Pharmaceutical composition for prevention or treatment of degenerative brain diseases comprising selexipag

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of degenerative brain diseases, comprising selexipag or a pharmaceutically acceptable salt thereof as an active ingredient. Selexipag inhibits the expression and production of pro-inflammatory or inflammatory cytokines in brain tissue, inhibits the activity of microglia or astrocytes, inhibits NLRP3 inflammasome activity and CDK6 expression, reduces amyloid plaques, increases the expression of the amyloid beta-degrading enzyme IDE, and inhibits the hyperphosphorylation of tau protein and the expression of tau kinase, and thus can be effectively used as a novel therapeutic agent for the prevention or treatment of degenerative brain diseases, cognitive disorders, learning disabilities, or memory disorders.

Description

Pharmaceutical composition for preventing or treating degenerative brain diseases containing selexipax

[0001] The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

[0002]

[0003] Degenerative brain diseases are diseases that cause various symptoms such as motor and sensory impairment, memory, learning, and higher-order causal functions such as arithmetic reasoning due to degenerative changes in the nerve cells of the central nervous system. Types of degenerative brain diseases include Alzheimer's disease (AD), Parkinson's disease, and memory impairment. Degenerative brain diseases are characterized by the death of nerve cells through necrosis or apoptosis, which progress rapidly or slowly.

[0004] Inflammation in the central nervous system (CNS), also known as neuroinflammation, contributes to degenerative brain diseases and is mediated by the release of cytokines and chemokines by glial cells in the CNS. Glial cells are divided into two types: microglia and astrocytes, which play a crucial role in maintaining neurons and detecting and defending against pathogens.

[0005] Meanwhile, the activity of microglia and astrocytes has been reported to be related to the onset and progression of degenerative brain diseases, and microglia are immune cells that reside in the central nervous system and are known to be activated by external stimuli to induce immune and inflammatory responses. Microglia are cells that perform primary immune functions in the central nervous system, and maintain the shape of a thin cell body with long, thin branches, but when toxins that enter from the outside or are generated internally are present, they change into an activated shape with thick, short branches and a fat cell body to protect nerve cells from these toxins.

[0006] However, when microglia are activated by substances such as bacterial endotoxins, lipopolysaccharide (LPS), interferon-γ, beta-amyloid, or ganglioside, unlike normal microglia, they actively perform phagocytosis, proliferate, and express genes such as cytokines, chemokines, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) to produce inflammatory mediators. This activation of microglia has the effect of removing damaged cells and protecting neurons from invading bacteria or viruses, but nitric oxide (NO) produced by iNOS, prostaglandins produced by COX-2, and TNF-α are also toxic to neurons, so microglia activation ends up worsening the damage to neurons. Therefore, suppressing the appropriate activation of microglia may be another way to treat degenerative brain diseases.

[0007] In addition, degenerative brain diseases are known to occur when specific brain cells die or degenerate temporarily or over a long period of time. Since dead brain cells cannot regenerate, they ultimately lead to fatal loss of brain function. In particular, brain dysfunction accompanied by progressive decline in cognitive, sensory, motor, and systemic functions ultimately leads to changes in personality and behavior, and patients reach a point where they are unable to care for themselves. The main pathways of this brain cell death include oxidative toxicity due to oxidative stress, excitotoxicity, and apoptosis, and each induces cell death through a unique signaling process. Specifically, oxidative damage to proteins, nucleic acids, and lipids following the accumulation of reactive oxygen species has been suggested as the main cause of brain cell death in patients with stroke, brain injury, Alzheimer's disease, and Parkinson's disease. In particular, oxidative stress caused by free radicals has been reported to be the main cause of cell death in each tissue in the body, and has also been suggested as one of the cycles of cell death that appear in neurological diseases (Schapira, AH, Curr. Opin. Neurol., 9(4):260-264, 1996).

[0008] Furthermore, astrocytes play a crucial role not only in brain development but also in maintaining normal brain activity. Brain astrocytes have been shown to support neuronal activity by appropriately removing neurotransmitters secreted by neurons and regulating brain ion concentrations. Furthermore, they have been shown to play a crucial role in aiding the differentiation of neural stem cells into neurons.

[0009] However, when astrocytes are injured in the brain, they proliferate rapidly, swell, and become activated into reactive astrocytes, a phenomenon known as astrogliosis. These reactive astrocytes have been observed in conditions such as AIDS-related dementia, brain injury, ischemic brain disease, and Alzheimer's disease. Therefore, persistent astrocyte activation ultimately leads to neuronal death. Therefore, appropriately suppressing astrocyte activation may also be another approach to treating degenerative brain diseases.

[0010] Currently, treatments used to treat degenerative brain diseases include drug therapy, surgical therapy, and physical therapy. In the case of drug therapy, drugs are generally used to supplement dopamine deficiency in the brain, correct the imbalance of neurotransmitters caused by dopamine deficiency, prevent or delay the destruction of nerve cells, and control other symptoms such as depression.

[0011] However, these drugs have limitations in that they are not intended to cure the disease but rather to control symptoms, as they cannot revive dead nerve cells. Therefore, there is an urgent need to develop new treatments that can more effectively prevent or treat degenerative brain diseases.

[0012] Meanwhile, selexipag, currently marketed under the name Uptravi, is a treatment for pulmonary arterial hypertension (PAH). Selexipag and its active metabolite, ACT-333679 (or MRE-269), are known to be prostacyclin receptor agonists that induce vasodilation in the pulmonary circulation (see Patent Document 0001).

[0013] In the present invention, it was confirmed that selexipag, previously known as a pulmonary arterial hypertension treatment agent, inhibits the expression and production of pro-inflammatory or inflammatory cytokines in LPS-induced brain tissue, inhibits the activity of microglia or astrocytes, and inhibits NLRP3 inflammasome activity and CDK6 expression, and thus, the use of selexipag for the prevention or treatment of degenerative brain diseases was proposed.

[0014]

[0015] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising selexipag or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cognitive impairment, learning disability or memory impairment, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

[0017] Another object of the present invention is to provide a composition for protecting nerve cells damaged by excessive inflammatory response in nerve cells, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

[0018] Another object of the present invention is to provide a reagent composition for inhibiting the activation of microglia or astrocytes, comprising selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

[0019] Another object of the present invention is to provide a reagent composition for inhibiting tau phosphorylation, comprising selexipag or a pharmaceutically acceptable salt thereof as an active ingredient.

[0020] Another object of the present invention is to provide a health functional food for preventing or improving neuroinflammation or degenerative brain disease, comprising Selexipak or a food-related acceptable salt thereof as an active ingredient.

[0021] Another object of the present invention is to provide a health functional food for improving cognitive function, learning ability or memory, comprising Selexipak or a food-scientifically acceptable salt thereof as an active ingredient.

[0022]

[0023] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

[0024] In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive impairment, learning disability or memory impairment, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

[0025] Selexipag is a compound with the following structural formula, its chemical name is 2-[4-[(5,6-diphenylpyrazin-2-yl)-propan-2-ylamino]butoxy]-N-methylsulfonylacetamide, and it is a prostacyclin receptor agonist.

[0026]

[0027] Selexipag is currently marketed under the brand name Uptravi as a treatment for pulmonary arterial hypertension (PAH) by causing vasodilation in the pulmonary blood vessels.

[0028] The pharmaceutical composition of the present invention may use not only Selexipag as an active ingredient, but also a pharmaceutically acceptable salt thereof. The pharmaceutically acceptable salt should have low toxicity to humans and should not have any negative effects on the biological activity and physicochemical properties of the parent compound. For example, the pharmaceutically acceptable salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, or phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanedioates, aromatic acids, and aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, Contains terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.Specifically, the above selexipag can be used in the form of selexipag sodium, which is a sodium salt form.

[0029] In the present invention, Selectifac may have one or more of the following activities:

[0030] (i) suppression of the levels of pro-inflammatory cytokines or inflammatory cytokines in brain tissue;

[0031] (ii) inhibition of inflammasome formation or levels of cyclin-dependent kinases in brain tissue;

[0032] (iii) Inhibition of the activity of microglia or astrocytes in brain tissue;

[0033] (iv) reducing amyloid plaques or inhibiting amyloid beta protein accumulation in brain tissue; or

[0034] (v) Inhibition of expression or activity of tau kinase in brain tissue, or inhibition of hyperphosphorylation of tau protein.

[0035] In one specific example, selixipag may have anti-inflammatory activity, and specifically, may inhibit or alleviate an inflammatory response in brain tissue.

[0036] In one specific example, selexipag can inhibit the expression and production of pro-inflammatory cytokines or inflammatory cytokines in brain tissue. Specifically, selexipag can decrease LPS-induced inflammatory factors (e.g., COX-2, IL-6, IL-1β, or TNF-α) or increase anti-inflammatory cytokines (e.g., TGF-β or IL-10).

[0037] In an embodiment of the present invention, it was confirmed that Selexipag has an activity of effectively suppressing the expression and / or production of inflammatory cytokines and proinflammatory cytokines induced by LPS, known as an inflammatory factor, in brain tissue, and specifically, it was confirmed that it has an activity of suppressing the production of COX-2, IL-1β, IL-6, or TNF-α by LPS. Among various inflammatory factors, LPS, which is one of the endotoxins, promotes the secretion of proinflammatory cytokines, which causes the secretion of inflammatory mediators such as nitric oxide and prostaglandins, and various inflammatory diseases are caused by excessive inflammatory responses.

[0038] In one embodiment, selexipag may inhibit LPS-induced proinflammatory responses, thereby preventing, improving, or treating degenerative brain diseases that may be caused by excessive inflammatory responses in brain tissue.

[0039] Selexipag can inhibit the formation of inflammasomes or the level of cyclin-dependent kinase (CDK) in brain tissue. Specifically, Selexipag can inhibit the NLRP3 inflammasome pathway by reducing or suppressing the expression of NLRP3 (nucleotide-binding domain and leucine-rich repeat protein-3), which is produced by inflammatory responses induced by factors such as LPS, or act as an inhibitor of CDK4 / 6 by reducing or suppressing the expression of CDK6.

[0040] The inflammasome is a protein complex composed of several proteins, including NLRP3, NLRC4, AIM2, and NLRP6. In response to bacterial infection, stress, and damage signals, it directly activates caspase-1, inducing the secretion of potent pro-inflammatory cytokines (e.g., IL-1β or IL-18), and inducing pyroptosis, an inflammatory cell death. In addition, CDK4 / 6 is a cell cycle regulatory protein that can regulate DNA synthesis and division of cells in relation to the cell cycle. However, overactivation of CDK4 / 6 affects the survival and cell cycle control of neurons, affecting amyloid beta accumulation and inducing neuronal degeneration. Therefore, CDK4 / 6 inhibitors have been reported to have therapeutic effects in the treatment of inflammatory diseases, neurological and neurodegenerative diseases, and Alzheimer's disease.

[0041] In one embodiment, it was confirmed that Selexipag can prevent, improve or treat degenerative brain diseases that can be caused by excessive inflammatory response in brain tissue by inhibiting the expression or level of NLRP3 and CDK6 induced by LPS in brain tissue.

[0042] Selexipag can inhibit the activity of microglia or astrocytes. Specifically, it can inhibit the damage that activated microglia or astrocytes cause to neurons.

[0043] Microglia, which function as macrophages in the brain, are key effector cells that regulate immune responses in the central nervous system (CNS). Their activation plays a crucial role in maintaining CNS homeostasis by removing foreign substances caused by drugs or toxins and secreting nerve growth factors. However, exposure to harmful stressors, such as signals from damaged neurons, the accumulation of abnormally formed proteins altered by external stimuli, or the invasion of pathogens, can lead to excessive microglia activity, which can damage neurons and contribute to degenerative neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and cerebral infarction. Specifically, unlike normal microglia, overly activated microglia actively engage in phagocytosis, proliferate, and express genes such as cytokines (TNF-α, IL-1β, and IL-6), chemokines, and COX-2, producing inflammatory mediators. Microglial activation has the effect of removing damaged cells and protecting neurons from invading bacteria and viruses. However, nitric oxide (NO), prostaglandins produced by COX-2, and tumor necrosis factor (TNF)-α are toxic to neurons. Consequently, microglial activation worsens neuronal damage and can cause degenerative brain diseases or neurodegenerative disorders. In addition, astrocytes are also known to play an important role in maintaining normal brain activity, and in particular, they are known to play a role in the formation of synapses, regulation of synapse numbers, synaptic function, and differentiation of neural stem cells into neurons. However, when these astrocytes become overly reactive, that is, when they remain in an excessively activated state, they can cause the death of neurons and induce the death of neighboring neurons, which can become a cause of degenerative brain diseases.Substances that cause excessive activation of microglia and astrocytes include bacterial endotoxins such as LPS, interferon-γ, beta-amyloid, and gangliosides, which can induce inflammatory responses.

[0044] In one embodiment of the present invention, it was confirmed that selexipag can suppress excessive activation of microglia and / or astrocytes in brain tissue, and therefore selexipag can be useful for preventing, improving, or treating degenerative brain diseases that can be caused by excessive inflammatory response in brain tissue.

[0045] Selexipag and pharmaceutically acceptable salts thereof have excellent effects of inhibiting the aggregation of amyloid-beta or decomposing aggregates, inhibiting the aggregation of tau protein or decomposing aggregates, and inhibiting the phosphorylation of tau protein. Therefore, Selexipag or a pharmaceutical composition comprising the same can be useful for preventing or treating diseases related to the inhibition of amyloid-beta aggregation and / or decomposition of aggregates, the inhibition of tau protein aggregation and / or decomposition of aggregates, and / or the inhibition of phosphorylation of tau protein, such as degenerative brain diseases.

[0046] In one specific example, the pharmaceutical composition according to the present invention comprises:

[0047] (1) Individuals (patients) with or at risk of having higher than normal levels of amyloid-beta aggregation;

[0048] (2) Individuals (patients) with higher than normal or at risk of higher levels of tau protein aggregation;

[0049] (3) Individuals (patients) with higher than normal or at risk of higher phosphorylation levels of tau protein; and

[0050] (4) Individuals (patients) who fall under two or more of the above (1) to (3)

[0051] It may be intended for administration to an individual (patient) selected from a group consisting of:

[0052] In one embodiment of the present invention, it was confirmed that selexipag reduces the accumulation of amyloid beta by regulating the expression of insulin degrading enzyme (IDE), an amyloid beta degrading enzyme, in an amyloid-overexpressing Alzheimer's disease animal model. In addition, it was confirmed that selexipag inhibits tau hyperphosphorylation by inhibiting the expression and activity of DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) and p-CDK5, which are tau kinases, in a tau-overexpressing animal model, which is another Alzheimer's disease animal model.

[0053] In this specification, the term "prevention" means any action that inhibits or delays the occurrence, spread, and recurrence of the disease by administering a compound or pharmaceutical composition according to the present invention, and "treatment" means any action that improves or beneficially changes the symptoms of the disease by administering a compound or pharmaceutical composition according to the present invention.

[0054] In this specification, the term "normal" may refer to an individual of the same species as the individual to which the pharmaceutical composition is applied and does not have the "degenerative brain disease" defined above, or brain tissue or brain cells (brain nerve cells) isolated and / or cultured from the individual.

[0055] As used herein, the term “inhibition” means inhibition of any step of transcription, mRNA processing, translation, translocation and maturation of a gene, or inhibition of protein-protein binding, protein activation or signal transduction through the same.

[0056] The term "pharmaceutical composition" as used herein may refer to a molecule or compound that, when administered to a subject, imparts several beneficial effects. Beneficial effects may include enabling diagnostic determination; improving a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or condition.

[0057] In this specification, the term "degenerative brain disease" is used to comprehensively describe all diseases related to degenerative changes in the brain, and in particular, all diseases (brain diseases) that can be caused by factors such as aggregation of amyloid-beta in the brain and / or brain neurons.

[0058] In one specific example, the degenerative brain disease may be selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, senile dementia, multiple sclerosis, multiple myelopathy, epilepsy, encephalophathy, and frontotemporal dementia, but is not limited thereto, and any disease caused by aggregation of amyloid-beta, aggregation of tau protein, and / or phosphorylation of tau protein may be the target.

[0059] The term "cognition" herein refers to the mental activity or process of acquiring knowledge and understanding through thought, experience, and sensation. This may include processes such as knowledge, attention, memory and working memory, judgment and evaluation, reasoning and calculation, problem solving and decision making, and understanding and production of language. Cognitive impairment refers to a category of mental health disorders that primarily affect learning, memory, perception, and problem solving, and may include amnesia, dementia, and delirium.

[0060] The aforementioned memory improvement may refer to improved memory storage, retention, and retrieval as a result of damage to neuroanatomical structures. It may also refer to improved memory impairments that may be progressive (including Alzheimer's disease).

[0061] In the present invention, "memory improvement" and "cognitive ability improvement" refer to the effect of maintaining cognitive ability by controlling harmful substances that damage brain cells or improving reduced cognitive ability by controlling neurotransmitters in the brain when the brain atrophies and destroys brain nerve cells due to physical fatigue, lack of sleep, excessive alcohol consumption, dementia, etc., such as memory decline, memory impairment, or cognitive ability decline. The memory is the ability to receive necessary information, store it in the brain, and retrieve and use it when needed, and cognitive ability refers to the ability to recognize and discern objects.

[0062] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is one commonly used in the preparation of a formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0063] In addition to the active ingredient, the pharmaceutical composition may further comprise one or more auxiliary agents selected from the group consisting of pharmaceutically acceptable carriers, excipients, diluents, fillers, bulking agents, wetting agents, disintegrating agents, emulsifiers (surfactants), lubricants, sweeteners, flavoring agents, suspending agents, preservatives, etc. The auxiliary agents may be appropriately adjusted according to the dosage form to which the pharmaceutical composition is applied, and one or more auxiliary agents may be selected and used from among all auxiliary agents commonly used in the pharmaceutical field. In one specific example, the pharmaceutically acceptable carrier is one commonly used in the formulation of drugs, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and target organ-specific antibodies or other ligands can be combined with the carriers to specifically act on the target organs. Furthermore, the composition can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's literature (Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA).

[0064] The effective amount of the above active ingredient or the pharmaceutical composition can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration can mean administration through an administration route other than oral, such as rectal, intravenous, peritoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, ocular, or subcutaneous, and can be administered by local administration to the affected area, etc. When administered orally, the pharmaceutical composition can be formulated into a formulation that coats the active ingredient to prevent it from being decomposed in the stomach or protects it from being decomposed in the stomach. When the pharmaceutical composition of the present invention is used as a medicine, it can additionally contain one or more active ingredients having the same or similar function.

[0065] In addition, the term "active ingredient" in this specification may refer to a physiologically active substance used to achieve the pharmacological activity (e.g., treatment of degenerative brain disease) mentioned in this specification, and this is distinguished from administering the substance alone, in combination with other substances, or additionally to treat the disease mentioned in this specification. That is, the above-mentioned Selexipag may be administered as a sole active ingredient for the direct treatment effect of degenerative brain disease, or may be administered in combination with other drugs.

[0066] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup, or emulsion in an aqueous or oily medium, or in the form of a powder, granule, tablet, or capsule, and may additionally contain a dispersing agent or stabilizer for formulation. When formulating the pharmaceutical composition, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include Witepsol, Macrogol, Tween 61, cocoa butter, liurin butter, and glycerogelatin.

[0067] The above pharmaceutical composition may be used by mixing with various carriers acceptable as pharmaceuticals, such as saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers may be used as pharmaceuticals to increase stability or absorbability.

[0068] The pharmaceutical composition may be administered in a pharmaceutically effective amount. There is no particular restriction on the dosage, and may vary depending on the absorption rate in the body, body weight, patient's age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The pharmaceutical composition of the present invention is manufactured in consideration of the effective dosage range, and the unit dosage form prepared in this way may be administered multiple times at regular intervals using a specialized dosage method according to the judgment of a specialist who monitors or observes the administration of the drug and the individual's needs, as needed. The dosage of the pharmaceutical composition may be 1 ug / kg / day to 1,000 mg / kg / day, but is not limited thereto. The daily or single dose may be prepared as a single preparation in the form of a unit dose, formulated in an appropriate amount, or placed in a multi-dose container.

[0069] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of treatment for a degenerative brain disease.

[0070] Additionally, the present invention provides a use of selexipac or a pharmaceutically acceptable salt thereof for preventing or treating degenerative brain diseases.

[0071] The present invention provides a use of selexipac or a pharmaceutically acceptable salt thereof for preventing or treating cognitive impairment, learning disability or memory impairment.

[0072] The present invention provides a use of selexipax or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of degenerative brain diseases.

[0073] The present invention provides a use of Selexipak or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for preventing or treating cognitive impairment, learning disability or memory impairment.

[0074] The present invention provides a method for preventing or treating a degenerative brain disease, comprising administering selexipag or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0075] The present invention provides a method for preventing or treating cognitive impairment, learning disability or memory impairment, comprising administering selexipag or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0076]

[0077] Furthermore, the present invention can provide a composition for protecting nerve cells damaged by excessive inflammatory responses in nerve cells, comprising Selexipag or a pharmaceutically acceptable salt thereof as an active ingredient. As described above, Selexipag can suppress excessive inflammatory responses in nerve cells or brain tissue, thereby inhibiting damage to nerve cells caused by inflammatory responses.

[0078] The present invention provides a use of selexipac or a pharmaceutically acceptable salt thereof for inhibiting damage to nerve cells caused by an inflammatory response.

[0079] The present invention also provides a method for inhibiting damage to nerve cells caused by an inflammatory reaction, comprising administering selexipag or a pharmaceutically acceptable salt thereof to a subject in need of inhibition of nerve cell damage caused by an inflammatory reaction.

[0080]

[0081] Furthermore, the present invention provides a reagent composition for inhibiting the activation of microglia or astrocytes, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

[0082] In another aspect, the present invention provides a health functional food for preventing or improving neuroinflammation or degenerative brain disease, comprising Selexipak or a food-wise acceptable salt thereof as an active ingredient.

[0083] In another aspect, the present invention provides a health functional food for improving cognitive function, learning ability or memory, comprising Selexipak or a food-wise acceptable salt thereof as an active ingredient.

[0084] In another aspect, the present invention provides a health functional food for inhibiting tau protein aggregation, decomposing tau protein aggregates, and / or inhibiting tau protein phosphorylation, comprising selexipac or a food-scientifically acceptable salt thereof as an active ingredient.

[0085] The above health functional foods are foods manufactured using nutrients that are easily deficient in daily meals or raw materials or ingredients (hereinafter referred to as "functional raw materials") that have beneficial functions for the human body. They refer to all foods that help maintain health or prevent and / or improve certain diseases or symptoms. There are no specific restrictions on the final product form. For example, the above health functional foods may be selected from the group consisting of various foods, beverage compositions, food additives, etc., but are not limited thereto.

[0086] The content of the active ingredient contained in the above health functional food is not particularly limited, depending on the type of food, desired use, etc. The above health functional food may be used alone or in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The content of the active ingredient may be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less relative to the raw material.

[0087] The above health functional food may additionally contain at least one selected from the group consisting of various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents or natural flavoring agents, coloring agents, thickening agents (cheese, chocolate, etc.), pectic acid or its salt, alginic acid or its salt, organic acid, protective colloid thickener, pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The proportion of such additives is generally selected in the range of 0.001 to about 20 parts by weight per 100 parts by weight of the entire health functional food, but is not limited thereto.

[0088]

[0089] In the present invention, Selexipag inhibited the expression and production of pro-inflammatory or inflammatory cytokines in brain tissue induced by LPS, suppressed the activity of microglia or astrocytes, and suppressed NLRP3 inflammasome activity and CDK6 expression. In addition, it was confirmed that in an amyloid-overexpressing Alzheimer's disease animal model administered with Selexipag, the accumulation of amyloid beta was reduced by regulating the expression of IDE, an amyloid beta-degrading enzyme, and at the same time, the activity of microglia and astrocytes was suppressed in the Alzheimer's disease animal model. In another Alzheimer's disease animal model, a tau-overexpressing animal model, it was confirmed that tau hyperphosphorylation was suppressed by inhibiting the expression and activity of DYRK1A and p-CDK5, which are tau kinases. In light of these experimental results, Selexipag may be useful in the prevention or treatment of degenerative brain diseases, cognitive impairment, learning disabilities, or memory impairment.

[0090]

[0091] Figure 1 is a graph analyzing the cytotoxicity of microglial cells BV2 according to the treatment concentration of Selexipag using an MTT assay.

[0092] Figures 2a and 2b are graphs showing the expression levels of proinflammatory cytokines in BV2 microglia induced by LPS following selexipag treatment ((a) 1 μM and (b) 5 μM) using real-time PCR.

[0093] Figures 3a and 3b are images and graphs analyzing the activity level of microglia induced by inflammation by LPS following selexipag treatment through immunofluorescence staining (anti-Iba-1 antibody) (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0094] Figures 4a and 4b are images and graphs analyzing the activity level of astrocytes induced by inflammation by LPS following selexipag treatment through immunofluorescence staining (anti-GFAP antibody) (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0095] Figures 5a and 5b are images and graphs analyzing the level of inhibition of COX-2 expression induced by LPS according to selexipag treatment through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0096] Figures 6a and 6b are images and graphs analyzing the level of inhibition of IL-6 expression induced by LPS according to selexipag treatment through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0097] Figures 7a and 7b are images and graphs analyzing the level of inhibition of IL-1β expression induced by LPS according to selexipag treatment through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0098] Figures 8a and 8b are images and graphs analyzing the level of inhibition of TNF-α expression induced by LPS according to selexipag treatment through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0099] Figures 9a and 9b are images and graphs analyzing the level of suppression of NLRP3 expression induced by LPS according to selexipag treatment through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0100] Figures 10a and 10b are images and graphs analyzing the level of inhibition of CDK6 expression induced by LPS according to selexipag treatment through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0101] Figures 11a, 11b and 11c are images and graphs analyzing the level of amyloid plaques (CA1: Cornu Ammonis 1) in an amyloid-overexpressing dementia animal model (5xFAD) treated with selexipag using immunofluorescence staining.

[0102] Figures 12a, 12b and 12c are images and graphs analyzing the expression level of IDE through immunofluorescence staining in amyloid-overexpressing dementia animals (5xFAD) treated with selexipag (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0103] Figures 13a and 13b are images and graphs analyzing the activity level of microglial cells through immunofluorescence staining (anti-Iba-1 antibody) in amyloid-overexpressing dementia animals (5xFAD) treated with selexipag (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0104] Figures 14a and 14b are images and graphs analyzing the expression level of GFAP through immunofluorescence staining in amyloid-overexpressing dementia animals (5xFAD) treated with selexipag (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0105] Figures 15a and 15b are images and graphs analyzing the expression level of IL-1β in amyloid-overexpressing dementia animals (5xFAD) treated with selexipag through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0106] Figures 16a and 16b are images and graphs analyzing the level of NLRP3 expression inhibition in amyloid-overexpressing dementia animals (5xFAD) treated with selexipag through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0107] Figures 17a and 17b are images and graphs analyzing the degree of tau hyperphosphorylation in tau overexpressing dementia animals (PS19) treated with selexipag through immunofluorescence staining using AT8 antibody (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0108] Figures 18a and 18b are images and graphs analyzing the degree of tau hyperphosphorylation in tau overexpressing dementia animals (PS19) treated with selexipag through immunofluorescence staining using AT180 antibody (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0109] Figures 19a and 19b are images and graphs analyzing the level of expression of tau kinase DYRK1A through immunofluorescence staining in tau overexpressing dementia animals (PS19) treated with selexipag (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0110] Figures 20a and 20b are images and graphs analyzing the level of p-CDK5 expression of tau kinase in an Alzheimer's disease animal model (PS19) treated with selexipag through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0111]

[0112] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0113]

[0114] Experimental method

[0115] 1. Reagent

[0116] LPS (Sigma, Cat no: L2630, Escherichia coli) reagent was dissolved in phosphate-buffered saline (PBS) for in vivo experiments. To induce central nervous system immune responses, including neuroinflammation, 10 mg / kg of LPS was administered at 8-hour intervals. In addition, selexipag (Invivochem, Cat no: V8461, Libertyville, IL, USA) was dissolved in dimethyl sulfoxide (DMSO) for in vitro experiments and in vehicle (saline containing 1% DMSO) for in vivo experiments.

[0117]

[0118] 2. Cell lines and culture conditions

[0119] BV2 microglial cells (obtained from Dr. Kyung-Ho Suk) were cultured in high glucose DMEM (Invitrogen, Carlsbad, CA, USA) medium containing 5% fetal bovine serum (FBS, Invitrogen), 100 U / mL of penicillin, and 100 μg / mL of streptomycin, in an incubator maintained at 5% CO2 and 37°C.

[0120]

[0121] 3. Immunofluorescence staining

[0122] Wild-type C57BL6 mice (male, 8 weeks old) were housed in a pathogen-free environment at 22±2℃, 50±5% humidity, and a 12-h light / dark cycle with free access to food and water. The mice were randomly assigned to three experimental groups: control (Veh), LPS-treated group (LPS), and 1 mg / kg selexipag plus LPS-treated group (Sel+LPS). Mice in each experimental group were administered selexipag (1 mg / kg) or vehicle (1% DMSO in saline) intraperitoneally (ip) daily for 7 days. On day 7, 30 minutes after the final administration, 10 mg / kg LPS or PBS was administered intraperitoneally, and 8 hours later, the mice were perfused and fixed with PBS and 4% paraformaldehyde, respectively.

[0123] In addition, 5xFAD mice (amyloid-overexpressing dementia mice) and PS19 mice (male, 4 months old), which are animal models of Alzheimer's disease, were housed under pathogen-free conditions of 22±2℃, 50±5% humidity, and a 12-h light / dark cycle with free access to food and water. The mice were randomly assigned to two experimental groups: a control group (Veh) and 1 mg / kg selexipag. Mice in each experimental group were intraperitoneally (ip) administered selexipag (1 mg / kg) or vehicle (1% DMSO in saline) daily for 14 days. On the 14th day, 2 hours after the final administration, the mice were perfused and fixed with PBS and 4% paraformaldehyde, respectively. To obtain brain sections for immunofluorescence staining, brain tissues stored in 4% paraformaldehyde for 24 h at 4°C were immersed in 30% sucrose-containing PBS for 72 h and then sectioned to 30 μm thickness using a cryostat (Leica CM1850, Wetzlar, Germany). Brain sections were blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature and then immunostained with primary antibodies overnight at 4°C. Brain sections were then washed with PBST buffer and reacted with Alexa 594- or Alexa 488-conjugated secondary antibodies for 2 h at room temperature. After mounting on glass slides using Vectashield mounting solution containing DAPI (Vector Labs, Burlingame, CA), fluorescence microscopy images of immunostained tissues were obtained (DMi8, Leica Microsystems, Wetzlar, Germany), and imaging analysis was performed (ImageJ, US National Institutes of Health, Bethesda, MD, USA).

[0124]

[0125] 4. Statistical analysis

[0126] Data analysis was performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Comparisons between two experimental groups were performed using the t-test, and comparisons between three experimental groups were performed using one-way ANOVA and Tukey's test. Values ​​showing p<0.05 were considered significant, and the results are expressed as the mean ± standard deviation (SD) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

[0127]

[0128] Example 1. Cytotoxicity analysis of Selexipag

[0129] To determine whether Selexipag had cytotoxicity against the microglial BV2 cell line, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed.

[0130] Specifically, BV2 microglial cells were treated with vehicle or selexipag at different concentrations (1, 5, 10, 25, or 50 μM) for 24 h to analyze the cell viability of selexipag at different concentrations. For this, BV2 microglial cells were first seeded in a 96-well plate (2x10) for 1 h. 5 (cells / well) were cultured in FBS-free medium, and then treated with 1, 5, 10, 25, and 50 μM selexipag at various concentrations for 24 hours. A group treated with 1% DMSO (vehicle) was used as a control group. The cells were then cultured with MTT solution for 2 hours, and then treated with DMSO to dissolve the formazan product. After 20 minutes, the absorbance was measured at 570 nm (reference wavelength 660 nm) using a SPECTROstar Nano microplate reader (BMG Labtech, Germany). The results are shown in Fig. 1.

[0131] Figure 1 is a graph analyzing the cytotoxicity of microglial cells BV2 according to the treatment concentration of Selexipag using an MTT assay.

[0132] As shown in Figure 1, no cytotoxicity was induced in BV2 microglial cells treated with selectifac at various concentrations. Therefore, it was confirmed that selectifac does not induce harmful toxicity in cells regardless of the treatment concentration.

[0133]

[0134] Example 2. Reduction of LPS-induced proinflammatory cytokine levels by selexipag in BV2 microglia.

[0135] To determine whether selexipag could modulate the levels of LPS (Lipopolysaccharid)-mediated proinflammatory cytokines, BV2 microglia were treated with selexipag (1 μM, 5 μM) or 1% DMSO (vehicle) for 30 min, and then treated with 200 ng / mL LPS or PBS for 5.5 h. The expression levels of proinflammatory cytokines COX-2, IL-1β, IL-6, and TNF-α were analyzed by real-time PCR. Specifically, cDNA synthesized with Superscript cDNA Premix Kit II (GeNetBio) was analyzed by real-time PCR at 40 cycles using Fast SYBR Green Master Mix (Thermo Fisher Scientific, CA, USA) and QuantStudio 5 system (Thermo Fisher Scientific). The values ​​were normalized to the cycle threshold (Ct) value of GAPDH (housekeeping gene). The fold change in the LPS-only and LPS-plus-Selexipag co-treatment groups was calculated relative to the control group (vehicle treatment). The results are shown in Figures 2a and 2b.

[0136] Figures 2a and 2b are graphs showing the expression levels of proinflammatory cytokines in BV2 microglia induced by LPS following selexipag treatment ((a) 1 μM and (b) 5 μM) using real-time PCR.

[0137] As shown in Figures 2a and 2b, in the groups treated with 1 μM or 5 μM selexipag, respectively, the expression levels of LPS-induced proinflammatory cytokines COX-2, IL-1β, IL-6, and TNF-α were all reduced compared to the vehicle-treated control group, and it was confirmed that the levels of proinflammatory cytokines were further reduced in the 5 μM selexipag-treated group compared to the 1 μM selexipag-treated group.

[0138]

[0139] Example 3. Inhibition of LPS-mediated microglia and astrocyte activation by selexipag in wild-type mice.

[0140] Next, we conducted an experiment to analyze the efficacy of selexipag on LPS-induced microgliosis and astrogliosis in a mouse animal model.

[0141] Specifically, 8-week-old wild-type mice were intraperitoneally administered selexipag (1 mg / kg) or vehicle daily for 7 days, and on the 7th day, LPS (10 mg / kg) or PBS was intraperitoneally administered 30 minutes after the intraperitoneal administration of selexipag or vehicle. Then, 8 hours after the LPS injection, brain sections obtained from the mice were subjected to immunofluorescence staining with anti-Iba-1 (Wako, 019-19741, 1:500, rabbit) or anti-GFAP antibody (Invitrogen, 13-0300, 1:500, rat) according to the method described in the experimental method above. The results are shown in Figures 3a and 3b and Figures 4a and 4b, respectively.

[0142] Figures 3a and 3b are images and graphs analyzing the activity level of microglia induced by inflammation by LPS following selexipag treatment through immunofluorescence staining (Iba-1 antibody) (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0143] Figures 4a and 4b are images and graphs analyzing the activity level of astrocytes induced by inflammation by LPS following selexipag treatment through immunofluorescence staining (GFAP antibody) (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0144] As shown in Figures 3a and 3b, in the 1 mg / kg selexipag-treated group, the fluorescence intensity of Iba-1, an indicator of microglial activation, and the percentage area of ​​Iba-1 were significantly reduced in the cerebral cortex and hippocampal CA1 region. However, no change was observed in the number of Iba-1-positive cells in the cerebral cortex and hippocampus of the group treated with 1 mg / kg selexipag.

[0145] As shown in Figures 4a and 4b, in the 1 mg / kg selexipag-treated group, the fluorescence intensity of GFAP, an indicator of astrocyte activation, and the area percentage of GFAP were significantly reduced in the cerebral cortex and hippocampal CA1 region. In addition, treatment with 1 mg / kg selexipag was shown to reduce the number of GFAP-positive cells in the cerebral cortex and hippocampal DG region.

[0146] From this, it was found that Selexipag could regulate LPS-mediated activation of microglia and astrocytes in wild-type mice, and in particular, it was confirmed that it could improve and alleviate microgliosis and astrogliosis by inhibiting the activation of these cells induced by LPS.

[0147]

[0148] Example 4. Inhibition of LPS-mediated COX-2 and IL-6 expression by selexipag in wild-type mice.

[0149] To determine whether selexipag could inhibit the expression of LPS-induced proinflammatory cytokines COX-2 and IL-6, 8-week-old wild-type mice were intraperitoneally administered selexipag (1 mg / kg) or vehicle daily for 7 days. On day 7, LPS (10 mg / kg) or PBS was intraperitoneally administered 30 minutes after the intraperitoneal administration of selexipag or vehicle. Then, 8 hours after LPS injection, immunofluorescence staining was performed on isolated mouse brain sections with anti-COX-2 (Abcam, ab15191, 1:200, rabbit) or anti-IL-6 antibody (Santa Cruz, SC57315, 1:50, mouse). The results are shown in Figures 5a and 5b and 6a and 6b.

[0150] Figures 5a and 5b are images and graphs analyzing the level of inhibition of COX-2 expression induced by LPS according to selexipag treatment by immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0151] Figures 6a and 6b are images and graphs analyzing the level of inhibition of IL-6 expression induced by LPS according to selexipag treatment by immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0152] As shown in Figures 5a and 5b and Figures 6a and 6b, in the case of the 1 mg / kg selexipag treatment group, it was confirmed that LPS-induced COX-2 and IL-6 expression in the cerebral cortex and hippocampus of wild-type mice was effectively inhibited.

[0153] From the above results, it can be seen that Selexipag can inhibit the expression of COX-2 and IL-6, which are pro-inflammatory cytokines induced by LPS in vivo.

[0154]

[0155] Example 5. Inhibition of LPS-mediated IL-1β and TNF-α expression by Selexipag in wild-type mice.

[0156] To determine whether selexipag could inhibit the expression of LPS-induced proinflammatory cytokines IL-1β and TNF-α, 8-week-old wild-type mice were intraperitoneally administered selexipag (1 mg / kg) or vehicle daily for 7 days. On day 7, LPS (10 mg / kg) or PBS was administered intraperitoneally 30 minutes after the intraperitoneal administration of selexipag or vehicle. Then, 8 hours after LPS injection, immunofluorescence staining was performed on isolated brain sections from the mice with anti-IL-1β (Abcam, ab9722, 1:200, rabbit) or anti-TNF-α antibody (Novus, NBP-19532, 1:200, rabbit). The results are shown in Figures 7a and 7b and 8a and 8b, respectively.

[0157] Figures 7a and 7b are images and graphs analyzing the level of inhibition of IL-1β expression induced by LPS according to selexipag treatment using immunofluorescence staining.

[0158] Figures 8a and 8b are images and graphs analyzing the level of inhibition of TNF-α expression induced by LPS according to treatment with Selexipak using immunofluorescence staining.

[0159] As shown in Figures 7a and 7b, in the 1 mg / kg selexipag treatment group, it was confirmed that LPS-induced IL-1β expression in the cerebral cortex and hippocampus of wild-type mice was effectively suppressed.

[0160] As shown in Figures 8a and 8b, in the 1 mg / kg selexipag treatment group, it was confirmed that LPS-induced TNF-α expression in the hippocampal CA1 region of wild-type mice was effectively suppressed.

[0161] The above results indicate that selexipag can regulate LPS-induced IL-1β and TNF-α expression in vivo, suggesting that it can suppress the expression of these proinflammatory cytokines.

[0162]

[0163] Example 6. Reduction of LPS-induced NLRP3 / CDK6 expression levels by selexipag in wild-type mice.

[0164] We conducted an experiment to determine whether selexipag could control brain inflammation by promoting NLRP3 inflammasome formation and inhibiting CDK6 expression following LPS-induced proinflammatory cytokine production.

[0165] Specifically, 8-week-old wild-type mice were intraperitoneally administered selexipag (1 mg / kg) or vehicle daily for 7 days, and on day 7, LPS (10 mg / kg) or PBS was intraperitoneally administered 30 minutes after the intraperitoneal administration of selexipag or vehicle. Then, 8 hours after the LPS injection, brain tissue sections isolated from the mice were immunofluorescently stained with anti-NLRP3 antibody (Novus, NBP-12446, 1:500, rabbit) or anti-CDK6 antibody (Invitrogen, PA5-27978, 1:200, rabbit). The results are shown in Figures 9a and 9b and 10a and 10b.

[0166] Figures 9a and 9b are images and graphs analyzing the level of inhibition of NLRP3 expression induced by LPS according to selexipak treatment using immunofluorescence staining.

[0167] Figures 10a and 10b are images and graphs analyzing the level of inhibition of CDK6 expression induced by LPS according to selexipag treatment using immunofluorescence staining.

[0168] As shown in Figures 9a and 9b and Figures 10a and 10b, treatment with selexipag (1 mg / kg, ip) significantly reduced LPS-induced NLRP3 expression in the cerebral cortex of wild-type mice, as well as significantly inhibited LPS-stimulated CDK6 expression in the cerebral cortex and hippocampus regions.

[0169] This suggests that selexipag can also inhibit LPS-induced NLRP3 inflammasome formation and CDK6 expression in vivo.

[0170]

[0171] Example 7. Reduction of amyloid plaques by selexipag in amyloid-overexpressing dementia animals.

[0172] We conducted an experiment to analyze the efficacy of Selexipag confirmed in wild-type mice by reducing the level of amyloid beta expression, a representative indicator of Alzheimer's disease, using an Alzheimer's disease animal model.

[0173] Specifically, 3-4 month old Alzheimer's disease animal model mice (5xFAD) were intraperitoneally administered selexipag (1 mg / kg) or vehicle daily for 14 days, and selexipag was administered on the 14th day. After 2 hours, immunofluorescence staining was performed using anti-6E10 antibody (Biolegend, 803002, 1:500, mouse), and the results are shown in Figures 11a, 11b, and 11c.

[0174] Figures 11a, 11b and 11c are images and graphs analyzing the level of amyloid plaques (6E10) in amyloid-overexpressing animals treated with selexipag through immunofluorescence staining (CA1: Cornu Ammonis 1).

[0175] As shown in Figures 11a, 11b, and 11c, treatment with selexipag (1 mg / kg, ip) significantly reduced the number of amyloid plaques (6E10) in the cerebral cortex and subiculum of amyloid-overexpressing animals. This suggests that selexipag can inhibit the expression of 6E10-labeled amyloid plaques in vivo in an animal model of Alzheimer's disease.

[0176]

[0177] Example 8. Increased expression of amyloid beta-degrading enzyme IDE by selexipag in amyloid-overexpressing animals.

[0178] Next, experiments were conducted to elucidate the molecular mechanism by which Selexpax inhibits amyloid plaques in an amyloid-overexpressing animal model.

[0179] Specifically, selexipag (1 mg / kg) or vehicle was administered intraperitoneally daily for 14 days to 3-4 month-old amyloid-overexpressing animals (5xFAD). On the 14th day, 2 hours after selexipag administration, brain sections obtained from the mice were subjected to immunofluorescence staining using an IDE antibody (Abcam, ab32216, 1:200, rabbit). The results are shown in Figures 12a, 12b, and 12c.

[0180] Figures 12a, 12b and 12c are images and graphs analyzing the expression level of IDE in amyloid-overexpressing animals treated with selexipag through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0181] As shown in Figures 12a, 12b, and 12c, treatment with selexipag (1 mg / kg, ip) significantly increased the expression of IDE in the CA1 and DG regions of the hippocampus of amyloid-overexpressing animals.

[0182] This suggests that selexipag may reduce amyloid plaques by increasing the expression of IDE, an amyloid beta-degrading enzyme, in amyloid-overexpressing animals.

[0183]

[0184] Example 9. Inhibition of microglia and astrocyte activity by selexipag in amyloid-overexpressing animals.

[0185] Next, we conducted experiments to analyze the efficacy of selexipag on microgliosis and astrogliosis in amyloid-overexpressing animals.

[0186] Specifically, selexipag (1 mg / kg) or vehicle was administered intraperitoneally daily for 14 days to 3-4 month-old amyloid-overexpressing animals (5xFAD). On the 14th day, 2 hours after selexipag administration, brain sections obtained from the mice were subjected to immunofluorescence staining using Iba-1 (Wako, 019-19741, 1:500, rabbit) or GFAP antibody (Invitrogen, 13-0300, 1:500, rat). The results are shown in Figures 13a and 13b and Figures 14a and 14b, respectively.

[0187] Figures 13a and 13b are images and graphs analyzing the activity level of microglial cells in amyloid-overexpressing animals treated with selexipag through immunofluorescence staining (Iba-1 antibody) (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0188] Figures 14a and 14b are images and graphs analyzing the activity level of astrocytes in amyloid-overexpressing animals treated with selexipag through immunofluorescence staining (GFAP antibody) (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0189] As shown in Figures 13a and 13b, in the 1 mg / kg selexipag-treated group, the fluorescence intensity of Iba-1, an activation indicator of microglial cells, the percentage area of ​​Iba-1, and the number of Iba-1-positive cells were reduced in the CA1 and DG regions of the cerebral cortex and hippocampus.

[0190] In addition, as shown in Figures 14a and 14b, in the 1 mg / kg selexipag-treated group, the fluorescence intensity of GFAP, which is an activation indicator of astrocytes, the percentage area of ​​GFAP, and the number of GFAP-positive cells were significantly reduced in the CA1 and DG regions of the cerebral cortex and hippocampus.

[0191] From this, it was confirmed that Selexipag can regulate the activation of microglia and astrocytes in amyloid-overexpressing animals, and in particular, it can improve and alleviate microgliosis and astrogliosis by inhibiting the activation of microglia and astrocytes in amyloid-overexpressing animals.

[0192]

[0193] Example 10. Reduction of IL-1β and NLRP3 expression levels by selexipag in amyloid-overexpressing animals.

[0194] Next, we conducted experiments to analyze the efficacy of selexipag in reducing the expression of IL-1β, a proinflammatory cytokine known to be closely associated with neurodegenerative diseases, and the formation of NLRP3 inflammasomes that activate IL-1β in amyloid-overexpressing animals.

[0195] Specifically, selexipag (1 mg / kg) or vehicle was administered intraperitoneally daily for 14 days to 3-4 month-old amyloid-overexpressing animals (5xFAD), and on the 14th day, 2 hours after selexipag administration, brain sections obtained from the mice were subjected to immunofluorescence staining using IL-1β (Abcam, ab9722, 1:200, rabbit) and NLRP3 (Adipogen, AG-20B-0014-100c, 1:200, mouse) antibodies. The results are shown in Figs. 15a and 15b and Figs. 16a and 16b.

[0196] Figures 15a and 15b are images and graphs analyzing the expression level of IL-1β, a proinflammatory cytokine, in amyloid-overexpressing animals treated with selexipag through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0197] Figures 16a and 16b are images and graphs analyzing the expression level of NLRP3 inflammasome in amyloid-overexpressing animals treated with selexipag through immunofluorescence staining (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0198] As shown in Figures 15a and 15b, treatment with selexipag (1 mg / kg, ip) significantly reduced the expression of IL-1β, a proinflammatory cytokine, in the CA1 and DG regions of the cerebral cortex and hippocampus of amyloid-overexpressing animals.

[0199] As shown in Figures 16a and 16b, treatment with selexipag (1 mg / kg, ip) significantly reduced the expression of NLRP3 inflammasome in the cerebral cortex of amyloid-overexpressing animals.

[0200] This suggests that selexipag can inhibit the expression of IL-1β and NLRP3 inflammasome, which are proinflammatory cytokines, in vivo in amyloid-overexpressing animals.

[0201]

[0202] Example 11. Reduction of AT8 and AT180 expression levels, indicators of tau hyperphosphorylation, by selexipag in tau-overexpressing animals.

[0203] To verify the efficacy of selexipag in wild-type mice, we conducted an experiment to analyze the efficacy of selexipag on the expression levels of AT8 and AT180, which are indicators of tau hyperphosphorylation, in another Alzheimer's disease animal model, a tau hyperphosphorylation mouse model (PS19).

[0204] Specifically, 3-4 month old tau overexpressing animals (PS19) were intraperitoneally administered selexipag (1 mg / kg) or vehicle daily for 14 days, and on the 14th day, 2 hours after selexipag administration, brain sections obtained from the mice were immunofluorescently stained with anti-AT8 (Invitrogen, mn1020, 1:200, mouse) and anti-AT180 (Invitrogen, mn1040, 1:200, mouse) according to the method described in the experimental method above. The results are shown in Figures 17a and 17b and Figures 18a and 18b.

[0205] Figures 17a and 17b are images and graphs analyzing the expression level of AT8, an indicator of tau hyperphosphorylation, through immunofluorescence staining in tau overexpressing animals treated with selexipag (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0206] Figures 18a and 18b are images and graphs analyzing the expression level of AT180, an indicator of tau hyperphosphorylation, through immunofluorescence staining in tau overexpressing animals treated with selexipag (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0207] As shown in Figures 17a and 17b, treatment with selexipag (1 mg / kg, ip) significantly reduced the expression of AT8, an indicator of tau hyperphosphorylation, in the hippocampal CA1 and DG regions of tau overexpressing animals (PS19).

[0208] Additionally, as shown in Figures 18a and 18b, treatment with selexipag (1 mg / kg, ip) significantly reduced the expression of AT180, an indicator of tau hyperphosphorylation, in the CA1 and DG regions of the cerebral cortex and hippocampus of tau-overexpressing animals.

[0209] From this, it can be seen that Selexipag inhibits the expression of AT8 and AT180, which are indicators of tau hyperphosphorylation, in tau overexpressing animals.

[0210]

[0211] Example 12. Reduction of the expression levels of DYRK1A and p-CDK5, tau kinases, by selexipag in tau-overexpressing animals.

[0212] Next, we conducted an experiment to analyze the efficacy of selexipag on changes in the expression of tau kinases DYRK1A and p-CDK5 in tau-overexpressing animals.

[0213] Specifically, 3-4 month old tau overexpressing animals (PS19) were intraperitoneally administered selexipag (1 mg / kg) or vehicle daily for 14 days, and on the 14th day, 2 hours after selexipag administration, brain sections obtained from the mice were immunofluorescently stained with anti-DYRK1A (Abcam, ab180910, 1:200, rabbit) and anti-p-CDK5 (LSbio, LS-c354604-100, 1:200, rabbit) according to the method described in the experimental method above. The results are shown in Figs. 19a and 19b and Figs. 20a and 20b.

[0214] Figures 19a and 19b are images and graphs analyzing the expression level of DYRK1A, a tau kinase, through immunofluorescence staining in tau-overexpressing animals treated with selexipag (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0215] Figures 20a and 20b are images and graphs analyzing the expression level of p-CDK5, a tau kinase, through immunofluorescence staining in a tau overexpression animal model following selexipag treatment (CA1: Cornu Ammonis 1, DG: Dentate Gyrus).

[0216] As shown in Figures 19a and 19b, treatment with selexipag (1 mg / kg, ip) significantly reduced the expression of DYRK1A, a tau kinase, in the CA1 and DG regions of the cerebral cortex and hippocampus of tau-overexpressing animals.

[0217] Additionally, as shown in Figures 20a and 20b, treatment with selexipag (1 mg / kg, ip) significantly reduced the expression of p-CDK5, a tau kinase, in the CA1 region of the cerebral cortex and hippocampus of tau-overexpressing animals.

[0218] This suggests that selexipag can inhibit the expression of tau kinases DYRK1A and p-CDK5 in tau-overexpressing animals.

[0219]

[0220] Through the above results, Selexipag inhibits the expression and production of pro-inflammatory or inflammatory cytokines in brain tissue induced by LPS, inhibits the activity of microglia or astrocytes, and inhibits NLRP3 inflammasome activity and CDK6 expression. In addition, in amyloid-overexpressing animals (5xFAD) administered Selexipag, it suppresses amyloid plaques, increases the expression of amyloid beta-degrading enzyme IDE, inhibits the expression and production of pro-inflammatory cytokines, and inhibits the activity of microglia or astrocytes. In addition, it suppresses tau hyperphosphorylation and the expression of tau kinase in tau-overexpressing animals (PS19) administered Selexipag. Based on the above results, Selexipag may be useful as a new therapeutic agent for the prevention or treatment of degenerative brain diseases.

[0221]

[0222] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating a degenerative brain disease, comprising selexipag or a pharmaceutically acceptable salt thereof as an active ingredient.

2. A pharmaceutical composition for preventing or treating a degenerative brain disease, having at least one of the following activities in claim 1: (i) suppression of the levels of pro-inflammatory cytokines or inflammatory cytokines in brain tissue; (ii) inhibition of inflammasome formation or levels of cyclin-dependent kinases in brain tissue; (iii) Inhibition of the activity of microglia or astrocytes in brain tissue; (iv) reducing amyloid plaques or inhibiting amyloid beta protein accumulation in brain tissue; or (v) Inhibition of expression or activity of tau kinase in brain tissue, or inhibition of hyperphosphorylation of tau protein.

3. A pharmaceutical composition for preventing or treating a degenerative brain disease according to claim 1 or claim 2, wherein the degenerative brain disease is selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, senile dementia, frontotemporal dementia, multiple sclerosis, multiple myelopathy, epilepsy, and encephalophathy.

4. A pharmaceutical composition for preventing or treating cognitive impairment, learning disability or memory impairment, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

5. A composition for protecting nerve cells damaged by excessive inflammatory response in nerve cells, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

6. A reagent composition for inhibiting the activation of microglia or astrocytes, comprising Selexipak or a pharmaceutically acceptable salt thereof as an active ingredient.

7. A health functional food for the prevention or improvement of neuroinflammation or degenerative brain disease, containing Selexipak or a food-scientifically acceptable salt thereof as an active ingredient.

8. A health functional food for preventing or improving a degenerative brain disease according to claim 7, wherein the degenerative brain disease is selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, senile dementia, multiple sclerosis, multiple atrophy, epilepsy, encephalophathy, and frontotemporal dementia.

9. A health functional food for improving cognitive function, learning ability or memory, containing Selexipak or a food-scientifically acceptable salt thereof as an active ingredient.

10. A method for preventing or treating a degenerative brain disease, comprising administering selexipag or a pharmaceutically acceptable salt thereof to a subject.

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