Composition for preventing or treating neuroinflammatory diseases containing didanosine

Abstract

The present invention relates to a composition for preventing or treating a neuroinflammatory disease, and more specifically to a composition for preventing or treating a neuroinflammatory disease, which comprises didanosine or a pharmaceutically acceptable salt thereof.

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing or treating a neuroinflammatory disease, and more specifically to a composition for preventing or treating a neuroinflammatory disease, which comprises didanosine or a pharmaceutically acceptable salt thereof. [Background technology]

[0002] The central nervous system consists of neurons and glial cells. Glial cells account for approximately 90% of all brain cells and occupy approximately 50% of the brain's total volume. Glial cells are further classified into three types: astrocytes, microglia, and oligodendrocytes. Of these, microglia are a type of specialized macrophage that are widely distributed throughout the brain. Microglia not only act as phagocytes that engulf tissue debris and dead cells, but also play a role in the brain's biological defense activities.

[0003] Neuroinflammation, a type of immune response in the nervous system, is closely associated with many degenerative neurological diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis, and is currently considered a typical hallmark of degenerative neurological diseases. Neuroinflammatory responses involve the activation of innate immune cells (microglia), the release of inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines, and the infiltration of macrophages, which induce neuronal death. Inflammatory activation of microglia and astrocytes is considered an important mechanism in the pathological markers and progression of degenerative neurological diseases. Because strict regulation of microglial activity is essential for maintaining brain homeostasis and preventing infection and inflammatory diseases, it is necessary to identify substances that can modulate microglial activity and mitigate neuroinflammation.

[0004] Didanosine is a nucleoside reverse transcriptase inhibitor approved by the FDA for use in the treatment of HIV / AIDS. It inhibits HIV reverse transcriptase by competing with natural dATP and acts as a viral DNA chain terminator by inhibiting the 5' to 3' phosphodiester linkage in viral DNA lacking a 3'-OH group. Didanosine easily crosses the blood-brain barrier and can easily affect the brain. However, didanosine's ability to suppress neuroinflammation or its effects on the brain is unknown, and further research is needed. Summary of the Invention [Problem to be solved by the invention]

[0005] In order to solve the above problems, an object of the present invention is to provide a composition for preventing, ameliorating or treating a neuroinflammatory disease, which comprises didanosine or a pharmaceutically acceptable salt thereof.

[0006] The present invention also provides a method for preventing or treating a neuroinflammatory disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising didanosine or a pharmaceutically acceptable salt thereof.

[0007] The present invention also provides didanosine, or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a neuroinflammatory disease in a subject.

[0008] In one embodiment of the present invention, the composition is capable of promoting the degradation of amyloid beta.

[0009] In another embodiment of the present invention, the composition can suppress neuroinflammation and restore memory.

[0010] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] One embodiment of the present invention provides a pharmaceutical composition for preventing or treating a neuroinflammatory disease, comprising didanosine or a pharmaceutically acceptable salt thereof.

[0012] Another embodiment of the present invention provides a composition for promoting the degradation of amyloid beta in microglia, comprising didanosine or a pharmaceutically acceptable salt thereof.

[0013] Yet another embodiment of the present invention provides an anti-inflammatory composition for nervous system inflammation, comprising didanosine or a pharmaceutically acceptable salt thereof.

[0014] Yet another embodiment of the present invention provides a composition for improving memory, comprising didanosine or a pharmaceutically acceptable salt thereof.

[0015] Yet another embodiment of the present invention provides a method for preventing or treating a neuroinflammatory disease, comprising administering to an individual a pharmaceutical composition comprising didanosine or a pharmaceutically acceptable salt thereof.

[0016] Yet another embodiment of the present invention provides a use of a pharmaceutical composition comprising didanosine or a pharmaceutically acceptable salt thereof for the prevention or treatment of a neuroinflammatory disease.

[0017] Yet another embodiment of the present invention provides a use of didanosine or a pharmaceutically acceptable salt thereof for producing a pharmaceutical composition for the prevention or treatment of a neuroinflammatory disease.

[0018] Yet another embodiment of the present invention provides a pharmaceutical composition comprising didanosine or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of a neuroinflammatory disease.

[0019] The present invention will be described in more detail below.

[0020] One embodiment of the present invention provides a pharmaceutical composition for preventing or treating a neuroinflammatory disease, comprising didanosine or a pharmaceutically acceptable salt thereof.

[0021] The "didanosine" of the present invention has the chemical formula C 10 H 12 N4, which is named as 9-[(2R,5S)-5-(hydroxymethyl)oxolan-2-yl]-1H-purin-6-one according to the IUPAC nomenclature system, and has the structure of the following chemical formula 1. The active ingredient of the pharmaceutical composition for preventing, ameliorating, or treating a neuroinflammatory disease according to one embodiment of the present invention may be one or more selected from the group consisting of didanosine, its derivatives, metabolites, and pharmaceutically acceptable salts.

[0022] [Chemical formula 1] [ka]

[0023] The didanosine represented by Chemical Formula 1 according to the present invention can be used in the form of a salt. Examples of the salt include acid addition salts formed with various pharmaceutically or food-grade acceptable organic or inorganic acids. Acid addition salts can be 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. Such non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfate, bisulfate, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propionic acid, oxalic acid, malonic acid, succinic acid, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate. The compound can be prepared using, for example, benzoic acid, chlorobenzoic acid, methylbenzoic acid, dinitrobenzoic acid, hydroxybenzoate, methoxybenzoic acid, phthalic acid, terephthalate, benzenesulfonic acid, toluenesulfonic acid, chlorobenzenesulfonic acid, xylenesulfonic acid, phenylacetic acid, phenylpropionic acid, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, trifluoroacetic acid, and the like.

[0024] The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of Formula 1 in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, the mixture can be dried after evaporating the solvent and excess acid, or the precipitated salt can be filtered by suction.

[0025] In addition, didanosine represented by Chemical Formula 1 according to the present invention can be prepared into a pharmaceutically or food-grade acceptable metal salt using a base. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. From an agrochemical perspective, it is preferable to prepare lithium, sodium, potassium, or calcium salts as the metal salt. The corresponding silver salt can also be obtained by reacting the alkali metal or alkaline earth metal salt with an appropriate silver salt (e.g., silver nitrate).

[0026] The term "neuroinflammatory disease" as used herein may include any disease caused by inflammation of the nervous system, for example, multiple sclerosis, neuroblastoma, stroke, dementia, Alzheimer's disease, cognitive impairment, memory impairment, attention disorder, Parkinson's disease, Ruggerick's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, neuropathic pain, and amyotrophic lateral sclerosis, but is not limited thereto.

[0027] Alzheimer's disease (AD) may include, for example, sporadic Alzheimer's disease (SAD) or familial Alzheimer's disease (FAD), etc. Familial Alzheimer's disease is known to be primarily caused by the PSEN gene, which expresses the transmembrane protein presenilin and the catalytic site of gamma-secretase.

[0028] According to one embodiment of the present invention, the disease may be hereditary dementia or familial Alzheimer's disease (FAD). More specifically, the disease may be Alzheimer's disease or Alzheimer's-related dementia with one or more genetic mutations selected from the group consisting of amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2). The presenilin 2 genetic mutation may include one or more selected from the group consisting of A85V, N141Y, M174I, G212V, A237V, M239I, and M239V in addition to PSEN2 N141I.

[0029] The disease is cognitive impairment, memory loss, Alzheimer's disease or related symptoms caused by neuroinflammation, and may occur or worsen with aging, genetic mutations, head trauma, depression, or complications from high blood pressure.

[0030] The neuroinflammation may be induced by one or more of the following: genetic mutation, infection, brain trauma, and alcoholism. The genetic mutation may be one or more of the following: amyloid precursor protein (APP), presenilin 1 (Psen1), and presenilin 2 (Psen2).

[0031] The term "neuroinflammatory-induced Alzheimer's disease" as used herein refers to dementia induced by artificially generating a neuroinflammatory response, and unlike aging-induced dementia models, dementia can be manifested within a short period of time. The term "neuroinflammatory-induced Alzheimer's disease" as used herein refers to dementia induced by artificially generating a neuroinflammatory response.

[0032] More specifically, the subject and / or disease of the present invention may have one or more gene mutation characteristics selected from the group consisting of Amyloid Precursor Protein (APP), Presenilin 1 (Psen1), and Psenilin 2, compared to a normal subject or normal disease.

[0033] The composition according to one embodiment of the present invention may suppress the expression of pro-inflammatory cytokines in the central nervous system. The pro-inflammatory cytokines may be neuroinflammatory cytokines.

[0034] The term "inflammatory cytokine" as used herein refers to a cytokine involved in inflammatory responses due to bacterial or viral infection, tissue damage, etc. The composition of the present invention containing didanosine or a salt thereof inhibits the expression of the inflammatory cytokine. The inflammatory cytokine inhibited by the present invention may be, but is not limited to, IL-6. Without being limited to a particular theory, inflammatory cytokines may increase in neuroinflammatory diseases, and inhibiting inflammatory cytokines such as IL-6 can treat and improve neuroinflammatory diseases such as Alzheimer's. Therefore, didanosine or a salt thereof according to the present invention inhibits the expression of inflammatory cytokines by repairing damage to transcriptional activators, and can be useful in the treatment of neuroinflammatory diseases including Alzheimer's.

[0035] In this embodiment, a composition containing didanosine or a salt thereof as an active ingredient suppresses the expression of neuroinflammatory cytokines that induce neuroinflammation in microglia, and its anti-inflammatory effect in animal models has been confirmed to be effective in preventing, ameliorating, or treating Alzheimer's disease.

[0036] According to one embodiment of the present invention, didanosine treatment significantly reduced IL-6 secretion in primary microglia of an animal model of Alzheimer's disease (see Example 6), with minimal cytotoxicity (see Example 5).

[0037] In this embodiment, it was confirmed that treatment of microglia derived from a mouse model of neuroinflammatory disease, e.g., neuroinflammatory Alzheimer's disease, with didanosine inhibits the expression of inflammatory cytokines, thereby exhibiting anti-inflammatory and memory-restoring effects. Therefore, a composition according to one embodiment of the present invention may inhibit the expression of neuroinflammatory cytokines in microglia. The microglia may have one or more gene mutations selected from the group consisting of amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2). Furthermore, a composition according to one embodiment of the present invention may be a composition for improving memory.

[0038] Specifically, the present inventors conducted research into substances that can treat neuroinflammatory diseases by regulating the expression of inflammatory cytokines that cause inflammatory responses in microglia. As a result, they confirmed that when didanosine was administered to microglia in a PSEN2 N141I KI / + mouse model with Alzheimer's disease, it exhibited anti-inflammatory effects by suppressing the expression of the inflammatory cytokine IL-6 and promoted amyloid beta degradation, thereby completing the present invention. They also confirmed that didanosine improved impaired cognitive function in 5XFAD mice, another Alzheimer's disease model.

[0039] As used herein, the term "PSEN2 gene" refers to a gene encoding a PSEN2 polypeptide. The PSEN2 gene is located in the NCBI Reference Sequence at NC_000001.11 (226870594..226903829) for the human PSEN2 gene and at NC_000067.7 for the mouse PSEN2 gene, and includes these sequences as well as known orthologs. The term "PSEN2 polypeptide" refers to the NCBI Reference Sequence at NP_000438.2 for the human PSEN2 protein and at NP_001122077 for the mouse PSEN2 protein, and includes these sequences as well as known orthologs.

[0040] Known mutations in the PSEN2 gene associated with neuroinflammation and / or Alzheimer's disease according to the present invention include A85V, N141I, N141Y, M174I, G212V, A237V, M239I, and M239V, and the present invention may include one or more of these mutations (Jiang et al., "A Review of the Familial Alzheimer's Disease Locus PRESENILIN 2 and Its Relationship to PRESENILIN 1." Journal of Alzheimer's Disease 66 (2018) 1223-1339).

[0041] According to this embodiment, it was confirmed that when didanosine was treated in a Psen2 N141I knock-in (KI) animal model, which is a familial Alzheimer's disease mutation in which the 141st amino acid N (arginine) of the presenilin2 gene is replaced with I (isoleucine), the expression of the inflammatory cytokine IL-6 (Interleuckine-6) was suppressed, thereby exhibiting anti-inflammatory effects and memory recovery effects.

[0042] Thus, yet another embodiment of the present invention provides an anti-inflammatory composition for neuroinflammation, comprising didanosine or a pharmaceutically acceptable salt thereof.

[0043] Yet another embodiment of the present invention provides a composition for improving memory, comprising didanosine or a pharmaceutically acceptable salt thereof.

[0044] From the results of the above embodiments, it was confirmed that when the didanosine of the present invention is treated in an animal model or its microglia, neuroinflammatory responses are alleviated. Since didanosine is known to pass through the blood-brain barrier, the composition of the present invention can be usefully used as a pharmaceutical composition or a functional health food composition for improving or treating neuroinflammatory diseases including Alzheimer's disease.

[0045] Yet another embodiment of the present invention provides a composition for promoting amyloid beta degradation in microglia, comprising didanosine or a pharmaceutically acceptable salt thereof. Alternatively, yet another embodiment of the present invention relates to the use of a pharmaceutical composition comprising didanosine or a pharmaceutically acceptable salt thereof for promoting amyloid beta degradation in microglia. Alternatively, yet another embodiment of the present invention provides a pharmaceutical composition comprising didanosine or a pharmaceutically acceptable salt thereof for use in promoting amyloid beta degradation in microglia.

[0046] The microglia may have a reduced ability to degrade amyloid beta due to neuroinflammation.

[0047] The microglia may have one or more gene mutations selected from the group consisting of amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2). For example, the microglia may be microglia with a presenilin 2 gene mutation, specifically, Psen2 N141I KI / + microglia.

[0048] In this embodiment, treatment of microglial cells with didanosine can restore their reduced amyloid beta decomposition ability, and therefore a composition according to one example of the present invention can restore the amyloid beta decomposition ability of microglial cells.

[0049] Another embodiment of the present invention provides a food composition for preventing or ameliorating a neuroinflammatory disease, comprising didanosine or a nutrient-acceptable salt thereof. The food may be a functional health food. The didanosine and the neuroinflammatory disease are as described above.

[0050] When the composition of the present invention is in the form of a functional health food composition, it can be manufactured into a food for specified health uses, a food with high medical and therapeutic effects that is processed to efficiently exhibit bioregulatory functions in addition to providing nutrients, and the food can be mixed as a functional food, health food, or health supplement depending on the case, and can be manufactured into various forms such as tablets, capsules, powder, granules, liquid, pills, etc. to obtain useful effects.

[0051] The health functional food of the present invention may contain additional ingredients commonly used in food compositions to improve aroma, taste, visual appearance, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may also be included. Amino acids such as lysine, tryptophan, cysteine, and valine may also be included. In addition, food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium D-bitartrate, etc.), strengtheners, emulsifiers, thickeners (thickeners), coating agents, gum bases, foam inhibitors, solvents, and improvers can be added. These additives are selected based on the type of food and can be used in appropriate amounts.

[0052] When the health functional food of the present invention is used as a food additive, it can be added as it is or used together with other foods or food ingredients, and can be used appropriately in a conventional manner.

[0053] In the health functional food of the present invention, the content of didanosine is not particularly limited and can be varied depending on the condition of the subject, the specific type and progression of the disease, etc. If necessary, didanosine can be included in the total content of the food.

[0054] Yet another embodiment of the present invention provides a method for treating a neuroinflammatory disease, comprising administering the pharmaceutical composition to an individual. The pharmaceutical composition, the neuroinflammatory disease, etc. are as described above.

[0055] In the present invention, an "individual" may be a mammal, including a rat, livestock, mouse, or human, and specifically may be a companion dog, racehorse, or human in need of treatment for a neuroinflammatory disease, such as Alzheimer's disease, preferably a human.

[0056] The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the condition and weight of the subject, the degree of disease, the drug form, the route and time of administration, but can be appropriately selected by those skilled in the art.

[0057] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or multiple times. It is important to administer an amount that can achieve maximum efficacy with minimal side effects, taking all of the above factors into consideration, and this can be determined by one skilled in the art.

[0058] Specifically, the effective amount of the pharmaceutical composition of the present invention varies depending on the subject's age, sex, condition, weight, absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, type of disease, and concomitant drugs, and is generally 0.001 to 4 mg per kg of body weight administered daily or every other day, or in 1 to 3 divided doses per day. However, since the dosage may increase or decrease depending on the route of administration, sex, weight, age, etc., the above dosage does not in any way limit the scope of the present invention.

[0059] For example, a composition according to one embodiment of the present invention can be administered at less than 1, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less of the effective dose of didanosine administered in an HIV treatment. In this case, even if the lower limit of the dosage is not specified, a person skilled in the art can clearly practice the present invention for the prevention or treatment of neuroinflammatory diseases. For example, the lower limit of the dosage may be, but is not limited to, 0.0001 times or more, 0.0005 times or more, 0.001 times or more, 0.005 times or more, 0.01 times or more, 0.05 times or more, or 0.1 times or more of the effective dose of didanosine administered as an HIV therapeutic agent.

[0060] For example, compositions according to one embodiment of the present invention may be administered in the form of a 0.001 to 4 mg / kg, 0.001 to 3 mg / kg, 0.001 to 2.5 mg / kg, 0.001 to 2 mg / kg, 0.001 to 1.5 mg / kg, 0.001 to 1 mg / kg, 0.001 to 0.9 mg / kg, 0.001 to 0.8 mg / kg, 0.001 to 0.7 mg / kg, 0.001 to 0.6 mg / kg, 0.001 to 0.5 mg / kg, or 0.001 to 0.45 mg / kg, 0.001~0.4mg / kg, 0.005~4mg / kg, 0.005~3mg / kg, 0.005~2.5mg / kg, 0.005~2mg / kg, 0.005~1.5mg / kg, 0.005~1mg / kg, 0.005~0.9mg / kg, 0.005~0.8mg / kg, 0.005~0.7mg / kg, 0.005~0.6mg / kg, 0.005~0.5mg / kg, 0.005~0.45mg / kg, 0.005~0.4mg / kg, 0.01~4mg / kg, 0.01~3mg / kg, 0.01~2.5mg / kg, 0.01~2mg / kg, 0.01~1.5mg / kg, 0.01~1mg / kg, 0.01~0.9mg / kg, 0.01~0.8mg / kg, 0.01~0.7mg / kg, 0.01~0.6mg / kg, 0.01~0.5mg / kg, 0.01~0.45mg / kg, 0.01~0.4mg It can be administered at a daily dosage of 0.1-4 mg / kg, 0.1-3 mg / kg, 0.1-2.5 mg / kg, 0.1-2 mg / kg, 0.1-1.5 mg / kg, 0.1-1 mg / kg, 0.1-0.9 mg / kg, 0.1-0.8 mg / kg, 0.1-0.7 mg / kg, 0.1-0.6 mg / kg, 0.1-0.5 mg / kg, 0.1-0.45 mg / kg, or 0.1-0.4 mg / kg.

[0061] The term "prevention" as used in the present invention means any action of suppressing or delaying the onset of a neuroinflammatory disease by administering the composition of the present invention.

[0062] The term "amelioration" as used herein means any action that reduces the severity of symptoms of a neuroinflammatory disease by administering the composition of the present invention.

[0063] The term "treatment" as used herein refers to any action in which the symptoms of a neuroinflammatory disease are improved or favorably altered by administering a pharmaceutical composition according to the present invention. Specifically, the term "treatment" as used herein includes the reduction or alleviation of at least one symptom associated with or caused by the condition, disorder, or disease being treated. Treated subjects may exhibit partial or total alleviation of symptoms (e.g., Alzheimer's disease or related conditions), or symptoms may remain static after treatment with the methods of the present invention. The term "treatment" is intended to include prophylaxis, therapy, and cure.

[0064] When the composition of the present invention is in the form of a pharmaceutical composition, it may contain a pharmaceutically effective amount of didanosine alone or one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, but are not limited to, commonly used pharmaceutical carriers such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the composition may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0065] Yet another embodiment of the present invention relates to the use of the pharmaceutical composition for the prevention or treatment of a neuroinflammatory disease. The pharmaceutical composition, the neuroinflammatory disease, etc. are as described above.

[0066] Yet another embodiment of the present invention relates to use of the pharmaceutical composition for producing a pharmaceutical composition for preventing or treating a neuroinflammatory disease, wherein the pharmaceutical composition, the neuroinflammatory disease, etc. are as described above.

[0067] Yet another embodiment of the present invention relates to the pharmaceutical composition for use in the prevention or treatment of a neuroinflammatory disease. The pharmaceutical composition, the neuroinflammatory disease, etc. are as described above. [Effects of the Invention]

[0068] The composition according to one embodiment of the present invention can suppress the expression of neuroinflammatory cytokines, is effective in treating neuroinflammatory diseases, and has been shown to improve cognitive function in mice with Alzheimer's disease. It is therefore expected to be useful in the development of pharmaceutical and quasi-drug materials and in related industries.

[0069] A composition according to one embodiment of the present invention has been shown to suppress the expression of neuroinflammatory cytokines, promote the degradation of amyloid beta, and improve cognitive function in an animal model of Alzheimer's disease, and has been shown to be effective against neuroinflammatory diseases. It is expected that the composition will be useful in the development of pharmaceutical and quasi-drug materials and in related industries.

[0070] However, it should be understood that the effects of the present invention are not limited to the above effects, but include all effects that can be inferred from the configuration of the invention described in the detailed description of the present invention or the claims. [Brief explanation of the drawings]

[0071] [Figure 1] Regarding the construction of a Psen2 N141I mutant Alzheimer's disease mouse model according to one embodiment of the present invention, Figure 1a shows a schematic diagram for targeted insertion of N141I, and Figure 1b shows the Sanger sequencing chromatograms of normal (wild-type), KI / +, and KI / KI mice. [Figure 2]According to one embodiment of the present invention, we confirmed that Psen2 N141I mutant Alzheimer's disease mouse models exhibit an exaggerated inflammatory response compared to normal (wild-type) mice. Figure 2a shows the blood levels of IL-6 in animals in which neuroinflammation was induced by intraperitoneal injection of various concentrations of lipopolysaccharide (LPS). Following intraperitoneal injection of various concentrations of LPS, IL-6 was overexpressed in Psen2 mutant Alzheimer's disease mice at all LPS concentrations, with the difference in expression between normal mice and Alzheimer's disease mice becoming increasingly pronounced at lower concentrations. Figure 2b shows the production of TNF-α following intraperitoneal injection of LPS. At all LPS concentrations, TNF-α levels were similar in normal mice and Psen2 N141I mutant Alzheimer's disease mice. Figure 2c shows the changes in the blood levels of the inflammatory cytokines IL-6, CXCL1, CCL2, and CCL5. We show that when a low concentration of LPS (0.35 μg / kg) was injected, which does not induce an inflammatory response in wild-type mice, a significant increase in inflammatory cytokines was observed only in Psen2 N141I mutant mice. [Figure 3] Figure 3a shows Iba-1 (microglia-labeling antibody) staining images in the hippocampus of normal mice and Psen2 N141I mutant Alzheimer's disease mice. Figure 3b shows 3D filament images of Iba-1 signals captured using IMARIS software. Figure 3c shows dendrite length and branch number data obtained by FilamentTracker analysis in IMARIS software. This indicates that Psen2 N141I mutant Alzheimer's disease mice produce inflammatory cytokines and mount an excessive immune response even in response to low concentrations of inflammatory stimuli. [Figure 4]Figure 4a shows the results of confirming that a Psen2 N141I mutant Alzheimer's disease mouse model exhibits impaired memory following intraperitoneal injection of low concentrations of LPS. Figure 4a shows the Y-maze analysis confirming that Psen2 N141I mutant mice with induced neuroinflammation exhibit impaired memory. Figure 4b shows the Y-maze analysis demonstrating no difference in mouse mobility. Figure 4c is a schematic diagram of the T-maze analysis method. Figure 4d shows the T-maze analysis confirming that Psen2 N141I mutant mice with induced neuroinflammation exhibit a significantly reduced success rate and impaired memory. [Figure 5] This shows the results of observing the cell death rate after treating microglial cells derived from wild-type mice with didanosine. [Figure 6] These are the results of measuring the amount of IL-6 secreted, an inflammatory factor, in microglial cells derived from wild-type (WT) and Psen2 N141I KI / + mice (KI / +), an animal model of Alzheimer's disease, after treatment with LPS and didanosine. [Figure 7] This figure confirms that didanosine restores the reduced amyloid beta-degrading ability of microglia derived from wild-type (WT) and Psen2 N141I KI / + mice (KI / +), an animal model of Alzheimer's disease. [Figure 8] This figure confirms that the amount of IL-6 hypersecreted by neuroinflammation is significantly reduced in wild-type (WT) and Psen2 N141I KI / + mice (KI / +), an animal model of Alzheimer's disease. [Figure 9a] This figure confirms that didanosine administration does not affect motility. [Figure 9b] FIG. 1 is a graph confirming that didanosine has the effect of recovering memory loss. [Figure 10] FIG. 10 is a graph confirming that didanosine has the effect of reducing the increased amount of IL-6 secretion from microglial cells derived from Psen2 N141I KI / + mice in which neuroinflammation was induced by LPS. [Figure 11a]FIG. 10 confirms that didanosine administration does not affect motility in wild-type mice and in an animal model of 5XFAD disease. [Figure 11b] This figure confirms that didanosine has the effect of reversing memory loss in an animal model of 5XFAD disease. [Figure 12] This figure confirms that didanosine has the effect of reversing brain inflammation in an animal model of 5XFAD disease. DETAILED DESCRIPTION OF THE INVENTION

[0072] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided merely to facilitate understanding of the present invention, and the content of the present invention is not limited to the following examples. [Example]

[0073] Example 1: Preparation of an animal model of disease

[0074] All procedures for the care and use of animals were approved by the Institutional Animal Care and Use Committee of DGIST. Animals were maintained in a pathogen-free environment under a 12-hour light / 12-hour dark cycle at the DGIST animal facility.

[0075] Heterozygous Psen2 to more accurately recapitulate human neuroinflammatory diseases, such as Alzheimer's disease, and maintain endogenous expression levels N141I / + (KI / +) mice were used. N141I / + Mice were generated using homologous recombination.

[0076] Specifically, we constructed a Psen2 N141I knock-in (KI) animal model in which the 141st amino acid in the presenilin2 gene, N (arginine) to I (isoleucine), is a familial Alzheimer's disease mutation. The targeting vector mediated the I141 mutation in exon 4 and the Neo r -loxp sequence. The homology site of the targeting vector was inserted into the wild-type (WT) allele of Psen2. N141I / N141I ;loxp-Neo r -loxp mice were crossed with Cre mice using the Cre-loxp system to generate the Psen2 N141I mutation knock-in mouse model.

[0077] In this example, "Psen2 N141I" refers to a substitution of the normal Psen2 gene in an animal model to express the same mutation as a dementia mutation reported in humans, more specifically, a substitution of amino acid 141 from N to I in the mouse Presenilin2 gene. In the present invention, the polynucleotide of SEQ ID NO: 1 is used as the Psen2 N141I gene, and the wild-type Psen2 gene is represented by SEQ ID NO: 2.

[0078] As shown in Figure 1a, the Psen2 N141I allele (Psen2 N141I / + and Psen2 N141I / N141I As shown in Figure 1b, the substitution of AAC and ATC in the asparagine (N) consisting of the AAC sequence was confirmed by genome sequence analysis of DNA extracted from the tails of the KI / + model, which has all asparagine (N) and isoleucine (I), and the KI / KI model, which has all I141 substituted with ATC.

[0079] Example 2: Induction of inflammation in animal models of disease by LPS

[0080] This example was carried out to confirm that the Psen2 N141I mutant Alzheimer's disease mouse model exhibits an exaggerated inflammatory response compared to normal (wild-type) mice.

[0081] 2-1: Analysis of inflammation-inducing LPS concentration

[0082] Psen2 N141I / + To confirm that the immune response of mouse-derived microglia worsens, animals tend to exhibit inflammation and cognitive decline. We performed immunizations of wild-type and Psen2 mice at various LPS concentrations. N141I / + The immune responses between the mice were compared.

[0083] The immune response in mice tends to peak within a few hours of the onset of activity. N141I / + Mice were intraperitoneally (ip) injected with LPS at 18:00 (Zeitgeber time 11:00, lights on at 07:00, lights off at 19:00). 20 hours later, the inflammatory response was monitored at 14:00 the following day. LPS was derived from Escherichia coli 0111:B4 strain and acts as a ligand for Toll-like receptor 4 to induce cellular immune responses. 100 μL of LPS diluted to the appropriate concentration in phosphate-buffered saline (PBS) was injected intraperitoneally into mice.

[0084] Specifically, neuroinflammation was induced in the Psen2 N141I mutant Alzheimer's disease mouse model obtained in Example 1 above by intraperitoneal injection of LPS at concentrations of 1.4, 3.6, 4.0, 25, and 5,000 μg / kg.

[0085] As control experiments, wild-type mice were used instead of the Alzheimer's disease mouse model, and neuroinflammatory induction tests were performed in the same manner. Additionally, wild-type mice without LPS injection and a Psen2 N141I mutant Alzheimer's disease mouse model were prepared as controls. Group 1 consisted of wild-type mice without LPS injection (WT(LPS(-))), Group 2 consisted of wild-type mice with various LPS injections (WT(LPS(+))), Group 3 consisted of an Alzheimer's disease mouse model without LPS injection (KI / +(LPS(-))), and Group 4 consisted of an Alzheimer's disease mouse model with various LPS injections (KI / +(LPS(+))). Five to eight mice were used per group.

[0086] To investigate neuroinflammation caused by injection of various concentrations of LPS, approximately 20 hours after LPS injection, blood was extracted from the buccal vein of wild-type mice (Group 2) and Alzheimer's disease mouse models (Group 4). The blood was centrifuged and the resulting serum was assayed for the amount of each protein by ELISA using IL-6, TNFα, CCL2, CXCL1, and CCL5 ELISA kits (R&D Systems) according to the manufacturer's instructions. Serum was also extracted from wild-type mice (Group 1) that did not receive LPS injection and the Psen2 N141I mutant Alzheimer's disease mouse model (Group 3), and the amount of each protein was assayed by ELISA according to the kit manufacturer's instructions.

[0087] The IL-6 and TNF-α concentrations analyzed in the serum of the mice in Groups 1 to 4 are shown in Table 1 below.

[0088] [Table 1]

[0089] Table 1 shows the mean (mean ± SEM) blood levels of IL-6 and TNF-α in each group. In the LPS-injected wild-type mice (Group 2) and the Alzheimer's disease mouse model (Group 4), intraperitoneal injection of various concentrations of LPS resulted in overexpression of IL-6, with the difference in expression between the wild-type mice and the Alzheimer's disease mouse model becoming increasingly pronounced at lower concentrations. LPS-induced TNF-α showed similar blood levels in wild-type mice and the Alzheimer's disease mouse model. In the wild-type mice (Group 1) not injected with LPS and the Psen2 N141I mutant Alzheimer's disease mouse model (Group 3), both IL-6 and TNF-α were secreted at very low levels and were similar.

[0090] As a result, as shown in Figure 2a, KI / + Alzheimer's disease mice had higher circulating levels of IL-6 than wild-type mice at all LPS doses tested, with the difference between the genotypes being more pronounced at lower doses.As shown in Figure 2b, blood levels of TNF-α were similar between genotypes at all doses.

[0091] 2-2: Inflammation induction in diseased animals by treatment with low concentrations of LPS

[0092] Animals in groups 1 to 4 were prepared in the same manner as in Example 2-1, except that instead of treatment with various concentrations of LPS, groups 2 and 4 were treated with a low concentration of LPS (0.35 μg / kg) that does not induce an inflammatory response in wild-type mice.

[0093] The same serum was extracted from the mouse model, and the amount of each protein was measured using ELISA according to the kit manufacturer's guidelines. The blood concentrations of inflammatory cytokines IL-6, CXCL1, CCL2, and CCL5 analyzed in the serum of mice from Groups 1 to 4 are shown in Table 2 below.

[0094] [Table 2]

[0095] Table 2 above shows the changes (mean ± SEM) in the blood levels of the inflammatory cytokines IL-6, CXCL1, CCL2, and CCL5. When a low concentration of LPS (0.35 μg / kg) that does not induce an inflammatory response in wild-type mice was injected in Group 2, a significant increase in inflammatory cytokines, unlike TNF-α, was observed only in Psen2 Alzheimer's disease mice in Group 4.

[0096] As shown in Figure 2c, the lowest LPS dose (0.35 μg / kg body weight) did not induce inflammation in wild-type mice, but significantly increased the blood levels of inflammatory cytokines (IL-6, CXCL1, CCL2, and CCL5) in Psen2 Alzheimer's disease mice.

[0097] Example 3: Confirmation of inflammation exacerbation in a neuroinflammation animal model using microglial morphology analysis

[0098] Microglial morphology is closely related to its function, and microglial activation is characterized by changes in shape. To determine whether increased production of inflammatory cytokines is reflected in microglial morphology, we investigated the shape of microglia in the hippocampus of wild-type and Psen2 mutant Alzheimer's disease mice by immunohistochemical analysis using an antibody against the microglia-specific marker Iba-1.

[0099] Immunohistochemical analysis and confocal analysis were performed on the mice of Groups 1 to 4 prepared in Example 2-2. The LPS concentration injected into Groups 2 and 4 was a low concentration (0.35 μg / kg) that does not induce an inflammatory response in wild-type mice.

[0100] For immunohistochemistry, mice were anesthetized with a mixture of Zoletil (Virbac, 50 mg / kg) and Rompun (Bayer, 10 mg / kg). Then, mice were perfused with PBS and fixed with 4% paraformaldehyde (PFA). Brains were harvested and fixed in 4% PFA for 16 hours. Then, they were transferred to 30% sucrose until they reached the bottom of the tube and stored in frozen solution. Brain samples were sliced coronally at 50 μm thickness and subjected to antigen retrieval at 95°C. Then, they were treated with IBA-1 antibody (1:250) in PBS containing 3% bovine serum albumin for 24 hours at 4°C, followed by secondary antibody treatment for 2 hours at room temperature. Images were captured using LSM7 and LSM700 confocal laser scanning microscopes.

[0101] As shown in Figure 3a, hippocampal microglia from wild-type mice in group 1 had small cell bodies with highly differentiated processes, and consistent with the lack of cytokine release induction, low doses of LPS did not alter the morphology of hippocampal microglia from wild-type mice in group 2. However, even without LPS injection, hippocampal microglia from Psen2 mutant Alzheimer's disease mice in group 3 showed enlarged somata with a shorter, rounder shape, and these morphological characteristics were further enhanced by LPS injection in group 4.

[0102] As shown in Figure 3b, the histological confocal images of hippocampal microglia from mice in Groups 1 to 4 were reconstructed into 3D morphologies, and various morphological parameters were measured using IMARIS software. Specifically, images were obtained by aligning the entire Z axis of randomly selected fields using a confocal microscope, and then 3D imaging was performed using IMARIS software (v9.2.1, bitplane AG).

[0103] [Table 3]

[0104] Table 3 above summarizes the quantitative values (mean ± SEM) of dendrite length and dendrite branch number.

[0105] As a result, as shown in Figure 3c, the total dendrite length and dendritic terminal point of each microglia were confirmed to be further reduced in Alzheimer's disease mice in groups 3 and 4 and by LPS injection compared to wild-type mice in groups 1 and 2. These results confirmed that morphologically based microglial activation was clearly demonstrated in Psen2 mutant Alzheimer's disease mice and was further induced by mild LPS injection.

[0106] Example 4: Confirmation of memory impairment in an animal model of neuroinflammation

[0107] 4-1.Y-maze analysis

[0108] To examine the spatial learning and memory abilities of the mice in Groups 1 to 4 prepared in Example 2-2, a Y-maze test was performed 20 hours after LPS injection. The LPS injected into Groups 2 and 4 was a low concentration (0.35 μg / kg) that does not induce an inflammatory response in wild-type mice.

[0109] Specifically, the Y-maze was used to assess spatial working memory. Mice were placed in the white plastic arms of a Y-maze and allowed to explore freely within the arms for 5 minutes. Experiments were recorded using EthoVision software (Noldus). The number of entries and triads was analyzed, and alternation was calculated by dividing the number of three consecutive entries by the number of possible triads × 100 (total arm entries minus 2).

[0110] [Table 4]

[0111] Table 4 shows the mean (mean ± SEM) of arm alternation rates and arm entries in the Y-maze for each group. As shown in Figures 4a and 4b, arm alternation in the Y-maze was proportional to the secretion of inflammatory cytokines, with no difference in memory ability between wild-type mice (Group 1, n=13) and wild-type mice (Group 2, n=13) injected with LPS. There was also no difference in memory ability between wild-type mice (Group 1, n=12) not injected with LPS and Psen2 mutant Alzheimer's disease mice (Group 3, n=12). However, there was a significant decrease in memory ability in Psen2 mutant Alzheimer's disease mice (Group 4, n=15) injected with LPS. The total number of arm entries was the same in all groups, confirming normal motor function.

[0112] 4-2.T-maze analysis

[0113] To further analyze the learning and memory abilities of the mice in Groups 1 to 4 prepared in Example 2-2, a T-maze test with food reward was performed 20 hours after LPS injection. The LPS concentration injected into Groups 2 and 4 was a low concentration (0.35 μg / kg) that does not induce an inflammatory response in wild-type mice.

[0114] Specifically, we assessed spatial learning and memory for reward using a T-maze. As shown in Figure 4c, the experiment was conducted in the white plastic arms of a T-maze. Mice were allowed to adapt to the maze and food reward for 5 minutes before the experiment. In subsequent trials, one arm of each arm was blocked, and the other arm was rewarded. Mice were required to reach the open arm to confirm the reward. In the next trial, the previously closed arm was opened, and the mice were allowed to depart again. They could then select the newly opened arm to confirm the reward. If the mice incorrectly selected the previously visited arm, they would not receive the reward. The number of trials in which the correct arm was visited across multiple trials was calculated as a percentage of the total trials.

[0115] [Table 5] Table 5 shows the mean (mean ± SEM) success rate for the T-maze performance for each group. As shown in Figure 4d, there was no difference in learning and memory ability between wild-type mice (Group 1, 11 mice) and LPS-injected mice (Group 2, 11 mice), in proportion to the secretion of inflammatory cytokines. There was also no difference in learning and memory ability between wild-type mice (Group 1, not injected with LPS) and Alzheimer's disease mice (Group 3, 10 mice), but there was a significant decrease in learning and memory ability in the LPS-injected Alzheimer's disease mice (Group 4, 10 mice). These results demonstrate that low doses of LPS induce hyperactivation of the immune response and memory loss through the overproduction of inflammatory cytokines, including IL-6, in Psen2 N141I KI / + Alzheimer's mice, whereas the same dose of LPS is harmless to wild-type mice.

[0116] Example 5. Confirmation of cytotoxicity by didanosine treatment

[0117] 5-1. Preparation of wild-type mouse microglia

[0118] To obtain primary cultured microglia from wild-type mice, brains were extracted from 1- to 3-day-old newborn wild-type mice, followed by dissociation and culture in Dulbecco's modified Eagle's medium (DMEM, Corning) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Hyclone) and 1% penicillin-streptomycin (Hyclone). Primary microglia were isolated after 12 days by in vitro tapping. The purity of primary microglia was estimated by immunostaining with anti-Iba-1 antibody, a microglial marker.

[0119] 5-2.Cytotoxicity confirmation

[0120] To confirm the cytotoxicity of didanosine, we treated wild-type (WT) mouse microglia with didanosine at concentrations of 1, 5, or 10 μM and measured the cell death rate. Didanosine (CCL-D1-000017-G06) was provided by Korea Chemical Bank and used in the experiments.

[0121] Specifically, 5×10 microglial cells prepared in Example 5-1 were 4 Cells were seeded into 96-well plates at a density of 0 μM. The next day, the seeded cells were treated with didanosine at concentrations of 0, 1, 5, or 10 μM for 12 hours. Cell death was then measured by cavity staining with Hoechst 33342 (Invitrogen, H3570) and propidium iodide (PI; Sigma-Aldrich, P4170). Images of the stained cells were captured using a fluorescence microscope (Axiovert 40 CFL; Carl Zeiss), and the number of Hoechst- and PI-positive cells was calculated using NIH ImageJ software. The percentage of cell death was calculated as (number of PI-positive [dead] cells / number of Hoechst-positive [total] cells) × 100.

[0122] As shown in FIG. 5 and Table 6, it was confirmed that didanosine did not exhibit toxicity to cells.

[0123] [Table 6]

[0124] Example 6. Neuroinflammatory inhibitory effects of drugs using microglia from diseased animals

[0125] The brains of Psen2 N141I KI / + mice and wild-type mice prepared in Example 1 were isolated on postnatal days 1 to 3. Primary microglial cells derived from wild-type and Psen2 N141I KI / + mice were cultured as described in Example 5-1.

[0126] The microglial cells prepared above were pretreated with didanosine at 0, 5, or 10 μM for 30 minutes, and then treated with Escherichia coli O111:B4 (L4391)-derived LPS at 1 μg / mL. After 12 hours, the amount of cytokine IL-6 secreted into the culture medium was measured by ELISA. A mouse IL-6 ELISA kit was purchased from R&D Systems and used to measure cytokines in the culture medium according to the manufacturer's instructions.

[0127] Specifically, the groups in which primary cultured microglia were treated with didanosine were divided into the following groups:

[0128] [Table 7] As shown in Table 7 and FIG. 6, didanosine significantly reduced the amount of IL-6 secretion that was increased in microglial cells derived from Psen2 N141I KI / + mice treated with LPS.

[0129] Example 7: Confirmation of the recovery effect of didanosine treatment on amyloid beta degradation ability reduced by Psen2 N141I mutation in microglia

[0130] Brains were isolated from Psen2 N141I KI / + mice and wild-type mice prepared in Example 1 on postnatal days 1 to 3. Primary microglial cells from wild-type and Psen2 N141I KI / + mice were cultured as described in Example 5-1. The microglial cells prepared above were isolated and cultured and seeded onto 24-well plates with cover glasses.

[0131] Specifically, amyloid beta 1-42 conjugated with FITC signal was prepared according to the following reference (Cho, M.-H. et al., "Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome." Autophagy 10, 1761-1775 (2014)).

[0132] FITC-conjugated amyloid beta oligomers were fibrillated in the medium for 24 hours.

[0133] The microglial cells were pretreated with didanosine at a concentration of 10 μM for 30 minutes, and then the fibrilized amyloid beta 1-42 (fAβ42) prepared above was added directly to the microglial culture medium at a concentration of 4 μM. The aggregated amyloid beta was then treated, and after 2 hours, the medium was washed to remove any amyloid beta that had not been phagocytosed and remained in the medium.

[0134] After 24 hours of continuous treatment with didanosine, the cells were fixed and mounted on glass slides, and the amount of aggregated amyloid beta remaining in the cells was measured and compared to the degree of degradation. The amount of amyloid beta was quantified by measuring the relative fluorescence intensity of the FITC-labeled amyloid beta signal present in the cells using a confocal laser scanning microscope (LSM700) by capturing images of the entire z-axis of randomly selected cells at 2 μm intervals and calculating the number of pixels in the signal using ZEN (black edition; Carl Zeiss) software.

[0135] As shown in Figure 7 and Table 8, didanosine restored the reduced amyloid beta degrading ability in Psen2 N141I KI microglia and demonstrated the effect of treating neuroinflammatory diseases, such as Alzheimer's disease.

[0136] [Table 8]

[0137] Example 8. Cytokine expression analysis using Psen2 N141I KI model mice (KI / +)

[0138] Wild-type mice (WT) and Alzheimer's disease-induced Psen2 N141I KI model mice (KI / +) prepared in Example 1 were intraperitoneally injected with didanosine at a concentration of 5 mg / kg, and 4 hours later, LPS was intraperitoneally injected at a concentration of 0.35 μg / kg.

[0139] As shown in Figure 8, LPS at a concentration of 0.35 μg / kg corresponds to a very low concentration that does not induce an inflammatory response or increase blood levels of IL-6 cytokines in wild-type mice. In contrast, it induced neuroinflammation and overproduction of IL-6 in Psen2 N141I KI / + mice. Furthermore, didanosine at a concentration of 5 mg / kg corresponds to a concentration of 0.4 mg / kg (24 mg / 60 kg) in humans, which is approximately 0.1 times lower than the daily dose of 250 mg prescribed for HIV treatment.

[0140] Twenty-four hours after didanosine administration, blood was collected from the mice, serum was separated, and the amount of secreted cytokine IL-6 was analyzed by ELISA. As shown in Figure 8 and Table 9, didanosine significantly reduced the amount of IL-6 secreted by neuroinflammation.

[0141] [Table 9]

[0142] Example 9. Experiments to evaluate motor and memory ability using Psen2 N141I KI model mice (KI / +)

[0143] Animal experiments were conducted using the Psen2 N141I KI / + disease animal model. Using the same method as in Example 5, Psen2 N141I KI model mice (KI / +) obtained in Example 1 and wild-type mice were administered didanosine. Four hours later, LPS was intraperitoneally injected at a concentration of 0.35 μg / kg. As shown in Figure 9b and Table 11, LPS at a concentration of 0.35 μg / kg is a very low concentration that does not induce memory impairment in wild-type mice. In contrast, it induced neuroinflammation and memory impairment in Psen2 N141I KI / + mice.

[0144] To assess motility, we analyzed the movement speed and total distance traveled using the open field test 24 hours after didanosine administration. As shown in Figure 9a and Table 10, intraperitoneal injection of didanosine did not affect motility.

[0145] [Table 10]

[0146] To assess memory recovery, spatial memory ability was assessed using a Y-maze analysis 24 hours after didanosine administration. Specifically, mice were placed in the nearest arm of a Y-maze and allowed to wander freely for 5 minutes. Each arm of the Y was named A, B, and C, and the name of the arm into which the mouse entered was recorded. The number of times the mouse entered a new arm that it had not entered during the recording period was calculated and analyzed as follows: Alternation (%) = (three consecutive other arms / (total number of entries - 2) × 100. The total number of entries into each arm was the same, confirming that the mobility of each group was similar.

[0147] As shown in Figure 9b and Table 11, didanosine restored the memory loss in Alzheimer's disease-induced Psen2 N141I KI model mice to the memory level of wild-type mice. Specifically, as confirmed in Example 4-1, low concentrations of LPS did not induce memory loss or affect motor function in the normal group, and the LPS-administered normal group showed no difference in memory or motor function compared with the LPS-unadministered normal group. Low concentrations of LPS induced memory loss only in the Alzheimer's disease-induced group. In other words, didanosine restored the reduced memory of LSP-injected Psen2 N141I KI mice with Alzheimer's disease to the level of wild-type mice without LPS injection.

[0148] [Table 11]

[0149] Example 10. Efficacy of drugs in treating neuroinflammation using microglia from diseased animals

[0150] The brains of Psen2 N141I KI / + mice and wild-type mice prepared in Example 1 were isolated on postnatal days 1 to 3. Primary microglial cells derived from wild-type and Psen2 N141I KI / + mice were cultured as described in Example 5-1.

[0151] To observe the therapeutic effects of didanosine, the microglial cells prepared above were first treated with LPS at a concentration of 1 μg / mL, followed one hour later by treatment with didanosine at a concentration of 10 μM. After 11 hours, the amount of cytokine IL-6 secreted into the culture medium was measured by ELISA. A mouse IL-6 ELISA kit was purchased from R&D System and used to measure cytokines in the culture medium according to the manufacturer's instructions.

[0152] The primary cultured microglial cells were treated with didanosine and divided into the following groups. As shown in Figure 10 and Table 12, treatment of microglial cells from Psen2 N141I KI / + mice with LPS-induced neuroinflammation with didanosine significantly reduced the increased IL-6 secretion. This confirmed the therapeutic effect of didanosine on neuroinflammation.

[0153] [Table 12]

[0154] Example 11. Experiments to evaluate motor and memory abilities using the 5xFAD mouse model

[0155] One of the animal models used in Alzheimer's disease research is the 5XFAD mouse model, which expresses five AD-associated mutations in the APP and PSEN1 genes (APP; Swedish (K670N / M671L), Florida (I716V), and London (V717I) mutations; and PSEN1; M146L and L286V mutations). The APP and PSEN1 mutant genes are expressed under the Thy1 (mature neuron-specific marker) promoter, and even hemizygous mice exhibited severe amyloid pathology and behavioral deficits (Jawhar S. et al., "Motor deficits, neuron loss, and reduced anxiety coincident with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease." Neurobiology of Aging 196, p. 29-40 (2012)).

[0156] Six-month-old 5xFAD mice and wild-type mice were injected intraperitoneally with didanosine at a dose of 0.5 mg / kg / day, five days a week for a total of four weeks. The 0.5 mg / kg dose corresponds to a human dose of 0.04 mg / kg (2.4 mg / 60 kg), which is approximately 0.01 times lower than the prescribed daily dose of 250 mg for HIV treatment.

[0157] To evaluate motility, didanosine was administered, and then the movement speed and total distance were analyzed using the open field test in the same manner as in Example 9. As shown in Figure 11a and Table 13, intraperitoneal injection of didanosine did not affect motility.

[0158] [Table 13]

[0159] To evaluate memory recovery, didanosine was administered, and then the memory recovery effect was evaluated in the same manner as in Example 9. As shown in Figure 11b and Table 14, it was confirmed that didanosine restored the memory impairment induced in 5XFAD (6-month-old) Alzheimer's disease model mice.

[0160] [Table 14]

[0161] Example 12. Evaluation of neuroinflammation recovery using the 5xFAD mouse model

[0162] After completing the experiment in Example 11, hippocampal tissues from 5xFAD mice were isolated and IL-6 (IL-6) gene expression was examined. Specifically, IL-6 mRNA expression levels were measured using quantitative RT-PCR (qRT-PCR). RNA was isolated from the isolated hippocampal tissues, and cDNA was synthesized using the ImProm-II Reverse Transcriptase kit (Promega). PCR primers were commercially synthesized (Cosmo Genetech). qRT-PCR was performed using Taq Polymerase (Invitrogen) specific for mouse cDNA and the primers listed in Table 15 below. TOPreal TM qPCR2xPreMIX (SYBR Green with low ROX) (Enzynomics) was used, and 50-cycle amplification was applied to all primers using a CFX96 Real-Time System (Bio-Rad). Actb was used as a reference gene for normalization.

[0163] [Table 15] As shown in Figure 12 and Table 16, didanosine reduced increased IL-6 expression in the brain tissue of 5XFAD mice, confirming that didanosine has the effect of relieving brain inflammation, specifically inflammation in the hippocampal tissue, thereby demonstrating its effectiveness in treating neuroinflammatory diseases.

[0164] [Table 16]

[0165] Example 13. Statistical Analysis

[0166] In this example, data are presented as means ± standard error of the mean (SEM) from a minimum of three independent experiments. Statistical analysis was performed using unpaired student t-tests, one-way analysis of variance (ANOVA), or two-way ANOVA, and statistical significance was analyzed using GraphPad Prism.

[0167] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A pharmaceutical composition for preventing or treating Alzheimer's disease in a subject, comprising didanosine or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition of claim 1, wherein the composition inhibits the expression of neuroinflammatory cytokines in the central nervous system.

3. The pharmaceutical composition of claim 1 , wherein the composition inhibits the expression of neuroinflammatory cytokines in microglial cells.

4. The pharmaceutical composition of claim 1, wherein the composition restores the amyloid beta degrading ability of microglial cells.

5. 10. The pharmaceutical composition of claim 1, wherein the composition is administered at a daily dosage of 0.001 to 4 mg / kg.

6. 10. The pharmaceutical composition of claim 1, wherein the composition is administered at a daily dosage of 0.001 to 2.5 mg / kg.

7. 2. The pharmaceutical composition of claim 1, wherein the Alzheimer's disease is familial Alzheimer's disease.

8. 2. The pharmaceutical composition of claim 1, wherein the Alzheimer's disease is Alzheimer's disease with one or more gene mutations selected from the group consisting of Amyloid Precursor Protein (APP), Presenilin 1 (PSEN1), and Presenilin 2 (PSEN2).

9. 9. The pharmaceutical composition of claim 8, wherein the presenilin 2 gene mutation comprises one or more selected from the group consisting of PSEN N141I, A85V, N141Y, M174I, G212V, A237V, M239I, and M239V.

10. The pharmaceutical composition of claim 1 , wherein the Alzheimer's disease is caused by amyloid beta.

11. The pharmaceutical composition of claim 1, wherein the Alzheimer's disease is caused by a decrease in the ability of microglia to degrade amyloid beta.

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