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Home , Dobutamine, Droxidopa, Hexoprenaline, Isoprenaline

(19) TZZ ¥_T

(11) EP 2 764 863 A1

(12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC

(43) Date of publication: (51) Int Cl.: 13.08.2014 Bulletin 2014/33 A61K 31/137 (2006.01) A61K 31/472 (2006.01) A61P 25/28 (2006.01) A61P 43/00 (2006.01) (21) Application number: 12837822.1 (86) International application number: (22) Date of filing: 03.10.2012 PCT/JP2012/006363

(87) International publication number: WO 2013/051266 (11.04.2013 Gazette 2013/15)

(84) Designated Contracting States: • NAGATA, Hiroyuki AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Nishitokyo-shi GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Tokyo 202-0004 (JP) PL PT RO RS SE SI SK SM TR • IHARA, Yasuo Kyotanabe-shi (30) Priority: 03.10.2011 JP 2011219059 Kyoto 610-0394 (JP) • MIYASAKA, Tomohiro (71) Applicants: Kyotanabe-shi • National Center for Geriatrics and Gerontology Kyoto 610-0394 (JP) Obu-shi, Aichi 474-8511 (JP) • SUGIMOTO, Hachiro • The Doshisha Kyotanabe-shi Kyoto 602-8580 (JP) Kyoto 610-0394 (JP)

(72) Inventors: (74) Representative: Chevalier, Renaud Philippe et al • TAKASHIMA, Akihiko Cabinet Germain & Maureau Wako-shi BP 6153 Saitama 351-0104 (JP) 69466 Lyon Cedex 06 (FR) • SOEDA, Yoshiyuki Shiki-shi Saitama 353-0004 (JP)

(54) TAU AGGREGATION INHIBITOR

(57) A tau aggregation inhibitor that can sufficiently reduce tau aggregation in cells is provided. The tau aggregation inhibitor includes a structure-containing compound or a salt thereof, and the catechol structure-containing compound is selected from the group consisting of , , , levodopa, levodopa/ , trimetoquinol, , , and . The catechol structure-containing compound is preferably isoprenaline. Tauopathies to be prevented or treated by the inhibitor include AD, Down’s syndrome, Pick’s disease, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). EP 2 764 863 A1

Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 764 863 A1

2 1 EP 2 764 863 A1 2

Description Application) No. 2004-534854

TECHNICAL FIELD SUMMARY OF THE INVENTION

[0001] The present invention relates to inhibitors for 5 TECHNICAL PROBLEM tau aggregation that causes neurologic deficits and syn- aptic losses. [0009] The tau aggregation inhibitor described above, however, does not sufficiently inhibit tau aggregation in BACKGROUND ART cells, and is insufficient for treatment of tauopathies in- 10 cluding AD. [0002] Alzheimer’s disease (AD) is a type of dementia [0010] It is therefore an object of the present invention whose main symptoms are cognitive decline and person- to provide a tau aggregation inhibitor that can sufficiently ality change. Dementia is a common disorder, and about reduce tau aggregation in cells. 25% of Japanese aged 85 years or older develop, and about a half of dementia cases are caused by AD. In15 SOLUTION TO THE PROBLEM Japan, there are about 1.6 to 1.8 million AD patients in 2011, and the number of AD patients has been increasing [0011] An example of a tau aggregation inhibitor ac- with aging of the population. This is a serious issue es- cording to the present disclosure includes isoprenaline pecially in Japan which has a decreasing birth rate and or a salt thereof. Isoprenaline included in the tau aggre- an aging population. 20 gation inhibitor may be d-enantiomer. Alternatively, iso- [0003] An acetylcholinesterase inhibitor that is consid- prenaline included in the tau aggregation inhibitor may ered as a most effective inhibitor for prevention and treat- be d/l-racemic mixture. It should be noted that d-enanti- ment of AD is effective only for patients with mild to mod- omer or d-isoprenaline refers to (S)-(+)-isoprenaline and erate symptoms, and many researchers find no effec- d/l-racemic mixture or d/l-isoprenaline refers to a mixture tiveness of the acetylcholinesterase inhibitor for patients 25 of (S)-(+)-isoprenaline and (R)-(-)-isoprenaline. with advanced cases. [0012] Another example of the tau aggregation inhibi- [0004] Although neuropathological findings on AD pa- tor according to the present invention includes a catechol tients have two features: senile plaques ofβ -amyloid; structure-containing compound or a salt thereof, and the and neurofibrillary tangles (NFTs) formed by abnormal catechol structure-containing compound is selected from accumulation of tau, a mainstream of current AD re-30 the group consisting of dopamine, dobutamine, levo- search is based on the amyloid- β hypothesis that abnor- dopa, levodopa/carbidopa, trimetoquinol, hexoprena- mal accumulation of amyloid-β peptide eventually leads line, methyldopa, and droxidopa. to AD. [0005] However, it has been revealed that mutation of ADVANTAGES OF THE INVENTION the tau gene promotes NFT formation and causes de- 35 mentia in frontotemporal dementia and parkinsonism [0013] According to the present invention, tau aggre- (FTDP) and that only aggregation and accumulation of gation in cells can be sufficiently reduced. Thus, patients tau in the cause neuronal abnormalities. Thus, the suffering from tauopathies including AD, for which no ef- correlation between tau aggregation and AD occurrence fective therapies have not been discovered yet, can be has attracted attention in recent years. 40 cured. In the current era of aging society, the technique [0006] Neuronal cells in CNS contain a large amount disclosed herein can achieve more effective social con- of tau, which is essential for the function of axons con- tributions by, for example, improving quality of life in eld- stituting the neural network of brain. Insoluble aggrega- erly population, alleviating the burden of cares, and re- tion of tau in cells hinders axonal transport, leading to ducing medical expenses. neuronal death. 45 [0007] PATENT DOCUMENT 1 describes a drug con- BRIEF DESCRIPTION OF THE DRAWINGS taining, as a main component, a naphthoquinone-type compound that inhibits tau aggregation for improving AD [0014] symptoms. This drug reduces tau aggregation in cells to some extent so that NFT formation is reduced and AD 50 [FIG. 1] FIGS. 1A to 1E show thioflavine T activity, symptoms are alleviated. FIG. 1A shows the effect of inhibiting thioflavine T activity by (R)-(-)-epinephrine, FIG. 1B shows the CITATION LIST effect of inhibiting thioflavine T activity by levodopa, FIG. 1C shows the effect of inhibiting thioflavine T PATENT DOCUMENT 55 activity by dopamine, FIG. 1D shows the effect of inhibiting thioflavine T activity by , [0008] [PATENT DOCUMENT 1] Japanese Unexam- and FIG. 1E shows the effect of inhibiting thioflavine ined Patent Publication (Japanese Translation of PCT T activity by isoprenaline.

3 3 EP 2 764 863 A1 4

[FIG. 2] FIGS. 2A and 2B show results of sodium [FIG. 9] FIGS. 9A and 9B show an increase in de- dodecyl sulfate (SDS)-PAGE western blotting indi- phosphorylation of TBS-soluble tau by isoprenaline cating tau aggregation inhibition effects detected by in mice (WT tau Tg mice) overexpressing wild-type sucrose-density gradient centrifugation, FIG. 2A tau, FIG. 9A shows detection of dephosphorylation shows results for (R)-(-)-epinephrine, and FIG. 2B 5 in a TBS-soluble fraction with taul antibody, and FIG. shows results for isoprenaline. 9B shows the proportion of dephosphorylated tau to [FIG. 3] FIGS. 3A and 3B show that isoprenaline re- total tau. duces SDS-insoluble tau aggregation, FIG. 3A [FIG. 10] FIGS. 10A and 10B show a change in thio- shows detection of tau in an SDS-insoluble fraction, flavine T activity by d-isoprenaline, FIG. 10A shows and FIG. 3B is a graph corresponding to FIG. 3A and 10 the effect of inhibiting thioflavine T activity by d-iso- shows that isoprenaline reduces the amount of SDS- prenaline, and FIG. 10B shows the effect of inhibiting insoluble tau. thioflavine T activity by d/l-isoprenaline. [FIG. 4] FIGS. 4A and 4B show the inhibition of tau [FIG. 11] FIGS. 11A and 11B show results of SDS- phopsophorylation by isoprenaline, FIG. 4A shows PAGE western blotting indicating that inhibition of detection of phosphorylated tau (AT8 site) and tau 15 tau aggregation by d-isoprenaline detected by su- in an RIPA-soluble fraction, and FIG. 4B is a graph crose-density gradient centrifugation, FIG. 11A corresponding to FIG. 4A and shows that isoprena- shows results for d-isoprenaline, and FIG. 11B line reduces the amount of phosphorylated tau in the shows results for d/l-isoprenaline. RIPA-soluble fraction. [FIG. 12] FIGS. 12A to 12C show morphological [FIG. 5] FIGS. 5A to 5C show effects of isoprenaline 20 changes of tau aggregation by d-isoprenaline ob- to a tau conformational change (that can be detected served with an atomic force microscope, FIG. 12A with MC1 antibody) observed in AD brain, FIG. 5A is a photograph showing aggregated tau without shows detection of MC1 antibody-labeled tau, tau compound, control, FIG. 12B is a photograph show- phosphorylation (AT8 site), total tau, and GAPDH ing an effect of d-isoprenaline on a tau aggregation (loading control) in a TBS-soluble fraction, FIG. 5B 25 sample, and FIG. 12C is a graph of measured major shows the proportion of MC1 antibody-labeled tau axes of tau aggregations and shows that d-isopren- to total tau, and FIG. 5C shows the proportion of aline reduces the numbers of granular and filamen- phosphorylated tau to total tau. tous tau aggregations. [FIG. 6] FIGS. 6A to 6F show increases in amount [FIG. 13] FIGS. 13A and 13B show a decrease in of tau and acetylated tubulin in microtubule fractions 30 the amounts of sarkosyl-insoluble tau by d-isopren- of isoprenaline, FIG. 6A shows detection of tau in a aline in cerebral cortex of P301L tau Tg mice, FIG. microtubule fraction, FIG. 6B shows detection of tau 13A shows detection of tau in a sarkosyl-insoluble in a total fraction, FIG. 6C is a graph corresponding fraction, and FIG. 13B shows detection of tau and to FIG. 6A and shows that isoprenaline increases GAPDH (loading control) in a TBS-soluble fraction. the amount of tau in the microtubule fraction, FIG. 35 [FIG. 14] FIGS. 14A and 14B show a decrease in 6D shows detection of acetylated tubulin in a micro- amount of sarkosyl-insoluble tau by d-isoprenaline tubule fraction, FIG. 6E shows detection of acetylat- in cerebral cortex of P301L tau Tg mice, FIG. 14A ed tubulin in a total fraction, FIG. 6F is a graph cor- shows the proportion of sarkosyl-insoluble tau to responding to FIG. 6D and shows that isoprenaline TBS-soluble tau, and FIG. 14B shows the proportion increases the amount of acetylated tubulin in the mi- 40 of tau to GAPDH in a TBS-soluble fraction. crotubule fraction. [FIG. 15] FIGS. 15A and 15B show a decrease in [FIG. 7] FIGS. 7A and 7B show decreases in amount amount of sarkosyl-insoluble tau by d-isoprenaline of sarkosyl-insoluble tau in the of isoprenaline in hippocampus of P301L tau Tg mice, FIG. 15A administered mice (P301L tau Tg mice), which over- shows detection of tau in a sarkosyl-insoluble frac- expressing human P301L mutant tau, FIG. 7A shows 45 tion, and FIG. 15B shows detection of tau and GAP- detection of a sarkosyl-insoluble fraction, and FIG. DH (loading control) in a TBS-soluble fraction. 7B is a graph corresponding to FIG. 7A and shows [FIG. 16] FIGS. 16A and 16B show a decrease in that isoprenaline decreases the amount of sarkosyl- amount of sarkosyl-insoluble tau by d-isoprenaline insoluble tau in the brains of P301L tau Tg mice. in hippocampus of P301L tau Tg mice, FIG. 16A [FIG. 8] FIGS. 8A to 8C show that isoprenaline in- 50 shows the proportion of sarkosyl-insoluble tau to hibits a decrease in the numbers of neurons in P301L TBS-soluble tau, and FIG. 16B shows the proportion tau Tg mice, FIG. 8A shows a brain region in which of tau to GAPDH in a TBS-soluble fraction. the number of neurons was counted and also shows a method for counting the number, FIG. 8B shows DESCRIPTION OF EMBODIMENTS that isoprenaline inhibited a decrease in number of 55 cells of the entorhinal cortex, and FIG. 8C shows that [0015] An embodiment of the present invention will be isoprenaline inhibited a decrease in number of cells specifically described with reference to the attached fig- in the temporal cortex. ures. The embodiment below is intended to facilitate un-

4 5 EP 2 764 863 A1 6 derstanding of the principle of the invention. The scope sulfate, nitrate, and phosphate; and organic acid salts of the invention is not limited to the embodiment below, such as formate, acetate, propionate, oxalate, malonate, and includes other embodiments expected by those succinate, fumarate, maleate, lactate, malate, citrate, tar- skilled in the art by making replacements or modifications trate, citrate, carbonate, picrate, methanesulfonate, and to the embodiment when necessary. 5 glutamate. [0016] Inventors of the present invention have inten- [0020] The tau aggregation inhibitor of this embodi- sively investigated, to find for the first time that catechol ment may contain an effective amount of at least one structure-containing compounds are effective for preven- selected from the group consisting of catechol structure- tion or therapy of tauopathies. Based on this finding, the containing compounds and salts thereof, together with a inventors have achieved the invention. A catechol struc- 10 pharmacologically acceptable carrier. The carrier may ture-containing compound herein refers to a compound be a solid such as an excipient or liquid such as a diluent. containing a catechol structure in its constitutional for- Specifically, examples of the carrier include magnesium mula. The catechol structure refers to a structure of cat- stearate, lactose, starch, gelatin, agar, talc, pectin, gum echol that is a compound in which two of its substituents arabic, olive oil, sesame oil, cacao butter, ethylene glycol, are hydroxyl groups and these two hydroxyl groups are 15 and distilled water. in ortho-positions each other. [0021] Tauopathies are neurodegenerative diseases [0017] Specifically, the catechol structure-containing in which accumulation of phosphorylated tau occurs in compound is selected from the group consisting of iso- neuronal cells and glia cells. Tauopathies are, for exam- prenaline, dopamine, dobutamine, levodopa, levo- ple, AD, Down’s syndrome, Pick’s disease, corticobasal dopa/carbidopa, trimetoquinol, hexoprenaline, methyl- 20 degeneration (CBD), and progressive supranuclear pal- dopa, and droxidopa. A tau aggregation inhibitor accord- sy (PSP). ing to this embodiment may be a catechol structure-con- [0022] Prevention of tauopathies means preventing taining compound alone or a combination of some or all occurrence of tauopathy disorder. Therapy of tauopa- of the catechol structure-containing compounds de- thies means preventing or improving/reducing progress scribed above. 25 of tauopathy disorder. [0018] The catechol structure-containing compound is [0023] The tau aggregation inhibitor of this embodi- preferably isoprenaline. Isoprenaline may be preferably ment may contain, if necessary, one or more additives used in any one of l-enantiomer (R-configuration), d- selected from the group consisting of pharmaceutically enantiomer (S-configuration), or d/l-racemic mixture. acceptable tonicity adjusting agents, buffers, solubiliz- Side effects of isoprenaline include palpitation and my- 30 ers, preservatives, and pH adjusters. ocardial ischemia. In isoprenaline, l-enantiomer has [0024] Examples of the tonicity adjusting agents in- greater effects than d-enantiomer, and side effects there- clude potassium chloride, sodium chloride, boric acid, of decrease in the order of l-enantiomer, d/l-racemic mix- mannitol, glycerol, propylene glycol, polyethylene glycol, ture, and d-enantiomer. On the other hand, as described maltose, sucrose, sorbitol, and glucose. below, d-isoprenaline exhibits a tau aggregation inhibi- 35 [0025] Examples of the buffers include organic acids tion effect to a degree similar to that of d/l-isoprenaline. such as and succinic acid, inorganic acids Thus, between optical isomers, d-enantiomer is consid- such as boric acid and phosphoric acid, and pharmaceu- ered to be most preferable for use in a therapeutic agent tically acceptable salts thereof. for dementia because d-enantiomer has a similar tau ag- [0026] Examples of the solubilizers include: polymers gregation inhibition effect but has smaller side effects 40 such as polyethylene glycol, polyvinylpyrrolidone, poly- than l-enantiomer. vinyl alcohol, and hydroxypropyl methylcellulose; sur- [0019] Salts of these catechol structure-containing factants such as polysorbate, polyoxyethylene hydro- compounds are pharmacologically acceptable salts. Ex- genated castor oil, and polyoxyethylene polyoxypropyl- amples of the salts include: inorganic basic salts such as ene; polyhydric alcohol such as propylene glycol; organic metal salts including alkali metal salts (e.g., potassium 45 acids such as benzoic acid and sorbic acid; and amino salt and sodium salt) and alkali-earth metal salts (e.g., acids such as aspartic acid, histidine, glycine, and lysine. magnesium salt and calcium salt), alkali metal carbon- [0027] Examples of the preservatives include: quater- ates (e.g., lithium carbonate, potassium carbonate, so- nary ammonium salts such as benzethonium, benzalko- dium carbonate, and cesium carbonate), alkali metal hy- nium, and benzododecinium; cation compound salts drogen carbonates (e.g., lithium hydrogen carbonate, so- 50 such as chlorhexidine; parahydroxybenzoic acid esters dium hydrogen carbonate, and potassium hydrogen car- such as methyl parahydroxybenzoate and propyl parahy- bonate), and alkali metal hydroxides (e.g., sodium hy- droxybenzoate; and alcohol compounds such as chlo- droxide and potassium hydroxide); organic basic salts robutanol and benzyl alcohol. such as trialkylamine (e.g., and triethyl- [0028] Examples of the pH adjusters include sulfuric amine), pyridine, quinoline, , imidazole, pico- 55 acid, hydrochloric acid, acetic acid, lactic acid, calcium line, dimethylamino pyridine, dimethylaniline, N-alkyl- hydroxide, potassium hydroxide, sodium hydroxide, morpholine, DBN, and DBU; inorganic acid salts such as magnesium hydroxide, monoethanolamine, trieth- hydrochloric acid salt, hydrobromide, hydriodic acid salt, anolamine, diisopropanolamine, and triisopropa-

5 7 EP 2 764 863 A1 8 nolamine. sultant pellets were suspended in an HEPES solution to [0029] The dose of the tau aggregation inhibitor of this obtain a fraction Fr6. Thereafter, binding capacities be- embodiment is not specifically limited as long as appro- tween tau included in Fr 1, 3, and 5 with predetermined priate effects are obtained, and is determined in consid- 6600 compounds were analyzed by a surface plasmon eration of the degree of symptoms, sexuality, and ages 5 resonance technique. The surface plasmon resonance of patients to be treated. For example, the dose of the technique is a technique for analyzing an intermolecular tau aggregation inhibitor can be 0.0001 to 1000 mg per interaction between two molecules by monitoring a day for an adult. This dose of the inhibitor per day may change in refractive index caused by, for example, a be administered once daily or may be divided for several change in molecular mass fixed on a thin gold film. The administrations daily. 10 surface plasmon resonance technique can be performed [0030] The tau aggregation inhibitor of this embodi- with a commercially available surface plasmon reso- ment can be prepared in formulation types in accordance nance system, e.g., BIAcore 2000 (produced by Phar- with the manner of administration. Examples of oral ad- macia Biosensor). As a result, it was found that 111 com- ministration types include solid formulations and solution pounds out of the 6600 compounds were bound to tau. formulations including granules, balls, tablets, capsules, 15 [0033] Then, it was analyzed, by thioflavine T stain, powders, and solutions. Examples of parenteral admin- whether the 111 compounds inhibit tau aggregation. For istration types include injections such as intravenous in- this analysis, 10-mM tau, a compound (1 mM, 10 mM, or jection and intramuscular injection. 100 mM), and thioflavine T (a beta-pleated sheet struc- [0031] When phosphorylated, tau-tau association oc- ture-specific detection reagent) were mixed together. curs and tau oligomers are formed. When these tau oli- 20 Thereafter, heparin, which is a tau aggregation inducer, gomers grow to have a beta-pleated sheet structure, was added to the mixture, and the resulting mixture was spherical granular tau aggregation is formed. The gran- incubated at 37°C, thereby allowing tau to aggregate. ular tau aggregation is considered to be constituted by Subsequently, the thioflavine T activity in an incubation about 40 tau molecules. The granular tau aggregations sample was measured at various times to investigate tau are joined together to form neurofibrillary tangles (NFTs) 25 aggregation inhibition effects by the compounds. As a called paired helical filaments (PHFs). Recent research- result, it was observed that nine out of the 111 com- es using mouse models shows that inhibition of tau over- pounds noticeably inhibited the thioflavine T activity in a expression in the period of NFT formation improves low concentration of 1 mM. memory learning of mice but formation of NFTs contin- [0034] To further investigate the effects of these nine ues. This suggests that neuronal dysfunction occurs30 compounds on tau aggregation specifically, samples that mainly in the process of formation of NFTs, rather than had been incubated at 37°C were centrifuged at 250000 being caused by NFTs themselves. NFTs themselves xg for 2 h, the resultant pellets were obtained so that the are not toxic, and products formed in the process of NFT amount of insoluble tau were quantified. As a result, it formation is considered to be a major cause of neurotox- was found that (R)-(-)-epinephrine and pyrocatechol vi- icity. The tau aggregation inhibitor of this embodiment 35 olet reduced the amount of insoluble tau in a concentra- inhibits not only tau aggregation in the process of PHF tion dependent manner. These two compounds, i.e., formation by joined granular tau aggregation but also tau (R)-(-)-epinephrine and pyrocatechol violet, were found aggregation in the process of formation of spherical gran- to have the same skeleton of catechol nucleus. ular tau aggregations. Neurodegeneration in brain oc- curs by not only accumulation of mutant tau protein but 40 (Example 2) also accumulation of wild-type tau. The tau aggregation inhibitor of the present invention can inhibit aggregation [0035] Then, it was analyzed, by thioflavine T stain, of wild-type tau. Thus, tauopathy symptoms including AD whether compounds whose structures resemble to can be prevented or treated. (R)-(-)-epinephrine and pyrocatechol violet inhibit tau ag- 45 gregation. As a result, as shown in FIG. 1, levodopa, [Examples] dopamine, norepinephrine, and isoprenaline significantly reduced thioflavine T activity, similar to (R)-(-)-epine- (Example 1) phrine. FIG. 1A shows a change in thioflavine T activity by (R)-(-)-epinephrine, FIG. 1B shows a change in thio- [0032] Compounds that can be bound to tau were50 flavine T activity by levodopa, FIG. 1C shows a change screened. First, 10 mM of 2N4R tau (TAU-441 HUMAN) in thioflavine T activity by dopamine, FIG. 1D shows a and 10 mM of heparin were mixed together and incubated change in thioflavine T activity by norepinephrine, and at 37°C to form tau aggregates. This aggregated tau sam- FIG. 1E shows a change in thioflavine T activity by iso- ple (1 ml) was loaded onto a sucrose-density gradient prenaline. solution (consisting of 1 ml layers 20%, 30%, 40%, and 55 [0036] Among these samples, the (R)-(-)-epinephrine 50%), and was centrifuged (at 200000 xg for 2 h at 20°C). sample and the isoprenaline samples were divided into Then, the solution was collected in units of 1 ml from the fractions Fr1-Fr6 by sucrose-density gradient centrifuga- top layer to obtain samples of fractions (Fr) 1-5. The re- tion, and tau was detected by SDS-PAGE western blot-

6 9 EP 2 764 863 A1 10 ting. The concentration of the compounds used was 100 out separation through electrophoresis and specifically mM. As a result, as shown in FIG. 2, aggregated tau was quantitating the protein amount with enzyme-labeled an- detected in fractions Fr3, 4, 5, and 6 in Control, whereas tibodies. As a result, as shown in FIGS. 5A and 5B, iso- 100-mM of compounds reduced the amount of tau in frac- prenaline reduced the tau conformational change detect- tions Fr3, 4, 5, and 6. FIG. 2A shows tau aggregation 5 ed with the MC1 antibody. FIG. 5A shows detections of inhibition effects of (R)-(-)-epinephrine, and FIG. 2B MC1 antibody-positive tau, tau phosphorylations (AT8 shows tau aggregation inhibition effects of isoprenaline. sites), total tau, and GAPDH (loading control) in a TBS- From the foregoing results, a novel common structure soluble fraction. FIG. 5B shows the proportion of MC1 that inhibits tau aggregation in vitro was found. Among antibody-positive tau with respect to total tau. As shown these compounds, isoprenaline is an existing drug, and 10 in FIG. 5C, isoprenaline also reduced phosphorylation is considered to have higher extents of safety than other under similar conditions. FIG. 5C shows the proportion drugs of . Thus, isoprenaline was used of phosphorylated tau with respect to total tau. in the following analyses. (Example 5) (Example 3) 15 [0040] It was investigated how isoprenaline affects [0037] Then, it was investigated whether isoprenaline binding between microtubules and tau. First, 10m M of inhibits tau aggregation in cultured cells. As cells, isoprenaline was added to COS-7 cells expressing WT Neuro2a cell lines in which human P301L mutant tau tau, and the cells were left for 24 hours and then homog- 20 (i.e., mutant tau in which 301st proline of tau is changed enized with RA buffer (0.1- mM MES, 0.5-mM MgSO 4, 1- to leucine) is expressed in stable were used. To these mM EGTA, 2-mM DTT, 0.1% TritonX-100, 20-mM taxol, cells, isoprenaline was added in concentrations of 0.01, and 2-mM GTP). The homogenates were then centri- 0.1,and 1 mM for48 hours. Then,SDS-insoluble fractions fuged at 3000 xg for 5 min at 25°C, and the supernatant were obtained so that a change in the amounts of tau was obtained as a total fraction. The total fraction was was observed. As a result, as shown in FIG. 3, isopren- 25 further centrifuged at 100000 xg for 20 min at 20°C, and aline reduced the amounts of SDS-insoluble tau similarly a pellet(a microtubule fraction) was obtained. Inthis man- to lithium chloride, a positive control, which is a glycogen ner, the microtubule fraction was taken from the COS-7 synthase kinase 3β (GSK3β) inhibitor (e.g., a material cells expressing WT tau, and acetylated tubulin serving that physically or chemically inhibits the function of as an index of tau and microtubule stabilization was de- GSK3β). FIG. 3A shows detection of tau in SDS-insoluble 30 tected. fractions, and FIG. 3B is a graph corresponding to FIG. [0041] As a result, as shown in FIG. 6, in WT tau-ex- 3A, and shows that isoprenaline reduces the amount of pressing COS-7 cells, 10- mM isoprenaline increased the SDS-insoluble tau. amount of tau in the microtubule fraction. FIG. 6A shows [0038] In addition, changes of tau phosphorylation in detectionof tau inthe microtubule fraction. FIG. 6Bshows radio-immunoprecipitation assay (RIPA) buffer-soluble 35 detection of tau in the total fraction. FIG. 6C is a graph fractions obtained from similar cells were analyzed. As corresponding to FIG. 6A. a result, as shown in FIG. 4, it was found that isoprenaline [0042] In the WT tau-expressing COS-7 cells, the reduced tau phosphorylation labeled by anti-phosphor- amount of acetylated tubulin increased as compared to ylated tau antibody (AT8). The composition of the RIPA vector-expressing cells. Addition of isoprenaline to these buffer was 50mM Tris-HCl (pH7.4), 150mM sodium chlo- 40 cells further increased the amount of acetylated tubulin, ride, 0.25 w/v% sodium deoxycholate, 1mM EGTA, and as shown in FIGS. 6D, 6E, and 6F. Thus, the results 1.0 w/v% NP-40 substitute. Thus, it was found that iso- suggest the possibility that isoprenaline stabilizes of mi- prenaline also inhibits tau phosphorylation as well as tau crotubules by increasing the amount of tau bound to the aggregation. FIG. 4A shows detection of phosphorylated microtubules. FIG. 6D shows acetylated tubulin in the tau (AT8 site) and total tau in RIPA-soluble fractions, and 45 microtubule fraction. FIG. 6E shows acetylated tubulin in FIG. 4B shows proportions of phosphorylated tau to total the total fraction. FIG. 6F is a graph corresponding to tau. FIG. 6D.

(Example 4) (Example 6) 50 [0039] Tau phosphorylations at AT8 sites are known [0043] It wasinvestigated whetherisoprenaline inhibits to induce a tau conformational change (that can be de- tau aggregation in mice. First, P301L tau Tg mice were tected with the MC1 antibody) observed in AD brains. given isoprenaline (1.5 mg/g fed) mixed in mash for three Thus, WT tau was expressed in COS-7 cells (derived months. Then, cerebral cortex and hippocampus were from African green monkey ), and a tau conforma- 55 excised from the mice, and stored at -80°C. To obtain tional change was detected by dot blotting using the MC1 fractions including soluble tau and insoluble tau from antibody. The dot blotting is a technique of fixing protein these tissues, a frozen tissues were homogenized in TBS to a nitrocellulose membrane or a PVDF membrane with- solution, centrifuged (at 23000 rpm for 15 min at 4°C),

7 11 EP 2 764 863 A1 12 and then fractionated into the supernatant and the pellet. (see Example 2) and dopamine, dobutamine, levodopa, The supernatant was used as a TBS-soluble fraction (in- levodopa/carbidopa, trimetoquinol, hexoprenaline, cluding soluble tau). In addition, 0.32M of sucrose was methyldopa, and droxidopa have catechol amine struc- added to the pellet, and the pellet was homogenized tures similar to that of isoprenaline, these materials ap- again, centrifuged (at 23000 rpm for 15 min at 4°C), and 5 pear to have the effect of inhibiting tau aggregation in then fractionated into the supernatant (including tau ag- cultured cells and animals, similarly to isoprenaline. gregation) and the pellet (including nuclei). Thereafter, surfactant (1% sarkosyl) was added to the supernatant, (Example 9) and the resulting supernatant was incubated (at 37°C for 1 h) and centrifuged (at 200000 xg for 1 h at 4°C). A pellet 10 [0047] Tau (10 mM), d- and d/l-isoprenaline (1-100 dissolved with Laemmli buffer (containing 2-mercap- mM), and thioflavine T were mixed together. Then, toethanol) was used as a sarkosyl-insoluble fraction. Tau heparin was added to the mixture, and the resulting mix- in the TBS-soluble fraction and the sarkosyl-insoluble ture was incubated at 37°C, thereby allowing tau to ag- fraction was detected by SDS-PAGE western blotting. gregate. In the period shown in the figures, the thioflavine As a result, as shown in FIG. 7, isoprenaline reduced the 15 T activity in incubation samples were analyzed to inves- amount of sarkosyl-insoluble tau in the brains of the tigate tau aggregation inhibition effects of compounds. P301L tau Tg mice. FIG. 7A shows detection of tau in FIG. 10A is a graph showing the effect of d-isoprenaline the sarkosyl-insoluble fraction, and FIG. 7B is a graph in on the thioflavine T activity. FIG. 10B is a graph showing which the result of FIG. 7A is quantitated. In the graph, the effect of d/l-isoprenaline on the thioflavine T activity. Ntg means Non-transgenic mice. 20 As shown in FIGS. 10A and 10B, d-isoprenaline exhibited an inhibition of thioflavine T activity similar to that of d/l- (Example 7) isoprenaline. [0048] Then, to analyze a change of tau aggregation [0044] In the brains of P301L tau Tg mice, the numbers by d- and d/l-isoprenaline biochemically, an incubated of neurons are decreased accompanying tau aggregates 25 tau aggregation sample was fractionated into Fr1-Fr6 by formation. Thus, there is the possibility that isoprenaline sucrose-density gradient centrifugation, and tau was de- having a tau aggregation inhibition function can suppress tected by SDS-PAGE western blotting. FIG. 11 shows decreases in the number of neuronal cells. Thus, brain results of SDS-PAGE western blotting indicating tau ag- slices were prepared from isoprenaline-administered gregation inhibition effects detected by sucrose-density mice, and the number of neuronal cells was counted. To 30 gradient centrifugation. FIG. 11A shows results of d-iso- measure the number of neuronal cells, 0.1-mm2 boxes prenaline,and FIG. 11B shows resultsof d/l-isoprenaline. as shown in FIG. 8A were drawn in entorhinal cortex or As shown in FIGS. 11A and 11B, similarly to d/l-isopre- temporal area, and the number of cells in each of the naline, d-isoprenaline apparently reduced the amount of boxes was counted. Then, the average number of cells tau (granular and filamentous tau aggregates) fraction- was used as a number for one slice. Two slices were 35 ated into Fr3 and subsequent fractions. prepared from one individual. As a result, as shown in [0049] Thereafter, to analyze a change of tau aggre- FIGS. 8B and 8C, isoprenaline suppressed a decrease gation by d-isoprenaline morphologically, the sample in number of cells in entoehinal cortex and temporal area was investigated by using an atomic force microscope. as indicated in the P301L tau Tg mice. This suggests that A tau aggregation sample that had been incubated for isoprenaline can suppress a decrease in number of neu- 40 120 hours was loaded on a mica board and adsorbed ronal cells by inhibiting tau aggregation. In FIGS. 8B and thereon. After removal of the sample, the mica board was 8C, "Mice" refers to the number of mice used, and "Slice" filled with milliQ water, and the aggregated tau was ob- refers to the number of slices per one individual in which served with an atomic force microscope. FIG. 12 shows the number of cells was counted. morphological change of tau aggregation observed with 45 an atomic force microscope. FIG. 12A is a photograph (Example 8) of a control, FIG. 12B is a photograph showing effects of d-isoprenaline on a tau aggregation sample, and FIG. [0045] A change in tau phosphorylation in TBS-soluble 12C is a graph of measured major axes of tau aggrega- fractions from WT tau Tg mice was investigated. As a tion. As shown in FIGS. 12A, 12B, and 12C, d-isopren- result, as shown in FIG. 9, isoprenaline induced dephos- 50 aline apparently reduced the number of tau aggregates phorylation of tau labeled by monoclonal antibody tau1. (granular and filamentous tau aggregates) having major FIG. 9A shows a TBS-soluble fraction, and FIG. 9B axes of 20 nm or more. shows the proportion of dephosphorylated tau to tau. The [0050] To investigate tau aggregation inhibition effects results suggest that isoprenaline also inhibits tau aggre- of d-isoprenaline in vivo P301L tau Tg mice were used gation and phosphorylation of total tau in mice. 55 for analysis. First, P301L tau Tg mice aged 20-21 months [0046] The above examples show effects of isoprena- were given d-isoprenaline (2.168 mg/g fed) mixed in line. However, since a catechol amine moiety was found mash for three months. Then, cerebral cortices and hip- as a novel common structure that inhibits tau aggregation pocampi were excised from the mice and stored at -80°C.

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Thereafter, a TBS-soluble fraction and a sarkosyl-insol- 5. The tau aggregation inhibitor of claim 1, further in- uble fraction were prepared from these brain tissues, and cluding at least one additive selected from the group tau was analyzed by SDS-PAGE western blotting. consisting of a pharmaceutically acceptable tonicity [0051] FIG.13 shows decreases in amount of sarkosyl- adjusting agent, a buffer, a solubilizer, a preserva- insoluble tau in cerebral cortices of d-isoprenaline-treat- 5 tive, and a pH adjuster. ed P301L tau Tg mice. FIG. 13A shows detection of tau in the sarkosyl-insoluble fraction. FIG. 13B shows detec- 6. A tau aggregation inhibitor for use in prevention tion of tau in the TBS-soluble fraction and GAPDH in a and/or treatment of tauopathy, wherein loading control. FIG. 14 shows decreases in amount of the tau aggregation inhibitor includes a catechol sarkosyl-insoluble tau by d-isoprenaline in cerebral cor- 10 structure-containing compound or a salt thereof, and tex of P301L tau Tg mice. FIG. 14A shows the proportion the catechol structure-containing compound is se- of sarkosyl-insoluble tau to TBS-soluble tau. FIG. 14B lected from the group consisting of dopamine, dob- shows the proportion of tau to GAPDH in the TBS-soluble utamine, levodopa, levodopa/carbidopa, trimetoqui- fraction. As shown in FIGS. 13 and 14, d-isoprenaline nol, hexoprenaline, methyldopa, and droxidopa. apparently reduced the amount of insoluble tau in cere- 15 bral cortices of P301L tau Tg mice. [0052] In a manner similar to the above-described ex- amination ofthe amount ofinsoluble tau incerebral cortex of P301L tau Tg mice, the amount of insoluble tau in hippocampus of P301L tau Tg mice were obtained. FIG. 20 15 shows decreases in amount of sarkosyl-insoluble tau by d-isoprenaline in hippocampus of P301L tau Tg mice. FIG. 15A shows detection of tau in a sarkosyl-insoluble fraction. FIG. 15B showsdetection of tauin a TBS-soluble fraction and GAPDH in a loading control. FIG. 16 shows 25 decreases in amount of sarkosyl-insoluble tau by d-iso- prenaline in hippocampus of P301L tau Tg mice. FIG. 16A shows the proportion of sarkosyl-insoluble tau to TBS-soluble tau. FIG. 16B shows the proportion of tau to GAPDH in the TBS-soluble fraction. As shown in FIGS. 30 15and 16,d-isoprenaline apparently reducedthe amount of insoluble tau in hippocampi of P301L tau Tg mice.

INDUSTRIAL APPLICABILITY 35 [0053] The present invention is useful for treatment of tauopathies.

Claims 40

1. A tau aggregation inhibitor for use in prevension and/or treatment of tauopathy, the tau aggregation inhibitor including isoprenaline or a salt thereof. 45 2. The tau aggregation inhibitor of claim 1, wherein iso- prenaline included in the tau aggregation inhibitor is d-enantiomer.

3. The tau aggregation inhibitor of claim 1, wherein iso- 50 prenaline included in the tau aggregation inhibitor is d/l-racemic mixture.

4. The tau aggregation inhibitor of claim 1, wherein tauopathy is one of AD, Down’s syndrome, Pick’s 55 disease, corticobasal degeneration (CBD), or pro- gressive supranuclear palsy (PSP).

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• JP 2004534854 PCT [0008]

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