Pharmaceutical composition comprising aminoaromatic compound and liver function protecting agent, and use thereof

Abstract

The present invention relates to a pharmaceutical composition comprising an aminoaromatic compound and a liver function protecting agent, and uses thereof. Specifically, the aminoaromatic compound is a drug that interacts with various molecular targets to exhibit a therapeutic effect, but may cause a significant increase in liver enzymes (AST and ALT), which may pose a potential risk of hepatotoxicity. By co-administering the compound KDS12025 and a bile acid, it is thus possible in the present invention to effectively control the increase in liver enzyme levels by the aminoaromatic compound according to one embodiment.

Description

Pharmaceutical composition comprising an aminoaromatic compound and a hepatoprotective agent and uses thereof

[0001] The present invention relates to a pharmaceutical composition comprising an aminoaromatic compound and a hepatoprotective agent, and to the use thereof.

[0002] Neurodegenerative disease refers to a condition in which the decline or loss of nerve cell function causes abnormalities in motor control, cognitive function, perceptual function, sensory function, and autonomic nervous system function. Representative examples include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and various dementia-related diseases. These neurodegenerative diseases generally follow a chronic and progressive course, and since no fundamental cure has been established to date, the development of effective treatment methods is continuously required.

[0003] One of the major pathological mechanisms of neurodegenerative diseases is oxidative stress resulting from the overproduction of reactive oxygen species (ROS). Oxidative stress refers to a phenomenon in which the balance between the body's antioxidant system and oxidative reactions is disrupted, leading to an excessive accumulation of ROS. It is reported that this increase in ROS causes lipid peroxidation, protein denaturation, and DNA damage, thereby inducing apoptosis and leading to the decline in neuronal function and death.

[0004] In particular, the brain is an organ highly vulnerable to oxidative stress because it has high oxygen saturation, is rich in polyunsaturated fatty acids and metal ions that are direct targets of oxidative stress, and neurotransmitters undergo auto-oxidation; consequently, when subjected to oxidative stress caused by reactive oxygen species (ROS), the content of unsaturated fatty acids decreases while the production of neurotoxic oxidation products increases. Furthermore, the brain has limited antioxidant and recovery capabilities against oxidative stress. Due to these characteristics, oxidative stress acts as a key mechanism in the development and progression of neurodegenerative diseases.

[0005] Meanwhile, aminoaromatic compounds are known to act as scavengers that remove hydrogen peroxide (H2O2), one of the reactive oxygen species. Specifically, it has been reported that aminoaromatic compounds interact with heme-containing enzymes to selectively scavenge H2O2 produced in excess within cells, thereby regulating ROS concentrations to physiologically acceptable levels. Accordingly, these compounds have the advantage of mitigating cell damage caused by oxidative stress by selectively regulating only pathologically excessive reactive oxygen species, rather than indiscriminately removing ROS. Furthermore, some aminoaromatic compounds possess the characteristic of effectively crossing the blood-brain barrier (BBB), making them a subject of interest as candidate substances for the treatment of central nervous system diseases.

[0006] However, some of these aminoaromatic compounds have been reported to increase the levels of liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which may lead to liver toxicity. Therefore, despite their excellent neuroprotective effects, there is a problem that there are limitations in terms of safety regarding the clinical application of these compounds.

[0007] Accordingly, the inventors of the present invention completed the present invention by researching a method to effectively alleviate liver toxicity while maintaining the efficacy of amino-aromatic compounds, and confirming that when ursodeoxycholic acid (UDCA) is administered in combination, liver toxicity, including elevated liver enzymes induced by amino-aromatic compounds, can be significantly suppressed and controlled.

[0008] [Prior Art Literature]

[0009] [Patent Literature]

[0010] Republic of Korea Registered Patent No. 10-2024361

[0011] One aspect provides a pharmaceutical composition comprising an amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof; and a bile acid.

[0012] Another aspect provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases, comprising an amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is co-administered with a bile acid.

[0013] Another aspect provides a health functional food for the prevention or improvement of neurodegenerative diseases, comprising an amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is administered in combination with a bile acid.

[0014] Another aspect provides a method for preventing, treating, or improving a neurodegenerative disease comprising the step of administering an effective amount of an amino-aromatic compound represented by Formula 1 or a pharmaceutically acceptable salt thereof; and a bile acid to an individual in need thereof.

[0015] Another aspect provides a method for inhibiting or alleviating hepatotoxicity in an individual with a neurodegenerative disease, comprising the step of administering an effective amount of an aminoaromatic compound represented by Formula 1 or a pharmaceutically acceptable salt thereof; and a bile acid to an individual in need thereof.

[0016] Another aspect is to provide amino-aromatic compounds or pharmaceutically acceptable salts thereof for the treatment of neurodegenerative diseases; and the use of bile acids.

[0017] Another aspect is to provide an amino-aromatic compound or a pharmaceutically acceptable salt thereof for manufacturing a therapeutic agent for neurodegenerative diseases; and a use of a bile acid.

[0018] One aspect provides a pharmaceutical composition comprising an aminoaromatic compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof; and a bile acid:

[0019] [Chemical Formula 1]

[0020]

[0021] In the above chemical formula 1,

[0022] Ar is C6-C 20 It is arylene, and the arylene of the above Ar is C1-C 10 Alkyl, C1-C 10 Alkoxy, amino, mono- or di-C1-C 10 Alkylamino, Halo-C1-C 10 Alkyl, halo-C1-C 10 It may be further substituted with one or more selected from alkoxy and hydroxyl groups;

[0023] R 1 and R 2 Each independently consists of hydrogen or C1-C 10 It is alkyl;

[0024] R 3 is a halogen, C1-C 10 Alkoxy, halo-C1-C 10 Alkyl or halo-C1-C 10 It is an alkoxy;

[0025] n is an integer of 1 or 2;

[0026] Single R 3 If it is a halogen, n is an integer of 1.

[0027] In one embodiment, the amino-aromatic compound may be a compound represented by the following chemical formula 2:

[0028] [Chemical Formula 2]

[0029]

[0030] In the above chemical formula 2,

[0031] R 1 and R 2 Each is independently hydrogen or C1-C7 alkyl;

[0032] R 3 is a halogen, C1-C7 alkoxy, haloC1-C7 alkyl;

[0033] R' is a C1-C7 alkyl, C1-C7 alkoxy, amino, or hydroxyl;

[0034] a is an integer from 0 to 4;

[0035] n is an integer of 1 or 2.

[0036] In one embodiment, the amino-aromatic compound may be any one selected from the group consisting of the following chemical formulas 3 to 6:

[0037] [Chemical Formula 3]

[0038]

[0039]

[0040] [Chemical Formula 4]

[0041]

[0042]

[0043] [Chemical Formula 5]

[0044]

[0045]

[0046] [Chemical Formula 6]

[0047]

[0048]

[0049]

[0050] The above bile acid may be a secondary or tertiary bile acid produced by modifying a primary bile acid by intestinal microorganisms, etc.

[0051] The bile acid may be ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), glycoursodeoxycholic acid (GUDCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), or tauro-deoxycholic acid (TDCA).

[0052] Ursodeoxycholic acid (UDCA) is one of the major components of bile acids and has efficacy such as hepatoprotection, antioxidant effects, and improvement of bile flow within the liver, and is clinically used in the treatment of liver disease.

[0053] The above pharmaceutical composition may be a complex formulation comprising an aminoaromatic compound and a bile acid. The above complex formulation is intended for "combined administration" of the aminoaromatic compound and the bile acid, and may be achieved by administering the individual components of the therapeutic regimen simultaneously, sequentially, or individually. A combination therapeutic effect is obtained by administering two or more drugs simultaneously or sequentially, or by administering them alternately at regular or indeterminate intervals. The combination therapy is not limited thereto, but may be defined as providing a synergistic effect in which the efficacy, measured by, for example, the degree of response, the rate of response, the time to disease progression, or the survival period, is therapeutically and toxicologically superior to the efficacy obtainable by administering one or the rest of the components of the combination therapy at a normal dose.

[0054] In one embodiment, the complex formulation may suppress the rise in liver enzyme levels. Specifically, the complex formulation may effectively control the rise in liver enzyme levels caused by the amino-aromatic compound while maintaining the effect of the amino-aromatic compound represented by Chemical Formula 1.

[0055] The above liver enzyme levels refer to values ​​that can confirm whether liver function is impaired, such as AST, ALT, γP, ALP, bilirubin, albumin, protein, and PT (prothrombin time).

[0056] In one embodiment, the liver enzyme level may be ALT or AST.

[0057]

[0058] Another aspect provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases comprising an amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is co-administered with a bile acid.

[0059] Another aspect provides a method for preventing, treating, or improving a neurodegenerative disease comprising the step of administering an effective amount of an amino-aromatic compound represented by Formula 1 or a pharmaceutically acceptable salt thereof; and a bile acid to an individual in need thereof.

[0060] Another aspect provides a method for inhibiting or alleviating hepatotoxicity in an individual with a neurodegenerative disease, comprising the step of administering an effective amount of an aminoaromatic compound represented by Formula 1 or a pharmaceutically acceptable salt thereof; and a bile acid to an individual in need thereof.

[0061] Another aspect provides the use of amino-aromatic compounds or pharmaceutically acceptable salts thereof for the treatment of neurodegenerative diseases; and bile acids.

[0062] Another aspect provides the use of an aminoaromatic compound or a pharmaceutically acceptable salt thereof for preparing a therapeutic agent for neurodegenerative diseases; and a bile acid.

[0063] The contents regarding the above chemical formula 1 and bile acid are as described in the above 'complex preparation'.

[0064] The above amino-aromatic compound may be administered at a dose of 0.0001 mg / kg to 1000 mg / kg. Specifically, 0.0001 mg / kg, 0.001 mg / kg, 0.001 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, It may be administered at doses of 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg, 500 mg / kg, or 1000 mg / kg.

[0065] In one embodiment, the dosage range of KDS12025 of the present invention can be set based on the toxicity limit observed in preclinical tests. Specifically, when KDS12025 was administered to a monkey model at doses of 0.03 mg, 0.1 mg, and 10 mg per kg of body weight, respectively, toxic findings including hepatotoxicity were observed in the 10 mg dose group per kg of body weight, and no significant toxic findings were observed at lower doses. Based on these results, the present invention may set the possible upper limit for human administration based on the dosage range in which no toxicity was observed.

[0066] In establishing a dosage range for humans according to the present invention, it is preferable to apply a Human Equivalent Dose (HED) conversion method based on body surface area. Specifically, the conversion method may be calculated according to the following formula by referring to the body surface area-based conversion presented in the clinical dosage setting guidelines provided by the U.S. Food and Drug Administration (FDA), but is not limited thereto:

[0067] HED (mg / kg) = Animal Dose (mg / kg) × (Animal Species Km / Human Km)

[0068] The above Km value represents the body surface area correction factor, and the Km value for monkeys is applied as 12, and the Km value for adults as 37.

[0069] According to the above conversion formula, when 10 mg is administered per kg of body weight in monkeys, the human equivalent dose is calculated to be approximately 3.2 mg / kg, and in the present invention, this value can be set as a reference standard for the limit range of toxicity occurrence in humans. Accordingly, the dosage of KDS12025 administered to humans according to the present invention is preferably set in the range of approximately 3 mg or less per kg of body weight, more preferably in the range of approximately 0.001 mg to 2 mg per kg of body weight, and most preferably in the range of approximately 0.003 mg to 1 mg or less per kg of body weight, but is not limited thereto.

[0070] In one embodiment, the dosage of KDS12025 of the present invention may be in the range of about 0.005 mg to about 0.5 mg per kg of body weight, which is the limit of toxicity observed in preclinical tests, and more preferably in the range of about 0.01 mg to about 0.1 mg per kg of body weight.

[0071] Meanwhile, the dosage according to the present invention may be adjusted considering the patient's age, weight, gender, concomitant drugs, route of administration, underlying disease, and other clinical factors, and a person skilled in the art may set an appropriate dosage range by taking into account the preclinical toxicity limit and the results of the human equivalent dose conversion.

[0072] In addition, the above neurodegenerative disease may be one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, post-traumatic stress disorder, multiple sclerosis, cerebral ischemic disease, and amyotrophic lateral sclerosis.

[0073] The above co-administration may involve co-administering the above amino-aromatic compound with bile acid simultaneously, sequentially, or in reverse order, as described in 'Combination Preparations'.

[0074] The above concomitant administration involves administering the aminoaromatic compound via the routes of intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, intranasal administration, intrapulmonary administration, or rectal administration; and

[0075] The above bile acid may be administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, local, nasal, pulmonary, or rectal administration routes.

[0076] In one embodiment, the bile acid may be administered at a dose of 50 mg / kg or less. Specifically, it may be administered at a dose of 50 mg / kg, 40 mg / kg, 35 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 15 mg / kg, 10 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg.

[0077]

[0078] The term "salt" above refers to a salt prepared using a specific compound according to one aspect and a relatively non-toxic acid or base. Additionally, in this specification, the "salt" may be a "pharmaceutically acceptable salt."

[0079] The term "pharmaceuticalally acceptable" above means exhibiting characteristics that are not toxic to cells or humans exposed to a specific compound according to one aspect.

[0080] When the above compound contains relatively acidic functional groups, a base addition salt can be obtained by contacting a sufficient amount of base with the neutral form of the compound in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include salts of sodium, potassium, calcium, ammonium, organic amines, or magnesium, or similar salts. When the above compound contains relatively basic functional groups, an acid addition salt can be obtained by contacting a sufficient amount of acid with the neutral form of the compound in a pure solution or a suitable inert solvent. Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromide, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, or phosphoric acid, and salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, souveric acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid, and further include salts of amino acids (e.g., arginine) and salts of organic acids such as glucuronic acid.

[0081] The above-mentioned pharmaceutically acceptable salts can be synthesized by conventional chemical methods from parent compounds containing an acidic or basic portion. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometrically appropriate amount of base or acid in water, an organic solvent, or a mixture of both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0082] The above term "prevention" may refer to any act of suppressing or delaying neurodegenerative diseases in an individual by administering a pharmaceutical composition according to one aspect.

[0083] The term "treatment" above may refer to any act in which the symptoms of a neurodegenerative disease in an individual are improved or beneficially altered by the administration of a pharmaceutical composition according to one aspect.

[0084] Additionally, the above pharmaceutical composition may be provided as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0085] Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano-spherical particle. These may form a complex with or be associated with a transport means and may be transported in vivo using a transport system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption-enhancing substances, or fatty acids.

[0086] When the above pharmaceutical composition is formulated, it may be prepared using diluents or excipients such as commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as bases for suppositories, and known diluents or excipients may be used when manufactured in the form of ophthalmic preparations.

[0087]

[0088] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitantly used drugs, and other factors well known in the medical field. The administration may be given once a day or divided into several doses. For example, it may be given every other day or once a week.

[0089] The term "administration" above refers to the introduction of a specific substance into an individual by an appropriate method, and "individual" refers to all living organisms, including rats, mice, pigs, horses, cattle, and livestock, including humans, that may harbor neurodegenerative diseases. Specific examples may include mammals, including humans.

[0090]

[0091] Another aspect provides a health functional food for the prevention or improvement of neurodegenerative diseases, comprising an amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the amino-aromatic compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is administered in combination with a bile acid.

[0092] The contents regarding the above chemical formula 1, bile acid, neurodegenerative disease, and concomitant administration are as described in the above 'Pharmaceutical Composition'.

[0093]

[0094] The term "improvement" above may refer to any action that at least reduces parameters related to the condition being treated, such as the severity of symptoms. In this case, the health functional food may be used for the prevention or improvement of neurodegenerative diseases, either simultaneously with or separately from medications for treatment, either before or after the onset of the disease.

[0095] The term "functional food" above is synonymous with "food for special health use (FosHu)," and refers to a food with high medical or health effects that is processed to efficiently exhibit bio-regulatory functions in addition to providing nutrition. Here, "functional" means obtaining useful effects for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body.

[0096] In addition, "health food" refers to a food that has active effects on maintaining or promoting health compared to general food, and "health supplement food" refers to a food intended for the purpose of health support. The above "health functional food" includes health functional foods, health foods, and health supplement foods.

[0097] In the above-mentioned health functional food, the compound of Formula 1 or its food-grade acceptable salt may be added directly to the food or mixed with other foods or food ingredients and used appropriately according to conventional methods. The amount of the compound of Formula 1 or its food-grade acceptable salt mixed may be appropriately determined according to the purpose of its use (for prevention or improvement). Generally, when manufacturing food or beverages, the compound of Formula 1 or its food-grade acceptable salt may be added in an amount of about 15% by weight or less, specifically about 10% by weight or less, with respect to the raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0098] The above-mentioned health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, by further including one or more of a carrier, a diluent, an excipient, and an additive. Foods to which a compound according to one aspect may be added include various types of food, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

[0099] Specific examples of the above carrier, excipient, diluent, and additive may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0100] The above-mentioned health functional food may contain other ingredients as essential components without special limitations, in addition to containing the compound of Formula 1 or a food-grade acceptable salt thereof. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, such as in ordinary beverages. Examples of the above-mentioned natural carbohydrates may be monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., common sugars; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by the choice of a person skilled in the art.

[0101] In addition to the above, a health functional food according to one aspect may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the proportion of these additives may also be appropriately selected by a person skilled in the art.

[0102] In addition, in one aspect, the compound of Formula 1 or a food-grade acceptable salt thereof may be consumed at a concentration of 0.1 mg / kg to 100 mg / kg. Specifically, it may be consumed at a concentration of 1 mg / kg to 50 mg / kg, and more specifically, at a concentration of 5 mg / kg to 10 mg / kg. For example, the compound of Formula 1 or a food-grade acceptable salt thereof may be consumed at a concentration of 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 10 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 10 mg / kg, 5 mg / kg to 100 mg / kg, 5 mg / kg to 50 mg / kg, or 5 mg / kg to 10 mg / kg.

[0103] In one aspect, the above health functional food may further include other health functional foods for the prevention or improvement of neurodegenerative diseases in addition to the compound of Formula 1 or a food-grade acceptable salt thereof.

[0104] The above-mentioned health functional food for the prevention or improvement of other neurodegenerative diseases may be included in the above-mentioned health functional food in a minimum amount that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0105] In addition, in one aspect, the above-mentioned health functional food may be consumed alone or in combination with the above-mentioned health functional food for the prevention or improvement of neurodegenerative diseases. That is, the above-mentioned health functional food for the prevention or improvement of neurodegenerative diseases may be consumed in conjunction with other health functional foods for the prevention or improvement of neurodegenerative diseases, and may be consumed simultaneously, separately, or sequentially, and may be consumed as a single or multiple times.

[0106] When consumed in combination with other health functional foods for the prevention or improvement of the aforementioned neurodegenerative diseases, it may be consumed in an amount or ratio that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0107] The above-mentioned health functional food for the prevention or improvement of other neurodegenerative diseases may be a health functional food for the prevention or improvement of neurodegenerative diseases that is conventionally known or a health functional food for the prevention or improvement of neurodegenerative diseases that is newly developed.

[0108] Amino-aromatic compounds are drugs that exhibit therapeutic effects by interacting with various molecular targets. However, they can cause a significant increase in liver enzymes (AST and ALT), which may pose a potential risk of liver toxicity.

[0109] In the present invention, by administering KDS12025 and bile acid together, the increase in liver enzyme levels caused by an amino-aromatic compound according to one embodiment can be effectively controlled.

[0110] Figure 1 is a figure confirming the effect of the KDS12025 compound of the present invention in a neurodegenerative disease model.

[0111] Figures 2 and 3 show the results of liver function evaluation following the combined administration of the KDS12025 compound of the present invention and ursodeoxycholic acid (UDCA).

[0112] Figure 4 is a figure showing the results of liver tissue pathology examination following the combined administration of the KDS12025 compound of the present invention and UDCA.

[0113] The following examples will be explained in more detail. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0114]

[0115] Example 1. Preparation of amino-aromatic compounds

[0116] The amino-aromatic compound of the present invention was prepared by the method disclosed in Korean Registered Patent No. 10-2643653, and among the said compounds, the experiment of the present invention was performed using the compound (KDS12025) described in [Chemical Formula 4] below.

[0117] [Chemical Formula 4]

[0118]

[0119] 400 MHz 1 H NMR (MeOD-d4) δ7.34 (s, 4H), 7.25 (m, 1H), 7.17 (m, 1H), 6.97 (d, 2H, J = 7.88), 6.90 (m, 1H), 3.85 (s, 3H), 3.52 (t, 2H, J = 7.68), 3.01 (s, 3H), 3.01 (t, 2H, J = 7.88)

[0120]

[0121] Example 2. Mouse rearing, preparation of an Alzheimer's disease (AD) mouse model

[0122] To confirm the efficacy of KDS12025 in an AD mouse model, mouse rearing conditions and drug administration methods were performed as follows.

[0123] Specifically, all mice (female and male) used in the experiment were housed in a temperature and humidity-controlled environment under conditions of a 12-hour light cycle and a 12-hour dark cycle, and all animal experiments were conducted with the approval of the Animal Ethics Committee of the Institute for Basic Science (IBS).

[0124] In addition, APP / PS1 transgenic mice aged 10 to 18 months were used as an animal model for Alzheimer's disease, and the mice were randomly assigned to groups to evaluate the therapeutic effect of KDS12025. KDS12025 was prepared by dissolving it in 200 μl of saline, and the dosage was calculated based on the mouse body weight (kg). The dose was set at 3 mg / kg / day or 10 mg / kg / day and administered once daily via intraperitoneal injection (ip) for 1 or 2 weeks. In addition, to evaluate the effect of oral administration of KDS12025, a dose of 3 mg / kg / day of KDS12025 was administered via ad libitum for 2 weeks. To minimize differences in intake among individuals during oral administration, a baseline was established by measuring the daily water intake of each mouse for 3 days prior to drug administration, and the concentration of KDS12025 in the drinking water was adjusted based on this data. Subsequently, water intake was monitored for 3 days after providing drinking water containing the drug to confirm whether intake remained stable. As a result, it was confirmed that KDS12025 has high water solubility and excellent palatability, with no significant change in water intake before and after drug administration.

[0125]

[0126] Example 3. Induction of astrocyte-specific lesions using viruses and diphtheria toxin

[0127] To induce astrocyte-derived lesions in APP / PS1 mice, local manipulation using viral vectors and diphtheria toxin was performed. Mice were deeply anesthetized with vaporized 1% isoflurane and fixed to a stereotaxic frame (RWD Life Science, China). An incision was made along the midline of the scalp, and a hole was perforated in the skull above the hippocampus. Subsequently, AAV-GFAP104-GFP or AAV-GFAP104-DTR-GFP was bilaterally microinjected into the hippocampal CA1 region (AP -2.0 mm, ML ±1.5 mm, DV -1.65 mm, bregma reference) to induce astrocyte-specific expression of the diphtheria toxin receptor (DTR). Following the viral injection, diphtheria toxin was prepared at a concentration of 2 mg / ml and administered continuously for 16 days to induce the selective elimination of DTR-expressing astrocytes. Through this, APP / PS1 mice (fGiD) with astrocyte-derived focal lesions were prepared. Meanwhile, the virus injection procedure was designed to be completed within 5 weeks prior to the behavioral analysis and the initiation of drug administration in this experiment. All viral vectors used in this example were manufactured at the IBS virus facility (IBS virus facility, South Korea).

[0128]

[0129] Example 4. Passive Avoidance Test (PAT)

[0130] PAT was performed to verify the cognitive function improvement effect of KDS12025.

[0131] Specifically, prior to conducting the experiment, mice were acclimatized to the experimental environment through sufficient handling and placed in a two-compartment shuttle chamber (light and dark compartments, Ugo Basile, Italy) equipped with a shock generator. During the acquisition phase on the first day of the experiment, mice were placed in the light compartment and allowed to explore freely for 60 seconds; afterward, the door separating the light and dark compartments was lifted to allow the mice to move into the dark compartment. Once the mice had fully entered the dark compartment, the separation door was immediately closed, and an electrical stimulus (foot shock, 0.5 mA, duration 2 seconds) was applied via a floor grid. Subsequently, the mice were returned to their home cages, and the reception phase test was conducted 24 hours after the acquisition phase. During the reception phase, mice were placed back in the light compartment and allowed to wait for 60 seconds before the separation door was opened. Learning and memory abilities were evaluated by recording the latency to step-through (up to 540 seconds) until the mice stepped into the dark compartment.

[0132]

[0133] Example 5. Novel Place Recognition (NPR)

[0134] To verify the effect of KDS12025 on improving spatial recognition memory, a novel place recognition (NPR) test was performed.

[0135] Specifically, prior to conducting the experiment, mice were subjected to sufficient handling and then habituated to an open field test (OFT), which was performed in a square chamber (40 × 40 × 40 cm). After habituation was complete, the mice were placed in the test apparatus with two identical objects positioned in the first and second quadrants of the open field cage, respectively, and observed for 10 minutes to allow them to freely explore the two objects. Subsequently, the mice were returned to their home cages to rest for 1 hour. Then, to evaluate spatial awareness memory, one of the two previously placed objects was moved to a new location (quadrant), and the mice were placed back into the open field chamber to observe their behavior for 10 minutes. Novel location recognition ability was evaluated using the discrimination index (DI), which was calculated as the percentage of time spent at the novel location relative to the total time spent at the novel location and the existing location. All behavioral analysis and frequency measurement were performed using EthoVision XT software (Noldus, Netherlands).

[0136]

[0137] Example 6. Preparation of a human α-synuclein overexpressing Parkinson's disease (PD) mouse model

[0138] To confirm the efficacy of KDS12025 in a PD mouse model, a mouse model overexpressing human α-synuclein was constructed.

[0139] Specifically, AAV-CMV-A53T virus or PBS as a control was injected into the right substantia nigra pars compacta (SNpc) of mice using a stereotactic fixation device, and the injection coordinates were set to AP -3.2 mm, ML -1.3 mm, and DV -4.0 mm relative to bregma. Virus injection was performed under general anesthesia using 1% isoflurane, the injection rate was set to 0.2 μl / min, and the total injection volume was 1 μl.

[0140] The above mice were sequentially circulated on a rotarod device at speeds of 5 rpm, 10 rpm, and 15 rpm, respectively, and their motor coordination ability and endurance were evaluated by measuring the time maintained for up to 3 minutes at a speed of 20 rpm. At this time, the elapsed time from the moment they were placed on the rotating rod until they fell (latency to fall) was used as an indicator to evaluate motor dysfunction due to Parkinson's disease and the therapeutic effect of KDS12025.

[0141]

[0142] Example 7. Preparation of an amyotrophic lateral sclerosis (ALS) mouse model

[0143] To confirm the efficacy of KDS12025 in an ALS mouse model, SOD1 G93A A mouse (B6SJL-Tg(SOD1*G93A)1Gur / J) was purchased from Jackson Laboratory (strain number 002726) and used as an ALS mouse model.

[0144] The mice were classified into four groups: a control group, an SOD1 group, a KDS12025 (1 mg / kg / day) administration group, and a KDS12025 (10 mg / kg / day) administration group. KDS12025 was administered via free drinking water starting when the mice reached 10 weeks of age, and was provided diluted in water to achieve a daily dose of 1 mg / kg / day or 10 mg / kg / day, respectively. The control group and the SOD1 group were provided with regular drinking water that did not contain the drug.

[0145]

[0146] Motor function was evaluated weekly using a rotarod device. The running times of the mice were normalized against the values ​​of the littermate control group to compare and analyze changes in motor coordination ability and endurance over time. Mice were followed up for up to 24 weeks, and the median survival time was calculated for each group.

[0147]

[0148] Example 8. Immunohistochemistry analysis

[0149] Immunohistochemical analysis was performed to confirm pathological changes following the administration of KDS12025.

[0150] Specifically, mice were anesthetized with 2% isoflurane and fixed to an operating table; the abdomen was opened to expose the heart, which was then perfused with physiological saline and further perfused with a 4% paraformaldehyde (PFA) solution dissolved in 0.1 M PBS. Subsequently, the mouse heads were desecrated and the brains were excised from the skulls; the excised brains were post-fixed in a 4% PFA solution overnight at 4°C and dehydrated in a 30% sucrose solution for 48 hours. Coronal hippocampal sections of the hippocampus were prepared to a thickness of 30 μm using a cryostat and stored in a preservation solution.

[0151] For the ALS experiment, lumbar spinal cord (L2–L5) sections were isolated and post-fixed by immersion in 4% PFA solution. The prepared sections were washed with 0.1 M PBS and incubated in a blocking solution (0.3% Triton X-100, 2% donkey serum, and 2% goat serum in 0.1 M PBS) at room temperature for 1 hour. Subsequently, immunostaining was performed overnight on a shaker at 4°C using a primary antibody mixture diluted in the blocking solution. Afterward, the sections were washed three times with PBS; following washing, fluorescently labeled secondary antibodies corresponding to each primary antibody were added and incubated at room temperature for 1 hour. DAPI staining was performed during the second washing step by adding DAPI solution (1:2000 dilution). After all washing was completed, the sections were fixed to cover glasses using mounting medium. Fluorescence images were obtained using an LSM900 confocal microscope or a Lattice SIM Elyra 7 system (both Zeiss, Germany).

[0152] Z-stack images in the 15-22 μm range acquired at 1-2 μm intervals were analyzed using the ImageJ program (NIH, USA) and the ZEN optical microscope digital imaging system (Zeiss, Germany). Meanwhile, the primary antibodies used in the present invention were chicken anti-GFAP (1:500, Millipore, ab5541), rabbit anti-amyloid beta (1:500, Abcam, ab2539), mouse anti-NeuN (1:200, Millipore, ab377), rabbit anti-Hbβ (1:200, Abcam, ab214049), mouse anti-Hbβ (1:200, Santa Cruz, sc-21757), mouse anti-MAOB (1:200, Santa Cruz, sc-515354), mouse anti-8-OHdG (1:500, Santa Cruz, sc-393871), rabbit anti-TH (1:500, Pelfreez, p40101), and mouse anti-TH (1:200, Sigma, T2928), and the secondary antibodies were Jackson's products prepared at a 1:500 concentration in a blocking solution. It was diluted and used (donkey-anti-chicken 405, donkey-anti-chicken 647, donkey-anti-rabbit 488, donkey-anti-rabbit 594, donkey-anti-rabbit 647, donkey-anti-guinea pig 488, donkey-anti-guinea pig 647, donkey-anti-mouse 594, donkey-anti-mouse 647).

[0153] GFAP-immunolabeled serial confocal slices were synthesized into maximal projection images to evaluate GFAP signals within the hippocampus (Z-stack images ranging from 15 to 22 μm acquired at 1-2 μm intervals from the brain slices). Sholl analysis was performed using an ImageJ plugin, which automatically generated concentric circles at 10 μm intervals extending from the soma to the farthest astrocyte process. This analysis quantified the number of crossings of GFAP-labeled processes within each concentric circle and calculated the ramification index and ending radius, thereby enabling the evaluation of astrocyte hypertrophy and morphological changes as previously reported. Additionally, in the fGiD mouse model, the number of CA1 pyramidal neurons was quantified using ImageJ by quantifying the number of NeuN-positive CA1 pyramidal neurons within a 50 × 50 μm² area.

[0154]

[0155] Example 9. Combined administration of KDS12025 and UDCA

[0156] 9.1 Preparation of Primates

[0157] The non-human primate used in this example was the crab-eating monkey (Cynomolgus macaque, Macaca fascicularis), and the study was conducted under contract at the K-MEDI hub preclinical center.

[0158] - Gender and Age: Male, approximately 4-6 years old

[0159] - Weight range: Approx. 3.5-7.0 kg

[0160] - Number of animals: 1 per group, a total of 4

[0161] - Vehicle control group;

[0162] KDS12025 10 mg / kg administration group;

[0163] KDS12025 0.1 mg / kg administration group + UDCA 0.03 mg / kg combination administration group;

[0164] KDS12025 0.03 mg / kg + UDCA 0.03 mg / kg combination therapy group

[0165] All primate experiments were conducted in a dedicated primate facility within the Preclinical Center of the Daegu Gyeongbuk Advanced Medical Industry Promotion Foundation (K-MEDI hub, Daegu, South Korea) after obtaining approval from the institution's Institutional Animal Care and Use Committee (IACUC). Animals were housed individually in stainless steel cages and reared under conditions of a temperature of 22±2°C, relative humidity of 40-70%, and a 12-hour light-dark cycle (lighting from 07:00 to 19:00). Commercial primate feed and fruit were fed twice daily, and water was available for free intake.

[0166]

[0167] 9.2 Surgical method to induce stroke (cerebral infarction) (Photothrombosis, PT)

[0168] Anesthesia and preparation

[0169] After pretreatment with atropine sulfate (approx. 0.02 mg / kg, IM), anesthesia was induced using a combination of ketamine hydrochloride (5-10 mg / kg, IM) and xylazine (0.5-1.0 mg / kg, IM). Subsequently, endotracheal intubation was performed, and maintenance anesthesia was administered with isoflurane (1-2% in O2). Body temperature, electrocardiogram, oxygen saturation, and blood pressure were continuously monitored during surgery.

[0170]

[0171] Surgical approach and cranial incision

[0172] After anesthesia, the animal was secured to a head fixation device, and the scalp was incised to expose the fronto-parietal skull. A craniotomy of approximately 10-15 mm in diameter was performed according to coordinates based on the target cortical region (primary motor cortex / sensorimotor cortex or internal capsule region), and the cortical surface was exposed while preserving the dura mater.

[0173]

[0174] Induction of localized cerebral infarction by photothrombosis

[0175] Rose bengal was dissolved in 0.9% physiological saline and slowly administered intravenously at a dose of approximately 10-20 mg / kg. Immediately after administration, a local intravascular thrombus was induced by irradiating the target cortical area with a diameter of approximately 3-5 mm for 10-20 minutes using a 532 nm green laser (or a high-intensity green light source). After irradiation, the reduction in blood flow and color change at the target area were visually confirmed, the skull was restored with artificial bone or bone wax, and the periosteum, muscle, and skin were sutured in that order.

[0176]

[0177] Post-operative care

[0178] After the surgery, anesthesia was restored, and analgesics (e.g., buprenorphine) and antibiotics were administered as needed. Neurological status, behavior, and feeding status were monitored daily to check for complications.

[0179]

[0180] 9.3 Formulation and Administration Routes of Test Substances (KDS12025 and UDCA)

[0181] test substance

[0182] KDS12025 is a water-soluble astrocytic hemoglobin peroxidase enhancer that exhibits neuroprotective effects upon oral administration in oxidative stress-related brain disease models, as previously reported.

[0183] UDCA (ursodeoxycholic acid) is a bile acid class drug that is widely used to protect hepatocellularity and promote bile excretion in various hepatobiliary diseases.

[0184]

[0185] Solvents and formulations

[0186] KDS12025 and UDCA were dissolved or suspended in an aqueous solution of purified water for oral administration. The final concentration of the solution was adjusted to be within 5 mL / kg of body weight.

[0187]

[0188] Route of administration and method of administration

[0189] All test substances were administered via the oral (gavage) route. After lightly immobilizing the monkeys, a flexible gastric tube was inserted through the mouth-esophagus into the stomach, and the pre-prepared suspension of the test substance was slowly infused. The vehicle control group was administered the same volume of solvent (aqueous purified water solution) in the same manner.

[0190]

[0191] 9.4 Dosage Schedule and UDCA Dosage

[0192] Capacity by group

[0193] In this embodiment, the dosage and combination relationship of KDS12025 and UDCA is as follows.

[0194] - Vehicle Control Group: No test substance administered, solvent only 5 mL / kg orally

[0195] - KDS12025 10 mg / kg monotherapy group

[0196] - KDS12025 + UDCA combination therapy group:

[0197] KDS12025 0.1 mg / kg + UDCA 20 mg / kg, once daily, total once-daily oral co-administration

[0198] KDS12025 0.03 mg / kg + UDCA 20 mg / kg, orally administered once daily for a total of 3 days.

[0199]

[0200] The UDCA 20 mg / kg dose was set to minimize the potential burden on the hepatobiliary system while maintaining the neuroprotective effect against H2O2-induced brain injury when used in combination with KDS12025. UDCA has previously been reported to have hepatoprotective effects and reduce serum liver enzymes (ALT, AST) in cholestatic liver disease, and based on this, it was selected as a co-administered drug in this example.

[0201]

[0202] Time and duration of administration

[0203] On Day 0, a cerebral infarction was induced through photothrombosis surgery.

[0204] The first administration of the test substance began within approximately 1 hour after PT induction (Day 0), and was repeated once a day depending on the group.

[0205] Blood biochemical tests, including AST and ALT, were measured using an automated biochemical analyzer with serum collected from a jugular vein at a set time after the last administration (e.g., the day after the last administration) (see Example 10).

[0206]

[0207] Example 10. AST and ALT Measurement

[0208] 10.1 Blood Collection and Serum Separation

[0209] In the control treatment (PT only) and oral administration groups of the test substance, blood was collected at baseline before PT induction, immediately after PT induction, and at the end of test substance administration (e.g., 3, 7, and 14 days after PT induction). Animals were appropriately sedated under isoflurane inhalation anesthesia, and approximately 2-3 mL of whole blood was collected from the femoral vein or radial vein using a 21-23 G needle. The collected whole blood was placed in a serum separator tube containing a coagulation promoter. Subsequently, the tube was left at room temperature (20-25°C) for approximately 30 minutes to allow for complete coagulation, and then centrifuged at 4°C, 3,000 rpm, for 10 minutes to separate the supernatant serum.

[0210] The isolated serum was stored at 4°C for a short period (within 24 hours) until analysis, and

[0211] If long-term storage was required, frozen storage was performed at -80°C. Thawing was performed slowly at 4°C, and repeated freezing and thawing were avoided.

[0212]

[0213] 10.2 Analytical Equipment and Reagents

[0214] AST and ALT activity measurements were performed using an automated blood biochemistry analyzer (e.g., automated clinical chemistry analyzer, set to 37°C) equipped at the Daegu Gyeongbuk Advanced Medical Industry Promotion Foundation Preclinical Center (K-MEDI hub). The equipment was calibrated and precision verified before analysis using a standard calibration program provided by the manufacturer and quality control serum (two levels of low and high concentrations).

[0215] Both AST and ALT were measured using commercial reagents (e.g., IFCC standardized AST / ALT reagents) that utilize the kinetic UV method of continuous absorbance measurement in accordance with the 37°C enzyme activity measurement standard method recommended by the International Federation of Clinical Chemistry (IFCC).

[0216]

[0217] 10.3 Principle and Reaction Mechanism of AST Measurement (IFCC Method)

[0218] AST activity was measured using an enzymatic reaction based on the IFCC recommended method. First, in the primary reaction, L-aspartate and 2-oxoglutarate react to produce oxaloacetate and L-glutamate. In the subsequent secondary reaction, malate dehydrogenase (MDH) acts to convert the produced oxaloacetate into L-malate and NAD by reacting with NADH and H.

[0219] - First-order reaction (AST reaction)

[0220] L-aspartate + 2-oxoglutarate → oxaloacetate + L-glutamate

[0221] - Secondary binding reaction (by malate dehydrogenase, MDH)

[0222] oxaloacetate + NADH + H+ → L-malate + NAD+

[0223] For the analysis, the decrease in absorbance (340 nm) of NADH in the reaction solution was continuously measured at 37°C for a set period of time, and AST activity (unit: U / L) was calculated using the rate of decrease in absorbance per unit time. Measurement parameters (reaction time, lag time, measurement interval, etc.) followed the recommended conditions of the automated analyzer and reagent manufacturer used.

[0224]

[0225] 10.4 ALT Measurement Principle

[0226] ALT activity was measured using an enzymatic reaction based on the IFCC recommended method. In the primary reaction, L-alanine and 2-oxoglutarate react to produce pyruvate and L-glutamate. Subsequently, in the secondary reaction, pyruvate produced by the action of lactate dehydrogenase (LDH) reacts with NADH and H to be converted into L-lactate and NAD.

[0227] - First-order reaction (ALT reaction)

[0228] L-alanine + 2-oxoglutarate → pyruvate + L-glutamate

[0229] - Secondary binding reaction (by lactate dehydrogenase, LDH)

[0230] pyruvate + NADH + H+ → L-lactate + NAD+

[0231] Similarly, the decrease in NADH absorbance (340 nm) in the reaction system was continuously measured at 37°C to calculate ALT activity (unit: U / L). As with AST, the measurement conditions followed the protocols of the equipment and reagent manufacturers (ensuring traceability to IFC standards).

[0232]

[0233] 10.5 Calibration and Quality Control (QC)

[0234] The equipment was calibrated using enzyme calibrator serum traceable to IFCC standards, and analysis was performed only within acceptable ranges after verifying the linearity and correlation coefficient (r) of the calibration curve. Quality control serums at two levels—low and high concentrations—were measured before, during, and after the analysis to monitor intra-assay CV and inter-assay CV. If the QC value deviated from the manufacturer's suggested acceptable range or the self-set acceptable range, the equipment was inspected and recalibrated, after which the samples from the corresponding batch were reanalyzed.

[0235]

[0236] 10.6 Result Processing

[0237] AST and ALT were quantified in U / L units for each individual, and comparisons between groups (control vs. test substance administration group) were summarized as mean ± standard deviation. If necessary, the change from baseline (ΔAST, ΔALT) or percentage change (% change) was calculated and used as an indicator to evaluate the hepatoprotective effect or hepatotoxicity of the test substance.

[0238]

[0239] Example 11. Pathological examination of liver tissue

[0240] 11.1. Tissue Sampling and Fixation

[0241] After the experiment was completed, the experimental animals were anesthetized with 2% isoflurane, laparotomy was performed, and liver tissue was excised. The excised liver tissue was immediately immersed in a 10% neutral formalin solution and fixed at room temperature for 24-48 hours.

[0242]

[0243] 11.2. Tissue Processing and Paraffin Embedding

[0244] Fixed liver tissue was dehydrated and cleared using an automatic tissue processor and then embedded in paraffin. Specifically, stepwise dehydration was performed with 70%, 80%, 95%, and 100% ethanol, cleared with xylene, and then infiltrated into 60°C paraffin to produce a paraffin block.

[0245]

[0246] 11.3. Tissue Section Preparation and Staining

[0247] The paraffin blocks were sectioned to a thickness of 4–5 μm using a rotary microtome. The sections were attached to slide glasses and stained with Hematoxylin & Eosin (H&E). The staining procedure is as follows:

[0248] - Deparaffinization: Xylene 3 times, 5 minutes each

[0249] - Function: Sequential treatment of 100%, 95%, 80%, and 70% ethanol and distilled water

[0250] - Hematoxylin staining: 5-10 minutes

[0251] - Washing and Separation: 1% Acidic Alcohol

[0252] - Blueing: Ammonia water or Scott's tap water

[0253] - Eosin Dyeing: 1-3 minutes

[0254] - Dehydration, clearing, and encapsulation

[0255]

[0256] 11.4. Histopathological examination

[0257] The severity of the lesions on the stained tissue slides was examined using a light microscope.

[0258]

[0259]

[0260] Experimental Example 1. Confirmation of the therapeutic effect of KDS12025 on neurodegenerative diseases

[0261] To confirm the therapeutic effect of KDS12025 on neurodegenerative diseases, efficacy evaluations were performed using animal models of Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS).

[0262] Specifically, an astrocyte model (fGiD) in which the diphtheria toxin receptor (DTR) is locally expressed in APP / PS1 mice was used as the animal model for AD. The fGiD model is designed to promote neuronal death by inducing locally severely reactive astrocytes to compensate for the limitations of the APP / PS1 model, in which amyloid lesions appear but overall neuronal degeneration is observed only to a limited extent. To evaluate the efficacy of KDS12025, behavioral analyses including the passive avoidance test (PAT) and the novel place recognition test (NPR) were performed. As a result, a decline in memory and cognitive function was observed in fGiD mice, and it was confirmed that this was significantly improved when KDS12025 was administered intraperitoneally a total of 16 times at a dose of 3 mg per kg of body weight (Figs. 1a to 1c).

[0263] In addition, it was confirmed that the excessive increase in reactive astrocytes and the decrease in the number of NeuN-positive neurons observed in fGiD mice were restored to the control (fGcon) level by treatment with KDS12025 (Figs. 1d to 1f). Through the above results, it was confirmed that KDS12025 of the present invention effectively improves the major pathological features of AD, including neurodegeneration, astrogliosis, and memory impairment.

[0264]

[0265] For the PD model, a mouse model overexpressing human A53T α-synuclein was used. This model is characterized by the induction of dopaminergic neuronal loss and motor abnormalities through the progressive degeneration of the substantia nigra pars compacta (SNpc). As a result of oral administration of KDS12025 at a dose of 1 mg per kg of body weight for 4 weeks (free drinking method) (Fig. 1g), it was confirmed that the decline in motor function observed in A53T mice was significantly improved (Fig. 1h).

[0266] In addition, similar to the AD model, it was confirmed that KDS12025 restored the reduction of tyrosine hydroxylase (TH)-positive dopaminergic neurons and astrocyte proliferation observed in the ipsilateral (Ipsi.) SNpc of A53T mice to control levels compared to the contralateral (contralateral, contra.) side (Figs. 1j to 1l).

[0267]

[0268] Meanwhile, the ALS model is SOD1 G93A Mice were used (Fig. 1m). The above model is a severe ALS model in which significant motor neuron degeneration is observed along with reactive astrocytosis. As a result of administering KDS12025 of the present invention via free drinking at doses of 1 mg and 10 mg per kg of body weight, it was confirmed that the onset of motor impairment was delayed by more than 7 weeks, and the median survival time was extended from 140 days to 168 days (Figs. 1n to 1o).

[0269]

[0270] In addition, to confirm histological changes, SOD1 G93AImmunohistochemical analysis was performed on the ventral lumbar spinal cord (L2–L5) of mice. As a result, GFAP signal intensity was higher compared to SOD1 in WT mice G93A Although it increased significantly in mice, it was confirmed that the said increase was significantly reduced by KDS12025 treatment (Figs. 1p to 1r). At the same time, NeuN signal intensity was SOD1 G93A It was confirmed that although it decreased in mice, the decrease was restored by the KDS12025 treatment of the present invention (Figs. 1p to 1r).

[0271]

[0272] In conclusion, KDS12025 of the present invention demonstrated an effect of improving pathological and behavioral indicators in various neurodegenerative disease models, including AD, PD, and ALS, and confirmed its potential as a therapeutic agent for neurodegenerative diseases.

[0273]

[0274] Experimental Example 2. Confirmation of the effect of co-administration of KDS12025 and ursodeoxycholic acid (UDCA) on controlling liver toxicity

[0275] To determine whether hepatotoxicity occurred with the administration of KDS12025 and to confirm the hepatoprotective effect of concomitant use of UDCA, blood liver enzyme levels were compared and analyzed between the group administered KDS12025 alone and the group administered KDS12025 and UDCA in combination. KDS12025 was administered to test animals at doses of 10 mg, 0.1 mg, and 0.03 mg per kg of body weight, and UDCA was administered in combination in some test groups. Liver function was evaluated by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels over a certain period.

[0276] As a result, as shown in Figures 2 and 3, when KDS12025 was administered at a dose of 10 mg / kg, AST and ALT levels rose sharply, showing signs of severe liver damage, and it was observed that the individual reached the end of life (expire) (Figures 2 and 3). Meanwhile, in the groups administered KDS12025 alone at doses of 0.1 mg / kg and 0.03 mg / kg, an increase in AST and ALT levels exceeding the normal range was observed, and in some individuals, blood levels tended to increase to over approximately 100 IU / L.

[0277] In contrast, in the group administered with KDS12025 and UDCA, the upward trend in liver enzyme levels observed in the monotherapy group was significantly alleviated, and it was confirmed that AST and ALT levels remained within a relatively stable range. In particular, when UDCA was administered in combination at a dose of 0.03 mg / kg, it was confirmed that the increase in liver enzyme levels observed with KDS12025 monotherapy was significantly suppressed, and the fluctuation range of levels over time also decreased.

[0278]

[0279] In conclusion, it was confirmed that liver dysfunction caused by the administration of KDS12025 can be effectively alleviated or controlled by the concomitant administration of UDCA, and it was confirmed that the combination therapy of the present invention has the effect of reducing the risk of liver toxicity associated with the administration of KDS12025 and improving safety.

[0280]

[0281] Experimental Example 3. Hepatic histopathological examination following combined administration of KDS12025 and UDCA

[0282] To determine the effects of the combined administration of KDS12025 and UDCA on liver tissue, animals were divided into a group administered KDS12025 alone (10 mg per kg body weight) and a group administered KDS12025 and UDCA in combination (0.03 mg KDS12025 + UDCA per kg body weight). After the administration was completed, liver tissues were excised and pathologically analyzed. The excised liver tissues were fixed, embedded, and sectioned according to standard histological procedures, then stained with hematoxylin and eosin (H&E) and observed under a light microscope.

[0283] As a result, as shown in Figure 4, irregularity in hepatocyte arrangement, changes in cell density, and heterogeneity in tissue structure were observed in the group administered KDS12025 alone at 10 mg / kg, confirming that liver tissue abnormalities occurred (left side of Figure 4).

[0284]

[0285] On the other hand, in the group administered KDS12025 and UDCA in combination, the hepatocyte structure remained relatively uniform, and the hepatic lobule structure also showed histological findings similar to those of the normal control group (Figure 4, right). From the above results, it was confirmed that liver tissue abnormalities occurring with KDS12025 alone are improved and controlled by the combination of UDCA.

[0286]

[0287] In conclusion, as a result of administering KDS12025 of the present invention in combination with UDCA, the elevation of liver enzyme levels of KDS12025 can be significantly improved; thus, it was confirmed that the combination formulation can be usefully used to confirm various clinical efficacy as well as neurodegenerative diseases of KDS12025.

Claims

1. A pharmaceutical composition for the prevention and treatment of neurodegenerative diseases comprising, as an active ingredient, an aminoaromatic compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, wherein the aminoaromatic compound represented by the following Chemical Formula 1 or the pharmaceutically acceptable salt thereof is co-administered with a bile acid: [Chemical Formula 1] In the above chemical formula 1, Ar is C6-C 20 It is arylene, and the arylene of the above Ar is C1-C 10 Alkyl, C1-C 10 Alkoxy, amino, mono- or di-C1-C 10 Alkylamino, Halo-C1-C 10 Alkyl, halo-C1-C 10 It may be further substituted with one or more selected from alkoxy and hydroxyl groups; R 1 and R 2 Each independently consists of hydrogen or C1-C 10 It is alkyl; R 3 is a halogen, C1-C 10 Alkoxy, halo-C1-C 10 Alkyl or halo-C1-C 10 It is an alkoxy; n is an integer of 1 or 2; Single R 3 If it is a halogen, n is an integer of 1.

2. A pharmaceutical composition according to claim 1, wherein the amino-aromatic compound is a compound represented by the following chemical formula 2: [Chemical Formula 2] In the above chemical formula 2, R 1 and R 2 Each is independently hydrogen or C1-C7 alkyl; R 3 is a halogen, C1-C7 alkoxy, haloC1-C7 alkyl; R' is a C1-C7 alkyl, C1-C7 alkoxy, amino, or hydroxyl; a is an integer from 0 to 4; n is an integer of 1 or 2.

3. A pharmaceutical composition according to claim 1, wherein the amino-aromatic compound is any one selected from the group consisting of the following chemical formulas 3 to 6: [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] .

4. A pharmaceutical composition according to claim 1, wherein the bile acid is one or more selected from the group consisting of ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), glycoursodeoxycholic acid (GUDCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), and tauro-deoxycholic acid (TDCA).

5. A pharmaceutical composition according to claim 1, wherein the amino-aromatic compound is administered at a dose of 3 mg / kg or less.

6. A pharmaceutical composition according to claim 1, wherein the amino-aromatic compound is administered at a dose of 0.001 mg / kg to 2 mg / kg.

7. A pharmaceutical composition according to claim 1, wherein the neurodegenerative disease is one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, post-traumatic stress disorder, multiple sclerosis, cerebral ischemic disease, and amyotrophic lateral sclerosis.

8. A pharmaceutical composition according to claim 1, wherein the combined administration is the combined administration of the amino-aromatic compound with the bile acid simultaneously, sequentially, or in reverse order.

9. The amino-aromatic compound of claim 1 is administered via intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, nasal administration, pulmonary administration, or rectal administration; and A pharmaceutical composition wherein the above bile acid is administered via intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, nasal administration, pulmonary administration, or rectal administration.

10. A health functional food for the prevention or improvement of neurodegenerative diseases comprising, as an active ingredient, an amino-aromatic compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, wherein the amino-aromatic compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof is co-administered with a bile acid: [Chemical Formula 1] In the above chemical formula 1, Ar is C6-C 20 It is arylene, and the arylene of the above Ar is C1-C 10 Alkyl, C1-C 10 Alkoxy, amino, mono- or di-C1-C 10 Alkylamino, Halo-C1-C 10 Alkyl, halo-C1-C 10 It may be further substituted with one or more selected from alkoxy and hydroxyl groups; R 1 and R 2 Each independently consists of hydrogen or C1-C 10 It is alkyl; R 3 is a halogen, C1-C 10 Alkoxy, halo-C1-C 10 Alkyl or halo-C1-C 10 It is an alkoxy; n is an integer of 1 or 2; Single R 3 If it is a halogen, n is an integer of 1.

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