Preventive or therapeutic agent for neurodegenerative disease

Abstract

The purpose of the present invention is to provide a drug that exhibits the effect of inhibiting aggregation of a causative protein of an HRE-related neurodegenerative disease such as ALS. According to the present invention, rifampicin or a related substance selected from the group consisting of rifampicin, a derivative thereof, and salts thereof, and / or resveratrol or a related substance selected from the group consisting of resveratrol and a derivative thereof is an active ingredient of a preventive or therapeutic agent for a neurodegenerative disease caused by TDP-43 accumulation, or an active ingredient of a preventive or therapeutic agent for ALS.

Description

Preventive or therapeutic drug for neurodegenerative diseases

[0001] The present invention relates to a pharmaceutical agent useful for the prevention or treatment of neurodegenerative diseases.

[0002] Frontotemporal dementia (FTD) primarily impairs cognitive function, while amyotrophic lateral sclerosis (ALS) primarily impairs motor function. The estimated prevalence of FTD is 15-22 per 100,000, compared with 5-6 per 100,000 for ALS. Approximately 30% of FTD cases and 5-10% of ALS cases are familial, meaning they are hereditary, and the causative genes for both diseases share common features. The most common genetic cause is a mutation in the untranslated region of the C9orf72 gene, resulting in an abnormal expansion of a hexanucleotide repeat (GGGGCC) in the untranslated region (HRE) (Non-Patent Document 1). While healthy individuals have fewer than 30 repeats of the GGGGCC sequence, patients experience 100-5,000 repeats (Non-Patent Document 2).

[0003] Possible mechanisms by which HREs cause disease include loss of C9orf72 protein function and the acquisition of toxicity of HRE-derived RNA and proteins (Non-Patent Documents 3 and 4). In the first mechanism, HREs likely form G-quadruplex structures in the promoter region, inhibiting C9orf72 gene transcription by acting as a steric block to the transcriptional machinery (Non-Patent Document 5). C9orf72 protein is expressed in the brain, spinal cord, and immune cells and has been reported to regulate autophagy and vesicular transport (Non-Patent Document 4). Reduced C9orf72 expression leads to haploinsufficiency, resulting in autophagy dysfunction (Non-Patent Document 6) and neurodegeneration (Non-Patent Document 7). In the second mechanism, HRE-derived sense and antisense RNAs form hairpin structures with G-quadruplexes (Non-Patent Documents 8 and 9). These structures trap RNA-binding proteins, such as heterogeneous nuclear ribonucleoprotein (hnRNP) H, in inclusion bodies, or RNA foci, within the nucleus and sometimes the cytoplasm (Non-Patent Documents 9-11). This trapping is thought to inhibit RNA processing, leading to RNA-mediated cytotoxicity (NPL 12). In the third mechanism, dipeptide repeat proteins (DPRs), such as polyGA, polyGP, polyGR, polyAP, and polyPR, are synthesized from HRE-derived sense and antisense RNAs by repeat-associated non-ATG (RAN) translation (NPLs 13-17).

[0004] These DPRs self-aggregate to form inclusions in the cytoplasm and sometimes in the nucleus, where they associate with autophagy and the ubiquitin-proteasome-associated protein p62 (Non-Patent Documents 13, 14, 16, 18, 19). Furthermore, HRE-derived RNAs and DPRs promote abnormal cytoplasmic liquid-liquid phase separation (LLPS), forming membraneless organelles called stress granules, which condense RNA, RNA-binding proteins such as TDP-43, and the translation machinery (Non-Patent Documents 20-22). Disease-associated LLPS was first suggested by the FUS protein (Non-Patent Documents 23, 24), but many proteins with low-complexity domains (LCDs), including TDP-43 and other RNA-binding proteins, are now believed to be involved in this phenomenon (Non-Patent Document 25). Furthermore, HRE-derived RNAs and DPRs form nuclear pore complexes, inhibiting nucleocytoplasmic transport (Non-Patent Documents 22, 26, 27). These alterations accelerate TDP-43 cytoplasmic accumulation and self-aggregation in stress granules, leading to the formation of cytoplasmic TDP-43 inclusions and nuclear depletion of TDP-43, resulting in loss of protein function (Non-Patent Documents 22, 28). The presence of cytoplasmic inclusions of phosphorylated TDP-43 (pTDP-43) and loss of TDP-43 nuclear localization are characteristic of C9orf72-associated FTD / ALS (Non-Patent Document 28). Furthermore, TDP-43 forms toxic oligomers in brain cells of FTD and ALS patients (Non-Patent Documents 28-30). It is TDP-43 inclusions, rather than RNA foci or DPR inclusions, that are associated with neurodegeneration (Non-Patent Documents 2, 19), suggesting that TDP-43 is the ultimate effector of C9orf72 HRE mutations.

[0005] These studies suggest that inhibiting G-quadruplex formation and RAN translation may prevent C9orf72-associated diseases. RAN translation has been shown to be highly regulated by double-stranded RNA-dependent protein kinase (PKR), which is activated by disease-associated repeat expansion RNAs via phosphorylation of Thr446 and Thr451 (Non-Patent Document 31). Furthermore, metformin, a drug used to treat type 2 diabetes, has been reported to suppress RAN translation by inhibiting PKR phosphorylation, thereby improving the phenotype of C9-500 mice (Non-Patent Document 31). Thus, inhibiting PKR phosphorylation may lead to the suppression of RAN translation.

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[0007] There is currently no effective treatment for HRE-associated neurodegenerative diseases such as ALS. If a drug that inhibits the aggregation of proteins that cause these neurodegenerative diseases (DPR, TDP-43, etc.) could be found, it could become a promising candidate for a preventive drug for HRE-associated neurodegenerative diseases.

[0008] Therefore, an object of the present invention is to provide a drug that exhibits an effect of suppressing the aggregation of proteins that cause HRE-associated neurodegenerative diseases such as ALS.

[0009] The present inventors administered rifampicin to FTD / ALS model mice, generated by introducing the human full-length C9orf72 gene containing approximately 500 repeats of the GGGGCC sequence, and examined the effect of rifampicin on the formation of RNA foci and cytoplasmic inclusions consisting of DPR or TDP-43 in the brain. They found that intranasal administration of rifampicin for one month prevented HRE-associated neuropathology (aggregation of DPR, TDP-43, etc.) and improved memory in the FTD / ALS model mice from 5.5 to 6 months. Furthermore, when the FTD / ALS model mice were administered rifampicin alone, in combination with rifampicin and resveratrol, or resveratrol alone, all treatments improved cognitive function. Furthermore, they found that resveratrol significantly improved cognitive function in the FTD / ALS model mice. Based on these findings, further investigations led to the completion of the present invention.

[0010] That is, the present invention provides the following aspects. Item 1. A prophylactic or therapeutic agent for neurodegenerative diseases caused by TDP-43 accumulation, comprising, as an active ingredient, a resveratrol selected from the group consisting of resveratrol and its derivatives. Item 2. A prophylactic or therapeutic agent for amyotrophic lateral sclerosis (ALS), comprising, as an active ingredient, a resveratrol selected from the group consisting of resveratrol and its derivatives. Item 3. A prophylactic or therapeutic agent for neurodegenerative diseases caused by C9orf72 genetic abnormality and / or TDP-43 genetic abnormality, comprising, as an active ingredient, a resveratrol selected from the group consisting of resveratrol and its derivatives. Item 4. The prophylactic or therapeutic agent according to any one of Items 1 to 3, which is used for nasal administration. Item 5. The prophylactic or therapeutic agent according to any one of Items 1 to 4, wherein the dose of the resveratrol is 3.75 mg / kg / day or less. Item 6. Item 7. A prophylactic or therapeutic agent for neurodegenerative diseases caused by TDP-43 accumulation, comprising a combination of a rifampicin selected from the group consisting of rifampicin, its derivatives, and salts thereof, and a resveratrol selected from the group consisting of resveratrol and its derivatives. Item 8. A prophylactic or therapeutic agent for amyotrophic lateral sclerosis (ALS), comprising, as an active ingredient, a rifampicin selected from the group consisting of rifampicin, its derivatives, and salts thereof. Item 9. A prophylactic or therapeutic agent for neurodegenerative diseases caused by C9orf72 genetic abnormality and / or TDP-43 genetic abnormality, comprising, as an active ingredient, a rifampicin selected from the group consisting of rifampicin, its derivatives, and salts thereof. Item 10. The prophylactic or therapeutic agent according to any one of Items 7 to 9, which is used for nasal administration. Item 11. Item 11. The preventive or therapeutic drug according to any one of Items 7 to 10, wherein the dose of the rifampicin analogue is 3.75 mg / kg·day or less.

[0011] The preventive or therapeutic agent of the present invention provides a drug that exhibits an effect of suppressing the aggregation of proteins that cause HRE-associated neurodegenerative diseases such as ALS.

[0012] Cognitive and motor function in C9-500 mice. Transgenic and non-transgenic littermates were tested at 4.5 and 12 months (mo) in the Morris water maze (A), rotarod (B), and inverted screen test (C). Transgenic mice showed cognitive impairment in both memory acquisition and retention (probe trial) tests at 4.5 months, but no motor impairment at 12 months. *p = 0.0497 vs. non-transgenic littermates. The number of mice analyzed was: n = 12 (5 males, 7 females) 4.5-month-old transgenic mice; n = 8 (4 males, 4 females) age-matched non-transgenic littermates; n = 9 (7 males, 2 females) 12-month-old transgenic mice; and n = 9 (5 males, 4 females) age-matched non-transgenic littermates. C9orf72 HRE-associated pathology in C9-500 mice. (A) Brain sections showing the prefrontal cortex (PFC), motor cortex (MC), hippocampus (HC), and entorhinal cortex (EC) were prepared at 3, 6, and 12 months of age. Sections were stained for G-quadruplex (B), poly(GA) (C), poly(GR) (D), poly(GP) (E), and pTDP-43 (F). G-quadruplex and DPR accumulation were detected in all regions at 3 months. pTDP-43 accumulation appeared in the PFC and EC at 3 months and in the MC and HC at 6 months. n = 3 mice (2 males, 1 female) were analyzed per group. Age-dependent pathology in each of these brain regions was observed in all mice tested. Synaptic loss, neuronal loss, and microglial activation in C9-500 mice. Brain sections were stained for synaptophysin (A), NeuN (B), and Iba1 (C). Synaptophysin intensity was measured in a fixed area (50 × 50 μm) of the hippocampal mossy fiber (HC mf). Levels began to decrease at 6 months. NeuN-positive areas and Iba1-positive cells were quantified in fixed areas (280 × 370 μm) of the PFC, MC, HC, and EC. Neuronal loss was detected in the PFC and EC at 6 months and in the MC at 12 months, but not in the HC. Microglial activation was observed in the PFC, MC, and EC at 6 months, but not in the HC at 12 months. Each group consisted of three mice (n = 3, two males and one female). Effects of rifampicin on cognitive function in C9-500 mice.Intranasal administration of rifampicin (RFP) at 0.1 mg / day for one month significantly improved cognitive function in both memory acquisition (A) and retention (B) tests in 5.5-6-month-old transgenic mice. * p = 0.0155 between the transgenic and non-transgenic groups, † p = 0.0271 between the transgenic and non-transgenic groups. The number of mice analyzed was n = 9 (6 males, 3 females) for rifampicin-treated transgenic mice, n = 10 (6 males, 4 females) for CMC-treated transgenic mice, and n = 8 (5 males, 3 females) for non-transgenic littermates. Effect of rifampicin on HRE-associated pathology in C9-500 mice. After behavioral testing, brain sections were prepared and stained with antibodies against G-quadruplex (A), poly-GA (B), poly-GR (C), poly-GP (D), and pTDP-43 (E). These pathologies were assessed in the PFC and MC by counting immunopositive puncta within a fixed area (280 × 370 μm). Intranasal administration of rifampicin (RFP) significantly reduced the levels of G-quadruplex, DPR, and pTDP-43 puncta. n = 5 mice (3 males, 2 females) were analyzed per group. Effect of rifampicin on RNA foci and cytoplasmic inclusions in C9-500 mice. Brain sections were stained with a combination of antibodies against G-quadruplex and hnRNP H1 for RNA foci (A), DPR and p62 for DPR inclusions (B-D), and pTDP-43 and amyloidogenic protein oligomers (F11G3) for TDP-43 oligomer inclusions (E). The nuclear dye DAPI was used. These pathologies were assessed in the PFC and MC by counting cells with double-positive puncta (green + red = yellow) in a fixed area (280 × 370 μm) of all cells (blue DAPI positive). In Tg mice, RNA foci and the formation of DPR and TDP-43 inclusions were observed. Intranasal administration of rifampicin (RFP) significantly attenuated these pathologies. Effects of rifampicin on synaptic loss, neuronal loss, and microglial activation in C9-500 mice. Brain sections were stained with antibodies for synaptophysin (A), NeuN (B), and Iba1 (C). Synaptophysin intensity was measured in a fixed area (50 × 50 μm) of hippocampal mossy fibers (HC mf), and NeuN-positive areas and Iba1-positive cells were quantified in a fixed area (280 × 370 μm) of the PFC and MC.Intranasal administration of rifampicin (RFP) significantly reversed synaptic loss in HCs, neuronal loss in PFCs, and microglial activation in PFCs and MCs. Effects of rifampicin on G-quadruplex formation and PKR phosphorylation. (A) In vitro effects of rifampicin on DNA G-quadruplex formation. The left and right photographs are fluorescent and colorimetric images of the same gel, respectively. Sense (GGGGCC)4 oligonucleotides formed G-quadruplexes in the presence of 100 mM KCl, whereas antisense (GGCCCC)4 or control (ATGC)6 oligonucleotides did not. Addition of 1 mM rifampicin (RFP) did not affect these observations. XC, xylene cyanol FF; BPB, bromophenol blue; OG, orange G. The apparent molecular sizes of XC and BPB in a 20% native gel are 45 bp and 12 bp, respectively. (B) Effects of rifampicin on PKR phosphorylation in C9-500 mice. Brain sections were stained with anti-phospho-PKR (Thr446) antibody. PFC refers to the prefrontal cortex, and MC refers to the motor cortex. Phospho-PKR intensity was measured in fixed areas (280 × 370 μm) of the PFC and MC. Transgenic mice showed significantly increased phospho-PKR levels, accompanied by dense punctate staining (arrows), in the PFC and MC compared with non-transgenic siblings. Intranasal administration of rifampicin (RFP) significantly reduced phospho-PKR levels. The C9orf72 HRE-mediated cytotoxic pathway and the effects of rifampicin were verified. C9orf72 HRE mutations are presumed to cause disease through loss-of-function and gain-of-toxicity of related proteins. The HRE forms a G-quadruplex structure in the promoter region, inhibiting C9orf72 gene transcription, leading to haploinsufficiency of the C9orf72 protein and resulting autophagy dysfunction. HREs also generate aberrant sense and antisense transcripts that form G-quadruplexes and hairpin structures, trapping RNA-binding proteins in RNA foci. This trapping inhibits RNA processing and can lead to RNA-mediated cytotoxicity. HRE-derived sense and antisense RNAs generate five DPRs via RNA translation.These DPRs self-aggregate to form inclusion bodies, involving autophagy and the ubiquitin-proteasome-associated protein p62. Furthermore, HRE-derived RNA and DPRs promote cytoplasmic LLPS, forming stress granules containing RNA, RNA-binding proteins with LCDs such as TDP-43, and the translation machinery. These changes promote the formation of TDP-43 inclusions in the cytoplasm and the depletion of TDP-43 in the nucleus, resulting in loss of protein function. Rifampicin (RFP) does not inhibit G-quadruplex formation (T-shaped line with an X), but it inhibits RAN translation by inhibiting PKR phosphorylation, preventing the formation of DPRs and TDP-43 inclusions (T-shaped line). Rifampicin also attenuates the formation of RNA foci through an unidentified mechanism. Nasal administration of 0.04 mg / day of resveratrol (Resv) significantly improved cognitive function in FTD / ALS model mice. Synaptophysin levels were assessed in the hippocampal CA2-3 region and dentate gyrus (DG). Resveratrol administration significantly reversed synaptic loss in the CA2-3 region and DG. G-quadruplex levels were assessed in the prefrontal cortex. This pathology was significantly reduced by rifampicin, resveratrol, and their combination, with resveratrol showing the strongest effect, and the combination showing an intermediate effect between resveratrol and rifampicin. Phosphorylated PKR levels were assessed in the prefrontal cortex. This pathology was significantly reduced by rifampicin, resveratrol, and their combination, with resveratrol showing the strongest effect, and the combination showing an intermediate effect between resveratrol and rifampicin. Poly(GA) and poly(GP) levels were assessed in the prefrontal cortex. This pathology was significantly reduced by rifampicin, resveratrol, and their combination, with resveratrol showing the strongest effect, and the combination showing an intermediate effect between resveratrol and rifampicin. Levels of phosphorylated TDP-43 were assessed in the prefrontal cortex. This pathology was significantly reduced by rifampicin, resveratrol, and their combination, with resveratrol showing the strongest effect and the combination showing an intermediate effect between resveratrol and rifampicin.

[0013] [Rifampicins] The pharmaceutical of the present invention may contain, as an active ingredient, a rifampicin selected from the group consisting of rifampicin, its derivatives, and salts thereof. Rifampicin is a known antibiotic.

[0014] Rifampicin is generally a compound represented by the following formula (I):

[0015]

[0016] The rifampicin derivative is not particularly limited as long as it has a naphthohydroquinone or naphthoquinone structure and is pharmaceutically acceptable, and examples thereof include 3-Folmyl-rifamycin SV, rifamysin S, rifamycin B, rifamycin SV, and the main active metabolite 25-desacetyl-RFP. Among the rifampicin derivatives, derivatives that do not have a 3-position substituent in the 1,4-dihydroxynaphthalene structure that is responsible for antibiotic activity, such as rifamycin SV, are preferred from the viewpoint of suppressing the induction of resistant bacteria due to long-term administration. These rifampicin derivatives may be used alone or in combination of two or more.

[0017] The salt of rifampicin is not particularly limited as long as it forms a salt with rifampicin or a rifampicin derivative and is pharmaceutically acceptable. Examples include salts of alkali metals (potassium, sodium, etc.), salts of alkaline earth metals (calcium, magnesium, etc.), ammonium salts, salts of pharmaceutically acceptable organic amines (tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)aminomethane, lysine, arginine, N-methyl-D-glucamine, etc.), inorganic acid salts (hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, etc.), and organic acid salts (acetate, lactate, tartrate, benzoate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, gluconate, etc.). These salts may be used alone or in combination of two or more.

[0018] As the rifampicins, one may be selected from rifampicin, salts of rifampicin, derivatives of rifampicin, and salts of derivatives of rifampicin, or two or more may be used in combination.

[0019] Among the above-mentioned rifampicins, rifampicin and rifamycin SV are preferred.

[0020] The content of rifampicins in the pharmaceutical preparation of the present invention is not particularly limited and is appropriately adjusted so as to be administered at the doses described below. For example, the content of rifampicins in the pharmaceutical preparation of the present invention may be 0.19 w / v% or more, preferably 0.4 w / v% or more, and more preferably 0.5 w / v% or more. Furthermore, from the viewpoint of efficiently administering a predetermined dose with a small number of administrations, the content of rifampicins in the pharmaceutical preparation of the present invention may be preferably 2 w / v% or more, 2.5 w / v% or more, 5 w / v% or more, or 30 w / v% or more. Furthermore, the content of rifampicins in the pharmaceutical preparation of the present invention may be 95 w / v% or less, preferably 85 w / v% or less, or 50 w / v% or less. When the pharmaceutical preparation of the present invention is prepared for nasal administration, the content is preferably 85 w / v% or less, or 50 w / v% or less, from the viewpoint of obtaining good sprayability of the nasally administered drug.

[0021] [Resveratrols] The pharmaceutical of the present invention may contain, as an active ingredient, a resveratrol selected from the group consisting of resveratrol and its derivatives. The resveratrols may be combined with rifampicins.

[0022] In the present invention, it is preferable that the active ingredient contains a resveratrol, and it is particularly preferable that the active ingredient contains a resveratrol but does not contain a rifampicin.

[0023] Resveratrol is 3,5,4'-trihydroxystilbene. Examples of resveratrol in the present invention include the cis isomer of resveratrol, the trans isomer of resveratrol, and a mixture of these isomers, and preferably the trans isomer of resveratrol. The trans isomer of resveratrol is a compound represented by the following formula (II):

[0024]

[0025] Resveratrol may be purified from plant extracts such as lingonberry extract, grape extract, bilberry extract, Japanese knotweed extract, and melinjo extract, or may be obtained by chemical synthesis, genetic engineering, or microbiological methods.

[0026] Resveratrol derivatives are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include those having, as derivative groups, protecting groups such as N-phenylacetyl and 4,4'-dimethoxytrityl (DMT) groups; biopolymers such as proteins, peptides, sugars, lipids, and nucleic acids; synthetic polymers such as polystyrene, polyethylene, polyvinyl, and polyester; and functional groups such as ester groups. Examples of the ester groups include aliphatic ester groups such as methyl ester and ethyl ester groups, and aromatic ester groups.

[0027] As the resveratrols, one kind may be selected and used from resveratrol and resveratrol derivatives, or two or more kinds may be used in combination.

[0028] Among these resveratrols, resveratrol is preferred, and the trans isomer of resveratrol (3,5,4'-trihydroxy-trans-stilbene) is more preferred.

[0029] The content of resveratrols in the pharmaceutical preparation of the present invention is not particularly limited and is appropriately adjusted so as to be administered at the doses described below. For example, the content of resveratrols in the pharmaceutical preparation of the present invention may be 0.19 w / v% or more, preferably 0.4 w / v% or more, and more preferably 0.5 w / v% or more. From the viewpoint of efficiently administering a predetermined dose with a small number of administrations, the content of resveratrols in the pharmaceutical preparation of the present invention may be preferably 2 w / v% or more, 2.5 w / v% or more, 5 w / v% or more, or 30 w / v% or more. Furthermore, the content of resveratrols in the pharmaceutical preparation of the present invention may be 95 w / v% or less, preferably 85 w / v% or less, or 50 w / v% or less. When the pharmaceutical preparation of the present invention is prepared for nasal administration, from the viewpoint of obtaining good sprayability of the nasally administered drug, the content may be preferably 85 w / v% or less, or 50 w / v% or less.

[0030] When the pharmaceutical composition of the present invention contains a combination of rifampicins and resveratrols, the ratio of rifampicins to resveratrols is not particularly limited and can be determined depending on the content of each of the above-mentioned components. Furthermore, when the pharmaceutical composition of the present invention is prepared for nasal administration, the content of resveratrols may be prepared to be greater than the predetermined effective amount, taking into account the difference in the water solubility of rifampicins (e.g., 2.5 mg / mL at 25°C for rifampicin) and the water solubility of resveratrols (e.g., 0.03 mg / mL at 25°C for resveratrol), and the tendency of components with lower water solubility to be more easily passed into the digestive tract due to the movement of the mucus layer by ciliated cells in the nasal cavity. For example, when the pharmaceutical composition of the present invention is prepared for absorption through the paranasal sinuses at a weight ratio of approximately 1:1, the content of resveratrols may be prepared to be greater than 1 part by weight per part by weight of rifampicins. Examples of cases where the difference in solubility in water between rifampicins and resveratrols is taken into consideration include when the pharmaceutical of the present invention is prepared as a combination of rifampicins and resveratrols, and when it is prepared as a kit containing a drug containing rifampicins and a drug containing resveratrols, and both drugs are prepared using the same base containing at least water.

[0031] From these viewpoints, the lower limit of the content of resveratrols per part by weight of rifampicins is, for example, 1 / 500 part by weight or more, preferably 1 / 300 part by weight or more, more preferably 1 / 200 part by weight or more, even more preferably 1 / 100 part by weight or more, still more preferably 0.05 part by weight or more, even more preferably 0.1 part by weight or more, and particularly preferably 0.2 part by weight or more. Preferably, the content of resveratrols per part by weight of rifampicins is 0.5 part by weight or more, more preferably 0.8 part by weight or more, and even more preferably 1 part by weight or more. Furthermore, the content of resveratrols per 1 part by weight of rifampicin may be more than 1 part by weight, for example, 1.2 parts by weight or more, 1.5 parts by weight or more, 2 parts by weight or more, 3 parts by weight or more, 4 parts by weight or more, 5 parts by weight or more, 6 parts by weight or more, 8 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 40 parts by weight or more, 60 parts by weight or more, 80 parts by weight or more, 100 parts by weight or more, 200 parts by weight or more, or 300 parts by weight or more.

[0032] Furthermore, the upper limit of the content of resveratrols per part by weight of rifampicins may be, for example, 500 parts by weight or less, 300 parts by weight or less, 200 parts by weight or less, 100 parts by weight or less, 75 parts by weight or less, 50 parts by weight or less, 30 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, 3 parts by weight or less, 2 parts by weight or less, 1.5 parts by weight or less, or 1.2 parts by weight or less.

[0033] [Dosage Form] The pharmaceutical of the present invention is a pharmaceutical composition containing the above-mentioned rifampicins and / or resveratrols and formulated by a method known per se. The pharmaceutical of the present invention may or may not contain a pharmacologically acceptable base and / or additive as appropriate, depending on the administration method and / or use of the pharmaceutical.

[0034] Examples of pharmacologically acceptable bases and / or additives that may or may not be contained in the pharmaceutical of the present invention include excipients, thickeners, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, emulsifiers, isotonicity agents, buffers, soothing agents, stabilizers, preservatives (antiseptics), pH adjusters, refreshing agents, antioxidants, humectants, adhesives, and odor masking agents.

[0035] Examples of excipients that may or may not be contained in the pharmaceutical product of the present invention include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc. Examples of thickeners include polyhydric alcohols such as glycerin and macrogol, celluloses such as methylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose (preferably sodium carboxymethylcellulose), sodium alginate, chondroitin sulfate, cyclodextrin, d-α-tocopheryl polyethylene glycol 1000 succinate, and polyethylene glycol. Examples of lubricants include magnesium stearate, calcium stearate, talc, and colloidal silica. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, and sodium carboxymethylcellulose. Examples of disintegrants include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, sodium carboxymethylstarch, and L-hydroxypropylcellulose. Examples of solvents include water, ethanol, isopropyl alcohol, acetone, propylene glycol, macrogol, sesame oil, and corn oil, and preferably contain at least water. Examples of solubilizers include celluloses such as methylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose; polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, polyvinylpyrrolidone, and macrogol.Examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, polyoxyethylene hydrogenated castor oil, and polysorbate, polyhydric alcohols such as glycerin and macrogol, sugars such as sorbitol, mannitol, and sucrose, celluloses such as methylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose, and chondroitin sulfate. Examples of isotonic agents include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, potassium chloride, concentrated glycerin, propylene glycol, and sucrose. Examples of buffering agents include phosphates (sodium hydrogen phosphate, sodium dihydrogen phosphate, etc.), boric acid, borax, acetates (sodium acetate, etc.), carbonates (sodium carbonate, calcium carbonate, potassium carbonate, etc.), citric acid, sodium L-glutamate, etc. Examples of soothing agents include benzyl alcohol, chlorobutanol, propylene glycol, ethyl aminobenzoate, lidocaine, etc.Examples of stabilizers include sulfur compounds such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, Rongalite, thioglycerol, thioglycolic acid, thiolactic acid, cysteine, glutathione, thioacetic acid, methionine, thiosorbitol, thioglucose, and thiourea; inorganic acids and salts thereof such as boric acid, borax, phosphoric acid, metaphosphoric acid, sodium carbonate, and sodium bicarbonate; organic acids and salts thereof (e.g., sodium edetate) such as formic acid, oxalic acid, tartaric acid, citric acid, and edetic acid; acid amides such as acetamide, diethylacetamide, nicotinamide, urea, and barbital; urea derivatives; polyhydric alcohols such as glycol, propylene glycol, glycerin, polyethylene glycol, glucose, and ascorbic acid; sugars; phenols such as thymol, quinone, coumarone, and isocumarone; amino acids such as dibutylhydroxytoluene, glycine, glutamic acid, lysine, phenylalanine, casein, and edestin; and proteins. Examples of emulsifiers include glycerin esters (glycerin monooleate), saponins (Sophora saponin, Quillaja extract, soybean saponin, etc.), sucrose fatty acid esters, lecithins (vegetable lecithin, egg yolk lecithin, soybean lecithin, etc.), polyhydric alcohols (oleyl alcohol, stearyl alcohol, cetyl alcohol, etc.), fatty esters (octyldodecyl myristate, etc.), medium-chain fatty acid triglycerides (MCT), various surfactants (alkylbenzenesulfonate-type emulsifiers, benzalkonium chloride, sorbitan sesquioleate, dodecylbenzenesulfonic acid, etc.), and triethanolamine. Examples of preservatives (antiseptics) include parahydroxybenzoic acid esters such as propyl parahydroxybenzoate and butyl parahydroxybenzoate; parabens such as methylparaben, ethylparaben, propylparaben and butylparaben; cationic soaps such as benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate and cetylpyridium chloride; alcohol derivatives such as chlorobutanol, benzyl alcohol and phenethyl alcohol; organic acids and salts thereof such as sodium dehydroacetate, sorbic acid and sodium sorbate; and phenols such as parachloromethoxyphenol and parachlorometacresol.Examples of pH adjusters include sodium hydroxide, potassium hydroxide, trisodium phosphate, disodium hydrogen phosphate, hydrochloric acid, nitric acid, citric acid, boric acid, and acetic acid. Examples of freshening agents include 1-menthol, camphor, and peppermint water. Examples of antioxidants include sulfites, ascorbic acid, citric acid, and sodium edetate. Examples of humectants include propylene glycol, polysorbate, macrogol, and glycerin. Examples of adhesives include hydroxypropyl cellulose, hydroxypropyl methylcellulose 2208, carboxyvinyl polymer, propylene glycol, and polysorbate 80. Examples of flavoring agents include trehalose, malic acid, maltose, potassium gluconate, anise essential oil, vanilla essential oil, cardamom essential oil, and herbal ingredients.

[0036] The pharmaceutical of the present invention may be either a liquid or a solid, preferably a liquid. A liquid can be prepared by mixing rifampicins and / or resveratrols with, as needed, a solvent, a solubilizer, a suspending agent, an isotonicity agent, a buffer, a soothing agent, etc., followed by dissolution, suspension, or emulsification. When the pharmaceutical of the present invention is prepared as a nasal drug, it is also preferable to add a thickener to increase viscosity and provide retention. A solid can be prepared by uniformly mixing rifampicins and / or resveratrols with, as needed, an excipient, a binder, a disintegrant, or other appropriate additives, obtaining granules by an appropriate granulation method, and then drying the granules to form a powder or fine particles.

[0037] When the pharmaceutical of the present invention is prepared as a nasal drug, it can be filled in a container for nasal administration and used. Any commercially available container for nasal administration can be used.

[0038] A more specific embodiment of the pharmaceutical of the present invention containing a combination of rifampicins and resveratrols is a combination of rifampicins and resveratrols. This combination is a pharmaceutical composition containing rifampicins and resveratrols in a mixed state. This combination allows rifampicins and resveratrols to be administered simultaneously in any dosage form. Another specific embodiment of the pharmaceutical of the present invention in this case is a kit containing a drug containing rifampicins and a drug containing resveratrols. Each drug may be prepared using the same base and / or additives, or may be prepared using bases and / or additives selected respectively for the rifampicins and resveratrols. This kit allows rifampicins and resveratrols to be administered separately in any dosage form. Furthermore, when applied to a nasal administration device that includes a cartridge filled with a drug containing rifampicins and a separate cartridge filled with a drug containing resveratrols, both components can be administered simultaneously even though it is a kit.

[0039] [Dosage and Administration] The pharmaceutical of the present invention may be prepared for any of oral, subcutaneous, and nasal administration. Among these, the pharmaceutical of the present invention is more preferably prepared for nasal administration, from the viewpoint of non-invasiveness, more favorable effect of reducing side effects, and therefore, administration at a smaller dose and / or for a longer period of time.

[0040] Regarding the dosage of the pharmaceutical of the present invention to humans, the lower limit of the dosage of rifampicins, from the viewpoint of efficacy expression, is, for example, 0.15 mg / kg-day or more, preferably 0.3 mg / kg-day or more, more preferably 0.75 mg / kg-day or more, and even more preferably 1 mg / kg-day or more. The upper limit of the dosage of rifampicins, from the viewpoint of suppressing side effects, is, for example, 3.75 mg / kg-day or less, preferably 2.5 mg / kg-day or less, more preferably 2 mg / kg-day or less, even more preferably 1 mg / kg-day or less, still more preferably 0.5 mg / kg-day or less, and even more preferably 0.4 mg / kg-day or less.

[0041] In addition, when the pharmaceutical of the present invention contains a combination of rifampicins and resveratrols, a lower dose of rifampicins to be administered to a human can be tolerated. In this case, the lower limit of the dose of rifampicins to be administered to a human can be, for example, 0.001 mg / kg-day or more, preferably 0.002 mg / kg-day or more, more preferably 0.003 mg / kg-day or more, even more preferably 0.005 mg / kg-day or more, and even more preferably 0.01 mg / kg-day or more, from the viewpoint of the manifestation of pharmacological efficacy. In addition, the lower limit of the dose of rifampicins to be administered to a human can be, for example, 0.025 mg / kg-day or more, preferably 0.05 mg / kg-day or more, and even more preferably 0.1 mg / kg-day or more, from the viewpoint of the manifestation of even more preferable pharmacological efficacy. Furthermore, from the viewpoint of further suppressing side effects, the upper limit of the dose of rifampicins administered to humans can be 1.5 mg / kg-day or less, preferably 1 mg / kg-day or less, more preferably 0.5 mg / kg-day or less, even more preferably 0.1 mg / kg-day or less, and even more preferably 0.07 mg / kg-day or less.

[0042] Regarding the dosage of the pharmaceutical of the present invention to humans, the dosage of resveratrols (when combined with rifampicins and when not combined with rifampicins) may be, from the viewpoint of the efficacy of the drug, for example, 0.001 mg / kg-day or more, preferably 0.002 mg / kg-day or more, more preferably 0.003 mg / kg-day or more, even more preferably 0.005 mg / kg-day or more, and even more preferably 0.01 mg / kg-day or more, 0.05 mg / kg-day or more, or 0.1 mg / kg-day or more. The upper limit of the dosage for humans is not particularly limited, and examples include 3.75 mg / kg-day or less, preferably 2.5 mg / kg-day or less, more preferably 2 mg / kg-day or less, even more preferably 1.5 mg / kg-day or less or 1 mg / kg-day or less, even more preferably 0.5 mg / kg-day or less, even more preferably 0.4 mg / kg-day or less, 0.3 mg / kg-day or less, 0.2 mg / kg-day or less, or 0.15 mg / kg-day or less, particularly preferably 0.1 mg / kg-day or less, and most preferably 0.07 mg / kg-day or less.

[0043] The pharmaceutical of the present invention can be administered at a small dose and is therefore suitable for continuous administration. The administration period of the pharmaceutical of the present invention to humans can be, for example, one month or more, preferably three months or more. Because the pharmaceutical of the present invention can be administered for even longer periods, more preferred examples of the administration period to humans include, for example, six months or more, preferably one year or more, more preferably 1.5 years or more, even more preferably two years or more, and even more preferably 2.5 years or more. The administration period may also be three years or more. The upper limit of the administration period to humans is not particularly limited, but examples include 10 years or less, eight years or less, six years or less, or four years or less. The upper limit of the administration period may also be three years or less. The administration interval can be daily, every other day, or once or twice a week, preferably every other day or every day, and more preferably every day.

[0044] [Subjects for Administration] As a first use, the pharmaceutical product of the present invention can be used for the prevention or treatment of neurodegenerative diseases caused by TDP-43 accumulation (TDP-43 proteinopathy). Neurodegenerative diseases caused by TDP-43 accumulation include familial neurodegenerative diseases and sporadic neurodegenerative diseases, and specific examples of such diseases include amyotrophic lateral sclerosis (ALS), which is a TDP-43 proteinopathy, and frontotemporal dementia (FTD), which is a TDP-43 proteinopathy.

[0045] Furthermore, as a second use, the pharmaceutical product of the present invention can be used for the prevention or treatment of amyotrophic lateral sclerosis (ALS). Such amyotrophic lateral sclerosis (ALS) includes TDP-43 proteinopathy, FUS proteinopathy, and SOD1 type. In particular, a form of the pharmaceutical product of the present invention containing resveratrols as an active ingredient (most preferably a form containing resveratrols as an active ingredient but not rifampicins as an active ingredient) is preferably used for the prevention or treatment of ALS caused by TDP-43 proteinopathy.

[0046] Examples of the first and second uses include neurodegenerative diseases caused by C9orf72 genetic abnormalities and / or TDP-43 genetic abnormalities, preferably neurodegenerative diseases caused by TDP-43 genetic abnormalities.

[0047] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples.

[0048] Test Example 1 1. Materials and Methods 1.1 Mice FVB / NJ-Tg(C9orf72)500Lpwr / J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). This mouse strain was generated as a C9orf72-linked FTD / ALS model (hereinafter also referred to as "FTD / ALS model mice") by expressing the human C9orf72 gene with approximately 500 hexanucleotide (GGGGCC) repeats using a bacterial artificial chromosome (BAC) vector. Transgenic (Tg) mice were crossed with wild-type FVB / NJc1 mice and maintained as heterozygotes for the transgene in our animal facility.

[0049] 1.2. Behavioral Tests Cognitive function of mice was examined using the Morris water maze test at 4.5 months, and motor function was examined using the rotarod test and the inverted screen test at 4.5 and 12 months, as described above.

[0050] 1.3. Immunohistochemical Analysis. To study age-dependent neuropathology in mice, brain sections from transgenic and non-transgenic littermates were prepared at 3, 6, and 12 months of age as previously described. For staining of pTDP-43, synaptophysin, and NeuN, sections were boiled in 10 mM citrate buffer (pH 6) for 30 min to expose antigens. After overnight blocking with 10% calf serum, sections were stained with antibodies against DNA / RNA G-quadruplex (BG4; Absolute Antibody, Cleveland, UK), poly(GA), poly(GP), and poly(GR) (all from Cosmo Bio, Tokyo, Japan), pSer409 / 410-TDP-43 (Cosmo Bio), synaptophysin (SVP-38; Sigma-Aldrich, St. Louis, MO, USA), NeuN (Chemicon, Temecula, CA, USA), and Iba1 (Fujifilm-Wako, Osaka, Japan). After staining, biotin-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA, USA), horseradish peroxidase (HRP)-conjugated avidin-biotin complex (Vector Laboratories), and the HRP substrate diaminobenzidine (DAB), or FITC-conjugated secondary antibodies for synaptophysin alone (Jackson Laboratory) were used. Stained specimens were observed under a BZ-X800 fluorescence microscope (Keyence, Osaka, Japan), and images of specific brain regions were photographed. Neuropathology was assessed by measuring the staining intensity or area of ​​each photograph or by counting positive puncta or cells within a given area using NIH ImageJ software (ImageJ bundled with 64-bit Java 1.8.0_172; https: / / imagej.nih.gov / ij / , accessed on 27 March 2022).

[0051] To examine the formation of nuclear and cytoplasmic inclusions, sections were double-stained with antibodies against G-quadruplexes and hnRNP H1 (Proteintech, Rosemont, IL, USA) for RNA foci without pretreatment, and with antibodies against pSer409 / 410-TDP-43 and amyloidogenic protein oligomers (F11G3; Sigma-Aldrich) for TDP-43 oligomer inclusions after pretreatment at pH 6. For DPR inclusions, sections were double-stained with antibodies against polyGA, polyGP, polyGR, and SQSTM1 / p62 (A-6; Santa Cruz Biotechnology, Dallas, TX, USA) without pretreatment. After staining, FITC- and rhodamine-conjugated secondary antibodies (Jackson Laboratory) were used. The specimens were then treated with TrueBlack Plus Lipofuscin (Biotium, Fremont, CA, USA) to quench autofluorescence and mounted with Vectashield vibrance antifade mounting medium containing DAPI (Vector Laboratories). Images of the stained sections were taken with a BZ-X800 fluorescence microscope, and inclusion-bearing cells were counted in a fixed area.

[0052] 1.4. Rifampicin Treatment. FTD / ALS model mice aged 4.5 to 5 months were divided into two groups. One group received rifampicin, and the other group received carboxymethylcellulose (CMC) daily, Monday through Friday, for 4 weeks. Rifampicin (RFP; Sigma-Aldrich, Rifampicin ≥97% (HPLC), powder, also known as 3-(4-methylpiperazinyliminomethyl)rifamycin SV, rifamycin AMP, rifampin, R3501) was dissolved in 0.5% low-viscosity CMC (Sigma-Aldrich) at 10 mg / mL. 10 μL of rifampicin (i.e., 0.1 mg) or CMC solution was administered intranasally. Non-Tg littermate mice were treated with CMC alone. After 1 month of treatment, cognitive function of the mice was assessed using a water maze test, during which rifampicin administration was continued. After behavioral testing, each group was divided into two groups: one for immunohistochemical analysis, and the other for further biochemical analysis. For immunohistochemical analysis, brain sections were prepared as described above. For biochemical analysis, whole brains were removed and frozen at -80°C until use.

[0053] 1.5. G-Quadruplex Gel Shift Assay A gel shift assay was performed to detect G-quadruplex formation. Sense (GGGGCC)4, antisense (GGCCCC)4, and control (ATGC)6 oligonucleotides were synthesized and dissolved at 100 μM in 0.89 M Tris-borate buffer, pH 8.3, containing 0.02 M EDTA (TBE). KCl was dissolved at 400 mM in TBE, and rifampicin was dissolved at 100 mM in DMSO. 5 μL of DNA solution, 25 μL of KCl or TBE alone, and 1 μL of rifampicin or DMSO alone were mixed with 69 μL of TBE to achieve final concentrations of 5 μM DNA, 100 mM KCl, and 1 mM rifampicin. The mixture was incubated at 98°C for 3 minutes and then slowly cooled to room temperature. The samples were mixed with Blue / Orange Loading Dye (Promega, Madison, WI, USA) and subjected to native PAGE using a 20% polyacrylamide gel (Novex TBE Gels, Invitrogen, Carlsbad, CA, USA) in TBE running buffer containing 50 mM KCl. The gel was stained with SYBR Gold nucleic acid gel stain (Invitrogen) for 10 minutes, and images were acquired using an ImageQuant LAS 500 image analyzer (GE Healthcare, Hino, Japan).

[0054] 1.6. Immunohistochemical Study of Phosphorylated PKR To examine the possibility that rifampicin affects RAN translation, we examined the levels of phosphorylated PKR, a regulator of RAN translation, by immunohistochemistry. Brain sections were boiled in 10 mM citrate buffer (pH 6) for 30 minutes to expose the antigen. After blocking, sections were stained with anti-phospho-PKR (Thr446) antibody (MilliporeSigma, Burlington, MA, USA), followed by staining with a biotin-conjugated secondary antibody, HRP-conjugated avidin-biotin complex, and DAB. Stained specimens were observed under a BZ-X800 fluorescence microscope, and the levels of phosphorylated PKR were measured by quantifying the staining intensity in a fixed area using NIH ImageJ software.

[0055] 1.7 Statistical Analysis All experiments and data analyses were performed under open-label conditions. Comparisons of means between two or more groups were performed using ANOVA or two-factor repeated measures ANOVA (for the Morris water maze test) followed by Fisher's PLSD test. Differences with a p-value of <0.05 were considered significant.

[0056] 2. Results First, we investigated the pathological phenotype of FTD / ALS model mice. The cognitive function of the mice was assessed using the Morris water maze test at 4.5 months, and the motor function was assessed using the rotarod test and the inverted screen test at 4.5 and 12 months. Compared with non-Tg littermates, Tg mice showed significantly reduced memory at 4.5 months, but no motor deficits were observed (Figure 1). Furthermore, the motor function of Tg mice remained normal at 12 months. These mice are considered to have a slowly progressive form of the disease.

[0057] We next examined the neuropathology of the mice using immunohistochemistry. Brain sections were prepared at 3, 6, and 12 months and stained with antibodies for DNA / RNA G-quadruplexes, poly(GA), poly(GR), poly(GP), and pTDP-43. We examined the prefrontal cortex (PFC), motor cortex (MC), hippocampus (HC), and entorhinal cortex (EC), which are vulnerable regions in FTD and ALS (Fig. 2A). G-quadruplexes appeared in all brain regions tested at 3 months (Fig. 2B). The three DPRs also began to accumulate in all brain regions at 3 months (Fig. 2C–E). pTDP-43 was detected in the PFC and EC at 3 months and in the MC and HC at 6 months (Fig. 2F).

[0058] Brain sections were then stained with antibodies against synaptophysin, the neuronal marker NeuN, and the microglial marker Iba1. Synaptophysin levels were measured in the hippocampal mossy fibers. Significant synaptic loss was detected in Tg mice at 6 months (Figure 3A). NeuN-positive areas and Iba1-positive cells were quantified in the PFC, MC, HC, and EC. Significant neuronal loss was observed in the PFC and EC at 6 months and in the MC at 12 months, but was almost absent in the HC even at 12 months (Figure 3B). Furthermore, significant microglial activation was observed in the PFC, MC, and EC at 6 months, with a similar but non-significant trend in the HC (Figure 3C). These results suggest that G-quadruplex formation and DPR accumulation appear as early pathologies in FTD / ALS model mice, followed by pTDP-43 accumulation. Furthermore, neurodegeneration was associated with pTDP-43 accumulation, with the PFC and EC being the most affected brain regions in FTD / ALS model mice.

[0059] We investigated the therapeutic potential of rifampicin for C9orf72-associated FTD in 4.5- to 5-month-old mice. Transgenic mice were divided into two groups: one group received intranasal administration of 0.1 mg / day of rifampicin for one month, and the other group received CMC alone. Non-transgenic littermates were treated with CMC. After treatment, cognitive function of the mice was evaluated using a water maze test. Nasal administration of rifampicin significantly improved memory in the transgenic mice, to the same level as that of their non-transgenic littermates (Figure 4).

[0060] After behavioral testing, each group was divided into two groups: one for immunohistochemical analysis and the other for future biochemical analysis. For immunohistochemistry, brain sections were prepared and stained with antibodies for G-quadruplex, poly-GA, poly-GR, poly-GP, and pTDP-43. These pathologies were assessed in the PFC and MC. Intranasal administration of rifampicin significantly reduced the levels of G-quadruplex, DPR, and pTDP-43 puncta (Figure 5).

[0061] We further investigated whether these pathological molecules constitute RNA foci and cytoplasmic inclusions. RNA foci are primarily formed in the nucleus, but occasionally in the cytoplasm, and are composed of RNA G-quadruplexes and RNA-binding proteins such as hnRNP H. DPR inclusions are typically formed in the cytoplasm, but occasionally in the nucleus, and involve autophagy and the ubiquitin-proteasome-associated p62 protein. TDP-43 inclusions form in the cytoplasm and are positive for aggregated pTDP-43.

[0062] Therefore, brain sections were stained for RNA foci with a combination of antibodies against G-quadruplexes and hnRNP H, for DPR inclusions with DPR and p62, and for TDP-43 oligomer inclusions with pTDP-43 and an amyloidogenic protein oligomer (F11G3). In Tg mice, RNA foci and cytoplasmic inclusions of DPR and TDP-43 were formed in the PFC and MC (Fig. 6). Intranasal administration of rifampicin significantly attenuated these pathologies.

[0063] Brain sections were then stained with antibodies against synaptophysin, NeuN, and Iba1. Nasal administration of rifampicin significantly suppressed hippocampal mossy fiber synapse loss, PFC neuronal loss, and microglial activation in the PFC and MC (Figure 7). These results suggest the therapeutic potential of intranasal administration of rifampicin in preventing C9orf72-related diseases.

[0064] Next, we investigated the mechanism by which rifampicin suppresses C9orf72 HRE-associated pathology. We hypothesized that rifampicin interacts with DNA / RNA to inhibit G-quadruplex formation. To test this hypothesis, we examined the effect of rifampicin on G-quadruplex formation induced by a synthetic (GGGGCC)4 oligonucleotide in vitro. Gel shift assays demonstrated that DNA formed G-quadruplexes in the presence of 100 mM KCl, and the addition of 1 mM rifampicin failed to inhibit this formation (Figure 8A). Therefore, we speculated that rifampicin, like metformin, may suppress RAN translation by inhibiting PKR phosphorylation. We examined the levels of phosphorylated PKR in the mouse brain using immunohistochemistry. Transgenic mice showed significantly increased levels of phosphorylated PKR in the PFC and MC compared with their non-transgenic siblings (Figure 8B). Intranasal administration of rifampicin significantly attenuated the levels of phosphorylated PKR. These results suggest that rifampicin inhibits PKR phosphorylation, thereby suppressing RAN translation and preventing brain pathology.

[0065] 3. Discussion The FTD / ALS model mice used in this study were originally generated as a model for C9orf72-linked FTD / ALS. According to the authors, these mice can be divided into two subsets: acutely progressive and slowly progressive. Acutely progressive mice (accounting for approximately 30–35% of all female mice) developed widespread neuronal loss in layers II / III of the entire cerebral cortex, layer V of the MC, HC, cerebellum, and spinal cord within 2–5 months, exhibited hindlimb gait abnormalities within 4 months, and survival rates declined dramatically between 5–20 months. In contrast, slowly progressive mice exhibited spinal motor neuron loss, focal neurodegeneration in the neocortex, and mild degeneration in the cerebellum at 18 months, but no degeneration in the HC. Male mice showed no decline in survival even at 1 year, but the majority (approximately 43–45%) developed the phenotype seen in slowly progressive female mice by 1 year. Sense and antisense RNA foci were detected in the MC, HC, cerebellum, and spinal cord as early as 2 months of age. Poly(GA) aggregates were detected throughout the brain at 2 months, first appearing in the occipital cortex and increasing with age and disease. Poly(GP) aggregates were also detected in the neocortex and thalamus in acute end-stage mice. Finally, TDP-43 aggregates were detected in degenerated neurons throughout the brain, including layers II / III and V of the HC, MC, and MC in acute end-stage mice.

[0066] In this example, we first characterized the pathological phenotype of FTD / ALS model mice purchased from the Jackson Laboratory and bred in our animal facility. Cognitive dysfunction was observed at 4.5 months, but motor dysfunction was not observed at 12 months. From 3 months onward, clear neuropathology was observed in the PFC, MC, HC, and EC regions in an age-dependent manner, including RNA foci, DPR and TDP-43 inclusions, synaptic loss, neuronal loss, and microglial activation.

[0067] Neurodegenerative diseases are generally thought to be caused by toxic gain-of-function of amyloidogenic proteins. These proteins are known to exert neurotoxicity by aggregating into oligomers. For example, AD is initiated by the formation of synaptic-toxic Aβ oligomers and progresses through toxic aggregates of tau protein. FTD is induced by the oligomerization of tau and TDP-43, and TDP-43 inclusions are a hallmark of most ALS cases. DLB and Parkinson's disease are associated with α-synuclein oligomers. Meanwhile, C9orf72 HRE mutations have been suggested to cause both toxic gain-of-function and loss-of-function. C9orf72 HRE generates RNA foci and inclusions of DPR and TDP-43, all of which have cytotoxic effects. At the same time, this mutation negatively affects C9orf72 expression by forming G-quadruplex structures in the promoter region, leading to protein haploinsufficiency. Furthermore, RNA foci can sequester several RNA-binding proteins, resulting in the loss of function of these proteins. Furthermore, HRE-derived RNAs and DPRs promote the formation of cytoplasmic LLPS and stress granules, in which TDP-43 tends to self-aggregate and form cytoplasmic inclusions, which may deplete TDP-43 in the nucleus and cause loss of protein function.

[0068] As described above, we demonstrated that intranasal administration of rifampicin inhibits the formation of RNA foci, DPR, and TDP-43 inclusions in FTD / ALS model mice. These results suggest that rifampicin attenuates not only the gain-of-function but also the loss-of-function, at least partially, of the toxic effects of the C9orf72 HRE mutation. Several mechanisms by which rifampicin attenuates the pathology have been proposed (Figure 9). We initially speculated that rifampicin might interact with DNA / RNA to inhibit G-quadruplex formation. However, rifampicin failed to inhibit this formation. Therefore, we hypothesized that rifampicin, like metformin, might suppress RAN translation by inhibiting PKR phosphorylation. Immunohistochemistry showed that rifampicin administration significantly reduced phosphorylated PKR levels in the mouse brain, supporting our hypothesis. This explains why rifampicin was effective in reducing DPR and TDP-43 inclusions.

[0069] Here, we demonstrate that RNA derived from the C9orf72 HRE forms multivalent base pairs, causing RNA gelation without the need for protein components, leading to the formation of RNA foci. Notably, this RNA gelation and RNA foci formation are inhibited in vitro by doxorubicin or ammonium acetate. Because rifampicin and doxorubicin share structural similarities, it is possible that rifampicin inhibits RNA foci formation via a similar mechanism to doxorubicin.

[0070] In neurodegenerative diseases, the pathology of aggregated proteins is thought to spread within the brain via a cell-to-cell transmission mechanism. DPR and TDP-43 have also been suggested to be transmitted between cells. Previously, the inventors demonstrated that intranasal administration of rifampicin inhibits the propagation of tau oligomers in model mice. It is thought that rifampicin attenuates the neuropathology of FTD / ALS model mice by inhibiting the intercellular transmission of DPR and TDP-43.

[0071] In this example, rifampicin was administered at a dose of 0.1 mg / day for one month to FTD / ALS model mice. This administration period of one month (31 days) corresponds to approximately three years or more in humans. (The lifespan of a mouse is generally said to be approximately 2 to 2.2 years. (For example, Yuichi Yamashita et al., "Induction of prolonged natural lifespans in mice exposed to acoustic environmental enrichment," 2018, Scientific Reports volume 8, Article number: 7909, shows that when C57BL / 6J rats (four males and four females) were kept in a standard laboratory animal breeding environment, their average lifespan was approximately 700 days (approximately two years).) However, according to the World Health Organization's WORLD HEALTH STATISTICS OVERVIEW 2019 MONITORING HEALTH FOR THE SDGs, the average human lifespan in high-income countries is approximately 80 years. Considering this, the lifespan of a laboratory mouse is approximately 36 to 40 times longer than that of a human.)

[0072] Expansion of short nucleotide repeats in both coding and non-coding regions is known to underlie more than 50 human diseases. Although the causative genes and pathological phenotypes of each disease are different, the pathogenic mechanisms are thought to be common.

[0073] For example, in Huntington's disease (HD), spinocerebellar ataxia (SCA), dentatorubral-pallidoluysian atrophy, and spinobulbar muscular atrophy, CAG repeats in coding regions are expanded by 20-300-fold or more, resulting in long polyQ sequences in translated proteins that tend to self-aggregate and form inclusion bodies, leading to cytotoxicity. In contrast, in nucleotide repeat expansion disorders such as myotonic dystrophy (DM), benign adult familial myoclonic epilepsy, fragile X syndrome, fragile X-associated tremor and ataxia syndrome, and several SCAs, short nucleotide repeats (e.g., CTG, CCTG, and TTTCA) are expanded by 30-10,000-fold or more in noncoding regions, resulting in the generation of abnormal RNAs and proteins that cause cytotoxicity through both gain- and loss-of-function, as in C9orf72-associated FTD / ALS. Recently, it has been demonstrated that RAN translation also occurs in HD, and that four novel RAN proteins, poly-A, poly-S, poly-L, and poly-C, in addition to poly-Q, are synthesized from sense and antisense repeat RNAs and accumulate in the brain. Furthermore, metformin has been shown to have beneficial effects in HD model mice and in human patients with HD and DM type 1.

[0074] Given the commonality of these conditions and the metformin-like effects of rifampicin, it is likely that rifampicin will be effective in treating these coding and noncoding repeat disorders.

[0075] These results suggest that intranasal administration of rifampicin suppressed pathological phenotypes such as RNA foci, DPR and TDP-43 inclusions, neurodegeneration, and cognitive impairment in FTD / ALS model mice, demonstrating that rifampicin has therapeutic effects against the TDP-43 proteinopathies FTD and ALS.

[0076] [Test Example 2] 1. Materials and Methods 1.1. Mice FVB / NJ-Tg(C9orf72)500Lpwr / J mice (FTD / ALS model mice) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). This mouse strain was generated as a model of C9orf72-associated FTD / ALS by expressing the human C9orf72 gene with approximately 500 hexanucleotide (GGGGCC) repeats using a bacterial artificial chromosome (BAC) vector. Transgenic (Tg) mice were crossed with wild-type FVB / NJc1 mice and maintained in our animal facility as heterozygotes for the transgene.

[0077] 1.2. Rifampicin and Resveratrol Administration. Male and female FTD / ALS model mice aged 7–8 months were divided into four groups based on drug administration: rifampicin alone (0.04 mg / day), resveratrol alone (0.04 mg / day), their combination (0.02 mg rifampicin + 0.02 mg resveratrol / day), and carboxymethylcellulose (CMC) as a control. Rifampicin (Sigma-Aldrich, St. Louis, MO) and trans-resveratrol (Sigma-Aldrich) were dissolved at 4 mg / mL in 0.5% low-viscosity CMC (Sigma-Aldrich). Equal volumes of the rifampicin and resveratrol solutions were mixed to obtain a combinatorial drug containing the two drugs at 2 mg / mL each. Ten μL of rifampicin (0.04 mg), resveratrol (0.04 mg), a combination drug (0.02 mg each), or CMC solution was administered bilaterally via microchip, 5 days a week, Monday through Friday, for 1 month. Age-matched non-Tg rats received only CMC.

[0078] 1.3. Behavioral Testing After one month of treatment, the cognitive function of the mice was examined using the Morris water maze test as previously described.

[0079] 1.4. Immunohistochemical Analysis. After behavioral testing, each group was divided into two groups: one for immunohistochemical analysis and the other for further biochemical analysis. Immunohistochemical staining was performed as previously described. For staining of phosphorylated double-stranded RNA-dependent protein kinase (PKR), phosphorylated TDP-43, and synaptophysin, sections were boiled in 10 mM citrate buffer (pH 6) for 30 min to expose the antigens. After overnight blocking with 10% calf serum, sections were incubated with DNA / RNA G-quadruplex (BG4; Absolute antibody, Cleveland, UK), phospho-PKR (Thr446) (MilliporeSigma, Burlington, MA, USA), poly-GA, poly-GP (both Cosmo Bio, Tokyo, Japan), pSer409 / 410-TDP-43 (Cosmo Bio), and synaptophysin (SVP-38; Sigma-Aldrich, St. Louis, MO, USA). Staining was performed with synaptophysin alone, biotin-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA, USA), horseradish peroxidase (HRP)-conjugated avidin-biotin complex (Vector Laboratories), HRP substrate, diaminobenzidine (DAB), or FITC-conjugated secondary antibodies (Jackson Laboratory). Stained specimens were observed under a BZ-X800 fluorescence microscope (Keyence, Osaka, Japan), and images of specific brain regions were captured. Neuropathological evaluation was performed using NIH ImageJ software by measuring the staining intensity or area of ​​each photograph or by counting positive stipples or cells within a certain area.

[0080] 1.5 Statistical Analysis All experiments and data analyses were performed under an open-label approach. Comparisons of means between two or more groups were performed using ANOVA or two-factor repeated measures ANOVA (for the Morris water maze test) followed by Fisher's PLSD test. Differences with a p-value of <0.05 were considered significant.

[0081] 2. Results Intranasal administration of 0.04 mg / day of resveratrol (Resv) significantly improved cognitive function in FTD / ALS model mice (Figure 10). Administration of the same dose of rifampicin (RFP) also improved cognitive function in mice, but the effect was incomplete and not significant. The combination (0.02 mg rifampicin + 0.02 mg resveratrol / day) showed an effect intermediate between that of resveratrol and rifampicin.

[0082] Synaptophysin levels were assessed in the hippocampal CA2-3 region and dentate gyrus (DG). Resveratrol administration significantly reversed synaptic loss in the CA2-3 region and DG, but rifampicin administration did not (Figure 11). The combination of the two drugs was effective only in the DG. In the prefrontal cortex, levels of G-quadruplex (Figure 12), phosphorylated PKR (Figure 13), poly(GA) and poly(GP) (Figure 14), and phosphorylated TDP-43 (Figure 15) were assessed. These pathologies were significantly reduced by rifampicin, resveratrol, and their combination, with resveratrol showing the strongest effect and the combination showing an intermediate effect between resveratrol and rifampicin.

[0083] 3. Discussion In this study, rifampicin, resveratrol, and their combination improved cognition and C9orf72 hexanucleotide repeat expansion (HRE)-associated neuropathology in FTD / ALS model mice, with resveratrol showing stronger effects than rifampicin. The difference in efficacy between rifampicin and resveratrol is likely due to their different mechanisms of action. Both rifampicin and resveratrol have antioxidant properties and are effective against amyloid oligomerization. We previously demonstrated that rifampicin inhibits Aβ and tau oligomerization more efficiently than resveratrol in a mouse model, suggesting that rifampicin can inhibit TDP-43 oligomerization more efficiently than resveratrol. However, in C9orf72-associated FTD / ALS, TDP-43 oligomerization is downstream of the pathological cascade, and its inhibition may only provide partial therapeutic benefit. Further upstream pathologies, such as C9orf72 haploinsufficiency, RNA foci formation, RNA translation, and stress granule formation, may cause more extensive and severe damage to cells, which may be ameliorated by resveratrol more effectively than rifampicin. Resveratrol, but not rifampicin, induced BDNF expression in mouse brain. Furthermore, only resveratrol has been reported to activate Sirt1, a gene associated with longevity. These activities of resveratrol may contribute to its stronger effect on HRE-associated neuropathology.

[0084] These results suggest that resveratrol is a promising therapeutic candidate for FTD / ALS.

[0085] 4. Conclusions: Intranasal administration of 0.04 mg / day rifampicin (monotherapy), 0.04 mg / day resveratrol (monotherapy), and a combination of 0.02 mg / day rifampicin and 0.02 mg / day resveratrol improved neuropathological and cognitive impairment in FTD / ALS model mice. Therefore, rifampicin and resveratrol were shown to have therapeutic effects against the TDP-43 proteinopathies FTD and ALS. Furthermore, resveratrol showed the strongest effect, while the combined effect was intermediate between that of resveratrol and rifampicin, demonstrating that resveratrol has a particularly strong therapeutic effect against the TDP-43 proteinopathies FTD and ALS. Therefore, even if the dose of resveratrol (single agent) is half of the above (specifically, 0.02 mg / day), it can be inferred that it will naturally improve neuropathological and cognitive dysfunction, and ultimately have a therapeutic effect on FTD and ALS, which are TDP-43 proteinopathies.

Claims

1. A preventive or therapeutic agent for neurodegenerative diseases caused by accumulation of TDP-43, comprising as an active ingredient a resveratrol selected from the group consisting of resveratrol and its derivatives.

2. A preventive or therapeutic drug for amyotrophic lateral sclerosis (ALS) comprising, as an active ingredient, a resveratrol selected from the group consisting of resveratrol and its derivatives.

3. A preventive or therapeutic agent for neurodegenerative diseases caused by C9orf72 genetic abnormalities and / or TDP-43 genetic abnormalities, comprising as an active ingredient a resveratrol selected from the group consisting of resveratrol and its derivatives.

4. A prophylactic or therapeutic drug according to any one of claims 1 to 3, which is administered intranasally.

5. A preventive or therapeutic drug according to any one of claims 1 to 3, wherein the dose of the resveratrol derivative is 3.75 mg / kg / day or less.

6. A preventive or therapeutic agent for neurodegenerative diseases caused by accumulation of TDP-43, comprising a combination of a rifampicin selected from the group consisting of rifampicin, its derivatives, and salts thereof, and a resveratrol selected from the group consisting of resveratrol and its derivatives.

7. A preventive or therapeutic drug for neurodegenerative diseases caused by accumulation of TDP-43, comprising, as an active ingredient, a rifampicin selected from the group consisting of rifampicin, its derivatives, and salts thereof.

8. A preventive or therapeutic drug for amyotrophic lateral sclerosis (ALS), comprising as an active ingredient a rifampicin-based compound selected from the group consisting of rifampicin, its derivatives, and salts thereof.

9. A preventive or therapeutic drug for neurodegenerative diseases caused by C9orf72 genetic abnormality and / or TDP-43 genetic abnormality, comprising as an active ingredient a rifampicin analog selected from the group consisting of rifampicin, its derivatives, and salts thereof.

10. A preventive or therapeutic drug according to any one of claims 7 to 9, which is administered intranasally.

11. The preventive or therapeutic drug according to any one of claims 7 to 9, wherein the dose of the rifampicin is 3.75 mg / kg / day or less.

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