KR20190129000A - Composition for preventing or treating tau related disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating tau-related diseases. More especially, the composition contains levosimendan and rivastigmine together so that, compared to when using levosimendan alone, a tau aggregation inhibitory effect is remarkably excellent, the tau aggregation inhibitory effect is superior to the combination of other compounds, and there is no cytotoxicity, thereby being able to be effectively used for preventing and/or treating tau-related diseases including neurodegenerative diseases.

Description

Composition for preventing or treating tau related disease

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.

In healthy nerves, tau stabilizes microtubules by promoting growth from axons and polarization of nerve cells. In the case of pathological hyperphosphorylation, 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). Human full-length tau comprises a microtubule binding domain consisting of four conserved sequence repeats. Positively charged residues during this sequence repetition play an important role in binding to negatively charged microtubules (20-30 electrons per αβ-tubulin dimer) to a high degree (Minoura I, Muto E (2006) Dielectric measurement of individual microtubules using the 5 electroorientation method.Biophys. J. 90: 3739-3748 .; Mukrasch MD, Biernat J, von Bergen M, Griesinger C, Mandelkow E, et al. (2005) Sites of tau important for aggregation populate beta -structure and bind to microtubules and polyanions.J. Biol. Chem. 280: 24978-24986). 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. 270: 7679- 7688 .; Biernat J, Gustke N, Drewes G, Mandelkow EM, Mandelkow E (1993) Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding. Neuron 11: 153-163).

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. Although it is not known exactly how the abnormal tau protein aggregation in tau disease plays a role in aggravating disease, it is similar to that commonly found in degenerative brain diseases.

Representative 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. Despite active research in the field, the mechanisms by which tau protein-mediated diseases specifically signal and are toxic have not been elucidated. In addition, there is no clear treatment or treatment for tau-related diseases.

Accordingly, 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.

In addition, 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. To provide a composition.

One embodiment of the present invention is a composition for the prevention or treatment of tau aggregation-related diseases, including levosimendan and rivastigmine.

Here, it is possible that the lebocymendan is a lebocymendan compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.

And, it is possible that the rivastigmine is a rivastigmine compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.

In addition, the 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.

In addition, the composition according to the present invention may further comprise an active agent selected from the group consisting of antioxidants, cholinesterase inhibitors and combinations thereof.

In addition, 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 It may be selected from the group consisting of glaucoma.

In addition, 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. Among them, it 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. For example, it is possible that 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.

In addition, 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.
4 is a result of measuring the effect of reducing the tau phosphorylation of the levocimendane compound according to an embodiment of the present invention.
5 is a result of measuring the effect of protecting the neuronal cell damage 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. FIG.
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.
8 is a result of measuring the tau flocculation inhibitory effect of the levocimendane compound derivative according to the embodiment of the present invention.
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.
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.
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 showing.
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.

As the inventive concept allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, the terms "comprise" or "have" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Terms such as 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. This drug increases the contractile force by combining the Troponin C of the heart muscle increases the Ca ^{2 +} glass fiber-reactive. In addition, by opening the K _ATP pathway of cardiomyocytes, it acts as a vasodilator with anti-ischemic and antistunning effects (In-Chul Choi (2006), a new contractile promoter-Levosimendan.Korean J Anesthesiol 51: 519-27).

However, it is not known to date whether the 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.

To date, agents and mechanisms that may affect tau aggregation and phosphorylation have not been elucidated.Therapeutic targets under study are CDK5 (Cyclin-dependent kinase 5) inhibitors, propentofylline (PPE), N-phenylamines, quinoxalines And methylene blue. Among them, 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).

[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.

Specifically, according to an embodiment of the present invention described below, 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). In addition, in real-time measurement of tau aggregation in vitro, 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). Furthermore, in the Novel object recognition test using an animal model (mouse), administration of levocimendan showed a high recognition index for a new object (see FIG. 9), and in the passive avoidance test. Administration of levosimendan had an effect of improving cognitive abilities (see FIG. 10), and even when checking brain tissue sections of the animal model, it was confirmed that administration of lebocimendan had an effect of inhibiting neuronal cell degeneration due to the higher number of neurons. (See FIG. 11).

Meanwhile, 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).

[Formula 2]

Here, it is possible that the lebocymendan is a lebocymendan compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound. It is also possible that the rivastigmine is also a rivastigmine compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.

While the present inventors have studied various dozens of compounds and their combinations to select new tau aggregation inhibitors, the combination of levocimendane and rivastigmine reduces tau aggregation and phosphorylation than combinations of other compounds. After confirming that remarkably excellent, the present invention was completed.

Specifically, as shown in the embodiments of the present invention described below, with the levosimendan compound, donepezil (donepezil), tacrine (Tacrine), rivastigmine (Rivastigmine), galantamine (galantamine), One compound selected from the group consisting of memantine, dopamine, documin, curcumin, and lithium chloride (LiCl) was treated in combination to confirm the tau aggregation inhibitory effect.

As a result, the combination of the levosimendan (Levo.) (3 μM) 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 μM) and the lithium chloride compound (5, 10, 20 mM) Was determined according to the cytotoxicity of the lithium chloride compound (see FIG. 13). In addition, 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).

According to the present invention, 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. In addition, 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.

Furthermore, the 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.

Basically, the present invention is characterized by including levocimendane and rivastigmine together, and having excellent tau aggregation inhibitory effect and no cytotoxicity. In addition, 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).

According to this, it 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.

On the other hand, 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.

The 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. .

Furthermore, the present invention may further comprise an activator selected from the group consisting of antioxidants, cholinesterase inhibitors and combinations thereof.

The antioxidants include α-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 (Rivastigmin), Galantamine (Galantamine), Butylcholinesterase, Penserine (Phenserine), Huperzin (Huperzin A) can be used in combination of one or more.

In addition, 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. It may be one or more selected from the group consisting of. 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.

In addition, the present invention relates to a method for inhibiting tau aggregation, which comprises treating the compound or a derivative thereof.

For example, 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.

Furthermore, 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. For example, 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. In the preparation, it may additionally contain additional raw materials and components. 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In addition, the following examples were performed based on the Tau-BiFC cell model. 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. , And more detailed definitions and methods are as described in Republic of Korea Patent 10-1546485.

[ Example  One : Lebocymendan  Screening and Checking Effect]

Example  1-1: Intracellular Tau  Confirmation of agglutination inhibitory effect

To select new tau aggregation inhibitors, an FDA approved drug library (1018 compounds in total) was processed in a tau-BiFC cell model that facilitated the observation of tau oligomer formation in live cells. As shown in Figure 1, Tau-BiFC cells were first plated on 384 well plates. The following day, FDA-approved drugs (treatment concentration 10 uM) were treated with Tau-BiFC cells 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 the cell nuclear staining using Hoechst, along with the degree of inhibition of tau aggregation of the drugs. Among the 1,018 drugs, the drug Levosimendan, which shows the cell 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).

To confirm the tau aggregation inhibitory effect of selected Levosimendan drugs, DMSO, MB, Levosimendan (treatment concentration 10 uM) together with Forskolin (treatment concentration 30 uM), a compound that activates the tau kinase PKA to induce tau aggregation Was treated to Tau-BiFC cells. At this time, Methylene blue (MB), which is known to show tau aggregation inhibition effect, was used as a comparative compound (positive control group). After 48 hours, the cell viability was confirmed by the tau aggregation inhibitory effect and the cell nuclear staining using Hoechst.

As a result, as shown in FIG. 2, relative fluorescence units (RFU) were reduced in the Levosimendnan group compared to the negative control group (DMSO) while the Tau-BiFC cell model was operating (Tau-BiFC ^ON ). . In addition, when compared to MB used as a positive control group, it was confirmed that the fluorescence value similarly decreased. And when the numbers of Nuclei stained with hoechst were used to evaluate cytotoxicity, the number of negative control group (DMSO) and positive control group (MB) decreased, while the experimental group (Levosimendnan) had a similar number to that of the normal control group (Basal). Seemed.

Accordingly, it was confirmed that 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.

In addition, in order to examine in detail the inhibitory effect of the selected Levosimendan compound on the tau aggregation in living cells, the concentration of 1, 3, 10, 30 100 uM with Levosimendan compound together with Forskolin (treatment concentration of 30 uM), a tau aggregation inducer Rho-BiFC cells were treated. Tau-BiFC response analysis after 48 hours was used to analyze the cell survival according to the intracellular tau aggregation response and compound toxicity.

As a result, as shown in Figure 3, the positive control group (MB) and the experimental group (Levosimendan) was confirmed that the fluorescence value decreases with concentration, IC50 was 2.2 uM. Levosimendan compound (treatment concentration 10 uM) showed similar tau aggregation inhibitory effect and cell viability as the cells treated with nothing.

Example  1-2: Tau  Confirmation of phosphorylation inhibitory effect

In order to determine whether Levosimendan compound showed tau aggregation inhibition inhibition effect by tau phosphorylation, 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.

As a result, as shown in FIG. 4, it was confirmed that tau phosphorylation was decreased in the positive control group (+, MB) and the experimental group (+, Levosimendan) compared to the cells treated with Forskolin (+). The experimental group (+, Levosimendan) showed a reduction of tau phosphorylation by 34% at tau phosphorylation residues Ser199 and 35% at Ser396.

Example  1-3: Check the neuronal protective effect

It was confirmed that the tau aggregation inhibitory effect of Levosimendan compound leads to neuroprotective effect in the situation of neuronal damage. Levosimendan compound (treatment concentration 10 uM) was first treated with rat cerebral cortical neurons (E18, DIV8), and then after 2 hours, Tau protein TauK18P301L (treatment concentration 20ug / mL) showed neuronal cell damage and intracellular tau aggregation effect ) Was treated.

As a result, as shown in FIG. 5, the experimental group (Levosimendan) had less nerve cell and neurite length damage than the negative control group (DMSO) and the positive control group (MB). In contrast, 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.

Example  1-4: in in vitro Levosimendan  Compound Tau  Confirmation of agglutination inhibitory effect

Levosimendan compounds that inhibited tau aggregation inhibited tau phosphorylation but were not involved in the increase or decrease of tau kinase. Therefore, the aim of this study was to determine whether Levosimendan compounds are directly involved in the tau agglutination process. In vitro, the inhibitory effect of Levosimendan compound on the tau agglutination process was confirmed, and it was confirmed whether it plays a role in releasing the coagulation by preventing the further coagulation of the tau already coagulated. In the tau agglutination process in vitro (Tau pre-Agg + 100 uM DTT + 0.1mg / mL Heparin, 37 degrees incubation) was treated with Levosimendan compound by concentration, and the degree of tau aggregation was confirmed by ThS (Thioflavin S). The ThS is a dye that can measure tau aggregation in real time in the invitro, and is used as a fluorescent probe for sensing β-sheet aggregation.

As a result, as shown in the graph shown in Figure 6, the Levosimendan compound confirmed a direct tau aggregation inhibitory effect in vitro. In vitro, the IC50 of the Levosimendan compound was 4.71 uM.

In addition, the treatment of Levosimendan compound with tau aggregates (Tau-Agg + 100 uM DTT + 0.1mg / mL Heparin, 37 ° C culture) was confirmed by treating the tau aggregates which had already been aggregated in vitro (Tau-Agg + 100 uM DTT + 0.1 mg / mL Heparin). 7).

Example  1-5: Levosimendan  Of compound derivatives Tau  Confirmation of coagulation inhibitory effect

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.

As a result, as shown in Figure 8, the experimental group (Levosimendan) reduced tau aggregation similar to the positive control group (MB). In other words, Levosimendan compound showed a concentration-dependent tau aggregation inhibitory effect.

In contrast, 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: Levosimendan  Identify the effect of improving the cognitive function of the 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.

For Novel object recognition test, two groups of the same shape and size (O) were fixed in a box (40 x 40 x 40 cm) manufactured so that the outside was not visible from the inside, and then the mouse of each group Let the object learn for 10 minutes. After 24 hours, one of the two objects is replaced with a new object (N), and the time of searching for the previously learned object (O) and the new object (N) for 10 minutes is recorded and recognized. It was quantified by the index (Recognition index = individual object (O or N) search time / total object search time).

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).

For the passive avoidance test, 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. When the mouse enters the B space, 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. After 24 hours, 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.

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. As a result, as shown in 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). Through the above results, it was confirmed that the Levosimendan compound has an cognitive improvement effect.

Example 1-7 confirming the neuronal degeneration of the Levosimendan compound derivative

We confirmed whether the tau aggregation inhibitory effect of Levosimendan compound also affects neuronal degeneration in tau disease animal model. In order to secure the brain tissue, the mice administered with the drug administration and behavior evaluation experiment (the experiment of Example 1-6) were over anesthetized using 2% Evertin, and perfusion was performed through cardiac perfusion using saline. After perfusion, tissues were fixed using 4% paraformaldehyde and brains were extracted. The extracted brain was further fixed in 4% paraformaldehyde for one day, and then stored in 30% sucrose solution to prepare frozen section samples. Thereafter, a mold for cryosection was prepared using an OCT compound, and a tissue fragment having a thickness of 40 μm was obtained using cryosection.

In order to stain neuronal bodies, 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 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 showing. As a result, as shown in FIG. 11, 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. In other words, it was confirmed that the Levosimendan compound is effective in suppressing neuronal degeneration caused by tau disease.

[ Example  2 : Lebosimendan and  Confirm effect of combination treatment of 8 compounds]

Example  2-1: intracellular Tau  Confirmation of coagulation inhibitory effect

Donepezil, Tacrine, Rivastigmine, galantamine, Memantine, Dopamine, Curcumin, and Lithium chloride with Levosimendan When one compound selected from the group consisting of LiCl) was treated in combination, the effect of inhibiting tau aggregation in cells was confirmed.

Purified metastatic tau protein TauK18P301L (treatment concentration: 5 μg / ml) in Tau-BiFC cells capable of monitoring tau aggregation was treated with tau aggregation derivatives. At the same time, levosimendan (Levo.) Compounds (treatment concentration 3 μM) and 8 compounds were donepezil (treatment concentrations 3, 10, 30 μM), tacrine (treatment concentration 2.5, 5, 10 μM), Rivastigmine (treatment concentrations 1, 10, 100 μM), galantamine (treatment concentrations 2, 10, 50 μM), Memantine (treatment concentration 2.5, 5, 10 μM), Dopamine (treatment concentrations 1, 10, 100 μM), Curcumin (treatment concentrations 1, 5 μM), and lithium chloride (LiCl) (treatment concentrations 5, 10, 20 mM) Each concentration was combined. At this time, methylene blue (MB) (treatment concentration 3 μM), which is known to exhibit tau aggregation inhibition effect, was used as a comparative compound (positive control group). 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). As a result, as shown in Figure 12, when combined treatment with the levosimendan (Levosimendan, Levo.) Compounds and the eight compounds, respectively, it inhibits tau aggregation (100%) induced by the treatment of the metastatic tau protein TauK18P301L We confirmed that we could.

Among them, the combination of the levosimendan (Levo.) (3 μM) 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.

In comparison, methylene blue (MB), when combined with each of the eight compounds, was able to inhibit tau aggregation (100%) induced by the treatment of the metastatic tau protein TauK18P301L. When using blue alone, there was no combination showing a better concentration-dependent effect.

Example  2-2: Cytotoxicity Check

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.

46 hours after drug treatment in the concentration conditions of Example 2-1, Hoechst (2 μg / 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). As a result, as shown in Figure 13, 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.

However, when co-treated with dopamine (Dopamine) and lithium chloride (LiCl) compounds, 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 μM) and the lithium chloride compound (5, 10, 20 mM) is due to the cytotoxicity of the lithium chloride compound.

In addition, in the case of methylene blue, when treated alone or in combination with each of the eight compounds, it was confirmed that the cell viability between 25% -60%. Therefore, the inhibitory effect of intracellular tau aggregation upon the co-administration of methylene blue and the eight compounds was determined to be due to the cytotoxicity of the compounds.

Example  2-3: Intracellular by concentration Tau  Inhibition of aggregation and cell survival

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 μg / ml), levosimendan (treatment concentrations 1, 3, 10 μM) and ribastik. Rivastigmine (treatment concentrations 1, 10, 100 μM) was treated 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.

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. As a result, as shown in Figure 14, when the combination of levocimendane and rivastigmine, Tau-BiFC together with the cell image that the tau aggregation inhibitory effect increases in a concentration-dependent manner according to the concentration of each of the levocimendan and rivastigmine The decrease in intensity was confirmed. According to this, when combined 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.

In comparison, for methylene blue (MB), methylene blue (treatment concentrations 1, 3, 10 μM) and single treatment with rivastigmine (RV) (treatment concentrations 1, 10) , 100 μM), the degree of inhibition of intracellular tau aggregation seen in combination with the compound is believed to be due to the decrease in cell number due to the cytotoxicity of the methylene blue compound by concentration.

While the invention has been shown and described with respect to certain preferred embodiments thereof, it will be understood that the invention may be modified and modified in various ways without departing from the spirit or scope of the invention provided by the following claims. It will be apparent to one of ordinary skill in the art.

Claims (8)

Levosimendan (Levosimendan) and rivastigmine (Rivastigmine), comprising a composition for the prevention or treatment of tau aggregation-related diseases.
The method of claim 1,
Wherein the levosimendan compound, pharmaceutically acceptable salts, stereoisomers or solvates of the compound, characterized in that the composition for the prevention or treatment of tau (tau) aggregation-related diseases.
The method of claim 1,
The rivastigmine is a rivastigmine compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound, characterized in that the composition for the prevention or treatment of tau (tau) aggregation-related diseases.
The method of claim 1,
At least one compound selected from the group consisting of donepezil, galantamine, memantine, tacrine, curcumin, dopamine and lithium chloride (LiCl) It characterized in that it further comprises, tau (tau) composition for the prevention or treatment of aggregation-related diseases.
The method according to any one of claims 1 to 4,
An anti-oxidant, a cholinesterase inhibitor, and a combination thereof, further comprising an active agent selected from the group consisting of, a composition for preventing or treating tau aggregation-related diseases.
The method according to any one of claims 1 to 4,
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. A composition for the prevention or treatment of tau agglutination related diseases, characterized in that selected from the group consisting of.
The method of claim 6,
The tau disease is 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, related to tau aggregation Composition for preventing or treating a disease.
A dietary supplement for the prevention or amelioration of tau aggregation-related diseases, including levosimendan and rivastigmine.

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