Agent for suppressing aggregation of TDP-43, agent for suppressing cell death of cells in which TDP-43 is overexpressed, and agent for preventing or treating diseases associated with aggregation of TDP-43

Abstract

To provide an agent for suppressing aggregation of TDP-43.SOLUTION: An agent for suppressing aggregation of TDP-43 comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells in which TDP-43 is overexpressed, and an agent for treating or preventing a disease accompanied by TDP-43 aggregation. [Background technology]

[0002] TDP-43 (TAR DNA-binding protein of 43kDa) is a type of heterogeneous nuclear ribonucleoprotein. TDP-43 has been identified as a major component of ubiquitin-positive inclusions that appear in degenerated neurons and glial cells in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 is a nuclear-localized protein, but in cells in which ubiquitin-positive inclusions are formed, TDP-43 translocates from the nucleus to the cytoplasm, aggregates, becomes insoluble, and accumulates in the cytoplasm.

[0003] TDP-43, which accumulates in the brains and spinal cords of ALS and FTLD patients, is abnormally phosphorylated at multiple serine residues at the C-terminus. In ALS and FTLD patients, the full-length TDP-43 molecule does not necessarily aggregate, but rather the C-terminal fragment of TDP-43 aggregates. Previous research has strongly suggested that TDP-43 aggregation causes abnormalities in RNA metabolism and cytotoxicity, leading to the onset and progression of various diseases.

[0004] An example of a disease involving the aggregation of TDP-43 is TDP-43 proteinopathy. Patent Document 1 proposes the use of N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis(methanesulfonate) as a therapeutic or preventive agent for TDP-43 proteinopathy. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5898701 Summary of the Invention [Problem to be solved by the invention]

[0006] One of the objects of the present invention is to provide an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells in which TDP-43 is overexpressed, and an agent for treating or preventing diseases accompanied by TDP-43 aggregation. [Means for solving the problem]

[0007] According to an aspect of the present invention, there is provided an inhibitor of TDP-43 aggregation, comprising at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0008] The above-mentioned inhibitor of TDP-43 aggregation may contain cycloserine or a salt thereof.

[0009] In the above-mentioned inhibitor of TDP-43 aggregation, cycloserine may be D-cycloserine.

[0010] In the above-mentioned inhibitor of TDP-43 aggregation, cycloserine may be L-cycloserine.

[0011] According to an aspect of the present invention, there is provided use of at least one selected from the group consisting of cycloserine and terizidone, and salts thereof in the manufacture of an inhibitor of TDP-43 aggregation.

[0012] In the above uses, cycloserine or a salt thereof may be selected.

[0013] In the above uses, the cycloserine may be D-cycloserine.

[0014] In the above uses, the cycloserine may be L-cycloserine.

[0015] According to an aspect of the present invention, there is provided a method for treating TDP-43 aggregation, comprising administering to a patient suffering from TDP-43 aggregation at least one selected from the group consisting of cycloserine and terizidone, and salts thereof.

[0016] In the above method for treating TDP-43 aggregation, cycloserine or a salt thereof may be selected.

[0017] In the above-mentioned method for treating TDP-43 aggregation, cycloserine may be D-cycloserine.

[0018] In the above-mentioned method for treating TDP-43 aggregation, cycloserine may be L-cycloserine.

[0019] According to an aspect of the present invention, there is provided an agent for suppressing cell death of a cell in which TDP-43 is overexpressed, comprising at least one selected from the group consisting of cycloserine, terizidone, and a salt thereof.

[0020] The above-mentioned inhibitor of cell death of cells in which TDP-43 is overexpressed may contain cycloserine or a salt thereof.

[0021] In the above-mentioned agent for suppressing cell death of cells in which TDP-43 is overexpressed, cycloserine may be D-cycloserine.

[0022] In the above-mentioned agent for suppressing cell death of cells in which TDP-43 is overexpressed, cycloserine may be L-cycloserine.

[0023] According to an aspect of the present invention, there is provided use of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof in the manufacture of an agent for suppressing cell death of cells in which TDP-43 is overexpressed.

[0024] In the above uses, cycloserine or a salt thereof may be selected.

[0025] In the above uses, the cycloserine may be D-cycloserine.

[0026] In the above uses, the cycloserine may be L-cycloserine.

[0027] According to an aspect of the present invention, there is provided a method for treating cell death in cells in which TDP-43 is overexpressed, comprising administering to a patient in which TDP-43 is overexpressed at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0028] In the above-mentioned method for treating cell death in cells overexpressing TDP-43, cycloserine or a salt thereof may be selected.

[0029] In the above-mentioned method for treating cell death of a cell in which TDP-43 is overexpressed, cycloserine may be D-cycloserine.

[0030] In the above-mentioned method for treating cell death of a cell in which TDP-43 is overexpressed, cycloserine may be L-cycloserine.

[0031] According to an aspect of the present invention, there is provided an agent for treating or preventing a disease accompanied by aggregation of TDP-43, comprising at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0032] The above-mentioned therapeutic or prophylactic agent for a disease accompanied by aggregation of TDP-43 may contain cycloserine or a salt thereof.

[0033] In the above-mentioned agent for treating or preventing a disease accompanied by aggregation of TDP-43, cycloserine may be D-cycloserine.

[0034] In the above-mentioned agent for treating or preventing a disease accompanied by aggregation of TDP-43, cycloserine may be L-cycloserine.

[0035] In the above-mentioned therapeutic or prophylactic agent for a disease accompanied by aggregation of TDP-43, the disease accompanied by aggregation of TDP-43 may be TDP-43 proteinopathy.

[0036] In the above-mentioned therapeutic or preventive agent for a disease accompanied by aggregation of TDP-43, the TDP-43 proteinopathy may be at least one selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive amyotrophy.

[0037] In the above-mentioned therapeutic or prophylactic agent for a disease accompanied by aggregation of TDP-43, the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.

[0038] According to an aspect of the present invention, there is provided use of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof in the manufacture of an agent for treating or preventing a disease associated with TDP-43 aggregation.

[0039] In the above uses, cycloserine or a salt thereof may be selected.

[0040] In the above uses, the cycloserine may be D-cycloserine.

[0041] In the above uses, the cycloserine may be L-cycloserine.

[0042] In the above use, the disease associated with TDP-43 aggregation may be a TDP-43 proteinopathy.

[0043] In the above use, the TDP-43 proteinopathy may be at least one selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive muscular dystrophy.

[0044] In the above use, the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.

[0045] According to an embodiment of the present invention, there is provided a method for treating a disease associated with TDP-43 aggregation, comprising administering to a patient suffering from a disease associated with TDP-43 aggregation at least one selected from the group consisting of cycloserine, terizidone, and a salt thereof.

[0046] In the above-mentioned method for treating a disease associated with TDP-43 aggregation, cycloserine or a salt thereof may be selected.

[0047] In the above-mentioned method for treating a disease associated with TDP-43 aggregation, cycloserine may be D-cycloserine.

[0048] In the above-mentioned method for treating a disease associated with TDP-43 aggregation, cycloserine may be L-cycloserine.

[0049] In the above-mentioned method for treating a disease accompanied by TDP-43 aggregation, the disease accompanied by TDP-43 aggregation may be TDP-43 proteinopathy.

[0050] In the above-mentioned method for treating a disease accompanied by aggregation of TDP-43, the TDP-43 proteinopathy may be at least one selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive amyotrophy.

[0051] In the above-mentioned method for treating a disease accompanied by aggregation of TDP-43, the TDP-43 proteinopathy may be amyotrophic lateral sclerosis. Effect of the Invention

[0052] According to the present invention, it is possible to provide an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells in which TDP-43 is overexpressed, and an agent for treating or preventing a disease accompanied by TDP-43 aggregation. [Brief description of the drawings]

[0053] [Figure 1] FIG. 1 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Reference Example 1. [Diagram 2] FIG. 2 is a graph showing the relationship between the time elapsed from transfection of the TDP-43 gene in Reference Example 1 and the proportion of the insoluble fraction of TDP-43 in cells. [Diagram 3] FIG. 3 is a photograph of the cells according to Example 1. [Figure 4] FIG. 4 is a graph showing the relationship between the concentration of the compound according to Example 1 and the relative survival rate of cells. [Diagram 5] FIG. 5 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 2. [Figure 6] FIG. 6 is a graph showing the relationship between the compound of Example 2 and the proportion of the insoluble fraction of TDP-43 in cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The following describes embodiments of the present invention. However, it should not be understood that the following embodiments limit the present invention. From this disclosure, various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0055] The therapeutic or preventive agent for a disease accompanied by aggregation of TDP-43 (TAR DNA-binding protein of 43 kDa), the inhibitor of TDP-43 aggregation, or the inhibitor of cell death of cells in which TDP-43 is overexpressed according to the embodiments includes at least one selected from the group consisting of cycloserine and physiologically acceptable salts thereof. The cycloserine may be D-cycloserine or L-cycloserine.

[0056] Examples of diseases involving the aggregation of TDP-43 include TDP-43 proteinopathies, neurodegenerative diseases, and muscular diseases.

[0057] Examples of TDP-43 proteinopathies include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), primary lateral sclerosis (PLS), and progressive myelopathy (PMA).

[0058] Examples of neurodegenerative diseases include dementia with Lewy bodies (DLB), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Guam-type ALS / Parkinson's complex dementia, hippocampal sclerosis, Pick's disease, Alzheimer's disease, Huntington's disease, Parkinson's disease, argyrophilic grain disease (AGD), chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), and limbic age-related TDP-43 encephalopathy.

[0059] Examples of muscle diseases include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia, sporadic IBM, myofibrillar myopathy, oculopharyngeal muscular dystrophy, and distal myopathy with rimmed vacuoles.

[0060] The IUPAC name of D-cycloserine (CAS number: 68-41-7) is (4R)-4-Amino-1,2-oxazolidin-3-one. The chemical formula of D-cycloserine is C3H6N2O2. The chemical structural formula of D-cycloserine is as follows: [ka]

[0061] The IUPAC name of L-cycloserine (CAS number: 339-72-0) is (4S)-4-amino-1,2-oxazolidin-3-one. The chemical formula of L-cycloserine is C3H6N2O2. The chemical structural formula of L-cycloserine is: [ka]

[0062] According to the embodiments, the therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation, the inhibitor of TDP-43 aggregation, or the inhibitor of cell death of a cell in which TDP-43 is overexpressed may contain at least one prodrug of the above-mentioned compound, or a physiologically acceptable salt thereof.

[0063] For example, terizidone is known as a prodrug of D-cycloserine. The IUPAC name of terizidone (CAS number: 25683-71-0) is 4,4'-{1,4- Phenylenebis[(E)methylylidene(E)azanylylidene]}bis(1,2-oxazolidin-3-one). The chemical formula of terizidone is C 14 H 14 N4O4. The chemical structure of terizidone is: Terizidone is broken down in the body and exerts effects similar to those of D-cycloserine. [ka]

[0064] The therapeutic or preventive agent for a disease accompanied by the aggregation of TDP-43, the inhibitor of the aggregation of TDP-43, or the inhibitor of cell death in cells in which TDP-43 is overexpressed according to the embodiment can be formulated into a pharma- ceutically acceptable dosage form. For example, the therapeutic or preventive agent for a disease accompanied by the aggregation of TDP-43, the inhibitor of the aggregation of TDP-43, or the inhibitor of cell death in cells in which TDP-43 is overexpressed can be formulated into an injection, a solution, a suspension, a tablet, a capsule, a pill, a granule, a syrup, a suppository, an inhalant, and a spray. The injection includes, for example, an injection solution, a sterile powder for injection, and a concentrated solution for injection.

[0065] As described above, it is strongly suggested that the aggregation of TDP-43 causes abnormalities in RNA metabolism and cytotoxicity, leading to the onset and progression of various diseases. The therapeutic or preventive agent for a disease accompanied by the aggregation of TDP-43 according to the embodiment can treat or prevent a disease accompanied by the aggregation of TDP-43 by suppressing the aggregation of TDP-43. Alternatively, the therapeutic or preventive agent for a disease accompanied by the aggregation of TDP-43 according to the embodiment can treat or prevent a disease accompanied by the aggregation of TDP-43 by suppressing cell death of cells in which TDP-43 is overexpressed.

[0066] Although the present invention has been described above by way of the embodiment, the description and drawings forming part of this disclosure should not be understood as limiting the present invention. From this disclosure, various alternative embodiments, examples and operating techniques should become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0067] (Reference example 1) Mouse neuroblastoma (Neuro2a) was seeded on a dish. Neuro2a cells were cultured in a medium of DMEM + 10% FBS at 37°C in the presence of 5% CO2. One day after seeding, differentiation into neurons was initiated. Differentiation into neurons was performed in a medium of DMEM + 2% FBS + 20 μmol / L retinoic acid. Four days after seeding, cells were transfected with TDP-43mRNA synthesized by lipofection. Cells were sampled 6, 9, 12, 15, 18, and 21 hours after transfection. Cell death was observed 21 hours after transfection. For sampling, cells were lysed in RIPA buffer and centrifuged at 22000×g for 30 minutes at 4°C. The supernatant was taken as the soluble fraction, and the precipitate was taken as the insoluble fraction. An equal amount of 2×SDS sample buffer was added to each of them, and the mixture was heated at 95°C for 5 minutes to prepare electrophoretic samples. After each electrophoresis sample was electrophoresed using a polyacrylamide gel, TDP-43 was transferred to a PVDF membrane, and TDP-43 was detected by chemiluminescence using an anti-TDP-43 antibody as the primary antibody and an HRP-conjugated anti-rabbit antibody as the secondary antibody. The results are shown in Figures 1 and 2. It was confirmed that the insoluble fraction of TDP-43 increased in the cells from the time of transfection with the TDP-43 gene until cell death. The insoluble fraction of TDP-43 indicates that TDP-43 is aggregated.

[0068] Example 1 Neuro2a were seeded on dishes. On the first day after seeding, differentiation into neurons was initiated. On the fourth day after seeding, the cells were transfected with the TDP-43 gene. Five hours after transfection, 10 nmol / L, 50 nmol / L, 100 nmol / L, 500 nmol / L, 1000 nmol / L, 5000 nmol / L, or 10,000 nmol / L of D-cycloserine or L-cycloserine was added to the medium. As a control, 10 nmol / L, 50 nmol / L, 100 nmol / L, 500 nmol / L, 1000 nmol / L, 5000 nmol / L, or 10,000 nmol / L of ropinirole was added to the medium. Ropinirole has been reported to have a therapeutic effect on ALS.

[0069] The percentage of dead cells was evaluated on the 7th day after the cells were transfected with the TDP-43 gene. As shown in FIG. 3A, cell death was not observed in cells that were not transfected with the TDP-43 gene. As shown in FIG. 3B, cell death was observed in cells that were transfected with the TDP-43 gene and did not receive D-cycloserine or edaravone. As shown in FIG. 3C, cell death was suppressed in cells that were transfected with the TDP-43 gene and received D-cycloserine.

[0070] As shown in FIG. 4, D-cycloserine and L-cycloserine exhibited a concentration-dependent effect of suppressing cell death. The EC 50 The EC value of L-cycloserine was 128 nmol / L. 50 The EC value for ropinirole was 237 nmol / L. 50 The relative survival rate is the ratio of the survival rate of cells that overexpressed TDP-43 to the survival rate of cells that did not overexpress TDP-43, which was taken as 100%.

[0071] Example 2 Neuro2a were seeded on a dish. On the first day after seeding, differentiation into neurons was initiated. On the fourth day after seeding, the cells were transfected with the TDP-43 gene. Six hours after transfection, 10 μmol / L of D-cycloserine, L-cycloserine, ropinirole, or DMSO was added to the cells, and the cells were cultured for 12 hours. The results of analyzing the soluble and insoluble fractions of TDP-43 in the cells are shown in Figure 5. As shown in Figure 5, in cells treated with DMSO as a control, it was confirmed that the insoluble fraction of TDP-43 was greater than the soluble fraction of TDP-43. In contrast, in cells treated with D-cycloserine and L-cycloserine, it was confirmed that the soluble fraction of TDP-43 was greater than the insoluble fraction of TDP-43.

[0072] The ratio of the insoluble fraction to the total of the soluble and insoluble fractions of TDP-43 in cells was quantified, and the results are shown in Figure 6. In cells treated with D-cycloserine and L-cycloserine, the ratio of the insoluble fraction was less than half.

Claims

1. An inhibitor of TDP-43 aggregation, comprising at least one member selected from the group consisting of cycloserine, terizidone, and salts thereof.

2. The TDP-43 aggregation inhibitor according to claim 1, comprising cycloserine or a salt thereof.

3. The TDP-43 aggregation inhibitor according to claim 1 or 2, wherein the cycloserine is D-cycloserine.

4. The TDP-43 aggregation inhibitor according to claim 1 or 2, wherein the cycloserine is L-cycloserine.

5. An agent for suppressing cell death in cells in which TDP-43 is overexpressed, comprising at least one member selected from the group consisting of cycloserine, terizidone, and salts thereof.

6. The inhibitor of cell death in a cell in which TDP-43 is overexpressed according to claim 5, comprising cycloserine or a salt thereof.

7. The inhibitor of cell death in a cell in which TDP-43 is overexpressed according to claim 5 or 6, wherein the cycloserine is D-cycloserine.

8. The inhibitor of cell death in cells in which TDP-43 is overexpressed according to claim 5 or 6, wherein the cycloserine is L-cycloserine.

9. An agent for treating or preventing a disease accompanied by aggregation of TDP-43, comprising at least one member selected from the group consisting of cycloserine, terizidone, and salts thereof.

10. The therapeutic or preventive agent for a disease accompanied by aggregation of TDP-43 according to claim 9, comprising cycloserine or a salt thereof.

11. The therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation according to claim 9 or 10, wherein the cycloserine is D-cycloserine.

12. The therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation according to claim 9 or 10, wherein the cycloserine is L-cycloserine.

13. The therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation according to any one of claims 9 to 12, wherein the disease accompanied by TDP-43 aggregation is TDP-43 proteinopathy.

14. The therapeutic or preventive agent for a disease accompanied by aggregation of TDP-43 according to claim 13, wherein the TDP-43 proteinopathy is at least one selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive muscular atrophy.

15. The therapeutic or preventive agent for a disease accompanied by aggregation of TDP-43 according to claim 13 or 14, wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis.

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