USE OF VINPOCETINE DERIVATIVES FOR INHIBITING PRODUCTION OR SECRETION OF AMYLOID BETA PROTEIN

Abstract: A pharmaceutical composition for inhibiting production or secretion of amyloid beta protein, which comprises a compound of formula (I), wherein ring A is an optionally substituted benzene ring, R represents OR<1>, (a) or SR<1>, wherein R<1>, R<2> and R<3> are the same or different and each is selected from a hydrogen atom, an optionally substituted hydrocarbon group or R<2> and R<3>, taken together with the adjacent nitrogen atom, form an optionally substituted nitrogen-containing heterocyclic group, and Y is an optionally substituted alkyl group, or a pharmaceutically acceptable salt thereof, if necessary, with a pharmaceutically acceptable excipient, carrier or diluent.

Description: USE OF VINPOCETINE DERIVATIVES FOR INHIBITING PRODUCTION OR SECRETION OF AMYLOID BETA PROTEIN TECHNICAL FIELD The present invention relates to a composition, particularly a pharmaceutical composition, for inhibiting production or secretion of an amyloid β protein. The composition is effective in treating degenerative brain disorders such as senile dementia, Alzheimer's disease, Down's syndrome, etc., amyloid angiopathy, brain disorders caused by amyloid β protein in cerebrovascular diseases, etc. BACKGROUND OF THE INVENTION Alzheimer's disease is a neurodegenerative disease characterized by senile plague and neurofibrillar tangles as well as degeneration and loss of neurons. The senile plagues, which are most characteristic of the disease, are deposits composed of amyloid β protein (hereinafter sometimes abbreviated as Aβ) derived from β-amyloid precursor protein (hereinafter sometimes abbreviated as βAPP) as a major component (Biochem. Biophys. Res. Commun., 122, 1131 (1984)), apolipoprotein E (Brain Res., 541, 163 (1991)) and heparan sulfate proteoglycan (Am. J. Pathol., 133, 456 (1988)), etc. Aβ of 40 or 42 amino acids exhibits toxicity to neurons (Science, 250, 279 (1990)), and induces neurofibrillar changes (Proc. Natl. Acad. Sci., 90, 7789 (1993)). patients with familial Alzheimer's disease, mutations on t βAPP gene are observed (Lancet, 337, 978 (1991); Nature, 34 704 (1991); Science, 254, 97 (1991); Nature, 354, 844 (1991) Cells containing such mutated gene produce and secrete increased amount of Aβ (Nature, 360, 672 (1992); Science, 25 514 (1993); Science, 264, 1336 (1994)). Patients with Down's syndrome showing cerebr changes pathologically similar to those of Alzheimer's disea (Proc. Natl. Acad. Sci., 82, 4245 (1985)) have trisomy (i.e., three chromosome 21s containing the βAPP gene) a promoted expression of the βAPP gene and βAPP protein in t brain (Science, 235, 880 (1987); N. Eng. J. Med., 320, 14 (1989)). Based on these findings, the participation of t abnormal production or secretion of Aβ in the onset Alzheimer's disease is considered to be highly important. It is believed that βAPP is metabolized wi proteases called - and β-secretases through two pathwa (Science, 248, 492 (1990); Science, 248, 1122 (1990); Science 255, 726 (1992); Science, 255, 728 (1992); Nature, 357, 50 (1992)). When βAPP is metabolized with α-secretase, βAPP i cleaved at position 16 of Aβ, Aβ is not produced, and the N ter inal fragment of βAPP is released out of the cells a secreted βAPP, which acts as a neurotrophic factor (Neuron 10, 243-254 (1993)). On the other hand, when βAPP is cleaved with β-secretase, a C-terminal fragment of βAPP containing Aβ is produced. However, it is unclear where the C-terminal fragment or Aβ is produced in the cells and how Aβ is secreted out of the cells. In addition, although candidate enzymes for α- and β-secretases have been reported (J. Biol. Chem., 268, 16699 (1993); Biochemistry, 33, 192 (1994)), they have not identified. It has been reported that some compounds, phorbol esters (Proc. Natl. Acad. Sci., 89, 3055 (1992)) and Ml muscarinic receptor agonist, carbacol (Science, 258, 302 (1992)) increase the secreted βAPP and inhibit Aβ production or secretion in in vitro various cells. However, these compounds are unsatisfactory in terms of efficacy, safety, etc. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing combinations of antibodies used in the EIA system. HRP means horseradish peroxidase. Fig. 2 shows a standard curve of the EIA system against Aβ (1-40) and Aβ (1-42). Fig. 3 represents graphs showing inhibitory activity of vinpocetine (i.e. the compound of the formula (II) wherein X is hydrogen and R* is ethyl) against Aβ production or secretion in human neuroblastoma IMR-32 cells. Each bar gra indicates the mean value with respect to 4 wells. Fig. 4 is a graph showing inhibitory activity vinpocetine against Aβ production or secretion in guinea p primary culture neurons. Each bar graph indicates the me value with respect to 4 wells. DISCLOSURE OF THE INVENTION The present inventors have intensively studied find an inhibitor of Aβ production or secretion. As a resul it has been found that vinpocetine (CALAN ) or a derivati thereof has inhibitory activity against Aβ production secretion. Vinpocetine or its derivative is a known compou disclosed in JP-B 51-32640 (U.S. Patent No. 4035370) and JP 2-27352 (U.S. Patent No. 4400514). However, the publications only disclose cerebro-vasodilative, systemi vasodilative and hypotensive activities of the compound, a fails to teach or suggest inhibitory activity against production or secretion. The present invention provides a pharmaceutic composition for inhibiting production or secretion of amylo β protein, particularly for preventing or treating Alzheimer' disease, which comprises a compound of the formula (I): wherein ring A is an optionally substituted benzene ring, R represents OR 1 , R 2 - or SR 1 , N< . ) wherein R 1 , R and R 3 are the same or different and each is selected from a hydrogen atom, an optionally substituted hydrocarbon group or R 2 and R 3 , taken together with the adjacent nitrogen atom, form an optionally substituted nitrogen-containing heterocyclic group, and Y is an optionally substituted alkyl group, or a pharmaceutically acceptable salt thereof, if necessary, with a pharmaceutically acceptable excipient, carrier or diluent. "Hydrocarbon group" of the term "optionally substituted hydrocarbon group" used in this specification include, among others, aliphatic hydrocarbon groups, monocyclic saturated hydrocarbon groups and aromatic hydrocarbon groups. The carbon number of the hydrocarbon group is preferably 1 to 16. An alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group and an aryl gro are exemplified. "Alkyl group" is preferably a lower alkyl group, f example, Cj. 6 alkyl groups such as methyl, ethyl, propy isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl a hexyl are used. "Alkenyl group" is preferably a lower alkenyl grou for example, C 2 . 6 alkenyl groups such as vinyl, 1-propeny allyl, isopropenyl, butenyl and isobutenyl are used. "Alkynyl group" is preferably a lower alkynyl grou for example, C 2 . 6 alkynyl groups such as ethynyl and 1-propyn are used. "Cycloalkyl group" is preferably a lower cycloalky group, for example, C 3 -. 6 cycloalkyl groups such as cyclopropyl cyclobutyl, cyclopentyl and cyclohexyl are used. "Aryl group" is preferably C 6 -. aryl groups such a phenyl, xylyl, 1-naphthyl, 2-naphthyl, biphenylyl, 2-indeny and 2-anthlyl. Among others, a phenyl group, for example, i used. Examples of the substituents, which "hydrocarbo group" of "optionally substituted hydrocarbon group" ma optionally have, include halogen atoms, (e.g. fluorine chlorine, bromine and iodine), a nitro group, a cyano group a hydroxyl group, optionally halogenated C*. 6 alkyl group (e.g. methyl, chloromethyl, difluoromethyl, trichloromethyl trifluoromethyl, ethyl, 2-bromoethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, propyl, 3,3,3-trifluoropropyl, isopropyl, butyl, 4,4,4-trifluorobutyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 5,5,5-trifluoropentyl, hexyl and 6,6,6-trifluorohexyl) , lower alkoxy groups (e.g. C*^ alkoxy groups such as methoxy, ethoxy, propoxy, isopopoxy, butoxy, isobutoxy, pentyloxy and hexyloxy) , an amino group, mono-lower alkylamino groups (e.g. mono-Cj. o alkylamino groups such as methylamino and ethylamino), di-lower alkylamino groups (e.g. alkylamino groups such as dimethylamino and diethylamino) , a carboxyl group, lower alkylcarbonyl groups (e.g. Cj. 6 alkyl-carbonyl groups such as acetyl and propionyl), lower alkoxycarbonyl groups (e.g. Cχ. 6 alkoxy-carbonyl groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl and butoxycarbonyl) , a carbamoyl group, mono-lower-alkylcarbamoyl groups ( e . g . mono-C* , -. 6 alkylcarbamoyl groups such as methylcarbamoyl and ethylcarbamoyl) , di-lower alkylcarbamoyl groups (e-g- alkylcarbamoyl groups such as dimethylcarbamoyl and diethylcarbamoyl) , arylcarbamoyl groups (e»g- C 6 -ιo arylcarbamoyl groups such as phenylcarbamoyl and naphthylcarbamoyl), aryl groups (e.g. C 6 . 10 aryl groups such as phenyl and naphthyl) and aryloxy groups (e.g. C 6 . 10 aryloxy groups such as phenyloxy and naphthyloxy) , optionally halogenated lower alkylcarbonylamino groups (e.g. optionally halogenated Cj_ 6 alkyl-carbonylamino groups such acetylamino, trifluoroacetylamino) . "Hydrocarbon group" "optionally substituted hydrocarbon group" may optionally ha 1 to 5, preferably 1 to 3, of these substituents. When t number of the substituents is two or more, they may be t same one or different from one another. Examples of "nitrogen-containing heterocyclic grou formed by R 2 , R 3 and the adjacent nitrogen atom include 4- 8-membered heterocyclic groups each of which contains at lea one nitrogen atom and may contain 1 to 3, preferably 1 to oxygen atoms, sulfur atoms, etc. in addition to carbon ato as the ring-constituting atoms, and their benzo-condens groups. Specific examples of the nitrogen-containi heterocyclic groups include aromatic heterocyclic groups suc as 1-pyrrolyl, 1-imidazolyl, 1-indolyl, 1-pyrazolyl, 2 isoindolyl, 1-indazolyl, etc.; cyclic amino groups such a morpholino, piperidino, 1-piperazinyl optionally having substituent on the nitrogen atom at the 4-position, 1 pyrrolidinyl, 1-pyrazolidinyl, 1-azepinyl, etc.; or thei benzo-condensed groups (e.g. 1-indolinyl, 2-isoindolinyl 1,2,3 , 4-tetrahydroquinolin-l-yl, 1,2,3 ,4-tetrahydro isoquinolin-2-yl, 3-benzazepin-3-yl) ; lactam or i ido group such as phthalimido, succinimido, 2-pyrrolidon-l-yl, 2 pyridon-1-yl, 2-quinolon-l-yl, etc. "Nitrogen-containing heterocyclic group" formed by R , R and the adjacent nitrogen atom may have the same substituents as those described above for "optionally substituted hydrocarbon group" . The benzo-condensed group may have one or more substituents at any possible position on the benzene ring. The substituents are selected from halogen atoms (e.g. fluorine, chlorine, bromine, iodine, etc.), lower alkylendioxy groups (e.g. C 1 alkylenedioxy groups such as methylenedioxy, ethylenedioxy, etc.), a nitro group, a cyano group, optionally halogenated lower alkyl groups (e.g. optionally halogenated C^ alkyl groups such as chloromethyl, difluoromethyl, trichloromethyl, trifluoro ethyl, ethyl, 2- bromoethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, propyl, 3,3,3-trifluoropropyl, isopropyl, butyl, 4,4,4-trifluorobutyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 5,5,5-trifluoropentyl, hexyl and 6,6,6-trifluorohexyl) , optionally halogenated lower alkoxy groups (e.g. optionally halogenated Cι_ 6 alkoxy groups such as chloromethoxy, difluoromethoxy, trichloromethoxy, trifluoromethoxy, ethoxy, 2-bromoethoxy, 2,2,2-trifluoroethoxy, pentafluoroethoxy, propoxy, 3,3,3-trifluoropropoxy, isopropoxy, butoxy, 4,4,4- trifluorobutoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy, 5,5,5- trifluoropentyloxy, hexyloxy and 6,6,6-trifluorohexyloxy) , optionally halogenated lower alkylthio groups (e.g. optionally halogenated C^ alkylthio groups such as chloromethylthi difluoromethylthio, trichloromethylthio, trifluoromethylthi ethylthio, 2-bromoethylthio, 2,2,2-trifluoroethylthi pentafluoroethylthio, propylthio, 3,3,3-trifluoropropylthi isopropylthio, butylthio, 4,4 ,4-trifluorobutylthi isobutylthio, sec-butylthio, tert-butylthio, pentylthi isopentylthio, neopentylthio, 5,5,5-trifluoropentylthi hexyl hio and 6,6,6-trifluorohexylthio) , a hydroxyl group, amino group, mono-lower alkylamino groups (e.g. mono-Cj alkylamino groups such as methylamino, ethylamin propylamino, isopropylamino, butylamino, etc.), di-low alkylamino groups (e.g., di-C^ alkylamino groups such dimethylamino, diethylamino, dipropylamino, dibutylamin etc.), a carboxyl group, lower alkoxycarbonyl groups (e.g. C- , , 6 alkoxycarbonyl groups such as methoxycarbonyl ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, etc. ) and carbamoyl group. "Alkyl group" of the term "optionally substitute alkyl group" used in this specification includes, for example C j .-.-. j alkyl groups such as methyl, ethyl, propyl, isopropyl butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl dodecyl, tridecyl, tetradecyl, pentadecyl, preferably lowe (C L6 ) alkyl groups such as methyl, ethyl, propyl, isopropyl butyl, isobutyl, sec-butyl and tert-butyl. The alkyl grou may optionally have 1 to 3 substituents which, for example, the above-mentioned "hydrocarbon group" may optionally have. The term "optionally substituted benzene ring" used in this specification means a benzene ring which optionally have, at any possible position, one to three substituents selected from halogen atoms (e.g. fluorine, chlorine, bromine and iodine), optionally substituted hydrocarbon groups, optionally substituted hydroxyl groups (preferably, optionally substituted lower (Cj. 6 ) alkoxy groups such as methoxy, ethoxy, propoxy and isopropoxy), a hydroxyl group, an optionally substituted amino group, amido groups (e.g. C _ 6 acylamino groups such as acetamido), and lower alkylenedioxy groups (e.g. Ci.β alkylenedioxy groups such as methylenedioxy, ethylenedioxy) . The term "optionally substituted amino group" used in this specification means an amino group which may optionally have, as the substituents, one or two of the above- mentioned "optionally substituted hydrocarbon group" for example. Preferable examples of the substituents, which this "amino group" may optionally have, include an optionally substituted Cι_ 6 alkyl group and an optionally substituted C 6 . 10 aryl group. The substituents of the alkyl or aryl group are, for example, the same substituents which above-mentioned "hydrocarbon group" may optionally have. The term "optionally substituted hydroxyl grou used in this specification means a hydroxyl group which m have, in place of the hydrogen atom of the hydroxyl group, o "optionally substituted hydrocarbon group" mentioned abov preferable examples of "substituted hydroxyl group" inclu a hydroxyl group substituted with one lower alkyl group Examples of the "lower alkyl group" include C 6 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl sec-butyl and tert-butyl. The substituents which "lower alky group" may optionally have are, for example, the same ones a the above-mentioned "hydrocarbon group" may optionally have The "optionally substituted hydrocarbon group includes the same ones as those mentioned above. When th number of the substituents is two or more, they may be th same one or different from one another. In the above formulae, ring A is an optionall substituted benzene ring. Preferable examples of ring include an unsubstituted benzene ring or benzene rings havin 1 to 3 substituents selected from halogen atoms (e.g. fluorin and chlorine), Cj_ 6 alkyl groups (e.g. methyl and ethyl), C-._ alkoxy groups (e.g. methoxy and ethoxy) which may have a C 6 . 1 aryl group, hydroxyl group and mono-C-._.,-•, alkylamino group especially preferable one being, for example, a benzene rin substituted with one, for example, C* . _ 6 alkoxy group (e.g methoxy) . Among others, ring A is preferably an unsubstituted benzene ring or benzene rings having 1 to 3 halogen atoms (e.g. fluorine and chlorine). More preferably, ring A is a benzene ring. In the above formulae, R represents OR 1 , R 2 - λ N< i R 3 ^ , or SR , wherein R , R and R are the same or different and each is selected from a hydrogen atom, an optionally substituted hydrocarbon group or R 2 and R 3 , taken together with the adjacent nitrogen atom, • form an optionally substituted nitrogen-containing heterocyclic group. Preferable examples of "hydrocarbon group" shown by R , R and R include alkyl groups (e.g., C* , . 6 alkyl groups such as methyl, ethyl, propyl and isopropyl), alkenyl groups (e.g. C 2 _ 6 alkenyl groups such as vinyl), alkynyl groups (e.g. C 2 -β alkynyl groups such as ethynyl), cycloalkyl groups (e.g. C 3 . 6 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl) and aryl groups (e.g. C 6 _ι « aryl groups such as phenyl). Among others, alkyl groups (e.g. C-._ 6 alkyl groups such as methyl) and cycloalkyl groups (e.g. C 3 . 6 cycloalkyl groups such as cyclopropyl) are preferably used. The "alkyl group", "alkenyl group", "alkynyl group", "cycloalkyl group", and "aryl group" may optionally have, for example, 1 to 5, preferably 1 to 3 substituents, which the above-mentioned "hydrocarbon group" may optionally have, (preferably halog atoms such as fluorine). A preferable example of R is OR 1 wherein R 1 is alkyl group, more preferably, R is ethyl. In the above formulae, Y is an optional substituted alkyl group. Preferable examples of Y are low (C LS ) alkyl groups (e.g. methyl, ethyl, propyl, isopropy butyl, isobutyl, sec-butyl and tert-butyl). More preferab examples of Y include ethyl. Preferable examples of the compound (I) include t compound of the formula (II): wherein X is hydrogen or halogen and R is alkyl or pharmaceutically acceptable salt thereof, and the preferabl compounds may be, if necessary, used with a pharmaceuticall acceptable excipient, carrier or diluent. The halogen represented by X in the formula (II includes fluorine, chlorine, bromine, and iodine. The alk represented by R* includes, for example, alkyl having 1 to carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, and hexyl. Methyl and ethyl are preferred. In particular, vinpocetine, i.e. the compound of the formula (II) wherein X is hydrogen and R* is ethyl, is preferred. The pharmaceutically acceptable salts of the compound of the formula (I) include, for example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid, salts with organic acids such as formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p- toluenesulfonic acid. The compound of the formula (I) or a pharmaceutically acceptable salt thereof can be prepared by known methods disclosed in, for example, the above publications and their corresponding DE Patent 2,253,750 and U.S. Patent No. 4,035,370. The compound of the formula (I) or a pharmaceutically acceptable salt thereof can be used by per se known methods as an inhibitor of Aβ production or secretion, in particular as an agent for preventing or treating Alzheimer's disease. For example, it can be administered orally or parenterally to mammals (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey and ma in various conventional dosage forms, such as tablet granules, fine granules, capsules, injections, a suppositories. The dose varies with the subject diseas administration route, symptoms, etc. In general, it is 0 mg to 500 mg, preferably 1 mg to 100 mg, more preferably 1 to 20 mg, per day for a human adult. Vinpocetine has been used as a medicament, and has no toxic problems and can safely be administered. As described above, the inhibitor of Aβ secreti or production of the invention is useful for treating preventing brain disorders in mammals including humans. T subject diseases include, for example, senile dementi Alzheimer's disease, cerebrovascular dementia, amylo angiopathy, and Down's syndrome. EXAMPLES The following experiments and examples furth illustrate the present invention in detail, but are not to construed to limit the scope thereof. Experiment 1 Inhibitory Activity against Aβ Production o Secretion in Human Neuroblastoma IMR-32 Cells 1) Materials and Methods Studies on Aβ production or secretion in human- derived neurons and gliocytes are very interesting because of the relation to Alzheimer's dementia. However, human primary culture neurons and gliocytes cannot generally be used for experimental materials. IMR-32 cells were therefore used. The IMR-32 cells are human-derived neuroblastoma cells, and produce and secrete Aβ in the culture supernatant (Shinkei Kagaku (Neuroche istry) 33, 232 (1994)). In addition, Dr. Hironobu Suzuki et al. (Science, 264, 1336 (1994)) had developed an enzyme immunoassay method that can determine and distinguish between Aβ of 40 amino acids (Aβ (1-40)) and Aβ of 42 amino acids (Aβ (1-42)). This method was used to determine inhibitory activity of the test compound against Aβ production or secretion. a) Materials Human neuroblastoma IMR-32 cells were purchased from American Type Culture Collection (ATCC). Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical Co., Ltd., Japan. Fetal calf serum (hereinafter sometimes abbreviated as FCS) and a mixed solution of penicillin (5000 U/ml) and streptomycin (5 mg/ml) were purchased from Biowhittaker Inc. Phosphate buffered saline (PBS) was purchased from Flow Laboratories. Block Ace was purchased from Dainippon Pharmaceutical Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma. Flasks for culture and 48- well multi well plates were those manufactured by Falcon a Coster Corporation, respectively. Standard samples of Aβ ( 40) and Aβ (1-42) were purchased from Bachem. The oth reagents used were commercially available highest quali goods. b) Methods Culture of IMR-32 Cells IMR-32 cells were cultured at 37°C in 10% carb dioxide / 90% air using 10% FCS/DMEM culture solution in flask (Falcon, 750 ml) until the confluent growth of the cel was obtained. Then, the cells were inoculated in a 48-we multi well plate at a density of 2.5 x 10 cells/well, a cultured for 5 days. The culture solution was sucked off, an then DMEM / 0.5% BSA (0.5 ml for the assay of Aβ (1-40) o 0.25 ml for the assay of Aβ (1-42)) was added. The cells wer cultured for 24 hours. The supernatant was sampled and store at below -20°C until use for the assay of Aβ. Enzvme Immunoassay (EIA of Aβ Fig. 1 shows combinations of antibodies for th assay of Aβ (1-40) and Aβ (1-42). In each case, the primar antibody was BAN-50 antibody that recognizes the N-termina region of Aβ (1-40). BA-27 is an antibody prepared using A (1-40) as an antigen, and recognizes the C-terminal region o Aβ (1-40). On the other hand, BC-05 antibody is an antibod prepared using Aβ (35-43) as an antigen, and recognizes th C-terminal region of Aβ (1-42) but not Aβ (1-40). Therefore, Aβ (1-40) can be assayed with a combination of BAN-50 antibody with BA-27 antibody, and Aβ (1-42) can be assayed with a combination of BAN-50 antibody with BC-05 antibody. A solution (100 μl/well) of BAN-50 antibody (15 μg/ml) in 0.1M carbonic acid buffer (pH 9.6) was added to a polyethylene microtiter plate, and the plate was allowed to stand at 4°C overnight. The plate was rinsed with PBS three times, and a block solution (25% Block Ace / 0.1% sodium azide / PBS) (200 μl) was added. The plate thus prepared was stored at 4°C until use. The plate was rinsed with PBS three times, and a buffer for the primary reaction (20 mM phosphate buffer, pH 7.0; 400 mM NaCl; 2 mM EDTA; 10% Block Ace; 0.2% BSA; 0.05% sodium azide) (50 μl) was added. In addition, the standard samples (1000, 200, 40 and 8 pg/ml) (each 100 μl) diluted with the buffer for the primary reaction or the culture supernatant (100 μl) were added, and the mixture was allowed to stand at 4°C overnight. After removal with suction, the plate was rinsed with PBS three times. A solution (100 μl) of a secondary antibody (BA-27 or BC-05 antibody) labeled with horseradish peroxidase (HRP) in a buffer for the secondary reaction (20 mM phosphate buffer, pH 7.0; 400 mM NaCl; 2mM EDTA; 1% BSA) . The reaction was carried out for 6 hours at room temperature, the plate was rinsed seven times, and a coloring reagent (TMB Peroxidase Substrate, Kirkegaard & Perry Lab.) (100 μl) was added. The reaction was carried out room temperature for 8 to 10 minutes, and then stopped wi 1M phosphate solution (100 μl). The mixture was subjected colorimetry (wavelength: 450 nm) on a plate reader (Coron MTP-32 Micro Plate Reader). Evaluation of the Cvtotoxicity (1) MTT Method The culture supernatant was sampled, and DM culture solution (500 μl) containing 1.2 mM MTT {3-(4, dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide} w added. The reaction was carried out for 1 hour at 37°C, a then an equal amount of 10% sodium dodecyl sulfate soluti was added. The mixture was gently stirred overnight dissolve the resulting formazan, and then subjected colorimetry (wavelength: 550 nm) with a plate reader. (2) Lactate Dehydrogenase (LDH) Method The culture supernatant (10 μl) obtained after t assay of Aβ (1-40) was sampled, and the amount of LDH releas from the cells was determined with a LDH assay kit (LDH C- manufactured by Wako Pure Chemical Industries, Ltd.). Statistical Analysis Four wells were used per dose of the drug. T value in the data was represented as a ratio (%) to t control group. Dunnett's t test was used for the statistic analysis. 2) Results a) Standard Curve of Aβ (1-40) and Aβ (1-42) Fig. 2 shows a standard curve of the EIA system against the concentrations of Aβ (1-40) and Aβ (1-42). The assay was carried out in 8 pg/ml to 1000 pg/ml. In this range, the absorbance increased in proportion to the concentrations of Aβ (1-40) and Aβ (1-42). The detection limit of this method was about 10 pg/ml. b) Effects of the Compound of the Formula (I) on the Production and Secretion of Aβ (1-40) and Aβ (1-42) from IMR- 32 Cells IMR-32 cells constantly produce and secrete Aβ. The amount of Aβ in the culture supernatant was measured. It was confirmed that Aβ (1-40) in not less than 500 pg/ml and Aβ (1- 42) in an amount of not more than one tenth times the amount of Aβ (1-40) were produced and secreted under the culture conditions used in this experiment. Thus, the effects of the compound on the production and secretion were able to be evaluated by the determination of the amount of Aβ in the supernatant obtained after adding the compound. Fig. 3 shows inhibitory activity of vinpocetine against production or secretion of Aβ. Vinpocetine in 3 x 10 " , 1 x 10 " , 3 x 10 " and 1 x 10 " * M inhibited Aβ production or secretion by 10, 30, 40 and 45%, respectively, based on the control group. Aβ (1-42) production and secretion showed a tendency to decrease at x 10 " and 1 x 10 " M compared to the control group, but it w not a significant change. Presumably, this is partly becau the amount of Aβ (1-42) production and secretion was very l and varied with the wells. In this case, the amount formazan formed from MTT and LDH activity in the cultu supernatant were determined as an indication of t cytotoxicity. The results showed only slight cytotoxicity 1 x 10 " , the maximum concentration used. Experiment 2 Inhibitory Activity against Aβ Production a Secretion in Guinea Pig Primary Culture Neurons 1) Materials and Methods Cultivation of Neurons Fetal brains were taken out from guinea pigs (SL Hartley) that was 28 days pregnant. The cerebral cortex a hippocampus were taken out under a stereoscopic microscop and, after removing the meninges, were treated in Hank' Balanced Salt Solution (20 ml) containing 0.25% trypsin 0.01% DNase I for 10 minutes. FCS was added to stop t reaction, and the mixture was centrifuged. The cells we dispersed in DMEM culture solution (DMEM/N2 culture solutio containing insulin 5 μg/ml, transferrin 100 μg/ml progesterone 20 nM, putrescine 100 μM and sodium selenite 3 nM. The dispersion was filtered through double lens pape and the filtrate was centrifuged to collect the cells. The cells were dispersed in DMEM/N2 culture solution, and inoculated in a 24-well plate at a density of 1 x 10 δ cells/well. On the second day from the beginning of the cultivation, the culture solution was replaced with DMEM/N2 culture solution containing cytosine arabinoside (1 μM) . After the treatment for 3 days, the culture solution was replaced with DMEM/N2 culture solution. Thereafter, the culture solution was replaced every 2 to 3 days. On the 12th day from the beginning of the cultivation, DMEM/N2 culture solution containing vinpocetine was added. After 24 hours, the culture supernatant was collected and stored at below -20°C until use for the quantification of Aβ. Enzyme Immunoassay of Aβ fEIA) Aβ (1-40) and Aβ (1-42) in the culture supernatant were quantified in the same manner as that described in Experiment 1. Evaluation of the Cytotoxicity After the culture supernatant was collected, DMEM culture solution (300 μl) containing 1.2 mM MTT was added. After the reaction was carried out at 37°C for 1 hour, an equal amount of 10% sodium dodecyl sulfate solution was added. Thereafter, the same procedure as that described in Experiment 1 was followed. 2) Results Fig. 4 shows the results of the test for inhibitory activity of vinpocetine against Aβ (1- production and secretion in neurons. Vinpocetine inhibi production or secretion of Aβ (1-40) in 3 x 10 "5 M without cytotoxicity. Because Aβ (1-42) was produced or secreted a small amount, it was impossible to evaluate the inhibito activity of vinpocetine against Aβ (1-42). Example 1 (1) Vinpocetine 5.0 g (2) Lactose 82.5 g (3) Hydroxypropylcellulose 2.8 g (4) Magnesium stearate 0.4 g (5) Hydroxypropylmethylcellulose 2910 2.994 g (6) Corn starch 19.3 g (7) Macrogol 6000 0.6 g (8) Titanium oxide 0.4 g (9) Iron sesquioxide 0.006 g The ingredients (1) to (6) were mixed. From t mixture, 1000 raw tablets each containing 5 mg of vinpoceti and being 6.5 mm in diameter were obtained. The tablets we coated with the ingredients (7) to (9) to obtain film-coat tablets of 6.6 cm in diameter. Example 2 (1) Vinpocetine 5 g (2) Lactose-crystalline cellulose 330 g (granules) (3) D-mannitol 29 g (4) Low substituted hydroxypropylcellulose 20 g (5) Talc 25 g (6) Hydroxypropylcellulose 50 g (7) Aspartame 3 g (8) Dipotassium glycyrrhizinate 3 g (9) Hydroxypropylmethylcellulose 2910 30 g (10) Titanium oxide 3.5 g (11) Yellow iron sesquioxide 0.5 g (12) Light anhydrous silicic acid 1 g The ingredients (1), (3), (4), (5), (6), (7) and (8) were suspended or dissolved in purified water, and the nucleus granules of the ingredient (2) were coated with the suspension or solution to obtain raw fine granules. The raw fine granules were coated with the ingredients (9) to (11) to obtain coated fine granules. They were mixed with the ingredient (12) to obtain vinpocetine fine granules (1%, 500 g). The fine granules are wrapped so that each wrapper contains about 500 mg of the fine granules.

Claims: CLAIMS 1. A pharmaceutical composition for inhibiti production or secretion of amyloid β protein, which compris a compound of the formula (I): wherein ring A is an optionally substituted benzene ring, represents OR , R ** N or SR , N< j R 3 -' wherein R 1 , R 2 and R 3 are the same or different and each i selected from a hydrogen atom, an optionally substitute hydrocarbon group or R 2 and R , taken together with th adjacent nitrogen atom, form an optionally substitute nitrogen-containing heterocyclic group, and Y is an optionall substituted alkyl group, or a pharmaceutically acceptable sal thereof, if necessary, with a pharmaceutically acceptabl excipient, carrier or diluent. 2. A composition according to claim 1, wherein th ring A is a benzene ring which may have 1 to 3 substituent selected from halogen, R is OR 1 wherein R 1 is alkyl, and Y is ethyl. 3. A composition according to claim 2, wherein the ring A is a benzene ring, and R 1 is ethyl. 4. A pharmaceutical composition for preventing or treating Alzheimer's disease, which comprises a compound of the formula (I) : wherein ring A is an optionally substituted benzene ring, R represents OR 1 , R 2 - or SR 1 , N< . R 3 ~' wherein R 1 , R 2 and R 3 are the same or different and each is selected from a hydrogen atom, an optionally substituted hydrocarbon group or R and R , taken together with the adjacent nitrogen atom, form an optionally substituted nitrogen-containing heterocyclic group, and Y is an optionally substituted alkyl group, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable excipient, carrier or diluent. 5. A composition according to claim 4, wherein t ring A is a benzene ring which may have 1 to 3 substituen selected from halogen, R is OR wherein R 1 is alkyl, and Y ethyl 6. A composition according to claim 5, wherein t ring A is a benzene ring, and R ,ι is ethyl. 7. A method of inhibiting production or secreti of amyloid β protein in a mammal, which compris administering to said mammal an effective amount of a compou of the formula (I): wherein ring A is an optionally substituted benzene ring, represents OR 1 , R 2 - N or SR , N< / R 3 -' wherein R 1 , R 2 and R 3 are the same or different and each i selected from a hydrogen atom, an optionally substitute hydrocarbon group or R 2 and R , taken together with th adjacent nitrogen atom, form an optionally substitute nitrogen-containing heterocyclic group, and Y is an optionall substituted alkyl group, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable excipient, carrier or diluent. 8. A method according to claim 7, wherein the ring A is a benzene ring which may have 1 to 3 substituents selected from halogen, R is OR 1 wherein R 1 is alkyl, and Y is ethyl. 9. A method according to claim 8, wherein the ring A is a benzene ring, and R 1 is ethyl. 10. A method of preventing or treating Alzheimer's disease in a mammal, which comprises administering to said mammal an effective amount of a compound of the formula (I) : wherein ring A is an optionally substituted benzene ring, R represents OR , R -» N or SR , N< R' wherein R 1 , R 2 and R 3 are the same or different and each is selected from a hydrogen atom, an optionally substituted hydrocarbon group or R 2 and R , taken together with the adjacent nitrogen atom, form an optionally substitut nitrogen-containing heterocyclic group, and Y is an optional substituted alkyl group, or a pharmaceutically acceptable sa thereof, or a pharmaceutically acceptable excipient, carri or diluent. 11. A method according to claim 10, wherein t ring A is a benzene ring which may have 1 to 3 substituen selected from halogen, R is OR wherein R is alkyl, and Y ethyl. 12. A method according to claim 11, wherein t ring A is a benzene ring, and R is ethyl. 13. Use of a compound of the formula (I) : wherein ring A is an optionally substituted benzene ring, represents OR 1 , R — or SR , N< ; R 3 -' wherein R 1 , R 2 and R 3 are the same or different and each selected from a hydrogen atom, an optionally substitut hydrocarbon group or R 2 and R , taken together with t adjacent nitrogen atom, form an optionally substituted nitrogen-containing heterocyclic group, and Y is an optionally substituted alkyl group, or a pharmaceutically acceptable salt thereof, for the manufacture of a composition for inhibiting production or secretion of amyloid β protein. 14. Use according to claim 13, wherein the ring A is a benzene ring which may have 1 to 3 substituents selected from halogen, R is OR 1 wherein R 1 is alkyl, and Y is ethyl. 15. Use according to claim 14, wherein the ring A is a benzene ring, and R l is ethyl. 16. Use of a compound of the formula (I): wherein ring A is an optionally substituted benzene ring, R represents OR 1 , R 2 - s or SR 1 , N< ' R 3 -' wherein R 1 , R 2 and R 3 are the same or different and each is selected from a hydrogen atom, an optionally substituted hydrocarbon group or R 2 and R , taken together with the adjacent nitrogen atom, form an optionally substituted nitrogen-containing heterocyclic group, and Y is an optionall substituted alkyl group, or a pharmaceutically acceptable sal thereof, for the manufacture of a composition for preventin or treating Alzheimer's disease. 17. Use according to claim 16, wherein the ring is a benzene ring which may have 1 to 3 substituents selecte from halogen, R is OR wherein R is alkyl, and Y is ethyl. 18. Use according to claim 17, wherein the ring is a benzene ring, and R is ethyl.

Inventors: GOTO GIICHI, KAKIHANA MITSURU, KOSAKA TAKUO, NAGAOKA AKINOBU, GOTO, GIICHI, KAKIHANA, MITSURU, KOSAKA, TAKUO, NAGAOKA, AKINOBU
Applicants: GOTO GIICHI, KAKIHANA MITSURU, KOSAKA TAKUO, NAGAOKA AKINOBU, TAKEDA CHEMICAL INDUSTRIES LTD, GOTO, GIICHI, KAKIHANA, MITSURU, KOSAKA, TAKUO, NAGAOKA, AKINOBU, TAKEDA CHEMICAL INDUSTRIES, LTD.
Application year: 1996
Main classification: A61K31
IPC codes: A61K31