EP0778773A1 - Procedes permettant de traiter et/ou de prevenir la maladie d'alzheimer a l'aide de phenothiazines et/ou de thioxanthenes - Google Patents

Procedes permettant de traiter et/ou de prevenir la maladie d'alzheimer a l'aide de phenothiazines et/ou de thioxanthenes

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Publication number
EP0778773A1
EP0778773A1 EP95929441A EP95929441A EP0778773A1 EP 0778773 A1 EP0778773 A1 EP 0778773A1 EP 95929441 A EP95929441 A EP 95929441A EP 95929441 A EP95929441 A EP 95929441A EP 0778773 A1 EP0778773 A1 EP 0778773A1
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group
hydrogen
lower alkyl
carbon atoms
compound
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German (de)
English (en)
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Peter Davies
Inez J. Vincent
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Albert Einstein College of Medicine
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Albert Einstein College of Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame

Definitions

  • This invention relates to methods for preventing or treating Alzheimer's Disease. More specifically, the methods comprise administering to a patient an amount of a phenothiazine or a thioxanthene effective to prevent or diminish the accumulation of abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease.
  • Alzheimer's Disease is a progressive neurodegenerative disorder affecting 7% of the population over
  • Alzheimer's Disease is characterized by the accumulation of abnormally phosphorylated, paired helical filaments (PHFs) , which accumulate as neurofibrillary tangles in neuronal cell bodies and in the neurites of the neuritic plaques. PHFs also are present in neuronal processes, such as axons and dendrites.
  • Alzheimer case is present in the neuronal processes rather than in the plaque or tangle ( olozin, B.L. and Davies, P.
  • MAPs neurofilaments and other microtubule associated proteins
  • Monoclonal antibodies have been generated by the inventors of the present invention which are reactive with PHFs in the brain tissue of Alzheimer's Disease patients, and in most cases, minimally or not cross reactive with proteins in the normal adult brain. These antibodies react with abnormally phosphorylated epitopes on PHF proteins in the Alzheimer's brain which are modified as a result of the disease process.
  • the ability to inhibit the production of abnormally phosphorylated PHF proteins including tau, other MAPs and neurofilament proteins by blocking epitopes recognized by these antibodies is expected to interfere or prevent the formation and acccumulation of PHF, and therefore inhibit or prevent the progression of Alzheimer's disease. To date, however, it has not been possible to consistently induce the formation of abnormally phosphorylated PHFs epitopes in abundance either in vivo or in vitro.
  • Shea, T.B., et al. reported that aluminum salts induce the accumulation of neurofilaments in neuroblastoma cells (Shea, T.B., et al. Brain Res. 492:53-64 (1989)).
  • the neurofilaments produced by Shea, et al. have not been shown to correlate with PHFs associated with Alzheimer's Disease.
  • Ko, Li-wen, et al. reported the alleged expression of epitopes associated with neurons in Alzheimer's Disease in the human neuroblastoma cell line IMR32 grown in a differentiation medium using polyclonal antibodies to PHF epitopes (Ko, Li-wen, et al. Am. J. Path. 136(4) :867-879 (1990) ) .
  • the present invention which is directed to a tissue culture model in which Alzheimer's Disease epitopes are continually and abundantly expressed, fulfills a great need in this field.
  • the present invention provides an in vitro model cell system which expresses abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease, which comprises neuroblastoma cells which have been treated with an amount of a protein phosphatase inhibitor effective to render homogenate from the cells treated therewith immunoreactive with antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes.
  • the model system of this invention may be used for determining whether an agent is capable of preventing or diminishing the accumulation of abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease.
  • the present invention also provides a method for treating or preventing Alzheimer's Disease which comprises the administration of a drug which blocks the accumulation of abnormally phosphorylated, paired helical epitopes associated with Alzheimer's Disease.
  • Figure 1 represents the time course for OkA treatment.
  • Confluent layers of MSN cells were incubated at 37°C for 0 minutes and 4 hours with the KRP containing 0.05% DMSO (Controls), or 30 minutes, 90 minutes, 4 hours, 10 hours, and 24 hours with 1 ⁇ M OkA.
  • the cells were harvested and boiled to generate heat-stable fractions. Twenty ⁇ g of protein per lane was resolved by SDS-PAGE, and gels were electrophoretically transferred to nitrocellulose. Blots were immunostained with the Alz-50, Tau-1, 188, PHF-1, NP-8, and T3-P antibodies. The arrow beside the PHF-1 blot points to the 68-kDa protein, which is only apparent after OkA treatment.
  • Figure 2 represents the dose-response curve for OkA treatment.
  • MSN cells were incubated with 5 nM, 50 nM, 1 ⁇ M, and 50 ⁇ M OkA in KRP for 4 hours at 37°C Heat-stable fractions were prepared and used for western blot analysis as in Figure 1. Note that the 50 ⁇ M OkA-treated sample in the Alz-50 blot represents only 13 ⁇ g of protein.
  • Figure 3 represents the effects of protein synthesis inhibition. MSN cells were incubated at 37°C for 90 minutes with 1 ⁇ M OkA in the absence or presence of 100 ⁇ M anisomycin. Heat-stable fractions were prepared, and 30 ⁇ g of protein was analyzed by western blotting as in Figure 1.
  • Figure 4 represents the treatment of MSN cell lysates with OkA: Effects of alkaline phosphatase.
  • MSN cells were collected by centrifugation and resuspended in KRP containing 4 M PMSF, 25 ⁇ M leupeptin, and 2 inM EGTA. The cell suspension was sonicated for 2 seconds, and the resulting lysate was incubated at 37°C for 60 minutes in the absence or presence of 10 ⁇ M OkA. Additional samples were treated with 50 nM OkA. Thirty ⁇ g of the respective heat-stable fractions were loaded per lane.
  • Duplicate blots were incubated at 37°C for 24 hours with 20 units of alkaline phosphatase per ml of pH 8.0 buffer in the presence of 10 mM PMSF (+) . The blots were then immunized with the Alz-50, Tau-1, 188, PHF-1, T3P, and NP-8 antibodies.
  • Figure 5A represents the 32 P-labeling of tau using pulse-chase.
  • MSN cells were preincubated at 37°C for one hour with 2.5 mCi of 32 P-orthophosphoric acid in phosphate-free medium. The cells were then chased for 90 minutes with KRP in the absence (control) or presence of 1 ⁇ M OkA. Heat-stable supernatants were further enriched for tau by perchloric acid (PCA) treatment and methanol precipitation as described in the Experimental Details Section. Blots were stained with Alz-50 and then subjected to autoradiography.
  • PCA perchloric acid
  • Figure 5B represents the 32 P-labeling of tau using pulse.
  • Cells were preincubated for 30 minutes with labeled orthophosphoric acid at the end of which time, 1 ⁇ M OkA acid was introduced into one sample.
  • heat-stable supernatants were isolated and subjected to PCA and methanol precipitation. Blots were stained with Alz-50 and subsequently subjected to autoradiography.
  • Figure 6 represents the 35 S-methionine labelling of tau.
  • MSN cells were pulse labeled overnight in methionine- free medium containing 200 ⁇ Ci per ml isotopic methionine. The cells were subsequently chased for 90 minutes with KRP containing 2 mM unlabelled methionine with the indicated additions. Heat-stable supernatants were treated with PCA, and the PCA soluble protein methanol-precipitated for gel- loading. Blots were stained with Alz-50, and subjected to autoradiography.
  • Figure 7 represents the immunoreactivity of antibodies Alz-50, PHF-1, TG3, TG4, MC2, MC6, and MC15 with normal and Alzheimer's Disease brain, and with MSN cells before (MSN-) and after treatment (MSN+) with okadaic acid.
  • the data from brain tissue are the mean values derived from studies of mid-temporal cortex from five normal cases and five cases of Alzheimer's Disease.
  • Values from MSN cells are from a single experimental series in which 1 micromolar okadaic acid was added to triplicate MSN cell cultures for 90 minutes prior to harvest of the cells. Each sample (tissue or cell ho ogenate) was assayed at 8 dilutions, in duplicate.
  • Figure 8 represents the immunoblot of neuroblastoma (MSN-A) cells expressing paired helical filament epitopes as a control, and after treatment with trifluoperazine and chlorpromazine.
  • Figure 9A represents relative ADAP concentrations in Broadman area 10 in Alzheimer's Disease patients (AD), normal controls (NC) , and schizophrenic patients on chlorpromazine (RX) .
  • Figure 9B represents relative ADAP concentrations in Broadman area 38 in Alzheimer's Disease patients (AD), normal controls (NC) , and schizophrenic patients on chlorpromazine (RX) .
  • the present invention provides an in vitro model cell system which expresses abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease, said system comprising neuroblastoma cells which have been treated with an amount of a protein phosphatase inhibitor effective to render homogenate from the cells treated therewith immunoreactive with antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes.
  • the model system of the present invention is produced by treating neuroblastoma cells with an amount of a protein phosphatase inhibitor effective to render homogenate from the cells treated therewith immunoreactive with antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes.
  • the neuroblastoma cells useful in the present invention are neuroblastoma cells isolated from the nervous system tumors of children which contain tau, neurofilament proteins, and other neuronal proteins. These cells express express little, if any, abnormally phosphorylated, paired helical filament epitopes until treated with the phosphatase inhibitor.
  • the neuroblastoma cells are human cells deposited with the American Type Culture Collection under ATTC Accession No. CRL 11253.
  • the protein phosphatase inhibitor is any inhibitor which induces the expression of abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease in neuroblastoma cells treated therewith.
  • the protein phosphatase inhibitor is okadaic acid, calyculin A, microcystin-LR, nodularin and phenylarsine oxide.
  • the protein phosphatase inhibitor is okadaic acid.
  • the amount of protein phosphastase inhibitor used to treat the neuroblastoma cells is an amount effective to induce expression of abnormally phosphorylated, paired helical filament epitopes in the cells treated with that inhibitor.
  • the concentration of the inhibitor is between 0.1 and 10 ⁇ M. Most preferably, the concentration is 1.0 ⁇ M. However, the particular optimal concentration will depend upon the inhibitor and cells used.
  • the expression of abnormally phosyphorylated, paired helical filament epitopes may be detected using antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes. The epitopes are detected by Western blot analyses or by quantitative ELISA as described in the Experimental Details Section.
  • the antibodies may be polyclonal or monoclonal, and are preferably monoclonal.
  • the antibodies may be prepared from PHFs isolated from Alzheimer's patients using known procedures.
  • the PHFs are isolated and purified using immunoaffinity as described in Vincent, I.J. and Davies, P. Proc. Natl. Acad. Sci. USA 89:2878-2882 (1992) .
  • immunoaffinity as described in Vincent, I.J. and Davies, P. Proc. Natl. Acad. Sci. USA 89:2878-2882 (1992) .
  • twenty grams of cortical tissue from postmortem Alzheimer's patients is homogenized in 10 vol. of Tris-buffered saline (TBS; 0.1 M Tris base/0.14 M NaCl, pH 7.4) using a Polytron operating at a setting of 5 for two 30 second bursts.
  • the homogeneate is centrifuged at 27,000 x g for 30 minutes at 4°C, and the supernatant is applied to the immunoaffinity column.
  • a monoclonal antibody useful for affinity purification is Ab42 (IgG) , a class of switch clone of Alz-50 produced according to published protocol (Spira, G. , et al. J. Immunol. Methods 74:307-313 (1984).
  • Ab42 IgG
  • Approximately 20 mg of protein A-purified Ab42 is dialyzed into 0.1 M sodium phosphate buffer (pH 8.1) and mixed with 10 ml of washed Affi- Gel 10 (Bio-Rad) . The mixture is incubated on a rotary shaker for 30 minutes at room temperature.
  • a volume of 0.1 ethanolamine (pH 8.1) equal to that of the gel/antibody mixture is added and the incubation is continued for 1 hour.
  • the coupling efficiency is generally >90%.
  • the immunoadsorbent is poured into a column and washed with TBS.
  • the column is maintained at 4°C and all chromatography steps are conducted at this temperature.
  • the column is treated with at least 2 bed vol. of eluting buffer (3 M potassium thiocyanate) followed by 5 bed vol. of TBS.
  • the 27,000 x g supernatant is loaded onto the immunoaffinity column at a flow rate of approximately 25 ml/hr.
  • Nonspecific binding is reduced by washing the immunoadsorbent with at least 30 bed vol. of TBS. Subsequently, adsorbed protein is eluted with the eluting buffer. Fractions are assayed for protein concentration with the Quantigold protein reagent (Bio-Rad) . Peak fractions are dialyzed against TBS and aliquots are stored at -70°C.
  • mice are immunized with the antigen purified as above by intraperitoneal injection of 10 to 20 micrograms of protein per mouse per injection. Mice are immunized 4 or 5 times before removal of spleens for the production of hybridoma cells by standard protocols. Hybridomas are tested for the production of specific antibodies which react to Alzheimer's brain tissue by ELISA and immunocytochemistry.
  • the antibodies have high reactivity with Alzheimer's brain tissue and little or no reactivity with brain tissue from normal individuals.
  • the antibodies are selected from the group consisting of Alz-50, PHF-1, TG3, TG4, MC2, MC6, and MC15.
  • hybridomas secreting these antibodies have been deposited with the American Type Culture Collection, Rockville, Maryland: Alz-50 is secreted from the hybridoma deposited under ATCC Accession Number HB9205; PHF-1 is secreted from the hybridoma deposited under ATCC Accession Number 11743; TG3 is secreted from the hybridoma deposited under ATCC Accession Number 11744; TG4 is secreted from the hybridoma deposited under ATCC Accession Number 11745; MC2 is secreted from the hybridoma deposited under ATCC Accession Number 11737; MC6 is secreted from the hybridoma deposited under ATCC Accession Number 11740; and MC15 is secreted from the hybridoma deposited under ATCC Accession Number 11741.
  • the model system of the present invention and the antibodies above may be used for determining whether an agent or drug is capable of preventing or decreasing Alzheimer's Disease activity, namely the accumulation of abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease. Accordingly, the present invention also provides a method for determining whether an agent is capable of preventing or diminishing the accumulation of abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease.
  • That method comprises (a) treating neuroblastoma cells with an amount of a protein phosphatase inhibitor effective to permit expression of abnormally phosphorylated, paired helical filament epitopes in the treated cells, thereby rendering homogeneate from the cells immunoreactive with antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes; (b) either concomitantly or thereafter adding to the treated cells an agent suspected of being capable of preventing or diminishing the accumulation of abnormally phosphorylated, paired helical filament epitopes; (c) adding to the treated cells antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease; (d analyzing homogenate from the cells for immunoreactivity with the antibodies; and (e) determining whether the agent has caused the level of immmunoreactivity between homogenate from the neuroblastoma cells and the antibodies to decrease, said decrease being indicative that the agent has prevented or -diminished the accumulation of abnormal
  • the present invention also provides a screening kit for use in determining whether an agent is capable of preventing or diminishing the accumulation of abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease, said kit comprising (a) neuroblastoma cells; (b) a protein phosphatase inhibitor which is capable of causing the neuroblastoma cells to express abnormally phosphorylated, paired helical filament epitopes when the cells are treated therewith, thereby rendering homogenate from the treated cells immunoreactive with antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes; and (c) antibodies which specifically bind to abnormally phosphorylated, paired helical filament epitopes expressed by the neuroblastoma cells treated with the protein phosphatase inhibitor.
  • phenothiazine compounds i.e. trifluoperazine and chlorpromazine (CPZ)
  • CPZ chlorpromazine
  • chlorpromazine is effective in decreasing Alzheimer's activity
  • the phenothiazines and structurally similar compounds such as thioxanthenes described below display the same profile of activity in the model system and assays described herein as chlorpromazine
  • the phenothiazines and thioxanthenes are also effective in preventing or decreasing Alzheimer's activity, and therefore are effective in treating or preventing Alzheimer's Disease.
  • the present invention also provides a method for treating or preventing Alzheimer's Disease in a subject in need of such treatment or prevention which comprises administering an amount of a phenothiazine or a thioxanthene effective to prevent or diminish the accumulation of abnormally phosphorylated, paired helical filament epitopes associated with Alzheimer's Disease.
  • the phenothiazines useful in the invention have the structural skeleton shown in formula I:
  • A represents divalent alkylene or alkenylene group having from about 2-8 carbon atoms; and R ⁇ and R 2 independently represent hydrogen, or organic groups optionally substituted with a variety of moieties such as, for example, halogen, amino, carbonyl, sulfonyl, trifluoromethyl, lower alkyl, lower alkoxyl, etc.; or R and R 2 together with the nitrogen to which they are attached form a five to seven-membered heterocyclic ring, such as, for example, pyrrolidinyl, piperazinyl, lower alkyl piperazinyl, piperidyl, thiomorpholinyl, morpholinyl or hexahydroazepinyl.
  • the tricyclic ring system may optionally be substituted with a variety of groups such as, for example, halogen, amino, carbonyl, sulfonyl, perfluoroalkyl, such as trifluoromethyl, lower alkyl, and lower alkoxyl, etc.
  • the sulfur atom in formula I above may optionally be present as a sulfoxide.
  • the thioxanthenes useful in the invention have the structural skeletons shown in formulas II and III:
  • R' is hydrogen or an alkyl group
  • R and R 1 independently represent hydrogen or alkyl, or R and R 1 together form a 5, 6, or 7-membered heterocyclic ring such as, for example, pyrrolidino, piperidino, morpholino or piperazino
  • A is a 5, 6, or 7-membered heterocyclic ring such as, for example, pyrrolidino, piperidino, morpholino or piperazino.
  • the 5, 6, or 7-membered heterocyclic ring may optionally be substituted with a variety of moieties such as halogen, amino, carbonyl, sulfonyl, perfluoroalkyl, lower alkyl, and lower alkoxy, etc.
  • the tricyclic ring system may optionally be substituted with a variety of groups such as, for example, halogen, amino, carbonyl, sulfonyl, perfluoroalkyl, such as trifluoromethyl, lower alkyl, and lower alkoxyl, etc.
  • Compounds suitable for use in the methods of the invention include single enantiomers, racemates and geometric isomers of compounds having the structural skeletons of Formulas I, II, or III, and the structures and compounds described below. It further includes mixtures of enantiomers and geometric isomers of these structures and compounds.
  • Examples of drugs in the Phenothiazine family which are effective in blocking the appearance of paired helical filament epitopes include Promazine, Triflupromazine, Methotri eprazine, Acetophenazine, Fluphenazine, Perphenzine, Prochloroperazine, Mesoridazine and Thoridazine. These and other similar phenothiazine compounds are described in U.S. Patent Nos. 2,921,069; 2,837,518; 2,860,138; 2,902,484; 3,194,733; 3,084,161; 2,519,886; 2,645,640; and 2,985,654, which are hereby incorporated by reference.
  • Examples of drugs in the thioxanthene family that are effective in blocking the appearance of paired helical filament epitopes are Chlorprothixene and Thiothixene.
  • Representative thioxanthenes are described in U.S. Patent Nos. 3,310,553 and 2,951,082, which also are hereby incorporated by reference.
  • Representative phenothiazine compounds useful in practicing the method of this invention are:
  • U.S. Patent No. 2.921.069 Compounds of the general formula: wherein Z is -S- or -SO-; Y is trifluoromethyl; R is hydrogen, halogen, trifluoromethyl, lower alkyl or lower alkoxyl; A represents a divalent, straight or branched aliphatic chain containing 2 to 6 carbon atoms; and R and R 2 are either hydrogen, lower alkyl or divalent groups which together with the nitrogen to which they are attached form a five to seven-membered heterocyclic ring, such as pyrrolidinyl, piperazinyl, lower alkyl piperazinyl, piperidyl, thiomorpholinyl, morpholinyl or hexahydroazepinyl.
  • Z is -S- or -SO-
  • Y is trifluoromethyl
  • R is hydrogen, halogen, trifluoromethyl, lower alkyl or lower alkoxyl
  • A represents a divalent, straight or
  • Advantageous compounds for use in this invention are represented by the above structural formula wherein: Z is -S-; Y is a trifluoromethyl in the 2 or 4 position; R is hydrogen, halogen, trifluoromethyl, lower alkyl or lower alkoxy in the 6 or 8 position; A is a divalent, straight or branched aliphatic chain containing 2 to 5 carbon atoms; and R and R 2 represent either hydrogen, lower alkyl or divalent groups which together with the nitrogen to which they are attached form a five to six-membered heterocyclic ring, such as pyrrolidinyl, piperazinyl, lower alkyl piperazinyl, piperidyl, thiomorpholinyl or morpholinyl.
  • Still more advantageous compounds of this invention are represented by the above structural formula wherein: Z is -S-; Y is trifluoromethyl in the 2 or 4 position; R is hydrogen; A is either a divalent, straight aliphatic chain containing 2 to 4 carbon atoms or a divalent, branched aliphatic chain containing 2 to 5 carbon atoms; and R 1 and R represent either hydrogen, lower alkyl or divalent groups which together with the nitrogen to which they are attached form a five to six-membered heterocyclic ring, such as pyrrolidinyl, piperazinyl, lower alkyl piperazinyl, piperidyl, thiomorpholinyl or morpholinyl.
  • the most preferred compounds are represented by the structural formula wherein: Z is -S-; Y is trifluoromethyl in the 2 position; R is hydrogen; A is a chain represented by the structure: —CH 2 -CH—CH,
  • R 3 wherein: R 3 is hydrogen, methyl or ethyl; and R ⁇ and R 2 represent lower mono-or dialkyl or divalent groups which taken together with the nitrogen to which they are attached form a five to six-membered heterocyclic ring, such as pyrrolidinyl, piperazinyl, lower alkyl piperazinyl, piperidinyl, thiomorpholinyl or morpholinyl.
  • lower alkyl or lower alkoxyl refer to aliphatic groups having not more than 4 carbon atoms and preferably not more than 2 carbon atoms, as indicated.
  • Y is selected from the class consisting of a sulphur atom and the SO and S0 2 groups
  • X is selected from the class consisting of a hydrogen atom, a methyl, a ethoxy, an ethyl and an ethoxy group
  • Z is selected from the class consisting of the monomethylamino, monoethylamino, dimethylamino, diethylamino, pyrrolidino and piperidino groups.
  • X is a halogen atom having an atomic number greater than 9 but less than 53; R is a member of the group consisting of hydrogen and lower alkyl groups whose combined number of carbon atoms is less than 6; and n is a natural number less than 3.
  • A is a divalent saturated aliphatic hydrocarbon radical with a straight or branched chain containing from 2 to 6 carbon atoms;
  • R is a hydrogen atom, or a lower alkyl, aryl or arylalkyl group;
  • Y and Y 2 represent a hydrogen or halogen atom, or a lower alkyl, alkoxy or aryl or aryloxy group, preferably (in the case of Y) in the 1- or 3- position.
  • the phenothiazine ring may carry a substituent in the form a methyl group.
  • lower alkyl means an alkyl group containing not more than 6 and preferably not more than 4 carbon atoms.
  • r is 1 or 2;
  • X is hydrogen, halogen (preferably chloro) , trifluoromethyl, lower alkyl, lower alkoxy, lower alkanoyl, lower alkyl mercapto, trifluoromethylmercapto, and lower alkysulfonyl (preferably methylsulfonyl) ; and
  • Y is higher alkyl, higher alkenyl, higher alkynyl, aryl, ⁇ -carboalkoxy (higher alkyl) or diphenyl (hydroxymethyl) .
  • the terms "higher alkyl,” “higher alkenyl” and “higher alkynyl” as employed herein include both straight and branched chain radicals of more than five carbon atoms.
  • ⁇ -carboalkoxy (higher alkyl)
  • substituents derived from hydrocarbon carboxylic acids of more than 6 carbon atoms include substituents derived from hydrocarbon carboxylic acids of more than 6 carbon atoms, and may be represented by the formula:
  • n is a positive integer of more than 6 and is preferably a positive integer of from 7 to 12.
  • aryl as employed herein includes substitutents derived from moncyclic and bicyclic aryl carboxylic acids, and may be substituted or unsubstituted and further may be represented by the formulae:
  • each R is hydrogen, lower alkyl, lower alkoxy or halogen (e.g. chloro or bromo) .
  • R is preferably hydrogen, lower alkyl or lower alkoxy, and most preferably, is hydrogen or lower alkyl.
  • aryl carboxylic acids which may be employed include benzoic, o-toluic, 2,6-dimethylbenzoic, 2,6-dimethylanisic, o-bromobenzoic, o-chlorobenzoic, 2,6-dichlorobenzoic, naphthoic acid, dimethylnaphthoic acid, and other like acids.
  • the preferred compounds are those wherein X is chloro or trifluoromethyl and Y is a higher alkyl, higher alkenyl or higher alkynyl radical of from six to fourteen carbon atoms, lower alkyl- of lower alkoxy-substituted aryl, or ⁇ -carboaloxy (higher alkyl) or less than 13 carbon atoms.
  • Particularly preferred are those compounds wherein X is tifluoromethyl and Y is a higher alkyl radical of from nine to fourteen carbon atoms.
  • X is S, SO, or S0 2 ; and each of " R and R is a member selected from the group consisting of alkyl radicals containing from 1 to 4 carbon atoms; and n is an integer from 1 to 2.
  • ⁇ and R 2 are members of the class consisting of hydrogen atoms and alkyl groups (for example, methyl, ethyl and propyl) ; R 3 and R 4 represent alkyl groups (for example, methyl, ethyl, propyl, butyl) ; and n represents an integer greater than 1 (n may conveniently represent, for example, 2, 3, 4 or 5).
  • the benzene nuclei may be substituted by alkyl or alkoxy groups. It should be understood that the designation:
  • CR 1 R 2 groups are identical, branched aliphatic chains in which successive CR ⁇ R groups are identical, and branched aliphatic chains in which successive CR ! R 2 groups may be different.
  • the CR ⁇ R designation includes the branched chain:
  • R is a hydrogen, chlorine or bromine atom, or a methyl or methoxy group in the 6- or 8- position
  • X is either a chlorine or a bromine atom in the 1- of 3- position
  • A is a divalent, straight or branched aliphatic chain containing from 2 to 5 carbon atoms
  • R ⁇ and R 2 are either individual methyl or ethyl groups or divalent groups which together with the adjacent nitrogen atom form a mononuclear heterocyclic ring.
  • A represents an alkylene group containing from 2 to 5 carbon atoms and R ⁇ ⁇ and R 2 each represent a methyl or ethyl group or together represent the atoms necessary to complete a pyrrolidine, piperidine or morpholine nucleus.
  • Y is a bivalent hydrocarbon radical of from 2 to 5 carbon atoms;
  • R is a lower alkyl group;
  • R' is a hydrogen or lower alkyl;
  • A is a member of the group consisting of saturated and unsaturated aliphatic hydrocarbon groups and hydroxyalkyl groups including ethers and esters of the hydroxy group.
  • Typical examples of the group represented by R are methyl, ethyl and propyl.
  • Representative of A are groups such as methyl, ethyl, allyl, propragyl, butenyl, 2-hydroxyethyl,
  • 2-carbamoylethyl and the like. Included in the group Y are ethylene, propylene, isopropylene, butylene, and the like. Methods for preparing these compounds are described in U.S. Patent No. 2,985,654. Representative thioxanthene compounds useful in practicing the methods of this invention are:
  • R is hydrogen or, when taken together with R , forms a single bond
  • R is selected from the group consisting of hydrogen and lower alkyl or, when taken together with R forms a single bond
  • R 2 is selected from the group consisting of hydrogen and lower alkyl
  • R 3 and R 4 when taken separately are selected from the group consisting of hydrogen and lower alkyl, and R 3 and R 4 when taken together with the nitrogen atom to which they are attached form a cyclic member selected from the group consisting of pyrrolidino, piperidino, morpholino and 4-lower alkylpiperazino
  • A is selected from the group consisting of dialkylamino , 4-alkylpiperaz inyl ,
  • X is a hydrogen, a halogen selected from the group consisting of chlorine, bromine, fluorine and iodine, a straight or branched chain alkyl radical having from 1 to about 4 carbon atoms, and a straight or branched chain alkoxy radical having with from 1 to 4 carbon atoms; and
  • R 1 is a tertiary amino group which advantageously is a dilower-alkylamino, 1-piperidyl, 1-pyrrolidyl or 4-morpholinyl group.
  • One or more of the hydrogens attached to the trimethylene side chain may be substituted by a lower alkyl group provided the total number of carbons in all substituent alkyl groups does not exceed 4.
  • one or more of the trimethylene hydrogens is substituted by an alkyl group, one of the alkyl substituents can be linked with R 1 to form a heterocyclic ring including the nitrogen atom.
  • This invention also includes salts of the -above defined bases formed with non-toxic " organic and inorganic acids.
  • Such salts are easily prepared by methods known to the art.
  • the base is reacted with either the calculated amount of organic or inorganic acid in aqueous miscible solvent, such as acetone or ethanol, with isolation of the salt by concentration and cooling or an excess of the acid in aqueous immiscible solvent, such as thyl ether or chloroform, with the desired salt separating directly.
  • aqueous miscible solvent such as acetone or ethanol
  • organic salts are those with maleic, enesalicyclic, methysulfonic, ethansesulfonic, acetic, propionic, artaric, salicyclic, citric, gluconic, lactic, malic, mandelic, cinnamic, citraconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzane sulfonic and theophylline acetic acids as well as with the 8-halotheophyllines, for example, 8-chlorotheophylline and 8-bromotheophylline.
  • 8-halotheophyllines for example, 8-chlorotheophylline and 8-bromotheophylline.
  • inorganic salts are those with hydrochloric, hydrobromic, sufuric, sulfamic, phosphoric and nitric acids.
  • these salts may also be prepared by the classical method of dobule decomposition of appropriate salts which is well known in the art.
  • this invention includes the use of enantiomers, homologs and isomers of the above-identified compounds.
  • These compounds may be formulated into pharmaceutical dosage forms by well recognized methods.
  • the compounds described above may be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • a pharmaceutical formulation comprising one of the above compounds and a pharmaceutically acceptable carrier.
  • One or more of these compounds may be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants and if desired other active ingredients.
  • compositions containing these compounds may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and dinintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose , sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylen oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorb
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, olive oil, sesame oil or coconut oil, or in a mineral such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations.
  • These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
  • Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate.
  • the emulsions may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or-diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the above described compounds may also be administered in the form of suppositories for rectal administration of the drug-.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the above-identified compounds may be administered parenterally in a sterile medium.
  • The"drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
  • Dosage levels of the order of from about 0.1 mg to about 2500 mg per day are useful in the treatment of the above-indicated.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage for will vary depending upon the host treated and the particular mode of administration.
  • Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.
  • Preferred compounds are: Compound Name
  • Triflupromazine N,N-Dimethyl-2- (trifluoromethyl) -1 OH- phenothiazine-10-propanamine
  • Chlorpromazine 2-Chloro-N,N-dimethyl-10H- phenothiazine-10-propanamine
  • Okadaic acid was obtained from Moana Biochemical (Honolulu) , anisomycin was obtained from Sigma, and alkaline phosphatase was obtained from Boehringer Mannheim. 32 P-orthophosphoric acid was purchased from NEN, and 35 S-l-methionine was purchased from ICN Biomedicals.
  • RPMI 1640 medium for routine growth of MSN cells, and phosphate-free and methionine-free RPMI media were obtained from GIBCO BRL.
  • Antibodies T3P and tau46 were provided by Dr. Virginia Lee of the University of Pennsylvania. Monoclonal antibodies 188 and 147 were raised against highly enriched pair helical filament preparations and were provided by Abbott Laboratories. Ell is a polyclonal antiserum against the human exon 11 peptide of tau and was provided by Dr. Hana Kseizak-Reding of Albert Einstein College of Medicine of Yeshiva University. Tau-l was obtained from Dr. Lester Binder of UAB.
  • SMS-MSM-A human neuroblastoma cell line was cloned from a population of SMS-MSN cells obtained from the laboratory of Dr. June Biedler (Sloan-Kettering, New York, NY) , and was deposited with the American Type Culture Collection in Rockville, Maryland on February 2, 1993, and catalogued as ATCC # CRL 11253.
  • the deposited human neuroblastoma cell line was tested for viability and determined viable on February 9, 1993.
  • the neuroblastoma cells were grown in T75 flasks in RPMI 1640 medium supplemented with 15% fetal calf serum.
  • OkA was dissolved in dimethyl sulfoxide (DMSO) and aliquots were added to Krebs-Ringer- phosphate buffer (KRP) containing 0.122 M NaCl, 4.9 mM KC1, 1.3 mM CaCl 2 , 1.6 mM Na 2 HP0 4 , and 10 mM dextrose (pH 7.4) to produce the required concentration. Control samples were incubated with similar aliquots of the DMSO vehicle, and the final concentration of DMSO was maintained below 0.1%. Before incubation, the growth medium was aspirated off from each flask, and replaced with KRP containing either vehicle or OkA. The flasks were incubated at 37°C for the appropriate durations.
  • KRP Krebs-Ringer- phosphate buffer
  • MSN cells were pelleted, resuspended in 250 ⁇ l cold Tris-buffered saline (TBS) containing 4 mM phenylmethylsulfonyl fluoride (PMSF) , 2 mM EGTA, and 25 ⁇ M leupeptin, and disrupted by sonicating with a 2 second pulse.
  • PMSF phenylmethylsulfonyl fluoride
  • 2 mM EGTA 2 mM EGTA
  • 25 ⁇ M leupeptin 25 ⁇ M leupeptin
  • the flasks were rinsed with cold Tris buffered saline (TBS) , and the entire cell suspension was centrifuged at 3000 g for 5 minutes in order to collect the cells.
  • TBS Tris buffered saline
  • the cell pellets were resuspended in 250 ⁇ l TBS containing 4 mM PMSF and 2 mM EGTA, and boiled for 10 minutes.
  • the heat stable proteins were assayed for protein by a micro BioRad procedure.
  • Electrophoresis and Blotting Aliquots of heat-treated supernatant containing 20 ⁇ g protein were boiled with electrophoresis sample buffer and loaded onto mini 10% SDS-PAGE gels. The gels were electrophoretically transferred to nitrocellulose membrane and immunolabelled as previously described (Vincent, I. and Davies, P. PNAS USA 87:4840-4844 (1990) ) .
  • MSN cells were preincubated at 37°C for 1 hour with KRP in the absence or presence of 100 mM aniso ycin. At the end of this time, 1 ⁇ M OkA was introduced into the appropriate flasks, and the samples incubated a further 90 minutes. Samples were then prepared for Western blot analysis according to routine protocol.
  • the human neuroblastoma cell line SMS-MSN expresses tau protein as a doublet of apparent molecular weights 57 and 60 kDa. These proteins were detected with antibodies Alz-50, Tau-l, PHF-1, NP8 and T3P when 20 ⁇ g of heat-stable protein was utilized for SDS-PAGE and Western blot analysis (0 minute control, Figure 1) .
  • MSN cells with OkA As a first step in investigating the role of protein phosphatases on these various epitopes, MSN cells with OkA, a cell- permeable protein phosphatase inhibitor (Bialojan, C. and Takai, A. J. Biochem. 256:283-290 (1988)).
  • the monoclonal antibody 188 which recognizes primary sequence in tau was used as a negative control in these experiments. While the 0.1% dimethyl sulfoxide (DMSO) vehicle produced no changes (see 4 hour control, Figure 1) , a 30 minute exposure to 1 ⁇ M OkA resulted in a marked induction of the phosphorylated tau epitopes, PHF-1, NP8, and T3P. In addition, OkA treatment induced the appearance of PHF-1, NP8, and T3P epitopes on several higher molecular weight proteins. Both PHI and T3P reactivities were observed with a 68-kDa protein ( Figure 1) .
  • DMSO dimethyl sulfoxide
  • Figure 3 shows the effects of 100 ⁇ M anisomycin, a protein synthesis inhibitor, on the OkA-induced effects on tau. Consistent with their rapidity, the increases in immunoractivity of Alz-50, 188, PHF-1, NP8, or T3P and the shift in molecular weight do not require protein synthesis. Moreover, the disappearance of Tau-l immunoreactivity that occurred as a result of OkA treatment was also observed in the presence of anisomycin ( Figure 3) .
  • MSN cells were labelled with 32 P-orthophosphate according to two independent protocols.
  • the cells were pulse-labeled and subsequently incubated with KRP with or without OkA ( Figure 5A) .
  • the presence of OkA in the chase produced a substantial accumulation of label in tau in comparison with cells chased with KRP alone.
  • the corresponding immunoblot reveals that Alz-50 immunoreactivity with tau was barely detected when the label was lost in the chase, but was greatly enhanced when incorporated phosphate was retained in the presence of OkA.
  • phosphatase 1 0.1-0.5 mM
  • phosphatase 2A is 1 nM
  • the IC-50 for phosphatase 2B i.e., the Ca 2+ /CAM-dependent phosphatase, Calcineurin
  • phosphatase 2C i.e., the Mg 2+ -dependent phosphatase
  • Optimal inductions in PHF-1, NP8, and T3P immunoreactivities occur at 50 nM, a concentration 50-fold that of the IC-50 for phosphatase 2A, making it likely that phosphatase 2A plays an important role in turnover of these epitopes.
  • this concentration is 2-10 fold less than the IC-50 for phosphatase 2B, it is unlikely that either of these phosphatases play role in regulation of these epitopes.
  • Phosphorylation at the Tau-l site is favored at or over 1 ⁇ M, although some inhibition in Tau-l immunoreactivity is observed at 50 nM OkA.
  • it is more likely that phosphatase 1 is involved in turnover of the phosphatase at the Tau-l site, although a role for phosphatase 2A cannot be excluded.
  • phosphatase 2A The suggested role of phosphatase 2A in the formation of the PHF-1, T3P and NP8 epitopes in MSN cells indicates that this phosphatase is crucial in determining the phosphorylation state of both normal and paired helical filament-like epitopes in tau, and may be an important enzyme in tau metabolism.
  • the immunoreactivity of monoclonal antibodies Alz- 50, PHF-1, TG3, TG4, MC2, MC6, and MC15 with normal and Alzheimer's brain tissue, and with MSN cells before (MSN-) and after treatment (MSN+) with okadaic acid was examined, and the results are presented in Figure 7.
  • the data from brain tissue are the mean values derived from studies of mid-temporal cortex from five normal cases and five cases of Alzheimer's Disease.
  • Values from MSN cells are from a single experimental series in which 1 micromolar okadaic acid was added to triplicate MSN cell cultures for 90 minutes prior to harvest of the cells. Each sample (tissue or cell homogenate) was assayed at 8 dilutions in duplicate.
  • the units on the Y axis are arbitrary units of immunoreactivity. In all cases, it is clear that okadaic acid produces increases in immunoreactivity in MSN cells, and that this increase is similar in extent to the difference between normal and Alzheimer's brain tissue.
  • MSN-A cells expressing PHF epitopes were incubated with 100 ⁇ M Trifluoperazine (TFP) , 100 ⁇ M Chlorpromazine (CPZ) or 0.2% DMSO vehicle (control) for 2 hours at 37°C.
  • the cells were isolated by centrifugation, boiled for 10 minutes and 25 ⁇ g protein from the resulting heat stable supernatants were loaded per lane on an SDS-PAGE gel. The gel was electrophoretically transferred to nitrocellulose membrane and then immunostained with PHF-1.
  • both TFP and CPZ greatly decreased the production of paired PHF epitopes by the neuroblastoma cells.
  • the control shows that without the addition of TFP or CPZ, there was production of PHF epitopes by the neuroblastoma cells.
  • Frontal and cortical brain tissue specimens were obtained at autopsy from chronic schizophrenic patients chronically treated with chlorpromazine and analyzed using the
  • the brain tissue from approximately the same number of (age matched) normal controls and
  • Alzheimer's disease patients were also analyzed and are presented in the Figures as negative and positive controls

Abstract

On décrit des procédés qui permettent de prévenir ou traiter la maladie d'Alzheimer et consistent à administrer à un patient une quantité d'une phénothiazine ou d'un thioxanthène efficace pour prévenir ou diminuer l'accumulation d'épitopes anormalement phosphorylés, à filaments hélicoïdaux appariés, qui sont associés à la maladie d'Alzheimer.
EP95929441A 1994-08-08 1995-08-07 Procedes permettant de traiter et/ou de prevenir la maladie d'alzheimer a l'aide de phenothiazines et/ou de thioxanthenes Withdrawn EP0778773A1 (fr)

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PCT/US1995/010110 WO1996004915A1 (fr) 1994-08-08 1995-08-07 Procedes permettant de traiter et/ou de prevenir la maladie d'alzheimer a l'aide de phenothiazines et/ou de thioxanthenes

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GB9506197D0 (en) 1995-03-27 1995-05-17 Hoffmann La Roche Inhibition of tau-tau association.
GB9713484D0 (en) * 1997-06-27 1997-09-03 Smithkline Beecham Plc Neuroprotective vanilloid compounds
DE19842416A1 (de) * 1998-09-16 2000-04-13 Max Planck Gesellschaft Sekundäre Amine zur Prävention und Therapie von Erkrankungen, die durch Oxidationsprozesse verursacht oder verstärkt werden
GB0017060D0 (en) * 2000-07-11 2000-08-30 Hunter Fleming Ltd Production, stabilisation and use of reduced forms of pharmaceutical compounds
GB0100119D0 (en) 2001-01-03 2001-02-14 Univ Aberdeen Materials and methods relating to protein aggregation in neurodegenerative disease
GB0101049D0 (en) 2001-01-15 2001-02-28 Univ Aberdeen Materials and methods relating to protein aggregation in neurodegenerative disease
GB0106953D0 (en) * 2001-03-20 2001-05-09 Univ Aberdeen Neufofibrillary labels
GB0117326D0 (en) 2001-07-16 2001-09-05 Univ Aberdeen Napthoquinone-type inhibitors of protein aggregation
PT1470818E (pt) * 2003-04-25 2006-11-30 Neuro3D Utilização de derivados de fenotiazina piperazina no fabrico de um medicamento com efeitos neuroprotectores e/ou neurotróficos no cns e/ou pns
JP5186212B2 (ja) 2004-09-23 2013-04-17 ウィスタ ラボラトリーズ リミテッド メチルチオニニウム塩化物（ｍｔｃ）などのジアミノフェノチアジニウム化合物を化学合成および精製する方法
EP1836161B1 (fr) 2004-12-22 2016-07-20 BHI Limited Partnership Methodes et compositions de traitement de maladies liees a l'amyloide
CA2645919C (fr) * 2006-03-29 2016-05-31 Wista Laboratories Ltd. Composes de thioninium et utilisation
ATE478058T3 (de) 2006-03-29 2010-09-15 Wista Lab Ltd 3,7-diamino-10h-phenothiazinsalze und ihre verwendung
DK2004155T3 (en) 2006-03-29 2018-04-30 Wista Lab Ltd PROTEIN AGGREGATION INHIBITORS
PT3851447T (pt) 2006-10-12 2023-12-11 Bellus Health Inc Métodos, compostos, composições e veículos para administrar ácido 3-amino-1-propanossulfónico
GB0701970D0 (en) * 2007-02-01 2007-03-14 Wilson Stuart Treatment of protein aggregation diseases
ES2331282B1 (es) * 2008-06-25 2010-10-21 Consejo Superior De Investigaciones Cientificas (Csic) (45%) Hidrazidas de sistemas heterociclicos y su uso en el tratamiento de enfermedades neurodegenerativas.
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