Pharmaceutical composition comprising histone deacetylase inhibitor for preventing or treating polyglutamine diseases

Abstract

The present invention relates to a composition for treating polyglutamine diseases, particularly Huntington's disease. The composition according to one aspect activates the expression of proteins related to autophagy so as to activate an autophagy process and reduce the number of mutant Huntington protein aggregates, and can inhibit the activation of microglia so as to alleviate neuroinflammation.

Description

Pharmaceutical composition for preventing or treating multiple glutamine diseases comprising a histone deacetylase inhibitor

[0001] It relates to the therapeutic use of histone deacetylase inhibitors.

[0002] This invention is a technology developed with the support of the Global Innovation Special Zone Innovation Business Promotion (R&D) Overseas Joint Demonstration R&D Project (RS-2024-00488530) supported by the Korean government (Ministry of SMEs and Startups).

[0003] Polyglutamine (polyQ) diseases are a group of neurodegenerative diseases caused by mutations that result in the repetitive presence of glutamine (Q). Among them, Huntington's disease (HD) is a representative example. It is caused by the repetitive presence of a CAG sequence in exon 1 of the huntingtin (HTT) gene, or by damage to medium spiny neurons in the striatum due to the accumulation of mutant huntingtin protein aggregates. It typically presents with symptoms such as chorea, psychosis, and dementia. In North America, approximately 30,000 HD patients have been reported, resulting in a prevalence of 5.7 per 100,000. The prevalence can vary by more than tenfold depending on geographic location, which is likely closely related to variations in patient identification and diagnostic criteria. In general, Asian countries have a lower prevalence than Europe, North America, and Australia. Furthermore, the incidence in children is relatively lower than in adults, accounting for approximately 5-10% of all cases. Huntington's disease is a dominantly inherited disease, with approximately 90% of cases being caused by genetic factors.

[0004] There is currently no fundamental cure for Huntington's disease. However, symptomatic medications such as tetrabenazine (Xenazine) from Lundbeck and deutetrabenazine (Austedo) from Teva have been approved by the U.S. Food and Drug Administration (FDA) and are currently on the market. Among the candidate drugs currently in development, representative examples include tominersen, which is being jointly researched by Roche and Ionis, and pridopidine from Prilenia Therapeutics, and several institutions are actively conducting research. Tominersen, which is being jointly developed by Roche and Ionis, is an anti-sense oligonucleotide (ASO)-based treatment for Huntington's disease. The phase 3 clinical trial was discontinued due to safety issues, and a new phase 2 clinical trial is currently underway for patients in the early stage. Next, in the case of Prilenia Therapeutics' pridopidine, it is a sigma-1 receptor (S1R) agonist that has a mechanism of alleviating the symptoms of Huntington's disease by activating various signaling pathways essential for neurological function and survival through S1R activation, and is currently in phase 3 clinical trials.

[0005] Despite the clear identification of the cause of Huntington's disease, there is currently no fundamental treatment, and clinical trials for potential therapeutic agents continue to face challenges. To address these issues, the inventors sought to discover the potential use of histone deacetylase (HDAC) inhibitors, specifically CXD 101, as a treatment for Huntington's disease.

[0006] Histones are basic proteins that bind to the DNA of cells, and can regulate the transcriptional activity of specific genes depending on the degree of DNA binding. Reversible acetylation reactions can occur at specific lysine residues of histone proteins, and the transcriptional activity of specific genes is regulated depending on the degree of acetylation. Histones exist in six types according to their amino acid sequences: histone 1, 2A, 2B, 3, 4, and archaeal histone. Histone deacetylases (HDACs) are enzymes that remove acetyl groups from lysine residues of histone proteins, and HDAC inhibitors are a general term for compounds that inhibit the activity of HDACs. HDACs are divided into five subgroups (Class I, IIa, IIb, III, and IV) based on amino acid sequence similarity, and each group has different characteristics such as subcellular location and tissue-specific distribution.

[0007] Several studies have reported that increased HDAC activity, leading to increased expression of deacetylated histone proteins, is characteristic of cancer and neurodegenerative disease models. For this reason, HDACs have been studied as therapeutic targets for these diseases, and research into the development of HDAC inhibitors is actively underway.

[0008] CXD101 (Zabadinostat), the IPB-H01 used in this patent, is a Class I HDAC inhibitor, and is currently undergoing phase 1 and 2 clinical trials as a treatment for lymphoma (SW Booth et al, 2021), and several patents are pending in relation to cancer or immune checkpoint inhibitors (WO-2021058974-A1, US-2021093625-A1), but it is not known whether it can exhibit effective effects as a treatment for Huntington's disease. The present researchers completed the present invention by confirming the efficacy of CXD101 in reducing mutant protein huntingtin aggregates and alleviating neuronal inflammation caused by them.

[0009] One aspect is to provide a pharmaceutical composition for preventing or treating polyglutamine diseases, comprising a compound represented by the following chemical formula 1 (CXD 101) or a pharmaceutically acceptable salt thereof:

[0010] [Chemical Formula 1]

[0011] .

[0012] Another aspect is to provide a pharmaceutical composition for preventing or treating neuroinflammation comprising CXD 101 or a pharmaceutically acceptable salt thereof.

[0013] Another aspect is to provide a health functional food for preventing or improving polyglutamine disease, comprising CXD 101 or a salt thereof.

[0014] Another aspect is to provide a health functional food for preventing or improving neuroinflammation, comprising CXD 101 or a salt thereof.

[0015] Another aspect is to provide a feed composition for preventing or improving polyglutamine disease comprising CXD 101 or a salt thereof.

[0016] Another aspect is to provide a feed composition for preventing or improving neuroinflammation comprising CXD 101 or a salt thereof.

[0017] Another aspect provides a method for preventing, ameliorating or treating polyglutamine disease comprising administering to a subject in need thereof an effective amount of CXD 101 or a pharmaceutically acceptable salt thereof.

[0018] Another aspect provides the use of CXD101 or a pharmaceutically acceptable salt thereof for the manufacture of a formulation for preventing, ameliorating or treating polyglutamine diseases.

[0019] Another aspect provides a method for preventing, ameliorating or treating neuroinflammation comprising administering to a subject in need thereof an effective amount of CXD 101 or a pharmaceutically acceptable salt thereof.

[0020] Another aspect provides the use of CXD101 or a pharmaceutically acceptable salt thereof for the manufacture of a formulation for preventing, ameliorating or treating neuroinflammation.

[0021] One aspect provides the use of CXD 101 for preventing, ameliorating or treating polyglutamine diseases and / or neuroinflammation.

[0022] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition of the present invention to a subject. For preventive purposes, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0023] As used herein, the term "treatment" refers to any action that improves the symptoms of a disease or provides benefit by administering the composition of the present invention to a subject. As used herein, the terms "treatment," "palliation," and "improvement" may be used interchangeably. A therapeutic benefit refers to any therapeutically significant improvement or effect on one or more diseases, conditions, or symptoms under treatment.

[0024] The term “CXD 101” in this specification refers to N-(2-aminophenyl)-4-(1-[(1,3-dimethyl-1H-pyrazol-4-yl)methyl]piperidin-4-yl)benzamide, a compound represented by the following chemical formula 1. It is also named Zabadinostat and was developed as a Class I HDAC inhibitor. It was reported that when taken orally, it actually functions as an HDAC inhibitor, resulting in increased expression of acetylated histone proteins (Blaszczak W., Liu G., et al. Immune modulation underpins the anti-cancer activity of HDAC inhibitors. Molecular Oncology. 2021. 15: 3280-3298.) In this specification, IPB-H01, a drug used by the inventors of the present invention, refers to CXD 101.

[0025] [Chemical Formula 1]

[0026] .

[0027] The term "neuroinflammation" used herein refers to an immune response to harmful stimuli in the nervous system, and is considered a hallmark feature closely associated with many neurodegenerative diseases. Neuroinflammatory responses may include the activation of innate immune cells (microglia), the release of inflammatory mediators such as nitric oxide (NO), cytokines, and / or chemokines, and the infiltration of macrophages, which may induce neuronal cell death.

[0028] The term “polyglutamine disease” in this specification refers to a degenerative brain disease caused by the repetitive appearance of glutamine (Q) due to mutations, etc., and can be used interchangeably with “polyglutamine disease” or “polyQ disease”. In polyglutamine diseases, the pathology is commonly induced by misfolded protein aggregates, and it has been reported that the related pathology can be improved or alleviated by degrading the aggregates through autophagy activation or the ubiquitin-proteasome system (UPS), which are part of the protein quality control (PQC) system of eukaryotes: Nat Rev Neurosci. 2017 Oct; 18(10): 613-626; Cell Death Differ 2010 Oct; 17(10): 1577-87; Mol Cell Neurosci. 2015 May; 66(0 0): 53-61.

[0029] In one specific example, the polyglutamine disease may be caused by the accumulation of protein aggregates due to glutamine repeat mutations.

[0030] In one specific example, the polyglutamine disease may be any one selected from the group consisting of Huntington's disease, polyglutamine expansion disease, spinocerebellar ataxias (SCA), Machado-Joseph disease (MJD / SCA3), dentatorubro-Pallidoluysian atrophy (DRPLA), and X-linked spinal and bulbar muscular atrophy (SMAX).

[0031] In one specific example, the polyglutamine disease may preferably be Huntington's disease.

[0032] The term “Huntington’s disease” as used herein refers to a degenerative brain disease caused by a mutation in which a CAG sequence is repeatedly present in exon 1 of the huntingtin protein gene (Huntingtin, HTT), resulting in the presence of repeated glutamine (Q). The Huntington’s disease may be caused by damage to medium spiny neurons (medium spiny neurons) in the striatum due to the accumulation of aggregates of mutant huntingtin protein.

[0033] According to one specific example, the compound can prevent or treat damage or death of nerve cells. Specifically, it can prevent or treat damage or death of nerve cells caused by the accumulation of protein aggregates.

[0034] In one embodiment, the compound may inhibit or reduce the accumulation of protein aggregates.

[0035] In one specific example, the compound may inhibit or reduce the accumulation of huntingtin protein and / or its aggregates. The compound may reduce the expression of mutant huntingtin protein markers. Specifically, the compound may reduce the expression of 1C2, MW1, HTT, and / or MW8.

[0036] According to one specific example, the compound can inhibit or reduce the accumulation of protein aggregates by inducing autophagy of protein aggregates. The compound can increase the expression of proteins involved in autophagy.

[0037] In this specification, "autophagy" refers to a naturally occurring regenerative process occurring at the cellular level in the body. It is a crucial pathway for maintaining protein homeostasis and preserving proper cellular function by selectively degrading damaged cells and unnecessary or dysfunctional cellular components during the regulatory process. Autophagy can be strategically utilized in cancer prevention by enhancing tumor suppression and inducing cell death.

[0038] In one specific example, the compound can increase the expression of heat shock proteins (HSPs). More specifically, it can significantly increase the acetylation of HSP90 and the expression of HSP70. HSPs are well known as proteins involved in autophagy, and among them, HSP90 and HSP70 are known to be important factors. Furthermore, it is known that increased acetylation of HSP90 increases the expression of HSP70.

[0039] In one specific example, the compound can increase the expression of a protein involved in the formation of autophagosomes of protein aggregates. More specifically, it can increase the expression of p62 and LC-3II. “p62” is known to increase in expression in the early stage of the autophagy process and is subsequently degraded together with protein aggregates. Since it has a ubiquitin binding site, it can bind to protein aggregates that have not properly conjugated proteins, such as mutant huntingtin protein. In addition, p62 binds to LC-3 bound to the autolysosome, and ultimately, proteins and organelles in the autophagosome, including p62, are degraded in the lysosome. “LC-3” is known to be cleaved at the carboxy-terminal proximal region by the protease Atg4 during the autophagy process to become LC-3I, and LC-3I is known to be modified into LC-3II by phosphatidylethanolamine (PE).

[0040] In one specific example, the compound may reduce the activity of microglia. By reducing the activity of microglia, the compound may alleviate neuroinflammation induced by microglia in the cerebral cortex.

[0041] The compound may reduce the expression of proteins involved in microglial activation. In one specific example, the compound may reduce the expression of Iba-1. "Iba-1" is a marker indicating microglial activation.

[0042] The compound can induce changes in the phenotype of microglia. The compound can decrease the size of the microglia cell body or increase the number of branches extending from the cell body compared to the normal microglia. Typically, when microglia are activated, the number of branches extending from the cell body decreases and the cell body size increases compared to microglia in a homeostatic state. Activated microglia induce neuroinflammation.

[0043] According to one specific example, a compound can increase the number of intermediate dendritic neurons. The compound can increase the expression of a protein involved in the survival of intermediate dendritic neurons. More specifically, the compound can decrease the expression of DARPP32. "DARPP32" is a representative protein marker of intermediate dendritic neurons. Huntington's disease patients are characterized by a decrease in intermediate dendritic neurons in the striatum.

[0044]

[0045] Another aspect provides a composition for preventing, improving or treating a disease comprising CXD 101 or a pharmaceutically acceptable salt thereof.

[0046] The composition is present in an amount of 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 It may comprise from 0.1 wt% to 30 wt%, from 0.1 wt% to 20 wt%, from 0.1 wt% to 10 wt%, or from 0.1 wt% to 5 wt% of the compound or its salt.

[0047] The above composition “comprising” a compound or a salt thereof means that the compound of the present specification or a pharmaceutically acceptable salt thereof is added to an extent capable of exhibiting the above-mentioned effect, and includes formulation in various forms by adding various components as auxiliary components for drug delivery and stabilization, etc.

[0048] As used herein, the term “pharmaceutically acceptable” means physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal upset, dizziness, or similar reactions when administered to humans.

[0049] As used herein, the term “pharmaceutically acceptable salt” refers to a salt according to one aspect of the present invention that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. Salts of the parent compound can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base, such as sodium, calcium, magnesium, or potassium, or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. These reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Typically, when practical, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used. The pharmaceutically acceptable salts include both acid or base addition salts and stereochemically isomeric forms thereof, and may be, for example, addition salts of organic or inorganic acids. The above salt includes any salt that maintains the activity of the parent compound in the subject of administration and does not cause undesirable effects, and is not particularly limited thereto.

[0050] These salts include inorganic and organic salts, for example, acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idet, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluceptic acid, esilinic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, mandelic acid, mucic acid, It can be naphthylic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilinic acid, or methanesulfonic acid.

[0051] In addition, the salt forms include salts of alkali and alkaline earth metals, such as ammonium salts, lithium salts, sodium salts, potassium salts, magnesium salts, and calcium salts, salts with organic bases, such as benzathine, N-methyl-D-glucamine, and hydrabamine salts, and salts with amino acids, such as arginine and lysine. In addition, the salt forms can be converted into free forms by treatment with a suitable base or acid.

[0052] The compound or its salt can be combined by reacting in the presence of a base and a solvent for 5 to 20 hours, for example, 10 to 20 hours, 15 to 20 hours, 15 to 19 hours, or 15 to 18 hours. The reaction can be performed at room temperature, and those skilled in the art can appropriately change the reaction time depending on the temperature.

[0053] The base may be NaH, lithium diisopropylamide (LDA), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), pyridine, ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, dipropylamine, diisopropylamine, trimethylamine, tripropylamine, triisopropylamine, aniline, methylaniline, dimethylaniline, pyridine, azazurolidine, benzylamine, methylbenzylamine, dimethylbenzylamine, 2,6-lutidine, morpholine, piperidine, piperazine, proton-sponge, ammonium hydroxide, triethanolamine, ethanolamine, or trizmal.

[0054] The solvent may be dimethylacetamide (DMAc), dichloromethane (DCM), tetrahydrofuran (THF), dimethylformamide (DMF), acetonitrile (ACN), DMAP, water, acetic acid, acetone, dioxane, benzene, 1-butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexane, diethyl ether, dimethyl sulfoxide (DMSO), ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, pentane, pyridine, toluene, hydrochloric acid, and triethyl amine.

[0055]

[0056] The above composition may be a pharmaceutical composition.

[0057] The pharmaceutical composition of the present invention may be in any form suitable for the intended method of administration. In the pharmaceutical composition of the present invention, "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the pharmaceutical composition may be administered through any common route as long as the drug can reach the target tissue. Administration may be by a method known in the art, and examples thereof include, but are not limited to, topical ocular administration (e.g., periocular (e.g., subTenon's), subconjunctival, intraocular, intravitreal, intracameral, subretinal, suprachoroidal, and retrobulbar administration), intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. In addition, the active ingredient may be administered by any device capable of transporting to target cells, and the route of administration is preferably determined depending on the type of disease to which it is applied.

[0058] The above administration is 0.00001 mg to 1,000 mg of the composition according to one specific example per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, Alternatively, it may be administered in doses of 10 mg to 25 mg.

[0059] However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects, and the administration may be done in one or more sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage as for humans per kg may be used, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (e.g., heart) of the target animal and the human.

[0060] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., formulated according to conventional methods, or parenteral formulations such as suspensions, emulsions, lyophilized preparations, external preparations, suppositories, sterile injection solutions, and implantable preparations. The pharmaceutical composition may further comprise, in addition to the active ingredient, a pharmaceutically acceptable excipient that can be used in formulation.

[0061] The above excipients include carriers, vehicles, diluents, solvents, for example, monohydric alcohols, for example, ethanol, isopropanol, and polyhydric alcohols, for example, glycerol, and edible oils, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters, for example, ethyl oleate, isopropyl myristate; It may include at least one selected from the group consisting of binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, glidants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, coloring agents, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starches, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, ion exchange resins, etc., but is not limited thereto.

[0062] The carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0063] The pharmaceutical composition of the present invention may be formulated in the form of an oral administration dosage form, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc. These oral administration dosage forms may, in addition to the active ingredient according to the typical composition of each dosage form, contain pharmaceutically acceptable carriers, such as diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine, or lubricants such as silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol.

[0064] If the oral dosage form is a tablet, it may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidine, and in some cases, a disintegrating agent such as starch, agar, alginic acid or its sodium salt, an effervescent mixture and / or an absorbent, a coloring agent, a flavoring agent or a sweetening agent.

[0065] The pharmaceutical composition of the present invention being formulated in the form of a parenteral administration dosage form may mean that it is administered by a method such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In this case, in order to formulate the pharmaceutical composition in the parenteral administration dosage form, the active ingredient is mixed with a stabilizer or buffer in water to prepare a solution or suspension, and this solution or suspension can be prepared in a unit dosage form of an ampoule or vial.

[0066] In addition, the pharmaceutical composition may be sterilized or may further contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying agents, salts for osmotic pressure control and / or buffers, and may further contain other therapeutically useful substances, and may be formulated according to conventional methods of mixing, granulating or coating.

[0067] The content of the compound or salt containing the compound in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the purpose of use of the pharmaceutical composition, the form of the formulation, etc., and may be, for example, 0.001 to 99 wt%, 0.001 to 90 wt%, 0.001 to 50 wt%, 0.01 to 50 wt%, 0.1 to 50 wt%, or 1 to 50 wt% based on the total weight of the pharmaceutical composition.

[0068] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and may be adjusted according to factors including the type of the patient's disease, the severity of the disease, the type of active ingredient administered, the type of formulation, the patient's age, sex, weight, health condition, diet, sensitivity, the time and method of drug administration, the combination of the composition or concurrently used drugs, and other factors well known in the medical field.

[0069] The pharmaceutical composition of the present invention can prevent or treat a disease in a subject, including a step of administering to the subject an amount effective to prevent or treat the disease.

[0070] The dosage of the pharmaceutical composition for the prevention or treatment of diseases according to the present invention may range from 0.01 ug / kg to 10 g / kg per day, specifically from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0071] The above entity may be a mammal. The mammal may be a human, a dog, a cat, a cow, a goat, or a pig.

[0072] The terms and methods described for the above compounds and diseases also apply to the above pharmaceutical compositions.

[0073]

[0074] The above composition may be a health functional food.

[0075] The term "health functional food" as used herein refers to a food manufactured or processed for the purpose of health supplementation using a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, or other methods of specific ingredients contained in food raw materials. It refers to a food designed and processed so that the above-mentioned ingredients can sufficiently exert bioregulatory functions on the body, such as biological defense, biological rhythm regulation, disease prevention, and recovery, and can be used for the purpose of preventing or improving polyglutamine disease or neuroinflammation.

[0076] There are no specific restrictions on the types of the above foods. Examples of the above foods include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks, and include all health foods in the conventional sense.

[0077] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0078] The terms and methods described for the above compounds and diseases also apply to the above health functional foods.

[0079]

[0080] The above composition may be a feed composition.

[0081] The feed composition can be prepared by adding the compound or its salt in an appropriate effective concentration range according to various feed preparation methods known in the art, and can be used for the purpose of preventing or improving polyglutamine disease or neuroinflammation.

[0082] The above "feed" may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are suitable for or intended for an animal to eat, ingest, or digest. The type of feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feed such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, or grain by-products; animal feed such as proteins, fat-free substances, oils, minerals, oils, single-cell proteins, zooplankton, or food.

[0083] The terms and methods described for the above compounds and diseases also apply to the above feed composition.

[0084]

[0085] Another aspect provides a method of preventing, ameliorating, or treating a condition in a subject comprising the step of treating or administering to the subject an effective amount of CXD 101 or a pharmaceutically acceptable salt thereof.

[0086] The condition of the above entity may be a condition associated with polyglutamine disease, or a condition associated with neuroinflammation.

[0087] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of an improvement effect for a condition associated with a polyglutamine disease or a condition associated with neuroinflammation.

[0088] The terms and methods described for the above compounds and diseases also apply to the above method.

[0089]

[0090] Another aspect provides the use of CXD101 or a pharmaceutically acceptable salt thereof for the manufacture of a formulation for preventing, ameliorating or treating polyglutamine disease and / or neuroinflammation.

[0091] The terms and methods described for the above compounds and diseases also apply to the above method.

[0092]

[0093] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0094] The composition according to one aspect has the activity of activating the expression of proteins associated with autophagy, thereby activating the autophagy process and reducing or eliminating the number of mutant huntingtin protein aggregates. Furthermore, it can alleviate neuroinflammation by suppressing microglial activation. Therefore, the composition is effective as a treatment for Huntington's disease and, furthermore, polyglutamine disease.

[0095] Figure 1 is a Western blotting image showing changes in the expression of mutant huntingtin protein markers (1C2, HTT) according to treatment with excipients or IPB-H01.

[0096] Figure 2 is an immunofluorescence staining image showing changes in the expression of mutant huntingtin protein markers (1C2, HTT) according to treatment with an excipient or IPB-H01.

[0097] Figure 3 is a graph showing Western blotting images and quantitative comparisons of the degree of acetylation of histone H3 and α-tubulin according to treatment with excipients, IPB-H01, or TSA.

[0098] Figures 4 and 5 are Western blotting images showing changes in the expression of mutant huntingtin protein markers according to treatment with excipients, IPB-H01, or TSA, and graphs comparing the quantitative results. Figure 4 shows changes in the expression of MW1, and Figure 5 shows changes in the expression of HTT.

[0099] Figures 6 to 8 are Western blotting images showing changes in the expression of autophagy markers according to treatment with excipients, IPB-H01, or TSA, and graphs comparing the quantitative results. Figure 6 shows changes in the expression of HSP90 and HSP70, Figure 7 shows changes in the expression of p62, and Figure 8 shows changes in the expression of LC-3I and LC-3II.

[0100] Figures 9 and 10 are immunohistochemical staining images and quantitative graphs comparing the changes in expression of mutant huntingtin protein markers in the brain cortex and striatum of wild-type and R6 / 2 mice following treatment with a vehicle or IPB-H01. Figure 9 shows changes in EM48 expression, and Figure 10 shows changes in MW8 expression.

[0101] Figures 11 and 12 are immunohistochemical staining images showing changes in the expression of a microglial marker (Iba-1) in the brain cortex and striatum of wild-type mice and R6 / 2 mice following treatment with a vehicle or IPB-H01, and graphs comparing the quantitative results thereof.

[0102] Figure 13 is a graph comparing the immunohistochemical staining images and quantitative comparisons showing changes in the expression of a striatal medium spiny neuron marker (DARPP-32) according to treatment with a vehicle or IPB-H01 in the brain cortex and striatum of wild-type mice and R6 / 2 mice.

[0103] Figure 14 is a graph showing the survival rate over time of wild-type mice and R6 / 2 mice administered with excipients or IPB-H01.

[0104] Figure 15 is a graph showing the results of rotarod behavioral evaluation of wild-type mice and R6 / 2 mice administered with an excipient or IPB-H01.

[0105] Figure 16 is a graph showing the results of the clasping test of wild-type mice and R6 / 2 mice administered with an excipient or IPB-H01.

[0106] Figure 17 is a graph showing the results of the rotarod behavioral evaluation of wild-type mice and YAC128 mice administered with an excipient or IPB-H01.

[0107] Figure 18 is a graph showing the results of an open space behavioral evaluation of wild-type mice and YAC128 mice administered with an excipient or IPB-H01, and an image showing the movement path of the mice.

[0108] Figure 19 is a graph showing the results of the Forced Swimming Test of wild-type mice and YAC128 mice administered with an excipient or IPB-H01.

[0109] Figure 20 is a graph showing the results of a grip strength test of wild-type mice and YAC128 mice administered with an excipient or IPB-H01.

[0110] Figure 21 is a graph comparing the immunohistochemical staining images and quantitative results showing changes in the expression of a mutant huntingtin protein marker (EM48) in the brain cortex and striatum of wild-type mice and YAC128 mice following treatment with a vehicle or IPB-H01.

[0111] Figure 22 is a graph comparing the immunohistochemical staining images and quantitative comparisons showing changes in the expression of a medium spiny neuron marker (DARPP-32) in the striatum of wild-type mice and YAC128 mice according to treatment with a vehicle or IPB-H01.

[0112] Figure 23 is a graph comparing the immunohistochemical staining images and quantitative results showing changes in the expression of microglial markers (Iba-1) in the brain cortex of wild-type mice and YAC128 mice following treatment with a vehicle or IPB-H01.

[0113] Figure 24 is a graph comparing the immunohistochemical staining images and quantification thereof showing changes in the expression of microglial marker (Iba-1) in the striatum of wild-type mice and YAC128 mice following treatment with a vehicle or IPB-H01.

[0114] Figure 25 is a graph showing the BBB-PAMPA (Blood Brain Barrier-Parallel Artificial Membrane Permeability Assay) values ​​of IPB-H01.

[0115] Figure 26 is a graph and the results of an in vivo pharmacokinetic test on female and male wild-type mice administered a single oral dose of IPB-H01.

[0116] Figures 27 to 29 are images showing the results of hematoxylin and eosin (H&E) staining of the liver, heart, and kidney of wild-type mice and YAC128 mice that were repeatedly administered orally with excipients or IPB-H01 for 3 months.

[0117] Figure 30 is a graph quantifying the differences in genes related to autophagy using RNA-seq results performed using RNA extracted from the striatum of control mice and YAC128 mice administered with excipient or IPB-H01.

[0118] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0119]

[0120] Example 1. Verification of efficacy in a Huntington's disease patient-derived neuronal cell model.

[0121] The disease-modifying effect of IPB-H01 treatment was verified in a Huntington's disease patient-derived neuronal cell model.

[0122]

[0123] 1.1 Culturing of neural cells derived from Huntington's disease patients

[0124] The Q109 NPC cell line used in the experiment is a neural progenitor cell (NPC) derived from induced pluripotent stem cells (iPSC) in which the CAG base sequence, which is translated into glutamine (Q), is repeated 109 times. Neural progenitor cells (Q109 NPCs) were cultured on cell culture plates double-coated with 15 μg / ml Poly-L-Ornithine hydrobromide (PLO) (Sigma-Aldrich, Cat No. P3655) and 1 μg / ml Laminin (Sigma-Aldrich, Cat No. L2020) supplemented with 1% Antibiotic antimycotic solution (Welgene, Cat No. LS203-01), 1% Non-essential amino acid (Gibco, Cat No. GIB-11140-050), 0.1% β-mercaptoethanol (Gibco, Cat No. GIB-21985-023), 2% B27 supplement without Vit A (Gibco, Cat No. GIB-12587-010), 1% MEM sodium pyruvate solution (Gibco, Cat No. GIB-11360-070), Culture was initiated by dissolving in DMEM / F12 (Gibco, Cat No. GIB-11320-082) medium supplemented with 1% D-glucose (Sigma-Aldrich, Cat No. G7021), 1% L-glutamine (Corning, Cat No. 25-005-Cl), 20 ng / ml Recombinant human FGF-basic (bFGF) (Peprotech, Cat No. 100-188-1MG), and 10 μM Y-27632 ROCKi (Peprotech, Cat No. 1293823).

[0125] Since neural progenitor cells do not adhere well to the plate, the condition of the cells was observed daily using an optical microscope to remove cells with relatively weak adhesion, and a light physical force was applied to the plate about 10 times to detach cells that were weakly attached to the bottom of the plate. After that, the existing medium was removed and replaced with a new medium with the same composition as above but without Y-27632 ROCKi. This process was repeated for approximately 5 to 6 days until the amount of cells attached to the plate was about 80-90% of the plate. After that, the cells were subcultured at 5-day intervals by treating with 0.5X TrypLE Select (Gibco, Cat No. 12563-029), and the cells were used in the experiment after stabilizing.

[0126]

[0127] 1.2 Manufacturing of IPB-H01

[0128] IPB-H01 (CXD101, CAS No: 934828-12-3) was prepared by adding Dimethylsulfoxide (DMSO) and dissolving it to a concentration of 10 mM.

[0129]

[0130] 1.3 Confirmation of decreased huntingtin protein expression

[0131] To confirm the change in expression of mutant huntingtin protein according to IPB-H01 treatment, the following experiments were performed.

[0132] First, Western blotting was performed. Specifically, the neural cells obtained in Example 1.1 were treated with IPB-H01 of Example 1.2, and collected in an appropriate container using RIPA buffer (Biosesang, Cat No. RC2022-050-00) containing PhosSTOP EASYpack (Phosphatase inhibitor cocktail tablets) (Roche, Cat No. 04-906-837-001) and cOmplete Mini, EDTA-free (protease inhibitor cocktail tablets) (Sigma, Cat No. 11836170001). Afterwards, the cells were reacted for 5 minutes in an ultrasonic disperser (Hwashin technology, Powersonic 610), and the layers were separated by centrifugation at 13,000 rpm for 15 minutes to isolate the supernatant. Afterwards, the amount of protein contained in the supernatant was quantified and electrophoresis was performed using a 6–13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and then blocked with a TBS-T solution containing 5% albumin (Biosesang, Cat No. AC1025-050-00) to prevent non-specific reactions other than the target protein. The target protein on the membrane was reacted with primary antibodies against mutant huntingtin protein markers 1C2 (Millipore, MAB1574, mouse) and HTT (Sigma-aldrich, H7540, rabbit) at 4°C for 24 hours, and then reacted with anti-(mouse, rabbit) IgG secondary antibodies corresponding to each antibody for 1 hour at room temperature, followed by incubation with SuperSignal West Pico Plus Chemiluminescent substrate (Thermo fisher, Cat No.The proteins on the membrane surface reacted using (34577, 34578) were visualized using a chemidoc machine (Thermo fisher, iBright1500), and the relative expression levels of the proteins were quantified and compared, and the results are shown in Figure 1.

[0133]

[0134] As shown in Fig. 1, it was confirmed that the expression of mutant huntingtin protein markers 1C2 and HTT (huntingtin) protein decreased in the range of 0.5 uM to 2.5 uM when treated with IPB-H01.

[0135]

[0136] In addition, immunofluorescence staining was performed. The neural cells obtained in Example 1.1 were treated with IPB-H01 of Example 1.2, the cell medium was removed, and the cells were rinsed once with phosphate buffered saline (PBS, pH 7.4), and then fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes. Thereafter, the permeability of the cells was increased using a PBS solution containing 0.1% Triton X-100 and 2% albumin. After blocking non-specific binding other than the target protein by reacting in a PBS solution containing 10% albumin and 0.1% Triton X-100 at room temperature for 1 hour, the primary antibodies against the mutant huntingtin marker 1C2 (Millipore, MAB1574, mouse) and the neural stem cell marker musashi (Millipore, AB5977, rabbit) were reacted at 4℃ for 24 hours, and then the secondary antibodies IgG (H+L), Alexa Fluor 594 Goat-anti rabbit IgG (H+L), and Alexa Fluor 488 were reacted at 4℃ for 24 hours in a light-protected state. After that, 30 minutes before observation, the cells were reacted with DAPI for 5 minutes at room temperature, rinsed with PBS, and the expression of specific markers was confirmed through a fluorescence microscope, and the results are shown in Fig. 2.

[0137] As shown in Fig. 2, the results of tissue analysis using immunofluorescence staining after IPB-H01 treatment also confirmed that 1C2 expression was reduced in the range of 0.1 uM to 5 uM. Scale bar = 200 μm.

[0138]

[0139] Example 2. Verification of efficacy in a neural cell model transformed with the huntingtin gene.

[0140] The disease-modifying effect of IPB-H01 treatment was verified in a neuronal cell model transgenic to express the huntingtin gene, which contains high amounts of glutamine.

[0141]

[0142] 2.1 Preparation of STHdhQ111 / Q111 neural cell line

[0143] The STHdhQ111 / Q111 cell line used in the experiment is a striatal neuronal cell line (STHdhQ111 / Q111, Accession: CVCL_M591) transformed to express the huntingtin gene containing 111 glutamines. Neurons were cultured in DMEM, high glucose (Gibco, Cat No. 11965-092) medium supplemented with 10% fetal bovine serum (Gibco, Cat No. 10082-147) and 1% penicillin-streptomycine (Gibco, Cat No. 25200072). Subculture was then performed every two days by treating with 0.25% trypsin-EDTA (Gibco, Cat No. 15140122). The medium used in the process after the first subculture was DMEM medium with the above composition, to which Geneticin selective antibiotic (G418 sulfate) (Gibco, #10131-035) was added at a final concentration of 0.4 mg / ml. Cells were maintained by subculture at two-day intervals, and after three subcultures, the cells were stabilized and used in the experiment.

[0144]

[0145] 2.2 Preparation of IPB-H01 and TSA (Tubastatin A Hydrochloride)

[0146] IPB-H01 (CXD101, CAS No: 934828-12-3) was prepared by adding Dimethylsulfoxide (DMSO) and dissolving it to a concentration of 10 mM.

[0147] Additionally, Tubastatin A (TSA), a Class II HDAC inhibitor, was used as a control for comparison. Class I HDACs are generally known to be observed primarily in the nucleus, while Class II HDACs are found primarily in the cytoplasm. Histone H3 and α-tubulin are known to be representative nuclear and cytoplasmic factors, respectively.

[0148] Tubastatin A Hydrochloride (TSA, CAS No: 1310693-92-5) was prepared by dissolving it in dimethylsulfoxide (DMSO) to a concentration of 10 mM.

[0149]

[0150] 2.3. Confirmation of decreased huntingtin protein expression

[0151] To confirm the change in expression of mutant huntingtin protein according to IPB-H01 treatment, the following experiments were performed.

[0152] First, to confirm whether IPB-H01 works well in the cell line, IPB-H01 and TSA were treated for 24 hours or 48 hours, respectively, in the same manner as the Western blotting of Example 1.3, and the changes in the expression of acetylated proteins were compared. To confirm the degree of acetylation, primary antibodies against Acetyl-Histone H3 (Santa Cruz, sc-56616, mouse), Acetyl-alpha-tubulin (Sigma-aldrich, T7451, mouse), Histone H3 (Cell signaling, 9175, Rabbit), and alpha-tubulin (Sigma-aldrich, T6074, mouse) were reacted at 4℃ for 24 hours, and then anti-(mouse, rabbit) IgG secondary antibodies corresponding to each antibody were reacted at room temperature for 1 hour, and the expression levels were confirmed. The Western blotting results and the relative expression levels of the proteins are shown in Fig. 3. (ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001)

[0153] As shown in Fig. 3, IPB-H01, a Class I HDAC inhibitor, was confirmed to increase acetylation of histone H3, and TSA, a Class II HDAC inhibitor, was confirmed to increase acetylation of α-tubulin. This means that IPB-H01 works well for the cell lines used in the experiment.

[0154]

[0155] After confirming that IPB-H01 works well for the cell line, IPB-H01 and TSA were treated for 24 or 48 hours, respectively, in the same manner as the Western blotting of Example 1.3, and then the change in the expression of the mutant huntingtin protein was confirmed. After reacting with primary antibodies against MW1 (DSHB, MW1, mouse) and HTT (Sigma-aldrich, H7540, rabbit) as mutant huntingtin markers at 4℃ for 24 hours, the expression level was confirmed after reacting with the corresponding anti-(mouse, rabbit) IgG secondary antibody at room temperature for 1 hour, and the results are shown in Figs. 4 and 5.

[0156] As shown in Figures 4 and 5, the expression of mutant huntingtin protein markers MW1 and HTT was significantly reduced 48 hours after IPB-H01 treatment. (ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001)

[0157]

[0158] These results indicate that IPB-H01 reduces and inhibits the accumulation of huntingtin protein.

[0159]

[0160] 2.4 Confirmation of autophagy of protein aggregates

[0161] HDAC inhibitors are known to induce autophagy as part of the protein quality control (PQC) system, a system inherent to eukaryotic cells. Therefore, we aimed to determine whether autophagy was activated by IPB-H01 treatment.

[0162] Here, the expression changes of chaperone proteins (Ac-HSP90, HSP90, HSP70) were compared when IPB-H01 and TSA were treated for 48 hours in the same manner as the Western blotting of Example 1.3. After reacting with primary antibodies against Acetyl-HSP90 (Thermo fisher, 600-401-981, rabbit), HSP90 (abcam, ab13492, mouse), and HSP70 (Santa cruz, sc-66048, mouse) as chaperone protein markers at 4℃ for 24 hours, the expression levels were confirmed after reacting with anti-(mouse, rabbit) IgG secondary antibodies corresponding to each antibody at room temperature for 1 hour, and the Western blotting results and relative expression levels of the proteins are shown in Figure 6.

[0163] As shown in Figure 6, IPB-H01 significantly increased the acetylation of HSP90, and consequently, the expression of HSP70 was also confirmed to increase. On the other hand, it was confirmed that it had no effect on the expression of HSP90. (ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001)

[0164]

[0165] Additionally, we confirmed the effect on the expression of other key autophagy-related markers besides heat shock proteins.

[0166] Specifically, the expression changes of p62 and LC-3, which are major markers related to autophagy, were compared when IPB-H01 and TSA were treated for 24 or 48 hours in the same manner as the Western blotting of Example 1.3. After reacting with primary antibodies against p62 / SQSTM1 (Sigma-aldrich, P0067, rabbit) and LC-3B (Cell signaling, 2775S, rabbit) at 4℃ for 24 hours, the expression levels were confirmed after reacting with anti-(mouse, rabbit) IgG secondary antibodies corresponding to each antibody at room temperature for 1 hour, and the Western blotting results and relative expression levels of proteins are shown in Figures 7 and 8.

[0167] As shown in Figures 7 and 8, it was confirmed that the expression of p62 and LC-3II increased 24 hours after administration by IPB-H01. (ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001)

[0168]

[0169] These results indicate that IPB-H01 activates the autophagy process of aggregated proteins.

[0170]

[0171] Example 3. Verification of efficacy in the R6 / 2 mouse model

[0172] The disease treatment effect of IPB-H01 treatment was verified in an in vivo mouse model.

[0173]

[0174] 3.1 Preparation of the mouse model

[0175] The R6 / 2 mouse is a representative Huntington's disease mouse model that expresses a portion of the exon 1 region of the Huntington's disease gene. The R6 / 2 mouse is a genetically recombinant transgenic mouse in which a huntingtin gene with 145 CAG repeats has been inserted into the exon 1 region of the Huntington's disease gene. This mouse exhibits a characteristic of Huntington's disease, characterized by increased glutamine sequences at the amino-terminus of the huntingtin protein. R6 / 2 (Strain: B6CBA) mice were maintained on a 12:12 h light / dark cycle and fed ad libitum at a constant room temperature for 2 weeks of acclimation. Five-week-old R6 / 2 mice were orally administered the IPB-H01 composition (50 mg / kg) or vehicle once daily at 24-hour intervals for a total of 5 weeks, with the composition administered for 5 days followed by 2 days of rest. Mice were sacrificed 5 weeks after the start of administration.

[0176] All animals were handled strictly in accordance with the National Institute of Health (NIH) Guide for the Humane Care and Use of Laboratory Animals, and experiments were performed in accordance with the Institute for Laboratory Research (ILAR) Guide for the Care and Use of Laboratory Animals.

[0177]

[0178] 3.2 Manufacturing of IPB-H01

[0179] IPB-H01 (CXD101, CAS No:934828-12-3) was dissolved in 10% Dimethylsulfoxide (DMSO), 40% Polyethyleneglycol 300 (PEG300), 5% Tween 80, and 45% Phosphate buffered saline (PBS, pH7.4) to prepare a concentration of 50 mg / kg / 10 ml.

[0180]

[0181] 3.3 Confirmation of decreased huntingtin protein expression

[0182] To confirm the changes in huntingtin protein expression, survival level of intermediate dendritic neurons in the striatum, and activity of microglial cells in an animal model following IPB-H01 treatment, the following experiments were performed.

[0183] Specifically, immunohistochemical staining was performed. The mouse of Example 3.1 was anesthetized using 100 mg / kg of ketamine and 10 mg / kg of xylazine, and then perfused whole body with phosphate buffered saline (PBS, pH 7.4) through the heart to remove blood in the body. After that, the brain was fixed by perfusion of 4% paraformaldehyde (PFA), and the brain was removed from the mouse. The removed brain was immersed in the PFA solution and refrigerated for 24 hours, then immersed in a 30% sucrose solution and refrigerated until the brain sank. After the brain sank, the 30% sucrose solution was replaced once more and refrigerated until the brain sank completely. After that, the brain was cut horizontally into 30 μm-thick sections using a cryotome (microtome) and used in the experiment. After washing the tissue sections with PBS, non-specific binding other than the target protein was blocked with normal serum. Afterwards, the primary antibodies against the mutant huntingtin markers EM48 (Sigma-Aldrich, #MAB5374, mouse) and MW8 (DSHB, #MW8, mouse) were reacted at 4℃ for 48 hours, and then the secondary antibodies Goat-anti mouse IgG (H+L) and Alexa Fluor 488 were reacted for 24 hours at 4℃ in a light-protected state. Afterwards, the tissues were attached to slides and the expression of specific markers was confirmed through a microscope. The results of immunohistochemical staining are shown in Figures 9 and 10.

[0184] As shown in Figures 9 and 10, it was confirmed that the expression of EM48 was significantly reduced by IPB-H01 in the cerebral cortex of R6 / 2 mice. Scale bar = 100 μm.

[0185] These results indicate that IPB-H01 reduces and inhibits the accumulation of huntingtin protein.

[0186]

[0187] 3.4 Confirmed relief of neuroinflammation

[0188] In Huntington's disease, activated microglia recognize and activate neuronal damage signals induced by the mutant huntingtin protein. Activated microglia secrete inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and produce reactive oxygen species (ROS) and nitrous oxide species (RNS). Furthermore, complement proteins such as C1q are activated, ultimately inducing neuroinflammation. Because the phenotype of activated microglia varies depending on the degree of activation, we investigated the effects of IPB-H01 administration on microglia phenotype.

[0189] Specifically, the expression changes of the Iba-1 marker associated with the activation of microglia upon IPB-H01 treatment were compared in a manner similar to the immunohistochemical staining of Example 3.3. After reacting with the primary antibody against Iba-1 (abcam, ab178846, goat) at 4℃ for 24 hours, the biotinylated anti-goat IgG secondary antibody was reacted for 1 hour at room temperature, and then reacted with the ABC kit (VECTOR, #PK-4000) for 1 hour. The solution was prepared immediately before the reaction using the DAB kit (VECTOR, #SK-4100), and the tissue sections were immersed in the solution to confirm color development. After that, the tissue sections were washed using PBS, and the tissues were attached to slides, and the expression of Iba-1 was confirmed through a microscope, and the results of the immunohistochemical staining are shown in Figs. 11 and 12.

[0190] As shown in Figures 11 and 12, the number of branches and the degree of intersection of branches (Junction) were significantly increased and the size of the cell body was significantly decreased in the cerebral cortex and striatum of R6 / 2 mice (R6 / 2 + IPB-H01) administered with IPB-H01. Scale bar = 100 μm.

[0191] These results indicate that IPB-H01 alleviates neuroinflammation by reducing the degree of microglial activation.

[0192]

[0193] 3.5 Confirmation of increased survival of intermediate dendritic neurons in the striatum

[0194] Since Huntington's disease patients are characterized by a decrease in the number of intermediate dendritic neurons in the striatum, the effect of IPB-H01 treatment on the survival of intermediate dendritic neurons in the striatum was confirmed.

[0195] Specifically, the expression changes of DARPP32, a representative marker of intermediate dendritic neurons, were compared when treated with IPB-H01 in a manner similar to the immunohistochemical staining of Example 3.3. After reacting with the primary antibody against DARPP32 (Santa cruz, #sc-135877, mouse) at 4℃ for 24 hours, the secondary antibodies Goat-anti rabbit IgG (H+L) and Alexa Fluor 488 were reacted for 24 hours at 4℃ in a light-protected state, and the results of immunohistochemical staining are shown in Fig. 13.

[0196] As shown in Figure 13, it was confirmed that DARPP32 expression was significantly low in the striatum of transgenic (Tg) mice, and that the expression of DARPP32 significantly increased after IPB-H01 treatment. Scale bar = 100 μm.

[0197]

[0198]

[0199] 3.6 Survival rate assessment

[0200] We compared the survival rates before and after the experiment to determine whether IPB-H01 improved the survival rate of R6 / 2 mice. We compared the differences between the model group (R6 / 2) administered with vehicle (R6 / 2 + Vehicle) and the group administered IPB-H01 (R6 / 2 + IPB-H01). During the experiment, mice were checked daily for death, and then Kaplan-Meier survival analysis was performed.

[0201] As shown in Figure 14, it was confirmed that the survival rate tended to improve when treated with IPB-H01 compared to mice administered only the excipient (R6 / 2 + Vehicle).

[0202]

[0203] 3.7 Rotarod Test

[0204] The rotarod test assesses motor coordination and balance in animals by forcing them to walk on a rotating cylinder. This test was used to assess whether IPB-H01 treatment improved motor skills and balance.

[0205] Specifically, the rotarod equipment used in the experiment was Technolab's Rotarod-Rod. The rotarod essentially consists of a long rotating drum divided by partitions into several independent lanes. A control device controls the drum's rotational speed, and each lane is equipped with an individual stopwatch to record the time it takes the animal to fall.

[0206] The mice to be used in the experiment were brought out to the experimental location in advance and allowed to acclimate to the environment for approximately one hour to minimize errors due to anxiety. To allow the mice to adapt to the rotarod equipment, a three-day training period was conducted before the main experiment. During training, the animals were placed on the rotating cylinder and slowly acclimated to it at a low speed of 5 to 10 rpm. The maximum rotational speed of the cylinder was then gradually increased by 5 rpm until the animals were finally trained at a speed of 5 to 20 rpm. This process continued for three days before the main test. This test, also conducted over three days, was conducted in the opposite direction to the direction in which the cylinder rotated during the training period, and the rotational speed was set to 5 to 40 rpm. The time it took the animals to fall from the rotating cylinder was measured. The average of the three measured values ​​was used for statistical analysis, and the results are shown in Figure 15.

[0207] As shown in Figure 15, in mice administered only the excipient (R6 / 2 + Vehicle), as the disease worsened over time, motor ability declined and the time taken to fall from the rotating cylinder decreased, whereas in mice administered IPB-H01 (R6 / 2 + IPB-H01), the time taken to fall from the cylinder remained constant at a level similar to that at the beginning of the experiment. This confirmed that the motor ability (motor function) of R6 / 2 mice was improved by IPB-H01.

[0208]

[0209] 3.8 Limb Clasping Test

[0210] The fixation test is used to assess neurological abnormalities in laboratory animals. It is useful for simply identifying motor function, balance, and nervous system abnormalities. While healthy animals generally tend to maintain balance by extending their legs, animals with neurological abnormalities tend to pull their limbs toward the body and exhibit a clasping posture. The degree of clasping is scored to determine the degree of motor or neurological abnormalities. This test was used to determine whether IPB-H01 administration improved motor function in R6 / 2 mice.

[0211] The mice to be used in the experiment were brought out to the experimental location in advance and allowed to acclimate to the environment for approximately one hour to minimize errors due to anxiety. With a camera set up to observe the mouse's behavior, the mouse was carefully lifted by the tail and suspended in mid-air, its limb movements observed for 15 seconds. The results were quantified by assigning a score based on the degree of grasping, and the results are shown in Figure 16.

[0212] As shown in Figure 16, in the case of mice administered only the excipient (R6 / 2 + Vehicle), the limb clasp score continuously increased, whereas in the case of mice administered IPB-H01 (R6 / 2 + IPB-H01), the limb clasp score began to show a difference compared to the vehicle administration group from the 4th week and decreased from the 7th week. This confirmed that the motor disorder of R6 / 2 mice was improved by IPB-H01.

[0213]

[0214] Example 4. Verification of efficacy in the YAC128 mouse model

[0215] The disease treatment effect of IPB-H01 treatment was verified in an in vivo mouse model.

[0216]

[0217] 4.1 Preparation of the mouse model

[0218] The YAC128 mouse is a representative Huntington's disease mouse model in which the huntingtin gene, which contains 128 CAG repeats, is inserted into exon 1 of the Huntington's disease gene. It well represents the characteristics of Huntington's disease patients, which are characterized by an increase in the glutamine base sequence at the amino-terminus of the huntingtin protein. YAC128 (Strain: FVB / N) mice were maintained on a 12:12 h light / dark cycle and fed ad libitum at a constant room temperature for 2 weeks of acclimation. Seven-month-old YAC128 mice were orally administered IPB-H01 (20 mg / kg) or vehicle twice daily at 12-h intervals for a total of 13 weeks. Mice were sacrificed 13 weeks after the start of administration.

[0219] All animals were handled strictly in accordance with the National Institute of Health (NIH) Guide for the Humane Care and Use of Laboratory Animals, and experiments were performed in accordance with the Institute for Laboratory Research (ILAR) Guide for the Care and Use of Laboratory Animals.

[0220]

[0221] 4.2 Manufacturing of IPB-H01

[0222] IPB-H01 (CXD101, CAS No:934828-12-3) was dissolved in 10% Dimethylsulfoxide (DMSO), 40% Polyethyleneglycol 300 (PEG300), 5% Tween 80, and 45% Phosphate buffered saline (PBS, pH7.4) to prepare a concentration of 50 mg / kg / 10 ml.

[0223]

[0224] 4.3 Rotarod Test

[0225] To assess whether motor skills and balance were improved by IPB-H01 treatment, a rotarod test was performed. Specifically, the rotarod test was performed in the same manner as in Example 3.7, and the results are shown in Fig. 17.

[0226] As shown in Figure 17, in mice administered only the excipient (YAC128 + Vehicle), the time to fall from the cylinder remained constant, whereas in mice administered IPB-H01 (YAC128 + IPB-H01), the time to fall from the cylinder gradually increased. This confirmed that the motor function of YAC128 mice was improved by IPB-H01.

[0227]

[0228] 4.4 Spontaneous Locomotion Assessment or Open Field Test

[0229] The Open Field Test (OPT) is a test used to assess an animal's general level of locomotor activity. To examine behavioral changes in animals after a treatment, it is necessary to examine their basic level of movement. While analysis typically focuses on movement, the act of movement itself is influenced by a variety of factors, including not only the animal's motor skills but also its level of anxiety, circadian rhythms, and the severity of injury. Therefore, this test can also be used to indirectly observe changes in these indicators. This test was used to evaluate whether IPB-H01 administration improved motor skills and anxiety disorders in YAC128 mice.

[0230] The mice to be used in the experiment were brought out to the experimental location in advance and allowed to acclimate to the environment for approximately 1 hour to minimize errors due to anxiety. The experiment was conducted in a rectangular open box (male or acrylic) that allowed the animals to move around sufficiently. Four boxes, each measuring 45 cm in length, width, and height, were prepared, and an observation camera was fixed vertically using a tripod to observe all four boxes. After that, the mice were placed in the center of the boxes one by one, and their movements were observed for 17 minutes. Afterwards, the results of the indices related to the mouse movement (Track, Total Distance Traveled, Frequency in the central zone, Cumulative duration) were extracted using the Ethovision program from Noldus and used for statistical analysis. The results are shown in Figure 18.

[0231] As shown in Fig. 18, it was confirmed that the total distance traveled and the frequency in the central zone increased when IPB-H01 was administered compared to YAC128 mice administered only the vehicle (YAC128 + IPB-H01). This confirmed that the motor function of YAC128 mice was improved by IPB-H01.

[0232]

[0233] 4.5 Forced Swimming Test

[0234] The forced swim test is useful for observing depression-related behaviors in animals and examining the effects and neurobiological mechanisms of various antidepressants. Using this test, we evaluated whether IPB-H01 administration improved anxiety in YAC128 mice.

[0235] The mice to be used in the experiment were brought out to the experimental location in advance and allowed to acclimate to the environment for approximately 1 hour to minimize errors due to anxiety. With a camera set up to observe the mouse's behavior, lukewarm water was poured into an open-top cylindrical cylinder to a height of 15 cm from the bottom of the cylinder. The animals were forced to swim for 5 minutes to observe their behavior. Afterwards, the immobility time (the time the animals remained immobile, floating on the water without struggling and with no movement other than their heads above the water's surface) was measured, and the results are shown in Figure 19.

[0236] As shown in Figure 19, YAC128 mice administered only the vehicle (YAC128 + Vehicle) showed a tendency for the immobility time to increase 12 weeks after administration compared to before administration, whereas YAC128 mice administered IPB-H01 (YAC128 + IPB-H01) showed a tendency for the immobility time to decrease. This confirmed that depressive disorder in YAC128 mice was improved by IPB-H01.

[0237]

[0238] 4.6 Grip Strength Test

[0239] The grip test is an experimental method used to assess muscle strength and neuromuscular function in animals. It is used to assess musculoskeletal and nervous system function, or to study neuromuscular disorders, drug effects, or genetic mutations. This test was used to evaluate whether IPB-H01 administration improved neuromuscular function in YAC128 mice.

[0240] The experiment was conducted using force measurement equipment from Chatillon. The mice were brought out to the experimental location in advance and allowed to acclimate to the environment for approximately one hour to minimize errors due to anxiety. Prior to the actual measurement, the animals were trained to hold their tails and touch only their forelimbs to an acrylic plate. Then, with both forelimbs fully extended, they were held against a grip strength meter and the measured values ​​were recorded. Measurements were conducted twice a day for three days. Three values ​​were measured per measurement, and after a rest period of approximately 30 minutes to an hour, a second measurement was conducted. Eighteen results were obtained over the three days, and the results are shown in Figure 20.

[0241] As shown in Figure 20, the grip strength of YAC128 mice administered only the vehicle (YAC128 + Vehicle) was maintained at a constant level, whereas that of YAC128 mice administered IPB-H01 (YAC128 + IPB-H01) tended to increase over time. This confirmed that the neuromuscular function of YAC128 mice was improved by IPB-H01.

[0242]

[0243] 4.7 Confirmation of decreased huntingtin protein expression

[0244] To examine changes in huntingtin protein expression, striatal intermediate dendritic neuron survival, and microglial activity following IPB-H01 treatment in an animal model, the following experiments were performed. Specifically, immunohistochemical staining was performed in the same manner as in Example 3.3, and the results are shown in Figure 21.

[0245] As shown in Fig. 21, it was confirmed that the expression of EM48 was significantly reduced by IPB-H01 in the cerebral cortex of YAC128 mice. Scale bar = 100 μm.

[0246]

[0247] 4.8 Increased survival of intermediate dendritic neurons in the striatum confirmed

[0248] Since Huntington's disease patients are characterized by a decrease in intermediate dendritic neurons in the striatum, the effect of IPB-H01 treatment on the survival of intermediate dendritic neurons in the striatum was investigated. Specifically, immunohistochemical staining was performed in the same manner as in Example 3.5, and the results are shown in Figure 22.

[0249] As shown in Figure 22, it was confirmed that DARPP-32 expression was significantly low in the striatum of YAC128 mice, and an increase in DARPP-32 expression was observed after IPB-H01 treatment. Scale bar = 100 μm.

[0250]

[0251] 4.9 Confirmed relief of neuroinflammation

[0252] Since neuroinflammation is induced by activated microglia, the effects of IPB-H01 administration on microglia phenotype were investigated. Specifically, immunohistochemical staining was performed in the same manner as in Example 3.4, and the results are shown in Figures 23 and 24.

[0253] As shown in Figure 23, in the brain cortex of YAC128 mice (YAC128 + IPB-H01) administered with IPB-H01, the number of branches and the degree of intersection of branches (Junction) tended to increase, and the size of the cell body was confirmed to significantly decrease. Scale bar = 100 μm.

[0254] As shown in Figure 24, it was confirmed that the number of branches and the degree of intersection of branches (Junction) in the striatum of YAC128 mice (YAC128 + IPB-H01) administered with IPB-H01 significantly increased, and the size of the cell body tended to decrease. Scale bar = 100 μm.

[0255]

[0256] Example 5. Verification of blood-brain barrier permeability using brain-derived lipids.

[0257] To verify the blood-brain barrier (BBB) ​​permeability of IPB-H01, a BBB-PAMPA (Parallel artificial membrane permeability assay) test was performed using a product from Pion to verify the blood-brain barrier permeability.

[0258] First, to prepare a donor buffer, PRISMA HT solution (Pion, #110151) was diluted 1:40 in distilled water and the pH was adjusted to 7.4. IPB-H01 was diluted in donor buffer to a concentration of 50 μM and used as an initial sample, and brain sink buffer (Pion, #110674) was used as an acceptor buffer.

[0259] To confirm the blank value, the donor buffer prepared above was dispensed at 150 μl / well on a UV plate, and the absorbance was measured. Similarly, the initial value was also measured by dispensing 150 μl / well of the diluted IPB-H01 above. After that, the prepared initial sample was dispensed at 200 μl / well on the donor plate of a Stirwell PAMPA sandwich (Pion, #110243), and the brain sink buffer was dispensed at 200 μl / well on the acceptor plate whose membrane was coated with BBB-1-lipid (Pion, #110672). To confirm the permeability, the donor plate and acceptor plate were combined and reacted at 25°C for 4 hours. After that, the donor plate and acceptor plate were separated and transferred to a UV plate, and the absorbance was measured, which was used as the donor value and the acceptor value. Reference is the absorbance of the initial sample, Donor is the absorbance of the sample remaining on the donor plate after 4 hours of reaction, and Acceptor is the absorbance of the sample transmitted through the acceptor plate after 4 hours of reaction. After measuring the absorbance of the sample, the transmittance value (P) was calculated using the PAMPA explorer program. e, 10 -6 cm / sec) was analyzed, and the results and graphs of the average and standard deviation for three repeated experimental values ​​are shown in Table 1 and Figure 25.

[0260]

[0261] SampleConcentration(μM)pHP e (10 -6 cm / sec)IPB-H01507.418.5718.9819.50

[0262]

[0263] As shown in Table 1 and Figure 25, IPB-H01 was confirmed to have a very high possibility of penetrating the blood-brain barrier with a permeability of 10 or higher, and also a very high possibility of diffusing into the central nervous system.

[0264]

[0265] Example 6. In vivo pharmacokinetic validation in the FVB mouse model

[0266] To predict the blood concentration and drug efficacy in humans, wild-type FVB mice, which are identical to YAC128 mice, were used. The plasma and brain tissues of mice that received a single oral dose of IPB-H01 were used to determine the residual concentration in the body and pharmacokinetic parameters (PK parameters).

[0267] For plasma, 90 μl of acetonitrile was added to 10 μl, and for brain tissue, 90 μl of acetonitrile was added to 10 μl, and the brain tissue was homogenized with 4 times the tissue weight of PBS. Then, 180 μl of acetonitrile was added to 20 μl of the brain tissue homogenate, vortexed, and centrifuged at 15,000 rpm for 5 minutes at 4°C. The supernatant was separated and analyzed using LC-MS / MS. For samples exceeding the upper limit of quantification (ULOQ), the concentration was diluted with blank tissue and analyzed so that the concentration value was included within the linear range of the calibration curve. Pharmacokinetic indices were calculated using the Phoenix WinNolin 6.4 version (Pharsight, USA) program, and the calculations were performed using a non-compartmental analysis model. Table 2 shows the PK parameters in plasma after drug administration, Table 3 shows the PK parameters in brain, and the analysis results are shown in Table 4 and Figure 26.

[0268]

[0269] SubjectT 1 / 2(hr)Tmax(hr)Cmax(ng / ml)AUClast(hr*ng / ml)AUCINF-obs (hr*ng / ml)%AUC ExtrapMRTINF_obs (hr)11.41.05781.114491.014782.12.02.121.61.05282.612205.512603.63.22.131.21.06059.811433.411539.40.91.6Mean1.41.05707.812710.012975.02.01.9SD0.20.0393.71590.01653.01.10.3

[0270]

[0271] SubjectT 1 / 2 (hr)Tmax(hr)Cmax(ng / g)AUClast(hr*ng / g)AUCINF-obs (hr*ng / g)%AUC ExtrapMRTINF_obs (hr)12.21.01068.63703.44137.310.53.622.71.0851.93128.23700.615.54.232.11.0804.32796.23075.69.13.5Mean2.41.0908.23209.33637.811.73.8SD0.30.0140.9459.0533.63.40.4

[0272]

[0273] Time (hr)Subject 1Subject 2Subject 3MeanSD0.250.090.110.070.090.0210.180.160.130.160.0320.280.240.240.250.0240.420.711.180.770.3880.910.841.451.070.34

[0274] * C t / C p : Tissue[C t ] / Plasma[C t ]

[0275] ([C t ]: Concentration at sampling time)

[0276]

[0277] As shown in Tables 2 to 4 and Figure 26, IPB-H01 was confirmed to be well delivered to the brain through the blood-brain barrier. Furthermore, similar to other histone deacetylase inhibitors (HDAC inhibitors) currently under study, it was confirmed to exhibit a short half-life and Tmax value in plasma and brain tissue.

[0278]

[0279] Example 7. Histopathological examination using a mouse model

[0280] To evaluate the safety and efficacy of IPB-H01 and identify potential toxicities and adverse reactions, histopathological changes were observed using hematoxylin-eosin (H&E) staining. Pathological changes in major target organs (liver, heart, and kidney) were analyzed to determine the toxicity of IPB-H01.

[0281] Mice were anesthetized with 100 mg / kg of ketamine and 10 mg / kg of xylazine, and the heart, liver, and kidneys were removed from the mice. The removed organs were stored in a paraformaldehyde (PFA) solution at room temperature for 24 hours. After wrapping the organs in paraffin to create paraffin blocks, 8 μm-thick sections were prepared using a microtome. The tissue sections were washed with phosphate-buffered saline (PBS), attached to coated slides, and dried for 2 hours. Afterwards, the slides were immersed in a 1:1 mixture of xylene and CHCl3 to remove paraffin. The slides were dehydrated by immersing them in 100%, 95%, and 70% ethanol solutions in that order, and then immersed in distilled water. Afterwards, staining was completed by reacting with Hematoxylin and Eosin in that order, and pathological changes were observed through a microscope, and the results are shown in Figures 27 to 29.

[0282] As shown in Figures 27 to 29, the findings such as fatty changes, inflammatory cell infiltration and necrosis observed in the liver, and basophilic tubules, tubular dilatation, tubular hypertrophy, cylindrical structures and cysts observed in the kidney were generally observed background lesions, and it was confirmed that the frequency and extent of occurrence of the lesions were weak.

[0283]

[0284] Example 8. Confirmation of regulation of autophagy-related gene expression

[0285] We performed transcriptome sequencing of YAC128 mouse brain tissue to investigate genes involved in autophagy. RNA was extracted from the striatum of brain tissue using the Ribo-Zero rRNA Removal Kit to prepare total RNA-seq. The purified RNA was randomly fragmented for short-read sequencing. The fragmented RNA was reverse transcribed into cDNA, and different adapters were ligated to both ends of the cDNA fragments. The ligated cDNA fragments were amplified by PCR to obtain sufficient quantities for sequencing.

[0286] Raw reads underwent quality control (QC) analysis, including assessment of read quality, total base count, total reads, and GC content. To avoid bias, preprocessing was performed to remove low-quality reads, adapter sequences, contaminating DNA, and PCR duplicates. Preprocessed reads were aligned to the reference genome to generate aligned reads. Read counts, transcript length, and coverage depth were considered to generate normalized expression profiles. Differentially expressed genes (DEGs) were analyzed, and gene ontology (GO) analysis was performed for significant genes (log2FC ≥ |1| and P-value < 0.05). Gene annotation was performed specifically for GO:0006914, which is related to autophagy.

[0287] As shown in Figure 30, eight autophagy-related genes were found to have significantly reduced expression in YAC128 (Transgenic) animals compared to wild-type (WT) animals, which was restored after treatment with IPB-H01.

[0288]

[0289] Additionally, pathway analysis of significantly altered genes was performed using the KEGG database (Kyoto Encyclopedia of Genes and Genomes, https: / www.genome.jp / pathway / mmu04140) focusing on the autophagy pathway.

[0290]

[0291] Gene annotation results showed that significantly altered genes (IRS, PI3K, ULK1 / 2, WIPI, and DAPK) were involved in the autophagy process, particularly within the insulin signaling pathway and the ULK complex. These results suggest that IPB-H01 has the potential to contribute to the restoration of autophagy function in the YAC128 mouse model by regulating the expression of autophagy-related genes.

[0292]

[0293] In summary, compositions containing IPB-H01 according to one aspect have the activity of reducing or eliminating the number of mutant huntingtin protein aggregates, activating the autophagy process, and suppressing the activation of microglial cells, thereby alleviating neuroinflammation. In other words, IPB-H01 is useful as a treatment for Huntington's disease and diseases with similar aspects.

[0294]

[0295] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating polyglutamine diseases, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

2. A pharmaceutical composition according to claim 1, wherein the polyglutamine disease is any one selected from the group consisting of Huntington's disease, polyglutamine expansion disease, spinocerebellar ataxias (SCA), Machado-Joseph disease (MJD / SCA3), dentatorubro-Pallidoluysian atrophy (DRPLA), and X-linked spinal and bulbar muscular atrophy (SMAX).

3. A pharmaceutical composition according to claim 1, wherein the polyglutamine disease is caused by accumulation of protein aggregates due to glutamine repeat mutations.

4. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition prevents or treats damage or death of nerve cells.

5. In claim 1, the pharmaceutical composition has one or more of the following characteristics: - Inhibits or reduces the accumulation of protein aggregates; - Induces autophagy of protein aggregates; - Reduces the activity of microglia; and - Increases the number of intermediate dendritic neurons.

6. In claim 1, the pharmaceutical composition has one or more of the following characteristics: (i) reducing the expression of any one or more selected from the group consisting of 1C2, MW1, HTT and MW8; (ii) increasing the expression of any one or more selected from the group consisting of HSP70, p62 and LC-3II; and (iii) Reduces the expression of Iba-1.

7. A pharmaceutical composition for preventing or treating neuroinflammation comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

8. A pharmaceutical composition according to claim 7, wherein the neuroinflammation is caused by accumulation of protein aggregates due to glutamine repeat mutations.

9. A health functional food for preventing or improving polyglutamine disease, comprising a compound represented by the following chemical formula 1 or a salt thereof.

10. A health functional food for preventing or improving neuroinflammation, comprising a compound represented by the following chemical formula 1 or a salt thereof: [Chemical Formula 1] .

11. Feed composition for preventing or improving polyglutamine disease, comprising a compound represented by the following chemical formula 1 or a salt thereof: [Chemical Formula 1] .

12. A feed composition for preventing or improving neuroinflammation, comprising a compound represented by the following chemical formula 1 or a salt thereof: [Chemical Formula 1] .

13. A method for preventing, improving or treating polyglutamine disease, comprising administering to a subject in need thereof an effective amount of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

14. Use of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof for the manufacture of a preparation for preventing, improving or treating polyglutamine disease: [Chemical Formula 1] .

15. A method for preventing, improving or treating neuroinflammation, comprising administering to a subject in need thereof an effective amount of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

16. Use of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof for the manufacture of a preparation for preventing, improving or treating neuroinflammation: [Chemical Formula 1] .

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