Therapeutic or prophylactic agent for amyotrophic lateral sclerosis

Abstract

Disclosed is 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. The active ingredient may comprise at least one substance selected from the group consisting of cycloserine, terizidone, and salts thereof. The cycloserine may be a D-cycloserine. The cycloserine may also be an L-cycloserine.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent or a preventive agent for amyotrophic lateral sclerosis. Background Art

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

[0003] TDP-43 accumulated in the brain and spinal cord of ALS and FTLD patients has multiple serine residues at the C-terminus that are abnormally phosphorylated. In ALS and FTLD patients, the full-length TDP-43 molecule does not need to aggregate, but the C-terminal fragment of TDP-43 does. Based on the research so far, it is strongly suggested that the aggregation of TDP-43 may cause abnormal RNA metabolism or cytotoxicity, leading to the onset and development of various diseases.

[0004] As an example of a disease accompanied by TDP-43 aggregation, TDP-43 proteinopathy is known. Patent Document 1 proposes the use of N,N,N',N',-tetramethyl-10H-phenothiazine-3,7-diamine bis(mesylate) as a therapeutic agent or preventive agent for TDP-43 proteinopathy.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent No. 5898701

[0008] Patent Document 2: Japanese Patent Application No. 2022-500397

[0009] Patent Document 3: Japanese Patent Application No. 2018-526345

[0010] Patent Document 4: U.S. Patent Application Publication No. 2011 / 0160260

[0011] Patent Document 5: Japanese Patent Application No. 2002-511409 Summary of the invention

[0012] Technical issues

[0013] One object of the present invention is to provide a therapeutic or preventive agent for amyotrophic lateral sclerosis.

[0014] Technical Solution

[0015] According to an aspect of the present invention, there is provided a therapeutic or preventive agent for amyotrophic lateral sclerosis, wherein the active ingredient consists essentially of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0016] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, the active ingredient may be composed of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0017] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, the active ingredient may consist essentially of cycloserine or a salt thereof.

[0018] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, the active ingredient may consist of cycloserine or a salt thereof.

[0019] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, cycloserine may be D-cycloserine.

[0020] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, cycloserine may be L-cycloserine.

[0021] The therapeutic or preventive agent for amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of the active ingredient.

[0022] The therapeutic or preventive agent for amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of D-cycloserine.

[0023] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, the active ingredient may not be in a cationic form.

[0024] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, the active ingredient may not be combined with the compound in an anionic form.

[0025] In the above-mentioned therapeutic or preventive agent for amyotrophic lateral sclerosis, the active ingredient may not be combined with acamprosate.

[0026] 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 for producing a therapeutic or preventive agent for amyotrophic lateral sclerosis.

[0027] In the above use, the active ingredient may be composed of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0028] In the above use, the active ingredient may consist essentially of cycloserine or a salt thereof.

[0029] In the above use, the active ingredient may consist of cycloserine or a salt thereof.

[0030] In the above use, the cycloserine may be D-cycloserine.

[0031] In the above use, the cycloserine may be L-cycloserine.

[0032] In the above use, the therapeutic or preventive agent for amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of the active ingredient.

[0033] In the above use, the therapeutic or preventive agent for amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of D-cycloserine.

[0034] In the above uses, the active ingredient may not be in cationic form.

[0035] In the above-mentioned uses, the active ingredient may not be combined with the compound in an anionic form.

[0036] In the above uses, the active ingredient may not be combined with acamprosate.

[0037] According to the present invention, a method for treating or preventing amyotrophic lateral sclerosis is provided, the method comprising administering a therapeutic or preventive agent for amyotrophic lateral sclerosis to a patient with amyotrophic lateral sclerosis, wherein the active ingredient of the therapeutic or preventive agent is essentially composed of at least one selected from cycloserine and terizidone and a salt thereof.

[0038] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis may be composed of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0039] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis may consist essentially of cycloserine or a salt thereof.

[0040] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis may consist of cycloserine or a salt thereof.

[0041] 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.

[0042] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the therapeutic or preventive agent for amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of the active ingredient.

[0043] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the therapeutic or preventive agent for amyotrophic lateral sclerosis may contain 15 mg or more and 100 mg or less of D-cycloserine.

[0044] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis may not be in a cationic form.

[0045] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis may not be combined with the compound in an anionic form.

[0046] In the above-mentioned method for treating or preventing amyotrophic lateral sclerosis, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis may not be combined with acamprosate.

[0047] Technical Effects

[0048] According to the present invention, a therapeutic agent or a preventive agent for amyotrophic lateral sclerosis can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a photograph showing the TDP-43 soluble fraction and the TDP-43 insoluble fraction of Reference Example 1.

[0050] Figure 2 This is a graph showing the relationship between the elapsed time after TDP-43 gene transfection in Reference Example 1 and the ratio of the TDP-43 insoluble fraction in cells.

[0051] Figure 3 This is a photograph of the cells of Example 1.

[0052] Figure 4 This is a graph showing the relationship between the concentration of the compound of Example 1 and the relative survival rate of cells.

[0053] Figure 5 The photographs show the TDP-43 soluble fraction and the TDP-43 insoluble fraction of Example 2.

[0054] Figure 6This is a graph showing the relationship between the compound of Example 2 and the ratio of the insoluble component of cellular TDP-43.

[0055] Figure 7 The photographs show the TDP-43 soluble fraction and the TDP-43 insoluble fraction of Example 3.

[0056] Figure 8 This is a graph showing the ratio of the insoluble component of TDP-43 in Example 3. DETAILED DESCRIPTION

[0057] The following describes the embodiments of the present invention. However, it should not be understood that the following embodiments limit the present invention. Based on this disclosure, a person skilled in the art should understand various alternative embodiments, examples, and application techniques. It should be understood that the present invention includes various embodiments not described herein.

[0058] The active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis (ALS) of the embodiment consists essentially of at least one selected from the group consisting of cycloserine and physiologically acceptable salts thereof. In the present disclosure, "consisting essentially of" 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.

[0059] Alternatively, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment is composed of at least one selected from the group consisting of cycloserine and physiologically acceptable salts thereof. The cycloserine may be D-cycloserine or L-cycloserine.

[0060] Amyotrophic lateral sclerosis (ALS) is a disease accompanied by the aggregation of TDP-43 and is classified as a TDP-43 proteinopathy.

[0061] The IUPAC name of D-cycloserine (CAS number: 68-41-7) is (4R)-4-amino-1,2-oxazolin-3-one. The chemical formula of D-cycloserine is C3H6N2O2. The chemical structure of D-cycloserine is as follows.

[0062] [Chemical formula 1]

[0063]

[0064] The IUPAC name of L-cycloserine (CAS No.: 339-72-0) is (4S)-4-amino-1,2-oxazolin-3-one. The chemical formula of L-cycloserine is C3H6N2O2. The chemical structure of L-cycloserine is as follows.

[0065] [Chemical formula 2]

[0066]

[0067] The active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment may consist essentially of at least one prodrug of the above-mentioned compound or a physiologically acceptable salt thereof. Alternatively, the active ingredient of the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment may also consist of at least one prodrug of the above-mentioned compound or a physiologically acceptable salt thereof.

[0068] For example, terizidone is known as a prodrug of D-cycloserine. The IUPAC name of terizidone (CAS No.: 25683-71-0) is 4,4'-{1,4-phenylbis[(E)methylene(E)nitro]}bis(1,2-oxazolin-3-one). The chemical formula of terizidone is C 14 H 14 N4O4. The chemical structure of terizidone is as follows. Terizidone is decomposed in the body and exerts the same effect as D-cycloserine.

[0069] [Chemical formula 3]

[0070]

[0071] The therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment can be formulated into a pharmaceutically 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. Injections include, for example, injections, sterile powders for injection, and concentrated solutions for injection. The therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment can contain, for example, 15 mg or more and 100 mg or less, 20 mg or more and 80 mg or less, or 25 mg or more and 60 mg or less of an active ingredient. The therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment can contain, for example, 15 mg or more and 100 mg or less, 20 mg or more and 80 mg or less, or 25 mg or more and 60 mg or less of D-cycloserine.

[0072] It has been reported that the maximum blood concentration of D-cycloserine (C max) is about 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 HealthySubjects. 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 HealthySubjects. Neuropsychopharmacol 17,116(1997). https: / / doi.org / 10.1016 / S0893-133X(97)00082-1).

[0073] It has been reported that the brain transfer rate of D-cycloserine is 95%. Therefore, it is estimated that the maximum concentration of D-cycloserine in the brain is about 5.3 μmol / L. The concentration of about 5.3 μmol / L exceeds the EC value of D-cycloserine for inhibiting cell death of neurons forced to express TDP-43 as shown in Example 1 below. 50 128nmol / L. In addition, it has been reported that the half-life of D-cycloserine is about 10 hours. Therefore, it is speculated that the trough value of D-cycloserine when 15mg is orally administered twice a day also exceeds the EC 50 Therefore, the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment can fully exert the therapeutic or preventive effect for human amyotrophic lateral sclerosis by containing 15 mg or more of D-cycloserine.

[0074] In addition, it is reported that when D-cycloserine is used for the treatment of tuberculosis, if 250 mg is administered to a human, serious side effects such as epileptic seizures and mental confusion occur. In addition, there are reports that if 150 mg is administered to a human, a headache occurs. Therefore, the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment can suppress the side effects caused by D-cycloserine by containing 100 mg or less of D-cycloserine.

[0075] It should be noted that in the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment, the active ingredient may not be in a cationic form. In addition, the active ingredient may not be combined with a compound in an anionic form. The chemical structural formula of the compound in an anionic form is shown below, for example.

[0076] [Chemical formula 4]

[0077]

[0078] In the chemical formula of the compound in anionic form,

[0079] X is -NH2 or -OH,

[0080] L1 and L2 are each independently C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkyne, or, if valence permits, one of L1 and L2 is N, O or S and the other is C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkyne group. The compound in anionic form is, for example, succinic acid, D-tartaric acid, L-tartaric acid, mesotartaric acid, fumaric acid, maleic acid or malic acid. The combination of the active ingredient in cationic form and the compound in anionic form may cause the active ingredient to be free and not easily soluble in water, thereby reducing the quality of the active ingredient.

[0081] In addition, in the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment, the active ingredient may not be combined with acamprosate. Acamprosate is a derivative of homotaurine, also known as 3-(acetylamino)propylsulfonic acid or N-acetylhomotaurine. If acamprosate is administered to a human, an allergic reaction accompanied by symptoms such as systemic rash, eruption, urticaria, stomatitis, laryngospasm and asthma may occur as a side effect. In addition, if acamprosate is administered to a human, angioedema accompanied by symptoms such as tongue swelling and lymph node swelling may occur as a side effect. Since the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment may not be combined with acamprosate, these side effects will not occur.

[0082] The therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment can treat or prevent amyotrophic lateral sclerosis by inhibiting the aggregation of TDP-43. Alternatively, the therapeutic or preventive agent for amyotrophic lateral sclerosis of the embodiment can treat or prevent amyotrophic lateral sclerosis by inhibiting cell death of cells overexpressing TDP-43.

[0083] As described above, the present invention is described by the embodiments, but it should not be understood that the description and drawings constituting a part of the disclosure limit the present invention. According to the present disclosure, a person skilled in the art should understand various alternative embodiments, examples and application technologies. It should be understood that the present invention includes various embodiments not described herein.

[0084] (Reference Example 1)

[0085] Mouse neuroblastoma (Neuro2a) was inoculated in a culture dish. Neuro2a cells were cultured in a medium of DMEM+10% FBS at 37°C and 5% CO2. Differentiation into neurons began on the first day from inoculation. Differentiation into neurons was carried out in a medium of DMEM+2% FBS+20μmol / L retinoic acid. On the fourth day from inoculation, the cells were transfected with synthetic TDP-43 mRNA by liposome transfection. The cells were sampled 6 hours, 9 hours, 12 hours, 15 hours, 18 hours and 21 hours after transfection. Cell death was observed 21 hours after transfection. During the sampling process, the cells were dissolved with RIPA buffer, centrifuged at 22000×g, 30 minutes, and 4°C, and the supernatant was used as the soluble component and the precipitate was used as the insoluble component. Equal amounts of 2×SDS sample buffer were added, heated at 95°C for 5 minutes, and used as electrophoresis samples. After electrophoresis of each electrophoresis sample using 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-labeled anti-rabbit antibody as the secondary antibody. The results are shown in Figure 1 and Figure 2 It was confirmed that the amount of insoluble TDP-43 increased in the cells from the time of TDP-43 gene transfection to the time of cell death. The amount of insoluble TDP-43 indicates that TDP-43 is aggregated.

[0086] (Example 1)

[0087] Neuro2a was inoculated in a culture dish. From the first day after inoculation, differentiation into neurons began. From the fourth day after inoculation, the TDP-43 gene was transfected into the cells. Five hours after transfection, 10nmol / L, 50nmol / L, 100nmol / L, 500nmol / L, 1000nmol / L, 5000nmol / L or 10000nmol / L of D-cycloserine or L-cycloserine was added to the culture medium. In addition, as a control, 10nmol / L, 50nmol / L, 100nmol / L, 500nmol / L, 1000nmol / L, 5000nmol / L, or 10000nmol / L of ropinirole was added to the culture medium. There are reports showing that ropinirole has a therapeutic effect on ALS.

[0088] The proportion of dead cells was evaluated on day 7 from the time the cells were transfected with the TDP-43 gene. Figure 3 As shown in Figure A, no cell death was observed in cells not transfected with the TDP-43 gene. Figure 3 As shown in FIG. 2B , cell death was observed in cells transfected with the TDP-43 gene and without the addition of D-cycloserine or edaravone. Figure 3 As shown in C, in cells transfected with the TDP-43 gene and supplemented with D-cycloserine, cell death was suppressed.

[0089] like Figure 4 As shown, D-cycloserine and L-cycloserine showed an inhibitory effect on cell death depending on the concentration. The EC 50 The EC of L-cycloserine is 128 nmol / L. 50 The EC of ropinirole is 237 nmol / L. 50 It is 198 nmol / L. The relative survival rate indicates the ratio of the number of surviving cells overexpressing TDP-43 to the number of surviving cells not overexpressing TDP-43 as 100%.

[0090] (Example 2)

[0091] Neuro2a was inoculated in a culture dish. On the first day after inoculation, differentiation into neurons began. On the fourth day after inoculation, the TDP-43 gene was transfected into the cells. 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. Then, the soluble and insoluble components of TDP-43 in the cells were analyzed, and the results are shown in FIG. Figure 5 .like Figure 5As shown in the figure, in cells treated with DMSO as a control, more TDP-43 insoluble fractions were found than TDP-43 soluble fractions. In contrast, in cells treated with D-cycloserine and L-cycloserine, more TDP-43 soluble fractions were found than TDP-43 insoluble fractions.

[0092] The results of quantitative processing of the ratio of the insoluble fraction of TDP-43 in cells to the total of the soluble fraction and the insoluble fraction are shown in FIG. Figure 6 In cells treated with D-cycloserine and L-cycloserine, the proportion of insoluble components was less than half.

[0093] (Example 3)

[0094] 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.) and mixed, and added to a 24-well plate at 600 μL / well. After the plate was left at room temperature for 2 hours, the solution was removed from the wells. Next, DMEM was added at 600 μL / well, and the plate was left at room temperature for 2 hours.

[0095] Motor neurons derived from iPS cells of healthy humans and motor neurons derived from iPS cells of ALS patients were obtained from iXCells Biotechnologies. Motor neurons that were rapidly thawed in a thermostatic chamber at 37°C were suspended in motor neuron maintenance medium (iXCells Biotechnologies). The medium was centrifuged at 600Xg for 5 minutes, and after collecting the motor neurons, the medium was removed and the motor neurons were suspended in motor neuron maintenance medium.

[0096] The density of motor neurons in the culture medium was 3 × 10 5 After diluting the cells at 10 cells / mL, 600 μL of the medium containing motor neurons was added to each well of the matrix-coated plate, and the motor neurons were cultured at 37°C in the presence of 5% CO 2. Every 2 or 3 days, 300 μL of the medium was removed from each well of the wells, and 300 μL of new medium was added to each well of the wells.

[0097] On the 7th day from the start of culture, 300 μL of the culture medium was removed from each of the wells, and 300 μL of a culture medium containing 0.2% DMSO, 300 μL of a culture medium containing 20 μmol / L D-cycloserine (Cayman Chemical), or 300 μL of a culture medium containing 20 μmol / L ropinirole (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each of the wells. 300 μL of the culture medium was removed from each of the wells every 2 or 3 days, and a culture medium containing the same compound as the culture medium removed from each of the wells was added, i.e., 300 μL of a culture medium containing 0.1% DMSO, 300 μL of a culture medium containing 10 μmol / L D-cycloserine, or 300 μL of a culture medium containing 10 μmol / L ropinirole was added.

[0098] 5 μL of a mixture of protease inhibitors and phosphatase inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, 100X, registered trademark: Thermo Fisher Scientific) was added to 500 μL of a buffer (RIPA buffer, Nacalai Tesque) containing a surfactant to prepare a cell extract. 50 μL of 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 recovered in a 1.5 mL tube by pipetting. The cell extract was centrifuged at 4°C and 21000Xg for 30 minutes.

[0099] After taking 45 μL of the supernatant after centrifugation, 45 μL of electrophoresis buffer (AE-1430EzApply, ATTO) was added to the supernatant and heated at 95°C for 5 minutes to prepare a protein soluble fraction. In addition, the precipitate after centrifugation was washed twice with 50 μL of RIPA buffer, and then 45 μL of RIPA buffer and 45 μL of electrophoresis buffer were added to the precipitate and heated at 95°C for 5 minutes to prepare a protein insoluble fraction.

[0100] SDS-PAGE of the soluble and insoluble components of the protein was performed, and the protein was transferred to a PVDF membrane. The PVDF membrane was immersed in a blocking solution and shaken at room temperature for 1 hour to block the PVDF membrane. The PVDF membrane was immersed in a solution containing a TDP-43 polyclonal antibody (PGI Proteintech Group) and shaken at 4°C overnight. Then, after washing the PVDF membrane three times with a buffer solution, the PVDF membrane was immersed in a solution containing an 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, after washing the PVDF membrane three times with a buffer solution, 1 mL of a luminescent reagent (ImmunoStar LD, Fujifilm Wako Pure Chemical Industries, Ltd.) was used to make the labeled secondary antibody emit light, and the PVDF membrane was photographed. The photographed PVDF membrane is shown in Figure 7 .

[0101] like Figure 7 As shown, as a control, in the motor neurons derived from iPS cells from healthy human beings cultured in the presence of DMSO, 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 from healthy human beings 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 from healthy human beings 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.

[0102] like Figure 7 As shown, as a control, in the motor neurons derived from iPS cells from ALS patients cultured in the presence of DMSO, the band of the soluble component of TDP-43 was clearly observed, and the band of the insoluble component was also clearly confirmed. In the motor neurons derived from iPS cells from ALS patients cultured in the presence of D-cycloserine, the band of the soluble component of TDP-43 was clearly observed, and the band of the insoluble component was faint. In the motor neurons derived from iPS cells from ALS patients cultured in the presence of ropinirole, the band of the soluble component of TDP-43 was clearly observed, and the band of the insoluble component was faint.

[0103] The image of the PVDF membrane was opened with Image J 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 with boxes of the same size in sequence, and the band intensity was quantified with the Measure command. Based on the band intensity of the TDP-43 soluble component and the band intensity of the TDP-43 insoluble component of each sample, the ratio of the TDP-43 insoluble component was calculated according to the following formula.

[0104] R={I1 / (I1+I S )}×100

[0105] In the above formula, R represents the ratio (%) of the insoluble component of TDP-43, I1 represents the band intensity of the insoluble component of TDP-43, and I S Indicates the band intensity of the soluble fraction of TDP-43.

[0106] The experiment was performed three times, and the average value of the ratio of the insoluble component of TDP-43 was calculated. Figure 8 In the figure, the proportion of the insoluble components of TDP-43 in each test is indicated by a dot, and the average value is indicated by a horizontal line (bar). In the motor neurons derived from iPS cells of healthy humans, the average value of the proportion of the insoluble components of TDP-43 is about 7.4%. On the other hand, in the motor neurons derived from iPS cells of ALS patients, the average value of the proportion of the insoluble components of TDP-43 is about 46.1%. However, in the motor neurons derived from iPS cells of ALS patients in the presence of D-cycloserine, the average value of the proportion of the insoluble components of TDP-43 is reduced to about 17.4%. In addition, in the motor neurons derived from iPS cells of ALS patients in the presence of ropinirole, the average value of the proportion of the insoluble components of TDP-43 is reduced to about 19.3%.

Claims

1. A therapeutic or preventive agent for amyotrophic lateral sclerosis, characterized in that: The active ingredient consists essentially of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

2. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that The active ingredient consists of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

3. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that The active ingredient consists essentially of cycloserine or a salt thereof.

4. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that The effective ingredient consists of cycloserine or its salt.

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

6. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that: The cycloserine is L-cycloserine.

7. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that The active ingredient is contained in an amount of 15 mg or more and 100 mg or less.

8. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that The invention contains 15 mg or more and 100 mg or less of D-cycloserine.

9. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that: The active ingredient is not in cationic form.

10. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that The active ingredient is not combined with the compound in anionic form.

11. The therapeutic or preventive agent for amyotrophic lateral sclerosis according to claim 1, characterized in that: The active ingredient is not combined with acamprosate.

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