KR102317080B1 - Composition for preventing or treating tau related disease - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of tau-related diseases, in particular, it is characterized in that it contains levosimendan and rivastigmine together, so that it is better than when levosimendan is used alone. The 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, so it can be effectively used for the prevention and/or treatment of tau-related diseases, including neurodegenerative diseases.

Description

Composition for preventing or treating tau related disease

To a pharmaceutical composition comprising a compound, an optically active isomer thereof, or a pharmaceutically acceptable salt thereof, used for the prevention and/or treatment of a disease caused by aggregation or phosphorylation of tau.

Tau is a microtubule-associated protein (MAP) that is mainly expressed in the axons of neurons with a molecular weight of 50,000 to 70,000, and plays a role in stabilizing microtubules (MT). indicates In humans, 6 isoforms of tau are generated by insertion of 29 or 58 amino acid residues at the N-terminus, and alternative splicing of 3 or 4 repeats at the C-terminus (referred to as microtubule binding site) mRNA splicing. do.

In healthy neurons, tau stabilizes microtubules by promoting growth from axons and neuronal polarization. In the case of pathological hyperphosphorylation, tau dissociates from microtubules and induces insoluble aggregation (Gendron TF, Petrucelli L (2009) The role of tau in neurodegeneration. Mol. Neurodegener. 4: 13). A structural framework that induces tau aggregation has been proposed. Evidence has been presented that insoluble filaments are formed from ten soluble monomers, and that these filaments bind into higher-order structures called neurofibrillary tangles (NFTs). Human full-length tau contains a microtubule binding domain consisting of four conserved sequence repeats. Positively charged residues in these sequence repeats play an important function in binding to highly negatively charged microtubules (20-30 electrons per αβ-tubulin dimer) (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. -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 results in a dynamic rearrangement of the microtubule network. Abnormally excessive phosphorylation of tau disrupts the balance of this dynamic rearrangement and sharply 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).

Tau disease (tauopathy) is a degenerative neurological disease caused by abnormal accumulation of tau protein hyperphosphorylation and aggregation in nerve cells, and is pointed out as the cause of various degenerative brain diseases. Tau protein aggregates seen in patients with tau disease are mainly found in the cell body and dendrites of nerve cells, and are called Neurofibrillary tangles (NFT) and neuropil threads. Looking at neurofibrillary tangles, the tau protein is composed of paired helical filaments (PHFs) that are tangled like fine threads, which is aggregated and hyperphosphorylated differently from normal tau protein. Although it is not known exactly what the role of abnormal tau protein aggregation in tauopathy plays in the advanced stage of the disease, it is similar to the aggregation phenomenon commonly seen in degenerative brain diseases.

Representative tau-related diseases include Alzheimer's disease, in addition to Parkinson's disease, tauopathy, vascular dementia, acute stroke, trauma, cerebrovascular disease, brain cord trauma, spinal cord trauma, peripheral neuropathy, retinopathy, glaucoma, These include progressive supranuclear palsy, cortical-basal ganglia degeneration, Argyrophilic Grain Disease, Peak disease and hereditary frontotemporal dementia. Despite active research in the field, it has not been clearly identified by what mechanism specifically transmits signals and exhibits toxicity in diseases mediated by tau protein. In addition, there is still no clear treatment or therapeutic agent for tau-related diseases.

Accordingly, the present inventors were studying various tens of compounds and combinations thereof in order to select a new tau aggregation inhibitor, a combination of compounds having a different combination of Levosimendan and Rivastigmine After confirming that the effect of reducing tau aggregation and phosphorylation is significantly superior, the present invention was completed.

The present invention is to solve the above problems, and an object of the present invention is to provide a combination of compounds having significantly superior tau aggregation inhibitory effect than when levosimendan is used alone.

In addition, the present invention provides a new compound combination that has excellent tau aggregation inhibitory effect and no cytotoxicity among combinations of various compounds, 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 preventing or treating tau aggregation-related diseases, including levosimendan and rivastigmine.

Here, the levosimendan may be a levosimendan compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.

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

In addition, the composition according to the present invention, donepezil (donepezil), galantamine (galantamine), memantine (Memantine), tacrine (Tacrine), curcumin (Curcumin), dopamine (Dopamine) and lithium chloride (LiCl) It may further include at least one compound selected from the group.

In addition, it is possible that the composition according to the present invention further comprises an active agent selected from the group consisting of antioxidants, cholinesterase inhibitors and combinations thereof.

In addition, the tau (tau) aggregation-related disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, tauopathy, and vascular dementia.

In addition, the tauopathy may be selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy, cortical-basal ganglia degeneration, Argyrophilic Grain Disease, peak disease, and hereditary frontotemporal dementia.

Another embodiment of the present invention is a health functional food for preventing or improving tau aggregation-related diseases, including levosimendan and rivastigmine.

Another embodiment of the present invention is a method comprising administering Levosimendan and Rivastigmine. Among them, it may be a method for preventing or treating a disease related to tau aggregation. Administration to mammals, animals, humans, and/or non-humans is possible. The order in which levosimendan and rivasticmine are administered is not relevant, and it is possible to administer them sequentially 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 present invention is a compound for preventing or treating tau aggregation-related diseases, including Levosimendan and Rivastigmine. Since the compound containing levosimendan and rivasticmine has an excellent tau aggregation inhibitory effect, it is suitable for use as a compound for preventing or treating tau aggregation-related diseases.

Another embodiment of the present invention is the use of Levosimendan and Rivastigmine compounds as tau aggregation inhibitors. For example, it is possible that the present invention is the use of a compound of levosimendan and rivasticmine for the manufacture of a therapeutic agent for a disease related to tau aggregation. The present invention may be used for the manufacture of a medicament for the prophylaxis or treatment of tau aggregation-related diseases of levosimendan and rivasticmine compounds.

The present invention is characterized in that it contains levosimendan and rivastigmine together, so that the tau aggregation inhibitory effect is significantly better than when levosimendan is used alone.

In addition, the combination of levosimendan and rivasticmine according to the present invention has a superior tau aggregation inhibitory effect than the combination of other compounds and has no cytotoxicity, so it can be effectively used for the prevention and/or treatment of tau-related diseases, including neurodegenerative diseases. can

1 is a drug library screening result of a levosimendan compound according to an embodiment of the present invention.
2 is a result of measuring the intracellular tau aggregation inhibitory effect and cytotoxicity of the levosimendan compound according to an embodiment of the present invention.
3 is a result of measuring the concentration-dependent intracellular tau aggregation inhibitory effect of the levosimendan compound according to an embodiment of the present invention.
4 is a result of measuring the tau phosphorylation reduction effect of the levosimendan compound according to an embodiment of the present invention.
5 is a result of measuring the protective effect of the levosimendan compound according to an embodiment of the present invention against nerve cell damage.
6 is a result of measuring the direct tau aggregation inhibitory effect of the levosimendan compound in vitro according to an embodiment of the present invention.
7 is a result of measuring the tau aggregate resolution of the levosimendan compound in vitro according to an embodiment of the present invention.
8 is a result of measuring the tau aggregation inhibitory effect of the levosimendan compound derivative according to an embodiment of the present invention.
9 is a result of a novel object recognition test confirming the cognitive function improvement effect of the levosimendan compound using an animal model of tauopathy according to an embodiment of the present invention.
10 is a result of a passive avoidance test confirming the cognitive function improvement effect of the levosimendan compound using an animal model of tauopathy according to an embodiment of the present invention.
11 is an optical microscopic photograph of a brain tissue section confirming the neurodegeneration inhibitory effect of the levosimendan compound using an animal model of tauopathy, and the number of neurons included in the brain tissue section according to an embodiment of the present invention. It is a graph representing
12 shows, according to an embodiment of the present invention, donepezil, tacrine, rivastigmine, galantamine (donepezil), tacrine, and galantamine ( galantamine), memantine (Memantine), dopamine (Dopamine), curcumin (Curcumin), and lithium chloride (LiCl), respectively, after the combined treatment, the results of measuring the inhibition of intracellular tau aggregation.
13 is, according to an embodiment of the present invention, for each of levosimendan and methylene blue, donepezil, tacrine, rivastigmine, galantamine ( galantamine), memantine, dopamine, curcumin, and lithium chloride (LiCl), respectively, were treated in combination, and cell viability was measured.
Figure 14 is, according to an embodiment of the present invention, with respect to each concentration of the levosimendan compound and methylene blue, rivastigmine (Rivastigmine) for each concentration, after the combination treatment, intracellular tau aggregation inhibition and cells It is the result of measuring toxicity.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above 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 of the compound for use in a method for preventing or treating tau pathology or its symptoms will be.

The levosimendan (Levosimendan) is known as a drug used for the treatment of congestive heart failure as a calcium sensitiser (calcium sensitiser). This drug increases the contractile force by combining the Troponin C of the heart muscle increases the Ca ^{2 +} glass fiber-reactive. In addition, it _{acts as a vasodilator exhibiting anti-ischemic and anti-stunning effects by opening the K-ATP} pathway of cardiomyocytes (Choi In-cheol (2006) A new contractility promoter-Levosimendan. Korean J Anesthesiol 51: 519-27).

However, it is not known precisely whether the levosimendan compound is useful for the prevention and/or treatment of diseases caused by aggregation or phosphorylation of tau to date. Drug repositioning refers to an attempt to apply new indications (diseases) to new indications (diseases) by reviewing existing drugs and drugs. It is used as a strategy for new drug development. The present invention is characterized by applying a levosimendan compound to the aggregation or phosphorylation of tau as part of the re-creation of such a new drug.

To date, agents and mechanisms that can affect tau aggregation and phosphorylation have not been clearly elucidated. , methylene blue, and the like. 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 Examples below, and it was found to have higher cytotoxicity than the Levosimendan compound according to the present invention (see FIG. 2 ).

The levosimendan compound may be represented by the following formula (1).

[Formula 1]

The compound is used as a concept including all of the compound itself, pharmaceutically acceptable salts, hydrates, solvates, isomers, and prodrugs thereof.

The compound is characterized in that it inhibits tau aggregation and phosphorylation, and does not affect the increase or decrease in the activity of tau kinase. In addition, it is characterized in that it prevents further aggregation of the aggregation-progressed tau and decomposes the aggregation of the agglomerated tau.

Specifically, according to an embodiment of the present invention to be described later, when a cell line treated with a tau aggregation-inducing compound (Forskolin) is treated with a levosimendan compound, a tau aggregation inhibitory effect (see FIG. 3 ), a tau phosphorylation reduction effect (See FIG. 4), and it was confirmed that there is a neuroprotective effect (see FIG. 5). In addition, as a result of measuring tau aggregation in vitro in real time, levosimendan was directly involved in the tau aggregation process and showed an inhibitory effect (see FIGS. It was confirmed that it does not have (see FIG. 8). Furthermore, in a novel object recognition test using an animal model (mouse), administration of levosimendan showed a high recognition index for a new object (see FIG. 9 ), and also in the passive avoidance test When levosimendan was administered, there was an effect of improving cognitive ability (see Fig. 10), and even when the brain tissue section of the animal model was checked, it was confirmed that administration of levosimendan had an effect of inhibiting neuronal degeneration because the number of nerve cells was higher. (see FIG. 11).

On the other hand, 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, the levosimendan may be a levosimendan compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound. In addition, it is possible that the rivasticmine is also a rivasticmine compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound.

While the present inventors were studying various tens of compounds and their combinations in order to select novel tau aggregation inhibitors, the combination of levosimendan and rivasticmine reduces tau aggregation and phosphorylation than the combination of other compounds After confirming the fact that is remarkably excellent, the present invention was completed.

Specifically, as shown in the Examples of the present invention to be described later, donepezil, tacrine, rivastigmine, galantamine together with a levosimendan compound, Memantine (Memantine), dopamine (Dopamine), curcumin (Curcumin), and one type of compound selected from the group consisting of lithium chloride (LiCl) was treated in combination, respectively, the tau aggregation inhibitory effect was confirmed.

As a result, when the levosimendan (Levo.) (3 μM) compound and rivastigmine or lithium chloride (LiCl) were treated in combination, tau was superior to that when the levosimendan compound was treated alone. It was confirmed that it has an aggregation inhibitory effect (see FIG. 12). And, as a result of confirming whether the tau aggregation inhibitory effect is due to cytotoxicity, the synergistic effect of tau aggregation inhibition shown in the combined treatment of a levosimendan compound (3 μM) and a lithium chloride compound (5, 10, 20 mM) could be determined according to the cytotoxicity of the lithium chloride compound (see FIG. 13). In addition, with respect to each concentration of the levosimendan compound, ribastikmine was co-treated for each concentration, and the inhibition of intracellular tau aggregation and cytotoxicity were measured. It was confirmed that the aggregation inhibitory effect increased in a concentration-dependent manner. Therefore, when levosimendan and rivastigmine were treated in combination, it was confirmed that the tau inhibitory effect was significantly better than when administered alone (see FIG. 14 ) by having a synergistic effect.

According to this, the present invention is characterized in that it contains both levosimendan and rivasticmine, and thus the tau aggregation inhibitory effect is significantly superior to that when levosimendan is used alone. In addition, the combination of levosimendan and rivasticmine according to the present invention has a superior tau aggregation inhibitory effect than the combination of other compounds and has no cytotoxicity, so it can be effectively used for the prevention and/or treatment of tau-related diseases, including neurodegenerative diseases. can

Furthermore, the composition according to the present invention, donepezil (donepezil), galantamine (galantamine), memantine (Memantine), tacrine (Tacrine), curcumin (Curcumin), dopamine (Dopamine) and lithium chloride (LiCl) It may further include at least one compound selected from the group.

The present invention is characterized in that it has excellent tau aggregation inhibitory effect and has no cytotoxicity by including levosimendan and rivasticmine together. In addition, when donepezil, galantamine, memantine, tacrine, curcumin, dopamine and lithium chloride (LiCl) are treated together with levosimendan, each , was able to inhibit tau aggregation induced by treatment with the metastatic tau proteomic TauK18P301L (see Fig. 12).

According to this, it basically contains levosimendan and rivasticmine, plus donepezil, galantamine, memantine, tacrine, curcumin, dopamine ( Dopamine) and lithium chloride (LiCl), even when further comprising at least one compound selected from the group consisting of, it is shown that it is possible to effectively inhibit tau aggregation.

On the other hand, the present invention for the prevention or treatment of tau (tau) aggregation-related diseases, comprising the above compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound, and at least one pharmaceutically acceptable carrier to the composition.

The composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-mentioned 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, bacteriostats, fragrances, and vitamins may be added as needed. In addition, it can be formulated as a solution, suspension, powder, granule, pill, capsule, or tablet, and a target organ-specific antibody or other ligand can be used in combination with the carrier so that it can specifically act on a target organ. .

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

The antioxidant is α-tocopherol, vitamin C (Ascorbic aicd), vitamin C palmitate, butylhydroxyanisole, dibutylhydroxytoluene, citric acid, erythorbic acid, fumaric acid, malic acid Acid, maltodextrin, potassium potassium metabisulfide, sodium metabisulfate, propionic acid, propyl gallate, sodium ascorbate, sodium sulfate, Tymol, cyclodextrin or sulfo One or more types from the group consisting of butyl ether β-cyclodextrin and the like may be mixed and used.

The cholinesterase inhibitor is a substance that inhibits the action of cholinesterase and inhibits the hydrolysis of acetylcholine in vivo, and is involved in promoting donation of cholinergic nerves (parasympathetic nerves, motor nerves), for example, One or more from the group consisting of donepezil (Donepezil), rivastigmin (Rivastigmin), galantamine (Galantamine), Butylcholinesterase, phenserine (Penserine), huperzin (Huperzin A) and the like can be used by mixing.

In addition, the present invention is a composition for preventing or treating tau aggregation-related diseases, comprising the above compound, 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 preventing and/or treating 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 tauopathy may be selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy, cortical-basal ganglia degeneration, Argyrophilic Grain Disease, peak disease, and hereditary frontotemporal dementia.

In addition, the present invention relates to a method for inhibiting tau aggregation, comprising treating the compound or a derivative thereof as described above.

For example, it may be characterized in that the hyperphosphorylation of tau is inhibited by treating the cell line in vitro with a compound of levosimendan and rivasticmine.

The present inventors first confirmed that tau hyperphosphorylation was inhibited when the levosimendan compound was treated in a cell line in vitro, and the combined administration of levosimendan and rivasticmine was more effective than levosimendan alone administration. was newly found to be excellent.

Furthermore, the present invention relates to a health functional food for preventing or improving tau aggregation-related diseases, comprising the above compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound. That is, another embodiment of the present invention is a health functional food for preventing or improving tau aggregation-related diseases, including levosimendan and rivastigmine.

The health functional food may be formulated in the formulation of a conventional health functional food known in the art. For example, various foods, beverages, gum, tea, vitamin complexes, dietary supplements, powders, powders, granules, tablets, capsules, suspensions, emulsions, syrups, teas, jellies, or beverages may be prepared in the form of. During the preparation, raw materials and components commonly added may be additionally contained. For example, disaccharides, polysaccharides, dextrins, cyclodextrins, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents and flavor enhancers, pectic acid and its salts, alginic acid and its salts, 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 through examples. These examples are only for illustrating the present invention, and 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 reliable system for monitoring oligomeric tau interactions, and is a Venus protein-based BiFC method in which N-terminal and C-terminal non-fluorescent portions of Venus protein are fused to tau. , more detailed definitions and methods are as described in Korean Patent Registration No. 10-1546485.

[ Example One : Levosimendan's Screening and Effect Confirmation]

Example 1-1: intracellular tau Confirmation of aggregation inhibitory effect

In order to select novel tau aggregation inhibitors, an FDA-approved drug library (a total of 1018 compounds) was applied to the tau-BiFC cell model, which facilitates observation of tau oligomer formation in living cells. As shown in the schematic diagram of Figure 1, first, Tau-BiFC cells were plated in a 384 well plate. The next day, tau-BiFC cells were treated with FDA-approved drugs (treatment concentration: 10 uM) together with Forskolin (treatment concentration 30 uM), a compound that induces tau aggregation by activating tau kinase PKA. After 46 hours, along with the degree of inhibition of tau aggregation of the drugs, cell viability by the compound was analyzed through cell nuclear staining using Hoechst. Out of a total of 1,018 drugs, Levosimendan, a drug that shows tau aggregation inhibitory effect to the extent of Methylene blue (MB), which is known to have an aggregation inhibitory effect, and shows cell viability at a level of ^{normal state (tau-BiFC off ) without drug treatment.} were selected ( FIG. 1 ).

In order to confirm the tau aggregation inhibitory effect of the selected Levosimendan drug, DMSO, MB, Levosimendan (treatment concentration 10 uM) together with Forskolin (treatment concentration 30 uM), a compound that induces tau aggregation by activating the tau kinase PKA were treated with Tau-BiFC cells. At this time, Methylene blue (MB), which is known to have an inhibitory effect on tau aggregation, was used as a comparative compound (positive control). After 48 hours, the tau aggregation inhibitory effect and cell viability by the compound were confirmed through cell nuclear staining using Hoechst.

As a result, as shown in Figure 2, the experimental group (Levosimendnan) compared to the negative control group (DMSO) in the ^{operating state (Tau-BiFC ON) of the Tau-BiFC cell model fluorescence value (relative fluorescence units, RFU) was reduced.} . In addition, it was confirmed that the fluorescence value was similarly decreased when compared to MB used as a positive control. And when the number of nuclei stained with hoechst was confirmed to evaluate the cytotoxicity, the negative control group (DMSO) and the positive control group (MB) decreased the number, whereas the experimental group (Levosimendnan) showed a similar number to the normal control group (Basal). seemed

Accordingly, it was confirmed that Levosimendnan, a compound with low cytotoxicity while exhibiting an inhibitory effect on intracellular tau aggregation similar to that of MB, was selected as a tau aggregation inhibitor.

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

As a result, as shown in FIG. 3 , in the positive control group (MB) and the experimental group (Levosimendan), it was confirmed that the fluorescence value decreased according to the concentration, and the IC50 was 2.2 uM. Levosimendan compound (treated concentration of 10 uM) showed similar tau aggregation inhibitory effect and cell viability to cells untreated.

Example 1-2: tau Confirmation of phosphorylation inhibitory effect

In order to confirm whether the Levosimendan compound exhibits an inhibitory effect on tau aggregation according to the inhibition of tau phosphorylation, Tau-BiFC cells were treated with Forskolin, a tau aggregation inducer (treatment concentration: 30 uM) and Levosimendan compound (treatment concentration: 15 uM). After 36 hours, tau phosphorylation Western blotting was performed with the tau-BiFC cell lysate.

As a result, as shown in FIG. 4 , it was confirmed that tau phosphorylation was reduced 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 in tau phosphorylation of about 34% at Ser199 and 35% at Ser396 at the tau phosphorylation residue.

Example 1-3: Confirm the neuroprotective effect

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

As a result, as shown in FIG. 5 , the experimental group (Levosimendan) had less damage to nerve cells and neurite length than the negative control group (DMSO) and the positive control group (MB). In contrast, the shape of the nerve cells and the length of the neurites were similar to those of the control group. That is, it was confirmed that the Levosimendan compound has a protective effect on nerve cell damage caused by TauK18P301L.

Example 1-4 : in in vitro Levosimendan compound tau Confirm the aggregation inhibitory effect

The Levosimendan compound, which exhibits an inhibitory effect on tau aggregation, inhibited tau phosphorylation, but was not involved in the increase or decrease in the activity of tau phosphorylation enzyme. Therefore, it was attempted to determine whether the Levosimendan compound was directly involved in the tau aggregation process and showed an inhibitory effect. It was confirmed that the inhibitory effect of the Levosimendan compound on the tau aggregation process in vitro and whether it plays a role in releasing the aggregation by preventing further aggregation of tau that has already been agglomerated. In the in vitro tau aggregation process (Tau pre-Agg + 100 uM DTT + 0.1mg/mL Heparin, 37 degrees incubation), Levosimendan compounds were treated with each concentration, and the degree of tau aggregation was confirmed as ThS (Thioflavin S). The ThS is a dye that can measure tau aggregation in vitro in real time, and is used as a fluorescent probe for sensing β-sheet aggregation.

As a result, as shown in the graph shown in FIG. 6 , the Levosimendan compound directly inhibited tau aggregation in vitro was confirmed. The IC50 of Levosimendan compound in vitro was 4.71 uM.

In addition, by treating the previously aggregated tau aggregates with the Levosimendan compound in vitro (Tau-Agg + 100 uM DTT + 0.1mg/mL Heparin, incubated at 37°C), the decomposition effect of the Levosimendan compound on the tau aggregates was confirmed (Fig. 7).

Example 1-5: Levosimendan of compound derivatives tau Confirmation of aggregation inhibitory effect

Levosimendan compounds have two inactivated (OR-1855) or activated (OR-1896) metabolite forms. It is known that the OR-1896 compound, which has undergone the acetylation process of the OR-1855 compound, remains in the body for a long time and shows the effect of the drug. Therefore, by treating tau-BiFC cells with Levosimendan compound and two metabolites, it was confirmed which form showed more tau aggregation inhibitory effect in the cells. MB, Levosimendan, OR-1855, OR-1896 compounds were treated with forskolin (treatment concentration 30 uM), a tau aggregation-inducing substance, in tau-BiFC cells by concentration. The degree of tau aggregation after 48 hours was analyzed.

As a result, as shown in FIG. 8, the experimental group (Levosimendan) reduced tau aggregation similarly to the positive control group (MB). That is, the Levosimendan compound showed a concentration-dependent tau aggregation inhibitory effect.

On the other hand, OR-1855 and OR-1896 compounds did not show tau aggregation inhibitory effect. Therefore, the metabolite form of Levosimendan in the cell does not show a tau aggregation inhibitory effect.

Example 1-6: Levosimendan Confirm the cognitive function improvement effect of compound derivatives

Whether the tau aggregation inhibitory effect of the Levosimendan compound also affects the improvement of cognitive function was confirmed using an animal model. The animal model was performed based on the Tau-P301L BiFC transgenic mouse exhibiting tauopathy, specifically, the mouse described in Korean Patent No. 10-1876423 (Title of the Invention: Dementia model transgenic mouse and screening method using the same) was used.

Levosimendan compound, positive control (MB) and negative control (DMSO) were dissolved in 30% PEG 400 physiological saline, and this was administered to 9-month-old mice at a dose of 5 mg/kg (body weight) 3 times a week for 3 months. It was administered intraperitoneally. In order to measure the degree of cognitive function improvement due to the administration of the Levosimendan compound, 3 months after drug administration, a novel object recognition test and a passive avoidance test were performed.

For the novel object recognition test, after fixing two objects (O) of the same shape and size in a box (40 x 40 x 40 cm) made so that the outside cannot be seen from the inside, the mice in each group was allowed to learn the object for 10 minutes. After 24 hours, replace one of the two objects with a new object (N), record the time of searching for the previously learned object (O) and the new object (N) for 10 minutes, and recognize it It was quantified as an index (Recognition index = individual object (O or N) search time/total object search time).

9 is a result of a novel object recognition test confirming the cognitive function improvement effect of the levosimendan compound using an animal model of tauopathy according to an embodiment of the present invention. As a result, as shown in FIG. 9 , it was confirmed that the experimental group administered with the Levosimendan compound had a higher recognition index for a new object compared to the negative control group or the positive control group (MB).

For the passive avoidance test, place the mouse in the bright space A in the passive avoidance test device, and after about 30 seconds of search time elapses, open the door to enter the dark space B. made it possible When the mouse enters the B space, the door is closed, and an electric shock of 3 mA is passed through the grid on the floor for 2 seconds, so that the mouse can learn to memorize the dark space B and the electric shock. After 24 hours, the mouse was placed in the A space again, and when the door was opened after 30 seconds, the time taken to cross to the dark side was measured up to 540 seconds.

10 is a result of a passive avoidance test confirming the cognitive function improvement effect of the levosimendan compound using an animal model of tauopathy according to an embodiment of the present invention. As a result, as shown in FIG. 10 , it was confirmed that the experimental group administered with the Levosimendan compound remembered the stimulus in the dark space and took a longer time to move to the dark side 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 effect of improving cognitive ability.

Example 1-7: Confirmation of neuronal degeneration inhibitory effect of Levosimendan compound derivatives

In an animal model of tauopathy, it was confirmed whether the tau aggregation inhibitory effect of the Levosimendan compound had an effect on the inhibition of neuronal degeneration. In order to secure brain tissue, mice after drug administration and behavioral evaluation experiments (experiments in Examples 1-6 above) were overanesthetized using 2% Evertin, and perfusion was performed through cardiac perfusion using physiological saline. After perfusion, the tissue was fixed using 4% paraformaldehyde and the brain was extracted. The extracted brain was additionally fixed in 4% paraformaldehyde for about one day, and then stored in 30% sucrose solution to prepare frozen section samples. Thereafter, a mold for frozen sectioning was made using OCT compound, and tissue sections with a thickness of 40 μm were obtained using cryosection.

To stain the neuronal cell body, the brain tissue sections of each administration group were stained using 1% cresyl violet and imaged using an optical microscope. The number of CA1 cells in the hippocampus was measured.

11 is an optical microscopic photograph of a brain tissue section confirming the neurodegeneration inhibitory effect of the levosimendan compound using an animal model of tauopathy, and the number of neurons included in the brain tissue section according to an embodiment of the present invention. It is a graph representing As a result, as shown in FIG. 11 , in the brain tissue section of the experimental group to which the Levosimendan compound was administered, the number of neurons was observed in the cortex by about 42.4% and the hippocampus by about 36.0% more than in the negative control group. That is, it was confirmed that the Levosimendan compound was effective in inhibiting neuronal degeneration caused by tauopathy.

[ Example 2 : Levosimendan and Confirmation of the effect of combined treatment of 8 types of compounds]

Example 2-1: intracellular tau Confirmation of aggregation inhibitory effect

donepezil, tacrine, rivastigmine, galantamine, memantine, dopamine, curcumin, and lithium chloride ( LiCl), when one type of compound selected from the group consisting of each was treated in combination, the inhibitory effect on tau aggregation in cells was confirmed.

Tau-BiFC cells capable of monitoring tau aggregation were treated with purified metastatic tau proteomic TauK18P301L (treatment concentration 5 μg/ml) with a tau aggregation derivative. At the same time, levosimendan (Levo.) compound (treatment concentration 3 μM), donepezil (treatment concentration 3, 10, 30 μM), and tacrine (treatment concentration 2.5, 5, 10 μM), Rivastigmine (treatment concentration 1, 10, 100 μM), galantamine (treatment concentration 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 treated in combination. At this time, methylene blue (MB) (treatment concentration of 3 μM), which is known to have an inhibitory effect on tau aggregation, was used as a comparative compound (positive control). After 46 hours of drug treatment, the degree of intracellular tau aggregation was quantified by Tau-BiFC fluorescence intnesity.

12 shows, according to an embodiment of the present invention, donepezil, tacrine, rivastigmine, galantamine (donepezil), tacrine, and galantamine ( galantamine), memantine (Memantine), dopamine (Dopamine), curcumin (Curcumin), and lithium chloride (LiCl), respectively, after the combined treatment, the results of measuring the inhibition of intracellular tau aggregation. As a result, as shown in FIG. 12, when the levosimendan (Levosimendan, Levo.) compound and the eight compounds were treated in combination, the metastatic tau proteomic TauK18P301L-induced tau aggregation (100%) was inhibited. confirmed that it can be done.

Among them, when the levosimendan (Levo.) (3 μM) compound and rivastigmine or lithium chloride (LiCl) were treated in combination, tau was superior to that when the levosimendan compound was treated alone. It showed an aggregation inhibitory effect. Furthermore, the combination of levosimendan and rivasticmine and the combination of levosimendan and lithium chloride showed a significantly better tau aggregation inhibitory effect in a concentration-dependent manner as the concentration of rivastikmin and lithium chloride increased, so that the inhibition of tau aggregation It was confirmed once again that the effect was a synergistic effect according to the combination of levosimendan and rivasticmine and the combination of levosimendan and lithium chloride.

In comparison, in the case of methylene blue (MB), when co-treated with the 8 compounds, respectively, it was possible to inhibit the tau aggregation (100%) induced by the metastatic tau proteomic TauK18P301L treatment, but methylene It was confirmed that there was no combination showing an excellent effect in a concentration-dependent manner than when blue was used alone.

Example 2-2: Confirmation of cytotoxicity

Levosimendan compound and the 8 kinds of donepezil, tacrine, rivastigmine, galantamine, memantine, dopamine, curcumin (Curcumin) and lithium chloride (LiCl) It was confirmed whether the inhibitory effect of tau aggregation seen when one compound selected from the group consisting of compounds was treated in combination with cytotoxicity.

The number of cells was quantified by treatment with Hoechst (2 μg/ml), which stains cell nuclei 46 hours after drug treatment under the concentration conditions of Example 2-1, which reflects the degree of cytotoxicity according to compound treatment.

13 is, according to an embodiment of the present invention, for each of levosimendan and methylene blue, donepezil, tacrine, rivastigmine, galantamine ( galantamine), memantine, dopamine, curcumin, and lithium chloride (LiCl), respectively, were treated in combination, and cell viability was measured. As a result, as shown in FIG. 13 , the levosimendan compound alone, and of the eight compounds, donepezil, tacrine, rivastigmine, and galantamine , It was confirmed that more than 90% of all cells were alive when treated in combination with memantine.

However, when levosimendan was treated in combination with dopamine and lithium chloride (LiCl) compounds, respectively, the cell viability was 75% or less and 50% or less at high concentrations. It can be judged that the synergistic effect of inhibiting tau aggregation in the combined treatment of the levosimendan 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 the 8 types of compounds, it was confirmed that the cell viability was between 25% and 60%. Therefore, it was determined that the intracellular tau aggregation inhibitory effect when methylene blue and the eight compounds were co-administered was due to the cytotoxicity of the compound.

Example 2-3: Intracellular by concentration tau Confirmation of aggregation inhibitory effect and cell survival

The tau aggregation inhibitory effect of the levosimendan compound and the rivastigmine (RV) compound was confirmed for each concentration in the cell. Tau aggregation monitoring cell line Tau-BiFC cells were treated with the metastatic tau protein TauK18P301L (treatment concentration 5 μg/ml), a tau aggregation derivative, and Levosimendan (treatment concentration 1, 3, 10 μM) and Rivastik. Min (Rivastigmine) (treatment concentrations 1, 10, 100 μM) was treated in combination with each concentration. 46 hours after drug treatment, the degree of intracellular tau aggregation was quantified by Tau-BiFC intensity, and cell viability was quantified by counting the number of cells according to cell nuclear staining using Hoechst.

Figure 14 is, according to an embodiment of the present invention, with respect to each concentration of the levosimendan compound and methylene blue, rivastigmine (Rivastigmine) for each concentration, after the combined treatment, intracellular tau aggregation inhibition and cells It is the result of measuring toxicity. As a result, as shown in FIG. 14 , when levosimendan and rivastigmine were treated in combination, the concentration-dependent increase in the tau aggregation inhibitory effect according to the respective concentrations of levosimendan and rivastigmine was observed together with the cell image of Tau-BiFC. It was confirmed by the decrease in intensity. According to this, it was confirmed that when levosimendan and rivastigmine were treated in combination, the tau inhibitory effect had a synergistic effect and was significantly superior to when administered alone.

In comparison, in the case of methylene blue (MB), when methylene blue (treatment concentrations 1, 3, 10 μM) was treated alone, and rivastigmine (RV) (treatment concentrations 1, 10) , 100 μM), the degree of inhibition of intracellular tau aggregation when treated in combination with the compound appears to be due to a decrease in the number of cells according to the cytotoxicity of the methylene blue compound by concentration.

Although the present invention has been shown and described in relation to specific preferred embodiments, the present invention may be modified and changed in various ways without departing from the technical features or fields of the present invention provided by the following claims. It will be apparent to those of ordinary skill in the art.

Claims (8)

As a composition for preventing or treating tau (tau) aggregation-related diseases, including levosimendan and rivastigmine,
The tau (tau) aggregation-related disease is Alzheimer's disease, Parkinson's disease, tauopathy (tauopathy) and vascular dementia, characterized in that selected from the group consisting of, tau (tau) aggregation-related disease prevention or treatment composition.
The method of claim 1,
The levosimendan is a levosimendan compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound, tau (tau) aggregation-related disease prevention or treatment composition.
The method of claim 1,
The rivastiqmin is a rivasticmine compound, a pharmaceutically acceptable salt, stereoisomer or solvate of the compound, tau (tau) aggregation-related disease prevention or treatment composition.
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) A composition for preventing or treating tau aggregation-related diseases, characterized in that it further comprises.
5. The method according to any one of claims 1 to 4,
A composition for preventing or treating tau aggregation-related diseases, characterized in that it further comprises an active agent selected from the group consisting of antioxidants, cholinesterase inhibitors, and combinations thereof.
delete The method of claim 1,
The tauopathy is Alzheimer's disease, progressive supranuclear palsy, cortical-basal nuclear degeneration, Argyrophilic Grain Disease, Peak disease and hereditary frontotemporal dementia, characterized in that selected from the group consisting of, tau aggregation-related A composition for preventing or treating a disease.
As a health functional food for the prevention or improvement of tau (tau) aggregation-related diseases, including levosimendan and rivastigmine,
The tau (tau) aggregation-related disease is Alzheimer's disease, Parkinson's disease, tauopathy (tauopathy) and vascular dementia, characterized in that selected from the group consisting of, tau (tau) aggregation-related disease prevention or improvement health functional food.

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