KR102601028B1 - Pharmaceutical composition for the prevention or treatment of neurodegenerative disease comprising abemaciclib an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of degenerative brain diseases containing abemaciclib as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of degenerative brain diseases containing abemaciclib or a salt thereof as an active ingredient. It relates to pharmaceutical compositions for the prevention or treatment of and health functional foods for the prevention or improvement of degenerative brain diseases. Abemaciclib according to the present invention has excellent activity in suppressing inflammatory cytokines COX-2, IL-1β, IL-6 or iNOS in brain neurons, and inhibits signaling of TLR4 and p-STAT3 related to inflammatory response. It has excellent activity in suppressing the level of LPS and can suppress the activity of microglia or astrocytes by LPS. It also has the activity of suppressing amyloid plaques, increasing the number of dendrites, and suppressing the phosphorylation of tau protein. , has the characteristic of improving long-term memory in mice suffering from degenerative brain disease. Therefore, abemaciclib of the present invention can be used as an active ingredient in medicines and health functional foods to prevent, improve, or treat degenerative brain diseases or neuroinflammatory diseases.

Description

Pharmaceutical composition for the prevention or treatment of neurodegenerative disease comprising abemaciclib an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases containing abemaciclib as an active ingredient.

Degenerative brain disease refers to a disease that occurs in the brain among degenerative diseases that occur with age. It can be classified based on the main symptoms and affected brain area, and representative examples include Alzheimer's disease and Parkinson's disease. . Degenerative brain diseases are known to be caused by neurodegeneration due to aging and the death of nerve cells due to protein aggregation due to genetic and environmental factors.

In addition, degenerative brain diseases include the death or deterioration of specific brain cells that occurs temporarily or over a long period of time. Since brain cells that die once cannot be regenerated, they are known to eventually lead to fatal loss of brain function, especially , Brain dysfunction accompanied by a progressive decline in cognitive, sensory, motor, and systemic functions eventually leads to changes in personality and behavior, reaching a point where patients are unable to take care of themselves. The main pathways of brain cell death include oxidative toxicity due to oxidative stress, excitatory toxicity, and apoptosis, and each causes cell death through a unique signaling process. Specifically, oxidative damage to proteins, nucleic acids, and lipids following accumulation of reactive oxygen species was suggested as the main cause of brain cell death in patients with stroke, brain damage, Alzheimer's disease (AD), and Parkinson's disease. , In particular, oxidative stress caused by free radicals has been reported as the main cause of cell death in each tissue in the body, and has also been suggested as one of the cell death cycles that occurs in brain and nerve diseases (Schapira, A.H., Curr. Opin. Neurol., 9(4):260-264, 1996).

In addition, the activity of microglia and astrocytes has been reported to be related to the onset and progression of degenerative cranial nerve diseases, and microglia are immune cells residing in the central nervous system (CNS). It is known to be activated by external stimuli and cause immune and inflammatory reactions. Microglia are cells that perform the primary immune function in the central nervous system. They maintain the shape of long, thin branches and a thin cell body, and when toxins introduced from the outside or generated internally are present, they attack the nerves from these toxins. To protect the cell, it changes into an activated shape with thick, short branches and a fat cell body.

However, when microglia are activated by substances such as bacterial endotoxin lipopolysaccharide (LPS), interferon-γ, beta-amyloid, or ganglioside, they actively engage in phagocytosis, unlike microglial cells in a normal state. It proliferates cells and expresses genes such as cytokines such as TNF-α, IL-1β, and IL-6, chemokines, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2), thereby eliminating inflammatory mediators. Create. This activation of microglial cells removes damaged cells and protects nerve cells from invading bacteria or viruses, but also includes nitric oxide (NO) produced by iNOS, prostaglandins produced by COX-2, and TNF. Since -α and other substances are also toxic to nerve cells, the activation of microglial cells ultimately worsens the damage to nerve cells. Therefore, inhibiting the appropriate activation of microglia may be another way to treat degenerative brain diseases.

Additionally, astrocytes play an important role not only in the brain development process but also in maintaining normal brain activity. Astrocytes in the brain have been shown to play a role in supporting neuronal activity by appropriately removing neurotransmitters secreted by nerve cells or regulating ion concentration in the brain. In addition, neural stem cells differentiate into nerve cells. It has been found to play a decisive role.

However, when astrocytes are injured in the brain, proliferation becomes active, swelling occurs, and they become activated into reactive astrocytes such as astrogliosis. These reactive astrocytes are observed in AIDS-related dementia, brain damage, ischemic brain disease, and Alzheimer's disease. Therefore, continuous activation of astrocytes eventually leads to neuronal death. Therefore, suppressing the appropriate activation of astrocytes can also be another way to treat degenerative brain diseases.

Additionally, it is known that degenerative brain diseases, including Alzheimer's, are caused by the accumulation of beta-amyloid, and research is ongoing to remove plaques formed by the aggregation of these proteins. Recently, research has been published showing that the tau protein has an abnormal structure and forms inclusion bodies, which causes degenerative brain diseases, so tau protein is emerging as a target for these diseases.

Meanwhile, treatments currently being used to treat degenerative brain diseases include drug therapy, surgery, and physical therapy. In the case of drug therapy, it generally supplements the dopamine that is lacking in the brain and improves nerve transmission due to the lack of dopamine. Drugs are being used for the purpose of correcting the imbalance of substances, preventing or delaying the destruction of nerve cells, and controlling other symptoms such as depression, but there is still no specific mechanism for signal transmission for degenerative brain diseases. As it is not yet clearly known whether it is toxic or not, there is no fundamental and effective treatment.

Meanwhile, abemaciclib is a drug currently used as a treatment for breast cancer and is known to have inhibitory activity on cyclin-dependent kinase (CDK) 4 and 6, but its relationship with degenerative brain disease has not yet been studied. There is no bar.

US published patent US20190076425

Accordingly, the present inventors found that abemaciclib, known as a conventional breast cancer treatment, can effectively suppress inflammation of brain neurons, inhibits phosphorylation of tau protein, improves long-term memory, and inhibits amyloid plaque formation, so it can prevent or treat degenerative brain diseases. The present invention was completed by confirming that it can be used as a therapeutic agent.

Therefore, the object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning disability, or memory impairment, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving degenerative brain diseases, containing abemaciclib or a salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving cognitive impairment, learning disability, or memory impairment, containing abemaciclib or a salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neuroinflammatory diseases, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving neuroinflammatory diseases, which contains abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the composition can inhibit the expression and production of inflammatory cytokines COX-2, IL-1β, IL-6, or iNOS.

In one embodiment of the present invention, the composition can inhibit the activity of Toll-like receptor 4 (TLR4) and suppress the level of p-STAT3.

In one embodiment of the present invention, the composition inhibits the activity of microglia or astrocytes; Inhibits amyloid plaques; Increased number of dendrites; Inhibition of phosphorylation of tau protein; Alternatively, it may have an activity that increases memory and cognition.

In one embodiment of the present invention, the degenerative brain disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, multiple neural atrophy, epilepsy, encephalopathy, and stroke.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive impairment, learning disability, or memory impairment, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving degenerative brain diseases, comprising abemaciclib or a salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving cognitive impairment, learning disability, or memory impairment, containing abemaciclib or a salt thereof as an active ingredient.

In one embodiment of the present invention, abemaciclib or a salt thereof can inhibit the expression and production of proinflammatory cytokines COX-2, IL-1β, IL-6 or iNOS.

In one embodiment of the present invention, abemaciclib or a salt thereof can inhibit the activity of Toll-like receptor 4 (TLR4) and suppress the level of p-STAT3.

In one embodiment of the present invention, abemaciclib or a salt thereof inhibits the activity of microglia or astrocytes; Inhibits amyloid plaques; Increased number of dendrites; Inhibition of phosphorylation of tau protein; Alternatively, it may have an activity that increases memory and cognition.

In one embodiment of the present invention, the degenerative brain disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, multiple neural atrophy, epilepsy, encephalopathy, and stroke.

The present invention also provides a pharmaceutical composition for preventing or treating neuroinflammatory diseases, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the neuroinflammatory disease may be selected from the group consisting of neuroblastoma, Lou Gehrig's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, and amyotrophic lateral sclerosis.

In addition, the present invention provides a health functional food for preventing or improving neuroinflammatory diseases, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the neuroinflammatory disease may be selected from the group consisting of neuroblastoma, Lou Gehrig's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, and amyotrophic lateral sclerosis.

Abemaciclib according to the present invention has excellent activity in suppressing inflammatory cytokines COX-2, IL-1β, IL-6 or iNOS in brain neurons, and inhibits signaling of TLR4 and p-STAT3 related to inflammatory response. It has excellent activity in suppressing the level of LPS and can suppress the activity of microglia or astrocytes by LPS. It also has the activity of suppressing amyloid plaques, increasing the number of dendrites, and suppressing the phosphorylation of tau protein. , has the characteristic of improving long-term memory in mice suffering from degenerative brain disease. Therefore, abemaciclib of the present invention can be used as an active ingredient in medicines and health functional foods to prevent, improve, or treat degenerative brain diseases or neuroinflammatory diseases.

Figure 1 shows the results of analyzing the levels of pro-inflammatory cytokines and anti-inflammatory cytokines induced by LPS according to abemaciclib treatment in microglial cells. (A) to (B) show the results after treatment with abemaciclib at various concentrations. These are the results of the MTT assay, (C) to (G) are the results of analyzing the levels of pro-inflammatory cytokines after pretreatment with abemaciclib and LPS treatment through RT-PCR, and (H) to (L) are the results of the MTT assay. is the result of analyzing the level of pro-inflammatory cytokines after pretreatment with LPS and treatment with abemaciclib by RT-PCR, and (M) to (O) are the levels of anti-inflammatory cytokines after pretreatment with abemaciclib and treatment with LPS. This is the result of analysis through Q-PCR, and (P) to (R) show the results of LPS treatment followed by abemaciclib treatment and then analysis of the level of anti-inflammatory cytokines through Q-PCR.
Figure 2 shows the results of confirming whether the effect of abemaciclib on changes in pro-inflammatory cytokines induced by LPS is dependent on TLR4. (A) to (E) show microglial cells treated with TAK-242, After treatment with abemaciclib and subsequent treatment with LPS, the levels of COX-2, IL-6, IL-1β, and COX-2 in cells were measured through RT-PCR, (F) to (H) ) shows that microglial cells were treated with abemaciclib, then treated with LPS, and the levels of p-AKT and AKT were measured through Western blot. (I)~(M), microglial cells were treated with MK2206 (AKT inhibitor), then treated with abemaciclib, and then treated with LPS, and COX-2, IL-6, IL- These are the results of measuring the levels of 1β and COX-2, and (N) to (Q) are the nuclear and cytoplasmic fractions of cell lysates obtained by treating microglial cells with abemaciclib and then treating them with LPS. After processing, the level of p-STAT3 in each fraction was measured by Western blot.
Figure 3 shows the results of treating primary microglial cells and primary astrocytes with abemaciclib, followed by treatment with LPS, and analyzing the levels of pro-inflammatory cytokines by Q-PCR. (A) shows the results measured in primary microglial cells, and (B) shows the results measured in primary astrocytes.
Figure 4 is an analysis of the effect of abemaciclib on cognitive decline caused by LPS in wild-type mice. Abemaciclib was administered to wild-type mice every day for 7 days, then LPS was administered 30 minutes later, and behavioral experiments were conducted on days 8 and 9. Shows the results of performing , (A) shows the results of Y maze analysis on the 8th day, and (B) shows the results of the NOR test analysis on the 8th and 9th days.
Figure 5 is an analysis of the effect of abemaciclib on the level of inflammatory cytokines caused by LPS in wild-type mice. After daily injection of abemaciclib for 3 days in wild-type mice, LPS was administered for 6 hours, and then the brain cortex was injected with abemaciclib. This shows the results of immunohistochemistry on the cortex and hippocampus. (A) and (C) used anti-Iba-1 antibodies, and (B) and (C) used anti-Iba-1 antibodies. This is the result using -GFAP antibody.
Figure 6 is an analysis of the effect of abemaciclib on changes in the levels of IL-1β and IL-6 inflammatory cytokines caused by LPS in wild-type mice. After daily injection of abemaciclib mesylate for 3 days in wild-type mice, (A) and (C) show the results of immunohistochemistry on the brain cortex and hippocampus after administering LPS for 6 hours. Anti-IL-1β antibody was used in (A) and (C). , (B), (C) are the results using anti-IL-6 antibody.
Figure 7 shows the results of immunohistochemistry on the brain cortex and hippocampus after treating Alzheimer's disease-induced 5xFAD mice with abemaciclib daily for 2 weeks, (A) , (D) is the result using an anti-Iba-1 antibody, (B) and (D) are the results using an anti-GFAP antibody, and (C) and (D) are the results using an anti-IL-1β antibody.
Figure 8 shows the analysis of cognitive and memory improvement activity and dendrite number increase activity with the control group administered vehicle after abemaciclib was administered to Alzheimer's disease-induced 5xFAD mice daily for 2 weeks, (A) ~ ( B) shows the results of Y-maze, NOR Training, and NOR test, and (C) to (E) show the results of measuring the number of dendrites in the cortex and hippocampus tissue.
Figure 9 shows the results of measuring tau phosphorylation levels in each of the cortex and hippocampus tissues in Alzheimer's disease-induced 5xFAD mice administered abemaciclib, (A) and (D) using anti-AT180 antibody, ( B) and (D) show the results of immunohistochemical analysis using an anti-AT100 antibody, and (C) and (D) show the results of immunohistochemical analysis using an anti-tau5 antibody.
Figure 10 shows the amyloid plaque inhibition effect of abemaciclib treatment in Alzheimer's disease-induced 5xFAD mice in the cortex and hippocampus tissue. (A)-(B) show immunohistochemical analysis using anti-4G8 antibody. It shows the results.
Figure 11 shows the analysis of cognitive and memory improvement activity in Alzheimer's disease-induced 5xFAD mice after daily administration of abemaciclib or donepezil for 2 weeks, together with the control group administered vehicle, (A) Y-maze, (B) ) is the result of measuring NOR Training and NOR test.
Figure 12 shows immunohistochemistry on the brain cortex and hippocampus together with the control group administered vehicle after abemaciclib or donepezil was administered to Alzheimer's disease-induced 5xFAD mice daily for 2 weeks. Showing the results, (A)-(B) are the results using anti-GFAP antibody and anti-6E10 antibody.
Figure 13 shows immunohistochemistry on the brain cortex and hippocampus together with the control group administered vehicle after abemaciclib or donepezil was administered to Alzheimer's disease-induced 5xFAD mice daily for 2 weeks. Showing the results, (A)-(B) are the results using anti-phospho-GSK-3β (Y216) antibody or anti-DYRK1A antibody.
Figure 14 shows improvement in cognition and memory along with the control group administered vehicle after abemaciclib mesylate was administered intraperitoneally (30 mg/kg) or orally (60 mg/kg) daily to Alzheimer's disease-induced PS19 mice for 2 weeks. As a result of analyzing the activity, (A) is the result of measuring the Y-maze, and (B) is the result of analysis through NOR Training and NOR test.

The present invention is characterized by providing a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

As previously mentioned in the prior art, abemaciclib is known as a treatment for breast cancer, but has not yet been studied for its potential in treating degenerative brain diseases.

The abemaciclib according to the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid can be used. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, as well as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodine. Ide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methylbenzoate Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol nitrate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

In one embodiment of the present invention, abemaciclib methylate (abemaciclib mesylate) was used.

Accordingly, the present invention demonstrated for the first time that abemaciclib can be used as a treatment for degenerative brain diseases.

According to one embodiment of the present invention, it was confirmed that abemaciclib can effectively inhibit the expression and production of inflammatory cytokines and pro-inflammatory cytokines induced by LPS, a known inflammatory factor, in brain nerve cells. It was confirmed that the production of COX-2, IL-1β, IL-6, or iNOS by LPS was effectively inhibited. Among various inflammatory factors, LPS (lipopolysaccharide), one of the endotoxins, enhances the secretion of pro-inflammatory cytokines, which leads to the secretion of inflammatory mediators such as nitric oxide and prostaglandins, and various inflammatory diseases due to excessive inflammatory responses. It occurs.

Therefore, the present inventors found that abemaciclib can suppress the pro-inflammatory response caused by LPS, thereby preventing, improving, or treating inflammatory diseases and brain inflammatory diseases that can be caused by excessive inflammatory responses.

In another embodiment of the present invention, an experiment was performed to determine whether the inhibition of inflammatory cytokines by abemaciclib was related to the TLR4/AKT/STAT3 signaling system.

TLR4 is known to induce the pathology of inflammatory diseases by producing various inflammatory factors through activation of NF-kB when cells are stimulated by LPS. AKT is known to activate NF-kB, and AKT is known to activate NF-kB. Signal transduction is known to be related to the immune response induced by LPS through the TLR4 signaling pathway, and the level of pro-inflammatory cytokines is known to be reduced in human liver cancer cell lines when AKT is knocked out. Additionally, activated TLR4 is known to affect STAT3 and other transcription factors, and continuously activated STAT3 is known to be closely related to the onset of inflammatory diseases.

According to one embodiment of the present invention, it was confirmed that abemaciclib inhibits the signaling activation of TLR4/AKT/STAT3, and LPS-induced inflammatory cytokines COX-2, IL-1β, IL-6, and iNOS. The level of was found to be reduced in both microglia and astrocytes by abemaciclib treatment, and the phosphorylation level of p-AKT was also found to be reduced.

Therefore, it was found that abemaciclib can inhibit the activation of TLR4/AKT/STAT3 signaling, which is involved in causing inflammation in brain neurons.

In addition, abemaciclib of the present invention has the effect of inhibiting the damage caused by activated microglia or activated astrocytes to nerve cells by inhibiting the activity of microglia or astrocytes caused by LPS. It was confirmed that it exists.

Microglial cells, which play the role of macrophages in the brain, are important effector cells that regulate immune responses in the central nervous system (CNS). Their activation plays an important role in maintaining CNS homeostasis by removing foreign substances caused by drugs or toxins and secreting nerve growth factors. However, when exposed to harmful stress such as signals generated from damaged neurons, accumulation of abnormal proteins modified by external stimuli, or invasion of pathogens, the activity of microglial cells increases excessively, causing damage to nerve cells, leading to Alzheimer's disease. It can cause degenerative neurological diseases such as Parkinson's disease, multiple sclerosis, and cerebral infarction. In addition, recent research results have revealed that excessive inflammatory responses in microglia or astrocytes are the cause of degenerative brain diseases.

Specifically, hyperactivated microglia, unlike normal microglia, actively engage in phagocytosis, proliferate, and produce cytokines such as TNF-α, IL-1β, and IL-6, chemokines, and iNOS. Genes such as (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) are expressed to produce inflammatory mediators. Activation of microglial cells removes damaged cells and protects nerve cells from invading bacteria or viruses. However, nitric oxide (NO) produced by iNOS, prostaglandins produced by COX-2, and TNF- Since α and the like are also toxic to nerve cells, the resulting activation of microglial cells worsens damage to nerve cells and acts as a cause of degenerative brain disease or neurodegenerative disease.

In addition, astrocytes are also known to play an important role in maintaining normal brain activity. In particular, they are known to play a role in neuronal synapse formation, synapse number control, synaptic function, and differentiation of neural stem cells into neurons. . However, when these astrocytes become excessively reactive, that is, when they remain in an excessively activated state, they cause death of nerve cells and induce death of neighboring nerve cells, thereby acting as a cause of degenerative brain disease. Therefore, suppressing excessive astrocyte activation can also be a new treatment method for degenerative brain diseases.

Causes of excessive activation of microglia and astrocytes include lipopolysaccharide (LPS), interferon-γ, beta-amyloid, and ganglioside, which are bacterial endotoxins that can induce inflammatory responses.

In addition, in another embodiment of the present invention, it was confirmed that abemaciclib can increase dendritic spines in hippocampal neurons, and further, abemaciclib was administered to mice suffering from Alzheimer's disease, a degenerative brain disease. As a result of administration, it was confirmed that it had the activity of suppressing amyloid plaques, suppressing beta amyloid, and suppressing phosphorylation of Tau protein.

Beta-amyloid (Aβ) is derived from amyloid precursor protein (APP), which is broken down by beta-secretase and gamma-secretase to produce Aβ, and is mainly found in the brains of patients with brain diseases such as Alzheimer's disease. It is the main ingredient and if abnormally accumulated, it can cause various brain diseases.

Additionally, excessively accumulated beta-amyloid causes an inflammatory response in the hippocampus, cerebral cortex, and perioral cells, which can damage nerve cells and damage the neural network that maintains normal brain function.

Additionally, tau protein is a class of microtubule-binding proteins with a molecular weight of 50,000 to 70,000 Da and is involved in the tangle of nerve fibers. Tau protein is one of the microtubule-associated proteins present in axons of normal nerve cells. When these tau proteins accumulate excessively, microtubules collapse and the network of normal nerve cells disintegrates. It is known that the cause of tau protein accumulation is that phosphorus is excessively attached to tau protein, forming a heavy body due to phosphorylation, and the accumulation of these tau proteins and aggregation of nerve cells are known to cause various degenerative brain diseases. .

In this regard, since abemaciclib of the present invention has the activity of inhibiting beta amyloid and phosphorylation of Tau protein, it is used as a treatment for degenerative brain diseases caused by phosphorylation of Tau protein in nerve cells or brain cells. I found out that it was available.

In addition, in one embodiment of the present invention, Y-maze and NOR tests for cognitive and memory analysis were performed on mice with Alzheimer's disease in a group administered abemaciclib and a control group not administered abemaciclib. It was confirmed that mice with Alzheimer's disease treated with abemaciclib had improved long-term memory compared to control mice.

Therefore, abemaciclib of the present invention can be used as a treatment for the prevention, improvement or treatment of degenerative brain diseases, and the degenerative brain diseases are not limited thereto, but include Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and multiple neuropathy. It may be selected from the group consisting of atrophy, epilepsy, encephalopathy, and stroke.

Furthermore, it was confirmed in a mouse animal model with degenerative brain disease that the abemaciclib of the present invention has the effect of improving memory, cognitive ability, and learning ability. Abemaciclib of the present invention or a pharmaceutically acceptable salt thereof A composition containing as an active ingredient can also be used as a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning disability, or memory impairment.

In the present invention, "improvement of memory" and "improvement of cognitive ability" are memory loss, memory impairment, or cognitive ability that occurs when the brain atrophies and brain nerve cells are destroyed due to physical fatigue, lack of sleep, excessive alcohol consumption, dementia, etc. Reduction refers to the effect of maintaining cognitive ability by controlling harmful substances that damage brain cells, or improving deteriorated cognitive ability by controlling neurotransmitters in the brain. The above-mentioned memory is the ability to receive necessary information, store it in the brain, and retrieve it for use when necessary, and cognitive ability refers to the ability to discern and recognize objects.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose. ), gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition may be any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. It can have one dosage form.

Additionally, as described above, the pharmaceutical compositions of the present invention can be administered to treat various diseases, including symptoms associated with degenerative brain diseases.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, and It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects. The general dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or rectally, depending on the purpose, but is not limited thereto. Additionally, the composition can be administered by any device capable of transporting the active agent to target cells.

The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response regulators for the prevention and treatment of inflammatory diseases.

Furthermore, the present invention can provide a health functional food for preventing or improving degenerative brain diseases containing abemaciclib (abemaciclib mesylate) as an active ingredient.

In addition, the present invention can provide a health functional food for preventing or improving cognitive impairment, learning disability, or memory impairment containing abemaciclib (abemaciclib mesylate) as an active ingredient.

The health functional food of the present invention may additionally contain a foodologically acceptable carrier.

The health functional food includes the form of pills, powders, granules, precipitates, tablets, capsules or liquids, and there is no particular limitation on the type of food to which the active ingredient of the present invention can be added, for example, various beverages, These include gum, tea, vitamin complexes, and health supplements.

In addition to the active ingredients of the present invention, other ingredients that do not interfere with the prevention and improvement of degenerative brain diseases can be added to the health functional food, and the types are not particularly limited. For example, like regular foods, it may contain various herbal extracts, foodologically acceptable food supplements, or natural carbohydrates as additional ingredients.

The term “health functional food” used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, functionality means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.

There is no particular limitation on the type of health functional food of the present invention, and specific examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, There are beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and can include all health functional foods in the conventional sense.

Furthermore, the present invention can provide a pharmaceutical composition for preventing or treating neuroinflammatory diseases, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

Neuroinflammatory responses include activation of innate immune cells (microglia), release of inflammatory mediators such as nitric oxide (NO), cytokines and chemokines, and macrophage infiltration, which leads to neuronal cell death. Inflammatory activation of microglia and astrocytes is thought to be a pathological marker and an important mechanism in the progression of neurodegenerative diseases. Tight regulation of microglial activity is essential to maintain brain homeostasis and prevent infectious and inflammatory diseases.

Meanwhile, in one embodiment of the present invention, it was confirmed that abemaciclib can effectively suppress the levels of pro-inflammatory cytokines induced by LPS in microglia and astrocytes, and can increase the expression of anti-inflammatory factors. Not only that, but it was also confirmed through experiments that it can inhibit the activation of microglia and astrocytes.

Therefore, abemaciclib of the present invention can suppress inflammation in brain cells and has the effect of preventing or treating neuroinflammatory diseases by suppressing the activity of microglia and astrocytes.

In the present invention, the neuroinflammatory disease is not limited thereto, but may be selected from the group consisting of neuroblastoma, Lou Gehrig's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, and amyotrophic lateral sclerosis.

In addition, the present invention can provide a health functional food for preventing or improving neuroinflammatory diseases, which contains abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Preparation example and experiment method>

All experiments performed in the examples of the present invention were conducted with the approval of the Korea Brain Research Institute's Animal Experiment Ethics Committee (IACUC) (KBRI, approval numbers 19-00049, 19-00042).

Cell lines and culture conditions

BV2 microglial cells (obtained from Dr. Kyung-Ho Suk) were cultured in DMEM/high glucose (Invitrogen, Carlsbad, CA, USA) containing 5% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). , USA) medium was used and cultured in a 5% CO2 incubator. Additionally, the results obtained from all in vitro experiments were analyzed semi-automatically using Image J software, and the results were confirmed by an independent researcher who was not involved in the current experiment. Additionally, in the following examples of the present invention, abemaciclib mesylate, commercially available as abemaciclib, was used.

MTT assay

Cell viability was measured using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. BV2 microglial cells were distributed in 96-well plates and treated with various concentrations of abemaciclib mesylate (0.1, 1, 5, 10, 25, 50 μM) in the absence of FBS for 24 hours. The cells were then treated with 0.5 mg/ml MTT and cultured in a 5% CO2 incubator at 37°C for 3 hours, and then the absorbance was measured at 580 nm.

RT-PCR (Reverse transcription-polymerase chain reaction)

Total RNA was extracted from cells using TriZol (Invitrogene). Afterwards, total RNA was reverse transcribed into cDNA using Superscript cDNA Premix Kit II containing oligoDT (GeNetBio, Korea). RT-PCR was performed using Prime Taq Premix (GeNetBio, Korea), and RT-PCR was performed on BV microglial cells using the following primers.

IL-1β Forward (F): 5'-AGC TGG AGA GTG TGG ATC CC-3'

IL-1β Reverse (R): 5'-CCT GTC TTG GCC GAG GAC TA-3'

IL-6 Forward (F): 5'-CCA CTT CAC AAG TCG GAG GC-3'

IL-6 Rorward (R): 5'-GGA GAG CAT TGG AAA TTG GGG T-3'

COX-2 Forward (F): 5'-GCC AGC AAA GCC TAG AGC AA-3'

COX-2 Rorward (R): 5'-GCC TTC TGC AGT CCA GGT TC-3'

iNOS Forward (F): 5'-CCG GCA AAC CCA AGG TCT AC-3'

iNOS Rorward (R): 5'-GCA TTT CGC TGT CTC CCC AA-3'

GAPDH Forward (F): 5'-CAG GAG CGA GAC CCC ACT AA-3'

GAPDH Rorward (R): 5'-ATC ACG CCA CAG CTT TCC AG-3'

RT-PCR products were then separated by electrophoresis on a 1.5% agarose gel containing Eco Dye (1:5000, Korea). Electrophoresed RT-PCR was subjected to image analysis using Image J (NIH) and Fusion (Korea).

Real-time PCR (Q-PCR)

To determine the effect of abemaciclib mesylate on the levels of pro- and anti-inflammatory cytokines induced by LPS, BV2 microglia and mouse primary microglia were treated with LPS (1 μg/ml) for 30 min. Alternatively, after treatment with PBS, the cells were treated with abemaciclib mesylate (5 μM) or vehicle (1% DMSO) for 5.5 hours. At this time, in another experiment, abemaciclib mesylate was treated first, and then LPS was treated later.

Afterwards, total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol, and reverse transcribed into cDNA using Superscript cDNA Premix Kit II (GeNetBio, Korea) with oligo (dT) primers. Then, real-time PCR was performed using Fast SYBR Green Master Mix (Thermo Fisher Scientific, CA, USA) and QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, San Jose, CA, USA). Cycle threshold (Ct) values for the mRNAs of inflammatory mediators were normalized to the Ct values for Gapdh, and data were quantified as fold change compared to controls.

Antibodies and Inhibitors

The primary antibodies used to perform Western blot and immunohistochemistry (IHC) were rabbit anti-AKT (1:1000 for WB, Santa Cruz Biotechnology), rabbit anti-p-AKT (Ser473) (1:1000 for WB, Cell Signaling Technology), rabbit anti-STAT3 (Y705, 1:1000 for WB, Cell Signaling Technology), rabbit anti-Iba-1 (1:500 for IHC, Wako, Japan), rabbit anti-GFAP (1: 500 for IHC, Neuromics, U.S.A), rabbit anti-IL-1beta (1:100 for IHC, Abcam, UK), mouse anti-IL-6 (1:100 for IHC, Santacruz Technology, U.S.A), mouse anti-AT100 (1:200, Invitrogen, U.S.A), mouse anti-AT180 (1:200, Invitrogen, U.S.A), mouse anti-Tau5 (1:200, Invitrogen, U.S.A), rabbit anti-DYRK1A (1:200, abcam, UK ), mouse anti-4G8 (1:500, Biolegend, U.S.A), mouse anti-6E10 (1:500, Biolegend, U.S.A), rabbit anti-phospho-GSK-3β(Y216) (1:200, Abcam) did. Additionally, TLR4 inhibitor (TAK-242, 500 nM; Calbiochem) and AKT inhibitor (MK2206, 10 mM; Selleckchem) were used as inhibitors. Additionally, LPS was derived from E. coli O111:B4 and was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Western blotting

To determine whether abemaciclib mesylate affects AKT signaling, BV2 microglia were treated with abemaciclib mesylate (5 μM) or vehicle (1% DMSO) for 45 min, followed by LPS (1 μg/ml). Or treated with PBS for 45 minutes.

The cells were then lysed using RIPA buffer containing protease and phosphatase inhibitors (Roche, USA). Western blot was performed using a conventional Western blot method, and images were analyzed using Fusion software.

Cytosol and nuclear fractionation

To determine whether abemaciclib mesylate affects the levels of p-STAT3 in the cytoplasm and nucleus induced by LPS, BV2 microglia were cultured in serum free media for 1 hour and then incubated with abemaciclib mesylate (5 μM). ) or vehicle (1% DMSO) for 30 minutes, and then treated with LPS (1 μg/ml) or PBS for 5.5 hours. The cells were then lysed using buffer for cytoplasmic fractionation (10 mM HEPES pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 300 mM sucrose, 0.1 % NP-40, and 0.5 mM PMSF). 5 minutes after adding the solution, the cell lysate was centrifuged at 10,000 rpm at 4°C for 1 minute, and the supernatant was separated into a cytosolic fraction and stored. The pellet was solubilized with nuclear fractionation buffer (10 mM HEPES pH 8.0, 20% glycerol, 100 mM KCl, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF) for 15 min on ice. Afterwards, it was centrifuged at 10,000 rpm at 4°C for 15 minutes, and Western blot analysis was performed using anti-STAT3 (Y705), PCNA, and β-actin antibodies and Fusion software.

Culture of mouse primary microglia and astrocytes

To determine whether abemaciclib mesylate modulates the levels of LPS-induced inflammatory cytokines in primary glial cells, mixed mouse cultures (a mixture of primary microglia and primary astrocytes) were prepared. Specifically, whole brains from 1-day postnatal day-old C57BL/6 mice were minced and mechanically disrupted using a 70 μm nylon mesh. Mixed glial cells were seeded into 75 T culture flasks and grown in low-glucose DMEM medium supplemented with 10% FBS (containing 100 unit/mL penicillin and 100 μg/mL streptomycin). Then, to obtain mouse primary astrocytes, the mixed glial cells were shaken at 250 rpm overnight. The next day, the conditioned medium was discarded, and the remaining cells were separated using trypsin-EDTA and centrifuged (1200 rpm, 30 minutes). After centrifugation, the conditioned medium was removed, the cell pellet (primary astrocytes) was collected, and analysis of the effect of abemaciclib mesylate on neuroinflammation in primary astrocytes was performed.

For obtaining mouse primary microglia, mixed glial cells were subjected to mild trypsinization (5-fold dilution of 0.25% trypsin in serum-free DMEM) for 40 min in a 5% CO incubator at 37 °C. Afterwards, all of the upper astrocyte layer was discarded, low-glucose DMEM containing 10% FBS was added, and the remaining primary microglial cells were used in the experiment. Microglia or astrocytes were cultured in serum free media for 1 hour, treated with abemaciclib (5 μM) or vehicle (1% DMSO) for 30 minutes, and then treated with LPS (200 ng/ml) or PBS for 5.5 hours. After processing, real-time PCR was performed.

wild type mice

All experiments were performed in accordance with the animal testing and guidelines approved by the Korea Brain Research Institute (IACUC-2016-0013). C57BL6/N mice were purchased from OrientBio Company, and male C57BL6/N (8 weeks, 25-30g) mice were raised in a pathogen-free facility at an ambient temperature of 22°C under a 12-hour light/dark cycle. To confirm the efficacy of abemiciclib on cognitive dysfunction caused by LPS, wild-type mice were administered LPS (30 mg/kg, intraperitoneal administration) or vehicle (10% DMSO) 30 minutes daily for 7 days. 250 ㎍/ml, intraperitoneal administration) or PBS was administered, and Y maze and NOR test were performed on the 8th and 9th days. During the behavioral test period (days 8 and 9), abemaciclib and LPS were administered after the behavioral test. To determine the effect of abemaciclib mesylate on LPS-induced neuroinflammation, wild-type mice were intraperitoneally administered abemaciclib mesylate (30 mg/kg, i.p.) or vehicle (10% DMSO) daily for 3 days. Then, LPS (Sigma, Escherichia coli, 10 mg/kg, ip) or PBS was continuously administered for 6 hours, and brain tissue was analyzed. Data were also analyzed in a semi-automated manner using Image J software, and the results were confirmed by an independent researcher not involved in the current experiment.

Production of Alzheimer's-induced mouse animal model (5x FAD mice)

F1 generation male 5x FAD mice (stock # 34848-JAX, B6Cg-Tg APPSwFlLon, PSEN1 * M146L * L286V6799Vas/Mmjax) were purchased from Jackson Laboratory and used. 5x FAD mice (3 months old) were housed in a germ-free facility with 12 hours of light/dark control per day at an ambient temperature of 22°C. After administering abemaciclib (30 mg/kg, intraperitoneal administration) or vehicle (10% DMSO) to 5xFAD mice daily for 14 days, perform Y maze test on day 15 and NOR test on day 16-17, followed by fluorescent tissue staining or Golgi staining was performed. In addition, to compare the efficacy of abemaciclib in controlling degenerative brain disease symptoms with donepezil, a currently commercially available dementia treatment drug, abemaciclib (30 mg/kg, intraperitoneal administration) and donepezil (1 mg/kg) were administered to 5xFAD mice. , intraperitoneal administration) or vehicle (10% DMSO) every day for 14 days, a Y maze test was performed on the 15th day, a NOR test was performed on the 16th and 17th days, and fluorescent tissue staining was performed.

Production of Alzheimer's-induced mouse animal model (PS19 mice)

Male PS19 mice (stock # 008169-JAX, B6;C3-Tg (Prnp-MAPT * P301S) PS19Vle/J) were purchased from Jackson Laboratory and used. PS19 mice (6 months old) were housed in a germ-free facility at an ambient temperature of 22°C with 12 hours of light/dark conditions per day. After administering abemaciclib (30 mg/kg, intraperitoneal administration or 60 mg/kg, oral administration) or vehicle (10% DMSO) to PS19 mice daily for 14 days, Y maze test on day 15 and NOR test on days 15-16. was carried out.

Immunofluorescence staining

To determine the effect of abemaciclib mesylate on LPS-induced neuroinflammation in vivo, wild-type mice were treated with abemaciclib mesylate (30 mg/kg, i.p.) or vehicle (10% DMSO) daily for 3 days. was administered intraperitoneally, followed by continuous administration of LPS (Sigma, Escherichia coli, 10 mg/kg, ip) or PBS for 6 hours. Then, after injecting LPS or PBS, the mice were perfused and fixed with 4% paraformaldehyde (PFA) solution, and the brain tissue was quickly frozen and sectioned using a cryostat (35㎛ thickness). For another immunohistochemical experiment, abemaciclib mesylate (30 mg/kg, i.p.) or vehicle (10% DMSO) was intraperitoneally administered to Alzheimer's-induced mice (5x FAD mice) daily for 2 weeks, and after 14 days, Brain tissue was sectioned in the same manner as above.

Each brain section was then processed for immunohistochemical staining. Brain sections were rinsed with PBS and permeabilized using PBS containing 0.2% Triton X-100 and 1% BSA for 1 hour at room temperature. Afterwards, the primary antibody was diluted in PBST (0.2% Triton The reaction was allowed for 2 hours. Afterwards, the brain slices were washed three times with PBS, placed on a slide, and observed under a fluorescence microscope.

Golgi staining

To determine the effect of abemaciclib mesylate on dendritic spinogesis in vivo, Golgi staining was performed using the FD Rapid GolgiStain Kit (FD Neurotechnologies, Ellicott City, MD, USA). Specifically, animals injected with vehicle or abemaciclib mesylate were immersed in solutions A and B, respectively, in a dark room for 15 days and then transferred to solution C for 24 hours. Solution C was then replaced 24 h later, and the brain of each mouse was sliced at 150-μm thickness using a VT1000S Vibratome (Leica, Bannockburn, IL, USA). Dendritic images were taken by bright field microscopy using Axioplan 2 (Zeiss, Oberkochen, Germany). Dendritic spine density was measured using 8 to 10 sections of each mouse brain measuring -1.70 mm to -2.30 mm relative to bregma. All data were analyzed using Image J software, and all experimental results were confirmed by an independent researcher not involved in the current experiment.

Y maze test

To verify the ability to improve short-term memory when administering abemaciclib mesylate to normal animals (wild type mice) or Alzheimer's disease-induced animal models (5xFAD mice and PS19 mice), each arm was 42 cm long, 3 cm wide, and 12 cm high. The animal was placed at the end of one arm of a Y-shaped maze with three arms at an angle of 120 degrees, and the arms visited for 5 minutes were recorded. Afterwards, Percent alteration = (number of alterations / number of triads) x 100 was calculated and used as a measure of short-term memory.

alteration: 1 point is awarded if it enters three different arms one after another.

triads: total visits - 2

Novel object recognition test

To verify the ability to improve long-term memory when administering abemaciclib mesylate to normal animals (wild type mice) or Alzheimer's-induced animal models (5xFAD mice and PS19 mice), two cells of the same shape and size were placed in a box measuring 42 x 42 x 25 cm. After placing the dog's object in a corner, the animal was started from the center of the box, and the time the animal showed interest in the object was recorded for 5 minutes. After 24 hours, one of the two objects was replaced with a new one, and the time taken to approach the old and new objects was measured, and the preference for the new object was analyzed and used as an index for long-term memory analysis.

Statistical processing

All data were analyzed using two-tailed T-test or ANOVA using Graphpad Prism 6 software. Post-hoc analysis was performed with Tukey's Multiple Comparison test, and significance was set at p <0.05. Data are presented as mean ± S.E.M (* p < 0.05, ** p < 0.01, *** p < 0.001).

<Example 1>

Analysis of cytotoxicity of abemaciclib on BV2 microglial cells and reduction of pro-inflammatory cytokine levels

To confirm whether abemaciclib is cytotoxic to BV2 microglial cells, the present inventors treated BV2 microglial cells with abemaciclib at various concentrations (0.1, 1, 5, 10, 25, 50uM), MTT analysis was performed.

As a result of the analysis, as shown in Figures 1A and 1B, abemaciclib did not appear to cause cytotoxicity to BV2 microglial cells.

Additionally, to determine whether abemaciclib affects the expression levels of pro-inflammatory cytokines induced by LPS, the present inventors administered abemaciclib (5 μM) or vehicle (1% DMSO) to BV2 microglial cells for 30 minutes. After pretreatment for 5.5 hours and treatment with LPS (1 μg/ml) or PBS for 5.5 hours, the mRNA levels of inflammatory cytokines were analyzed. At this time, in another experiment, BV2 microglial cells were pretreated with LPS (1 μg/ml) or PBS for 30 minutes, treated with abemaciclib (5 μM) or vehicle (1% DMSO) for 5.5 hours, and then treated with inflammatory cells. The mRNA levels of the cytokines IL-6, IL-1beta, iNOS, and COX-2 were analyzed.

As a result, as shown in Figures 1C to 1G, when inflammation was induced with LPS after pretreatment with abemaciclib, the production of inflammatory cytokines IL-6, IL-1beta, iNOS, and COX-2 was shown to be effectively suppressed. When inflammation was induced with LPS and then treated with abemaciclib, the production of inflammatory cytokines IL-6, IL-1beta, iNOS, and COX-2 was found to be effectively suppressed (Figures 1H-1L).

Therefore, these results mean that abemaciclib can inhibit the production of pro-inflammatory cytokines by acting both before and after the inflammatory response is induced. Additionally, to determine whether abemaciclib also affects the anti-inflammatory response, the present inventors pretreated BV2 microglial cells with abemaciclib (5 μM) or vehicle (1% DMSO) for 30 minutes, and then incubated with LPS ( After treatment with 1 μg/mL) or PBS for 5.5 hours, the mRNA levels of IL-4, IL-10, and MRC-1 were measured.

Also, in the group where BV2 microglial cells were pretreated with LPS (1 μg/mL) or PBS for 30 minutes and then treated with abemaciclib (5 μM) or vehicle (1% DMSO) for 5.5 hours, IL-4 , the mRNA levels of IL-10 and MRC-1 were measured.

As a result, as shown in Figures 1M to 1R, abemaciclib was found to significantly increase IL-4, IL-10, and MRC-1 mRNA levels in LPS-treated BV2 microglial cells. Even when pretreated with abemaciclib first and then treated with LPS, the mRNA levels of anti-inflammatory factors IL-4, IL-10, and MRC-1 were found to be significantly increased.

Therefore, through these results, the present inventors demonstrated that abemaciclib can not only effectively suppress the expression and production of pro-inflammatory cytokines induced by LPS, but also has the activity of promoting the expression and production of anti-inflammatory cytokines, thereby reducing excessive It was found that brain inflammation-related diseases and degenerative brain diseases caused by brain inflammation that can be caused by inflammatory reactions can be prevented, improved, or treated.

<Example 2>

Analysis of the impact of abemaciclib on TLR4/AKT/STAT3 signaling

To determine whether abemaciclib can regulate LPS-induced inflammatory response through any signaling system, the effect of abemaciclib on TLR4 signaling was analyzed. For this purpose, BV2 microglial cells were first cultured, maintained in starvation for 1 hour, then treated with TAK-242 (TLR4 inhibitor, 500 nM) or vehicle (1% DMSO) for 30 minutes, and then abemaciclib (5 μM). ) or vehicle (1% DMSO) for 30 minutes, and then treated with LPS (1 μg/ml) or PBS for 5 hours. Afterwards, the levels of inflammatory cytokines for each experimental group were analyzed through RT-PCR.

As a result, the group treated with abemaciclib significantly reduced the mRNA levels of IL-6, IL-1β, iNOS, and COX-2, which are LPS-induced inflammatory cytokines, especially TAK-242 and abemaciclib. In the combination treatment group, the level of LPS-induced pro-inflammatory cytokine iNOS mRNA was found to be not significantly changed (see Figures 2A-2E). Therefore, it was found that abemaciclib inhibits the expression of iNOS, the final product of the inflammatory response caused by LPS, depending on the TLR4 signaling pathway.

In addition, in order to confirm the intracellular signaling pathway through which abemaciclib is involved in suppressing LPS-mediated inflammatory response, the present inventors cultured BV2 microglial cells, maintained starvation for 1 hour, and then added abemaciclib (5 μM). ) or vehicle (1% DMSO) for 45 minutes, followed by treatment with LPS (1 μg/ml) or PBS for 45 minutes, and then the levels of AKT and p-AKT (phosphorylated) were analyzed by Western blot. .

As a result, abemaciclib was found to significantly reduce the level of p-AKT induced by LPS in treated BV2 microglial cells, while having no effect on the total expression level of AKT protein (Figure 2F see ~2H).

Next, to determine whether abemaciclib suppresses the levels of LPS-mediated proinflammatory cytokines in an AKT-dependent manner, BV2 microglia were incubated with MK2206 (AKT inhibitor, 10 uM) or vehicle (1% DMSO) for 30 min. After treatment, abemaciclib (5 μM) or vehicle (1% DMSO) was treated for 30 minutes, followed by LPS (1 μg/ml) or PBS for 5 hours.

After analyzing the levels of pro-inflammatory cytokines, the group treated with MK2206, abemaciclib, and LPS showed no significant change in the mRNA levels of iNOS, IL-1beta, and COX-2 induced by LPS compared to the other groups. It was confirmed that this was not the case (see Figures 2I-2M). These results mean that abemaciclib can inhibit brain inflammation by inhibiting AKT signaling of LPS-induced pro-inflammatory cytokines iNOS, IL-1beta, and COX-2.

Additionally, to determine whether abemaciclib can regulate p-STAT3 levels in the cytoplasm and nucleus, BV2 microglia were treated with abemaciclib (5 μM) or vehicle (1% DMSO) for 30 min and incubated with LPS. After treatment for 5.5 hours, cell lysates were obtained from each experimental group, fractionated into cytoplasmic and nuclear fractions, and then analyzed for the level of p-STAT3 through Western blot.

As a result, as shown in Figures 2N to 2Q, the level of p-STAT3 in the nucleus was found to be significantly reduced by abemaciclib, while there was little change in the level of p-STAT3 in the cytosol. appear. Therefore, the abemaciclib of the present invention inhibits the level of p-STAT3 in the nucleus, which means that it can ultimately inhibit the activation of STAT3, which means that phosphorylation of STAT3 (p-STAT3) is a factor related to inflammatory response in the nucleus. It is known to play a role in inducing the expression of p-STAT3, which means that suppressing p-STAT3 levels can inhibit the expression of inflammatory factors and ultimately suppress excessive inflammatory responses.

<Example 3>

Analysis of the inhibitory effect of LPS on pro-inflammatory cytokines following abemaciclib treatment in microglia and astrocytes

To analyze whether abemaciclib is related to LPS-induced neuroinflammation in microglial cells, primary microglial cells were cultured under low glucose conditions, and primary microglial cells were treated with abemaciclib (5 μM) or After pretreatment with vehicle (1% DMSO) for 30 minutes and treatment with LPS (200 ng/ml) or PBS for 5.5 hours, real-time PCR was performed to measure the levels of pro-inflammatory cytokines.

As a result, as shown in Figure 3A, the levels of LPS-induced pro-inflammatory cytokines were significantly reduced in primary microglial cells treated with abemaciclib.

In addition, the present inventors confirmed whether abemaciclib can regulate the neuroinflammatory response caused by LPS in primary astrocytes under the same drug treatment conditions. As a result, the LPS-mediated effect was significantly observed in primary astrocytes treated with abemaciclib. The mRNA levels of inflammatory cytokines COX-2, IL-1β, IL-6, TNF-a, and iNOS were found to be decreased (Figure 3B).

These results mean that abemaciclib can effectively inhibit the neuroinflammatory response caused by LPS in both microglia and astrocytes.

<Example 4>

Analysis of the effect of abemaciclib on LPS-induced cognitive decline in wild-type mice

To determine whether abemaciclib improves the decline in memory function caused by repetitive inflammatory stimulation in wild-type mice, wild-type mice were administered abemaciclib (30 mg/kg, intraperitoneal administration) or vehicle (10% DMSO) daily for 7 days. ) 30 minutes after administration, LPS (250 ㎍/ml, intraperitoneal administration) or PBS was administered. On the 8th day, Y maze and NOR training were performed, and on the 9th day, the NOR test was performed. During the behavioral test period (days 8 and 9), abemaciclib and LPS were administered after the behavioral test.

As a result, as shown in Figures 4A-4B, treatment with abemaciclib improved spatial memory (analyzed by Y maze performance) and recognition memory (analyzed by NOR test performance) in LPS-induced memory-deficient mice. It appeared that it did.

<Example 5>

Analysis of the effects of abemaciclib on LPS-induced microglial/astrocyte activation and pro-inflammatory cytokine levels in wild-type mice.

We also analyzed in vivo whether abemaciclib could control LPS-induced neuroinflammation. For this purpose, wild-type mice (normal mice) were injected daily with abemaciclib (30 mg/kg, i.p.) or vehicle (10% DMSO) for 3 days, and LPS (10 mg/kg, i.p.) or PBS for 6 h. processed for a while. Afterwards, the cortex and hippocampus were each isolated from the mouse, and immunohistochemical staining was performed using anti-Iba-1, anti-GFAP antibodies, and anti-IL-1β antibodies.

As a result, as shown in Figures 5A and 5C, in the abemaciclib treatment group, LPS-induced Iba-1 was found to be significantly reduced in the cortex, while in the hippocampus, the level of abemaciclib was significantly reduced. The decrease in Iba-1 following treatment appeared to be more subtle than in the cortex. Therefore, through these results, the present inventors were able to see that abemaciclib can inhibit cell activation of microglial cells.

In addition, it was confirmed whether abemaciclib could inhibit the activation of astrocytes induced by LPS. As a result, as shown in Figures 5B to 5C, GFAP, a marker of astrocyte activation induced by LPS, was detected in the cortex. was found to be significantly decreased, while the decrease in GFAP in the hippocampus was found to be less significant than in the cortex.

In addition, as a result of confirming whether abemaciclib affects the level of pro-inflammatory cytokines induced by LPS, as shown in Figures 6A and 6C, abemaciclib treatment in the cortex and hippocampus CA1 region (hippocampus) LPS-induced IL-1β was found to be significantly reduced, and as shown in Figures 6B and 6C, LPS-induced IL-6 was also significantly reduced in the cortex of wild-type mice. I was able to.

Therefore, through these results, the present inventors demonstrated that abemaciclib can suppress the activation of microglia and astrocytes by LPS even in normal nerve cells, and can suppress the production and expression of pro-inflammatory cytokines, It was found that inflammation of brain nerve cells can be prevented in advance.

<Example 6>

Analysis of the effect of abemaciclib on the activation of microglia/astrocytes and pro-inflammatory cytokine levels induced by amyloid beta in mice with degenerative brain disease.

As it was confirmed through Example 5 that abemaciclib has the activity of suppressing brain inflammation in the brain neurons of normal mice, the present inventors developed mice (5x FAD) induced with Alzheimer's disease, one of the degenerative brain diseases. An experiment was performed on mice) to determine whether treatment with abemaciclib had an inhibitory activity on brain inflammation. For this purpose, abemaciclib (30 mg/kg, ip) or vehicle (10% DMSO) was intraperitoneally administered to Alzheimer's-induced 5x FAD mice daily for 2 weeks, followed by immunohistochemical staining in the same manner as in Example 5. was carried out.

As a result, as shown in Figures 7A and 7D, Iba-1, an indicator of microglial activity, was found to be significantly reduced in the cortex and hippocampus in the abemaciclib treatment group, Figures 7B and 7D As shown, in the cortex, the abemaciclib-treated group showed a significant decrease in GFAP, a marker of astrocyte activation, compared to the control group, while in the hippocampus, the decrease in GFAP was greater than in the cortex. It was found to be insufficient.

Additionally, as shown in Figures 7C and 7D, IL-1β, an inflammatory cytokine, was found to be significantly reduced in the cortex of the abemaciclib-treated group compared to the control group.

Therefore, abemaciclib can suppress the activation of microglia and astrocytes in the neurons of the brain caused by degenerative brain disease, and can suppress the production and expression of pro-inflammatory cytokines, making it a new treatment for degenerative brain disease. It was found that it could be used as a treatment.

<Example 7>

Analysis of increase in cognitive function and dendritic spine density in brain tissue of mice induced with degenerative brain disease caused by abemaciclib

To determine whether abemaciclib affects the formation of dendritic spines and the promotion of learning and memory, abemaciclib (30 mg/kg, ip) or vehicle (10% DMSO) was administered to Alzheimer's-induced mice. After intraperitoneal administration, the Y maze experiment was performed on the 15th day, NOR training was performed on the 16th day, and the NOR test was performed on the 17th day. Additionally, the number of dendritic branches was measured for the cortical and hippocampal neurons of the mice.

As a result, as shown in Figures 8A and 8B, treatment with abemaciclib was found to improve short-term memory and long-term memory in mice suffering from degenerative brain disease, and as shown in Figures 8C to 8E, abemaciclib treatment The treatment group was found to have an increased number of dendritic branches in the AO area of the cerebral cortex and hippocampus tissue compared to the control group. These results were also confirmed in the Golgi staining photographic results.

Therefore, abemaciclib of the present invention can increase the number of dendrite branches in brain tissue caused by degenerative brain disease, and it has been shown to improve reduced cognitive ability and memory due to degenerative brain disease, and abemaciclib can be used to treat degenerative brain disease. It was found that it could be used as a treatment for diseases.

<Example 8>

Analysis of tau phosphorylation inhibition activity of abemaciclib

To determine the effect of abemaciclib on tau phosphorylation, 3-month-old 5x FAD mice with Alzheimer's disease were administered abemaciclib (30 mg/kg, ip) or vehicle (10% DMSO) daily for 2 weeks. Afterwards, immunohistochemistry was performed using anti-AT100 antibody, anti-AT180 antibody, and anti-Tau-5 antibody that can detect phosphorylated tau protein.

As a result, AT100, a phosphorylated tau protein, was found to be significantly reduced in the cerebral cortex of 5x FAD mice with degenerative brain disease (Alzheimer's disease) injected with abemaciclib compared to the control group that was not injected with abemaciclib. , the levels of both AT180 and Tau-5 were found to be significantly reduced in the cerebral cortex and hippocampus of 5x FAD mice compared to the control group not injected with abemaciclib (see Figures 9A-9D).

Therefore, these results showed that abemaciclib can prevent or treat degenerative brain diseases such as Alzheimer's disease by effectively inhibiting tau phosphorylation, which is known to be the cause of degenerative brain diseases.

<Example 9>

Analysis of Aβ plaque reduction effect by abemaciclib in mice with degenerative brain disease

To analyze whether abemaciclib affects the pathogenesis of amyloid plaques (Aβ), 3-month-old Alzheimer's disease-induced mice, 5x FAD mice, were treated with abemaciclib (30 mg/kg, i.p.) or vehicle (10% DMSO). was injected every day for 2 weeks, and then the cortex and hippocampus tissues were separated from the mice, and changes in the level of amyloid plaques (Aβ) were measured.

As a result, as shown in Figure 10, the level of amyloid plaques (Aβ) in the hippocampal CA1 region of Alzheimer's-induced mice administered abemaciclib was found to be significantly reduced compared to the vehicle-treated control group.

<Example 10>

Efficacy of cognitive function improvement by abemaciclib and comparison of efficacy with donepezil in mice with degenerative brain disease

To compare the efficacy of abemaciclib in controlling degenerative brain disease symptoms with donepezil, a currently commercially available dementia treatment drug, abemaciclib (30 mg/kg, intraperitoneally administered) and donepezil (1 mg/kg, intraperitoneally administered) to 5xFAD mice. After administering) or vehicle (10% DMSO) every day for 14 days, the Y maze test was performed on the 15th day and the NOR test was performed on the 16th and 17th days.

As a result, as shown in Figures 11A and 11B, the group administered donepezil improved short-term memory and long-term memory in mice with degenerative brain disease, and treatment of abemaciclib mesylate was at a similar level to donepezil. It has been shown to improve short-term and long-term memory in mice suffering from degenerative brain disease.

<Example 11>

Comparison of the efficacy of abemaciclib in inhibiting astrocyte activity and its efficacy with donepezil in mice with degenerative brain disease

To compare the efficacy of abemaciclib in controlling degenerative brain disease symptoms with donepezil, a currently commercially available dementia treatment drug, abemaciclib (30 mg/kg, intraperitoneal administration) and donepezil (1 mg/kg, intraperitoneally) were administered to 5xFAD mice. After administration) or vehicle (10% DMSO) every day for 14 days, behavioral experiments were performed on days 15 and 17, and immunohistochemical analysis was performed.

As a result, as shown in Figures 12A and 12B, administration of donepezil reduced astrocyte activity and proliferation in the cerebral cortex of mice suffering from degenerative brain disease, and astrocytes directly interacted with amyloid plaques. The number was also found to have decreased. In addition, the group administered amemaciclib was shown to suppress astrocyte activity and proliferation to a similar level as the group administered donepezil, and to reduce the number of astrocytes interacting with amyloid plaques.

<Example 12>

Analysis of inhibition of tau phosphatase activity by abemaciclib and comparison of efficacy with donepezil in mice with degenerative brain disease

To compare the efficacy of abemaciclib in controlling degenerative brain disease symptoms with donepezil, a currently commercially available dementia treatment drug, abemaciclib (30 mg/kg, intraperitoneal administration) and donepezil (1 mg/kg, intraperitoneally) were administered to 5xFAD mice. After administration) or vehicle (10% DMSO) every day for 14 days, behavioral experiments were performed on days 15 and 17, and immunohistochemical analysis was performed.

As a result, as shown in Figure 13A, administration of donepezil decreased the expression of phospho-GSK-3β, a tau phosphorylation enzyme, in the hippocampus of mice suffering from degenerative brain disease, and administration of abemaciclib decreased the expression of phospho-GSK-3β in the hippocampus of mice suffering from degenerative brain disease. The efficacy of inhibiting the expression of phospho-GSK-3β, a tau phosphorylation enzyme, was found to be significantly higher than that of donepezil administration. In addition, as shown in Figure 13B, administration of donepezil decreased the expression of DYRK1A, a tau phosphorylation enzyme, in the cortex and hippocampus of mice suffering from degenerative brain disease, and administration of abemaciclib decreased the expression of donepezil in the cortex and hippocampus. The efficacy of inhibiting the expression of DYRK1A, a tau phosphorylation enzyme, was found to be significantly higher than that of Fezil administration.

<Example 13>

Confirmation of cognitive function improvement effect by abemaciclib in mice with degenerative brain disease overexpressing tau

Since abemaciclib showed efficacy in reducing tau phosphorylation and inhibiting tau phosphorylation enzyme activity in 5xFAD mice, an animal model of degenerative brain disease that overexpresses APP, abemaciclib has been shown to improve cognitive function in PS19 mice, an animal model of degenerative brain disease that overexpresses tau. To determine whether 6-month-old PS19 mice were administered abemaciclib (30 mg/kg, intraperitoneal administration or 60 mg/kg, oral administration) or vehicle (10% DMSO) daily for 14 days, then on the 15th day. Y maze test and NOR test were performed on days 15-16.

As a result, as shown in Figures 14A and 14B, administration of abemaciclib was found to improve the spatial perception ability and recognition memory function of mice with degenerative brain disease caused by overexpression of tau.

Through the above results, the present inventors demonstrated that abemaciclib can effectively suppress the expression and production of pro-inflammatory cytokines induced by LPS and the activation of microglia and astrocytes induced by LPS, while simultaneously suppressing amyloid Since it has all the activities of inhibiting plaque, increasing the number of dendrites, improving long-term memory, and inhibiting phosphorylation of tau protein, it was found that abemaciclib mesylate can be used as a new treatment to prevent, improve, or treat degenerative brain diseases. .

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (16)

Brain degeneration selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple neural atrophy, epilepsy, encephalopathy, and stroke, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutical composition for preventing or treating diseases. According to paragraph 1,
The composition is characterized by inhibiting the expression and production of inflammatory cytokines COX-2, IL-1β, IL-6 or iNOS, Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple neural atrophy, epilepsy, and encephalopathy. ) and a pharmaceutical composition for preventing or treating degenerative brain diseases selected from the group consisting of stroke. According to paragraph 1,
The composition inhibits the activity of TLR4 (Toll-like receptor 4) and inhibits p-STAT3 levels, treating Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple neural atrophy, epilepsy, encephalopathy and stroke. A pharmaceutical composition for preventing or treating degenerative brain diseases selected from the group consisting of. According to paragraph 1,
The composition is,
Inhibits the activity of microglia or astrocytes;
Inhibits amyloid plaques;
Increased number of dendrites;
Inhibition of phosphorylation of tau protein; or
A pharmaceutical composition for the prevention or treatment of a degenerative brain disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple neural atrophy, epilepsy, encephalopathy, and stroke, characterized by improving long-term memory. delete Brain degeneration selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple neural atrophy, epilepsy, encephalopathy, and stroke, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient. Health functional food for preventing or improving disease. According to clause 6,
The abemaciclib or a salt thereof is characterized by inhibiting the expression and production of inflammatory cytokines COX-2, IL-1β, IL-6 or iNOS, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and multiple sclerosis. A health functional food for preventing or improving degenerative brain diseases selected from the group consisting of neural atrophy, epilepsy, encephalopathy, and stroke. According to clause 6,
The abemaciclib or its salt inhibits the activity of TLR4 (Toll-like receptor 4) and inhibits the level of p-STAT3, Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple neural atrophy, A health functional food for preventing or improving degenerative brain diseases selected from the group consisting of epilepsy, encephalopathy, and stroke. According to clause 6,
The abemaciclib or a salt thereof,
Inhibits the activity of microglia or astrocytes;
Inhibits amyloid plaques;
Increased number of dendrites;
Inhibition of phosphorylation of tau protein; or
A health functional food for preventing or improving degenerative brain diseases selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple neural atrophy, epilepsy, encephalopathy and stroke, characterized by having long-term memory increasing activity. delete delete delete A neuroinflammatory drug selected from the group consisting of Lou Gehrig's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, and amyotrophic lateral sclerosis, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutical composition for preventing or treating diseases. delete A neuroinflammatory drug selected from the group consisting of Lou Gehrig's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, and amyotrophic lateral sclerosis, comprising abemaciclib or a pharmaceutically acceptable salt thereof as an active ingredient. Health functional food for preventing or improving disease. delete