KR20240097396A - Pharmaceutical composition for the prevention or treatment of neuroinflammatory disease or neurodegenerative disease comprising ribociclib an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases containing ribociclib as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition containing ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutical compositions for preventing or treating inflammatory or degenerative brain diseases, compositions for protecting nerve cells damaged by excessive inflammatory reactions in nerve cells, reagent compositions for inhibiting the activation of microglia or astrocytes, and ribosytes It relates to a health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, comprising clip or a foodologically acceptable salt thereof as an active ingredient.

Description

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

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

Degenerative brain disease refers to a disease that occurs in the brain among the degenerative diseases that occur with age. It can be classified based on the main symptoms and the brain area affected. Representative examples include Alzheimer's disease (AD) and Parkinson's disease. Included. 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, each of which causes cell death through a unique signaling process. Specifically, oxidative damage to proteins, nucleic acids, and lipids after accumulation of reactive oxygen species has been suggested as the main cause of brain cell death in patients with stroke, brain damage, Alzheimer's disease, and Parkinson's disease, and in particular, free radicals (free radicals). Oxidative stress caused by 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 nerve diseases (Schapira, A.H., Curr. Opin. Neurol., 9 (4):260-264, 1996).

Additionally, inflammation in the central nervous system (CNS), that is, neuroinflammation, causes degenerative brain diseases and is mediated by the release of cytokines and chemokines by glial cells (glia) of the central nervous system. Glial cells are divided into two types: microglia and astrocytes, and they play important roles in maintaining neurons and detecting and defending against pathogens.

Meanwhile, the activity of microglia and astrocytes has been reported to be related to the onset and progression of degenerative cranial nerve diseases. Microglia are immune cells residing in the central nervous system and are activated by external stimuli to initiate immune and inflammatory reactions. It is known to cause . Microglial cells 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 protect nerve cells from toxins introduced from the outside or generated internally when these toxins are present. To do this, 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. and cell proliferation, and by expressing genes such as cytokines such as TNF-α, IL-1β, and IL-6, chemokines, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2), inflammatory mediators are removed. 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 in maintaining normal brain activity as well as the brain development process. Astrocytes in the brain have been found to assist nerve cell activity by appropriately removing neurotransmitters secreted by nerve cells or regulating ion concentration in the brain. In addition, they play a critical role in helping neural stem cells differentiate into nerve cells. It has been found to do so.

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.

The treatments currently being used to treat degenerative brain diseases include drug therapy, surgery, and physical therapy. In the case of drug therapy, it generally replenishes dopamine that is lacking in the brain and reduces neurotransmitter loss due to the lack of dopamine. Drugs are used to balance the imbalance, prevent or delay the destruction of nerve cells, and control other symptoms such as depression.

However, since these drugs cannot revive dead nerve cells, they have the limitation that they are aimed at controlling symptoms rather than a cure, so there is an urgent need to develop new treatments that can prevent or treat degenerative brain diseases more effectively. am.

Meanwhile, ribociclib is used in combination with an aromatase inhibitor to treat hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer in postmenopausal women. Although it is known as a drug, there has yet to be an example of ribociclib's impact on the pathology of neuroinflammatory and degenerative brain diseases and the possibility of its use as a treatment for these diseases.

Republic of Korea Open Patent 10-2021-0126653

Accordingly, the present inventors completed the present invention by confirming that the drug ribociclib, which was conventionally used as an anticancer agent, can be used as a new therapeutic agent that can prevent or treat neuroinflammation or degenerative brain diseases.

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

Another object of the present invention is to provide a composition for protecting nerve cells damaged by excessive inflammatory response in nerve cells, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a reagent composition for inhibiting the activation of microglia or astrocytes, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient. .

Another object of the present invention is to provide a health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, which contains ribociclib or a foodologically 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 neuroinflammatory or degenerative brain diseases, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the composition inhibits the activity of microglia or astrocytes; or inhibiting the production of pro-inflammatory cytokines; It may have an effect.

In one embodiment of the present invention, the composition may inhibit inflammatory responses within nerve cells.

In one embodiment of the present invention, the pro-inflammatory cytokine may be IL-1β, IL-6, or COX-2.

In one embodiment of the present invention, the inflammatory response may be induced by LPS (Lipopolysaccharide).

In one embodiment of the present invention, the degenerative brain disease includes Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivary nucleus-pontine-cerebellar atrophy (OPCA), Shay-Drager syndrome, and striatal-substantia nigra degeneration. , Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortico-basal gangrene, diffuse Lewy body disease, Parkinson-ALS-dementia complex, Niemann-Pick disease, Pick's disease, cerebral ischemia, and cerebral infarction. It could be.

The present invention also provides a composition for protecting nerve cells damaged by excessive inflammatory response in nerve cells, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a reagent composition for inhibiting the activation of microglia or astrocytes, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the activation of the microglial cells or astrocytes may be due to an excessive inflammatory response of nerve cells.

In addition, the present invention provides a health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, comprising ribociclib or a foodologically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the composition inhibits the activity of microglia or astrocytes; or inhibiting the production of pro-inflammatory cytokines; It may have an effect.

In one embodiment of the present invention, the composition may inhibit inflammatory responses within nerve cells.

In one embodiment of the present invention, the pro-inflammatory cytokine may be IL-1β, IL-6, or COX-2.

In one embodiment of the present invention, the inflammatory response may be induced by LPS (Lipopolysaccharide).

In one embodiment of the present invention, the degenerative brain disease includes Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivary nucleus-pontine-cerebellar atrophy (OPCA), Shay-Drager syndrome, and striatal-substantia nigra degeneration. , Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortico-basal gangrene, diffuse Lewy body disease, Parkinson-ALS-dementia complex, Niemann-Pick disease, Pick's disease, cerebral ischemia, and cerebral infarction. It could be.

The ribociclib of the present invention can suppress the expression and production of inflammatory cytokines in nerve cells caused by LPS, and can suppress the activity of microglia or astrocytes, preventing or preventing neuroinflammation or degenerative brain disease. It can be useful as a new treatment for treatment.

Figure 1 shows the results confirming the inhibition of microglial activation by LPS following ribociclib treatment in wild-type mice. (A) shows vehicle or ribociclib (10 or 30 mg/kg, ip) administered daily for 7 days. After intraperitoneal administration for 1 day, on the 7th day, LPS (10 mg/kg, ip) or PBS was administered intraperitoneally 30 minutes after drug administration, and 8 hours later, the brain was removed and immunofluorescent staining was performed with anti-Iba-1 antibody. It shows one result, and (B) shows the result of (A) in a quantitative graph.
Figure 2 shows the results confirming the inhibition of astrocyte activation by LPS following ribociclib treatment in the hippocampal CA1 region of wild-type mice. (A) shows vehicle or ribociclib (10 or 30 mg/kg, ip). After intraperitoneal administration every day for 7 days, on the 7th day, LPS (10 mg/kg, ip) or PBS was administered intraperitoneally 30 minutes after drug administration, and 8 hours later, the brain was extracted and immunofluorescent staining was performed with anti-GFAP antibody. It shows the results of the performance, and (B) shows the results of (A) in a quantitative graph.
Figure 3 confirms the effect of reducing the expression of COX-2 inflammatory factor induced by LPS according to ribociclib treatment in wild-type mice. (A) shows vehicle or ribociclib (10 or 30 mg/kg, ip) administered daily. After intraperitoneal administration for 7 days, on the 7th day, LPS (10 mg/kg, ip) or PBS was administered intraperitoneally 30 minutes after drug administration, and 8 hours later, the brain was extracted and immunofluorescent staining was performed with anti-COX-2 antibody. It shows the results of the performance, and (B) shows the results of (A) in a quantitative graph.
Figure 4 confirms the effect of reducing the expression of IL-6 pro-inflammatory cytokine induced by LPS according to ribociclib treatment in wild-type mice. (A) shows vehicle or ribociclib (10 or 30 mg/kg, ip). After intraperitoneal administration every day for 7 days, on the 7th day, LPS (10 mg/kg, ip) or PBS was intraperitoneally administered 30 minutes after drug administration, and 8 hours later, the brain was extracted and immunofluorescent with anti-IL-6 antibody. It shows the results of staining, and (B) shows the results of (A) in a quantitative graph.
Figure 5 confirms the effect of reducing the expression of IL-β pro-inflammatory cytokine induced by LPS according to ribociclib treatment in wild-type mice. (A) shows vehicle or ribociclib (10 or 30 mg/kg, ip). After intraperitoneal administration every day for 7 days, on the 7th day, LPS (10 mg/kg, ip) or PBS was intraperitoneally administered 30 minutes after drug administration, and 8 hours later, the brain was removed and immunofluorescent with anti-IL-β antibody. It shows the results of staining, and (B) shows the results of (A) in a quantitative graph.

The present invention identifies a new use of ribociclib as a treatment for neuroinflammation or degenerative brain diseases. Specifically, the present invention is a drug containing ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient. , is characterized by providing a pharmaceutical composition for preventing or treating neuroinflammation or degenerative brain diseases.

Ribociclib is known to be a drug used in combination with an aromatase inhibitor to treat breast cancer, but no studies have yet been conducted on its potential use as a treatment for neuroinflammation or degenerative brain diseases.

Accordingly, the present inventors conducted an experiment to confirm the possibility of ribociclib, which was used as an anticancer drug, as a new medicinal use as a treatment for neuroinflammation or degenerative brain diseases.

According to one embodiment of the present invention, wild-type mice are pretreated with the ribociclib of the present invention, then treated with LPS to induce neuroinflammation, and then have an inhibitory effect on the activity of microglia and astrocytes in the brain tissue of the mouse. was confirmed.

As a result, in the case of the group pretreated with ribociclib of the present invention, the activation of microglia and astrocytes caused by LPS was shown to be suppressed.

Through these results, the present inventors found that ribociclib of the present invention can prevent, improve, and treat neuroinflammation or degenerative brain diseases caused by excessive activation of microglia or astrocytes.

Microglial cells, which play the role of macrophages in the brain, are important effector cells that regulate immune responses in the central nervous system. Their activation plays an important role in maintaining homeostasis in the central nervous system 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. Additionally, recent research has revealed that excessive inflammatory responses in microglia or astrocytes are the cause of neurodegenerative 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 the formation of neuronal synapses, regulation of synapse number, 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.

According to another embodiment of the present invention, to determine whether ribociclib can suppress inflammatory factors in brain tissue or nerve cells, wild-type mice were treated with ribociclib, then treated with LPS, and brain cells were treated with ribociclib. The expression levels of inflammatory cytokines were analyzed in tissues.

As a result, it was confirmed that the expression of COX-2, IL-1β, and IL-6 was effectively suppressed in the group treated with ribociclib compared to the group not treated with ribociclib.

These results mean that the ribociclib drug of the present invention can effectively suppress excessive inflammatory responses in nerve cells caused by LPS.

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 causes neuroinflammation and degeneration due to excessive inflammatory responses. Brain disease develops.

Therefore, the present invention provides a pharmaceutical composition for preventing or treating neuroinflammatory or degenerative brain diseases, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition according to the present invention inhibits the production of pro-inflammatory cytokines; and inhibiting the activity of microglia or astrocytes; neuroinflammation or degenerative brain diseases can be prevented, improved, and treated.

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 inflammatory responses.

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 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 to prevent and treat neuroinflammation or degenerative brain diseases.

In the present invention, the neuroinflammation refers to inflammatory diseases in the central nervous system mediated by glial cells, and preferably refers to 'cranial nerve inflammatory disease'.

The neuroglia include, but are not limited to, oligodendrocytes, astrocytes, ependymal cells, microglia in the central nervous system, and Schwann in the peripheral nervous system. It may include Schwann cells and satellite cells.

In the present invention, the degenerative brain diseases that the composition containing ribociclib is intended to treat are not limited thereto, but include Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olive nucleus-pontine-cerebellar atrophy (OPCA), Shay-Drager syndrome, striato-substantia nigra degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortico-basal ganglia degeneration, diffuse Lewy body disease, Parkinson-ALS-dementia complex, Niemann-Pick disease, It may be selected from the group consisting of Pick's disease, cerebral ischemia, and cerebral infarction.

Furthermore, the present invention provides a health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, comprising ribociclib or a foodologically acceptable salt thereof as an active ingredient.

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

The health functional food may have a function that helps improve neuroinflammation or degenerative brain disease, and the food may be in the form of pills, powders, granules, needles, tablets, capsules, or liquids, and may be effective in the present invention. There are no particular restrictions on the types of foods to which ingredients can be added, for example, various beverages, gum, tea, vitamin complexes, health supplements, etc.

In addition to the active ingredients of the present invention, other ingredients that do not interfere with the inhibitory activity of neuroinflammation or degenerative brain disease 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.

Additionally, in the present invention, “pharmacologically or foodologically acceptable” means that the composition is not toxic to cells or humans exposed to it.

The salt may be used in the form of either a pharmaceutically or foodologically acceptable basic salt or an acid salt. Basic salts can be used in the form of either organic base salts or inorganic base salts, including sodium salts, potassium salts, calcium salts, lithium salts, magnesium salts, cesium salts, amidium salts, ammonium salts, triethylaminium salts, and pyrilicate salts. In the group consisting of dinium salts

can be selected. A useful acid salt is an acid addition salt formed by free acid. Inorganic acids and organic acids can be used as free acids. Hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, double phosphoric acid, and nitric acid can be used as inorganic acids, and citric acid, acetic acid, maleic acid, malic acid, and fumaric acid can be used as organic acids. , glucolic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, Glutamic acid, citric acid, aspartic acid, stearic acid, etc. may be used, but are not limited thereto and may include all salts formed using various inorganic acids and organic acids commonly used in the art.

In addition, the ribociclib compound or its salt of the present invention may include not only pharmaceutically or foodologically acceptable salts, but also all salts, hydrates, solvates, derivatives, etc. that can be prepared by conventional methods. . Addition salts can be prepared by conventional methods, by dissolving them in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, adding an excess amount of organic base, or adding an aqueous base solution of an inorganic base and then precipitating or crystallizing. It can be manufactured by ordering. Alternatively, an addition salt can be obtained by evaporating the solvent or excess base from this mixture and drying it, or it can be prepared by suction filtration of the precipitated salt.

Furthermore, the present invention can provide a method for inhibiting the inflammatory activity of cells in vitro, comprising the step of treating cells in which inflammatory activity is induced with ribociclib or a salt thereof, wherein the inflammatory activity is achieved by LPS. It may be induced by (Lipopolysaccharide) treatment.

In addition, the present invention can provide a composition for protecting nerve cells damaged by excessive inflammatory response in nerve cells, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient.

As described above, ribociclib can suppress excessive inflammatory responses in nerve cells or brain tissue, thereby suppressing damage to nerve cells caused by inflammatory responses.

Furthermore, the present invention can provide a reagent composition for inhibiting the activation of microglia or astrocytes, comprising ribociclib 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 merely 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 present invention were approved by the Institutional Biological Safety Committee (IBC) and were performed in accordance with the animal protocol approved by the Korea Brain Research Institute (KBRI, approval number IACUC-19-00049).

Ribociclib preparation

Ribociclib succinate hydrate was purchased from InvivoChem (V3926, Libertyville, IL, USA). Ribociclib was used dissolved in a solvent containing 5% DMSO, 40% PEG, 5% Tween80, and 50% saline at a dose of 10 mg/kg or 30 mg/kg, and administered intraperitoneally for in vivo experiments.

Wild-type mouse preparation

Male C57BL6/N mice (3 months old, 24-26 g; Koatech, Gyeonggi-do, Korea) were randomly divided into experimental groups and used for in vivo experiments. Mice were allowed to freely consume food and water under germ-free conditions in a 12-hour photoperiod in a cage that accommodated 3-4 mice each. All experimental procedures were approved by the Institutional Biological Safety Committee (IBC) and were performed in accordance with the approved animal protocols and guidelines of the Korea Brain Research Institute (KBRI, approval number IACUC-19-00049).

Immunofluorescence staining (IF)

Immunofluorescence staining experiments to analyze the effect of ribociclib on LPS-induced neuroinflammation were performed as follows. 10 mg/kg or 30 mg/kg ribociclib (i.p.) or vehicle (5% DMSO + 40% PEG + 5% Tween80 + 50% saline) was administered intraperitoneally to wild-type mice for 7 consecutive days. On the 7th day of drug administration, LPS (Sigma Aldrich, St. Louis, MO, USA, Escherichia coli, 10 mg/kg, i.p.) or PBS was administered intraperitoneally 30 minutes after drug administration. Then, after 8 hours, the mouse was anesthetized with 2,2,2-tribromoethanol (Sigma Aldrich, St. Louis, MO, USA, 2.5% v/v, 150 mg/kg, i.p.) and then administered with PBS. After transcardial perfusion, it was fixed with 4% PFA (Chembio, Seoul, Korea). Brains were then isolated from mice, postfixed overnight in 4% paraformaldehyde at 4°C, and then dehydrated in 30% sucrose solution for 2 days. Sections were sectioned to a thickness of 35 μm using a cryostat (Leica CM1850, Wetzlar, Germany), and permeabilized with PBS containing 0.2% Triton X-100 (PBST) and 10% normal goat serum for 1 hour at room temperature. . Afterwards, mouse brain sections were immunostained with anti-Iba-1, anti-GFAP, anti-IL-1β, anti-IL-6, or anti-COX-2 antibodies diluted in PBST for 48-72 hours at 4°C. was carried out. After reaction with the primary antibody, the brain sections were washed three times with PBST and reacted with the secondary antibody at room temperature for 2 hours. After completion of the secondary antibody reaction, the cells were washed three times with PBS and mounted in anti-discoloration mounting medium using DAPI (Vector Laboratories, Burlingame, CA, USA). Images of sections were acquired with a fluorescence microscope (DMi8, Leica Microsystems, Wetzlar, Germany) and analyzed with ImageJ software (version 1.53a, National Institutes of Health, Bethesda, MD, USA). Table 1 below shows information on the primary and secondary antibodies used in the above experiments.

<Example 1>

Confirmation of inhibition of LPS-mediated microglial activity by ribociclib in wild-type mice

To determine whether ribociclib affects LPS-induced microglial activation, wild-type mice were administered vehicle or ribociclib (10 or 30 mg/kg, i.p.) daily for 7 days. On the 7th day, 30 minutes after drug administration, LPS (10 mg/kg, ip) or PBS was administered intraperitoneally, and 8 hours later, the brain was extracted and immunofluorescent staining was performed with anti-Iba-1 antibody.

As a result of the analysis, in the group treated with 30 mg/kg ribociclib, LPS-mediated Iba-1 fluorescence intensity, Iba-1 positive area, and number of Iba-1 positive cells were significantly reduced in the cortical area of the brain. appeared (Figure 1A). In addition, in the CA1 region of the hippocampus, when treated with 30 mg/kg ribociclib, LPS-mediated Iba-1 fluorescence intensity and the number of Iba-1 positive cells were significantly reduced (Figures 1A, 1B). The positive area of LPS-induced Iba-1 was also found to be reduced in the hippocampal DG region by treatment with 30 mg/kg ribociclib (Figure 1A, 1B). Meanwhile, the decrease in Iba-1 fluorescence intensity and number of Iba-1 positive cells induced by LPS by treatment with 30 mg/kg ribociclib appeared to be somewhat insignificant (Figures 1A, 1B).

Through these results, the present inventors were able to see that the ribociclib drug of the present invention affects the activation of microglial cells induced by LPS in wild-type mice, and specifically, the ribociclib drug affects the activation of microglial cells. It was found that it could be suppressed.

<Example 2>

Confirmation of inhibition of LPS-mediated astrocyte activity by ribociclib in wild-type mice

In addition, to determine whether ribociclib can also regulate the activation of astrocytes caused by LPS, we injected wild-type mice with vehicle or ribociclib (10 or 30 mg/kg, i.p.) every day for 7 days. On the 7th day, 30 minutes after drug administration, LPS (10 mg/kg, ip) or PBS was administered intraperitoneally, and 8 hours later, the brain was removed and immunofluorescent staining was performed with an anti-GFAP antibody. GFAP is an indicator that can confirm the activation of astrocytes, and in the present invention, it was confirmed whether ribociclib can affect the activation of astrocytes through immunofluorescence staining.

As a result of the analysis, there was no significant change in LPS-mediated GFAP fluorescence intensity, GFAP-positive area, and number of GFAP-positive cells in the cortex and hippocampal DG region of the brain tissue of wild-type mice treated with ribociclib (10 or 30 mg/kg). appeared (Figures 2A, 2B). In the CA1 region of the hippocampus, the LPS-mediated GFAP fluorescence intensity was found to be downregulated in the group treated with 30 mg/kg ribociclib, but the GFAP-positive area and the number of GFAP-positive cells showed no significant changes (Figure 2A, 2B).

Through these results, the present inventors were able to see that ribociclib can inhibit the activation of astrocytes by ribociclib treatment in the CA1 hippocampus region of wild-type mice (Figures 2A, 2B). In addition, it was found that the ribociclib of the present invention had a greater effect on suppressing the activation of microglial cells than on suppressing the activation of astrocytes.

<Example 3>

Analysis of the inhibitory effect of LPS-mediated pro-inflammatory cytokines following ribociclib treatment in wild-type mice

Since it was confirmed through the experiments of the above examples that ribociclib can regulate LPS-mediated microgliosis, the present inventors confirmed whether ribociclib also affects the level of pro-inflammatory cytokines induced by LPS in vivo. An experiment was performed to do this. For this purpose, vehicle or ribociclib (10 or 30 mg/kg, i.p.) was administered to wild-type mice every day for 7 days, and on the 7th day, LPS (10 mg/kg, ip) or PBS was administered intraperitoneally 30 minutes after drug administration. Then, 8 hours later, the brain was removed, and immunofluorescence staining was performed with anti-COX-2, anti-IL-6, and anti-IL-1b antibodies.

As a result, in the group treated with 10 and 30 mg/kg ribociclib, the LPS-mediated expression level of COX-2 was significantly suppressed in the brain cortex and hippocampus (Figures 3A, 3B). In addition, wild-type mice administered ribociclib (30 mg/kg) also showed significantly reduced LPS-stimulated IL-6 levels in the cortex and hippocampus (Figures 4A, 4B). Furthermore, the level of IL-1beta, an inflammatory cytokine caused by LPS, was significantly suppressed in the cortex and hippocampus CA1 region of wild-type mice administered ribociclib (30 mg/kg) (Figures 5A, 5B).

Through the above results, the present inventors have confirmed that the ribociclib of the present invention can effectively inhibit the activation of microglia and astrocytes induced by LPS and can suppress the pro-inflammatory response induced by LPS. , it was found that ribociclib can be used as a new treatment for the prevention, improvement, and treatment of neuroinflammation and 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 (15)

A pharmaceutical composition for preventing or treating neuroinflammatory or degenerative brain diseases, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient. According to paragraph 1,
The composition is,
Inhibition of activity of microglia or astrocytes; or
Inhibition of production of pro-inflammatory cytokines; A pharmaceutical composition for preventing or treating neuroinflammatory or degenerative brain diseases, characterized by having an effect. According to paragraph 1,
The composition is a pharmaceutical composition for preventing or treating neuroinflammation or degenerative brain disease, characterized in that it inhibits inflammatory reactions in nerve cells. According to paragraph 2,
A pharmaceutical composition for preventing or treating neuroinflammatory or degenerative brain diseases, wherein the pro-inflammatory cytokine is IL-1β, IL-6, or COX-2. According to paragraph 3,
A pharmaceutical composition for preventing or treating neuroinflammation or degenerative brain disease, wherein the inflammatory response is induced by LPS (Lipopolysaccharide). According to paragraph 1,
The degenerative brain diseases include Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivary nucleus-pontine-cerebellar atrophy (OPCA), Shay-Drager syndrome, striatal-substantia nigra degeneration, Huntington's disease, and amyotrophic lateral sclerosis ( ALS), essential tremor, cortico-basal degeneration, diffuse Lewy body disease, Parkinson-ALS-dementia complex, Niemann Pick disease, Pick disease, cerebral ischemia, and cerebral infarction. Pharmaceutical composition for preventing or treating brain diseases. A composition for protecting nerve cells damaged by excessive inflammatory response in nerve cells, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient. A reagent composition for inhibiting the activation of microglia or astrocytes, comprising ribociclib or a pharmaceutically acceptable salt thereof as an active ingredient. According to clause 8,
A reagent composition for inhibiting the activation of microglia or astrocytes, wherein the activation of the microglia or astrocytes is caused by an excessive inflammatory response of nerve cells. A health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, comprising ribociclib or a foodologically acceptable salt thereof as an active ingredient. According to clause 10,
The composition is,
Inhibition of activity of microglia or astrocytes; or
Inhibition of production of pro-inflammatory cytokines; A health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, which is characterized by having an effect. According to clause 10,
The composition is a health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, characterized in that it inhibits inflammatory reactions in nerve cells. According to clause 11,
A health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, wherein the pro-inflammatory cytokine is IL-1β, IL-6, or COX-2. According to clause 12,
A health functional food composition for preventing or improving neuroinflammation or degenerative brain disease, wherein the inflammatory response is induced by LPS (Lipopolysaccharide). According to clause 10,
The degenerative brain diseases include Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivary nucleus-pontine-cerebellar atrophy (OPCA), Shay-Drager syndrome, striatal-substantia nigra degeneration, Huntington's disease, and amyotrophic lateral sclerosis ( ALS), essential tremor, cortico-basal degeneration, diffuse Lewy body disease, Parkinson-ALS-dementia complex, Niemann Pick disease, Pick disease, cerebral ischemia, and cerebral infarction. Health functional food composition for preventing or improving brain disease.