Agent for treating or preventing amyotrophic lateral sclerosis

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 an agent for treating or preventing amyotrophic lateral sclerosis. [Background technology]

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

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

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

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

[0006] [Patent Document 2] Special Publication No. 2022-500397 [Patent Document 3] Special Publication No. 2018-526345 [Patent Document 4] US Patent Application Publication No. 2011 / 0160260 [Patent Document 5] Special Publication No. 2002-511409 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an agent for treating or preventing amyotrophic lateral sclerosis. [Means for solving the problem]

[0008] According to an aspect of the present invention, there is provided an agent for treating or preventing amyotrophic lateral sclerosis, the active ingredient of which essentially consists of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

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

[0010] In the above-mentioned 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 agent for treating or preventing amyotrophic lateral sclerosis, cycloserine may be D-cycloserine.

[0013] In the above-mentioned agent for treating or preventing amyotrophic lateral sclerosis, cycloserine may be L-cycloserine.

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

[0015] The above-mentioned 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-mentioned agent for treating or preventing amyotrophic lateral sclerosis, the active ingredient does not have to be in a cationic form.

[0017] In the above-mentioned agent for treating or preventing amyotrophic lateral sclerosis, the active ingredient does not need to be combined with a compound in an anionic form.

[0018] In the above-mentioned therapeutic or prophylactic agent for amyotrophic lateral sclerosis, the active ingredient does not need to be combined with acamprosate.

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

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

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

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

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

[0024] In the above uses, the cycloserine may be L-cycloserine.

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

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

[0027] In the above uses, the active ingredient may not be in cationic form.

[0028] In the above uses, the active ingredient does not have to be combined with a compound in anionic form.

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

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

[0031] In the above-mentioned 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-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the agent for treating or preventing amyotrophic lateral sclerosis may consist essentially of cycloserine or a salt thereof.

[0033] In the above-mentioned 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-mentioned method for treating or preventing amyotrophic lateral sclerosis, cycloserine may be D-cycloserine.In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, cycloserine may be L-cycloserine.

[0035] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the agent for treating or preventing amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of an active ingredient.

[0036] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the agent for treating or preventing amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of D-cycloserine.

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

[0038] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the agent for treating or preventing amyotrophic lateral sclerosis does not need to be combined with a compound in an anionic form.

[0039] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the agent for treating or preventing amyotrophic lateral sclerosis does not need to be combined with acamprosate. Effect of the Invention

[0040] According to the present invention, it is possible to provide an agent for treating or preventing amyotrophic lateral sclerosis. [Brief description of the drawings]

[0041] [Figure 1]FIG. 1 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Reference Example 1. [Diagram 2] FIG. 2 is a graph showing the relationship between the time elapsed from transfection of the TDP-43 gene in Reference Example 1 and the proportion of the insoluble fraction of TDP-43 in cells. [Diagram 3] FIG. 3 is a photograph of the cells according to Example 1. [Figure 4] FIG. 4 is a graph showing the relationship between the concentration of the compound according to Example 1 and the relative survival rate of cells. [Diagram 5] FIG. 5 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 2. [Figure 6] FIG. 6 is a graph showing the relationship between the compound of Example 2 and the proportion of the insoluble fraction of TDP-43 in cells. [Figure 7] FIG. 7 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 3. [Figure 8] FIG. 8 is a graph showing the ratio of the insoluble fraction of TDP-43 according to Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The following describes embodiments of the present invention. However, it should not be understood that the following embodiments limit the present invention. From this disclosure, various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0043] In the embodiment, the agent for treating or preventing amyotrophic lateral sclerosis (ALS) contains an active ingredient essentially consisting of at least one selected from the group consisting of cycloserine and physiologically acceptable salts thereof. In the present disclosure, "essentially consisting" means that the active ingredient does not contain other ingredients, and does not exclude the presence of ingredients other than the active ingredient, such as excipients and / or lubricants.

[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. The cycloserine may be D-cycloserine or L-cycloserine.

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

[0046] The IUPAC name of D-cycloserine (CAS number: 68-41-7) is (4R)-4-Amino-1,2-oxazolidin-3-one. The chemical formula of D-cycloserine is C 3 H 6 N 2 O 2 The chemical structure of D-cycloserine is: [ka]

[0047] 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 structure of L-cycloserine is: [ka]

[0048] The active ingredient of the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment may essentially consist of at least one prodrug of the above-mentioned compound or a physiologically acceptable salt thereof. Alternatively, the active ingredient of the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment may consist of at least one prodrug of the above-mentioned 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 N 4 O 4 The chemical structure of terizidone is: Terizidone is broken down in the body and exerts effects similar to those of D-cycloserine. [ka]

[0050] The therapeutic or preventive agent for amyotrophic lateral sclerosis according to the embodiment can be formulated into a pharma- ceutically acceptable dosage form. For example, the therapeutic or preventive agent for amyotrophic lateral sclerosis can be formulated into an injection, a solution, a suspension, a tablet, a capsule, a pill, a granule, a syrup, a suppository, an inhalant, and a spray. The injection includes, for example, an injection solution, a sterile powder for injection, and a concentrated solution for injection. The therapeutic or preventive agent for amyotrophic lateral sclerosis 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 an active ingredient. The therapeutic or preventive agent for amyotrophic lateral sclerosis 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 maximum blood concentration (Cmax) of D-cycloserine when 15 mg of D-cycloserine was orally administered to humans was reported to be 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 rate of D-cycloserine transfer into the brain has been reported to be 95%. Therefore, the maximum concentration of D-cycloserine in the brain is estimated to be about 5.3 μmol / L. The concentration of about 5.3 μmol / L is the EC value of D-cycloserine that suppresses cell death in neurons forced to express TDP-43, as shown in Example 1 below. 50 The half-life of D-cycloserine is reported to be approximately 10 hours. Therefore, the trough value when 15 mg of D-cycloserine is orally administered twice daily is also EC 50 It is estimated that the concentration of D-cycloserine in the treatment or prevention of amyotrophic lateral sclerosis in humans is more than 128 nmol / L. Therefore, the treatment or prevention agent for amyotrophic lateral sclerosis according to the embodiment contains 15 mg or more of D-cycloserine, and therefore can fully exert the effect of treating or preventing amyotrophic lateral sclerosis in humans.

[0053] In addition, it has been reported that when D-cycloserine is administered to humans at 250 mg for use in the treatment of tuberculosis, it causes severe side effects such as epileptiform convulsions and mental confusion. In addition, it has been reported that when D-cycloserine is administered to humans at 150 mg, it causes headaches. Therefore, the therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to the embodiment contains 100 mg or less of D-cycloserine, thereby making it possible to suppress side effects caused by D-cycloserine.

[0054] In the treatment or prevention agent for amyotrophic lateral sclerosis according to the embodiment, the active ingredient may not be in cationic form. In addition, the active ingredient may not be combined with an anionic compound. The chemical structure of the anionic compound is, for example, as shown below. [ka]

[0055] In the chemical structure of the compound in anionic form: X is -NH 2 or -OH, Each of L1 and L2 is independently selected from the group consisting of C 1 ~ 6 Alkylene, C 2 ~ 6 Alkenylene or C 2 ~ 6 Alkynylene or, as valences permit, one of L and L is N, O, or S and the other is C 1 ~ 6 Alkylene, C 2 ~ 6 Alkenylene or C 2 ~ 6 The anionic compound is, for example, succinic acid, D-tartaric acid, L-tartaric acid, mesotartaric acid, fumaric acid, maleic acid, or malic acid. The combination of a cationic active ingredient with an anionic compound may cause the active ingredient to become a free form, which makes it difficult to dissolve in water, resulting in a decrease in the quality of the active ingredient.

[0056] In addition, in the treatment or prevention agent for amyotrophic lateral sclerosis according to the embodiment, the active ingredient may not 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 a human, side effects may include anaphylaxis accompanied by symptoms such as generalized rash, rash, urticaria, stomatitis, laryngeal spasm, and shortness of breath. In addition, when acamprosate is administered to a human, side effects may include angioedema accompanied by symptoms such as tongue swelling and lymph node swelling. The treatment or prevention agent for amyotrophic lateral sclerosis according to the embodiment may not be combined with acamprosate, and therefore these side effects do not occur.

[0057] The therapeutic or preventive agent for amyotrophic lateral sclerosis according to the embodiment can treat or prevent amyotrophic lateral sclerosis by suppressing aggregation of TDP-43. Alternatively, the therapeutic or preventive agent for amyotrophic lateral sclerosis according to the embodiment can treat or prevent amyotrophic lateral sclerosis by suppressing cell death of cells in which TDP-43 is overexpressed.

[0058] Although the present invention has been described above by way of the embodiment, the description and drawings forming part of this disclosure should not be understood as limiting the present invention. From this disclosure, various alternative embodiments, examples and operating techniques should become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0059] (Reference example 1) Mouse neuroblastoma (Neuro2a) was seeded on the dish. Neuro2a cells were cultured in DMEM + 10% FBS medium at 37°C and 5% CO 2The cells were cultured in the presence of 1000 mM NaCl. Differentiation into neurons was initiated on the first day after seeding. 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 TDP-43 mRNA synthesized by lipofection. The cells were sampled 6, 9, 12, 15, 18, and 21 hours after transfection. Cell death was observed 21 hours after transfection. For sampling, the cells were lysed in RIPA buffer and centrifuged at 22000 × g, 30 minutes, and 4 ° C. The supernatant was taken as the soluble fraction, and the precipitate was taken as the insoluble fraction. An equal amount of 2 × SDS sample buffer was added to each of them, and the mixture was heated at 95 ° C. for 5 minutes to prepare electrophoretic samples. After each electrophoresis sample was electrophoresed using a polyacrylamide gel, TDP-43 was transferred to a PVDF membrane, and TDP-43 was detected by chemiluminescence using an anti-TDP-43 antibody as the primary antibody and an HRP-conjugated anti-rabbit antibody as the secondary antibody. The results are shown in Figures 1 and 2. It was confirmed that the insoluble fraction of TDP-43 increased in the cells from the time of transfection with the TDP-43 gene until cell death. The insoluble fraction of TDP-43 indicates that TDP-43 is aggregated.

[0060] Example 1 Neuro2a were seeded on dishes. On the first day after seeding, differentiation into neurons was initiated. On the fourth day after seeding, the cells were transfected with the TDP-43 gene. Five hours after transfection, 10 nmol / L, 50 nmol / L, 100 nmol / L, 500 nmol / L, 1000 nmol / L, 5000 nmol / L, or 10,000 nmol / L of D-cycloserine or L-cycloserine was added to the medium. As a control, 10 nmol / L, 50 nmol / L, 100 nmol / L, 500 nmol / L, 1000 nmol / L, 5000 nmol / L, or 10,000 nmol / L of ropinirole was added to the medium. Ropinirole has been reported to have a therapeutic effect on ALS.

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

[0062] As shown in FIG. 4, D-cycloserine and L-cycloserine exhibited a concentration-dependent effect of suppressing cell death. The EC 50 The EC value of L-cycloserine was 128 nmol / L. 50 The EC value for ropinirole was 237 nmol / L. 50 The relative survival rate is the ratio of the number of surviving cells that overexpressed TDP-43 to the number of surviving cells that did not overexpress TDP-43, which was taken as 100%.

[0063] Example 2 Neuro2a were seeded on a dish. On the first day after seeding, differentiation into neurons was initiated. On the fourth day after seeding, the cells were transfected with the TDP-43 gene. Six hours after transfection, 10 μmol / L of D-cycloserine, L-cycloserine, ropinirole, or DMSO was added to the cells, and the cells were cultured for 12 hours. The results of analyzing the soluble and insoluble fractions of TDP-43 in the cells are shown in Figure 5. As shown in Figure 5, in cells treated with DMSO as a control, it was confirmed that the insoluble fraction of TDP-43 was greater than the soluble fraction of TDP-43. In contrast, in cells treated with D-cycloserine and L-cycloserine, it was confirmed that the soluble fraction of TDP-43 was greater than the insoluble fraction of TDP-43.

[0064] The ratio of the insoluble fraction to the total of the soluble and insoluble fractions of TDP-43 in cells was quantified, and the results are shown in Figure 6. In cells treated with D-cycloserine and L-cycloserine, the ratio of the insoluble fraction was less than half.

[0065] 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, FUJIFILM Wako Pure Chemical Industries, Ltd.), mixed, and added to a 24-well plate at 600 μL / well. The plate was left at room temperature for 2 hours, and then the solution was removed from the wells. Next, 600 μL / well of DMEM was added, and the plate was left at room temperature for 2 hours.

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

[0067] Motor neuron density is 3X10 5 After diluting the cells in culture medium to 100 cells / mL, 600 μL of the medium containing motor neurons was placed in each of multiple wells of the matrix-coated plate and incubated at 37°C and 5% CO. 2 Motor neurons were cultured in the presence of 0.1% EDTA and 0.1% EDTA. Every 2-3 days, 300 μL of 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 seventh day after the start of the culture, 300 μL of medium was removed from each of the wells, and 300 μL of medium containing 0.2% DMSO, medium containing 20 μmol / L D-cycloserine (Cayman Chemical), or medium containing 20 μmol / L ropinirole (Fujifilm Wako Pure Chemical Industries) was added to each of the wells. Every 2 to 3 days, 300 μL of medium was removed from each of the wells, and 300 μL of medium containing the same compound as the removed medium, i.e., medium containing 0.1% DMSO, medium containing 10 μmol / L D-cycloserine, or medium containing 10 μmol / L ropinirole, was added to each of the wells.

[0069] A cell extract 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 detergent-containing protein extraction buffer (RIPA buffer, Nacalai Tesque). 50 μL of the cell extract 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 21,000Xg for 30 minutes.

[0070] After centrifugation, 45 μL of the supernatant was collected, 45 μL of electrophoresis buffer (AE-1430 EzApply, Atto) was added to the supernatant, and the mixture was heated at 95°C for 5 minutes to prepare a soluble protein fraction. After washing the precipitate 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 prepare an insoluble protein fraction.

[0071] SDS-PAGE of the soluble and insoluble fractions of proteins was performed, and the proteins were 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 washing the PVDF membrane three times with buffer, the PVDF membrane 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 luminescently stimulated using 1 mL of luminescence reagent (Immunostar LD, Fujifilm Wako Pure Chemical Industries, Ltd.), and the PVDF membrane was photographed. The photographed PVDF membrane is shown in Figure 7.

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

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

[0074] The captured PVDF membrane image was opened in ImageJ software and inverted with the Invert command. After removing the background with the Subtract Background command, the TDP-43 bands in each lane were surrounded by square frames of the same size, and the band intensities were quantified with the Measure command. The proportion of the insoluble fraction of TDP-43 was calculated from the band intensities of the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 for each sample according to the following formula: R={I I / (I I +I S )}×100 In the above formula, R is the percentage of insoluble fraction of TDP-43 (%), I I is the band intensity of the insoluble fraction of TDP-43, I S represents the band intensity of the soluble fraction of TDP-43.

[0075] Three tests were performed, and the average value of the insoluble fraction of TDP-43 was calculated. In Figure 8, the percentage of the insoluble fraction of TDP-43 in each test is shown by dots, and the average value is shown by bars. In motor neurons derived from iPS cells from healthy subjects, the average value of the insoluble fraction of TDP-43 was about 7.4%. In motor neurons derived from iPS cells from ALS patients, the average value of the insoluble fraction of TDP-43 was about 46.1%. However, in motor neurons derived from iPS cells from ALS patients in the presence of D-cycloserine, the average value of the insoluble fraction of TDP-43 decreased to about 17.4%. In motor neurons derived from iPS cells from ALS patients in the presence of ropinirole, the average value of the insoluble fraction of TDP-43 decreased to about 19.3%.

Claims

1. 1. A therapeutic or preventive agent for amyotrophic lateral sclerosis, comprising an active ingredient essentially consisting of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

2. 2. The therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to claim 1, wherein the active ingredient comprises at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

3. The method for treating or preventing amyotrophic lateral sclerosis according to claim 1 , wherein the active ingredient essentially consists of cycloserine or a salt thereof.

4. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1 , wherein the active ingredient comprises cycloserine or a salt thereof.

5. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, wherein the cycloserine is D-cycloserine.

6. The agent for treating or preventing amyotrophic lateral sclerosis according to claim 1, wherein the cycloserine is L-cycloserine.

7. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, comprising 15 mg or more and 100 mg or less of the active ingredient.

8. The therapeutic or prophylactic agent for amyotrophic lateral sclerosis according to claim 1, comprising 15 mg to 100 mg of D-cycloserine.

9. The method for treating or preventing amyotrophic lateral sclerosis according to claim 1 , wherein the active ingredient is not in a cationic form.

10. The method for treating or preventing amyotrophic lateral sclerosis according to claim 1 , wherein the active ingredient is not combined with a compound in an anionic form.

11. The method for treating or preventing amyotrophic lateral sclerosis according to claim 1, wherein the active ingredient is not combined with acamprosate.

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