Preventive or therapeutic agent for Alzheimer's disease, composition for preventing or treating Alzheimer's disease, and method

Abstract

To provide a prophylactic agent or therapeutic agent for Alzheimer's disease, a screening method therefor, and a composition for preventing or treating alzheimer's disease, based on new action mechanisms.SOLUTION: A prophylactic agent or therapeutic agent for Alzheimer's disease is 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 is characterized by using a screening method for a GM1 gangliosidosis prophylactic agent or therapeutic agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a preventive or therapeutic agent for Alzheimer's disease, a screening method therefor, and a composition for preventing or treating Alzheimer's disease. This application claims priority based on Japanese Patent Application No. 2017-254887, filed on December 28, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] Alzheimer's disease (hereinafter referred to as "AD") accounts for 60-70% of dementia cases and is the disease with the highest financial cost in developed countries. The most widely accepted pathogenesis of AD is the amyloid cascade hypothesis, which begins with the accumulation of amyloid beta (hereinafter referred to as "Aβ") due to increased production or insufficient degradation.

[0003] Aβ is a small protein with a molecular weight of approximately 4 kD, consisting of 38-43 amino acids, and is produced by secretase cleavage from the amyloid precursor protein (APP). There are two major Aβ molecular species, Aβ40 and Aβ42, which differ in the number of amino acids. Of these, Aβ42 is known to have stronger pathogenicity due to its high aggregation tendency.

[0004] Various efforts have been made to develop therapeutic agents for AD (see Non-Patent Document 1). For example, Non-Patent Document 1 describes a method for screening anti-Aβ drugs using neurons that have been differentiated from human iPS cells and express a forebrain marker. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] PLoS One. 2011;6(9):e25788. doi: 10.1371 / journal.pone.0025788. Epub 2011 Sep 30. Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease. Yahata N., et al. Summary of the Invention [Problem to be solved by the invention]

[0006] To date, no fundamental cure for AD has been found, and only symptomatic treatment is available. Clinical research has been conducted on secretase inhibitors and anti-Aβ immunotherapy as fundamental treatments for AD. However, all of these have been discontinued due to insufficient efficacy and strong side effects in subjects. Therefore, to develop a fundamental treatment for AD, it is necessary to conduct drug discovery from a new perspective and target molecules.

[0007] Therefore, an object of the present invention is to provide a preventive or therapeutic agent for Alzheimer's disease, a screening method therefor, and a composition for preventing or treating Alzheimer's disease, which are based on a new mechanism of action. [Means for solving the problem]

[0008] The inventors discovered that a screening system using neural stem cells differentiated from GM1 ganglioside (hereinafter also referred to as "GM1") patient-derived iPS cells can be used in a screening method for agents for preventing or treating Alzheimer's disease, and completed the present invention. That is, the present invention is as follows. [1] A preventive or therapeutic agent for Alzheimer's disease, comprising a preventive or therapeutic agent for GM1 gangliosidosis as an active ingredient. [2] Amodiaquine, Acacetin, Sulfamerazine, Ungerine, Amiodarone, Sertindole, Delcorine, Perphenazine, Althiazide, Diethylstilbestrol, Thiethylperazine A preventive or therapeutic agent for Alzheimer's disease, characterized by containing at least one active ingredient selected from the group consisting of Perazine, Harmol, Skimmianine, Succinylsulfathiazole, Fillalbin, Canavanine, Harmaline, Trihexyphenidyl, Fluoxetine, Lovastatin, Haloperidol, Prenylamine lactate, Bromperidol, Convolamine, and Miglustat, as well as pharmaceutically acceptable salts thereof, or solvates thereof. [3] A preventive or therapeutic agent for Alzheimer's disease, comprising, as an active ingredient, a compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof:

[0009] [ka]

[0010] [In general formula (1), R 1 and R 2 each independently represents 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 3represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group. 4 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. 5 represents a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 5 carbon atoms. 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 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, comprising 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 for screening for a preventive or therapeutic agent for Alzheimer's disease, comprising using the method for screening for a preventive or therapeutic agent for GM1 gangliosidosis. [6] A method for screening an agent for preventing or treating Alzheimer's disease according to [5], comprising the step of contacting a compound to be evaluated with cultured GM1 gangliosidosis neural stem cells and evaluating changes in the amount of GM1 ganglioside accumulated in the neural stem cells. [7] The method for screening for a preventive or therapeutic agent for Alzheimer's disease according to [6], wherein the GM1 gangliosidosis neural stem cells are cells differentiated from iPS cells derived from a patient with GM1 gangliosidosis. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide an agent for preventing or treating Alzheimer's disease having a new mechanism of action. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram comparing GM1 accumulation in neural stem cells derived from healthy individuals and GM1 patients. [Figure 2](A) A diagram comparing β-gal activity in neural stem cells derived from healthy individuals, GM1 patients, and Alzheimer's disease patients. (B) A diagram comparing β-gal activity in neural stem cells derived from healthy individuals transfected with a control vector or an APP overexpression vector. (C) A diagram comparing GM1 accumulation in whole neural stem cells derived from healthy individuals and Alzheimer's disease patients. (D) A diagram comparing GM1 accumulation in lipid raft fractions of neural stem cells derived from healthy individuals and Alzheimer's disease patients. [Figure 3] (A) A graph comparing the amount of Aβ40 in neural stem cells from healthy individuals and Alzheimer's disease patients overexpressing β-gal protein. (B) A graph comparing the amount of Aβ42 in neural stem cells from healthy individuals and Alzheimer's disease patients overexpressing β-gal protein. (C) A graph comparing the Aβ42 / Aβ40 ratio in neural stem cells from healthy individuals and Alzheimer's disease patients overexpressing β-gal protein. (D) A graph comparing the total amount of Aβ in neural stem cells from healthy individuals and Alzheimer's disease patients overexpressing β-gal protein. (E) A graph comparing the sensitivity to Aβ42 in neural stem cells from healthy individuals and neural stem cells from patients with GM1 gangliosidosis. [Figure 4](A) A comparison of the quantification results of soluble Aβ40 in TBS (Tris-buffered saline) extract fractions from the brains of WT, BKO, 5×FAD, and BKO / 5×FAD mice. (B) A comparison of the quantification results of soluble Aβ42 in TBS (Tris-buffered saline) extract fractions from the brains of WT, BKO, 5×FAD, and BKO / 5×FAD mice. (C) A comparison of the quantification results of insoluble Aβ40 in guanidin-HCl extract fractions from the brains of WT, BKO, 5×FAD, and BKO / 5×FAD mice. (D) A comparison of the quantification results of insoluble Aβ42 in guanidin-HCl extract fractions from the brains of WT, BKO, 5×FAD, and BKO / 5×FAD mice. (E) A graph comparing the amount of amyloid deposition in brain sections from WT mice, BKO mice, 5×FAD mice, and BKO / 5×FAD mice. [Figure 5] FIG. 1 shows the state of GM1 accumulation when a GM1-suppressing compound is used. [Figure 6] FIG. 1 shows the state of GM1 accumulation when a GM1-suppressing compound is used. [Figure 7] FIG. 1 shows the results of comparing the amount of Aβ and the Aβ42 / Aβ40 ratio in the medium when a GM1-inhibiting compound was used. [Figure 8] FIG. 1 shows the results of comparing the amount of Aβ produced in cells and the Aβ42 / Aβ40 ratio when a GM1 inhibitory compound was used. [Figure 9] FIG. 1 shows the effect of intraperitoneal administration of a GM1-suppressing compound on GM1 accumulation in brain slices from GM1 model mice. [Figure 10] FIG. 1 shows the results of comparing the amount of GM1 ganglioside in the brain of GM1 model mice when a GM1-inhibitory compound was administered intraperitoneally. [Figure 11](A) A comparison of the quantitative results of soluble Aβ40 in TBS (Tris Buffered Saline) extract fractions from the brains of 5×FAD mice administered PBS, amodiaquine, and thiethylperanzine. (B) A comparison of the quantitative results of soluble Aβ42 in TBS (Tris Buffered Saline) extract fractions from the brains of 5×FAD mice administered PBS, amodiaquine, and thiethylperanzine. (C) A comparison of the quantitative results of insoluble Aβ40 in guanidin-HCl extract fractions from the brains of 5×FAD mice administered PBS, amodiaquine, and thiethylperanzine. (D) A comparison of the quantitative results of insoluble Aβ42 in guanidin-HCl extract fractions from the brains of 5×FAD mice administered PBS, amodiaquine, and thiethylperanzine. (E) A graph comparing the amount of amyloid deposition in brain sections from 5×FAD mice administered PBS, amodiaquine, and thiethylperanzine. [Figure 12] FIG. 1 shows the synthesis and degradation processes of gangliosides. [Figure 13] This shows the results of an analysis of the expression levels of each enzyme gene in neural stem cells obtained by differentiation from normal (201B7) and disease-derived (A138 #1-3) iPS cells to which amodiaquine or thiethylperanzine was added. DETAILED DESCRIPTION OF THE INVENTION

[0013] [Method for screening preventive or therapeutic agents for Alzheimer's disease] In recent years, it has become clear that GM1 ganglioside, present in lipid rafts on the surface of neuronal membranes in AD patients, promotes the polymerization of Aβ in AD and is deeply involved in the pathogenesis of the disease (see Hoshino T, Mahmood MI, Mori K, Matsuzaki K. J Phys Chem B. 2013 Jul 11;117(27):8085-94.).

[0014] GM1 ganglioside is a type of glycolipid and a molecule involved in cell signaling, etc. GM1 gangliosidosis is known as a disease in which GM1 is involved.

[0015] GM1 gangliosidosis is a lysosomal storage disorder, a congenital metabolic disorder in which GM1 accumulates, particularly in the nervous system (brain), due to a deficiency or abnormality of the enzyme involved in the hydrolysis of GM1 ganglioside. GM1 gangliosidosis is classified into infantile, juvenile, and adult forms depending on the time of onset and clinical course. In particular, infantile forms show developmental delays by 3-6 months of age, and most patients develop severe neurological disorders such as decerebrate posturing by the time they are one year old, and usually die by the age of 3-4 years.

[0016] As described below in the Examples, the inventors have discovered that there is a relationship between GM1 gangliosidosis and Alzheimer's disease. That is, the screening method for a preventive or therapeutic agent for Alzheimer's disease of the present invention uses a screening method for a preventive or therapeutic agent for GM1 gangliosidosis.

[0017] (First embodiment) In one embodiment, the present invention provides a method for screening for an agent for preventing or treating Alzheimer's disease, comprising the steps of contacting a compound to be evaluated with cultured GM1 gangliosidosis neural stem cells and evaluating changes in the amount of GM1 ganglioside accumulated in the neural stem cells.

[0018] In this embodiment, for example, a compound library is added to the medium of cultured neural stem cells with GM1 gangliosidosis to examine the effect on GM1 ganglioside in the neural stem cells. More specifically, for example, the neural stem cells are seeded on a well plate and cultured in the presence of the compound library for approximately 1 to 5 days.

[0019] Because it is difficult to collect neural cells from the brain of a patient, the GM1 gangliosidosis neural stem cells are preferably cells differentiated from iPS cells derived from a patient with GM1 gangliosidosis. Cells derived from GM1 patients include adipocytes, chondrocytes, osteoblasts, blood cells, fibroblasts, etc., with fibroblasts being preferred. Methods for reprogramming GM1 patient-derived cells into iPS cells and methods for differentiating iPS cells into neural stem cells follow standard methods.

[0020] As a method for evaluating changes in the amount of GM1 ganglioside accumulation, as described below in the Examples, a qualitative and quantitative evaluation method using a substance that has affinity for GM1 ganglioside, such as flow cytometry or Western blotting, can be used.

[0021] The method may further include a step of evaluating the therapeutic effect of the compound found by the above step on AD, such as evaluating Aβ expression. Aβ expression can be evaluated by culturing neural stem cells derived from AD patients together with a GM1-inhibiting compound and quantifying the ratio of Aβ42 to Aβ40 in the culture medium.

[0022] (Second embodiment) In one embodiment, the present invention provides a method for screening for a preventive or therapeutic agent for Alzheimer's disease, which comprises administering a compound to be evaluated to an Alzheimer's disease model animal described below.

[0023] In this embodiment, for example, a compound to be evaluated is administered parenterally, such as orally or intraperitoneally, to an Alzheimer's disease model animal, which will be described later, and then amyloid deposition in brain tissue is evaluated.

[0024] The preventive or therapeutic agent for Alzheimer's disease obtained using the screening method of the present invention is preferably a compound that can exert a therapeutic effect for AD by directly or indirectly suppressing GM1 accumulation. That is, the therapeutic agent for AD that contains such a candidate compound for treating AD as an active ingredient preferably exerts a therapeutic effect for AD by directly or indirectly suppressing GM1 accumulation through the action of directly removing accumulated GM1, suppressing GM1 aggregation, inhibiting GM1 synthesis, promoting GM1 degradation, or the like.

[0025] [Alzheimer's disease preventive or therapeutic agent] In one embodiment, the present invention provides a steroid drug, such as Amodiaquine, Acacetin, Sulfamerazine, Ungerine, Amiodarone, Sertindole, Delcorine, Perphenazine, Althiazide, Diethylstilbestrol, Thiethylperazin Provided is an agent for preventing or treating Alzheimer's disease, which contains, as an active ingredient, at least one member selected from the group consisting of Thiethylperazine, Harmol, Skimmianine, Succinylsulfathiazole, Fillalbin, Canavanine, Harmaline, Trihexyphenidyl, Fluoxetine, Lovastatin, Haloperidol, Prenylamine lactate, Bromperidol, Convolamine, and Miglustat, as well as pharmaceutically acceptable salts thereof, or solvates thereof. These preventive or therapeutic agents for Alzheimer's disease can also be provided as preventive or therapeutic agents for GM1 gangliosidosis.

[0026] In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, comprising, as an active ingredient, a compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof: This agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.

[0027] [ka]

[0028] [In general formula (1), R 1 and R 2 each independently represents 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 having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group. 4 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. 5 represents a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 5 carbon atoms. 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group.]

[0029] R 1 and R 2 Specific examples of the alkyl group having 1 to 5 carbon atoms in the formula (I) include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, and a neopentyl group. R 1 and R 2The aryl group in the formula (I) is preferably one having 6 to 18 carbon atoms, more preferably one having 6 to 10 carbon atoms, and specifically, a phenyl group is particularly preferred. R 1 and R 2 The aralkyl group in R 1 and R 2 In the above, the aryl group is preferably bonded to the aryl group. R 1 and R 2 The cycloalkyl group in the formula (I) 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, and cyclooctane.

[0030] Examples of the divalent linking group for X include an alkylene group, -O-, -C(=O)-, -NH-, -S-, -S(=O)2-, or a combination thereof.

[0031] R 3 In the above, the alkyl group, aralkyl group, aryl group, and cycloalkyl group having 1 to 5 carbon atoms are R 1 and R 2 Examples of the above-mentioned examples include: R 4 As the alkyl group having 1 to 5 carbon atoms in R 1 and R 2 Examples of the above-mentioned examples include:

[0032] R 5 As the alkyl group having 1 to 5 carbon atoms in R 1 and R 2 Examples of the above-mentioned examples include: R 5 As the alkoxy group having 1 to 5 carbon atoms, the R part of -OR is R 1 and R 2 Examples of the alkyl group include the same alkyl groups having 1 to 5 carbon atoms as those mentioned above.

[0033] R 6In the formula, the alkyl group, aralkyl group, aryl group, and cycloalkyl group having 1 to 5 carbon atoms are R 1 and R 2 Examples of the above-mentioned examples include: R 6 As the alkoxy group having 1 to 5 carbon atoms in R 5 Examples of the above-mentioned examples include:

[0034] Among the above, X is preferably a single bond, n is preferably 1, and R 3 ~R 5 is preferably a hydrogen atom.

[0035] The preventive or therapeutic agent for Alzheimer's disease of this embodiment preferably contains, as an active ingredient, a compound represented by the following general formula (1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0036] [ka]

[0037] [In general formula (1-1), R 1 and R 2 each independently represents 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. R 6 represents a hydrogen atom, a hydroxyl group, a cyano group, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group.]

[0038] Among the above, R 6 is preferably a hydroxyl group, and R 1 and R 2 As the alkyl group, an alkyl group having 1 to 5 carbon atoms is preferred.

[0039] It is particularly preferable that the Alzheimer's disease preventive or therapeutic agent of this embodiment contains, as an active ingredient, a compound represented by the following formula (1-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0040] [ka]

[0041] In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, comprising, as an active ingredient, a compound represented by the following general formula (2), a pharmaceutically acceptable salt thereof, or a solvate thereof: This agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.

[0042] [ka]

[0043] [In general formula (2), R 10 represents a single bond or a divalent linking group. 11 represents a hydrogen atom, an alkyl 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, an aralkyl group, an aryl group, or a cycloalkyl group. 12 and R 13 each independently 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, a hydroxyalkyl group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group. 10 and n 11 R each independently represents an integer of 0 to 4. 12 and R 13 When there are multiple of each, they may be the same or different. 14 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, or an alkoxy group having 1 to 5 carbon atoms. n 12represents an integer from 0 to 4. 14 When there are multiple of them, they may be the same or different.

[0044] R 10 Examples of the divalent linking group in include the same groups as those described above for X. R 11 In the above, examples of the alkyl group having 1 to 5 carbon atoms, the alkoxy group having 1 to 5 carbon atoms, the aralkyl group, the aryl group, and the cycloalkyl group include R 5 Examples of the above-mentioned examples include: R 11 In the case of the hydroxyalkyl group having 1 to 5 carbon atoms, the alkyl group is 1 and R 2 Examples of the above-mentioned examples include: R 12 and R 13 In the above, examples of the alkyl group having 1 to 5 carbon atoms, the alkoxy group having 1 to 5 carbon atoms, the hydroxyalkyl group having 1 to 5 carbon atoms, the aralkyl group, the aryl group, and the cycloalkyl group include R 11 Examples of the above-mentioned examples include: R 12 and R 13 Examples of the halogen atom in the formula include a fluorine atom, a chlorine atom, and a bromine atom. R 12 and R 13 In the case of the thioalkyl group having 1 to 5 carbon atoms, the alkyl group portion is R 1 and R 2 Examples of the above-mentioned examples include: R 14 In the formula (I), examples of the halogen atom, 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 include R 12 and R 13 Examples of the above-mentioned examples include:

[0045] Among the above, R 13 and R 14 is preferably a hydrogen atom, and n 10is preferably 1.

[0046] The preventive or therapeutic agent for Alzheimer's disease of this embodiment preferably contains, as an active ingredient, a compound represented by the following general formula (2-1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0047] [ka]

[0048] [In general formula (2-1), R 10 represents a single bond or a divalent linking group. 11 represents a hydrogen atom, an alkyl 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, an aralkyl group, an aryl group, or a cycloalkyl group. 12 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, a hydroxyalkyl group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group.]

[0049] Among the above, R 10 As R, an alkylene group is preferred. 10 The alkylene group in the formula (I) is preferably a group in which one hydrogen atom has been removed from a linear or branched alkyl group having 1 to 5 carbon atoms. As the alkyl group having 1 to 5 carbon atoms, R 1 and R 2 Examples of the above-mentioned examples include:

[0050] It is more preferable that the Alzheimer's disease preventive or therapeutic agent of this embodiment contains, as an active ingredient, a compound represented by the following general formula (2-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0051] [ka]

[0052] [In general formula (2-1-1), R 11 represents a hydrogen atom, an alkyl 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, an aralkyl group, an aryl group, or a cycloalkyl group. 12 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, a hydroxyalkyl group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group.]

[0053] Among the above, R 11 is preferably an alkyl group having 1 to 5 carbon atoms or a hydroxyalkyl group having 1 to 5 carbon atoms. 12 is preferably a halogen atom or a thioalkyl group having 1 to 5 carbon atoms.

[0054] It is particularly preferable that the Alzheimer's disease preventive or therapeutic agent of this embodiment contains, as an active ingredient, a compound represented by the following formula (2-1-1-1) or (2-1-1-2), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0055] [ka]

[0056] In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, comprising, as an active ingredient, a compound represented by the following general formula (3), a pharmaceutically acceptable salt thereof, or a solvate thereof: This agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.

[0057] [ka]

[0058] [In general formula (3), R 20represents 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, a hydroxyalkyl group having 1 to 5 carbon atoms, an aralkyl group, an aryl group, or a cycloalkyl group. 21 and R 24 R each independently represents a single bond or a divalent linking group. 22 and R 23 each independently represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms, a haloalkyl 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, or a hydroxyalkyl group having 1 to 5 carbon atoms.]

[0059] R 20 In the above, examples of the halogen atom, the alkyl group having 1 to 5 carbon atoms, the thioalkyl group having 1 to 5 carbon atoms, the alkoxy group having 1 to 5 carbon atoms, the hydroxyalkyl group having 1 to 5 carbon atoms, the aralkyl group, the aryl group, and the cycloalkyl group include R 12 Examples of the above-mentioned examples include: R 21 and R 24 Examples of the divalent linking group in include the same groups as those described above for X. R 22 and R 23 In the above, examples of the halogen atom, the alkyl group having 1 to 5 carbon atoms, the thioalkyl group having 1 to 5 carbon atoms, the alkoxy group having 1 to 5 carbon atoms, and the hydroxyalkyl group having 1 to 5 carbon atoms include R 12 Examples of the above-mentioned examples include: R 22 and R 23 In the formula, examples of the haloalkyl group having 1 to 5 carbon atoms include alkyl fluoride, alkyl chloride, and alkyl bromide. 1 and R 2 Examples of the alkyl groups include those in which at least one hydrogen atom has been replaced with a halogen atom.

[0060] Among the above, R 20 R is preferably an alkyl group having 1 to 5 carbon atoms or an aralkyl group.20 The aralkyl group in the formula (I) is preferably an aralkyl group in which an alkylene group having 1 to 5 carbon atoms is bonded to a phenyl group. R 21 and R 24 is preferably a single bond or —O—. R 22 and R 23 is preferably a hydrogen atom or a trifluoromethyl group.

[0061] It is particularly preferable that the Alzheimer's disease preventive or therapeutic agent of this embodiment contains, as an active ingredient, a compound represented by the following formula (3-1) or (3-2), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0062] [ka]

[0063] In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, comprising, as an active ingredient, a compound represented by the following general formula (4), a pharmaceutically acceptable salt thereof, or a solvate thereof: This agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.

[0064] [ka]

[0065] [In general formula (4), R 30 and R 31 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, 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, or an aralkyl group. The double line consisting of a solid line and a dotted line represents a single bond or a double bond.]

[0066] R 30 and R 31In the above, examples of the halogen atom, the alkyl group having 1 to 5 carbon atoms, the thioalkyl group having 1 to 5 carbon atoms, the alkoxy group having 1 to 5 carbon atoms, the hydroxyalkyl group having 1 to 5 carbon atoms, and the aralkyl group include R 12 Examples of the above-mentioned examples include:

[0067] Among the above, R 30 and R 31 As the alkyl group, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, and a hydroxyl group are preferred.

[0068] It is particularly preferable that the Alzheimer's disease preventive or therapeutic agent of this embodiment contains, as an active ingredient, a compound represented by the following formula (4-1) or (4-2), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0069] [ka]

[0070] In one embodiment, the present invention provides an agent for preventing or treating Alzheimer's disease, comprising, as an active ingredient, a compound represented by the following general formula (5), a pharmaceutically acceptable salt thereof, or a solvate thereof: This agent for preventing or treating Alzheimer's disease can also be provided as an agent for preventing or treating GM1 gangliosidosis.

[0071] [ka]

[0072] [In general formula (5), R 40 represents a single bond or a divalent linking group. 41 ~R 43 each independently represents 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, a hydroxyalkyl group having 1 to 5 carbon atoms, or an aralkyl group.

[0073] R 40 Examples of the divalent linking group in include the same groups as those described above for X. R 41 ~R 43 In the formula (I), examples of the halogen atom, the alkyl group having 1 to 5 carbon atoms, the thioalkyl group having 1 to 5 carbon atoms, the alkoxy group having 1 to 5 carbon atoms, the hydroxyalkyl group having 1 to 5 carbon atoms, and the aralkyl group include R 12 Examples of the above-mentioned examples include:

[0074] Among the above, R 40 As R, an alkylene group is preferred. 40 The alkylene group in is preferably a group in which one hydrogen atom has been removed from a linear or branched alkyl group having 1 to 5 carbon atoms. Examples of the linear or branched alkyl group having 1 to 5 carbon atoms include R 1 and R 2 Examples of the above-mentioned examples include: R 41 ~R 43 is preferably a hydrogen atom or a halogen atom.

[0075] It is particularly preferable that the Alzheimer's disease preventive or therapeutic agent of this embodiment contains, as an active ingredient, a compound represented by the following formula (5-1) or (5-2), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0076] [ka]

[0077] 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 a pharmaceutically acceptable salt, or may be used in the form of a solvate of the free form or a solvate of the salt.

[0078] The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include hydrochloride, sulfate, hydrobromide, hydroiodide, phosphate, nitrate, benzoate, methanesulfonate, 2-hydroxyethanesulfonate, p-toluenesulfonate, acetate, propanoate, oxalate, malonate, succinate, glutarate, adipate, tartrate, maleate, fumarate, malate, mandelate, etc. The solvate is not particularly limited as long as it is a pharmaceutically acceptable solvate, and examples thereof include hydrates, organic solvents, etc.

[0079] [Composition for preventing or treating Alzheimer's disease] In one embodiment, the present invention provides a composition for preventing or treating Alzheimer's disease, comprising the above-mentioned agent for preventing or treating Alzheimer's disease and a pharmaceutically acceptable carrier. This composition for preventing or treating Alzheimer's disease can also be provided as a composition for preventing or treating GM1 gangliosidosis.

[0080] The composition for preventing or treating Alzheimer's disease of this embodiment can be administered orally in the form of, for example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., or parenterally in the form of injections, suppositories, topical preparations for skin, etc.

[0081] As the pharmaceutically acceptable carrier, those usually used in the preparation of pharmaceutical compositions can be used without any particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, etc.; excipients such as starch, crystalline cellulose, etc.; swelling agents such as alginic acid, etc.; solvents for injections such as water, ethanol, glycerin, etc.; adhesives such as rubber-based adhesives, silicone-based adhesives, etc. The pharmaceutically acceptable carriers can be used alone or in combination of two or more.

[0082] The composition for preventing or treating Alzheimer's disease of this embodiment may further contain additives, such as lubricants such as calcium stearate and magnesium stearate, sweeteners such as sucrose, lactose, saccharin, and maltitol, flavorings such as peppermint and rhizome oil, stabilizers such as benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, solubilizers such as benzyl benzoate and benzyl alcohol, antioxidants, and preservatives. The additives can be used alone or in combination of two or more.

[0083] (Administration method) The method of administration of an agent for preventing or treating Alzheimer's disease or a composition for preventing or treating Alzheimer's disease is not particularly limited and may be determined appropriately depending on the patient's symptoms, body weight, age, sex, etc. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc. are administered orally. Injections are administered intravenously alone or mixed with common replacement fluids such as glucose and amino acids, and may also be administered intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed. Suppositories are administered rectally. External skin preparations are applied, pasted, or sprayed onto the affected area.

[0084] (Dosage) The dosage of an agent for preventing or treating Alzheimer's disease or a composition for preventing or treating Alzheimer's disease varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined in general. However, for oral administration, for example, 1 μg to 10 g of active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. For injections, for example, 0.1 μg to 1 g of active ingredient may be administered per day, for example, 0.001 to 200 mg per day. For suppositories, for example, 1 μg to 10 g of active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. For topical skin preparations, for example, 1 μg to 10 g of active ingredient may be administered per day, for example, 0.01 to 2000 mg per day.

[0085] [Other embodiments] In one embodiment, the present invention provides a pharmaceutical composition comprising Amodiaquine, Acacetin, Sulfamerazine, Ungerine, Amiodarone, Sertindole, Delcorine, Perphenazine, Althiazide, Diethylstilbestrol, Thiet for the prevention or treatment of Alzheimer's disease. Provided is a compound selected from the group consisting of hylperazine, Harmol, Skimmianine, Succinylsulfathiazole, Fillalbin, Canavanine, Harmaline, Trihexyphenidyl, Fluoxetine, Lovastatin, Haloperidol, Prenylamine lactate, Bromperidol, Convolamine, Miglustat, and compounds represented by the general formulae (1) to (5), or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0086] In one embodiment, the present invention provides a compound selected from the group consisting of Amodiaquine, Acacetin, Sulfamerazine, Ungerine, Amiodarone, Sertindole, Delcorine, Perphenazine, Althiazide, Diethylstilbestrol, Thiethylperazin The present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to a patient in need thereof an effective amount of a compound selected from the group consisting of Thiethylperazine, Harmol, Skimmianine, Succinylsulfathiazole, Fillalbin, Canavanine, Harmaline, Trihexyphenidyl, Fluoxetine, Lovastatin, Haloperidol, Prenylamine lactate, Bromperidol, Convolamine, Miglustat, and the compounds represented by the general formulae (1) to (5), or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0087] In one embodiment, the present invention provides a method for producing a preventive or therapeutic agent for Alzheimer's disease, or a composition for the prevention or treatment of Alzheimer's disease, comprising administering to a subject a compound selected from the group consisting of Amodiaquine, Acacetin, Sulfamerazine, Ungerine, Amiodarone, Sertindole, Delcorine, Perphenazine, Althiazide, Diethylstilbestrol, and the like. The present invention provides use of a compound selected from the group consisting of Rubestrol, Thiethylperazine, Harmol, Skimmianine, Succinylsulfathiazole, Fillalbin, Canavanine, Harmaline, Trihexyphenidyl, Fluoxetine, Lovastatin, Haloperidol, Prenylamine lactate, Bromperidol, Convolamine, Miglustat, and compounds represented by the general formulas (1) to (5), or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0088] [Alzheimer's disease model animal] The Alzheimer's disease model animal used in the evaluation of the present invention is a non-human mammal in which the expression of the β-galactosidase (hereinafter also referred to as β-gal) gene is suppressed or lost, or the function of the β-gal protein encoded by the β-gal gene is suppressed or lost, and a mutation has been introduced into the β-gal gene or the expression regulatory region of the β-gal gene.

[0089] β-gal protein is an enzyme present in lysosomes that plays a role in the hydrolysis of various biomolecules. GM1 ganglioside is a substrate of β-gal protein and is abundant in the brain. Therefore, inhibition or loss of β-gal leads to the accumulation of GM1 ganglioside.

[0090] "Loss of β-gal protein function" refers to a state in which the original function of β-gal protein is completely lost, and "repressed β-gal protein function" refers to a state in which the original function of β-gal protein is partially lost. When the original function of β-gal protein is partially lost, the degree of inhibition of β-gal protein function should be such that, when comparing the model animal with a control animal such as a wild-type animal, the model animal exhibits a condition recognizable as Alzheimer's disease (a difference from the control). Indicators of a condition recognized as Alzheimer's disease include deposition of amyloid proteins (Aβ40 and 42) in the brain.

[0091] Suppression or loss of β-gal protein function can also occur through suppression or loss of β-gal gene expression. Loss of β-gal gene expression refers to the loss of the β-gal gene product in a model animal. Suppression of β-gal gene expression refers to the suppression of the amount of β-gal gene product in a model animal compared to a control animal, such as a wild-type animal. When the amount of the β-gal gene product is suppressed, the degree of suppression may be such that the model animal exhibits a condition recognizable as Alzheimer's disease as described above, compared with a control animal such as a wild-type animal. β-gal gene expression can be suppressed by gene knockdown or the like by introducing into the model animal a nucleic acid sequence that induces expression of an RNAi-inducing nucleic acid, antisense nucleic acid, aptamer, ribozyme, or the like against the β-gal gene.

[0092] The model animals used in the evaluation of the present invention are preferably those into which a mutation has been introduced into the β-gal gene or the expression regulatory region of the β-gal gene. The mutation to be introduced may be any mutation that suppresses or eliminates the expression of the β-gal gene, or that suppresses or eliminates the function of the β-gal protein encoded by the β-gal gene.

[0093] The above-mentioned β-gal gene includes not only exons, which are the regions that encode the β-gal protein, but also introns if they can suppress or eliminate the expression of the β-gal gene or suppress or eliminate the function of the β-gal protein encoded by the β-gal gene. 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, silencer, enhancer, or response element.

[0094] Loss of β-gal protein function can be achieved, for example, by introducing a mutation into the β-gal gene to disrupt the β-gal gene, while suppression or loss of β-gal gene expression can be achieved, for example, by introducing a mutation into the expression regulatory region of the β-gal gene. Mutations can be introduced into the β-gal gene or the expression regulatory region of the β-gal gene by gene modification, a known genetic engineering technique. Mutations can be generated by partial or complete deletion, substitution, or insertion of any sequence in the β-gal gene or the expression regulatory region of the β-gal gene. When a portion of the β-gal gene is deleted, the deletion is preferably one or more exons. These mutations can be introduced using techniques such as treatment with mutagen (Mutagen), ultraviolet irradiation, gene targeting using homologous recombination techniques, gene knockout, gene knockdown, and conditional knockout using the Cre-loxP system.

[0095] Examples of β-gal gene mutations in human β-gal protein include E186A, P10L, R201C, C127Y, W161G, Q255H, R351X, S532G, and R148S.

[0096] As will be shown in the Examples below, it has been confirmed that amyloid deposition is accelerated in mice that are a cross between β-gal knockout mice and Alzheimer's disease model mice. Therefore, it is preferable that the Alzheimer's disease model animal used in the evaluation of the present invention further has at least one mutation selected from the group consisting of a deletion of exon 9 in human presenilin 1 (PS1), an M146L mutation, an L285V mutation, an N141I mutation in human PS2, a KM670 / 671NL Swedish mutation, an I716V Florida mutation, and a V717I London mutation in human amyloid precursor protein (APP), and it is more preferable that the animal has five mutations: an M146L mutation and an L285V mutation in human presenilin 1 (PS1), and a KM670 / 671NL Swedish mutation, an I716V Florida mutation, and a V717I London mutation in human amyloid precursor protein (APP).

[0097] The non-human mammal is not particularly limited, but is preferably an animal classified as a rodent, including a mouse, rat, guinea pig, hamster, rabbit, goat, pig, dog, and cat.

[0098] The model animals used in the evaluation of the present invention can be used to elucidate the mechanism of Alzheimer's disease, to search for substances useful for its treatment, and to develop treatment methods. [Example]

[0099] The present invention will be explained in more detail below by way of experimental examples, but the present invention is not limited to these examples.

[0100] [Experimental Example 1] [Establishment of skin-derived fibroblasts] Fibroblasts were established from skin biopsy explants from patients with GM1 gangliosidosis and healthy controls under an ethical committee-approved protocol and with informed consent. Skin samples from patients and healthy volunteers were minced and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). After confirming the appearance of fibroblasts, the fibroblasts were expanded to introduce reprogramming genes.

[0101] [Establishment and maintenance of iPS cells] iPS cells were established from the established human fibroblasts using the method described in N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, M. Hasegawa, Proc. Jpn. Acad. Ser., B. Phys. Biol. Eci., 85, 348 (2009). Specifically, 1 day before infection, 5 × 10 cells were cultured per well in a 6-well plate. 5 Human fibroblasts were seeded onto the cells and then infected with a Sendai virus (SeV) vector containing the Oct3 / 4, Sox2, K1f4, and c-Myc genes at a multiplicity of infection (MOI) of 3. The SeV vector containing the Oct3 / 4, Sox2, K1f4, and c-Myc genes was prepared according to the method described in N. Fusaki, H. Ban, A. Nishiyama, K. Saeki, and M. Hasegawa, Proc. Jpn. Acad. Ser., B. Phys. Biol. Eci., 85, 348 (2009). Seven days after infection, the infected fibroblasts were harvested using trypsin and plated at 5.4 × 10 cells per 60-mm dish. 4 cells, or 1-2 x 10 per 100 mm dish 5 The cells were seeded onto MMC-treated MEF feeder cells. The next day, the medium was replaced with human iPS cell medium, and the culture was continued for 30 days after infection, and colonies were collected.

[0102] 20% Knockout™ Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine, 1×10 -4 M non-essential amino acids (NEAA, Sigma), 1×10 -4 M 2-mercaptoethanol (Sigma), 0.5% penicillin and streptomycin (Nacalai Tesque, Japan), and 5 ng / mL basic fibroblast growth factor (bFGF, Wako, Japan) were added to DMEM / F12 (Sigma) to contain a human iPS medium, and human iPS cells were maintained on mitomycin C (MMC)-treated MEF feeder cells.

[0103] [Differentiation into Neural Stem Cells (Neural Stem Cell: NSC)] One day before the start of differentiation induction, human iPS cells were seeded at a density of 2.5×10 5 ~3×10 5 cells / well on a 6-well plate coated with Geltrex (Thermo Fisher Scientific) and cultured in human iPS medium. The next day (the start day of induction: Day 0), the human iPS medium was removed and replaced with PSC Neural Induction Medium (Thermo Fisher Scientific). Subsequently, the medium was changed with PSC Neural Induction Medium once every two days, and the cells were cultured until Day 7 after the start of culture to induce NSC. The induced NSC was detached using StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific) except for the medium, suspended in Neural Expansion Medium (Thermo Fisher Scientific), and seeded onto a 100 mm cell culture dish coated with Geltrex. The cells at this point were designated as P0 NSC (NSC at passage 0), and thereafter, NSC was appropriately expanded in this medium and used for experiments.

[0104] [Experimental Example 2] [Visualization of GM1 Accumulation Using FITC-CTB] Neural stem cells induced from iPS cells derived from healthy individuals and patients with GM1 gangliosidosis were fixed with 4% paraformaldehyde, permeabilized with 0.1% TritonX-100 / PBS, and blocked with 1% BSA solution. Subsequently, multiple staining was performed using FITC-CTB (a reagent that specifically stains GM1 ganglioside) and anti-nestin antibody (a molecule specifically expressed in neural stem cells: a neural stem cell marker). The results are shown in Figure 1. Nestin staining showed strong staining in NSCs from both healthy individuals and GM patients, confirming their differentiation into neural stem cells (Figure 1, bottom).On the other hand, FITC-CTB staining was barely observed in healthy individuals, but strong staining was observed in GM1 patients (Figure 1, top). These results confirmed that GM1 accumulates in neural stem cells derived from GM1 patients.

[0105] [Experimental Example 3] It has been reported that the amount of GM1 ganglioside in the brains of Alzheimer's disease patients is higher than that in the brains of healthy individuals. The inventors investigated the metabolic state of GM1 ganglioside in neural stem cells derived from Alzheimer's disease patients. First, we examined the β-gal activity of neural stem cells derived from patients and confirmed that the β-gal activity of neural stem cells derived from Alzheimer's disease patients was reduced compared to that of neural stem cells derived from healthy individuals (see Figure 2A). In Figure 2A, 201B7 and 409B2 represent neural stem cells derived from healthy individuals, A138 and A154 represent neural stem cells derived from GM1 gangliosidosis patients, and A232#3-1 and A232#2-2 represent neural stem cells derived from Alzheimer's disease patients. In Figure 2A, Sol represents the insoluble fraction, and Sup represents the soluble fraction. This experiment shows that there is no difference in β-gal activity between the insoluble and soluble fractions.

[0106] Furthermore, the inventors investigated the effect of APP overexpression on β-gal activity. A control vector or an APP overexpression vector was transfected into neural stem cells derived from healthy individuals, and β-gal activity was measured. It was confirmed that β-gal activity was reduced in neural stem cells transfected with the APP overexpression vector compared to neural stem cells transfected with the control vector (see Figure 2B).

[0107] Furthermore, the inventors investigated the accumulation of GM1 ganglioside in patient-derived neural stem cells (see Figures 2C and 2D). Figure 2C is a graph showing the amount of GM1 ganglioside in the whole cell, and Figure 2D is a graph showing the amount of GM1 ganglioside in the lipid raft fraction. The amount of GM1 ganglioside in neural stem cells derived from Alzheimer's disease was confirmed to be higher than that in neural stem cells derived from healthy individuals. The difference was particularly large in the lipid raft fraction. Given that β-gal is known to be involved in the degradation of GM1 ganglioside, these findings suggest that β-gal activity is reduced in neural stem cells derived from Alzheimer's disease, leading to the accumulation of GM1 ganglioside.

[0108] [Experimental Example 4] To determine whether the reduction in GM1 ganglioside affects Aβ metabolism, we overexpressed β-gal protein in neural stem cells (see Figures 3A-D). In Figure 3, "Control" indicates neural stem cells transfected with a control vector, and "GLB1 OE" indicates neural stem cells transfected with a β-gal protein overexpression vector. As shown in Figure 3B, overexpression of β-gal protein in Alzheimer's disease-derived neural stem cells reduced the amount of Aβ42.

[0109] It has been reported that treatment with Aβ42 induces cell death. Therefore, the inventors compared the sensitivity of neural stem cells derived from healthy individuals and those derived from patients with GM1 gangliosidosis to Aβ42 (see Figure 3E). In the legend of Figure 3E, 201B7NSC indicates neural stem cells derived from healthy individuals, A138NSC indicates neural stem cells derived from patients with GM1 gangliosidosis, and 201B7+GM1 indicates neural stem cells derived from healthy individuals treated with GM1 ganglioside. Cell viability assays confirmed that neural stem cells derived from patients with GM1 gangliosidosis were more sensitive to Aβ42 than neural stem cells derived from healthy individuals. Furthermore, the sensitivity of neural stem cells derived from healthy individuals treated with GM1 ganglioside to Aβ42 was similar to that of neural stem cells derived from patients with GM1 gangliosidosis. These findings suggest that GM1 ganglioside affects Aβ production in neural stem cells.

[0110] [Experimental Example 5] After birth, Aβ was extracted from the brains of GM1 gangliosidosis model mice (BKO mice: β-gal-deficient mice, β-gal(- / -)) on a C57BL / 6NCr Slc background and quantified (see Figure 4A–D). In Figure 4, WT represents a normal mouse, BKO represents the GM1 gangliosidosis model mouse described above, 5×FAD represents an Alzheimer's disease model mouse harboring five mutations in human presenilin 1 (PS1): M146L and L285V, and in human amyloid precursor protein (APP): KM670 / 671NL Swedish, I716V Florida, and V717I London. BKO / 5×FAD represents a cross between BKO and 5×FAD. 4A and 4B show the results of quantification of soluble Aβ in the TBS (Tris Buffered Saline) extracted fraction, and FIGS. 4C and 4D show the results of quantification of insoluble Aβ in the guanidin-HCl extracted fraction. Compared to WT, BKO mice showed a tendency to increase Aβ40 and 42. Furthermore, in the guanidin-HCl extract fraction, Aβ40 and 42 were significantly increased in BKO / 5×FAD compared to 5×FAD, confirming that crossbreeding BKO mice with 5×FAD promotes amyloid deposition.

[0111] After birth, brain sections were prepared from C57BL / 6NCr Slc mice (WT, BKO, 5xFAD, and BKO / 5xFAD). The sections were immunostained with anti-Aβ antibody (clone: ​​4G8). The results are shown in Figure 4E. Similar to the results in Figures 4A-D, the order of amyloid deposition was confirmed to be BKO / 5xFAD, 5xFAD, BKO, and WT.

[0112] [Experimental Example 6] <Evaluation of compounds through drug screening using an imaging cytometer> Screening for therapeutic agents for Alzheimer's disease was carried out as follows. After coating a 96-well plate with Geltrex, 5 × 10 neural stem cells induced from iPS cells derived from healthy individuals and GM1 gangliosidosis patients suspended in Neural Expansion Medium were added to the plate. 4 Cells were seeded at a density of 1000 / well. Compounds from the known drug library were then added to each well to a final concentration of 5 μM, and the cells were cultured for 72 hours. After fixation with 4% paraformaldehyde, permeabilization with 0.1% Triton X-100 / PBS, and blocking with 1% BSA solution, the cells were fluorescently stained using FITC-CTB (a reagent that specifically stains GM1 ganglioside), Hoechst 33342 (a reagent that stains cell nuclei), and CellMask (a reagent that stains cell membranes). The fluorescence intensity of each well was measured using an imaging cytometer (IN Cell Analyzer 6000) to obtain images, and the amount of GM1 ganglioside per cell was calculated using the IN Cell Developer Toolbox protocol. Table 1 shows the results of the compounds investigated.

[0113]

Table 1

[0114] Among the compounds examined, the stained images of neural stem cells after drug treatment in several compounds that hit as GM1 inhibitory compounds are shown in FIGS. 5 and 6. In the hit compounds, it was confirmed that the fluorescence of CTB decreased and the accumulation of GM1 was suppressed as compared with the examples without adding the drug (lower row in FIG. 5b and FIG. 6e) (FIGS. 5c and d, FIGS. 6f to g, all lower rows).

[0115] [Experimental Example 7] <Effect of Hit Drugs That Reduce GM1 Accumulation on Extracellular Aβ in the Medium> Neural stem cells derived from iPS cells of Alzheimer's disease patients were seeded at a density of 2×10 6 cells / well on a 6-well plate coated with Geltrex, and the hit drug was added to a final concentration of 5 μM and cultured for 72 hours. After 72 hours, the cell supernatant was collected, and the supernatant centrifuged at 400 g for 10 minutes was used as a sample for analysis. These samples for analysis were measured for Aβ42 and Aβ40 respectively by sandwich ELISA using Human / Rat βAmyloid(42)ELISA Kit wako, High Sensitive (WAKO), and Human / Rat βAmyloid(40)ELISA Kit wako II (WAKO). From these measurement results, the Total Aβ (Aβ42 + Aβ40) and the Aβ42 / Aβ40 ratio (Aβ42÷Aβ40) were calculated. The results are shown in FIGS. 7 and 8.

[0116] The Aβ42 / Aβ40 in the medium of neural stem cells derived from Alzheimer's disease patients and the Aβ42 / Aβ40 in the cells showed higher values compared with those of healthy subjects (lower rows in FIGS. 7 and 8). Treatment with the GM1 inhibitory compound was confirmed to reduce the levels of total Aβ and the Aβ42 / Aβ40 ratio in the culture medium and cells of neural stem cells derived from Alzheimer's disease patients (Figure 7c, Figure 8f).

[0117] [Experimental Example 8] <Changes in the amount of GM1 ganglioside in the brains of GM1 gangliosidosis model mice after administration of the GM1 inhibitory compound> For GM1 gangliosidosis model mice (BKO mice: mice with complete deficiency of β-Galactosidase, β-Gal(- / -)), Amodiaquine (40 mg / kg) and Thiethylperanzine (6 mg / kg) were intraperitoneally administered twice a day for 6 days from postnatal P9 to P15. For the positive control, PBS was used as the drug solution and the same procedure was performed. For the negative control, PBS was used as the drug solution and β-Gal(+ / -) mice were used, and the same procedure was performed. Then, the mouse brains were embedded in OCT compound and sectioned at a thickness of 5 μm. The sections were fixed with 4% paraformaldehyde, blocked with 1% BSA solution, and fluorescently stained with Alexa Fluor488-CTB and Hoechst 33342. In addition, the mouse brains were fractionated and purified for sphingolipids based on the extraction method of Svennerholm and Fredman, and these samples were analyzed by Agilent6460 Triple Quadrupole LC / MS to quantify GM1 in the brain. The results of the fluorescent staining are shown in Figure 9.

[0118] In the negative control, almost no fluorescence of CTB was observed (upper panel of Figure 9a). On the other hand, in the positive control, strong fluorescence of CTB was observed and accumulation of GM1 was confirmed (upper panel of Figure 9b). On the other hand, in all cases treated with the GM1 inhibitory compound, the fluorescence of CTB was strongly suppressed, and it was confirmed that the accumulation of GM1 was suppressed (both upper panels of Figure 9c and d). The quantitative results of GM1 are shown in Figure 10.

[0119] An increase in the amount of GM1 ganglioside was confirmed in the positive subjects compared to the negative subjects. On the other hand, in any of the chemical solution treatments, it was confirmed that the amount of GM1 ganglioside was decreased compared to the positive subjects.

[0120] [Experimental Example 9] <Change in the amount of Aβ in the brain of Alzheimer's disease model mice after administration of GM1 inhibitory compound> 5×FAD mice at 3 months after birth were intraperitoneally administered Amodiaquine (40 mg / kg) and Thiethylperanzine (6 mg / kg) twice a day for 3 months. As positive subjects, the same operation was performed using PBS in the chemical solution. Thereafter, Aβ was extracted and quantified from the brains of each mouse (see Figures 11A to D). Figures 11A and B show the quantification results of soluble Aβ in the TBS (Tris Buffered Saline) extraction fraction, and Figures 11C and D show the quantification results of insoluble Aβ in the Guanidin-HCl extraction fraction. It was confirmed that Aβ40 and 42 were decreased in the Amodiaquine administration group and the Thiethylperanzine administration group compared to the PBS administration group.

[0121] In addition, sections were prepared from the brains of each mouse after continuous administration. The sections were immunostained using an anti-Aβ antibody (clone: 4G8). The results are shown in Figure 11E. Similar to the results of Figures 11A to D, it was confirmed that Aβ40 and 42 were decreased in the Amodiaquine administration group and the Thiethylperanzine administration group.

[0122] [Experimental Example 10] <Effect on autophagy by addition of GM1 inhibitory compound> The synthesis and degradation processes of gangliosides are shown in Figure 12. Each ganglioside of GM1, GM2, and GM3 is generated by sequential addition of monosaccharides and N-acetylneuraminic acid. In addition, the degradation of gangliosides is carried out by the stepwise action of glycosidases in lysosomes. (1) to (7) in Figure 12 indicate the enzymes acting in each reaction. Figure 13 shows that the expression of NEU1 and β-GLU increases upon treatment with candidate compounds. Neural stem cells differentiated from normal (201B7) and disease-derived (A138 #1-3) iPS cells were seeded into wells, and amodiaquine or thiethylperanzine was added to a concentration of 5 μM. The cells were cultured for 72 hours. After culture, RNA was isolated and the expression levels of each enzyme gene were analyzed. In Figure 13, - indicates a sample without addition, amo indicates a sample with addition of amodiaquine, and thie indicates a sample with addition of thiethylperanzine. These results suggest that the action of GM1 inhibitors promotes the degradation of gangliosides in lysosomes and activates autophagy. [Industrial Applicability]

[0123] According to the present invention, it is possible to provide an agent for preventing or treating Alzheimer's disease having a new mechanism of action.

Claims

1. A preventive or therapeutic agent for Alzheimer's disease, which is administered to a patient having intracellular accumulation of GM1 ganglioside, comprising: A preventive or therapeutic agent for Alzheimer's disease, characterized by containing amodiaquine as an active ingredient.

2. A composition for preventing or treating Alzheimer's disease, comprising the agent for preventing or treating Alzheimer's disease according to claim 1 and a pharmaceutically acceptable carrier.

3. A method for assisting in determining the therapeutic effect of amodiaquine on Alzheimer's disease associated with intracellular accumulation of GM1 ganglioside, comprising: A method comprising measuring the amount of intracellular accumulation of GM1 ganglioside before and after administration in cells derived from a patient who has been administered a therapeutic agent for Alzheimer's disease containing amodiaquine as an active ingredient, and evaluating that the therapeutic effect is likely to be achieved if the amount of accumulation is reduced.

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