WO2023171106A1 - 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 - Google Patents

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 Download PDF

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WO2023171106A1
WO2023171106A1 PCT/JP2023/000180 JP2023000180W WO2023171106A1 WO 2023171106 A1 WO2023171106 A1 WO 2023171106A1 JP 2023000180 W JP2023000180 W JP 2023000180W WO 2023171106 A1 WO2023171106 A1 WO 2023171106A1
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tdp
cycloserine
aggregation
agent
cells
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勝久 堀本
貴之 木卜
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ソシウム株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells overexpressing TDP-43, and a therapeutic or preventive agent for diseases accompanied by TDP-43 aggregation.
  • TDP-43 (TAR DNA-binding protein of 43kDa) is a type of heterogeneous nuclear ribonucleic acid protein. TDP-43 was identified as a major constituent protein of ubiquitin-positive inclusion bodies 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 inclusion bodies are formed, TDP-43 moves from the nucleus to the cytoplasm, aggregates, becomes insolubilized, and accumulates in the cytoplasm.
  • ALS amyotrophic lateral sclerosis
  • FTLD frontotemporal lobar degeneration
  • TDP-43 which accumulates in the brains and spinal cords of ALS and FTLD patients, has multiple serine residues at its C-terminus abnormally phosphorylated. In ALS and FTLD patients, the full-length TDP-43 molecule does not need to be aggregated, but the C-terminal fragment of TDP-43 is aggregated. Previous studies have strongly suggested that aggregation of TDP-43 causes abnormalities in RNA metabolism and cell toxicity, leading to the onset and progression of various diseases.
  • TDP-43 proteinopathy is known as an example of a disease accompanied by TDP-43 aggregation.
  • 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. are doing.
  • the present invention aims to provide an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells overexpressing TDP-43, and an agent for treating or preventing diseases accompanied by TDP-43 aggregation. be one of the.
  • an inhibitor of TDP-43 aggregation which comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.
  • the above-mentioned TDP-43 aggregation inhibitor may contain cycloserine or a salt thereof.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • cycloserine or a salt thereof may be selected.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • the aggregation of TDP-43 comprises administering to a patient with agglutination of TDP-43 at least one selected from the group consisting of cycloserine and terizidone, and salts thereof.
  • a method of treatment is provided.
  • cycloserine or a salt thereof may be selected.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • an agent for suppressing cell death of cells overexpressing TDP-43 which comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof. .
  • the above-mentioned inhibitor of cell death of cells overexpressing TDP-43 may contain cycloserine or a salt thereof.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • cycloserine or a salt thereof may be selected.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • the method of administering to a patient overexpressing TDP-43 at least one selected from the group consisting of cycloserine and terizidone, and salts thereof.
  • Methods of treating cell death of overexpressing cells are provided.
  • cycloserine or a salt thereof may be selected.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • a therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.
  • the therapeutic or preventive agent for a disease accompanied by aggregation of TDP-43 described above may contain cycloserine or a salt thereof.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • the disease accompanied by TDP-43 aggregation may be TDP-43 proteinopathy.
  • the TDP-43 proteinopathy is associated with amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive muscular atrophy. It may be at least one selected from the group consisting of:
  • the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.
  • cycloserine or a salt thereof may be selected.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • the disease accompanied by TDP-43 aggregation may be TDP-43 proteinopathy.
  • 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 amyotrophy. There may be.
  • the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.
  • the method of producing TDP-43 comprises administering at least one selected from the group consisting of cycloserine, terizidone, and salts thereof to a patient with a disease accompanied by aggregation of TDP-43.
  • Methods of treating diseases involving aggregation are provided.
  • Cycloserine or a salt thereof may be selected in the above-mentioned method for treating a disease accompanied by TDP-43 aggregation.
  • cycloserine may be D-cycloserine.
  • cycloserine may be L-cycloserine.
  • the disease accompanied by TDP-43 aggregation may be TDP-43 proteinopathy.
  • TDP-43 proteinopathy is caused by amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive muscular atrophy. It may be at least one selected from the group consisting of:
  • the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.
  • an inhibitor of TDP-43 aggregation an inhibitor of cell death of cells overexpressing TDP-43, and an agent for treating or preventing diseases accompanied by TDP-43 aggregation.
  • FIG. 1 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Reference Example 1.
  • FIG. 2 is a graph showing the relationship between the elapsed time from transfection of the TDP-43 gene according to Reference Example 1 and the proportion of the insoluble fraction of TDP-43 in cells.
  • FIG. 3 is a photograph of cells according to Example 1.
  • 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.
  • FIG. 5 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 2.
  • FIG. 6 is a graph showing the relationship between the compound according to Example 2 and the proportion of the insoluble fraction of TDP-43 in cells.
  • FIG. 7 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 3.
  • FIG. 8 is a graph showing the percentage of insoluble fraction of TDP-43 according to Example 3.
  • An agent for treating or preventing a disease accompanied by aggregation of TDP-43 (TAR DNA-binding protein of 43 kDa), an inhibitor of aggregation of TDP-43, or cell death of cells overexpressing TDP-43 according to an embodiment
  • the inhibitor includes at least one selected from the group consisting of cycloserine and physiologically acceptable salts thereof. Cycloserine may be D-cycloserine or L-cycloserine.
  • TDP-43 aggregation examples include TDP-43 proteinopathy, neurodegenerative diseases, and muscle diseases.
  • TDP-43 proteinopathies include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), primary lateral sclerosis (PLS), and progressive muscular atrophy (PMA). Can be mentioned.
  • ALS amyotrophic lateral sclerosis
  • FTLD frontotemporal lobar degeneration
  • PLS primary lateral sclerosis
  • PMA progressive muscular atrophy
  • neurodegenerative diseases include dementia with Lewy bodies (DLB), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Guam-type ALS/Parkinson complex dementia, and hippocampal sclerosis.
  • DLB dementia with Lewy bodies
  • CBD corticobasal degeneration
  • PSP progressive supranuclear palsy
  • Guam-type ALS/Parkinson complex dementia and hippocampal sclerosis.
  • Pick's disease Alzheimer's disease, Huntington's disease, Parkinson's disease, argyrophilic granular disease (AGD), chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), and limbic age-related TDP-43 encephalopathy.
  • muscle diseases include inclusion body myopathy with bone Paget disease and frontotemporal dementia, sporadic IBM, myofibrillar myopathy, oculopharyngeal muscular dystrophy, and distal myopathy with fringed vacuoles. It will be done.
  • D-cycloserine (CAS number: 68-41-7) is (4R)-4-Amino-1,2-oxazolidin-3-one.
  • the chemical formula of D-cycloserine is C 3 H 6 N 2 O 2 .
  • the chemical structural formula of D-cycloserine is as follows.
  • L-cycloserine (CAS number: 339-72-0) is (4S)-4-amino-1,2-oxazolidin-3-one.
  • the chemical formula of L-cycloserine is C 3 H 6 N 2 O 2 .
  • the chemical structural formula of L-cycloserine is as follows.
  • the therapeutic or preventive agent for diseases accompanied by TDP-43 aggregation, the inhibitor of TDP-43 aggregation, or the inhibitor of cell death of cells in which TDP-43 is overexpressed according to the embodiments is a compound of the above. It may contain at least one prodrug, or a physiologically acceptable salt thereof.
  • 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) It is.
  • the chemical formula of terizidone is C 14 H 14 N 4 O 4 .
  • the chemical structural formula of terizidone is as follows. Terizidone is broken down in the body and produces effects similar to D-cycloserine.
  • 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 cells overexpressing TDP-43 is a pharmaceutically acceptable agent. It can be formulated into the following dosage form.
  • therapeutic or preventive agents for diseases accompanied by TDP-43 aggregation, inhibitors of TDP-43 aggregation, or inhibitors of cell death of cells overexpressing TDP-43 can be used as injections, solutions, or suspensions. It can be formulated into suspensions, tablets, capsules, pills, granules, syrups, suppositories, inhalants, and sprays.
  • injectables include, for example, injectable solutions, injectable sterile powders, and injectable concentrates.
  • the agent for treating or preventing a disease accompanied by TDP-43 aggregation according to the embodiment can treat or prevent a disease accompanied by TDP-43 aggregation by inhibiting TDP-43 aggregation.
  • the therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation according to an embodiment can treat a disease accompanied by TDP-43 aggregation by suppressing cell death of cells overexpressing TDP-43. Or prevention is possible.
  • Neuro2a Mouse neuroblastoma (Neuro2a) was seeded in a dish. Neuro2a cells were cultured in a medium of DMEM + 10% FBS at 37° C. in the presence of 5% CO 2 . On the first day after seeding, differentiation into neurons was initiated. Differentiation into neurons was performed in a medium of DMEM + 2% FBS + 20 ⁇ mol/L retinoic acid. On the fourth day after seeding, the cells were transfected with the synthesized TDP-43 mRNA by lipofection. Cells were sampled 6, 9, 12, 15, 18, and 21 hours after transfection. Cell death was observed 21 hours after transfection.
  • Neuro2a was seeded in a dish. On the first day after seeding, differentiation into neurons was initiated. Four days after seeding, 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 10000 nmol/L of D-cycloserine or L-cycloserine was added to the medium. .
  • the percentage of dead cells was evaluated 7 days after transfecting the cells with the TDP-43 gene. As shown in Figure 3A, no cell death was observed in cells that were not transfected with the TDP-43 gene. As shown in FIG. 3B, cell death was observed in cells transfected with the TDP-43 gene and to which D-cycloserine or edaravone was not added. As shown in FIG. 3C, cell death was suppressed in cells transfected with the TDP-43 gene and supplemented with D-cycloserine.
  • D-cycloserine and L-cycloserine exhibited a concentration-dependent effect of suppressing cell death.
  • the EC 50 of D-cycloserine was 128 nmol/L.
  • the EC 50 of L-cycloserine was 237 nmol/L.
  • the EC 50 of ropinirole was 198 nmol/L. Note that the relative survival rate represents the ratio of the number of surviving cells that overexpressed TDP-43 to the number of surviving cells that did not overexpress TDP-43 as 100%.
  • Example 2 Neuro2a was seeded in a dish. On the first day after seeding, differentiation into neurons was initiated. Four days after seeding, cells were transfected with the TDP-43 gene. Six hours after transfection, 10 ⁇ mol/L of D-cycloserine, L-cycloserine, ropinirole, or DMSO was added to the cells and cultured for 12 hours. Thereafter, the soluble and insoluble fractions of TDP-43 in the cells were analyzed, and the results are shown in FIG. As shown in FIG. 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.
  • Figure 6 shows the results of quantifying the ratio of the insoluble fraction to the total of the soluble and insoluble fractions of TDP-43 in cells. In cells treated with D-cycloserine and L-cycloserine, the proportion of insoluble fraction was less than half.
  • Example 3 100 ⁇ L of EHS gel basement membrane matrix (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 12.5 mL of DMEM (Dulbecco's Modified Eagle Medium, Fuji Film Wako Pure Chemical Industries, Ltd.) and mixed, and 600 ⁇ L/well was added to a 24-well plate. After allowing the plate to stand at room temperature for 2 hours, the solution was removed from the wells. Next, 600 ⁇ L/well of DMEM was added, and the plate was left standing at room temperature for 2 hours.
  • DMEM Dynamic Eagle Medium
  • Motor neurons derived from iPS cells derived from healthy individuals and motor neurons derived from iPS cells derived from ALS patients were obtained from iX Cells Biotechnologies. Motor neurons were quickly thawed in a thermostat at 37°C and suspended in motor neuron maintenance medium (iX Cells Biotechnologies). After centrifuging the medium at 600 ⁇ g for 5 minutes to collect motor neurons, the medium was removed and the motor neurons were suspended in motor neuron maintenance medium.
  • each of the multiple wells of the matrix-coated plate was filled with 600 ⁇ L of the medium containing the motor neurons and incubated at 37°C in the presence of 5% CO2. Motor neurons were cultured under every few days, 300 ⁇ L of medium was removed from each of the wells and 300 ⁇ L of fresh medium was added to each of the wells.
  • protease inhibitor and phosphatase inhibitor cocktail (Halt Protease and Phosphatase Inhibitor Cocktail, 10OX, registered trademark, Thermo Fisher Scientific) to 500 ⁇ L of protein extraction buffer containing surfactant (RIPA buffer, Nacalai Tesque). was added to prepare a cell extraction solution.
  • 50 ⁇ L of cell extraction solution was added to motor neurons cultured for 20 days in the presence of DMSO, D-cycloserine, or ropinirole, and the cell extract was collected into a 1.5 mL tube by pipetting. Cell extracts were centrifuged at 21,000 ⁇ g for 30 minutes at 4°C.
  • the photographed image of the PVDF membrane was opened using ImageJ software, and black and white was inverted using the Invert command.
  • the TDP-43 bands in each lane were successively surrounded by square frames of the same size, and the band intensities were quantified using the Measure command.
  • the ratio of the insoluble fraction of TDP-43 was calculated from the band intensity of the soluble fraction of TDP-43 and the band intensity of the insoluble fraction of TDP-43 of each sample according to the following formula.
  • R ⁇ I I /(I I +I S ) ⁇ 100
  • R represents the proportion (%) of the insoluble fraction of TDP-43
  • II represents the band intensity of the insoluble fraction of TDP-43
  • IS represents the band intensity of the soluble fraction of TDP-43.
  • the test was conducted three times, and the average value of the proportion of the insoluble fraction of TDP-43 was calculated.
  • the percentage of the insoluble fraction of TDP-43 in each test is shown as a dot, and the average value is shown as a bar.
  • the average percentage of TDP-43 insoluble fraction was approximately 7.4%.
  • the average percentage of TDP-43 insoluble fraction in motor neurons derived from iPS cells derived from ALS patients was approximately 46.1%.
  • the average percentage of the insoluble fraction of TDP-43 decreased to about 17.4% in motor neurons derived from iPS cells derived from ALS patients.
  • the average percentage of the insoluble fraction of TDP-43 decreased to about 19.3% in motor neurons derived from iPS cells derived from ALS patients.

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Abstract

An agent for suppressing aggregation of TDP-43, the agent comprising at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

Description

ＴＤＰ－４３の凝集の抑制剤、ＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤、及びＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤An inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells overexpressing TDP-43, and a therapeutic or preventive agent for diseases accompanied by TDP-43 aggregation.
  本発明は、ＴＤＰ－４３の凝集の抑制剤、ＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤、及びＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤に関する。 The present invention relates to an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells overexpressing TDP-43, and a therapeutic or preventive agent for diseases accompanied by TDP-43 aggregation.
　ＴＤＰ－４３（ＴＡＲ　ＤＮＡ－ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎ　ｏｆ　４３ｋＤａ）は、不均一核リボ核酸タンパク質の一種である。ＴＤＰ－４３は、筋萎縮性側索硬化症（ＡＬＳ）及び前頭側頭葉変性症（ＦＴＬＤ）の変性神経細胞やグリア細胞に出現するユビキチン陽性封入体の主要構成タンパク質として同定された。ＴＤＰ－４３は核局在タンパク質であるが、ユビキチン陽性封入体が形成されている細胞では、ＴＤＰ－４３が核から細胞質へ移行し、凝集、不溶化して、細胞質に蓄積する。 TDP-43 (TAR DNA-binding protein of 43kDa) is a type of heterogeneous nuclear ribonucleic acid protein. TDP-43 was identified as a major constituent protein of ubiquitin-positive inclusion bodies 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 inclusion bodies are formed, TDP-43 moves from the nucleus to the cytoplasm, aggregates, becomes insolubilized, and accumulates in the cytoplasm.
　ＡＬＳ患者及びＦＴＬＤ患者の脳や脊髄に蓄積するＴＤＰ－４３は、Ｃ末端の複数のセリン残基が異常にリン酸化されている。ＡＬＳ患者及びＦＴＬＤ患者において、ＴＤＰ－４３の全長分子が凝集している必要はなく、ＴＤＰ－４３のＣ末端断片が凝集している。これまでの研究により、ＴＤＰ－４３の凝集により、ＲＮＡの代謝に異常が生じたり、細胞毒性を招いたりして、様々な病気が発症及び進行することが強く示唆されている。 TDP-43, which accumulates in the brains and spinal cords of ALS and FTLD patients, has multiple serine residues at its C-terminus abnormally phosphorylated. In ALS and FTLD patients, the full-length TDP-43 molecule does not need to be aggregated, but the C-terminal fragment of TDP-43 is aggregated. Previous studies have strongly suggested that aggregation of TDP-43 causes abnormalities in RNA metabolism and cell toxicity, leading to the onset and progression of various diseases.
　ＴＤＰ－４３の凝集を伴う疾患の例としては、ＴＤＰ－４３プロテイノパチーが知られている。特許文献１は、ＴＤＰ－４３プロテイノパチーの治療又は予防剤として、Ｎ，Ｎ，Ｎ’，Ｎ’，－テトラメチル－１０Ｈ－フェノチアジン－３，７－ジアミニウムビス（メタンスルホネート）を用いることを提案している。 TDP-43 proteinopathy is known as an example of a disease accompanied by TDP-43 aggregation. 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. are doing.
特許第５８９８７０１号Patent No. 5898701
　本発明は、ＴＤＰ－４３の凝集の抑制剤、ＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤、及びＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤を提供することを目的の一つとする。 The present invention aims to provide an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells overexpressing TDP-43, and an agent for treating or preventing diseases accompanied by TDP-43 aggregation. be one of the.
　本発明の態様によれば、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを含む、ＴＤＰ－４３の凝集の抑制剤が提供される。 According to an aspect of the present invention, an inhibitor of TDP-43 aggregation is provided, which comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.
　上記のＴＤＰ－４３の凝集の抑制剤が、サイクロセリン又はその塩を含んでいてもよい。 The above-mentioned TDP-43 aggregation inhibitor may contain cycloserine or a salt thereof.
　上記のＴＤＰ－４３の凝集の抑制剤において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above-mentioned TDP-43 aggregation inhibitor, cycloserine may be D-cycloserine.
　上記のＴＤＰ－４３の凝集の抑制剤において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above-mentioned TDP-43 aggregation inhibitor, cycloserine may be L-cycloserine.
　本発明の態様によれば、ＴＤＰ－４３の凝集の抑制剤の製造における、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つの使用が提供される。 According to an aspect of the present invention, there is provided the use of at least one selected from the group consisting of cycloserine and terizidone, and salts thereof, in the manufacture of an inhibitor of aggregation of TDP-43.
　上記の使用において、サイクロセリン又はその塩が選択されてもよい。 In the above use, cycloserine or a salt thereof may be selected.
　上記の使用において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above use, cycloserine may be D-cycloserine.
　上記の使用において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above use, cycloserine may be L-cycloserine.
　本発明の態様によれば、ＴＤＰ－４３が凝集している患者に、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを投与することを含む、ＴＤＰ－４３の凝集の治療方法が提供される。 According to an aspect of the invention, the aggregation of TDP-43 comprises administering to a patient with agglutination of TDP-43 at least one selected from the group consisting of cycloserine and terizidone, and salts thereof. A method of treatment is provided.
　上記のＴＤＰ－４３の凝集の治療方法において、サイクロセリン又はその塩が選択されてもよい。 In the above method for treating TDP-43 aggregation, cycloserine or a salt thereof may be selected.
　上記のＴＤＰ－４３の凝集の治療方法において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above method for treating TDP-43 aggregation, cycloserine may be D-cycloserine.
　上記のＴＤＰ－４３の凝集の治療方法において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above method for treating TDP-43 aggregation, cycloserine may be L-cycloserine.
　本発明の態様によれば、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを含む、ＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤が提供される。 According to an aspect of the present invention, there is provided an agent for suppressing cell death of cells overexpressing TDP-43, which comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof. .
　上記のＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤が、サイクロセリン又はその塩を含んでいてもよい。 The above-mentioned inhibitor of cell death of cells overexpressing TDP-43 may contain cycloserine or a salt thereof.
　上記のＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above-mentioned inhibitor of cell death of cells overexpressing TDP-43, cycloserine may be D-cycloserine.
　上記のＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above-mentioned inhibitor of cell death of cells overexpressing TDP-43, cycloserine may be L-cycloserine.
　本発明の態様によれば、ＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤の製造における、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つの使用が提供される。 According to an aspect of the present invention, there is provided the use of at least one selected from the group consisting of cycloserine and terizidone, and salts thereof, in the manufacture of an inhibitor of cell death of cells overexpressing TDP-43. be done.
　上記の使用において、サイクロセリン又はその塩が選択されてもよい。 In the above use, cycloserine or a salt thereof may be selected.
　上記の使用において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above use, cycloserine may be D-cycloserine.
　上記の使用において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above use, cycloserine may be L-cycloserine.
　本発明の態様によれば、ＴＤＰ－４３が過剰発現している患者に、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを投与することを含む、ＴＤＰ－４３が過剰発現している細胞の細胞死の治療方法が提供される。 According to an aspect of the present invention, the method of administering to a patient overexpressing TDP-43 at least one selected from the group consisting of cycloserine and terizidone, and salts thereof. Methods of treating cell death of overexpressing cells are provided.
　上記のＴＤＰ－４３が過剰発現している細胞の細胞死の治療方法において、サイクロセリン又はその塩が選択されてもよい。 In the method for treating cell death in cells overexpressing TDP-43, cycloserine or a salt thereof may be selected.
　上記のＴＤＰ－４３が過剰発現している細胞の細胞死の治療方法において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above method for treating cell death in cells overexpressing TDP-43, cycloserine may be D-cycloserine.
　上記のＴＤＰ－４３が過剰発現している細胞の細胞死の治療方法において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above method for treating cell death in cells overexpressing TDP-43, cycloserine may be L-cycloserine.
　本発明の態様によれば、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを含む、ＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤が提供される。 According to an aspect of the present invention, a therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation is provided, which comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤が、サイクロセリン又はその塩を含んでいてもよい。 The therapeutic or preventive agent for a disease accompanied by aggregation of TDP-43 described above may contain cycloserine or a salt thereof.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation, cycloserine may be D-cycloserine.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation, cycloserine may be L-cycloserine.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤において、ＴＤＰ－４３の凝集を伴う疾患が、ＴＤＰ－４３プロテイノパチーであってもよい。 In the above therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation, the disease accompanied by TDP-43 aggregation may be TDP-43 proteinopathy.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤において、ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症、前頭側頭葉変性症、原発性側索硬化症、及び進行性筋萎縮症からなる群から選択される少なくとも１つであってもよい。 In the treatment or prevention agent for a disease accompanied by TDP-43 aggregation, the TDP-43 proteinopathy is associated with amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive muscular atrophy. It may be at least one selected from the group consisting of:
　上記のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤において、ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症であってもよい。 In the above therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation, the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.
　本発明の態様によれば、ＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤の製造における、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つの使用が提供される。 According to an aspect of the present invention, there is provided the use of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof in the production of a therapeutic or preventive agent for a disease involving aggregation of TDP-43.
　上記の使用において、サイクロセリン又はその塩が選択されてもよい。 In the above use, cycloserine or a salt thereof may be selected.
　上記の使用において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above use, cycloserine may be D-cycloserine.
　上記の使用において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above use, cycloserine may be L-cycloserine.
　上記の使用において、ＴＤＰ－４３の凝集を伴う疾患が、ＴＤＰ－４３プロテイノパチーであってもよい。 In the above use, the disease accompanied by TDP-43 aggregation may be TDP-43 proteinopathy.
　上記の使用において、ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症、前頭側頭葉変性症、原発性側索硬化症、及び進行性筋萎縮症からなる群から選択される少なくとも１つであってもよい。 In the above use, 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 amyotrophy. There may be.
　上記の使用において、ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症であってもよい。 In the above use, the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.
　本発明の態様によれば、ＴＤＰ－４３の凝集を伴う疾患の患者に、サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを投与することを含む、ＴＤＰ－４３の凝集を伴う疾患の治療方法が提供される。 According to an aspect of the present invention, the method of producing TDP-43 comprises administering at least one selected from the group consisting of cycloserine, terizidone, and salts thereof to a patient with a disease accompanied by aggregation of TDP-43. Methods of treating diseases involving aggregation are provided.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療方法において、サイクロセリン又はその塩が選択されてもよい。 Cycloserine or a salt thereof may be selected in the above-mentioned method for treating a disease accompanied by TDP-43 aggregation.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療方法において、サイクロセリンが、Ｄ－サイクロセリンであってもよい。 In the above-mentioned method for treating a disease accompanied by aggregation of TDP-43, cycloserine may be D-cycloserine.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療方法において、サイクロセリンが、Ｌ－サイクロセリンであってもよい。 In the above-mentioned method for treating a disease accompanied by aggregation of TDP-43, cycloserine may be L-cycloserine.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療方法において、ＴＤＰ－４３の凝集を伴う疾患が、ＴＤＰ－４３プロテイノパチーであってもよい。 In the above method for treating a disease accompanied by TDP-43 aggregation, the disease accompanied by TDP-43 aggregation may be TDP-43 proteinopathy.
　上記のＴＤＰ－４３の凝集を伴う疾患の治療方法において、ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症、前頭側頭葉変性症、原発性側索硬化症、及び進行性筋萎縮症からなる群から選択される少なくとも１つであってもよい。 In the above method for treating a disease accompanied by TDP-43 aggregation, TDP-43 proteinopathy is caused by amyotrophic lateral sclerosis, frontotemporal lobar degeneration, primary lateral sclerosis, and progressive muscular atrophy. It may be at least one selected from the group consisting of:
　上記のＴＤＰ－４３の凝集を伴う疾患の治療方法において、ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症であってもよい。 In the above method for treating a disease involving TDP-43 aggregation, the TDP-43 proteinopathy may be amyotrophic lateral sclerosis.
  本発明によれば、ＴＤＰ－４３の凝集の抑制剤、ＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤、及びＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤を提供可能である。 According to the present invention, it is possible to provide an inhibitor of TDP-43 aggregation, an inhibitor of cell death of cells overexpressing TDP-43, and an agent for treating or preventing diseases accompanied by TDP-43 aggregation. be.
図１は、参考例１に係るＴＤＰ－４３の可溶フラクションとＴＤＰ－４３の不溶フラクションを示す写真である。FIG. 1 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Reference Example 1. 図２は、参考例１に係るＴＤＰ－４３遺伝子のトランスフェクションからの経過時間と、細胞におけるＴＤＰ－４３の不溶フラクションの割合と、の関係を示すグラフである。FIG. 2 is a graph showing the relationship between the elapsed time from transfection of the TDP-43 gene according to Reference Example 1 and the proportion of the insoluble fraction of TDP-43 in cells. 図３は、実施例１に係る細胞の写真である。FIG. 3 is a photograph of cells according to Example 1. 図４は、実施例１に係る化合物の濃度と、細胞の相対生存率と、の関係を示すグラフである。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. 図５は、実施例２に係るＴＤＰ－４３の可溶フラクションとＴＤＰ－４３の不溶フラクションを示す写真である。FIG. 5 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 2. 図６は、実施例２に係る化合物と、細胞におけるＴＤＰ－４３の不溶フラクションの割合と、の関係を示すグラフである。FIG. 6 is a graph showing the relationship between the compound according to Example 2 and the proportion of the insoluble fraction of TDP-43 in cells. 図７は、実施例３に係るＴＤＰ－４３の可溶フラクションとＴＤＰ－４３の不溶フラクションを示す写真である。FIG. 7 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 3. 図８は、実施例３に係るＴＤＰ－４３の不溶フラクションの割合を示すグラフである。FIG. 8 is a graph showing the percentage of insoluble fraction of TDP-43 according to Example 3.
　以下に本発明の実施形態を説明する。ただし、以下の実施形態が本発明を限定するものであると理解するべきではない。本開示から当業者には様々な代替実施形態、実施例及び運用技術が明らかになるはずである。本発明はここでは記載していない様々な実施形態等を包含するということを理解すべきである。 Embodiments of the present invention will be described below. However, it should not be understood that the following embodiments limit the present invention. Various alternative embodiments, implementations, and operational techniques will be apparent to those skilled in the art from this disclosure. It should be understood that the invention encompasses various embodiments and the like that are not described herein.
　実施形態に係るＴＤＰ－４３（ＴＡＲ　ＤＮＡ－ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎ　ｏｆ　４３ｋＤａ）の凝集を伴う疾患の治療又は予防剤、ＴＤＰ－４３の凝集の抑制剤、又はＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤は、サイクロセリン及びその生理学的に許容される塩からなる群から選択される少なくとも１つを含む。サイクロセリンは、Ｄ－サイクロセリンであってもよいし、Ｌ－サイクロセリンであってもよい。 An agent for treating or preventing a disease accompanied by aggregation of TDP-43 (TAR DNA-binding protein of 43 kDa), an inhibitor of aggregation of TDP-43, or cell death of cells overexpressing TDP-43 according to an embodiment The inhibitor includes at least one selected from the group consisting of cycloserine and physiologically acceptable salts thereof. Cycloserine may be D-cycloserine or L-cycloserine.
　ＴＤＰ－４３の凝集を伴う疾患の例としては、ＴＤＰ－４３プロテイノパチー、神経変性疾患、及び筋疾患が挙げられる。 Examples of diseases involving TDP-43 aggregation include TDP-43 proteinopathy, neurodegenerative diseases, and muscle diseases.
　ＴＤＰ－４３プロテイノパチーの例としては、筋萎縮性側索硬化症（ＡＬＳ）、前頭側頭葉変性症（ＦＴＬＤ）、原発性側索硬化症（ＰＬＳ）、及び進行性筋萎縮症（ＰＭＡ）が挙げられる。 Examples of TDP-43 proteinopathies include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), primary lateral sclerosis (PLS), and progressive muscular atrophy (PMA). Can be mentioned.
　神経変性疾患の例としては、レビー小体型認知症（ＤＬＢ）、大脳皮質基底核変性症（ＣＢＤ）、進行性核上性麻痺（ＰＳＰ）、グアム型ＡＬＳ／パーキンソン複合体認知症、海馬硬化症、ピック病、アルツハイマー病、ハンチントン病、パーキンソン病、嗜銀顆粒性病（ＡＧＤ）、慢性外傷性脳症（ＣＴＥ）、多系統萎縮症（ＭＳＡ）、及び大脳辺縁型加齢性ＴＤＰ－４３脳症が挙げられる。 Examples of neurodegenerative diseases include dementia with Lewy bodies (DLB), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Guam-type ALS/Parkinson complex dementia, and hippocampal sclerosis. , Pick's disease, Alzheimer's disease, Huntington's disease, Parkinson's disease, argyrophilic granular disease (AGD), chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), and limbic age-related TDP-43 encephalopathy. Can be mentioned.
　筋疾患の例としては、骨Ｐａｇｅｔ病及び前頭側頭型認知症を伴う封入体ミオパチー、散発性ＩＢＭ、筋原線維性ミオパチー、眼咽頭型筋ジストロフィー、及び縁取り空胞を伴う遠位型ミオパチーが挙げられる。 Examples of muscle diseases include inclusion body myopathy with bone Paget disease and frontotemporal dementia, sporadic IBM, myofibrillar myopathy, oculopharyngeal muscular dystrophy, and distal myopathy with fringed vacuoles. It will be done.
　Ｄ－サイクロセリン（ＣＡＳ番号：６８－４１－７）のＩＵＰＡＣ名は、（４Ｒ）－４－Ａｍｉｎｏ－１，２－ｏｘａｚｏｌｉｄｉｎ－３－ｏｎｅである。Ｄ－サイクロセリンの化学式は、Ｃ3Ｈ6Ｎ2Ｏ2である。Ｄ－サイクロセリンの化学構造式は、以下である。
Figure JPOXMLDOC01-appb-C000001
 The IUPAC name for D-cycloserine (CAS number: 68-41-7) is (4R)-4-Amino-1,2-oxazolidin-3-one. The chemical formula of D-cycloserine is C 3 H 6 N 2 O 2 . The chemical structural formula of D-cycloserine is as follows.
Figure JPOXMLDOC01-appb-C000001
　Ｌ－サイクロセリン（ＣＡＳ番号：３３９－７２－０）のＩＵＰＡＣ名は、（４Ｓ）－４－ａｍｉｎｏ－１，２－ｏｘａｚｏｌｉｄｉｎ－３－ｏｎｅである。Ｌ－サイクロセリンの化学式は、Ｃ3Ｈ6Ｎ2Ｏ2である。Ｌ－サイクロセリンの化学構造式は、以下である。
Figure JPOXMLDOC01-appb-C000002
 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 C 3 H 6 N 2 O 2 . The chemical structural formula of L-cycloserine is as follows.
Figure JPOXMLDOC01-appb-C000002
　実施形態に係るＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤、ＴＤＰ－４３の凝集の抑制剤、又はＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤は、上記の化合物の少なくとも１つのプロドラッグ、又はそれの生理学的に許容される塩を含んでいてもよい。 The therapeutic or preventive agent for diseases accompanied by TDP-43 aggregation, the inhibitor of TDP-43 aggregation, or the inhibitor of cell death of cells in which TDP-43 is overexpressed according to the embodiments is a compound of the above. It may contain at least one prodrug, or a physiologically acceptable salt thereof.
　例えば、Ｄ－サイクロセリンのプロドラッグとして、テリジドンが知られている。テリジドン（ＣＡＳ番号：２５６８３－７１－０）のＩＵＰＡＣ名は、４，４'－｛１，４－Ｐｈｅｎｙｌｅｎｅｂｉｓ［（Ｅ）ｍｅｔｈｙｌｙｌｉｄｅｎｅ（Ｅ）ａｚａｎｙｌｙｌｉｄｅｎｅ］｝ｂｉｓ（１，２－ｏｘａｚｏｌｉｄｉｎ－３－ｏｎｅ）である。テリジドンの化学式は、Ｃ14Ｈ14Ｎ4Ｏ4である。テリジドンの化学構造式は、以下である。テリジドンは体内で分解され、Ｄ－サイクロセリンと同様の効果を発する。
Figure JPOXMLDOC01-appb-C000003
 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) It is. The chemical formula of terizidone is C 14 H 14 N 4 O 4 . The chemical structural formula of terizidone is as follows. Terizidone is broken down in the body and produces effects similar to D-cycloserine.
Figure JPOXMLDOC01-appb-C000003
　実施形態に係るＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤、ＴＤＰ－４３の凝集の抑制剤、又はＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤は、薬学的に許容される剤形に製剤化することができる。例えば、ＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤、ＴＤＰ－４３の凝集の抑制剤、又はＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤は、注射剤、溶液、懸濁液、錠剤、カプセル、ピル、顆粒、シロップ、坐剤、吸入剤、及びスプレーに製剤化することができる。注射剤は、例えば、注射液、注射用滅菌粉末、及び注射用濃縮液を含む。 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 cells overexpressing TDP-43 according to the embodiments is a pharmaceutically acceptable agent. It can be formulated into the following dosage form. For example, therapeutic or preventive agents for diseases accompanied by TDP-43 aggregation, inhibitors of TDP-43 aggregation, or inhibitors of cell death of cells overexpressing TDP-43 can be used as injections, solutions, or suspensions. It can be formulated into suspensions, tablets, capsules, pills, granules, syrups, suppositories, inhalants, and sprays. Injectables include, for example, injectable solutions, injectable sterile powders, and injectable concentrates.
　上述したように、ＴＤＰ－４３の凝集により、ＲＮＡの代謝に異常が生じたり、細胞毒性を招いたりして、様々な病気が発症及び進行することが強く示唆されている。実施形態に係るＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤は、ＴＤＰ－４３の凝集を抑制することにより、ＴＤＰ－４３の凝集を伴う疾患の治療又は予防をすることが可能である。あるいは、実施形態に係るＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤は、ＴＤＰ－４３が過剰発現している細胞の細胞死を抑制することにより、ＴＤＰ－４３の凝集を伴う疾患の治療又は予防をすることが可能である。 As mentioned above, it has been strongly suggested that aggregation of TDP-43 causes abnormalities in RNA metabolism and cell toxicity, leading to the onset and progression of various diseases. The agent for treating or preventing a disease accompanied by TDP-43 aggregation according to the embodiment can treat or prevent a disease accompanied by TDP-43 aggregation by inhibiting TDP-43 aggregation. Alternatively, the therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation according to an embodiment can treat a disease accompanied by TDP-43 aggregation by suppressing cell death of cells overexpressing TDP-43. Or prevention is possible.
　上記のように本発明を実施形態によって記載したが、この開示の一部をなす記述及び図面はこの発明を限定するものであると理解するべきではない。この開示から当業者には様々な代替実施形態、実施例及び運用技術が明らかになるはずである。本発明はここでは記載していない様々な実施形態等を包含するということを理解すべきである。 Although the present invention has been described by way of embodiments as described above, it should not be understood that the descriptions and drawings that form part of this disclosure limit the present invention. Various alternative embodiments, implementations, and operational techniques will become apparent to those skilled in the art from this disclosure. It should be understood that the invention encompasses various embodiments and the like that are not described herein.
　（参考例１）
　マウス神経芽細胞腫（Ｎｅｕｒｏ２ａ）をディッシュに播種した。Ｎｅｕｒｏ２ａ細胞をＤＭＥＭ＋１０％ＦＢＳの培地で３７℃、５％ＣＯ2存在下で培養した。播種してから１日目に、ニューロンへの分化を開始させた。ニューロンへの分化は、ＤＭＥＭ＋２％ＦＢＳ＋２０μｍｏｌ／Ｌレチノイン酸の培地で行った。播種してから４日目に、細胞に合成したＴＤＰ－４３ｍＲＮＡをリポフェクション法によりトランスフェクトした。トランスフェクションから６時間後、９時間後、１２時間後、１５時間後、１８時間後、及び２１時間後に、細胞をサンプリングした。トランスフェクションから２１時間後、細胞死が観察された。サンプリングにおいて、細胞をＲＩＰＡバッファーで溶解し、２２０００×ｇ、３０分、４℃で遠心分離し、上清を可溶性画分、沈殿物を不溶性画分として、それぞれ等量の２×ＳＤＳサンプルバッファーを加えて９５℃で５分加熱し、泳動サンプルとした。各泳動サンプルを、ポリアクリルアミドゲルを用いて電気泳動した後、ＴＤＰ－４３をＰＶＤＦ膜に転写し、一次抗体として抗ＴＤＰ－４３抗体、二次抗体としてＨＲＰコンジュゲート抗ウサギ抗体を使用し、化学発光によりＴＤＰ－４３を検出した。それらの結果を図１及び図２に示す。ＴＤＰ－４３遺伝子をトランスフェクトしてから、細胞死までの間に、細胞内においてＴＤＰ－４３の不溶フラクションが増加したことが確認された。ＴＤＰ－４３の不溶フラクションは、ＴＤＰ－４３が凝集していることを示している。 (Reference example 1)
Mouse neuroblastoma (Neuro2a) was seeded in a dish. Neuro2a cells were cultured in a medium of DMEM + 10% FBS at 37° C. in the presence of 5% CO 2 . On the first 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. On the fourth day after seeding, the cells were transfected with the synthesized TDP-43 mRNA 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 with RIPA buffer, centrifuged at 22,000 x g for 30 minutes at 4°C, the supernatant was used as the soluble fraction, and the precipitate was used as the insoluble fraction, and an equal volume of 2x SDS sample buffer was added to each. In addition, it was heated at 95° C. for 5 minutes to prepare an electrophoresis sample. After each electrophoresis sample was electrophoresed using polyacrylamide gel, TDP-43 was transferred to a PVDF membrane, anti-TDP-43 antibody was used as the primary antibody, HRP-conjugated anti-rabbit antibody was used as the secondary antibody, and chemical TDP-43 was detected by luminescence. The results are shown in FIGS. 1 and 2. It was confirmed that the insoluble fraction of TDP-43 increased within the cells between the time of transfection of the TDP-43 gene and the time of cell death. The insoluble fraction of TDP-43 indicates that TDP-43 is aggregated.
　（実施例１）
　Ｎｅｕｒｏ２ａをディッシュに播種した。播種してから１日目に、ニューロンへの分化を開始させた。播種してから４日目に、細胞にＴＤＰ－４３遺伝子をトランスフェクトした。トランスフェクションから５時間後に、培地に１０ｎｍｏｌ／Ｌ、５０ｎｍｏｌ／Ｌ、１００ｎｍｏｌ／Ｌ、５００ｎｍｏｌ／Ｌ、１０００ｎｍｏｌ／Ｌ、５０００ｎｍｏｌ／Ｌ、又は１００００ｎｍｏｌ／ＬのＤ－サイクロセリン又はＬ－サイクロセリンを添加した。また、コントロールとして、培地に１０ｎｍｏｌ／Ｌ、５０ｎｍｏｌ／Ｌ、１００ｎｍｏｌ／Ｌ、５００ｎｍｏｌ／Ｌ、１０００ｎｍｏｌ／Ｌ、５０００ｎｍｏｌ／Ｌ、又は１００００ｎｍｏｌ／Ｌのロピニロールを添加した。ロピニロールはＡＬＳに対して治療効果を有すると報告されている。 (Example 1)
Neuro2a was seeded in a dish. On the first day after seeding, differentiation into neurons was initiated. Four days after seeding, 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 10000 nmol/L of D-cycloserine or L-cycloserine was added to the medium. . Furthermore, as a control, 10 nmol/L, 50 nmol/L, 100 nmol/L, 500 nmol/L, 1000 nmol/L, 5000 nmol/L, or 10000 nmol/L of ropinirole was added to the medium. Ropinirole has been reported to have therapeutic effects on ALS.
　細胞にＴＤＰ－４３遺伝子をトランスフェクトしてから、７日目に死細胞の割合を評価した。図３Ａに示すように、ＴＤＰ－４３遺伝子をトランスフェクションしなかった細胞では、細胞死は観察されなかった。図３Ｂに示すように、ＴＤＰ－４３遺伝子をトランスフェクションし、Ｄ－サイクロセリン又はエダラボンを添加されなかった細胞では、細胞死が観察された。図３Ｃに示すように、ＴＤＰ－４３遺伝子をトランスフェクションし、Ｄ－サイクロセリンを添加された細胞では、細胞死が抑制された。 The percentage of dead cells was evaluated 7 days after transfecting the cells with the TDP-43 gene. As shown in Figure 3A, no cell death was observed in cells that were not transfected with the TDP-43 gene. As shown in FIG. 3B, cell death was observed in cells transfected with the TDP-43 gene and to which D-cycloserine or edaravone was not added. As shown in FIG. 3C, cell death was suppressed in cells transfected with the TDP-43 gene and supplemented with D-cycloserine.
　図４に示すように、Ｄ－サイクロセリン及びＬ－サイクロセリンは、濃度に依存して、細胞死を抑制する効果を示した。Ｄ－サイクロセリンのＥＣ50は、１２８ｎｍｏｌ／Ｌであった。Ｌ－サイクロセリンのＥＣ50は、２３７ｎｍｏｌ／Ｌであった。ロピニロールのＥＣ50は、１９８ｎｍｏｌ／Ｌであった。なお、相対生存率とは、ＴＤＰ－４３を過剰発現していない細胞の生存数を１００％としたときの、ＴＤＰ－４３を過剰発現した細胞の生存数の割合を表す。 As shown in FIG. 4, D-cycloserine and L-cycloserine exhibited a concentration-dependent effect of suppressing cell death. The EC 50 of D-cycloserine was 128 nmol/L. The EC 50 of L-cycloserine was 237 nmol/L. The EC 50 of ropinirole was 198 nmol/L. Note that the relative survival rate represents the ratio of the number of surviving cells that overexpressed TDP-43 to the number of surviving cells that did not overexpress TDP-43 as 100%.
　（実施例２）
　Ｎｅｕｒｏ２ａをディッシュに播種した。播種してから１日目に、ニューロンへの分化を開始させた。播種してから４日目に、細胞にＴＤＰ－４３遺伝子をトランスフェクトした。トランスフェクションから６時間後に、細胞に１０μｍｏｌ／ＬのＤ－サイクロセリン、Ｌ－サイクロセリン、ロピニロール、又はＤＭＳＯを添加し、１２時間培養した。その後、細胞内におけるＴＤＰ－４３の可溶フラクションと不溶フラクションを分析した結果を図５に示す。図５に示すように、コントロールとしてＤＭＳＯで処理した細胞においては、ＴＤＰ－４３の可溶フラクションよりＴＤＰ－４３の不溶フラクションのほうが多いことが確認された。これに対し、Ｄ－サイクロセリン及びＬ－サイクロセリンで処理した細胞においては、ＴＤＰ－４３の不溶フラクションよりＴＤＰ－４３の可溶フラクションのほうが多いことが確認された。 (Example 2)
Neuro2a was seeded in a dish. On the first day after seeding, differentiation into neurons was initiated. Four days after seeding, 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 cultured for 12 hours. Thereafter, the soluble and insoluble fractions of TDP-43 in the cells were analyzed, and the results are shown in FIG. As shown in FIG. 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.
　細胞内におけるＴＤＰ－４３の可溶フラクションと不溶フラクションの合計に対する不溶フラクションの割合を定量した結果を図６に示す。Ｄ－サイクロセリン及びＬ－サイクロセリンで処理した細胞においては、不溶フラクションの割合は半分未満であった。 Figure 6 shows the results of quantifying the ratio of the insoluble fraction to the total of the soluble and insoluble fractions of TDP-43 in cells. In cells treated with D-cycloserine and L-cycloserine, the proportion of insoluble fraction was less than half.
　（実施例３）
　１００μＬのＥＨＳゲル基底膜マトリックス（富士フイルム和光純薬）を１２．５ｍＬのＤＭＥＭ（Dulbecco’s Modified Eagle Medium、富士フイルム和光純薬）に加えて混合し、２４ウェルプレートに６００μＬ／ウェルずつ添加した。室温で２時間プレートを静置した後、ウェルから溶液を除去した。次に、ＤＭＥＭを６００μＬ／ウェルずつ添加し、室温で２時間プレートを静置した。 (Example 3)
100 μL of EHS gel basement membrane matrix (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 12.5 mL of DMEM (Dulbecco's Modified Eagle Medium, Fuji Film Wako Pure Chemical Industries, Ltd.) and mixed, and 600 μL/well was added to a 24-well plate. After allowing the plate to stand at room temperature for 2 hours, the solution was removed from the wells. Next, 600 μL/well of DMEM was added, and the plate was left standing at room temperature for 2 hours.
　健常者由来のｉＰＳ細胞由来の運動ニューロンと、ＡＬＳ患者由来のｉＰＳ細胞由来の運動ニューロンと、をｉＸＣｅｌｌｓ　Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓより入手した。３７℃の恒温槽で素早く解凍した運動ニューロンを、運動ニューロン維持培地（ｉＸＣｅｌｌｓ　Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ）に懸濁した。６００Ｘｇで培地を５分間遠心し運動ニューロンを集めた後、培地を除去し、運動ニューロンを運動ニューロン維持培地に懸濁した。 Motor neurons derived from iPS cells derived from healthy individuals and motor neurons derived from iPS cells derived from ALS patients were obtained from iX Cells Biotechnologies. Motor neurons were quickly thawed in a thermostat at 37°C and suspended in motor neuron maintenance medium (iX Cells Biotechnologies). After centrifuging the medium at 600×g for 5 minutes to collect motor neurons, the medium was removed and the motor neurons were suspended in motor neuron maintenance medium.
　運動ニューロンの密度が３Ｘ１０5ｃｅｌｌｓ／ｍＬになるように培地に希釈した後、マトリックスでコーティングしたプレートの複数のウェルのそれぞれに６００μＬの運動ニューロンを含む培地を入れ、３７℃、５％ＣＯ２存在下で運動ニューロンを培養した。２、３日毎に複数のウェルのそれぞれから３００μＬの培地を除去し、複数のウェルのそれぞれに新たな３００μＬの培地を加えた。 After diluting the motor neurons in the medium to a density of 3×10 5 cells/mL, each of the multiple wells of the matrix-coated plate was filled with 600 μL of the medium containing the motor neurons and incubated at 37°C in the presence of 5% CO2. Motor neurons were cultured under Every few days, 300 μL of medium was removed from each of the wells and 300 μL of fresh medium was added to each of the wells.
　培養開始から７日目、複数のウェルのそれぞれから３００μＬの培地を除去し、複数のウェルのそれぞれに、０．２％のＤＭＳＯを含む培地、２０μｍｏｌ／ＬのＤ－サイクロセリン（Cayman Chemical）を含む培地、又は２０μｍｏｌ／Ｌのロピニロール（富士フィルム和光純薬）を含む培地を３００μＬ加えた。２、３日毎に複数のウェルのそれぞれから３００μＬの培地を除去し、複数のウェルのそれぞれに除去された培地と同じ化合物を含む、即ち０．１％のＤＭＳＯを含む培地、１０μｍｏｌ／ＬのＤ－サイクロセリンを含む培地、又は１０μｍｏｌ／Ｌのロピニロールを含む培地を３００μＬ加えた。 Seven days after the start of culture, 300 μL of the medium was removed from each of the multiple wells, and a medium containing 0.2% DMSO and 20 μmol/L of D-cycloserine (Cayman Chemical) were added to each of the multiple wells. 300 μL of a medium containing the same or 20 μmol/L of ropinirole (Fuji Film Wako Pure Chemical Industries, Ltd.) was added. Every few days, 300 μL of medium was removed from each of the wells, and each of the wells was treated with medium containing the same compounds as the removed medium, i.e., 0.1% DMSO, 10 μmol/L D - 300 μL of a medium containing cycloserine or a medium containing 10 μmol/L ropinirole was added.
　界面活性剤を含んだタンパク質抽出用の緩衝液（ＲＩＰＡバッファー、ナカライテスク）５００μＬに、プロテアーゼ阻害剤及びホスファターゼ阻害剤のカクテル（Halt Protease and Phosphatase Inhibitor Cocktail、10OX、登録商標、Thermo Fisher Scientific）５μＬを添加して細胞抽出用液を作製した。ＤＭＳＯ、Ｄ－サイクロセリン、又はロピニロールの存在下で２０日間培養された運動ニューロンに、５０μＬの細胞抽出用液を添加し、ピペッティングにより細胞抽出液を１．５ｍＬのチューブに回収した。細胞抽出液を、４℃、２１，０００Ｘｇで３０分遠心分離した。 Add 5 μL of protease inhibitor and phosphatase inhibitor cocktail (Halt Protease and Phosphatase Inhibitor Cocktail, 10OX, registered trademark, Thermo Fisher Scientific) to 500 μL of protein extraction buffer containing surfactant (RIPA buffer, Nacalai Tesque). was added to prepare a cell extraction solution. 50 μL of cell extraction solution was added to motor neurons cultured for 20 days in the presence of DMSO, D-cycloserine, or ropinirole, and the cell extract was collected into a 1.5 mL tube by pipetting. Cell extracts were centrifuged at 21,000×g for 30 minutes at 4°C.
　遠心後の上清を４５μＬ分取後、上清に電気泳動用緩衝液（ＡＥ－１４３０　ＥｚＡｐｐｌｙ、アトー）を４５μＬ添加し、９５℃で５分加熱して、タンパク質の可溶フラクションを作製した。また、遠心後の沈殿物を５０μＬのＲＩＰＡバッファーで２回洗浄した後、沈殿物に４５μＬのＲＩＰＡバッファーと４５μＬの電気泳動用緩衝液を添加し、９５℃で５分加熱して、タンパク質の不溶フラクションを作製した。 After collecting 45 μL of the supernatant after centrifugation, 45 μL of electrophoresis buffer (AE-1430 EzApply, ATTO) was added to the supernatant and heated at 95° C. for 5 minutes to prepare a soluble fraction of proteins. In addition, after washing the precipitate after centrifugation twice with 50 μL of RIPA buffer, 45 μL of RIPA buffer and 45 μL of electrophoresis buffer were added to the precipitate, and the mixture was heated at 95°C for 5 minutes to insoluble proteins. Fractions were prepared.
　タンパク質の可溶フラクションと不溶フラクションのＳＤＳ－ＰＡＧＥを実施し、タンパク質をＰＶＤＦ膜に転写した。ＰＶＤＦ膜をブロッキング溶液に浸して室温で１時間振盪して、ＰＶＤＦ膜をブロッキングした。ＰＶＤＦ膜をＴＤＰ－４３ポリクローナル抗体(PGI Proteintech Group)を含む溶液に浸して、４℃で一晩振盪した。その後、緩衝液でＰＶＤＦ膜を３回洗浄した後、ＰＶＤＦ膜をＨＲＰ（ホースラディッシュ・ペルオキシダーゼ）標識二次抗体（Anti-Rabbit IgG, HRP-Linked F（ab’）2 Fragment Donkey, Cytiva）を含む溶液に浸して、室温で１時間振盪した。次に、緩衝液でＰＶＤＦ膜を３回洗浄した後、１ｍＬの発光試薬（イムノスターＬＤ、富士フイルム和光純薬）を用いて標識二次抗体を発光させ、ＰＶＤＦ膜を撮影した。撮影したＰＶＤＦ膜を図７に示す。 SDS-PAGE of the soluble and insoluble fractions of the protein was performed, and the protein was transferred to a PVDF membrane. The PVDF membrane was blocked by immersing it in a blocking solution and shaking it at room temperature for 1 hour. The PVDF membrane was immersed in a solution containing TDP-43 polyclonal antibody (PGI Proteintech Group) and shaken overnight at 4°C. After that, the PVDF membrane was washed three times with a buffer solution, and then the PVDF membrane was washed with an HRP (horseradish peroxidase)-labeled secondary antibody (Anti-Rabbit IgG, HRP-Linked F(ab')2 Fragment Donkey, Cytiva). It was immersed in the solution and shaken for 1 hour at room temperature. Next, after washing the PVDF membrane three times with a buffer solution, the labeled secondary antibody was caused to emit light using 1 mL of a luminescent reagent (Immunostar LD, Fujifilm Wako Pure Chemical Industries, Ltd.), and the PVDF membrane was photographed. The photographed PVDF membrane is shown in FIG.
　図７に示すように、コントロールとしてＤＭＳＯ存在下で培養された健常者由来のｉＰＳ細胞由来の運動ニューロンにおいては、ＴＤＰ－４３の可溶フラクションのバンドが明確に観察され、不溶フラクションのバンドは薄かった。Ｄ－サイクロセリン存在下で培養された健常者由来のｉＰＳ細胞由来の運動ニューロンにおいては、ＴＤＰ－４３の可溶フラクションのバンドが明確に観察され、不溶フラクションのバンドは薄かった。ロピニロール存在下で培養された健常者由来のｉＰＳ細胞由来の運動ニューロンにおいては、ＴＤＰ－４３の可溶フラクションのバンドが明確に観察され、不溶フラクションのバンドは薄かった。 As shown in Figure 7, in motor neurons derived from iPS cells derived from healthy subjects cultured in the presence of DMSO as a control, the band of the soluble fraction of TDP-43 was clearly observed, and the band of the insoluble fraction was faint. Ta. In motor neurons derived from iPS cells derived from healthy subjects cultured in the presence of D-cycloserine, a band of the soluble fraction of TDP-43 was clearly observed, and a band of the insoluble fraction was faint. In motor neurons derived from iPS cells from healthy subjects cultured in the presence of ropinirole, a band of the soluble fraction of TDP-43 was clearly observed, and a band of the insoluble fraction was faint.
　図７に示すように、コントロールとしてＤＭＳＯ存在下で培養されたＡＬＳ患者由来のｉＰＳ細胞由来の運動ニューロンにおいては、ＴＤＰ－４３の可溶フラクションのバンドが明確に観察され、不溶フラクションのバンドも明確に確認された。Ｄ－サイクロセリン存在下で培養されたＡＬＳ患者由来のｉＰＳ細胞由来の運動ニューロンにおいては、ＴＤＰ－４３の可溶フラクションのバンドが明確に観察され、不溶フラクションのバンドは薄かった。ロピニロール存在下で培養されたＡＬＳ患者由来のｉＰＳ細胞由来の運動ニューロンにおいては、ＴＤＰ－４３の可溶フラクションのバンドが明確に観察され、不溶フラクションのバンドは薄かった。 As shown in Figure 7, in motor neurons derived from iPS cells derived from ALS patients cultured in the presence of DMSO as a control, a band of the soluble fraction of TDP-43 was clearly observed, and a band of the insoluble fraction was also clearly observed. was confirmed. In motor neurons derived from iPS cells derived from ALS patients cultured in the presence of D-cycloserine, a band of the soluble fraction of TDP-43 was clearly observed, and a band of the insoluble fraction was faint. In motor neurons derived from iPS cells derived from ALS patients cultured in the presence of ropinirole, a band of the soluble fraction of TDP-43 was clearly observed, and a band of the insoluble fraction was faint.
　撮影したＰＶＤＦ膜の画像をＩｍａｇｅＪ　ソフトウェアで開き、Ｉｎｖｅｒｔコマンドで白黒反転を行った。Ｓｕｂｔｒａｃｔ　Ｂａｃｋｇｒｏｕｎｄ　コマンドを用いてバックグラウンドを除去した後、各レーンのＴＤＰ－４３のバンドを同じ大きさの四角の枠で順次囲い、Ｍｅａｓｕｒｅコマンドでバンド強度を定量した。各サンプルのＴＤＰ－４３の可溶フラクションのバンド強度と、ＴＤＰ－４３の不溶フラクションのバンド強度と、から下記式に従って、ＴＤＰ－４３の不溶フラクションの割合を算出した。
　　Ｒ＝｛ＩＩ／（ＩＩ＋ＩＳ）｝×１００
　上記式において、ＲはＴＤＰ－４３の不溶フラクションの割合（％）、ＩＩはＴＤＰ－４３の不溶フラクションのバンド強度、ＩＳはＴＤＰ－４３の可溶フラクションのバンド強度を表す。 The photographed image of the PVDF membrane was opened using ImageJ software, and black and white was inverted using the Invert command. After removing the background using the Subtract Background command, the TDP-43 bands in each lane were successively surrounded by square frames of the same size, and the band intensities were quantified using the Measure command. The ratio of the insoluble fraction of TDP-43 was calculated from the band intensity of the soluble fraction of TDP-43 and the band intensity of the insoluble fraction of TDP-43 of each sample according to the following formula.
R={I I /(I I +I S )}×100
In the above formula, R represents the proportion (%) of the insoluble fraction of TDP-43, II represents the band intensity of the insoluble fraction of TDP-43, and IS represents the band intensity of the soluble fraction of TDP-43.
　３回の試験を行い、ＴＤＰ－４３の不溶フラクションの割合の平均値を算出した。図８に、各試験におけるＴＤＰ－４３の不溶フラクションの割合をドットで示し、平均値をバーで示した。健常者由来のｉＰＳ細胞由来の運動ニューロンにおいては、ＴＤＰ－４３の不溶フラクションの割合の平均値は約７．４％だった。―方、ＡＬＳ患者由来のｉＰＳ細胞由来の運動ニューロンにおいて、ＴＤＰ－４３の不溶フラクションの割合の平均値は約４６．１％だった。しかし、Ｄ－サイクロセリン存在下におけるＡＬＳ患者由来のｉＰＳ細胞由来の運動ニューロンにおいて、ＴＤＰ－４３の不溶フラクションの割合の平均値は約１７．４％に減少した。また、ロピニロール存在下におけるＡＬＳ患者由来のｉＰＳ細胞由来の運動ニューロンにおいて、ＴＤＰ－４３の不溶フラクションの割合の平均値は約１９．３％に減少した。 The test was conducted three times, and the average value of the proportion of the insoluble fraction of TDP-43 was calculated. In FIG. 8, the percentage of the insoluble fraction of TDP-43 in each test is shown as a dot, and the average value is shown as a bar. In motor neurons derived from iPS cells from healthy subjects, the average percentage of TDP-43 insoluble fraction was approximately 7.4%. - On the other hand, the average percentage of TDP-43 insoluble fraction in motor neurons derived from iPS cells derived from ALS patients was approximately 46.1%. However, in the presence of D-cycloserine, the average percentage of the insoluble fraction of TDP-43 decreased to about 17.4% in motor neurons derived from iPS cells derived from ALS patients. Furthermore, in the presence of ropinirole, the average percentage of the insoluble fraction of TDP-43 decreased to about 19.3% in motor neurons derived from iPS cells derived from ALS patients.

Claims (15)

  1. 　サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを含む、ＴＤＰ－４３の凝集の抑制剤。 An inhibitor of TDP-43 aggregation, comprising at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.
  2. 　サイクロセリン又はその塩を含む、請求項１に記載のＴＤＰ－４３の凝集の抑制剤。 The inhibitor of aggregation of TDP-43 according to claim 1, comprising cycloserine or a salt thereof.
  3. 　前記サイクロセリンが、Ｄ－サイクロセリンである、請求項１又は２に記載のＴＤＰ－４３の凝集の抑制剤。 The inhibitor of aggregation of TDP-43 according to claim 1 or 2, wherein the cycloserine is D-cycloserine.
  4. 　前記サイクロセリンが、Ｌ－サイクロセリンである、請求項１又は２に記載のＴＤＰ－４３の凝集の抑制剤。 The inhibitor of aggregation of TDP-43 according to claim 1 or 2, wherein the cycloserine is L-cycloserine.
  5. 　サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを含む、ＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤。 A cell death inhibitor of cells overexpressing TDP-43, which comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.
  6. 　サイクロセリン又はその塩を含む、請求項５に記載のＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤。 The inhibitor of cell death of cells overexpressing TDP-43 according to claim 5, which comprises cycloserine or a salt thereof.
  7. 　前記サイクロセリンが、Ｄ－サイクロセリンである、請求項５又は６に記載のＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤。 The inhibitor of cell death of cells overexpressing TDP-43 according to claim 5 or 6, wherein the cycloserine is D-cycloserine.
  8. 　前記サイクロセリンが、Ｌ－サイクロセリンである、請求項５又は６に記載のＴＤＰ－４３が過剰発現している細胞の細胞死の抑制剤。 The inhibitor of cell death of cells overexpressing TDP-43 according to claim 5 or 6, wherein the cycloserine is L-cycloserine.
  9. 　サイクロセリン及びテリジドン、並びにこれらの塩からなる群から選択される少なくとも１つを含む、ＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤。 A therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation, comprising at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.
  10. 　サイクロセリン又はその塩を含む、請求項９に記載のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤。 The agent for treating or preventing a disease accompanied by TDP-43 aggregation according to claim 9, which comprises cycloserine or a salt thereof.
  11. 　前記サイクロセリンが、Ｄ－サイクロセリンである、請求項９又は１０に記載のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤。 The agent for treating or preventing a disease accompanied by TDP-43 aggregation according to claim 9 or 10, wherein the cycloserine is D-cycloserine.
  12. 　前記サイクロセリンが、Ｌ－サイクロセリンである、請求項９又は１０に記載のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤。 The agent for treating or preventing a disease accompanied by TDP-43 aggregation according to claim 9 or 10, wherein the cycloserine is L-cycloserine.
  13. 　前記ＴＤＰ－４３の凝集を伴う疾患が、ＴＤＰ－４３プロテイノパチーである、請求項９から１２のいずれか１項に記載のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤。 The agent for treating or preventing 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. 　前記ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症、前頭側頭葉変性症、原発性側索硬化症、及び進行性筋萎縮症からなる群から選択される少なくとも１つである、請求項１３に記載のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤。 4. 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 amyotrophy. 14. A therapeutic or preventive agent for a disease accompanied by TDP-43 aggregation according to item 13.
  15. 　前記ＴＤＰ－４３プロテイノパチーが、筋萎縮性側索硬化症である、請求項１３又は１４に記載のＴＤＰ－４３の凝集を伴う疾患の治療又は予防剤。
          The agent for treating or preventing a disease accompanied by TDP-43 aggregation according to claim 13 or 14, wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis.
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