WO2006077954A1 - Remede pour maladie neurologique - Google Patents

Remede pour maladie neurologique 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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neural stem
substance
promotes
stem cell
acceptable salt
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Japanese (ja)
Inventor
Hideyuki Onodera
Michio Ichimura
Kazuhiro Sakurada
Ayako Kawabata
Toshio Ota
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KH Neochem Co Ltd
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Kyowa Hakko Kogyo Co Ltd
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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.

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Abstract

L'invention concerne un remède destiné au traitement d'une maladie neurologique ; un promoteur de la neurogenèse ou de la régénération nerveuse ; un aliment ou une boisson destinés à la prévention d'une baisse d'une fonction nerveuse ou à la promotion de la fonction nerveuse et un additif alimentaire destiné à la prévention d'une baisse d'une fonction nerveuse ou à la promotion de la fonction nerveuse ; un promoteur de croissance de cellules souches nerveuses ; une méthode de production de cellules souches nerveuses, de neurones ou de cellules gliales ; des neurones ou des cellules gliales produites selon cette méthode ; ou une méthode de criblage d'un promoteur de croissance de cellules souches nerveuses. L'invention concerne plus particulièrement un remède destiné au traitement d'une maladie neurologique, contenant, comme ingrédient actif, une substance augmentant l'activité de mTOR ; un promoteur de la neurogenèse ou de la régénération nerveuse ; un aliment ou une boisson destinés à la prévention d'une baisse d'une fonction nerveuse ou à la promotion de la fonction nerveuse et un additif alimentaire destiné à la prévention d'une baisse d'une fonction nerveuse ou à la promotion de la fonction nerveuse ; un promoteur de croissance de cellules souches nerveuses ; une méthode de production de cellules souches nerveuses, de neurones ou de cellules gliales ; des neurones ou des cellules gliales produites selon cette méthode ; ou une méthode de criblage d'un promoteur de croissance de cellules souches nerveuses.
PCT/JP2006/300821 2005-01-21 2006-01-20 Remede pour maladie neurologique Ceased WO2006077954A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008044691A1 (fr) * 2006-10-10 2008-04-17 Otsuka Pharmaceutical Factory, Inc. Agent antidépresseur
WO2009096445A1 (fr) * 2008-01-29 2009-08-06 Kyowa Hakko Kirin Co., Ltd. Accélérateur de la propagation de cellules du tronc nerveux
JP2012515166A (ja) * 2009-01-09 2012-07-05 ザ・ボード・オブ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・テキサス・システム 神経新生促進化合物
JP2013541495A (ja) * 2010-07-07 2013-11-14 ザ・ボード・オブ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・テキサス・システム 神経新生促進化合物

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