KR20190081385A - Composition for preventing the ischemia stroke disease containing oxcarbazepine - Google Patents

Abstract

The present invention relates to a composition containing oxcarbazepine as an active ingredient for preventing ischemic cerebral diseases. The oxcarbazepine is not only a material having safety secured as a drug used as an existing agent for treating epilepsy, but also a material having a remarkably excellent protective effect of nerve cells and inhibitory effect of nerve cell death compared to the existing agent for treating epilepsy while having an excellent activation inhibitory effect of neuroglia cell. Further, according to a result of experiment on animals, the composition has a remarkably excellent effect of preventing cerebral ischemia through administration before cerebral ischemia, compared to administration for treatment after cerebral ischemia occurs, and is capable of effectively preventing neural damage of a CA1 area of hippocampal brain tissue known to be sensitive to cerebral ischemia, thereby being useful for effectively preventing ischemic cerebral diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient,

The present invention relates to a pharmaceutical composition for the prevention of ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient.

With the recent improvement in the economic level, the elderly population is rapidly increasing worldwide due to the development of medical and life sciences. According to the 'Social Indicators of Korea in 2015' published by the National Statistical Office in 2016, it is said that Korea will enter the 'super aging society' over 52 years of age by 2040, The ratio is expected to increase steadily to 14% this year, 24.3% in 2030, 32.3% in 2040 and 40.1% in 2060.

With the aging of the population, the incidence and prevalence of various geriatric diseases due to aging are increasing. Especially, the central nervous system (CNS) diseases including brain or spinal injuries are continuously increasing. The most prevalent CNS disease is cerebrovascular disease, which is the number one disease death rate, and 60,000 cases are occurring in Korea every year.

The cerebrovascular disease is a typical geriatric disease. It is also called a stroke, and is a disease caused by clogging or bursting of blood vessels in the brain. The cerebrovascular disease is characterized in that blood vessels in the brain are clogged or burst, and the circulation of a certain part of the brain suddenly stops due to abnormal cerebral blood flow, resulting in loss of function of the corresponding nerve in the corresponding part.

The cerebrovascular disease is considered to be the first cause of permanent disability such as muscle paralysis, consciousness disorder, visual impairment, and cognitive dysfunction, unlike other diseases in which survival deficit is restored. Although the demand for development is increasing, it is known that a large number of patients are recurred after treatment, resulting in huge social and economic losses.

The cerebrovascular disease is largely caused by a hemorrhagic stroke caused by a blood vessel supplying blood to the brain due to an impact such as a traffic accident, a temporary blood flow is blocked by a thrombus, a decrease in perfusion, There are two types of ischemic stroke that occur because the supply of nutrients is not done.

The stroke can cause hemiplegic paralysis, speech disturbances, loss of memory and reasoning ability, coma depending on the area of the damaged brain, and even death in severe cases.

In recent years, the incidence of ischemic cerebrovascular disease has been continuously increasing in Korea compared with hemorrhagic cerebrovascular disease due to westernization and lifestyle of dietary habits. According to one study, ischemic cerebrovascular disease accounts for about 80% of all cerebrovascular disease types.

As described above, an ischemic stroke having a high incidence of cerebrovascular disease has a problem that clots generated in the cardiovascular system, such as thrombosis and other wastes, are occluded in the cerebral blood vessels, or blood is not supplied to the brain in a pathological condition such as cardiac arrest, (Mongolian gerbil). In the transient forebrain ischemic model, 5-minute induction of cerebral ischemia was observed in the hippocampus (4 days after induction of cerebral ischemia) (Kirino, Brain Res., 239, pp. 57-69, 1982), which is known to be a neuronal apoptosis in CA1 (conus ammonis 1) region of hippocampus.

Previously, many mechanisms have been suggested for neuronal death due to cerebral ischemia. Specific mechanisms include excitotoxicity due to excessive increase of glutamate, oxidative stress due to increased reactive oxygen species, activation of neuroglial cells and invasion of peripheral inflammatory cells Inflammatory response is known (Mitani et al., Neuroscience., 42, pp 661-670, 1991, Chuetal., JNeuroinflammation., 9, pp 69, 2012; Wang et al., FreeRadicBiolMed., 43, 43 - 47, 2016-05-04 pp1048 - 1060, 2007), but the precise mechanism of neuronal death due to cerebral ischemia has not been elucidated, and the hypothesis about the new mechanism is being continuously presented.

Therefore, studies for treating ischemic cerebrovascular disease are currently proceeding in a direction to effectively inhibit neuronal cell death induced by cerebral ischemia based on the above-described mechanism. In this respect, It is distinguished from the degenerative cerebrovascular disease treatment which treats the loss of nerve cell or the disappearance or inhibition of neural differentiation.

At present, nimodipine, flunarizine and glutamate antagonist MK-801, dextraphan, and tirilazad, a free radical scavenger, are the calcium channel antagonists. Although it has been developed as a protective agent, it has been proved that nerve cell protection efficacy has been confirmed through preclinical studies, but it is difficult to be used as a drug for clinical use as a cerebral ischemia treatment agent due to lack of efficacy and side effects in large clinical trials.

In order to develop a drug that can effectively inhibit neuronal cell death due to a safe cerebral ischemia, it is necessary to go through the preclinical and final clinical trial stages starting from the search for candidate substances, so that time and cost in terms of time and cost Is required.

Therefore, there is an increasing need for studies on substances that can inhibit or delay the death of brain cells due to ischemia, among the substances that are recognized as safe until now.

KR 10-1129303B KR 10-1132378B KR 10-1246169B KR 10-2007-0085309A KR 10-2017-0048602A

It is an object of the present invention to provide a pharmaceutical composition or a health food containing an effective ingredient for inhibiting or delaying ischemic neuronal cell death and effective for preventing ischemic cerebrovascular disease.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention of ischemic cerebrovascular disease. Specifically, the pharmaceutical composition for preventing ischemic cerebrovascular disease may contain oxcarbazepine as an active ingredient. The oxcarbazepine may be a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

The pharmaceutical composition for preventing ischemic cerebrovascular disease may be a composition for preventing ischemic cerebrovascular disease, which is selected from the group consisting of thrombosis, embolism, transient ischemic attack, subarachnoid hemorrhage, and small infarction.

The oxcarbazepine, which is an active ingredient of the pharmaceutical composition for the prevention of ischemic cerebrovascular disease, may be characterized in that it protects neurons in the brain hippocampal CA1 region.

In addition, the pharmaceutical composition for preventing ischemic cerebrovascular disease may contain 0.001 to 99.999% by weight of the oxcarbazepine, which is an active ingredient, based on the total weight of the composition.

Further, the present invention can be a food composition for preventing ischemic cerebrovascular disease using oxcarbazepine as an active ingredient. The oxcarbazepine may be a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

The food composition for preventing ischemic cerebrovascular disease may be a health functional food.

In addition, the food composition for preventing ischemic cerebrovascular disease may contain 0.001 to 49.999% by weight of the oxcarbazepine, which is an active ingredient, based on the total weight of the composition.

Oxcarbazepine, which is an active ingredient of the present invention, has been used as a therapeutic agent for brain tumors and has been confirmed to have no side effects or resistance through strict clinical trials. Therefore, not only is safety recognized, but also, The oxcarbazepine is effective in preventing or ameliorating ischemic cerebrovascular disease, specifically preventing ischemic cerebrovascular disease due to protection of cerebral nerve cells and delayed death by ischemic conditions, And can be widely used to prevent death or disorder caused by ischemic cerebrovascular disease in a modern society in which circulatory diseases due to nutritional excess are soaring.

FIG. 1 is a photograph showing a result of staining a hippocampal tissue of a cerebral ischemia-inducing animal with a cresyl violet in order to examine the effect of oxcarbazepine to prevent ischemic brain disease according to an embodiment of the present invention.
FIG. 2 is a photograph showing a CA1 region of a hippocampal tissue of a cerebral ischemia-inducing animal to be stained with a cresyl violet in order to examine the effect of oxcarbazepine to prevent ischemic brain disease according to an embodiment of the present invention .
FIG. 3 is a graph showing the effect of a mouse anti-neuronal nuclei (NeuN) antibody, which is known as a neuron marker, on the hippocampal tissue of an experimental animal for inducing cerebral ischemia in order to examine the effect of oxcarbazepine to prevent ischemic brain disease according to an embodiment of the present invention Immunohistochemistry This is a photograph of the result of staining by staining method.
FIG. 4 is a graph showing the results of immunohistochemical staining of the CA1 region of hippocampal tissues of brain ischemia-induced experimental animals using NeuN antibody in order to examine the effect of oxcarbazepine to prevent ischemic brain diseases according to an embodiment of the present invention .
FIG. 5 is a graph showing the results of counting neurons showing immunoreactivity to NeuN antibodies in the CA1 region of hippocampal tissues of cerebral ischemia-induced experimental animals in order to examine the effect of oxcarbazepine to prevent ischemic brain diseases according to an embodiment of the present invention. Is a graph showing the relative values for the normal group (Sham).
FIG. 6 is a graph showing the effect of oxcarbazepine on the inhibition of ischemic cerebropathy of the present invention by measuring the CA1 region of the hippocampal tissue of the cerebral ischemia- This is a photograph of the result of staining with tissue fluorescence using fluoro-jade B (FJ B).
FIG. 7 is a graph showing the results of counting fluoro-jade B-labeled neurons in the CA1 region of the hippocampal tissue of a cerebral ischemia-induced experimental animal in order to examine the effect of oxcarbazepine to prevent ischemic brain diseases according to an embodiment of the present invention. Is a graph showing the relative values for the normal group (Sham).
FIG. 8 is a graph showing the effect of oxcarbazepine on the ischemic cerebrovascular proliferation according to an embodiment of the present invention. In order to examine the effect of oxcarbazepine on the ischemic stroke, Immunohistochemical staining method using antibody. This photograph shows the result of staining.
9 is a graph showing the results of counting cells showing immunoreactivity to GFAP antibody in the CA1 region of hippocampal tissues of cerebral ischemia-induced experimental animals in order to examine the effect of oxcarbazepine to prevent ischemic cerebrovascular disease according to an embodiment of the present invention. Is a graph showing relative numerical values for the sham.
FIG. 10 is a graph showing the effect of oxcarbazepine on the ischemic cerebrovascular prophylaxis according to an embodiment of the present invention. In order to examine the effect of oxcarbazepine on the ischemic stroke, (Iba-1) antibody for immunohistochemical staining.
11 is a graph showing the results of counting cells showing immunoreactivity to Iba-1 antibody in the CA1 region of hippocampal tissues of cerebral ischemia-induced experimental animals in order to examine ischemic cerebrovascular prophylaxis effect of oxcarbazepine according to an embodiment of the present invention As a relative value to the normal group (Sham).

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless otherwise specified herein, a composition is meant to encompass a product that contains the specified ingredients in the specified amounts, as well as the product that results, directly or indirectly, from the combination of the specified ingredients in the specified amounts.

As used herein, the term "effective ingredient" refers to a composition, specifically a pharmaceutical composition or food composition, that is effective in preventing or ameliorating ischemic cerebrovascular disease, specifically, ischemic or delayed neuronal cell death and activation of neuroglial cells Means a component that can be used.

As used herein, unless otherwise stated, an effective amount means an amount of a compound or composition of the present invention effective to produce the desired therapeutic, alleviating, inhibiting or preventive effect.

In the present invention, ischemic brain disease refers to a term collectively referred to as a cerebral ischemic disease, which is a term that occurs when blood vessels of the brain are blocked for some reason and the blood required for the brain is not supplied. The ischemic brain disease is a disease caused by delayed neuronal cell death in the neuron of the hippocampal CA1 region, which is known to be sensitive to cerebral ischemia in brain cells because the blood flow of the brain is decreased and oxygen and glucose are not normally supplied. . Examples of ischemic cerebrovascular diseases include ischemic cerebrovascular disease, ischemic cerebral infarction, and the like.

The inventors of the present invention have found that a substance having excellent neuronal cell protection and excellent inhibitory activity on neurite outgrowth is excellent in treating or preventing damage to nerve cells and is useful for treating diseases related thereto, specifically, ischemic brain diseases such as ischemic cerebrovascular disease Or preventive effects, and that the substances used as conventional drugs are substances that have been confirmed to be safe for side effects or immunity through strict clinical trials. In studying substances that inhibit glutamate activation, drugs used as drugs for treatment of epidemic diseases through animal experiments, specifically, Perampanel, Levetiracetam, Retigabine, Oxcarbazepine Oxcarbazepine), particularly oxcarbazepine, Animal studies have confirmed that neuroprotective effect and neuroprotective effect of neurons are superior in the CA1 region of brain hippocampal tissue which is known to be sensitive to cerebral ischemia through inhibition of delayed neuronal cell death. It has been confirmed that administration of a prophylactic agent prior to the occurrence of an ischemic condition is remarkably superior to administration of a therapeutic agent after ischemia to prevent neuronal damage due to cerebral ischemia. It was completed.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a pharmaceutical composition for the prevention of ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient.

The oxcarbazepine may be a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

Specifically, the oxcarbazepine is known to inactivate the sodium channel as an antiepileptic agent or an antiepileptic agent, like other antiepileptics. In addition, the oxcarbazepine has a function of inhibiting the generation of action potential by inactivating the sodium channel involved in excitable cells.

In the present invention, nebulis administered with the above-mentioned oxcarbazepine anthelmintic agents pelampanel, levitiracetam and retigavine for a certain period of time are anesthetized to induce cerebral ischemia by ligation of the common carotid artery, and then the surviving neurons are stained and killed As a result of conducting an experiment to measure the efficacy through the staining of neurons, it was found that oxcarbazepine was remarkably superior in protecting neurons when administered before cerebral ischemia, , And it was confirmed to protect neurons in the CA1 region of the brain hippocampal tissue. From these results, it was estimated that oxcarbazepine could effectively prevent ischemic cerebrovascular disease.

The composition for preventing ischemic stroke comprising oxcarbazepine as an active ingredient can be directly applied to animals including humans.

The composition for preventing ischemic stroke comprising the oxcarbazepine as an active ingredient may further comprise a substance having oxcarbazepine as an active ingredient alone or a substance having an effect of treating or preventing ischemic cerebrovascular disease in the oxcarbazepine as an active ingredient And may further comprise additional components, that is, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or subcomponents, depending on the formulation, the method of use and the intended use.

More specifically, the composition for preventing ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient may further comprise, in addition to the active ingredient, a nutritional agent, a vitamin, an electrolyte, a flavoring agent, a colorant, a thickening agent, a pectic acid or a salt thereof, And a salt thereof, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like. The carrier, excipient or diluent may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acicia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline Wherein the surfactant is selected from the group consisting of cellulose, cellulose, cellulose, cellulose, cellulose, cellulose, cellulose, cellulose, cellulose acetate, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, But not limited thereto, and any conventional carrier, excipient or diluent can be used. The components may be added to the active ingredient, i. E., Oxcarbazepine, either alone or in combination.

The composition for preventing ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient may contain 0.001% by weight to 99.999% by weight, preferably 0.1% by weight to 10% by weight, of the effective ingredient, specifically, the oxcarbazepine, 99% by weight, and more preferably 1% by weight to 50% by weight. The content of the additional component may be added in the range of 0.1% by weight to 20% by weight based on the total weight of the composition for preventing ischemic cerebrovascular disease, but is not limited thereto.

When the composition for the prevention of ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient is weakly formulated, it may be formulated with conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, Preservatives, and the like, and they can be used either orally or parenterally.

Particularly, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by mixing the above active ingredients, that is, oxcarbazepine with at least one excipient such as starch, calcium carbonate (calcium carbonate), sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations 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.

The formulation of a composition for the prevention of ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient may be in a form suitable for the method of use and may provide an immediate, sustained or delayed release of the active ingredient after administration to a mammal By a method known in the art. Examples of the specific formulations include granules, powders, syrups, liquids, suspensions, tablets, injections, main tablets, cataplasma, capsules, soft or hard gelatin capsules and the like.

Furthermore, a composition for the prevention of ischemic cerebrovascular disease comprising the above oxcarbazepine as an active ingredient can be prepared by using a suitable method known in the art or by a method described in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA ). ≪ / RTI >

The dose of the composition for preventing ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient according to the present invention may be suitably selected by those skilled in the art in consideration of the administration method, the age, sex, and weight of the recipient, . For example, the composition for preventing ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient may be used in an amount of 0.0001 mg / kg to 1000 mg / kg based on the above-mentioned effective ingredient per weight of an administration subject, 0.01 mg / kg to 100 mg / kg or 30 mg / kg to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In addition, the composition for preventing ischemic cerebrovascular disease comprising the oxcarbazepine of the present invention as an active ingredient may further comprise a compound having a therapeutic or preventive effect for known ischemic cerebrovascular disease or an extract for a natural product, in addition to the above- And may be included in an amount of 5 to 200 parts by weight based on 100 parts by weight of the active ingredient.

The composition for preventing ischemic cerebrovascular disease according to another embodiment of the present invention contains oxcarbazepine as an active ingredient.

In this respect, the present invention relates to a food composition for the prevention of ischemic brain diseases comprising oxcarbazepine as an active ingredient.

The term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, Beverages, food additives, beverage additives, and the like. In this respect, the food composition for the prevention of ischemic brain diseases may be a health functional food for the prevention of ischemic brain diseases.

Such foods include, for example, various foods, beverages, gums, tea, vitamin complexes, and health functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, fruits, vegetables, beverages, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.).

The food, the health functional food, the beverage, the food additive and the beverage additive can be produced by a usual production method.

The health functional food refers to a food group that imparts added value to the function of the food by using physical, biochemical, biotechnological, and the like to control the biofunctional rhythm of the food composition, prevention and recovery of the disease Means a food which is processed and designed so that the body controlling function of the body is sufficiently expressed to the living body.

The health functional food may include food-acceptable food supplementary additives, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

The drink refers to a generic term for eliminating thirst or drinking to enjoy a taste, and is intended to include a health functional drink. The beverage is not particularly limited to the ingredients other than the oxcarbazepine as an active ingredient in the indicated ratio as an essential ingredient and may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient have.

Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and Xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate may be generally about 1 to 20 g, preferably 5 to 12 g, per 100 ml of the food composition of the present invention. In addition, the composition of the present invention may be used for natural fruit juice, fruit juice drink, And may further contain pulp for the production of beverages.

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 2,000 parts by weight per 100 parts by weight of the oxcarbazepine of the present invention.

The health-functional beverage is used to control the bio-defense rhythm of the beverage group or beverage composition to which the added value is imparted so that the function of the beverage functions for a specific purpose by physical, biochemical, biotechnological, or the like, Means a beverage which has been designed so as to sufficiently express the body controlling function related to the living body.

The health functional beverage is not particularly limited to the ingredients other than oxcarbazepine of the present invention as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages .

Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol , Sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention.

The active ingredient may be used in an amount of 0.001 to 49.999% by weight or 0.01% to 25% by weight or 0.1% by weight, based on the total weight of the whole food composition, of the composition for preventing ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient, To 10% by weight, and the beverage composition may be included in a proportion of 0.02 g to 5 g, or 0.3 g to 1 g based on 100 ml.

The oxcarbazepine of the present invention significantly inhibits neuronal cell death in the CA1 region of the hippocampus caused by cerebral ischemia induced by pre-administration of ischemic cerebrovascular disease, in particular, before cerebral ischemia induction, It has been experimentally confirmed that the oxcarbazepine is able to inhibit or prevent neuronal damage due to cerebral ischemia and thus is remarkably excellent in the effect of preventing ischemic cerebrovascular disease.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein.

Example  1: through experimental animals Oxcabazepine Cerebral ischemia  Nerve cell protective efficacy Measure 1

(Mongolian gerbil, Harlan, USA), weighing 65g to 75g, to induce cerebral ischemia after ligation of the common carotid artery, followed by staining and observing the hippocampal tissues of Mongolian jerville The effect of oxcarbazepine on neuronal death due to cerebral ischemia was confirmed.

Example  1-1. Breeding of experimental animals

Sixty-six male Mongolian gerbils (Meriones unguiculatus Harlan, USA) weighing between 65 g and 75 g at 6 months of age were irradiated with a constant light period from 7 am to 7 pm, a temperature of 21 ° C to 25 ° C, And 65% relative humidity. For the experimental animal feed, the usual pellet dried feed (Korean Experimental Animals, Korea) was used, and feed and water were made available at all times.

Examples 1-2. Measurement of neuroprotective effect of oxcarbazepine due to cerebral ischemia

In order to confirm neuroprotective effect of oxcarbazepine, more specifically, to prevent or prevent neuronal cell death due to cerebral ischemia, pelampanel, levitiracetam, retigavine and the like were administered to the experimental animals raised in Example 1-1 Oxcarbazepine was intraperitoneally administered in a physiological saline containing 10% Tween 80 so as to be 100 mg or 200 mg per 1 kg body weight of the experimental animals. The duration of administration of the drugs was administered daily for 3 days before or after induction of cerebral ischemia.

Before and after administration of the drugs, the animals were anesthetized with a gas mixture of 3% isoflurane (Baxtor, USA) in a gas mixture of 7: 3 nitrogen and oxygen. The animals were operated with the gas mixture of nitrogen and oxygen mixed with 2.5% isoflurane while maintaining the anesthetic state of the experimental animals.

The anesthesia was performed with an aneurysm clip (Staelting, USA) for 5 minutes to induce cerebral ischemia, and then the aneurysm clip And then reperfusion was performed. At this time, ophthalmoscope was used to observe the blood circulation of the central artery of the retina and confirm the complete ocular artery ligation.

The body temperature was measured by inserting a rectal thermometer during the operation of the experimental animals, and the body temperature was kept constant at 36.7 ° C. to 37.3 ° C., which is a normal body temperature, using a thermostatic pad automatically controlled according to the body temperature of the experimental animal .

The animals were anesthetized by intraperitoneal injection of thiopental sodium (Yuhan, Korea) at a dose of 30 mg per kg of body weight 5 days after induction of cerebral ischemia and then 1000 IU of heparin per 1000 ml 4 ° C of physiological saline was injected into the left ventricle and perfused. The perfusion-washed animals were perfused with 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4 ° C.

The brain of the experimental animals was perfused using a bone cutter. The brain of the excised animal was fixed with 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4 ° C for 4 hours. The post-fixed brain was placed in a 30% sucrose solution (0.1 M phosphate buffer, pH 7.4), settled to sink to the bottom, and then applied to a slide microtome (Reicert-Jung, Germany) The tissue was cut to a thickness of 30 탆 to prepare a tissue section. The tissue section was placed in a 6-well plate containing a storing solution and stored at 4 캜 until the staining was performed.

Among the prepared tissue sections, tissues in which hippocampal formation appeared well were selected and washed three times for 10 minutes with 0.01M PBS to remove the preservation solution on the tissue sections. The washed tissue sections were plated on gelatin coated slides and dried well at 37 < 0 > C. The well-dried tissue sections were immersed briefly in distilled water and then stained with a solution of 2% cresyl violet acetate (Sigma, USA) for 1 minute to stain tissue sections. The dyed tissue sections were thoroughly washed in flowing water to remove excess dyestuffs from the slides and immersed in distilled water for a short time. Subsequently, 50%, 70%, 80%, 90%, 95% and 100% To effect dehydration and excess cresyl violet washing. After confirming the appearance of the Nissl body in the tissue section, the tissue was immersed in xylene (Junsei, Japan) and made transparent and then sealed with Canadian Balsam (Kanto, Japan).

The control group (vehicle administration group, Vehicle) subjected to ischemia-reperfusion was prepared in the same manner as above except that only the physiological saline solution used to dissolve the oxcarbazepine was administered. The normal group (Sham) No ischemia-reperfusion was performed, and tissue sections of the normal group were prepared in the same manner as described above.

The tissues of the sham, control and experimental groups were covered with an AxioM1 microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Cal Zeiss, Germany) The CA1 area was enlarged by 400 times to photograph each tissue section. Photographs of the entire hippocampal area were shown in A to R in Fig. 1, and photographs of CA1 area in hippocampus in A to R in Fig. Respectively.

As can be seen from FIGS. 1 and 2, the normal group (Sham) stained the neurons intensively with purple in the whole area of the hippocampus (FIG. 1 A) (Fig. 2 (A)). However, neuronal cell death was not observed in the control group (Vehicle) which was administered with only the solvent and neuron death in the CA1 region of the hippocampus due to delayed neuronal death due to cerebral ischemia 1 B), it was confirmed that most of the neurons disappeared into the pyramidal layer of the CA1 region (FIG. 2B). In addition, in the test group administered with the above-mentioned drugs at 100 mg / kg before or after induction of cerebral ischemia, neuronal apoptosis occurred in the CA1 region of the hippocampus similar to the control group, K, M, O, and Q in FIG. 1), and most of the neurons disappeared in the pyramidal layer of the CA1 region (C, E, G, I, K, . Similarly, in the experimental group administered with the drugs other than oxcarbazepine 200 mg / kg before or after inducing cerebral ischemia, neuronal cell death was observed in the CA1 region of the hippocampus, and nerve cell staining was hardly observed (Fig. 1 (D, F, H, J, L, and P in FIG. 2) were found in the pyramidal layer of the CA1 region. However, when compared with the control group and the oxcarbazepine-administered group of 100 mg / kg, the test group administered with 200 mg / kg of oxcarbazepine before or after cerebral ischemia caused a large number of nerve cells (N or R in FIG. 1), and neurons were dense in the pyramidal layer of the CA1 region (N or R in FIG. 2). Based on the above results, it was confirmed that 200 mg / kg of oxcarbazepine could effectively inhibit or prevent neuronal cell death caused by cerebral ischemia, unlike other antiepileptic drugs.

Example  2: Through experimental animals Oxcabazepine Cerebral ischemia  Nerve cell protective efficacy Measure 2

To further confirm the neuroprotective effect of oxcarbazepine, which was found to be excellent in Example 1 above. Immunohistochemical staining was performed using an antibody against NeuN (a neuronal specific nuclear protein), a neuron marker, and fluorescence staining was performed using Fluoride-Jade B, a marker for apoptotic neurons or apoptotic neurons Respectively.

More specifically, among the tissue slices prepared in Example 1, a tissue slice showing a good hippocampal complex was selected and used in the experiment. The selected tissue slice was used to remove the endogenous peroxidase present in the tissue And reacted with 0.3% H ₂ O ₂ (in 0.01 MPBS) for 20 minutes. To prevent nonspecific immune responses, the reacted tissue sections were reacted with 5% normal horse serum (0.05 M PBS) for 30 minutes.

The tissue sections reacted with 5% normal horse serum were reacted with mouse anti-NeuN (mouse anti-NeuN, Chemicon, USA) diluted 1: 800 at 4 ° C for 24 hours. After the reaction, the sections were reacted with anti-mouse IgG (anti-mouse IgG, Vector), a secondary antibody diluted 1: 250 for 2 hours at room temperature. Then, ABC (avidin-biotin complex, Vector) solution for 1 hour, and 3, 3'-DAB (diaminobenzidine) was developed as a substrate. In performing each step of the above procedure, the tissue sections were washed three times for 10 minutes each using 0.01 M PBS.

The tissue sections that had undergone color development with the 3, 3'-DAB substrate were plated on a slide glass coated with gelatin and then dried at room temperature for 12 hours. %, 80%, 90%, 95% and 100% ethanol. After the dehydration process was completed, the tissue slice was dipped in xylene (Junsei, Japan) and sealed in canada balsam (Canada Balsam, Kanto, Japan).

The tissues of the sham, control and experimental groups were covered with an AxioM1 microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Cal Zeiss, Germany) The CA1 area was enlarged by 400 times, and each tissue section was photographed. The photographs of the entire hippocampal area were shown in the AR of FIG. 3, and the photographs of the CA1 area of the hippocampus were shown in the AR of FIG.

Neurons of the tissue sections stained with NeuN antibody were counted by using an image analyzer (Optimas 6.5, USA) program. One-way analysis of variance (ANOVA) The results of the counting are shown in FIG.

As shown in FIGS. 3 to 5, in the hippocampal CA1 region of the normal group, many neurons showing immunoreactivity to NeuN were observed (FIG. 3A), and in particular, neurons exhibiting immunoreactivity in the pyramidal layer of the CA1 region (Fig. 4 (A)). On the other hand, in the control group, nerve cells showing immunoreactivity to NeuN were hardly observed in the hippocampal CA1 region (Fig. 3B, Fig. 4B and Fig. 5).

In addition, in the experimental group to which 100 mg / kg of the above drugs were administered before or after the cerebral ischemia induction, neurons showing immunoreactivity to NeuN were hardly observed in the CA1 region of the hippocampus similar to the control group (C, E, G, I, K, M, O, and Q), and neurons showing immunoreactivity to most of NeuN in the pyramidal layer of CA1 region were lost (C, E, M, O and Q, Figure 5). Similarly, in the experimental group administered with the drugs other than oxcarbazepine 200 mg / kg before or after inducing cerebral ischemia, neuron apoptosis occurred in the CA1 region of the hippocampus, and neuron-immunoreactive neurons were hardly observed (D, F, H, J, L and P in FIG. 3), it was confirmed that neurons showing immunoreactivity to most of NeuN in the pyramidal layer among CA1 regions were lost , J, L and P, Fig. 5). However, in the experimental group in which 200 mg / kg of oxcarbazepine was administered before or after cerebral ischemia induction, neurons exhibiting a large number of NeuN-immunoreactive cells were observed in the whole region of the hippocampus (N and R in FIG. 3) It was observed that the neurons showing immunoreactivity to NeuN in the pyramidal layer among CA1 regions were dense (N and R in Fig. 4).

From the above results, it was confirmed that 200 mg / kg of oxcarbazepine effectively inhibited neuronal cell death caused by cerebral ischemia when compared with other antiepileptic drugs.

Tissue fluorescent staining was also performed using Fluoride-Jade B, a marker of neuronal cells in the process of death or death. The tissue sections were washed three times for 5 minutes each using distilled water, and then reacted in 0.06% potassium permanganate solution for 20 minutes. After that, the plate was washed with distilled water for 2 minutes and stained with 0.001% fluorine-Jade B (Histochem, Jefferson, AR, USA) solution containing 0.1% acetic acid for 40 minutes. The stained tissue was washed with distilled water three times for 1 minute, dried in a slide warmer at 50 ° C for 20 minutes, transparent to xylene (Junsei, Japan) and then sealed with DPX (Sigma, USA). The encapsulated tissue was observed using a fluorescence microscope (Ex: 385 nm, Em: 425 nm), and the CA1 region was enlarged 400 times to photograph each tissue section. The photograph of the CA1 region of the hippocampus was taken as AR Respectively.

Neurons of the tissue sections stained with fluorine-jade B were counted using an image analyzer (Optimas 6.5, USA) on the photographed images, and the counted results are shown in FIG.

As shown in FIG. 6, no nerve cells stained by fluorine-iodine B were observed in the CA1 region of the hippocampus of the normal group (FIG. 6A), whereas the fluorine-iodine B (Fig. 6B). ≪ tb > < TABLE >

In the experimental group administered with the above-mentioned drugs at 100 mg / kg before or after induction of cerebral ischemia, many nerve cells stained with fluoro-jade B in the hippocampal CA1 region were observed similarly to the control group (C, E, G, I, K, M, O and Q, FIG. 7). Similarly, in the experimental group administered with the drugs other than oxcarbazepine 200 mg / kg before or after inducing cerebral ischemia, a number of fluoro-jade B-stained nerve cells were observed in the hippocampal CA1 region D, F, H, J, L and P of FIG. 7). On the other hand, in the experimental group administered with 200 mg / kg of oxcarbazepine before or after the cerebral ischemia induction, nerve cells stained with fluoro-jade B were scarcely observed (N and R in FIG.

Example  3: Through experimental animals Oxcabazepine Cerebral ischemia  Nerve cell protective efficacy Measure 3 (Measurement of glutamatergic cell activity change)

In order to confirm the change of neuroglia activity following the cerebral ischemia of oxcarbazepine, we used glial fibrillary acidic protein (GFAP), which is a mark of astrocyte, and Ionized calcium-binding adapter molecule (Iba-1), which is a marker of microglia -1) antibody was used for immunohistochemical staining.

In the immunohistochemical staining process, mouse anti-GFAP (Chemicon, USA) diluted 1: 1000 was used as a primary antibody to confirm the change of astrocytes, (Rabbit anti-Iba-1, Wako, Japan) diluted 1: 1000 was used as the primary antibody to identify the anti-Iba-1 antibody. Immunohistochemical staining was performed using the primary antibody described in Example 2, and the encapsulated tissue slice was observed using a microscope. Photographs taken by observation of GFAP were shown in AR of FIG. 8 , And the immunoreactivity of astrocytes stained with GFAP antibody was measured using an image analyzer program as described in Example 2. The measured results are shown in FIG. 9 as relative values to the normal group. In addition, photographs taken by Iba-1 were shown in AR of FIG. 10, and immunoreactivity of microglia stained with Iba-1 antibody was measured using an image analyzer program as described in Example 2 , And the measured results are shown in Fig. 11 as relative values to the normal group.

As shown in FIG. 8, astrocytes showing immunoreactivity to GFAP in the normal group were observed in a dormant state (FIG. 8A). In contrast, astrocytes showing immunoreactivity to GFAP in the control group were hypercellular , And was observed in an activated form (Fig. 8B).

In the experimental group administered with the above-mentioned drugs at 100 mg / kg before or after induction of cerebral ischemia, astrocyte cells showing immunoreactivity to GFAP in the hippocampal CA1 region were observed similarly to the control group (C, E, G, I, K, M, O and Q, FIG. 9). Similarly, in the experimental group administered with the drugs other than oxcarbazepine 200 mg / kg before or after inducing cerebral ischemia, asthma cells showing immunoreactivity to GFAP were observed in the hippocampal CA1 region 8 D, F, H, J, L and P, FIG. 9). On the other hand, in the experimental group administered with 200 mg / kg of oxcarbazepine before cerebral ischemia and in the experimental group administered with 200 mg / kg of oxcarbazepine after cerebral ischemia (N and R in FIG. 8, 9).

As shown in Fig. 10, microglia showing immunoreactivity to Iba-1 in the normal group were uniformly distributed in the dormant state in the CA1 region of the hippocampus (Fig. 10A). On the other hand, microglial cells showing immunoreactivity to Iba-1 in the control group were observed in the activated form and increased in number compared to the normal group, and they were clustered in the pyramidal cell layer (Stratum pyramidale, SP) of the hippocampal CA1 region (Fig. 10B).

In the experimental group administered with 100 mg / kg of oxcarbazepine before or after induction of cerebral ischemia, microglial cells showing immunoreactivity to Iba-1 were collected in the pyramidal cell layer (Stratum pyramidale, SP) of the hippocampal CA1 region, (C, E, G, I, K, M, O and Q in FIG. 10, FIG. 11). Similarly, in the experimental group administered with the drugs other than oxcarbazepine 200 mg / kg before or after inducing cerebral ischemia, the number of microglial cells showing immunoreactivity to Iba-1 in the hippocampal CA1 region increased, (FIG. 10 D, F, H, J, L and P, FIG. 11). On the other hand, in the experimental group in which 200 mg / kg of oxcarbazepine was administered before cerebral ischemia and in the control group of 200 mg / kg of oxcarbazepine after cerebral ischemia, the control group and the oxcarbazepine group administered with 100 mg / Were observed similarly to the normal group (N and R in Fig. 10, Fig. 11).

From the above results, it can be seen that other anti-epileptic drugs do not protect neurons in CA1 region of hippocampus, whereas oxcarbazepine has excellent neuronal protective effect, Were confirmed by cresyl violet staining, anti-NeuN immunohistochemical staining and fluoro-zid B fluorescent staining. In addition, oxcarbazepine was immunohistochemically stained with anti-GFP and anti-Iba-1 antibody. As a result, it was confirmed that oxcarbazepine was able to inhibit the activation of glial cells remarkably.

As described above, oxcarbazepine has an excellent effect for nerve cell protection and is also excellent in inhibiting the activation of neuroglial cells. Thus, it has been proved that nerve cell damage due to cerebral ischemia can be prevented or improved.

Claims (9)

A pharmaceutical composition for the prevention of ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient. The method according to claim 1,
Wherein the oxcarbazepine is a compound of the following formula 1 or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

The method according to claim 1,
The pharmaceutical composition for preventing ischemic cerebrovascular disease according to claim 1, wherein the oxcarbazepine is contained in an amount of 0.001 to 99.999% by weight based on the total weight of the composition. The method according to claim 1,
Wherein the ischemic cerebrovascular disease is any one selected from the group consisting of cerebral infarction, thrombosis, embolism, transient ischemic attack, subarachnoid hemorrhage, and small infarction. The method according to claim 1,
Wherein the oxcarbazepine protects neurons in the hippocampal CA1 region of the brain hippocampus. A composition for preventing ischemic cerebrovascular disease comprising oxcarbazepine as an active ingredient. The method according to claim 6,
Wherein the oxcarbazepine is a compound of the following formula 1 or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

The method according to claim 6,
Wherein the food composition for preventing ischemic cerebrovascular disease is a health functional food. The method according to claim 6,
Wherein the food composition for preventing ischemic cerebrovascular disease comprises 0.001 to 49.999% by weight of the oxcarbazepine based on the total weight of the composition.

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