Composition for treating kidney diseases comprising metixene or pharmaceutically acceptable salt thereof

Abstract

One embodiment of the present invention relates to a composition for treating kidney diseases, comprising metixene or a pharmaceutically acceptable salt thereof and, more specifically, to metixene which inhibits the expression level of a gene or protein of a renal fibrosis factor.

Description

Composition for treating kidney disease comprising methixene or a pharmaceutically acceptable salt thereof

[0001] The present invention was made under the support of the Ministry of Science and ICT under the project identification number 1711177331 and project number 2023010277. The research management specialized institution of the project is the Daegu Gyeongbuk Institute of Science and Technology, the research project name is “DGIST Institutional Project”, the research project name is “Development of Diagnosis and Treatment Technology for Intractable Diseases”, the lead institution is the Daegu Gyeongbuk Institute of Science and Technology, and the research period is from January 1, 2023 to December 31, 2023.

[0002] The present invention was made with the support of the Ministry of Science and ICT, under the grant number 1711182466 and grant number 2023030010. The research management organization of the above-mentioned project is the Daegu Gyeongbuk Institute of Science and Technology, the research project name is “(Type 1-1) Mid-career Research”, the research project title is “Discovery of Preventive Agents for Chronic Renal Disease Related to Metabolic Diseases Using a 3D Micro-tissue-Microfluid Chip-Based Drug Screening Platform”, the lead organization is the Daegu Gyeongbuk Institute of Science and Technology, and the research period is from 2023.03.01 to 2024.02.29.

[0003] The present invention relates to a composition for treating kidney disease comprising Metixene or a pharmaceutically acceptable salt thereof, and more particularly, to Metixene that suppresses the gene or protein expression level of a renal fibrosis factor.

[0004] The kidneys are one of the most crucial organs in the human body, performing a variety of functions, including metabolism and waste removal, maintaining electrolyte balance, regulating blood pressure, and producing and secreting various hormones. When kidney function is impaired, these vital functions are disrupted, potentially leading to significant health problems.

[0005] Kidney diseases are broadly divided into congenital and acquired diseases. Among these, renal fibrosis and renal failure are considered very significant. Renal fibrosis occurs when kidney cells are damaged and replaced by fibrous tissue. This reduces the kidney's filtering function and, in severe cases, can lead to renal failure. Renal failure refers to a condition in which kidney function is significantly reduced, leading to the accumulation of waste products in the body. It can be divided into acute and chronic types, and delayed treatment can be life-threatening.

[0006] While various treatments for renal fibrosis and renal failure are currently being studied, most focus on symptom relief or slowing progression. In the case of renal failure in particular, alternative treatments such as dialysis and kidney transplantation are primarily used rather than fundamental treatment.

[0007] These existing treatments have several limitations. First, kidney transplantation is plagued by a shortage of organ donors, and even after transplantation, there is a risk of complications such as blood rejection. Second, dialysis reduces the patient's quality of life, and long-term dialysis treatment can lead to other side effects.

[0008] Recent research is actively pursuing small molecule compounds that target the fundamental causes of kidney disease. These compounds are known to have the effect of repairing kidney cell damage or inhibiting kidney cell fibrosis.

[0009] Small molecule compounds hold great potential because they offer an approach that directly addresses the underlying cause of disease, rather than relying on existing alternative treatments. However, the efficacy, safety, and long-term effects of these compounds still need to be verified.

[0010] Against this backdrop of the importance of kidney disease and the need for development of new treatments to overcome the limitations of existing treatments, the present invention proposes an effective treatment method for kidney disease centered on small molecule compounds.

[0011] The present inventors have diligently researched and developed a treatment for kidney disease using Metixene. As a result, they confirmed that Metixene has therapeutic effects on kidney disease, including suppressing the expression of renal fibrogenic factors such as Plasminogen Activator Inhibitor-1 (PAI-1), without causing significant side effects in the body. This led to the completion of the present invention.

[0012] Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, comprising methixene or a pharmaceutically acceptable salt thereof.

[0013] Another object of the present invention is to provide a food composition for alleviating, suppressing or improving kidney disease, comprising methixene or a pharmaceutically acceptable salt thereof.

[0014] Another object of the present invention is to provide a use of methixene or a pharmaceutically acceptable salt thereof for treating kidney disease.

[0015] The present invention relates to a composition for treating renal disease, comprising Metixene or a pharmaceutically acceptable salt thereof. The composition according to the present invention is a substance that has been proven to be safe while effectively reducing factors associated with renal fibrosis, and can be effectively used for treating renal disease, including renal fibrosis.

[0016] Hereinafter, the present invention will be described in more detail.

[0017]

[0018] One embodiment of the present invention is a pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, comprising methixene represented by chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0019] [Chemical Formula 1]

[0020]

[0021] The term “renal disease” in this specification means a condition in which the normal structure or function of the kidney is damaged or abnormal.

[0022] In one embodiment of the present invention, the renal disease may be one or more selected from the group consisting of renal failure, glomerulonephritis, pyelonephritis, renal cancer, or renal fibrosis, but is not limited thereto.

[0023] The term “renal fibrotic disease” as used herein refers to a pathological change characterized by the accumulation of excessive collagen and other soft tissue components in the renal tissue, which interferes with the normal function of the kidney and often contributes significantly to the progression of chronic kidney disease (CKD). During the renal fibrotic process, when renal cells are damaged or die, excessive production and accumulation of extracellular matrix (ECM) components occur. This excessive accumulation of ECM negatively affects the vascular and filtering structures of the kidney, leading to a decrease in the glomerular filtration rate (GFR), proteinuria, and other renal dysfunction. Renal fibrosis can be caused by various factors, including chronic hypertension, diabetes, glomerular nephritis, and exposure to certain drugs or toxins. These factors cause stress or damage to the cells within the kidney, thereby promoting a fibrotic response.

[0024] In one embodiment of the present invention, the renal fibrotic disease may be at least one disease selected from the group consisting of renal failure, glomerulonephritis, pyelonephritis, and renal cancer, and may be, for example, renal failure, but is not limited thereto.

[0025] The term “methixene” in this specification refers to a compound conventionally used as a Parkinson’s treatment, mainly a compound used as an anticholinergic, and is a compound that provides effective treatment for nervous system diseases by blocking acetylcholine receptors, a neurotransmitter, or inhibiting the action of cholinesterase, an enzyme that breaks down acetylcholine. As described above, the methixene according to the present invention has been conventionally used as a Parkinson’s treatment, and its safety has been continuously proven. Accordingly, the composition for treating kidney disease according to the present invention has the advantage of effectively improving the condition without causing side effects.

[0026] The term “pharmaceutically acceptable salt” as used herein means a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The above pharmaceutically acceptable salts are obtained by reacting the compound of the present invention with inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, and phosphoric acid, sulfonic acids (bisulfite) such as methanesulfonic acid, ethanesulphonic acid, and p-toluenesulfonate, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and salicylic acid. It can be obtained by reacting with organic carbonic acids such as (salicylice acid).In addition, the compound of the present invention can be obtained by reacting it with a base to form salts such as an ammonium salt, an alkali metal salt such as a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, salts of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl) methylamine, and salts of amino acids such as arginine and lysine.

[0027] In one embodiment of the present invention, the pharmaceutically acceptable salt of metixene may be, for example, but is not limited to, metixene hydrochloride hydrate.

[0028] In addition, the above-mentioned metixene and pharmaceutically acceptable salts of metixene may include not only pharmaceutically acceptable salts but also all salts, hydrates and solvates that can be prepared by conventional methods.

[0029] In the present invention, treatment means any act of improving or alleviating kidney disease or symptoms thereof by administering a pharmaceutical composition according to the present invention, or inhibiting or delaying the progression of kidney disease and thereby inhibiting the worsening of kidney disease.

[0030] In the present invention, treatment includes reducing mortality due to kidney disease or delaying the start of dialysis.

[0031] In the present invention, prevention means any act of preventing or suppressing the occurrence of kidney disease or symptoms thereof or delaying the onset thereof by administering a pharmaceutical composition according to the present invention.

[0032] The composition of the present invention was confirmed to not exhibit cytotoxicity as cell viability did not significantly change upon treatment with 0 μM, 0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM of Methixene, and it was confirmed to suppress the expression of renal fibrosis factors upon treatment with 1 μM, 2 μM and 5 μM of Methixene, and therefore can be applied as a drug for treating renal fibrosis that occurs due to chronic renal failure.

[0033] The pharmaceutical composition according to the present invention has been confirmed to inhibit the expression of renal fibrosis factors as described above, and such renal fibrosis factors may be, but are not limited to, Plasminogen Activator Inhibitor-1 (PAI-1), Collagen Type 1, Fibronectin, Alpha-Smooth Muscle Actin (α-SMA), p-Smad3, and Smad3.

[0034] In one embodiment of the present invention, the pharmaceutical composition may additionally contain pharmaceutically suitable and physiologically acceptable carriers, excipients, and diluents, and other auxiliary agents.

[0035] Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, methyl, cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talcum, magnesium stearate. and mineral oil, but is not limited thereto.

[0036] When formulating, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants commonly used in the art can be used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into a pharmaceutical composition, and in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used, but are not limited thereto.

[0037] Oral preparations include suspensions, solutions, emulsions, syrups, and ointments, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included, but are not limited to these.

[0038] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and transdermal preparations. Non-aqueous solutions and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0039] Suppository formulations that may be used include, but are not limited to, witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerinated gelatin.

[0040] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0041] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective amount may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0042] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0043] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitantly administered drugs.

[0044] Another aspect of the present invention is a food composition for alleviating, suppressing or improving kidney disease, comprising a methixene represented by chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0045] [Chemical Formula 1]

[0046]

[0047] The food composition according to the present invention may include ingredients commonly added during food manufacturing, and may include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings, but is not limited thereto.

[0048] Carbohydrates that may be included in the food composition according to the present invention include, but are not limited to, monosaccharides such as glucose and fructose, disaccharides such as maltose, sucrose, and oligosaccharides, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0049] Flavoring agents that may be included in the food composition according to the present invention may include, but are not limited to, natural flavoring agents such as thaumatin and stevia extract, and synthetic flavoring agents such as saccharin and aspartame.

[0050] Another aspect of the present invention relates to a method for treating kidney disease, comprising administering to a subject a methixene represented by chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0051] [Chemical Formula 1]

[0052]

[0053] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow.

[0054] As used herein, the term "administration" refers to providing a given substance to a subject by any suitable method. The composition of the present invention for alleviating, suppressing, treating, or preventing kidney disease may be administered orally or parenterally via any conventional route, as long as it can reach the target tissue. Furthermore, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

[0055] In one embodiment of the present invention, the pharmaceutical composition according to the present invention may be administered alone, but may generally be administered mixed with a pharmaceutical carrier selected in consideration of the administration method and standard pharmaceutical practice.

[0056] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist.

[0057] For example, the daily dosage may be 1 to 1000 μg / ml based on the active ingredient content, but this is an average case and the dosage may be higher or lower depending on individual differences.

[0058] Another aspect of the present invention is the use of methixene or a pharmaceutically acceptable salt thereof for the treatment of renal disease.

[0059] The present invention relates to a composition for treating kidney disease, comprising Metixene or a pharmaceutically acceptable salt thereof. The pharmaceutical composition comprising Metixene or a pharmaceutically acceptable salt thereof according to the present invention can effectively treat, alleviate, suppress, or prevent kidney disease. The pharmaceutical composition according to the present invention can prevent or suppress the onset of kidney disease in advance, or improve the kidney disease, or suppress the progression or worsening of the kidney disease.

[0060] The effects of the present invention are not limited to the above-described effects, and it should be understood that the description of the present invention includes all effects that can be inferred from the composition of the invention described in the claims.

[0061] Figure 1 is a graph showing the results of toxicity confirmation according to administration of a composition containing Metixene, which is an example of the present invention. (* on the graph indicates the probability of significance (P value), and *P<0.01 vs. 0μM.

[0062] Figure 2A is a graph showing the results of evaluating the gene expression of Plasminogen Activator Inhibitor-1 (PAI-1) according to the dosage of a composition containing Methixene in rat kidney fibroblast cells (NRK-49F). (*, **, and # above the graph indicate the probability of significance (P value), with *P<0.001 vs. the control group and **P<0.001, #P<0.01 vs. TGF-β.)

[0063] Figure 2B is a diagram and graph showing the results of evaluating the protein expression of PAI-1 according to the dosage of a composition containing meticixene in renal fibroblasts. (* and ** above the graph indicate the probability of significance (P value), with *P<0.001 vs. the control group and **P<0.001 vs. TGF-β.)

[0064] Figure 2C is a graph showing the secretion level of PAI-1 according to the dose of a composition containing methixene in renal fibroblasts. (*, **, and # above the graph indicate the probability of significance (P value), with *P<0.001 vs. the control group and **P<0.001, #P<0.01 vs. TGF-β.)

[0065] Figure 3 is a graph showing the results of PAI-1 promoter activity evaluation according to administration of a composition containing Methixene, which is an example of the present invention. *, **, and # above the graph indicate the probability of significance (P value), and *P<0.01 vs. control group and **P<0.01, #P<0.05 vs. TGF-β.)

[0066] Figure 4A is a graph showing the results of evaluating the gene expression of collagen type 1 according to the dosage of a composition containing methicen in renal fibroblasts. (**, ##, and $ above the graph indicate the probability of significance (P value), and **P<0.01 vs. the control group, ##P<0.01, and $P<0.05 vs. TGF-β.)

[0067] Figure 4B is a graph showing the results of evaluating the gene expression of fibronectin according to the dosage of a composition containing methixene in renal fibroblasts. (The * and ## above the graph indicate the probability of significance (P value), with *P<0.001 vs. the control group and ##P<0.01 vs. TGF-β.)

[0068] Figure 4C is a graph showing the results of evaluating the gene expression of alpha-smooth muscle actin (α-SMA) according to the dosage of a composition containing methixene in renal fibroblasts. (*, #, and ## above the graph indicate the probability of significance (P value), with *P<0.001 vs. the control group, #P<0.001, and ##P<0.01 vs. TGF-β.)

[0069] Figure 5A is a diagram showing the results of evaluating the protein expression of Collagen Type 1, Fibronectin, α-SMA, and beta-actin (β-actin) antibodies according to the dosage of a composition containing methixene in renal fibroblasts.

[0070] Figure 5B is a graph showing the results of evaluating the protein expression of Collagen Type 1 according to the dosage of a composition containing Methixene in renal fibroblasts. (*, ##, and $ above the graph indicate the probability of significance (P value), with *P<0.01 vs. the control group, ##P<0.01, and $P<0.05 vs. TGF-β.)

[0071] (C) is a graph showing the results of evaluating the protein expression of fibronectin according to the dosage of a composition containing methixene in renal fibroblasts. (*, ##, and $ on the graph indicate the probability of significance (P value), with *P<0.01 vs. the control group, ##P<0.01, and $P<0.05 vs. TGF-β.)

[0072] (D) is a graph showing the results of evaluating α-SMA protein expression according to the dosage of a composition containing methixene in renal fibroblasts. (**, ##, and $ above the graph indicate the probability of significance (P value), **P<0.001 vs. the control group, ##P<0.01, and $P<0.05 vs. TGF-β.)

[0073] Figure 6 is a drawing and graph showing the results of evaluating the expression level of phospho-Smad3 (p-Smad3) or Smad3 and SMAD activity according to administration of a composition containing Methixene, which is an example of the present invention. (* and ** above the graph indicate the probability of significance (P value), and *P<0.01 vs. the control group and **P<0.05 vs. TGF-β.)

[0074] Metixene represented by chemical formula 1 or a pharmaceutically acceptable salt thereof;

[0075] A pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, comprising:

[0076] [Chemical Formula 1]

[0077]

[0078] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples. These examples are provided to more fully explain the present invention to those of ordinary skill in the art.

[0079]

[0080] Example 1: Materials and Methods

[0081] 1-1. Cell culture

[0082] Rat kidney fibroblast cell line (NRK-49F) purchased from the American Type Culture Collection (ATCC) was cultured in animal cell culture medium (Dulbeco's Modified Eagle's Medium; DMEM) containing 5% fetal bovine serum (FBS) and 1% antibiotics at 37°C under 5% CO2 conditions. NRK-49F was cultured in 100 mm dishes at 70-80% confluency with the culture medium and subcultured once every two days.

[0083]

[0084] 1-2. Toxicity Check

[0085] NRK-49F was cultured in 96-well plates at 80% confluency for 18 hours, and then treated with various concentrations of Methixene. After 24 hours, the cells were reacted with 10 μL of Dong-In Biotech's Cell Counting Kit for 2 hours, and the absorbance was measured at 450 nm. The cell viability of Methixene was expressed as a percentage of the 0 μM group.

[0086]

[0087] 1-3. Enzyme-linked immunosorbent assay (ELISA)

[0088] NRK-49F was cultured in 96-well plates at 80% confluency for 18 hours, and then reacted with culture medium containing 0.5% FBS for 24 hours. Methixene was treated for 1 hour at various concentrations, and fibrosis was induced by reacting with transforming growth factor-beta (TGF-β) for 24 hours.

[0089]

[0090] 1-4. Measurement of PAI-1 promoter activity

[0091] NRK-49F was cultured in 12-well plates at 90% confluency for 18 hours. NRK-49F was transfected with the Plasminogen Activator Inhibitor-1 Luciferase reporter plasmid (PAI-1 Luciferase reporter plasmid) and the beta-galactosidase plasmid (β-galactosidase plasmid) using Lipofectamine 2000 (Thermo Fisher Scientific, USA). The transfected NRK-49F was pretreated with methixene at various concentrations for 1 hour, and then reacted with TGF-β for 24 hours to induce fibrosis. The dissolved NRK-49F was reacted with the substrate luciferin to confirm luciferase activity, and beta-galactosidase (β-galactosidase) was measured to correct the luciferase activity.

[0092]

[0093] 1-5. Western blot

[0094] NRK-49F was cultured in a 6-well plate at 80% density for 18 hours, and then treated with culture medium containing 0.5% FBS for 24 hours. Methixene was treated at various concentrations, and after 1 hour of culture, TGF-β was treated to induce fibrosis. The protein samples of the cells were electrophoresed, and Western blotting was performed with antibodies against alpha-smooth muscle actin (α-SMA), phospho-Smad3 (p-Smad3), and Smad3 purchased from Cellsignaling, USA, PAI-1, Collagen Type 1, and Fibronectin purchased from Abcam, UK, and beta-actin (β-actin) purchased from Santa Cruz, USA. The Western blotting was performed with ChemiDocTMXRS, a chemiluminescent material from Bio-Rad Laboratories, USA. + Protein expression was confirmed by reaction with and the expression concentration of Western blot was analyzed using Image J, a Java-based image processing program of the National Institutes of Health (NIH).

[0095]

[0096] 1-6. Quantitative real-time polymerase chain reaction (QRT)

[0097] Ribonucleic acid (RNA) extracted from NRK-49F treated with methixene was used to synthesize complementary deoxyribonucleic acid (cDNA) using a reverse transcription kit from Applied Biosystems, USA. The expression levels of messenger ribonucleic acid (mRNA) of PAI-1, Collagen Type 1, Fibronectin, α-SMA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the cells were measured using an ABI 7500 Quantitative-Polymerase Chain Reaction System (qPCR system).

[0098]

[0099] Example 2: Confirmation of toxicity following administration of Methixene

[0100] After culturing NRK-49F at 80% density in a 96-well plate for 18 hours, methixene was treated at concentrations of 0 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, and 10 μM, and the cell viability was confirmed accordingly, and the results are shown in Figure 1.

[0101] As can be seen in Fig. 1, when cells were treated with methixene at doses of 0 μM, 0.5 μM, 1 μM, and 2 μM, it was confirmed that cell viability was maintained without cytotoxicity, and when cells were treated with methixene at doses of 5 μM and 10 μM, it was confirmed that cell viability decreased by 18% and 21%, respectively. Accordingly, cells were treated with methixene at a concentration of 5 μM or less that did not decrease cell viability by more than 20%.

[0102]

[0103] Example 3: Evaluation of gene and protein expression and secretion of PAI-1 following administration of Methixene

[0104] NRK-49F was treated with methyxene at concentrations of 1 μM, 2 μM, and 5 μM, cDNA was synthesized from the RNA extracted therefrom, and the PAI-1 mRNA expression level was confirmed, and the results are shown in Figure 2A.

[0105] As can be confirmed in Fig. 2A, the PAI-1 mRNA expression level was confirmed to increase by the reaction of NRK-49F and TGF-β. When the cells were treated with 1 μM of Methixene, it was confirmed that the level decreased by approximately 28% compared to the control group. When the cells were treated with 2 μM of Methixene, it was confirmed that the level decreased by approximately 50% compared to the control group. When the cells were treated with 5 μM of Methixene, it was confirmed that the level decreased by approximately 53% compared to the control group.

[0106] NRK-49F was cultured in 6-well plates at 80% confluency for 18 hours, and then cultured for 24 hours in a medium containing 0.5% FBS. Afterwards, NRK-49F reacted with FBS was treated with methixene at concentrations of 1 μM, 2 μM, and 5 μM, and reacted with TGF-β to induce fibrosis. Afterwards, protein samples of the dissolved NRK-49F were subjected to electrophoresis, reacted with PAI-1 and β-actin antibodies, and Western blotting was performed to confirm the level of protein expression, and the results are shown in Fig. 2B.

[0107] As can be seen in Fig. 2B, the PAI-1 protein expression level was confirmed to increase by the reaction between NRK-49F and TGF-β. When the cells were treated with 1 μM of Methixene, a decrease of approximately 2% was confirmed compared to the control group, and when the cells were treated with 2 μM, a decrease of approximately 30% was confirmed compared to the control group. When the cells were treated with 5 μM, a decrease of approximately 46% was confirmed compared to the control group.

[0108] NRK-49F cells were treated with methyxene at concentrations of 1 μM, 2 μM, and 5 μM, and then reacted with TGF-β to induce fibrosis. To confirm the secretion of PAI-1, cell culture fluid was obtained and measured using PAI-1 ELISA, and the results are shown in Figure 2C.

[0109] As can be confirmed in Fig. 2C, the secretion of PAI-1 was confirmed to increase by the reaction of NRK-49F and TGF-β. When the cells were treated with 1 μM and 2 μM of Methixene, the inhibition of PAI-1 secretion was confirmed to decrease by approximately 32% compared to the control group, and when the cells were treated with 5 μM of Methixene, the inhibition of PAI-1 secretion was confirmed to decrease by approximately 39% compared to the control group.

[0110] Accordingly, it was confirmed that treatment with Methixene affects the mRNA and protein expression levels of PAI-1 and the secretion of PAI-1, and that Methixene is effective in preventing and treating renal fibrosis through the significant decrease in the mRNA and protein expression levels and secretion of PAI-1, which are significantly increased in the process of renal fibrosis.

[0111]

[0112] Example 4: Evaluation of PAI-1 promoter activity following metichen administration

[0113] After culturing NRK-49F cells at 90% confluency in 12-well plates for 18 hours, they were transfected with the PAI-1 luciferase reporter plasmid and β-galactosidase plasmid using Lipofectamine 2000. The transfected NRK-49F cells were treated with methixene at concentrations of 2 μM and 5 μM, and fibrosis was induced by reaction with TGF-β. Luciferase activity was then confirmed by reaction with luciferin, and the results are shown in Figure 3.

[0114] As can be confirmed in Fig. 3, it was confirmed that PAI-1 promoter activity increased by the reaction of NRK-49F and TGF-β. When the cells were treated with 2 μM of methixene, it was confirmed that PAI-1 promoter activity was suppressed by approximately 42% compared to the control group, and when the cells were treated with 5 μM of methixene, it was confirmed that PAI-1 promoter activity was suppressed by approximately 47%.

[0115] Accordingly, it was confirmed that treatment with Methixene affects PAI-1 promoter activity, and that Methixene is effective in preventing and treating renal fibrosis through a significant decrease in PAI-1 promoter activity, the secretion of which is significantly increased during the renal fibrosis process.

[0116]

[0117] Example 5: Evaluation of gene expression of renal fibrosis factors following administration of Methixene

[0118] NRK-49F was treated with methyxene at concentrations of 1 μM, 2 μM, and 5 μM, and cDNA was synthesized from the RNA extracted therefrom, and the expression levels of Collagen Type 1, Fibronectin, and α-SMA mRNA were confirmed. The results are shown in Figures 4A, 4B, and 4C.

[0119] As can be confirmed in Fig. 4a, the expression level of Collagen Type 1 mRNA was confirmed to increase by the reaction of NRK-49F and TGF-β. When the cells were treated with 1 μM of Methixene, the expression level of Collagen Type 1 mRNA was confirmed to be suppressed by about 14% compared to the control group, when the cells were treated with 2 μM of Methixene, the expression level of Collagen Type 1 mRNA was confirmed to be suppressed by about 36% compared to the control group, and when the cells were treated with 5 μM of Methixene, the expression level of Collagen Type 1 mRNA was confirmed to be suppressed by about 44% compared to the control group.

[0120] As can be confirmed in Fig. 4B, the Fibronectin mRNA expression level was confirmed to increase by the reaction of NRK-49F and TGF-β. When the cells were treated with 1 μM of Methixene, the Fibronectin mRNA expression level was confirmed to be suppressed by about 2% compared to the control group, when the cells were treated with 2 μM of Methixene, the Fibronectin mRNA expression level was confirmed to be suppressed by about 16% compared to the control group, and when the cells were treated with 5 μM of Methixene, the Fibronectin mRNA expression level was confirmed to be suppressed by about 22% compared to the control group.

[0121] As can be confirmed in Fig. 4C, the α-SMA mRNA expression level was confirmed to increase by the reaction of NRK-49F and TGF-β. When the cells were treated with 1 μM of Methixene, the α-SMA mRNA expression level was confirmed to be suppressed by about 11% compared to the control group, when the cells were treated with 2 μM of Methixene, the α-SMA mRNA expression level was confirmed to be suppressed by about 46% compared to the control group, and when the cells were treated with 5 μM of Methixene, the α-SMA mRNA expression level was confirmed to be suppressed by about 70% compared to the control group.

[0122] Accordingly, it was confirmed that treatment with Methixen affected the expression levels of Collagen Type 1, Fibronectin, and α-SMA mRNA, and that Methixen was effective in preventing and treating renal fibrosis through a significant decrease in the expression levels of Collagen Type 1, Fibronectin, and α-SMA mRNA, whose secretion is significantly increased during the renal fibrosis process.

[0123]

[0124] Example 6: Evaluation of protein expression of renal fibrosis factors following administration of Methixene

[0125] After culturing NRK-49F at 80% confluency in 6-well plates for 18 hours, the cells were treated with culture medium containing 0.5% FBS and allowed to react. The NRK-49F reacted with FBS was treated with methixene at concentrations of 1 μM, 2 μM, and 5 μM, and allowed to react with TGF-β to induce fibrosis. Afterwards, the protein sample of the dissolved NRK-49F was subjected to electrophoresis and reacted with Collagen Type 1, Fibronectin, α-SMA, and β-actin antibodies to perform Western blotting, and the protein expression level was confirmed, and the results are shown in Figures 5A, 5B, 5C, and 5D.

[0126] As shown in Figure 5A, the expression levels of Collagen type 1, Fibronectin, and α-SMA proteins increased in response to the reaction between NRK-49F and TGF-β. Subsequently, when the cells were treated with 2 μM and 5 μM of Methixene, the expression levels of Collagen type 1, Fibronectin, and α-SMA proteins decreased compared to the control group.

[0127] As can be confirmed in Figure 5B, the expression level of Collagen type 1 protein was confirmed to increase by the reaction of NRK-49F and TGF-β. When the cells were treated with 2 μM of Methixene, the expression level of Collagen type 1 protein was confirmed to be suppressed by approximately 11% compared to the control group, and when the cells were treated with 5 μM of Methixene, the expression level of Collagen type 1 protein was confirmed to be suppressed by approximately 20% compared to the control group.

[0128] As shown in Figure 5C, the expression level of Fibronectin protein increased due to the reaction between NRK-49F and TGF-β. When the cells were treated with 2 μM of Methixene, the expression level of Fibronectin protein was suppressed by approximately 23% compared to the control group. When the cells were treated with 5 μM of Methixene, the expression level of Fibronectin protein was suppressed by approximately 42% compared to the control group.

[0129] As can be confirmed in Figure 5D, the α-SMA protein expression level was confirmed to increase by the reaction between NRK-49F and TGF-β. When the cells were treated with 2 μM of Methixene, the α-SMA protein expression level was confirmed to be suppressed by approximately 7% compared to the control group, and when the cells were treated with 5 μM of Methixene, the α-SMA protein expression level was confirmed to be suppressed by approximately 51% compared to the control group.

[0130] Accordingly, it was confirmed that treatment with Methixen affected the expression levels of Collagen type 1, Fibronectin, and α-SMA proteins, and that Methixen was effective in preventing and treating renal fibrosis through a significant decrease in the expression levels of Collagen type 1, Fibronectin, and α-SMA proteins, whose secretion significantly increases during the renal fibrosis process.

[0131]

[0132] Example 7: Evaluation of SMAD activity

[0133] After culturing NRK-49F at 80% confluency in 6-well plates for 18 hours, the cells were treated with culture medium containing 0.5% FBS and allowed to react. The NRK-49F cells reacted with FBS were treated with methixene at concentrations of 1 μM, 2 μM, and 5 μM, and allowed to react with TGF-β to induce fibrosis. Afterwards, the protein samples of the dissolved NRK-49F were subjected to electrophoresis and reacted with p-Smad3 and Smad3 to perform Western blotting, and the protein expression levels were confirmed, and the results are shown in Fig. 6.

[0134] As shown in Fig. 6, the Smad3 signaling pathway was activated by the reaction of NRK-49F and TGF-β, which confirmed that the expression level of p-Smad3 increased. Afterwards, when the cells were treated with 2 μM of Methixene, it was confirmed that the inhibition was about 10% compared to the control group, and when the cells were treated with 5 μM of Methixene, it was confirmed that the inhibition was about 20% compared to the control group. Accordingly, it was confirmed that Methixene treatment inhibited the activation of the Smad3 signaling pathway, significantly reducing the expression level of p-Smad3, whose secretion is significantly increased during the renal fibrosis process, and thus Methixene is effective in the prevention and treatment of renal fibrosis.

[0135] The present invention relates to a composition for treating kidney disease comprising methixene or a pharmaceutically acceptable salt thereof, and more particularly, to methixene that suppresses the gene or protein expression level of a renal fibrosis factor.

Claims

1. Metixene represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof; A pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, comprising: [Chemical Formula 1] 2. In paragraph 1, A pharmaceutical composition for treating, alleviating, suppressing or preventing a kidney disease, wherein the kidney disease is a kidney fibrosis disease.

3. In paragraph 2, A pharmaceutical composition for treating, alleviating, suppressing or preventing renal disease, wherein the renal fibrotic disease is at least one disease selected from the group consisting of renal failure, glomerulonephritis, pyelonephritis and renal cancer.

4. In paragraph 1, The above-mentioned Methixene is a pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, which suppresses the gene or protein expression level of a renal fibrosis factor.

5. In paragraph 4, A pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, wherein the renal fibrosis factor is one of Plasminogen Activator Inhibitor-1 (PAI-1), Collagen Type 1, Fibronectin, Alpha-Smooth Muscle Actin (α-SMA), phospho-Smad3 (p-Smad3), and Smad3.

6. In paragraph 1, A pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, wherein the pharmaceutically acceptable salt of the above metixene is metixene hydrochloride hydrate.

7. In paragraph 1, A pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, wherein the composition further comprises a pharmaceutically acceptable additive.

8. In paragraph 1, A pharmaceutical composition for treating, alleviating, suppressing or preventing kidney disease, wherein the composition is for oral administration.

9. Metixene represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof; A food composition for alleviating, suppressing or improving kidney disease, comprising: [Chemical Formula 1] 10. In paragraph 9, A food composition for alleviating, suppressing or improving kidney disease, wherein the kidney disease is a kidney fibrosis disease.

11. In paragraph 10, A food composition for alleviating, suppressing or improving renal disease, wherein the renal fibrotic disease is at least one disease selected from the group consisting of renal failure, glomerulonephritis, pyelonephritis and renal cancer.

12. In paragraph 10, The above-mentioned metichen is a food composition for alleviating, suppressing or improving renal disease, which suppresses the gene or protein expression level of a renal fibrosis factor.

13. In paragraph 12, A food composition for alleviating, suppressing or improving renal disease, wherein the renal fibrosis factor is one of Plasminogen Activator Inhibitor-1 (PAI-1), Collagen Type 1, Fibronectin, Alpha-Smooth Muscle Actin (α-SMA), p-Smad3, and Smad3.

14. In paragraph 9, A food composition for alleviating, suppressing or improving kidney disease, wherein the pharmaceutically acceptable salt of the above metixene is metixene hydrochloride hydrate.

Images