KR20230095368A - Phatmaceutical composition comprising apigenin and antioxidant for preventing or treating brain disease - Google Patents

Abstract

The present invention relates to a pharmaceutical complex composition for treating brain diseases containing: apigenin or a pharmaceutically acceptable salt thereof; and an antioxidant and, more specifically, to a pharmaceutical complex composition for preventing or treating brain diseases containing apigenin and an antioxidant, exhibiting synergistic effects in protecting neurons/promoting differentiation of neurons and alleviating neuroinflammation. A composition according to the present invention can exhibit effects of increased prevention and treatment of ischemic brain diseases and neurodegenerative diseases compared to a single administration and can reduce side effects that may be caused by excessive or long-term administration of each drug by coadminister: apigenin or a pharmaceutically acceptable salt thereof; and an antioxidant.

Description

Pharmaceutical composite composition for treating brain diseases containing apigenin and antioxidants

The present invention relates to apigenin or a pharmaceutically acceptable salt thereof; And it relates to a pharmaceutical composite composition for treating brain diseases containing an antioxidant, and more particularly, to prevention or treatment of brain diseases including apigenin and an antioxidant that exhibit synergistic effects in protecting/promoting differentiation of neurons and alleviating neuroinflammation. It relates to a composite composition for

According to data released by the National Statistical Office in 2010, Korea's elderly population aged 65 or older accounted for 11.4% of the total population in 2011, and it is expected to enter a super-aged society by reaching 37.4% in 2050. As the aging problem has recently emerged as a social issue, the public's interest in the characteristics of the elderly population, housing, health, culture, leisure, and welfare for the elderly is increasing, and the demand for statistics on this is also increasing.

The core of this change is that chronic degenerative diseases are emerging as a bigger problem than acute infectious diseases, which have been the main cause of death for the past 50 years due to the increase in the aging population. In particular, among chronic degenerative diseases, death due to cerebrovascular disease is a very important disease that ranks second among deaths caused by a single disease.

These cerebrovascular diseases can be largely classified into two types. One is a hemorrhagic brain disease seen in cerebral hemorrhage, etc., and the other is an ischemic brain disease caused by occlusion of cerebral blood vessels. BACKGROUND OF THE INVENTION Hemorrhagic encephalopathy is mainly caused by a traffic accident, and ischemic encephalopathy is a disease that often appears in the elderly.

When temporary cerebral ischemia is induced in the cerebrum, the supply of oxygen and glucose is blocked, resulting in decreased ATP and edema in nerve cells, resulting in extensive damage to the brain. Neuronal death occurs after a considerable period of time after cerebral ischemia, which is referred to as delayed neuronal death. Delayed neuronal cell death In an experiment using a transient forebrain ischemic model using a Mongolian gerbil, neuronal cell death was observed in the CA1 region of the hippocampus after 4 days of induction of cerebral ischemia for 5 minutes. It has been reported (Kirino T, Sano K. Acta Neuropathol., 62: 201-208, 1984; Kirino T. Brain Res., 239: 57-69, 1982).

There are two most widely known mechanisms of neuronal cell death by cerebral ischemia. One is that cerebral ischemia causes excessive glutamate to accumulate outside cells, and this increased glutamate causes excessive intracellular calcium accumulation to induce neuronal cell death (Kang TC, et al., J. Neurocytol., 30: 945-955, 2001), and the other is oxidative neuronal cell death, which is caused by damage to DNA and cytoplasm due to an increase in radicals in vivo due to sudden oxygen supply during ischemia-reperfusion (Won MH , et al., Brain Res., 836: 70-78, 1999;Sub AY., Chen YM., J. Biomed. Sci., 5: 401-414, 1998; Flowers F, Zimmerman JJ. New Horiz.6 : 169-180, 1998).

Based on these mechanistic studies, many studies have been conducted to search for substances that effectively suppress neuronal cell death during cerebral ischemia or to reveal the mechanism of substances. However, there are few substances that effectively suppress neuronal cell death caused by cerebral ischemia.

Tissue plasminogen activator, approved by the FDA as the only treatment for cerebral ischemia, is a thrombolytic agent that induces rapid supply of oxygen and glucose by dissolving blood clots that cause cerebral ischemia. Therefore, since it does not directly protect nerve cells, it is necessary to use it quickly, and because of its characteristics as a thrombolytic agent, excessive or frequent use causes thinning of the blood vessel walls, eventually causing hemorrhagic cerebrovascular disease. In addition, in the case of MK-801, which is a calcium channel blocker for effectively suppressing the initial calcium influx, clinical tests have been conducted, but the drug has been discarded due to its side effects. In Japan, Mitsubishi's synthetic antioxidant, edaravone, succeeded in clinical trials in Japan and is prescribed only in Japan as the world's only paralytic treatment. It can be said that it is being used because of .

Meanwhile, it has recently been revealed that the inflammatory response is one of the main mechanisms that induce neurodegeneration. In other words, microglia, an immune cell present in the central nervous system, can be activated by various exogenous and endogenous substances, and activated microglia are inflammatory cytokines TNF-α and IL-1β, nitric oxide, prostaglandin, and superoxide. (Gao et al., J Neurochem, 81, 1285-97, 2002; Nelson, PT. et al., Ann Med, 34, 491-500, 2002; Griffin, W.S. et al., J Neuroinflammation, 3, 5, 2006). The production of these substances induces an immune response in the short term, but excessive production or continuous production induces the death of adjacent nerve cells and eventually causes neurodegeneration. In addition, since the substances released by dying nerve cells trigger the activation of microglia again, neurodegeneration falls into a continuous vicious cycle. In fact, it has been reported that the activity of microglial cells is related to various degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfelt-Jakob disease (CJD), and multiple sclerosis.

Considering the importance of the neuroinflammatory response in degenerative cranial nerve diseases, it is possible to treat neuroinflammation and degenerative cranial diseases that may result from it by reducing the expression level of pro-inflammatory mediators in these microglia. .

Accordingly, the present inventors have conducted intensive research to develop a therapeutic combination that exhibits a synergistic effect in the treatment of ischemic brain disease and neurodegenerative disease among known substances whose safety has been confirmed clinically. The present invention was completed after discovering that it exhibits remarkably improved neuronal cell protection and differentiation promoting effects compared to the drug alone.

Accordingly, an object of the present invention is apigenin or a pharmaceutically acceptable salt thereof; And to provide a pharmaceutical composition for preventing or treating ischemic brain disease comprising an antioxidant as an active ingredient.

Another object of the present invention is apigenin or a pharmaceutically acceptable salt thereof; And to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an antioxidant as an active ingredient.

Another object of the present invention is apigenin or a pharmaceutically acceptable salt thereof; And to provide a food composition for preventing or improving ischemic brain disease comprising an antioxidant as an active ingredient.

Another object of the present invention is apigenin or a pharmaceutically acceptable salt thereof; And to provide a food composition for preventing or improving neurodegenerative diseases comprising an antioxidant as an active ingredient.

Another object of the present invention is apigenin or a pharmaceutically acceptable salt thereof; and simultaneously (simultaneously), separately (separate) or sequentially (sequentially) administration, to provide a method for treating ischemic brain disease in animals other than humans.

Another object of the present invention is apigenin or a pharmaceutically acceptable salt thereof; and simultaneously (simultaneously), separately (separate) or sequentially (sequential) administration, to provide a method for treating neurodegenerative diseases of animals other than humans.

In order to achieve the above object of the present invention, the present invention is apigenin (apigenin) or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating ischemic brain disease comprising an antioxidant as an active ingredient.

In order to achieve another object of the present invention, the present invention is apigenin (apigenin) or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an antioxidant as an active ingredient.

In order to achieve another object of the present invention, the present invention is apigenin (apigenin) or a pharmaceutically acceptable salt thereof; And it provides a food composition for preventing or improving ischemic brain disease comprising an antioxidant as an active ingredient.

In order to achieve another object of the present invention, the present invention is apigenin (apigenin) or a pharmaceutically acceptable salt thereof; And it provides a food composition for preventing or improving neurodegenerative diseases comprising an antioxidant as an active ingredient.

In order to achieve another object of the present invention, the present invention is apigenin (apigenin) or a pharmaceutically acceptable salt thereof; and simultaneously (simultaneously), separately (separate) or sequentially (sequentially) administering, it provides a method for treating ischemic brain disease in animals other than humans.

In order to achieve another object of the present invention, the present invention is apigenin (apigenin) or a pharmaceutically acceptable salt thereof; and simultaneously (simultaneously), separately (separate) or sequentially (sequential) administration, providing a method for treating neurodegenerative diseases of animals other than humans.

As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the present invention with a pharmaceutically acceptable carrier. A pharmaceutical composition facilitates administration of the compound to a patient or subject. A variety of techniques exist in the art for administering compounds including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration.

As used herein, the terms "combination therapy" or "in combination" refer to the administration of two or more therapeutic agents to treat the conditions or disorders described herein (ie, ischemic brain disease or neurodegenerative disease). do. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed proportion of the active ingredients. Alternatively, such administration includes co-administration in multiple or separate containers (eg, capsules, powders, and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to the desired dosage prior to administration. In addition, such administration includes sequential use of each type of therapeutic agent at about the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in the treatment of the conditions or disorders described herein.

Combination therapy can provide a "synergistic effect" and can prove to be "synergistic." That is, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by using the compounds individually. A synergistic effect occurs when the active ingredients are (1) co-formulated and administered or delivered simultaneously in a combined unit dosage form; (2) when delivered alternately or in parallel as separate dosage forms; or (3) when delivered by some other initiative. When delivered in alternating therapy, a synergistic effect can be obtained when the compounds are administered or delivered sequentially, eg by different injections in separate syringes. Generally, during alternation therapy, effective doses of each active ingredient are administered sequentially, i.e. consecutively, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. Synergistic effect as used herein refers to the action of two therapeutic agents to produce an effect greater than the simple sum of the effects of each drug administered alone. Synergistic effects are described, for example, by the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additive equation (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)), and the median effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). can be calculated. Each of the equations mentioned above can be applied to experimental data to generate corresponding graphs that help evaluate the effects of drug combinations. Corresponding graphs related to the above-mentioned equations are concentration-effect curves, isobologram curves, and combination index curves, respectively.

Hereinafter, the present invention will be described in detail.

The present invention relates to apigenin or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating ischemic brain disease comprising an antioxidant as an active ingredient.

The present invention also relates to apigenin or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an antioxidant as an active ingredient.

In the present invention, apigenin is a flavonoid having a structure represented by Chemical Formula 1 below and contained in various fruits and vegetables, and has an antioxidant function related to skin aging, diseases of the digestive system including gastrointestinal diseases, and inflammation. , it has been reported to have an effect on hypertension. In particular, apigenin is a metabolite having a function of suppressing the expression of cancer cells by inducing cell death (apoptosis) or inhibiting angiogenesis. In addition, apigenin exhibits antioxidant activity and antiviral activity. In particular, it increases the antiviral activity of acyclovir, an antiviral agent used for the treatment of herpes infection, and prevents immunodeficiency virus (HIV-1). It is known to act to prevent activation.

[Formula 1]

As used herein, the term "pharmaceutically acceptable" means that it does not interfere with the biological activity or properties of a compound and is relatively non-toxic, that is, a substance has an undesirable biological effect or any component of a composition it contains. Refers to a substance, such as a carrier or diluent, that can be administered to an individual without interacting in a detrimental way with the component.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound to be administered prepared from non-toxic acids including pharmaceutically acceptable inorganic acids, organic acids, solvates, hydrates or clathrates thereof. indicate As the pharmaceutically acceptable salt of apigenin, an acid addition salt formed from a pharmaceutically acceptable free acid may be useful. Acid addition salts are formed with inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanedios. It is obtained from non-toxic organic acids such as oxalates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulphate, sulphite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol Late, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate may be used, but is not limited thereto.

In the present invention, the "antioxidant" is a substance that helps protect the human body from oxidative stress by removing active oxygen generated in the body, and its type is well known in the art. By way of example, the antioxidant is N-acetylcysteine, ascorbic acid, alpha-lipoic acid, scopoletin, forsythin, isoferul Isoferulic acid, gamma-oryzanol, trans-anethol, thioctic acid, cysteamine, gallic acid, salvianolic acid ( salvianolic acid), vitamin E, rappaconitine, L-selenomethionine, selenium, coenzyme Q10, vitamin A, catechin, daltro, etc. , but not limited thereto, preferably N-acetylcysteine, alpha-lipoic acid, daltron or a salt thereof, and most preferably N-acetylcysteine or a salt thereof.

The N-acetylcysteine was developed about 50 years ago and is mainly used as an expectorant or used for the treatment of addiction to acetaminophen, an analgesic, and is a drug for inhibiting oxidative stress having the structure of Formula 2 below. N-acetylcysteine reduces oxidative stress by directly reacting with peroxide, hydrogen peroxide, hydroxyl radical, etc., or indirectly increases glutathione synthesis by providing cysteine, a raw material for glutathione biosynthesis.亢) It is known to show an oxidizing effect. N-acetylcysteine has been used for decades in various clinical fields such as treatment of acute respiratory distress syndrome, treatment of acute pulmonary oxygen toxicity, and prevention of acute renal failure by contrast agents, and is known as a safe drug that does not cause side effects.

[Formula 2]

According to one embodiment of the present invention, it was confirmed that the combination preparation containing apigenin and N-acetylcysteine had remarkably excellent effects of protecting nerve cells, alleviating neuroinflammation, and promoting nerve cell differentiation compared to each single substance. The same effect was confirmed to have a synergistic effect compared to each single substance.

Therefore, since the composition of the present invention containing apigenin and N-acetylcysteine can exhibit more improved effects with a lower dose compared to using each drug alone, side effects are reduced and the treatment effect of brain diseases is improved. There is an advantage of being, that is, a synergic effect.

The Bliss independence combined response C for two single compounds with effects A and B is C = A + B - A*B, where each effect is expressed as a fractional inhibition from 0 to 1. (See Bliss (1939) Annals of Applied Biology). The Bliss value, defined as the difference between the experimental response and the calculated independent Bliss value, indicates whether the effect of the two components in combination is additive or synergistic.

A bliss value of zero (0) is considered additive. The term "additive" means that the result of combining two types of target agents is the sum of each of the individual drugs.

The term "synergy" or "synergistic" is used to mean that the response of the combination of the two agents is greater than the sum of the responses of the individual agents. More particularly, in an in vitro setting, one measure of synergism is known as “Bliss synergy”. The Bliss synergistic effect means "exceeding the Bliss independent value" determined by the previously defined Bliss value. If the bliss value is greater than zero, it is considered an indicator of synergy. Of course, the concept of “synergy” as used in the present invention includes in vitro synergy measured by additive and/or alternative methods.

In the present invention, the fact that the biological effect of the combination of apigenin and antioxidants in vitro is greater than or equal to the sum of the individual components of the combination can be correlated with the Bliss value. In addition, "synergy", which includes cases where a combination of components exhibits an activity equal to or greater than the sum of the individual components, as used herein, can be measured by additional and/or alternative methods.

In one aspect of the present invention, the composition of the present invention is for treating brain diseases, and contains an effective amount of apigenin or a pharmaceutically effective salt, derivative or metabolite thereof, an effective amount of an antioxidant or a pharmaceutically effective amount thereof. The salt can be combined in an amount sufficient to achieve a synergistic effect.

In one aspect of the present invention, apigenin and antioxidants may be combined in a molar ratio of 1:0.1 to 100, preferably in a molar ratio of 1:0.1 to 50, more preferably in a molar ratio of 1:0.1 to 10, and more More preferably, they may be combined at a molar ratio of 1:0.1 to 5, and most preferably at a molar ratio of 1:0.3 to 5.

In the present invention, the ischemic brain disease is also called cerebrovascular disease, and any pathological abnormality in the blood vessels supplying blood to the brain due to thrombosis, embolism, cerebrovascular thickening, cerebrovascular occlusion, etc. may be included without limitation. Non-limiting examples of the ischemic brain disease may include stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disorder, vascular dementia, and cerebral functional coma.

In the present invention, the degenerative neurological diseases include Alzheimer's disease, Parkinson's disease, dementia, cognitive dysfunction, progressive supranuclear palsy, multiple system atrophy, olive-pons-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatal-substantia nigra degeneration It may be selected from the group consisting of dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortico-basal ganglia degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Niemann-Pick's disease and Pick's disease, It is not limited thereto, and may preferably mean a neurodegenerative disease caused by neuroinflammation.

The cognitive dysfunction is a disease in which the death of abnormal nerve cells in a part of the nervous system or the entire brain rapidly occurs, unlike the normal aging process, and the function of the brain and spinal cord is lost, resulting in a decrease in cognitive ability while being deeply related to aging. can be included without limitation. Non-limiting examples of the cognitive dysfunction include mild cognitive impairment, Alzheimer's disease, frontotemporal dementia, Lewy body disease, cortico-basal ganglia degeneration, learning disabilities, agnosia, amnesia, aphasia, apraxia and delirium.

The "prevention" means any action that reduces the frequency or severity of pathological phenomena. Prevention can be complete or partial. In this case, it may mean a phenomenon in which the symptoms of carcinogenesis in the subject are reduced compared to the case where the composition is not used.

The "treatment" means any action that clinically intervenes to change the natural process of a target or cell to be treated, and can be performed while a clinical pathology is progressing or to prevent it.

The desired therapeutic effect is to prevent the onset or recurrence of the disease, alleviate the symptoms, reduce any direct or indirect pathological consequences of the disease, slow the rate of disease progression, alleviate or temporarily relieve the disease state. alleviating or improving the prognosis.

In the composition of the present invention, the apigenin and N-acetylcysteine may be administered simultaneously, separately, or sequentially, and the apigenin and N-acetylcysteine act synergistically in the body to the brain. It may exhibit disease preventive or therapeutic activity.

Typically, the apigenin (apigenin) or a pharmaceutically acceptable salt thereof; And antioxidants will generally be administered simultaneously as a composition, but even if each active ingredient is administered to the human body with a time difference, an equivalent level of therapeutic activity can be achieved by acting simultaneously in the body as each individually administered active ingredient.

Specifically, 'simultaneous administration' means administration of the two active ingredients through the same route of administration, or administration through the same or different routes of administration at substantially the same time (for example, 15 minutes or less between administration times). it means to do The individual administration means administration of the two active ingredients through the same or different administration routes at a predetermined time interval (eg, 3 days apart). The sequential means that the two active ingredients are administered through the same or different administration routes according to the patient's disease state with a certain ordering rule.

As the route of administration, it may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraneural, intraventricular (subventricular area), intracerebrovascular, intraperitoneal It can be intranasal, intranasal, enteral, topical, sublingual or rectal administration.

The pharmaceutical composition according to the present invention may contain only pharmaceutically effective amounts of apigenin and N-acetylcysteine, or may additionally contain a pharmaceutically acceptable carrier. The 'pharmaceutically effective amount' refers to an amount that exhibits a higher response than that of the negative control group, and preferably increases lifespan, improves motility, and suppresses neuroinflammation by administering the two active ingredients in combination in the treatment or prevention of brain diseases. , It means an amount sufficient to exhibit the effect of inhibiting neuronal cell death and promoting neuronal differentiation.

As used herein, the term "pharmaceutically acceptable carrier" refers to a liquid or solid filler, stabilizer, A pharmaceutically acceptable substance, composition or carrier such as a dispersing agent, suspending agent, diluent, additive, thickening agent, solvent or encapsulating material. Typically, these components are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formula, including compounds useful within the present invention, and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose used in pharmaceutical formulations; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; additives such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; Surfactants; alginic acid; pyrogen-free water; isotonic solution; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other non-toxic compatible substances. In addition, as used in the present invention, "pharmaceutically acceptable carrier" is compatible with the activity of compounds useful within the present invention, and is physiologically acceptable to patients in part or in whole coating agent, antiviral and antibacterial agent, and absorption delaying agent. Include etc. Supplementary active compounds may also be incorporated into the compositions. Further "pharmaceutically acceptable carriers" may further include pharmaceutically acceptable salts of compounds useful within the present invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co. , 1985, Easton, PA).

The pharmaceutical composition of the present invention may include apigenin or a pharmaceutically acceptable salt thereof; And antioxidants can be formulated in various ways according to the route of administration by a method known in the art, together with a pharmaceutically acceptable carrier to exhibit a synergistic effect according to the combined use. 'Pharmaceutically acceptable' refers to a non-toxic composition that is physiologically acceptable, does not inhibit the action of active ingredients when administered to humans, and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions. . Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes. Pharmaceutically acceptable carriers included in the composition are those commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Specifically, for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colors or flavors, etc. may be used. In the case of injections, buffers, preservatives, pain relievers, solubilizers , isotonic agents, stabilizers, etc. may be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. may be used.

In addition, the composition of the present invention can be used in the form of general pharmaceutical preparations. Parenteral preparations include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions or freeze-dried preparations, injections, transdermal preparations, nasal inhalations, etc. For oral administration, tablets, troches, capsules, elixirs, suspensions, syrups or wafers, etc. It can be manufactured in the form of In the case of an injection, it can be prepared in a unit dosage ampoule or multiple dosage form. In the case of the injection, it must be sterilized and must be protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof, and/or solvents or dispersion media containing vegetable oils. can More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with triethanolamine or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, and thimerosal may be further included. Also, in most cases, the injection may further include an isotonic agent such as sugar or sodium chloride.

In addition, the pharmaceutical composition of the present invention can be administered by any device capable of moving an active substance to a target site. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, or intravenous infusions. Injections include aqueous solvents such as physiological saline or IV, vegetable oils, higher fatty acid esters (eg, ethyl oleate, etc.), non-aqueous solvents such as alcohols (eg, ethanol, benzyl alcohol, propylene glycol, or glycerin). Stabilizers for preventing deterioration (eg, ascorbic acid, sodium hydrogensulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, and antimicrobial growth Preservatives (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included. A method of treating or preventing neurodegenerative diseases using the composition of the present invention includes administering an effective amount (pharmaceutically effective amount) of the composition for treatment of the present invention to a subject in need thereof. The pharmaceutically effective amount is well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, the route of administration, the method of administration, the number of times of administration, the duration of treatment, drugs used in combination or concurrently, and the like. Depending on the factors, it can be easily determined by a person skilled in the art.

In addition, the pharmaceutical composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The total effective amount of the composition of the present invention can be administered to the patient in a single dose or by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per kg of patient body weight per day. However, the dose of the pharmaceutical composition is determined by considering various factors such as the formulation method, administration route, and number of treatments as well as the patient's age, weight, health condition, sex, severity of disease, diet, and excretion rate. Therefore, considering this point, those skilled in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it exhibits the effects of the present invention.

Depending on the administration method and route of administration, the composition of the present invention may be formulated so that apigenin and antioxidants, which are components, are simultaneously included in one formulation, or each component is individually formulated and administered daily or once. Depending on the dosage unit, it may be included in one package. Formulations of individually formulated apigenin and antioxidants may or may not be the same. The specific formulation method of the pharmaceutical composition of the present invention and the pharmaceutically acceptable carrier that may be included in the formulation are as described above in the pharmaceutical composition, and reference may be made to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The present invention also relates to apigenin or a pharmaceutically acceptable salt thereof; And it provides a food composition for preventing or improving ischemic brain disease comprising an antioxidant as an active ingredient.

The present invention also relates to apigenin or a pharmaceutically acceptable salt thereof; And it provides a food composition for preventing or improving neurodegenerative diseases comprising an antioxidant as an active ingredient.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, but not limited thereto, as a health food, apigenin or a pharmaceutically acceptable salt thereof; And antioxidants can be ingested by preparing them in the form of tea, juice, and drink, liquefied, granulated, encapsulated, and powdered so that they can be consumed. In addition, it can be prepared in the form of a composition by mixing apigenin and antioxidants with known active ingredients known to be effective against infectious diseases. In addition, functional foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled products, jams, marmalades, etc.), fish, meat, and their processed foods (e.g., ham, sausages). Corned beef, etc.), bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable apigenin or a pharmaceutically acceptable salt thereof; And it can be prepared by adding an antioxidant. In addition, apigenin or a pharmaceutically acceptable salt thereof; And in order to use the antioxidant in the form of an additive, it can be prepared and used in the form of a powder or concentrate.

Preferred contents of apigenin and antioxidants in the food composition of the present invention are not limited thereto, but are preferably 0.1 to 90% by weight of the finally prepared food. More preferably, apigenin of the present invention or a pharmaceutically acceptable salt thereof; And a food composition containing an antioxidant as an active ingredient may be prepared in the form of a health functional food or dietary supplement by mixing with an active ingredient known to be effective against infectious diseases.

The present invention also relates to apigenin or a pharmaceutically acceptable salt thereof; and simultaneously (simultaneously), separately (separate) or sequentially (sequentially) administering, it provides a method for treating ischemic brain disease in animals other than humans.

The present invention also relates to apigenin or a pharmaceutically acceptable salt thereof; and simultaneously (simultaneously), separately (separate) or sequentially (sequential) administration, providing a method for treating neurodegenerative diseases of animals other than humans.

The composition according to the present invention may include apigenin or a pharmaceutically acceptable salt thereof; And antioxidants can exhibit increased preventive and therapeutic effects of ischemic brain disease and neurodegenerative diseases compared to single administration, and can reduce side effects that can be caused by excessive or long-term administration of each drug. It works.

1 shows the NO concentration generated after apigenin alone, N-acetylcysteine (NAC) alone, or a combination thereof (NDC-001) together with LPS (lipopolysaccharide), an inflammatory response-inducing substance, in microglia (BV2 cells). This is the result of evaluating the anti-neuroinflammatory effect by measuring.
Figure 2 measures the level of IL-6 (Interleukin-6) generated after apigenin alone, NAC alone or a combination thereof (NDC-001) together with LPS, an inflammatory response-inducing substance, in microglia to measure anti-neuro-inflammation This is the result of evaluating the effect.
Figure 3 shows that after culturing neural precursor cells (ReNcell VM) with apigenin alone, NAC alone, or a combination thereof (NDC-001), the expression level of HB9, a motor neuron marker, was evaluated to evaluate the differentiation promoting effect of the drug. This is the result.
Figure 4 shows the expression of Tuj-1, a neuronal differentiation marker, in cerebral cortical tissue after intravenous administration of apigenin alone, NAC alone, or apigenin + NAC combination (NDC-001) immediately after induction of a stroke-reperfusion animal model for 21 days. It is the result of measuring the quantity.
Figure 5 shows the expression level of Caspase 3, a marker of apoptosis around the cerebral infarct, after intravenous administration of apigenin alone, NAC alone, or apigenin + NAC complex (NDC-001) immediately after induction of stroke-reperfusion animal model for 21 days is the result of measuring
6 is a result of analyzing the degree of brain damage after intravenous administration of apigenin alone, NAC alone, or apigenin + NAC combination (NDC-001) immediately after induction of a stroke-reperfusion animal model for 21 days.
Figure 7 shows the intravenous administration of apigenin alone, NAC alone, or apigenin + NAC combination (NDC-001) immediately after induction of a stroke-reperfusion animal model for 21 days, on days 1, 3, 7, and 14 after induction of stroke. This is the result of performing the Rotarod latency test.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1: Apigenin + N-acetylcysteine (NAC) Anti-inflammatory effect of the complex ( in vitro )

In the present invention, microglia (BV2 cells) are treated with LPS to cause an inflammatory response, and at the same time, nitrogen monoxide (NO) generated after apigenin alone, NAC alone, or apigenin + NAC combined treatment (NDC-001) Alternatively, the neuroinflammation alleviating effect of the drug was confirmed by measuring the expression level of an inflammatory marker (IL-6).

As a result, as shown in FIGS. 1 and 2, a significant reduction in NO or IL-6 was confirmed in the drug-treated group compared to the LPS control group, and the reduction effect was found to be greatest in the apigenin + NAC complex-treated group (NDC-001). In addition, as a result of calculation by applying the Bliss independence model, the synergistic effect of the two drugs was confirmed. (** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA)

Example 2: Effect of Apigenin + NAC Complex on Promoting Neural Progenitor Cell Differentiation ( in vitro )

ReNcell VM (immortalized human neural progenitor cell line), a neural progenitor cell capable of differentiating into neurons and glial cells, was treated with apigenin alone, NAC alone, or apigenin + NAC complex treatment (NDC-001). Immunostaining was performed using an antibody (anti-HB9 antibody) capable of selectively binding to motor neurons. Thereafter, by analyzing the number of HB9 positive signals and confirming the number of motor neurons, the degree of differentiation of neural precursor cells into motor neurons was measured.

As a result, as shown in FIG. 3, a significant increase in HB-9 expression level was confirmed in the apigenin and NAC combined treatment group (NDC-001) compared to the control group. This increase did not appear in the group treated with apigenin or NAC alone, and it was confirmed that the combined treatment effect was greater in the predicted results through the Bliss independence model, indicating that the combined treatment of apigenin and NAC had a synergistic effect in promoting the differentiation of neural progenitor cells. confirmed what was seen. (** P < 0.01, **** P < 0.0001, one-way ANOVA)

Example 3: Apigenin + NAC complex promotes neuronal differentiation ( in vivo )

To verify the efficacy of the drug in vivo , a stroke model was constructed using a cerebral infarction-reperfusion model using Sprague-Dawley rats (SD rats), and apigenin alone, NAC alone, and apigenin + NAC combination (NDC- 001) was administered IV for 21 days immediately after induction of cerebral infarction-reperfusion injury, and the experiment was conducted.

Immediately after cerebral infarction-reperfusion modeling, apigenin alone, NAC alone, or apigenin + NAC combined drug was administered for 21 days, and then the expression level of Tuj-1 protein, a neuronal differentiation marker, was confirmed by Western Blot in cortical tissue.

As a result, as shown in FIG. 4, it was confirmed that the Tuj-1 expression level was significantly increased in the NAC alone or NDC-001 complex administration group. In addition, the synergistic effect of the combination drug was confirmed by showing a significantly increased Tuj-1 expression level compared to each single treatment group in the NDC-001 combination treatment group. (*** P < 0.001, **** P < 0.0001, one-way ANOVA)

Example 4: Neuronal cell protective effect of apigenin + NAC complex ( in vivo )

Immediately after cerebral infarction-reperfusion modeling, apigenin alone, NAC alone, or apigenin + NAC combination drug was administered for 21 days, and then brain tissue sections were stained by immunohistochemical staining with Caspase 3 antibody, a cell death marker, and the area around the damaged area was stained. Caspase 3 expression levels in the penumbra region were analyzed.

As a result, as shown in FIG. 5, it was confirmed that cell death increased compared to the normal control group in the cerebral infarction-reperfusion model tissue, and cell death could be reduced through NDC-001 combined drug administration. In addition, the synergistic effect of the combined use of the two drugs was confirmed through the fact that these effects did not appear in the group treated with apigenin or NAC alone. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA)

Immediately after cerebral infarction-reperfusion modeling, apigenin alone, NAC alone, or apigenin + NAC combination drug (NDC-001) was administered for 21 days, and brain tissue slices were analyzed using neuronal (NeuN), microglia (GFAP) marker antibody, and cell nuclei. As a result of analyzing the degree of brain damage by immunohistochemical staining through staining solution (DAPI), as shown in Figure 6, only the NDC-001 combined administration group showed a significant decrease in the degree of brain damage compared to the Vehicle administration group (* P < 0.05, one-way ANOVA).

Example 5: Effect of restoring motor function of apigenin + NAC complex ( in vivo )

In order to confirm the effect of restoring motor function through the NDC-001 complex, apigenin alone, NAC alone, and apigenin + NAC combined drug (NDC-001) were administered after stroke induction through a cerebral infarction-reperfusion model, and representative motor coordination evaluations The motor function of the animals was measured on days 1, 3, 7, and 14 after cerebral infarction-reperfusion modeling using the Rotarod latency test.

As a result, as shown in FIG. 7, only the NDC-001 administration group showed a significant motor function improvement compared to the Vehicle administration group, confirming the motor function recovery effect of the NDC-001 combination drug and the synergistic effect of the combination (** P < 0.01, * ** P < 0.001, one-way ANOVA).

The composition according to the present invention may include apigenin or a pharmaceutically acceptable salt thereof; And the combined administration of antioxidants can exhibit increased preventive and therapeutic effects of brain diseases compared to single administration, and can reduce side effects that can be caused by excessive or long-term administration of each drug. Availability is high.

Claims (13)

apigenin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating ischemic brain disease comprising an antioxidant as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the ischemic brain disease is selected from the group consisting of stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disorder, vascular dementia, and cerebral functional coma.
The method of claim 1, wherein the antioxidant is N-acetylcysteine, ascorbic acid, alpha-lipoic acid, scopoletin, forsythin , isoferulic acid, gamma-oryzanol, trans-anethol, thioctic acid, cysteamine, gallic acid, sal Salvianolic acid, vitamin E, lappaconitine, selenium, coenzyme Q10, vitamin A, catechin, daltron and L- A pharmaceutical composition, characterized in that at least one selected from the group consisting of selenomethionine (L-selenomethionine) or a salt thereof.
apigenin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an antioxidant as an active ingredient.
The method of claim 5, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, dementia, cognitive dysfunction, progressive supranuclear palsy, multiple system atrophy, olive-pons-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatal- Substances selected from the group consisting of substantia nigra degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortico-basal ganglia degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Niemann-Pick's disease and Pick's disease Characterized by the pharmaceutical composition.
The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition exhibits a neuronal differentiation promoting effect.
The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition exhibits an effect of alleviating neuroinflammation.
The method according to any one of claims 1 to 5, wherein the apigenin or a pharmaceutically acceptable salt thereof; and the antioxidant is administered simultaneously (simultaneously), separately (separately) or sequentially (sequential).
apigenin or a pharmaceutically acceptable salt thereof; And a food composition for preventing or improving ischemic brain disease comprising an antioxidant as an active ingredient.
apigenin or a pharmaceutically acceptable salt thereof; And a food composition for preventing or improving neurodegenerative diseases comprising an antioxidant as an active ingredient.
The food composition according to claim 9 or 10, wherein the food is a health functional food.
apigenin or a pharmaceutically acceptable salt thereof; and simultaneously (simultaneously), separately (separate) or sequentially (sequentially) administration, a method for treating ischemic brain disease in animals other than humans.
apigenin or a pharmaceutically acceptable salt thereof; and simultaneously, separately or sequentially administering an antioxidant.

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