JP7318948B2 - Prophylactic, therapeutic or diagnostic agent for Alzheimer's disease using microbial compounds - Google Patents

Description

TECHNICAL FIELD The present invention relates to a preventive, therapeutic, or diagnostic agent for Alzheimer's disease using a microorganism-derived substance.

Alzheimer's disease (AD) is a type of dementia whose main symptoms are cognitive decline and personality changes. Alzheimer's disease brain lesions are characterized by degeneration and loss of nerve cells accompanied by cerebral atrophy, multiple senile plaques, and multiple neurofibrillary tangles (NFTs). Among these, senile plaques are known to be aggregated accumulations of amyloid β (Aβ) peptides. Aβ is a 38-43 amino acid peptide produced as a result of β- and γ-secretase cleavage of the amyloid precursor protein (APP). Among these, Aβ42 is known to have high aggregation ability and neuronal toxicity, and it has been reported that an increase in the Aβ42/40 ratio is observed in cells with causative mutations of familial Alzheimer's disease (FAD). . Therefore, it is widely recognized that reducing the amount of Aβ42 and the Aβ42/40 ratio are key points for suppressing the onset of Alzheimer's disease.

It has been suggested that the onset of Alzheimer's disease is contributed not only by the genome information possessed by individuals but also by the microbiome, which is composed of microorganisms in the body. However, the detailed mechanism is not known, and no knowledge has been obtained about microorganisms associated with the onset of Alzheimer's disease and microorganism-derived compounds associated with the onset of Alzheimer's disease.

At present, several therapeutic agents for Alzheimer's disease, including donepezil, have appeared on the market. Drug discovery of a new therapeutic agent having

In addition, it is known that pathological changes in Aβ are already progressing before mild cognitive impairment (MCI) occurs, and early detection and early treatment are considered necessary. It is However, the diagnostic agents currently on the market cannot sufficiently detect pathological changes in Aβ at an early stage, and there is a strong desire to develop new diagnostic agents that can detect pathological changes in Aβ at an early stage. rice field.

By the way, the disease-causing cells induced to differentiate from patient-derived iPS cells (disease iPS cells) are expected to reproduce the patient's pathology in vitro, and are expected to be a powerful drug efficacy evaluation system. there is Recent reports on human iPS cell-derived neurons emphasize the importance of human neurons as a tool for evaluating drug responsiveness (Non-Patent Documents 1-3).

PLoS One 6, e25788 (2011) JAMA Neurol. 71, 1481-9 (2014) Stem Cell Reports 1, 491-498 (2013)

As described above, in the treatment of Alzheimer's disease, no satisfactory therapeutic drug has been found yet, and new drug discovery has been expected. The present invention screens microorganism-derived compounds to discover and provide new prophylactic or therapeutic agents for Alzheimer's disease, with the aim of drug discovery using compounds with new structures that have not been studied as therapeutic agents for Alzheimer's disease. intended to Another object of the present invention is to find and provide a diagnostic agent for Alzheimer's disease using a microorganism-derived compound.

The present inventors conducted intensive research to solve the above problems, and as a result of screening various microorganism-derived compounds using neurons differentiated from iPS cells derived from Alzheimer's disease patients, some specific microorganisms The inventors have found that the compound changes the amount of Aβ42 and the Aβ42/40 ratio in the nerve cells, and have completed the present invention.

That is, the present invention relates to the following [1] to [16-2].
[1]
one or more compounds selected from the group consisting of Mer-A2026A, velcarin A, kalafungin, concanamycin A, efomycin A, brefeldin A, eupenifeldin, elaiophyllin, and boromycin, or a pharmaceutically acceptable A prophylactic or therapeutic agent for Alzheimer's disease, comprising a salt of
[2]
The agent of [1], wherein the compound is one or more compounds selected from the group consisting of Mer-A2026A and Bercalin A or a pharmaceutically acceptable salt thereof;
[3]
The agent of [1], wherein the compound is Mer-A2026A or a pharmaceutically acceptable salt thereof;
[3-1]
The agent of [1], wherein the compound is carafungin or a pharmaceutically acceptable salt thereof;
[4]
1 selected from the group consisting of Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, brefeldin A, eupenifeldin, elaiophyllin, and boromycin in the manufacture of a prophylactic or therapeutic agent for Alzheimer's disease use of one or more compounds or pharmaceutically acceptable salts thereof;
[4-1]
The use of [4], wherein the compound is one or more compounds selected from the group consisting of Mer-A2026A and Bercalin A, or a pharmaceutically acceptable salt thereof;
[4-2]
The use according to [4], wherein the compound is Mer-A2026A or a pharmaceutically acceptable salt thereof;
[5]
1 selected from the group consisting of Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, brefeldin A, eupenifeldin, elaiophyllin, and boromycin for the prevention or treatment of Alzheimer's disease a compound of the above or a pharmaceutically acceptable salt thereof;
[5-1]
one or more compounds selected from the group consisting of Mer-A2026A and velcarin A, or a pharmaceutically acceptable salt thereof, for the prevention or treatment of Alzheimer's disease;
[5-2]
Mer-A2026A or a pharmaceutically acceptable salt thereof for the prevention or treatment of Alzheimer's disease;
[5-3]
one or more compounds selected from the group consisting of Mer-A2026A, velcarin A, kalafungin, concanamycin A, efomycin A, brefeldin A, eupenifeldin, elaiophyllin, and boromycin, or a pharmaceutically acceptable a method of preventing or treating Alzheimer's disease comprising administering to a patient a therapeutically effective amount of a salt of
[5-4]
The method of prevention or treatment of [5-3], wherein the compound is one or more compounds selected from the group consisting of Mer-A2026A and velcarin A, or a pharmaceutically acceptable salt thereof;
[5-5]
The method of prevention or treatment of [5-3], wherein the compound is Mer-A2026A or a pharmaceutically acceptable salt thereof;
[6]
One or more compounds selected from the group consisting of eupeniferdin, elaiophylline, boromycin, Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, and brefeldin A in the sample or pharmaceutical a diagnostic agent for Alzheimer's disease, comprising a substance for detecting a salt acceptable to , a microorganism producing said compound or a pharmaceutically acceptable salt thereof, or an extract of said microorganism;
[7]
A substance for detecting a compound or a pharmaceutically acceptable salt thereof, a microorganism producing the compound or a pharmaceutically acceptable salt thereof, or an extract of the microorganism is eupeniferdin, elaiophylline, boromycin , Mer-A2026A, Vercarin A, Karafungin, Concanamycin A, Efomycin A, and Brefeldin A, or one or more compounds selected from the group consisting of, or a pharmaceutically acceptable salt thereof, the compound or a pharmaceutically The diagnostic agent of [6], which is an antibody that binds to an acceptable salt-producing microorganism or an extract of the microorganism;
[8]
The diagnostic agent of [6] or [7], wherein the compound is one or more compounds selected from the group consisting of eupenifergin, elaiophylline, and boromycin, or a pharmaceutically acceptable salt thereof;
[9]
The diagnostic agent according to any one of [6] to [8], wherein the compound is eupeniferzine or a pharmaceutically acceptable salt thereof;
[9-1]
One or more selected from the group consisting of eupenifelgin, elaiophylline, boromycin, Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, and brefeldin A in the manufacture of a diagnostic agent for Alzheimer's disease or a pharmaceutically acceptable salt thereof, a microorganism producing said compound or a pharmaceutically acceptable salt thereof, or an antibody that binds to an extract of said microorganism;
[9-2]
The use of [9-1], wherein the compound is one or more compounds selected from the group consisting of eupenifergin, elaiophylline, and boromycin, or a pharmaceutically acceptable salt thereof;
[9-3]
The use of [9-1], wherein the compound is eupeniferzine or a pharmaceutically acceptable salt thereof;
[10]
one or more selected from the group consisting of eupeniferdin, elaiophyllin, boromycin, Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, and brefeldin A for diagnosing Alzheimer's disease an antibody that binds to a compound or a pharmaceutically acceptable salt thereof, a microorganism producing said compound or a pharmaceutically acceptable salt thereof, or an extract of said microorganism;
[10-1]
The antibody of [10], wherein the compound is one or more compounds selected from the group consisting of eupenifergin, elaiophyllin, and boromycin, or a pharmaceutically acceptable salt thereof;
[10-2]
One or more compounds selected from the group consisting of eupeniferdin, elaiophylline, boromycin, Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, and brefeldin A in the sample or pharmaceutical a method of diagnosing Alzheimer's disease comprising a process of detecting an acceptable salt of the compound or a microorganism producing said compound or a pharmaceutically acceptable salt thereof, or an extract of said microorganism;
[10-3]
producing one or more compounds selected from the group consisting of eupenifergin, elaiophyllin, and boromycin or a pharmaceutically acceptable salt thereof, the compound or a pharmaceutically acceptable salt thereof in the sample A method of diagnosing Alzheimer's disease comprising a process of detecting a microorganism, or an extract of said microorganism;
[10-4]
Diagnosis of Alzheimer's disease comprising a process of detecting eupeniferdin or a pharmaceutically acceptable salt thereof, a microorganism producing said compound or a pharmaceutically acceptable salt thereof, or an extract of said microorganism in a specimen Method;
[11]
one or more compounds or pharmaceutically acceptable salts thereof selected from the group consisting of roridin A, leucicidin, bafilomycin A1, and bafilomycin B1 in the sample, the compound or a pharmaceutically acceptable salt thereof A diagnostic agent for Alzheimer's disease comprising a substance for detecting a salt-producing microorganism, or an extract of said microorganism;
[12]
A substance for detecting a compound or a pharmaceutically acceptable salt thereof, a microorganism producing the compound or a pharmaceutically acceptable salt thereof, or an extract of the microorganism is roridin A, leucanicidin, bafilomycin A1, and bafilomycin B1, or a pharmaceutically acceptable salt thereof, a microorganism producing the compound or a pharmaceutically acceptable salt thereof, or an extract of the microorganism The diagnostic agent of [11], which is a binding antibody;
[13]
The diagnostic agent of [11] or [12], wherein the compound is Roridin A or a pharmaceutically acceptable salt thereof;
[13-1]
One or more compounds selected from the group consisting of roridin A, leucicidin, bafilomycin A1, and bafilomycin B1, or a pharmaceutically acceptable salt thereof, the compound, or a compound thereof, in the manufacture of a diagnostic agent for Alzheimer's disease use of an antibody that binds to a microorganism producing a pharmaceutically acceptable salt, or to an extract of said microorganism;
[13-2]
The use of [13-1], wherein the compound is Roridin A or a pharmaceutically acceptable salt thereof;
[14]
One or more compounds selected from the group consisting of roridin A, leucicidin, bafilomycin A1, and bafilomycin B1, or pharmaceutically acceptable salts thereof, the compound, or a medicament thereof, for diagnosing Alzheimer's disease an antibody that binds to a microorganism producing a biologically acceptable salt, or to an extract of said microorganism;
[14-1]
the antibody of [14], wherein the compound is Roridin A or a pharmaceutically acceptable salt thereof;
[15]
The specimen is selected from the group consisting of blood, plasma, serum, interstitial fluid, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, lymph, saliva, urine, and stool [6] to [9] or The diagnostic agent according to any one of [11] to [13];
[16]
one or more compounds or pharmaceutically acceptable salts thereof selected from the group consisting of roridin A, leucicidin, bafilomycin A1, and bafilomycin B1 in the sample, the compound or a pharmaceutically acceptable salt thereof A method of diagnosing Alzheimer's disease comprising a process of detecting a salt-producing microorganism, or an extract of said microorganism;
[16-1]
A method of diagnosing Alzheimer's disease comprising a process of detecting Roridin A or a pharmaceutically acceptable salt thereof, a microorganism producing said compound or a pharmaceutically acceptable salt thereof, or an extract of said microorganism in a specimen;
[16-2]
The specimen is selected from the group consisting of blood, plasma, serum, interstitial fluid, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, lymph, saliva, urine, and stool, [10-2] to [10 -4] or the diagnostic method according to any one of [16] to [16-1].

The present invention also relates to the following [17] to [45].
[17]
one or more compounds selected from the group consisting of Mer-A2026A, velcarin A, kalafungin, concanamycin A, efomycin A, brefeldin A, eupenifeldin, elaiophyllin, and boromycin, or a pharmaceutically acceptable an agent for altering the amount of Aβ42 and/or the Aβ42/40 ratio, comprising a salt of
[18]
an agent for reducing the amount of Aβ42 and/or the ratio of Aβ42/40, comprising one or more compounds selected from the group consisting of Mer-A2026A and velcarin A, or pharmaceutically acceptable salts thereof;
[19]
an agent for reducing the amount of Aβ42 and/or the Aβ42/40 ratio comprising Mer-A2026A or a pharmaceutically acceptable salt thereof;
[20]
An agent for increasing the amount of Aβ42 and/or the Aβ42/40 ratio, comprising one or more compounds selected from the group consisting of eupenifergin, elaiophyllin, and boromycin, or pharmaceutically acceptable salts thereof ;
[21]
agents for increasing the amount of Aβ42 and/or the Aβ42/40 ratio, comprising eupeniferdin or a pharmaceutically acceptable salt thereof;
[22]
Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, brefeldin A, eupeniferdin, elaiophyllin, and boromycin in the manufacture of agents for altering Aβ42 amount and/or Aβ42/40 ratio use of one or more compounds selected from the group consisting of or a pharmaceutically acceptable salt thereof;
[23]
use of one or more compounds selected from the group consisting of Mer-A2026A and velcarin A, or a pharmaceutically acceptable salt thereof, in the manufacture of an agent for reducing Aβ42 amount and/or Aβ42/40 ratio;
[24]
Use of Mer-A2026A or a pharmaceutically acceptable salt thereof in the manufacture of an agent for reducing Aβ42 amount and/or Aβ42/40 ratio;
[25]
One or more compounds selected from the group consisting of eupeniferdin, elaiophylline, and boromycin, or pharmaceutically acceptable thereof, in the manufacture of an agent for increasing the amount of Aβ42 and/or the ratio of Aβ42/40 use of salt;
[26]
use of eupeniferdin or a pharmaceutically acceptable salt thereof in the manufacture of an agent for increasing the amount of Aβ42 and/or the Aβ42/40 ratio;
[27]
from the group consisting of Mer-A2026A, velcarin A, carafungin, concanamycin A, efomycin A, brefeldin A, eupenifeldin, elaiophyllin, and boromycin for altering Aβ42 amount and/or Aβ42/40 ratio one or more selected compounds or pharmaceutically acceptable salts thereof;
[28]
one or more compounds selected from the group consisting of Mer-A2026A and velcarin A, or a pharmaceutically acceptable salt thereof, for decreasing the amount of Aβ42 and/or the Aβ42/40 ratio;
[29]
Mer-A2026A or a pharmaceutically acceptable salt thereof for decreasing the amount of Aβ42 and/or the Aβ42/40 ratio;
[30]
one or more compounds selected from the group consisting of eupenifergin, elaiophylline, and boromycin, or a pharmaceutically acceptable salt thereof, for increasing the amount of Aβ42 and/or the Aβ42/40 ratio;
[31]
eupeniferdin or a pharmaceutically acceptable salt thereof for increasing the amount of Aβ42 and/or the Aβ42/40 ratio;
[32]
one or more compounds selected from the group consisting of Mer-A2026A, velcarin A, kalafungin, concanamycin A, efomycin A, brefeldin A, eupenifeldin, elaiophyllin, and boromycin, or a pharmaceutically acceptable a method of altering the amount of Aβ42 and/or the Aβ42/40 ratio comprising administering to the patient an effective amount of a salt of
[33]
reducing the amount of Aβ42 and/or the ratio of Aβ42/40 comprising administering to the patient an effective amount of one or more compounds selected from the group consisting of Mer-A2026A and velcarin A, or a pharmaceutically acceptable salt thereof how to make
[34]
A method of reducing Aβ42 amount and/or Aβ42/40 ratio comprising administering to a patient an effective amount of Mer-A2026A or a pharmaceutically acceptable salt thereof;
[35]
Aβ42 amount and/or Aβ42/40 ratio comprising adding to the sample one or more compounds selected from the group consisting of eupenifergin, elaiophyllin, and boromycin, or a pharmaceutically acceptable salt thereof a method of increasing
[36]
A method of increasing Aβ42 amount and/or Aβ42/40 ratio comprising adding eupeniferdin or a pharmaceutically acceptable salt thereof to a specimen;
[37]
increasing Aβ42 amount and/or Aβ42/40 ratio comprising one or more compounds selected from the group consisting of roridin A, leucicidin, bafilomycin A1, and bafilomycin B1, or pharmaceutically acceptable salts thereof agent for causing;
[38]
an agent for increasing the amount of Aβ42 and/or the Aβ42/40 ratio, comprising Roridin A or a pharmaceutically acceptable salt thereof;
[39]
One or more compounds selected from the group consisting of roridin A, leucanicidin, bafilomycin A1, and bafilomycin B1 in the manufacture of an agent for increasing the amount of Aβ42 and/or the ratio of Aβ42/40; use of acceptable salts for
[40]
Use of Roridin A or a pharmaceutically acceptable salt thereof in the manufacture of an agent for increasing the amount of Aβ42 and/or the Aβ42/40 ratio;
[41]
one or more compounds selected from the group consisting of roridin A, leucicidin, bafilomycin A1, and bafilomycin B1 for increasing the amount of Aβ42 and/or the Aβ42/40 ratio, or pharmaceutically acceptable thereof salt;
[42]
use of Roridin A or a pharmaceutically acceptable salt thereof to increase the amount of Aβ42 and/or the Aβ42/40 ratio;
[43]
Aβ42 amount and/or comprising adding to the sample one or more compounds selected from the group consisting of roridin A, leucicidin, bafilomycin A1, and bafilomycin B1, or a pharmaceutically acceptable salt thereof A method of increasing the Aβ42/40 ratio;
[44]
A method of increasing Aβ42 amount and/or Aβ42/40 ratio comprising adding Roridin A or a pharmaceutically acceptable salt thereof to a specimen;
[45]
the specimen is selected from the group consisting of blood, plasma, serum, interstitial fluid, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, lymph, saliva, urine, and stool [35], [36]; The method of any one of [43], or [44].

The microorganism-derived compound used in the present invention has the effect of altering the amount of Aβ42 and the Aβ42/40 ratio, and is therefore useful for preventing, treating, or diagnosing Alzheimer's disease. In particular, the preventive or therapeutic compound found in the present invention is a compound of an unknown structural group as a therapeutic drug for Alzheimer's disease, and is expected as a drug that overcomes the problems of existing therapeutic drugs for Alzheimer's disease. . In addition, the diagnostic compounds found in the present invention belong to an unknown structural group as Alzheimer's disease biomarkers, and are expected to overcome the problems of existing Alzheimer's disease biomarkers.

FIG. 1 shows the results of the first screening using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease having the G384A mutation of PSEN1. The vertical axis indicates the percent change relative to the DMSO control for Aβ40, Aβ42, and the Aβ42/40 ratio when 1 μM or 50 nM of each compound was added. The horizontal axis indicates the compound number. FIG. 10 shows the results of the second screening using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease having the G384A mutation of PSEN1. Correlation plots of the first and second screenings are shown for studies performed at 1 μM compound using cortical neurons derived from iPS cells derived from a familial Alzheimer's disease patient with the G384A mutation of PSEN1. The vertical axis indicates the value in the first screening, and the horizontal axis indicates the value in the second screening. The results of screening using cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease are shown. The vertical axis indicates the percent change relative to the DMSO control for Aβ40, Aβ42, and the Aβ42/40 ratio when 1 μM or 50 nM of each compound was added. The horizontal axis indicates the compound number. FIG. 4 shows the results of plotting the Aβ42/40 ratio in screening using cerebral cortical neurons induced from iPS cells derived from familial and sporadic Alzheimer's disease patients for each screened microorganism-derived compound. The vertical axis shows the rate of change in the Aβ42/40 ratio against DMSO when using cerebral cortical neurons induced from iPS cells derived from sporadic Alzheimer's disease, and the horizontal axis shows the rate of change from iPS cells derived from familial Alzheimer's disease. Fig. 10 shows the rate of change of Aβ42/40 ratio against DMSO when using cerebral cortical neurons. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and the concentration dependence of leucicidin (compound 01) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of bafilomycin A1 (compound 02) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of Roridin A (compound 03) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of bafilomycin B1 (compound 04) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and the concentration dependence of carafungin (compound 05) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of concanamycin A (compound 06) on toxicity. Fig. 2 shows concentration dependence of velcarin A (compound 07) on Aβ40 and Aβ42 production, Aβ42/40 ratio, and toxicity using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease. 2 shows two graphs with different Y-axis scales for the same data. Fig. 2 shows concentration dependence of velcarin A (compound 07) on Aβ40 and Aβ42 production, Aβ42/40 ratio, and toxicity using cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease. 2 shows two graphs with different Y-axis scales for the same data. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of eupeniferdin (compound 08) on toxicity. FIG. 4 shows concentration dependence of Mer-A2026A (compound 09) on Aβ40 and Aβ42 production, Aβ42/40 ratio, and toxicity using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease. 2 shows two graphs with different Y-axis scales for the same data. Concentration dependence of Mer-A2026A (compound 09) on Aβ40 and Aβ42 production, Aβ42/40 ratio, and toxicity using cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease. . 2 shows two graphs with different Y-axis scales for the same data. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of efomycin A (compound 10) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of Brefeldin A (compound 11) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of elaiophyllin (compound 12) on toxicity. Aβ40 and Aβ42 production amounts, Aβ42/40 ratio, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, and concentration dependence of boromycin (compound 13) on toxicity.

The microbial compounds used in the present invention are useful for prevention, treatment, or diagnosis of Alzheimer's disease. The term "Alzheimer's disease" as used herein includes familial Alzheimer's disease (FAD) and sporadic Alzheimer's disease (SAD). In one embodiment, the microbial compounds used in the present invention are used to prevent, treat, or diagnose familial Alzheimer's disease. In another embodiment, the microbial compounds used in the invention are used to prevent, treat, or diagnose sporadic Alzheimer's disease.

Preventive or therapeutic agent The preventive or therapeutic agent of the present invention includes kalafungin (CAS number 11048-5-0; molecular weight 300.3) and concanamycin A (CAS number 80890-47-7; molecular weight 866.1). ), verrucarin A (CAS number 3148-09-2; molecular weight 502.6), eupenifeldin (CAS number 151803-45-1; molecular weight 548.7), Mer-A2026A (CAS number 144357 -08-4; molecular weight 413.6), efomycin A (CAS number 106387-82-0; molecular weight 1039.3), brefeldin A (CAS number 20350-15-6; molecular weight 280.4 ), elaiophylin (CAS number 37318-06-2; molecular weight 1025.3), and boromycin (CAS number 34524-20-4; molecular weight 879.9) one or more selected from the group consisting of or a pharmaceutically acceptable salt thereof (hereinafter also referred to as "component of the present invention").

A compound selected from the group consisting of carafungin, concanamycin A, velcarin A, eupeniferdin, Mer-A2026A, efomycin A, brefeldin A, elaiophyllin, and boromycin, or a pharmaceutically acceptable salt thereof, is Aβ42 Since it has the effect of changing the amount and Aβ42/40 ratio, it can be used for the prevention or treatment of Alzheimer's disease. These may be used alone, or may be used in combination of two or more. The compound used for the prevention or treatment of Alzheimer's disease is preferably one or more compounds selected from the group consisting of Mer-A2026A, velcarin A, carafungin, and concanamycin A, or pharmaceutically acceptable salts thereof. -A2026A and one or more compounds selected from the group consisting of velcarin A or a pharmaceutically acceptable salt thereof are more preferred, with Mer-A2026A or a pharmaceutically acceptable salt thereof being especially preferred.

The component of the present invention may be a commercially available product, may be produced from a commercially available product by a known method, or may be extracted from microorganisms. The prophylactic or therapeutic agent in the present invention may contain the component of the present invention itself, or may contain microorganisms that produce the component of the present invention. In the prophylactic or therapeutic method of the present invention, the component of the present invention itself may be administered, or a microorganism that produces the component of the present invention may be administered. The microorganism is not particularly limited as long as it is readily available to those skilled in the art and produces at least one component of the present invention.

The components of the present invention may exist in tautomeric forms or mixtures thereof. Components of the present invention may also exist in stereoisomeric forms, such as enantiomers and diastereomers, or in mixtures thereof. When the components of the present invention are obtained in diastereomeric or enantiomeric forms, these may be separated by conventional methods well known in the art, such as chromatography and fractional crystallization.

The components of the present invention are compounds labeled with isotopes (e.g., ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³² P, ³⁵ S, ¹²⁵ I, etc.) and deuterium converters. encompasses

The components of the present invention can exist in free form or in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts include, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, formate, acetate, propionate, fumarate, oxalate, malonate, succinate, methanesulfonate, ethanesulfonate, benzenesulfonate, maleate, lactate, malate, tartrate, citrate, and trifluoroacetate metal salts such as lithium, potassium, calcium, magnesium, sodium, zinc, and aluminum salts; and ammonium, diethanolamine, ethylenediamine, triethanolamine, and triethylamine salts. and base addition salts such as Pharmaceutically acceptable salts include any of their inner salts, adducts, their solvates, hydrates, and the like.

In one embodiment, the present invention provides the use of the composition of the invention in the manufacture of a prophylactic or therapeutic agent for Alzheimer's disease.

In another embodiment, the invention provides a method of preventing or treating Alzheimer's disease comprising administering to a patient a therapeutically effective amount of a component of the invention.

In another embodiment, the invention provides compositions of the invention for the prevention or treatment of Alzheimer's disease.

As used herein, the term "patient" is an individual, preferably a mammal, more preferably a human, to be prevented, treated or diagnosed.

The components of the present invention can be administered either orally or parenterally, either alone or in admixture with pharmaceutically acceptable excipients. Pharmaceutically acceptable additives may be additives commonly used in the art, such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.). , excipients (lactose, sucrose, cornstarch, potassium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (carboxymethylcellulose, carboxymethylcellulose calcium , and potato starch, etc.), emulsifiers (polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters, etc.), stabilizers (methylparaben, propylparaben, etc.), and diluents. In addition, the dosage form of the formulation of the present invention is not particularly limited, and tablets, pills, granules, capsules, powders, syrups, emulsions, suspensions, injections, inhalants, suppositories, etc. can be used as a conventional pharmaceutical formulation of

The components of the present invention may be formulated singly or in combination of two or more. When two or more components are combined, they may be formulated as a single formulation or as separate formulations. When two or more components of the present invention are formulated as separate formulations, the dosage form of each formulation may be the same or different.

In addition, the component of the present invention may be used in combination with other therapeutic agents for Alzheimer's disease. Other Alzheimer's disease therapeutic agents include, for example, donepezil, memantine, galantamine, and rivastigmine.

The therapeutically effective amount of each component of the present invention varies depending on the type of component, administration method, patient age, body weight, condition, etc. Preferably 0.5 to 500 mg/kg, and for parenteral administration, 0.01 to 100 mg per day, preferably 0.05 to 50 mg/kg, once daily Or it can be administered several times. When the formulations of the invention contain more than one component, they may be administered simultaneously or separately.

Diagnostic Agents and Diagnostic Methods Components of the Invention and Leucanicidin (CAS No. 91021-66-8; Molecular Weight 783.0), Bafilomycin A1 (CAS No. 88899-55-2; Molecular Weight 622.8), A compound selected from the group consisting of roridin A (CAS number 14729-29-4; molecular weight 532.6) and bafilomycin B1 (CAS number 88899-56-3; molecular weight 816.0) Or a pharmaceutically acceptable salt thereof (hereinafter also referred to as "biomarker of the present invention") has the effect of changing the amount of Aβ42 and the Aβ42/40 ratio, and thus can be used for diagnosis of Alzheimer's disease and the like. In the component of the present invention, the compound to be diagnosed is preferably one or more compounds selected from the group consisting of eupeniferdin, elaiophyllin, and boromycin, or a pharmaceutically acceptable salt thereof. Peniferdin or a pharmaceutically acceptable salt thereof is more preferred. In addition, in the biomarker of the present invention, the compound to be diagnosed is preferably Roridin A or a pharmaceutically acceptable salt thereof. The diagnostic agent for Alzheimer's disease of the present invention includes a substance for detecting the component of the present invention or the biomarker of the present invention, a microorganism that produces the component or the biomarker, or an extract of the microorganism in a specimen. The microorganism is not particularly limited as long as it is readily available to those skilled in the art and produces at least one of the component of the present invention or the biomarker of the present invention. In addition, the extract of the microorganism is not particularly limited as long as it is a substance other than the component of the present invention or the biomarker of the present invention present in the microorganism and is characteristic of the microorganism. Examples include DNA and RNA that are characteristic of microorganisms. In one embodiment, the diagnostic agent for Alzheimer's disease of the present invention comprises a substance for detecting the component of the present invention or the biomarker of the present invention in a specimen.

The term "specimen" as used herein means a biological sample taken from a patient to be diagnosed, such as blood, plasma, serum, interstitial fluid, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, Examples include lymph, saliva, urine, and stool. You may use these in combination of 2 or more types. Preferably blood, plasma, serum or cerebrospinal fluid is used.

Substances for detecting the component of the present invention or the biomarker of the present invention, the microorganism that produces the component or biomarker, or the extract of the microorganism are not particularly limited, and for example, receptors, antibodies, etc. can be used. , antibodies are preferred, more preferably antibodies that specifically bind to a component of the invention or a biomarker of the invention, a microorganism producing said component or biomarker, or an extract of said microorganism. In addition, the method for detecting the component of the present invention or the biomarker of the present invention, the microorganism producing the component or biomarker, or the extract of the microorganism is not particularly limited. When using the component of the present invention or the biomarker of the present invention, a microorganism that produces the component or biomarker, or an antibody against an extract of the microorganism, an immunological method such as an enzyme-linked immunosorbent assay (ELISA) is used. The component of the present invention or the biomarker of the present invention, the microorganism producing the component or biomarker, or the extract of the microorganism can be detected, preferably by ELISA using direct adsorption, sandwich, or competitive methods. Used.

For example, a component of the invention or a biomarker of the invention, a microorganism that produces the component or biomarker, or an extract of the microorganism produces an antibody that produces the component of the invention or the biomarker of the invention, the component or biomarker. The component of the present invention or the biomarker of the present invention bound to the antibody after being bound to the microorganism or the extract of the microorganism, the microorganism producing the component or the biomarker, or the extract of the microorganism is the antibody. Detected by an antibody that is another antibody (primary detection antibody) and labeled with a labeling substance, or bound to the primary detection antibody and detected by an antibody that is labeled with a labeling substance (secondary detection antibody). As the labeling substance, substances well known in the art can be used, such as radioactive isotopes such as ³² P, ¹⁴ C and ¹²⁵ I, enzymes such as horseradish peroxidase, and the like. When ELISA is used, it is preferable to use an enzyme such as horseradish peroxidase as a labeling substance. In this case, by adding a fluorescent or chromogenic substrate of the enzyme such as 3,3′,5,5′-tetramethylbenzidine (TMB) and detecting the fluorescence or color development using a fluorometer or the like, the present Invention components or biomarkers of the invention, microorganisms producing said components or biomarkers, or extracts of said microorganisms can be detected.

The component of the invention or the biomarker of the invention, the microorganism producing the component or biomarker, or the antibody against the extract of the microorganism is the component of the invention or the biomarker of the invention, the microorganism producing the component or biomarker, Alternatively, it is not particularly limited as long as it can specifically bind to the extract of the microorganism, and non-human antibodies such as human antibodies, humanized antibodies, chimeric antibodies, and mouse antibodies can be mentioned, and polyclonal It may be an antibody or a monoclonal antibody. Antibodies can be produced by methods known in the art, for example, by known methods using hybridomas of antibody-producing cells and myeloma cells.

In one embodiment, the invention provides a diagnostic agent for Alzheimer's disease comprising an antibody that binds to a component of the invention or a biomarker of the invention, a microorganism producing said component or biomarker, or an extract of said microorganism. . In another embodiment, the invention provides a diagnostic agent for Alzheimer's disease comprising an antibody that binds to a component of the invention or a biomarker of the invention. The diagnostic agent may be in kit form. In one embodiment, the diagnostic agent may be a diagnostic agent for ELISA measurements, for example solid phases such as microplates or beads adsorbed with said antibody; a primary detection antibody that binds to the microorganism producing the marker, or an extract of said microorganism, and that recognizes a different epitope than said antibody; A subsequent detection antibody; a chromogenic substrate for the enzyme, such as TMB (3,3',5,5'-tetramethylbenzidine); a dilution buffer; and a wash buffer, as appropriate.

Alzheimer's disease by comparing the concentration of the component of the present invention or the biomarker of the present invention, the microorganism producing the component or the biomarker, or the extract of the microorganism detected using the above diagnostic agent with the reference value. can diagnose. The reference value is appropriately determined by a physician, clinician, veterinarian, or the like having ordinary knowledge in the art. Only one of the components of the present invention or the biomarkers of the present invention, microorganisms that produce the components or biomarkers, or extracts of the microorganisms may be detected. Accuracy can be improved.

In one embodiment, the present invention relates to the use of a substance that binds to a component of the invention or a biomarker of the invention, a microorganism producing said component or biomarker, or an extract of said microorganism in the manufacture of a diagnostic agent for Alzheimer's disease. I will provide a. In another embodiment, the invention provides the use of an antibody that binds to a component of the invention or a biomarker of the invention, a microorganism producing said component or biomarker, or an extract of said microorganism in the manufacture of a diagnostic for Alzheimer's disease. I will provide a. In another embodiment, the invention provides the use of a substance that binds to a component of the invention or a biomarker of the invention in the manufacture of a diagnostic for Alzheimer's disease. In another embodiment, the invention provides the use of an antibody that binds to a component of the invention or a biomarker of the invention in the manufacture of a diagnostic for Alzheimer's disease.

In one embodiment, the invention provides a substance that binds to a component of the invention or a biomarker of the invention, a microorganism producing said component or biomarker, or an extract of said microorganism for diagnosing Alzheimer's disease. . In another embodiment, the invention provides an antibody that binds to a component of the invention or a biomarker of the invention, a microorganism producing said component or biomarker, or an extract of said microorganism for diagnosing Alzheimer's disease. . In another embodiment, the invention provides a substance that binds to a component of the invention or a biomarker of the invention for diagnosing Alzheimer's disease. In another embodiment, the invention provides an antibody that binds to a component of the invention or a biomarker of the invention for diagnosing Alzheimer's disease.

In one embodiment, the invention provides a method of diagnosing Alzheimer's disease comprising a process of detecting a component of the invention or a biomarker of the invention, a microorganism producing said component or biomarker, or an extract of said microorganism in a specimen. I will provide a. In another embodiment, the invention provides a method of diagnosing Alzheimer's disease comprising a process of detecting a component of the invention or a biomarker of the invention in a specimen.

EXAMPLES The present invention will be more specifically described below with reference to examples, but the following examples are provided for the purpose of explaining the present invention and are not intended to limit the present invention. In the examples, each term shall have the meaning commonly used in the technical field.

Example 1
Screening Using Cerebral Cortical Neurons Derived from iPS Cells Derived from Familial Alzheimer's Disease Patients Having PSEN1 G384A Mutation Screening of compounds was performed according to the method described in WO2017/115873A1. That is, cerebral cortical neurons were prepared using iPS cells transfected with the human Neurogenin 2 gene (NGN2) linked to a doxycycline-inducible promoter using PiggyBac. iPS cells are described in Okita K et al. , Nature. 2007, vol. 448, pp313-317. The NGN2 gene was introduced into these iPS cells, and doxycycline was added for transient expression for 5 days. Familial Alzheimer's disease (FAD) patient-derived iPS cells with a high Aβ42/40 ratio PSEN1 G384A mutation and sporadic Alzheimer's disease (SAD) patient-derived iPS cells used for high-throughput screening (HTS) iPS cells were selected to generate cerebral cortical neurons.

Subsequently, using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease, a microbial compound library consisting of 123 compounds listed in Table 1 below provided by Microbiopharma Japan Co., Ltd. was screened. to search for preventive, therapeutic, or diagnostic agents for Alzheimer's disease.

10 μL of each microorganism-derived compound diluted with DMSO to 1 μM or 50 nM was added to 100,000/well cerebral cortical neurons dispensed into a 96-well plate and cultured at 37°C. After 48 hours, the culture supernatant was collected, and the amounts of Aβ40 and Aβ42 produced in the supernatant were measured by ECL-ELISA. Also, the Aβ42/40 ratio was calculated from the measured values of Aβ40 and Aβ42 obtained. Using 0.1% v/v DMSO as a negative control, using 1 μM β-secretase inhibitor IV (BSI) as a positive control that reduces Aβ40 or Aβ42, and using 1 μM JNJ-40418677 (GSM) as a positive control that reduces the Aβ42/40 ratio ) was used. FIG. 1 shows the screening results obtained using cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease having the PSEN1 G384A mutation. In addition, the values of the components of the present invention and the biomarkers of the present invention are shown in Tables 2 and 3, respectively.

Furthermore, in order to examine the reproducibility of the screening, a second screening was performed. The results are shown in FIG. In addition, the values of the components of the present invention and the biomarkers of the present invention are shown in Tables 4 and 5, respectively. In addition, correlation plots for the first and second screens are shown in FIG. 3 for tests performed at 1 μM compound.

Example 2
Screening using cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease The same procedure as in Example 1 was performed except that cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease were used. screened by the method. The results are shown in FIG. In addition, the values of the components of the present invention and the biomarkers of the present invention are shown in Tables 6 and 7, respectively. Furthermore, the results of plotting the Aβ42/40 ratio in the second screening of Example 1 and the Aβ42/40 ratio in the screening of Example 2 for each microorganism-derived compound are shown in FIG. Names of microorganism-derived compounds that are the risk of sporadic Alzheimer's disease, which have a particularly large rate of change, and microorganism-derived compound names that are therapeutic agents for sporadic Alzheimer's disease are shown.

As described above, a compound selected from the group consisting of carafungin, concanamycin A, velcarin A, eupeniferdin, Mer-A2026A, efomycin A, brefeldin A, elaiophylline, and boromycin can be used in patients with familial Alzheimer's disease Aβ42/40 was significantly altered compared to DMSO in cerebral cortical neurons derived from iPS cell-derived cells, especially in studies performed at 1 μM compound. Karafungin also significantly reduced Aβ42/40 in cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease. In addition, concanamycin A and velcarin A significantly reduced Aβ42/40 even at low concentrations (50 nM) in cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease. Therefore, a compound selected from the group consisting of carafungin, concanamycin A, velcarin A, eupeniferdin, Mer-A2026A, efomycin A, brefeldin A, elaiophylline, and boromycin is useful for preventing or treating Alzheimer's disease, etc. was shown to be useful for

A compound selected from the group consisting of leucanicidin, bafilomycin A1, roridin A, and bafilomycin B1, compared to DMSO, particularly in cerebral cortical neurons derived from iPS cells derived from patients with sporadic Alzheimer's disease. , significantly increased Aβ42/40. Accordingly, compounds selected from the group consisting of leucanicidin, bafilomycin A1, roridin A, and bafilomycin B1 have been shown to be useful in diagnosing Alzheimer's disease, particularly sporadic Alzheimer's disease.

Example 3
Concentration-dependent evaluation of the component of the present invention or the biomarker compound of the present invention For the component of the present invention or the biomarker compound of the present invention, cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and sporadic Alzheimer's disease Concentration dependence of Aβ production and cytotoxicity was evaluated using cerebral cortical neurons induced from patient-derived iPS cells.

DMSO-added group as a negative control, and dissolution in DMSO for cerebral cortical neurons induced from iPS cells derived from patients with familial Alzheimer's disease and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease Under the conditions of a total of 8 groups of 7 concentrations of each compound addition group (0.32 nM, 1.6 nM, 8 nM, 40 nM, 0.2 μM, 1 μM, and 5 μM), the production amount of Aβ40 and Aβ42, the Aβ42/40 ratio (Aβ42/40 ratio), as well as concentration dependence for toxicity were evaluated. Aβ amount measurement (ECL-ELISA method) in the culture supernatant was evaluated using the same method as the screening in Examples 1 and 2. Toxicity was also evaluated by measuring adenylate kinase activity in the culture supernatant using ToxiLight™ (manufactured by Lonza). For each compound, the degree of change (Fold change (vs. DMSO control)) was plotted for each different concentration, with the DMSO added group as a negative control set to 1. The results are shown in Figures 6-1 to 6-13. In these figures, fold change of Aβ species (vs. DMSO control) is shown on the left Y-axis for changes in Aβ, and fold change from DMSO control for toxicity. is shown on the right Y-axis (Fold change of toxicity (vs. DMSO control)). In addition, numerical values such as "-10" in the drug concentration (Drug concentration (M)) on the x-axis ^of FIGS. 6-1 to 6-13 indicate indices. (M)”.

As is clear from FIGS. 6-7-1, 6-7-2, 6-9-1, and 6-9-2, familial Alzheimer's disease for Berculin A (compound 07) and Mer-A2026A (compound 09) In the concentration range (0.32 to 40 nM) where toxicity is not observed in cerebral cortical neurons induced from iPS cells derived from disease patients and/or cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease Aβ42 level lowering effect was also observed. In addition, as is clear from FIGS. 6-8, 6-12, and 6-13, eupeniferdin (compound 08), elaiophyllin (compound 12), and boromycin (compound 13) are associated with familial Alzheimer's disease In both cerebral cortical neurons induced from patient-derived iPS cells and cerebral cortical neurons induced from iPS cells derived from patients with sporadic Alzheimer's disease, Aβ42 production is suppressed particularly in the low concentration range (0.32 to 8 nM). It was shown to be a factor for accumulation of Aβ42 in the brain, which causes Alzheimer's disease.

Since the microorganism-derived compound used in the present invention has the effect of altering the amount of Aβ42 and the Aβ42/40 ratio, it can be used for the prevention, treatment, or diagnosis of Alzheimer's disease.

Claims (4)

A prophylactic or therapeutic agent for Alzheimer's disease comprising Mer-A2026 A or a pharmaceutically acceptable salt thereof. A prophylactic or therapeutic agent for Alzheimer's disease, comprising Mer-A2026A. Use of Mer-A2026A or a pharmaceutically acceptable salt thereof in the manufacture of a prophylactic or therapeutic agent for Alzheimer's disease. Use of Mer-A2026A in the manufacture of a prophylactic or therapeutic agent for Alzheimer's disease.

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