Use of ROCK and Syk inhibitors

Abstract

To provide a new agent for inhibiting cleavage of tau protein, a new agent for inhibiting phosphorylation of tau protein, and a new agent for activating autophagy, and a new agent for treating Alzheimer's disease by using these new agents.SOLUTION: The present invention discloses a new agent for inhibiting cleavage of tau protein, a new agent for inhibiting phosphorylation of tau protein, and a new agent for activating autophagy, and a new agent for treating Alzheimer's disease by using these new agents, each of which contains an ROCK inhibitor and an Syk inhibitor as active ingredients.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the use of ROCK inhibitors and Syk inhibitors. [Background technology]

[0002] Rho kinase (ROCK: Rho-associated coiled coil protein kinase), a small G protein, is a serine-threonine phosphorylating enzyme with a molecular weight of about 160 kDa. It is known that ROCK in the brain increases with aging and / or smoking. ROCK inhibitors that inhibit the activity of ROCK are used to prevent post-vasospasm baracnoid hemorrhage and to treat glaucoma. In addition, ROCK inhibitors are known to suppress the formation of tau protein oligomers, suppress the phosphorylation of tau protein, affect the degradation of tau protein, and activate autophagy (e.g., Non-Patent Document 1).

[0003] Tau protein is known to be related to Alzheimer's disease. Pathological findings of Alzheimer's disease include senile plaques (SP) and neurofibrillary tangles (NFT). The main cause of NFT is tau protein. Tau protein binds to microtubules and stabilizes them. When tau protein is phosphorylated, it is released from the microtubules and polymerizes to form oligomers, which form NFTs. When NFTs are formed, neuronal cell death occurs, resulting in the onset of Alzheimer's disease. Phosphorylation of tau protein is carried out by a specific kinase. Furthermore, polymerization of tau protein is promoted by cleavage of the C-terminus of tau protein.

[0004] Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase. Syk inhibitors that inhibit Syk activity have been suggested to have therapeutic effects on allergic rhinitis, rheumatoid arthritis, immune thrombocytopenia, IgA nephropathy, and autoimmune hemolytic anemia. Syk has also been suggested to activate glycogen synthase kinase 3β, which inhibits the phosphorylation enzyme of tau protein (e.g., Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hamano et al. "Rho-kinase ROCK inhibitors reduce oligomeric tau protein" Neurobiology of Aging 89(2020) 41-54 [Non-Patent Document 2] Paris D., "The spleen tyrosine kinase(Syk) regulates Alzheimer amyloid-beta production and Tau hyperphosphorylation" J. Biol. Chem., 2014 Dec, 5;289(49), 33927-33944 Summary of the Invention [Problem to be solved by the invention]

[0006] The above-mentioned ROCK inhibitors have the effects of inhibiting the cleavage of tau protein, inhibiting the phosphorylation of tau protein, and activating autophagy, but there is a demand for the development of drugs with even stronger effects.

[0007] One aspect of the present invention has been made in consideration of the above, and aims to provide a new cleavage inhibitor of tau protein, a phosphorylation inhibitor of tau protein, and an autophagy activator, as well as a therapeutic agent for Alzheimer's disease using these. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have conducted extensive research and found that the combined use of a ROCK inhibitor and a Syk inhibitor provides a much more excellent therapeutic effect than the use of each inhibitor alone, and have completed the present invention. That is, the present invention includes the following features. <1> A tau protein cleavage inhibitor that contains a ROCK inhibitor and a Syk inhibitor as active ingredients. <2> the molar ratio of the ROCK inhibitor to the Syk inhibitor (ROCK inhibitor / Syk inhibitor) is 1 / 0.5 to 1 / 1; <1> An inhibitor of cleavage of tau protein described in <3> A tau protein phosphorylation inhibitor that contains a ROCK inhibitor and a Syk inhibitor as active ingredients. <4> the molar ratio of the ROCK inhibitor to the Syk inhibitor (ROCK inhibitor / Syk inhibitor) is 1 / 0.5 to 1 / 1; <3> 2. A phosphorylation inhibitor of tau protein according to claim 1. <5> An autophagy activator that contains a ROCK inhibitor and a Syk inhibitor as active ingredients. <6> the molar ratio of the ROCK inhibitor to the Syk inhibitor (ROCK inhibitor / Syk inhibitor) is 1 / 0.5 to 1 / 0.5; <5> 2. An autophagy activator according to claim 1 . <7> <1> Or <2> A cleavage inhibitor for tau protein according to any one of claims 1 to 5, <3> Or <4> or <5> Or <6> 1. A therapeutic agent for Alzheimer's disease, comprising the autophagy activator described in claim 1 as an active ingredient. Effect of the Invention

[0009] According to one embodiment of the present invention, a drug having superior effects (effect of inhibiting the cleavage of tau protein, effect of inhibiting the phosphorylation of tau protein, effect of activating autophagy, effect of treating Alzheimer's disease) compared to conventional drugs can be provided. [Brief description of the drawings]

[0010] [Figure 1] 1 shows the results of Western blot showing the expression levels of caspase-cleaved tau protein when a ROCK inhibitor and a Syk inhibitor were used, and a graph quantifying the results. [Diagram 2] 1 shows the results of Western blot analysis indicating the expression level of phosphorylated tau protein when a ROCK inhibitor and a Syk inhibitor were used, and a graph in which the results were quantified. [Diagram 3] FIG. 1 shows the results of Western blot analysis indicating the expression levels of LC3I and LC3II, which are marker proteins for activation of autophagy, when a ROCK inhibitor and a Syk inhibitor were used, and a graph quantifying the results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] An embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to each of the configurations described below, and various modifications are possible within the scope of the claims. The embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples are also included in the technical scope of the present invention. In addition, all of the documents described in this specification are incorporated herein by reference. In this specification, when "A to B" is described with respect to a numerical range, the description intends "A or more and B or less."

[0012] [1. Tau protein cleavage inhibitors] A tau protein cleavage inhibitor according to one embodiment of the present invention (hereinafter also referred to as the present cleavage inhibitor) contains a ROCK inhibitor and a Syk inhibitor as active ingredients.

[0013] With the above configuration, the combined use of a ROCK inhibitor and a Syk inhibitor results in a significantly stronger effect of inhibiting the cleavage of tau protein than when each is used alone, as shown in the Examples described below.

[0014] The ROCK inhibitor is not particularly limited as long as it can inhibit the ROCK, and known drugs known as ROCK inhibitors can be used. As the ROCK inhibitor, for example, one or more selected from Y-276732 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.dihydrochloride), H-1152 (4-Methyl-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline dihydrochloride), fasudil (hexahydro-1-(5-isoquinolinesulfonyl)-1H-1,4-diazepinemonohydrochloride hemihydrate), and pitavastatin may be used. As the ROCK inhibitor, a commercially available product may be used.

[0015] The Syk inhibitor is not particularly limited as long as it can inhibit the activity of Syk, and known drugs known as Syk inhibitors can be used. As the Syk inhibitor, for example, one or more selected from BAY61-3606 (2-[[7-(3,4-Dimethoxyphenyl)imidazo[1,2-c]pyrimidin-5-yl]amino]pyridine-3-carboxamide hydrochloride), fostamatinib, and R406 (6-(5-fluoro-2-(3,4,5-trimethoxyphenylamino)pyrimidin-4-ylamino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one) may be used. As the Syk inhibitor, a commercially available product may be used.

[0016] The combination of the ROCK inhibitor and the Syk inhibitor is not particularly limited. In addition, the number of types of compounds corresponding to the "ROCK inhibitor" contained in the present cleavage inhibitor and the number of types of compounds corresponding to the "Syk inhibitor" may be the same or different. The present cleavage inhibitor may contain, for example, Y-276732, H-1152, and fostamatinib as active ingredients, or may contain Y-276732, fostamatinib, and R406. From the viewpoint of obtaining advantageous effects such as reduction of caspase-cleaved tau and reduction of phosphorylated tau, it is preferable to use fasudil as the ROCK inhibitor and BAY61-3606 as the Syk inhibitor.

[0017] The mechanism of action by which the present cleavage inhibitor inhibits the cleavage of tau protein is not particularly limited, and for example, by inhibiting the activity of ROCK and Syk, it may (i) inhibit the activity of an enzyme that cleaves tau protein, or (ii) activate an enzyme that inhibits the cleavage of tau protein.

[0018] The enzyme that cleaves tau protein is preferably an enzyme that removes the C-terminal amino acid sequence from tau protein by cleaving the amino acid sequence near the C-terminus of tau protein. The amino acid sequence near the C-terminus of tau protein has the effect of suppressing tau protein aggregation (polymerization of tau protein, formation of tau oligomers). Therefore, removing the C-terminal amino acid sequence from tau protein promotes tau protein aggregation. On the other hand, inhibiting the activity of the enzyme that cleaves the amino acid sequence near the C-terminus of tau protein suppresses tau protein aggregation, and as a result, the formation of NFTs can be suppressed.

[0019] Examples of the enzyme that cleaves the amino acid sequence near the C-terminus of the tau protein include caspase. On the other hand, examples of the protein that inhibits the activity of the enzyme that cleaves the amino acid sequence near the C-terminus of the tau protein include Z-VAD-FMK. From the viewpoint that the inhibitor can effectively inhibit the cleavage of tau protein, it is preferable that the enzyme that cleaves the amino acid sequence near the C-terminus of the tau protein is caspase.

[0020] The caspase is not particularly limited as long as it is a caspase that cleaves tau protein. Examples of such caspases include caspase 2, caspase 3, and caspase 7. From the viewpoint that the present cleavage inhibitor can effectively inhibit the cleavage of tau protein, the caspase is preferably caspase 3.

[0021] The amount of active ingredients (specifically, ROCK inhibitor and Syk inhibitor) contained in the present cleavage inhibitor is not particularly limited, but may be 0.001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, 0.1% by weight to 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to 70% by weight, 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight, 0.1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight.

[0022] The molar ratio of the ROCK inhibitor to the Syk inhibitor contained in the present cleavage inhibitor (ROCK inhibitor / Syk inhibitor) is preferably 1 / 0.5 to 1 / 1, more preferably 1 / 0.7 to 1 / 1, even more preferably 1 / 0.8 to 1 / 1, and most preferably 1 / 0.9 to 1 / 1. With this configuration, the effect of the present cleavage inhibitor in inhibiting the cleavage of tau protein is further improved.

[0023] The cleavage inhibitor may contain components other than the active ingredient. The components other than the active ingredient may be pharma- ceutically acceptable components, such as buffers, pH adjusters, isotonicity agents, preservatives, excipients, carriers, diluents, solvents, solubilizers, stabilizers, antioxidants, high molecular weight polymers, fillers, binders, surfactants, and stabilizers.

[0024] The buffering agent includes phosphoric acid, phosphates, boric acid, borates, citric acid, citrates, acetic acid, acetates, carbonic acid, carbonates, tartaric acid, tartrates, ε-aminocaproic acid, and trometamol. The phosphates include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. The borates include borax, sodium borate, and potassium borate. The citrates include sodium citrate, disodium citrate, and trisodium citrate. The acetates include sodium acetate and potassium acetate. The carbonates include sodium carbonate and sodium bicarbonate. The tartrates include sodium tartrate and potassium tartrate.

[0025] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0026] The isotonicity agent includes ionic isotonicity agents (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.) and non-ionic isotonicity agents (glycerin, propylene glycol, sorbitol, mannitol, etc.).

[0027] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0028] The antioxidants include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0029] Examples of the high molecular weight polymers include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, and polyethylene glycol.

[0030] The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

[0031] Examples of the solvent and solubilizing agent include glycerin, DMSO, DMA, N-methylpyrrolidone, ethanol, benzyl alcohol, isopropyl alcohol, polyethylene glycols of various molecular weights (such as PEG300 and PEG400), and propylene glycol.

[0032] Such surfactants include nonionic, anionic, cationic, zwitterionic, polymeric, and amphoteric surfactants.

[0033] The stabilizers include fatty acids, fatty alcohols, alcohols, long chain fatty acid esters, long chain ethers, hydrophilic derivatives of fatty acids, polyvinylpyrrolidones, polyvinyl ethers, polyvinyl alcohols, hydrocarbons, hydrophobic polymers, hygroscopic polymers, and amide analogs.

[0034] The amount of ingredients other than the active ingredient contained in the present cleavage inhibitor is not particularly limited, but preferably, when the entire present cleavage inhibitor is taken as 100% by weight, it may be 0% by weight to 99.999% by weight, may be 0% by weight to 99.99% by weight, may be 0% by weight to 99.9% by weight, may be 0% by weight to 99.9% by weight, may be 5% by weight to 99.9% by weight, may be 10% by weight to 99.9% by weight, may be 20% by weight to 99.9% by weight, may be 30% by weight to 99.9% by weight, may be 40% by weight to 99.9% by weight, may be 50% by weight to 99.9% by weight, may be 60% by weight to 99.9% by weight, may be 70% by weight to 99.9% by weight, may be 80% by weight to 99.9% by weight, or may be 90% by weight to 99.9% by weight.

[0035] The cleavage inhibitor may be administered to a subject. The subject may be a human or a non-human subject (e.g., an animal). Examples of the non-human animal include non-human mammals (cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, rats, etc.). The cleavage inhibitor may also be administered to a non-living organism (e.g., an artificially constructed expression system for tau protein).

[0036] The cleavage inhibitor can be administered to a subject by any administration route. Examples of the administration route include oral administration, parenteral administration, transdermal administration, transmucosal administration, and intravenous administration. Therefore, the dosage form of the cleavage inhibitor can be oral medication, topical medication, injection, suppository, inhalant, eye drop, etc.

[0037] The cleavage inhibitor may be administered into a blood vessel, more specifically, into a vein. Methods for administering into a blood vessel include administration by drip infusion and administration by syringe.

[0038] When administering the present cleavage inhibitor, there is no limit to the administration interval as long as the desired effect can be obtained. The administration interval is, for example, once every hour to six months, once every hour to one month, once every hour to ten days, once every hour to five days, once every hour to one day, once every hour to 12 hours, once every hour to 6 hours, or once every hour to two hours.

[0039] Alternatively, the compound may be administered continuously without an administration interval. The administration period is, for example, 1 hour to 6 months, 1 hour to 1 month, 1 hour to 10 days, 1 hour to 5 days, 1 hour to 1 day, 1 hour to 12 hours, 1 hour to 6 hours, or 1 hour to 2 hours.

[0040] [2. Tau protein phosphorylation inhibitors] The phosphorylation inhibitor of tau protein according to one embodiment of the present invention (hereinafter also referred to as the present phosphorylation inhibitor) contains a ROCK inhibitor and a Syk inhibitor as active ingredients. Note that the details of the ROCK inhibitor and the Syk inhibitor already explained in the above [1. Tau protein cleavage inhibitor] will not be explained again.

[0041] With the above configuration, the combined use of a ROCK inhibitor and a Syk inhibitor results in a significantly stronger inhibitory effect on the phosphorylation of tau protein than when each inhibitor is used alone, as shown in the Examples described below.

[0042] The mechanism of action by which the present phosphorylation inhibitor inhibits the phosphorylation of tau protein is not particularly limited, and for example, by inhibiting the activity of ROCK and Syk, it may (i) inhibit an enzyme that phosphorylates tau protein, or (ii) activate an enzyme that inhibits the phosphorylation of tau protein.

[0043] When tau protein is phosphorylated, tau oligomers are more likely to form. On the other hand, if tau protein is not phosphorylated, tau oligomers are less likely to form. Therefore, by inhibiting the phosphorylation of tau protein, the aggregation of tau protein (polymerization of tau protein, formation of tau oligomers) is suppressed, and as a result, the formation of NFTs can be suppressed.

[0044] Examples of the enzyme that phosphorylates the tau protein include activated major tau kinase. Examples of the activated major tau kinase include glycogen synthase kinase 3β, cyclin-dependent kinase 5, P38 mitogen-activated protein kinase (P38MAPK), and Stress-activated protein kinase (SAPK) / Jun amino terminal kinase (JNK). On the other hand, examples of the enzyme that inhibits the phosphorylation of tau protein include inactivated tau phosphatase. Examples of the inactivated tau phosphatase include protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B), and protein phosphatase 1 (PP1).

[0045] The amounts of the active ingredients (specifically, the ROCK inhibitor and the Syk inhibitor) contained in this phosphorylation inhibitor can be the same as those in [1. Tau protein cleavage inhibitor].

[0046] The molar ratio of the ROCK inhibitor to the Syk inhibitor contained in the phosphorylation inhibitor (ROCk inhibitor / Syk inhibitor) is preferably 1 / 0.5 to 1 / 1, more preferably 1 / 0.7 to 1 / 1, even more preferably 1 / 0.8 to 1 / 1, and most preferably 1 / 0.9 to 1 / 1. With this configuration, the effect of the phosphorylation inhibitor in inhibiting the phosphorylation of tau protein is further improved.

[0047] This phosphorylation inhibitor may contain ingredients other than the active ingredients described in [1. Tau protein cleavage inhibitors] above, at the concentrations described in [1. Tau protein cleavage inhibitors] above.

[0048] The subjects, administration route, and administration intervals of the phosphorylation inhibitor may be the same as those described in the above section [1. Tau protein cleavage inhibitor]. These have already been described, so the description will be omitted here.

[0049] [3. Autophagy activators] An autophagy activator according to one embodiment of the present invention (hereinafter also referred to as the present activator) contains a ROCK inhibitor and a Syk inhibitor as active ingredients. Note that the details of the ROCK inhibitor and the Syk inhibitor already explained in the above section [1. Tau protein cleavage inhibitor] will not be explained here.

[0050] With the above configuration, the combined use of a ROCK inhibitor and a Syk inhibitor has a significantly stronger effect of activating autophagy than the use of each inhibitor alone.

[0051] Autophagy (also called self-eating) is a process in which cells degrade their own proteins, intracellular organelles (e.g., mitochondria or endoplasmic reticulum), and other components. Autophagy can be classified into, for example, macrophagy, microautophagy, and chaperone-mediated autophagy. The autophagy that the present activator activates can be any of the above-mentioned autophagy.

[0052] The mechanism of action by which the present activator activates autophagy is not particularly limited, and for example, by inhibiting ROCK and Syk, (i) the activity of a protein that activates autophagy may be improved, or (ii) the activity of a protein that inactivates autophagy may be reduced. Activating autophagy suppresses the formation of tau oligomers, and as a result, the formation of NFTs can be suppressed.

[0053] Examples of substances that activate autophagy include lithium, rapamycin, metformin, and carbamazepine, while examples of substances that inactivate autophagy include chloroquine and hydroxychloroquine.

[0054] The amounts of the active ingredients (specifically, the ROCK inhibitor and the Syk inhibitor) contained in the present activator can be the same as those in [1. Tau protein cleavage inhibitor].

[0055] The molar ratio of the ROCK inhibitor to the Syk inhibitor contained in the present activator (ROCK inhibitor / Syk inhibitor) is preferably 1 / 0.5 to 1 / 1, more preferably 1 / 0.7 to 1 / 1, even more preferably 1 / 0.8 to 1 / 1, and most preferably 1 / 0.9 to 1 / 1. With this configuration, the effect of the present activator in activating autophagy is further improved.

[0056] The present activator may contain ingredients other than the active ingredients described in the above section [1. Tau protein cleavage inhibitors] at the concentrations described in the above section [1. Tau protein cleavage inhibitors].

[0057] The subjects, administration route, and administration intervals of the present activator may be the same as those described in the above section [1. Tau protein cleavage inhibitor]. These have already been described, so the description will be omitted here.

[0058] 4. Drugs for treating Alzheimer's disease The therapeutic drug for Alzheimer's disease according to one embodiment of the present invention (hereinafter also referred to as the therapeutic drug) contains the cleavage inhibitor, the phosphorylation inhibitor, or the activator as an active ingredient. Regarding the ROCK inhibitor and the Syk inhibitor, the matters already explained in the above [1. Tau protein cleavage inhibitor] will not be explained again.

[0059] With the above configuration, the combined use of the ROCK inhibitor and the Syk inhibitor provides a more significant therapeutic effect against Alzheimer's disease than the use of each of them. With the above configuration, the amount of the ROCK inhibitor and the amount of the Syk inhibitor can be reduced compared to the use of the ROCK inhibitor or the Syk inhibitor alone. By reducing the amount of the ROCK inhibitor and the Syk inhibitor, the side effects caused by them can be reduced. The effect of reducing the side effects can also be obtained by the above-mentioned tau protein cleavage inhibitor, tau protein phosphorylation inhibitor, and autophagy activator.

[0060] As described above, the cleavage inhibitor can inhibit the cleavage of tau protein, thereby suppressing the formation of NFTs that cause Alzheimer's disease. The phosphorylation inhibitor can inhibit the phosphorylation of tau protein, thereby suppressing the formation of NFTs that cause Alzheimer's disease. Furthermore, the activator can activate autophagy, thereby suppressing the formation of NFTs that cause Alzheimer's disease. The therapeutic agent can suppress the formation of NFTs, which are causative substances of Alzheimer's disease, by containing the cleavage inhibitor, the phosphorylation inhibitor, or the activator. Therefore, the therapeutic agent can be used as a therapeutic agent for Alzheimer's disease.

[0061] Examples of Alzheimer's disease include familial Alzheimer's disease, sporadic Alzheimer's disease, and early-onset Alzheimer's disease.

[0062] As used herein, the term "therapeutic agent" refers to a drug that provides a therapeutic effect. The therapeutic effect includes, but is not limited to, the effects exemplified below.

[0063] (1) The severity of one or more symptoms associated with Alzheimer's disease is reduced when the therapeutic agent is administered compared to when the therapeutic agent is not administered.

[0064] (2) Preventing an increase in severity or a progression of severity of one or more symptoms associated with Alzheimer's disease when the therapeutic agent is administered compared to when the therapeutic agent is not administered.

[0065] (3) The administration of the therapeutic agent reduces the rate of increase in severity or the rate of progression of severity of one or more symptoms associated with Alzheimer's disease when compared to when the therapeutic agent is not administered.

[0066] The one or more symptoms associated with Alzheimer's disease may be systemic or localized.

[0067] The therapeutic agent may also be used as a prophylactic agent for preventing Alzheimer's disease. In this specification, the term "prophylactic agent" refers to a drug that provides a prophylactic effect. The prophylactic effect refers to, but is not limited to, the following examples:

[0068] (1) When the prophylactic drug is administered, the onset of one or more symptoms associated with Alzheimer's disease is prevented or the risk of onset is reduced, compared to when the prophylactic drug is not administered.

[0069] (2) When the prophylactic drug is administered, the recurrence of one or more symptoms associated with Alzheimer's disease is prevented or the risk of recurrence is reduced compared to when the prophylactic drug is not administered.

[0070] (3) Administration of the prophylactic agent prevents the onset of, or reduces the risk of the onset of, one or more symptoms of Alzheimer's disease compared to not administering the prophylactic agent.

[0071] The one or more symptoms associated with Alzheimer's disease may be systemic or localized.

[0072] This therapeutic agent may contain ingredients other than the active ingredients described in [1. Tau protein cleavage inhibitors] above, at the concentrations described in [1. Tau protein cleavage inhibitors] above.

[0073] The subjects, administration route, and administration intervals of this therapeutic agent may be the same as those described in the above section [1. Tau protein cleavage inhibitor]. These have already been described, so the description will be omitted here.

[0074] [5. Other] The present invention may also be configured as follows.

[0075] <1> A method for inhibiting the cleavage of tau protein, comprising the step of administering a tau protein cleavage inhibitor containing a ROCK inhibitor and a Syk inhibitor as active ingredients to a subject or a non-human subject (e.g., a mammal, a non-human mammal, a primate, an artiodactyl, a perissodactyl, a rodent, or a lagomorph).

[0076] <2> A method for inhibiting the phosphorylation of tau protein, comprising the step of administering a tau protein phosphorylation inhibitor containing a ROCK inhibitor and a Syk inhibitor as active ingredients to a subject or a non-human subject (e.g., a mammal, a non-human mammal, a primate, an artiodactyl, a perissodactyl, a rodent, or a lagomorph).

[0077] <3> A method for activating autophagy, comprising the step of administering an autophagy activator containing a ROCK inhibitor and a Syk inhibitor as active ingredients to a subject or a non-human subject (e.g., a mammal, a non-human mammal, a primate, an artiodactyl, a perissodactyl, a rodent, or a lagomorph).

[0078] <4> A method for treating Alzheimer's disease, comprising the step of administering a therapeutic agent for Alzheimer's disease containing a ROCK inhibitor and a Syk inhibitor as active ingredients to a subject or a non-human subject (e.g., a mammal, a non-human mammal, a primate, an artiodactyl, a perissodactyl, a rodent, or a lagomorph).

[0079] <5> Use of a ROCK inhibitor and a Syk inhibitor for producing an inhibitor of tau protein cleavage.

[0080] <6> Use of a ROCK inhibitor and a Syk inhibitor for producing an inhibitor of phosphorylation of tau protein.

[0081] <7> Use of a ROCK inhibitor and a Syk inhibitor to produce an autophagy activator.

[0082] <8> Use of a ROCK inhibitor and a Syk inhibitor for the manufacture of a therapeutic agent for Alzheimer's disease. EXAMPLES

[0083] An embodiment of the present invention will now be described.

[0084] 〔material and method〕 Unless otherwise stated, the materials and methods used in the experiments are as follows.

[0085] [Experimental materials] Human neuroblastoma cell line (M1C cells) [Introduction of TetOff induction system] The TetOff-Neo gene (Clontech) was introduced into human neuroblastoma cells BE(2)-M17D using Tfx reagent (Promega), and the cells into which the TetOff-Neo gene was introduced were selected using G418. The cells obtained were then transfected with wild-type tau pTRE / Tau(4R0N) and pBABE-puro, and the cells into which these plasmids were introduced were selected using puromycin. Furthermore, the cells obtained were transfected with BstETi-linealized pVgRXR (Invitrogen), and the cells into which the plasmid was introduced were selected using Zeocin. Furthermore, the cells obtained were transfected with FspI-linearized pIND-Hydrogen / tau[IN+10(1N)], and the cells into which the plasmid was introduced were selected using hygromycin, to produce M1C cells.

[0086] [Creation of wild-type tau protein expressing cells] The above M1C cells were added to 1.5 × 10 6 ~2×10 6 The cells were cultured to a density of cells / culture dish. The culture medium used was Dulbecco's modified Eagle medium containing 10% bovine serum, supplemented with G418 (400 μg / mL), tetracycline (2 μg / mL), Zeocin (100 μg / mL), hygromycin (100 μg / mL), and puromycin (1 μg / mL). Approximately 24 hours after the start of culture, the culture medium was replaced with a medium having the same composition as the above culture medium except that it did not contain tetracycline. TetOff induction was initiated by replacing the medium. Five days after the start of TetOFF induction, the cells were harvested and proteins were extracted.

[0087] [Test 1. Detection of caspase-cleaved tau protein] Neuronal cells (M1C cells) expressing wild-type tau protein (4R0N) were cultured in a medium without tetracycline for 5 days to express tau protein. 24 hours before the end of the culture, (i) compound fasudil as a ROCK inhibitor was administered at 1 μM in the medium, (ii) compound BAY61-3606 as a Syk inhibitor was administered at 0.5 μM in the medium, or (iii) compound fasudil as a ROCK inhibitor was administered at 1 μM in the medium and compound BAY61-3606 as a Syk inhibitor was administered at 1 μM in the medium.

[0088] After the culture was completed, the cells corresponding to each of the above tests (i) to (iii) were collected. Tau protein was extracted from the collected cells, and tau protein cleaved by caspase was detected by Western blotting. Note that TauC3 antibody manufactured by Santa Cruz was used as the primary antibody for Western blotting.

[0089] The test results are shown in Figure 1. The "control" in Figure 1 corresponds to a test in which neither a ROCK inhibitor nor a Syk inhibitor was added to the medium. The left side of Figure 1 shows the results detected by the Western blotting method. The right side of Figure 1 is a graph in which the results obtained by the Western blotting method were quantified using a densitometer. As is clear from Figure 1, the amount of tau protein cleaved by caspases (e.g., caspase 3) was significantly smaller in cells treated with a ROCK inhibitor or a Syk inhibitor than in cells treated with either inhibitor alone.

[0090] [Test 2. Detection of phosphorylated tau protein] As in Test 1, neural cells (M1C cells) expressing wild-type tau protein (4R0N) were cultured. After the culture was completed, cells corresponding to each of the tests (i) to (iii) above were collected. Tau protein was extracted from the collected cells, and phosphorylated tau protein was detected by Western blotting. Note that Tau (Phospho Ser199 / Ser202) antibody manufactured by Gene Tex was used as the primary antibody for Western blotting.

[0091] The test results are shown in Figure 2. The "control" in Figure 2 corresponds to a test in which neither the ROCK inhibitor nor the Syk inhibitor was added to the medium. The left side of Figure 2 shows the results detected by the Western blotting method. The right side of Figure 2 is a graph in which the results obtained by the Western blotting method were quantified using a densitometer. As is clear from Figure 2, the amount of phosphorylated tau protein was significantly lower in cells treated with a combination of the ROCK inhibitor and the Syk inhibitor than in cells treated with either one alone.

[0092] [Test 3. Detection of autophagy activation] As in Test 1, neural cells (M1C cells) expressing wild-type tau protein (4R0N) were cultured. After the culture was completed, cells corresponding to each of the tests (i) to (iii) above were collected. Tau protein was extracted from the collected cells, and LC3I and LC3II, which are marker proteins that indicate the activation of autophagy, were detected by Western blotting. Note that anti-LC3 antibody manufactured by Cell signaling was used as the primary antibody for Western blotting.

[0093] The test results are shown in Figure 3. The "-" in Figure 3 corresponds to a test in which neither the ROCK inhibitor nor the Syk inhibitor was added to the medium. The left side of Figure 3 shows the results detected by the Western blotting method. The right side of Figure 3 is a graph in which the results obtained by the Western blotting method were quantified using a densitometer. As is clear from Figure 3, the amount of LC3II, an autophagy activation marker (specifically, the ratio of the amount of LC3II to the amount of LC3I), was significantly higher in cells treated with a combination of a ROCK inhibitor and a Syk inhibitor than in cells treated with either one alone. [Industrial Applicability]

[0094] The present invention can be used as a cleavage inhibitor of tau protein, a phosphorylation inhibitor of tau protein, an autophagy activator, or a therapeutic agent for Alzheimer's disease, etc.

Claims

1. Contains a ROCK inhibitor and a Syk inhibitor as active ingredients, The ROCK inhibitor is one or more selected from Y-276732, H-1152, fasudil, and pitavastatin; The Syk inhibitor is one or more selected from BAY61-3606, fostamatinib, and R406; An inhibitor of tau protein cleavage.

2. The tau protein cleavage inhibitor according to claim 1, wherein the molar ratio of the ROCK inhibitor to the Syk inhibitor (ROCK inhibitor / Syk inhibitor) is 1 / 0.5 to 1 / 1.

3. Contains a ROCK inhibitor and a Syk inhibitor as active ingredients, The ROCK inhibitor is one or more selected from Y-276732, H-1152, fasudil, and pitavastatin; The Syk inhibitor is one or more selected from BAY61-3606, fostamatinib, and R406; Inhibitor of tau protein phosphorylation.

4. The tau protein phosphorylation inhibitor according to claim 3, wherein the molar ratio of the ROCK inhibitor to the Syk inhibitor (ROCK inhibitor / Syk inhibitor) is 1 / 0.5 to 1 / 1.

5. Contains a ROCK inhibitor and a Syk inhibitor as active ingredients, The ROCK inhibitor is one or more selected from Y-276732, H-1152, fasudil, and pitavastatin; The Syk inhibitor is one or more selected from BAY61-3606, fostamatinib, and R406; Activator of autophagy.

6. The autophagy activator according to claim 5, wherein the molar ratio of the ROCK inhibitor to the Syk inhibitor (ROCK inhibitor / Syk inhibitor) is 1 / 0.5 to 1 / 1.

7. A therapeutic agent for Alzheimer's disease comprising, as an active ingredient, a tau protein cleavage inhibitor according to claim 1 or 2, a tau protein phosphorylation inhibitor according to claim 3 or 4, or an autophagy activator according to claim 5 or 6.

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