KR20190081386A - Composition for preventing the ischemia stroke disease containing rufinamide - Google Patents

Abstract

The present invention relates to a composition containing rufinamide as an active ingredient for preventing ischemic cerebral diseases. The rufinamide 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 prevention of ischemic cerebrovascular disease comprising lupinamide as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing ischemic cerebrovascular disease comprising lupinamide 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. In addition, the life expectancy of Koreans was 82.1, which was significantly higher than that of 1970, which was 61.9 years old.

However, in terms of quality of health, the health life span of Koreans is estimated to be 65.1 years in 2015, 0.1 years less than the 65.2 years old in 3 years ago, and 16.9 years in 20.6% of life expectancy.

According to the increase of life expectancy of Koreans and the tendency of aging of Korean society, the number of patients with geriatric diseases is increasing year by year, and the pain and social cost are expected to increase.

As described above, the incidence and prevalence of various geriatric diseases due to aging are increasing along with the aging of the population, and the central nervous system (CNS) disease including damage to the brain or vertebra is continuously increased . 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. .. al, FreeRadicBiolMed, 43, 43-47 2016-05-04 pp 1048 - 1060, 2007), while there are not disclosed the precise mechanism of nerve cell death due to cerebral ischemia are two hypothesis on the new mechanism is presented as a continuous .

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 a growing need for studies on substances that can inhibit or delay the death of ischemic neuronal cells among the substances that are considered safe until now.

KR 10-1129303B KR 10-1132378B KR 10-1246169B KR 10-2011-0067022A

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 preventing cerebrovascular diseases. Specifically, the pharmaceutical composition for preventing ischemic cerebrovascular disease may contain lupine amide as an active ingredient. The lupine amide 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 lupine amide, 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 area of CA1 of the brain hippocampal tissue.

In addition, the pharmaceutical composition for the prevention of ischemic cerebrovascular disease may contain 0.001 to 99.999% by weight of the active ingredient lupine amide, based on the total weight of the composition.

Further, the present invention can be a food composition for preventing ischemic cerebrovascular disease using lupinamide as an active ingredient. The lupine amide 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 the prevention of ischemic cerebrovascular disease may contain 0.001 to 49.999% by weight of the active ingredient lupine amide, based on the total weight of the composition.

Lupinamide, 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 lupine amide has an excellent effect of preventing or ameliorating ischemic cerebrovascular disease due to the prevention or amelioration effect of ischemic cerebrovascular disease, specifically the protection of neuronal cell in ischemic condition and delayed death, Can be widely used to prevent death or disability due to ischemic cerebrovascular disease in a modern society in which circulatory diseases are rapidly increasing.

FIG. 1 is a photograph of a result of dying the hippocampal tissue of a cerebral ischemia-inducing animal with a cresyl violet in order to examine the effect of lupinamide 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 lupinamide 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 CA1 region of the hippocampal tissue of a brain ischemia-inducing experimental animal in order to examine the effect of lupinamide against the ischemic brain disease according to an embodiment of the present invention (Immunohistochemistry) using a staining method.
FIG. 4 is a graph showing the results of counting neurons showing immunoreactivity to NeuN antibody in the CA1 region of the hippocampal tissues of the brain ischemia-inducing experimental animals in order to examine the effect of lupinamide to prevent ischemic brain diseases according to an embodiment of the present invention. Is a graph showing relative numerical values for the sham.
FIG. 5 is a graph showing the effect of lupine amide on the inhibition of ischemic brain diseases according to an embodiment of the present invention. In order to examine the effect of lupine amide on ischemic brain diseases, B (fluoro-jade B, FJ B).
FIG. 6 is a graph showing the results of counting fluorine-iodine-labeled neurons in the CA1 region of the hippocampal tissues of the cerebral ischemia-induced experimental animals in order to examine the effect of lupinamide against ischemic brain diseases according to an embodiment of the present invention. Is a graph showing relative numerical values for the sham.
FIG. 7 is a graph showing the effect of lupine amide on the ischemic cerebrovascular proliferation according to one embodiment of the present invention. In order to examine the effect of lupinamide on the ischemic cerebral blood vessel, And the results of immunohistochemical staining.
FIG. 8 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 ischemic cerebrovascular prophylaxis effect of lupinamide according to one embodiment of the present invention. (Sham).
FIG. 9 is a graph showing the effect of lupine amide on the ischemic cerebrovascular proliferation according to one embodiment of the present invention. In order to examine the effect of lupinamide on the ischemic cerebrovascular proliferation, Iba-1) antibody, and immunohistochemical staining.
10 shows the results of counting cells showing immunoreactivity to Iba-1 antibody in CA1 region of hippocampal tissues of cerebral ischemia-induced experimental animals in order to examine ischemic cerebrovascular prophylaxis effect of lupinamide according to one embodiment of the present invention Is a graph showing the relative values for 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. Materials with inhibitory activity of glutamate activation were studied.

In the course of research on the above-mentioned conventional drugs, drugs used as drugs for treating epidemic diseases such as Perampanel, Levetiracetam, Retigabine, RUFINAMIDE, In particular, it has been shown through animal experiments that the neuroprotective effect of neuroprotective effect of neuronal cell death inhibition of CA1 region of brain hippocampal tissue, which is known to be sensitive to cerebral ischemia due to cerebral ischemia induced by lupinamide, In addition, it has been confirmed that the above-mentioned lupulin amide has remarkably excellent effect as compared with administration for therapeutic use after ischemia, and that prevention of neuronal damage due to cerebral ischemia And the present invention has been completed on the basis thereof.

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

The present invention relates to a pharmaceutical composition for preventing ischemic cerebrovascular disease comprising lupinamide as an active ingredient.

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

[Chemical Formula 1]

Specifically, the lupine amide is known to block the sodium channel as an antiepileptic drug or an antiepileptic drug, like other antiepileptics. In addition, the above-mentioned lupine amide has a function of inhibiting the action potential by inactivating the sodium passage involved in excitable cells, and it has a function of inhibiting the action potential such as Lennox-Gastaut syndrome, .

According to the existing studies, it is known that the sodium channel inactivation mechanism of the sodium channel blockers is related to the role of protecting the nerve cells from killing due to cerebral ischemia. However, according to the experiment conducted by the inventors of the present invention, It has been found that most of the therapeutic agents for brain stem with the activation function do not have the nerve cell protection effect or have no significant effect.

Specifically, in the present invention, an animal is anesthetized by administering prampanel, levitraacetam, and retigavine, which are anti-epilepsy drugs including lupinamide, for a certain period of time, to induce cerebral ischemia by ligation of the common carotid artery, As a result of the experiment to measure the efficacy through the staining of the dead neurons, it was found that the lupine amide was significantly superior to the neuroprotective effect when administered before the cerebral ischemia, And it was confirmed to protect neurons in the CA1 region of brain hippocampal tissue. From the above results, it was estimated that the lupinamide could effectively prevent ischemic cerebrovascular disease.

The composition for the prevention of ischemic stroke comprising the lupine amide as an active ingredient can be directly applied to animals including humans.

The composition for preventing ischemic stroke comprising the lupine amide as an active ingredient may further comprise the lupine amide alone as an active ingredient or a substance having an effect of treating or preventing ischemic cerebrovascular disease in the lupine amide as an active ingredient , And may further contain 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 the above-mentioned lupinamide as an active ingredient may further comprise, in addition to the above-mentioned active ingredients, a nutritional supplement, a vitamin, an electrolyte, a flavoring agent, a colorant, a heavy stabilizer, a pectic acid and its salt, A salt thereof, an organic acid, a protective colloid thickener, a pH adjuster, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated beverage. 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., lupine amide, either alone or in combination.

The composition for preventing ischemic cerebrovascular disease comprising the lupinamide as an active ingredient may contain 0.001 to 99.999% by weight, preferably 0.1 to 99% by weight, of the active ingredient, specifically, the lupulin amide, %, More preferably from 1 wt% to 50 wt%. 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 the lupine amide as an active ingredient is weakly formulated, it may be formulated with conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives And the like, and either orally or parenterally may be used.

Particularly, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and these solid preparations can be prepared by mixing the active ingredient, i.e., lupulinamide, with at least one excipient such as starch, calcium carbonate calcium carbonate, sucrose or lactose, and gelatin. 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 the above-mentioned lupinamide as an active ingredient may be in a preferable form depending on the method of use, and in particular, may be formulated so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. And may be formulated by employing methods 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-mentioned lupinamide as an active ingredient can be obtained 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 lupinamide as an active ingredient according to the present invention is appropriately selected by a person skilled in the art in consideration of the administration method, the age, sex and weight of the recipient, the severity of the disease and the like . For example, the composition for preventing ischemic cerebrovascular disease comprising the above-mentioned lupinamide as an active ingredient may be administered at a dose of 0.0001 mg / kg to 1000 mg / kg, 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 lupine amide 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 5 parts by weight to 200 parts by weight with respect to 100 parts by weight of the active ingredient.

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

In this respect, the present invention relates to a food composition for the prevention of ischemic brain diseases comprising lupinamide 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 has no particular limitation on the ingredients other than the lupine amide as an active ingredient in the indicated ratios as an essential ingredient and may contain various flavors or natural carbohydrates as an additional ingredient 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 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 lufinamide 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 other components except for containing the lupine amide of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient 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.

In the food composition for preventing ischemic cerebrovascular disease, the active ingredient is contained in an amount of 0.001 to 49.999% by weight or 0.01% by weight to 25% by weight or 0.1% by weight or less, 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 lupine amide of the present invention remarkably inhibits neuronal cell death in the CA1 region of the hippocampus caused by cerebral ischemia through prophylactic or ameliorative effect of ischemic cerebrovascular disease, particularly, prior to induction of cerebral ischemia, It has been experimentally confirmed that the lupine amide can 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 Lupinamide Cerebral ischemia  Nerve cell protective efficacy Measure 1

(Mongolian gerbil, Harlan, USA), weighing 75g to 80g, to induce cerebral ischemia by ligation after exposure to common carotid artery, followed by staining and observing hippocampal tissues of Mongolian jerville The effect of inhibiting neuronal cell death due to cerebral ischemia of lupinamide was confirmed.

Example  1-1. Breeding of experimental animals

56 male Mongolian gerbils (Meriones unguiculatus Harlan, USA) weighing between 75 g and 85 g at 6 months of age were exposed to a constant light period from 7 am to 7 pm, a temperature of 21 ° C to 25 ° C, and a temperature of 45% 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 lupinamide by cerebral ischemia

In order to confirm neuroprotective effect of lupinamide, more specifically, to prevent or prevent neuronal cell death due to cerebral ischemia, an experimental animal raised in Example 1-1 was administered pelampanel, levitiracetam, retigavine and lupine Amide was dissolved in physiological saline to give 50 mg or 100 mg per kg body weight of the experimental animals, and was administered intraperitoneally. 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 drug was administered. The normal group (Sham) The ischemia-reperfusion procedure was not performed, and tissue sections of the normal group were also prepared in the same manner as 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, in the case of sham treated with 50 mg / kg and 100 mg / kg of drug, including vehicle-sham, (Fig. 1, A, C, E, G, I, K, M, O and Q), and in particular in the CA1 region, neuronal cells were concentrated in the pyramidal layer (A, C, E, G, I, K, M, O and Q in FIG. However, in the case of vehicle-isch treated with solvent alone, neuronal cell death was observed in the CA1 region of the hippocampus due to delayed neuronal cell death due to cerebral ischemia, (FIG. 1B), it was confirmed that most of the neurons disappeared in the pyramidal layer of the CA1 region (FIG. 2B). Similarly, before inducing cerebral ischemia, neuronal cell death was observed in the CA1 region of the hippocampus in the experimental group (isch) in which all drugs except lupinamide were administered (100 mg / kg) 2, D, F, H, J, L, N, and P of FIG. 2), most of the neurons disappeared in the pyramidal layer ). However, in the experimental group in which 100 mg / kg of rufinamide was administered before inducing cerebral ischemia, a large number of neurons in the hippocampus were densely stained purple to purple (R in FIG. 1) (R in Fig. 2). Based on the above results, it was confirmed that 200 mg / kg of lupine amide effectively inhibited neuronal cell death caused by cerebral ischemia when compared with other cerebrovascular treatment drugs.

Example  2. Through experimental animals Lupinamide Cerebral ischemia  Nerve cell protective efficacy Measure 2

To further confirm the neuroprotective effect of lupinamide, which was found to be superior 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% H2O2 (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: 1000 for 24 hours at 4 ° C. 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).

Each tissue of the normal group (Sham), control group and experimental group was irradiated with an AxioM1 microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Cal Zeiss, Germany) Each of the tissue sections was photographed. The photographed photographs are shown in FIG.

Neurons of the tissue sections stained with NeuN antibody were counted by using an image analyzer (Optimas 6.5, USA) on the photographed images. One-way analysis ANOVA test) was performed, and the results of the counting are shown in FIG.

As shown in FIG. 3 and FIG. 4, immunohistochemical staining was performed using NeuN, a neuron marker, in order to confirm neuroprotection or neuroprotective effect of lupinamide administration in cerebral ischemia. As a result, (Fig. 3 (a)). In the hippocampal CA1 region of the control (sham) treated with 50 mg / kg and 100 mg / kg of drugs including Vehicle-sham, many neurons showing immunoreactivity to NeuN were observed A, C, E, G, I, K, M, O and Q). On the other hand, in the hippocampal CA1 region of the control (Vehicle-isch), neurons showing immunoreactivity to NeuN were rarely observed (Fig. 3B). In addition, a small number of NeuN immunoreactive neurons were observed throughout the hippocampal CA1 region, similar to the control (Vehicle-isch), except for the experimental group to which 100 mg / kg of rufinamide was administered (D, F , H, J, L, N and P, FIG. 4). However, in the experimental group to which 100 mg / kg of rufinamide was administered, a number of NeuN immunoreactive neurons were observed compared to the control group (R in Fig. 3, Fig. 4).

From the above results, it was confirmed that 100 mg / kg of rufinamide effectively inhibited neuronal cell death induced 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 magnified 200 times to photograph each tissue section. The photographs are shown in FIG.

The photographs of the photographs were also counted by using an image analyzer (Optimas 6.5, USA) program to count the neurons of the tissue sections stained with fluoro-jade B. The counted results are shown in FIG.

As shown in FIGS. 5 and 6, in order to confirm neuroprotection or neuroprotective effect of lupinamide administration in brain ischemia, fluoro-jade B, which is a marker of neuron during death or death, In the control group (sham) treated with 50 mg / kg and 100 mg / kg of the drugs in the normal group, the neurons stained with Fluorozide B in CA1 region of the hippocampus were observed (A, C, E, G, I, K, M, O and Q in FIG. 5). On the other hand, a number of neurons stained by fluoro-iodine B were observed in the hippocampal CA1 region of the control (Vehicle-isch) (Fig. 5B). In addition, neurons stained with fluoro-jade B in all drug test groups (isch) except lupinamide at 100 mg / kg before inducing cerebral ischemia were observed similarly to the control group (D, F, H, J, L, N and P, FIG. 6). However, nerve cells stained with fluoro-jade B were scarcely observed in the experimental group in which 100 mg / kg rufinamide was administered and in which cerebral ischemia was caused (R in Fig. 5).

From the above results, it was confirmed that 100 mg / kg of rufinamide effectively inhibited neuronal cell death induced by cerebral ischemia when compared with other antiepileptic drugs.

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

In order to confirm the change of neuroglia activity by the cerebral ischemia of lupinamide, glial fibrillary acidic protein (GFAP) and microglia (Iba-1), which are markers of astrocyte, Immunohistochemical staining was performed using 1) antibodies.

In the immunohistochemical staining process, mouse anti-GFAP (Chemicon, USA) diluted 1: 800 was used as a primary antibody for confirming 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 carried out using the primary antibody described in Example 1-2, and the encapsulated tissue slice was observed using a microscope. Photographs taken by observation of GFAP were shown in FIG. 7 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. 8 as relative values to the normal group. In addition, photographs taken by Iba-1 were shown in FIG. 9, and immunoreactivity of microglia stained with Iba-1 antibody was measured using an image analyzer program as described in Example 2, The results are shown in Fig. 10 as a relative value to the normal group.

As shown in FIGS. 7 and 8, in order to confirm the activity of astrocyte cells upon administration of lupinamide in the case of cerebral ischemia, immunohistochemical staining was performed using GFAP, a marker of astrocytic cells, (Fig. 7A, B, C, and D), asthmatic cells showing immunoreactivity to GFAP were observed in the control group (sham) after the administration of 50 mg / kg and 100 mg / C, E, G, I, K, M, O, and Q). On the other hand, in the case of the control (Vehicle-isch), the astrocytes showing immunoreactivity to GFAP were observed as hypertrophied and activated cytoplasm (FIG. 7B).

Also, astrocytes showing immunoreactivity to GFAP in all drug test groups (isch) except for 100 mg / kg of lupine amide before cerebral ischemia were observed similar to the control (Vehicle-isch) (D, F, H, J, L, N, and P, FIG. 8). However, astrocyte cells immunoreactive with GFAP in the experimental group that induced cerebral ischemia after administration of 100 mg / kg of lupinamide were found to be much less active compared with the control group (R, 8).

9 and 10, immunohistochemical staining was performed using Iba-1, a marker of microglial cells, in order to confirm the activity of microglia as a result of lupinamide administration in cerebral ischemia , And vehicle-sham (50 mg / kg and 100 mg / kg), the microglial cells showing immunoreactivity to Iba-1 in the experimental group underwent false-operation, (A, C, E, G, I, K, M, O and Q in FIG. 9). On the other hand, microglia showing immunoreactivity to Iba-1 in the control group (Vehicle-isch) were observed in an activated form and increased in number compared to the normal group, and the pyramidal cell layer (Stratum pyramidale, SP) (FIG. 9B).

In addition, astrocyte cells showing immunoreactivity to Iba-1 in all drug test groups (isch) except for 100 mg / kg of lupine amide before cerebral ischemia were observed similar to the control group (Vehicle-isch) F, H, J, L, N, and P, FIG. 10). However, astrocyte cells immunoreactive with GFAP in experimental groups that induced cerebral ischemia after administration of 100 mg / kg of rufinamide showed a much smaller degree of activation than those of the control group (R, 10).

From the above results, it has been shown that 100 mg / kg of rufinamide significantly inhibits neuronal cell death in the hippocampal CA1 region due to cerebral ischemia induced by cresyl violet staining, anti-NeuN immunohistochemical staining and fluoro-zid B fluorescent staining And it was confirmed through the anti-GFP and anti-Iba-1 antibodies that the activation of neuroblastoma cells can be suppressed very well. Therefore, the above-mentioned lupine amide has an excellent effect for nerve cell protection, It was also proved that neuronal cell damage due to cerebral ischemia can be prevented or improved because it is possible to effectively inhibit delayed neuronal cell death and activation of glial cells in response to cerebral ischemia.

As described above, although other therapeutic agents for brain hematopoiesis have little effect of preventing or inhibiting neuronal damage due to cerebral ischemia, lupine amide has excellent effect for nerve cell protection, . This suggests that the neuronal effect of lupinamide on cerebral ischemia is not related to the blockade of sodium channel. This is due to the fact that lupinamide has its own preventive effect on cerebral ischemia Was proven to be proven.

Claims (9)

A pharmaceutical composition for preventing ischemic cerebrovascular disease comprising lupinamide as an active ingredient. The method according to claim 1,
The pharmaceutical composition for the prevention of ischemic cerebrovascular diseases according to claim 1, wherein the lupine amide is a compound represented by 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 diseases according to claim 1, wherein the composition comprises 0.001 to 99.999 wt% of the lupine amide 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 lupine amide protects neurons in the hippocampal CA1 region of the brain hippocampus. A composition for preventing ischemic cerebrovascular disease comprising lupinamide as an active ingredient. The method according to claim 6,
Wherein the lupine amide 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 the prevention of ischemic cerebrovascular disease comprises 0.001 to 49.999% by weight of the lupine amide relative to the total weight of the composition.

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