WO2019216589A1 - Composition pour la prévention ou le traitement d'une maladie liée à tau - Google Patents

Composition pour la prévention ou le traitement d'une maladie liée à tau Download PDF

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WO2019216589A1
WO2019216589A1 PCT/KR2019/005180 KR2019005180W WO2019216589A1 WO 2019216589 A1 WO2019216589 A1 WO 2019216589A1 KR 2019005180 W KR2019005180 W KR 2019005180W WO 2019216589 A1 WO2019216589 A1 WO 2019216589A1
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tau
compound
aggregation
levosimendan
treatment
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Korean (ko)
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배애님
임성수
김도희
임상민
김윤경
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한국과학기술연구원
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Priority claimed from KR1020190050228A external-priority patent/KR102317080B1/ko
Publication of WO2019216589A1 publication Critical patent/WO2019216589A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • 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
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • a pharmaceutical composition comprising a compound, an optically active isomer thereof, or a pharmaceutically acceptable salt thereof, for use in the prophylaxis and / or treatment of a disease caused by aggregation or phosphorylation of tau.
  • Tau is a microtubule-associated protein (MAP) mainly expressed in axons of neurons with molecular weights of 50,000 to 70,000. It is responsible for stabilizing microtubules (MT) and molecular diversity by phosphorylation. Indicates. In humans, tau produces six isoforms by insertion of 29 or 58 amino acid residues at the N-terminus, or by mRNA splicing of three or four repeats (called microtubule-binding sites) at the C terminus. do.
  • MAP microtubule-associated protein
  • tau stabilizes microtubules by promoting growth from axons and polarization of nerve cells.
  • tau separates from microtubules and induces insoluble aggregation (Gendron TF, Petrucelli L (2009) The role of tau in neurodegeneration. Mol. Neurodegener. 4: 13).
  • Structural frameworks that induce tau aggregation have been proposed.
  • Evidence has been provided that insoluble filaments form from the ten soluble monomers and that these filaments bind into a high dimensional structure called neurofibrillary tangles (NFTs).
  • NFTs neurofibrillary tangles
  • the binding affinity of tau to microtubules is also actively regulated by phosphorylation, which leads to dynamic rearrangement of the microtubule network. Abnormally excessive phosphorylation of tau hinders the balancing of these dynamic rearrangements and dramatically reduces affinity for microtubules (Drewes G, Trinczek B, Illenberger S, Biernat J, Schmitt-Ulms G, et al. (1995) Microtubule-associated protein / microtubule affinity-regulating kinase (p110mark) .A novel protein kinase that regulates taumicrotubule interactions and dynamic instability by phosphorylation at the Alzheimer-specific site serine 262. J. Biol. Chem.
  • Tauopathy is a neurodegenerative disorder caused by abnormal accumulation of tau protein in the phosphorylation and aggregation of tau protein. It is regarded as a cause of various degenerative brain diseases. Aggregates of tau protein found in patients with tau disease are found primarily in the cell bodies and dendrites of neurons, which are called neurofibrillary tangles (NFTs) and neuropil threads. In neurofibrillary vines, the tau protein is composed of paired helical filaments (PHFs), which are aggregated and hyperphosphorylated unlike normal tau protein.
  • PHFs paired helical filaments
  • tau-related diseases include Alzheimer's disease, Parkinson's disease, tauopathy, vascular dementia, acute stroke, trauma, cerebrovascular disease, brain cord trauma, spinal cord trauma, peripheral neuropathy, retinopathy, glaucoma, Progressive nucleus palsy, cortical-basal nucleus degeneration, Argyrophilic Grain Disease, peak disease and hereditary frontal lobe dementia.
  • tau protein-mediated diseases specifically signal and are toxic have not been elucidated.
  • tau protein-mediated diseases specifically signal and are toxic have not been elucidated.
  • tau-related diseases include Alzheimer's disease, Parkinson's disease, tauopathy, vascular dementia, acute stroke, trauma, cerebrovascular disease, brain cord trauma, spinal cord trauma, peripheral neuropathy, retinopathy, glaucoma, Progressive nucleus palsy, cortical-basal nucleus degeneration, Argyrophilic Grain Disease, peak disease and hereditary frontal lobe dementia.
  • the present inventors are studying various dozens of compounds and combinations thereof in order to select new tau aggregation inhibitors, and a combination of compounds having different combinations of levosimendan and rivastigmine After confirming that the effect of reducing tau aggregation and phosphorylation is significantly better, the present invention was completed.
  • the present invention has been made to solve the above problems, and an object of the present invention is to provide a combination of compounds having a significantly superior tau aggregation inhibitory effect than when levocymendan is used alone.
  • the present invention provides a combination of new compounds having excellent tau aggregation inhibitory effect and no cytotoxicity among various compound combinations, which can be effectively used for the prevention and / or treatment of tau related diseases including neurodegenerative diseases.
  • tau related diseases including neurodegenerative diseases.
  • One embodiment of the present invention is a composition for the prevention or treatment of tau aggregation-related diseases, including levosimendan and rivastigmine.
  • the lebocymendan is a lebocymendan compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.
  • the rivastigmine is a rivastigmine compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.
  • composition according to the present invention consisting of donepezil (donepezil), galantamine (galantamine), memantine (Memantine), tacrine (Tacrine), curcumin (Curcumin), dopamine (Dopamine) and lithium chloride (LiCl) It may further comprise at least one compound selected from the group.
  • composition according to the present invention may further comprise an active agent selected from the group consisting of antioxidants, cholinesterase inhibitors and combinations thereof.
  • tau aggregation-related diseases include Alzheimer's disease, Parkinson's disease, tauopathy, vascular dementia, acute stroke, trauma, cerebrovascular disease, brain cord trauma, spinal cord trauma, peripheral neuropathy, retinopathy, and It may be selected from the group consisting of glaucoma.
  • the tau disease may be selected from the group consisting of Alzheimer's disease, advanced nuclear palsy, cortical-basal nucleus degeneration, Argyrophilic Grain Disease, peak disease and hereditary prefrontal dementia.
  • Another embodiment of the present invention is a dietary supplement for the prevention or amelioration of tau agglutination related diseases, including levosimendan and rivastigmine.
  • Another embodiment of the invention is a method comprising administering Levosimendan and Rivastigmine.
  • Levosimendan and Rivastigmine may be a method for preventing or treating tau aggregation-related diseases.
  • Administration to mammals, animals, humans, and / or non-humans is possible.
  • the order of administration of the lebocymendan and rivastigmine is irrelevant and can be administered continuously or simultaneously.
  • the administration method is also not particularly limited, and methods such as oral administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intraperitoneal injection and intramuscular injection can be used.
  • Another embodiment of the invention is a compound for the prophylaxis or treatment of tau aggregation related diseases, including Levosimendan and Rivastigmine.
  • Compounds containing lebocymendan and rivastigmine are suitable for use as a compound for the prevention or treatment of tau aggregation-associated diseases because of their excellent tau aggregation inhibitory effect.
  • Another embodiment of the invention is the use of levosimendan and Rivastigmine compounds as tau aggregation inhibitors.
  • the present invention is intended for use in the preparation of therapeutic agents for tau aggregation related diseases of levocimendane and rivastigmine compounds.
  • the present invention may be used for the manufacture of a medicament for the prevention or treatment of diseases associated with tau aggregation of levocimendan and rivastimin compounds.
  • the present invention is characterized by including both levosimendan (Levosimendan) and rivastigmine (Rivastigmine), the tau aggregation inhibitory effect is remarkably superior than when using the levosimendan alone.
  • the combination of levocimendane and ribastikimin according to the present invention has better tau aggregation inhibitory effect and no cytotoxicity than the combination of other compounds, and thus can be effectively used for the prevention and / or treatment of tau related diseases including neurodegenerative diseases. Can be.
  • 1 is a drug library screening result of the levocimendane compound according to an embodiment of the present invention.
  • Figure 2 is a result of measuring the tau aggregation inhibitory effect and cytotoxicity of the levocimendane compound according to an embodiment of the present invention.
  • Figure 3 is the result of measuring the concentration-dependent intracellular tau aggregation inhibitory effect of the levocimendane compound according to an embodiment of the present invention.
  • FIG. 6 shows the results of measuring the effects of direct tau aggregation inhibition of levocimendane compounds in vitro, according to an embodiment of the present invention.
  • Figure 7 is the result of measuring the tau aggregate resolution of the levocimendane compound in vitro, in accordance with an embodiment of the present invention.
  • Passive avoidance test results confirmed the effect of improving the cognitive function of the levosimendan compound using a tauopathy animal model according to an embodiment of the present invention.
  • FIG. 11 is an optical micrograph of a brain tissue section confirming neurodegeneration inhibitory effect of the lebocymendan compound using a tauopathy animal model and the number of neurons included in the brain tissue section according to an embodiment of the present invention. It is a graph.
  • FIG. 12 shows donepezil, tacrine, rivastigmine, galantamine (concentration) for each of levocimendan and methylene blue, according to an embodiment of the present invention.
  • Intracellular tau aggregation inhibition was measured after a combination of galantamine, memantine, dopamine, documin, curcumin, and lithium chloride (LiCl).
  • FIG. 13 illustrates donepezil, tacrine, rivastigmine, and galantamine (concentration) for each of levocymendan and methylene blue, according to an embodiment of the present invention.
  • Cell viability was measured after co-treatment with galantamine, memantine, memantine, dopamine, curcumin, and lithium chloride (LiCl).
  • Figure 14 according to an embodiment of the present invention, for each of the concentration of levocimendane compound and methylene blue (Rivastigmine) in combination with concentration, after intracellular tau aggregation inhibition and cells Toxicity is measured.
  • first and second may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.
  • the present invention relates to a compound such as Levosimendan, a pharmaceutically acceptable salt, stereoisomer or solvate, derivative thereof for use in a method for preventing or treating tau pathology or its symptoms. will be.
  • Levosimendan is a calcium sensitiser known as a drug used to treat congestive heart failure.
  • the drug increases contractility by binding to Troponin C in the heart and increasing Ca 2+ muscle residue.
  • K ATP pathway of cardiomyocytes it acts as a vasodilator with anti-ischemic and antistunning effects (In-Chul Choi (2006) 519-27).
  • Levosimendan compound is useful for the prevention and / or treatment of diseases caused by aggregation or phosphorylation of tau.
  • Drug repositioning refers to attempts to review existing drugs and drugs and apply them to new indications (diseases), which can reduce the time and cost of drug development, and drug toxicity and clinical research failures. Is low and is being used as a new drug development strategy.
  • the present invention is characterized in that the levosimendan compound applied to the aggregation or phosphorylation of tau as part of the new drug production.
  • CDK5 Cyclin-dependent kinase 5
  • PPE propentofylline
  • N-phenylamines N-phenylamines
  • quinoxalines methylene blue.
  • methylene blue is known to be effective in inhibiting tau protein aggregation.
  • the methylene blue was used as a comparative example in the following examples, it was found that the cytotoxicity is higher than the levosimendan compound according to the present invention (see Figure 2).
  • the levosimendan compound may be represented by the following formula (1).
  • the compound is used in the sense of including the compound itself, pharmaceutically acceptable salts, hydrates, solvates, isomers and prodrugs thereof.
  • the compound is characterized by inhibiting tau aggregation and phosphorylation, and does not affect the increase or decrease of the activity of tau kinase. In addition, it is characterized in that it prevents further coagulation of the tau in which coagulation proceeds and decomposes coagulation of the tau in which coagulation is advanced.
  • the levocymendan compound when the levocymendan compound is treated to a cell line treated with a compound (Forskolin) that induces tau aggregation, the tau aggregation inhibitory effect (see FIG. 3), tau phosphorylation reduction effect (See FIG. 4), and neuronal protective effect (see FIG. 5).
  • a compound Forskolin
  • tau aggregation inhibitory effect see FIG. 3
  • tau phosphorylation reduction effect See FIG. 4
  • neuronal protective effect see FIG. 5
  • levosimendan was directly involved in the tau aggregation process and showed an inhibitory effect (see FIGS. 6 and 7). It was confirmed that it does not have (see Fig. 8).
  • one embodiment of the present invention is a composition for preventing or treating tau aggregation-related diseases, including levosimendan and rivastigmine.
  • the rivastigmine compound may be represented by the following formula (2).
  • the lebocymendan is a lebocymendan compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.
  • the rivastigmine is also a rivastigmine compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.
  • the combination of the levosimendan (Levo.) (3 ⁇ M) compound with rivastigmine or lithium chloride (LiCl) was superior to that of the levosimendane compound alone. It was confirmed that it had an aggregation inhibitory effect (see FIG. 12). As a result of confirming that the tau aggregation inhibitory effect is due to cytotoxicity, the synergistic effect of tau aggregation inhibition shown in the combination treatment of the levocymendan compound (3 ⁇ M) and the lithium chloride compound (5, 10, 20 mM) was determined according to the cytotoxicity of the lithium chloride compound (see FIG. 13).
  • the concentration of each of the levocimendane compound, after treatment with ribastikimin for each concentration, after measuring the inhibition of tau aggregation and cytotoxicity in the cell the tau according to the concentration of each of the levocimendan and ribastigmine It was confirmed that the aggregation inhibitory effect increased in a concentration dependent manner. Therefore, when the combination treatment with levosimendan and rivastigmine, it was confirmed that the tau inhibitory effect has a synergistic effect, which is significantly better than when administered alone (see Fig. 14).
  • the present invention is characterized by including both levocimendane and rivastigmine, and the tau aggregation inhibitory effect is remarkably superior to when levocimendan is used alone.
  • the combination of levocimendane and ribastikimin according to the present invention has better tau aggregation inhibitory effect and no cytotoxicity than the combination of other compounds, and thus can be effectively used for the prevention and / or treatment of tau related diseases including neurodegenerative diseases. Can be.
  • composition according to the present invention is made of donepezil, galantamine, memantine, tacrine, curcumin, dopamine and lithium chloride (LiCl). It may further comprise at least one compound selected from the group.
  • the present invention is characterized by including levocimendane and rivastigmine together, and having excellent tau aggregation inhibitory effect and no cytotoxicity.
  • levocimendane when treated with levocimendane in combination with donepezil, galantamine, memantine, tacrine, curcumin, dopamine and lithium chloride (LiCl) , It was possible to inhibit tau aggregation induced by the treatment of the metastatic tau protein TauK18P301L (see FIG. 12).
  • the present invention basically includes levocimendane and rivastigmin, plus donepezil, galantamine, memantine, tacrine, curcumin, dopamine ( Dopamine) and lithium chloride (LiCl) even in the case of further comprising at least one compound selected from the group, it is shown that it is possible to effectively suppress tau aggregation.
  • the present invention is for the prevention or treatment of tau aggregation-related diseases comprising the compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound and at least one pharmaceutically acceptable carrier. It relates to a composition.
  • composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above compounds as an active ingredient.
  • Pharmaceutically acceptable carriers include diluents, lubricants, lubricants, binders, disintegrants, sweeteners, dispersants, surfactants, preservatives, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol , Liposomes and one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, bacteriostatics, flavorings, vitamins and the like may be added as necessary. It may also be formulated into solutions, suspensions, powders, granules, pills, capsules, or tablets, and target organ specific antibodies or other ligands may be used in combination with the carrier to specifically act on the target organ. .
  • the present invention may further comprise an activator selected from the group consisting of antioxidants, cholinesterase inhibitors and combinations thereof.
  • the antioxidants include ⁇ -tocopherol, vitamin C (Ascorbic aicd), vitamin C palmitate, butylhydroxyanisole, dibutylhydroxytoluene, citric acid, erythorbic acid, fumaric acid, malic Acids, Maltodextrins, Potassium Metabisulfide, Sodium Metabiasulfate, Propionic Acid, Propyl Gallate, Sodium Ascorbate, Sodium Sulfate, Tymol, Cyclodextrin or Sulfo
  • One or more types can be mixed and used from the group which consists of butyl ether (beta) -cyclodextrin, etc.
  • the cholinesterase inhibitor is a substance that inhibits the action of cholinesterase and inhibits hydrolysis of acetylcholine in vivo, and is involved in causing donation enhancement of choline working nerves (parasympathetic nerves, motor nerves), for example.
  • Donepezil Donepezil
  • rivastigmin Rostigmin
  • Galantamine Galantamine
  • Butylcholinesterase Penserine (Phenserine)
  • Huperzin Huperzin A
  • Huperzin A can be used in combination of one or more.
  • the present invention is a composition for the prevention or treatment of tau agglutination related diseases, comprising the above compounds, having excellent tau aggregation inhibitory activity, Alzheimer's disease, Parkinson's disease, tauopathy, vascular dementia, It is useful as an active ingredient of a medicament for the prophylaxis and / or treatment of one or more diseases selected from the group consisting of acute stroke, trauma, cerebrovascular disease, brain cord trauma, spinal cord trauma, peripheral neuropathy, retinopathy, and glaucoma.
  • the tau aggregation-related diseases include Alzheimer's disease, Parkinson's disease, tauopathy, vascular dementia, acute stroke, trauma, cerebrovascular disease, brain cord trauma, spinal cord trauma, peripheral neuropathy, retinopathy, and glaucoma.
  • the tau disease may be selected from the group consisting of Alzheimer's disease, advanced nuclear palsy, cortical-basal nucleus degeneration, Argyrophilic Grain Disease, peak disease, and hereditary prefrontal dementia.
  • the present invention relates to a method for inhibiting tau aggregation, which comprises treating the compound or a derivative thereof.
  • the levocymendan and rivastigmine compounds may be treated with cell lines in vitro to inhibit hyperphosphorylation of tau.
  • the present inventors first confirmed that the treatment of the levocymendan compound in vitro inhibits the overphosphorylation of the tau, and the effect of the combination of the lebocymendan and ribastikimin compound is more effective than the lebocymendan alone administration. Newly learned that it is excellent.
  • the present invention relates to a dietary supplement for the prevention or amelioration of tau agglutination related diseases, comprising the compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound. That is, another embodiment of the present invention is a dietary supplement for preventing or ameliorating tau aggregation-related diseases, including levosimendan and rivastigmine.
  • the dietary supplement may be formulated in the form of a conventional dietary supplement known in the art.
  • a conventional dietary supplement known in the art.
  • it may be prepared in the form of various foods, beverages, gums, teas, vitamin complexes, dietary supplements, powders, powders, granules, tablets, capsules, suspensions, emulsions, syrups, teas, jelly, or beverages.
  • it may additionally contain additional raw materials and components.
  • disaccharides for example, disaccharides, polysaccharides, dextrins, cyclodextrins, various nutrients, vitamins, electrolytes, flavors, colorants and flavor enhancers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusters, stabilizers, preservatives, glycerin, carbonation agents and the like.
  • the Tau-BiFC cell model is a Venus protein-based BiFC method in which the N-terminal and C-terminal non-fluorescent portions of Venus protein are fused to tau as a reliable system for monitoring tau interactions in oligomeric form.
  • the Tau-BiFC cell model is a Venus protein-based BiFC method in which the N-terminal and C-terminal non-fluorescent portions of Venus protein are fused to tau as a reliable system for monitoring tau interactions in oligomeric form.
  • tau-BiFC cells were first plated in 384 well plates. The following day, tau-BiFC cells were treated with FDA-approved drugs (treatment concentration 10 uM) along with Forskolin (treatment concentration 30 uM), a compound that activates tau kinase PKA to induce tau aggregation. After 46 hours, the cell viability was analyzed by compound nuclear staining using Hoechst, along with the degree of inhibition of tau aggregation of the drugs.
  • the drug Levosimendan which exhibits the cell-like viability of the normal state (Tau-BiFC off ) without the drug treatment, has been shown to suppress the tau aggregation effect of Methylene blue (MB), which is known to exhibit the aggregation inhibitory effect. Selected (FIG. 1).
  • DMSO, MB, Levosimendan treatment concentration 10 uM
  • Forskolin treatment concentration 30 uM
  • MB Methylene blue
  • the compound Levosimendnan with low cytotoxicity was selected as a tau aggregation inhibitor while showing an intracellular tau aggregation inhibitory effect similar to that of MB.
  • Tau-BiFC cells were treated with Forskolin (treatment concentration 30 uM) and Levosimendan compound (treatment concentration 15 uM), which are tau aggregation inducers. After 36 hours, tau phosphorylated Western blot was performed with tau-BiFC cell lysate.
  • Example 1-3 confirming neuronal protective effect
  • Levosimendan compound leads to neuroprotective effect in the situation of neuronal damage.
  • Levosimendan compound treatment concentration 10 uM
  • Tau protein TauK18P301L treatment concentration 20ug / mL
  • Tau protein TauK18P301L treatment concentration 20ug / mL
  • the experimental group (Levosimendan) had less nerve cell and neurite length damage than the negative control group (DMSO) and the positive control group (MB).
  • the shape of the neurons and the neurite length were similar to those of the control. That is, it was confirmed that the Levosimendan compound has a protective effect on neuronal cell damage caused by TauK18P301L.
  • ThS Thioflavin S
  • the Levosimendan compound confirmed a direct tau aggregation inhibitory effect in vitro.
  • the IC50 of the Levosimendan compound was 4.71 uM.
  • Levosimendan compounds have two inactivated (OR-1855) or activated (OR-1896) metabolite forms. It is known that OR-1896 compound, which has undergone the acetyl process of OR-1855 compound, remains in the body for a long time and shows effects of drugs. Therefore, the Levosimendan compound and two metabolites were treated with tau-BiFC cells to confirm which cells showed more tau aggregation inhibitory effect.
  • MB Levosimendan, OR-1855, OR-1896 compounds were treated in tau-BiFC cells with forskolin (treatment concentration 30 uM), which is a tau aggregation inducer for each concentration. The degree of tau aggregation after 48 hours was analyzed.
  • the experimental group (Levosimendan) reduced tau aggregation similar to the positive control group (MB).
  • Levosimendan compound showed a concentration-dependent tau aggregation inhibitory effect.
  • OR-1855 and OR-1896 compounds did not show tau aggregation inhibition. Therefore, the metabolite form of Levosimendan does not show tau aggregation inhibitory effect in cells.
  • Example 1-6 confirming the effect of improving the cognitive function of the Levosimendan compound derivative
  • Animal model was used to determine whether the tau aggregation inhibitory effect of Levosimendan compound also affects the improvement of cognitive function.
  • the animal model was performed based on a Tau-P301L BiFC transgenic mouse showing tau disease, and specifically, a mouse described in Korean Patent Registration No. 10-1876423 (name of the invention: a dementia model transgenic mouse and a screening method using the same). Was used.
  • Levosimendan compound positive control (MB) and negative control (DMSO) were dissolved in physiological saline of 30% PEG 400, which was then administered to a 9-month mouse at a dose of 5 mg / kg (body weight) three times a week for 3 months. Intraperitoneal administration. In order to measure the degree of cognitive improvement due to the administration of the Levosimendan compound, a novel object recognition test and a passive avoidance test were performed three months after drug administration.
  • FIG. 9 is a novel object recognition test (Novel object recognition test) results confirmed the effect of improving the cognitive function of the levosimendan compound using a tau disease animal model according to an embodiment of the present invention. As a result, as shown in Figure 9 it was confirmed that the experimental group administered the Levosimendan compound has a high recognition index for the new object compared to the negative control group or the positive control group (MB).
  • MB positive control group
  • the mouse is placed in the brightly spaced A space in the passive avoidance test device, and after about 30 seconds of search time, the door is opened to enter the space B where the mouse remains dark. Made it possible.
  • the entrance is closed and 3mA electric shock is flowed through the bottom grid for 2 seconds so that the mouse can learn the dark B space and the electric shock.
  • the mouse was placed in the A space again and after 30 seconds the time taken to cross over to the dark side when the door was opened was measured up to 540 seconds.
  • FIG. 10 is a passive avoidance test (Passive avoidance test) results confirmed the effect of improving the cognitive function of the levosimendan compound using a tauopathy animal model according to an embodiment of the present invention.
  • Passive avoidance test results confirmed the effect of improving the cognitive function of the levosimendan compound using a tauopathy animal model according to an embodiment of the present invention.
  • FIG. 10 it was confirmed that the experimental group to which the Levosimendan compound was administered took longer to remember the stimulation in the dark space and move to the dark side as compared to the negative control group or the positive control group (MB).
  • MB positive control group
  • brain tissue sections of each dose group were stained using 1% cresyl violet and imaged by light microscopy. Somatosensory cortex in brain tissue sections was checked to confirm neuronal degeneration. The cell number of CA1 in the dorsal hippocampus region was measured.
  • FIG. 11 is an optical micrograph of the brain tissue section confirmed the neurodegeneration inhibitory effect of the levocimendane compound using the tau disease animal model according to an embodiment of the present invention and the number of neurons included in the brain tissue section It is a graph.
  • the number of neurons in the cortical sections of the experimental group to which the Levosimendan compound was administered was about 42.4% in the cortex and about 36.0% in the hippocampus compared to the negative control group. That is, it was confirmed that the Levosimendan compound is effective in inhibiting neuronal degeneration caused by tau disease.
  • Purified metastatic tau protein TauK18P301L (treatment concentration: 5 ⁇ g / ml) in Tau-BiFC cells capable of monitoring tau aggregation was treated with tau aggregation derivatives.
  • levosimendan (Levo.) Compounds (treatment concentration 3 ⁇ M) and 8 compounds were donepezil (treatment concentrations 3, 10, 30 ⁇ M), tacrine (treatment concentration 2.5, 5, 10 ⁇ M), Rivastigmine (treatment concentrations 1, 10, 100 ⁇ M), galantamine (treatment concentrations 2, 10, 50 ⁇ M), Memantine (treatment concentration 2.5, 5, 10 ⁇ M), Dopamine (treatment concentrations 1, 10, 100 ⁇ M), Curcumin (treatment concentrations 1, 5 ⁇ M), and lithium chloride (LiCl) (treatment concentrations 5, 10, 20 mM) Each concentration was combined.
  • methylene blue (MB) treatment concentration 3 ⁇ M
  • Tau-BiFC fluorescence intnesity 46 hours after drug treatment, intracellular tau aggregation was quantified by Tau-BiFC fluorescence intnesity.
  • FIG. 12 shows donepezil, tacrine, rivastigmine, galantamine (concentration) for each of levocimendan and methylene blue, according to an embodiment of the present invention.
  • Intracellular tau aggregation inhibition was measured after a combination of galantamine, memantine, dopamine, documin, curcumin, and lithium chloride (LiCl).
  • LiCl lithium chloride
  • the combination of the levosimendan (Levo.) (3 ⁇ M) compound and rivastigmine or lithium chloride (LiCl) together was superior to the treatment of the levocimendan compound alone. It showed an inhibitory effect on aggregation. Furthermore, the combination of levocimendane and rivastigmine and the combination of levocimendane and lithium chloride showed a significantly more excellent tau aggregation inhibitory effect in a concentration dependent manner as the concentration of rivastigmine and lithium chloride increased, thereby inhibiting the tau aggregation. It was confirmed once again that the effect is synergistic effect of the combination of levocimendane and rivastigmine and the combination of levocimendane and lithium chloride.
  • Levosimendan compound and the eight types of donepezil, tacrine, rivastigmine, galantamine, memantine, dopamine, curcumin (Curcumin) and lithium chloride (LiCl) compound in the group consisting of one compound selected from the combination treatment was confirmed whether the tau aggregation inhibitory effect is due to cytotoxicity.
  • Example 2-1 46 hours after drug treatment in the concentration conditions of Example 2-1, Hoechst (2 ⁇ g / ml) to stain the cell nuclei was treated to quantify the number of cells, which reflects the degree of cytotoxicity according to the compound treatment.
  • FIG. 13 illustrates donepezil, tacrine, rivastigmine, and galantamine (concentration) for each of levocymendan and methylene blue, according to an embodiment of the present invention.
  • Cell viability was measured after co-treatment with galantamine, memantine, memantine, dopamine, curcumin, and lithium chloride (LiCl).
  • the levocimendane compound alone, and of the eight compounds of the dopeezil (donepezil), tacrine (Tacrine), Rivastigmine (galivamin) (galantamine) When used in combination with Memantine, respectively, 90% or more of the cells were confirmed to be alive.
  • the levocimendan when co-treated with the dopamine (Dopamine) and lithium chloride (LiCl) compound, respectively, the levocimendan showed cell viability of less than 75% and less than 50% at high concentrations. It can be judged that the synergistic effect of tau aggregation inhibition shown in the combination treatment of the lebocymendan compound (3 ⁇ M) and the lithium chloride compound (5, 10, 20 mM) is due to the cytotoxicity of the lithium chloride compound.
  • the tau aggregation inhibitory effect of the levosimendan compound and the rivastigmine (RV) compound was confirmed by concentration in the cells.
  • Tau-BiFC cells a tau aggregation monitoring cell line, were treated with the tau agglutination derivative TauK18P301L (treatment concentration 5 ⁇ g / ml), levosimendan (treatment concentrations 1, 3, 10 ⁇ M) and ribastik. Rivastigmine (treatment concentrations of 1, 10, 100 ⁇ M) was used in combination by concentration. After 46 hours of drug treatment, intracellular tau aggregation was quantified by Tau-BiFC intensity, and cell viability was quantified by counting cells according to cell nuclear staining using Hoechst.
  • FIG. 14 in an embodiment of the present invention, for each of the concentration of levocimendane compound and methylene blue (Rivastigmine) in combination with concentration, after intracellular tau aggregation inhibition and cells Toxicity is measured.
  • methylene blue Rostigmine
  • Tau-BiFC together with the cell image showed that the tau aggregation inhibitory effect increased concentration-dependently with the concentrations of the levocimendan and rivastigmine, respectively.
  • the decrease in intensity was confirmed.
  • the tau inhibitory effect has a synergistic effect, which is significantly better than when administered alone.

Abstract

La présente invention concerne une composition pour la prévention ou le traitement d'une maladie liée à tau. La composition, qui est en particulier caractérisée en ce qu'elle comprend du lévosimendan et de la rivastigmine en association, présente un effet remarquablement plus important d'inhibition de l'agrégation de la protéine tau en comparaison avec l'utilisation du levosimendan seul, et un effet plus important d'inhibition de l'agrégation de la protéine tau en comparaison avec l'utilisation d'autres composés en association, et ne présente pas de cytotoxicité, lui permettant ainsi de trouver des applications intéressantes dans la prévention et/ou le traitement de maladies associées à tau, dont les maladies neurodégénératives.
PCT/KR2019/005180 2018-05-09 2019-04-30 Composition pour la prévention ou le traitement d'une maladie liée à tau WO2019216589A1 (fr)

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KR20080020930A (ko) * 2004-05-12 2008-03-06 오리온 코포레이션 혈전색전증 증후군들의 예방방법
KR20090015038A (ko) * 2006-03-29 2009-02-11 벨라코르 테라퓨틱스 피티와이 엘티디 신경변성 질환의 치료
US20120270836A1 (en) * 2009-11-03 2012-10-25 Pharnext Therapeutic approaches for treating alzheimer's disease
KR20160137807A (ko) * 2015-05-22 2016-12-01 단국대학교 천안캠퍼스 산학협력단 알츠하이머성 치매 치료를 위한 뇌 조직 재생용 약제학적 조성물
US9962384B1 (en) * 2017-09-07 2018-05-08 Korea Institute Of Science And Technology Levosimendan compound for preventing or treating tau-related diseases

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KR20080020930A (ko) * 2004-05-12 2008-03-06 오리온 코포레이션 혈전색전증 증후군들의 예방방법
KR20090015038A (ko) * 2006-03-29 2009-02-11 벨라코르 테라퓨틱스 피티와이 엘티디 신경변성 질환의 치료
US20120270836A1 (en) * 2009-11-03 2012-10-25 Pharnext Therapeutic approaches for treating alzheimer's disease
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