter containing a substance that promotes mTOR activity as an active ingredient.
(8) mTORの活性を促進する物質が化合物 (I)またはその薬理学的に許容される塩 である上記（7)の神経新生または再生促進剤。  (8) The neurogenesis or regeneration promoter according to (7) above, wherein the substance that promotes mTOR activity is compound (I) or a pharmacologically acceptable salt thereof.
(9) mTORの活性を促進する物質がビニグロールまたはその薬理学的に許容される 塩である上記（7)の神経新生または再生促進剤。  (9) The neurogenesis or regeneration promoter according to (7) above, wherein the substance that promotes mTOR activity is biniglol or a pharmacologically acceptable salt thereof.
(10) mTORの活性を促進する物質が分岐鎖アミノ酸またはその薬理学的に許容さ れる塩である上記（7)の神経新生または再生促進剤。  (10) The neurogenesis or regeneration promoter according to (7) above, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
( 11 ) mTORの活性を促進する物質がホスファチジン酸またはその薬理学的に許容 される塩である上記（7)の神経新生または再生促進剤。  (11) The neurogenesis or regeneration promoter according to (7) above, wherein the substance that promotes mTOR activity is phosphatidic acid or a pharmacologically acceptable salt thereof.
(12) mTORの活性を促進する物質を有効成分として含有する神経機能低下防止も しくは増進用飲食品または飲食品添加剤。  (12) A food / beverage product or food / beverage additive for preventing deterioration of nerve function or promoting food containing a substance that promotes mTOR activity as an active ingredient.
(13) mTORの活性を促進する物質が化合物 (I)またはその薬理学的に許容される 塩である上記（12)の飲食品または飲食品添加剤。  (13) The food or drink or food and drink additive according to (12) above, wherein the substance that promotes mTOR activity is compound (I) or a pharmacologically acceptable salt thereof.
( 14) mTORの活性を促進する物質がビニグロールまたはその薬理学的に許容され る塩である上記（12)の飲食品または飲食品添加剤。  (14) The food / beverage product or food / beverage product additive according to the above (12), wherein the substance that promotes mTOR activity is biniglol or a pharmacologically acceptable salt thereof.
(15) mTORの活性を促進する物質が分岐鎖アミノ酸またはその薬理学的に許容さ れる塩である上記（12)の飲食品または飲食品添加剤。  (15) The food or drink or food and drink additive according to (12) above, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
( 16) mTORの活性を促進する物質がホスファチジン酸またはその薬理学的に許容 される塩である上記（12)の飲食品または飲食品添加剤。  (16) The food or drink or food and drink additive according to (12) above, wherein the substance that promotes mTOR activity is phosphatidic acid or a pharmacologically acceptable salt thereof.
(17) mTORの活性を促進する物質を有効成分として含有する神経幹細胞増殖促 進剤。 (18) mTORの活性を促進する物質が化合物 (I)またはその薬理学的に許容される 塩である上記（17)の神経幹細胞増殖促進剤。 (17) A neural stem cell proliferation promoter containing a substance that promotes mTOR activity as an active ingredient. (18) The neural stem cell proliferation promoter according to (17) above, wherein the substance that promotes mTOR activity is compound (I) or a pharmacologically acceptable salt thereof.
(19) mTORの活性を促進する物質がビニグロールまたはその薬理学的に許容され る塩である上記（17)の神経幹細胞増殖促進剤。  (19) The neural stem cell proliferation promoter according to the above (17), wherein the substance that promotes mTOR activity is biniglol or a pharmaceutically acceptable salt thereof.
(20) mTORの活性を促進する物質が分岐鎖アミノ酸またはその薬理学的に許容さ れる塩である上記（17)の神経幹細胞増殖促進剤。  (20) The neural stem cell proliferation promoter according to (17) above, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
(21) mTORの活性を促進する物質がホスファチジン酸またはその薬理学的に許容 される塩である上記（17)の神経幹細胞増殖促進剤。  (21) The neural stem cell proliferation promoter according to (17) above, wherein the substance that promotes mTOR activity is phosphatidic acid or a pharmacologically acceptable salt thereof.
(22)神経幹細胞が成体神経幹細胞である上記（17)〜（21)のいずれか 1つの神経 幹細胞増殖促進剤。  (22) The neural stem cell proliferation promoter according to any one of the above (17) to (21), wherein the neural stem cell is an adult neural stem cell.
(23)上記（17)〜（22)のいずれか 1つの神経幹細胞増殖促進剤の存在下、神経幹 細胞を培養して神経幹細胞を増殖させ、該培養物中より神経幹細胞を採取すること を特徴とする神経幹細胞の製造方法。  (23) In the presence of any one of the above-described neural stem cell proliferation promoters, neural stem cells are cultured to proliferate neural stem cells, and neural stem cells are collected from the culture. A method for producing a neural stem cell, which is characterized.
(24)上記（23)の製造方法により製造される神経幹細胞を培養してニューロンへ分 化させ、該培養物中より-ユーロンを採取することを特徴とする-ユーロンの製造方法  (24) A method for producing euron, characterized in that neural stem cells produced by the production method of (23) are cultured and differentiated into neurons, and euron is collected from the culture.
(25)上記（24)の製造方法により製造されるニューロン。 (25) A neuron produced by the production method of (24) above.
(26)上記（23)の製造方法により製造される神経幹細胞を培養してグリア細胞へ分 化させ、該培養物中よりグリア細胞を採取することを特徴とするグリア細胞の製造方 法。  (26) A method for producing glial cells, comprising culturing neural stem cells produced by the production method according to (23) above to differentiate into glial cells, and collecting glial cells from the culture.
(27)上記（26)の製造方法により製造されるグリア細胞。  (27) A glial cell produced by the production method of (26) above.
(28) (i) mTOR、 mTORによりリン酸化されるペプチド、および ATPを共存させた場 合にぉ 、て、リン酸ィ匕ペプチドの量を測定する工程、  (28) (i) a step of measuring the amount of a phosphorylated peptide when coexisting with mTOR, a peptide phosphorylated by mTOR, and ATP,
(ii)被験物質、 mTOR、 mTORによりリン酸ィ匕されるペプチド、および ATPを共存さ せた場合において、リン酸ィ匕ペプチドの量を測定する工程、  (ii) a step of measuring the amount of phosphate peptide in the presence of test substance, mTOR, peptide phosphorylated by mTOR, and ATP;
(iii) (i)および (ii)の測定量から、被験物質の、該ペプチドに対するリン酸化促進活 性を評価する工程、および  (iii) evaluating the phosphorylation promoting activity of the test substance against the peptide from the measured amounts of (i) and (ii); and
(iv)該リン酸化促進活性を有する物質を神経幹細胞増殖促進物質として選択する 工程、 (iv) A substance having the phosphorylation promoting activity is selected as a neural stem cell proliferation promoting substance Process,
力 なる神経幹細胞増殖促進物質のスクリーニング方法。  A powerful screening method for neural stem cell proliferation promoting substances.
(29)神経幹細胞が成体神経幹細胞である上記（28)のスクリーニング方法。  (29) The screening method according to the above (28), wherein the neural stem cell is an adult neural stem cell.
発明の効果  The invention's effect
[0014] 本発明により、 mTORの活性を促進する物質を有効成分として含有する神経疾患 治療剤、神経新生もしくは再生促進剤、神経機能低下防止もしくは増進用飲食品お よび飲食品添加剤、神経幹細胞増殖促進剤、神経幹細胞、ニューロンもしくはグリア 細胞の製造方法、該製造方法により製造されるニューロンもしくはグリア細胞、または 神経幹細胞増殖促進物質のスクリーニング方法を提供することができる。  [0014] According to the present invention, a therapeutic agent for neurological disease, a neurogenesis or regeneration promoter, a food or drink for preventing or promoting neurological function and a food additive, a neural stem cell, comprising a substance that promotes mTOR activity as an active ingredient A method for producing a proliferation promoter, neural stem cell, neuron or glial cell, a method for screening a neuron or glial cell produced by the production method, or a neural stem cell proliferation promoting substance can be provided.
図面の簡単な説明  Brief Description of Drawings
[0015] [図 1]図 1は、ビニグロールの神経幹細胞増殖促進活性に対する FK506、ラパマイシ ンの影響を示す図である。グラフ横軸の数値は、培地中における FK506、ラパマイ シン、ビニグロールの終濃度を表す。また、グラフの縦軸は、細胞を播種しないゥエル の測定値を 0%、陰性コントロールの測定値を 100%としたときの薬剤添加群におけ る相対値を表す。 *： DMSOコントロールに対する有意差 P< 0. 01、 * *：ビ- グロールに対する有意差 P< 0. 01, * * *：ラパマイシン +ビニグロールに対す る有意差 P< 0. 01 (Unpaired Student 's t— test)  [0015] FIG. 1 is a graph showing the effects of FK506 and rapamycin on the neural stem cell proliferation promoting activity of biniglol. The values on the horizontal axis of the graph represent the final concentrations of FK506, rapamycin, and vinylogol in the medium. In addition, the vertical axis of the graph represents the relative value in the drug addition group when the measured value of the well without cell seeding is 0% and the measured value of the negative control is 100%. *: Significant difference with respect to DMSO control P <0.01, * *: Significant difference with bigol P <0.01, * * *: Significant difference with rapamycin + binigrol P <0.001 (Unpaired Student 'st — Test)
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0016] 1. mTORの活性を促進する物質  [0016] 1. Substances that promote mTOR activity
本発明の神経疾患治療剤、神経新生または再生促進剤、神経機能低下防止もしく は増強用飲食品または飲食品添加剤、ある!、は神経幹細胞増殖促進剤に含有され る mTORの活性を促進する物質としては、 mTORの活性を促進する物質であれば いずれでもよいが、例えばィ匕合物 (1)、分岐鎖アミノ酸、もしくはホスファチジン酸、ま たはそれらの薬理学的に許容される塩等があげられる。  The therapeutic agent for neurological diseases, the neurogenesis or regeneration promoter of the present invention, the prevention of nerve function or the food or drink additive for enhancement, or the food additive, is! Promotes the activity of mTOR contained in the neural stem cell proliferation promoter Any substance may be used as long as it promotes mTOR activity. For example, compound (1), branched chain amino acid, or phosphatidic acid, or a pharmacologically acceptable salt thereof. Etc.
[0017] 化合物 (I)としては、具体的には、例えば上記の式 (Π)で表されるビニグロール、式  As the compound (I), specifically, for example, vinylogol represented by the above formula (Π), formula
(III)〜 (VI)で表わされる化合物等があげられる。  Examples thereof include compounds represented by (III) to (VI).
[0018] [化 3] [9^ ] [Ϊ200] [0018] [Chemical 3] [9 ^] [Ϊ200]
Figure imgf000010_0001
Figure imgf000010_0001
TZ800C/900Zdf/X3d 6 1?56..0/900Ζ OAV TZ800C / 900Zdf / X3d 6 1? 56..0 / 900Ζ OAV
OH OH
[0022] (式中、 、 ぉよび
Figure imgf000011_0001
R2および R3と同義である力 少なくとも R2が保護されたヒドロキシであるカゝ R3が保護されたヒドロキシメチルである） [0022] (In the formula,
Figure imgf000011_0001
A force that is synonymous with R 2 and R 3 At least R 2 is protected hydroxy R 3 is protected hydroxymethyl)
a a  a a
ビニグロールは、例えば特公平 8— 2818記載の方法に従い、ヴィルガリア属に属 するビ-グロール生産菌を培地に培養し、得られる培養物力 ビニグロールを単離す ること〖こより製造することができる。  For example, according to the method described in JP-B-8-2818, biniglol can be produced by culturing a bigol-producing bacterium belonging to the genus Virgaria in a medium and isolating the resulting culture strength biniglol.
[0023] 式 (III)〜 (V)で表わされる化合物は、ビニグロールを常法によりジヨーンズ酸ィ匕す ること〖こよって得ることができる。 [0023] The compounds represented by the formulas (III) to (V) can be obtained by subjecting vinylol to diionates by a conventional method.
式 (VI)で表わされる化合物は、ビニグロール、式 (IV)で表される化合物または式（ The compound represented by the formula (VI) is vinylogol, the compound represented by the formula (IV) or the formula (
V)で表される化合物の 3位のヒドロキシメチル基におけるヒドロキシル基および Zまた は 4位のヒドロキシル基へ常法によりヒドロキシ保護基を導入することにより得ることが できる。 The compound represented by V) can be obtained by introducing a hydroxy protecting group into the hydroxyl group in the hydroxymethyl group at the 3-position and the hydroxyl group at the Z-position or 4-position by a conventional method.
[0024] その際、導入するヒドロキシ保護基の種類、反応試剤等の条件によって、 3位のヒド ロキシメチル基のヒドロキシル基および 4位のヒドロキシル基の片方のみにヒドロキシ 保護基が導入される場合もあり、また、このようにして得られたィ匕合物に再度別のヒド ロキシ保護基が導入される場合もある。さら〖こ、 3位のヒドロキシメチル基のヒドロキシ ル基および 4位のヒドロキシル基の双方にヒドロキシ保護基が導入された化合物を、 常法によりヒドロキシ保護基の脱離反応に付すことによって、片方のヒドロキシル基の みにヒドロキシ保護基が導入されたィ匕合物を得ることもできる。  [0024] In this case, depending on the conditions of the hydroxy protecting group to be introduced, the reaction reagent, etc., the hydroxy protecting group may be introduced into only one of the hydroxyl group of the hydroxyl group at the 3-position and the hydroxyl group at the 4-position. In addition, another hydroxy protecting group may be introduced again into the thus obtained compound. Furthermore, by subjecting a compound having a hydroxy protecting group introduced to both the hydroxyl group of the hydroxymethyl group at the 3-position and the hydroxyl group at the 4-position to the elimination reaction of the hydroxy protecting group by a conventional method, It is also possible to obtain a compound in which a hydroxy protecting group is introduced only at the hydroxyl group.
[0025] 保護されたヒドロキシとしては、通常ヒドロキシル基の保護基として使用される基 (例 えば、 THEODORA W. GREEN, PETER G. M. WUTS著、「PROTECTIV E GROUPS in ORGANIC SYNTHESIS」、第 3版、 WILEY— INTERSCI ENCE社、 1999年、参照）で保護されたヒドロキシであれば特に限定されないが、好 ましくはァセトキシ、プロピオニルォキシ、ブチリルォキシ、イソブチリルォキシ、 tーブ チリルォキシ等の低級アルカノィルォキシ、 t プチルジメチルシリルォキシ等の低級 アルキルシリルォキシ、ベンゾィルォキシ等のァロイルォキシ等があげられる。 [0025] Protected hydroxy is a group usually used as a protecting group for a hydroxyl group (eg, THEODORA W. GREEN, PETER GM WUTS, “PROTECTIV E GROUPS in ORGANIC SYNTHESIS”, 3rd edition, WILEY— INTERSCI ENCE, 1999)), but is not particularly limited, but is preferably a lower alkanoyloxy such as acetoxy, propionyloxy, butyryloxy, isobutyryloxy, tert-tyryloxy, t Lower alkylsilyloxy such as ptyldimethylsilyloxy, and allylooxy such as benzoyloxy.
[0026] また、保護されたヒドロキメチルとしては、通常ヒドロキシメチル基の保護基として使 用される基（例えば、 THEODORA W. GREEN, PETER G. M. WUTS著、「 PROTECTIVE GROUPS in ORGANIC SYNTHESIS」、第 3版、 WILEY — INTERSCIENCE社、 1999年、参照）で保護されたヒドロキシメチルであれば特 に限定されないが、好ましくはァセトキシメチル、プロピオ-ルォキシメチル、ブチリル ォキシメチル、イソブチリルォキシメチル、 tーブチリルォキシメチル等の低級アルカノ ィルォキシメチル、 t ブチルジメチルシリルォキシメチル等の低級アルキルシリルォ キシメチル、ベンゾィルォキシメチル等のァロイルォキシメチル等があげられる。  [0026] Further, as protected hydroxymethyl, a group usually used as a protective group for hydroxymethyl group (for example, “PROTECTIVE GROUPS in ORGANIC SYNTHESIS”, 3rd edition, written by THEODORA W. GREEN, PETER GM WUTS, , WILEY — INTERSCIENCE, 1999)), but not particularly limited, preferably acetoxymethyl, propio-oxymethyl, butyryloxymethyl, isobutyryloxymethyl, tert-butyryloxymethyl, etc. And lower alkylsilyloxymethyl such as t-butyldimethylsilyloxymethyl, allylooxymethyl such as benzoyloxymethyl, and the like.
[0027] 分岐鎖アミノ酸としては、パリン、ロイシンもしくはイソロイシンのそれぞれ単独、また はノ《リン、ロイシンおよびイソロイシンの 2〜3種混合物が好ましぐ該混合物における 各成分の重量比は 0. 5〜1. 5 : 1〜3 : 0. 5〜1. 5であるのが好ましい。  [0027] As the branched chain amino acid, palin, leucine or isoleucine alone, or a mixture of two or three of phosphorus << phosphorus, leucine and isoleucine is preferred. The weight ratio of each component in the mixture is 0.5 to It is preferable that the ratio is 1.5: 1 to 3: 0.5 to 1.5.
また、ノ リン、ロイシンおよびイソロイシンは、それぞれ L体、 D体、および L体と D体 の混合物の 、ずれを用いてもょ 、が、好ましくは L体を用いる。  Norin, leucine and isoleucine may be used in the L-form, D-form, and a mixture of L-form and D-form, respectively, but preferably the L-form.
[0028] 分岐鎖アミノ酸の製造方法は特に限定されず、発酵法、合成法、精製法等で製造 することができ、例えば協和発酵工業株式会社や株式会社協和ウェルネス等から購 人することちでさる。  [0028] The method for producing a branched chain amino acid is not particularly limited, and can be produced by a fermentation method, a synthesis method, a purification method, or the like. For example, by purchasing from Kyowa Hakko Kogyo Co., Ltd. or Kyowa Wellness Co., Ltd. Monkey.
ホスファチジン酸とは、 1, 2 ジァシルグリセロール 3 リン酸をいう。本発明で使用 するホスファチジン酸としては、 mTORの活性を促進するホスファチジン酸であれば いずれでもよいが、例えば、 1 パルミトイルー 2—ォレオイルホスファチジン酸等が あげられる。  Phosphatidic acid refers to 1,2 diacylglycerol triphosphate. The phosphatidic acid used in the present invention may be any phosphatidic acid that promotes the activity of mTOR, and examples thereof include 1 palmitoyl 2-oleoylphosphatidic acid.
[0029] ホスファチジン酸は、卵黄や大豆等力も精製して得ることができ、市販品としてまた は化学合成 (例えば米国特許第 3, 423, 440号）によって得ることもできる。  [0029] Phosphatidic acid can be obtained by purifying egg yolk, soybean, etc., and can also be obtained as a commercial product or by chemical synthesis (for example, US Pat. No. 3,423,440).
化合物 (1)、分岐鎖アミノ酸、またはホスファチジン酸の薬理学的に許容される塩と しては、毒性のない水溶性のものが好ましぐ例えば塩酸塩、硫酸塩、硝酸塩、リン 酸塩などの無機酸塩、酢酸塩、マレイン酸塩、フマル酸塩、クェン酸塩などの有機酸 塩があげられ、薬理学的に許容される金属塩としては、ナトリウム塩、カリウム塩など のアルカリ金属塩、マグネシウム塩、カルシウム塩などのアルカリ土類金属塩、アルミ ニゥム塩、亜鉛塩などがあげられ、薬理学的に許容されるアンモ-ゥム塩としては、ァ ンモ-ゥム、テトラメチルアンモ -ゥムなどの塩があげられ、薬理学的に許容される有 機ァミン付加塩としては、モルホリン、ピぺリジンなどの付加塩等があげられる。 As the pharmacologically acceptable salt of compound (1), branched chain amino acid, or phosphatidic acid, a non-toxic water-soluble salt is preferable, for example, hydrochloride, sulfate, nitrate, phosphorus Examples include inorganic acid salts such as acid salts, organic acid salts such as acetates, maleates, fumarate salts, and citrate salts. Examples of pharmacologically acceptable metal salts include sodium salts and potassium salts. Alkali earth metal salts such as alkali metal salts, magnesium salts, and calcium salts, aluminum salts, zinc salts, and the like. Examples of pharmacologically acceptable ammonium salts include ammonium salts, tetra salts. Examples thereof include salts such as methylammonium, and pharmacologically acceptable organic amine addition salts include addition salts such as morpholine and piperidine.
2.神経疾患治療剤、神経新生または再生剤  2. Neural disease therapeutic agent, neurogenesis or regenerative agent
本発明の神経疾患治療剤が使用される神経疾患としては特に限定されないが、好 ましくは、パーキンソン病、アルツハイマー病、ダウン症、脳血管障害、脳卒中、脊髄 損傷、トリプレットリピート病、多発性硬化症、筋萎縮性側索硬化症、多発-ユーロパ チー、てんかん、不安障害、統合失調症、鬱病、躁鬱病等をあげることができる。  The neurological disease for which the therapeutic agent for neurological diseases of the present invention is not particularly limited, but preferably Parkinson's disease, Alzheimer's disease, Down's syndrome, cerebrovascular disorder, stroke, spinal cord injury, triplet repeat disease, multiple sclerosis , Amyotrophic lateral sclerosis, multiple-europathy, epilepsy, anxiety disorder, schizophrenia, depression, manic depression and the like.
[0030] 本発明の神経新生または再生剤は、ヒトまたは動物の脳内の神経幹細胞に直接作 用することで神経幹細胞の増殖を促進し、その結果、神経の新生または再生を促進 する作用を有する。ここで、神経の新生とは、神経幹細胞の増殖分化によりニューロ ンゃグリア細胞が新生することをいい、神経の再生とは、新生した-ユーロンゃグリア 細胞により上記の神経疾患において脱落した神経回路が再構築され、その機能が 再生されることをいう。 [0030] The neurogenesis or regenerative agent of the present invention promotes the proliferation of neural stem cells by acting directly on the neural stem cells in the brain of humans or animals, and as a result, has the effect of promoting neurogenesis or regeneration. Have. Here, neurogenesis refers to the formation of neuronal glia cells by proliferation and differentiation of neural stem cells, and nerve regeneration refers to neural circuits that have fallen in the above-mentioned neurological diseases due to the born-euron glia cells. Is reconstructed and its functions are regenerated.
[0031] mTORの活性を促進する物質またはその薬理学的に許容される塩は、神経疾患 治療剤、または神経新生もしくは再生剤として、そのまま単独で投与することも可能で あるが、通常各種の医薬製剤として提供するのが望ましい。また、それら医薬製剤は 、動物および人に使用されるものである。  [0031] The substance that promotes mTOR activity or a pharmacologically acceptable salt thereof can be administered alone as it is as a therapeutic agent for neurological diseases, or as a neurogenesis or regenerative agent. It is desirable to provide it as a pharmaceutical formulation. These pharmaceutical preparations are used for animals and humans.
本発明の神経疾患治療剤、または神経新生もしくは再生剤は、活性成分として mT ORの活性を促進する物質またはその薬理学的に許容される塩を単独で、または任 意の他の治療のための有効成分との混合物として含有することができる。また、それ ら医薬製剤は、活性成分を薬理学的に許容される一種またはそれ以上の担体と一 緒に混合し、製剤学の技術分野にぉ 、てよく知られて 、る任意の方法により製造さ れる。  The therapeutic agent for neurological disease or neurogenesis or regenerative agent of the present invention is a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof as an active ingredient, alone or for any other treatment. It can contain as a mixture with this active ingredient. These pharmaceutical preparations can be prepared by any method well known in the technical field of pharmaceutics by mixing the active ingredient together with one or more pharmaceutically acceptable carriers. Manufactured.
[0032] 投与経路は、治療に際し最も効果的なものを使用するのが望ましぐ経口または、 例えば静脈内等の非経口をあげることができる。 [0032] The route of administration is preferably oral or the one that is most effective in treatment, or For example, parenteral such as intravenous can be mentioned.
投与形態としては、錠剤、散剤、顆粒剤、シロップ剤、注射剤等があげられる。 経口投与に適当な、例えばシロップ剤のような液体調製物は、水、蔗糖、ソルビット Examples of the dosage form include tablets, powders, granules, syrups, injections and the like. Liquid preparations suitable for oral administration, such as syrups, are water, sucrose, sorbit
、果糖等の糖類、ポリエチレングリコール、プロピレングリコール等のダリコール類、ご ま油、ォリーブ油、大豆油等の油類、 P—ヒドロキシ安息香酸エステル類等の防腐剤 、ストロベリーフレーバー、ペパーミント等のフレーバー類等を使用して製造できる。 また、錠剤、散剤および顆粒剤等は、乳糖、ブドウ糖、蔗糖、マンニット等の賦形剤、 澱粉、アルギン酸ソーダ等の崩壊剤、ステアリン酸マグネシウム、タルク等の滑沢剤、 ポリビニールアルコール、ヒドロキシプロピルセルロース、ゼラチン等の結合剤、脂肪 酸エステル等の界面活性剤、グリセリン等の可塑剤等を用いて製造できる。 , Sugars such as fructose, Daricols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, preservatives such as P-hydroxybenzoate esters, flavors such as strawberry flavor and peppermint Etc. can be used. Tablets, powders and granules are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy It can be produced using a binder such as propylcellulose and gelatin, a surfactant such as a fatty acid ester, and a plasticizer such as glycerin.
[0033] 非経口投与に適当な製剤は、好ましくは受容者の血液と等張である活性ィ匕合物を 含む滅菌水性剤からなる。例えば、注射剤の場合は、塩溶液、ブドウ糖溶液または 塩水とブドウ糖溶液の混合物カゝらなる担体等を用いて注射用の溶液を調製する。 また、これら非経口剤においても、経口剤で例示した希釈剤、防腐剤、フレーバー 類、賦形剤、崩壊剤、滑沢剤、結合剤、界面活性剤、可塑剤等から選択される 1種ま たはそれ以上の補助成分を添加することもできる。  [0033] Formulations suitable for parenteral administration preferably comprise a sterile aqueous preparation containing an active compound which is isotonic with the blood of the recipient. For example, in the case of an injection, a solution for injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of salt water and a glucose solution. Also in these parenteral agents, one kind selected from diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc., exemplified for oral agents. Or more auxiliary ingredients can be added.
[0034] mTORの活性を促進する物質またはその薬理学的に許容される塩の投与量およ び投与回数は、投与形態、患者の年齢、体重、治療すべき症状の性質または重篤 度により異なるが、経口投与の場合、通常、成人一人当り 0. 01mg〜20g、好ましく は 0. 05mg〜10gを一日一回ないし数回投与する。静脈内投与等の非経口投与の 場合、通常、成人一人当り 0. 001mg〜20g、好ましくは 0. Olmg〜： LOgを一日一回 ないし数回投与する。  [0034] The dose and frequency of administration of a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof depends on the dosage form, patient age, body weight, nature or severity of the condition to be treated. Although different, in the case of oral administration, usually 0.01 mg to 20 g, preferably 0.05 mg to 10 g per adult is administered once to several times a day. In the case of parenteral administration such as intravenous administration, 0.001 mg to 20 g per adult, preferably 0. Olmg to: LOg is administered once to several times a day.
3.神経機能低下防止または増強用飲食品および飲食品添加物  3. Food / beverage products and food / drink additives for prevention or enhancement of neurological function
本発明の飲食品としては、飲食品に mTORの活性を促進する物質またはその薬理 学的に許容される塩を添加したものをあげることができる。  Examples of the food and drink of the present invention include food and drink obtained by adding a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof.
[0035] 本発明の飲食品は、飲食品に mTORの活性を促進する物質またはその薬理学的 に許容される塩を添加する以外は、一般的な飲食品の製造方法を用いることにより、 加工製造することができる。 本発明の飲食品は、例えば流動層造粒、攪拌造粒、押し出し造粒、転動造粒、気 流造粒、圧縮成形造粒、解砕造粒、噴霧造粒、噴射造粒等の造粒方法、パンコーテ イング、流動層コーティング、ドライコーティング等のコーティング方法、パフドライ、過 剰水蒸気法、フォームマット方法、マイクロ波加熱方法等の膨化方法、押出造粒機や エキストルーダー等の押出方法等を用いて製造することもできる。 [0035] The food / beverage product of the present invention is processed by using a general method for producing a food / beverage product, except that a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof is added to the food / beverage product. Can be manufactured. The food and drink of the present invention are, for example, fluidized bed granulation, stirring granulation, extrusion granulation, rolling granulation, gas flow granulation, compression molding granulation, pulverization granulation, spray granulation, spray granulation, etc. Granulation method, pan coating, fluidized bed coating, coating method such as dry coating, puff drying, excess steam method, foam mat method, expansion method such as microwave heating method, extrusion granulator, extrusion method such as extruder, etc. It can also be manufactured using.
[0036] 本発明の飲食品は、ジュース類、清涼飲料水、茶類、乳酸菌飲料、発酵乳、冷菓、 バター、チーズ、ヨーグルト、加工乳、脱脂乳等の乳製品、ハム、ソーセージ、ハンバ ーグ等の畜肉製品、蒲鋅、竹輪、さつま揚げ等の魚肉練り製品、だし巻き、卵豆腐等 の卵製品、クッキー、ゼリー、チューインガム、キャンデー、喉飴、スナック菓子等の菓 子類、パン類、麵類、漬物類、燻製品、干物、佃煮、塩蔵品、スープ類、調味料等、 V ずれの形態のものであってもよ!/ \  [0036] The food and drink of the present invention includes juices, soft drinks, teas, lactic acid bacteria beverages, fermented milk, frozen desserts, butter, cheese, yogurt, processed milk, skim milk and other dairy products, ham, sausage, hamburger Livestock meat products such as fish, fish paste such as bamboo shoots, bamboo rings, and fish cakes, egg products such as soup rolls, egg tofu, cookies, jelly, chewing gum, candy, throat candy, snacks and other confectionery, breads, potatoes , Pickles, salmon products, dried fish, boiled fish, salted products, soups, seasonings, etc.
[0037] また、本発明の飲食品は、例えば粉末食品、シート状食品、瓶詰め食品、缶詰食 品、レトルト食品、カプセル食品、タブレット状食品、流動食品、ドリンク剤等の形態の ものであってもよい。  [0037] The food and drink of the present invention are in the form of, for example, powdered food, sheet-like food, bottled food, canned food, retort food, capsule food, tablet food, liquid food, and drink. Also good.
本発明の飲食品添加剤は、上記 2で説明した経口剤と同様な方法により調製する ことができる。当該飲食品添加剤は、通常、必要に応じて他の飲食品添加剤を混合 または溶解し、例えば粉末、顆粒、ペレット、錠剤、各種液剤の形態に加工製造され る。  The food and drink additive of the present invention can be prepared by the same method as the oral preparation described in 2 above. The food / beverage food additive is usually processed and manufactured in the form of powder, granules, pellets, tablets, or various liquids by mixing or dissolving other food / beverage food additives as necessary.
[0038] 本発明の飲食品または飲食品添加剤には、一般的に飲食品に用いられる飲食品 添加剤、例えば甘味料、着色料、保存料、増粘安定剤、酸化防止剤、発色料、漂白 料、防かび剤、ガムベース、苦味料、酵素、光沢剤、酸味料、調味料、乳化剤、強化 剤、製造用剤、香料、香辛料抽出物等が添加されてもよい。  [0038] The food / beverage products or food / beverage product additives of the present invention include food / beverage product additives generally used for food / beverage products, such as sweeteners, colorants, preservatives, thickening stabilizers, antioxidants, colorants. Bleaching agents, fungicides, gum bases, bittering agents, enzymes, brighteners, acidulants, seasonings, emulsifiers, fortifiers, manufacturing agents, fragrances, spice extracts and the like may be added.
本発明の飲食品は、神経機能低下防止もしくは増強用健康食品または機能性食 品等の飲食品として用いることができる。  The food or drink of the present invention can be used as a food or drink such as a health food for preventing or enhancing neurological function or a functional food.
[0039] 本発明の飲食品への mTORの活性を促進する物質またはその薬理学的に許容さ れる塩、または本発明の飲食品添加剤の添加量は、飲食品の種類、当該飲食品の 摂取により期待する効果等に応じて適宜選択されるが、 mTORの活性を促進する物 質またはその薬理学的に許容される塩として、通常は 0. 1〜： LOO重量％、好ましくは 1〜70重量％、特に好ましくは 5〜50重量％含有するように添加される。 [0039] The amount of the substance that promotes mTOR activity to the food or drink of the present invention or a pharmacologically acceptable salt thereof, or the food or drink additive of the present invention depends on the type of the food or drink, Although it is appropriately selected depending on the expected effect of ingestion, etc., as a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof, usually 0.1 to: LOO% by weight, preferably It is added so as to contain 1 to 70% by weight, particularly preferably 5 to 50% by weight.
[0040] 本発明の飲食品の摂取量は、摂取形態、摂取者の年齢、体重等に応じて異なるが 、 mTORの活性を促進する物質またはその薬理学的に許容される塩として、通常、 成人一人当たり 0. 01mg〜20g、好ましくは 0. 05mg〜： LOgを一曰一回ないし数回 摂取する。 [0040] The intake of the food and drink of the present invention varies depending on the intake form, the age of the user, the body weight, etc., but as a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof, For adults, 0.01 mg to 20 g, preferably 0.05 mg or more: Take LOg once or several times.
4.神経幹細胞増殖促進剤  4. Neural stem cell proliferation promoter
本発明の神経幹細胞増殖促進剤は、 in vivoまたは in vitroにおいて、神経幹細 胞と接触させたとき、該神経幹細胞の増殖を促進することができる。  The neural stem cell proliferation promoter of the present invention can promote proliferation of neural stem cells when contacted with neural stem cells in vivo or in vitro.
[0041] 幹細胞とは、複数の異なる種類の細胞に分化する多分化能と、対称的または非対 称的な分裂によって新たな幹細胞を生み出す自己複製能とを有する細胞をいう。こ れに対し、ある分ィ匕の系譜に入り、限られた回数の分裂の後に分化を遂げるように運 命づけられた細胞は前駆細胞と呼ばれる。しかし、神経幹細胞と神経前駆細胞また はグリア前駆細胞とを厳密に区別することは困難であるため、本発明では、神経幹細 胞、神経前駆細胞、およびグリア前駆細胞を合わせて神経幹細胞と称する。  [0041] Stem cells refer to cells having multipotency to differentiate into a plurality of different types of cells and self-renewal ability to generate new stem cells by symmetrical or asymmetric division. On the other hand, a cell that has entered a lineage of a certain amount and is destined to undergo differentiation after a limited number of divisions is called a progenitor cell. However, since it is difficult to strictly distinguish neural stem cells from neural progenitor cells or glial progenitor cells, in the present invention, neural stem cells, neural progenitor cells, and glial progenitor cells are collectively referred to as neural stem cells. .
[0042] 神経幹細胞は、神経幹細胞であれば特に限定されな 、が、脳の成体神経幹細胞 が好ましい。  [0042] The neural stem cell is not particularly limited as long as it is a neural stem cell, but is preferably an adult neural stem cell of the brain.
脳は、いずれの動物の脳であってもよいが、好ましくは哺乳動物、より好ましくはラッ ト、マウス、サル、ヒト等の脳をあげることができる。  The brain may be the brain of any animal, preferably a mammal, more preferably a rat, mouse, monkey, human or the like.
mTORの活性を促進する物質またはその薬理学的に許容される塩は、神経幹細 胞増殖促進剤として、そのまま単独で用いることも可能であるが、通常各種の医薬製 剤として提供するのが望ましい。また、それら医薬製剤は、動物および人に使用され るものである。  A substance that promotes mTOR activity or a pharmacologically acceptable salt thereof can be used alone as a neural stem cell proliferation promoter, but it is usually provided as various pharmaceutical products. desirable. These pharmaceutical preparations are used for animals and humans.
[0043] 本発明の神経幹細胞増殖促進剤は、活性成分として mTORの活性を促進する物 質またはその薬理学的に許容される塩を単独で、または任意の他の治療のための有 効成分との混合物として含有することができる。それら医薬製剤は、上記 2の神経疾 患治療薬の製剤と同様の方法により製造することができ、同様の投与方法により投与 することができる。  [0043] The neural stem cell proliferation promoter of the present invention is a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof as an active ingredient, or an active ingredient for any other treatment. And a mixture thereof. These pharmaceutical preparations can be produced by the same method as that for the preparation of the therapeutic agent for neurological diseases described above, and can be administered by the same administration method.
本発明の神経幹細胞増殖促進剤は、 in vitroにおいて、神経幹細胞と接触させ、 該神経幹細胞を培養することにより、神経幹細胞の増殖を促進させ、培養物から該 神経幹細胞を採取することを特徴とする神経幹細胞の製造法に用いることができる。 The neural stem cell proliferation promoter of the present invention is brought into contact with neural stem cells in vitro, By culturing the neural stem cell, the proliferation of the neural stem cell is promoted, and the neural stem cell can be collected from the culture, and can be used for a method for producing a neural stem cell.
[0044] 本発明の神経幹細胞増殖促進剤を i2__idimで用いる場合、 mTORの活性を促進 する物質またはその薬理学的に許容される塩を、該物質または該塩を溶解すること ができる溶液に溶解して用いることが好ましい。該溶液としては、水、 DMSO等をあ げることができる。  [0044] When the neural stem cell proliferation promoter of the present invention is used in i2_idim, a substance that promotes mTOR activity or a pharmacologically acceptable salt thereof is dissolved in a solution that can dissolve the substance or the salt. And preferably used. Examples of the solution include water and DMSO.
5.神経幹細胞増殖促進剤による神経幹細胞の増殖方法  5. Neural stem cell proliferation method using neural stem cell proliferation promoter
本発明の神経幹細胞増殖促進剤の存在下、動物の神経幹細胞を培養すること〖こ より、該神経幹細胞の増殖を積極的に促進させることができる。  By culturing animal neural stem cells in the presence of the neural stem cell proliferation promoter of the present invention, proliferation of the neural stem cells can be actively promoted.
[0045] 動物の神経幹細胞は、 、ずれの動物の神経幹細胞であってもよぐ好ましくは哺乳 動物、より好ましくはラット、マウス、サル、ヒト由来の神経幹細胞をあげることができ、 神経幹細胞としては、脳由来の神経幹細胞をあげることができる。神経幹細胞は、い ずれの週齢、または年齢の動物由来の細胞でもよいが、好ましくは成体神経幹細胞 をあげることができる。 [0045] The neural stem cells of an animal may be neural stem cells of any animal, preferably mammals, more preferably rat, mouse, monkey, or human-derived neural stem cells. Can include brain-derived neural stem cells. The neural stem cells may be cells derived from animals of any age or age, but preferably adult neural stem cells.
[0046] 動物力 成体神経幹細胞を取得する方法としては、 ["ザ ·ジャーナル ·ォブ 'ニュー 口サイエンス（The Journal of Neuroscinece) "1999年、 19卷、 p. 8487— 84 97]、 ["ジーンズ'アンド'デベロップメント（Genes and Development) "1996年 、 10卷、 p. 3129— 3140]等に記載の方法に準じて、外科的手法によって成体動 物から脳を摘出して、脳細胞粗抽出液を調製し、該粗抽出液から成体幹細胞を濃縮 する方法をあげることがでさる。  [0046] Animal power As a method for obtaining adult neural stem cells, ["The Journal of Neuroscinece" 1999, 19th, p. 8487—84 97], [" Jeans 'and' Development (Genes and Development) 1996, 10 卷, p. 3129-3140], etc., brains are extracted from adult animals by surgical technique, and rough extraction of brain cells It is possible to prepare a solution and concentrate adult stem cells from the crude extract.
[0047] また、ヒトから成体神経幹細胞を取得する方法としては、 ["ェタスペリメンタル ·セル •リサーチ（Experimental Cell Research) "2003年、 289卷、 p. 378- 383]記 載の方法に準じて、ノ ィォプシ一によつて神経疾患患者の側脳室壁力も組織を採取 して、脳細胞粗抽出液を調製し、該抽出液から成体幹細胞を濃縮する方法をあげる ことができる。  [0047] As a method for obtaining adult neural stem cells from humans, the method described in ["Experimental Cell Research" 2003, p. 378-383] is used. Similarly, a method of collecting a tissue of the lateral ventricular wall force of a neurological patient by using neuropsi, preparing a crude brain cell extract, and concentrating adult stem cells from the extract can be mentioned.
[0048] 本発明の神経幹細胞増殖促進剤存在下で成体神経幹細胞を培養する場合、 6. 2 5 X 104個 Zcm2程度の成体神経幹細胞に対して、該神経幹細胞増殖促進剤を 10 nm0lZl〜lmmolZlの濃度で作用させることが好まし ヽ。成体神経幹細胞と本発明 の神経幹細胞の増殖促進剤を接触させ、 37°Cで 5%CO雰囲気下、 1 [0048] When culturing adult neural stem cells in the presence of the neural stem cell proliferation promoter of the present invention, the neural stem cell proliferation promoter is applied to an adult neural stem cell of about 6.25 × 10 4 Zcm 2 at 10 nm. It is preferable to work at a concentration of 0 lZl to lmmolZl. Adult neural stem cells and the present invention Contacted with a neural stem cell growth promoter, at 37 ° C in a 5% CO atmosphere, 1
2 〜14日間、 2 日おきに全量または部分量培地交換しながら静置培養することで神経幹細胞の増 殖を促進させることができる。  The growth of neural stem cells can be promoted by culturing for 2 to 14 days every two days while changing the whole amount or a partial amount of the medium.
[0049] 該培地は、神経幹細胞の増殖促進を妨げな 、培地であれば!/、ずれの培地でもよ V、が、 1%の N— 2添カ卩物（インビトロジェン社製）を含む DMEMZF12培地（インビト ロジェン社製)等を用いるのが好まし 、。 [0049] The medium contains 1% N-2 supplement (made by Invitrogen Corp.) as long as the medium does not hinder the promotion of neural stem cell growth! It is preferable to use a medium (Invitrogen) or the like.
また、上記培養により取得される神経幹細胞を、本発明の神経幹細胞増殖促進剤 を含有せず lnM〜lmMのオールトランスレチノイン酸、 lnM〜lmMのフオルスコリ ン、または 0. lngZml〜lmgZmlの血小板由来成長因子（PDGF)等を含有する 培地で、 37°Cで 5%CO雰囲気下、 1  In addition, the neural stem cells obtained by the culture described above do not contain the neural stem cell proliferation promoter of the present invention, lnM to lmM all-trans retinoic acid, lnM to lmM forskolin, or 0.lngZml to lmgZml platelet-derived growth. Medium containing factor (PDGF), etc., at 37 ° C, 5% CO atmosphere, 1
2 〜14日間、 2日おきに全量または部分量培地 交換しながら静置培養することで-ユーロンへ分化させることができる。  It can be differentiated into -euron by culturing for 2 to 14 days every two days while changing the whole or partial medium.
[0050] 該培地は、ニューロンへの分化を妨げな 、培地であれば 、ずれの培地でもよ！/、が 、 1%の N— 2添カ卩物（インビトロジェン社製）を含む DMEMZF12培地（インビトロジ ェン社製)等を用いるのが好ま 、。 [0050] The medium may be a misaligned medium as long as it does not prevent differentiation into neurons! However, it is preferable to use DMEMZF12 medium (Invitrogen) or the like containing 1% N-2 supplement (Invitrogen).
さらに、上記培養により取得される神経幹細胞を、本発明の神経幹細胞増殖促進 剤を含有せず、 0. IngZml〜： LmgZmlの白血病阻止因子（LIF)、または 0. lng Zml〜lmgZmlの骨形成因子ー2 (BMP— 2)等を含有する培地で、 37°Cで 5%C O雰囲気下、 1〜14日間、 2日おきに全量または部分量培地交換しながら静置培養 Furthermore, the neural stem cells obtained by the above culture do not contain the neural stem cell proliferation promoter of the present invention, and 0. IngZml to: LmgZml of leukemia inhibitory factor (LIF), or 0. lng Zml to lmgZml of osteogenic factor -2 Medium containing (BMP-2), etc., static culture at 37 ° C in 5% CO atmosphere for 1-14 days, changing the whole or partial volume every 2 days
2 2
することでグリア細胞へ分ィ匕させることができる。  By doing so, it can be separated into glial cells.
[0051] 該培地は、グリア細胞への分ィ匕を妨げな 、培地であれば 、ずれの培地でもよ！/、が 、 1%の N— 2添カ卩物（インビトロジェン社製）を含む DMEMZF12培地（インビトロジ ェン社製)等を用いるのが好ま 、。 [0051] As long as the medium does not interfere with the separation of glial cells, any medium may be used! However, it is preferable to use DMEMZF12 medium (Invitrogen) or the like containing 1% N-2 supplement (Invitrogen).
上記培養により取得される神経幹細胞、ニューロン、またはグリア細胞は、培地から 回収し、神経疾患患者の障害部位へ外科的手法で移植することにより該神経疾患の 治療に用いることができる。該神経疾患としては、パーキンソン病、アルツハイマー病 、ダウン症、脳血管障害、脳卒中、脊髄損傷、トリプレットリピート病、多発性硬化症、 筋萎縮性側索硬化症、多発ニューロバチ一、てんかん、不安障害、統合失調症、鬱 病、躁鬱病等をあげることができる。 6.神経幹細胞増殖促進物質のスクリーニング方法 Neural stem cells, neurons, or glial cells obtained by the above culture can be collected from a medium and transplanted to a damaged site of a neurological disease patient by a surgical technique to be used for treatment of the neurological disease. The neurological diseases include Parkinson's disease, Alzheimer's disease, Down's syndrome, cerebrovascular disorder, stroke, spinal cord injury, triplet repeat disease, multiple sclerosis, amyotrophic lateral sclerosis, multiple neurobaty, epilepsy, anxiety disorder, integration Examples include ataxia, depression, manic depression. 6. Screening method for neural stem cell proliferation promoter
本発明の神経幹細胞増殖促進物質のスクリーニング方法としては、（i) mTOR、 m TORによりリン酸ィ匕されるペプチド、および ATPを共存させた場合において、リン酸 化ペプチドの量を測定する工程、（ii)被験物質、 mTOR、 mTORによりリン酸ィ匕され るペプチド、および ATPを共存させた場合において、リン酸化ペプチドの量を測定す る工程、 (iii) (i)および (ii)の測定量から、被験物質の、該ペプチドに対するリン酸化 促進活性を評価する工程、 (iv)該リン酸化促進活性を有する物質を神経幹細胞増 殖促進物質として選択する工程、カゝらなる方法をあげることができる。  The method for screening a neural stem cell proliferation promoting substance of the present invention includes (i) a step of measuring the amount of a phosphopeptide in the presence of mTOR, a peptide phosphorylated by mTOR, and ATP, (Ii) a step of measuring the amount of phosphorylated peptide in the presence of test substance, mTOR, peptide phosphorylated by mTOR, and ATP; (iii) measurement of (i) and (ii) A step of evaluating the phosphorylation-promoting activity of the test substance against the peptide from the amount; (iv) a step of selecting the substance having the phosphorylation-promoting activity as a neural stem cell proliferation-promoting substance; Can do.
[0052] 神経幹細胞は、神経幹細胞であれば特に限定されな 、が、脳の成体神経幹細胞 が好ましい。脳は、いずれの動物の脳であってもよいが、好ましくは哺乳動物、より好 ましくはラット、マウス、サル、ヒト等の脳をあげることができる。  [0052] The neural stem cell is not particularly limited as long as it is a neural stem cell, but is preferably an adult neural stem cell of the brain. The brain may be the brain of any animal, but is preferably a mammal, more preferably a rat, mouse, monkey, human or the like.
被験物質は、特に限定されないが、例えば、ペプチド、蛋白質、細胞抽出液ゃ該抽 出液由来の精製物、細胞培養上清や該上清由来の精製物、血清などの生体試料や 該生体試料由来の精製物、微生物の菌体抽出液ゃ該抽出液由来の精製物、微生 物培養上清や該上清由来の精製物、化合物、コンビナトリアルケミストリーを用いて 合成された化合物等をあげることができる。  The test substance is not particularly limited. For example, peptides, proteins, cell extracts, purified products derived from the extracts, cell culture supernatants, purified products derived from the supernatants, biological samples such as serum, and the biological samples Purified products derived from microorganisms, purified cells derived from microorganisms, purified products derived from these extracts, purified supernatants from microorganisms, purified products derived from these supernatants, compounds, compounds synthesized using combinatorial chemistry, etc. Can do.
[0053] mTORとしては、 mTORの活性を有するものであれば特に限定されな!、が、好まし くは哺乳類、より好ましくはラット、マウス、サルまたはヒト由来の mTORをあげることが でき、具体的には ["サイエンシズ ·シグナル ·トランスダクシヨン'ナリッジ ·エンバイロメ ント (science s signal Transduction Knowledge Environment) '2003年 、 212卷、 pp. rel5]に記載の蛋白質をあげることができる。  [0053] The mTOR is not particularly limited as long as it has mTOR activity !, but preferably mTOR can be mentioned from mammals, more preferably rat, mouse, monkey or human. For example, the protein described in ["Sciences Signal Transduction 'Narrange Environment (Sciences Signal Transduction Knowledge Environment)' 2003, 212 卷, pp. Rel5] can be mentioned.
[0054] mTORは mTORを発現して!/、る細胞から免疫沈降法により取得するすることがで きる。該細胞は mTORを発現している細胞であれば特に限定されないが、好ましくは 哺乳類、より好ましくはラット、マウス、サルまたはヒト由来の神経幹細胞をあげること ができる。  [0054] mTOR can be obtained from cells expressing mTOR! /, By immunoprecipitation. The cells are not particularly limited as long as they express mTOR, but preferably include neural stem cells derived from mammals, more preferably rats, mice, monkeys or humans.
mTORによりリン酸化されるペプチドとしては、リボゾ一マル S6キナーゼ（S6K)、 e ukaryotic initiation factor— 4E binding protein 1 (4E— BP1)等をめげ ことができ、具体的には ["ユーロビアン'ジャーナル ·ォブ ·バイオケミストリ一 (Europ ean Journal of Biochemistry) "2002年、 269卷、 p. 5338— 5349]に記載の ペプチドをあげることができる。 Examples of peptides phosphorylated by mTOR include ribozomal S6 kinase (S6K) and eukaryotic initiation factor—4E binding protein 1 (4E—BP1). · Ob · Biochemistry (Europ ean Journal of Biochemistry) "2002, 269 卷, p. 5338-5349].
[0055] mTORの活性を測定する方法としては、例えばリン酸の供与体である ATPとして [  [0055] As a method for measuring the activity of mTOR, for example, ATP which is a donor of phosphate [
Ύ 32 P]ATPを用い、被験物質存在下、または被験物質非存在下において、免疫 沈降法で取得した mTORもしくは mTORを含有する複合体による p70S6Kまたは 4 E— BP1、もしくは該酵素のリン酸ィ匕部位を含むペプチドのリン酸ィ匕反応を行い、該 酵素またはペプチドに取り込まれた32 Pの量を液体シンチレーシヨンカウンタ一等を用 いて測定する方法をあげることができる。また、放射性物質で標識されていない ATP を使用する場合は、リン酸化反応の後、リン酸化された基質酵素またはペプチドを特 異的に認識する抗体を用い、蛍光や化学発光などで標識した二次抗体を使用して 柳』定することができる。 Using Ύ 32 P] ATP, a test substance the presence or in the subject matter absence phosphorus Sani of p70S6K or 4 E- BP1 or enzyme, by complex containing mTOR or mTOR obtained by immunoprecipitation An example is a method in which a phosphoric acid-containing reaction of a peptide containing a sputum site is performed, and the amount of 32 P incorporated into the enzyme or peptide is measured using a liquid scintillation counter or the like. In addition, when using ATP that is not labeled with a radioactive substance, an antibody that specifically recognizes the phosphorylated substrate enzyme or peptide is used after the phosphorylation reaction and labeled with fluorescence or chemiluminescence. The following antibody can be used to determine “willow”.
7.神経疾患治療剤の評価法  7. Evaluation methods for therapeutic agents for neurological diseases
本発明の神経疾患治療剤が、 ίΩϋΩにおいて神経幹細胞を増殖させ、神経疾患 を治療することができることは、以下の方法により確認することができる。  It can be confirmed by the following method that the therapeutic agent for neurological diseases of the present invention can proliferate neural stem cells at ίΩϋΩ to treat neurological diseases.
[0056] 上述の本発明の神経疾患治療剤を、ラットまたはサル等の実験動物に投与する。  [0056] The therapeutic agent for a neurological disease of the present invention described above is administered to a laboratory animal such as a rat or monkey.
実験動物は、傷害を有していない健康な動物であってもよいが、海馬虚血傷害を与 えることにより神経の新生または再生を効果的に観察することができるので ["セル (C ell)，，2002年、 110卷、 p. 429- 441]、虚血、 6 - hydroxydopamine (6 - OHDA )投与またはカイニン酸投与等の方法により、脳に傷害を与えた動物が好ましい。投 与経路としては、経口、口腔内、皮下、筋肉内、静脈内または脳室内等への投与を あげることができる。投与量、投与方法としては、例えば体重 lkg当り 100ng〜500 mg、好ましくは 500ng〜200mgを一日一回ないし数回投与する。静脈内投与等の 非経口投与の場合、体重 lkg当り 10ng〜500mg、好ましくは 100ng〜200mgを一 日一回ないし数回投与する。  The experimental animal may be a healthy animal that does not have an injury, but it is possible to effectively observe the neurogenesis or regeneration by giving hippocampal ischemic injury. ), 2002, 110 卷, p. 429-441], preferred is an animal whose brain is damaged by ischemia, 6-hydroxydopamine (6-OHDA) administration or kainic acid administration. As an example, oral, buccal, subcutaneous, intramuscular, intravenous or intracerebroventricular administration, etc. The dosage and administration method are, for example, 100 ng to 500 mg, preferably 500 ng to 200 mg per kg body weight. In the case of parenteral administration such as intravenous administration, 10 ng to 500 mg, preferably 100 ng to 200 mg per kg body weight is administered once to several times a day.
[0057] 増殖した神経幹細胞は以下の方法により検出することができる。  [0057] Proliferated neural stem cells can be detected by the following method.
増殖細胞を標識することができる 5—プロモー 2'—デォキシゥリジン (BrdU)、また は Green Fluorescent Protein (GFP)やベータガラクトシダーゼ等の細胞標識 可能な遺伝子を発現できるレトロウイルスベクターを該物質の最初の投与と同時、投 与前または投与後に該実験動物に投与した後、該物質を一日一回ないし数回投与 して 10〜20日間飼育する。その後、該実験動物の脳を摘出し、脳の凍結切片を調 製して蛍光顕微鏡を用いて観察し、例えば増殖細胞を標識する薬剤として BrdUを 用いた場合は、単位面積当たりの BrdU陽性細胞数を、陰性コントロールと比較する The first administration of the substance is a retroviral vector that can express cell-labelable genes such as 5-promo 2'-deoxyuridine (BrdU) or Green Fluorescent Protein (GFP) or beta-galactosidase that can label proliferating cells. At the same time After administration to the experimental animals before or after administration, the substance is administered once to several times a day and kept for 10 to 20 days. Thereafter, the brain of the experimental animal is removed, and a frozen section of the brain is prepared and observed using a fluorescence microscope. For example, when BrdU is used as a drug for labeling proliferating cells, BrdU positive cells per unit area Compare numbers with negative controls
[0058] 以上の方法により、本発明の神経疾患治療薬の神経幹細胞増殖促進作用および 神経疾患治療効果を評価することができる。 [0058] According to the above method, the neural stem cell proliferation promoting action and the therapeutic effect on the neurological disease of the therapeutic agent for the neurological disease of the present invention can be evaluated.
8.神経幹細胞増殖促進剤の評価法  8. Evaluation method of neural stem cell proliferation promoter
本発明の神経幹細胞増殖促進剤が、 in vitroにお 、て神経幹細胞の増殖を促進 することができることは、以下の方法により確認することができる。  It can be confirmed by the following method that the neural stem cell proliferation promoter of the present invention can promote the proliferation of neural stem cells in vitro.
[0059] 上記 5の方法で取得した神経幹細胞を、 1%の N— 2添加物を含む DMEMZF12 培地に 2. 0 X 104{@/0. 1mlの濃度で懸濁し、ポリオル-チンおよびラミニン等で表 面加工した 96穴プレート等に播種して、 37°Cで 5%CO雰囲気下、インキュベートす [0059] The neural stem cells obtained by the above method 5 are suspended in DMEMZF12 medium containing 1% N-2 additive at a concentration of 2.0 X 10 4 {@ / 0. 1 ml, and polyortin and laminin Seed in a 96-well plate, etc. that has been surface-treated, etc., and incubate at 37 ° C in a 5% CO atmosphere.
2  2
る。細胞が底面に接着した後、 PBSまたは DMSOに溶解した被験物質を上記培地 または PBSで 0. 01nmolZl〜100mmolZlの濃度範囲で段階的に希釈し、添加す る。陰性コントロールとして同容量の該培地または PBSを添加する。また、陽性コント ロールとしては FGF2や EGF等を用いることが好ましい。その後、 5%CO雰囲気下  The After the cells adhere to the bottom surface, the test substance dissolved in PBS or DMSO is diluted stepwise with the above medium or PBS in a concentration range of 0.01 nmolZl to 100 mmolZl and added. Add the same volume of the medium or PBS as a negative control. In addition, it is preferable to use FGF2 or EGF as a positive control. Then, under 5% CO atmosphere
2 2
、 37°Cで 1〜14日間、必要に応じて全量または部分量の培地を交換しながら静置培 養することで、神経幹細胞の増殖を促進させることができる。 The growth of neural stem cells can be promoted by culturing at 37 ° C for 1 to 14 days while changing the whole or part of the medium as necessary.
[0060] 増殖させた神経幹細胞は、直接計数法、 FDSS測定法、 XTT法、 BrdU取込み法 等の方法により計測することができる。以下に、 FDSS測定法について具体的に説明 する。 [0060] The proliferated neural stem cells can be measured by a method such as a direct counting method, an FDSS measurement method, an XTT method, or a BrdU incorporation method. Below, the FDSS measurement method is explained in detail.
増殖させた神経幹細胞をホルムアルデヒドやアルコール等で固定し、 0. 3% Trit onX— 100 (ナカライテスタ社製)を含む PBS (以下、 TBSTと、う)を用いて洗浄を繰 り返す。次に、 TBSTで希釈したスキムミルク等を用いて表面をブロッキングした後、 TBSTで 3. 3 μ molZlに調製したへキスト 33342 (ナカライテスタ社製)で神経幹細 胞の核を染色する。その後、 TBSTで洗浄を繰り返し、 FDSS6000 (浜松ホトニタス 社製)等を用いて蛍光強度を測定し、対照と比較する。 [0061] 以上の方法により、本発明の神経幹細胞増殖促進剤の神経幹細胞増殖促進作用 を評価することができる。 The grown neural stem cells are fixed with formaldehyde, alcohol, etc., and the washing is repeated using PBS containing 0.3% Triton X-100 (manufactured by Nacalai Testa) (hereinafter referred to as TBST). Next, after blocking the surface with skim milk diluted with TBST, stain the nucleus of neural stem cells with Hoechst 33342 (Nacalai Testa) prepared to 3.3 μmolZl with TBST. Thereafter, washing with TBST is repeated, and the fluorescence intensity is measured using FDSS6000 (manufactured by Hamamatsu Photonics), etc., and compared with the control. [0061] With the above method, the neural stem cell proliferation promoting action of the neural stem cell proliferation promoting agent of the present invention can be evaluated.
以下、本発明の神経幹細胞増殖促進剤の神経幹細胞増殖促進作用に関する実 験例を示す。  Hereinafter, experimental examples relating to the neural stem cell proliferation promoting action of the neural stem cell proliferation promoting agent of the present invention will be shown.
試験例 1：ビニグロールによる神経幹細胞の増殖促進活性（1) 直接計数法  Test Example 1: Neural stem cell proliferation-promoting activity by biniglol (1) Direct counting method
参考例 1記載の方法で取得したラット成体神経幹細胞株 ANSC— 7細胞を、 N- 2 添カ卩物 [5 μ g/ml インシュリン（シグマ社製）、 100 μ gZmlゥシアポトランスフェリン (シグマ社製）、 6. 3ngZmlプロゲステロン（シグマ社製）、 1. 6 gZmlプトレシン（ シグマ社製）、 5. 2ngZmlセレン酸ナトリウム (シグマ社製) ]および抗生物質混合液 [0. 05UZmlペニシリン、 0. 05 gZmlストレプトマイシン（インビトロジェン社製）] を添カ卩した DMEMZF12培地 (インビトロジェン社製）（以下、アツセィ培地と称する） に 2. 0 X 105個 Zmlの濃度で懸濁し、ポリオル-チンおよびラミニンで表面加工し た 96穴プレート（ヌンク社製）に 0. 1 mlずつ播種して、 37°Cで 5%CO雰囲気下、 Rat adult neural stem cell line ANSC-7 cells obtained by the method described in Reference Example 1 were mixed with N-2 supplement [5 μg / ml insulin (manufactured by Sigma), 100 μg Zml Usiapotransferrin (Sigma) ), 6.3 ngZml progesterone (manufactured by Sigma), 1.6 gZml putrescine (manufactured by Sigma), 5.2 ngZml sodium selenate (manufactured by Sigma)] and antibiotic mixture [0. 05 UZml penicillin, 0.05 GZml streptomycin (manufactured by Invitrogen)] suspended in DMEMZF12 medium (Invitrogen) (hereinafter referred to as Atssey medium) at a concentration of 2.0 × 10 5 Zml, and surfaced with polyortin and laminin Inoculate 0.1 ml each into a processed 96-well plate (Nunk), and at 37 ° C in a 5% CO atmosphere.
2  2
一晩培養した。その後、 50 1の培養上清を除去し、アツセィ培地で終濃度の 2倍に なるように段階希釈したビニグロール、 FGF2 (陽性コントロール）、または DMSO (陰 性コントロール)を 1ゥエルあたり 50 1添加した。 71時間培養後、 50 1の培養上清 を除去し、 4°Cに冷却した 15%中性緩衝ホルマリン液 (和光純薬工業社製)を 1ゥェ ルあたり 50 1加え、 1時間静置した。その後、 0. 3% TritonX— 100(ナカライテス ク社製）を含む PBS (以下、 TBSTと称する）を用いて 2回洗浄し、 0. 5% TritonX - 100を含む PBSで希釈した 5%スキムミルク溶液で 3. 3 μ mol/1に調製したへキ スト 33342 (ナカライテスタ社製)を 50 1Zゥエルで添加し、暗所、 4°Cで一晩、核を 標識した。 100 17ゥェルの丁 3丁で2回、続ぃて!383で1回洗浄した後、 100 1 Zゥエルの PBSに浸し、 0. 1445mm2あたりの核染色された細胞数を蛍光顕微鏡（ ニコン社製)で計測した。 Cultured overnight. Subsequently, 50 1 culture supernatant was removed, and binigrol, FGF2 (positive control), or DMSO (negative control) serially diluted to twice the final concentration in Atsy medium was added at 50 1 per well. . After incubation for 71 hours, remove 50 1 of the culture supernatant, add 50 1 of 15% neutral buffered formalin solution (Wako Pure Chemical Industries, Ltd.) cooled to 4 ° C per well, and let stand for 1 hour did. Then, it was washed twice with PBS containing 0.3% TritonX-100 (manufactured by Nacalai Tesque) (hereinafter referred to as TBST) and diluted with PBS containing 0.5% TritonX-100. Then, hex 33342 (manufactured by Nacalai Testa Co., Ltd.) prepared at 3.3 μmol / 1 was added with 50 1Z well, and the nucleus was labeled overnight at 4 ° C in the dark. 100 17 wells 3 times twice, followed by 3 83 times, then washed once with 100 1 Zwell PBS, and 0.14 445 mm 2 of nuclear stained cells per fluorescence microscope (Nikon) ).
[0062] 結果を第 1表に示す。ビニグロールは 0. 3〜： L0. Ο/z molZlの濃度域で神経幹細 胞の増殖を促進することが示された。  [0062] The results are shown in Table 1. Vinigrol was shown to promote proliferation of neural stem cells in the concentration range of 0.3 to: L0.Ο / z molZl.
[0063] [表 1] 第 1表 ピニグロールの神経幹細胞増殖促進活性 （1 ) 直接計数法 平均値（cellsノ 0.1445 m m2， n =3) 標準偏差 [0063] [Table 1] Table 1. Neuronal stem cell proliferation-promoting activity of piniglol (1) Direct counting method Average value (cells 0.1445 mm 2 , n = 3) Standard deviation
ビニグロ一ル (μ(Τ10!/Ι)  Binigrole (μ (Τ10! / Ι)
0.3 288 13  0.3 288 13
0.6 310 30  0.6 310 30
1.3 349 77  1.3 349 77
2.5 355 32  2.5 355 32
5.0 353 6  5.0 353 6
10.0 295 3  10.0 295 3
FGF2 (ng/ml)  FGF2 (ng / ml)
6.3 421 28  6.3 421 28
25.0 542 31  25.0 542 31
100.0 614 93  100.0 614 93
DMSO (%)  DMSO (%)
0.1 219 24  0.1 219 24
[0064] 試験例 2 :ビニグロールによる神経幹細胞の増殖促進活性（2) FDSS測定法 [0064] Test Example 2: Activity of promoting proliferation of neural stem cells by biniglol (2) FDSS measurement method
試験例 1と同様にして、 96穴プレートに播種し、培養した ANSC— 7細胞の核をへ キスト 33342で標識した。 100 1Zゥエルの TBSTで 2回、続いて PBSで 1回洗浄し た後、 100 /z lZゥエルの PBSに浸し、核染色された細胞の蛍光強度を FDSS6000 (浜松ホトニタス社製)で測定した。細胞を播種しないゥエルの測定値を 0%、陰性コ ントロールの測定値を 100%とし、ビニグロール添加群における相対値を算出した。  In the same manner as in Test Example 1, the nuclei of ANSC-7 cells seeded in a 96-well plate and cultured were labeled with Hoechst 33342. After washing twice with 100 1Z-well TBST and then once with PBS, the cells were immersed in 100 / zl-well PBS and the fluorescence intensity of the nuclear-stained cells was measured with FDSS6000 (Hamamatsu Photonics). The relative value in the binigrol-added group was calculated assuming that the measured value of the well without cell seeding was 0% and the measured value of the negative control was 100%.
[0065] 結果を第 2表に示す。ビニグロールは 0. 3〜： L0. 0 molZlの濃度域で神経幹細 胞の増殖を促進することが示された。  [0065] The results are shown in Table 2. Vinigrol was shown to promote neural stem cell proliferation in the concentration range of 0.3-: L0.
[0066] [表 2] [0066] [Table 2]
第 2表 ビニグロールの神経幹細胞増殖促進活性 （2 ) F D S S測定法 平均値（％ of control, π=3) 標準偏差 TABLE 2 Neural Stem Cell Proliferation Promoting Activity of Binigrol (2) FDSSS Measurement Method Mean Value (% of control, π = 3) Standard Deviation
ビニグロ一ル (μΠ10ΐ/1)  Binigroll (μΠ10ΐ / 1)
0.3 136.1 6.0  0.3 136.1 6.0
0.6 146.2 5.3  0.6 146.2 5.3
1.3 155.7 6.6  1.3 155.7 6.6
2.5 146.4 2.9  2.5 146.4 2.9
5.0 143.8 5.2  5.0 143.8 5.2
10.0 134.6 4.5  10.0 134.6 4.5
20.0 112.3 9.0  20.0 112.3 9.0
FGF2 (ng/ml)  FGF2 (ng / ml)
1.6 145.6 5.3  1.6 145.6 5.3
3.1 171.6 5.0  3.1 171.6 5.0
6.3 203.5 20.0  6.3 203.5 20.0
12.5 216.7 4.6  12.5 216.7 4.6
25.0 241 .7 12.9  25.0 241 .7 12.9
50.0 250.4 8.7  50.0 250.4 8.7
100.0 257.4 4.2  100.0 257.4 4.2
[0067] 試験例 3 :ビニグロールによる神経幹細胞の増殖促進活性（3) ΧΤΤ法 [0067] Test Example 3: Vinigrol promotes proliferation of neural stem cells (3) Acupuncture
ANSC— 7細胞を、アツセィ培地に 2. 0 X 105個 Zmlの濃度で懸濁し、ポリオル- チンおよびラミニンで表面カ卩ェした 96穴プレート (ヌンク社製）に 0. 1 mlずつ播種し て、 37°Cで 5%CO雰囲気下、ー晚培養した。その後、 50 1の培養上清を除去し、 ANSC—7 cells were suspended in Atsy medium at a concentration of 2.0 × 10 5 Zml and seeded in 0.1 ml each in a 96-well plate (manufactured by NUNK) that was surface-coated with polyortin and laminin. The cells were cultured at 37 ° C in a 5% CO atmosphere. Then remove 50 1 culture supernatant,
2  2
アツセィ培地で終濃度の 2倍になるように段階希釈したビニグロール、 FGF2 (陽性コ ントロール）、または DMSO (陰性コントロール）を 1ゥエルあたり 50 1添カ卩した。 72 時間培養後、終濃度 lmgZmlとなるように XTT(sodium 3' - [ 1 - (phenylamin ocarbonyl)—d, 4― tetrazolium]― bis (4― methoxy— 6— nitro) benzene su lfonic acid hydrate)標準試薬（ロシュ ·ダイァグノスティックス社製）を該培養液中 に添加した。 37°Cで 5. 5時間、 5%COインキュベーター内で培養後、 1分間撹拌し  Binigrol, FGF2 (positive control), or DMSO (negative control) serially diluted to twice the final concentration in Atsey medium was added at a rate of 501 per well. XTT (sodium 3 '-[1-(phenylamin ocarbonyl) -d, 4-tetrazolium) -bis (4-methoxy-6-nitro) benzene su lfonic acid hydrate) standard to a final concentration of lmgZml after 72 hours of culture A reagent (Roche Diagnostics) was added to the culture medium. Cultivate in a 5% CO incubator for 5.5 hours at 37 ° C, then stir for 1 minute
2  2
、マイクロプレート分光光度計 Emax (モレキユラ一'デバイス社製）を用いて 490nm の吸光度 (対照波長 655nm)を測定した。細胞を播種しな!ヽゥエルの測定値を 0%、 陰性コントロールの測定値を 100%とし、ビニグロール添加群における相対値を算出 した。  Then, the absorbance at 490 nm (control wavelength: 655 nm) was measured using a microplate spectrophotometer Emax (manufactured by Molecular Devices Corporation). The cells were not seeded! The relative value in the biniglol-added group was calculated with the measured value of the well being 0% and the measured value of the negative control being 100%.
[0068] 結果を第 3表に示す。ビニグロールは 0. 3〜： L0. 0 molZlの濃度域で神経幹細 胞の増殖を促進することが示された。 [0068] The results are shown in Table 3. Vinigrol is 0.3 ~: Nerve trunk in the concentration range of L0. It has been shown to promote follicle growth.
[0069] [表 3] 第 3表 ビニグロールの神経幹細胞増殖促進活性 （3 ) X T T法 平均値（％ of control, n=3) 準偏差  [0069] [Table 3] Table 3 Binigrol's neural stem cell proliferation-promoting activity (3) XTT method mean (% of control, n = 3) quasi-deviation
ビニグロール (μηιοΙ/Ι)  Vinigrol (μηιοΙ / Ι)
0.3 142.1 4.6  0.3 142.1 4.6
0.6 152.4 7.9  0.6 152.4 7.9
1 .3 159.1 3.6  1 .3 159.1 3.6
2.5 149.7 1 .8  2.5 149.7 1 .8
5.0 142.9 2.1  5.0 142.9 2.1
10.0 129.3 3.7  10.0 129.3 3.7
20.0 106.5 1 .5  20.0 106.5 1.5
FGF2 (ng/ml)  FGF2 (ng / ml)
1 .6 154.1 7.9  1 .6 154.1 7.9
3.1 174.7 14.1  3.1 174.7 14.1
6.3 197.8 10.3  6.3 197.8 10.3
12.5 208.6 7.5  12.5 208.6 7.5
25.0 213.6 4,6  25.0 213.6 4,6
50.0 215.4 3.0  50.0 215.4 3.0
100.0 214.8 1 .1  100.0 214.8 1 .1
[0070] 試験例 4 :ビニグロールによる神経幹細胞の増殖促進活性 (4) BrdU取込み法 [0070] Test Example 4: Growth promotion activity of neural stem cells by biniglol (4) BrdU incorporation method
ANSC— 7細胞を、アツセィ培地に 2. 0 X 105個 Zmlの濃度で懸濁し、ポリオル- チンおよびラミニンで表面カ卩ェした 96穴プレート (ヌンク社製）に 0. 1mlずつ播種し て、 37°Cで 5%CO雰囲気下、ー晚培養した。その後、 50 1の培養上清を除去し、 ANSC—7 cells were suspended in Atsy's medium at a concentration of 2.0 × 10 5 Zml, and 0.1 ml each was seeded in a 96-well plate (manufactured by NUNK) that was surface-coated with polyortin and laminin. The cells were cultured at 37 ° C in a 5% CO atmosphere. Then remove 50 1 culture supernatant,
2  2
アツセィ培地で 2 μ molZlに希釈したビニグロール、 FGF2 (陽性コントロール）、また は DMSO (陰性コントロール）を 1ゥエルあたり 50 1添カ卩した。 2日後、 1ゥエルあたり 50 μ 1の培養上清を除去し、 40 μ molZlの 5—ブロモ— 2，—デォキシゥリジン（Brd U、ロシュ'ダイァグノスティックス社製）を含む新たなアツセィ培地を 1ゥエルあたり 50 μ 1加え、さらに 2日間培養した。その後、 50 1の培養上清を除去し、 4°Cに冷却した 15%中性緩衝ホルマリン液 (和光純薬工業社製)を 1ゥエルあたり 50 μ 1加え、静置し た。 30分後、全量を除去し、 1ゥヱルあたり 80 1の 15%中性緩衝ホルマリン液を加 えて 6時間固定した。次に、 TBST(100 /z lZゥエル）で 2回洗浄し、 6molZl塩酸を TBSTで 3倍に希釈した溶液を 1ゥエルあたり 80 1添カ卩した。室温で 3時間静置後、 丁 3丁（100 17ゥヱル）で洗浄し、 118. 5に調整した 0. ImolZlホウ酸緩衝液（1 00 μ 1Ζゥエル）で中和した。 TBST(100 μ lZゥエル）で 2回洗浄後、抗ー BrdU抗 体溶液（ロシュ ·ダイァグノスティックス社製）を 1ゥエルあたり 50 1添カ卩し、 4°Cでー晚 静置した。その後、溶液を除去し、 TBSTdOO /z 1Zゥエル)で 2回洗浄し、 0. 5% TritonX- 100および 1%ゥシ血清アルブミン（シグマ社製）を含む PBSで希釈した Alexa Fluor 488 (モレキユラ一'プローブス社製）を 50 lZゥエルでカ卩え、 2時間 静置した。溶液を除去後、 100 1Zゥエルの TBSTで 1回、続いて PBSで 1回洗浄し た後、 100 1Zゥエルの PBSに浸し、 0. 1445mm2あたりの BrdUで標識された細 胞数を蛍光顕微鏡 (ニコン社製)で計測した。 Vinignol, FGF2 (positive control), or DMSO (negative control) diluted to 2 μmolZl with Atsey medium was added at a rate of 50 per well. Two days later, remove 50 μl of culture supernatant per well, and add a new Atsey medium containing 40 μmolZl of 5-bromo-2, -deoxyuridine (Brd U, Roche Diagnostics). 50 μl per well was added and cultured for another 2 days. Thereafter, 50 1 of the culture supernatant was removed, and 50 μl of 15% neutral buffered formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) cooled to 4 ° C. was added and allowed to stand. After 30 minutes, the entire amount was removed, and 80 1 of 15% neutral buffered formalin solution was added per tool and fixed for 6 hours. Next, the plate was washed twice with TBST (100 / zlZwell), and a solution obtained by diluting 6 molZl hydrochloric acid three times with TBST was added to 801 per well. After standing at room temperature for 3 hours, Washed with 3 dies (100 17 ul) and neutralized with 0. ImolZl borate buffer (100 μl uel) adjusted to 118.5. After washing twice with TBST (100 μl Zwell), add 50-per-anti-BrdU antibody solution (Roche Diagnostics) per well and let stand at 4 ° C. . Then, the solution was removed, washed twice with TBSTdOO / z 1Z well), and diluted with PBS containing 0.5% TritonX-100 and 1% ushi serum albumin (Sigma). Alexa Fluor 488 (Probes) was placed in a 50 lZ well and allowed to stand for 2 hours. After removing the solution, wash once with 100 1Z-well TBST, then once with PBS, then soak in 100 1Z-well PBS, and count the number of BrdU-labeled cells per 0.145 mm 2 with a fluorescence microscope. (Measured by Nikon).
[0071] その結果、 BrdU陽性細胞（2連の平均値）は、陰性コントロールが 82. 5個であつ たのに対し、 1 mol/1のビニグロール添加群では 165個であった。なお、陽性コン トロールとした FGF2 (lOngZml)添加群では 155. 5個であった。 [0071] As a result, the number of BrdU positive cells (average of duplicates) was 82.5 in the negative control, whereas it was 165 in the 1 mol / 1 vinylogol addition group. The number of FGF2 (lOngZml) added as a positive control was 155.5.
以上より、ビニグロールは神経幹細胞の増殖を促進することが示された。 試験例 5：ビニグロール処理によって増殖した細胞におけるネスチンの発現  From the above, it was shown that biniglol promotes proliferation of neural stem cells. Test Example 5: Nestin expression in cells grown by biniglol treatment
ビニグロール処理によって増殖した細胞にぉ 、て、神経幹細胞のマーカーである ネスチンが発現して 、ることを以下の通り確認した。  It was confirmed that nestin, a marker for neural stem cells, was expressed in cells proliferated by the treatment with biniglol as follows.
ANSC— 7細胞を、アツセィ培地に 1. 5 X 105個 Zmlの濃度で懸濁し、ポリオル- チンおよびラミニンで表面加工した 96穴プレートに 0. 1mlずつ播種して、 37°Cで 5 %CO雰囲気下、一晩培養した。その後、 50 1の培養上清を除去し、アツセィ培地ANSC—7 cells suspended in Atsy medium at a concentration of 1.5 x 10 5 Zml, seeded in 0.1 ml each in 96-well plates surface-treated with polyortin and laminin, 5% at 37 ° C Cultivated overnight in a CO atmosphere. After that, remove 50 1 culture supernatant and add Atsey medium.
2 2
で 5 μ molZlに希釈したビニグロールまたは DMSO (陰性コントロール）を 1ゥエルあ たり 50 1添加した。  Add 50 1 per well of Vinigrol or DMSO (negative control) diluted to 5 μmolZl.
[0072] 3日間培養後、 50 μ 1の培養上清を除去し、 4°Cに冷却した 15%中性緩衝ホルマリ ン液を 1ゥ ルあたり 50 1加え、 15分振盪した。同溶液で再度半量交換し、冷蔵庫 内でー晚静置した後、 TBSTを用いて 2回洗浄し、 0. 5% TritonX— 100を含む P BSで希釈した 5%スキムミルク溶液で 1Z2000希釈した抗—ネスチン抗体 (ベタトン ディッキンソン)を 50 1Zゥエル添加し、暗所、室温で 2. 5時間標識した。溶液を除 去し、 TBSTを用いて 2回洗浄後、 Alexa Fluor 488 (モレキュラ^ ~ ·プローブス、 1 Ζ2000希釈）およびへキスト 33342 (3. 3 μ mol/1)を 0. 5% TritonX- 100を 含む PBSで希釈した 5%スキムミルク溶液に溶解し、 50 1Zゥエル添カ卩して、暗所、 4°Cで 2晚、静置した。溶液を除去後、 100 1Zゥエルの TBST、続いて PBSで 1回 ずつ洗浄した後、 100 1Zゥエルの PBSを添カ卩した。 [0072] After culturing for 3 days, 50 μl of the culture supernatant was removed, 50 1 of a 15% neutral buffered formalin solution cooled to 4 ° C was added, and the mixture was shaken for 15 minutes. Replace half the volume again with the same solution, leave it in a refrigerator, wash it twice with TBST, and dilute it with 5% skim milk solution diluted with PBS containing 0.5% TritonX-100. —Nestin antibody (Betaton Dickinson) was added to 50 1Z wells and labeled for 2.5 hours in the dark at room temperature. After removing the solution and washing twice with TBST, Alexa Fluor 488 (Molecular ^ ~ · Probes, diluted 1 to 2000) and Hoechst 33342 (3.3 μmol / 1) were added to 0.5% TritonX-100. The It was dissolved in a 5% skim milk solution diluted with PBS, and added with 50 1Z well, and allowed to stand at 4 ° C for 2 minutes in the dark. After removing the solution, the plate was washed with 100 1Z well TBST and then PBS once, and then 100 1Z well PBS was added.
[0073] へキスト 33342で核染色された細胞（総細胞）の蛍光強度および Alexa Fluorで染 色された細胞 (ネスチン陽性神経幹細胞）の蛍光強度をそれぞれ FDSS6000で測 定した。細胞を播種しないゥエルの測定値を 0%、 DMSOを添カ卩した陰性コントロー ルの測定値を 100%とし、ビニグロール添加群における相対値を算出した。  [0073] The fluorescence intensity of cells stained with Hoechst 33342 (total cells) and the fluorescence intensity of cells stained with Alexa Fluor (nestin-positive neural stem cells) were measured with FDSS6000. The relative value in the vinylogol addition group was calculated with the measured value of the well without cell seeding as 0% and the negative control value with DMSO as 100%.
結果を第 4表に示す。ビニグロール処理による総細胞数の増加に伴い、ネスチンを 発現する神経幹細胞が増加した。すなわち、ビニグロール処理によって増加した細 胞が神経幹細胞であることが示された。  The results are shown in Table 4. Neural stem cells that express nestin increased as the total number of cells increased due to the treatment with biniglol. That is, it was shown that the cells increased by the treatment with vinylogol were neural stem cells.
[0074] [表 4] ビニグロール処理によって増殖した神経幹細胞におけるネスチンの発現 総細胞 ネスチン陽性神経幹細胞 平均値 平均値 » 厂  [0074] [Table 4] Nestin expression in neural stem cells grown by biniglol treatment Total cells Nestin-positive neural stem cells Average value Average
標準偏差 CD  Standard deviation CD
(% of control, n=18) (% of control , π=12) 标準1"1 ^ ビニグロ一ル 162.8 5.8 (% of control, n = 18) (% of control, π = 12) Standard 1 " 1 ^ Binigrole 162.8 5.8
DMS0コントロール 100.0 6.5 100.0 3.6 DMS0 control 100.0 6.5 100.0 3.6
[0075] 試験例 6：ラバマイシンによるビニグロールの神経幹細胞増殖促進活性の抑制（1) mTORの特異的阻害剤であるラパマイシン ["オンコジーン（Oncogene) "2000年 、 19卷、 p. 6680-6686]によるビニグロールの神経幹細胞増殖促進活性の抑制効 果を調べた。 [0075] Test Example 6: Inhibition of neural stem cell proliferation-promoting activity of Vinigrol by ravamycin (1) Rapamycin, a specific inhibitor of mTOR ["Oncogene" 2000, 19 卷, p. 6680-6686] The inhibitory effect of biniglol on the activity of promoting proliferation of neural stem cells was examined.
ANSC— 7細胞を、アツセィ培地に 2. 0 X 104個 ZO. 1mlの濃度で懸濁し、ポリオ ル-チンおよびラミニンで表面カ卩ェした 96穴プレート（ヌンク社製）に 0. 1mlずつ播 種して、 37°Cで 5%CO雰囲気下、ー晚培養した。その後、 50 1の培養上清を除 ANSC—7 cells are suspended in Atsy medium at a concentration of 2.0 × 10 4 ZO. 1 ml, 0.1 ml each in a 96-well plate (manufactured by NUNK) that is surface-coated with poliotin and laminin. The seeds were seeded and cultured at 37 ° C in a 5% CO atmosphere. Then remove 50 1 culture supernatant.
2  2
去し、アツセィ培地で終濃度の 2倍になるように希釈したラバマイシン (シグマ社製)お よび DMSO (陰性コントロール）を 1ゥエルあたり 50 1添カ卩した。 50分後、 50 1の培 養上清を除去し、上記と同濃度のラパマイシンまたは DMSOを含むアツセィ培地で 1 /z molZlに希釈したビニグロールを 1ゥエルあたり 50 1添カ卩した。 2日後、 1ゥエルあ たり 50 1の培養上清を除去し、上記と同濃度の薬剤をそれぞれ含む新たなアツセィ 培地を 1ゥエルあたり 50 μ 1加え、さらに 2日間培養した。その後、 50 μ 1の培養上清 を除去し、 4°Cに冷却した 15%中性緩衝ホルマリン液を 1ゥエルあたり 50 1カ卩え、静 置した。 15分後、全量を除去し、 1ゥエルあたり 80 1の 15%中性緩衝ホルマリン液 をカロえて 1時間固定した後、 TBSTを用いて 2回洗浄し（100 1Zゥエル）、 0. 5% TritonX- 100を含む PBSに溶解した 5%スキムミルク溶液で 3. 3 μ mol/1に調製 したへキスト 33342を 50 1/ゥエルで添カ卩して、暗所、 4°Cで 2晚、核を標識した。 1 00 μ 1Ζゥエルの TBSTで 2回、続いて PBSで 1回洗浄した後、 100 μ lZゥエルの Ρ BSに浸し、核染色された細胞の蛍光強度を FDSS6000で測定した。細胞を播種し ないゥエルの測定値を 0%、陰性コントロールの測定値を 100%とし、薬剤添加群に おける相対値を算出した。 Then, rabamycin (Sigma) and DMSO (negative control) diluted to twice the final concentration with Atsey medium and DMSO (negative control) were added at 50 1 per well. After 50 minutes, 50 1 of the culture supernatant was removed, and vinigrol diluted to 1 / z molZl in an Atsey medium containing rapamycin or DMSO at the same concentration as above was added to 50 1 per well. 2 days later, 1 50 1 of the culture supernatant was removed, and 50 µ1 of new Atsey medium containing each of the same concentrations of drugs as above was added, followed by further culturing for 2 days. Thereafter, 50 μl of the culture supernatant was removed, and 50% of a 15% neutral buffered formalin solution cooled to 4 ° C. was added to each well and allowed to stand. After 15 minutes, remove the entire volume, fix 80 1 15% neutral buffered formalin solution per well and fix for 1 hour, then wash twice with TBST (100 1Zwell), 0.5% TritonX -Add 50% / well of hexist 33342 prepared in 3.3 μmol / 1 with 5% skim milk solution in PBS containing 100, and add 2 nuclei at 4 ° C in the dark. Labeled. After washing twice with 100 μl wel TBST and then once with PBS, the cells were soaked in 100 μlZ well BS and the fluorescence intensity of the nuclear-stained cells was measured with FDSS6000. The relative value in the drug-added group was calculated assuming that the measured value of the wells without cell seeding was 0% and the measured value of the negative control was 100%.
[0076] 結果を第 5表に示す。ビニグロールの神経幹細胞増殖促進活性は、 mTOR特異的 阻害剤であるラバマイシンによって、濃度依存的に抑制された。  [0076] The results are shown in Table 5. Vinigrol's neural stem cell proliferation-promoting activity was suppressed in a concentration-dependent manner by the mTOR-specific inhibitor ravamycin.
この結果より、ビニグロールは mTORを介して神経幹細胞の増殖を促進して 、るこ とが示された。  From this result, it was shown that biniglol promotes proliferation of neural stem cells via mTOR.
[0077] [表 5]  [0077] [Table 5]
第 5表 ラバマイシンによるビニグロールの神経幹細胞増殖促進活性の抑制 平均値■(% of control, π=3) 標準偏差  Table 5. Inhibition of neural stem cell proliferation-promoting activity of Vinigrol by ravamycin Mean value (% of control, π = 3) Standard deviation
0.1 % DMSO  0.1% DMSO
100.0 2.9  100.0 2.9
コントロール  Control
0.5 μΐηοΙ/Ι 0.5 μΐηοΙ / Ι
159.8 5.7  159.8 5.7
ビニグロ一ル単剤  Biniglo single agent
0.5 μΐηοΙ/Ιビニグロ一ル 0.5 μΐηοΙ / Ι vinylogall
+ラパマイシン（nmol/l)  + Rapamycin (nmol / l)
0.2 127.8 1 .1  0.2 127.8 1.1
0.3 122.9 10.9  0.3 122.9 10.9
0.6 1 14.3 4.1  0.6 1 14.3 4.1
1 .3 1 10.7 2.7  1 .3 1 10.7 2.7
2.5 104.1 2.0  2.5 104.1 2.0
5.0 95.0 1 .9  5.0 95.0 1 .9
10.0 90.8 3.8  10.0 90.8 3.8
20.0 84.1 7.1 [0078] 試験例 7：ラバマイシンによるビニグロールの神経幹細胞増殖促進活性の抑制（2) ラパマイシンは FKBP— 12ィムノフィリンタンパク質と複合体を形成して mTORのリ ン酸化活性を阻害する。同様に FK506も FKBP— 12ィムノフィリンタンパク質に結 合するため、ラパマイシン一 FKBP— 12ィムノフィリンタンパク質複合体形成に拮抗 するが、 FK506 - FKBP 12ィムノフィリンタンパク質複合体は mTORを阻害しな!ヽ [ "エフィービーエス.レターズ (FEBS Letters) "2003年、 550卷、 p. 94-100]。そ こで、ラバマイシンによるビニグロールの神経幹細胞増殖促進活性の抑制効果を FK 506で解除できる力否かを解析した。 20.0 84.1 7.1 [0078] Test Example 7: Inhibition of neural stem cell proliferation-promoting activity of biniglol by ravamycin (2) Rapamycin forms a complex with FKBP-12 immunophilin protein and inhibits mTOR phosphorylation activity. Similarly, FK506 also binds to FKBP-12 immunophilin protein, thus antagonizing rapamycin-FKBP-12 immunophilin protein complex formation, but the FK506-FKBP 12 immunophilin protein complex does not inhibit mTOR. ! ヽ ["FEBS Letters" 2003, 550 卷, p. 94-100]. Therefore, we analyzed whether the ability of Fava 506 to cancel the inhibitory effect of rabamicin on the proliferation-promoting activity of Vinigrol on neural stem cells was analyzed.
[0079] ANSC— 7細胞を、アツセィ培地に 2. 2 X 104個 ZO. 1mlの濃度で懸濁し、ポリオ ル-チンおよびラミニンで表面カ卩ェした 96穴プレート（ヌンク社製）に 0. 1mlずつ播 種して、 37°Cで 5%CO雰囲気下、ー晚培養した。その後、 50 1の培養上清を除 [0079] ANSC—7 cells were suspended in Atsey medium at a concentration of 2.2 × 10 4 ZO. 1 ml and surface-coated with poliotin and laminin in a 96-well plate (Nunk). 1 ml each was inoculated and cultured at 37 ° C in 5% CO atmosphere. Then remove 50 1 culture supernatant.
2  2
去し、アツセィ培地で終濃度の 2倍になるように段階希釈した FK506を 1ゥエルあたり 50 μ 1添カ卩した。 30分後、 FK506を含むアツセィ培地で 2nmolZlに希釈したラパマ イシンを、培養上清の半量交換により添加した。さらに 30分後、 FK506、ラバマイシ ンを含むアツセィ培地で 1 μ molZlに希釈したビニグロールを培養上清の半量交換 により添カロした。  The FK506 was diluted serially to twice the final concentration in Atsy's medium, and 50 μl per well was added. After 30 minutes, rapamycin diluted to 2 nmolZl with Atsy medium containing FK506 was added by exchanging half the culture supernatant. After an additional 30 minutes, biniglol diluted to 1 μmolZl in Atsy medium containing FK506 and lavamycin was added by exchanging half the culture supernatant.
[0080] 2日後、 1ゥエルあたり 50 μ 1の培養上清を除去し、それぞれの薬剤を含む新たなァ ッセィ培地を 1ゥエルあたり 50 μ 1加え、さらに 2日間培養した。その後、 50 μ 1の培養 上清を除去し、 4°Cに冷却した 15%中性緩衝ホルマリン液を 1ゥエルあたり 50 1カロ え、静置した。 15分後、全量を除去し、 1ゥエルあたり 80 1の 15%中性緩衝ホルマリ ン液を加えて 1時間固定した後、 TBSTを用いて 2回洗浄し（100 1Zゥエル）、 0. 5 % TritonX- 100を含む PBSに溶解した 5%スキムミルク溶液で 3. 3 μ molZlに 調製したへキスト 33342を 50 1Zゥエルで添カ卩して、暗所、 4°Cで 2晚、核を標識し た。 100 1Zゥエルの TBSTで 2回、続いて PBSで 1回洗浄した後、 100 lZゥエル の PBSに浸し、核染色された細胞の蛍光強度を FDSS6000で測定した。細胞を播 種しないゥエルの測定値を 0%、陰性コントロールの測定値を 100%とし、薬剤添カロ 群における相対値を算出した。  [0080] Two days later, 50 µl of the culture supernatant per well was removed, and a new assay medium containing each drug was added at 50 µl per well, followed by further culturing for 2 days. Subsequently, 50 μl of the culture supernatant was removed, and 50% of 15% neutral buffered formalin solution cooled to 4 ° C. was added to each well and allowed to stand. After 15 minutes, remove the entire volume, add 80 1 15% neutral buffered formalin solution per well, fix for 1 hour, then wash twice with TBST (100 1Zwell), 0.5% Add 5% skim milk solution dissolved in PBS containing TritonX-100 to 3.3 μmolZl. Add hexane 33342 with 50 1Z well and label the nucleus for 2 days at 4 ° C in the dark. It was. After washing twice with 100 1Z-well TBST and then once with PBS, the cells were immersed in 100 lZ-well PBS and the fluorescence intensity of the nuclear-stained cells was measured with FDSS6000. The relative value in the drug-added caro group was calculated with the measured value of the wells without cell seeding as 0% and the measured value of the negative control as 100%.
[0081] 結果を第 1図に示す。 mTORの特異的阻害剤であるラパマイシンは 1. 0nmol/l で 500nmolZlのビニグロールによる神経幹細胞の増殖促進作用を完全に抑制した 。一方、ラパマイシンと FKBP— 12ィムノフィリンタンパク質との複合体形成を拮抗的 に阻害する薬剤 FK506は、 lOnmolZl以上で濃度依存的にラパマイシンの上記抑 制効果を解除した。 [0081] The results are shown in FIG. Rapamycin, a specific inhibitor of mTOR, is 1.0 nmol / l In addition, 500 nmolZl of vinylogol completely suppressed the proliferation promoting effect of neural stem cells. On the other hand, the drug FK506, which antagonistically inhibits the complex formation between rapamycin and FKBP-12 immunophilin protein, canceled the above-mentioned inhibitory effect of rapamycin in a concentration-dependent manner at lOnmolZl or higher.
[0082] 以上の結果より、ビニグロールは mTORを介して神経幹細胞の増殖を促進してい ることが示された。  [0082] From the above results, it was shown that biniglol promotes proliferation of neural stem cells via mTOR.
試験例 8 :ビニグロールによる細胞内 mTORリン酸化制御因子 4E— BP1および p70 S6キナーゼのリン酸化促進作用  Test Example 8: Cellular phosphorylation regulator 4E— BP1 and p70 S6 kinase
翻訳開始因子 eIF4Eに結合してその機能を抑制するタンパク質 4E— BP1およびリ ボゾーム S6タンパク質をリン酸化する p70S6キナーゼは、細胞内で mTORによるリ ン酸化を受けることが知られている ["ョ一口ビアン 'ジャーナル'ォブ 'バイオケミストリ 一（European Journal of Biochemistry) "2002年、 269卷、 p. 5338-5349] 。そこで、細胞内でビニグロールによってこれらの mTOR下流制御因子のリン酸ィ匕が 亢進されるかどうか解析した。  A protein that binds to the translation initiation factor eIF4E and suppresses its function 4E— phosphorylates BP1 and ribosomal S6 protein p70S6 kinase is known to be phosphorylated by mTOR in cells [" Bian 'Journal' of 'Biochemistry "2002, 269 卷, p. 5338-5349]. Therefore, we analyzed whether phosphorylation of these mTOR downstream regulators was enhanced by binigrol in cells.
[0083] ポリオル-チンおよびラミニンで表面加工した 6cmの培養皿 (イワキネ土製）に、 6. 7 1 X 106個の ANSC— 7細胞を播種し、アツセィ培地中で、 37°C、 5%CO雰囲気 [0083] A 6 cm culture dish (made of Iwakine soil) surface-treated with polyortin and laminin was seeded with 6. 7 1 X 10 6 ANSC—7 cells in Atsy medium at 37 ° C, 5% CO atmosphere
2 下、 17時間培養を行った。その後、ビニグロールを終濃度 2. 5 molZlになるように 添加し、さら〖こ 8時間、 37°C、 5%CO雰囲気下、培養した。培地を除去し、 5mlの P  2) The culture was performed for 17 hours. Then, Vinigrol was added to a final concentration of 2.5 molZl and cultured for 8 hours at 37 ° C in a 5% CO atmosphere. Remove the medium and add 5 ml P
2  2
BSで洗浄後、スクレイパー (スミロン社製)を用いて細胞を回収した。冷却した溶解用 緩衝液 [50mmol/l へぺス（HEPES) 水酸化ナトリウム（pH7. 4に調整）、 250 mmol/1 塩化ナトリウム、 ImmolZl エチレンジァミン四酢酸（EDTA)、 1%ノ- デット P— 40 (NP— 40)、 ImmolZl ジチオスレィトール（DTT)、 ImmolZl Pefa bloc SC (ロシュ社製）、 1当量のプロテアーゼ阻害剤カクテル（コンプリート、ロシュ 社製）、 lOmgZml ぺプスタチン A、 2mmolZl オルト—バナジン酸ナトリウム（Na VO )、 1 mmol/1 フッ化ナトリウム（NaF)、 10mmol/l β—グリセ口リン酸]を用 After washing with BS, the cells were collected using a scraper (manufactured by Sumilon). Cold lysis buffer [50 mmol / l HEPES sodium hydroxide (adjusted to pH 7.4), 250 mmol / 1 sodium chloride, ImmolZl ethylenediamin tetraacetic acid (EDTA), 1% nodette P—40 (NP-40), ImmolZl dithiothreitol (DTT), ImmolZl Pefa bloc SC (Roche), 1 equivalent of protease inhibitor cocktail (Complete, Roche), lOmgZml pepstatin A, 2mmolZl ortho-vanadate Sodium (Na VO), 1 mmol / 1 sodium fluoride (NaF), 10 mmol / l β-glycose phosphate]
3 4 3 4
い、 4°Cで細胞を 1時間溶解した後、遠心分離した。得られた上清の蛋白質濃度を測 定し、各レーンあたりの蛋白質量が同一になるよう試料を調製した後、ドデシル硫酸 ナトリウム ポリアクリルアミドゲル電気泳動（SDS— PAGE)により蛋白質の分離を 行なった。分離された蛋白質試料をポリビ-リデンジフルオライド (PVDF)膜 (ミリポ ァ社製）に転写した後、 1次抗体として、抗セリン 65リン酸化 4E— BP1抗体 (セルシ ダナリング社製）、または抗スレオニン 389リン酸化 p70S6キナーゼ抗体 (セルシグナ リング社製)を加え、膜上の蛋白質と反応させた。その後、 2次抗体として、西洋ヮサ ビペルォキシダーゼ（Horseradish Peroxidase)で標識された抗ゥサギ Ig抗体（セ ルシダナリング社製)を反応させた。その後、 ECL試薬 (ピアス社製)で発色を行い、 LAS - 1000ルミノ'イメージアナライザー（富士フィルム社製)で検出した。 The cells were lysed at 4 ° C for 1 hour and then centrifuged. Measure the protein concentration in the supernatant and prepare a sample so that the amount of protein in each lane is the same. Then, separate the protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). I did it. After the separated protein sample is transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), anti-serine 65 phosphorylated 4E-BP1 antibody (Celcidnering) or anti-serine is used as the primary antibody. Threonine 389 phosphorylated p70S6 kinase antibody (manufactured by Cel Signaling) was added and reacted with the protein on the membrane. Thereafter, as a secondary antibody, an anti-rabbit Ig antibody (manufactured by Celsidanaring) labeled with horseradish peroxidase was reacted. Then, color was developed with ECL reagent (Pierce) and detected with LAS-1000 Lumino 'image analyzer (Fuji Film).
[0084] その結果、ビニグロールの添カ卩により、細胞内において 4E— BP1、 p70S6キナー ゼのリン酸化が、それぞれ 4. 2倍、 1. 9倍に亢進した。 [0084] As a result, phosphorylation of 4E-BP1 and p70S6 kinase was increased 4.2-fold and 1.9-fold, respectively, by the addition of binigrol.
また、この活性は mTORの特異的阻害剤であるラパマイシンを終濃度 20nmolZl で添加することにより、完全に抑制された。  This activity was completely suppressed by adding rapamycin, a specific mTOR inhibitor, at a final concentration of 20 nmolZl.
以上の結果より、ビニグロールは mTORを介して神経幹細胞の増殖を促進して ヽ ることが示された。  From the above results, it was shown that biniglol promotes proliferation of neural stem cells via mTOR.
試験例 9 :ビニグロールによる試験管内での 4E— BP1の直接的リン酸ィ匕促進作用 Test Example 9: Direct phosphorylation promoting action of 4E—BP1 in vitro by vinylogol
ANSC— 7細胞力も免疫沈降法によって mTOR複合体を精製し、 4E— BP1のリン 酸ィ匕を試験管内で解析した。 The ANTOR-7 cell force was also purified by immunoprecipitation, and the 4E-BP1 phosphate was analyzed in vitro.
[0085] ポリオル-チンおよびラミニンで表面加工した 6cmの培養皿 (イワキネ土製）に ANSC [0085] ANSC on a 6 cm culture dish (Iwakine Earth) surface-treated with polyortin and laminin
7細胞を播種し、 28. 5ng/mlの FGF2を含むアツセィ培地で、 5%CO雰囲気下  7 Seed cells, and 28.5 g of Atsy medium containing 5 ng / ml FGF2 in 5% CO atmosphere
2 2
、 37°Cで 3日間培養した。 1. 5mlの PBSで 2回洗浄後、スクレイパー (イワキネ土製)で 2. 51 X 107個の細胞を回収した。該回収細胞を冷却した 2mlの細胞溶解用緩衝液 The cells were cultured at 37 ° C for 3 days. 1. After washing twice with 5 ml of PBS, 2.51 × 10 7 cells were recovered with a scraper (made by Iwakine Earth). 2 ml of cell lysis buffer in which the recovered cells are cooled
[20mmolZlトリス—水酸化ナトリウム（PH7. 4に調整）、 20mmolZl 塩化ナトリウ ム、 ImmolZl エチレンジァミン四酢酸（EDTA)、 5mmolZl エチレングリコール ビス（ 13—アミノエチルエーテル）四酢酸（EGTA)、 20mmol/l β—グリセ口リン酸 、 ImM ジチオスレィトール（DTT)、 ImmolZl Pefabloc SC, 2 μ g/ml ァプ 口チュン、 1 μ mol/1 ロイぺプチン]に懸濁後、氷冷下、超音波で細胞を破砕し、 4 °Cで遠心分離して細胞抽出液を得た。上記の細胞溶解用緩衝液で平衡ィ匕した 50 μ Lの Protein G Sepharose (フアルマシア社製）に該細胞抽出液をカ卩ぇ 1時間、 4 °Cで撹拌後、遠心分離して非特異吸着成分を除去した。上清を新しい容器に移し、 抗 mTOR抗体 (セル'シグナリング社製)をカ卩え、 4°Cで一晩撹拌した後、上記の細胞 溶解用緩衝液で平衡化した 50 Lの Protein G Sepharoseをカ卩え、さらに 4°Cで 1時間撹拌した。その後、遠心分離し、上清を除去後、 1mlの高塩濃度緩衝液 [20m mol/1 トリス—水酸化ナトリウム（pH7. 4に調整）、 500mmol/l 塩化ナトリウム、 lmmol/1 EDTA、 5mmol/l EGTA、 20mmol/l β—グリセ口リン酸、 lmmo 1/1 DTT、 lmmol/1 Pefabloc SC、 2 μ g/ml ァプロチュン、 1 μ mol/1 口 ィぺプチン]で 2回洗浄し、続いて lmlのアツセィ緩衝液 [lOmmolZl へぺス一水 酸化ナトリウム（PH7. 4に調整）、 50mmolZl 塩化ナトリウム、 50mmolZl β—グ リセ口リン酸]で 3回洗浄した。最終的に lmlのアツセィ緩衝液に懸濁し、 mTOR複合 体免疫沈降ビーズを調製した。 [20mmolZl tris-sodium hydroxide (adjusted to PH7.4), 20mmolZl sodium chloride, ImmolZl ethylenediamine tetraacetic acid (EDTA), 5mmolZl ethylene glycol bis (13-aminoethylether) tetraacetic acid (EGTA), 20mmol / l β- Suspended in glyceport phosphate, ImM dithiothreitol (DTT), ImmolZl Pefabloc SC, 2 μg / ml apical chun, 1 μmol / 1 leupeptin] Was disrupted and centrifuged at 4 ° C to obtain a cell extract. Non-specific adsorption by stirring the cell extract for 1 hour at 4 ° C in 50 µL Protein G Sepharose (manufactured by Pharmacia) equilibrated with the above cell lysis buffer Ingredients were removed. Transfer the supernatant to a new container Prepare anti-mTOR antibody (manufactured by Cell Signaling), stir at 4 ° C overnight, and then add 50 L of Protein G Sepharose equilibrated with the above-mentioned cell lysis buffer. Stir at C for 1 hour. Then, after centrifuging and removing the supernatant, 1 ml of high salt buffer (20 mMol / 1 tris-sodium hydroxide (adjusted to pH 7.4), 500 mmol / l sodium chloride, lmmol / 1 EDTA, 5 mmol / l EGTA, 20 mmol / l β-glyceport phosphate, lmmo 1/1 DTT, lmmol / 1 Pefabloc SC, 2 μg / ml aprotune, 1 μmol / 1 pipeptin], followed by Washed three times with lml of Atsey buffer [10 mmol Zl hepes monohydrate sodium hydroxide (adjusted to PH 7.4), 50 mmol Zl sodium chloride, 50 mmol Zl β-glycose phosphate]. Finally, the suspension was suspended in 1 ml of Atsey buffer, and mTOR complex immunoprecipitated beads were prepared.
[0086] 50 μ 1の mTOR複合体免疫沈降ビーズに、終濃度 2. 5 μ molZlになるようにアツ セィ緩衝液で希釈したビニグロール（10 μ 1)および 50 μ 1のリン酸化反応緩衝液 [10 mmol/1 へぺス—水酸化ナトリウム（pH7. 4に調整）、 50mmolZl 塩化ナトリウム 、 50mmol/l β—グリセ口リン酸、 lOmmolZl 塩化マンガン、 100 /z molZl ァ デノシン三リン酸]を加え、 30°Cで 30分間撹拌した。その後、リン酸化基質として 4E — BP1リコンビナントペプチド（ラット PHAS— 1、カルビオケム社製）を 1 μ g添加し、 さらに 30°Cで 50分間撹拌した。 3倍濃度の SDSサンプルバッファーを 50 1添カロし、 5分間煮沸して反応を停止した後、 SDS— PAGEにより蛋白質の分離を行なった。 分離された蛋白質試料を PVDF膜に転写した後、 1次抗体として、抗スレオニン 37 Z46リン酸化 4E— BP1抗体 (セルシグナリング社製)を加え、膜上の蛋白質と反応さ せた。その後、 2次抗体として、西洋ヮサビペルォキシダーゼ標識された抗ゥサギ Ig 抗体 (セルシグナリング社製)を反応させた。その後、 ECL試薬で発色を行い、 LAS - 1000ルミノ'イメージアナライザーで検出した。  [0086] 50 μl mTOR complex immunoprecipitation beads were diluted with Atsy buffer to a final concentration of 2.5 μmolZl (10 μ1) and 50 μl phosphorylation buffer [ 10 mmol / 1 hepes-sodium hydroxide (adjusted to pH 7.4), 50 mmol Zl sodium chloride, 50 mmol / l β-glycephosphate, lOmmolZl manganese chloride, 100 / z molZl adenosine triphosphate] Stir at 30 ° C for 30 minutes. Thereafter, 1 μg of 4E-BP1 recombinant peptide (rat PHAS-1, manufactured by Calbiochem) was added as a phosphorylated substrate, and the mixture was further stirred at 30 ° C. for 50 minutes. After adding 50 times 3 times SDS sample buffer and boiling for 5 minutes to stop the reaction, proteins were separated by SDS-PAGE. After the separated protein sample was transferred to a PVDF membrane, an anti-threonine 37 Z46 phosphorylated 4E-BP1 antibody (manufactured by Cell Signaling) was added as a primary antibody to react with the protein on the membrane. Thereafter, anti-rabbit Ig antibody (manufactured by Cell Signaling) labeled with horseradish rust peroxidase was reacted as a secondary antibody. After that, color was developed with ECL reagent and detected with LAS-1000 Lumino 'image analyzer.
[0087] その結果、ビニグロールは 4E— BP1基質ペプチドのスレオニン 37Z46位のリン酸 化を 1. 5倍に亢進した。  [0087] As a result, biniglol increased the phosphorylation of threonine 37Z46 position of the 4E-BP1 substrate peptide by 1.5 times.
この結果より、ビニグロールが mTOR複合体に直接作用することで、神経幹細胞の 増殖促進活性を発揮することが示された。  From these results, it was shown that biniglol exerts a proliferation promoting activity of neural stem cells by directly acting on the mTOR complex.
試験例 10 :分岐鎖アミノ酸による神経幹細胞の増殖促進 低インシュリン N— 2添カ卩物 [0. 1 μ g/ml インシュリン（シグマ社製）、 100 μ gZ ml ゥシアポトランスフェリン（シグマ社製）、 6. 3ngZmlプロゲステロン（シグマ社製） 、 1. 6 μ g/ml プトレシン（シグマ社製）、 5. 2ng/ml セレン酸ナトリウム（シグマ 社製）]および抗生物質混合液 [0. 05U/ml ペニシリン、 0. 05 ^ g/ml ストレプ トマイシン（インビトロジェン社製） ]を添カ卩した DMEMZF12培地（インビトロジェン社 製）（以下、低インシュリン培地と称する）に、 ANSC— 7細胞を 2. 0 X 105個/ mlの 濃度で懸濁し、ポリオル-チンおよびラミニンで表面カ卩ェした 96穴プレートに 0. lml ずつ播種して、 37°Cで 5%CO雰囲気下、ー晚培養した。その後、 50 1の培養上 Test Example 10: Growth promotion of neural stem cells by branched chain amino acids Low Insulin N—2-supplemented [0.1 μg / ml insulin (Sigma), 100 μgZ ml Ussiapotransferrin (Sigma), 6.3 ngZml progesterone (Sigma), 1. 6 μg / ml putrescine (Sigma), 5.2 ng / ml sodium selenate (Sigma)] and antibiotic mixture [0.05 U / ml penicillin, 0.05 ^ g / ml streptomycin (Invitrogen) The suspension of ANSC-7 cells at a concentration of 2.0 × 10 5 cells / ml in DMEMZF12 medium (manufactured by Invitrogen) (hereinafter referred to as low insulin medium) supplemented with Then, 0.1 ml each was seeded on a 96-well plate surface-coated with laminin and cultured at 37 ° C in a 5% CO atmosphere. Then on 50 1 culture
2  2
清を除去し、低インシュリン培地で終濃度の 2倍になるように段階希釈した分岐鎖アミ ノ酸（L—パリン、 L一口イシン、 L—イソロイシン）、 FGF2 (陽性コントロール）、または DMSO (陰性コントロール）を 1ゥエルあたり 50 1添カ卩した。 96時間培養後、 50 1 の培養上清を除去し、 4°Cに冷却した 15%中性緩衝ホルマリン液 (和光純薬工業社 製）を 1ゥエルあたり 50 1カ卩え、 30分間静置した。溶液を全量除去し、 80 1の 15% 中性緩衝ホルマリン液を添カ卩し、 1時間静置後、 TBSTで 2回洗浄し、 0. 5% Trito nX— 100を含む PBSで希釈した 5%スキムミルク溶液で 3. 3 μ mol/1に調製したへ キスト 33342 (ナカライテスタ社製)を 50 /z lZゥエルで添加し、暗所、 4°Cで一晩、核 を標識した。 100 1Zゥエルの TBSTで 2回、続いて PBSで 1回洗浄した後、 100 1 Zゥエルの PBSに浸し、核染色された細胞の蛍光強度を FDSS6000で測定した。 細胞を播種しな ヽゥエルの測定値を 0%、陰性コントロールの測定値を 100%とし、 分岐鎖アミノ酸添加群における相対値を算出した。 Branched-chain amino acid (L-parin, L-tipped isine, L-isoleucine), FGF2 (positive control), or DMSO (negative) serially diluted to a final concentration of 2 times in low-insulin medium. Control) was added at 50 1 per well. After culturing for 96 hours, remove 50 1 culture supernatant, and add 50% 15% neutral buffered formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) cooled to 4 ° C for 30 minutes. did. Remove the entire solution, add 80 1 15% neutral buffered formalin solution, leave it for 1 hour, wash twice with TBST, and dilute with PBS containing 0.5% Triton X-100 5 Hexest 33342 (manufactured by Nacalai Testa Co., Ltd.) prepared to 3.3 μmol / 1 with a% skim milk solution was added at 50 / z lZ well, and nuclei were labeled in the dark at 4 ° C overnight. After washing twice with 100 1Z-well TBST and then once with PBS, the cells were immersed in 100 1Z-well PBS and the fluorescence intensity of the nuclear-stained cells was measured with FDSS6000. The relative value in the branched chain amino acid addition group was calculated by setting the measured value of the fuel without seeding as 0% and the measured value of the negative control as 100%.
その結果、 20mmolZl L -パリン添カ卩群で 115%、 20mmol/l L-ロイシン添カロ 群で 120%、 20mmol/l L-イソロイシン添加群で 137% (いずれも 3連の平均値） 、 ANSC— 7細胞の増殖が促進された。  As a result, 115% in the 20 mmolZl L-palin-added potassium group, 120% in the 20 mmol / l L-leucine-added caro group, 137% in the 20 mmol / l L-isoleucine added group (both mean values in triplicate) — 7 cell proliferation was promoted.
参考例 1：ラット脳力 の成体神経幹細胞の単離と培養 Reference Example 1: Isolation and culture of adult neural stem cells from rat brain power
7週齢の Sprague Dawley ratをエーテル麻酔によって眠らせた後に断頭し、頭 頂部より頭蓋骨を切開して脳を摘出した。摘出した脳から脳室周囲部位を含む組織 を顕微鏡下で眼科用のはさみとピンセットを用いて分離した。脳室周囲部位を含む 組織は眼科用はさみとメスを用いて lmm3程度の断片にした後、 2. 5UZmlのパパ イン、 250U/mlの DNase (いずれも Worthington, Freehold, NJ社製）、 lU/m 1の中性プロテアーゼ（Dispase、ベーリンガ一'マンハイム社製）を含む 5mlの HBSS 緩衝液 (インビトロジヱン社製）中で 37°C、 30分間消化反応を行なった。該反応によ り得られた細胞と組織の混合物を 10%の胎仔牛血清 (ハイクローン社製)を含む DM EM (インビトロジェン社製)で 3回洗浄後、 10%の胎仔牛血清を含む DMEMに溶解 し、 10 mのナイロンメッシュを用いて未消化物を除去した。 Seven-week-old Sprague Dawley rats were decapitated after anesthesia by ether anesthesia, and the brain was removed by incising the skull from the top of the head. The tissue including the periventricular region was separated from the excised brain using ophthalmic scissors and tweezers under a microscope. The tissue including the periventricular region is segmented to about lmm 3 using ophthalmic scissors and a scalpel, and then 2.5UZml of papa In, 250 U / ml DNase (both manufactured by Worthington, Freehold, NJ), 5 ml HBSS buffer (made by Invitrogen) containing neutral protease (Dispase, manufactured by Boehringer Mannheim) of lU / m 1 The digestion reaction was performed at 37 ° C for 30 minutes. The cell and tissue mixture obtained by the reaction was washed 3 times with DMEM (Invitrogen) containing 10% fetal calf serum (Hyclone), and then DMEM containing 10% fetal calf serum. The undigested material was removed using a 10 m nylon mesh.
[0089] 得られた細胞粗抽出液を、 10cmの培養皿上で 10%の胎仔牛血清を含む DMEM ZF 12培地（インビトロジェン社製）を用いて 37°Cのインキュベータ一中で 1晚培養し た。翌日、培地を 1%の N— 2添カ卩物（インビトロジェン社製）と 20ng/mlの FGF2 ( ペブロテック社製)を含む DMEMZF 12に置換して培養を開始した。 3日に一度、 培地の半分を新 ヽ 1 %の N— 2添カ卩物と 20ng/mlの FGF2を含む DMEM/F 12 に置換し、培養を継続した。小型細胞の小さなコロニーが形成されたら 1%のトリプシ ンで 30秒から 1分間程度処理し、剥がれた細胞を回収した。該細胞は、 lO ^ g/ml のポリオル-チン（シグマ社製）を用いて室温でー晚、および 5 μ gZmlのマウス EH S腫瘍由来ラミニン (ベタトン'ディッキンソン社製)を用いて 37°Cで一晩コートしたマ ルチウエルの培養皿 (フィッシャー ·サイェンティフイク社製)上に撒き、培養を継続し た。上記培養を続けることで、小型の突起を有し、厚みのある小型細胞が濃縮された 。本細胞を成体神経幹細胞株 ANSC— 7として上記の実験に使用した。  [0089] The obtained crude cell extract was cultured on a 10 cm culture dish in a 37 ° C incubator using DMEM ZF 12 medium (Invitrogen) containing 10% fetal calf serum. It was. On the next day, the culture medium was replaced with DMEMZF 12 containing 1% N-2 supplemented product (manufactured by Invitrogen) and 20 ng / ml FGF2 (manufactured by Pebrotech), and culture was started. Once every 3 days, half of the medium was replaced with DMEM / F 12 containing 1% fresh N-2 supplement and 20 ng / ml FGF2, and the culture was continued. When small colonies of small cells were formed, they were treated with 1% trypsin for 30 seconds to 1 minute, and the detached cells were collected. The cells were cultured at room temperature using lO ^ g / ml polyortin (Sigma), and 37 ° C using 5 μgZml mouse EHS tumor-derived laminin (Bettaton's Dickinson). The culture was continued overnight on a Multiwell culture dish (Fischer-Scientific). By continuing the culture, small cells having small protrusions and thickness were concentrated. This cell was used as an adult neural stem cell line ANSC-7 in the above experiment.
[0090] 以下に、本発明の実施例を示す。  [0090] Examples of the present invention will be described below.
実施例 1  Example 1
[0091] 錠剤 [0091] tablets
常法により、次の組成からなる錠剤を調製する。 A tablet having the following composition is prepared by a conventional method.
: L  : L
ビニグローノレ 5 mg  Vinignonole 5 mg
乳糖 62 mg  Lactose 62 mg
馬鈴薯デンプン 30 mg  Potato starch 30 mg
ポリビュルアルコール 2 mg  Polybur alcohol 2 mg
ステアリン酸マグネシウム 1 mg 実施例 2 Magnesium stearate 1 mg Example 2
[0092] 飴 [0092] 飴
常法により、次の組成からなる飴を調製する。 ノ ジン 1. OOg  According to a conventional method, a koji made of the following composition is prepared. Nojin 1. OOg
ロイシン 3. OOg  Leucine 3.OOg
イソロイシン 1. OOg  Isoleucine 1. OOg
粉末ソルビトール 94. 75g  Powdered sorbitol 94.75g
香料 0. 20g  Fragrance 0. 20g
ソノレビトーノレシード 0. 05g  Sonorebitonoresed 0. 05g
実施例 3  Example 3
[0093] 神経幹細胞の増殖促進剤  [0093] Neural stem cell proliferation promoter
常法により、ビニグロールを 0. ImmolZlになるように DMSOに溶解し、ビニグロー ルを含む神経幹細胞の増殖促進剤を調製した。  By a conventional method, vinylogol was dissolved in DMSO to a concentration of 0.1 mmol and a neural stem cell proliferation promoter containing vinylogol was prepared.
産業上の利用可能性  Industrial applicability
[0094] 本発明によれば、 mTORの活性を促進する物質を有効成分として含有する神経疾 患治療剤、神経新生もしくは再生促進剤、神経機能低下防止もしくは増進用飲食品 および飲食品添加剤、神経幹細胞増殖促進剤、神経幹細胞、ニューロンもしくはダリ ァ細胞の製造方法、該製造方法により製造されるニューロンもしくはグリア細胞、また は神経幹細胞増殖促進物質のスクリーニング方法を提供することができる。 [0094] According to the present invention, a therapeutic agent for neurological disease containing a substance that promotes mTOR activity as an active ingredient, an agent for neurogenesis or regeneration, a food or drink for preventing or enhancing neurological function, and a food or drink additive, It is possible to provide a method for producing a neural stem cell proliferation promoter, a neural stem cell, a neuron or a Daria cell, a neuron or glial cell produced by the production method, or a screening method for a neural stem cell proliferation promoting substance.

Claims

請求の範囲 The scope of the claims
[1] マンマリアン 'ターゲット'ォブ'ラパマイシン (以下、 mTORと略す)の活性を促進す る物質を有効成分として含有する神経疾患治療剤。  [1] A therapeutic agent for neurological diseases containing as an active ingredient a substance that promotes the activity of Mammalian 'target' ob 'rapamycin (hereinafter abbreviated as mTOR).
[2] 神経疾患が、パーキンソン病、アルツハイマー病、ダウン症、脳血管障害、脳卒中、 脊髄損傷、トリプレットリピート病、多発性硬化症、筋萎縮性側索硬化症、多発ニュー ロバチ一、てんかん、不安障害、統合失調症、鬱病および躁鬱病力 なる群より選ば れる神経疾患である請求項 1記載の治療剤。 [2] Neurological diseases are Parkinson's disease, Alzheimer's disease, Down's syndrome, cerebrovascular disorder, stroke, spinal cord injury, triplet repeat disease, multiple sclerosis, amyotrophic lateral sclerosis, multiple neurobaties, epilepsy, anxiety disorder 2. The therapeutic agent according to claim 1, wherein the therapeutic agent is a neurological disease selected from the group consisting of schizophrenia, depression, and manic depression.
[3] mTORの活性を促進する物質が、式 (I) [3] Substances that promote mTOR activity are represented by the formula (I)
[化 7]  [Chemical 7]
Figure imgf000036_0001
Figure imgf000036_0001
(式中、 R1は水素を表し、 ITはヒドロキシまたは保護されたヒドロキシを表す力 または R1と R2が一緒になつてォキソを表わし、 R3はヒドロキシメチル、保護されたヒドロキシメ チルまたはホルミルを表す)で表される化合物 [以下、化合物 (I)という]またはその薬 理学的に許容される塩である請求項 1または 2記載の治療剤。 (Wherein R 1 represents hydrogen, IT represents a force representing hydroxy or protected hydroxy, or R 1 and R 2 together represent oxo, R 3 represents hydroxymethyl, protected hydroxymethyl or The therapeutic agent according to claim 1 or 2, which is a compound represented by (formyl) [hereinafter referred to as compound (I)] or a pharmaceutically acceptable salt thereof.
Figure imgf000036_0002
で表されるビニグロールまたはその薬理学的に許容される塩である請求項 1または 2 記載の治療剤。
Figure imgf000036_0002
The therapeutic agent according to claim 1 or 2, which is biniglol represented by the formula or a pharmacologically acceptable salt thereof.
[5] mTORの活性を促進する物質が、分岐鎖アミノ酸またはその薬理学的に許容され る塩である請求項 1または 2記載の治療剤。  [5] The therapeutic agent according to claim 1 or 2, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
[6] mTORの活性を促進する物質が、ホスファチジン酸またはその薬理学的に許容さ れる塩である請求項 1または 2記載の治療剤。 [6] The therapeutic agent according to claim 1 or 2, wherein the substance that promotes mTOR activity is phosphatidic acid or a pharmacologically acceptable salt thereof.
[7] mTORの活性を促進する物質を有効成分として含有する神経新生または再生促 進剤。 [7] A neurogenesis or regeneration-promoting agent containing a substance that promotes mTOR activity as an active ingredient.
[8] mTORの活性を促進する物質が化合物 (I)またはその薬理学的に許容される塩で ある請求項 7記載の神経新生または再生促進剤。  8. The neurogenesis or regeneration promoter according to claim 7, wherein the substance that promotes mTOR activity is compound (I) or a pharmacologically acceptable salt thereof.
[9] mTORの活性を促進する物質がビニグロールまたはその薬理学的に許容される塩 である請求項 7記載の神経新生または再生促進剤。 [9] The neurogenesis or regeneration-promoting agent according to claim 7, wherein the substance that promotes mTOR activity is biniglol or a pharmacologically acceptable salt thereof.
[10] mTORの活性を促進する物質が分岐鎖アミノ酸またはその薬理学的に許容される 塩である請求項 7記載の神経新生または再生促進剤。 10. The neurogenesis or regeneration promoter according to claim 7, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
[11] mTORの活性を促進する物質がホスファチジン酸またはその薬理学的に許容され る塩である請求項 7記載の神経新生または再生促進剤。 [11] The neurogenesis or regeneration promoter according to claim 7, wherein the substance that promotes mTOR activity is phosphatidic acid or a pharmacologically acceptable salt thereof.
[12] mTORの活性を促進する物質を有効成分として含有する神経機能低下防止もしく は増進用飲食品または飲食品添加剤。 [12] A food / beverage additive or food / beverage additive for preventing or enhancing neurological function, which contains a substance that promotes mTOR activity as an active ingredient.
[13] mTORの活性を促進する物質が化合物 (I)またはその薬理学的に許容される塩で ある請求項 12記載の飲食品または飲食品添加剤。 [13] The food / beverage product or food / beverage product additive according to claim 12, wherein the substance that promotes mTOR activity is compound (I) or a pharmacologically acceptable salt thereof.
[14] mTORの活性を促進する物質がビニグロールまたはその薬理学的に許容される塩 である請求項 12記載の飲食品または飲食品添加剤。 [14] The food / beverage product or food / beverage additive according to claim 12, wherein the substance that promotes mTOR activity is biniglol or a pharmacologically acceptable salt thereof.
[15] mTORの活性を促進する物質が分岐鎖アミノ酸またはその薬理学的に許容される 塩である請求項 12記載の飲食品または飲食品添加剤。 [15] The food or drink or food and drink additive according to claim 12, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
[16] mTORの活性を促進する物質がホスファチジン酸またはその薬理学的に許容され る塩である請求項 12記載の飲食品または飲食品添加剤。 [16] The food / beverage product or food / beverage additive according to claim 12, wherein the substance that promotes mTOR activity is phosphatidic acid or a pharmacologically acceptable salt thereof.
[17] mTORの活性を促進する物質を有効成分として含有する神経幹細胞増殖促進剤 [18] mTORの活性を促進する物質が化合物 (I)またはその薬理学的に許容される塩で ある請求項 17記載の神経幹細胞増殖促進剤。 [17] Neural stem cell proliferation promoter containing a substance that promotes mTOR activity as an active ingredient 18. The neural stem cell proliferation promoter according to claim 17, wherein the substance that promotes mTOR activity is compound (I) or a pharmacologically acceptable salt thereof.
[19] mTORの活性を促進する物質がビニグロールまたはその薬理学的に許容される塩 である請求項 17記載の神経幹細胞増殖促進剤。 [19] The agent for promoting proliferation of neural stem cells according to claim 17, wherein the substance that promotes mTOR activity is biniglol or a pharmacologically acceptable salt thereof.
[20] mTORの活性を促進する物質が分岐鎖アミノ酸またはその薬理学的に許容される 塩である請求項 17記載の神経幹細胞増殖促進剤。 [20] The neural stem cell proliferation promoter according to claim 17, wherein the substance that promotes mTOR activity is a branched chain amino acid or a pharmacologically acceptable salt thereof.
[21] mTORの活性を促進する物質がホスファチジン酸またはその薬理学的に許容され る塩である請求項 17記載の神経幹細胞増殖促進剤。 [21] The agent for promoting proliferation of neural stem cells according to claim 17, wherein the substance that promotes the activity of mTOR is phosphatidic acid or a pharmacologically acceptable salt thereof.
[22] 神経幹細胞が成体神経幹細胞である請求項 17〜21の 、ずれか 1項に記載の神 経幹細胞増殖促進剤。 [22] The agent for promoting proliferation of neural stem cells according to any one of claims 17 to 21, wherein the neural stem cells are adult neural stem cells.
[23] 請求項 17〜22のいずれか 1項に記載の神経幹細胞増殖促進剤の存在下、神経 幹細胞を培養して神経幹細胞を増殖させ、該培養物中より神経幹細胞を採取するこ とを特徴とする神経幹細胞の製造方法。  [23] In the presence of the neural stem cell proliferation promoter according to any one of claims 17 to 22, the neural stem cell is cultured to proliferate the neural stem cell, and the neural stem cell is collected from the culture. A method for producing a neural stem cell, which is characterized.
[24] 請求項 23記載の製造方法により製造される神経幹細胞を培養してニューロンへ分 ィ匕させ、該培養物中より-ユーロンを採取することを特徴とする-ユーロンの製造方法  [24] A method for producing euron, characterized in that neural stem cells produced by the production method according to claim 23 are cultured and differentiated into neurons, and -euron is collected from the culture.
[25] 請求項 24記載の製造方法により製造されるニューロン。 [25] A neuron produced by the production method according to claim 24.
[26] 請求項 23記載の製造方法により製造される神経幹細胞を培養してグリア細胞へ分 ィ匕させ、該培養物中よりグリア細胞を採取することを特徴とするグリア細胞の製造方 法。  [26] A method for producing a glial cell, comprising culturing a neural stem cell produced by the production method according to claim 23 and distributing it to a glial cell, and collecting the glial cell from the culture.
[27] 請求項 26記載の製造方法により製造されるグリア細胞。  [27] A glial cell produced by the production method according to claim 26.
[28] (i) mTOR、 mTORによりリン酸ィ匕されるペプチド、および ATPを共存させた場合に おいて、リン酸ィ匕ペプチドの量を測定する工程、  [28] (i) a step of measuring the amount of phosphate peptide in the presence of mTOR, peptide phosphorylated by mTOR, and ATP;
(ii)被験物質、 mTOR、 mTORによりリン酸ィ匕されるペプチド、および ATPを共存さ せた場合において、リン酸ィ匕ペプチドの量を測定する工程、  (ii) a step of measuring the amount of phosphate peptide in the presence of test substance, mTOR, peptide phosphorylated by mTOR, and ATP;
(iii) (i)および (ii)の測定量から、被験物質の、該ペプチドに対するリン酸化促進活 性を評価する工程、および  (iii) evaluating the phosphorylation promoting activity of the test substance against the peptide from the measured amounts of (i) and (ii); and
(iv)該リン酸化促進活性を有する物質を神経幹細胞増殖促進物質として選択する 工程、 (iv) A substance having the phosphorylation promoting activity is selected as a neural stem cell proliferation promoting substance Process,
からなる神経幹細胞増殖促進物質のスクリーニング方法。 神経幹細胞が成体神経幹細胞である請求項 28記載のスクリ A screening method for a neural stem cell proliferation promoting substance comprising: 30. A script according to claim 28, wherein the neural stem cells are adult neural stem cells.
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