Pharmaceutical composition for treating brain diseases, comprising a cholinesterase inhibitor and an antioxidant.

Abstract

The present invention relates to a pharmaceutical composition for treating brain diseases, comprising a cholinesterase inhibitor and an antioxidant, more particularly to a composition for preventing or treating brain diseases, comprising a cholinesterase inhibitor and an antioxidant that exhibit a synergistic effect on protecting neural cells and promoting their differentiation.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2021-0012659, filed on January 28, 2021, and the entire above-mentioned specification is a reference of this application.

[0002] The present invention relates to a pharmaceutical composition for treating brain diseases containing a cholinesterase inhibitor and an antioxidant. More specifically, it relates to a composite composition for preventing or treating brain diseases containing a cholinesterase inhibitor and an antioxidant that exhibit a synergistic effect on neuroprotection and promotion of differentiation.

Background Art

[0003] According to data published by Statistics Korea in 2010, in Korea, the proportion of the elderly population aged 65 and above in the total population reached 11.4% in 2011, and it is expected to reach 37.4% in 2050, entering a super-aged society. Recently, as the aging problem has emerged as a social issue, the public's interest in the characteristics, housing, health, culture, leisure, and other aspects of the elderly population, such as elderly welfare, has increased, and the demand for statistical data on this has also been growing.

[0004] The core of such changes is that due to the increase in the aging population, chronic degenerative diseases have emerged as a more significant problem than acute infectious diseases, which were the main cause of death in the past approximately 50 years. In particular, among chronic degenerative diseases, death due to cerebrovascular diseases is a very important disease that ranks second among the mortality rates due to single diseases.

[0005] Such cerebrovascular diseases can be broadly classified into two forms. One is hemorrhagic brain diseases such as cerebral hemorrhage, and the other is ischemic brain diseases that appear due to the occlusion of blood vessels in the brain. Hemorrhagic brain diseases mainly appear due to traffic accidents, etc., and ischemic brain diseases are mainly diseases that often appear in the elderly.

[0006] When transient cerebral ischemia is induced in the cerebrum, the supply of oxygen and glucose is cut off, leading to decreased ATP and edema in nerve cells, ultimately inducing widespread brain damage. Nerve cell death appears a considerable time after cerebral ischemia; this is called delayed neuronal death. Experiments using a transient forebrain ischemic model with Mongolian gerbils have shown that neuronal death is observed in the CA1 region of the hippocampus four days after five minutes of cerebral ischemia induction (Kirino T, Sano K. Acta Neuropathol., 62:201-208, 1984; Kirino T. Brain Res., 239:57-69, 1982).

[0007] There are two most widely known mechanisms of neuronal cell death due to cerebral ischemia. One is excitatory neuronal cell death (Kang TC, et al., J. Neurocytol., 30:945-955, 2001), in which excessive glutamate accumulates extracellularly due to cerebral ischemia, and this increased glutamate induces neuronal cell death through excessive intracellular calcium accumulation. The other is oxidative neuronal cell death (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 Hori z.6:169-180, 1998).

[0008] Based on these mechanistic studies, much research is being conducted to search for substances that effectively suppress neuronal cell death that occurs during cerebral ischemia, and to elucidate the mechanisms by which these substances work. However, the reality is that there are still very few substances that effectively suppress neuronal cell death caused by cerebral ischemia.

[0009] Tissue plasminogen activator, the only FDA-approved and marketed treatment for cerebral ischemia to date, is a thrombolytic agent that dissolves blood clots causing cerebral ischemia and induces a rapid supply of oxygen and glucose. Therefore, it does not directly protect nerve cells, requiring rapid use, and due to its clot-dissolving properties, excessive or frequent use can thin the blood vessel walls, ultimately inducing hemorrhagic cerebrovascular disease. In a different example, MK-801, a calcium channel blocker designed to effectively suppress initial calcium influx, underwent clinical trials, but the drug was discarded due to side effects. In Japan, edaravone, a synthetic antioxidant from Tanabe Mitsubishi Pharma, has successfully completed clinical trials in Japan and is the only stroke treatment drug in the world prescribed exclusively in Japan, generating annual sales of 300 billion won. However, despite concerns about side effects, it is being used due to the lack of other available drugs.

[0010] On the other hand, cognitive impairments such as Alzheimer's disease primarily affect the elderly population aged 60 and over, and given the rapidly progressing aging population in South Korea, and considering the serious psychological distress and economic burden they place not only on patients but also on their families, society, and the nation, research into the etiology of these diseases is essential. Furthermore, it is only through such research that we can recognize the urgent need for preventative measures and treatments that can improve the disease, which currently only offer mild symptom relief or slow disease progression.

[0011] The causes of Alzheimer's disease can be divided into external and internal factors. External causes include age, genetic factors, and toxic substances due to environmental influences. Internal causes include decreased neurotransmission due to reduced acetylcholine levels, neuroinflammation caused by cytokines, cytotoxicity due to the aggregation of amyloid-beta and excessively phosphorylated tau protein, and neuronal cell death caused by reactive oxygen species due to oxidative stress. Such neuronal cell death is a fundamental pathology common to several brain diseases, and research into the factors and molecular mechanisms that cause cell death is being conducted in depth. However, currently, existing proteolytic enzyme agents and broad-spectrum cell death inhibitors have decreased clinical efficacy along with side effects. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Kirino T,Sano K.Acta Neuropathol.,62:201-208,1984; Kirino T.Brain Res.,239:57-69,1982 [Non-Patent Document 2] Won MH, et al.,Brain Res.,836:70-78,1999;Sub AY.,Chen YM.,J.Biomed.Sci.,5:401-414,1998;FlowersF,Zimmerman JJ.New Horiz.6:169-180, 1998 [Overview of the project] [Problems that the invention aims to solve]

[0013] As a result of conducting numerous studies to develop a combination of therapeutic agents that would have a synergistic effect in treating ischemic brain disease and degenerative neurological disease among known substances whose safety has been confirmed in clinical trials, the inventors discovered that the combination of donepezil and N-acetylcysteine ​​shows significantly improved neuroprotective and differentiation-promoting effects compared to each drug alone, thus completing the present invention.

[0014] Therefore, the object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ischemic brain disease, comprising a cholinestrease inhibitor and an antioxidant as active ingredients. Furthermore, an object of the present invention is to provide a pharmaceutical composition comprising a cholinesterase inhibitor and an antioxidant for the prevention or treatment of ischemic brain disease. Furthermore, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ischemic brain disease, which is essential for cholinesterase inhibitors and antioxidants.

[0015] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, comprising a cholinesterase inhibitor and an antioxidant as active ingredients. Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases comprising a cholinesterase inhibitor and an antioxidant. Another object of the present invention is to provide pharmaceutical compositions for the prevention or treatment of degenerative neurological diseases, which are essential for cholinesterase inhibitors and antioxidants.

[0016] Another object of the present invention is to provide a food composition for the prevention or improvement of ischemic brain disease, comprising a cholinesterase inhibitor and an antioxidant as active ingredients. Another object of the present invention is to provide a food composition comprising a cholinesterase inhibitor and an antioxidant for the prevention or improvement of ischemic brain disease. Another object of the present invention is to provide a food composition for preventing or improving ischemic brain diseases that are essential for cholinesterase inhibitors and antioxidants.

[0017] Another object of the present invention is to provide a food composition for preventing or improving degenerative nerve diseases, containing a cholinesterase inhibitor and an antioxidant as active ingredients. Another object of the present invention is to provide a food composition for preventing or improving degenerative nerve diseases, comprising a cholinesterase inhibitor and an antioxidant. Another object of the present invention is to provide a food composition for preventing or improving degenerative nerve diseases that are essential for cholinesterase inhibitors and antioxidants.

[0018] Another object of the present invention is to provide a method for treating ischemic brain diseases in animals other than humans, which includes the step of administering a cholinesterase inhibitor and an antioxidant simultaneously, separately or sequentially.

[0019] Another object of the present invention is to provide a method for treating degenerative nerve diseases in animals other than humans, which includes the step of administering a cholinesterase inhibitor and an antioxidant simultaneously, separately or sequentially.

[0020] Another object of the present invention is to provide the use of a cholinesterase inhibitor and an antioxidant for manufacturing a preparation for treating ischemic brain diseases.

[0021] Another object of the present invention is to provide a method for treating ischemic brain diseases, which includes administering to an individual requiring an effective amount of a composition containing a cholinesterase inhibitor and an antioxidant.

[0022] Another object of the present invention is to provide the use of a cholinesterase inhibitor and an antioxidant for manufacturing a preparation for treating degenerative nerve diseases. harmful agent and an antioxidant.

[0023] Another object of the present invention is to provide a method for treating degenerative neurological diseases, including administering an effective amount of a composition comprising a cholinesterase inhibitor and an antioxidant to an individual who needs it.

Means for Solving the Problems

[0024] To achieve the above object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating ischemic brain diseases, comprising a cholinesterase inhibitor and an antioxidant as active ingredients. The present invention also provides a pharmaceutical composition for preventing or treating ischemic brain diseases, comprising a cholinesterase inhibitor and an antioxidant. The present invention also provides a pharmaceutical composition for preventing or treating ischemic brain diseases, which is essentially composed of a cholinesterase inhibitor and an antioxidant.

[0025] To achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating degenerative neurological diseases, comprising a cholinesterase inhibitor and an antioxidant as active ingredients. The present invention also provides a pharmaceutical composition for preventing or treating degenerative neurological diseases, comprising a cholinesterase inhibitor and an antioxidant. The present invention also provides a pharmaceutical composition for preventing or treating degenerative neurological diseases, which is essentially composed of a cholinesterase inhibitor and an antioxidant.

[0026] To achieve another object of the present invention, the present invention provides a food composition for preventing or improving ischemic brain diseases, comprising a cholinesterase inhibitor and an antioxidant as active ingredients. The present invention also provides a food composition for preventing or improving ischemic brain diseases, comprising a cholinesterase inhibitor and an antioxidant. The present invention also provides a food composition for preventing or improving ischemic brain diseases, which is essentially composed of a cholinesterase inhibitor and an antioxidant.

[0027] To achieve another objective of the present invention, the present invention provides a food composition for the prevention or improvement of degenerative neurological diseases, comprising a cholinesterase inhibitor and an antioxidant as active ingredients. Furthermore, the present invention provides a food composition comprising a cholinesterase inhibitor and an antioxidant for the prevention or improvement of degenerative neurological diseases. Furthermore, the present invention provides a food composition for the prevention or improvement of degenerative neurological diseases, which are essential for the use of cholinesterase inhibitors and antioxidants.

[0028] To achieve another objective of the present invention, the present invention provides a method for treating ischemic brain disease in animals other than humans, comprising the step of administering a cholinesterase inhibitor and an antioxidant simultaneously, separately, or sequentially.

[0029] To achieve another objective of the present invention, the present invention provides a method for treating degenerative neurological diseases in animals other than humans, comprising the step of administering a cholinesterase inhibitor and an antioxidant simultaneously, separately, or sequentially.

[0030] To achieve another objective of the present invention, the present invention provides the use of cholinesterase inhibitors and antioxidants for the production of therapeutic formulations for ischemic brain diseases.

[0031] To achieve another objective of the present invention, the present invention provides a method for treating ischemic brain disease, comprising administering an effective amount of a composition comprising a cholinesterase inhibitor and an antioxidant to an individual in need.

[0032] To achieve another objective of the present invention, the present invention provides the use of cholinesterase inhibitors and antioxidants for the production of therapeutic formulations for degenerative neurological diseases.

[0033] To achieve another objective of the present invention, the present invention provides a method for treating a degenerative neurological disease, comprising administering an effective amount of a composition comprising a cholinesterase inhibitor and an antioxidant to an individual in need. [Modes for carrying out the invention]

[0034] The present invention will be described in detail below.

[0035] The present invention provides a pharmaceutical composition for the prevention or treatment of ischemic brain disease, comprising a cholinesterase inhibitor and an antioxidant as active ingredients.

[0036] The present invention also provides a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases (degenerative neurological diseases) comprising a cholinesterase inhibitor and an antioxidant as active ingredients.

[0037] In this invention, the term "cholinesterase inhibitor" refers to a compound that inhibits the enzymatic degradation of the neurotransmitter acetylcholine, and therefore increases the duration and level of action of acetylcholine in the synaptic cleft. Two enzymes are primarily responsible for the degradation of acetylcholine, acetylcholinesterase, and butyrylcholinesterase. "Cholinesterase inhibitors" include substances that inhibit or reduce the action of one or both of these enzymes.

[0038] In the compositions and methods of the present invention, the cholinesterase inhibitors are considered pharmaceutically effective. In this specification, “pharmaceutically effective” means that the cholinesterase inhibitor is therapeutically useful in humans. Therefore, this term excludes cholinesterase inhibitors used as insecticides, such as ardicarb (2-methyl-2-(methylthio)propionaldehyde-O-methylcarbamoyloxime), carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranylmethylcarbamate), and carbaryl (1-naphthylmethylcarbamate), as well as cholinesterase inhibitors that are deadly to humans and used as chemical weapons, such as sarin (2-(fluoro-methylphosphoryl)oxypropane), VX (S-[2-(diisopropylamino)ethyl]-O-ethylmethylphosphonothioate), and soman (3-(fluoro-methyl-phosphoryl)oxy-2,2-dimethyl-butane). Most cholinesterase inhibitors, which are used as insecticides and chemical weapons, are either quasi-reversible or irreversible. Most pharmacochemically effective cholinesterase inhibitors are reversible.

[0039] Pharmaceutically effective cholinesterase inhibitors are widely known in this field. Examples include 7-methoxytacrine, alvameline, ambenonium, anseculin, arecoline, cevimeline, citicoline, demacarium, donepezil, edrophonium, and eptastigmine. fasciculin, heptyl-physostigmine, huperzine A and its analogues, icopezil, ipidacrine, linopiridine, metrifonate, milameline, neostigmine, nomeostigmine, This includes, but is not limited to, pyridostigmine, norpyridostigmine, tacrine, physostigmine, rivastigmine, subcomeline, suronacrine, tacrine analogs, tacrine, talsaclidine, velnacrine, xanomeline, zifrosilone, itopride, acotiamide, huperzin, galantamine, and their salts, and most preferably, donepezil may be the cholinesterase inhibitor.

[0040] Donepezil is an acetylcholinesterase (AChE) inhibitor with the structure shown in formula (1) below, and is used to treat mild to severe dementia in Alzheimer's disease. In Alzheimer's disease, where cholinergic nervous system dysfunction in the brain is reported, donepezil increases acetylcholine in the brain and activates cholinergic neurons in the brain. Currently, donepezil is available on the market in tablet form and is prescribed orally to patients with Alzheimer's disease. [ka]

[0041] As pharmaceutically acceptable salts of donepezil, acid addition salts formed with pharmaceutically acceptable free acids are useful. Acid addition salts can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrite, or phosphorous acid, and from non-toxic organic acids such as aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanediates, aromatic acids, and aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, disulfates, sulfates, bisulfates, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propioates, oxalic acid, malonates, succinic acid, sverate, sebacate, fumarate, mariate, buty-1,4-dioate, hexane-1,6-dioate, benzoic acid, and chlorobenzoic acid. Acids, methylbenzoic acid, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylate, β-hydroxybutyrate, glycolate, malate, tatelate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate may be used, but are not limited to these.

[0042] In this invention, the "antioxidant" is a substance that helps protect the human body from oxidative stress by removing reactive oxygen species that are generated in our bodies, and its types are well known in the industry. Examples of antioxidants include N-acetylcysteine, ascorbic acid, alpha-lipoic acid, scopoletin, forsythin, isoferulic acid, gamma-oryzanol, trans-anethol, thioctic acid, cysteamine, gallic acid, salvianolic acid, vitamin E, lappaconitine, L-selenomethionine, selenium, coenzyme Q10, vitamin A, and catechins. Dantron These include, but are not limited to, N-acetylcysteine, alpha-lipoic acid, Dantron or a salt thereof, most preferably N-acetylcysteine ​​or a salt thereof.

[0043] The aforementioned N-acetylcysteine ​​(N-acetylcysteine) is an oxidative stress inhibitor with the structure shown in formula (2) below, developed about 50 years ago and mainly used as an expectorant or to treat acetaminophen poisoning. N-acetylcysteine ​​is known to exert its antioxidant effect by directly reacting with peroxides, hydrogen peroxide, hydroxyl radicals, etc. to reduce oxidative stress, or by indirectly increasing glutathione synthesis by providing cysteine, a raw material for glutathione biosynthesis. N-acetylcysteine ​​has been used for decades in various clinical fields, including the treatment of acute respiratory distress syndrome, acute pulmonary oxygen toxicity, and the prevention of acute renal failure caused by contrast agents, and is known as a safe drug that does not induce side effects. [ka]

[0044] According to one embodiment of the present invention, a combination formulation containing donepezil and N-acetylcysteine ​​was found to have significantly superior neuroprotective and differentiation-promoting effects compared to each of the individual substances.

[0045] Therefore, the composition of the present invention, comprising donepezil and N-acetylcysteine, can exhibit improved effects at lower doses compared to the use of each drug alone, thus offering the advantages of reduced side effects and improved therapeutic efficacy for brain diseases, i.e., the benefits of a synergistic effect.

[0046] The Bliss independent combined response (BLISS) C for two single compounds having effects A and B is given by C = A + BA * B, where each effect is expressed as a fractional inhibition value between 0 and 1. (See reference [Bliss (1939) Annals of Applied Biology]). The BLISS value, defined as the difference between the experimental reaction and the calculated BLISS independence value, indicates whether the effects of the two combined components are additive or synergistic.

[0047] A Bliss value of zero (0) is considered additive. The term "additive" means that the result of combining two targeted therapies is the sum of the individual drugs.

[0048] The term "synergistic effect" or "synergy" is used to mean that the reaction of a combination of two formulations is greater than the sum of the individual reactions of each formulation. More specifically In an in vitro setting, one measure of synergy is known as the "Bliss synergy." The Bliss synergy means "exceeding the Bliss independent value," which is 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 this invention also includes in vitro synergies measured by additional and / or alternative methods.

[0049] The biological effect (including, but not limited to, anti-inflammatory effect) exhibited in vitro by the combination of cholinesterase inhibitor and antioxidant in the present invention may correlate with the Bliss value if it is greater than or equal to the sum of the individual components of the combination. Furthermore, "synergistic effects," including cases where the combination of components used in the present invention exhibits activity equal to or greater than the sum of the individual components, can be measured by additional and / or alternative methods.

[0050] In one aspect of the present invention, the composition of the present invention can be used for the treatment of brain diseases by combining an effective amount of a cholinesterase inhibitor or a pharmaceutically effective salt, derivative, or metabolite thereof with an effective amount of an antioxidant or a pharmaceutically effective salt thereof in an amount sufficient to achieve a synergistic effect.

[0051] In one aspect of the present invention, the cholinesterase inhibitor and the antioxidant can be combined in a molar ratio of 1:0.1 to 2000. Preferably, they can be combined in a molar ratio of 1:1 to 1000, more preferably 1:50 to 500, even more preferably 1:100 to 400, and most preferably 1:100 to 350.

[0052] In the present invention, the aforementioned ischemic brain disease is also called cerebrovascular disease and can be included without limitation as any disease in which a pathological abnormality occurs in the blood vessels that supply blood flow to the brain due to thrombosis, embolism, cerebral vascular occlusion, etc. Non-limiting examples of ischemic brain disease include stroke, cerebral hemorrhage, cerebral infarction, head injury (head trauma), cerebral circulatory and metabolic disorders, vascular dementia, and cerebral functional coma.

[0053] In the present invention, the aforementioned degenerative neurological diseases (neurodegenerative diseases, degenerative neurological diseases) can be selected from, but are not limited to, the group consisting of Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, progressive supranuclear palsy, multiple system atrophy, olivary nucleus-pontine cerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential progression, corticobasal degeneration, Lewy body disease, Parkins-ALS-dementia complex, Niemann-Pick disease, and Pick disease.

[0054] The aforementioned cognitive impairments are closely related to aging, but differ from the normal aging process in that they involve rapid abnormal death of nerve cells in part of the nervous system or throughout the brain, resulting in loss of brain and spinal cord function and a decline in cognitive ability. This can be included without limitation. Non-limiting examples of cognitive impairments include mild cognitive impairment, Alzheimer's disease, frontotemporal dementia, Lewy body disease, corticobasal degeneration, learning disabilities, spontaneity, forgetting, aphasia, apraxia, and delirium.

[0055] "Prevention" means all actions that reduce the frequency or severity of pathological phenomena. Prevention may be complete or partial. In this case, it may mean a reduction in carcinogenic symptoms within an individual compared to when the aforementioned composition was not used.

[0056] "Treatment" means any clinical intervention aimed at altering the inherent processes of the target or cells being treated, and can be performed during the progression of a clinical condition or for preventative purposes.

[0057] The desired therapeutic effects may include preventing the onset or recurrence of the disease, reducing symptoms, decreasing all direct or indirect pathological consequences associated with the disease, slowing the rate of disease progression, reducing or temporarily alleviating the disease state, or improving the prognosis.

[0058] The donepezil and N-acetylcysteine ​​in the composition of the present invention can be administered simultaneously, separately, or sequentially, and donepezil and N-acetylcysteine ​​can exhibit preventive or therapeutic activity for brain diseases through synergistic action in the body.

[0059] While cholinesterase inhibitors and antioxidants are typically administered simultaneously as a composition, even if the active ingredients are administered to the human body with a time lag, the individually administered active ingredients can act simultaneously in the body, resulting in an equivalent level of therapeutic activity.

[0060] Specifically, "simultaneous administration" means administering the two active ingredients together via the same route of administration, or administering them separately via the same or different routes at substantially the same time interval (e.g., an interval of 15 minutes or less). "Individual administration" means administering the two active ingredients via the same or different routes at a fixed time interval (e.g., a 3-day interval). "Sequential administration" means administering both active ingredients via the same or different routes of administration using a fixed order rule, depending on the patient's disease state.

[0061] The drug can be administered orally or parenterally. Parenteral administration methods are not limited to these, but may include intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraneuronal (subventricular), intracerebral, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration.

[0062] The pharmaceutical composition according to the present invention may contain only pharmaceutically effective amounts of donepezil and N-acetylcysteine, or may further contain a pharmaceutically acceptable carrier. The “pharmaceutically effective amount” refers to an amount that shows a greater response than that of a negative control group, and preferably means an amount sufficient to show effects such as increased lifespan, improved mobility, suppression of neuroinflammation, suppression of neuronal cell death, and promotion of neuronal cell differentiation when the two active ingredients are administered in combination in the treatment or prevention of brain diseases.

[0063] The pharmaceutical compositions of the present invention can be formulated in various ways according to the route of administration, using methods known in the art, together with pharmaceutically acceptable carriers to exhibit a synergistic effect by the combined use of a cholinesterase inhibitor and an antioxidant. "Pharmaceutically acceptable" means a composition that is physiologically acceptable and, when administered to humans, does not inhibit the action of the active ingredient and does not generally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions; it is non-toxic. Carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes. The pharmaceutically acceptable carriers included in the compositions are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Other pharmaceutically acceptable carriers can be considered based on those known in the art.

[0064] In addition to the ingredients, the pharmaceutical composition contains lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, and preservatives. It may further contain preservatives, etc. Specifically, for oral administration, conjugates, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes or fragrances, etc. may be used; for injectable preparations, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used; and for topical administration, bases, excipients, lubricants, preservatives, etc. may be used.

[0065] Furthermore, the compositions of the present invention can be used in the form of general pharmaceutical formulations. Parenteral formulations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions or lyophilized formulations, injections, transdermal formulations, and nasal inhalants. Oral administration can be in the form of tablets, Trotskyi, capsules, elixirs, suspensions, syrups or wafers. In the case of injections, they can be manufactured in the form of unit dose ampoules or multiple doses. In the case of injections, sterilization is essential to protect them from contamination by microorganisms such as bacteria and fungi. For injections, suitable carriers may include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or solvents or dispersion media containing vegetable oils. More preferably, suitable carriers may include Hanks' solution, Ringer's solution, phosphate buffered salt (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injectable preparation from microbial contamination, it may further contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Furthermore, the injectable preparation may, in most cases, further contain isotonic agents such as sugars or sodium chloride.

[0066] Furthermore, the pharmaceutical compositions of the present invention can be administered by any device that allows the active substance to move to a target site. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or drip infusion. Injectable preparations can be prepared using aqueous solvents such as physiological saline or Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate), or alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin), and may contain pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA), emulsifiers, buffers for pH adjustment, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol). A method for treating or preventing degenerative neurological diseases using the compositions of the present invention comprises administering an effective amount (pharmaceutically effective amount) of the therapeutic composition of the present invention to an individual in need. The aforementioned pharmaceutically effective dose can be easily determined by those skilled in the art, depending on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of doses, the duration of treatment, and the drugs that are combined or used concurrently.

[0067] Furthermore, the pharmaceutical compositions 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.

[0068] The total effective dose of the composition of the present invention can be administered to a patient in a single dose, or in divided doses over a long period of time. The pharmaceutical composition of the present invention can vary the content of the active ingredient depending on 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 per kg of patient body weight per day, most preferably 0.1 μg to 500 mg. However, the effective dose of the above-mentioned pharmaceutical composition is determined by considering various factors such as the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate, as well as the formulation method, route of administration, and number of treatments. Therefore, considering these points, prior knowledge in the art is required. Anyone capable of determining the appropriate effective dose of the composition of the present invention can do so. The pharmaceutical composition according to the present invention is not particularly limited in terms of its formulation, route of administration, or method of administration, as long as it exhibits the effects of the present invention.

[0069] The composition of the present invention can be formulated so that the constituents donepezil and N-acetylcysteine ​​are simultaneously contained in a single formulation, depending on the method and route of administration. Each constituent can be formulated separately and included in a single package in dose units such as daily or once a day. The individually formulated donepezil and N-acetylcysteine ​​formulations may or may not be the same. Specific formulation methods for the pharmaceutical composition of the present invention and pharmaceutically acceptable carriers that can be included in the formulation are as described above for the pharmaceutical composition, and reference can be made to what is known in the art.

[0070] The present invention also provides a food composition for the prevention or improvement of ischemic brain disease, comprising a cholinesterase inhibitor and an antioxidant as active ingredients.

[0071] The present invention also provides a food composition for preventing or improving degenerative neurological diseases, comprising a cholinesterase inhibitor and an antioxidant as active ingredients.

[0072] The food composition of the present invention includes all forms such as functional foods, nutritional supplements, health foods, and food additives.

[0073] This type of food composition can be manufactured in various forms according to conventional methods known in the art. However, for example, as a health food, cholinesterase inhibitors and antioxidants can be manufactured in the form of tea, juice, and drink, and then liquefied, granulated, encapsulated, and powdered for consumption. Furthermore, donepezil and N-acetylcysteine ​​can be manufactured in the form of compositions by mixing them with known active ingredients effective against infectious diseases. Furthermore, functional foods, though not limited to these, can be manufactured by adding cholinesterase inhibitors and antioxidants to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meats and their processed foods (e.g., ham, sausage, corned beef, etc.), breads, noodles (e.g., udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candies, dairy products (e.g., butter, cheese, etc.), edible vegetable fats, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., miso, soy sauce, sauces, etc.). In addition, cholinesterase inhibitors and antioxidants can be manufactured and used in the form of powders or concentrated liquids for use as additives.

[0074] The preferred content of donepezil and N-acetylcysteine ​​in the food composition of the present invention is not limited to this, but is preferably 0.1 to 90 weight percent [%] of the final food product. More preferably, the food composition containing the cholinesterase inhibitor and antioxidant of the present invention as active ingredients can be mixed with active ingredients known to be effective against infectious diseases in order to produce a health functional food or dietary supplement.

[0075] The present invention also provides a method for treating ischemic brain disease in animals other than humans, comprising the step of administering a cholinesterase inhibitor and an antioxidant simultaneously, separately, or sequentially.

[0076] The present invention also provides a method for treating degenerative neurological diseases in animals other than humans, comprising the step of administering a cholinesterase inhibitor and an antioxidant simultaneously, separately, or sequentially.

[0077] This invention provides the use of cholinesterase inhibitors and antioxidants for the production of therapeutic formulations for ischemic brain diseases.

[0078] The present invention provides a method for treating ischemic brain disease, comprising administering an effective amount of a composition containing a cholinesterase inhibitor and an antioxidant to an individual in need thereof.

[0079] This invention provides the use of cholinesterase inhibitors and antioxidants for the production of therapeutic formulations for degenerative neurological diseases.

[0080] The present invention provides a method for treating degenerative neurological diseases, comprising administering an effective amount of a composition containing a cholinesterase inhibitor and an antioxidant to an individual in need thereof.

[0081] The "effective amount" of the present invention refers to an amount that, when administered to an individual, shows an effect of improving, treating, preventing, detecting, diagnosing, or suppressing or reducing ischemic brain disease and degenerative neurological disease. The "individual" refers to an animal, preferably a mammal, and may include humans, or it may be an animal-derived cell, tissue, organ, etc. The individual may be a patient who requires the effect. The “treatment” of this invention comprehensively refers to improving ischemic cerebrovascular diseases and degenerative neurological diseases or the symptoms of a disease, which may include curing, substantially preventing, or improving the condition of the disease. These include, but are not limited to, the alleviation, cure, or prevention of one or most of the symptoms arising from the disease.

[0082] In this specification, the term "contains" is used synonymously with "contains" or "characterizes," and does not exclude additional components or steps of the process that are not specifically mentioned in the compositions or methods according to the present invention. The term "consists of" means that additional elements, processes, or components that are not separately described are excluded. The term "essentially made" means that, within the scope of the composition or method, it may include materials or processes that do not substantially affect its basic properties in addition to the materials or processes described.

[0083] [Effects of the invention] The composition according to the present invention, when administered in combination with a cholinesterase inhibitor and an antioxidant, can demonstrate enhanced preventive and therapeutic effects against ischemic cerebrovascular disease and degenerative neurological disease compared to administration alone, and can reduce potential side effects caused by overdose or long-term administration of each drug. [Brief explanation of the drawing]

[0084] [Figure 1] Figure 1 shows the results of evaluating the reduction in reactive oxygen species (antioxidant effect) after inducing oxidative stress in nerve cells (SH-SY5Y) and then treating them with donepezil alone, N-acetylcysteine ​​(NAC) alone, or a donepezil + NAC combination (NDC-002). [Figure 2] Figure 2 shows the results of evaluating the anti-inflammatory effect by measuring IL-6 (Interleukin-6) levels generated after treating microglial cells (BV2 cells) with donepezil or NAC, either alone or in combination, along with LPS (lipopolysaccharide), an inflammatory response inducer. [Figure 3] Figure 3 shows the results of measuring NO concentrations generated in microglial cells after treatment with LPS along with dodepezil alone, NAC alone, or donepezil + NAC combination. [Figure 4] Figure 4 shows the results of evaluating the anti-inflammatory effect of NAC-like drugs by measuring the amount of NO generated after complex treatment of microblasts with LPS, donepezil, and antioxidant drugs. [Figure 5] Figure 5 shows the results of evaluating the cytotoxicity of NAC-like drugs by measuring the number of living cells after treating microglial cells with a combination of LPS, donepezil, and an antioxidant drug. [Figure 6] Figure 6 shows the results of measuring NO concentrations generated in microblast cells after treatment with LPS along with donepezil alone, an antioxidant alone, or a combination of donepezil and an antioxidant. [Figure 7] Figure 7 shows the results of evaluating the expression level of HB9, a motor neuron marker, in neural progenitor cells (ReNcell VM) after treatment with donepezil alone, NAC alone, or donepezil + NAC combination. [Figure 8] Figure 8 shows the results of evaluating the differentiation-promoting effect and cytotoxicity of donepezil-like drugs by measuring the expression level of HB9, a motor neuron marker, and the number of living cells after combined treatment with choline ester lyse inhibitors and NAC in neural progenitor cells. [Figure 9] Figure 9 shows the results of evaluating the differentiation-promoting effect of NAC-like drugs by measuring the expression level of HB9, a motor neuron marker, after combined treatment of neural progenitor cells with donepezil and an antioxidant drug. [Figure 10] Figure 10 shows the results of evaluating the cytotoxicity of NAC-like drugs by measuring the number of viable cells after combined treatment of neural progenitor cells with donepezil and antioxidant drugs. [Figure 11] Figure 11 shows the results of measuring the expression differences of PSD-95 and pCREB, which are involved in synaptic plasticity regulation, after primary culture of nerve cells from the brains of mouse fetuses, treatment with donepezil alone, NAC alone, or donepezil + NAC combination. [Figure 12] Figure 12 shows the results of evaluating the motor function recovery effects of donepezil alone, NAC alone, or donepezil + NAC combination therapy after 21 days of treatment following induction of a stroke-reperfusion animal model, based on the improvement in the Rotarod latency test at days 1, 3, 7, 14, and 21. [Figure 13]Figure 13 shows the results of quantifying the extent of brain damage after 21 days of treatment with donepezil alone, NAC alone, or donepezil + NAC combination immediately following induction of a stroke-reperfusion animal model. [Examples]

[0085] The present invention will be described in detail below with reference to the following examples. However, the following examples are for illustrative purposes only and do not limit the present invention.

[0086] Example 1: Antioxidant effect of donepezil + NAC combination In ischemic stroke, a type of cerebrovascular disease, a large amount of free radicals are produced in the ischemic area as blood flow decreases, resulting in the accumulation of oxidative metabolites. These free radicals cause oxidative stress, which can affect various organs, including brain cells. To minimize damage from reactive oxygen species, cells activate antioxidants and antioxidant enzymes to remove free radical intermediates and suppress oxidative reactions, thereby protecting the cells and the body.

[0087] In this invention, nerve cells (SH-SY5Y) were treated with H2O2 to induce oxidative stress, and then the amount of reactive oxygen species (ROS) generated was measured in the control group, after treatment with donepezil alone (5 μM), N-acetylcysteine ​​(NAC) alone (5 μM), and after combined treatment with donepezil (2.5 μM) + NAC (2.5 μM).

[0088] As a result, as shown in Figure 1, ROS was significantly reduced in the combined treatment group compared to the control group, and the combined treatment group showed a significantly improved reduction rate compared to the donepezil and NAC monotherapy groups, confirming a synergistic effect in antioxidant activity due to the combination therapy (**P<0.01, ****P<0.0001, one-way ANOVA).

[0089] Example 2: Anti-inflammatory effect of donepezil + NAC combination In this invention, microglia cells (BV2 cells) are treated with LPS to induce an inflammatory response, and at the same time, nitric oxide (NO) or an inflammatory marker (IL-6) is generated after treatment with donepezil alone, NAC alone, or donepezil + NAC combination. The drug's neuroinflammatory effect was confirmed by measuring its expression levels.

[0090] When microglia cells were treated with LPS to induce an inflammatory response, and the expression levels of the inflammatory marker IL-6 were measured after donepezil alone, NAC alone, or donepezil + NAC combined treatment, the most significant anti-inflammatory effect was observed with donepezil + NAC combined treatment, as shown in Figure 2. (****P<0.0001, one-way ANOVA)

[0091] As shown in Figure 3, the decrease in NO production after LPS treatment of microglial cells along with donepezil alone, NAC alone, or donepezil + NAC combined treatment was quantified, confirming that the donepezil + NAC combination drug showed a significantly improved anti-inflammatory effect compared to the same concentration of a single drug. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA)

[0092] To confirm that the anti-inflammatory effect observed with combined donepezil and NAC treatment is also present in combinations of donepezil and NAC with similar drugs, the amount of NO generated and the number of viable cells were measured after combined treatment of microglial cells with LPS and 22 types of antioxidant drugs from the same series as donepezil + NAC. The anti-inflammatory effect was confirmed at concentrations that did not show cytotoxicity in the similar drug combinations.

[0093] As a result, as shown in Figures 4 and 5, the donepezil + antioxidant combined treatment combination also showed a significant decrease in NO compared to the group treated with LPS alone. Similar to Figure 6, it was confirmed that the effect of donepezil + antioxidant combined treatment showed a synergistic effect compared to a single drug at the same concentration. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA)

[0094] Example 3: Differentiation-promoting effect of donepezil + NAC combination on neural progenitor cells Immortalized human neural progenitor cell line (VM), a neural progenitor cell line capable of differentiating into neurons and glial cells, was treated with donepezil alone, NAC alone, and donepezil + NAC combination. Immunostaining was then performed using an antibody (anti-HB9 antibody) that selectively and specifically binds to motor neurons. Subsequently, the degree of differentiation of neural progenitor cells into motor neurons was measured by analyzing the HB9 positive signal level to confirm the number of motor neurons.

[0095] As a result, as shown in Figure 7, a synergistic effect was observed in promoting neural progenitor cell differentiation in the combined treatment group compared to the treatment groups of each substance alone. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA)

[0096] To confirm that motor neuron differentiation is possible when treated with a combination of acetylcholinesterase inhibitors other than donepezil + NAC, and when administered in combination with an antioxidant, HB-9 levels and the number of viable cells were measured after treating neuronal bulb cells with a combination of cholinesterase inhibitors (8 types) similar to donepezil + NAC, or antioxidants (15 types) similar to donepezil + NAC. The motor neuron differentiation-promoting effect and cytotoxicity of these similar drug combinations were then confirmed.

[0097] As a result, as shown in Figure 8, eight combinations of donepezil-like drugs + NAC treatment showed a significantly increased differentiation-promoting effect on motor neurons compared to the control group, as indicated by donepezil + NAC treatment. Furthermore, as shown in Figures 9 and 10, combination treatments of 15 types of donepezil + NAC-like drugs also showed a similar effect to donepezil + NAC treatment compared to the control group. The significant increase in the differentiation-promoting effect on motor neurons was confirmed. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA)

[0098] Example 4: Synaptic plasticity-enhancing effect of donepezil + NAC combination agent Mouse nerve cells (primary neuron culture) were treated with donepezil alone, NAC alone, or donepezil + NAC combination. The expression levels of PSD-95 and pCREB, biomarkers of synaptic plasticity, were then measured using a Western blot assay.

[0099] As a result, as shown in Figure 11, the donepezil + NAC combination treatment group showed a synergistic trend in the increase of PSD-95 expression and pCREB compared to the control group, confirming the synaptic plasticity-enhancing effect of donepezil + NAC combination. (**P<0.01, one-way ANOVA)

[0100] For reference, there are different types of biomarkers available to confirm the functionality of improving cognitive ability and memory. Depending on the type, these include neuroprotective effects by inhibiting neuronal cell death, inhibition of beta-amyloid formation and accumulation, suppression of neuroinflammation, regulation of neurotransmitter production, secretion and metabolism, and regulation of synaptic plasticity.

[0101] Synaptic plasticity regulation is an important type of study that observes cognitive function effects by involving signal transduction between nerve cells, and its biomarkers include Postsynaptic Density-95 (PSD-95), CREB, and BDNF.

[0102] Postsynaptic density-95 (PSD-95) is a group of essential functional molecules involved in postsynaptic thickening (PSD), playing a crucial role in inducing long-term potentiation (LTP) through NMDA receptors and CaMKII. It directly and indirectly binds to neuroligin, NMDA receptors, AMPA receptors, potassium channels, and other molecules, playing a vital role in synaptic plasticity and the safety of synaptic changes during LTP. (Ehrlich et al., PSD-95 is required for activity-driven synapse stabilization, PNAS 2007. / Nagara et al., Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding deficient PSD-95 knockin mice, Molecular Brain, 2012) On the other hand, cAMP response element-binding protein (CREB) is a transcription factor that binds to the promoter sites of various genes involved in memory and synaptic plasticity, and CREB activation is known to induce transcription of genes related to memory formation and enhancement (such as BDNF). (Mizuno et al., CREB phosphorylation as a molecular marker of memory processing in the hippocampus for spatial learning. Behavioural Brain Research, 2002. / Kida et al., Functional) roles of CREB as a positive regulator in the formation and enhance Brain Research (Bulletin, 2014)

[0103] Therefore, donepezil + NAC combination therapy, which shows a synergistic effect on increasing PSD-95 expression and pCREB expression, also showed a synergistic effect on improving cognitive ability and memory.

[0104] Example 5: Confirmation of the synergistic effect of donepezil + NAC combination drug against a single drug in an in vivo experimental model. A stroke model was created using a cerebral infarction-reperfusion model in Sprague-Dawley rats (SD rats). Donepezil alone, NAC alone, or donepezil + NAC combination (NDC-002) were administered intravenously for 21 days immediately after the cerebral infarction-reperfusion model, and the experiment was conducted. In a representative motor control assessment test for experimental animal models of stroke, motor function in animals was measured via Rota-rod latency testing on days 1, 3, 7, 14, and 21 after administration of donepezil alone, NAC alone, and donepezil + NAC combination drugs, and the recovery rate of motor function was analyzed based on the results on day 1.

[0105] As a result, as shown in Figure 12, only the NDC-002 administration group showed a significant increase in recovery rate compared to the vehicle administration group, confirming the motor function recovery effect of the NDC-002 compound drug (**P<0.01, one-way ANOVA).

[0106] Furthermore, the NDC-002 combination drug showed a synergistic effect in the recovery of motor function, with a significantly increased recovery rate in the donepezil + NAC combination drug group compared to the donepezil monotherapy and NAC monotherapy groups (###P<0.001, one-way ANOVA).

[0107] Immediately after a stroke-reperfusion model, donepezil alone, NAC alone, and donepezil + NAC combination were administered for 21 days. Brain tissue was then stained using immunohistochemistry via neuronal (NeuN), microglia (GFAP) marker antibodies and nuclear staining solution (DAPI), and the extent of brain damage was analyzed. As shown in Figure 13, a significant reduction in the extent of brain damage was observed only in the NDC-002 combination administration group compared to the vehicle administration group (*P<0.05, one-way ANOVA). [Industrial applicability]

[0108] The composition according to the present invention, when administered in combination with a cholinesterase inhibitor and an antioxidant, exhibits enhanced preventive and therapeutic effects against brain diseases compared to administration alone, and can reduce potential side effects caused by overdose or long-term administration of each drug, making it highly industrially applicable.

Claims

1. It contains a cholinesterase inhibitor and an antioxidant as active ingredients. The cholinesterase inhibitor is donepezil, and The antioxidant is N-acetylcysteine ​​or danthone. A pharmaceutical composition for the prevention or treatment of ischemic cerebrovascular disease.

2. The pharmaceutical composition according to claim 1, characterized in that the ischemic cerebral disease is selected from the group consisting of stroke, cerebral hemorrhage, cerebral infarction, head injury, cerebral circulatory and metabolic disorder, vascular dementia, and cerebral functional coma.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that it exhibits a neuronal cell differentiation promoting effect.

4. It contains a cholinesterase inhibitor and an antioxidant as active ingredients. The cholinesterase inhibitor is donepezil, and The antioxidant is N-acetylcysteine ​​or danthone. Food composition for the prevention or improvement of ischemic cerebrovascular disease.

5. The food composition according to claim 4, characterized in that the aforementioned food is a health functional food.

6. A method for treating ischemic brain disease in non-human animals, comprising the step of administering a cholinesterase inhibitor and an antioxidant simultaneously, separately, or sequentially. The cholinesterase inhibitor is donepezil, and The antioxidant is N-acetylcysteine ​​or danthone. method.

7. The use of cholinesterase inhibitors and antioxidants for the manufacture of formulations to treat ischemic brain diseases, The cholinesterase inhibitor is donepezil, and The antioxidant is N-acetylcysteine ​​or danthone. use.

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