Treatment or preventive agent for amyotrophic lateral sclerosis (ALS)

Abstract

To provide an agent for treating or preventing amyotrophic lateral sclerosis.SOLUTION: This agent for treating or preventing amyotrophic lateral sclerosis essentially comprises at least one active ingredient selected from the group consisting of cycloserine and terizidone, and salts thereof. The active ingredient may be at least one selected from the group consisting of cycloserine and terizidone, and salts thereof. The cycloserine may be D-cycloserine. The cycloserine may be L-cycloserine.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] The present invention relates to a therapeutic or prophylactic agent for amyotrophic lateral sclerosis (ALS). [Background technology]

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

[0003] TDP-43 that accumulates in the brains and spinal cords of ALS and FTLD patients has abnormal phosphorylation of multiple serine residues at its C-terminus. In ALS and FTLD patients, it is not necessary for the entire TDP-43 molecule to aggregate; only the C-terminal fragment of TDP-43 aggregates. Previous studies have strongly suggested that TDP-43 aggregation leads to abnormalities in RNA metabolism and cytotoxicity, causing the onset and progression of various diseases.

[0004] An example of a disease involving TDP-43 aggregation 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 prophylactic agent for TDP-43 proteinopathy. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent No. 5898701

[0006] [Patent Document 2] Special Publication No. 2022-500397 [Patent Document 3] Special Publication No. 2018-526345 [Patent Document 4] U.S. Patent Application Publication No. 2011 / 0160260 [Patent Document 5] Special Publication No. 2002-511409 [Overview of the project] [Problems that the invention aims to solve]

[0007] One of the objectives of this invention is to provide a therapeutic or prophylactic agent for amyotrophic lateral sclerosis (ALS). [Means for solving the problem]

[0008] According to aspects of the present invention, there is a therapeutic or prophylactic agent for amyotrophic lateral sclerosis (ALS) whose active ingredient essentially consists of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0009] In the above-described therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the active ingredient may consist of at least one selected from the group comprising cycloserine, terizidone, and salts thereof.

[0010] In the above-described therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the active ingredient may essentially consist of cycloserine or a salt thereof.

[0011] In the above-mentioned therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the active ingredient may consist of cycloserine or a salt thereof.

[0012] In the above-mentioned therapeutic or prophylactic agent for amyotrophic lateral sclerosis, cycloserine may be D-cycloserine.

[0013] In the above therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the cycloserine may be L-cycloserine.

[0014] The above therapeutic or prophylactic agent for amyotrophic lateral sclerosis may contain an active ingredient of 15 mg or more and 100 mg or less.

[0015] The above therapeutic or prophylactic agent for amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of D-cycloserine.

[0016] In the above therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the active ingredient may not be in the cationic form.

[0017] In the above therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the active ingredient may not be combined with an anionic form compound.

[0018] In the above therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the active ingredient may not be combined with acamprosate.

[0019] According to an aspect of the present invention, there is provided the use of an active ingredient substantially consisting of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof in the production of a therapeutic or prophylactic agent for amyotrophic lateral sclerosis.

[0020] In the above use, the active ingredient may consist of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0021] In the above use, the active ingredient may consist essentially of cycloserine or a salt thereof.

[0022] In the above use, the active ingredient may consist of cycloserine or a salt thereof.

[0023] In the above use, the cycloserine may be D-cycloserine.

[0024] In the above use, cycloserine may be replaced with L-cycloserine.

[0025] In the above use, the therapeutic or prophylactic agent for amyotrophic lateral sclerosis may contain an active ingredient in an amount of 15 mg to 100 mg.

[0026] In the above use, the therapeutic or prophylactic agent for amyotrophic lateral sclerosis may contain 15 mg to 100 mg of D-cycloserine.

[0027] In the above use, the active ingredient does not have to be in cationic form.

[0028] In the above use, the active ingredient does not necessarily have to be combined with an anionic compound.

[0029] In the above use, the active ingredient does not necessarily have to be combined with acamprosate.

[0030] According to an aspect of the present invention, a method for treating or preventing amyotrophic lateral sclerosis (ALS) is provided, comprising administering to a patient with ALS a therapeutic or prophylactic agent for ALS, the active ingredient of which essentially consists of at least one selected from the group consisting of cycloserine and terizidone, and salts thereof.

[0031] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the agent for treating or preventing amyotrophic lateral sclerosis may consist of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0032] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the agent for treating or preventing amyotrophic lateral sclerosis may essentially consist of cycloserine or a salt thereof.

[0033] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the agent for treating or preventing amyotrophic lateral sclerosis may consist of cycloserine or a salt thereof.

[0034] In the above-described method for treating or preventing amyotrophic lateral sclerosis, cycloserine may be D-cycloserine. In the above-described method for treating or preventing amyotrophic lateral sclerosis, cycloserine may be L-cycloserine.

[0035] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the treatment or preventive agent for amyotrophic lateral sclerosis may contain an active ingredient in an amount of 15 mg to 100 mg.

[0036] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the treatment or prophylactic agent for amyotrophic lateral sclerosis may contain 15 mg to 100 mg of D-cycloserine.

[0037] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the treatment or preventive agent for amyotrophic lateral sclerosis does not have to be in a cationic form.

[0038] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the treatment or preventive agent for amyotrophic lateral sclerosis does not necessarily have to be combined with an anionic compound.

[0039] In the above-described method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the treatment or preventive agent for amyotrophic lateral sclerosis does not necessarily have to be combined with acamprosate. [Effects of the Invention]

[0040] According to the present invention, it is possible to provide a therapeutic or prophylactic agent for amyotrophic lateral sclerosis (ALS). [Brief explanation of the drawing]

[0041] [Figure 1]Figure 1 is a photograph showing the soluble fraction and the insoluble fraction of TDP-43 related to Reference Example 1. [Figure 2] Figure 2 is a graph showing the relationship between the time elapsed since transfection of the TDP-43 gene in Reference Example 1 and the proportion of the insoluble fraction of TDP-43 in the cells. [Figure 3] Figure 3 is a photograph of the cells related to Example 1. [Figure 4] Figure 4 is a graph showing the relationship between the concentration of the compound in Example 1 and the relative viability of cells. [Figure 5] Figure 5 is a photograph showing the soluble fraction and the insoluble fraction of TDP-43 according to Example 2. [Figure 6] Figure 6 is a graph showing the relationship between the compound used in Example 2 and the proportion of the insoluble fraction of TDP-43 in cells. [Figure 7] Figure 7 is a photograph showing the soluble fraction and the insoluble fraction of TDP-43 according to Example 3. [Figure 8] Figure 8 is a graph showing the proportion of insoluble fractions of TDP-43 in Example 3. [Modes for carrying out the invention]

[0042] Embodiments of the present invention are described below. However, these embodiments should not be understood as limiting the present invention. Various alternative embodiments, examples, and operational techniques should become apparent to those skilled in the art from this disclosure. It should be understood that the present invention encompasses various embodiments and the like that are not described herein.

[0043] The therapeutic or prophylactic agent for amyotrophic lateral sclerosis (ALS) according to the embodiment comprises an active ingredient which is essentially one selected from the group consisting of cycloserine and its physiologically acceptable salts. In this disclosure, “essentially” means that the active ingredient is free from other ingredients, and the presence of ingredients other than the active ingredient, such as excipients and / or lubricants, is not excluded.

[0044] Alternatively, the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment comprises at least one active ingredient selected from the group consisting of cycloserine and physiologically acceptable salts thereof. Cycloserine may be D-cycloserine or L-cycloserine.

[0045] Amyotrophic lateral sclerosis (ALS) is a disease characterized by the aggregation of TDP-43 and is classified as a TDP-43 proteinopathy.

[0046] The IUPAC name for D-cycloserine (CAS number: 68-41-7) is (4R)-4-Amino-1,2-oxazolidin-3-one. The chemical formula for D-cycloserine is C3H6N2O2. The chemical structure of D-cycloserine is as follows: [ka]

[0047] The IUPAC name for L-cycloserine (CAS number: 339-72-0) is (4S)-4-amino-1,2-oxazolidin-3-one. The chemical formula for L-cycloserine is C3H6N2O2. The chemical structure of L-cycloserine is as follows: [ka]

[0048] The therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment may essentially consist of at least one prodrug of the above compound, or a physiologically acceptable salt thereof. Alternatively, the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment may consist of at least one prodrug of the above compound, or a physiologically acceptable salt thereof.

[0049] 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 It is N4O4. The chemical structure of therizidone is as follows. Therizidone is broken down in the body and produces the same effects as D-cycloserine. [ka]

[0050] The therapeutic or prophylactic agent for amyotrophic lateral sclerosis (ALS) according to the embodiment can be formulated into pharmaceutically acceptable dosage forms. For example, the therapeutic or prophylactic agent for ALS can be formulated into injections, solutions, suspensions, tablets, capsules, pills, granules, syrups, suppositories, inhalants, and sprays. Injectable agents include, for example, injection solutions, sterile powders for injection, and concentrates for injection. The therapeutic or prophylactic agent for ALS according to the embodiment may contain, for example, 15 mg to 100 mg, 20 mg to 80 mg, or 25 mg to 60 mg of the active ingredient. The therapeutic or prophylactic agent for ALS according to the embodiment may contain, for example, 15 mg to 100 mg, 20 mg to 80 mg, or 25 mg to 60 mg of D-cycloserine.

[0051] The reported maximum blood concentration (Cmax) of D-cycloserine after oral administration of 15 mg to humans is approximately 5.6 μmol / L (van Berckel, B., Lipsch, C., Timp, S. et al. Behavioral and Neuroendocrine Effects of the Partial NMDA Agonist D-cycloserine in Healthy Subjects. Neuropsychopharmacol 16, 317-324 (1997). https: / / doi.org / 10.1016 / S0893-133X(96)00196-0, and van Berckel, B. Erratum: Behavioral and Neuroendocrine Effects of the Partial NMDA Agonist D-cycloserine in Healthy Subjects. Neuropsychopharmacol 17, 116 (1997).). https: / / doi.org / 10.1016 / S0893-133X(97)00082-1).

[0052] The brain penetration rate of D-cycloserine has been reported to be 95%. Therefore, the maximum concentration of D-cycloserine in the brain is estimated to be approximately 5.3 μmol / L. This concentration of approximately 5.3 μmol / L corresponds to the EC of D-cycloserine that suppresses cell death in nerve cells forcibly expressing TDP-43, as shown in Example 1 described later. 50 It exceeds 128 nmol / L. Furthermore, the half-life of D-cycloserine is reported to be approximately 10 hours. Therefore, the trough value when 15 mg of D-cycloserine is administered orally twice daily is also higher than the EC2 value. 50 It is estimated that the concentration exceeds 128 nmol / L. Therefore, the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment can fully exert a therapeutic or prophylactic effect on human amyotrophic lateral sclerosis by containing 15 mg or more of D-cycloserine.

[0053] Furthermore, when D-cycloserine is used in the treatment of tuberculosis, it has been reported that administering 250 mg to humans can cause serious side effects such as epileptic seizures and mental confusion. It has also been reported that administering 150 mg to humans can cause headaches. Therefore, the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to this embodiment can suppress the side effects caused by D-cycloserine by containing 100 mg or less of D-cycloserine.

[0054] Furthermore, in the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to this embodiment, the active ingredient does not have to be in cationic form. Also, the active ingredient does not have to be combined with an anionic compound. The chemical structural formula of the anionic compound is shown below, for example. [ka]

[0055] In the chemical structural formula of anionic compounds, X is either -NH2 or -OH. Each of L1 and L2 is independently a C1-6 alkylene, C2-6 alkenylene, or C2-6 alkynylene, or, as the valence allows, one of L1 or L2 is N, O, or S, and the other is a C1-6 alkylene, C2-6 alkenylene, or C2-6 alkynylene. Examples of compounds in anionic form are succinic acid, D-tartaric acid, L-tartaric acid, mesotartaric acid, fumaric acid, maleic acid, or malic acid. Combining a cationic active ingredient with an anionic compound may cause the active ingredient to become free, making it less soluble in water and potentially degrading the quality of the active ingredient.

[0056] Furthermore, in the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment, the active ingredient does not necessarily have to be combined with acamprosate. Acamprosate is a derivative of homotaurine and is also called 3-(acetylamino)propylsulfonic acid or N-acetylhomotaurine. When acamprosate is administered to humans, anaphylaxis accompanied by symptoms such as systemic rash, rash, urticaria, stomatitis, laryngospasm, and shortness of breath may occur as a side effect. In addition, when acamprosate is administered to humans, angioedema accompanied by symptoms such as tongue swelling and lymph node swelling may occur as a side effect. Since the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment does not have to be combined with acamprosate, these side effects do not occur.

[0057] The therapeutic or prophylactic agent for amyotrophic lateral sclerosis (ALS) according to the embodiment can treat or prevent ALS by suppressing the aggregation of TDP-43. Alternatively, the therapeutic or prophylactic agent for ALS according to the embodiment can treat or prevent ALS by suppressing cell death in cells that overexpress TDP-43.

[0058] Although the present invention has been described above with reference to embodiments, the descriptions and drawings that constitute part of this disclosure should not be understood as limiting the invention. Various alternative embodiments, examples, and operational techniques should become apparent to those skilled in the art from this disclosure. It should be understood that the present invention encompasses various embodiments and the like that are not described herein.

[0059] (Reference example 1) Mouse neuroblastoma cells (Neuro2a) were seeded in a dish. Neuro2a cells were cultured in DMEM + 10% FBS medium at 37°C in the presence of 5% CO2. Differentiation into neurons was initiated on day 1 after seeding. Differentiation into neurons was performed in DMEM + 2% FBS + 20 μmol / L retinoic acid medium. On day 4 after seeding, cells were transfected with 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 in RIPA buffer, centrifuged at 22000 × g for 30 minutes at 4°C, and the supernatant was used as the soluble fraction and the precipitate as the insoluble fraction. Equal volumes of 2 × SDS sample buffer were added to each, and the samples were heated at 95°C for 5 minutes to prepare electrophoresis samples. Each electrophoretic sample was subjected to electrophoresis using a polyacrylamide gel, after which TDP-43 was transferred to a PVDF membrane. 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 intracellularly from the time of TDP-43 gene transfection until cell death. The insoluble fraction of TDP-43 indicates that TDP-43 is agglutinating.

[0060] (Example 1) Neuro2a cells were seeded in a dish. Differentiation into neurons was initiated on day 1 after seeding. On day 4 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 10000 nmol / L of D-cycloserine or L-cycloserine were added to the culture medium. As a control, 10 nmol / L, 50 nmol / L, 100 nmol / L, 500 nmol / L, 10000 nmol / L, or 10000 nmol / L of ropinirole was added to the culture medium. Ropinirole has been reported to have therapeutic effects against ALS.

[0061] The percentage of dead cells was evaluated on day 7 after transfecting 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 Figure 3B, cell death was observed in cells that were transfected with the TDP-43 gene but not treated with D-cycloserine or edaravone. As shown in Figure 3C, cell death was suppressed in cells that were transfected with the TDP-43 gene and treated with D-cycloserine.

[0062] As shown in Figure 4, D-cycloserine and L-cycloserine showed a concentration-dependent effect in suppressing cell death. The EC of D-cycloserine... 50 The EC2 level was 128 nmol / L. 50 The EC of ropinirole was 237 nmol / L. 50 The concentration was 198 nmol / L. Relative viability refers to the percentage of surviving cells that overexpress TDP-43, compared to 100% of surviving cells that do not overexpress TDP-43.

[0063] (Example 2) Neuro2a cells were seeded in a dish. Differentiation into neurons was initiated on day 1 after seeding. On day 4 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 they were cultured for 12 hours. Subsequently, the soluble and insoluble fractions of TDP-43 in the cells were analyzed, and the results are shown in Figure 5. As shown in Figure 5, in cells treated with DMSO as a control, the insoluble fraction of TDP-43 was found to be greater than the soluble fraction of TDP-43. In contrast, in cells treated with D-cycloserine and L-cycloserine, the soluble fraction of TDP-43 was found to be greater than the insoluble fraction of TDP-43.

[0064] Figure 6 shows the results of quantifying the proportion of the insoluble fraction to the total of the soluble and insoluble fractions of TDP-43 within cells. In cells treated with D-cycloserine and L-cycloserine, the proportion of the insoluble fraction was less than half.

[0065] (Example 3) 100 μL of EHS gel basement membrane matrix (Fujifilm Wako Pure Chemical Industries) was added to 12.5 mL of DMEM (Dulbecco's Modified Eagle Medium, Fujifilm Wako Pure Chemical Industries) and mixed. 600 μL of this mixture was added to each 24-well plate. After allowing the plates to stand at room temperature for 2 hours, the solution was removed from the wells. Next, 600 μL of DMEM was added to each well, and the plates were allowed to stand at room temperature for 2 hours.

[0066] Motor neurons derived from iPS cells from healthy individuals and motor neurons derived from iPS cells from ALS patients were obtained from iXCells Biotechnologies. The motor neurons quickly thawed in a 37°C thermostat were suspended in a motor neuron maintenance medium (iXCells Biotechnologies). After centrifuging the medium at 600Xg for 5 minutes to collect the motor neurons, the medium was removed, and the motor neurons were suspended in the motor neuron maintenance medium.

[0067] The density of the motor neurons was diluted in the medium to 3X10 5 cells / mL, and then 600 μL of the medium containing the motor neurons was added to each of multiple wells of a plate coated with a matrix. The motor neurons were cultured at 37°C in the presence of 5% CO 2 2. Every 2 or 3 days, 300 μL of the medium was removed from each of the multiple wells, and 300 μL of fresh medium was added to each of the multiple wells.

[0068] On the 7th day from the start of the culture, 300 μL of the medium was removed from each of the multiple wells, and 300 μL of a medium containing 0.2% DMSO, a medium containing 20 μmol / L D-cycloserine (Cayman Chemical), or a medium containing 20 μmol / L ropinirole (FUJIFILM Wako Pure Chemical Corporation) was added to each of the multiple wells. Every 2 or 3 days, 300 μL of the medium was removed from each of the multiple wells, and 300 μL of a medium containing the same compound as the removed medium, that is, a medium containing 0.1% DMSO, a medium containing 10 μmol / L D-cycloserine, or a medium containing 10 μmol / L ropinirole was added to each of the multiple wells.

[0069] A cell extract solution was prepared by adding 5 μL of a protease inhibitor and phosphatase inhibitor cocktail (Halt Protease and Phosphatase Inhibitor Cocktail, 10OX, registered trademark, Thermo Fisher Scientific) to 500 μL of a protein extraction buffer containing a surfactant (RIPA buffer, Nacalai Tesque). 50 μL of the cell extract 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 in a 1.5 mL tube by pipetting. The cell extract was centrifuged at 4°C and 21,000 Xg for 30 minutes.

[0070] After centrifugation, 45 μL of the supernatant was collected, and 45 μL of electrophoresis buffer (AE-1430 EzApply, Atto) was added to the supernatant. The mixture was then heated at 95°C for 5 minutes to prepare the soluble fraction of the protein. Additionally, the precipitate after centrifugation was washed twice with 50 μL of RIPA buffer. Then, 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 prepare the insoluble fraction of the protein.

[0071] SDS-PAGE was performed on soluble and insoluble fractions of the protein, and the protein was transferred to a PVDF membrane. The PVDF membrane was blocked by immersion in a blocking solution and shaking 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 washing the PVDF membrane three times with buffer, it was immersed in a solution containing HRP (horseradish peroxidase)-labeled secondary antibody (Anti-Rabbit IgG, HRP-Linked F(ab')2 Fragment Donkey, Cytiva) and shaken at room temperature for 1 hour. Next, the PVDF membrane was washed three times with buffer, and the labeled secondary antibody was made to fluoresce using 1 mL of luminescence reagent (Immunostar LD, Fujifilm Wako Pure Chemical Industries), and the PVDF membrane was imaged. The imaged PVDF membrane is shown in Figure 7.

[0072] As shown in Figure 7, in motor neurons derived from iPS cells of healthy individuals cultured in the presence of DMSO as a control, a band of the soluble fraction of TDP-43 was clearly observed, while the band of the insoluble fraction was faint. In motor neurons derived from iPS cells of healthy individuals cultured in the presence of D-cycloserine, a band of the soluble fraction of TDP-43 was clearly observed, while the band of the insoluble fraction was faint. In motor neurons derived from iPS cells of healthy individuals cultured in the presence of ropinirole, a band of the soluble fraction of TDP-43 was clearly observed, while the band of the insoluble fraction was faint.

[0073] As shown in Figure 7, in motor neurons derived from iPS cells 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 identified. In motor neurons derived from iPS cells from ALS patients cultured in the presence of D-cycloserine, a band of the soluble fraction of TDP-43 was clearly observed, and the band of the insoluble fraction was faint. In motor neurons derived from iPS cells from ALS patients cultured in the presence of ropinirole, a band of the soluble fraction of TDP-43 was clearly observed, and the band of the insoluble fraction was faint.

[0074] The captured images of the PVDF film were opened in ImageJ software, and the black and white inversion was performed using the Invert command. After removing the background using the Subtract Background command, the TDP-43 bands in each lane were sequentially enclosed with square frames of the same size, and the band intensity was quantified using the Measure command. The proportion 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 in each sample according to the following formula. R={I I / ( I I +I S )} × 100 In the above formula, R is the percentage of the insoluble fraction of TDP-43, II This is the band intensity of the insoluble fraction of TDP-43, I S This represents the band intensity of the soluble fraction of TDP-43.

[0075] Three trials were conducted, and the average percentage of the insoluble fraction of TDP-43 was calculated. Figure 8 shows the percentage of the insoluble fraction of TDP-43 in each trial as dots, and the average value as a bar. In motor neurons derived from iPS cells from healthy individuals, the average percentage of the insoluble fraction of TDP-43 was approximately 7.4%. On the other hand, in motor neurons derived from iPS cells from ALS patients, the average percentage of the insoluble fraction of TDP-43 was approximately 46.1%. However, in motor neurons derived from iPS cells from ALS patients in the presence of D-cycloserine, the average percentage of the insoluble fraction of TDP-43 decreased to approximately 17.4%. Furthermore, in motor neurons derived from iPS cells from ALS patients in the presence of ropinirole, the average percentage of the insoluble fraction of TDP-43 decreased to approximately 19.3%.

Claims

1. A therapeutic or prophylactic agent for amyotrophic lateral sclerosis mediated by TDP-43 aggregation in nerve cells, wherein the active ingredient essentially consists of at least one selected from the group comprising cycloserine and terizidone, and salts thereof.

2. A therapeutic or prophylactic agent for amyotrophic lateral sclerosis mediated by TDP-43 aggregation in nerve cells, as described in claim 1, wherein the active ingredient comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

3. A therapeutic or prophylactic agent for amyotrophic lateral sclerosis mediated by TDP-43 aggregation in nerve cells, as described in claim 1, wherein the active ingredient essentially consists of cycloserine or a salt thereof.

4. The therapeutic or prophylactic agent for amyotrophic lateral sclerosis induced by TDP-43 aggregation in nerve cells according to claim 1, wherein the active ingredient comprises cycloserine or a salt thereof.

5. The therapeutic or prophylactic agent for amyotrophic lateral sclerosis induced by TDP-43 aggregation in nerve cells according to claim 1, wherein the cycloserine is D-cycloserine.

6. The therapeutic or prophylactic agent for amyotrophic lateral sclerosis induced by TDP-43 aggregation in nerve cells according to claim 1, wherein the cycloserine is L-cycloserine.

7. A therapeutic or prophylactic agent for amyotrophic lateral sclerosis mediated by aggregation of TDP-43 in nerve cells, as described in claim 1, wherein the active ingredient is not in a cation form.

8. A therapeutic or prophylactic agent for amyotrophic lateral sclerosis mediated by TDP-43 aggregation in nerve cells, as described in claim 1, wherein the active ingredient is not combined with an anionic compound.

9. A therapeutic or prophylactic agent for amyotrophic lateral sclerosis mediated by TDP-43 aggregation in nerve cells, as described in claim 1, wherein the active ingredient is not combined with acamprosate.

Images