Therapeutic or prophylactic agent for frontotemporal lobar degeneration accompanied by accumulation of TDP-43 (FTLD-TDP)

Abstract

Provided is a therapeutic or prophylactic agent for frontotemporal lobar degeneration accompanied by the accumulation of TDP-43 (FTLD-TDP), wherein the active ingredient essentially consists of at least one component selected from the group consisting of cycloserine, terizidone, and salts thereof.

Description

Treatment or prevention agent for frontotemporal lobar degeneration associated with TDP-43 accumulation (FTLD-TDP)

[0001] The present invention relates to a therapeutic or preventive agent for frontotemporal lobar degeneration associated with accumulation of TDP-43 (FTLD-TDP).

[0002] TDP-43 (TAR DNA-binding protein of 43 kDa) is a 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 its C-terminus. In ALS and FTLD patients, it is not necessary for the full-length TDP-43 molecule to aggregate; rather, C-terminal fragments of TDP-43 aggregate. Research to date has strongly suggested that TDP-43 aggregation causes abnormalities in RNA metabolism and induces cytotoxicity, leading to the onset and progression of various diseases.

[0004] An example of a disease associated with 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 preventive agent for TDP-43 proteinopathy.

[0005] Patent No. 5898701

[0006] Snowden JS, Neary D, Mann D M. Fronto-temporal lobar degeneration: fronto-temporal dementia, progressive aphasia, semantic dementia. NY: Churchill Livingstone; 1996.Mackenzie IRA, Neumann M, Baborie A,et al., Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an update, Acta Neuropathol, 2010;119:1-4.

[0007] An object of the present invention is to provide an agent for treating or preventing frontotemporal lobar degeneration accompanied by accumulation of TDP-43 (FTLD-TDP).

[0008] According to an aspect of the present invention, there is provided an agent for treating or preventing frontotemporal lobar degeneration associated with accumulation of TDP-43 (FTLD-TDP), wherein the active ingredient essentially consists of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0009] In the present disclosure, "consisting essentially of" means that the active ingredient does not contain any other ingredients, and does not exclude the presence of ingredients other than the active ingredient, such as excipients and / or lubricants.

[0010] In the above-mentioned agent for treating or preventing FTLD-TDP, the active ingredient may be at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0011] In the above-mentioned agent for treating or preventing FTLD-TDP, the active ingredient may consist essentially of cycloserine or a salt thereof.

[0012] In the above-mentioned agent for treating or preventing FTLD-TDP, the active ingredient may consist of cycloserine or a salt thereof.

[0013] In the above-mentioned agent for treating or preventing FTLD-TDP, the cycloserine may be D-cycloserine.

[0014] In the above-mentioned agent for treating or preventing FTLD-TDP, the cycloserine may be L-cycloserine.

[0015] The above-mentioned therapeutic or preventive agent for FTLD-TDP may contain 15 mg or more and less than 250 mg of an active ingredient.

[0016] The above-mentioned therapeutic or prophylactic agent for FTLD-TDP may contain 15 mg or more and less than 250 mg of D-cycloserine.

[0017] In the above-mentioned agent for treating or preventing FTLD-TDP, the active ingredient may not be in a cationic form.

[0018] In the above-mentioned agent for treating or preventing FTLD-TDP, the active ingredient does not have to be combined with a compound in an anionic form.

[0019] In the above-mentioned agent for treating or preventing FTLD-TDP, the active ingredient does not necessarily have to be combined with acamprosate.

[0020] 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 treating or preventing frontotemporal lobar degeneration associated with TDP-43 accumulation (FTLD-TDP).

[0021] 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 the treatment or prevention of frontotemporal lobar degeneration associated with accumulation of TDP-43 (FTLD-TDP).

[0022] In the above use, the active ingredient may be at least one selected from the group consisting of cycloserine and terizidone, and salts thereof.

[0023] In the above uses, the active ingredient may consist essentially of cycloserine or a salt thereof.

[0024] In the above use, the active ingredient may consist of cycloserine or a salt thereof.

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

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

[0027] In the above use, the therapeutic or preventive agent for FTLD-TDP may contain 15 mg or more but less than 250 mg of an active ingredient.

[0028] In the above use, the therapeutic or prophylactic agent for FTLD-TDP may contain 15 mg or more and less than 250 mg of D-cycloserine.

[0029] In the above uses, the active ingredient may not be in cationic form.

[0030] In the above uses, the active ingredient may not be combined with a compound in anionic form.

[0031] In the above uses, the active ingredient does not have to be combined with acamprosate.

[0032] According to an aspect of the present invention, there is provided a method for treating or preventing frontotemporal lobar degeneration associated with accumulation of TDP-43 (FTLD-TDP), comprising administering to a patient with FTLD-TDP an agent for treating or preventing FTLD-TDP, the active ingredient of which consists essentially of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0033] In the above-mentioned method for treating or preventing FTLD-TDP, the active ingredient of the agent for treating or preventing FTLD-TDP may consist of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

[0034] In the above-mentioned method for treating or preventing FTLD-TDP, the active ingredient of the agent for treating or preventing FTLD-TDP may consist essentially of cycloserine or a salt thereof.

[0035] In the above-mentioned method for treating or preventing FTLD-TDP, the active ingredient of the agent for treating or preventing FTLD-TDP may consist of cycloserine or a salt thereof.

[0036] In the above-mentioned method for treating or preventing FTLD-TDP, the cycloserine may be D-cycloserine.

[0037] In the above-mentioned method for treating or preventing FTLD-TDP, the cycloserine may be L-cycloserine.

[0038] In the above-mentioned method for treating or preventing FTLD-TDP, the agent for treating or preventing FTLD-TDP may contain 15 mg or more and less than 250 mg of an active ingredient.

[0039] In the above-mentioned method for treating or preventing FTLD-TDP, the agent for treating or preventing FTLD-TDP may contain 15 mg or more and less than 250 mg of D-cycloserine.

[0040] In the above-mentioned method for treating or preventing FTLD-TDP, the active ingredient of the agent for treating or preventing FTLD-TDP may not be in a cationic form.

[0041] In the above-mentioned method for treating or preventing FTLD-TDP, the active ingredient of the agent for treating or preventing FTLD-TDP does not have to be combined with a compound in an anionic form.

[0042] In the above-mentioned method for treating or preventing FTLD-TDP, the active ingredient of the agent for treating or preventing FTLD-TDP does not have to be combined with acamprosate.

[0043] According to the present invention, it is possible to provide a therapeutic or preventive agent for frontotemporal lobar degeneration accompanied by accumulation of TDP-43 (FTLD-TDP).

[0044] FIG. 1 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Reference Example 1. FIG. 2 is a graph showing the relationship between the time elapsed since transfection of the TDP-43 gene and the proportion of the insoluble fraction of TDP-43 in cells according to Reference Example 1. FIG. 3 is a photograph of cells according to Example 1. FIG. 4 is a graph showing the relationship between the concentration of the compound according to Example 1 and the relative viability of cells. FIG. 5 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 2. FIG. 6 is a graph showing the relationship between the compound according to Example 2 and the proportion of the insoluble fraction of TDP-43 in cells. FIG. 7 is a photograph showing the soluble fraction of TDP-43 and the insoluble fraction of TDP-43 according to Example 3. FIG. 8 is a graph showing the proportion of the insoluble fraction of TDP-43 according to Example 3.

[0045] 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.

[0046] In one embodiment of the agent for treating or preventing frontotemporal lobar degeneration associated with TDP-43 accumulation (FTLD-TDP), the active ingredient essentially consists 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.

[0047] Classically, the diagnosis of frontotemporal lobar degeneration (FTLD) required degeneration of the frontal and temporal lobes, regardless of the type of accumulated material. Because FTLD is a disease concept based on anatomical or morphological observations, a wide variety of diseases were included under the FTLD umbrella. This led to confusion, as symptoms of Alzheimer's disease and dementia with Lewy bodies, where the primary lesion was located in the frontotemporal lobes, were classified as FTLD. In contrast, since 2010, there has been growing momentum to classify FTLD based on the protein that accumulates, and FTLD is currently classified into frontotemporal lobar degeneration associated with TDP-43 accumulation (FTLD-TDP), frontotemporal lobar degeneration associated with tau protein accumulation (FTLD-tau), frontotemporal lobar degeneration associated with FUS (fused in sarcoma) protein accumulation (FTLD-FUS), familial FTD associated with chromosome 3 (FTLD-UPS), and FTLD without accumulation (FTLD-ni) (see Non-Patent Documents 1 and 2). A therapeutic or prophylactic agent according to an embodiment targets FTLD-TDP.

[0048] The IUPAC name of D-cycloserine (CAS number: 68-41-7) is (4R)-4-Amino-1,2-oxazolidin-3-one. The chemical formula of D-cycloserine is C3H6N2O2. The chemical structural formula of D-cycloserine is as follows:

[0049] The IUPAC name of L-cycloserine (CAS number: 339-72-0) is (4S)-4-amino-1,2-oxazolidin-3-one. The chemical formula of L-cycloserine is C3H6N2O2. The chemical structural formula of L-cycloserine is as follows:

[0050] The therapeutic or prophylactic agent for FTLD-TDP according to the embodiment may contain at least one prodrug of the above-mentioned compound or a physiologically acceptable salt thereof.

[0051] For example, terizidone is known as a prodrug of D-cycloserine. The IUPAC name of terizidone (CAS number: 25683-71-0) is 4,4'-{1,4-phenylenebis[(E)methylylidene(E)azanylylidene]}bis(1,2-oxazolidin-3-one). The chemical formula of terizidone is C 14 H 14 N4O4. The chemical structure of terizidone is: Terizidone is broken down in the body and exerts effects similar to those of D-cycloserine.

[0052] The therapeutic or preventive agent for FTLD-TDP according to the embodiment can be formulated into a pharmaceutically acceptable dosage form. For example, the therapeutic or preventive agent for FTLD-TDP can be formulated into an injection, solution, suspension, tablet, capsule, pill, granule, syrup, suppository, inhalant, or spray. Injectable agents include, for example, an injection solution, a sterile powder for injection, and a concentrated solution for injection. The therapeutic or preventive agent for FTLD-TDP according to the embodiment may contain, for example, 15 mg or more but less than 250 mg, 15 mg or more but not more than 249.9 mg, 15 mg or more but not more than 249 mg, 15 mg or more but not more than 200 mg, 15 mg or more but not more than 150 mg, 15 mg or more but not more than 100 mg, 20 mg or more but not more than 80 mg, or 25 mg or more but not more than 60 mg of the active ingredient. Alternatively, the therapeutic or prophylactic agent for FTLD-TDP according to the embodiment may contain an active ingredient in an amount of 100 mg or more but less than 250 mg, 100 mg or more but less than 249.9 mg, 100 mg or more but less than 249 mg, 100 mg or more but less than 200 mg, or 100 mg or more but less than 150 mg. The therapeutic or prophylactic agent for FTLD-TDP according to the embodiment may contain, for example, 15 mg or more but less than 250 mg, 15 mg or more but less than 200 mg, 15 mg or more but less than 150 mg, 15 mg or more but less than 100 mg, 20 mg or more but less than 80 mg, or 25 mg or more but less than 60 mg of D-cycloserine. Alternatively, the therapeutic or prophylactic agent for FTLD-TDP according to the embodiment may contain 100 mg or more but less than 250 mg, 100 mg or more but less than 249.9 mg, 100 mg or more but less than 249 mg, 100 mg or more but less than 200 mg, or 100 mg or more but less than 150 mg of D-cycloserine.

[0053] The maximum blood concentration (Cmax) of D-cycloserine following oral administration of 15 mg of D-cycloserine to humans has been 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).1694420249316_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).

[0054] 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 approximately 5.3 μmol / L. This concentration of approximately 5.3 μmol / L is the EC value of D-cycloserine, which suppresses cell death in neurons forced to express TDP-43, as shown in Example 1 below. ５０ The trough value of 15 mg of D-cycloserine administered orally twice daily exceeds EC200 / C128 nmol / L. ５０ Therefore, by containing 15 mg or more of D-cycloserine, the therapeutic or prophylactic agent for FTLD-TDP according to the embodiment can sufficiently exert the therapeutic or prophylactic effect for FTLD-TDP in humans.

[0055] Furthermore, when used to treat tuberculosis, it has been reported that administration of 250 mg of D-cycloserine to humans causes severe side effects such as epileptic seizures and mental confusion. Therefore, by including less than 250 mg of D-cycloserine in the therapeutic or preventive agent for FTLD-TDP according to the embodiment, it is possible to suppress the side effects of D-cycloserine. It has also been reported that administration of 150 mg of D-cycloserine to humans causes headache. However, because FTLD-TDP is an extremely serious disease, treatment should be prioritized even if a headache occurs as a side effect. By including 15 mg or more and less than 250 mg of an active ingredient in the therapeutic or preventive agent for FTLD-TDP according to the embodiment, the administered active ingredient acts in an agonist-like manner on the NMDA receptor.

[0056] In the therapeutic or preventive agent for FTLD-TDP according to the embodiment, the active ingredient does not have to be in a cationic form. Furthermore, the active ingredient does not have to be combined with a compound in an anionic form. The chemical structural formula of the compound in an anionic form is, for example, as shown below.

[0057] In the chemical structure of the anionic form of the compound, X is -NH ２ or —OH, and each of L1 and L2 is independently 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 convert the active ingredient into a free form, making it less soluble in water and resulting in a decrease in the quality of the active ingredient.

[0058] Furthermore, in the therapeutic or preventive agent for FTLD-TDP according to the embodiment, the active ingredient does not need 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 a human, side effects may include anaphylaxis accompanied by symptoms such as generalized rash, rash, hives, stomatitis, laryngeal spasm, and shortness of breath. When acamprosate is administered to a human, side effects may include angioedema accompanied by symptoms such as tongue swelling and lymphadenopathy. Because the therapeutic or preventive agent for FTLD-TDP according to the embodiment does not need to be combined with acamprosate, these side effects do not occur.

[0059] As described above, it has been strongly suggested that TDP-43 aggregation causes abnormalities in RNA metabolism and leads to cytotoxicity, resulting in the onset and progression of various diseases. The therapeutic or preventive agent for FTLD-TDP according to the embodiment is capable of treating or preventing FTLD-TDP by inhibiting TDP-43 aggregation. Alternatively, the therapeutic or preventive agent for FTLD-TDP according to the embodiment is capable of treating or preventing FTLD-TDP by inhibiting cell death of cells in which TDP-43 is overexpressed.

[0060] Although the present invention has been described above by way of the embodiments, the descriptions and drawings that form part of this disclosure should not be understood to 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.

[0061] (Reference Example 1) Mouse neuroblastoma (Neuro2a) cells were seeded onto a dish. Neuro2a cells were cultured in a DMEM + 10% FBS medium at 37°C in the presence of 5% CO2. One day after seeding, differentiation into neurons was initiated. Neuronal differentiation was carried out in a DMEM + 2% FBS + 20 μmol / L retinoic acid medium. Four days after seeding, the 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 and centrifuged at 22,000 x g for 30 minutes at 4°C. The supernatant was used as the soluble fraction, and the precipitate was used as the insoluble fraction. An equal volume of 2x SDS sample buffer was added to each, and the mixture was heated at 95°C for 5 minutes to prepare electrophoresis samples. Each electrophoresis 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 in cells between transfection with the TDP-43 gene and cell death. The insoluble fraction of TDP-43 indicates that TDP-43 has aggregated.

[0062] Example 1 Neuro2a cells were seeded onto a dish. One day after seeding, differentiation into neurons was initiated. Four days 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.

[0063] The percentage of dead cells was assessed 7 days after transfection of the TDP-43 gene. As shown in Figure 3A, cell death was not 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 did not receive D-cycloserine. As shown in Figure 3C, cell death was suppressed in cells that were transfected with the TDP-43 gene and received D-cycloserine.

[0064] As shown in Figure 4, D-cycloserine and L-cycloserine exhibited a concentration-dependent effect of suppressing cell death. 50 The EC value of L-cycloserine was 128 nmol / L. 50 The EC value of ropinirole was 237 nmol / L. 50 The relative survival rate represents the ratio of the number of surviving cells that overexpressed TDP-43 to the number of surviving cells that did not overexpress TDP-43, which was set at 100%.

[0065] Example 2 Neuro2a cells were seeded onto a dish. One day after seeding, differentiation into neurons was initiated. Four days 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 soluble and insoluble fractions of TDP-43 in the cells were then 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 confirmed to be greater than the soluble fraction. In contrast, in cells treated with D-cycloserine and L-cycloserine, the soluble fraction of TDP-43 was confirmed to be greater than the insoluble fraction of TDP-43.

[0066] The ratio of the insoluble fraction to the total of the soluble and insoluble fractions of intracellular TDP-43 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.

[0067] 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.) and mixed, and the mixture was added to a 24-well plate at 600 μL / well. The plate was left at room temperature for 2 hours, after which the solution was removed from the wells. Next, 600 μL of DMEM was added to each well, and the plate was left at room temperature for 2 hours.

[0068] Motor neurons derived from iPS cells from healthy individuals and 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 600×g 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.

[0069] The density of motor neurons is 3x10 5 After diluting the 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, 5% CO ２ 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.

[0070] On the seventh day after the start of culture, 300 μL of medium was removed from each of the wells, and 300 μL of medium containing 0.2% DMSO, 20 μmol / L D-cycloserine (Cayman Chemical), or 20 μmol / L ropinirole (Fujifilm Wako Pure Chemical Industries) was added to each of the wells. Every 2–3 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., 0.1% DMSO, 10 μmol / L D-cycloserine, or 10 μmol / L ropinirole, was added to each of the wells.

[0071] Cell extracts were 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 detergent-containing protein extraction buffer (RIPA buffer, Nacalai Tesque). 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 collected in a 1.5 mL tube by pipetting. The cell extract was centrifuged at 21,000 × g for 30 minutes at 4°C.

[0072] After centrifugation, 45 μL of the supernatant was collected, and 45 μL of electrophoresis buffer (AE-1430 EzApply, Atto) was added to the supernatant and heated at 95°C for 5 minutes to prepare a soluble protein fraction. After centrifugation, the precipitate 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 an insoluble protein fraction.

[0073] SDS-PAGE of the soluble and insoluble protein fractions was performed, and the proteins were transferred to a PVDF membrane. The PVDF membrane was blocked by immersing it in blocking solution and shaking at room temperature for 1 hour. The PVDF membrane was then 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)-conjugated secondary antibody (Anti-Rabbit IgG, HRP-Linked F(ab')2 Fragment Donkey, Cytiva) and shaken at room temperature for 1 hour. After washing the PVDF membrane three times with buffer, the labeled secondary antibody was sensitized using 1 mL of luminescence reagent (Immunostar LD, Fujifilm Wako Pure Chemical Industries), and the PVDF membrane was photographed. The photograph of the PVDF membrane is shown in FIG.

[0074] As shown in Figure 7, in motor neurons derived from iPS cells from healthy subjects cultured in the presence of DMSO as a control, a band for the soluble fraction of TDP-43 was clearly observed, while a faint band for the insoluble fraction. In motor neurons derived from iPS cells from healthy subjects cultured in the presence of D-cycloserine, a band for the soluble fraction of TDP-43 was clearly observed, while a faint band for the insoluble fraction. In motor neurons derived from iPS cells from healthy subjects cultured in the presence of ropinirole, a band for the soluble fraction of TDP-43 was clearly observed, while a faint band for the insoluble fraction.

[0075] As shown in Figure 7, in motor neurons derived from iPS cells from an ALS patient cultured in the presence of DMSO as a control, a band for the soluble fraction of TDP-43 was clearly observed, and a band for the insoluble fraction was also clearly confirmed. In motor neurons derived from iPS cells from an ALS patient cultured in the presence of D-cycloserine, a band for the soluble fraction of TDP-43 was clearly observed, and a faint band for the insoluble fraction. In motor neurons derived from iPS cells from an ALS patient cultured in the presence of ropinirole, a band for the soluble fraction of TDP-43 was clearly observed, and a faint band for the insoluble fraction.

[0076] The captured image of the PVDF membrane was opened in ImageJ software and inverted using the Invert command. After removing the background using the Subtract Background command, the TDP-43 bands in each lane were successively enclosed in square frames of the same size, and the band intensities were quantified using 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 the 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.

[0077] Three experiments were performed, and the average value of the percentage of the insoluble fraction of TDP-43 was calculated. In Figure 8, the percentage of the insoluble fraction of TDP-43 in each experiment is indicated by dots, and the average value is indicated by bars. In motor neurons derived from iPS cells from healthy subjects, 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%.

[0078] FTLD-TDP and ALS have many things in common, including the accumulation of TDP-43. Therefore, the decrease in the average percentage of the insoluble fraction of TDP-43 in motor neurons derived from iPS cells from ALS patients in the presence of D-cycloserine indicates that administration of D-cycloserine can also suppress TDP-43 accumulation in cells from FTLD-TDP patients.

Claims

1. A therapeutic or preventive agent for frontotemporal lobar degeneration accompanied by accumulation of TDP-43 (FTLD-TDP), the active ingredient essentially consisting of at least one selected from the group consisting of cycloserine, terizidone, and salts thereof.

2. The therapeutic or prophylactic agent for FTLD-TDP 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 therapeutic or prophylactic agent for FTLD-TDP according to claim 1, wherein the active ingredient essentially consists of cycloserine or a salt thereof.

4. The therapeutic or prophylactic agent for FTLD-TDP according to claim 1, wherein the active ingredient comprises cycloserine or a salt thereof.

5. The therapeutic or prophylactic agent for FTLD-TDP according to claim 1, wherein the cycloserine is D-cycloserine.

6. The therapeutic or prophylactic agent for FTLD-TDP according to claim 1, wherein the cycloserine is L-cycloserine.

7. The therapeutic or prophylactic agent for FTLD-TDP according to claim 1, comprising 15 mg or more and less than 250 mg of the active ingredient.

8. The therapeutic or prophylactic agent for FTLD-TDP according to claim 1, which contains 15 mg or more and less than 250 mg of D-cycloserine.

9. The therapeutic or prophylactic agent for FTLD-TDP according to claim 1, wherein the active ingredient is not in a cationic form.

10. The method for treating or preventing FTLD-TDP 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 FTLD-TDP according to claim 1, wherein the active ingredient is not combined with acamprosate.

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