WO2019132004A1 - Prophylactic agent or therapeutic agent for alzheimer's disease, screening method therefor, and composition for preventing or treating alzheimer's disease - Google Patents

Prophylactic agent or therapeutic agent for alzheimer's disease, screening method therefor, and composition for preventing or treating alzheimer's disease Download PDF

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Publication number
WO2019132004A1
WO2019132004A1 PCT/JP2018/048471 JP2018048471W WO2019132004A1 WO 2019132004 A1 WO2019132004 A1 WO 2019132004A1 JP 2018048471 W JP2018048471 W JP 2018048471W WO 2019132004 A1 WO2019132004 A1 WO 2019132004A1
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group
disease
carbon atoms
preventing
alzheimer
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PCT/JP2018/048471
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French (fr)
Japanese (ja)
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択実 江良
隆太郎 梶原
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国立大学法人熊本大学
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Priority to JP2019562504A priority Critical patent/JP7445251B2/en
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Priority to JP2024020478A priority patent/JP2024040445A/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • 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
    • 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
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to an agent for preventing or treating Alzheimer's disease, a method for screening the same, and a composition for preventing or treating Alzheimer's disease.
  • Priority is claimed on Japanese Patent Application No. 2017-254887, filed Dec. 28, 2017, the content of which is incorporated herein by reference.
  • AD Alzheimer's disease
  • a ⁇ amyloid cascade hypothesis
  • a ⁇ is a small protein with a molecular weight of about 4 kD, composed of 38-43 amino acids, and is produced by being cleaved from amyloid precursor protein (APP) by secretase.
  • APP amyloid precursor protein
  • a ⁇ 40 and A ⁇ 42 exist due to the difference in the number of amino acids.
  • a ⁇ 42 is known to have stronger virulence because it has high aggregation.
  • Non-Patent Document 1 describes a method of screening for anti-A ⁇ drugs using neural cells that have expressed forebrain markers differentiated from human iPS cells.
  • the fundamental therapeutic agent for AD has not been found at present, and currently only symptomatic treatment.
  • clinical studies such as secretase inhibitors and anti-A ⁇ immunotherapy have been conducted as fundamental therapeutic agents for AD.
  • the present invention aims to provide an agent for preventing or treating Alzheimer's disease, a method for screening the same, and a composition for preventing or treating Alzheimer's disease based on a novel mechanism of action.
  • GM1 GM1 ganglioside
  • iPS cells GM1 ganglioside
  • An agent for preventing or treating Alzheimer's disease which comprises an agent for preventing or treating GM1 gangliosidosis as an active ingredient.
  • Amodiaquine Amodiaquine
  • Acacetin Acacetin
  • Sulfamerazine Sulphamelazine
  • Ungerine Ungelin
  • Amiodarone Amiodarone
  • Sertindol Sertindol
  • Delcorine Delcholine
  • Perphenazine Perphenazine
  • Althiazide Althiazide
  • Diethylstilbestrol diethylstilbestrol
  • Thiethylperazine thiethylperazine
  • Harmol Harmol
  • Skimmianine skimmianin
  • Succinylsulfathiazole Succinylsulfathiazole
  • Fillalbin filalbin
  • Canavanine Canavanine
  • Harmaline haluline Fluoxetine (fluoxetine)
  • Lovastatin lovastatin
  • Haloperidol Haloperidol
  • Haloperidol Haloperidol
  • R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aryl group, an aralkyl group or a cycloalkyl group.
  • X represents a single bond or a divalent linking group.
  • n represents 0, 1 or 2;
  • R 3 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group.
  • R 4 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.
  • R 5 represents a hydrogen atom, a hydroxyl group, an alkyl group of 1 to 5 carbon atoms, or an alkoxy group of 1 to 5 carbon atoms.
  • R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group.
  • a method of screening a preventive or therapeutic agent for Alzheimer's disease characterized by using a screening method of a preventive or therapeutic agent for GM1 gangliosidosis.
  • the method for preventing Alzheimer's disease or the agent for preventing Alzheimer's disease according to [5] which comprises the step of contacting a cultured compound with GM1 gangliosidosis neural stem cells to evaluate the change in accumulated amount of GM1 ganglioside in the neural stem cells.
  • GM1 gangliosidosis neural stem cell is a cell differentiated from a GM1 gangliosidosis patient-derived iPS cell.
  • an Alzheimer's disease preventive or therapeutic agent having a novel mechanism of action can be provided.
  • FIG. 1 It is a figure which compared GM1 accumulation in a healthy subject and a neural stem cell from GM1 patient.
  • A Comparison of ⁇ -gal activity in neural stem cells derived from healthy subjects, GM1 patients and Alzheimer's disease patients.
  • B It is the figure which introduce
  • C shows a comparison of GM1 accumulation in whole neural stem cells from healthy individuals and patients with Alzheimer's disease.
  • D It is a figure which compared GM1 accumulation in a lipid raft fraction of a healthy subject and a neural stem cell from a patient with Alzheimer's disease.
  • A It is the figure which compared the quantity of A (beta) 40 in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease.
  • B It is the figure which compared the quantity of A (beta) 42 in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease.
  • C It is the figure which compared the value of A (beta) 42 / A (beta) 40 in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease.
  • (D) It is the figure which compared the A (beta) total amount in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease.
  • (E) It is the figure which compared the sensitivity with respect to A (beta) 42 in the neural stem cell from a healthy person and the neural stem cell from GM1 gangliosidosis patient, respectively.
  • (A) It is the figure which compared the quantification result of soluble A (beta) 40 in the TBS (Tris Buffered Saline) extraction fraction of the brain of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse.
  • TBS Tris Buffered Saline
  • (B) It is the figure which compared the quantification result of soluble A (beta) 42 in the TBS (Tris Buffered Saline) extraction fraction of the brain of WT mouse, BKO mouse, 5 * FAD mouse, and BKO / 5 * FAD mouse.
  • (C) It is the figure which compared the quantification result of insoluble A (beta) 40 in the Guanidin-HCl extraction fraction of the brain of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse.
  • (D) It is the figure which compared the quantification result of insoluble A (beta) 42 in the Guanidin-HCl extraction fraction of the brain of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse.
  • (E) It is the figure which compared the deposition amount of the amyloid in the brain slice of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse. It is the figure which showed the mode of GM1 accumulation at the time of using a GM1 inhibitory compound. It is the figure which showed the mode of GM1 accumulation at the time of using a GM1 inhibitory compound.
  • FIG. 6 is a graph showing the results of comparison of the amount of A ⁇ produced in cells and the A ⁇ 42 / A ⁇ 40 ratio in the case of using a GM1 inhibitory compound. It is the figure which investigated the influence on GM1 accumulation
  • GM1 ganglioside is a kind of glycolipid and is a molecule involved in cell signal transduction and the like.
  • GM1 gangliosidosis is known as a disease in which GM1 is involved.
  • GM1 gangliosidosis is one of lysosomal diseases, which is a congenital metabolic disorder in which GM1 accumulates particularly in the nervous system (brain) due to a deficiency or abnormality of an enzyme involved in the hydrolysis of GM1 ganglioside.
  • GM1 gangliosidosis is classified into infant type, juvenile type and adult type according to onset time and clinical course. Especially in the infant type, developmental delay is seen by 3 to 6 months after birth, and by 1 year after birth most patients develop severe neuropathy such as decerebrate, usually by 3 to 4 years of age To die.
  • the screening method of the preventive or therapeutic agent for Alzheimer's disease of the present invention uses the screening method of the preventive or therapeutic agent for GM1 gangliosidosis.
  • the present invention relates to an agent for preventing or preventing Alzheimer's disease, which comprises the step of bringing a compound to be evaluated into contact with cultured GM1 gangliosidic neural stem cells to evaluate changes in the accumulation amount of GM1 ganglioside in the neural stem cells.
  • a compound library is added to the culture medium of cultured GM1 gangliosidic neural stem cells, and the influence on GM1 ganglioside in the neural stem cells is examined. More specifically, for example, the neural stem cells are seeded in a well plate and cultured in the presence of a compound library for about 1 to 5 days.
  • the GM1 gangliosidosis neural stem cells be cells differentiated from the GM1 gangliosidosis patient-derived iPS cells.
  • Examples of cells derived from GM1 patients include adipocytes, chondrocytes, osteoblasts, blood cells, fibroblasts and the like, with fibroblasts being preferred.
  • the method for reprogramming GM1 patient-derived cells to iPS cells and the method for differentiating iPS cells to neural stem cells follow the standard methods.
  • the compound found by such a step may have a step of evaluating the therapeutic effect of AD, such as evaluating the expression of A ⁇ .
  • evaluating the expression of A ⁇ there is a method of culturing AD patient-derived neural stem cells with a GM1 inhibitory compound and quantifying the ratio of A ⁇ 42 to A ⁇ 40 in the culture medium.
  • the present invention provides a screening method for an agent for preventing or treating Alzheimer's disease, which comprises administering a compound to be evaluated to an animal model for Alzheimer's disease described later.
  • a compound to be evaluated is administered by oral administration or parenteral administration such as intraperitoneal administration to an animal model of Alzheimer's disease described later. Subsequently, amyloid deposits in brain tissue are assessed.
  • the agent for preventing or treating Alzheimer's disease obtained using the screening method of the present invention is preferably a compound capable of exerting the therapeutic effect of AD by suppressing GM1 accumulation directly or indirectly. That is, an AD therapeutic agent containing such an AD therapeutic candidate compound as an active ingredient directly removes accumulated GM1, suppresses the aggregation of GM1, inhibits the synthesis of GM1, and promotes the degradation of GM1 etc. It is preferable that the accumulation of GM1 be suppressed directly or indirectly to exert the therapeutic effect of AD.
  • the present invention includes Amodiaquine (Amodiaquine), Acacetin (Acetetine), Sulfamerazine (Sulphamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindol), Delcorine (Delcholine), Perphenazine (Perphenazine) ), Althiazide (altiazide), Diethylstilbestrol (diethylstilbestrol), Thiethylperazine (thiethylperazine), Harmol (harmol), Skimmianine (skimmianin), Succinylsulfathiazole (succinylsulfathiazole), Fillalbin (filalbin), Canavanine (Canabanine armaline) ), Trihexyphenyl (trihexyphenidyl), Fluoxetine
  • the present invention relates to an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
  • the agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
  • R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aryl group, an aralkyl group or a cycloalkyl group.
  • X represents a single bond or a divalent linking group.
  • n represents 0, 1 or 2;
  • R 3 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group.
  • R 4 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.
  • R 5 represents a hydrogen atom, a hydroxyl group, an alkyl group of 1 to 5 carbon atoms, or an alkoxy group of 1 to 5 carbon atoms.
  • R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group.
  • alkyl group having 1 to 5 carbon atoms as R 1 and R 2 include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group and pentyl group , Isopentyl group, neopentyl group and the like.
  • the aryl group for R 1 and R 2 is preferably one having 6 to 18 carbon atoms, more preferably one having 6 to 10 carbon atoms, and particularly preferably a phenyl group.
  • the aralkyl group in R 1 and R 2 is preferably one in which an alkylene group having 1 to 5 carbon atoms is bonded to the aryl group in R 1 and R 2 .
  • the cycloalkyl group in R 1 and R 2 is preferably a group in which one hydrogen atom has been removed from a monocycloalkane having 3 to 8 carbon atoms, and specific examples include cyclopentane, cyclohexane, cyclooctane and the like. .
  • Alkyl group of 1 to 5 carbon atoms for R 3, an aralkyl group, an aryl group, and the cycloalkyl group include the same as described above in R 1 and R 2.
  • Examples of the alkyl group having 1 to 5 carbon atoms for R 4 include the same ones as described above for R 1 and R 2 .
  • Examples of the alkyl group of 1 to 5 carbon atoms for R 5 include the same as those described above for R 1 and R 2 .
  • Examples of the alkoxy group having 1 to 5 carbon atoms for R 5 include the same ones as the alkyl group having 1 to 5 carbon atoms described above for R 1 and R 2 in the R moiety of —OR.
  • Examples of the alkyl group having 1 to 5 carbon atoms, the aralkyl group, the aryl group and the cycloalkyl group in R 6 include the same as those described above in R 1 and R 2 .
  • Examples of the alkoxy group having 1 to 5 carbon atoms for R 6 include the same ones as described above for R 5 .
  • X is preferably a single bond
  • n is preferably 1
  • R 3 to R 5 are preferably hydrogen atoms.
  • a compound represented by the following general formula (1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof may be contained as an active ingredient ,preferable.
  • R 1 and R 2 each independently represent a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an aryl group, an aralkyl group or a cycloalkyl group.
  • X represents a single bond or a divalent linking group.
  • R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group.
  • R 6 is preferably a hydroxyl group
  • R 1 and R 2 are preferably an alkyl group having 1 to 5 carbon atoms.
  • the agent for preventing or treating Alzheimer's disease of the present embodiment the compound represented by the following formula (1-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof is contained as an active ingredient Is particularly preferred.
  • the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
  • the agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
  • R 10 represents a single bond or a divalent linking group.
  • R 11 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group.
  • R 12 and R 13 each independently represent a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, or 1 to 5 carbon atoms And a hydroxyalkyl group, an aralkyl group, an aryl group or a cycloalkyl group of n 10 and n 11 each independently represent an integer of 0 to 4.
  • R 12 and R 13 When a plurality of R 12 and R 13 exist, they may be the same or different.
  • R 14 represents a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, or an alkoxy group of 1 to 5 carbon atoms.
  • n 12 represents an integer of 0 to 4; When a plurality of R 14 are present, they may be the same or different.
  • Examples of the divalent linking group for R 10 include the same as those described above for X.
  • Alkyl group of 1 to 5 carbon atoms for R 11, an alkoxy group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, and the cycloalkyl group include the same as described above in R 5.
  • Examples of the hydroxyalkyl group having 1 to 5 carbon atoms for R 11 include the same alkyl groups as those described above for R 1 and R 2 .
  • Alkyl group of R 12 and carbon number of 1 to the R 13 5, an alkoxy group having 1 to 5 carbon atoms, hydroxyalkyl group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, and the cycloalkyl group, the R 11 The same as those described above can be mentioned.
  • Examples of the thioalkyl group having 1 to 5 carbon atoms as R 12 and R 13 include the same alkyl groups as those described above for R 1 and R 2 .
  • the alkyl group having 1 to 5 carbon atoms, the thioalkyl group having 1 to 5 carbon atoms, and the alkoxy group having 1 to 5 carbon atoms those similar to those described above for R 12 and R 13 It can be mentioned.
  • n 10 is preferably 1.
  • a compound represented by the following general formula (2-1), a pharmaceutically acceptable salt thereof, or a solvate thereof may be contained as an active ingredient ,preferable.
  • R 10 represents a single bond or a divalent linking group.
  • R 11 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group.
  • R 12 represents a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, It represents an aryl group or a cycloalkyl group.
  • an alkylene group is preferable as R 10 .
  • the alkylene group for R 10 a group in which one hydrogen atom has been removed from a linear or branched alkyl group having 1 to 5 carbon atoms is preferable.
  • Examples of the alkyl group having 1 to 5 carbon atoms include the same as those described above for R 1 and R 2 .
  • the agent for preventing or treating Alzheimer's disease of the present embodiment contains a compound represented by the following general formula (2-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient Is more preferred.
  • R 11 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, It represents an aryl group or a cycloalkyl group.
  • R 12 represents a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, It represents an aryl group or a cycloalkyl group.
  • R 11 an alkyl group of 1 to 5 carbon atoms or a hydroxyalkyl group of 1 to 5 carbon atoms is preferable.
  • R 12 a halogen atom or a thioalkyl group having 1 to 5 carbon atoms is preferable.
  • the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (3), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
  • the agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
  • R 20 represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms, a thioalkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, 1 to 5 carbon atoms And a hydroxyalkyl group, an aralkyl group, an aryl group or a cycloalkyl group of R 21 and R 24 each independently represent a single bond or a divalent linking group.
  • R 22 and R 23 each independently represent a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a haloalkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, or 1 to 5 carbon atoms And an alkoxy group of 1 to 5 carbon atoms and a hydroxyalkyl group having 1 to 5 carbon atoms.
  • R 20 is a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group, And as the cycloalkyl group, the same as described above for R 12 can be mentioned.
  • Examples of the divalent linking group for R 21 and R 24 include the same as those described above for X.
  • a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, and a hydroxyalkyl group of 1 to 5 carbon atoms include The same as described above for R 12 can be mentioned.
  • the haloalkyl group having 1 to 5 carbon atoms as R 22 and R 23 include a fluoroalkyl group, an alkyl chloride group and an alkyl bromide group, and at least one of the alkyl groups described above in R 1 and R 2 What has a hydrogen atom replaced by a halogen atom is mentioned.
  • R 20 an alkyl group having 1 to 5 carbon atoms or an aralkyl group is preferable.
  • the aralkyl group for R 20 one in which an alkylene group of 1 to 5 carbon atoms is bonded to a phenyl group is preferable.
  • R 21 and R 24 a single bond or —O— is preferable.
  • R 22 and R 23 a hydrogen atom or a trifluoromethyl group is preferable.
  • a compound represented by the following formula (3-1) or (3-2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an agent for preventing or treating Alzheimer's disease of the present embodiment is an active ingredient It is particularly preferable to contain it as
  • the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (4), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
  • the agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
  • each of R 30 and R 31 independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, or 1 to 5 carbon atoms]
  • 5 represents an alkoxy group, a hydroxyalkyl group having 1 to 5 carbon atoms, or an aralkyl group.
  • the solid and dotted double line portions represent single bonds or double bonds.
  • R 30 and R 31 each represent a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, and an aralkyl group And the same as those described above for R 12 can be mentioned.
  • R 30 and R 31 an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, and a hydroxyl group are preferable.
  • a compound represented by the following formula (4-1) or (4-2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an agent for preventing or treating Alzheimer's disease of the present embodiment is an active ingredient. It is particularly preferable to contain it as
  • the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (5), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient: I will provide a.
  • the agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
  • R 40 represents a single bond or a divalent linking group.
  • R 41 to R 43 each independently represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkyl group having 1 to 5 carbon atoms, a thioalkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, It represents a hydroxyalkyl group of -5 or an aralkyl group.
  • Examples of the divalent linking group for R 40 include the same as those described above for X.
  • a halogen atom for R 41 to R 43 an alkyl group having 1 to 5 carbon atoms, a thioalkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, a hydroxyalkyl group having 1 to 5 carbon atoms, and an aralkyl group are the same as described above for R 12 .
  • an alkylene group is preferable.
  • the alkylene group for R 40 a group in which one hydrogen atom has been removed from a linear or branched alkyl group having 1 to 5 carbon atoms is preferable.
  • Examples of the linear or branched alkyl group having 1 to 5 carbon atoms include the same as those described above for R 1 and R 2 .
  • R 41 to R 43 a hydrogen atom or a halogen atom is preferable.
  • a compound represented by the following formula (5-1) or (5-2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an agent for preventing or treating Alzheimer's disease of the present embodiment is an active ingredient. It is particularly preferable to contain it as
  • the above-mentioned compound may be used in the form of a free form or in the form of a pharmaceutically acceptable salt. Also, they may be used in the form of a free form solvate, or may be used in the form of a salt solvate.
  • the salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and, for example, hydrochloride, sulfate, hydrobromide, hydroiodide, phosphate, nitrate, benzoate, methanesulfone Acid, 2-Hydroxyethane Sulfonate, p-Toluene Sulfonate, Acetate, Propanoate, Oxalate, Malonate, Succinate, Glutarate, Adipate, Tartrate, Maleic Acid Salts, fumarates, malates, mandelates and the like can be mentioned.
  • the solvate is not particularly limited as long as it is a pharmaceutically acceptable solvate, and includes, for example, hydrate, organic solvate and the like.
  • the present invention provides a composition for the prophylaxis or treatment of Alzheimer's disease, which comprises the above-mentioned prophylactic or therapeutic agent for Alzheimer's disease, and a pharmaceutically acceptable carrier.
  • the composition for preventing or treating Alzheimer's disease can also be provided as a composition for preventing or treating GM1 gangliosidosis.
  • composition for preventing or treating Alzheimer's disease of the present embodiment may be, for example, orally in the form of a tablet, a coated tablet, a pill, a powder, a granule, a capsule, a solution, a suspension, an emulsion, etc.
  • the pharmaceutically acceptable carrier those generally used for the preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injection such as water, ethanol and glycerin;
  • the pressure-sensitive adhesive include rubber-based pressure-sensitive adhesives and silicone-based pressure-sensitive adhesives.
  • the pharmaceutically acceptable carrier can be used alone or in combination of two or more.
  • the composition for preventing or treating Alzheimer's disease of the present embodiment may further contain an additive.
  • additives such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavoring agents such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid Salts, buffers such as sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.
  • the additives may be used alone or in combination of two or more.
  • the administration method of the preventive or therapeutic agent for Alzheimer's disease or the composition for preventing or treating Alzheimer's disease is not particularly limited, and may be appropriately determined according to the condition, weight, age, sex and the like of the patient. For example, tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions and the like are orally administered.
  • the injections are intravenously administered alone or mixed with ordinary fluid replacements such as glucose and amino acids, and further, if necessary, intraarterially, intramuscularly, intradermal, subcutaneous or intraperitoneally. Suppositories are administered rectally.
  • the external skin preparation is applied, affixed or sprayed to the affected area.
  • the dose of the prophylactic or therapeutic agent for Alzheimer's disease, or the composition for the prophylaxis or treatment of Alzheimer's disease varies depending on the patient's condition, body weight, age, sex, etc. and can not be determined generally, but in the case of oral administration
  • 1 ⁇ g to 10 g per day for example, 0.01 to 2000 mg of the active ingredient per day may be administered.
  • 0.1 ⁇ g to 1 g per day for example, 0.001 to 200 mg of the active ingredient per day may be administered.
  • suppositories for example, 1 ⁇ g to 10 g per day, for example, 0.01 to 2000 mg of the active ingredient per day may be administered.
  • a skin external preparation for example, 1 ⁇ g to 10 g of active ingredient per day, for example, 0.01 to 2000 mg per day may be administered.
  • the present invention provides for the prevention or treatment of Alzheimer's disease, Amodiaquine (Amodiaquine), Acacetin (Acacetin), Sulfamerazine (Sulfamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindole) , Delcorine (Dercoline), Perphenazine (Perphenazine), Althiazole (Althiazide), Diethylstilbestrol (Diethylstilbestrol), Thiethylperazine (Thiethylperazine), Harmol (Halmol), Skimmianine (Skimmianin), Succinylsulfathiazole (Succinyl Sulfathiazole), ), Canavanine (Canavanine), Harmaline (Halmarin), Trihexyphenidyl (Trihexyphenidyl), Flu
  • the present invention includes Amodiaquine (Amodiaquine), Acacetin (Acacetin), Sulfamerazine (Sulphamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindol), Delcorine (Delcholine), Perphenazine (Perphenazine) ), Althiazide (altiazide), Diethylstilbestrol (diethylstilbestrol), Thiethylperazine (thiethylperazine), Harmol (harmol), Skimmianine (skimmianin), Succinylsulfathiazole (succinylsulfathiazole), Fillalbin (filalbin), Canavanine (Canabanine armaline) ), Trihexyphenyl (trihexyphenidyl), Fluoxetine (fluoxetine), Lovastatin (lovastatin), Halope
  • the present invention relates to Amodiaquine (Amodiaquine), Acacetin (acacetin), Sulfamerazine (sulfamelazine), Ungerine for producing a preventive or therapeutic agent for Alzheimer's disease, or a composition for preventing or treating Alzheimer's disease.
  • Amodiaquine Amodiaquine
  • Acacetin acacetin
  • Sulfamerazine sulfamelazine
  • Ungerine for producing a preventive or therapeutic agent for Alzheimer's disease, or a composition for preventing or treating Alzheimer's disease.
  • the animal model for Alzheimer's disease used in the evaluation of the present invention is a ⁇ -gal protein of which ⁇ -Galactosidase (hereinafter also referred to as ⁇ -gal) gene expression is suppressed or lost or which is encoded by the ⁇ -gal gene. It is a non-human mammal whose function has been suppressed or lost and a mutation has been introduced into the expression control region of the ⁇ -gal gene or the ⁇ -gal gene.
  • ⁇ -gal ⁇ -Galactosidase
  • the ⁇ -gal protein is an enzyme present in lysosomes that plays a role in the hydrolysis of various biomolecules.
  • GM1 ganglioside is a substrate for ⁇ -gal protein and is abundant in the brain. Therefore, suppression or loss of ⁇ -gal leads to accumulation of GM1 ganglioside.
  • the loss of the function of the ⁇ -gal protein refers to the state in which the function originally possessed by the ⁇ -gal protein is completely lost.
  • the fact that the function of ⁇ -gal protein is suppressed refers to a state in which the function originally possessed by ⁇ -gal protein is partially lost.
  • the degree of suppression of the function of the ⁇ -gal protein is determined by comparing the model animal and the wild-type animal of the animal model with the control. It should be such an extent that a condition (difference from control) appears in a model animal in comparison with the animal.
  • An indicator of a condition recognized as Alzheimer's disease includes deposition of amyloid proteins (A ⁇ 40 and 42) in the brain.
  • Suppression or loss of function of ⁇ -gal protein can also be caused by suppression or loss of expression of ⁇ -gal gene.
  • Loss of expression of the ⁇ -gal gene means loss of the ⁇ -gal gene product in a model animal.
  • the suppression of the expression of the ⁇ -gal gene means that the amount of the ⁇ -gal gene product is suppressed as compared to a control animal such as a wild-type animal of a model animal. .
  • the degree of suppression is compared to Alzheimer's disease as shown above in comparison with a control animal such as a wild-type animal of a model animal. It is sufficient if it is in such an extent that a recognized state appears.
  • a nucleic acid sequence that causes expression of RNAi-inducing nucleic acid, antisense nucleic acid, aptamer or ribozyme for ⁇ -gal gene is introduced into a model animal and generated by gene knockdown etc. Can.
  • the model animal used for the evaluation of the present invention preferably has a mutation introduced into the expression control region of the ⁇ -gal gene or ⁇ -gal gene.
  • the introduced mutation may be one that suppresses or loses the expression of the ⁇ -gal gene or suppresses or loses the function of the ⁇ -gal protein encoded by the ⁇ -gal gene.
  • the above-mentioned ⁇ -gal gene is not only an exon which is a region encoding ⁇ -gal protein, but also suppresses or loses the expression of ⁇ -gal gene or functions of ⁇ -gal protein encoded by the above-mentioned ⁇ -gal gene Introns are also included if they can be suppressed or lost.
  • the expression regulatory region of the ⁇ -gal gene is a region that regulates the expression of the ⁇ -gal gene, and is, for example, a promoter, a silencer, an enhancer, or a response element.
  • Loss of function of the ⁇ -gal protein can be produced, for example, by introducing a mutation into the ⁇ -gal gene and disrupting the ⁇ -gal gene.
  • the suppression or loss of the expression ability of the ⁇ -gal gene can be produced, for example, by introducing a mutation into the expression regulatory region of the ⁇ -gal gene.
  • the method for introducing a mutation into the expression control region of the ⁇ -gal gene or ⁇ -gal gene can be carried out by genetic modification which is a known genetic engineering technique.
  • the mutation can be generated by deletion or substitution of part or all of the expression control region of the ⁇ -gal gene or ⁇ -gal gene, insertion of any sequence, and the like.
  • the deletion is preferably a deletion of one or more exons.
  • Introduction of these mutations includes, for example, treatment with mutagenic substances (Mutagen), gene targeting by ultraviolet irradiation, homologous recombination technology etc., gene knockout, conditional knock out by gene knockdown, Cre-loxP system etc. It can be done using the method.
  • ⁇ -gal gene mutation examples include mutations in human ⁇ -gal protein such as E186A, P10L, R201C, C127Y, W161G, Q255H, R351X, S532G, R148S and the like.
  • the animal model for Alzheimer's disease used in the evaluation of the present invention further includes deletion of exon 9 of human prisenilin 1 (PS1), M146L mutation, L285V mutation, N141I mutation of human PS2, KM670 / of human amyloid precursor protein (APP) It is preferable to have at least one mutation selected from the group consisting of 671 NL Swedish type mutation, I716V Florida type mutation, and V717 I London type mutation, M146L mutation of human prisenilin 1 (PS1), and L285V mutation, and human amyloid precursor It is more preferable to have five types of mutations, KM670 / 671 NL Swedish type mutation, I716V Florida type mutation, and V717I London type mutation of body protein (APP).
  • the non-human mammal is preferably, but not limited to, an animal classified into rodents.
  • Non-human mammals include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs and cats.
  • the model animal according to the evaluation of the present invention can be used for elucidating the mechanism of Alzheimer's disease, searching for a substance useful for the treatment or the like, and developing a treatment method or the like.
  • Fibroblasts were established from skin biopsies explants of GM1 gangliosidosis patients and healthy persons under informed consent according to a protocol approved by the ethics committee. Skin samples from patients and healthy individuals were minced and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). After confirming that fibroblasts appeared, fibroblasts were expanded to introduce a reprogramming gene.
  • FBS fetal bovine serum
  • iPS cells were established from established human-derived fibroblasts. Specifically, 1 day before infection, 5 ⁇ 10 5 human fibroblasts are seeded per well in a 6-well plate, and then Oct3 / 4 gene, Sox2 gene, K1f4 gene, and c-Myc gene. The cells were infected with a Sendai virus (SeV) vector containing MOI (multiplicity of infection) 3.
  • SeV Sendai virus
  • NSCs neural stem cells
  • the induced NSCs were deprived of the medium, the cells were detached using StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific), the cells were suspended in Neural Expansion Medium (Thermo Fisher Scientific), and then 100 mm cell culture coated with Geltrex was performed. Sowing to petri dishes. The cells at this time point were designated as P0 NSCs (NSCs with zero passage number), and thereafter, NSCs were appropriately increased in this medium and used for experiments.
  • Example 3 The amount of GM1 ganglioside in the brains of Alzheimer's patients is reported to be greater than the amount of GM1 gangliosides in the brains of healthy individuals.
  • the inventors examined the metabolic state of GM1 ganglioside in neural stem cells derived from Alzheimer's disease. First, when ⁇ -gal activity of patient-derived neural stem cells was examined, it was found that ⁇ -gal activity of Alzheimer's disease-derived neural stem cells was decreased as compared to that of healthy individuals. See confirmed ( Figure 2A). ). In FIG.
  • 201B7 and 409B2 indicate neural stem cells derived from healthy persons
  • A138 and A154 indicate neural stem cells derived from GM1 gangliosidosis patients
  • A232 # 3-1 and A232 # 2-2 indicate Alzheimer's disease.
  • 1 shows neural stem cells derived from.
  • Sol means the insoluble fraction
  • Sup means the soluble fraction. It is shown that there is no difference in ⁇ -gal activity in the insoluble fraction / availability fraction in this experimental example.
  • the inventors examined the effect of overexpression of APP on ⁇ -gal activity.
  • a control vector or an APP overexpression vector was introduced into neural stem cells from healthy individuals, and ⁇ -gal activity was measured. It was confirmed that ⁇ -gal activity was reduced in neural stem cells into which the APP overexpression vector was transferred, as compared to neural stem cells into which the control vector was transferred (see FIG. 2B).
  • FIG. 2C is a graph showing the amount of GM1 ganglioside in the whole cell
  • FIG. 2D is a graph showing the amount of GM1 ganglioside in the lipid raft fraction. It has been confirmed that the amount of GM1 ganglioside in neural stem cells derived from Alzheimer's disease is greater than the amount in neural stem cells derived from healthy individuals. In particular, the difference in the lipid raft fraction was large. There is also a finding that ⁇ -gal is involved in the degradation of GM1 ganglioside. From these facts, in neural stem cells derived from Alzheimer's disease, ⁇ -gal activity is reduced, whereby GM1 ganglioside is accumulated. It was suggested that.
  • Example 4 To confirm whether GM1 ganglioside reduction affected A ⁇ metabolism, ⁇ -gal protein was overexpressed in neural stem cells (see FIG. 3A-D).
  • Control indicates a neural stem cell into which a control vector has been introduced
  • GLB1 OE indicates a neural stem cell into which a ⁇ -gal protein overexpression vector has been introduced.
  • FIG. 3B it was confirmed that the amount of A ⁇ 42 decreased due to the overexpression of ⁇ -gal protein in neural stem cells derived from Alzheimer's disease.
  • 201B7 NSC represents neural stem cells derived from healthy subjects
  • A138 NSC represents neural stem cells derived from GM1 gangliosidosis patients
  • 201 B7 + GM1 represents neural stem cells derived from healthy subjects treated with GM1 ganglioside.
  • neural stem cells derived from GM1 gangliosidosis patients are more sensitive to A ⁇ 42 than neural stem cells derived from healthy individuals. Furthermore, it was confirmed that the sensitivity to A ⁇ 42 in neural stem cells derived from GM1 ganglioside-treated healthy individuals shows a tendency similar to that of GM1 gangliosidosis-derived neural stem cells. From these facts, it was suggested that GM1 ganglioside affects the production of A ⁇ in neural stem cells.
  • FIGS. 4A and 4B show the results of quantification of soluble A ⁇ in TBS (Tris Buffered Saline) extract fractions, and FIGS.
  • Example 6 ⁇ Evaluation of compounds by drug screening using imaging cytometer> A screening agent for Alzheimer's disease was conducted as follows. After coating a 96-well plate with Geltrex, neural stem cells derived from healthy individuals and iPS cells from GM1 gangliosidosis patients suspended in Neural Expansion Medium were seeded at a density of 5 ⁇ 10 4 / well. Thereafter, compounds of the known drug library were added to each well to a final concentration of 5 ⁇ M, and culture was performed for 72 hours.
  • FIG. 5 and FIG. 5 staining images of neural stem cells after drug treatment with several compounds hit as GM1 inhibitory compounds are shown in FIG. 5 and FIG.
  • the hit compounds it is confirmed that the fluorescence of CTB is lower and the accumulation of GM1 is suppressed, as compared with the case where no drug is added (FIG. 5 b, lower, FIG. 6 e, lower). (FIG. 5 c and d, FIG. 6 f to g, each lower).
  • Example 7 ⁇ Effect on hit (extracellular) A ⁇ of a hit drug that reduces GM1 accumulation> Neural stem cells derived from Alzheimer's disease patient-derived iPS cells are seeded at a density of 2 ⁇ 10 6 / well in Geltrex-coated 6-well plates, hit drug is added to a final concentration of 5 ⁇ M, and culture is performed for 72 hours did. After 72 hours, the cell supernatant was collected, and the supernatant obtained by centrifugation at 400 g for 10 minutes was used as a sample for analysis.
  • a ⁇ 42 / A ⁇ 40 in the medium of neural stem cells derived from Alzheimer's disease patients and A ⁇ 42 / A ⁇ 40 in the cells showed high values as compared to healthy persons (lower row in FIGS. 7 and 8). It was confirmed that the treatment with the GM1 inhibitory compound reduced the total A ⁇ and A ⁇ 42 / A ⁇ 40 ratio in the medium and cells of neural stem cells derived from Alzheimer's disease patients (FIG. 7 c, FIG. 8 f).
  • mouse brain was embedded with OCT compound, and a section was prepared with a thickness of 5 ⁇ m. The section was fixed with 4% paraformaldehyde and then 1 % Blocked with BSA solution and fluorescently stained with Alexa Fluor 488-CTB, Hoechst 33342.
  • mouse brain was fractionated and purified based on the extraction method of Svennerholm and Fredman, and used as a sample. , These samples Agilent 64 Analysis was carried out with 60 Triple Quadrupole LC / MS to quantify GM1 in the brain The results of the fluorescent staining are shown in FIG.
  • FIG. 9a In negative subjects, little CTB fluorescence was seen (Fig. 9a, upper row). On the other hand, in the positive subjects, the fluorescence of CTB was strong, and the accumulation of GM1 was confirmed (Fig. 9b, upper row). On the other hand, it was confirmed that the fluorescence of CTB was strongly suppressed and the accumulation of GM1 was suppressed in any of the treatments with the GM1 inhibitory compound (FIG. 9 c, d, each upper row). The quantitative result of GM1 is shown in FIG.
  • 11C and 11C show the results of determination of insoluble A ⁇ in Guanidin-HCl extract fractions. It was confirmed that A ⁇ 40 and 42 decreased in the Amodiaquine administration group and Thiethylperanzine administration group, as compared with the PBS administration group.
  • FIG. 11E Similar to the results of FIGS. 11A to D, it was confirmed that A ⁇ 40 and 42 decrease in the Amodiaquine administration group and the Thiethylperanzine administration group.
  • Example 10 ⁇ Influence on autophagy by addition of GM1 inhibitory compound> The synthesis and degradation process of ganglioside is shown in FIG. Each ganglioside of GM1, GM2 and GM3 is produced by the sequential addition of monosaccharides and N-acetylneuraminic acid. Furthermore, the degradation of gangliosides is carried out by the stepwise action of glycosidases in lysosomes. (1) to (7) in FIG. 12 show enzymes acting in each reaction. FIG. 13 shows that expression of NEU1 and ⁇ -GLU is increased by treatment with candidate compounds.
  • Neural stem cells obtained by differentiating from normal (201B7) and disease-derived (A138 # 1-3) iPS cells are seeded in the wells, Amodiaquine or Thiethylperanzine is added to 5 ⁇ M, respectively, and culture is performed for 72 hours. The After culture, RNA was isolated, and the expression amount of each enzyme gene was analyzed. In FIG. 13,-indicates an additive-free sample, amo indicates an Amodiaquine-loaded sample, and thie indicates a Thiethylperanzine-loaded sample. The present results suggest that the action of the GM1 inhibitory compound promotes degradation of ganglioside in lysosome and activates autophagy.
  • an Alzheimer's disease preventive or therapeutic agent having a novel mechanism of action can be provided.

Abstract

A prophylactic agent or therapeutic agent for Alzheimer's disease characterized by containing a GM1 gangliosidosis prophylactic agent or therapeutic agent as an active ingredient. A screening method for prophylactic agents or therapeutic agents for Alzheimer's disease characterized by involving the use of a GM1 gangliosidosis prophylactic agent or therapeutic agent.

Description

アルツハイマー病予防剤又は治療剤、そのスクリーニング方法、及びアルツハイマー病予防用又は治療用組成物Agent for preventing or treating Alzheimer's disease, method for screening the same, and composition for preventing or treating Alzheimer's disease
　本発明は、アルツハイマー病予防剤又は治療剤、そのスクリーニング方法、及びアルツハイマー病予防用又は治療用組成物に関する。
本願は、２０１７年１２月２８日に、日本に出願された特願２０１７－２５４８８７号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to an agent for preventing or treating Alzheimer's disease, a method for screening the same, and a composition for preventing or treating Alzheimer's disease.
Priority is claimed on Japanese Patent Application No. 2017-254887, filed Dec. 28, 2017, the content of which is incorporated herein by reference.
　アルツハイマー病（以下、「ＡＤ」ともいう。）は、認知症の６０－７０％を占め、先進国において最も金銭的コストが高い疾患となっている。ＡＤの病態発症機構としては、アミロイドβ（以下、「Ａβ」ともいう。）の産出上昇又は分解不全による、Ａβ蓄積を開始点とするアミロイドカスケード仮説が最も支持されている。 Alzheimer's disease (hereinafter also referred to as "AD") accounts for 60-70% of dementia and is the most expensive disease in developed countries. As the pathogenesis mechanism of AD, the amyloid cascade hypothesis, which starts from Aβ accumulation due to increased production or degradation of amyloid β (hereinafter also referred to as “Aβ”), is most supported.
　Ａβは、分子量約４ｋＤの小さな蛋白質で３８－４３個のアミノ酸からなり、アミロイド前駆蛋白（ＡＰＰ）からセクレターゼにより切り出されて産出される。主要なＡβの分子種として、アミノ酸数の違いにより、Ａβ４０とＡβ４２が存在する。これらのうち、Ａβ４２は、高い凝集性を有することから、より強い病原性を有することが知られている。 Aβ is a small protein with a molecular weight of about 4 kD, composed of 38-43 amino acids, and is produced by being cleaved from amyloid precursor protein (APP) by secretase. As the main molecular species of Aβ, Aβ40 and Aβ42 exist due to the difference in the number of amino acids. Among these, Aβ42 is known to have stronger virulence because it has high aggregation.
　ＡＤに対する治療剤開発において、様々な取組みがなされている（非特許文献１参照。
）。例えば、非特許文献１では、ヒトｉＰＳ細胞から分化させた前脳マーカーを発現した神経細胞を用いた抗Ａβ薬のスクリーニング方法が記載されている。 Various approaches have been made in the development of therapeutic agents for AD (see Non-Patent Document 1).
). For example, Non-Patent Document 1 describes a method of screening for anti-Aβ drugs using neural cells that have expressed forebrain markers differentiated from human iPS cells.
　ＡＤに対する根本的治療剤は、現在のところ見つかっておらず、対症療法のみにとどまっているのが現状である。これまでＡＤの根本治療剤として、セクレターゼ阻害剤、抗Ａβ免疫療法等の臨床研究が行われてきた。しかしながら、被験者に対する不十分な有効性や強い副作用のため、いずれも中止に追い込まれている。
　したがって、ＡＤの根本的治療剤開発のためには、今までにはない、新たな観点・標的分子から創薬を行う必要がある。 The fundamental therapeutic agent for AD has not been found at present, and currently only symptomatic treatment. Until now, clinical studies such as secretase inhibitors and anti-Aβ immunotherapy have been conducted as fundamental therapeutic agents for AD. However, due to insufficient efficacy and strong side effects on subjects, both are forced to stop.
Therefore, in order to develop a fundamental therapeutic agent for AD, it is necessary to conduct drug discovery from new perspectives and target molecules that have never been available.
　そこで本発明は、新たな作用機序に基づく、アルツハイマー病予防剤又は治療剤、そのスクリーニング方法、及びアルツハイマー病予防用又は治療用組成物を提供することを目的とする。 Therefore, the present invention aims to provide an agent for preventing or treating Alzheimer's disease, a method for screening the same, and a composition for preventing or treating Alzheimer's disease based on a novel mechanism of action.
　発明者らは、ＧＭ１ガングリオシド（以下、「ＧＭ１」ともいう。）患者由来ｉＰＳ細胞から分化させた神経幹細胞を用いたスクリーニング系が、アルツハイマー病予防剤又は治療剤のスクリーニング方法に用いられることを見出し、本発明を完成させた。
　すなわち、本発明は以下のとおりである。
［１］ＧＭ１ガングリオシドーシス予防剤又は治療剤を有効成分として含有することを特徴とするアルツハイマー病予防剤又は治療剤。
［２］Amodiaquine（アモジアキン）、Acacetin（アカセチン）、Sulfamerazine（スルファメラジン）、Ungerine（ウンゲリン）、Amiodarone（アミオダロン）、Sertindole（セルチンドール）、Delcorine（デルコリン）、Perphenazine（ペルフェナジン）、Althiazide（アルチアジド）、Diethylstilbestrol（ジエチルスチルベストロール）、Thiethylperazine（チエチルペラジン）、Harmol（ハルモール）、Skimmianine（スキムミアニン）、Succinylsulfathiazole（スクシニルサルファチアゾール）、Fillalbin（フィラルビン）、Canavanine（カナバニン）、Harmaline（ハルマリン）、Trihexyphenidyl（トリヘキシフェニジル）、Fluoxetine（フルオキセチン）、Lovastatin（ロバスタチン）、Haloperidol（ハロペリドール）、Prenylamine lactate（プレニルアミン乳酸）、Bromperidol（ブロムペリドール）、Convolamine（コンボルバミン）、及びMiglustat（ミグルスタット）、並びに、これらの薬学的に許容される塩、又はそれらの溶媒和物からなる群から選ばれる少なくとも１種を有効成分として含有することを特徴とするアルツハイマー病予防剤又は治療剤。
［３］下記一般式（１）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することを特徴とするアルツハイマー病予防剤又は治療剤。 The inventors found that a screening system using neural stem cells differentiated from GM1 ganglioside (hereinafter also referred to as “GM1”) patient-derived iPS cells is used as a screening method for an agent for preventing or treating Alzheimer's disease. , Completed the present invention.
That is, the present invention is as follows.
[1] An agent for preventing or treating Alzheimer's disease, which comprises an agent for preventing or treating GM1 gangliosidosis as an active ingredient.
[2] Amodiaquine (Amodiaquine), Acacetin (Achacetin), Sulfamerazine (Sulphamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindol), Delcorine (Delcholine), Perphenazine (Perphenazine), Althiazide (Alchiazide) , Diethylstilbestrol (diethylstilbestrol), Thiethylperazine (thiethylperazine), Harmol (harmol), Skimmianine (skimmianin), Succinylsulfathiazole (succinylsulfathiazole), Fillalbin (filalbin), Canavanine (canavanine), Harmaline (haluline) (trihexyphenyl) Fluoxetine (fluoxetine), Lovastatin (lovastatin), Haloperidol (haloperidol), Prenylamine lactate (prenylamine lactic acid), Bromperidol (bromperidol), Convolamine (Convolamine) Alzheimer's disease characterized in that it contains at least one member selected from the group consisting of rubamine), Miglustat (Miglustat), and a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient Prophylactic or therapeutic agent.
[3] An agent for preventing or treating Alzheimer's disease, which comprises the compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
［一般式（１）中、Ｒ１及びＲ２は、それぞれ独立して、水素原子、炭素数１～５のアルキル基、アリール基、アラルキル基、又はシクロアルキル基を表す。Ｘは、単結合、又は２価の連結基を表す。ｎは、０、１、又は２を表す。Ｒ３は、水素原子、炭素数１～５のアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。Ｒ４は、水素原子、又は炭素数１～５のアルキル基を表す。Ｒ５は、水素原子、水酸基、炭素数１～５のアルキル基、又は炭素数１～５のアルコキシ基を表す。Ｒ６は、水素原子、水酸基、シアノ基、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、アラルキル基、アリール基、又はシクロアルキル基を表す。］
［４］［１］～［３］のいずれか一つに記載のアルツハイマー病予防剤又は治療剤及び薬学的に許容される担体を含有する、アルツハイマー病予防用又は治療用組成物。
［５］ＧＭ１ガングリオシドーシス予防剤又は治療剤のスクリーニング方法を用いることを特徴とするアルツハイマー病予防剤又は治療剤のスクリーニング方法。
［６］評価対象化合物を、培養されたＧＭ１ガングリオシドーシス神経幹細胞に接触させて、前記神経幹細胞におけるＧＭ１ガングリオシドの蓄積量の変化を評価する工程を含む［５］に記載のアルツハイマー病予防剤又は治療剤のスクリーニング方法。
［７］前記ＧＭ１ガングリオシドーシス神経幹細胞が、ＧＭ１ガングリオシドーシス患者由来ｉＰＳ細胞から分化させた細胞である［６］に記載のアルツハイマー病予防剤又は治療剤のスクリーニング方法。 [In the general formula (1), R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aryl group, an aralkyl group or a cycloalkyl group. X represents a single bond or a divalent linking group. n represents 0, 1 or 2; R 3 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. R 4 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. R 5 represents a hydrogen atom, a hydroxyl group, an alkyl group of 1 to 5 carbon atoms, or an alkoxy group of 1 to 5 carbon atoms. R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. ]
[4] A composition for preventing or treating Alzheimer's disease, which comprises the agent for preventing or treating Alzheimer's disease according to any one of [1] to [3] and a pharmaceutically acceptable carrier.
[5] A method of screening a preventive or therapeutic agent for Alzheimer's disease, characterized by using a screening method of a preventive or therapeutic agent for GM1 gangliosidosis.
[6] The method for preventing Alzheimer's disease or the agent for preventing Alzheimer's disease according to [5], which comprises the step of contacting a cultured compound with GM1 gangliosidosis neural stem cells to evaluate the change in accumulated amount of GM1 ganglioside in the neural stem cells. Methods of screening for therapeutic agents.
[7] The screening method of the agent for preventing or treating Alzheimer's disease according to [6], wherein the GM1 gangliosidosis neural stem cell is a cell differentiated from a GM1 gangliosidosis patient-derived iPS cell.
　本発明により、新たな作用機序を有するアルツハイマー病予防剤又は治療剤を提供することができる。 According to the present invention, an Alzheimer's disease preventive or therapeutic agent having a novel mechanism of action can be provided.
健常者及びＧＭ１患者由来の神経幹細胞におけるＧＭ１蓄積を比較した図である。It is a figure which compared GM1 accumulation in a healthy subject and a neural stem cell from GM1 patient. （Ａ）健常者、ＧＭ１患者、及びアルツハイマー病患者由来の神経幹細胞におけるβ－ｇａｌ活性を比較した図である。（Ｂ）健常者由来の神経幹細胞に、コントロールベクター又はＡＰＰ過剰発現ベクターを導入し、β－ｇａｌ活性を比較した図である。（Ｃ）健常者、及びアルツハイマー病患者由来の神経幹細胞全体におけるＧＭ１蓄積を比較した図である。（Ｄ）健常者、及びアルツハイマー病患者由来の神経幹細胞の脂質ラフト画分におけるＧＭ１蓄積を比較した図である。(A) Comparison of β-gal activity in neural stem cells derived from healthy subjects, GM1 patients and Alzheimer's disease patients. (B) It is the figure which introduce | transduced the control vector or the APP overexpression vector into the neural stem cell derived from a healthy subject, and compared (beta) -gal activity. (C) shows a comparison of GM1 accumulation in whole neural stem cells from healthy individuals and patients with Alzheimer's disease. (D) It is a figure which compared GM1 accumulation in a lipid raft fraction of a healthy subject and a neural stem cell from a patient with Alzheimer's disease. （Ａ）β－ｇａｌタンパク質を過剰発現させた健常者、及びアルツハイマー病由来の神経幹細胞におけるＡβ４０の量を比較した図である。（Ｂ）β－ｇａｌタンパク質を過剰発現させた健常者、及びアルツハイマー病由来の神経幹細胞におけるＡβ４２の量を比較した図である。（Ｃ）β－ｇａｌタンパク質を過剰発現させた健常者、及びアルツハイマー病由来の神経幹細胞におけるＡβ４２／Ａβ４０の値を比較した図である。（Ｄ）β－ｇａｌタンパク質を過剰発現させた健常者、及びアルツハイマー病由来の神経幹細胞におけるＡβ全量を比較した図である。（Ｅ）健常者由来の神経幹細胞及びＧＭ１ガングリオシドーシス患者由来の神経幹細胞、それぞれにおけるＡβ４２に対する感受性を比較した図である。(A) It is the figure which compared the quantity of A (beta) 40 in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease. (B) It is the figure which compared the quantity of A (beta) 42 in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease. (C) It is the figure which compared the value of A (beta) 42 / A (beta) 40 in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease. (D) It is the figure which compared the A (beta) total amount in the healthy subject which overexpressed (beta) -gal protein, and the neural stem cell from Alzheimer's disease. (E) It is the figure which compared the sensitivity with respect to A (beta) 42 in the neural stem cell from a healthy person and the neural stem cell from GM1 gangliosidosis patient, respectively. （Ａ）ＷＴマウス、ＢＫＯマウス、５×ＦＡＤマウス、及びＢＫＯ／５×ＦＡＤマウスの脳のＴＢＳ（Tris Buffered Saline）抽出画分における可溶性Ａβ４０の定量結果を比較した図である。（Ｂ）ＷＴマウス、ＢＫＯマウス、５×ＦＡＤマウス、及びＢＫＯ／５×ＦＡＤマウスの脳のＴＢＳ（Tris Buffered Saline）抽出画分における可溶性Ａβ４２の定量結果を比較した図である。（Ｃ）ＷＴマウス、ＢＫＯマウス、５×ＦＡＤマウス、及びＢＫＯ／５×ＦＡＤマウスの脳のGuanidin-HCl抽出画分における不溶性Ａβ４０の定量結果を比較した図である。（Ｄ）ＷＴマウス、ＢＫＯマウス、５×ＦＡＤマウス、及びＢＫＯ／５×ＦＡＤマウスの脳のGuanidin-HCl抽出画分における不溶性Ａβ４２の定量結果を比較した図である。（Ｅ）ＷＴマウス、ＢＫＯマウス、５×ＦＡＤマウス、及びＢＫＯ／５×ＦＡＤマウスの脳切片におけるアミロイドの沈着量を比較した図である。(A) It is the figure which compared the quantification result of soluble A (beta) 40 in the TBS (Tris Buffered Saline) extraction fraction of the brain of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse. (B) It is the figure which compared the quantification result of soluble A (beta) 42 in the TBS (Tris Buffered Saline) extraction fraction of the brain of WT mouse, BKO mouse, 5 * FAD mouse, and BKO / 5 * FAD mouse. (C) It is the figure which compared the quantification result of insoluble A (beta) 40 in the Guanidin-HCl extraction fraction of the brain of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse. (D) It is the figure which compared the quantification result of insoluble A (beta) 42 in the Guanidin-HCl extraction fraction of the brain of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse. (E) It is the figure which compared the deposition amount of the amyloid in the brain slice of WT mouse, BKO mouse, 5xFAD mouse, and BKO / 5xFAD mouse. ＧＭ１抑制化合物を用いた場合のＧＭ１蓄積の様子を示した図である。It is the figure which showed the mode of GM1 accumulation at the time of using a GM1 inhibitory compound. ＧＭ１抑制化合物を用いた場合のＧＭ１蓄積の様子を示した図である。It is the figure which showed the mode of GM1 accumulation at the time of using a GM1 inhibitory compound. ＧＭ１抑制化合物を用いた場合の培地中におけるＡβ量、及びＡβ４２／Ａβ４０比を比較した結果を示した図である。It is the figure which showed the result of having compared the amount of A (beta) in the culture medium at the time of using a GM1 inhibitory compound, and A (beta) 42 / A (beta) 40 ratio. ＧＭ１抑制化合物を用いた場合の細胞内におけるＡβ産出量及びＡβ４２／Ａβ４０比を比較した結果を示した図である。FIG. 6 is a graph showing the results of comparison of the amount of Aβ produced in cells and the Aβ42 / Aβ40 ratio in the case of using a GM1 inhibitory compound. ＧＭ１抑制化合物を腹腔内投与した場合のＧＭ１モデルマウスの脳切片におけるＧＭ１蓄積への影響を調べた図である。It is the figure which investigated the influence on GM1 accumulation | storage in the brain section of GM1 model mouse at the time of intraperitoneally administering GM1 inhibitory compound. ＧＭ１抑制化合物を腹腔内投与した場合のＧＭ１モデルマウスの脳におけるＧＭ１ガングリオシド量を比較した結果を示した図である。FIG. 6 shows the results of comparison of GM1 ganglioside amounts in the brain of GM1 model mice when a GM1 inhibitory compound was intraperitoneally administered. （Ａ）ＰＢＳ、Amodiaquine、及びThiethylperanzineをそれぞれ投与した５×ＦＡＤマウスの脳のＴＢＳ（Tris Buffered Saline）抽出画分における可溶性Ａβ４０の定量結果を比較した図である。（Ｂ）ＰＢＳ、Amodiaquine、及びThiethylperanzineをそれぞれ投与した５×ＦＡＤマウスの脳のＴＢＳ（Tris Buffered Saline）抽出画分における可溶性Ａβ４２の定量結果を比較した図である。（Ｃ）ＰＢＳ、Amodiaquine、及びThiethylperanzineをそれぞれ投与した５×ＦＡＤマウスの脳のGuanidin-HCl抽出画分における不溶性Ａβ４０の定量結果を比較した図である。（Ｄ）ＰＢＳ、Amodiaquine、及びThiethylperanzineをそれぞれ投与した５×ＦＡＤマウスの脳のGuanidin-HCl抽出画分における不溶性Ａβ４２の定量結果を比較した図である。（Ｅ）ＰＢＳ、Amodiaquine、及びThiethylperanzineをそれぞれ投与した５×ＦＡＤマウスの脳切片におけるアミロイドの沈着量を比較した図である。(A) It is the figure which compared the quantification result of soluble A (beta) 40 in the TBS (Tris Buffered Saline) extraction fraction of the brain of 5 * FAD mouse | mouth which each administered PBS, Amodiaquine, and Thiethylperanzine. (B) It is the figure which compared the quantification result of soluble A (beta) 42 in the TBS (Tris Buffered Saline) extraction fraction of the brain of 5 * FAD mouse | mouth which each administered PBS, Amodiaquine, and Thiethylperanzine. (C) It is the figure which compared the quantification result of insoluble A (beta) 40 in the Guanidin-HCl extraction fraction of the brain of 5 * FAD mouse | mouth which each administered PBS, Amodiaquine, and Thiethylperanzine. (D) It is the figure which compared the quantification result of insoluble A (beta) 42 in the Guanidin-HCl extraction fraction of the brain of 5 * FAD mouse | mouth which each administered PBS, Amodiaquine, and Thiethylperanzine. (E) It is the figure which compared the deposition amount of the amyloid in the brain section of 5xFAD mouse | mouth which each administered PBS, Amodiaquine, and Thiethylperanzine. ガングリオシドの合成及び分解過程を示した図である。It is a figure showing the synthesis and decomposition process of ganglioside. Amodiaquine又はThiethylperanzineを添加した、正常(201B7)及び疾患由来 (A138 #1-3)のiPS細胞から分化して得られた神経幹細胞における各酵素遺伝子の発現量の解析結果である。It is the analysis result of the expression level of each enzyme gene in neural stem cells obtained by differentiating from normal (201B7) and disease-derived (A138 # 1-3) iPS cells to which Amodiaquine or Thiethylperanzine is added.
［アルツハイマー病予防剤又は治療剤のスクリーニング方法］
　近年、ＡＤ患者の神経細胞膜表面における脂質ラフトに存在するＧＭ１ガングリオシドが、ＡＤにおけるＡβの重合を促進し、病態形成に深く関わっていることが明らかとなってきた（Hoshino T, Mahmood MI, Mori K, Matsuzaki K. J Phys Chem B. 2013 Jul 11;117(27):8085-94.参照。）。 [Method of screening for preventive or therapeutic agent for Alzheimer's disease]
Recently, it has been clarified that GM1 ganglioside present in lipid rafts on the nerve cell surface of AD patients promotes Aβ polymerization in AD and is deeply involved in the pathogenesis (Hoshino T, Mahmood MI, Mori K , Matsuzaki K. J Phys Chem B. 2013 Jul 11; 117 (27): 8085-94.).
　ＧＭ１ガングリオシドは、糖脂質の一種であり、細胞のシグナル伝達などに関与する分子である。このＧＭ１が関与する疾患として、ＧＭ１ガングリオシドーシスが知られている。 GM1 ganglioside is a kind of glycolipid and is a molecule involved in cell signal transduction and the like. GM1 gangliosidosis is known as a disease in which GM1 is involved.
　ＧＭ１ガングリオシドーシスはライソゾーム病の１つであり、ＧＭ１ガングリオシドの加水分解に関与する酵素の欠損・異常によって、ＧＭ１が特に神経系（脳）に蓄積してしまう先天性代謝異常症である。
　ＧＭ１ガングリオシドーシスは、発症時期と臨床経過により、乳児型、若年型、成人型に分類される。特に乳児型は生後３～６ヶ月までに発達の遅れが見られ、生後１年を過ぎるまでにほとんどの患児が、除脳硬直など重度の神経障害を示すようになり、通常３～４歳までに死亡する。 GM1 gangliosidosis is one of lysosomal diseases, which is a congenital metabolic disorder in which GM1 accumulates particularly in the nervous system (brain) due to a deficiency or abnormality of an enzyme involved in the hydrolysis of GM1 ganglioside.
GM1 gangliosidosis is classified into infant type, juvenile type and adult type according to onset time and clinical course. Especially in the infant type, developmental delay is seen by 3 to 6 months after birth, and by 1 year after birth most patients develop severe neuropathy such as decerebrate, usually by 3 to 4 years of age To die.
　実施例において後述するように、発明者らは、ＧＭ１ガングリオシドーシスとアルツハイマー病との間に関係性があることを見出した。
　即ち、本発明のアルツハイマー病予防剤又は治療剤のスクリーニング方法は、ＧＭ１ガングリオシドーシス予防剤又は治療剤のスクリーニング方法を用いる。 As described below in the Examples, the inventors have found that there is a relationship between GM1 gangliosidosis and Alzheimer's disease.
That is, the screening method of the preventive or therapeutic agent for Alzheimer's disease of the present invention uses the screening method of the preventive or therapeutic agent for GM1 gangliosidosis.
（第１実施形態）
　１実施形態において、本発明は、評価対象化合物を、培養されたＧＭ１ガングリオシドーシス神経幹細胞に接触させて、前記神経幹細胞におけるＧＭ１ガングリオシドの蓄積量の変化を評価する工程を含むアルツハイマー病予防剤又は治療剤のスクリーニング方法を提供する。 First Embodiment
In one embodiment, the present invention relates to an agent for preventing or preventing Alzheimer's disease, which comprises the step of bringing a compound to be evaluated into contact with cultured GM1 gangliosidic neural stem cells to evaluate changes in the accumulation amount of GM1 ganglioside in the neural stem cells. Provided are methods of screening for therapeutic agents.
　本実施形態において、例えば、化合物ライブラリーを、培養されたＧＭ１ガングリオシドーシス神経幹細胞の培地に添加し、前記神経幹細胞におけるＧＭ１ガングリオシドに対する影響を検討する。より具体的には、例えば、ウェルプレートに前記神経幹細胞を播種し、化合物ライブラリーの存在下で１～５日間程度培養する。 In the present embodiment, for example, a compound library is added to the culture medium of cultured GM1 gangliosidic neural stem cells, and the influence on GM1 ganglioside in the neural stem cells is examined. More specifically, for example, the neural stem cells are seeded in a well plate and cultured in the presence of a compound library for about 1 to 5 days.
　患者の脳から神経系細胞を採取することは困難であることから、前記ＧＭ１ガングリオシドーシス神経幹細胞が、ＧＭ１ガングリオシドーシス患者由来ｉＰＳ細胞から分化させた細胞であることが好ましい。
　ＧＭ１患者由来の細胞としては、脂肪細胞、軟骨細胞、骨芽細胞、血液細胞、線維芽細胞等が挙げられ、線維芽細胞が好ましい。ＧＭ１患者由来細胞からｉＰＳ細胞への初期化方法、及びｉＰＳ細胞から神経幹細胞への分化方法については定法に従う。 Since it is difficult to collect nervous system cells from the patient's brain, it is preferable that the GM1 gangliosidosis neural stem cells be cells differentiated from the GM1 gangliosidosis patient-derived iPS cells.
Examples of cells derived from GM1 patients include adipocytes, chondrocytes, osteoblasts, blood cells, fibroblasts and the like, with fibroblasts being preferred. The method for reprogramming GM1 patient-derived cells to iPS cells and the method for differentiating iPS cells to neural stem cells follow the standard methods.
　ＧＭ１ガングリオシドの蓄積量の変化を評価する方法としては、実施例にて後述するように、ＧＭ１ガングリオシドに対して親和性を有する物質を用いて、フローサイトメトリーやウエスタンブロット等により、定性・定量評価する方法が挙げられる。 As a method for evaluating changes in the accumulated amount of GM1 ganglioside, as described later in the Examples, qualitative / quantitative evaluation is performed by flow cytometry, western blot, etc. using a substance having affinity to GM1 ganglioside. Methods are included.
　係る工程により見いだされた化合物に対して、Ａβの発現を評価する等、ＡＤの治療効果を評価する工程を有していてもよい。
　Ａβの発現評価としては、ＡＤ患者由来神経幹細胞を、ＧＭ１抑制化合物ととともに培養を行い、培地中のＡβ４２とＡβ４０の比率を定量する方法が挙げられる。 The compound found by such a step may have a step of evaluating the therapeutic effect of AD, such as evaluating the expression of Aβ.
As the expression evaluation of Aβ, there is a method of culturing AD patient-derived neural stem cells with a GM1 inhibitory compound and quantifying the ratio of Aβ42 to Aβ40 in the culture medium.
（第２実施形態）
　１実施形態において、本発明は、後述するアルツハイマー病モデル動物に評価対象化合物を投与する、アルツハイマー病予防剤又は治療剤のスクリーニング方法を提供する。 Second Embodiment
In one embodiment, the present invention provides a screening method for an agent for preventing or treating Alzheimer's disease, which comprises administering a compound to be evaluated to an animal model for Alzheimer's disease described later.
　本実施形態において、例えば、後述するアルツハイマー病モデル動物に、経口投与又は腹腔内投与等の非経口投与で評価対象化合物を投与する。続いて、脳組織におけるアミロイド沈着を評価する。 In the present embodiment, for example, a compound to be evaluated is administered by oral administration or parenteral administration such as intraperitoneal administration to an animal model of Alzheimer's disease described later. Subsequently, amyloid deposits in brain tissue are assessed.
　本発明のスクリーニング方法を用いて得られるアルツハイマー病予防剤又は治療剤は、ＧＭ１蓄積を、直接もしくは間接的に抑制することによりＡＤの治療効果を発揮しうる化合物であることが好ましい。すなわち、かかるＡＤ治療候補化合物を有効成分とするＡＤ治療剤は、蓄積したＧＭ１を直接除去する、ＧＭ１の凝集を抑制する、ＧＭ１の合成を阻害する、ＧＭ１の分解を促進するなどの作用により、ＧＭ１の蓄積を、直接的又は間接的に抑制しＡＤの治療効果を発揮するものであることが好ましい。 The agent for preventing or treating Alzheimer's disease obtained using the screening method of the present invention is preferably a compound capable of exerting the therapeutic effect of AD by suppressing GM1 accumulation directly or indirectly. That is, an AD therapeutic agent containing such an AD therapeutic candidate compound as an active ingredient directly removes accumulated GM1, suppresses the aggregation of GM1, inhibits the synthesis of GM1, and promotes the degradation of GM1 etc. It is preferable that the accumulation of GM1 be suppressed directly or indirectly to exert the therapeutic effect of AD.
［アルツハイマー病予防剤又は治療剤］
　１実施形態において、本発明は、Amodiaquine（アモジアキン）、Acacetin（アカセチン）、Sulfamerazine（スルファメラジン）、Ungerine（ウンゲリン）、Amiodarone（アミオダロン）、Sertindole（セルチンドール）、Delcorine（デルコリン）、Perphenazine（ペルフェナジン）、Althiazide（アルチアジド）、Diethylstilbestrol（ジエチルスチルベストロール）、Thiethylperazine（チエチルペラジン）、Harmol（ハルモール）、Skimmianine（スキムミアニン）、Succinylsulfathiazole（スクシニルサルファチアゾール）、Fillalbin（フィラルビン）、Canavanine（カナバニン）、Harmaline（ハルマリン）、Trihexyphenidyl（トリヘキシフェニジル）、Fluoxetine（フルオキセチン）、Lovastatin（ロバスタチン）、Haloperidol（ハロペリドール）、Prenylamine lactate（プレニルアミン乳酸）、Bromperidol（ブロムペリドール）、Convolamine（コンボルバミン）、及びMiglustat（ミグルスタット）、並びに、これらの薬学的に許容される塩、又はそれらの溶媒和物からなる群から選ばれる少なくとも１種を有効成分として含有するアルツハイマー病予防剤又は治療剤を提供する。
これらのアルツハイマー病予防剤又は治療剤は、ＧＭ１ガングリオシドーシス予防剤又は治療剤としても提供できる。 [Alzheimer's disease preventive or therapeutic agent]
In one embodiment, the present invention includes Amodiaquine (Amodiaquine), Acacetin (Acetetine), Sulfamerazine (Sulphamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindol), Delcorine (Delcholine), Perphenazine (Perphenazine) ), Althiazide (altiazide), Diethylstilbestrol (diethylstilbestrol), Thiethylperazine (thiethylperazine), Harmol (harmol), Skimmianine (skimmianin), Succinylsulfathiazole (succinylsulfathiazole), Fillalbin (filalbin), Canavanine (Canabanine armaline) ), Trihexyphenyl (trihexyphenidyl), Fluoxetine (fluoxetine), Lovastatin (lovastatin), Haloperidol (haloperidol), Prenylamine lactate (prenylamine lactic acid), Bromperidol (Bromperidol) And Convolamine (Convolbamine), and Miglustat (Miglustat), or a pharmaceutically acceptable salt thereof, or at least one member selected from the group consisting of solvates thereof as an active ingredient. An agent for preventing or treating Alzheimer's disease is provided.
These agents for preventing or treating Alzheimer's disease can also be provided as agents for preventing or treating GM1 gangliosidosis.
　また、１実施形態において、本発明は、下記一般式（１）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有するアルツハイマー病予防剤又は治療剤を提供する。
このアルツハイマー病予防剤又は治療剤は、ＧＭ１ガングリオシドーシス予防剤又は治療剤としても提供できる。 Furthermore, in one embodiment, the present invention relates to an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
The agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
［一般式（１）中、Ｒ１及びＲ２は、それぞれ独立して、水素原子、炭素数１～５のアルキル基、アリール基、アラルキル基、又はシクロアルキル基を表す。Ｘは、単結合、又は２価の連結基を表す。ｎは、０、１、又は２を表す。Ｒ３は、水素原子、炭素数１～５のアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。Ｒ４は、水素原子、又は炭素数１～５のアルキル基を表す。Ｒ５は、水素原子、水酸基、炭素数１～５のアルキル基、又は炭素数１～５のアルコキシ基を表す。Ｒ６は、水素原子、水酸基、シアノ基、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、アラルキル基、アリール基、又はシクロアルキル基を表す。］ [In the general formula (1), R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aryl group, an aralkyl group or a cycloalkyl group. X represents a single bond or a divalent linking group. n represents 0, 1 or 2; R 3 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. R 4 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. R 5 represents a hydrogen atom, a hydroxyl group, an alkyl group of 1 to 5 carbon atoms, or an alkoxy group of 1 to 5 carbon atoms. R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. ]
　Ｒ１及びＲ２における、炭素数１～５のアルキル基としては、具体的には、メチル基、エチル基、プロピル基、イソプロピル基、ｎ－ブチル基、イソブチル基、ｔｅｒｔ－ブチル基、ペンチル基、イソペンチル基、ネオペンチル基等が挙げられる。
　Ｒ１及びＲ２におけるアリール基としては、炭素数６～１８であるものが好ましく、炭素数６～１０であるものがより好ましく、具体的にはフェニル基が特に好ましい。
　Ｒ１及びＲ２におけるアラルキル基としては、炭素数１～５のアルキレン基と上記Ｒ１及びＲ２におけるアリール基とが結合したものが好ましい。
　Ｒ１及びＲ２におけるシクロアルキル基としては、炭素数３～８のモノシクロアルカンから１個の水素原子を除いた基が好ましく、具体的には、シクロペンタン、シクロヘキサン、シクロオクタン等が挙げられる。 Specific examples of the alkyl group having 1 to 5 carbon atoms as R 1 and R 2 include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group and pentyl group , Isopentyl group, neopentyl group and the like.
The aryl group for R 1 and R 2 is preferably one having 6 to 18 carbon atoms, more preferably one having 6 to 10 carbon atoms, and particularly preferably a phenyl group.
The aralkyl group in R 1 and R 2 is preferably one in which an alkylene group having 1 to 5 carbon atoms is bonded to the aryl group in R 1 and R 2 .
The cycloalkyl group in R 1 and R 2 is preferably a group in which one hydrogen atom has been removed from a monocycloalkane having 3 to 8 carbon atoms, and specific examples include cyclopentane, cyclohexane, cyclooctane and the like. .
　Ｘにおける２価の連結基としては、アルキレン基、－Ｏ－、－Ｃ（＝Ｏ）－、－ＮＨ－、－Ｓ－、－Ｓ（＝Ｏ）２－、又はこれらの組み合わせが挙げられる。 Examples of the divalent linking group for X include an alkylene group, -O-, -C (= O)-, -NH-, -S-, -S (= O) 2- , or a combination thereof.
　Ｒ３における炭素数１～５のアルキル基、アラルキル基、アリール基、及びシクロアルキル基としては、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。
　Ｒ４における炭素数１～５のアルキル基としては、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。 Alkyl group of 1 to 5 carbon atoms for R 3, an aralkyl group, an aryl group, and the cycloalkyl group include the same as described above in R 1 and R 2.
Examples of the alkyl group having 1 to 5 carbon atoms for R 4 include the same ones as described above for R 1 and R 2 .
　Ｒ５における炭素数１～５のアルキル基としては、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。
　Ｒ５における炭素数１～５のアルコキシ基としては、－ＯＲのＲの部分が、Ｒ１及びＲ２において上述した炭素数１～５のアルキル基と同様のものが挙げられる。 Examples of the alkyl group of 1 to 5 carbon atoms for R 5 include the same as those described above for R 1 and R 2 .
Examples of the alkoxy group having 1 to 5 carbon atoms for R 5 include the same ones as the alkyl group having 1 to 5 carbon atoms described above for R 1 and R 2 in the R moiety of —OR.
　Ｒ６における炭素数１～５のアルキル基、アラルキル基、アリール基、シクロアルキル基としては、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。
　Ｒ６における炭素数１～５のアルコキシ基としては、Ｒ５において上述したものと同様のものが挙げられる。 Examples of the alkyl group having 1 to 5 carbon atoms, the aralkyl group, the aryl group and the cycloalkyl group in R 6 include the same as those described above in R 1 and R 2 .
Examples of the alkoxy group having 1 to 5 carbon atoms for R 6 include the same ones as described above for R 5 .
　上述した中でも、Ｘとしては、単結合が好ましく、ｎは１が好ましく、Ｒ３～Ｒ５は水素原子が好ましい。 Among the above, X is preferably a single bond, n is preferably 1, and R 3 to R 5 are preferably hydrogen atoms.
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記一般式（１－１）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、好ましい。 As the agent for preventing or treating Alzheimer's disease of the present embodiment, a compound represented by the following general formula (1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof may be contained as an active ingredient ,preferable.
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
［一般式（１－１）中、Ｒ１及びＲ２は、それぞれ独立して、水素原子、炭素数１～５のアルキル基、アリール基、アラルキル基、又はシクロアルキル基を表す。Ｘは、単結合、又は２価の連結基を表す。Ｒ６は、水素原子、水酸基、シアノ基、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、アラルキル基、アリール基、又はシクロアルキル基を表す。］ [In the general formula (1-1), R 1 and R 2 each independently represent a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an aryl group, an aralkyl group or a cycloalkyl group. X represents a single bond or a divalent linking group. R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. ]
　上述した中でも、Ｒ６としては、水酸基が好ましく、Ｒ１及びＲ２としては、炭素数１～５のアルキル基が好ましい。 Among the above, R 6 is preferably a hydroxyl group, and R 1 and R 2 are preferably an alkyl group having 1 to 5 carbon atoms.
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記式（１－１－１）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、特に好ましい。 As the agent for preventing or treating Alzheimer's disease of the present embodiment, the compound represented by the following formula (1-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof is contained as an active ingredient Is particularly preferred.
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
　また、１実施形態において、本発明は、下記一般式（２）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有するアルツハイマー病予防剤又は治療剤を提供する。
このアルツハイマー病予防剤又は治療剤は、ＧＭ１ガングリオシドーシス予防剤又は治療剤としても提供できる。 In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
The agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
［一般式（２）中、Ｒ１０は、単結合又は２価の連結基を表す。Ｒ１１は、水素原子、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。Ｒ１２及びＲ１３は、それぞれ独立して、水素原子、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。ｎ１０及びｎ１１は、それぞれ独立して、０～４の整数を表す。Ｒ１２及びＲ１３が、それぞれ複数存在する場合、互いに同じでも異なってもよい。Ｒ１４は、水素原子、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、又は炭素数１～５のアルコキシ基を表す。
ｎ１２は、０～４の整数を表す。Ｒ１４が、複数存在する場合、互いに同じでも異なってもよい。］ [In general formula (2), R 10 represents a single bond or a divalent linking group. R 11 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. R 12 and R 13 each independently represent a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, or 1 to 5 carbon atoms And a hydroxyalkyl group, an aralkyl group, an aryl group or a cycloalkyl group of n 10 and n 11 each independently represent an integer of 0 to 4. When a plurality of R 12 and R 13 exist, they may be the same or different. R 14 represents a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, or an alkoxy group of 1 to 5 carbon atoms.
n 12 represents an integer of 0 to 4; When a plurality of R 14 are present, they may be the same or different. ]
　Ｒ１０における２価の連結基としては、Ｘにおいて上述したものと同様のものが挙げられる。
　Ｒ１１における炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、アラルキル基、アリール基、及びシクロアルキル基としては、Ｒ５において上述したものと同様のものが挙げられる。
　Ｒ１１における炭素数１～５のヒドロキシアルキル基としては、アルキル基の部分が、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。
　Ｒ１２及びＲ１３における炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、及びシクロアルキル基としては、Ｒ１１において上述したものと同様のものが挙げられる。
　Ｒ１２及びＲ１３におけるハロゲン原子としては、フッ素原子、塩素原子、臭素原子が挙げられる。
　Ｒ１２及びＲ１３における炭素数１～５のチオアルキル基としては、アルキル基の部分が、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。
　Ｒ１４におけるハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、及び炭素数１～５のアルコキシ基としては、Ｒ１２及びＲ１３において上述したものと同様のものが挙げられる。 Examples of the divalent linking group for R 10 include the same as those described above for X.
Alkyl group of 1 to 5 carbon atoms for R 11, an alkoxy group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, and the cycloalkyl group include the same as described above in R 5.
Examples of the hydroxyalkyl group having 1 to 5 carbon atoms for R 11 include the same alkyl groups as those described above for R 1 and R 2 .
Alkyl group of R 12 and carbon number of 1 to the R 13 5, an alkoxy group having 1 to 5 carbon atoms, hydroxyalkyl group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, and the cycloalkyl group, the R 11 The same as those described above can be mentioned.
Examples of the halogen atom in R 12 and R 13, a fluorine atom, a chlorine atom, a bromine atom.
Examples of the thioalkyl group having 1 to 5 carbon atoms as R 12 and R 13 include the same alkyl groups as those described above for R 1 and R 2 .
As the halogen atom for R 14 , the alkyl group having 1 to 5 carbon atoms, the thioalkyl group having 1 to 5 carbon atoms, and the alkoxy group having 1 to 5 carbon atoms, those similar to those described above for R 12 and R 13 It can be mentioned.
　上述した中でも、Ｒ１３及びＲ１４としては、水素原子が好ましく、ｎ１０は１が好ましい。 Among the above, as R 13 and R 14 , a hydrogen atom is preferable, and n 10 is preferably 1.
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記一般式（２－１）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、好ましい。 As the agent for preventing or treating Alzheimer's disease of the present embodiment, a compound represented by the following general formula (2-1), a pharmaceutically acceptable salt thereof, or a solvate thereof may be contained as an active ingredient ,preferable.
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
［一般式（２－１）中、Ｒ１０は、単結合又は２価の連結基を表す。Ｒ１１は、水素原子、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。Ｒ１２は、水素原子、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。］ [In general formula (2-1), R 10 represents a single bond or a divalent linking group. R 11 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. R 12 represents a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, It represents an aryl group or a cycloalkyl group. ]
　上述した中でも、Ｒ１０としては、アルキレン基が好ましい。Ｒ１０におけるアルキレン基としては、炭素数１～５の直鎖状又は分岐鎖状のアルキル基から１個の水素原子を除いた基が好ましい。炭素数１～５のアルキル基としては、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。 Among the above, an alkylene group is preferable as R 10 . As the alkylene group for R 10, a group in which one hydrogen atom has been removed from a linear or branched alkyl group having 1 to 5 carbon atoms is preferable. Examples of the alkyl group having 1 to 5 carbon atoms include the same as those described above for R 1 and R 2 .
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記一般式（２－１－１）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、更に好ましい。 The agent for preventing or treating Alzheimer's disease of the present embodiment contains a compound represented by the following general formula (2-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient Is more preferred.
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
［一般式（２－１－１）中、Ｒ１１は、水素原子、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。Ｒ１２は、水素原子、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。］ [In general formula (2-1-1), R 11 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, It represents an aryl group or a cycloalkyl group. R 12 represents a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, It represents an aryl group or a cycloalkyl group. ]
　上述した中でも、Ｒ１１としては、炭素数１～５のアルキル基、又は炭素数１～５のヒドロキシアルキル基が好ましい。Ｒ１２としては、ハロゲン原子、又は炭素数１～５のチオアルキル基が好ましい。 Among the above, as R 11 , an alkyl group of 1 to 5 carbon atoms or a hydroxyalkyl group of 1 to 5 carbon atoms is preferable. As R 12 , a halogen atom or a thioalkyl group having 1 to 5 carbon atoms is preferable.
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記式（２－１－１－１）又は（２－１－１－２）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、特に好ましい。 As the agent for preventing or treating Alzheimer's disease of the present embodiment, a compound represented by the following formula (2-1-1-1) or (2-1-1-2), a pharmaceutically acceptable salt thereof, It is particularly preferable to contain the solvate as an active ingredient.
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
　また、１実施形態において、本発明は、下記一般式（３）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有するアルツハイマー病予防剤又は治療剤を提供する。
このアルツハイマー病予防剤又は治療剤は、ＧＭ１ガングリオシドーシス予防剤又は治療剤としても提供できる。 In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (3), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
The agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
［一般式（３）中、Ｒ２０は、水素原子、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。Ｒ２１及びＲ２４は、それぞれ独立して、単結合又は２価の連結基を表す。Ｒ２２及びＲ２３は、それぞれ独立して、水素原子、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のハロアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基を表す。］ [In the general formula (3), R 20 represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms, a thioalkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, 1 to 5 carbon atoms And a hydroxyalkyl group, an aralkyl group, an aryl group or a cycloalkyl group of R 21 and R 24 each independently represent a single bond or a divalent linking group. R 22 and R 23 each independently represent a hydrogen atom, a halogen atom, an alkyl group of 1 to 5 carbon atoms, a haloalkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, or 1 to 5 carbon atoms And an alkoxy group of 1 to 5 carbon atoms and a hydroxyalkyl group having 1 to 5 carbon atoms. ]
　Ｒ２０における、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、アラルキル基、アリール基、及びシクロアルキル基としては、Ｒ１２において上述したものと同様のものが挙げられる。
　Ｒ２１及びＲ２４における２価の連結基としては、Ｘにおいて上述したものと同様のものが挙げられる。
　Ｒ２２及びＲ２３における、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、及び炭素数１～５のヒドロキシアルキル基としては、Ｒ１２において上述したものと同様のものが挙げられる。
　Ｒ２２及びＲ２３における、炭素数１～５のハロアルキル基としては、フッ化アルキル基、塩化アルキル基、臭化アルキル基が挙げられ、Ｒ１及びＲ２において上述したアルキル基から、少なくとも１つの水素原子がハロゲン原子に置き換えられたものが挙げられる。 R 20 is a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group, And as the cycloalkyl group, the same as described above for R 12 can be mentioned.
Examples of the divalent linking group for R 21 and R 24 include the same as those described above for X.
As R 22 and R 23 , a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, and a hydroxyalkyl group of 1 to 5 carbon atoms include The same as described above for R 12 can be mentioned.
Examples of the haloalkyl group having 1 to 5 carbon atoms as R 22 and R 23 include a fluoroalkyl group, an alkyl chloride group and an alkyl bromide group, and at least one of the alkyl groups described above in R 1 and R 2 What has a hydrogen atom replaced by a halogen atom is mentioned.
　上述した中でも、Ｒ２０としては、炭素数１～５のアルキル基、又はアラルキル基が好ましい。Ｒ２０におけるアラルキル基としては、炭素数１～５のアルキレン基とフェニル基とが結合したものが好ましい。
　Ｒ２１及びＲ２４としては、単結合又は－Ｏ－が好ましい。
　Ｒ２２及びＲ２３としては、水素原子又はトリフルオロメチル基が好ましい。 Among the above, as R 20 , an alkyl group having 1 to 5 carbon atoms or an aralkyl group is preferable. As the aralkyl group for R 20, one in which an alkylene group of 1 to 5 carbon atoms is bonded to a phenyl group is preferable.
As R 21 and R 24 , a single bond or —O— is preferable.
As R 22 and R 23 , a hydrogen atom or a trifluoromethyl group is preferable.
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記式（３－１）又は（３－２）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、特に好ましい。 A compound represented by the following formula (3-1) or (3-2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an agent for preventing or treating Alzheimer's disease of the present embodiment is an active ingredient It is particularly preferable to contain it as
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
　また、１実施形態において、本発明は、下記一般式（４）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有するアルツハイマー病予防剤又は治療剤を提供する。
このアルツハイマー病予防剤又は治療剤は、ＧＭ１ガングリオシドーシス予防剤又は治療剤としても提供できる。 Furthermore, in one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (4), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient I will provide a.
The agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000012
［一般式（４）中、Ｒ３０及びＲ３１は、それぞれ独立して、水素原子、ハロゲン原子、水酸基、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、又はアラルキル基を表す。実線と点線の二重線部分は単結合又は二重結合を表す。］ [In general formula (4), each of R 30 and R 31 independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, or 1 to 5 carbon atoms] 5 represents an alkoxy group, a hydroxyalkyl group having 1 to 5 carbon atoms, or an aralkyl group. The solid and dotted double line portions represent single bonds or double bonds. ]
　Ｒ３０及びＲ３１において、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、及びアラルキル基としては、Ｒ１２において上述したものと同様のものが挙げられる。 R 30 and R 31 each represent a halogen atom, an alkyl group of 1 to 5 carbon atoms, a thioalkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, a hydroxyalkyl group of 1 to 5 carbon atoms, and an aralkyl group And the same as those described above for R 12 can be mentioned.
　上述した中でも、Ｒ３０及びＲ３１としては、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、水酸基が好ましい。 Among the above, as R 30 and R 31 , an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, and a hydroxyl group are preferable.
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記式（４－１）又は（４－２）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、特に好ましい。 A compound represented by the following formula (4-1) or (4-2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an agent for preventing or treating Alzheimer's disease of the present embodiment is an active ingredient. It is particularly preferable to contain it as
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
　また、１実施形態において、本発明は、下記一般式（５）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有するアルツハイマー病予防剤又は治療剤を提供する。
このアルツハイマー病予防剤又は治療剤は、ＧＭ１ガングリオシドーシス予防剤又は治療剤としても提供できる。 In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, which comprises a compound represented by the following general formula (5), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient: I will provide a.
The agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000014
［一般式（５）中、Ｒ４０は、単結合又は２価の連結基を表す。Ｒ４１～Ｒ４３は、それぞれ独立して、水素原子、水酸基、ハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、又はアラルキル基を表す。］ [In general formula (5), R 40 represents a single bond or a divalent linking group. R 41 to R 43 each independently represent a hydrogen atom, a hydroxyl group, a halogen atom, an alkyl group having 1 to 5 carbon atoms, a thioalkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, It represents a hydroxyalkyl group of -5 or an aralkyl group. ]
　Ｒ４０における２価の連結基としては、Ｘにおいて上述したものと同様のものが挙げられる。
　Ｒ４１～Ｒ４３におけるハロゲン原子、炭素数１～５のアルキル基、炭素数１～５のチオアルキル基、炭素数１～５のアルコキシ基、炭素数１～５のヒドロキシアルキル基、及びアラルキル基としては、Ｒ１２において上述したものと同様のものが挙げられる。 Examples of the divalent linking group for R 40 include the same as those described above for X.
As a halogen atom for R 41 to R 43 , an alkyl group having 1 to 5 carbon atoms, a thioalkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, a hydroxyalkyl group having 1 to 5 carbon atoms, and an aralkyl group Are the same as described above for R 12 .
　上述した中でも、Ｒ４０としては、アルキレン基が好ましい。Ｒ４０におけるアルキレン基としては、炭素数１～５の直鎖状又は分岐鎖状のアルキル基から１個の水素原子を除いた基が好ましい。炭素数１～５の直鎖状又は分岐鎖状のアルキル基としては、Ｒ１及びＲ２において上述したものと同様のものが挙げられる。
　Ｒ４１～Ｒ４３としては、水素原子、ハロゲン原子が好ましい。 Among the above, as R 40 , an alkylene group is preferable. As the alkylene group for R 40, a group in which one hydrogen atom has been removed from a linear or branched alkyl group having 1 to 5 carbon atoms is preferable. Examples of the linear or branched alkyl group having 1 to 5 carbon atoms include the same as those described above for R 1 and R 2 .
As R 41 to R 43 , a hydrogen atom or a halogen atom is preferable.
　本実施形態のアルツハイマー病予防剤又は治療剤としては、下記式（５－１）又は（５－２）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することが、特に好ましい。 A compound represented by the following formula (5-1) or (5-2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an agent for preventing or treating Alzheimer's disease of the present embodiment is an active ingredient. It is particularly preferable to contain it as
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000015
　本発明のアルツハイマー病予防剤又は治療剤としては、上記化合物をフリー体の形態で用いてもよく、薬学的に許容される塩の形態で用いてもよい。また、フリー体の溶媒和物の形態で用いてもよく、塩の溶媒和物の形態で用いてもよい。 As the agent for preventing or treating Alzheimer's disease of the present invention, the above-mentioned compound may be used in the form of a free form or in the form of a pharmaceutically acceptable salt. Also, they may be used in the form of a free form solvate, or may be used in the form of a salt solvate.
　塩としては、薬学的に許容される塩であれば特に制限されず、例えば、塩酸塩、硫酸塩、臭化水素酸塩、ヨウ化水素酸塩、燐酸塩、硝酸塩、安息香酸塩、メタンスルホン酸塩、２－ヒドロキシエタンスルホン酸塩、ｐ－トルエンスルホン酸塩、酢酸塩、プロパン酸塩、シュウ酸塩、マロン酸塩、コハク酸塩、グルタル酸塩、アジピン酸塩、酒石酸塩、マレイン酸塩、フマール酸塩、リンゴ酸塩、マンデル酸塩等が挙げられる。溶媒和物としては、薬学的に許容される溶媒和物であれば特に制限されず、例えば、水和物、有機溶媒和物等が挙げられる。 The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and, for example, hydrochloride, sulfate, hydrobromide, hydroiodide, phosphate, nitrate, benzoate, methanesulfone Acid, 2-Hydroxyethane Sulfonate, p-Toluene Sulfonate, Acetate, Propanoate, Oxalate, Malonate, Succinate, Glutarate, Adipate, Tartrate, Maleic Acid Salts, fumarates, malates, mandelates and the like can be mentioned. The solvate is not particularly limited as long as it is a pharmaceutically acceptable solvate, and includes, for example, hydrate, organic solvate and the like.
［アルツハイマー病の予防用又は治療用組成物］
　１実施形態において、本発明は、上記のアルツハイマー病の予防剤又は治療剤、及び薬学的に許容される担体を含有する、アルツハイマー病の予防用又は治療用組成物を提供する。
このアルツハイマー病の予防用又は治療用組成物は、ＧＭ１ガングリオシドーシス予防用又は治療用組成物としても提供できる。 [Composition for Prevention or Treatment of Alzheimer's Disease]
In one embodiment, the present invention provides a composition for the prophylaxis or treatment of Alzheimer's disease, which comprises the above-mentioned prophylactic or therapeutic agent for Alzheimer's disease, and a pharmaceutically acceptable carrier.
The composition for preventing or treating Alzheimer's disease can also be provided as a composition for preventing or treating GM1 gangliosidosis.
　本実施形態のアルツハイマー病の予防用又は治療用組成物は、例えば、錠剤、被覆錠剤、丸剤、散剤、顆粒剤、カプセル剤、液剤、懸濁剤、乳剤等の形態で経口的に、あるいは、注射剤、坐剤、皮膚外用剤等の形態で非経口的に投与することができる。 The composition for preventing or treating Alzheimer's disease of the present embodiment may be, for example, orally in the form of a tablet, a coated tablet, a pill, a powder, a granule, a capsule, a solution, a suspension, an emulsion, etc. Can be administered parenterally in the form of injections, suppositories, external preparations for skin and the like.
　薬学的に許容される担体としては、通常医薬組成物の製剤に用いられるものを特に制限なく用いることができる。より具体的には、例えば、ゼラチン、コーンスターチ、トラガントガム、アラビアゴム等の結合剤；デンプン、結晶性セルロース等の賦形剤；アルギン酸等の膨化剤；水、エタノール、グリセリン等の注射剤用溶剤；ゴム系粘着剤、シリコーン系粘着剤等の粘着剤等が挙げられる。薬学的に許容される担体は、１種を単独で又は２種以上を混合して用いることができる。 As the pharmaceutically acceptable carrier, those generally used for the preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injection such as water, ethanol and glycerin; Examples of the pressure-sensitive adhesive include rubber-based pressure-sensitive adhesives and silicone-based pressure-sensitive adhesives. The pharmaceutically acceptable carrier can be used alone or in combination of two or more.
　本実施形態のアルツハイマー病の予防用又は治療用組成物は、更に添加剤を含んでいてもよい。添加剤としては、ステアリン酸カルシウム、ステアリン酸マグネシウム等の潤滑剤；ショ糖、乳糖、サッカリン、マルチトール等の甘味剤；ペパーミント、アカモノ油等の香味剤；ベンジルアルコール、フェノール等の安定剤；リン酸塩、酢酸ナトリウム等の緩衝剤；安息香酸ベンジル、ベンジルアルコール等の溶解補助剤；酸化防止剤；防腐剤等が挙げられる。
　添加剤は、１種を単独で又は２種以上を混合して用いることができる。 The composition for preventing or treating Alzheimer's disease of the present embodiment may further contain an additive. As additives, lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavoring agents such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid Salts, buffers such as sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.
The additives may be used alone or in combination of two or more.
（投与方法）
　アルツハイマー病の予防剤又は治療剤、又はアルツハイマー病の予防用又は治療用組成物の投与方法は特に限定されず、患者の症状、体重、年齢、性別等に応じて適宜決定すればよい。例えば、錠剤、被覆錠剤、丸剤、散剤、顆粒剤、カプセル剤、液剤、懸濁剤、乳剤等は経口投与される。また、注射剤は、単独で、又はブドウ糖、アミノ酸等の通常の補液と混合して静脈内投与され、更に必要に応じて、動脈内、筋肉内、皮内、皮下又は腹腔内投与される。坐剤は直腸内投与される。皮膚外用剤は、患部に塗布、貼付又はスプレーされる。 (Method of administration)
The administration method of the preventive or therapeutic agent for Alzheimer's disease or the composition for preventing or treating Alzheimer's disease is not particularly limited, and may be appropriately determined according to the condition, weight, age, sex and the like of the patient. For example, tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions and the like are orally administered. In addition, the injections are intravenously administered alone or mixed with ordinary fluid replacements such as glucose and amino acids, and further, if necessary, intraarterially, intramuscularly, intradermal, subcutaneous or intraperitoneally. Suppositories are administered rectally. The external skin preparation is applied, affixed or sprayed to the affected area.
（投与量）
　アルツハイマー病の予防剤又は治療剤、又はアルツハイマー病の予防用又は治療用組成物の投与量は、患者の症状、体重、年齢、性別等によって異なり、一概には決定できないが、経口投与の場合には、例えば１日あたり１μｇ～１０ｇ、例えば１日あたり０．０１～２０００ｍｇの有効成分を投与すればよい。また、注射剤の場合には、例えば１日あたり０．１μｇ～１ｇ、例えば１日あたり０．００１～２００ｍｇの有効成分を投与すればよい。また、坐剤の場合には、例えば１日あたり１μｇ～１０ｇ、例えば１日あたり０．０１～２０００ｍｇの有効成分を投与すればよい。また、皮膚外用剤の場合には、例えば１日あたり１μｇ～１０ｇ、例えば１日あたり０．０１～２０００ｍｇの有効成分を投与すればよい。 (Dose)
The dose of the prophylactic or therapeutic agent for Alzheimer's disease, or the composition for the prophylaxis or treatment of Alzheimer's disease varies depending on the patient's condition, body weight, age, sex, etc. and can not be determined generally, but in the case of oral administration For example, 1 μg to 10 g per day, for example, 0.01 to 2000 mg of the active ingredient per day may be administered. In the case of injection, for example, 0.1 μg to 1 g per day, for example, 0.001 to 200 mg of the active ingredient per day may be administered. In the case of suppositories, for example, 1 μg to 10 g per day, for example, 0.01 to 2000 mg of the active ingredient per day may be administered. In the case of a skin external preparation, for example, 1 μg to 10 g of active ingredient per day, for example, 0.01 to 2000 mg per day may be administered.
［その他の実施形態］
　一実施形態において、本発明は、アルツハイマー病の予防又は治療のための、Amodiaquine（アモジアキン）、Acacetin（アカセチン）、Sulfamerazine（スルファメラジン）、Ungerine（ウンゲリン）、Amiodarone（アミオダロン）、Sertindole（セルチンドール）、Delcorine（デルコリン）、Perphenazine（ペルフェナジン）、Althiazide（アルチアジド）、Diethylstilbestrol（ジエチルスチルベストロール）、Thiethylperazine（チエチルペラジン）、Harmol（ハルモール）、Skimmianine（スキムミアニン）、Succinylsulfathiazole（スクシニルサルファチアゾール）、Fillalbin（フィラルビン）、Canavanine（カナバニン）、Harmaline（ハルマリン）、Trihexyphenidyl（トリヘキシフェニジル）、Fluoxetine（フルオキセチン）、Lovastatin（ロバスタチン）、Haloperidol（ハロペリドール）、Prenylamine lactate（プレニルアミン乳酸）、Bromperidol（ブロムペリドール）、Convolamine（コンボルバミン）、Miglustat（ミグルスタット）、及び前記一般式（１）～（５）で表される化合物からなる群から選ばれる化合物、その薬学的に許容される塩、又はそれらの溶媒和物を提供する。 Other Embodiments
In one embodiment, the present invention provides for the prevention or treatment of Alzheimer's disease, Amodiaquine (Amodiaquine), Acacetin (Acacetin), Sulfamerazine (Sulfamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindole) , Delcorine (Dercoline), Perphenazine (Perphenazine), Althiazole (Althiazide), Diethylstilbestrol (Diethylstilbestrol), Thiethylperazine (Thiethylperazine), Harmol (Halmol), Skimmianine (Skimmianin), Succinylsulfathiazole (Succinyl Sulfathiazole), ), Canavanine (Canavanine), Harmaline (Halmarin), Trihexyphenidyl (Trihexyphenidyl), Fluoxetine (Fluoxetine), Lovastatin (Lovastatin), Haloperidol (Haloperidol), Prenylamine lactate (Puramine) Compounds selected from the group consisting of lenylamine lactic acid), Bromperidol (Bromperidol), Convolamine (Convolbamine), Miglustat (Miglustat), and the compounds represented by the above-mentioned general formulas (1) to (5), and their pharmacological properties And salts thereof, or solvates thereof.
　一実施形態において、本発明は、Amodiaquine（アモジアキン）、Acacetin（アカセチン）、Sulfamerazine（スルファメラジン）、Ungerine（ウンゲリン）、Amiodarone（アミオダロン）、Sertindole（セルチンドール）、Delcorine（デルコリン）、Perphenazine（ペルフェナジン）、Althiazide（アルチアジド）、Diethylstilbestrol（ジエチルスチルベストロール）、Thiethylperazine（チエチルペラジン）、Harmol（ハルモール）、Skimmianine（スキムミアニン）、Succinylsulfathiazole（スクシニルサルファチアゾール）、Fillalbin（フィラルビン）、Canavanine（カナバニン）、Harmaline（ハルマリン）、Trihexyphenidyl（トリヘキシフェニジル）、Fluoxetine（フルオキセチン）、Lovastatin（ロバスタチン）、Haloperidol（ハロペリドール）、Prenylamine lactate（プレニルアミン乳酸）、Bromperidol（ブロムペリドール）、Convolamine（コンボルバミン）、Miglustat（ミグルスタット）、及び前記一般式（１）～（５）で表される化合物からなる群から選ばれる化合物、その薬学的に許容される塩、又はそれらの溶媒和物の有効量を、予防又は治療を必要とする患者に投与することを含む、アルツハイマー病の予防又は治療方法を提供する。 In one embodiment, the present invention includes Amodiaquine (Amodiaquine), Acacetin (Acacetin), Sulfamerazine (Sulphamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindol), Delcorine (Delcholine), Perphenazine (Perphenazine) ), Althiazide (altiazide), Diethylstilbestrol (diethylstilbestrol), Thiethylperazine (thiethylperazine), Harmol (harmol), Skimmianine (skimmianin), Succinylsulfathiazole (succinylsulfathiazole), Fillalbin (filalbin), Canavanine (Canabanine armaline) ), Trihexyphenyl (trihexyphenidyl), Fluoxetine (fluoxetine), Lovastatin (lovastatin), Haloperidol (haloperidol), Prenylamine lactate (prenylamine lactic acid), Bromperidol Compounds selected from the group consisting of Ridolol), Convolamine (Convolbamine), Miglustat (Miglustat), and the compounds represented by the above general formulas (1) to (5), or a pharmaceutically acceptable salt thereof, or those The present invention provides a method for preventing or treating Alzheimer's disease, which comprises administering an effective amount of a solvate of the present invention to a patient in need thereof.
　一実施形態において、本発明は、アルツハイマー病の予防剤又は治療剤、又はアルツハイマー病の予防用又は治療用組成物を製造するためのAmodiaquine（アモジアキン）、Acacetin（アカセチン）、Sulfamerazine（スルファメラジン）、Ungerine（ウンゲリン）、Amiodarone（アミオダロン）、Sertindole（セルチンドール）、Delcorine（デルコリン）、Perphenazine（ペルフェナジン）、Althiazide（アルチアジド）、Diethylstilbestrol（ジエチルスチルベストロール）、Thiethylperazine（チエチルペラジン）、Harmol（ハルモール）、Skimmianine（スキムミアニン）、Succinylsulfathiazole（スクシニルサルファチアゾール）、Fillalbin（フィラルビン）、Canavanine（カナバニン）、Harmaline（ハルマリン）、Trihexyphenidyl（トリヘキシフェニジル）、Fluoxetine（フルオキセチン）、Lovastatin（ロバスタチン）、Haloperidol（ハロペリドール）、Prenylamine lactate（プレニルアミン乳酸）、Bromperidol（ブロムペリドール）、Convolamine（コンボルバミン）、Miglustat（ミグルスタット）、及び前記一般式（１）～（５）で表される化合物からなる群から選ばれる化合物、その薬学的に許容される塩、又はそれらの溶媒和物の使用を提供する。 In one embodiment, the present invention relates to Amodiaquine (Amodiaquine), Acacetin (acacetin), Sulfamerazine (sulfamelazine), Ungerine for producing a preventive or therapeutic agent for Alzheimer's disease, or a composition for preventing or treating Alzheimer's disease. (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindole), Delcorine (Dercholine), Perphenazine (Perphenazine), Althiazide (Althiazide), Diethylstilbestrol (Diethylstilbestrol), Thiethylperazine (Thiethylperazine), Harmol (Halmol), Skimmianine (skimmianin), Succinylsulfathiazole (succinyl sulfathiazole), Fillalbin (filalbin), Canavanine (canavanine), Harmaline (halmarin), Trihexyphenidyl (trihexyphenidyl), Fluoxetine (fluoxetine), Lovastatin (lovastatin) (Vavastatin), Haloperidol (Haloperidol), Prenylamine lactate (Prenylamine Lactic Acid), Bromperidol (Bromperidol), Convolamine (Convolbamine), Miglustat (Miglustat), and Formulas (1) to (5) There is provided use of a compound selected from the group consisting of compounds, a pharmaceutically acceptable salt thereof, or a solvate thereof.
［アルツハイマー病モデル動物］
　本発明の評価に用いるアルツハイマー病モデル動物は、β-Galactosidase（以下、β－ｇａｌともいう。）遺伝子の発現が抑制若しくは喪失している、又は前記β－ｇａｌ遺伝子がコードするβ－ｇａｌタンパク質の機能が抑制若しくは喪失しており、前記β－ｇａｌ遺伝子又は前記β－ｇａｌ遺伝子の発現調節領域に変異が導入されている非ヒト哺乳動物である。 [Alzheimer's disease model animal]
The animal model for Alzheimer's disease used in the evaluation of the present invention is a β-gal protein of which β-Galactosidase (hereinafter also referred to as β-gal) gene expression is suppressed or lost or which is encoded by the β-gal gene. It is a non-human mammal whose function has been suppressed or lost and a mutation has been introduced into the expression control region of the β-gal gene or the β-gal gene.
　β－ｇａｌタンパク質は、様々な生体分子の加水分解の役割を果たすリソソームに存在する酵素である。ＧＭ１ガングリオシドは、β－ｇａｌタンパク質の基質であり、脳に豊富に存在する。そのため、β－ｇａｌの抑制又は喪失により、ＧＭ１ガングリオシドの蓄積を導く。 The β-gal protein is an enzyme present in lysosomes that plays a role in the hydrolysis of various biomolecules. GM1 ganglioside is a substrate for β-gal protein and is abundant in the brain. Therefore, suppression or loss of β-gal leads to accumulation of GM1 ganglioside.
　β－ｇａｌタンパク質の機能が喪失しているとは、β－ｇａｌタンパク質が本来有する機能が完全に失われている状態のことをいう。β－ｇａｌタンパク質の機能が抑制されているとは、β－ｇａｌタンパク質が本来有する機能が部分的に失われている状態のことをいう。
　β－ｇａｌタンパク質が本来有する機能が部分的に失われている場合には、β－ｇａｌタンパク質の機能の抑制の程度は、モデル動物と、モデル動物となる動物の野生型の動物等のコントロールとなる動物とを比較して、モデル動物にアルツハイマー病と認められる状態（コントロールとの差異）が表れる程度であればよい。
　アルツハイマー病と認められる状態の指標としては、アミロイドタンパク質（Ａβ４０及び４２）の脳における沈着が挙げられる。 The loss of the function of the β-gal protein refers to the state in which the function originally possessed by the β-gal protein is completely lost. The fact that the function of β-gal protein is suppressed refers to a state in which the function originally possessed by β-gal protein is partially lost.
In the case where the intrinsic function of the β-gal protein is partially lost, the degree of suppression of the function of the β-gal protein is determined by comparing the model animal and the wild-type animal of the animal model with the control. It should be such an extent that a condition (difference from control) appears in a model animal in comparison with the animal.
An indicator of a condition recognized as Alzheimer's disease includes deposition of amyloid proteins (Aβ 40 and 42) in the brain.
　β－ｇａｌタンパク質の機能の抑制又は喪失は、β－ｇａｌ遺伝子の発現が抑制もしくは喪失することによっても生じ得る。β－ｇａｌ遺伝子の発現が喪失しているとは、モデル動物となる動物において、β－ｇａｌ遺伝子産物が喪失していることをいう。β－ｇａｌ遺伝子の発現が抑制しているとは、モデル動物となる動物の野生型の動物等のコントロールとなる動物と比較して、β－ｇａｌ遺伝子産物の量が抑制されていることをいう。
　β－ｇａｌ遺伝子産物の量が抑制されている場合には、抑制の程度は、モデル動物となる動物の野生型の動物等のコントロールとなる動物と比較して、上記に示すようなアルツハイマー病と認められる状態が表れる程度であればよい。β－ｇａｌ遺伝子の発現の抑制は、β－ｇａｌ遺伝子に対するＲＮＡｉ誘導性核酸、アンチセンス核酸、アプタマー若しくはリボザイムなどの発現を生じさせる核酸配列をモデル動物に導入し、遺伝子ノックダウン等により生じさせることができる。 Suppression or loss of function of β-gal protein can also be caused by suppression or loss of expression of β-gal gene. Loss of expression of the β-gal gene means loss of the β-gal gene product in a model animal. The suppression of the expression of the β-gal gene means that the amount of the β-gal gene product is suppressed as compared to a control animal such as a wild-type animal of a model animal. .
When the amount of the β-gal gene product is suppressed, the degree of suppression is compared to Alzheimer's disease as shown above in comparison with a control animal such as a wild-type animal of a model animal. It is sufficient if it is in such an extent that a recognized state appears. In order to suppress the expression of β-gal gene, a nucleic acid sequence that causes expression of RNAi-inducing nucleic acid, antisense nucleic acid, aptamer or ribozyme for β-gal gene is introduced into a model animal and generated by gene knockdown etc. Can.
　本発明の評価に用いるモデル動物は、β－ｇａｌ遺伝子又はβ－ｇａｌ遺伝子の発現調節領域に変異が導入されてなることが好ましい。
　導入される変異は、β－ｇａｌ遺伝子の発現を抑制若しくは喪失させる、又は前記β－ｇａｌ遺伝子がコードするβ－ｇａｌタンパク質の機能を抑制若しくは喪失させるものであればよい。 The model animal used for the evaluation of the present invention preferably has a mutation introduced into the expression control region of the β-gal gene or β-gal gene.
The introduced mutation may be one that suppresses or loses the expression of the β-gal gene or suppresses or loses the function of the β-gal protein encoded by the β-gal gene.
　上記β－ｇａｌ遺伝子とは、β－ｇａｌタンパク質をコードする領域であるエクソンのほか、β－ｇａｌ遺伝子の発現を抑制若しくは喪失させる、又は前記β－ｇａｌ遺伝子がコードするβ－ｇａｌタンパク質の機能を抑制若しくは喪失させることができる場合にはイントロンも含まれる。
　β－ｇａｌ遺伝子の発現調節領域とは、β－ｇａｌ遺伝子の発現を調節する領域であって、例えば、プロモーター、サイレンサー、エンハンサー、応答配列である。 The above-mentioned β-gal gene is not only an exon which is a region encoding β-gal protein, but also suppresses or loses the expression of β-gal gene or functions of β-gal protein encoded by the above-mentioned β-gal gene Introns are also included if they can be suppressed or lost.
The expression regulatory region of the β-gal gene is a region that regulates the expression of the β-gal gene, and is, for example, a promoter, a silencer, an enhancer, or a response element.
　β－ｇａｌタンパク質の機能の喪失は、例えばβ－ｇａｌ遺伝子に変異を導入し、β－ｇａｌ遺伝子を破壊することにより生じさせることができる。β－ｇａｌ遺伝子の発現能の抑制又は喪失は、例えば、β－ｇａｌ遺伝子の発現調節領域に変異を導入することにより生じさせることができる。
　　β－ｇａｌ遺伝子又はβ－ｇａｌ遺伝子の発現調節領域に変異を導入する方法は、公知の遺伝子工学的手法である遺伝子改変により行うことができる。変異は、β－ｇａｌ遺伝子又はβ－ｇａｌ遺伝子の発現調節領域における一部又は全部の欠失、置換、任意の配列の挿入等により生じさせることができる。β－ｇａｌ遺伝子の一部が欠失している場合、欠失は１つ以上のエクソンの欠失であることが好ましい。これらの変異を導入することは、例えば、変異原性物質（Mutagen）による処理、紫外線照射、相同組み換え技術等による遺伝子ターゲッティング、遺伝子ノックアウト、遺伝子ノックダウン、Cre-loxP系等による条件的ノックアウト等の手法を用いて行うことができる。 Loss of function of the β-gal protein can be produced, for example, by introducing a mutation into the β-gal gene and disrupting the β-gal gene. The suppression or loss of the expression ability of the β-gal gene can be produced, for example, by introducing a mutation into the expression regulatory region of the β-gal gene.
The method for introducing a mutation into the expression control region of the β-gal gene or β-gal gene can be carried out by genetic modification which is a known genetic engineering technique. The mutation can be generated by deletion or substitution of part or all of the expression control region of the β-gal gene or β-gal gene, insertion of any sequence, and the like. When part of the β-gal gene is deleted, the deletion is preferably a deletion of one or more exons. Introduction of these mutations includes, for example, treatment with mutagenic substances (Mutagen), gene targeting by ultraviolet irradiation, homologous recombination technology etc., gene knockout, conditional knock out by gene knockdown, Cre-loxP system etc. It can be done using the method.
　β－ｇａｌ遺伝子変異としては、ヒトβ－ｇａｌタンパク質において、Ｅ１８６Ａ、Ｐ１０Ｌ、Ｒ２０１Ｃ、Ｃ１２７Ｙ、Ｗ１６１Ｇ、Ｑ２５５Ｈ，Ｒ３５１Ｘ、Ｓ５３２Ｇ、Ｒ１４８Ｓ等の変異が挙げられる。 Examples of the β-gal gene mutation include mutations in human β-gal protein such as E186A, P10L, R201C, C127Y, W161G, Q255H, R351X, S532G, R148S and the like.
　後述する実施例において示すように、β－ｇａｌノックアウトマウスとアルツハイマー病モデルマウスを掛けあわせたマウスでは、アミロイドの沈着が促進されていることが確認されている。
　したがって、本発明の評価に用いるアルツハイマー病モデル動物は、更に、ヒトプリセニリン１（ＰＳ１）のエクソン９の欠損、Ｍ１４６Ｌ変異、Ｌ２８５Ｖ変異、ヒトＰＳ２のＮ１４１Ｉ変異、ヒトアミロイド前駆体タンパク質（ＡＰＰ）のＫＭ６７０／６７１ＮＬスェーデン型変異、Ｉ７１６Ｖフロリダ型変異、及びＶ７１７Ｉロンドン型変異からなる群から選ばれる少なくとも１種の変異を有することが好ましく、ヒトプリセニリン１（ＰＳ１）のＭ１４６Ｌ変異、及びＬ２８５Ｖ変異、並びに、ヒトアミロイド前駆体タンパク質（ＡＰＰ）のＫＭ６７０／６７１ＮＬスェーデン型変異、Ｉ７１６Ｖフロリダ型変異、Ｖ７１７Ｉロンドン型変異の５種類の変異を有することがより好ましい。 As shown in Examples described later, it has been confirmed that amyloid deposition is promoted in mice in which a β-gal knockout mouse and an Alzheimer's disease model mouse are crossed.
Therefore, the animal model for Alzheimer's disease used in the evaluation of the present invention further includes deletion of exon 9 of human prisenilin 1 (PS1), M146L mutation, L285V mutation, N141I mutation of human PS2, KM670 / of human amyloid precursor protein (APP) It is preferable to have at least one mutation selected from the group consisting of 671 NL Swedish type mutation, I716V Florida type mutation, and V717 I London type mutation, M146L mutation of human prisenilin 1 (PS1), and L285V mutation, and human amyloid precursor It is more preferable to have five types of mutations, KM670 / 671 NL Swedish type mutation, I716V Florida type mutation, and V717I London type mutation of body protein (APP).
　非ヒト哺乳動物としては、特に制限されないが、げっ歯類に分類される動物であることが好ましい。非ヒト哺乳動物としては、マウス、ラット、モルモット、ハムスター、ウサギ、ヤギ、ブタ、イヌ、ネコが挙げられる。 The non-human mammal is preferably, but not limited to, an animal classified into rodents. Non-human mammals include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs and cats.
　本発明の評価に係るモデル動物は、アルツハイマー病のメカニズムの解明や、その治療等に有用な物質の探索、治療方法などの開発に用いることができる。 The model animal according to the evaluation of the present invention can be used for elucidating the mechanism of Alzheimer's disease, searching for a substance useful for the treatment or the like, and developing a treatment method or the like.
　以下、実験例により本発明をさらに詳細に説明するが、本発明はこれらの例によって限定されるものではない。 Hereinafter, the present invention will be described in more detail by way of experimental examples, but the present invention is not limited by these examples.
［実験例１］
［皮膚由来線維芽細胞の樹立］
　倫理委員会に承認されたプロトコールにより、インフォームドコンセントの下、ＧＭ１ガングリオシドーシス患者及び健常者の皮膚生検の外植片から線維芽細胞を樹立した。
　患者及び健常者からの皮膚試料を細かく刻み、１０％ウシ胎児血清（ＦＢＳ）を添加したＤＭＥＭ培地で培養した。線維芽細胞が出現したことを確認した後、初期化遺伝子を導入するために線維芽細胞を増殖させた。 [Experimental Example 1]
[Establishment of skin-derived fibroblasts]
Fibroblasts were established from skin biopsies explants of GM1 gangliosidosis patients and healthy persons under informed consent according to a protocol approved by the ethics committee.
Skin samples from patients and healthy individuals were minced and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). After confirming that fibroblasts appeared, fibroblasts were expanded to introduce a reprogramming gene.
［iPS細胞の樹立及び維持］
　N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, M. Hasegawa, Proc. Jpn. Acad. Ser., B. Phys. Biol. Eci., 85, 348 (2009)に記載される方法により、樹立したヒト由来線維芽細胞からｉＰＳ細胞を樹立した。
　具体的には、感染１日前に、６ウェルプレートにおいて、１ウェル当たり５×１０５個のヒト線維芽細胞を播種し、その後、Oct3/4遺伝子、Sox2遺伝子、K1f4遺伝子、及びc-Myc遺伝子を含むセンダイウイルス（ＳｅＶ）ベクターを、ＭＯＩ(multiplicity of infection)３にて、細胞に感染させた。Oct3/4遺伝子、Sox2遺伝子、K1f4遺伝子、及びc-Myc遺伝子を含むＳｅＶベクターについては、N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, M. Hasegawa, Proc. Jpn. Acad. Ser., B. Phys. Biol. Eci., 85, 348 (2009)に記載される方法に従い作製した。感染７日後、感染させた線維芽細胞を、トリプシンを用いて回収し、６０ｍｍのシャーレ当たり５．４×１０４個の細胞、又は１００ｍｍのシャーレ当たり１～２×１０５個の細胞をＭＭＣ処理したＭＥＦフィーダー細胞上に播種した。翌日、ヒトｉＰＳ細胞培地に置き換え、感染３０日後まで培養を継続し、コロニーを回収した。 [Establishment and maintenance of iPS cells]
N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, M. Hasegawa, Proc. Jpn. Acad. Ser., B. Phys. Biol. Eci., 85, 348 (2009) And iPS cells were established from established human-derived fibroblasts.
Specifically, 1 day before infection, 5 × 10 5 human fibroblasts are seeded per well in a 6-well plate, and then Oct3 / 4 gene, Sox2 gene, K1f4 gene, and c-Myc gene. The cells were infected with a Sendai virus (SeV) vector containing MOI (multiplicity of infection) 3. For SeV vectors containing Oct3 / 4 gene, Sox2 gene, K1f4 gene and c-Myc gene, see N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, M. Hasegawa, Proc. Jpn. Acad. , B. Phys. Biol. Eci., 85, 348 (2009). Seven days after infection, infected fibroblasts are harvested using trypsin and MMC treated with 5.4 × 10 4 cells per 60 mm petri dish or 1-2 × 10 5 cells per 100 mm petri dish Were seeded on the MEF feeder cells. The next day, human iPS cell culture medium was replaced, and culture was continued until 30 days after infection to recover colonies.
　２０％のＫＮＯＣＫＯＵＴ（商標）血清置換物（ＫＳＲ，インビトロゲン）、２ｍＭのＬ-グルタミン、１×１０－４Ｍの非必須アミノ酸（ＮＥＡＡ，シグマ）、１×１０－４Ｍの２－メルカプトエタノール（シグマ）、０．５％のペニシリンとストレプトマイシン（日本，ナカライテスク）、及び５ｎｇ／ｍＬの基本線維芽細胞増殖因子（ｂＦＧＦ，和光，日本）を添加したＤＭＥＭ／Ｆ１２（シグマ）を含有するヒトｉＰＳ培地を用いて、マイトマイシンＣ（ＭＭＣ）処理したＭＥＦフィーダー細胞上で、ヒトｉＰＳ細胞を維持した。 20% KNOCKOUTTM serum replacement (KSR, Invitrogen), 2 mM L-glutamine, 1 × 10 -4 M nonessential amino acids (NEAA, Sigma), 1 × 10 -4 M 2-mercaptoethanol (Sigma) Humans with DMEM / F12 (Sigma) supplemented with 0.5% penicillin and streptomycin (Nacalai Tesque, Japan), and 5 ng / mL of basic fibroblast growth factor (bFGF, Wako, Japan) Human iPS cells were maintained on mitomycin C (MMC) -treated MEF feeder cells using iPS medium.
［神経幹細胞（Neural Stem Cell:NSC）への分化］
　分化誘導開始の前日に、Geltrex(Thermo Fisher Scientific)をコーティングした６穴プレートに、ヒトｉＰＳ細胞を２．５×１０５～３×１０５個/ウェルの密度で播種し、ヒトｉＰＳ培地で培養した。翌日（誘導開始日：Day 0）、ヒトｉＰＳ培地を除去して、PSC Neural Induction Medium(Thermo Fisher Scientific）に入れ替えた。引き続き、２日に１回の頻度で、PSC Neural Induction Mediumにて培地交換を行いながら、培養開始後７日目(Day 7)まで培養を行い、ＮＳＣを誘導した。誘導されたＮＳＣは、培地を除き、StemPro Accutase Cell Dissociation Reagent(Thermo Fisher Scientific）を用いて細胞をはがし、Neural Expansion Medium(Thermo Fisher Scientific)に細胞を懸濁後、Geltrexにてコーティングした100mm細胞培養用シャーレへ播種した。この時点の細胞をP0NSC（継代数ゼロのNSC）とし、以後、適宜この培地にてNSCを増やし、実験に使用した。 [Differentiation into neural stem cells (NSCs)]
The day before differentiation induction starts, human iPS cells are seeded at a density of 2.5 × 10 5 to 3 × 10 5 cells / well in Geltrex (Thermo Fisher Scientific) coated 6-well plates and cultured in human iPS medium did. The next day (induction start date: Day 0), human iPS medium was removed and replaced with PSC Neural Induction Medium (Thermo Fisher Scientific). Subsequently, while changing the medium in PSC Neural Induction Medium at a frequency of once every two days, culture was performed until the seventh day (Day 7) after the start of culture to induce NSC. The induced NSCs were deprived of the medium, the cells were detached using StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific), the cells were suspended in Neural Expansion Medium (Thermo Fisher Scientific), and then 100 mm cell culture coated with Geltrex was performed. Sowing to petri dishes. The cells at this time point were designated as P0 NSCs (NSCs with zero passage number), and thereafter, NSCs were appropriately increased in this medium and used for experiments.
［実験例２］
＜FITC-CTBを用いたＧＭ１蓄積の可視化＞
　健常者およびＧＭ１ガングリオシドーシス患者由来ｉＰＳ細胞から誘導した神経幹細胞を、４％パラホルムアルデヒドにて固定後、０．１％ TritonX-100/PBSにて透過処理し、１％　ＢＳＡ溶液にてブロッキングを行った。その後、FITC-CTB（ＧＭ１ガングリオシドを特異的に染色する試薬）と抗Nestin抗体（神経幹細胞に特異的に発現している分子：神経幹細胞のマーカー）を用いて、多重染色を行った。図１に結果を示す。
　Nestin染色においては、健常者、ＧＭ患者、いずれのNSCにおいても強い染色が認められ、神経幹細胞への分化が確認された（図１，下段）。一方、FITC-CTBにおいては，健常者ではほとんど染色が認められなかったが、ＧＭ１患者においては，強い染色が認められた（図１，上段）。
　これらの結果から、ＧＭ１患者由来神経幹細胞では、ＧＭ１が蓄積していることが確認された。 [Experimental Example 2]
Visualization of GM1 accumulation using FITC-CTB
After fixation with 4% paraformaldehyde, neural stem cells derived from iPS cells from healthy subjects and GM1 gangliosidosis patients are permeabilized with 0.1% Triton X-100 / PBS and blocked with 1% BSA solution went. Thereafter, multiple staining was performed using FITC-CTB (a reagent for specifically staining GM1 ganglioside) and an anti-Nestin antibody (a molecule specifically expressed in neural stem cells: a marker for neural stem cells). The results are shown in FIG.
In Nestin staining, strong staining was observed in healthy individuals, GM patients, and all NSCs, and differentiation to neural stem cells was confirmed (FIG. 1, lower). On the other hand, in the case of FITC-CTB, almost no staining was observed in healthy subjects, but a strong staining was observed in GM1 patients (FIG. 1, upper stage).
From these results, it was confirmed that GM1 is accumulated in GM1 patient-derived neural stem cells.
［実験例３］
　アルツハイマー病患者の脳におけるＧＭ１ガングリオシドの量は、健常者の脳におけるＧＭ１ガングリオシドの量よりも多いと報告されている。発明者らは、アルツハイマー病由来の神経幹細胞におけるＧＭ１ガングリオシドの代謝状態を検討した。第一に、患者由来の神経幹細胞のβ－ｇａｌ活性を調べたところ、アルツハイマー病患者由来の神経幹細胞のβ－ｇａｌ活性は、健常者由来の神経幹細胞と比較して、低下していたことが確認された（図２Ａ）参照。）。図２Ａ中、２０１Ｂ７及び４０９Ｂ２は、健常者由来の神経幹細胞を示し、Ａ１３８及びＡ１５４は、ＧＭ１ガングリオシドーシス患者由来の神経幹細胞を示し、Ａ２３２＃３－１及びＡ２３２＃２－２は、アルツハイマー病由来の神経幹細胞を示す。また、図２Ａ中、Ｓｏｌは、不溶性画分を、Ｓｕｐは、可溶性画分を意味する。本実験例において不溶性画分・可用性画分におけるβ－ｇａｌ活性に差はないことを示している。 [Experimental Example 3]
The amount of GM1 ganglioside in the brains of Alzheimer's patients is reported to be greater than the amount of GM1 gangliosides in the brains of healthy individuals. The inventors examined the metabolic state of GM1 ganglioside in neural stem cells derived from Alzheimer's disease. First, when β-gal activity of patient-derived neural stem cells was examined, it was found that β-gal activity of Alzheimer's disease-derived neural stem cells was decreased as compared to that of healthy individuals. See confirmed (Figure 2A). ). In FIG. 2A, 201B7 and 409B2 indicate neural stem cells derived from healthy persons, A138 and A154 indicate neural stem cells derived from GM1 gangliosidosis patients, and A232 # 3-1 and A232 # 2-2 indicate Alzheimer's disease. 1 shows neural stem cells derived from. Also, in FIG. 2A, Sol means the insoluble fraction and Sup means the soluble fraction. It is shown that there is no difference in β-gal activity in the insoluble fraction / availability fraction in this experimental example.
　更に、発明者らは、ＡＰＰの過剰発現が、β－ｇａｌ活性に及ぼす影響を検討した。健常者由来の神経幹細胞に、コントロールベクター又はＡＰＰ過剰発現ベクターを導入し、β－ｇａｌ活性を測定した。コントロールベクターを導入した神経幹細胞と比較して、ＡＰＰ過剰発現ベクターを導入した神経幹細胞において、β－ｇａｌ活性が低下していたことが確認された（図２Ｂ参照。）。 Furthermore, the inventors examined the effect of overexpression of APP on β-gal activity. A control vector or an APP overexpression vector was introduced into neural stem cells from healthy individuals, and β-gal activity was measured. It was confirmed that β-gal activity was reduced in neural stem cells into which the APP overexpression vector was transferred, as compared to neural stem cells into which the control vector was transferred (see FIG. 2B).
　更に、発明者らは、患者由来の神経幹細胞における、ＧＭ１ガングリオシドの蓄積を検討した（図２Ｃ、図２Ｄ参照。）。図２Ｃは、細胞全体におけるＧＭ１ガングリオシド量を示したグラフであり、図２Ｄは、脂質ラフト画分におけるＧＭ１ガングリオシド量を示したグラフである。
　アルツハイマー病由来の神経幹細胞におけるＧＭ１ガングリオシドの量は、健常者由来の神経幹細胞における量より多いことが確認された。特に、脂質ラフト画分での差が大きかった。β－ｇａｌがＧＭ１ガングリオシドの分解に関与しているという知見もあり、これらのことから、アルツハイマー病由来の神経幹細胞においては、β－ｇａｌ活性が低下しており、これにより、ＧＭ１ガングリオシドが蓄積するものと示唆された。 Furthermore, the inventors examined accumulation of GM1 ganglioside in patient-derived neural stem cells (see FIG. 2C, FIG. 2D). FIG. 2C is a graph showing the amount of GM1 ganglioside in the whole cell, and FIG. 2D is a graph showing the amount of GM1 ganglioside in the lipid raft fraction.
It has been confirmed that the amount of GM1 ganglioside in neural stem cells derived from Alzheimer's disease is greater than the amount in neural stem cells derived from healthy individuals. In particular, the difference in the lipid raft fraction was large. There is also a finding that β-gal is involved in the degradation of GM1 ganglioside. From these facts, in neural stem cells derived from Alzheimer's disease, β-gal activity is reduced, whereby GM1 ganglioside is accumulated. It was suggested that.
［実験例４］
　ＧＭ１ガングリオシドの減少が、Ａβの代謝に影響するかどうかを確認するため、β－ｇａｌタンパク質を神経幹細胞内に過剰発現させた（図３Ａ～Ｄ参照。）。図３中、Ｃｏｎｔｒｏｌは、コントロールベクターを導入した神経幹細胞を示し、ＧＬＢ１ ＯＥは、β－ｇａｌタンパク質過剰発現ベクターを導入した神経幹細胞を示す。図３Ｂに示されるように、アルツハイマー病由来の神経幹細胞におけるβ－ｇａｌタンパク質の過剰発現より、Ａβ４２の量が減少したことが確認された。 [Experimental Example 4]
To confirm whether GM1 ganglioside reduction affected Aβ metabolism, β-gal protein was overexpressed in neural stem cells (see FIG. 3A-D). In FIG. 3, Control indicates a neural stem cell into which a control vector has been introduced, and GLB1 OE indicates a neural stem cell into which a β-gal protein overexpression vector has been introduced. As shown in FIG. 3B, it was confirmed that the amount of Aβ42 decreased due to the overexpression of β-gal protein in neural stem cells derived from Alzheimer's disease.
　Ａβ４２を処理することにより、細胞死が誘導されるという知見がある。そこで、発明者らは、健常者由来の神経幹細胞及びＧＭ１ガングリオシドーシス患者由来の神経幹細胞、それぞれにおけるＡβ４２に対する感受性を比較検討した（図３Ｅ参照。）。図３Ｅの凡例中、２０１Ｂ７ＮＳＣは、健常者由来の神経幹細胞を示し、Ａ１３８ＮＳＣは、ＧＭ１ガングリオシドーシス患者由来の神経幹細胞を示し、２０１Ｂ７＋ＧＭ１は、ＧＭ１ガングリオシドで処理された健常者由来の神経幹細胞を示す。
　細胞生存率アッセイの結果、ＧＭ１ガングリオシドーシス患者由来の神経幹細胞は、健常者由来の神経幹細胞よりもＡβ４２に対する感受性が高いことが確認された。更に、ＧＭ１ガングリオシドで処理された健常者由来の神経幹細胞におけるＡβ４２に対する感受性は、ＧＭ１ガングリオシドーシス患者由来の神経幹細胞と類似した傾向を示すことが確認された。
　これらのことから、ＧＭ１ガングリオシドは、神経幹細胞におけるＡβの生産に影響を及ぼすことが示唆された。 There is a finding that cell death is induced by treating Aβ42. Therefore, the inventors compared and examined the sensitivity to Aβ42 in each of neural stem cells from healthy individuals and neural stem cells from GM1 gangliosidosis patients (see FIG. 3E). In the legend of FIG. 3E, 201B7 NSC represents neural stem cells derived from healthy subjects, A138 NSC represents neural stem cells derived from GM1 gangliosidosis patients, and 201 B7 + GM1 represents neural stem cells derived from healthy subjects treated with GM1 ganglioside. .
As a result of the cell viability assay, it was confirmed that neural stem cells derived from GM1 gangliosidosis patients are more sensitive to Aβ42 than neural stem cells derived from healthy individuals. Furthermore, it was confirmed that the sensitivity to Aβ42 in neural stem cells derived from GM1 ganglioside-treated healthy individuals shows a tendency similar to that of GM1 gangliosidosis-derived neural stem cells.
From these facts, it was suggested that GM1 ganglioside affects the production of Aβ in neural stem cells.
［実験例５］
　出生後、C57BL/6NCr SlcをバックグランウドとしたＧＭ１ガングリオシドーシスモデルマウス（ＢＫＯマウス：β－ｇａｌの完全欠損マウス, β－ｇａｌ（－／－））の脳からＡβを抽出し定量した（図４Ａ～Ｄ参照。）。図４中、ＷＴは、正常マウスを示し、ＢＫＯは、前述したＧＭ１ガングリオシドーシスモデルマウスを示し、５×ＦＡＤは、ヒトプリセニリン１（ＰＳ１）のＭ１４６Ｌ変異、及びＬ２８５Ｖ変異、並びに、ヒトアミロイド前駆体タンパク質（ＡＰＰ）のＫＭ６７０／６７１ＮＬスェーデン型変異、Ｉ７１６Ｖフロリダ型変異、Ｖ７１７Ｉロンドン型変異の５種類の変異を有するアルツハイマー病モデルマウスを示し、ＢＫＯ／５×ＦＡＤは、ＢＫＯと５×ＦＡＤを掛けあわせたマウスを示す。
　図４Ａ及びＢは、ＴＢＳ（Tris Buffered Saline）抽出画分における可溶性Ａβの定量結果を示し、図４Ｃ及びＤは、Guanidin-HCl抽出画分における不溶性Ａβの定量結果を示す。
　ＷＴと比較して、ＢＫＯでは、Ａβ４０及び４２が増加傾向にあることが示された。また、Guanidin-HCl抽出画分において、５×ＦＡＤと比較して、ＢＫＯ／５×ＦＡＤでは、Ａβ４０及び４２が有意に増加しており、ＢＫＯマウスと５×ＦＡＤの掛け合わせにより、アミロイドの沈着が促進されていることが確認された。 [Experimental Example 5]
After birth, Aβ was extracted and quantified from the brain of GM1 gangliosidosis model mouse (BKO mouse: β-gal completely deficient mouse, β-gal (-/-)) with C57BL / 6NCr Slc as background. See Figures 4A-D). In FIG. 4, WT indicates a normal mouse, BKO indicates a GM1 gangliosidosis model mouse as described above, 5 × FAD indicates a M146L mutation of human prisenilin 1 (PS1) and a L285V mutation, and a human amyloid precursor BKO / 5 × FAD shows BKO / 5 × FAD multiplied by 5 × FAD, showing an Alzheimer's disease model mouse having 5 kinds of mutations of KM670 / 671NL Swedish type mutation, I716V Florida type mutation and V717I London type mutation of protein (APP) Shows a mouse.
FIGS. 4A and 4B show the results of quantification of soluble Aβ in TBS (Tris Buffered Saline) extract fractions, and FIGS. 4C and 4 show the results of quantification of insoluble Aβ in Guanidin-HCl extract fractions.
It was shown that Aβ 40 and 42 tend to increase in BKO as compared to WT. In addition, Aβ40 and 42 are significantly increased in BKO / 5 × FAD compared to 5 × FAD in the Guanidin-HCl-extracted fraction, and amyloid deposition is caused by crossing BKO mice and 5 × FAD. Was confirmed to be promoted.
　出生後、C57BL/6NCr SlcのＷＴ、ＢＫＯ、５×ＦＡＤ、ＢＫＯ／５×ＦＡＤの脳から切片を作製した。抗Ａβ抗体（クローン：4G8）を用いて、切片を免疫染色した。結果を図４Ｅに示す。図４Ａ～Ｄの結果と同様、アミロイドの沈着量の多さは、ＢＫＯ／５×ＦＡＤ、５×ＦＡＤ、ＢＫＯ、ＷＴの順であることが確認された。 After birth, sections were prepared from C57BL / 6NCr Slc WT, BKO, 5 × FAD, BKO / 5 × FAD brains. The sections were immunostained using an anti-Aβ antibody (clone: 4G8). The results are shown in FIG. 4E. Similar to the results shown in FIGS. 4A to 4D, the deposition amount of amyloid was confirmed to be in the order of BKO / 5 × FAD, 5 × FAD, BKO, and WT.
［実験例６］
＜イメージングサイトメーターを用いた薬剤スクリーニングによる化合物の評価＞
　以下の要領で、アルツハイマー病治療剤のスクリーニングを行った。
　９６ウェルプレートをＧｅｌｔｒｅｘにてコーティング後、Ｎｅｕｒａｌ Ｅｘｐａｎｓｉｏｎ Ｍｅｄｉｕｍに懸濁した、健常者およびＧＭ１ガングリオシドーシス患者由来ｉＰＳ細胞から誘導した神経幹細胞を、５×１０４／ウェルの密度で播種した。その後、既知薬剤ライブラリーの化合物を、各々のウェルに最終濃度５μＭになるように添加し、72時間培養を行った。その後、４％パラホルムアルデヒドにて固定、０．１％ＴｒｉｔｏｎＸ－１００／ＰＢＳにて透過処理、１％ ＢＳＡ溶液にてブロッキングを行い、ＦＩＴＣ－ＣＴＢ（ＧＭ１ガングリオシドを特異的に染色する試薬）、Ｈｏｅｃｈｓｔ ３３３４２（細胞の核を染色する試薬）、ＣｅｌｌＭａｓｋ（細胞膜を染色する試薬）を使い、蛍光染色した。
　各ウェルの蛍光強度を、イメージングサイトメーター（IN Cell Analyzer 6000）を用いて画像を取得し、IN Cell Developer Toolbox プロトコールを用いて、細胞当たりのGM1ガングリオシド量を算出した。
　表１に検討を行った化合物の結果を示す。 [Experimental Example 6]
<Evaluation of compounds by drug screening using imaging cytometer>
A screening agent for Alzheimer's disease was conducted as follows.
After coating a 96-well plate with Geltrex, neural stem cells derived from healthy individuals and iPS cells from GM1 gangliosidosis patients suspended in Neural Expansion Medium were seeded at a density of 5 × 10 4 / well. Thereafter, compounds of the known drug library were added to each well to a final concentration of 5 μM, and culture was performed for 72 hours. Then, fix with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100 / PBS, perform blocking with 1% BSA solution, FITC-CTB (reagent for specifically staining GM1 ganglioside), Hoechst Fluorescent staining was performed using 33342 (reagent for staining cell nuclei) and CellMask (reagent for staining cell membranes).
The fluorescence intensity of each well was imaged using an imaging cytometer (IN Cell Analyzer 6000), and the amount of GM1 ganglioside per cell was calculated using an IN Cell Developer Toolbox protocol.
Table 1 shows the results of the compounds examined.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　検討を行った化合物のうち、ＧＭ１抑制化合物としてヒットしたいくつかの化合物における薬剤処理後の神経幹細胞の染色像を、図５、図６に示す。
　ヒットした化合物においては，薬剤を添加していない例（図５ｂ・下段，図６ｅ・下段）と比較して、ＣＴＢの蛍光が低下しており、ＧＭ１の蓄積が抑制されていることが確認された（図５ｃならびにｄ，図６ｆからｇ、いずれも下段）。 Among the compounds examined, staining images of neural stem cells after drug treatment with several compounds hit as GM1 inhibitory compounds are shown in FIG. 5 and FIG.
In the hit compounds, it is confirmed that the fluorescence of CTB is lower and the accumulation of GM1 is suppressed, as compared with the case where no drug is added (FIG. 5 b, lower, FIG. 6 e, lower). (FIG. 5 c and d, FIG. 6 f to g, each lower).
［実験例７］
＜ＧＭ１蓄積を低下させるヒット薬剤の培地中（細胞外）Ａβに対する効果＞
　Geltrexをコーティングした６ウェルプレートに、アルツハイマー病患者由来ｉＰＳ細胞から誘導した神経幹細胞を、２×１０６個/ウェルの密度で播種し、ヒット薬剤を最終濃度５μＭになるように加え、７２時間培養した。７２時間後、細胞上清を回収し、４００ｇ・１０分間遠心した上清を解析用サンプルとした。これらの解析用サンプルを、Human/Rat βAmyloid（42）ELISA Kit wako、High Sensitive（WAKO）、Human/Rat βAmyloid（40）ELISA Kit wako II（WAKO）を用いたサンドイッチＥＬＩＳＡ法にて、Ａβ４２、Ａβ４０、それぞれを測定した。これらの測定結果より、Ｔｏｔａｌ　Ａβ（Ａβ４２+Ａβ４０）ならびにＡβ４２／Ａβ４０比（Ａβ４２÷Ａβ４０）を算出した。結果を図７、図８に示す。 [Experimental Example 7]
<Effect on hit (extracellular) Aβ of a hit drug that reduces GM1 accumulation>
Neural stem cells derived from Alzheimer's disease patient-derived iPS cells are seeded at a density of 2 × 10 6 / well in Geltrex-coated 6-well plates, hit drug is added to a final concentration of 5 μM, and culture is performed for 72 hours did. After 72 hours, the cell supernatant was collected, and the supernatant obtained by centrifugation at 400 g for 10 minutes was used as a sample for analysis. These samples for analysis were Aβ42 and Aβ40 by sandwich ELISA method using Human / Rat βAmyloid (42) ELISA Kit wako, High Sensitive (WAKO) and Human / Rat β Amyloid (40) ELISA Kit wako II (WAKO). , Each was measured. From these measurement results, Total Aβ (Aβ42 + Aβ40) and Aβ42 / Aβ40 ratio (Aβ42 ÷ Aβ40) were calculated. The results are shown in FIG. 7 and FIG.
　アルツハイマー病患者由来の神経幹細胞の培地中におけるＡβ４２／Ａβ４０及び細胞内におけるＡβ４２／Ａβ４０は、健常者に比較して高い値を示した（図７および８の下段）。
　ＧＭ１抑制化合物の処理により、アルツハイマー病患者由来の神経幹細胞の培地中及び細胞内におけるＴｏｔａｌ　ＡβならびにＡβ４２／Ａβ４０比を低下することが確認された（図７c，図８f）。 Aβ42 / Aβ40 in the medium of neural stem cells derived from Alzheimer's disease patients and Aβ42 / Aβ40 in the cells showed high values as compared to healthy persons (lower row in FIGS. 7 and 8).
It was confirmed that the treatment with the GM1 inhibitory compound reduced the total Aβ and Aβ42 / Aβ40 ratio in the medium and cells of neural stem cells derived from Alzheimer's disease patients (FIG. 7 c, FIG. 8 f).
［実験例８］
＜ＧＭ１抑制化合物投与後のＧＭ１ガングリオシドーシスモデルマウスの脳における、ＧＭ１ガングリオシド量の変化＞
　ＧＭ１ガングリオシドーシスモデルマウス（ＢＫＯマウス：β-Galactosidaseの完全欠損マウス, β－Ｇａｌ（－／－）へ、出生後P9からP15の6日間、Amodiaquine (40mg/kg)、Thiethylperanzine (6mg/kg)を、１日２回、腹腔内に投与した。なお、陽性対象として、薬液にＰＢＳを用い、同様の操作を行った。また、陰性対象として、薬液にＰＢＳ、マウスとしてβ－Ｇａｌ（＋/－）のものを用いて、同様の操作を行った。その後、マウス脳をOCTコンパウンドにて包埋し、５μｍの厚さで切片を作製した。切片は４％パラホルムアルデヒドにて固定後、１％　ＢＳＡ溶液にてブロッキングを行い、Alexa Fluor488-CTB、Hoechst 33342にて蛍光染色を行った。また、マウス脳を、Svennerholm and Fredmanの抽出法に基づいてスフィンゴ脂質を分画・精製し、試料とし、これらの試料をAgilent6460　Triple Quadrupole LC/MSにて解析し、脳内ＧＭ１を定量した。蛍光染色の結果を図９に示す。 [Experimental Example 8]
<Change in the amount of GM1 ganglioside in the brain of a GM1 gangliosidosis model mouse after administration of a GM1 inhibitory compound>
GM1 gangliosidosis model mouse (BKO mouse: β-Galactosidase completely deficient mouse, β-Gal (-/-), postnatal P9 to P15 for 6 days, Amodiaquine (40 mg / kg), Thiethylperanzine (6 mg / kg) Was administered twice a day into the abdominal cavity, and the same procedure was performed using PBS as a drug solution as a positive subject, and PBS as a drug solution as a negative subject, and β-Gal (+ / as a mouse. Then, the mouse brain was embedded with OCT compound, and a section was prepared with a thickness of 5 μm.The section was fixed with 4% paraformaldehyde and then 1 % Blocked with BSA solution and fluorescently stained with Alexa Fluor 488-CTB, Hoechst 33342. In addition, mouse brain was fractionated and purified based on the extraction method of Svennerholm and Fredman, and used as a sample. , These samples Agilent 64 Analysis was carried out with 60 Triple Quadrupole LC / MS to quantify GM1 in the brain The results of the fluorescent staining are shown in FIG.
　陰性対象では、CTBの蛍光がほとんど見られなかった（図９ａ・上段）。一方、陽性対象では、CTBの蛍光が強く、GM1の蓄積が確認された（図９ｂ・上段）。一方で、ＧＭ１抑制化合物による処理を行ったいずれにおいても、CTBの蛍光は強く抑制されており、ＧＭ１の蓄積が抑制されていることが確認された（図９c，d，いずれも上段）。ＧＭ１の定量結果を図１０に示す。 In negative subjects, little CTB fluorescence was seen (Fig. 9a, upper row). On the other hand, in the positive subjects, the fluorescence of CTB was strong, and the accumulation of GM1 was confirmed (Fig. 9b, upper row). On the other hand, it was confirmed that the fluorescence of CTB was strongly suppressed and the accumulation of GM1 was suppressed in any of the treatments with the GM1 inhibitory compound (FIG. 9 c, d, each upper row). The quantitative result of GM1 is shown in FIG.
　陰性対象と比較して、陽性対象では、ＧＭ１ガングリオシド量の増加が確認された。一方，薬液処理したいずれにおいても、陽性対象と比べて、ＧＭ１ガングリオシド量が低下していることが確認された。 An increase in the amount of GM1 ganglioside was confirmed in positive subjects as compared to negative subjects. On the other hand, it was confirmed that the amount of GM1 ganglioside decreased in any of the chemical treatment, as compared with the positive target.
［実験例９］
＜ＧＭ１抑制化合物投与後のアルツハイマー病モデルマウスの脳における、Ａβ量の変化＞
　出生後３か月の５×ＦＡＤマウスにAmodiaquine (40mg/kg) 、Thiethylperanzine (6mg/kg)を、1日2回、腹腔内に３か月間連続投与した。なお、陽性対象として、薬液にＰＢＳを用い、同様の操作を行った。その後、各マウスの脳からＡβを抽出し定量した（図１１Ａ～Ｄ参照。）。
　図１１Ａ及びＢは、ＴＢＳ（Tris Buffered Saline）抽出画分における可溶性Ａβの定量結果を示し、図１１Ｃ及びＤは、Guanidin-HCl抽出画分における不溶性Ａβの定量結果を示す。
　ＰＢＳ投与群と比較して、Amodiaquine 投与群、及びThiethylperanzine投与群では、Ａβ４０及び４２が減少していることが確認された。 [Experimental Example 9]
<Change in the amount of Aβ in the brain of Alzheimer's disease model mice after administration of a GM1 inhibitory compound>
Amodiaquine (40 mg / kg) and Thiethylperanzine (6 mg / kg) were continuously administered intraperitoneally twice a day for 3 months to the 3 months after birth 5 × FAD mice. The same operation was performed using PBS as a drug solution as a positive target. Thereafter, Aβ was extracted and quantified from the brain of each mouse (see FIGS. 11A to D).
11A and 11B show the results of quantification of soluble Aβ in TBS (Tris Buffered Saline) extract fractions, and FIGS. 11C and 11C show the results of determination of insoluble Aβ in Guanidin-HCl extract fractions.
It was confirmed that Aβ 40 and 42 decreased in the Amodiaquine administration group and Thiethylperanzine administration group, as compared with the PBS administration group.
　また、連続投与後の各マウスの脳から切片を作製した。抗Ａβ抗体（クローン：4G8）を用いて、切片を免疫染色した。結果を図１１Ｅに示す。図１１Ａ～Ｄの結果と同様、Amodiaquine 投与群、及びThiethylperanzine投与群では、Ａβ４０及び４２が減少していることが確認された。 In addition, sections were prepared from the brain of each mouse after continuous administration. The sections were immunostained using an anti-Aβ antibody (clone: 4G8). The results are shown in FIG. 11E. Similar to the results of FIGS. 11A to D, it was confirmed that Aβ 40 and 42 decrease in the Amodiaquine administration group and the Thiethylperanzine administration group.
［実験例１０］
＜ＧＭ１抑制化合物添加によるオートファジーへの影響＞
ガングリオシドの合成及び分解過程を図１２に示す。GM1、GM2、GM3の各ガングリオシドは、単糖及びN-アセチルノイラミン酸の連続的付加により生成される。また、ガングリオシドの分解は、ライソゾーム中のグリコシダーゼが段階的に作用することにより行われる。図１２における（１）～（７）は、各反応において作用する酵素を示している。
図１３は、NEU1 及びβ-GLUの発現が候補化合物による処理により増加することを示している。正常(201B7)及び疾患由来 (A138 #1-3)のiPS細胞から分化して得られた神経幹細胞をウェルに播種し、 Amodiaquine又はThiethylperanzineを5μMになるようにそれぞれ添加し、72時間培養を行った。培養後、RNAを単離し、各酵素遺伝子の発現量の解析を行った。図１３中、－は、無添加サンプルを示し、amoは、Amodiaquine添加サンプルを示し、thieは、Thiethylperanzine添加サンプルを示す。
本結果により、ＧＭ１抑制化合物の作用により、ライソゾーム中におけるガングリオシドの分解を促進し、オートファジーを活性化させることが示唆された。 [Experimental Example 10]
<Influence on autophagy by addition of GM1 inhibitory compound>
The synthesis and degradation process of ganglioside is shown in FIG. Each ganglioside of GM1, GM2 and GM3 is produced by the sequential addition of monosaccharides and N-acetylneuraminic acid. Furthermore, the degradation of gangliosides is carried out by the stepwise action of glycosidases in lysosomes. (1) to (7) in FIG. 12 show enzymes acting in each reaction.
FIG. 13 shows that expression of NEU1 and β-GLU is increased by treatment with candidate compounds. Neural stem cells obtained by differentiating from normal (201B7) and disease-derived (A138 # 1-3) iPS cells are seeded in the wells, Amodiaquine or Thiethylperanzine is added to 5 μM, respectively, and culture is performed for 72 hours. The After culture, RNA was isolated, and the expression amount of each enzyme gene was analyzed. In FIG. 13,-indicates an additive-free sample, amo indicates an Amodiaquine-loaded sample, and thie indicates a Thiethylperanzine-loaded sample.
The present results suggest that the action of the GM1 inhibitory compound promotes degradation of ganglioside in lysosome and activates autophagy.
本発明により、新たな作用機序を有するアルツハイマー病予防剤又は治療剤を提供することができる。 According to the present invention, an Alzheimer's disease preventive or therapeutic agent having a novel mechanism of action can be provided.

Claims (7)

  1. 　ＧＭ１ガングリオシドーシス予防剤又は治療剤を有効成分として含有することを特徴とするアルツハイマー病予防剤又は治療剤。 An agent for preventing or treating Alzheimer's disease, which comprises an agent for preventing or treating GM1 gangliosidosis as an active ingredient.
  2. 　Amodiaquine（アモジアキン）、Acacetin（アカセチン）、Sulfamerazine（スルファメラジン）、Ungerine（ウンゲリン）、Amiodarone（アミオダロン）、Sertindole（セルチンドール）、Delcorine（デルコリン）、Perphenazine（ペルフェナジン）、Althiazide（アルチアジド）、Diethylstilbestrol（ジエチルスチルベストロール）、Thiethylperazine（チエチルペラジン）、Harmol（ハルモール）、Skimmianine（スキムミアニン）、Succinylsulfathiazole（スクシニルサルファチアゾール）、Fillalbin（フィラルビン）、Canavanine（カナバニン）、Harmaline（ハルマリン）、Trihexyphenidyl（トリヘキシフェニジル）、Fluoxetine（フルオキセチン）、Lovastatin（ロバスタチン）、Haloperidol（ハロペリドール）、Prenylamine lactate（プレニルアミン乳酸）、Bromperidol（ブロムペリドール）、Convolamine（コンボルバミン）、及びMiglustat（ミグルスタット）、並びに、これらの薬学的に許容される塩、又はそれらの溶媒和物からなる群から選ばれる少なくとも１種を有効成分として含有することを特徴とするアルツハイマー病予防剤又は治療剤。 Amodiaquine (Amodiaquine), Acacetin (Achacetin), Sulfamerazine (Sulphamelazine), Ungerine (Ungelin), Amiodarone (Amiodarone), Sertindol (Sertindol), Delcorine (Delcoline), Perphenazine (Perphenazine), Althiazide (Alchiazide), Diethylstilbestrol), Thiethylperazine (Thethylperazine), Harmol (Halmol), Skimmianine (Skimmianin), Succinylsulfathiazole (Succinylsulfathiazole), Fillalbin (Filarbine), Canavanine (Canavanine), Harmaline (Halmarin), Trihexyphenidyl (Trihexyphenidyl) , Fluoxetine (fluoxetine), Lovastatin (lovastatin), Haloperidol (haloperidol), Prenylamine lactate (prenylamine lactic acid), Bromperidol (blomperidol), Convolamine (Convolamine) Alzheimer's disease characterized in that it contains at least one member selected from the group consisting of Min), Miglustat (Miglustat), and pharmaceutically acceptable salts thereof, or solvates thereof as an active ingredient. Prophylactic or therapeutic agent.
  3. 　下記一般式（１）で表される化合物、その薬学的に許容される塩、又はその溶媒和物を有効成分として含有することを特徴とするアルツハイマー病予防剤又は治療剤。
    Figure JPOXMLDOC01-appb-C000001
     ［一般式（１）中、Ｒ１及びＲ２は、それぞれ独立して、水素原子、炭素数１～５のアルキル基、アリール基、アラルキル基、又はシクロアルキル基を表す。Ｘは、単結合、又は２価の連結基を表す。ｎは、０、１、又は２を表す。Ｒ３は、水素原子、炭素数１～５のアルキル基、アラルキル基、アリール基、又はシクロアルキル基を表す。Ｒ４は、水素原子、又は炭素数１～５のアルキル基を表す。Ｒ５は、水素原子、水酸基、炭素数１～５のアルキル基、又は炭素数１～５のアルコキシ基を表す。Ｒ６は、水素原子、水酸基、シアノ基、炭素数１～５のアルキル基、炭素数１～５のアルコキシ基、アラルキル基、アリール基、又はシクロアルキル基を表す。］     An agent for preventing or treating Alzheimer's disease comprising the compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.
    Figure JPOXMLDOC01-appb-C000001
     [In the general formula (1), R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aryl group, an aralkyl group or a cycloalkyl group. X represents a single bond or a divalent linking group. n represents 0, 1 or 2; R 3 represents a hydrogen atom, an alkyl group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. R 4 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. R 5 represents a hydrogen atom, a hydroxyl group, an alkyl group of 1 to 5 carbon atoms, or an alkoxy group of 1 to 5 carbon atoms. R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group of 1 to 5 carbon atoms, an alkoxy group of 1 to 5 carbon atoms, an aralkyl group, an aryl group or a cycloalkyl group. ]
  4. 　請求項１～３のいずれか一項に記載のアルツハイマー病予防剤又は治療剤及び薬学的に許容される担体を含有する、アルツハイマー病予防用又は治療用組成物。 A composition for preventing or treating Alzheimer's disease, which comprises the agent for preventing or treating Alzheimer's disease according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.
  5. 　ＧＭ１ガングリオシドーシス予防剤又は治療剤のスクリーニング方法を用いることを特徴とするアルツハイマー病予防剤又は治療剤のスクリーニング方法。 A screening method for an agent for preventing or treating Alzheimer's disease, which comprises using a method for screening a preventive or therapeutic agent for GM1 gangliosidosis.
  6. 　評価対象化合物を、培養されたＧＭ１ガングリオシドーシス神経幹細胞に接触させて、前記神経幹細胞におけるＧＭ１ガングリオシドの蓄積量の変化を評価する工程を含む請求項５に記載のアルツハイマー病予防剤又は治療剤のスクリーニング方法。 The method for preventing or treating Alzheimer's disease according to claim 5, comprising the step of bringing a compound to be evaluated into contact with cultured GM1 gangliosidosis neural stem cells to evaluate a change in accumulated amount of GM1 ganglioside in the neural stem cells. Screening method.
  7. 　前記ＧＭ１ガングリオシドーシス神経幹細胞が、ＧＭ１ガングリオシドーシス患者由来ｉＰＳ細胞から分化させた細胞である請求項６に記載のアルツハイマー病予防剤又は治療剤のスクリーニング方法。 The screening method of the agent for preventing or treating Alzheimer's disease according to claim 6, wherein the GM1 gangliosidosis neural stem cells are cells differentiated from iPS cells derived from a GM1 gangliosidosis patient.
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