Pharmaceutical composition for preventing or treating degenerative brain diseases, comprising prasugrel as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases, the composition comprising prasugrel as an active ingredient. The composition containing prasugrel or a salt thereof according to the present invention has excellent neuroprotective activity and, in particular, can suppress MPP+-induced neurotoxicity and neuronal cell death, suppress LPS-induced microglial cell activation and cell migration, and has neuroinflammation inhibitory activity, and thus has the effect of preventing, alleviating, and treating various degenerative brain diseases including Parkinson's disease.

Description

Pharmaceutical composition for the prevention or treatment of degenerative brain diseases containing prasugrel as an active ingredient

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

[0002] Degenerative brain diseases, among the degenerative diseases that occur with age, refer to diseases that originate in the brain. They can be categorized based on their primary symptoms and the affected brain region. Representative examples include Alzheimer's disease and Parkinson's disease. Degenerative brain diseases are known to be caused by neurodegeneration due to aging, as well as by protein aggregation and neuronal cell death due to genetic and environmental factors.

[0003] 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 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 (AD), 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.

[0004] In addition, it has been reported that the activity of microglia is related to the onset and progression of degenerative brain diseases, and microglia are immune cells that reside in the central nervous system (CNS) and are known to be activated by external stimuli and induce immune and inflammatory responses. Microglia are cells that perform primary immune functions in the CNS, 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.

[0005] 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 ultimately worsens the damage to neurons. Therefore, suppressing the appropriate activation of microglia may be another way to treat degenerative brain diseases.

[0006] Among degenerative brain diseases, Parkinson's disease (PD) is a representative degenerative neurodegenerative disease that appears in old age, along with Alzheimer's disease. It is known that about 1% of the population over 65 years of age develops the disease, and the incidence rate increases with age.

[0007] Parkinson's disease is characterized by motor disturbances, including resting tremor, rigidity, bradykinesia, and postural instability. It is also characterized by microgliosis, astrogliosis, progressive degeneration of dopaminergic neurons, the presence of Lewy bodies in dopaminergic neurons, and the accumulation of alpha-synuclein in the substantia nigra pars compacta.

[0008] Currently, treatments for these 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.

[0009] 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.

[0010] Meanwhile, prasugrel is a thienopyridine ADP receptor antagonist that can be administered orally or parenterally and is currently used as a platelet inhibitor.

[0011] However, there are no studies yet demonstrating that prasugrel can be used as a treatment for degenerative brain diseases.

[0012] Accordingly, the inventors of the present invention completed the present invention by confirming that the drug prasugrel can be used as a therapeutic agent for preventing or treating degenerative brain diseases.

[0013] Therefore, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising prasugrel or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide a health functional food for preventing or improving degenerative brain diseases, comprising prasugrel or a salt thereof as an active ingredient.

[0015] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising prasugrel or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] In one embodiment of the present invention, the composition can suppress the activity of microglia and thereby suppress damage caused by activated microglia to nerve cells.

[0017] In one embodiment of the present invention, the composition can inhibit cell migration of microglial cells.

[0018] In one embodiment of the present invention, the activity of microglial cells or the cell migration of microglial cells may be induced by LPS (Lipopolysaccharides).

[0019] In one embodiment of the present invention, the composition comprises MPP + It can inhibit cytotoxicity and apoptosis of nerve cells induced by (1-Methyl-4-Phenylpyridine).

[0020] In one embodiment of the present invention, the composition may have anti-inflammatory activity in nerve cells.

[0021] In one embodiment of the present invention, the composition may decrease mRNA expression of Bax and increase mRNA expression of Bcl-2.

[0022] In one embodiment of the present invention, the degenerative brain disease may be selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, multiple neurotrophy, epilepsy, encephalopathy, and stroke.

[0023] In addition, the present invention provides a health functional food for preventing or improving degenerative brain diseases, comprising prasugrel or a salt thereof as an active ingredient.

[0024] In one embodiment of the present invention, the degenerative brain disease may be selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, multiple neurotrophy, epilepsy, encephalopathy, and stroke.

[0025] The present invention relates to the use of prasugrel as a therapeutic agent for degenerative brain diseases, and a composition containing prasugrel or a salt thereof according to the present invention has excellent neuroprotective activity, particularly MPP + It can suppress the toxicity and apoptosis of nerve cells induced by LPS, inhibit the activity and cell migration of microglia induced by LPS, and at the same time, it has neuroinflammation suppression activity, so it can be effective in preventing, improving, and treating various degenerative brain diseases including Parkinson's disease.

[0026] Figure 1 shows the neuroprotective effect of prasugrel treatment. (A) MPP was analyzed through CCK-8 analysis targeting an FDA-approved drug library.+ It shows that the drug that has the ability to recover cell viability from cytotoxicity by MPP was screened, and (B) is MPP + (C) shows the analysis of cell viability according to the treatment concentration of prasugrel in treated neurons, and (D) shows the cytotoxicity according to the treatment concentration of prasugrel, and MPP + It was confirmed that neuroprotective effect was achieved by inhibiting neuronal cell death by pretreatment with prasugrel, and (E) shows the results of measuring the neurite length of MAP2 according to the concentration of prasugrel treatment.

[0027] Figure 2 shows the results of confirming the apoptosis inhibitory effect of prasugrel in primary cultured neurons. (A) MPP in primary cortical neurons + This is a Western blot result confirming that JNK and p38 phosphorylation was reduced by prasugrel treatment, (B) is a graph converted from the result of (A), (C) shows the result of analyzing the mRNA expression level of apoptosis-related factors (Bax, Bcl-2) using RT-qPCR analysis, (D) shows the result of analyzing the level of reactive oxygen species production, and (E) shows the result of confirming the effect of prasugrel on preserving mitochondrial membrane potential.

[0028] Figure 3 shows the effect of prasugrel treatment on LPS-induced cell migration inhibition in BV2 microglial cells. (A) is a microscopic photograph showing that prasugrel inhibits cell migration of BV2 microglial cells, (B) is the result of measuring the number of migrated cells, and (C) is the result of measuring the scratch closure area.

[0029] Figure 4 shows the results of confirming the anti-inflammatory activity according to prasugrel treatment in BV2 microglial cells. (A) shows the results of Western blot confirming the inhibition of LPS-induced p38 phosphorylation in BV2 cells by prasugrel treatment, (B) shows a graph of the results of (A), (C) shows the results of RT-qPCR analysis confirming that the increase in mRNA expression of inflammatory cytokines and chemokines induced by LPS in BV2 microglial cells (n=3-4) was reduced by prasugrel treatment, (D) and (E) show the results of immunocytochemistry and Western blot confirming that the nuclear translocation of NF-kB induced by LPS was inhibited by prasugrel treatment, respectively.

[0030] Figure 5 shows the results of multiple proteomic analyses in primary cortical neurons and BV2 cells treated with prasugrel. (A) Venn diagrams for the identified proteomes in primary cortical neurons and BV2 cells, with volcano plots indicating downregulated proteins. (B) Comparative canonical pathway analysis of the PCN and BV2 proteomes using IPA, with orange and blue representing canonical pathways with positive or negative Z scores for pathway activation, respectively. (C) Protein-protein interactions in the proteome of primary cortical neurons, with increased and decreased protein levels indicated in red and green, respectively. (D) Protein-protein interactions in the proteome of BV2 cells, with increased and decreased protein levels indicated in red and green, respectively. (E) Apoptosis signaling in the proteome of primary cortical neurons, with red and green representing MPP. + MPP compared to the group treated with prasugrel + In the group treated with only prasugrel, the up-regulation and down-regulation were respectively shown.

[0031] Figure 6 shows the signaling pathway of neuroinflammation in the proteome of BV2 cells, with red and green indicating up-regulation and down-regulation, respectively, in response to prasugrel treatment in the LPS-only treated group compared to the LPS and prasugrel treated group.

[0032] Figure 7 shows the results of confirming the effect of prasugrel treatment on improving motor function in a mouse model of Parkinson's disease induced by MPTP. (A) shows the results of Rota-rod analysis, (B) shows the results of measuring the number of movements and duration through an open field test, and (C) shows the results of measuring the number of non-movements and duration.

[0033] Figure 8 shows the results of confirming the dopaminergic neuron protection effect according to prasugrel treatment in a mouse model of Parkinson's disease induced by MPTP. (A) is the result of confirming the expression level of tyrosine hydroxylase (TH) in the striatum by Western blot, and (B) is the result of quantifying the Western blot results in a graph.

[0034] The present invention is characterized by providing a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising prasugrel or a pharmaceutically acceptable salt thereof as an active ingredient.

[0035] As previously mentioned in the prior art, prasugrel is a drug currently used as a platelet inhibitor, and is used in combination with low-dose aspirin to prevent thrombosis in patients with acute coronary syndromes, including unstable angina, non-ST elevation myocardial infarction (NSTEMI), and ST elevation myocardial infarction (STEMI).

[0036] However, there has not yet been a report of research results showing that prasugrel can be used as a treatment for degenerative brain diseases, and the present invention has for the first time demonstrated that prasugrel can be used as a treatment for degenerative brain diseases.

[0037] According to one embodiment of the present invention, in order to determine whether the drug prasugrel has a neuroprotective effect, MPP, which causes toxicity and cell death in nerve cells, + In primary cortical neurons treated with the drug prasugrel, MPP + It was confirmed that neurotoxicity and neuronal cell death by prasugrel were effectively suppressed, and through MAP2 staining, a neuronal cell marker, the group treated with prasugrel showed a higher level of MPP compared to the group not treated with the drug. + It was confirmed that the length of the neurites reduced by was restored.

[0038] Through these results, the inventors were able to find out that prasugrel has the activity to protect nerve cells.

[0039] In another embodiment of the present invention, in order to determine through what activity in the cell the neuroprotective activity of prasugrel is induced, MPP + It is known that prussugrel activates MAPK (mitogen-activated protein kinase) in nerve cells, so prussugrel is known to activate MPP + We analyzed whether it could affect the activation of MAPK.

[0040] As a result, MPP + It was shown that phosphorylation of ERK was reduced and phosphorylation of JNK and p-38 MAPK was increased, whereas the group treated with prasugrel of the present invention was shown to effectively inhibit phosphorylation of JNK and p-38 MAPK.

[0041] Through this, the inventors of the present invention have found that the prasugrel of the present invention is MPP + It was found that it exhibited neuroprotective activity by effectively inhibiting neuronal cell death through activation of p-38 MAPK induced by .

[0042] In addition, the present invention confirmed that prasugrel has the effect of suppressing the activity of microglia and thus inhibiting the damage caused by activated microglia to nerve cells, and it was found that the onset of degenerative brain diseases caused by the cell migration of activated microglia can be suppressed by effectively suppressing the cell migration of activated microglia when treated with prasugrel.

[0043] Microglial cells, which function as macrophages in the brain, are crucial 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 mutated by external stimuli, or the invasion of pathogens, can lead to excessive microglial activity, which in turn damages neurons and contributes to degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and cerebral infarction. Therefore, methods to suppress excessive microglial activity could be a new treatment for degenerative brain diseases.

[0044] In addition, overly activated microglia, unlike normal microglia, actively engage in phagocytosis, proliferate, and express genes for inflammatory cytokines such as TNF-α, IL-1β, and CCL2, thereby producing inflammatory mediators, which ultimately can cause neuronal damage and lead to degenerative brain diseases.

[0045] Such substances that cause excessive activation of microglia may include bacterial endotoxin lipopolysaccharide (LPS), interferon-γ, beta-amyloid, and ganglioside, but in one embodiment of the present invention, lipopolysaccharide (LPS) is preferred.

[0046] Therefore, the present invention can provide a pharmaceutical composition for preventing or treating a degenerative brain disease, which comprises prasugrel or a pharmaceutically acceptable salt thereof as an active ingredient.

[0047] In addition, the prasugrel according to the present invention can be used in the form of a salt, preferably a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.

[0048] The term pharmaceutically acceptable salt means any organic or inorganic addition salt of the base compound of prasugrel, which is relatively non-toxic and has an effective action innocuous to the patient, and the side effects due to this salt do not diminish the beneficial efficacy of the base compound of prasugrel. These salts may use inorganic acids and organic acids as the free acids, and the inorganic acids may be hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc., and the organic acids may be citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid, etc. Additionally, these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, May contain tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts.

[0049] The degenerative brain disease according to the present invention is not limited thereto, but may be selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, multiple neurotrophy, epilepsy, encephalopathy, and stroke.

[0050] In one embodiment of the present invention, MPP + The prevention, improvement, and treatment effects of the disease according to the prasugrel treatment of the present invention were confirmed in a Parkinson's disease model induced by .

[0051] The composition of the present invention for the prevention or treatment of degenerative brain diseases may include a pharmaceutically acceptable carrier. The composition containing the pharmaceutically acceptable carrier may be administered orally or parenterally in various dosage forms. When formulated, it is prepared using commonly used diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0052] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0053] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0054] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

[0055] Additionally, these pharmaceutical compositions can be administered to treat various diseases, including neurodegeneration and / or symptoms associated therewith, as described above.

[0056] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0057] The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Considering all of the above factors, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. The typical dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg for adults.

[0058] The pharmaceutical composition may be administered via any conventional route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonary, or rectally, depending on the intended purpose, but is not limited thereto. Furthermore, the composition may be administered via any device capable of transporting the active ingredient to target cells.

[0059] The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the prevention and treatment of degenerative brain diseases.

[0060] In addition, the present invention can provide a health functional food for preventing or improving degenerative brain diseases, which contains prasugrel or a salt thereof as an active ingredient.

[0061] The above "health functional food" refers to a food manufactured using nutrients that are easily deficient in daily meals or raw materials or ingredients that have functions useful to the human body, and is used to mean a food that helps maintain human health, but is not limited thereto, and includes all health foods in the general sense.

[0062] The form and type of health functional foods are not particularly limited. Specifically, the health functional foods may be in the form of tablets, capsules, powders, granules, liquids, and pills. The health functional foods may include various flavoring agents, sweeteners, or natural carbohydrates as additional ingredients. The sweeteners may be natural or synthetic. Examples of natural sweeteners include thaumatin and stevia extracts. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides, and sugar alcohols.

[0063] In addition to the additional ingredients described above, the health functional food of the present invention may further include nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pexans and their salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and the like. These ingredients may be used independently or in combination. The proportion of the additives may be selected within the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0064] The active ingredient of the present invention, presugrel or its salt, can be added directly to food or used in combination with other foods or food ingredients. The amount of the added active ingredient can be determined depending on the intended use. Typically, the amount in a health functional food can range from 0.01 to 90 parts by weight of the total food weight. However, for long-term intake for health and hygiene purposes or for health control, the amount may be below the above range. However, if there are no safety concerns, the active ingredient may be used in amounts exceeding the above range.

[0065] Furthermore, the present invention can provide a method for inhibiting the activity of microglia, which comprises a step of treating brain nerve cells with prasugrel or a pharmaceutically acceptable salt thereof in a test tube.

[0066] The present invention can also provide a method for inhibiting LPS-induced microglia cell migration, comprising a step of treating brain neurons with prasugrel or a pharmaceutically acceptable salt thereof in a test tube.

[0067] The present invention will now be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0068]

[0069] <Preparation example and experimental method>

[0070] Primary cortical neuron culture

[0071] Primary cortical neurons (PCNs) were isolated from the cerebral cortex of Sprague-Dawley rats on embryonic days 17–18 (Coatech, Gyeonggi-do, South Korea). Briefly, the cortex was finely dissected in cold Hanks' balanced salt solution (HBSS; Welgene Inc., Daegu, South Korea). The cells were then treated with 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at 37°C, washed with HBSS, and plated on poly-L-lysine-coated plates in Neurobasal medium (Thermo Fisher Scientific) containing B-27 supplement and GlutaMax (Thermo Fisher Scientific). Cultured neurons were cultured in vitro for 7–10 days before use, and all animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Brain Research Institute (Approval No.: IACUC-22-00038).

[0072]

[0073] Cell Counting Kit-8 (CCK8) Assay

[0074] Primary cortical neurons (4 × 10 5 (10 cells / ml) were seeded in 96-well plates and cultured for 7 days. After pretreatment with several compounds from an FDA-approved drug library (Selleck Chemicals, Houston, TX, USA) for 4 h, the cells were co-treated with 200 μM MPP+ (Sigma-Aldrich) for 24 h. Afterwards, 10 μL of CCK-8 solution (Dojindo, Mashiki, Japan) was added to each well of the plate and incubated at 37°C for 3 h. The amount of formazan formed was quantified by measuring the absorbance at 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific).

[0075]

[0076] Immunocytochemistry

[0077] Primary cortical neurons (3×10 4 (cells / ml) were plated on poly-L-lysine-coated glass pretreated with prasugrel (Sigma-Aldrich, St. Louis, MO, USA) for 4 h, and MPP + BV2 cells (1 × 10 4 (cells / ml) were seeded in a 6-well plate equipped with glass and pretreated with prasugrel (1 μM and 10 μM) for 6 h, then MPP + were treated simultaneously for 30 min. Afterwards, the cells were washed with warm Dulbecco's phosphate-buffered saline (DPBS, Thermo Fisher Scientific) and fixed with 4% paraformaldehyde (Chembio, NY, USA) at 37°C for 20 min. The fixed primary cortical neurons were blocked with TBS-TS (Tris-buffered saline / 0.1% Triton X-100 / 3% goat serum) for 30 min at room temperature (RT; 25 ± 2°C). Primary cortical neurons were then incubated overnight at 4°C with primary antibodies against MAP2 (microtubule-associated protein 2, Sigma-Aldrich), washed with TBS-TS and TBS, and then incubated with anti-mouse IgG labeled with Alexa Fluor 568 (Invitrogen, Invitrogen, OR, USA) for 3 h at room temperature, followed by staining with 4',6-diamidino-2-phenylindole (DAPI) solution (Thermo Fisher Scientific) for 30 min at room temperature. Images of the stained cells were acquired using a STELLARIS 8 confocal microscope (Leica, Germany).

[0078]

[0079] Measurement of reactive oxygen species

[0080] Primary cortical neurons (3×104 (cells / ml) were seeded in black 96-well plates coated with poly-L-lysine and pretreated with 1 μM and 10 μM prasugrel for 6 h, followed by MPP + were co-treated for 6 h. Afterwards, the cells were incubated in 80 μM DCF-DA for 30 min at 37°C and washed twice with warm PBS. Fluorescence intensity changes were repeatedly measured at 10-min intervals using a FlexStation 3 fluorescence plate reader (Molecular Devices, CA, USA).

[0081]

[0082] Western blot

[0083] Cells were homogenized, and the protein concentration of the supernatant was measured using the PierceTM bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific) using bovine serum albumin (BSA) as a standard. The supernatant protein (10 μg protein per lane) was separated with Any KDTM Mini-PROTEAN TGXTM precast gels (Bio-Rad, Hercules, CA, USA) and transferred to Immobilon-PSQ membranes (Millipore, Burlington, MA, USA). The membranes were immediately incubated in 5% skim milk for 30 min, followed by the addition of primary antibodies (Table 1) in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T; pH 7.5) and incubated overnight at 4°C. The membrane was then washed with Tris-Bis-T, and secondary monoclonal anti-mouse and polyclonal anti-rabbit antibodies (1:10,000; GeneTex, Irvine, CA, USA) were added and incubated for 2 h. After incubation, horseradish peroxidase-conjugated secondary antibody labeling was detected by enhanced chemiluminescence (ECL) using a cooled CCD camera system (CELLGENTEK, Daejeon, Korea), and relative protein levels were quantified using a densitometer for total forms (ERK, JNK, or p38).

[0084]

[0085]

[0086] Measurement of mitochondrial membrane potential

[0087] Primary cortical neurons (3×10 4 (10 cells / ml) were seeded in Neurobasal medium in a confocal dish and cultured for 7 days, then pretreated with prasugrel at 1 μM and 10 μM for 6 hours, and then MPP +were co-treated for 6 h. Cells were then treated with tetramethylrhodamine (TMRE, Invitrogen) at 37°C for 20 min. Images were obtained using a STELLARIS 8 confocal microscope.

[0088]

[0089] RT-qPCR (Quantitative real-time polymerase chain reaction) analysis

[0090] RiboEX TM Cells were homogenized using a reagent (GeneAll, SEOUL, Korea), chloroform was added, and the mixture was shaken vigorously for 5 minutes. The aqueous phase was transferred to a new tube, isopropanol was added, and the mixture was incubated overnight at -80°C and centrifuged at 12,000 g for 15 minutes. The supernatant was removed, and the pellet was washed with 75% ethanol and centrifuged at 8,000 g for 5 minutes. The obtained RNA pellet was dried, dissolved in RNase-free water (Takara, Japan), and mRNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). mRNA was reverse transcribed into cDNA using SuPrimeScript RT Premix (Genetbio Inc., Daejeon, South Korea). Quantitative real-time polymerase chain reaction analysis was performed using SYBR Green Master Mix (BIOLINE, MA, USA) and CFX Duet (Bio-Rad) according to the manufacturer's instructions.

[0091]

[0092] Cell line culture

[0093] Microglial cells from BV2 mice were provided by Dr. Hyang-Sook Heo of the Korea Brain Research Institute and cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a humidified 5% CO2 atmosphere. The medium was replaced every 2–3 days, and cells were seeded onto cell culture plates for experiments.

[0094]

[0095] Migration assay

[0096] BV-2 cells were seeded into 6-well plates and cultured in growth medium at 37°C in a 5% CO2 atmosphere until 50% confluence was reached. Cells were pretreated with prasugrel for 4 h, and a scratch was created in the center of the monolayer of each well using a sterile pipette tip. Cells were washed twice with PBS, detached cells were removed, and immediately treated with 1 μg / ml lipopolysaccharide (LPS). The scratch area was monitored for 24 h. Images were obtained after scratching using a CKX53 microscope (Olympus, Tokyo, Japan) and 24 h after LPS treatment. These experiments were performed three independent times, and the scratch area was measured using HK Basic (Olympus).

[0097]

[0098] Experimental animals and drug administration

[0099] Four-week-old male C57BL / 6N mice were purchased from Coretech Co., Ltd. (Pyeongtaek, Korea) and used in the experiment after a two-week acclimatization period. The mice were randomly divided into five groups of 10 mice each: control group, MPTP treatment group, MPTP + prasugrel 1 mg / kg treatment group, MPTP + prasugrel 10 mg / kg treatment group, and MPTP + L-DOPA treatment group. Mice were housed under a 12-h light / dark cycle, with free access to food and water, and a temperature maintained at 21–23°C. Prasugrel was dissolved in 10% DMSO + corn oil at doses of 1 mg / kg and 10 mg / kg and administered orally once daily for 14 days. L-DOPA was used as a positive control, and was administered intraperitoneally (ip) at a dose of 10 mg / kg dissolved in phosphate-buffered saline (PBS) for the same period as prasugrel administration. On day 15, acute parkinsonism was induced by intraperitoneal administration of 20 mg / kg (dissolved in 0.1 M PBS) four times at 2-hour intervals. Behavioral experiments were performed 3 hours after the final MPTP administration.

[0100]

[0101] Rota-rod test

[0102] Mice were acclimated to the Rota-rod (LE8205, Panlab, Barcelona, ​​Spain) for 3 days before drug administration, running it four consecutive times daily at 5 rpm for 180 s. Testing was performed 3, 24, and 48 h after the final MPTP injection, and the time it took each mouse to walk on the rod without falling was measured.

[0103]

[0104] Open field testing

[0105] Mice were placed in a 30 cm x 30 cm chamber, and their locomotor activity was analyzed by recording the frequency and duration of immobility. Mouse movements were tracked using EthoVision XT video tracking software (Noldus, Wageningen, the Netherlands). After placing the mice in the chamber, they were allowed to acclimate to the new environment for 30 s, and their behavior was tracked for 5 min, at 4, 25, and 48 h after the final MPTP injection.

[0106]

[0107] Brain tissue preparation

[0108] Mice were anesthetized with isoflurane and perfused intracardially with 0.9% saline. For histological analysis, the perfused mice were fixed with 4% paraformaldehyde solution (Chembio, NY, USA), the skull and meninges were removed, and the brains were extracted and stored in the same fixative overnight at 4°C before being transferred to 30% sucrose. The cryofixed brains were serially sectioned at 40 μm in the coronal plane using a CM1850 Cryostat (Leica, Wetzlar, Germany), and the sections were stored in Dulbecco's phosphate-buffered saline (DPBS) containing 0.1% sodium azide at 4°C. For biochemical analysis, the brains were removed and dissected into the striatum and substantia nigra. The dissected tissues were placed in e-tubes, immediately flash-frozen in liquid nitrogen, and stored at -80°C until further use.

[0109]

[0110] Peptide production by in-solution digestion

[0111] Peptides were obtained from primary cortical neurons and BV2 cells by in-solution digestion. Cell pellets were lysed in lysis buffer (40 mM ammonium bicarbonate, pH 7.8) supplemented with 1% Protease MAX (Promega, Madison, WI, USA) and sonicated 10 times (30% amplitude, 3 s on and 10 s off), followed by incubation on ice for 30 min. The lysates were diluted fourfold with 40 mM ammonium bicarbonate buffer, 10 mM dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA) was added, and the mixture was incubated at 56°C for 20 min. The mixture was then treated with 20 mM iodoacetamide (Sigma-Aldrich) for 20 min at room temperature in the dark. Protein concentration was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific), and 100 μg of protein was used in subsequent experiments. Samples were mixed with a trypsin-Lys C mixture (Promega) at a ratio of 1:50 and treated at 50°C for 4 h. The reaction was stopped by adding 0.5% trifluoroacetic acid. Peptides digested by trypsin treatment were lyophilized and desalted using a desalting column (#89873, Thermo Fisher Scientific) according to the manufacturer's protocol.

[0112]

[0113] mass spectrometry

[0114] Trypsin-treated peptides are EASY-Spray TM Q Exactive interfaced with source (Thermo Fisher Scientific) TMThe peptides were analyzed using a Plus Hybrid Quadrupole-Orbitrap mass spectrometer (MS). An Acclaim™ PepMap™ 100 C18 high-performance liquid chromatography (HPLC) column (75 μm × 2 cm, 3 μm nanoviper; Thermo Fisher Scientific) was used as a loading column, and an EASY-Spray PepMap RSLC C18 column (75 μm × 50 cm, 2 μm, Thermo Fisher Scientific) was used as a separation column to perform chromatographic separation of the peptides. Peptides were injected from an RS autosampler and separated using a gradient of acetonitrile (ACN) / water containing 0.1% formic acid at a flow rate of 300 nL / min. The liquid chromatography eluent was electrosprayed from the separation column, and a voltage of 2.0 kV was applied through the liquid junction of the nanospray source. The peptide mixture was separated over a gradient of 10–50% ACN for 80 min. Analysis involved a full MS scan in the range of 350–2000 m / z and data-dependent tandem mass spectrometry (MS / MS) on the 10 most intense ions from the full MS scan. The mass spectrometer was programmed in data-dependent acquisition mode, and calibration was performed using the suggested calibration solution according to the manufacturer's instructions.

[0115] Tandem mass spectra were also processed using Proteome Discoverer software (Thermo Fisher Scientific) version 2.41 to perform database searches. Spectral data were searched against the Human UniProt database (release version 2023_07), and all identified proteins had a false discovery rate of less than 1%, calculated at the peptide level. Search parameters allowed for trypsin specificity of up to two missed cleavages, with methylthio-modification of cysteine ​​as a fixed modification and methionine oxidation as a dynamic modification. Mass search parameters for +1, +2, and +3 ions included a mass error tolerance of 20 ppm for precursor ions and 0.6 Da for fragment ions. The label-free quantification (LFQ) analysis sequence in Proteome Discoverer version 2.41 was used to calculate quantitative changes in the identified proteins between experimental groups.

[0116]

[0117] Bioinformatics analysis

[0118] IPA (Ingenuity Pathway Analysis) was used for GO (Gene Ontology)-based functional annotation and in-depth bioinformatics analysis. The UniProt protein accession numbers of the identified proteins were MPP + / The normalized fold change between LPS and M+prasugrel / L+prasugrel was combined and uploaded to IPA using protein expression criteria. The following criteria were used for quantitative pathway analysis: z-score cutoff = 0.5, -log(p-value) > 1.3.

[0119]

[0120] Statistical processing

[0121] All data are expressed as mean ± standard error of the mean (SEM), and the significance of differences between groups was determined using one-way analysis of variance (ANOVA) with Bonferroni's post hoc test in Prism version 9.0 (GraphPad Software Inc., San Diego, CA, USA), with statistical significance set at p-value <0.05.

[0122]

[0123] <Example 1>

[0124] Screening of prasugrel, a drug with excellent neuroprotective effects

[0125] We analyzed a candidate population for screening drugs with neuroprotective effects from a library of FDA-approved drugs in primary cortical neurons using the CCK-8 assay.

[0126] Analysis results showed that among FDA-approved drugs, E11 (prasugrel) was the most effective in inhibiting MPP in primary cortical neurons (PCNs). + It was found to most effectively inhibit the decrease in cell viability induced by (Fig. 1A).

[0127] Therefore, the present inventors conducted MPP to confirm whether prasugrel has a neuroprotective effect. + After treating damaged cells with prasugrel at various concentrations, cell viability was analyzed. The results confirmed that neuronal cell viability was restored in a concentration-dependent manner (Figure 1B). Furthermore, it was confirmed that prasugrel did not induce cytotoxicity at various treatment concentrations (Figure 1C).

[0128]

[0129] In addition, the inventors observed changes in neuronal dendrites by performing MAP2 immunostaining, and MPP + After treatment, the cell body was found to be condensed and the dendrites were shortened, whereas the group treated with prasugrel selected in the present invention showed MPP +It was confirmed that it had a neuroprotective effect in primary cortical neurons by significantly alleviating dendritic collapse induced by (Fig. 1D).

[0130]

[0131] <Example 2>

[0132] Inhibition of apoptosis by MPP+ in primary cortical neurons treated with prasugrel

[0133] To confirm the neuroprotective effect of prasugrel on primary cortical neurons, the following experiments were performed. MPP + It is known to inhibit mitochondrial electron transport chain complex I, thereby generating excessive reactive oxygen species (ROS) and causing mitochondrial dysfunction, and to induce apoptosis in neurons by activating mitogen-activated protein kinase (MAPK) in neurons.

[0134] Accordingly, the present inventors used Western blot analysis to confirm whether prasugrel affects the phosphorylation of MAPK in primary cortical neurons.

[0135] As a result, interestingly, MPP + was found to decrease phosphorylation of ERK and increase phosphorylation of JNK and p38 MAPK, whereas prasugrel was found to significantly decrease phosphorylation of JNK and p38 (Figures 2A and 2B).

[0136]

[0137] Additionally, the inventors performed RT-qPCR on these results to analyze whether prasugrel affects apoptosis gene expression.

[0138] As a result, MPP + When treated, the mRNA expression level of Bax, a gene that promotes apoptosis in primary cortical neurons, was found to increase significantly, while the mRNA expression level of Bcl2, a gene that inhibits apoptosis, was found to decrease. On the other hand, in the group treated with prasugrel, MPP+ The mRNA expression level of Bax increased by was found to be effectively suppressed, and the mRNA expression level of the apoptosis inhibitory gene Bcl2 was found to be increased (Fig. 2C).

[0139]

[0140] In addition, the inventors analyzed the effect of prasugrel on reactive oxygen species (ROS) production through DCF-DA analysis, and as a result, MPP + While the production of reactive oxygen species was found to increase excessively during treatment, the production of reactive oxygen species was found to be suppressed in a concentration-dependent manner by prasugrel treatment (Fig. 2D).

[0141]

[0142] In addition, the inventors analyzed whether prasugrel has the activity of preserving the membrane potential of mitochondria, and as a result, MPP + In the treatment group, the mitochondrial membrane potential was found to be destroyed, whereas in the prasugrel-treated group, the mitochondrial membrane potential was found to be restored (Fig. 2E).

[0143]

[0144] Through these results, the inventors of the present invention have shown that the prasugrel drug selected in the present invention inhibits MPP in primary cortical neurons. + It was found that it can effectively inhibit p38 MAPK-dependent apoptosis induced by , suppress the production of reactive oxygen species, and exert a neuroprotective effect by protecting the membrane potential of mitochondria.

[0145]

[0146] <Example 3>

[0147] Confirmation of the inhibitory effect of prasugrel on LPS-induced cell migration in BV2 microglia cells.

[0148] Excessive activation of glial cells is a hallmark of neurodegenerative diseases, including Parkinson's disease, and activated microglia typically migrate to lesions.

[0149] Therefore, the inventors of the present invention performed the following experiment to determine whether prasugrel can inhibit the activation of microglial cells. BV2 microglial cells were pretreated with 10 μM prasugrel for 4 hours, then co-treated with 1 μg / mL LPS for 24 hours, and phase images were observed to analyze the degree of microglial cell migration.

[0150]

[0151] As a result, as shown in Fig. 3, the group in which BV2 cells were stimulated with LPS showed increased cell migration compared to the control group, with cells moving and distributing to the scratched area, whereas the group treated with prasugrel showed significantly suppressed cell migration by LPS.

[0152] These results showed that prasugrel has the activity of significantly inhibiting the activation and cell migration of microglial cells.

[0153]

[0154] <Example 4>

[0155] Confirmation of the anti-inflammatory effect of prasugrel in BV2 microglia cells

[0156] MAPKs are known to be involved in the activation of microglia, and their activation is known to induce the NF-κB pathway, which induces an inflammatory response in microglia. Furthermore, recent reports suggest that neuroinflammatory responses occurring within neurons contribute to the development of degenerative diseases, and that increased p38 phosphorylation induced by LPS upregulates proinflammatory cytokines.

[0157] Accordingly, the inventors of the present invention analyzed by Western blot whether prasugrel affects the phosphorylation of ERK, JNK, and p38 MAPK by LPS, and whether it affects the production of proinflammatory cytokines by LPS.

[0158]

[0159] As a result, as shown in Figure 4, LPS treatment was shown to induce phosphorylation of ERK, JNK, and p38 MAPK, whereas the prasugrel treatment group was shown to reduce p38 phosphorylation in BV2 cells (Figures 4A and 4B).

[0160] Additionally, it was confirmed that the group treated with prasugrel effectively reduced the mRNA expression of LPS-induced inflammatory cytokines in BV2 cells (Fig. 4C).

[0161]

[0162] In addition, the inventors confirmed nuclear translocation of NF-κB dimer p65 through immunocytochemistry and Western blot, and confirmed that translocation to the nucleus was significantly inhibited by prasugrel treatment (Figures 4D and 4E).

[0163]

[0164] These results indicated that prasugrel exerts anti-inflammatory effects by inhibiting phosphorylation of p38 in BV2 microglia cells.

[0165]

[0166] <Example 5>

[0167] Multiplexed proteomics analysis in primary cortical neurons and BV2 cells treated with prasugrelor.

[0168] Furthermore, the present inventors used a reliable quantitative proteomics approach to MPP +We profiled proteomic changes in response to prasugrel treatment in primary cortical neurons and LPS-treated BV2 microglia. Proteomic analysis identified 2,918 and 3,115 proteins in the primary cortical neuron and BV2 proteomes, respectively ( Fig. 5A ). To visualize significantly altered proteins, volcano plots were plotted on the sides of each Venn diagram, indicating a general pattern of proteins exhibiting significant changes in response to prasugrel treatment.

[0169] IPA was performed on the proteomic analysis results to compare changes in signaling pathways after prasugrel treatment.

[0170]

[0171] *As a result, 21 pathways associated with neuroprotection and neuroinflammation were significantly altered by prasugrel treatment (Fig. 5B). These results demonstrated a similar correlation pattern between primary cortical neurons and the BV2 proteome. Interestingly, the MAPK signaling pathway and neuroinflammatory signaling were negatively correlated with prasugrel treatment.

[0172] Additionally, we investigated the top protein-protein interactions (PPIs) in each proteome.

[0173] As a result, caseinolytic peptidase P (CLPP) and leucine-rich pentatricopeptide repeat containing (LRPPRC) were shown to play important roles in PPI and are involved in neurotoxicity and PD pathogenesis (Fig. 5C). In addition to the primary cortical neuron proteome, ribosomal protein L35 (RPL35) was identified as a key PPI protein in the BV2 proteome (Fig. 5D).

[0174] Furthermore, to validate our proteomics results, we focused on apoptosis and neuroinflammatory signaling pathways. We analyzed the expression of key proteins in apoptotic signaling cascades (including RAS, RAF1, and MAPK) after prasugrel treatment in primary cortical neurons. These proteins were significantly reduced (Fig. 5E). Furthermore, we confirmed that IL1B and TNF levels were reduced by prasugrel treatment in BV2 cells, consistent with the RT-qPCR results.

[0175]

[0176] <Example 6>

[0177] Prasugrel treatment improves motor dysfunction in a mouse model of Parkinson's disease.

[0178] The neuroprotective and anti-inflammatory effects of prasugrel, confirmed in the in vitro experiments of the above examples, were verified in a mouse model of MPTP-induced Parkinson's disease. After administering L-DOPA and prasugrel for two weeks, the mice were subjected to an acute MPTP model, and their motor performance was assessed using the Rota-rod test.

[0179]

[0180] As a result, 3 hours after the final MPTP injection, the MPTP-treated group showed almost complete loss of motor function. On the other hand, the prasugrel-treated group showed gradual recovery of motor function loss caused by MPTP over time, and this effect was more excellent than that of the positive control group treated with L-DOPA (Fig. 7A). In addition, in the open field test, movement decreased in all MPTP-treated groups, but there was no significant effect in the prasugrel-treated group, suggesting that MPTP did not affect general experimental animal behavior, such as anxiety or exploratory behavior, which can be caused by dopamine reduction (Figs. 7B, 7C).

[0181] These results indicate that the prasugrel of the present invention can improve and treat the symptoms of Parkinson's disease.

[0182]

[0183] <Example 7>

[0184] Confirmation of the dopaminergic neuroprotective effect of prasugrel treatment in a mouse model of Parkinson's disease.

[0185] The nigrostriatal pathway is a key lesion in Parkinson's disease and is closely linked to motor function. To confirm the neuroprotective activity of prasugrel, we analyzed the expression level of tyrosine hydroxylase (TH), a marker of dopaminergic neurons in the striatum, using Western blot.

[0186]

[0187] As a result, the MPTP-treated group showed a significant decrease in the expression of tyrosine hydroxylase, a marker of dopaminergic neurons, whereas the prasugrel-treated group showed a significant increase in the expression of tyrosine hydroxylase, which had been reduced by MPTP. In particular, the group treated with 10 mg / kg of prasugrel showed a similar level of tyrosine hydroxylase expression to the normal control group (Figures 8A and 8B).

[0188]

[0189] Through the above results, the inventors of the present invention were able to determine that the drug prasugrel has neuroprotective activity, can suppress neuroinflammation, and can ultimately be used as a new therapeutic agent for the prevention, improvement, and treatment of degenerative brain diseases, including Parkinson's disease, by regulating the expression of proteins involved in such activity.

[0190]

[0191] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

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

2. In paragraph 1, A pharmaceutical composition for preventing or treating a degenerative brain disease, characterized in that the composition suppresses the activity of microglia and thereby suppresses damage caused to nerve cells by activated microglia.

3. In paragraph 1, A pharmaceutical composition for preventing or treating degenerative brain diseases, characterized in that the composition inhibits cell migration of microglial cells.

4. In paragraph 2 or 3, A pharmaceutical composition for preventing or treating a degenerative brain disease, characterized in that the activation of microglial cells or the cell migration of microglial cells is induced by LPS (Lipopolysaccharides).

5. In paragraph 1, The above composition is MPP + A pharmaceutical composition for the prevention or treatment of degenerative brain diseases, characterized in that it inhibits cytotoxicity and apoptosis of nerve cells induced by (1-Methyl-4-Phenylpyridine).

6. In paragraph 1, A pharmaceutical composition for preventing or treating degenerative brain diseases, characterized in that the composition has anti-inflammatory activity in nerve cells.

7. In paragraph 1, A pharmaceutical composition for preventing or treating a degenerative brain disease, characterized in that the composition reduces mRNA expression of Bax and increases mRNA expression of Bcl-2.

8. In paragraph 1, A pharmaceutical composition for preventing or treating a degenerative brain disease, characterized in that the degenerative brain disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, multiple atrophy, epilepsy, encephalopathy, and stroke.

9. A health functional food containing prasugrel or a salt thereof as an active ingredient for preventing or improving degenerative brain diseases.

10. In paragraph 9, A health functional food for preventing or improving degenerative brain diseases, characterized in that the above degenerative brain diseases are selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, multiple neurotrophy, epilepsy, encephalopathy, and stroke.

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