KR101711304B1 - A composition for preventing or treating status epilepticus comprising 3,3'-diindolylmethane, indole-3-carbinol, or mixture thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of epilepsy comprising 3,3'-diindolylmethane, its precursor indole-3-carbinol or a mixture thereof as an active ingredient; And food compositions for preventing or ameliorating epilepsy.
3,3'-diindolylmethane, indole-3-carbinol, or a mixture thereof, which is a precursor thereof, can be used without keratinocyte or pilocarpine-induced epileptic model in its own cytotoxicity It has neuroprotective effect and inhibits damage to synaptic contacts and nerve cell dendrites, thereby suppressing morphological changes of neurons. Therefore, it can be very useful for prevention and treatment of epilepsy.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating epilepsy, comprising 3,3'-diindolylmethane, indole-3-carbinol, or a precursor thereof, or a mixture thereof. diindolylmethane, indole-3-carbinol, or mixture thereof}

The present invention relates to a pharmaceutical composition for the prevention or treatment of epilepsy comprising 3,3'-diindolylmethane, its precursor indole-3-carbinol or a mixture thereof as an active ingredient; A method for treating epilepsy using the composition; To a food composition and a feed composition for preventing or ameliorating epilepsy.

Status epilepticus usually means that one episode lasts for more than 30 minutes, or that the episode is not recovered between episodes and that the episode repeats for 30 minutes or more, and that high mortality and permanent brain damage, (Epilepsy Behav., 2000, 1 (5), 301-14). Although epileptic seizures are stopped within minutes by the action of seizure suppression mechanism after seizure onset, the longer the epileptic seizure duration is, the higher the incidence of sequelae and the higher the mortality rate (Epilepsia, 1999, 40 (2), 164-9 ). Although more than 20% of epilepsy patients are refractory to the primary treatment of anticonvulsant drugs, the development of new treatments that can minimize effective treatments and neurological sequelae for the treatment of refractory epilepsy is still insufficient.

Among the epileptic patients, temporal lobe epilepsy is the largest part of the epileptic seizures compared with other local epilepsy. Pathological findings and therapeutic approach of temporal lobe epilepsy have been tried in many previous studies. Temporal lobe epilepsy models using chemoconvulsants such as kainic acid or pilocarpine have been studied (Epilepsy Res. 2002, 49, 109-120), it is known that epilepsy caused by kainic acid or pilocarpine induces neurotoxicity, gliosis, abnormally increased neurogenesis, and mossy fiber sprouting in the hippocampus (Brain Res., 1984,321, 237-253; Prog Brain Res., 2002, 135, 121-131).

Recently, research has been reported that novel nutrients that are being illuminated in food materials regulate various diseases including cancer. Among them, 3-carbinol (I3C) derived from plant chemical substance, 3 , 3'-diindoly methane (DIM) have been reported in the process of chewing and digesting cruciferous vegetables such as broccoli and cabbage, and anticancer activity, antioxidant activity, and anti-inflammatory activity have been reported , And epilepsy syndrome have not been reported.

Under these circumstances, the present inventors have made intensive efforts to find a substance having the effect of preventing and treating epilepsy caused by natural products. As a result, it has been found that 3,3'-diindolylmethane, its precursor indole- Or pilocarpine-induced epileptic seizure model, it has a neuroprotective effect without cytotoxicity itself, and inhibits morphological changes of neuronal cells, so that it can be very useful for prevention and treatment of epilepsy. And completed the present invention.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of epilepsy comprising 3,3'-diindolylmethane, its precursor indole-3-carbinol or a mixture thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or ameliorating epileptic seizure comprising 3,3'-diindolylmethane, indole-3-carbinol as its precursor or a mixture thereof as an active ingredient.

Another object of the present invention is to provide a feed composition for preventing or ameliorating epileptic seizure comprising 3,3'-diindolylmethane, indole-3-carbinol as its precursor or a mixture thereof as an active ingredient.

It is yet another object of the present invention to provide a method for treating epilepsy, comprising administering the pharmaceutical composition to a subject suspected of having epilepsy.

In order to achieve the above object, one aspect of the present invention is a pharmaceutical composition for preventing or treating epilepsy comprising 3,3'-diindolylmethane, its precursor indole-3-carbinol or a mixture thereof as an active ingredient Gt;

The above-mentioned 3,3'-diindolylmethane, indole-3-carbinol, or a mixture thereof has a neuronal cell protection effect without cytotoxicity itself in the epileptic neuropathy model, and inhibits damage to synapse contacts and nerve cell dendrites Inhibits morphological changes of neurons, and thus can be very useful for prevention and treatment of epilepsy.

The term "3,3'-diindolylmethane (DIM)" in the present invention refers to a compound represented by the following formula 1 to which two molecules of indole-3-carbinol are bonded or a pharmaceutically acceptable salt thereof: In the present invention, it is possible to protect neurons in the epileptic neuron model induced by kainic acid or pilocarpine and to inhibit morphological changes of neurons.

[Chemical Formula 1]

The term "indole-3-carbinol (I3C)" in the present invention means a compound represented by the following formula (2) as a precursor of the 3,3'-diindolylmethane or a pharmaceutically acceptable salt thereof In the present invention, it is possible to protect neurons in the epileptic seizure model induced by kainic acid or pilocarpine and to inhibit morphological changes of neurons.

(2)

The compositions of the present invention include all pharmaceutically acceptable salts of 3,3'-diindolylmethane and indole-3-carbinol as well as possible solvates and hydrates thereof which may be prepared therefrom, and all possible stereoisomers May also be included. Also, the solvates, hydrates and stereoisomers of the 3,3'-diindolylmethane and indole-3-carbinol can be prepared from the compounds shown in the above Formulas 1 and 2, respectively, using conventional methods.

The pharmaceutically acceptable salt is not particularly limited as it is commonly used in the art, such as an acid addition salt. Preferred pharmaceutically acceptable acid addition salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, orthophosphoric acid or sulfuric acid; Or organic acids such as, for example, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, acetic acid, propionic acid, lactic acid, citric acid, fumaric acid, malic acid, succinic acid, salicylic acid, maleic acid, glycerophosphoric acid or acetylsalicylic acid.

In addition, a pharmaceutically acceptable metal salt can be obtained by a conventional method using a base. For example, a compound represented by the formula (1) is dissolved in an excess of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, the non-soluble compound salt is filtered, and the filtrate is evaporated and dried to obtain a pharmaceutically acceptable metal salt. At this time, it is preferable to prepare metal salts, in particular, sodium salts, potassium salts or calcium salts, and these metal salts can be reacted with a suitable salt (for example, nitrate).

In the present invention, the above-mentioned 3,3'-diindolylmethane and indole-3-carbinol can be synthesized by a known synthesis method, separated or purified from plants, or obtained commercially .

The term "status epilepticus" in the present invention means a disease in which epileptic seizures appear repeatedly without restoring consciousness. Generally, one episode lasts for 30 minutes or more, Means a case having symptoms repeated for 30 minutes or more and can be induced by kainic acid or pilocarpine in an animal.

Specifically, the epileptic syndrome is accompanied by one or more seizures selected from the group consisting of simple partial seizures, complex partial seizures, secondary generalized seizures resulting from partial seizures, seizures, seizures, myoclonic seizures, and nonstress seizures But is not limited thereto.

The term "prophylactic" in the present invention refers to any act that inhibits or delays the onset of epilepsy by administering a composition according to the present invention.

The term "treatment" in the present invention refers to any action that improves or alters the symptoms of epilepsy by administration of the composition according to the present invention.

The composition may comprise a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not disturb the biological activity and properties of the compound being injected, without irritating the organism. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more.

The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

In particular, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition may be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, Activity, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. For example, the above-mentioned 3,3'-diindolylmethane, indole-3-carbinol or a pharmaceutically acceptable salt thereof may be used at a daily dose of 0.0001 to 1000 mg / kg, preferably 0.001 to 100 mg / kg Lt; / RTI >

Such administration means introducing the composition of the present invention to a patient in any appropriate manner, and the administration route of the composition can be administered through any conventional route as long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal administration.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or two or three times. When the two active ingredients are each monoglyceride, the number of administrations may be the same or different. In addition, the composition of the present invention can be used alone or in combination with other drug therapy for the prevention or treatment of epileptic seizures. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The term " individual " refers to all animals such as mice, mice, livestock and the like, including humans who have developed or are capable of developing epilepsy. Preferably, it may be a mammal, including a human.

In an embodiment of the present invention, in order to confirm neuronal protective effects of 3,3'-diindolylmethane and its precursor indole-3-carbinol in epileptic neuropathy model induced by kainic acid or pilocarpine, MTT The results of performing the assay (Figures 1 and 2), 3,3'-diindolylmethane, indole-3-carbinol, and mixtures thereof, show that in their epilepsy model, which is induced by chi- It was confirmed that the compound has a protective effect on nerve cells without toxicity, and thus it can be very useful for preventing or treating epilepsy.

Also, in one embodiment of the present invention, in order to confirm the therapeutic effect of 3,3'-diindolylmethane and indole-3-carbinol on epileptic seizure in an epileptic seizure model induced by kainic acid or pilocarpine, The results of the staining (Figs. 3 and 4), 3,3'-diindolylmethane, indole-3-carbinol and mixtures thereof show synaptic contacts and / or interactions in the epileptic seizure model induced by kainic acid or pilocarpine It has been confirmed that morphological changes of nerve cells are suppressed by inhibiting damage of nerve cell dendrites, and it can be very useful for prevention or treatment of epilepsy.

In one embodiment of the invention in vivo It was also found that pilocarpine induced epilepsy in the model (Fig. 5). In this model, 3,3'-diindolylmethane, indole-3-carbinol, and mixtures thereof have a neuroprotective effect and a morphological change inhibitory effect on nerve cells in a model in which epilepsy is induced by pilocarpine , It can be seen that it can be very useful for preventing or treating epilepsy.

Another embodiment of the present invention is a method for treating epilepsy comprising administering 3,3'-diindolylmethane, its precursor indole-3-carbinol, or a mixture thereof, to suspected epileptic children to provide. Specifically, the pharmaceutical composition of the present invention may be administered to a suspected epileptic disorder patient to treat epileptic seizure disorder.

The administration, epileptic syndrome and treatment are as described above.

Specifically, the method of treatment of the present invention comprises administering a pharmaceutically effective amount of 3,3'-diindolylmethane, its precursor indole-3-carbinol, or a mixture thereof, into suspected epileptic children. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by the above examples. The pharmaceutical compositions may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonary, and intranasally, and for localized immunosuppressive therapy, by a suitable method, including, if necessary, by intralesional administration. Non-oral injections may include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration, and specific intravenous, subcutaneous, intradermal, intramuscular and intradermal injections may be used. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

Another aspect of the present invention provides a food composition for preventing or ameliorating epilepsy comprising 3,3'-diindolylmethane, its precursor indole-3-carbinol, or a mixture thereof as an active ingredient.

The 3,3'-diindolylmethane, indole-3-carbinol, epileptic syndrome, and prevention are as described above.

The food composition may comprise a physiologically acceptable salt of 3,3'-diindolylmethane or indole-3-carbinol.

The term "physiologically acceptable " of the present invention is physiologically acceptable and when administered to an organism, the compound to be administered, without causing an allergic reaction such as gastrointestinal disorder, dizziness, or the like, Quot; means < / RTI >

The term "improvement" of the present invention means all actions in which administration of the composition of the present invention improves or alleviates epileptic seizure disorder.

The term "food" of the present invention is intended to encompass all kinds of foods, such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Vitamin complex, health functional food, and health food, all of which include foods in a conventional sense.

The term "functional food" as used herein is the same term as "food for special health use" (FoSHU). In addition to nutritional supplementation, functional foods are processed so as to efficiently show the biological control function, It means food. Here, the term "function (surname)" means that the structure and function of the human body have a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the formulations of the food can also be produced without restrictions as long as they are formulations recognized as food. The composition for food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has the advantage that there is no side effect that may occur when a drug is used for a long period of time, and is excellent in portability, Can be ingested as an adjuvant to enhance the effect of preventing or improving epilepsy.

The health food refers to a food having an active health promotion effect or a health promotion effect compared to a general food, and a health supplement food refers to a health supplement food. In some cases, the terms health functional foods, health foods, and health supplements are used.

Specifically, the health functional food may be prepared by adding 3,3'-diindolylmethane, indole-3-carbinol or a mixture thereof to a food material such as beverage, tea, spice, gum or confection, , Suspension, etc., which means that it has a certain health effect when ingested, but unlike general drugs, there is an advantage that there is no side effect that may occur when a drug is taken for a long time using a food as a raw material.

Since the food composition of the present invention can be routinely ingested, it is very useful because it can expect high epileptic seizure prevention or improvement effect.

The composition may further include a physiologically acceptable carrier. The carrier is not particularly limited and any carrier conventionally used in the art may be used.

In addition, the composition may contain additional ingredients which are commonly used in food compositions and which can improve odor, taste, vision and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like.

In addition, the composition can be used in combination with a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate), a disinfectant (such as bleaching powder and highly bleached white powder, sodium hypochlorite), an antioxidant (butylhydroxy anisole (BHA) (Sodium hypophosphate), bleach (sodium sulfite), seasoning (sodium MSG glutamate, etc.), sweeteners (hemicellulose, cyclamate, saccharin, A food additive such as a flavor (vanillin, lactones), a swelling agent (alum, D-tartrate, potassium hydrogen), an emulsifier, a thickening agent (glue), a covering agent, a gum base agent, a foam inhibitor, and food additives. The additives may be selected and used in appropriate amounts depending on the type of food.

When the 3,3'-diindolylmethane, indole-3-carbinol or a mixture thereof is used as a food additive, 3,3'-diindolylmethane, indole-3-carbinol or a mixture thereof Or may be used together with other food or food ingredients, and may be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). In general, the food composition of the present invention may be added in an amount of not more than 50 parts by weight, preferably not more than 20 parts by weight, based on the food or drink. However, in case of long-term ingestion for health and hygiene purposes, the active ingredient may be contained in an amount not exceeding the above range and there is no problem in terms of safety.

As an example of the food composition of the present invention, it can be used as a health beverage composition. In this case, various flavors or natural carbohydrates can be added as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjuster, stabilizer, Alcohols or carbonating agents, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The food composition of the present invention may be contained at various weight percentages as long as it can exhibit the effect of preventing or ameliorating epilepsy, but specifically, it may contain 3,3'-diindolylmethane, indole-3-carbinol, To 100% by weight or 0.01 to 80% by weight based on the total weight of the composition.

Another aspect of the present invention provides a feed composition for preventing or ameliorating epilepsy comprising 3,3'-diindolylmethane, indole-3-carbinol as a precursor thereof, or a mixture thereof as an active ingredient.

The 3,3'-diindolylmethane, indole-3-carbinol, epileptic syndrome, prevention and improvement are as described above.

Since the 3,3'-diindolylmethane or indole-3-carbinol has been used for a long time as a naturally-derived substance and has proven its safety, it is prepared in the form of a feedstuff which can prevent common epilepsy, It can be ingested.

In addition, the feed composition may comprise a physiologically acceptable salt of 3,3'-diindolylmethane or indole-3-carbinol.

In addition, the feed composition of the present invention may be a feed or feed additive for the purpose of preventing or improving epilepsy.

The term "feed" of the present invention is intended to mean any natural or artificial diet, single meal or the like, or a component of such an animal for eating, ingesting, digesting, The feed comprising the indole-3-carbinol as a precursor thereof or a mixture thereof as an active ingredient can be produced in various types of feed known in the art, and specifically, it can include a concentrated feed, a forage and / or a special feed have.

The above-mentioned 3,3'-diindolylmethane, its precursor, indole-3-carbinol, or a mixture thereof, contained in the feed composition of the present invention may be varied depending on the purpose and use conditions of the feed, May be included in an amount of 0.01 to 100% by weight, more specifically 1 to 80% by weight, based on the total weight of the composition.

The feed additive may be in a liquid or dry state, and in particular may be in the form of a dried powder. The drying method for preparing the feed additive of the present invention in the form of a dried powder is not particularly limited and a method commonly used in the art can be used. Non-limiting examples of the drying method include air drying, natural drying, spray drying, and freeze drying. These may be used alone or in a manner that uses two or more methods together.

The feed composition may further comprise other additives as required. Non-limiting examples of the above-mentioned usable additives include binders, emulsifiers, preservatives and the like to be added in order to prevent the deterioration of quality of the feed or feed additive; Flavoring agents, nonproteinaceous nitrogen compounds (non-protein nitrogenous compounds), silicates, buffers, coloring agents, extracting agents, or oligosaccharides, which are added for the purpose of increasing the efficiency of the feed or feed additive , A feed mixture, and the like. These may be used alone or two or more of them may be added together.

3,3'-diindolylmethane, its precursor indole-3-carbinol, or a mixture thereof, has neuroprotective effect without its own cytotoxicity in the epileptic neuritis model, and has the effect of damaging synaptic contacts and nerve cell dendrites And inhibit morphological changes of neurons, so that they can be very useful for the prevention and treatment of epilepsy.

FIG. 1 is a graph showing the effect of kainic acid (3'-diindolylmethane, DIM) or indole-3-carbinol (I3C) Lt; RTI ID = 0.0 >%)< / RTI >
FIG. 2 is a graph showing cell viability measured after Pilocarpine treatment on PC12 neurons pretreated with 3,3'-diindolylmethane (DIM) or indole-3-carbinol (I3C).
FIG. 3 shows the expression of PSD95 and MAP2 proteins by immunofluorescence staining after treatment with kainic acid in brain hippocampal neurons pretreated with 3,3'-diindolylmethane (DIM) or indole-3-carbinol (I3C) .
FIG. 4 shows the expression of PSD95 and MAP2 proteins by immunocytochemistry after treatment with phyllocarpine in brain hippocampal neurons pretreated with 3,3'-diindolylmethane (DIM) or indole-3-carbinol (I3C) .
FIG. 5 shows the induction of epileptic seizure by pilocarpine in vivo through the cresyl violet histochemical method (a) and the fluoro-jade B histochemical method (b), and the 3,3'-diindole Methane was pretreated to confirm the neuroprotective effect (DIM 125: 3,3'-diindolylmethane 125 mg / kg).
FIG. 6 is a graph showing the effect of 3,3'-diindole in the hilus portion of the hippocampus through the cresyl violet histochemical method (a) and the fluoro-jade B histochemical method (b) in an in vivo model of epileptic seizure by pilocarpine The neuroprotective effect of methane was also confirmed (DIM 125: 3,3'-diindolylmethane 125 mg / kg).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the embodiments and the like according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the above-described embodiments and the like. The embodiments of the present invention are provided by way of example in order to facilitate a specific understanding of the present invention.

Example  1. Cell culture

PC12 cells were cultured in RPMI 1640 medium, 10% FBS, penicillin / streptomycin / amphotericin B (100 U / ml, 100 ㎍ / ml, 0.025 ㎍ / ml, respectively)

Primary cultures of brain hippocampal neurons were obtained from 18-day-old Sprague-Dawley white rat embryos (Kim et al., J Neurosci. 28 (48): 12604-12613). First, the rats were anesthetized with 16.5% urethane (0.8 ml / 100 g) and the hippocampal tissues were incised in HEPES-buffered Hanks' salt solution (HHSS), pH 7.45, without Ca ^{2 +} and Mg ^{2 +} . The composition of HHSS was 20 mM HEPES, 137 mM NaCl, 1.3 mM CaCl 2, 0.4 mM MgSO 4, 0.5 mM MgCl 2, 5.0 mM KCl, 0.4 mM KHPO 4, 0.6 mM NaHPO 4, 3.0 mM NaHCO 3, and 5.6 mM glucose. After the incised horses were crushed with a flame-narrowed Pasteur pipette and then centrifuged, the pellet was washed with 2% B27 supplement, 0.25% Glutamax I, penicillin / streptomycin / amphotericin B (100 U / ml, 100 μg / ml, 0.025 μg / ml) containing Neurobasal medium (without L-glutamine) was added and mixed well. Separated hippocampal neurons were plated on a 25 mm round cover glass coated with Matrigel (0.2 mg / ml; BD Biocoscience) in an amount of 80,000. Then, neurons were cultured in an environment of 10% CO ₂ and 90% air, pH 7.4, 37 ° C, and 75% of the culture medium was changed after 3 days, 7 days and 10 days.

Example  2. MTT  Analysis method

Cells were seeded in 96 well plates and incubated in a cell incubator at 37 ° C in 5% CO ₂ and 95% air, pH 7.4. 3,3'-diindolylmethane (DIM) (10, 20, 40 μM) or a precursor thereof, indole-3-carbinol (I3C) 20, and 40 μM) containing DMEM containing 10% FBS and culture medium without DIM and I3C were pre-treated for 12 hours, respectively. Then, MTT (Choi et al., Br J Pharmacol, 162: 175-192, 2011) (0.5 mg / ml) was administered at 24 hours after treatment with 150 μM of kainic acid or 10 μM of pilocarpine, ml) was treated with 20 μl each. After incubation for 4 hours in a cell incubator, the absorbance was measured at 570 nm using a microplate reader (Bio-Rad).

Example  3. Immunofluorescent staining Immunohistochemistry )

Primary cultured brain hippocampal neurons were cultured and immunofluorescent staining (Choi et al., J Neurosci. 27 (11): 2999-3009, 2007) was performed 13 days later. Cells were first washed with phosphate-buffered saline (PBS) and fixed at -20 ° C with low-temperature methanol. Afterwards, the methanol was washed 3 times with PBS to wash, and permeabilized with 0.3% Triton X-100 for 5 minutes. After washing three times with PBS, the cells were blocked with 10% bovine serum albumin (BSA) at room temperature for one hour.

After blocking, the primary antibody was reacted at 4 ° C for 16 hours. The antibodies used were mouse anti-MAP2 (sigma-aldrich) to identify morphological changes in neurons and rabbit anti-PSD95 (Invitrogen) to identify synaptic contacts. In order to visualize immunostained neurons, they were reacted with Alexa Fluor 488 anti-rabbit IgG (in vitro) and Alexa Fluor 555 anti-mouse IgG (invitrogen) as secondary antibodies for 1 hour at room temperature. The neurons were then washed three times with PBS and coverslips were sealed using VECTASHIELD Mounting Medium (Vector Laboratories, Inc., Burlingame, Calif.). For cell observation, neurons stained with Alexa Fluor 488 (excitation, 488 nm; emission> 519 nm) and Alexa Fluor 555 (excitation, 555 nm; emission,> 565 nm) were observed using confocal microscopy.

Example  4. Confocal  Laser Microscope ( confocal imaging ) observe

The enclosed neurons were transferred to a retinometer of a confocal microscope (LSM 700, Carl Zeiss, Germany) and observed with a 20x objective lens (numerical aperture, 0.8). A total of 10 cross-sections were taken at 1 μm intervals on the z-axis of the cells using a z-stack imaging technique and one image was obtained by combining 10 cross sections. GFP is excited at 488 nm and emitted at 519 nm (10 nm bandpass) using an argon ion laser. RFP is excited at 555 nm and emitted at 565 nm using a HeNe laser.

Example  5. Animal experiments

Seven-week-old C57BL / 6J mice were orally administered 3, 3'-diindolylmethane 125 mg / kg three times at intervals of 8 hours, and after 1 hour of the last 3,3'-diindolylmethane treatment, Atropine 1.2 mg / kg < / RTI > for 30 minutes. Thereafter, pilocarpine 320 mg / kg was intraperitoneally administered, and the time for the behavioral change until the onset of epileptic seizure (Racine, 1972) was recorded between 4th and 5th stage and 6th stage. And the brain was excised 3 days after the onset of epileptic seizure.

Example  6. Cresyl  Violet( Cresyl violet ) Histochemical method

Cresyl violet experiments were performed to observe living cells. The tissue (PBS containing 30% sucrose) stored at -80 ° C was dissolved at 4 ° C, transferred to a gelatin-coated slide, and dried at room temperature for one day. After hydration with 95%, 90%, 80% and 70% ethanol, the cells were washed 3-4 times with a third order DW. Then, it was treated with 0.3% warm-up 0.3% cresyl violet (Sigma, C5042) solution for 20 minutes and then treated with 0.3% glacial acetic acid for 5 minutes. Dehydrated with 95% and 100% ethanol, treated with xylene and sealed with DPX (sigma-aldrich, 06522) using cover slip.

Example  7. Fluoro -jade B Histochemical method

Fluoro-jade B experiments were performed to observe dead or dying cells. The tissue (PBS containing 30% sucrose) stored at -80 ° C was dissolved at 4 ° C, transferred to a gelatin-coated slide, and dried at room temperature for one day. The fully dried tissue was hydrated to 100% and 70% and then treated with 80% EtOH containing 1% NaOH. Then 0.06% potassium permanganate was treated at room temperature for 10 minutes and 0.001% Fluoro-jade B (millipore, AG310) at room temperature for 20 minutes. After washing with 3-order DW, the tissue was dried in a dryer for 30 minutes and then sealed with cover slip with DPX (sigma-aldrich, 06522).

Experimental Example  One. In vitro Epilepsy  In the model, 3,3'- Diindolylmethane  And indole-3-carbinol

Experimental Example  1-1. On kainic acid  Induced by Epilepsy  In the model, 3,3'- Diindolylmethane  And indole-3- Carbinol PC12  Nerve cell protection effect

The effect of 3,3'-diindolylmethane and indole-3-carbinol on neuronal cell protection was confirmed by MTT assay in an epileptic overexpression model induced by kainic acid.

First, PC12 neurons were pretreated with 10, 20, and 40 μM of 3,3'-diindolylmethane or indole-3-carbinol, respectively, for 12 hours, treated with 150 μM of kainic acid, Survival rate (%) was measured (Fig. 1).

As a result, the survival rate of PC12 neurons was remarkably decreased by treatment with kainic acid, whereas when 3,3'-diindolylmethane or indole-3-carbinol was pretreated and treated with kainic acid, The survival rate was similar to that of the untreated case. That is, pretreatment of 3,3'-diindolylmethane or indole-3-carbinol inhibited the decrease of cell viability due to treatment with kainic acid.

In addition, even when 3,3'-diindolylmethane or indole-3-carbinol is treated, the survival rate of PC12 neurons is similar to that of 3,3'-diindolylmethane or indole- Or indeed increased, 3,3'-diindolylmethane or indole-3-carbinol protects nerve cells from cytotoxicity induced by kainic acid while not exhibiting cytotoxicity.

It has been confirmed that 3,3'-diindolylmethane, indole-3-carbinol and mixtures thereof have neuroprotective effects without cytotoxicity in their epileptic model induced by kainic acid, It can be very useful for prevention and treatment.

Experimental Example  1-2. Filocarpine  Induced by Epilepsy  In the model, 3,3'- Diindolylmethane  And indole-3- Carbinol PC12  Nerve cell effect

The protective effect of 3,3'-diindolylmethane and indole-3-carbinol on neurons in the pilocarpine-induced epileptic seizure model was confirmed by MTT assay.

First, PC12 neurons were pretreated with 10, 20, and 40 μM of 3,3'-diindolylmethane or indole-3-carbinol for 12 hours, treated with 10 μM of pilocarpine, Cell viability (%) was measured (Figure 2).

As a result, the survival rate of PC12 neurons was markedly decreased by pilocarpine, whereas pretreatment of 3,3'-diindolylmethane or indole-3-carbinol and treatment of pilocarpine resulted in the formation of pilocarpine The survival rate was similar to that of the untreated case. That is, pretreatment of 3,3'-diindolylmethane or indole-3-carbinol inhibited the decrease of cell viability due to the treatment with pilocarpine.

In addition, even when 3,3'-diindolylmethane or indole-3-carbinol is treated, the survival rate of PC12 neurons is similar to that of 3,3'-diindolylmethane or indole- Or indeed increased, 3,3'-diindolylmethane or indole-3-carbinol was found to protect nerve cells from cytotoxicity induced by pilocarpine without exhibiting cytotoxicity.

It has been confirmed that 3,3'-diindolylmethane, indole-3-carbinol and mixtures thereof have a neuronal cell protection effect on its own cytotoxicity in a pilocarpine-induced epileptic seizure model, It can be very useful for prevention and treatment of complications.

Experimental Example  1-3. On kainic acid  Induced by Epilepsy  In the model, 3,3'- Diindolylmethane  And indole-3- Carbinol Brain hippocampus  Nerve cell protection effect

The effect of 3,3'-diindolylmethane and indole-3-carbinol on neuronal cell protection was confirmed by immunofluorescence staining in an epileptic model induced by kainic acid.

First, brain hippocampal neurons cultured in primary culture were stained with immunofluorescent staining 4 hours after treatment with kainic acid, and the stained neurons were observed using a confocal microscope (FIG. 3).

As a result, it was confirmed that the expression of PSD95, a protein representing the synaptic contact, was reduced in the hippocampal neurons treated with kainic acid, so that synaptic damage occurred before neuronal death.

In addition, the expression pattern of MAP2, a dendritic protein of neurons in kainic acid-treated brain hippocampal neurons was observed. As a result, morphological analysis such as atrophy of dendrites, reduction of dendrites, pruning, The change was confirmed.

Accordingly, brain hippocampal neurons were pretreated with 20 μM of 3,3'-diindolylmethane or 20 μM of indole-3-carbinol for 12 hours, treated with 150 μM of kainic acid and observed neurons after 4 hours As a result, it was confirmed that the expression level of PSD95 was similar to that of control neurons, and the expression of MAP2 was inhibited by kainic acid, and compared with the control and kainic acid-treated groups, And morphological changes such as pruning were suppressed.

Thus, 3,3'-diindolylmethane, indole-3-carbinol, and mixtures thereof inhibit damage to synaptic contacts and neuronal dendrites in an epileptic overexpression model induced by kainic acid, And it is found that it can be very useful for prevention and treatment of epilepsy.

Experimental Example  1-4. Filocarpine  Induced by Epilepsy  In the model, 3,3'- Diindolylmethane Brain hippocampus  Nerve cell protection effect

The protective effect of 3,3'-diindolylmethane on neurons in the pilocarpine-induced epileptic seizure model was confirmed by immunofluorescence staining.

First, the primary cultured brain hippocampal neurons were stained with immunofluorescent staining 4 hours after the treatment with pilocarpine, and the stained neurons were observed using a confocal microscope (FIG. 4).

As a result, it was confirmed that the expression of PSD95, a protein representing the synaptic contact, was reduced in pilocarpine-treated brain hippocampal neurons, and it was found that synaptic damage occurred before neuronal death.

In addition, morphological changes such as atrophy of dendrites, decrease of dendrites, and pruning were observed by observing the expression patterns of MAP2, a dendritic protein of neurons, in the pilocarpine-treated brain hippocampal neurons.

In addition, brain hippocampal neurons were pretreated with 20 μM of 3,3'-diindolylmethane for 12 hours and treated with 10 μM of pilocarpine. After 4 hours, neurons were observed and the expression level of PSD95 was observed as a control Similar to normal neurons, damage to synaptic contacts by pilocarpine was suppressed. MAP2 expression patterns were compared with those of the control and pilocarpine-treated groups, indicating atrophy of dendrites, reduction of dendrites, and pruning Morphological changes were suppressed.

It has been confirmed that 3,3'-diindolylmethane inhibits neuronal morphological changes by inhibiting damage to synaptic contacts and nerve cell dendrites in a pilocarpine-induced epileptic model, And it can be very useful for preventing or treating complications.

Experimental Example  2. In vivo Epilepsy  In the model, 3,3'- Diindolylmethane  effect

Experimental Example  2-1. Filocarpine  Mouse over Epilepsy  Modeling

30 minutes after injection of 1.2 mg / ml of atropine methyl nitrate into male C57BL / 6J mice at 7 weeks of age, 320 mg / ml of pilocarpine hydrochloride was intraperitoneally injected.

Of the five seizures (Racine, 1972), four stages [rearing and falling], five stages (balance loss, sustained rise and fall); Or severe tonic-clonic seizures], and that the animals in which the above steps were observed were defined as animals exhibiting epileptic seizures.

To record the duration of epileptic syndrome and to help the recovery of experimental animals after the symptom ends, from the first day after the end of epileptogenesis in the optimal experimental environment, the saline containing 10% sucrose is dipped in the food depending on the condition of the experimental animals Respectively.

The cells of the mouse designated as the above-mentioned epileptic seizure-expressing animal were observed through cresyl violet histochemistry and Fluoro-jade B histochemistry, and as a result, in In vivo , neurotoxicity induced by pilocarpine - induced epilepsy was induced.

Experimental Example  2-2. Filocarpine  by in vivo Epilepsy  In the model, 3,3'- Diindolylmethane  effect

When oral administration of 3,3'-diindolylmethane (250 mg / kg) to mice is approximately 5-36.5 μM (Drug Metab Dispos., 2004, 32, 632-638). When pharmacokinetic considerations were taken, in vivo , 3, 3'-diindolyl methane 125-250 mg / kg was pretreated three times at intervals of 8 hours and 10-40 μM in vitro (Acta Pharmacol Sin., 2007, 28, 1274-1304). In this experiment, significant results were obtained in the group pretreated with 125 mg / kg of 3,3'-diindolylmethane, and the group treated with 125 mg / kg of 3,3'-diindolylmethane and the group treated with pilocarpine Were compared by cresyl violet histochemistry and Fluoro-jade B histochemistry.

As a result, it was confirmed that epileptic seizure was induced by pilocarpine in vivo and that 3,3'-diindolylmethane had a neuroprotective effect even in vitro (FIG. 5).

The viable cells of the CA1 and CA3 regions of the hippocampus shown in FIG. 5A, which were confirmed by the cresyl violet histochemical method, are shown in Table 1 below. As a result, it was confirmed that the group pretreated with 3,3'-diindolylmethane had more living cells in the CA1 and CA3 regions than the group treated with only pilocarpine.

The dead or dead cells of the hippocampal CA1 and CA3 regions as shown in Fig. 5B, which were confirmed by Fluoro-jade B histochemistry, are shown in Table 2. As a result, it was confirmed that the group pretreated with 3,3'-diindolylmethane had fewer dead or dying cells in the CA1 and CA3 regions than the group treated with pilocarpine alone.

In addition, the number of living cells (FIG. 6A) and dead or dying cells (FIG. 6B) in the hilus region of the hippocampus was confirmed, and as a result of the CA1 and CA3 regions, 3,3'-diindolylmethane was pretreated Group showed more viable cells and fewer dead or dying cells than the group treated with pilocarpine alone.

Thus, in vivo by pilocarpine In the epileptic seizure model, 3,3'-diindolylmethane was found to have protective effect on neurons, and it could be very useful for prevention and treatment of epilepsy.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (8)

3,3'-diindolylmethane, indole-3-carbinol as a precursor thereof, or a mixture thereof as an active ingredient.
8. The method of claim 1, wherein the epileptic syndrome is a secondary generalized seizure resulting from a simple partial seizure, a complex partial seizure, or a partial seizure; Sacking; Great seizure; Myocardial infarction, myocardial infarction, myocardial infarction seizure and nonstress seizure.
2. The pharmaceutical composition according to claim 1, wherein the epileptic syndrome is induced by kainic acid, pilocarpine or a combination thereof.
The pharmaceutical composition according to claim 1, wherein the composition inhibits damage to synapse contacts or nerve cell dendrites.
The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable diluent, excipient or carrier.
3,3'-diindolylmethane, indole-3-carbinol as a precursor thereof, or a mixture thereof as an active ingredient.
3,3'-diindolyl methane, its precursor indole-3-carbinol, or a mixture thereof as an active ingredient.
A method for treating epileptic seizure comprising administering a composition according to any one of claims 1 to 5 to a subject other than a human with suspected epileptic seizure.

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