KR102816429B1 - Composition for treating neurodegenerative diseases - Google Patents

Abstract

The present invention relates to a composition for treating neurodegenerative diseases, and more specifically, to a composition for treating neurodegenerative diseases comprising flibanserin, which is highly effective in inhibiting beta-amyloid deposition and phosphorylation of tau protein; and ripasudil or everolimus.
The composition provided by the present invention exhibits activity to inhibit accumulation of beta-amyloid in the brain and phosphorylation of tau protein through re-creation of an FDA-approved drug whose safety has been confirmed in humans, and thus can be very usefully utilized in the development of therapeutic agents for various degenerative neurodegenerative diseases including Alzheimer's.

Description

Composition for treating neurodegenerative diseases

The present invention relates to a composition for treating neurodegenerative diseases, and more specifically, to a composition for treating neurodegenerative diseases comprising flibanserin, which is highly effective in inhibiting beta-amyloid deposition and phosphorylation of tau protein; and ripasudil or everolimus.

Neurodegenerative diseases are mental dysfunctions caused by the gradual structural and functional loss of nerve cells (neurons). Neurodegenerative diseases are accompanied by symptoms such as dementia, extrapyramidal abnormalities, cerebellar abnormalities, sensory disorders, and motor disorders due to the progressive degeneration of nerve cells in specific parts of the nervous system. Symptoms may be complex because abnormalities may occur in multiple parts at the same time. Diseases are diagnosed based on the clinical manifestations of the patient, but since symptoms can vary widely and different diseases often show common clinical symptoms, diagnosis is difficult (Soc. Sci. Med. Vol. 40. No. 6, pp. 847-858, 1995).

Neurodegenerative diseases show symptoms gradually and often occur with aging. Once developed, the disease progresses continuously for years or even decades until death, resulting in a huge social burden. It is known that genetic influences based on family history have a significant impact on the cause of development, but acquired factors are also considered to play an important role. Depending on their clinical symptoms, neurodegenerative diseases are largely classified into progressive dementia (e.g., Alzheimer's disease), neurological abnormalities (e.g., Pick's disease), postural and motor abnormalities (e.g., Parkinson's disease), progressive ataxia, muscular atrophy and weakness, and sensory and motor disorders (International Journal of Engineering and Technology, Vol.2, No.4, August 2010 Classification of Neurodegenerative Disorders Based on Major Risk Factors Employing Machine Learning Techniques).

The cytotoxicity of beta-amyloid (Aβ) plaques and hyperphosphorylated tau tangles is attracting attention as a direct cause of Alzheimer's disease, a representative degenerative brain disease.

Aβ is produced from the precursor APP and is produced outside the nerve cell by the action of enzymes such as β-secretase and γ-secretase. When the concentration of Aβ exceeds a certain level, Aβ proteins bind to each other to form insoluble senile plaques. This substance can cause inflammation and neurotoxicity and destroy surrounding nerve cells. For example, it is known that neuronal cell death and microgliosis observed in patients with Alzheimer's disease are associated with senile plaques. In vitro experiments have shown that Aβ peptides can induce the activation of microglia (brain macrophages), supporting the hypothesis that this is the cause of microgliosis and brain inflammation found in the brains of patients with Alzheimer's disease. Once Aβ is produced and senile plaques are formed, there is currently no widely accepted therapy or treatment that is expected to significantly dissolve them or prevent the formation of deposits.

Tau is composed of four parts: an N-terminal protrusion, a proline aggregation domain, a microtubule-binding domain, and a C-terminus (Mandelkow et al., Acta. Neuropathol., 103, 26-35, 1996), and plays a role in connecting microtubules that form the physical structure of neurons. It is known that abnormal hyperphosphorylation or modification of tau in neurons of the central nervous system causes degenerative brain diseases such as tauopathy. Representative tauopathies include Alzheimer's disease, Picks disease, and frontotemporal dementia with Parkinson's disease on chromosome 17 (FTDP-17) (Lee et al., Annu. Rev. Neurosci., 24, 1121-1159, 2001; Bergeron et al., J. Neuropathol. Exp. Neurol., 56, 726-734, 1997; Bugiani et al., J. Neuropathol. Exp. Neurol., 58, 667-677, 1999; Delacourte et al., Ann. Neurol., 43, 193-204, 1998; Ittner and Gotz, Nat. Rev. Neurosci., 12, 65-72, 2011).

Meanwhile, Alzheimer's disease (AD) is a neurological disorder mainly thought to be caused by amyloid plaques, which are abnormal deposits of proteins in the brain. The most common type of amyloid found in the brains of diseased individuals is mainly composed of Aβ fibrils. Scientific evidence demonstrates that increased production and accumulation of beta-amyloid protein in plaques leads to neuronal cell death, which contributes to the development and progression of AD. In turn, loss of neurons in strategic brain regions leads to a decrease in neurotransmitters and memory impairment. Proteins mainly essential for plaque formation include amyloid precursor protein (APP) and two presenilins (presenilin I and presenilin II). Sequential cleavage of the amyloid precursor protein (APP), which is constitutively expressed and catabolized in most cells, by the enzymes β and γ secretases releases 39- to 43-amino acid Aβ peptides. Cleavage of APP likewise increases their tendency to aggregate into plaques. The Aβ(1-42) fragment is particularly prone to aggregate accumulation, which is due to two very hydrophobic amino acid residues at its C-terminus. Thus, the Aβ(1-42) fragment is thought to be primarily involved in and essential for the initiation of neutritic plaque formation in AD, and thus to have a high pathological potential. Therefore, there is a need for specific molecules that can target and disperse amyloid plaque formation.

Accordingly, the inventors of the present invention have conducted extensive research to search for drugs or combinations of drugs that have been approved by the FDA and have been verified for human compatibility, and can exhibit preventive or therapeutic effects on various degenerative neurodegenerative diseases by inhibiting the accumulation of beta-amyloid and phosphorylation of tau protein. As a result, they discovered that a combination preparation of flibanserin and ripasudil, or flibanserin and everolimus, exhibits such effects, and completed the present invention.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating a degenerative neurological disease, comprising as an active ingredient at least one selected from the group consisting of flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil, everolimus and pharmaceutically acceptable salts thereof.

Another object of the present invention is to provide a food composition for preventing or improving a degenerative neurological disease, comprising as an active ingredient at least one selected from the group consisting of flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil, everolimus and pharmaceutically acceptable salts thereof.

In order to achieve the above-mentioned object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a degenerative neurological disease, comprising as an active ingredient at least one selected from the group consisting of flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil, everolimus and pharmaceutically acceptable salts thereof.

In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving a degenerative neurological disease, comprising as an active ingredient at least one selected from the group consisting of flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil, everolimus and pharmaceutically acceptable salts thereof.

The term "treatment" or "treating" as used herein is defined as the application or administration of a therapeutic agent, i.e., a compound of the present invention (alone or in combination with other pharmaceutical agents) to a patient, or to an isolated tissue or cell line, for the purpose of treating, curing, alleviating, alleviating, altering, resolving, improving, enhancing or affecting HBV infection, symptoms of HBV infection or the likelihood of developing HBV infection in a patient (e.g., for diagnosis or ex vivo application). Such treatment may be tailored and modified, in particular based on knowledge gained from the field of genomic pharmacology.

As used herein, the term "prevent" or "prevention" means, if nothing had happened, that there would be no progression of a disorder or disease, or, if there had already been a disorder or disease progression, that there would be no further progression of the disorder or disease. Also contemplated is the ability to prevent some or all of the symptoms associated with a disorder or disease.

The terms "patient", "subject" or "individual" as used in the present invention refer to a human or a non-human mammal. For example, non-human mammals include domestic animals and pets such as mammals of the ovine, bovine, porcine, canine, feline and murine families. Preferably, the patient, subject or individual is a human.

The terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount," as used herein, refer to a non-toxic, sufficient amount of an agent that provides the desired biological result. That result may be a reduction and/or attenuation of a signal, symptom, or cause of a disease, or any other desired change in a biological system. The appropriate therapeutic amount for any individual can be determined by one of ordinary skill in the art using routine experimentation.

The term "pharmaceutically acceptable" as used herein refers to a material, such as a carrier or diluent, that does not inhibit the biological activity or properties of the compound and is relatively nontoxic, i.e., can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term "pharmaceutically acceptable salt" as used in the present invention refers to a salt of the compound to be administered prepared from a non-toxic acid including a pharmaceutically acceptable inorganic acid, organic acid, solvate, hydrate or clathrate thereof. Examples of such inorganic acids include hydrochloric acid, hydrobromic acid, iodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, hexafluorophosphoric acid, citric acid, gluconic acid, benzoic acid, propionic acid, butyric acid, sulfosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, tartaric acid, amsonic acid, pamoic acid, p-toluenesulfonic acid and mesylic acid. Suitable organic acids can be selected, for example, from the aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, including formic acid, acetic acid, propionic acid, succinic acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, isethionic acid, lactic acid, malic acid, mucic acid, tartaric acid, para-toluenesulfonic acid, glycolic acid, glucuronic acid, maleic acid, furoic acid, glutamic acid, benzoic acid, atranilic acid, salicylic acid, phenylacetic acid, mandelic acid, emphonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, pantothenic acid, benzenesulfonic acid (besilate), stearic acid, sulfanilic acid, alginic acid, galacturonic acid, and the like. Additionally, pharmaceutically acceptable salts include, but are not limited to, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium-dependent or potassium), and ammonium salts.

The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable substance, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, which is associated with the carrying or transportation of a useful compound within the invention to or from a patient so that the compound may perform its intended function within the patient. Typically, such compositions 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 components of the formula comprising the useful compound within the invention and not damaging to the patient. Some examples of substances which can act as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose, which are 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; 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 solutions; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances. In addition, the "pharmaceutically acceptable carrier" used in the present invention includes coating agents, antiviral and antibacterial agents and absorption delaying agents, which are all or part of the active compounds useful in the present invention and are physiologically acceptable to the patient. In addition, supplementary active compounds may be incorporated into the composition. Additionally, the "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of a compound 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), which is incorporated herein by reference.

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

As used herein, the term "combination therapy" or "in combination" refers to the administration of two or more therapeutic agents to treat a condition or disorder described herein (i.e., a metabolic disease and a fibrotic disease). Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having fixed ratios of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in multiple or separate containers (e.g., capsules, powders, and liquids). The powders and/or liquids may be reconstituted or diluted to the desired dosage prior to administration. Such administration also includes sequential use of each type of therapeutic agent, either at about the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in the treatment of the condition or disorder described herein.

Combination therapy can provide a "synergistic effect" and can be "synergistic." That is, the effect achieved when the active ingredients are used together is greater than the sum of the effects that would occur if the compounds were used individually. A synergistic effect can be achieved when the active ingredients are (1) co-formulated and administered or delivered simultaneously in a combined unit dosage form; (2) delivered alternately or in parallel as separate dosage forms; or (3) delivered by some other schedule. When delivered in alternation therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example, by injection from separate syringes. In general, during alternation therapy, effective doses of each active ingredient are administered sequentially, i.e., sequentially, whereas in combination therapy, effective doses of two or more active ingredients are administered together. As used herein, synergy refers to the action of two therapeutic agents to produce an effect that is greater than the simple sum of the effects of each agent when administered alone. The synergistic effect can be calculated using any suitable method, such as the sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additivity 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)). Each of the above-mentioned equations can be applied to experimental data to generate corresponding graphs that are helpful in evaluating the effect of the drug combination. The corresponding graphs associated with 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 provides a pharmaceutical composition for preventing or treating a degenerative neurological disease, comprising as an active ingredient at least one selected from the group consisting of flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil, everolimus and pharmaceutically acceptable salts thereof.

In the present invention, the flibanserin is a drug used for the treatment of premenopausal women with decreased sexual desire disorder, and is a compound represented by the following chemical formula 1.

[Chemical Formula 1]

In the present invention, the lipasudil is a derivative of fasudil and is a compound represented by the following chemical formula 2, used for the treatment of glaucoma and ocular hypertension.

[Chemical formula 2]

In the present invention, everolimus is a drug used as an immunosuppressant to prevent organ transplant rejection and to treat renal cell carcinoma and other tumors, and is a compound represented by the following chemical formula 3.

[Chemical Formula 3]

According to one embodiment of the present invention, the drug combination of flibanserin and rifasudil; or flibanserin and everolimus was confirmed to exhibit the effect of inhibiting the deposition of beta-amyloid protein and inhibiting the phosphorylation of tau protein in Alzheimer's brain organoids. In particular, the effect of such combination therapy was confirmed to have a synergistic or synergistic effect compared to the treatment effect of each single drug.

Human beta-amyloid is a peptide molecule containing about 36-43 amino acids, and is known to be involved in the development of Alzheimer's disease as a major component of amyloid plaques expressed in the brains of Alzheimer's patients. The beta-amyloid peptide molecule may be obtained by cleaving amyloid precursor protein with beta-secretase and gamma-secretase. Beta-amyloid peptide molecules aggregate to form neurotoxic oligomers, causing degenerative brain diseases.

Tau protein is composed of four parts: an N-terminal protrusion, a proline aggregation domain, a microtubule-binding domain, and a C-terminus. It is known that abnormal hyperphosphorylation or modification of tau in the nerve cells of the central nervous system causes degenerative brain diseases such as Parkinson's disease and tauopathy.

Therefore, in the present invention, the “neurodegenerative disease” may be at least one selected from the group consisting of Alzheimer’s dementia, Parkinson’s disease dementia, progressive supranuclear palsy, Shy-Drager syndrome, striatonigral dystrophy, Huntington’s disease, amyotrophic lateral sclerosis, essential tremor, corticobasal degeneration, Parkinson-ALS-dementia complex, Niemann-Pick disease, amnesia, Down syndrome, cerebral amyloid angiopathy, mild cognitive impairment, presenile dementia, senile dementia, liquefied dementia, corticobasal degenerative dementia, amyloid stroke, systemic amyloidosis, Dutch amyloidosis, spinocerebellar ataxia, dementia with Lewy bodies, and frontotemporal dementia, but is not limited thereto.

Each component included in the combination preparation provided by the present invention can be formulated to be used separately, simultaneously, or sequentially. That is, when administered sequentially, the combination preparation can be administered in two or more administrations. The combination administration can include co-administration using separate formulations or a single pharmaceutical formulation, and sequential administration in either order.

In the present invention, when flibanserin and ripasudil are used as a combination therapy or a combination preparation, these drugs may be administered or formulated in combination at a molar ratio of 1:0.1 to 5, preferably at a molar ratio of 1:0.1 to 3, more preferably at a molar ratio of 1:0.1 to 1, and most preferably at a molar ratio of 1:0.3 to 0.7.

In the present invention, when flibanserin and everolimus are used as combination therapy or a combination formulation, these drugs may be administered or formulated in combination at a molar ratio of 1:0.01 to 5, preferably at a molar ratio of 1:0.01 to 3, more preferably at a molar ratio of 1:0.01 to 2, and most preferably at a molar ratio of 1:0.03 to 0.7.

The pharmaceutical composition according to the present invention may be formulated in a suitable form containing only the above drug combination or together with a pharmaceutically acceptable carrier, and may additionally contain an excipient or diluent. The term "pharmaceutically acceptable" as used herein refers to a non-toxic composition that is physiologically acceptable and does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, etc., when administered to humans.

Pharmaceutically acceptable carriers may additionally include, for example, carriers for oral administration or carriers for parenteral administration.

Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like.

In addition, it may include various drug delivery materials used for oral administration. In addition, the carrier for parenteral administration may include water, suitable oils, saline solution, aqueous glucose and glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.

The pharmaceutical composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, etc. Other pharmaceutically acceptable carriers and preparations may be referred to as described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration depending on the administration route as described above.

In the case of preparations for oral administration, the composition of the present invention can be formulated into powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. For example, oral preparations can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule to obtain a tablet or dragee. Examples of suitable excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; cellulosics including cellulose, methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropylmethyl-cellulose; fillers such as gelatin and polyvinylpyrrolidone. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as disintegrating agents in some cases.

Furthermore, the pharmaceutical composition of the present invention may additionally include an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, and a preservative.

In the case of parenteral administration preparations, they can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalers by methods known in the art. These formulations are described in a generally known prescription book for all pharmaceutical chemistry (Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered for 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 dosage of the pharmaceutical composition of the present invention can be about 0.01 to 10,000 mg per 1 day of patient body weight, most preferably 0.1 to 500 mg. However, since the dosage 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, a person having ordinary knowledge in the art will be able to determine the appropriate effective dosage of the composition of the present invention considering these points. 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 effect of the present invention.

The present invention also provides a food composition for preventing or improving a degenerative neurological disease, comprising as an active ingredient at least one selected from the group consisting of flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil, everolimus and pharmaceutically acceptable salts thereof.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives, etc., and is intended for consumption by humans or animals including livestock. The food composition of the above type can be manufactured in various forms according to conventional methods known in the art.

The food composition of the above type can be manufactured in various forms according to conventional methods known in the art. As general foods, the above-mentioned complex preparation can be manufactured by adding the above-mentioned complex preparation to, but is not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible plant oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.). In addition, as nutritional supplements, the above-mentioned complex preparation can be manufactured by adding the above-mentioned complex preparation to, but is not limited to, capsules, tablets, pills, etc. In addition, as a health functional food, it is not limited to this, but for example, the above complex preparation itself can be manufactured in the form of tea, juice, and drink, and can be consumed by liquefying, granulating, encapsulating, and powdering so that it can be consumed (health drink). In addition, in order to use the above complex preparation in the form of a food additive, it can be manufactured in the form of a powder or concentrate and used. In addition, it can be manufactured in the form of a composition by mixing the above complex preparation with a known active ingredient known to have an effect of improving degenerative neurological diseases.

When the food composition of the present invention is used as a health beverage composition, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, like a typical beverage. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol. The sweetener may be a natural sweetener such as thaumatin and stevia extract; a synthetic sweetener such as saccharin and aspartame, etc. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

When the above-described complex preparation according to the present invention is contained as an effective ingredient of a food composition, the amount is not particularly limited to an amount effective to achieve symptom improvement action, but is preferably 0.01 to 100 wt% based on the total weight of the entire composition. The food composition of the present invention can be prepared by mixing the above-described complex preparation with other active ingredients known to have an effect of improving degenerative neurological diseases.

In addition to the above, when the food composition of the present invention is used as a health food, it may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, the health food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These components may be used independently or in mixtures. The ratio of these additives is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The composition provided by the present invention exhibits activity to inhibit accumulation of beta-amyloid in the brain and phosphorylation of tau protein through re-creation of an FDA-approved drug whose safety has been confirmed in humans, and thus can be very usefully utilized in the development of therapeutic agents for various degenerative neurodegenerative diseases including Alzheimer's.

Figure 1 shows the results of analyzing the degree of beta-amyloid accumulation after treating E4/E4 Alzheimer's brain organoids with flibanserin and ripasudil, alone or in combination, at the concentrations indicated in the figure.
Figure 2 shows the results of analyzing the degree of accumulation of phosphorylated-tau protein (p-tau) after treating E4/E4 Alzheimer's brain organoids with a combination of flibanserin and ripasudil at the concentrations indicated in the figure.
Figure 3 shows the results of analyzing the degree of accumulation of beta-amyloid and phosphorylated-tau protein (p-tau) after treating E4/E4 Alzheimer's brain organoids with a combination of flibanserin and everolimus at the concentrations indicated in the figure.

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

Materials and Methods

(1) Subject recruitment and brain amyloid imaging

Ten subjects were recruited for the generation of induced pluripotent stem cells (iPSCs) and iCOs. Information about the subjects is shown in Table 1 below.

For brain amyloid imaging, each subject underwent both 3D 11C-PiB-PET and MRI using a 3.0T PET-MR scanner (Siemens Healthineers). The methods used to acquire and process the subject's imaging data were described in our previous papers (Park, J.C., et al. Plasma tau/amyloid-beta1-42 ratio predicts brain tau deposition and neurodegeneration in Alzheimer's disease. Brain 142, 771-786 (2019). Byun, M.S., et al. Korean Brain Aging Study for the Early Diagnosis and Prediction of Alzheimer's Disease: Methodology and Baseline Sample Characteristics. Psychiatry Investig 14, 851-863 (2017)).

(2) Generation, maintenance, and characterization of human iPSCs

Human iPSCs were generated from subject-derived peripheral blood mononuclear cells (PBMCs) according to a previously published protocol (Seki, T., Yuasa, S. & Fukuda, K. Generation of induced pluripotent stem cells from a small amount of human peripheral blood using a combination of activated T cells and Sendai virus. Nat Protoc 7, 718-728 (2012)). Whole blood samples were obtained by venous blood collection into K2 ethylenediaminetetraacetic acid (EDTA) tubes (367525, BD Biosys). PBMCs were separated by gradient centrifugation with Ficoll Paque Plus (17-1440-03, GE Healthcare), washed with PBS, centrifuged at 300 g for 15 min, and resuspended in KBM502 medium (16025020, Kohin Bio) at 1 × 10 ⁶ cells per mL. Cells were plated at a density of 5 × 10 ⁵ cells per well in 24-well plates pre-coated with human CD3-specific antibody (555336, BD Biosys) and cultured at 37°C in 5% CO2 for 5 days after adding fresh KBM502 medium on the second day. On day 5, activated T cells were harvested, resuspended in fresh KBM502 medium, and infected with Sendai virus using CytoTune-iPS 2.0 Sendai Reprogramming Kit (A16517, Invitrogen) at an MOI of 8 per 5 × 10 ⁵ cells. The cells were then replated onto fresh CD3 antibody-coated plates. On day 6, infected T cells were harvested, resuspended in mTeSR1 medium (ST85850, Stemcell Technologies), and then plated on 100-pi dishes coated with Matrigel hESC-qualified matrix (354277, Corning). The medium was replaced 2 days after plating the cells, and the medium was replaced daily thereafter. iPSC colonies began to appear on day 15–20. The cells were passaged on average every 5 days using ReLeSR (ST05872, Stemcell Technologies) as a dissociation reagent. To confirm the characterization of iPSCs, karyotype analysis was performed at GenDix, Inc. (Seoul, Republic of Korea), and alkaline phosphatase (ALP) staining was performed using an alkaline phosphatase (ALP) detection kit (SCR004, Milipore) according to the manufacturer's instructions. ApoE ε3 parental and ApoE ε4 isogenic iPSC lines have been used and validated in previous publications.

(3) Generation of iCOs from iPSCs

To form embryoid bodies (EBs), hiPSCs seeded on Matrigel-coated plates were dissociated with ReLeSR, and intact colonies were dissociated into single cells in AggreWell EB formation medium (ST05893, Stemcell Technologies) supplemented with ROCK inhibitor, Y-27632 (ST72304, Stemcell Technologies). On day 0, 2 × 10 ⁶ cells per well were loaded into AggreWell800 24-well plates (34811, Stemcell Technologies), and the medium was replaced with EB formation medium lacking ROCK inhibitor the following day.

On days 2 to 5, the medium was replaced daily with DMEM/F-12 containing GlutaMAX (10565-018, Gibco), 20% KockOut Serum Replacement (A31181501, Gibco), 1% MEM Non-Essential Amino Acids Solution (1140050, Gibco), 0.1 mM 2-mercaptoethanol (21985023, Gibco), and 100 U/mL penicillin and 100 μg/mL streptomycin (P4333, Merck), and additionally the SMAD inhibitors dorsomorphin (10 μM; P5499, Merck) and SB-431542 (10 μM; 1614, TOCRIS). On day 6, the EBs were harvested, and the following day, the EBs were seeded one by one into each well of a 96-well ultra-low-attachment microplate (Corning, 7007). The EBs seeded into each well of the 96-well plate were cultured in cerebral differentiation medium consisting of Neurobasal-A Medium (10888-022, Gibco), B-27 Supplement minus vitamin A (12587010, Gibco), 100 U/㎖ penicillin and 100 μg/㎖ streptomycin, GlutaMAX (350-061, Gibco), and 0.5% (v/v) Matrigel Basement Membrane Matrix (354234, Corning). The above brain differentiation medium was prepared by mixing the above components in a cooled state at 4℃, and was used by heating before culturing the EBs. On days 6 to 15, the medium was replaced daily with neuron medium supplemented with 20 ng/㎖ Epidermal Growth Factor (EGF, 01-107, Merck) and 20 ng/㎖ Fibroblast Growth Factor basic (bFGF; 233-FB, R&D Systems). On days 16 to 24, the medium was replaced every other day with the same medium. On days 25–42, the medium was replaced every other day with medium supplemented with 20 ng/mL Brain Derived Neurotrophic Factor (BDNF, 450-02, Peprotech) and 20 ng/mL Neurotropin-3 (NT-3; 450-03, Peprotech) in which EGF and BFGF were replaced. After day 43, the medium was replaced every 4 days with neuron medium without growth factors.

(4) QC of iCOs for drug screening

On day 7, high-quality embryoid bodies (EBs) were selected and seeded into ultra-low attachment 96-well plates. Then, on day 60, high-quality (low size variation) iCOs were selected and re-seeded. Subsequently, automated QC was performed during HCS imaging applying the following exclusion criteria: i) shape factor for circularity score (F = 4ÐA/(P ² ); F, shape factor; Ð, pi; A, organoid area; P, organoid perimeter); ii) diameter <1000 µm or >1400 µm, and iii) iCOs average area <700,000 µm ² .

(5) Drug treatment

Organoids cultured on 96-well plates were washed with Opti-MEM Reduced Serum Medium (31985-070, Gibco) containing 100 U/mL penicillin and 100 μg/mL streptomycin (P433, Merck). For drug treatment, drugs were mixed into the medium and added to the organoids at 37°C for 24 h. Drugs were treated as follows: Everolimus (0.25–5 μM; SML2282, Merck), Flibanserin (1–20 μM; HY-A0095, MedChemExpress), and Ripasudil (0.25–10 μM; HY-15685, MedChemExpress).

The specific drug combination treatment concentrations are shown in each result graph.

Experimental Results

1. Effect of combined treatment with flibanserin and ripasudil

When E4/E4 Alzheimer's brain organoids were treated with flibanserin and ripasudil, alone or in combination, at the concentrations indicated in the figure, the degree of beta-amyloid accumulation was analyzed. In the single treatment group of flibanserin or ripasudil, no significant beta-amyloid reduction effect was observed compared to the untreated control group, but in the combination treatment group at the same concentration, a concentration-dependent reduction in beta-amyloid accumulation was observed (Fig. 1).

In addition, when the degree of accumulation of phosphorylated-tau protein (p-tau) was analyzed after treating E4/E4 Alzheimer's brain organoids with a combination of flibanserin and ripasudil at the concentrations indicated in the figure, a significant decrease in p-tau accumulation was confirmed in the combination treatment group compared to the untreated control group (Fig. 2).

2. Effect of combined treatment with flibanserin and everolimus

When E4/E4 Alzheimer's brain organoids were treated with a combination of flibanserin and everolimus at the concentrations indicated in the figure, the accumulation of beta-amyloid and phosphorylated-tau protein (p-tau) was analyzed. It was confirmed that the accumulation of beta-amyloid and p-tau was reduced in the group treated with the combination of these drugs (Fig. 3).

The composition provided by the present invention exhibits activity to inhibit accumulation of beta-amyloid in the brain and phosphorylation of tau protein through re-creation of an FDA-approved drug whose safety has been confirmed in humans, and thus can be very usefully utilized in the development of treatments for various degenerative neurodegenerative diseases including Alzheimer's disease, and thus has very high potential for industrial use.

Claims (10)

A pharmaceutical composition for preventing or treating a degenerative neurological disease, comprising flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil or a pharmaceutically acceptable salt thereof as active ingredients,
A pharmaceutical composition wherein the above-mentioned neurodegenerative disease is at least one selected from the group consisting of Alzheimer's dementia, Parkinson's disease dementia, Niemann-Pick disease, cerebral amyloid angiopathy, presenile dementia due to beta-amyloid deposition, senile dementia due to beta-amyloid deposition, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, and Lewy body dementia.
delete In claim 1, the pharmaceutical composition is characterized in that the flibanserin is represented by the following chemical formula 1:
[Chemical Formula 1]

In claim 1, the pharmaceutical composition is characterized in that the lipasudil is represented by the following chemical formula 2:
[Chemical formula 2]

delete A pharmaceutical composition characterized in that in claim 1, flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil or a pharmaceutically acceptable salt thereof are administered simultaneously, separately, or sequentially.
A pharmaceutical composition, characterized in that in claim 1, flibanserin or a pharmaceutically acceptable salt thereof; and ripasudil or a pharmaceutically acceptable salt thereof are administered together in a molar ratio of 1:0.1 to 5.
delete In claim 1, the composition is a pharmaceutical composition characterized in that it inhibits beta-amyloid accumulation and phosphorylation of tau protein.
A food composition for preventing or improving a degenerative neurological disease, comprising flibanserin or a pharmaceutically acceptable salt thereof and ripasudil or a pharmaceutically acceptable salt thereof as active ingredients,
A food composition wherein the above-mentioned neurodegenerative disease is at least one selected from the group consisting of Alzheimer's dementia, Parkinson's disease dementia, Niemann-Pick disease, cerebral amyloid angiopathy, premature dementia due to beta-amyloid deposition, senile dementia due to beta-amyloid deposition, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, and Lewy body dementia.