Composition for preventing or treating epilepsy comprising eleutherine

Abstract

The present invention relates to a use of eleutherine for preventing or treating epilepsy, seizures, or convulsions. A composition comprising eleutherine or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention reduces behavioral seizures, restores synaptic dysfunction, and inhibits the activation of the ferroptosis pathway, and thus exhibits a preventive or therapeutic effect on epilepsy, seizures, and / or convulsions.

Description

Composition for the prevention or treatment of epilepsy containing eruterin

[0001] The present invention relates to the use of Eleutherine for the prevention or treatment of epilepsy, seizures, or convulsions.

[0002] Epilepsy is a disease characterized by intermittent neurological disorders caused by paroxysmal discharges of brain nerve cells due to organic lesions or functional impairments, manifesting as neurological symptoms, loss of consciousness, seizures, and sensory disturbances. Epilepsy is the third most common neurological disease following Alzheimer's disease and stroke, affecting approximately 0.5% to 2% of the global population. Globally, about 45 new cases occur per 100,000 people annually, while it is estimated that there are approximately 300,000 to 400,000 epilepsy patients in Korea. Regarding the age distribution of patients, 70% of all cases begin in childhood and adolescence, with a particularly high incidence rate during infancy. Furthermore, the incidence and prevalence rates exhibit a U-shaped curve, peaking within the first year of life, then rapidly declining, and increasing sharply again in the elderly population aged 60 and older; the prevalence of experiencing seizures during one's lifetime reaches 10–15%.

[0003] With recent technological advancements enabling the observation of subtle pathological changes in the brain that were previously undetectable, the identification of the causes of epilepsy is gradually expanding. Some epidemiological studies have reported that more than one-third of patients have a history of pathological changes in the brain or brain injury, and major causes are known to include stroke, congenital malformations, head trauma, encephalitis, brain tumors, degenerative encephalopathy, genetics, prematurity, and perinatal injuries. Meanwhile, the treatment of epilepsy can be broadly classified into pharmacotherapy and non-pharmacological treatments such as the ketogenic diet and vagus nerve stimulation.

[0004] However, non-drug treatment is performed only for patients who show resistance to drugs, so drug therapy focused on brain nerve cells has been used as a major method for treating epilepsy. It is known that conventional drugs include Ganaxoione, a GABA receptor agonist capable of strengthening inhibitory synapses, and NS1209, an AMPA receptor antagonist capable of weakening excitatory synapses.

[0005] However, despite such drug treatments, more than 30% of epilepsy patients do not have their seizures controlled even with drugs of various mechanisms of action, and some drugs cause side effects such as skin rashes due to immune hypersensitivity reactions, gastrointestinal irritation caused by the drug, and dizziness, making effective treatment of epilepsy difficult.

[0006] Accordingly, the inventors confirmed that Eleutherine, a natural compound, exhibits excellent effects in the prevention and treatment of epilepsy, seizures, or convulsions, and thus completed the present invention.

[0007] One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of epilepsy, seizures, or convulsions comprising eruterin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0008] Another objective of the present invention is to provide a food composition for the prevention or improvement of epilepsy, seizures, or convulsions comprising eruterin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] Another object of the present invention is to provide a method for the prevention or treatment of epilepsy, seizures, or convulsions comprising the step of administering a therapeutically effective amount of eruterin or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0010] Another object of the present invention is to provide a use of eruterin or a pharmaceutically acceptable salt thereof for manufacturing a medicine for the prevention or treatment of epilepsy, seizures or convulsions.

[0011] In the following, redundant details will be omitted to prevent clutter. In other words, the content of the invention is not limited solely to the following, and should be interpreted in accordance with the overall context of the invention.

[0012] The present invention will be described in detail below.

[0013] The present invention provides a pharmaceutical composition for the prevention or treatment of epilepsy, seizures, or convulsions comprising eruterin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] More specifically, the present invention provides a pharmaceutical composition for the prevention or treatment of epilepsy comprising eruterin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] In the present invention, the term "Eleutherine" is used interchangeably with "Eleutherin" or "Eleucerin," and is the simplest and most major naphthopyran derivative containing a 1,4-naphthoquinone structure. As an example, it may be a compound isolated from the plants Cipura paludosa, Eleutherine bulbosa, and Eleutherine americana (Iridaceae), and can also be chemically synthesized.

[0016] The structure of this eruterin compound is represented by Chemical Formula 1 as shown below.

[0017] [Chemical Formula 1]

[0018]

[0019] In the present invention, the term "epilepsy" refers to a chronic disease characterized by two or more spontaneous seizures, and is a serious neurological disorder accompanied by neurobiological, mental, cognitive, and social changes. The disease is known to be caused by sudden, abnormal, excessive, and disordered electrical discharges in brain cells.

[0020] These types of epilepsy may be more specifically TLE (e.g., mTLE or lTLE), benign Roland epilepsy, frontal lobe epilepsy, infantile spasms, adolescent myoclonic epilepsy, adolescent nominal epilepsy, childhood nominal epilepsy (pycnolepsy), hot water epilepsy, Lennox-Gastout syndrome, Lando-Krepner syndrome, Dravet syndrome, progressive myoclonic epilepsy, reflex epilepsy, Rasmussen syndrome, limbic epilepsy, status epilepticus, abdominal epilepsy, megabilateral myoclonus, menstrual epilepsy, Sachsonian seizure disorder, Lafora syndrome, photosensitive epilepsy, etc.

[0021] A seizure refers to a condition in which a sudden change in the brain's normal electrical activity occurs, generating uncontrolled electrical signals in brain cells that temporarily affect a patient's behavior, movement, thoughts, and way of feeling. Such seizures may include motor seizures involving convulsions and non-motor seizures that do not involve convulsions (e.g., petit mal seizures).

[0022] In the present invention, the term "prevention" refers to any act of suppressing epilepsy or delaying its onset by administering a pharmaceutical composition according to the present invention.

[0023] In the present invention, the term "treatment" refers to any act in which symptoms of epilepsy are improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention.

[0024] In the present invention, pharmaceutically acceptable salts and / or foodically acceptable salts refer to salts commonly used in the pharmaceutical and food industries, for example, inorganic ionic salts prepared with calcium, potassium, sodium, magnesium, etc.; inorganic salts prepared with hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, sulfuric acid, etc.; organic salts prepared with acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, hydroiodic acid, etc.; and sulfonates prepared with methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid, etc. Amino acid salts prepared with glycine, arginine, lysine, etc.; and amine salts prepared with trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts referred to in the present invention are not limited by these listed salts.

[0025] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. In this case, the pharmaceutically acceptable carrier is one that is commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol,

[0026] It includes, but is not limited to, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, in addition to the above components, it may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0027] The pharmaceutical composition according to the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may be appropriately selected by a person skilled in the art, although it may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0028] The pharmaceutical composition according to the present invention may be administered to a subject in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above-mentioned factors, and this can be easily determined by a person skilled in the art.

[0029] Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, gender, condition, body weight, absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and concomitant drugs. Generally, 100 to 500 mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of obesity, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0030] The term "subject" in the present invention includes animals or humans whose symptoms may be improved by the administration of a composition according to the present invention. For example, a subject is a subject diagnosed with epilepsy or at risk of developing epilepsy. A subject that may be treated using the disclosed method and composition is, for example, a subject who has experienced one or more prior therapeutic interventions related to the treatment of epilepsy or a subject who has not experienced any prior therapeutic interventions for the treatment of epilepsy.

[0031] In the present invention, the term “administration” typically refers to an act of achieving delivery of a composition to a subject. Those skilled in the art will recognize various routes that may be used to administer to a subject, e.g., an animal or a human, in an appropriate context. Administration may be made through the bronchi (e.g., by bronchial instillation), buccal, dermis (e.g., dermis, which may be local, intradermal, intercutaneous, transdermal, etc., or may include one or more of these), intestinal, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intra-abdominal, intraspinal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosa, nose, oral cavity, rectum, subcutaneous, sublingual, local, tracheal, etc., and preferably may be administered in the form of oral administration.

[0032] According to one embodiment, it was confirmed that when a pharmaceutical composition according to the present invention was orally administered to an epilepsy model, the behavioral seizures of the mouse were significantly reduced, thereby effectively preventing or treating epilepsy.

[0033] The pharmaceutical composition according to the present invention may be characterized by enhancing the expression level of one or more selected from the group consisting of PSD-95, CRMP2, Synapsin-1, and GPX4.

[0034] The pharmaceutical composition according to the present invention may be characterized by reducing the expression level of one or more selected from the group consisting of HO-1, ACSL4, FTH1, LAMP2A, p62, and LC3B II / I.

[0035] According to one embodiment, the pharmaceutical composition according to the present invention can enhance the expression of synapse function-related markers PSD-95, CRMP2, and Synapsin-1, enhance the expression of GPX4, which is known to be associated with ferroptosis, while reducing the expression of HO-1, ACSL4, and FTH1.

[0036] According to one embodiment, the pharmaceutical composition according to the present invention can reduce the expression levels of LAMP2A, p62, and LC3B II / I, which are markers associated with chaperone-mediated autophagy (CMA).

[0037] The pharmaceutical composition according to the present invention can inhibit ferroptosis by inhibiting the degradation of GPX4 through the inhibition of the chaperone-mediated autophagy (CMA) pathway, and can play a role in restoring protein homeostasis and neuronal survival. Accordingly, the composition can effectively prevent or treat epilepsy.

[0038] In the present invention, the terms "eluterin" or "epilepsy" are as described above.

[0039] The term "individual" in this invention refers to any animal that has developed or may develop epilepsy, and typically may be an animal that can exhibit beneficial effects through treatment using the eluterin of this invention or a pharmaceutically acceptable salt thereof, but includes without limitation any individual that has symptoms of epilepsy or is likely to have such symptoms. As described above, said epilepsy can be effectively prevented or treated by administering the pharmaceutical composition of this invention to an individual.

[0040] Another aspect of the present invention provides a pharmaceutical composition comprising eruterin or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of epilepsy, seizures, or convulsions.

[0041] The present invention also provides a food composition for the prevention or improvement of epilepsy, seizures, or convulsions, comprising eruterin or a food-grade acceptable salt thereof as an active ingredient.

[0042] The term "improvement" as used in the present invention refers to any action that at least reduces parameters related to the condition being treated, such as the severity of symptoms. In this case, the health functional food composition may be used for the prevention or improvement of epilepsy, seizures, or convulsions, either simultaneously with or separately from a therapeutic agent, either before or after the onset of the disease.

[0043] The term "health functional food" used in the present invention refers to a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term "functionality" means consuming for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0044] The food composition of the present invention may include conventional food additives, and unless otherwise specified, suitability as a "food additive" is determined in accordance with the specifications and standards for the relevant item, in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety.

[0045] Examples of items listed in the above "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0046] In addition, the food composition according to the present invention is characterized by being formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, comprising one or more of a carrier, a diluent, an excipient, and an additive, for the purpose of preventing and / or improving epilepsy, seizures, or convulsions. Foods that can be added to the composition of the present invention include various types of food, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, health functional foods, etc. As additives that may be further included in the present invention, one or more components selected from the group consisting of natural carbohydrates, flavoring agents, nutritional agents, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers, pactic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonating agents, and fruit pulp may be used. Examples of the natural carbohydrates mentioned above are monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used as the above flavoring agents. In addition to the above, the composition according to the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents, pactic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, other compositions of the present invention may contain fruit pulp for the production of natural fruit juices and vegetable beverages. These ingredients may be used independently or in combination.Specific examples of the above carrier, excipient, diluent, and additive are not limited thereto, but it is preferable to use one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0047] Another aspect of the present invention provides a method for the prevention or treatment of epilepsy, seizures, or convulsions, comprising the step of administering a therapeutically effective amount of eruterin or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0048] Another aspect of the present invention provides the use of eruterin or a pharmaceutically acceptable salt thereof for manufacturing a medicine for the prevention or treatment of epilepsy, seizures or convulsions.

[0049] A composition comprising eruterin or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention reduces behavioral seizures and restores impaired synaptic function, thereby exhibiting a preventive or therapeutic effect against epilepsy, convulsions, and / or seizures.

[0050] Figure 1 is a figure confirming the anticonvulsant preventive or therapeutic effect of Eleutherine on a mouse model in which epilepsy was induced through KA and PTZ.

[0051] Figure 2 shows the recovery effect of Eleutherine (Eleu) treatment on the abnormal expression of synapse function-related markers and ferroptosis-related markers caused by KA induction.

[0052] (a) This is a diagram showing changes in the expression of synapse function-related markers (synapsin-1, CRMP-2, PSD-95) and ferroptosis-related markers (GPX4, HO-1, ACSL4, FTH1) through Western blotting.

[0053] (b) to (h) Figures showing the above experimental results analyzed into quantified data (**p < 0.01 vs vehicle, #p < 0.05 vs KA, ##p < 0.01 vs KA).

[0054] Figure 3 shows the recovery effect on the abnormal expression of synaptic function-related markers and ferroptosis-related markers caused by PTZ induction.

[0055] (a) This is a diagram showing changes in the expression of synapse function-related markers (synapsin-1, CRMP-2, PSD-95) and ferroptosis-related markers (GPX4, HO-1, ACSL4, FTH1) through Western blotting.

[0056] (b) to (h) Figures showing the above experimental results analyzed into quantified data (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle, #p < 0.05 vs PTZ).

[0057] Figure 4 shows the recovery effect of Eleutherine (Eleu) treatment on the abnormal expression of chaperone-mediated autophagy (CMA)-related markers caused by KA induction.

[0058] (a) This is a diagram showing changes in the expression of chaperone-mediated autophagy (CMA) related markers through Western blotting.

[0059] (b) and (c) are figures showing the results of the above experiment analyzed into quantified data (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle, ###p < 0.001 vs KA).

[0060] Figure 5 shows the recovery effect of Eleutherine (Eleu) treatment on chaperone-mediated autophagy (CMA) and the abnormal expression of related markers caused by PTZ induction.

[0061] (a) This is a figure showing changes in the expression of chaperone-mediated autophagy (CMA) related markers (LAMP2A) and autophagy related markers (p62 and LC3B II / I) through Western blotting.

[0062] (b) to (d) are figures showing the results of the above experiment analyzed into quantified data (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle, ***p < 0.001 vs vehicle, ##p < 0.01 vs KA, ###p < 0.001 vs KA).

[0063] Figure 6 is a figure confirming the inhibitory effect of Eleutherine on the chaperone-mediated autophagy (CMA) pathway that mediates the degradation of GPX4.

[0064] (a) Figure showing the results of immunofluorescence analysis of GPX4, LAMP2A, and DAPI following Eleutherine treatment in RSL3-treated HT-22 cells (scale bar: 20 μm).

[0065] (b) This figure shows the changes in expression of chaperone-mediated autophagy (CMA) related markers (LAMP2A) and autophagy related markers (p62 and LC3B II / I) through Western blotting.

[0066] (c) to (e) Figures showing the results of the above experiment analyzed into quantified data (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle, #p < 0.05 vs Fer-1, ##p < 0.01 vs Fer-1, ###p < 0.001 vs Fer-1).

[0067] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention.

[0068] Experimental Example 1. Epilepsy Animal Experiment Design and Behavioral Seizure Monitoring

[0069] 1-1. Preparation of an Epilepsy Animal Model

[0070] ICR mice (5 weeks old) were purchased from the Orient Bio Department or RAONBIO and were housed under a 12 / 12-hour light / dark cycle with free access to food and water. This experiment was conducted in accordance with ethical guidelines and with the approval of the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University (Approval No. Ewha-IACUC 2013-01-041&2021-066).

[0071] Eleutherine was prepared for oral administration by suspending it in 0.2% methylcellulose (MC).

[0072] Specifically, a Kainic Acid (KA)-induced epilepsy animal model was prepared, and mice were divided into the following four groups:

[0073] - Normal control group (Sal / 0.2% MC, CON): In the experiment confirming the preventive effect, 0.2% MC (Veh) was administered orally once a day for 5 days as a pretreatment, followed by the administration of Sal. In the experiment confirming the therapeutic effect, 0.2% MC was administered orally once a day for 3 days, 30 minutes after the administration of Sal.

[0074] - KA group (KA 40 mg / kg): In experiments confirming preventive and therapeutic effects, KA 40 mg / kg was injected intraperitoneally.

[0075] - Eleu2+KA group (KA+Eleu 2 mg / kg): In the experiment confirming the preventive effect, Eleutherine 2 mg / kg was administered orally once a day for 5 days prior to KA administration, and in the experiment confirming the therapeutic effect, the same dose of Eleutherine was administered orally once a day for 3 days after KA administration.

[0076] - VPA30+KA group (KA+VPA 30 mg / kg): In the experiment confirming the preventive effect, 30 mg / kg of VPA (Valproate) was administered intraperitoneally once a day for 5 days prior to KA administration, and in the experiment confirming the therapeutic effect, the same dose of VPA was administered intraperitoneally once a day for 3 days after KA administration.

[0077] In addition, a Pentylentetrazole (PTZ)-induced epilepsy animal model was prepared using the same method as above, and mice were divided into the following four groups:

[0078] - Normal control group (Sal / 0.2% MC, CON): In the experiment confirming the preventive effect, 0.2% MC (Veh) was administered orally once a day for 5 days as a pretreatment. In the experiment confirming the therapeutic effect, 0.2% MC was administered orally once a day 30 minutes after Sal was administered.

[0079] - PTZ group (PTZ 70 mg / kg): In experiments confirming preventive and therapeutic effects, PTZ 70 mg / kg was injected intraperitoneally.

[0080] - Eleu2+PTZ group (PTZ+Eleu 2 mg / kg): In the experiment confirming the preventive effect, Eleutherine 2 mg / kg was administered orally once a day for 5 days prior to PTZ administration, and in the experiment confirming the therapeutic effect, the same dose of Eleutherine was additionally administered orally once a day after PTZ administration.

[0081] - VPA30+PTZ group (PTZ+VPA 30 mg / kg): In the experiment confirming the preventive effect, VPA 30 mg / kg was administered intraperitoneally once a day for 5 days prior to PTZ administration, and in the experiment confirming the therapeutic effect, the same dose of VPA was additionally administered intraperitoneally once a day after PTZ administration.

[0082] 1-2. Behavioral Seizure Monitoring

[0083] Behavioral seizures were evaluated using the Racine classification system and scored into Grades 0 through 6 as follows:

[0084] - Grade 0: No change in behavior

[0085] - Grade 1: Facial muscle clonus

[0086] - Grade 2: Head nodding

[0087] - Grade 3: Facial muscle clonus

[0088] - Grade 4: Forelimb clonic seizures occurring along with realignment and falling

[0089] - Grade 5: Generalized tonic-clonic seizure

[0090] - Grade 6: Severe tonic-clonic seizures, including death

[0091] Experimental Example 2. Sample Preparation and Western Blotting

[0092] After collecting hippocampal tissue, it was lysed using a low-temperature RIPA lysis buffer containing 1% protease inhibitor and stored on ice for 20 minutes. Subsequently, the lysate was centrifuged at 13,000 xg for 20 minutes at 4°C. The protein concentration of the supernatant was measured using a protein quantification kit (Biomax).

[0093] Equal amounts of protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, United States). The membrane was blocked with a blocking buffer prepared by dissolving 5% non-fat dry milk in TBS buffer (TBST) supplemented with 0.1% Tween 20. Subsequently, the membrane was incubated overnight at 4°C with the following primary antibodies in a solution containing 5% bovine serum albumin (BSA) in TBST:

[0094] - Glutathione peroxidase 4 (GPX4) (1:1000; Abcam ab125066)

[0095] - CRMP2 (1:1000, CST, 9393s)

[0096] - Synapsin-1 (1:1000, CST, 5297s)

[0097] -PSD-95 (1:1000, Santa Cruz SC32290)

[0098] - HO-1 (1:1000, CST, 82206S)

[0099] - FTH1 (1:1000, CST, 4393)

[0100] - ACSL4 (1:1000, ABclonal Technology A20414)

[0101] - β-actin (1:5000, CST, 3700s).

[0102] After the first antibody reaction, the reaction was carried out with HRP (horseradish peroxidase)-conjugated second antibody (1:5000 dilution) at room temperature for 2 hours, and protein bands were detected by chemiluminescence using Chemidoc MP (Bio-rad, Hercules, CA, United States), and the detected bands were analyzed using Image J software.

[0103] Example 1. Confirmation of anticonvulsant preventive and therapeutic effects on KA and PTZ-induced epilepsy mouse models

[0104] 1-1. Confirmation of Anticonvulsant Preventive Effect

[0105] To verify the anticonvulsant preventive effect of Eleutherine, experiments were conducted on four KA mouse groups (control group, KA group, Eleu2+KA group, VPA30+KA group) and four PTZ mouse groups (control group, PTZ group, Eleu2+PTZ group, VPA30+PTZ group) prepared in Experimental Example 1 above (Figs. 1a and 1e). At this time, Eleutherine or VPA was administered once a day for 5 days before administering KA / PTZ.

[0106] As a result, as shown in Figures 1c and 1g, the behavioral seizure scores of the groups administered Eleutherine (Eleu2+KA group and Eleu2+PTZ group) were significantly reduced compared to the control group, and it was confirmed that the behavioral seizure scores were at a level almost similar to those of the groups administered VPA (VPA30+KA group and VPA30+PTZ group). This trend became more pronounced over time.

[0107] 1-2. Confirmation of Anticonvulsant Treatment Effect

[0108] To verify the anticonvulsant therapeutic effect of Eleutherine, experiments were conducted on four KA mouse groups (control group, KA group, Eleu2+KA group, VPA30+KA group) and four PTZ mouse groups (control group, PTZ group, Eleu2+PTZ group, VPA30+PTZ group) prepared in Experimental Example 1 (Figs. 1b and 1f). At this time, Eleutherine or VPA was administered 1 hour after inducing seizures by administering KA / PTZ.

[0109] As a result, as shown in Figures 1d and 1h, the behavioral seizure scores of the groups administered Eleutherine (Eleu2+KA group and Eleu2+PTZ group) were significantly reduced compared to the control group, and it was confirmed that the behavioral seizure scores were at a level nearly similar to those of the groups administered VPA (VPA30+KA group and VPA30+PTZ group). This trend became more pronounced over time.

[0110] In summary, the above experiment confirmed that Eleutherine can demonstrate a preventive or therapeutic effect against epilepsy by reducing behavioral seizures.

[0111] Example 2. Confirmation of recovery effects on abnormal expression of synaptic function and ferroptosis-related markers caused by KA and PTZ

[0112] To confirm the restorative effect of Eleutherine on the abnormal expression of synaptic function and ferroptosis-related markers caused by KA and PTZ, experiments were performed on four KA mouse groups (control group, KA group, Eleu2+KA group, VPA30+KA group) and four PTZ mouse groups (control group, PTZ group, Eleu2+PTZ group, VPA30+PTZ group) prepared in Experimental Example 1.

[0113] Specifically, after euthanizing the mice of each group, hippocampal tissue was collected, and changes in the expression of synapse function-related marker proteins (PSD-95, CRMP-2, Synapsin-1) and ferroptosis-related marker proteins (GPX4, HO-1, ACSL4, FTH1) were observed through Western blotting (Figs. 2a and 3a).

[0114] As a result, in the groups administered Eleutherine (Eleu2+KA group and Eleu2+PTZ group), the expression levels of synapse function-related markers (PSD-95, CRMP-2, Synapsin-1), which were reduced by treatment with KA or PTZ, were restored to levels close to those of the control group (Figs. 2b to 2d, Figs. 3b to 3d). In addition, it was confirmed that the expression level of GPX4, which was reduced by treatment with KA or PTZ, increased, while the expression levels of HO-1, ACSL4, and FTH1, which were increased by treatment with KA, were significantly suppressed (Figs. 2e to 2h, Figs. 3e to 3h).

[0115] Therefore, through the above experiment, it was confirmed that administering Eleutherine restores impaired synaptic function and inhibits the activation of the ferroptosis pathway, thereby demonstrating a therapeutic effect for epilepsy.

[0116] Example 3. Confirmation of inhibitory effect on the chaperone-mediated autophagy (CMA) pathway involved in GPX4 degradation

[0117] 3-1. Confirmation of Changes in Expression of Chaperone-Mediated Autophagy (CMA) Related Markers in KA and PTZ-Induced Epilepsy Mouse Models

[0118] To confirm the recovery effect of Eleutherine on epilepsy induced by KA and PTZ, experiments were conducted on four KA mouse groups (control group, KA group, Eleu2+KA group, VPA30+KA group) and four PTZ mouse groups (control group, PTZ group, Eleu2+PTZ group, VPA30+PTZ group) prepared in Experimental Example 1.

[0119] Specifically, the KA mouse group and the PTZ mouse group were euthanized after 3 days and 24 hours, respectively, and changes in the expression of chaperone-mediated autophagy (CMA) related proteins were observed via Western blotting (Figures 4a and 5a).

[0120] As a result, it was confirmed that the expression level of the chaperone-mediated autophagy (CMA) related marker (LAMP2A), which was increased by PTZ treatment, was significantly reduced in the Eleu2+PTZ group administered with Eleutherine (Fig. 5b). This means that the administration of Eleutherine can restore the GPX4 protein expression reduced by PTZ by inhibiting the activation of chaperone-mediated autophagy (CMA).

[0121] In addition, it was confirmed that the expression levels of autophagy-related proteins p62 and LC3B II / I, which were increased by treatment with KA or PTZ in both the Eleu2+KA group and the Eleu2+PTZ group, were significantly suppressed (Figs. 4b, 4c, 5c, and 5d).

[0122] In other words, through the above experiment, it was confirmed that Eleutherine inhibits the Chaperone-Mediated Autophagy (CMA) pathway, thereby inhibiting GPX4 degradation and playing a role in restoring protein homeostasis and neuronal survival.

[0123] 3-2. Confirmation of the Inhibitory Effect on Ferroptosis by Modulation of the Chaperone-Mediated Autophagy (CMA) Pathway

[0124] HT-22 cells were pretreated with various concentrations (1, 5, 10 μM) of Eleutherine or Ferrostatin-1 (Fer-1, 1 μM), then 200 nM of RSL3 was added and cultured for 24 hours, after which immunofluorescence analysis was performed.

[0125] As a result, after RSL3 treatment, GPX4 protein expression decreased and the expression of the chaperone-mediated autophagy (CMA) related marker (LAMP2A) increased, confirming that GPX4 is degraded through the CMA pathway. On the other hand, when Eleutherine was treated, the expression of the chaperone-mediated autophagy (CMA) related marker (LAMP2A), which had increased due to RSL3 treatment, decreased, and the expression of GPX4 was restored (Fig. 6a).

[0126] Subsequently, the expression levels of LAMP2A and autophagy-related markers (p62 and LC3B II / I) were analyzed by Western blotting, and it was confirmed that the autophagy pathway is activated under ferroptosis conditions through the increased expression of LAMP2A and autophagy-related proteins (p62 and LC3B II / I) after RSL3 treatment. On the other hand, when Eleutherine was treated, it was confirmed that the expression levels of LAMP2A and autophagy-related proteins (p62 and LC3B II / I), which were increased by RSL3 treatment, were significantly reduced (Figs. 6b to 6e).

[0127] In summary, Eleutherine was found to maintain the stability of the GPX4 protein and inhibit ferroptosis by suppressing the chaperone-mediated autophagy (CMA) pathway involved in GPX4 degradation. From this, it was confirmed that Eleutherine acts very effectively in the prevention or treatment of epilepsy.

Claims

1. A pharmaceutical composition for the prevention or treatment of epilepsy, seizures, or convulsions, comprising eluterin or a pharmaceutically acceptable salt thereof as an active ingredient.

2. The composition according to claim 1, characterized in that the composition enhances the expression level of one or more selected from the group consisting of PSD-95, CRMP2, Synapsin-1, and GPX4.

3. The composition according to claim 1, characterized in that the composition reduces the expression level of one or more selected from the group consisting of HO-1, ACSL4, FTH1, LAMP2A, p62 and LC3B II / I.

4. The composition of claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of epilepsy.

5. A food composition for the prevention or improvement of epilepsy, seizures, or convulsions, comprising eluterin or a food-grade salt thereof as an active ingredient.

6. The composition according to claim 5, characterized in that the composition enhances the expression level of one or more selected from the group consisting of PSD-95, CRMP2, Synapsin-1, and GPX4.

7. The composition according to claim 5, characterized in that the composition reduces the expression level of one or more selected from the group consisting of HO-1, ACSL4, FTH1, LAMP2A, p62 and LC3B II / I.

8. In paragraph 5, the composition is a food composition for the prevention or improvement of epilepsy.

9. A method for the prevention or treatment of epilepsy, seizures, or convulsions comprising the step of administering a therapeutically effective amount of eruterin or a pharmaceutically acceptable salt thereof to an individual in need thereof.

10. In paragraph 9, the above method is a method for the prevention or treatment of epilepsy.

11. Use of eruterin or pharmaceutically acceptable salts thereof for the manufacture of medicines for the prevention or treatment of epilepsy, seizures or convulsions.

12. In paragraph 11, the above use is for manufacturing medicine for the prevention or treatment of epilepsy.

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