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WO2024058527A1 - Pharmaceutical composition for preventing or treating hearing loss comprising esomeprazole or dexlansoprazole as active ingredient - Google Patents

Pharmaceutical composition for preventing or treating hearing loss comprising esomeprazole or dexlansoprazole as active ingredient Download PDF

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Publication number
WO2024058527A1
WO2024058527A1 PCT/KR2023/013653 KR2023013653W WO2024058527A1 WO 2024058527 A1 WO2024058527 A1 WO 2024058527A1 KR 2023013653 W KR2023013653 W KR 2023013653W WO 2024058527 A1 WO2024058527 A1 WO 2024058527A1
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Prior art keywords
hearing loss
esomeprazole
dexlansoprazole
pharmaceutical composition
cisplatin
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PCT/KR2023/013653
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French (fr)
Korean (ko)
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최준
이인균
허우영
전한울
Original Assignee
고려대학교 산학협력단
한국과학기술연구원
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Priority claimed from KR1020230119511A external-priority patent/KR20240036481A/en
Application filed by 고려대학교 산학협력단, 한국과학기술연구원 filed Critical 고려대학교 산학협력단
Publication of WO2024058527A1 publication Critical patent/WO2024058527A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • cisplatin cis-diamminedichloroplatinum II
  • cisplatin cis-diamminedichloroplatinum II
  • Cisplatin enters cells by diffusion or a transporter and is hydrated, thereby lowering the chloride concentration inside the cell and releasing chloride ions.
  • the hydrated form of cisplatin cross-links with nucleotides in nuclear and mitochondrial DNA to form adducts, inhibiting the unrestricted replication of cancer cells.
  • cisplatin causes nephrotoxicity, neurotoxicity, and ototoxicity as side effects in normal tissues.
  • ototoxicity induced by cisplatin can cause irreversible sensorineural hearing loss, or hearing loss, and is a more serious problem, especially in the pediatric population.
  • Hearing loss can greatly impair a patient's quality of life. In particular, it can have a significant impact on social development such as language acquisition and speech in pediatric patients.
  • cisplatin inhibits the death receptor pathway, endoplasmic reticulum-stress pathway, and mitochondrial reactive oxygen species in normal cells. It has been shown to activate the mitochondrial reactive oxygen species (ROS)-generating pathway, which ultimately leads to cell death.
  • ROS mitochondrial reactive oxygen species
  • Excessive ROS production is believed to be the main cause of cisplatin-induced ototoxic hearing loss, and is also considered to be a direct attack of cisplatin on DNA.
  • the organ of Corti, spiral ganglion and lateral wall within the cochlea have been suggested to be key targets of cisplatin for potent ROS generation.
  • the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • Another object of the present invention is to provide a health functional food composition for preventing or improving hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • Another object of the present invention is to provide a reagent composition for inhibiting cisplatin uptake of OCT2 (Organ Cation Transporter 2) protein in hair cells containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • OCT2 Organic Transporter 2
  • Another object of the present invention is one or more proton pump inhibitors selected from Esomeprazole or Dexlansoprazole; and a drug selected from anticancer drugs or aminoglucoside antibiotics, wherein the proton pump inhibitor inhibits the drug absorption by OCT2 (Organ Cation Transporter 2) protein in hair cells and improves hair cell survival.
  • OCT2 Organic Cation Transporter 2
  • the present invention provides a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • the present invention provides a health functional food composition for preventing or improving hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • the present invention provides a reagent composition for inhibiting cisplatin uptake of OCT2 (Organ Cation Transporter 2) protein in hair cells containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • OCT2 Organic Transporter 2
  • the present invention relates to one or more proton pump inhibitors selected from Esomeprazole or Dexlansoprazole; and a drug selected from anticancer drugs or aminoglucoside antibiotics, wherein the proton pump inhibitor inhibits the drug absorption by OCT2 (Organ Cation Transporter 2) protein in hair cells and improves hair cell survival.
  • OCT2 Organic Cation Transporter 2
  • a pharmaceutical composition containing Esomeprazole or Dexlansoprazole as an active ingredient is It can be used to prevent and treat hearing loss, a side effect of using the anticancer drug cisplatin.
  • Figure 1 shows the results of analyzing the cytotoxicity of Cisplatin in auditory cells. ns; not significant, ****p ⁇ 0.0001.
  • Figure 2 shows the results of a compound screening analysis to identify substances showing a cytoprotective effect against cytotoxicity caused by cisplatin in auditory cells.
  • Figure 3 shows the results of analyzing the cytoprotective effect against cytotoxicity caused by cisplatin in auditory cells of the eight primary active substances identified through the above compound screening. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 4 shows the results of analyzing the cytoprotective effect of Esomeprazole and Dexlansoprazole against cytotoxicity caused by cisplatin in auditory cells. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figures 5 and 6 show the results of analyzing the cytoprotective effect of esomeprazole and dexlansoprazole against apoptosis caused by cisplatin in auditory cells.
  • Figure 7 shows the results of analyzing the effect of esomeprazole and dexlansoprazole on OCT2 (Organ Cation Transporter 2). ***p ⁇ 0.001
  • Figure 8 shows the results of analyzing the cytoprotective effect of esomeprazole against cell death caused by cisplatin in an animal model. ****p ⁇ 0.0001.
  • the present invention provides a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • the hearing loss may be selected from ototoxic hearing loss, sudden hearing loss, noise-induced hearing loss, or age-related hearing loss, but is not limited thereto.
  • the ototoxic hearing loss may be caused by drugs selected from anticancer drugs or aminoglucoside antibiotics.
  • the anticancer agent may be cisplatin or carboplatin, but is not limited thereto.
  • the aminoglucoside antibiotics include amikacin, arbekacin, kanamycin, gentamicin, neomycin, netilmicin, and dibekacin. , sisomycin, streptomycin, tobramycin, ribodomycin, and paromomycin, but is not limited thereto.
  • Esomeprazole or Dexlansoprazole can inhibit the uptake of cisplatin by OCT2 (Organ Cation Transporter 2) protein in hair cells.
  • OCT2 Organic Transporter 2
  • the pharmaceutical composition may contain suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, etc. commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders, and lubricants.
  • carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline.
  • Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used.
  • Solid preparations for oral administration include tablets, pills, powders, granules, and capsules.
  • Such solid preparations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium styrate and talc can also be used.
  • Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • injectable ester such as ethyl oleate.
  • As a base for suppositories witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.
  • the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or It can be administered to a subject in a conventional manner via the intradermal route.
  • the dosage of the active ingredient according to the present invention may vary depending on the subject's condition and weight, type and degree of disease, drug form, administration route and period, and may be appropriately selected by a person skilled in the art, and the daily dosage is 0.01 mg.
  • Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.
  • the present invention provides a health functional food composition for preventing or improving hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • the health functional food includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable drinks. These ingredients can be used independently or in combination.
  • the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverages, tea, functional water, drink, alcohol, and vitamin complex. It can be.
  • the above-mentioned health functional food may additionally contain food additives, and its suitability as a “food additive” is determined according to the general provisions and general test methods of the Food Additives Code approved by the Food and Drug Administration, unless otherwise specified. Determination is made according to relevant standards and standards.
  • Items listed in the "Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as subchromic pigment, licorice extract, crystalline cellulose, cold pigment, and guar gum, L -Mixed preparations such as sodium glutamate preparations, noodle-added alkaline preparations, preservative preparations, and tar color preparations are included.
  • the content of the active ingredients added to the food can be appropriately adjusted as needed, and is preferably added in an amount of 1 to 90 parts by weight per 100 parts by weight of the food. .
  • the present invention provides a reagent composition for inhibiting cisplatin uptake of OCT2 (Organ Cation Transporter 2) protein in hair cells containing Esomeprazole or Dexlansoprazole as an active ingredient.
  • OCT2 Organic Transporter 2
  • the present invention relates to one or more proton pump inhibitors selected from Esomeprazole or Dexlansoprazole; and a drug selected from anticancer drugs or aminoglucoside antibiotics, wherein the proton pump inhibitor inhibits the drug absorption by OCT2 (Organ Cation Transporter 2) protein in hair cells and improves hair cell survival.
  • OCT2 Organic Cation Transporter 2
  • HEI-OC1 cells House Ear Institute, Los Angeles, CA, USA
  • DMEM Dulbecco modified eagle medium, Welgene, Korea
  • IFN- ⁇ interferon gamma, Peprotech, USA
  • HEK293T cells CL-1573; American Type Culture Collection, Rockville, MD
  • a cell line derived from human embryonic kidney were grown in DMEM medium supplemented with 1% penicillin-streptomycin solution (Welgene) and 8% FBS. , cultured at 37°C and 5% CO 2 conditions.
  • CIS a protective effect against cytotoxicity by cisplatin
  • 923 FDA-approved drugs purchased from Prestwick Chemical. 923 drugs were evenly distributed in 100 ⁇ L culture medium of a 96-well plate, and cells cultured for 18 hours were treated with 0.5 ⁇ L of each drug so that the final drug concentration was 50 ⁇ M. After 2 hours, 20 ⁇ M CIS was added.
  • a well containing only DMSO (Dimethyl sulfoxide) was set as a negative control, and a well containing only 2mM erdosteine (erdosteine, Sigma-Aldrich, St.
  • HEI-OC1 cells were evenly distributed at 100 ⁇ L each at 1,000 cells/well using medium without IFN- ⁇ in a 96-well plate.
  • the distributed HEI-OC1 cells were cultured at 33°C and 10% CO 2 for 16 hours before being treated with the effective substance of Experimental Example 2.
  • the cultured HEI-CO1 cells were first treated with the active substance at different concentrations and cultured for 2 hours. Then, to induce cytotoxicity, the medium was replaced with 20 ⁇ M CIS and cultured for 48 hours. The control group was treated with DMSO without treatment with the active substance and then cultured under the same conditions.
  • Cell viability was measured by measuring ATP levels using the CellTiter-Glo assay (Promega Corp., Madison, WI, USA). 50 ⁇ L of ATP detection solution was added to each well of the plate containing cells in 100 ⁇ L of culture medium, reacted in the dark at room temperature for 10 minutes, and then luminescence was measured using an automated EnVision plate reader (Perkin-Elmer, Waltham, MA, USA). did. The experiment was repeated three times independently, and the results were calculated from six independent wells.
  • V-FITC fluorescein isothiocyanate
  • PI propidium iodide
  • HEK293T cells were transiently transfected with a plasmid expressing human organic cation transporter (hOCT2).
  • hOCT2 human organic cation transporter
  • One day before transfection HEK293T cells were subcultured at a density of 1 ⁇ 10 6 for 24 hours to reach 70-80% cell confluency. It was transfected with polyethylenimine (PEI) and hOCT2-expressing plasmid.
  • PEI polyethylenimine
  • HEK293T cells transfected using an empty vector that does not express hOCT were set as a control group.
  • the transfected HEK293T cells were evenly distributed at 100,000 cells/well in black poly L-lysine-coated 96-well plates (Greiner Bio-One, Frickenhausen, Germany) and cultured for 18 hours. Afterwards, the cells were washed twice with assay buffer (Hanks' Balanced Salt SolutionL; HBSS) (containing 5.6mM D-glucose, pH 7.4) stored at room temperature, and the active substance (esomeprazole or dexlansoprazole) was added by concentration ( 0.0001 ⁇ 1000 ⁇ M) and reacted for 5 minutes.
  • assay buffer Horte' Balanced Salt SolutionL; HBSS
  • active substance esomeprazole or dexlansoprazole
  • Fluorescence uptake assay was started by adding uptake buffer containing 10 ⁇ M fluorescent substrate organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+). After reacting at room temperature for 3 minutes, the reaction mixture was aspirated and washed twice with assay buffer containing 500 ⁇ M cimetidine to terminate the reaction. Afterwards, 100 ⁇ L RIPA buffer was added and the cells were lysed at room temperature for 5 minutes. ASP+fluorescence was measured at 485 nm and 500 nm for excitation and emission, respectively, using an EnVision plate reader (Perkin-Elmer).
  • ASP+fluorescence was measured at 485 nm and 500 nm for excitation and emission, respectively, using an EnVision plate reader (Perkin-Elmer).
  • Transgenic zebrafish (Brn3C:EGFP) embryos were obtained through mating of adult zebrafish maintained at 28.5°C at the zebrafish facility at Korea University Ansan Hospital.
  • Naturally occurring green neuromasts from transgenic zebrafish (Brn3C:EGFP) which can be viewed without staining using a fluorescence microscope, were used to monitor ototoxicity.
  • Five days after fertilization, transgenic zebrafish embryos were fed live paramecia and plant-based food.
  • Embryos were maintained in embryo medium (1mM MgSO 4 , 120 ⁇ M KH 2 PO 4 , 74 ⁇ M Na 2 HPO 4 , 1mM CaCl 2 , 500 ⁇ M KCl, 15 ⁇ M NaCl, and 500 ⁇ M NaHCO 3 ) at a density of approximately 50 embryos per 100mm 2 Petri dish.
  • This animal experiment was conducted with approval from Korea University's Animal Experiment Practice Committee (approval number: KOREA-2022-0097). All protocols were performed in accordance with the guidelines of the Korea University Medical Center Animal Care and Ethics Committee and the National Institutes of Health.
  • CIS solution was prepared by adding pure CIS to embryo medium. At 5 days after zebrafish fertilization, 27 zebrafish embryos were incubated in solvent control (EtOH 1.36%), 1000 ⁇ M CIS (Sigma-Aldrich) and esomeprazole (Aldrich, Milwaukee, WI, USA) (2, 20 and 200 ⁇ M) for 4 days. exposed for a period of time. Afterwards, the embryos were washed three times with embryonic medium and anesthetized with tricaine (MS-222, Sigma-Aldrich, Darmstadt, Germany, A5040) for 5 minutes.
  • solvent control EtOH 1.36%
  • 1000 ⁇ M CIS Sigma-Aldrich
  • esomeprazole Aldrich, Milwaukee, WI, USA
  • Cells within the neurosphere of zebrafish were identified using an in-situ cell detection kit (Roche Molecular Biochemicals, Mannheim, Germany) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method (according to the manufacturer's instructions). Death was analyzed. Embryos were exposed to medium containing 1000 ⁇ M CIS and 200 ⁇ M esomeprazole for 4 h. Afterwards, the embryos were washed with PBS and fixed with 4% paraformaldehyde.
  • embryos were incubated in 50 mL of TUNEL reaction mixture (TdT and fluorescein-dUTP) at 37°C for 60 min. Zebrafish were analyzed using an AxioCam MRc5 fluorescence microscope (Nikon, Spinning Disc Confocal Microscope).
  • the primary effective substance was administered to HEI-OC1 cells at different concentrations (8, 20, and 100 ⁇ M).
  • all eight active substances showed significant cell protection effects at all treatment concentrations, of which five active substances (esomeprazole, dexlansoprazole, emtricitabine, metoprolol, and cimetidine) ) showed a dose-dependent cytoprotective effect.
  • the cell protection effect according to the dose was analyzed by increasing the treatment concentration for the most effective esomeprazole and dexlansoprazole.
  • TUNEL was positive in the group treated only with CIS (1000 ⁇ M) compared to the control group. While the number of cells (red) significantly increased, the number of TUNEL-positive cells (red) significantly decreased in the esomeprazole (200 ⁇ M) treated group compared to the CIS only treated group. From the above results, it was confirmed that esomeprazole inhibits apoptosis caused by CIS in zebrafish hair cells.

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Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hearing loss comprising esomeprazole or dexlansoprazole as an active ingredient. As the effects of esomeprazole or dexlansoprazole on protecting hair cells from apoptosis caused by cisplatin and inhibiting the uptake of cisplatin by organ cation transporter 2 (OCT2) protein from hair cells, were confirmed, a pharmaceutical composition comprising same as an active ingredient can be used to prevent and treat hearing loss that is a side effect of using cisplatin, which is an anticancer drug.

Description

에스오메프라졸 또는 덱스란소프라졸을 유효성분으로 포함하는 난청 예방 또는 치료용 약학 조성물Pharmaceutical composition for preventing or treating hearing loss containing esomeprazole or dexlansoprazole as an active ingredient
본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
1960년대에 항암 효과가 발견된 이후 시스플라틴(cis-diamminedichloroplatinum II)은 난소, 폐, 방광 등에서 발견되는 고형 종양에 유효한 화학 요법 약물로 널리 사용되었다.Since its anticancer effect was discovered in the 1960s, cisplatin (cis-diamminedichloroplatinum II) has been widely used as an effective chemotherapy drug for solid tumors found in the ovaries, lungs, bladder, etc.
시스플라틴(cisplatin)은 확산(diffusion) 또는 운반체(transporter)에 의해 세포로 들어가고 수화(hydrate)되어 세포 내부의 염화 농도(chloride ions)를 낮추기 때문에 염화 이온(chloride ions)을 방출하게 한다. 수화된 형태의 시스플라틴은 핵과 미토콘드리아 DNA의 뉴클레오타이드와 교차결합(cross-links)하여 부가물(adducts)을 형성하여 암세포의 무제한적인 복제를 억제시킨다.Cisplatin enters cells by diffusion or a transporter and is hydrated, thereby lowering the chloride concentration inside the cell and releasing chloride ions. The hydrated form of cisplatin cross-links with nucleotides in nuclear and mitochondrial DNA to form adducts, inhibiting the unrestricted replication of cancer cells.
그러나 시스플라틴을 이용한 임상 치료는 정상 조직의 부작용으로서 신독성(nephrotoxicity), 신경독성(neurotoxicity) 및 이독성(ototoxicity)을 유발하기 때문에 사용에 제한이 있다. 특히 시스플라틴에 의해 유발된 이독성(ototoxicity)은 돌이킬 수 없는 감각신경성 청력손실, 즉 난청(hearing loss)을 유발시킬 수 있으며, 특히 소아 집단에서 더욱 심각한 문제가 된다.However, clinical treatment using cisplatin has limitations because it causes nephrotoxicity, neurotoxicity, and ototoxicity as side effects in normal tissues. In particular, ototoxicity induced by cisplatin can cause irreversible sensorineural hearing loss, or hearing loss, and is a more serious problem, especially in the pediatric population.
난청(hearing loss)은 환자의 삶의 질을 크게 저해시킬 수 있다. 특히 언어 습득 및 소아 환자의 말하기와 같은 사회 발전에 상당한 영향을 줄 수 있다. 비록 시스플라틴의 이독성을 일으키는 근원적인 분자 기전이 완전하게 밝혀지지 않았지만, 이전 연구에서는 시스플라틴이 정상 세포에서 사멸 수용체 경로(death receptor pathway), 소포체 스트레스 경로(endoplasmic reticulum-stress pathway) 및 미토콘드리아 활성 산소종(ROS) 생성 경로(mitochondrial reactive oxygen species(ROS)-generating pathway)를 활성화시킴을 보여 주었고, 결국 세포 죽음으로 이어짐을 밝힌 바 있다. 과도한 ROS의 생성은 시스플라틴으로부터 유도된 이독성 난청의 주요 원인으로 여겨지며, 또한 이는 DNA에 대한 시스플라틴의 직접적인 공격으로 간주된다. 특히 달팽이관(cochlea) 내의 코르티(corti) 기관, 나선형 신경절(spiral ganglion) 및 측벽(lateral wall)은 강력한 ROS 생성을 위한 시스플라틴의 주요 표적이라고 제안되어 왔다.Hearing loss can greatly impair a patient's quality of life. In particular, it can have a significant impact on social development such as language acquisition and speech in pediatric patients. Although the underlying molecular mechanism causing cisplatin's ototoxicity has not been completely elucidated, previous studies have shown that cisplatin inhibits the death receptor pathway, endoplasmic reticulum-stress pathway, and mitochondrial reactive oxygen species in normal cells. It has been shown to activate the mitochondrial reactive oxygen species (ROS)-generating pathway, which ultimately leads to cell death. Excessive ROS production is believed to be the main cause of cisplatin-induced ototoxic hearing loss, and is also considered to be a direct attack of cisplatin on DNA. In particular, the organ of Corti, spiral ganglion and lateral wall within the cochlea have been suggested to be key targets of cisplatin for potent ROS generation.
현재까지 개발되어 임상시험이 진행 중인 이독성 난청 보호 약물 대부분은 시스플라틴 약물의 활성산소 차단을 기전으로 하고 있다. 활성산소 차단 기전의 약물은 강력한 친핵체를 가지고 있어 시스플라틴과 착화합물을 형성하여 활성산소의 생성을 억제하는 효능을 가지고 있으나 시스플라틴의 항암 효과 또한 이로 인하여 저하되며, 비특이적 기작으로 인하여 그 효과가 미미하다는 근본적 한계를 가지고 있다. 따라서, 시스플라틴의 항암 효과가 저해되지 않고 이독성 난청에 대한 치료효과가 있는 치료제의 개발이 필요한 실정이다.Most of the ototoxic hearing loss protection drugs that have been developed to date and are undergoing clinical trials are based on the mechanism of cisplatin drug blocking free radicals. Drugs with an active oxygen blocking mechanism have a strong nucleophile and form a complex with cisplatin, which has the effect of inhibiting the production of free radicals. However, the anticancer effect of cisplatin is also reduced due to this, and the fundamental limitation is that the effect is minimal due to the non-specific mechanism. has. Therefore, there is a need to develop a treatment that has a therapeutic effect for ototoxic hearing loss without inhibiting the anti-cancer effect of cisplatin.
본 발명의 목적은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 치료용 약학 조성물을 제공하는 데에 있다.The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
본 발명의 다른 목적은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 개선용 건강기능식품 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a health functional food composition for preventing or improving hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
본 발명의 또 다른 목적은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 시스플라틴 흡수 저해용 시약 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a reagent composition for inhibiting cisplatin uptake of OCT2 (Organ Cation Transporter 2) protein in hair cells containing Esomeprazole or Dexlansoprazole as an active ingredient.
본 발명의 또 다른 목적은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)에서 선택된 하나 이상의 프로톤 펌프 억제제; 및 항암제 또는 아미노글루코시드계 항생제에서 선택된 약물을 포함하며, 상기 프로톤 펌프 억제제가 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 상기 약물 흡수를 저해하고, 유모세포 생존율을 향상시키는 것을 특징으로 하는, 약제학적 복합 제제를 제공하는 데에 있다.Another object of the present invention is one or more proton pump inhibitors selected from Esomeprazole or Dexlansoprazole; and a drug selected from anticancer drugs or aminoglucoside antibiotics, wherein the proton pump inhibitor inhibits the drug absorption by OCT2 (Organ Cation Transporter 2) protein in hair cells and improves hair cell survival. The purpose is to provide pharmaceutical combination preparations.
상기 목적을 달성하기 위하여, 본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
또한, 본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
또한, 본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 시스플라틴 흡수 저해용 시약 조성물을 제공한다.Additionally, the present invention provides a reagent composition for inhibiting cisplatin uptake of OCT2 (Organ Cation Transporter 2) protein in hair cells containing Esomeprazole or Dexlansoprazole as an active ingredient.
또한, 본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)에서 선택된 하나 이상의 프로톤 펌프 억제제; 및 항암제 또는 아미노글루코시드계 항생제에서 선택된 약물을 포함하며, 상기 프로톤 펌프 억제제가 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 상기 약물 흡수를 저해하고, 유모세포 생존율을 향상시키는 것을 특징으로 하는, 약제학적 복합 제제를 제공한다.In addition, the present invention relates to one or more proton pump inhibitors selected from Esomeprazole or Dexlansoprazole; and a drug selected from anticancer drugs or aminoglucoside antibiotics, wherein the proton pump inhibitor inhibits the drug absorption by OCT2 (Organ Cation Transporter 2) protein in hair cells and improves hair cell survival. A pharmaceutical combination preparation is provided.
본 발명에 따르면, 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)이 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 시스플라틴의 흡수를 저해시키는 효과를 확인함에 따라, 이를 유효성분으로 포함하는 약학 조성물은 항암제인 시스플라틴을 사용함에 따른 부작용 난청을 예방 및 치료하는데 활용될 수 있다.According to the present invention, as the effect of Esomeprazole or Dexlansoprazole on inhibiting the absorption of cisplatin by OCT2 (Organ Cation Transporter 2) protein in hair cells was confirmed, a pharmaceutical composition containing Esomeprazole or Dexlansoprazole as an active ingredient is It can be used to prevent and treat hearing loss, a side effect of using the anticancer drug cisplatin.
도 1은 청각세포에서 시스플라틴(Cisplatin)의 세포독성을 분석한 결과이다. ns; not significant, ****p<0.0001.Figure 1 shows the results of analyzing the cytotoxicity of Cisplatin in auditory cells. ns; not significant, ****p<0.0001.
도 2는 청각세포에서 시스플라틴에 의한 세포독성에 대한 세포 보호 효과를 나타내는 물질을 확인하기 위해, 화합물 스크리닝 분석을 수행한 결과이다. Figure 2 shows the results of a compound screening analysis to identify substances showing a cytoprotective effect against cytotoxicity caused by cisplatin in auditory cells.
도 3은 상기 화합물 스크리닝을 통해 확인한 8종의 1차 유효물질의 청각세포에서 시스플라틴에 의한 세포독성에 대한 세포 보호 효과를 분석한 결과이다. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Figure 3 shows the results of analyzing the cytoprotective effect against cytotoxicity caused by cisplatin in auditory cells of the eight primary active substances identified through the above compound screening. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
도 4는 에스오메프라졸(Esomeprazole) 및 덱스란소프라졸(Dexlansoprazole)의 청각세포에서 시스플라틴에 의한 세포독성에 대한 세포 보호 효과를 분석한 결과이다. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Figure 4 shows the results of analyzing the cytoprotective effect of Esomeprazole and Dexlansoprazole against cytotoxicity caused by cisplatin in auditory cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
도 5 및 6은 에스오메프라졸 및 덱스란소프라졸의 청각세포에서 시스플라틴에 의한 세포사멸에 대한 세포 보호 효과를 분석한 결과이다.Figures 5 and 6 show the results of analyzing the cytoprotective effect of esomeprazole and dexlansoprazole against apoptosis caused by cisplatin in auditory cells.
도 7은 에스오메프라졸 및 덱스란소프라졸이 OCT2(Organ Cation Transpoter 2)에 미치는 영향을 분석한 결과이다. ***p<0.001Figure 7 shows the results of analyzing the effect of esomeprazole and dexlansoprazole on OCT2 (Organ Cation Transporter 2). ***p<0.001
도 8은 동물모델에서 에스오메프라졸의 시스플라틴에 의한 세포 사멸에 대한 세포 보호 효과를 분석한 결과이다. ****p<0.0001.Figure 8 shows the results of analyzing the cytoprotective effect of esomeprazole against cell death caused by cisplatin in an animal model. ****p<0.0001.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
상기 난청은 이독성 난청, 돌발성 난청, 소음성 난청 또는 노화성 난청에서 선택된 것일 수 있으나, 이에 한정되는 것은 아니다.The hearing loss may be selected from ototoxic hearing loss, sudden hearing loss, noise-induced hearing loss, or age-related hearing loss, but is not limited thereto.
상기 이독성 난청은 항암제 또는 아미노글루코시드계 항생제에서 선택된 약물에 의한 것일 수 있다.The ototoxic hearing loss may be caused by drugs selected from anticancer drugs or aminoglucoside antibiotics.
상기 항암제는 시스플라틴(cisplatin) 또는 카르보플라틴(carboplatin)인 것일 수 있으나, 이에 한정되는 것은 아니다.The anticancer agent may be cisplatin or carboplatin, but is not limited thereto.
상기 아미노글루코시드계 항생제는 아미카신(amikacin), 아르베카신(arbekacin), 카나마이신(kanamycin), 겐타마이신(gentamicin), 네오마이신(neomycin), 네틸마이신(netilmicin), 디베카신(dibekacin), 시소마이신(sisomycin), 스트렙토마이신(streptomycin), 토브라마이신(tobramycin), 리보도마이신(livodomycin) 및 파로모마이신(paromomycin)으로 구성된 군에서 선택될 수 있으나, 이에 한정되는 것은 아니다.The aminoglucoside antibiotics include amikacin, arbekacin, kanamycin, gentamicin, neomycin, netilmicin, and dibekacin. , sisomycin, streptomycin, tobramycin, ribodomycin, and paromomycin, but is not limited thereto.
상기 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)은 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 시스플라틴의 흡수를 저해시킬 수 있다.Esomeprazole or Dexlansoprazole can inhibit the uptake of cisplatin by OCT2 (Organ Cation Transporter 2) protein in hair cells.
본 발명의 다른 구체예에서, 약학 조성물은 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다. 구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 본 발명의 일실시예에 따르면, 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다. 본 발명에 따른 유효성분의 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있고, 1일 투여량이 0.01mg/kg 내지 200mg/kg, 바람직하게는 0.1mg/kg 내지 200mg/kg, 보다 바람직하게는 0.1mg/kg 내지 100mg/kg 일 수 있다. 투여는 하루에 한번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.In another embodiment of the present invention, the pharmaceutical composition may contain suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, etc. commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders, and lubricants. Specifically, carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agents, etc., and such solid preparations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used. According to one embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or It can be administered to a subject in a conventional manner via the intradermal route. The dosage of the active ingredient according to the present invention may vary depending on the subject's condition and weight, type and degree of disease, drug form, administration route and period, and may be appropriately selected by a person skilled in the art, and the daily dosage is 0.01 mg. /kg to 200mg/kg, preferably 0.1mg/kg to 200mg/kg, more preferably 0.1mg/kg to 100mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.
또한, 본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
상기 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일 주스, 합성 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 건강기능식품 조성물은 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알코올 및 비타민 복합제 중 어느 하나의 형태일 수 있다. 또한, 상기 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합 여부는 다른 규정이 없는 한 식품의약품안전청에 승인된 식품첨가물공전의 총칙 및 일반 시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. 상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀룰로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류 첨가 알칼리제, 보존료제제, 타르색소 제제 등의 혼합 제제류 등을 들 수 있다. 이때, 건강기능식품을 제조하는 과정에서 식품에 첨가되는 유효성분은 필요에 따라 그 함량을 적절히 가감할 수 있으며, 바람직하게는 식품 100 중량부에 1 중량부 내지 90 중량부 포함되도록 첨가될 수 있다.The health functional food includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable drinks. These ingredients can be used independently or in combination. In addition, the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverages, tea, functional water, drink, alcohol, and vitamin complex. It can be. In addition, the above-mentioned health functional food may additionally contain food additives, and its suitability as a “food additive” is determined according to the general provisions and general test methods of the Food Additives Code approved by the Food and Drug Administration, unless otherwise specified. Determination is made according to relevant standards and standards. Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as subchromic pigment, licorice extract, crystalline cellulose, cold pigment, and guar gum, L -Mixed preparations such as sodium glutamate preparations, noodle-added alkaline preparations, preservative preparations, and tar color preparations are included. At this time, in the process of manufacturing health functional foods, the content of the active ingredients added to the food can be appropriately adjusted as needed, and is preferably added in an amount of 1 to 90 parts by weight per 100 parts by weight of the food. .
또한, 본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 시스플라틴 흡수 저해용 시약조성물을 제공한다.In addition, the present invention provides a reagent composition for inhibiting cisplatin uptake of OCT2 (Organ Cation Transporter 2) protein in hair cells containing Esomeprazole or Dexlansoprazole as an active ingredient.
또한, 본 발명은 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)에서 선택된 하나 이상의 프로톤 펌프 억제제; 및 항암제 또는 아미노글루코시드계 항생제에서 선택된 약물을 포함하며, 상기 프로톤 펌프 억제제가 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 상기 약물 흡수를 저해하고, 유모세포 생존율을 향상시키는 것을 특징으로 하는, 약제학적 복합 제제를 제공한다.In addition, the present invention relates to one or more proton pump inhibitors selected from Esomeprazole or Dexlansoprazole; and a drug selected from anticancer drugs or aminoglucoside antibiotics, wherein the proton pump inhibitor inhibits the drug absorption by OCT2 (Organ Cation Transporter 2) protein in hair cells and improves hair cell survival. Provided is a pharmaceutical combination preparation.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.
[실험예 1] 세포 배양[Experimental Example 1] Cell culture
마우스 청각 세포주인 HEI-OC1 세포(House Ear Institute, Los Angeles, CA, USA)는 DMEM(Dulbecco modified eagle medium, Welgene, Korea)에 항생제 첨가 없이 5% FBS(Fetal bovine serum, Welgene) 및 50U/mL IFN-γ(interferon gamma, Peprotech, USA)를 첨가하고, 33℃ 및 10% CO2 조건에서 배양하였다. 인간 배아 신장에서 유래한 세포주인 HEK293T 세포(CRL-1573; American Type Culture Collection, Rockville, MD)는 DMEM 배지에 1% 페니실린-스트렙토마이신(penicillin-streptomycin) 용액(Welgene) 및 8% FBS를 첨가하고, 37℃ 및 5% CO2 조건에서 배양하였다.HEI-OC1 cells (House Ear Institute, Los Angeles, CA, USA), a mouse auditory cell line, were grown in DMEM (Dulbecco modified eagle medium, Welgene, Korea) without the addition of antibiotics, 5% FBS (Fetal bovine serum, Welgene) and 50 U/mL. IFN-γ (interferon gamma, Peprotech, USA) was added and cultured at 33°C and 10% CO 2 conditions. HEK293T cells (CRL-1573; American Type Culture Collection, Rockville, MD), a cell line derived from human embryonic kidney, were grown in DMEM medium supplemented with 1% penicillin-streptomycin solution (Welgene) and 8% FBS. , cultured at 37°C and 5% CO 2 conditions.
[실험예 2] 화합물 스크리닝 [Experimental Example 2] Compound screening
HEI-OC1 세포에서 시스플라틴(cisplatin; 이하 CIS라 함)에 의한 세포독성에 대한 보호 효과를 나타내는 물질을 찾기 위해, Prestwick Chemical 사로부터 구매한 923개의 FDA 승인된 약물 라이브러리를 사용하여 스크리닝을 수행하였다. 923개의 약물을 96-웰 플레이트의 100μL 배양배지에 균등히 분주하고, 18시간 동안 배양한 세포에 약물을 0.5μL씩 처리하여 최종 약물의 농도가 50μM이 되도록 처리하였다. 2시간 후 20μM의 CIS를 첨가하였다. 각 플레이트마다 DMSO(Dimethyl sulfoxide)만 넣은 웰을 음성대조군으로 설정하고, 2mM 에르도스테인(erdosteine, Sigma-Aldrich, St. Louis, MO, USA)만 넣은 웰을 양성대조군으로 설정하였다. 약물 스크리닝은 0.82±0.06 정도의 Z’값(Z’ factor)을 나타냈다. 세포 생존율은 CellTiter-Glo luminescent assay(Promega, USA) 이용하여 제조사의 지침에 따라 측정하였다. 각 웰에 50μL의 CellTiter-Glo 시약(reagent)을 첨가하여 15분간 상온의 암실에서 반응시킨 후, automated EnVision plate reader(Perkin-Elmer)로 발광을 측정하였다. DMSO만 첨가된 웰의 측정 신호 값을 기준(0%)으로 하고, 에르도스테인이 들어간 웰의 측정 신호 값을 100%로 설정하였다. 첫 번째 스크리닝에서 양성대조군보다 CIS에 의한 세포독성에 대한 보호효과가 우수한 물질을 유효물질로 정의하였다. 이후 식별된 유효물질들을 2차 스크리닝을 통해 용량-반응 평가를 확인하였다. 선별된 유효물질들은 다양한 농도에서(3~100μM) 검증되었으며, 세포 생존율 측정은 하기 실험예 3과 동일한 방식으로 수행하였다.To find substances showing a protective effect against cytotoxicity by cisplatin (hereinafter referred to as CIS) in HEI-OC1 cells, screening was performed using a library of 923 FDA-approved drugs purchased from Prestwick Chemical. 923 drugs were evenly distributed in 100 μL culture medium of a 96-well plate, and cells cultured for 18 hours were treated with 0.5 μL of each drug so that the final drug concentration was 50 μM. After 2 hours, 20 μM CIS was added. For each plate, a well containing only DMSO (Dimethyl sulfoxide) was set as a negative control, and a well containing only 2mM erdosteine (erdosteine, Sigma-Aldrich, St. Louis, MO, USA) was set as a positive control. Drug screening showed a Z’ value (Z’ factor) of about 0.82±0.06. Cell viability was measured using CellTiter-Glo luminescent assay (Promega, USA) according to the manufacturer's instructions. 50 μL of CellTiter-Glo reagent was added to each well and reacted in the dark at room temperature for 15 minutes, and then luminescence was measured using an automated EnVision plate reader (Perkin-Elmer). The measurement signal value of the well containing only DMSO was set as the standard (0%), and the measurement signal value of the well containing erdosteine was set to 100%. In the first screening, substances with a better protective effect against cytotoxicity caused by CIS than the positive control were defined as effective substances. Afterwards, the identified effective substances were confirmed for dose-response evaluation through secondary screening. The selected effective substances were verified at various concentrations (3 to 100 μM), and cell viability was measured in the same manner as Experimental Example 3 below.
[실험예 3] 세포생존율 측정 [Experimental Example 3] Measurement of cell viability
HEI-OC1 세포는 96-웰 플레이트에 IFN-γ가 들어있지 않은 배지를 사용하여 1,000 cells/well로 100μL씩 균등히 분주하였다. 분주된 HEI-OC1 세포는 상기 실험예 2의 유효물질 처리 전 16시간 동안 33℃ 및 10% CO2에서 배양하였다. 상기 유효물질의 CIS에 의한 세포독성에 대한 보호효과를 확인하기 위해, 상기 배양한 HEI-CO1 세포에 유효물질을 농도별로 먼저 처리하고, 2시간 동안 배양하였다. 그 후, 세포독성을 유도하기 위해, 20μM의 CIS가 들어있는 배지로 교체한 후 48시간 배양하였다. 대조군은 유효물질을 처리하지 않고 DMSO를 처리한 후, 동일한 조건에서 배양하였다. 세포 생존율 측정은 CellTiter-Glo assay(Promega Corp., Madison, WI, USA)을 이용하여 ATP 수준을 측정을 통해 수행하였다. 100μL의 배양액 내 세포를 함유하는 플레이트의 각 웰에 50μL의 ATP 검출 용액을 첨가하여 10분간 상온의 암실에서 반응시킨 후, automated EnVision plate reader(Perkin-Elmer, Waltham, MA, USA)로 발광을 측정하였다. 실험은 3번 독립적으로 반복 수행하였고, 결과의 수치는 6개의 독립적인 웰에서 수행한 값을 사용하였다.HEI-OC1 cells were evenly distributed at 100 μL each at 1,000 cells/well using medium without IFN-γ in a 96-well plate. The distributed HEI-OC1 cells were cultured at 33°C and 10% CO 2 for 16 hours before being treated with the effective substance of Experimental Example 2. In order to confirm the protective effect of the active substance against cytotoxicity caused by CIS, the cultured HEI-CO1 cells were first treated with the active substance at different concentrations and cultured for 2 hours. Then, to induce cytotoxicity, the medium was replaced with 20 μM CIS and cultured for 48 hours. The control group was treated with DMSO without treatment with the active substance and then cultured under the same conditions. Cell viability was measured by measuring ATP levels using the CellTiter-Glo assay (Promega Corp., Madison, WI, USA). 50 μL of ATP detection solution was added to each well of the plate containing cells in 100 μL of culture medium, reacted in the dark at room temperature for 10 minutes, and then luminescence was measured using an automated EnVision plate reader (Perkin-Elmer, Waltham, MA, USA). did. The experiment was repeated three times independently, and the results were calculated from six independent wells.
[실험예 4] CIS에 의한 세포사멸에 대한 보호 효과 분석([Experimental Example 4] Analysis of protective effect against apoptosis by CIS ( in vitroin vitro ))
상기 유효물질(에스오메프라졸 또는 덱스란소프라졸)이 CIS에 의한 세포사멸에 대한 보호효과를 나타내는지 확인하기 위해, V-FITC(fluorescein isothiocyanate)/PI(propidium iodide) 염색을 수행하였다. HEI-OC1 세포를 6-웰 배양 플레이트에 분주하고, 유효물질(에스오메프라졸 또는 덱스란소프라졸)을 2시간 동안 처리하였다. 그 후, 20μM의 CIS를 48시간 동안 처리하였다. DMSO만을 처리한 배양배지를 대조군으로 설정하였다. 그 후, PBS로 1회 세척하고, binding buffer[10mM HEPES/NaOH(pH 7.4), 140mM NaCl 및 2.5mM CaCl2]에 상기 유효물질을 재부유(resuspend)하였다. 그 후, 세포를 세포 분류 튜브로 옮기고, 제조사 지침에 따라 Annexin V-FITC apoptosis detection kit(BD Biosciences, San Diego, CA, USA)를 사용하여 Annexin V-FITC 및 PI를 첨가하여 15분간 반응시켜 염색한 후, 유세포 분석을 수행하였다. 세포 사멸율은 Annexin V-FITC 양성(positive) 세포의 백분율로 정량화하였다. 모든 실험은 3번 반복 수행하였다. To confirm whether the active substance (esomeprazole or dexlansoprazole) has a protective effect against cell death caused by CIS, V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) staining was performed. HEI-OC1 cells were distributed in a 6-well culture plate and treated with an effective substance (esomeprazole or dexlansoprazole) for 2 hours. Afterwards, 20 μM CIS was treated for 48 hours. Culture medium treated with DMSO only was set as the control. Afterwards, it was washed once with PBS, and the active substance was resuspended in binding buffer [10mM HEPES/NaOH (pH 7.4), 140mM NaCl, and 2.5mM CaCl 2 ]. Afterwards, the cells were transferred to a cell sorting tube and stained by adding Annexin V-FITC and PI and reacting for 15 minutes using the Annexin V-FITC apoptosis detection kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Afterwards, flow cytometry was performed. Cell death rate was quantified as the percentage of Annexin V-FITC positive cells. All experiments were repeated three times.
[실험예 5] 세포 형질주입 및 형광 정량분석 [Experimental Example 5] Cell transfection and quantitative fluorescence analysis
fluorescence uptake assay를 수행하기 위해, HEK293T 세포는 인간 유기 양이온 수송체(hOCT2)를 발현하는 플라스미드로 일시적인 형질주입을 하였다. 형질주입 하루 전, HEK293T 세포는 1×106 의 밀도로 cell confluency가 70~80%가 되도록 24시간 동안 계대배양하였다. 폴리에틸렌이민(polyethylenimine; PEI) 및 hOCT2-발현 플라스미드로 형질주입하였다. hOCT를 발현하지 않는 공벡터를 사용하여 형질주입한 HEK293T 세포를 대조군으로 설정하였다. 형질주입된 HEK293T 세포는 black poly L-lysine-coated 96-웰 플레이트(Greiner Bio-One, Frickenhausen, Germany)에 100,000 cells/well로 균등히 분주하고, 18시간 동안 배양하였다. 그 후, 세포는 상온에 보관된 assay buffer(Hanks’ Balanced Salt SolutionL; HBSS)(5.6mM D-glucose 함유, pH 7.4)로 2회 세척하고, 유효물질(에스오메프라졸 또는 덱스란소프라졸)을 농도별로(0.0001~1000μM) 처리한 후 5분 반응시켰다. Fluorescence uptake assay는 10μM fluorescent substrate organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide(ASP+)가 들어있는 uptake buffer를 넣어 반응을 시작하였다. 실온에서 3분간 반응시킨 후 반응 혼합물을 흡입하고(aspirating), 500μM 시메티딘(Cimetidine)이 들어간 assay buffer로 2회 세척하여 반응을 종결시켰다. 그 후 100μL RIPA buffer를 넣고 5분 간 상온에서 세포를 용해시켰다. ASP+fluorescence는 EnVision plate reader(Perkin-Elmer)를 사용하여 excitation 및 emission을 각각 485nm 및 500nm에서 흡광도를 측정하였다.To perform fluorescence uptake assay, HEK293T cells were transiently transfected with a plasmid expressing human organic cation transporter (hOCT2). One day before transfection, HEK293T cells were subcultured at a density of 1×10 6 for 24 hours to reach 70-80% cell confluency. It was transfected with polyethylenimine (PEI) and hOCT2-expressing plasmid. HEK293T cells transfected using an empty vector that does not express hOCT were set as a control group. The transfected HEK293T cells were evenly distributed at 100,000 cells/well in black poly L-lysine-coated 96-well plates (Greiner Bio-One, Frickenhausen, Germany) and cultured for 18 hours. Afterwards, the cells were washed twice with assay buffer (Hanks' Balanced Salt SolutionL; HBSS) (containing 5.6mM D-glucose, pH 7.4) stored at room temperature, and the active substance (esomeprazole or dexlansoprazole) was added by concentration ( 0.0001~1000μM) and reacted for 5 minutes. Fluorescence uptake assay was started by adding uptake buffer containing 10μM fluorescent substrate organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+). After reacting at room temperature for 3 minutes, the reaction mixture was aspirated and washed twice with assay buffer containing 500 μM cimetidine to terminate the reaction. Afterwards, 100 μL RIPA buffer was added and the cells were lysed at room temperature for 5 minutes. ASP+fluorescence was measured at 485 nm and 500 nm for excitation and emission, respectively, using an EnVision plate reader (Perkin-Elmer).
[실험예 6] 동물모델[Experimental Example 6] Animal model
형질전환 제브라피쉬(Brn3C:EGFP) 배아는 고려대학교 안산병원 제브라피쉬 시설에서 28.5℃로 유지된 성체 제브라피쉬의 짝짓기를 통해 획득하였다. 형광 현미경을 사용하여 염색하지 않고 볼 수 있는 형질전환 제브라피시(Brn3C:EGFP)에서 자연적으로 발생하는 녹색 신경구(neuromast)는 이독성을 모니터링하기 위해 사용하였다. 수정 5일 후, 형질전환 제브라피쉬 배아에게 살아있는 짚신벌레(live paramecia) 및 식물성 음식(plant-based food)을 먹였다. 배아는 배아 배지(1mM MgSO4, 120μM KH2PO4, 74μM Na2HPO4, 1mM CaCl2, 500μM KCl, 15μM NaCl 및 500μM NaHCO3)에서 100mm2 페트리 접시당 약 50개의 배아 밀도로 유지하였다. 본 동물실험은 고려대학교 동물실험실무위원회의 승인을 받고 수행하였다(승인번호: KOREA-2022-0097). 모든 프로토콜은 고려대학교 의료원 동물보호윤리위원회 및 국립보건원 지침에 따라 수행하였다.Transgenic zebrafish (Brn3C:EGFP) embryos were obtained through mating of adult zebrafish maintained at 28.5°C at the zebrafish facility at Korea University Ansan Hospital. Naturally occurring green neuromasts from transgenic zebrafish (Brn3C:EGFP), which can be viewed without staining using a fluorescence microscope, were used to monitor ototoxicity. Five days after fertilization, transgenic zebrafish embryos were fed live paramecia and plant-based food. Embryos were maintained in embryo medium (1mM MgSO 4 , 120μM KH 2 PO 4 , 74μM Na 2 HPO 4 , 1mM CaCl 2 , 500μM KCl, 15μM NaCl, and 500μM NaHCO 3 ) at a density of approximately 50 embryos per 100mm 2 Petri dish. This animal experiment was conducted with approval from Korea University's Animal Experiment Practice Committee (approval number: KOREA-2022-0097). All protocols were performed in accordance with the guidelines of the Korea University Medical Center Animal Care and Ethics Committee and the National Institutes of Health.
[실험예 7] 약물 투여 및 제브라피쉬의 준비[Experimental Example 7] Drug administration and preparation of zebrafish
순수한 CIS를 배아 배지에 첨가하여 CIS 용액을 제조하였다. 제브라피쉬 수정 후 5일째에 27마리의 제브라피쉬 배아를 용매 대조군(EtOH 1.36%), 1000μM CIS(Sigma-Aldrich) 및 에스오메프라졸(Aldrich, Milwaukee, WI, USA)(2, 20 및 200μM)에 4시간 동안 노출시켰다. 그 후, 배아 배지로 배아를 3회 세척하고, 트리카인(tricaine)(MS-222, Sigma-Aldrich, Darmstadt, Germany, A5040)으로 5분 동안 마취하였다.CIS solution was prepared by adding pure CIS to embryo medium. At 5 days after zebrafish fertilization, 27 zebrafish embryos were incubated in solvent control (EtOH 1.36%), 1000 μM CIS (Sigma-Aldrich) and esomeprazole (Aldrich, Milwaukee, WI, USA) (2, 20 and 200 μM) for 4 days. exposed for a period of time. Afterwards, the embryos were washed three times with embryonic medium and anesthetized with tricaine (MS-222, Sigma-Aldrich, Darmstadt, Germany, A5040) for 5 minutes.
[실험예 8] 제브라피쉬 신경구 분석[Experimental Example 8] Zebrafish neurosphere analysis
제브라피쉬의 신경구 분석을 위해, 제브라피쉬를 슬라이드에서 메틸셀룰로오스(methylcellulose)로 고정하고, 제브라피쉬의 안와상부(SO1 및 SO2), 귀(O1) 및 후두부(OC1) 측선(lateral line)의 신경구(neuromast) 내 유모세포(hair cell)를 분석하였다(n=27). 형광 현미경(ECLIPSE Ni-U; Nikon, Tokyo, Japan) 및 공초점 현미경(Nikon, Spinning Disc Confocal Microscope)을 사용하여 신경구에서 유모세포의 평균수를 측정하였다.To analyze zebrafish neurospheres, zebrafish were fixed on slides with methylcellulose, and neurospheres were isolated from the lateral lines of the zebrafish's supraorbital area (SO1 and SO2), ear (O1), and occipital area (OC1). Hair cells within neuromast were analyzed (n=27). The average number of hair cells in the neurosphere was measured using a fluorescence microscope (ECLIPSE Ni-U; Nikon, Tokyo, Japan) and a confocal microscope (Nikon, Spinning Disc Confocal Microscope).
[실험예 9] CIS에 의한 세포사멸에 대한 보호 효과 분석([Experimental Example 9] Analysis of protective effect against apoptosis by CIS ( in vivoin vivo ))
in-situ cell detection kit(Roche Molecular Biochemicals, Mannheim, Germany) 및 TdT(terminal deoxynucleotidyl transferase) 매개 dUTP-biotin nick-end labeling(TUNEL) 방법을 이용하여 (제조업체의 지침에 따라) 제브라피쉬의 신경구 내 세포사멸을 분석하였다. 배아를 1000μM CIS 및 200μM 에스오메프라졸이 함유된 배지에 4시간 동안 노출시켰다. 그 후, 배아를 PBS로 세척하고 4% 파라포름알데하이드(Paraformaldehyde)로 고정시켰다. 그 후, 배아를 TUNEL 반응 혼합물(TdT 및 fluorescein-dUTP) 50mL에서 37℃로 60분 동안 배양하였다. 제브라피쉬는 AxioCam MRc5 형광 현미경(Nikon, Spinning Disc Confocal Microscope)을 사용하여 분석하였다. Cells within the neurosphere of zebrafish were identified using an in-situ cell detection kit (Roche Molecular Biochemicals, Mannheim, Germany) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method (according to the manufacturer's instructions). Death was analyzed. Embryos were exposed to medium containing 1000 μM CIS and 200 μM esomeprazole for 4 h. Afterwards, the embryos were washed with PBS and fixed with 4% paraformaldehyde. Afterwards, embryos were incubated in 50 mL of TUNEL reaction mixture (TdT and fluorescein-dUTP) at 37°C for 60 min. Zebrafish were analyzed using an AxioCam MRc5 fluorescence microscope (Nikon, Spinning Disc Confocal Microscope).
[실험예 10] 통계 분석 [Experimental Example 10] Statistical analysis
통계 분석은 GraphPad Prism 9.0(GraphPad Software, Inc., La Jolla, CA, USA)을 사용하여 수행하였다. 데이터는 Student’s t-test 또는 one-way analysis of variance(ANOVA)를 사용하여 비교 및 분석하였다. 데이터는 최소 3번 이상의 독립적인 실험에서 구해진 값의 평균값±표준오차(SEM) 또는 평균±표준편차(SD)로 표현하였다. 구체적으로, 실시예 1에 대한 데이터(도 2~4)는 평균±표준편차(SD)로, 나머지 데이터는 평균값±표준오차(SEM)로 표현하였다. p-값은 *p<0.05, **p<0.01, ***p<0.001 및 ****p<0.0001로 표시하였다. IC50(half maximal inhibitory concentration) 값은 비선형 회귀(nonlinear regression) 분석을 통해 계산하였다. Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data were compared and analyzed using Student's t-test or one-way analysis of variance (ANOVA). Data were expressed as mean ± standard error (SEM) or mean ± standard deviation (SD) of values obtained from at least three independent experiments. Specifically, the data for Example 1 (FIGS. 2 to 4) were expressed as mean ± standard deviation (SD), and the remaining data were expressed as mean ± standard error (SEM). p-values are indicated as *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. IC 50 (half maximal inhibitory concentration) value was calculated through nonlinear regression analysis.
[실시예 1] 화합물 스크리닝[Example 1] Compound screening
상기 실험예 2에 따라, HEI-OC1 세포에서 CIS에 의한 세포독성에 대한 보호 효과를 나타낸 물질 확인을 위해 화합물 스크리닝을 수행하였다. 스크리닝을 위한 CIS의 최적 농도를 결정하기 위해, HEI-OC1 세포에 CIS를 농도별로(1~30μM) 48시간 동안 처리한 결과, 도 1A와 같이, 세포 생존력이 용량 의존적으로 감소하였고, CIS 20μM 처리군의 세포생존율은 대조군(DMSO 처리군)의 33% 수준으로 나타났다. 이에, 후속 약물 스크리닝을 위한 CIS의 최적의 농도를 20μM로 설정했다. 또한, 도 1B와 같이, CIS 처리군(20μM)에서는 세포 수축이 나타나고, 세포가 배지에서 떠다니는 모습을 보였다. 그 후, FDA에서 승인한 923개 약물 라이브러리를 사용하여 스크리닝을 수행하였다. 그 결과, 도 2와 같이, 8개의 1차 유효물질을 확인하였다. 상기 1차 유효물질을 하기와 같다.According to Experimental Example 2, compound screening was performed to identify substances that showed a protective effect against cytotoxicity caused by CIS in HEI-OC1 cells. To determine the optimal concentration of CIS for screening, HEI-OC1 cells were treated with CIS at various concentrations (1 to 30 μM) for 48 hours. As shown in Figure 1A, cell viability decreased in a dose-dependent manner, and treatment with 20 μM CIS resulted in a dose-dependent decrease. The cell survival rate of the group was 33% of that of the control group (DMSO-treated group). Accordingly, the optimal concentration of CIS for subsequent drug screening was set at 20 μM. Additionally, as shown in Figure 1B, in the CIS-treated group (20 μM), cell shrinkage occurred and cells appeared to float in the medium. Afterwards, screening was performed using a library of 923 drugs approved by the FDA. As a result, as shown in Figure 2, eight primary active substances were identified. The primary active substance is as follows.
1) 에스오메프라졸(Esomeprazole)1) Esomeprazole
2) 덱스란소프라졸(Dexlansoprazole)2) Dexlansoprazole
3) 오메프라졸(Omeprazole)3) Omeprazole
4) 디다노신(Didanosine) 4) Didanosine
5) 엠트리씨타빈(Emtricitabine)5) Emtricitabine
6) 시메티딘(Cimetidine)6) Cimetidine
7) 메토프롤롤(Metoprolol)7) Metoprolol
8) 프로게스테론(Progesterone)8) Progesterone
또한, 상기 실험예 3에 따라, 상기 1차 유효물질의 CIS에 의한 세포독성에 대한 보호효과를 확인하기 위해, HEI-OC1 세포에 상기 1차 유효물질을 농도별로(8, 20 및 100μM)로 처리한 결과, 도 3과 같이, 상기 8종의 유효물질 모두 모든 처리 농도에서 유의한 세포 보호효과를 나타냈고, 그 중 5종의 유효물질(에스오메프라졸, 덱스란소프라졸, 엠트리시타빈, 메토프롤롤 및 시메티딘)은 용량 의존적인 세포 보호효과를 나타냈다. 그 중 가장 효과가 우수한 에스오메프라졸 및 덱스란소프라졸에 대해 처리 농도를 증가시켜 용량에 따른 세포 보호효과를 분석한 결과, 도 4와 같이, 대조군(DMSO 처리군) 대비 상기 2종의 유효물질 처리군에서 각각 세포생존율이 유의하게 증가했고, 에스오메프라졸은 100μM 처리 농도에서, 덱스란소프라졸은 200μM 처리 농도에서 가장 우수한 세포 보호효과를 나타냈다.In addition, according to Experimental Example 3, in order to confirm the protective effect of the primary active substance against cytotoxicity caused by CIS, the primary effective substance was administered to HEI-OC1 cells at different concentrations (8, 20, and 100 μM). As a result of the treatment, as shown in Figure 3, all eight active substances showed significant cell protection effects at all treatment concentrations, of which five active substances (esomeprazole, dexlansoprazole, emtricitabine, metoprolol, and cimetidine) ) showed a dose-dependent cytoprotective effect. Among them, the cell protection effect according to the dose was analyzed by increasing the treatment concentration for the most effective esomeprazole and dexlansoprazole. As shown in Figure 4, in the group treated with the two types of active substances compared to the control group (DMSO treated group), Each cell viability significantly increased, and esomeprazole showed the best cell protection effect at a treatment concentration of 100 μM, and dexlansoprazole showed the best cell protection effect at a treatment concentration of 200 μM.
[실시예 2] CIS에 의한 세포사멸에 대한 보호 효과 분석([Example 2] Analysis of protective effect against apoptosis by CIS ( in vitroin vitro ))
상기 실험예 4에 따라, 에스오메프라졸 및 덱스란소프라졸의 CIS에 의한 세포독성에 대한 세포 보호 효과를 확인하기 위해, HEI-OC1 세포를 이용한 유세포 분석을 수행한 결과, 도 5A 및 5B와 같이, 대조군 대비 CIS 처리군에서 세포사멸이 유의하게 증가하였다. 반면, 도 5B 및 5C와 같이, 에스오메프라졸 100μM 처리군 및 덱스란소프라졸 200μM 처리군은 CIS 처리군 대비 세포사멸이 유의하게 감소하였다. 구체적으로, 도 6과 같이, 에스오메프라졸 100μM 처리군 및 덱스란소프라졸 200μM 처리군에서 세포사멸의 초기(Annexin V+/PI-) 및 후기(Annexin V+/PI+) 단계 모두에서 세포사멸 비율이 감소하였다. 상기 결과로부터, 에스오메프라졸 및 덱스란소프라졸이 CIS에 의한 세포사멸 억제 효과를 나타내는 것을 확인하였다.According to Experimental Example 4, in order to confirm the cytoprotective effect of esomeprazole and dexlansoprazole against cytotoxicity caused by CIS, flow cytometry was performed using HEI-OC1 cells. As a result, as shown in Figures 5A and 5B, compared to the control group Cell death was significantly increased in the CIS treated group. On the other hand, as shown in Figures 5B and 5C, cell death was significantly reduced in the esomeprazole 100 μM treatment group and the dexlansoprazole 200 μM treatment group compared to the CIS treatment group. Specifically, as shown in Figure 6, the rate of apoptosis decreased in both the early (Annexin V+/PI-) and late (Annexin V+/PI+) stages of apoptosis in the esomeprazole 100 μM treatment group and the dexlansoprazole 200 μM treatment group. From the above results, it was confirmed that esomeprazole and dexlansoprazole exhibit an inhibitory effect on apoptosis caused by CIS.
[실시예 3] OCT2 특이적 억제 효과 분석[Example 3] Analysis of OCT2 specific inhibition effect
상기 실험예 5에 따라, 에스오메프라졸 및 덱스란소프라졸의 CIS에 의한 세포독성에 대한 보호효과가 CIS을 세포질로 운반하는 것으로 알려진 OCT2를 차단하는 능력을 통해 매개되는지 확인하기 위해, OCT2의 특이적 기질인 ASP+의 흡수를 평가하여 에스오메프라졸 및 덱스란소프라졸이 OCT2가 과발현된 HEK293T 세포에서 OCT2를 억제하는지 분석한 결과, 도 7A와 같이, OCT2 과발현 HEK293T 세포(hOCT2)의 ASP+(10μM) 흡수는 공벡터를 발현하는 HEK293 세포(Mock) 대비 약 25배 더 높게 나타났고, 시메티딘 처리군에서는 ASP+ 흡수가 감소하였다. 또한, 도 7B와 같이, 에스오메프라졸 및 덱스란소프라졸은 농도 의존적으로(0.001~1mM) ASP+(10μM)의 흡수를 억제하였다. 에스오메프라졸 및 덱스란소프라졸을 매개로 한 ASP+ 축적(accumulation) 저해의 IC50 값은 각각 6.4 및 1.3μM로 나타났다. 상기 결과로부터, 에스오메프라졸 및 덱스란소프라졸이 OCT2 매개 수송을 특이적으로 억제하는 것을 확인하였다. According to Experimental Example 5 above, in order to confirm whether the protective effect of esomeprazole and dexlansoprazole against cytotoxicity caused by CIS is mediated through the ability to block OCT2, which is known to transport CIS to the cytoplasm, OCT2's specific substrate, By evaluating the uptake of ASP+, we analyzed whether esomeprazole and dexlansoprazole inhibit OCT2 in OCT2-overexpressing HEK293T cells. As shown in Figure 7A, ASP+ (10 μM) uptake by OCT2-overexpressing HEK293T cells (hOCT2) expressing the empty vector was confirmed. was approximately 25 times higher than in HEK293 cells (Mock), and ASP+ uptake was decreased in the cimetidine-treated group. Additionally, as shown in Figure 7B, esomeprazole and dexlansoprazole inhibited the absorption of ASP+ (10 μM) in a concentration-dependent manner (0.001-1mM). The IC 50 values for inhibition of ASP+ accumulation mediated by esomeprazole and dexlansoprazole were 6.4 and 1.3 μM, respectively. From the above results, it was confirmed that esomeprazole and dexlansoprazole specifically inhibit OCT2-mediated transport.
[실시예 4] 제브라피쉬 신경구 분석[Example 4] Zebrafish neurosphere analysis
상기 실험예 8에 따라, CIS 및 에스오메프라졸에 노출시킨 제브라피쉬 신경구를 분석한 결과, 도 8A 및 8B와 같이, 대조군 대비 CIS 단독 처리군에서는 생존 유모세포 평균 수가 유의하게 감소한 한면, CIS 단독 처리군 대비 에스오메프라졸 처리군에서 생존 유모세포 평균 수가 유의하게 증가하였다(n=27). 구체적으로 각 실험군 간 생존 유모세포 평균 수는 하기 표 1과 같다. 사후 분석을 통한 일원 분산 분석(Tukey 방법) 결과, 200μM 에스오메프라졸이 CIS에 의한 유모세포 손실 감소 효과가 가장 효과적이었다(p = 0.001).According to Experimental Example 8, as a result of analyzing zebrafish neurospheres exposed to CIS and esomeprazole, as shown in Figures 8A and 8B, the average number of surviving hair cells was significantly reduced in the CIS-only treatment group compared to the control group, on the one hand, the CIS-only treatment group In contrast, the average number of surviving hair cells in the esomeprazole-treated group significantly increased (n=27). Specifically, the average number of surviving hair cells among each experimental group is shown in Table 1 below. As a result of one-way analysis of variance (Tukey method) with post hoc analysis, 200 μM esomeprazole was most effective in reducing hair cell loss due to CIS (p = 0.001).
실험군experimental group 생존 유모세포 평균 수Average number of surviving hair cells
대조군(음성대조군)Control group (negative control) 41.7 ± 4.541.7 ± 4.5
CIS 단독 처리군CIS only treatment group 14.7 ± 5.614.7 ± 5.6
에스오메프라졸 2μM 처리군Esomeprazole 2μM treatment group 22.8 ± 6.522.8 ± 6.5
에스오메프라졸 20μM 처리군Esomeprazole 20μM treatment group 22.9 ± 4.822.9 ± 4.8
에스오메프라졸 200μM 처리군Esomeprazole 200μM treatment group 28.8 ± 5.628.8 ± 5.6
[실시예 5] CIS에 의한 세포사멸에 대한 보호 효과 분석([Example 5] Analysis of protective effect against apoptosis by CIS ( in vivoin vivo ))
상기 실험예 9에 따라, 상기 에소메프라졸의 CIS에 의한 세포(신경구)사멸에 대한 보호효과를 분석한 결과, 도 8C와 같이, 대조군(Control) 대비 CIS(1000μM) 단독 처리군에서 TUNEL 양성 세포(붉은색) 수가 유의하게 증가한 반면, CIS 단독 처리군 대비 에스오메프라졸(200μM) 처리군에서 TUNEL 양성 세포(붉은색) 수가 유의하게 감소하였다. 상기 결과로부터, 에스오메프라졸이 제브라피쉬의 유모세포에서 CIS에 의한 세포사멸을 억제하는 것을 확인하였다.According to Experimental Example 9, as a result of analyzing the protective effect of esomeprazole against cell (neurosphere) death caused by CIS, as shown in Figure 8C, TUNEL was positive in the group treated only with CIS (1000 μM) compared to the control group. While the number of cells (red) significantly increased, the number of TUNEL-positive cells (red) significantly decreased in the esomeprazole (200 μM) treated group compared to the CIS only treated group. From the above results, it was confirmed that esomeprazole inhibits apoptosis caused by CIS in zebrafish hair cells.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (9)

  1. 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  2. 제1항에 있어서, 상기 난청은 이독성 난청, 돌발성 난청, 소음성 난청 또는 노화성 난청에서 선택된 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 1, wherein the hearing loss is selected from ototoxic hearing loss, sudden hearing loss, noise-induced hearing loss, or age-related hearing loss.
  3. 제2항에 있어서, 상기 이독성 난청은 항암제 또는 아미노글루코시드계 항생제에서 선택된 약물에 의한 난청인 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 2, wherein the ototoxic hearing loss is hearing loss caused by a drug selected from anticancer drugs or aminoglucoside antibiotics.
  4. 제3항에 있어서, 상기 항암제는 시스플라틴(cisplatin) 또는 카르보플라틴(carboplatin)인 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 3, wherein the anticancer agent is cisplatin or carboplatin.
  5. 제3항에 있어서 상기 아미노글루코시드계 항생제는 아미카신(amikacin), 아르베카신(arbekacin), 카나마이신(kanamycin), 겐타마이신(gentamicin), 네오마이신(neomycin), 네틸마이신(netilmicin), 디베카신(dibekacin), 시소마이신(sisomycin), 스트렙토마이신(streptomycin), 토브라마이신(tobramycin), 리보도마이신(livodomycin) 및 파로모마이신(paromomycin)으로 구성된 군에서 선택되는 어느 하나 이상인 것을 특징으로 하는 약학 조성물. The method of claim 3, wherein the aminoglucoside antibiotics are amikacin, arbekacin, kanamycin, gentamicin, neomycin, netilmicin, D. Characterized by at least one selected from the group consisting of dibekacin, sisomycin, streptomycin, tobramycin, ribodomycin, and paromomycin. A pharmaceutical composition.
  6. 제1항에 있어서, 상기 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)은 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 시스플라틴의 흡수를 저해시키는 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 1, wherein Esomeprazole or Dexlansoprazole inhibits the uptake of cisplatin by OCT2 (Organ Cation Transporter 2) protein in hair cells.
  7. 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 난청 예방 또는 개선용 건강기능식품 조성물.A health functional food composition for preventing or improving hearing loss containing Esomeprazole or Dexlansoprazole as an active ingredient.
  8. 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)을 유효성분으로 포함하는 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 시스플라틴 흡수 저해용 시약 조성물.A reagent composition for inhibiting cisplatin uptake of OCT2 (Organ Cation Transporter 2) protein in hair cells containing Esomeprazole or Dexlansoprazole as an active ingredient.
  9. 에스오메프라졸(Esomeprazole) 또는 덱스란소프라졸(Dexlansoprazole)에서 선택된 하나 이상의 프로톤 펌프 억제제; 및 항암제 또는 아미노글루코시드계 항생제에서 선택된 약물을 포함하며, 상기 프로톤 펌프 억제제가 유모세포에서 OCT2(Organ Cation Transpoter 2) 단백질의 상기 약물 흡수를 저해하고, 유모세포 생존율을 향상시키는 것을 특징으로 하는, 약제학적 복합 제제.One or more proton pump inhibitors selected from Esomeprazole or Dexlansoprazole; and a drug selected from anticancer drugs or aminoglucoside antibiotics, wherein the proton pump inhibitor inhibits the drug absorption by OCT2 (Organ Cation Transporter 2) protein in hair cells and improves hair cell survival. Pharmaceutical combination preparation.
PCT/KR2023/013653 2022-09-13 2023-09-12 Pharmaceutical composition for preventing or treating hearing loss comprising esomeprazole or dexlansoprazole as active ingredient WO2024058527A1 (en)

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US20080242703A1 (en) * 2007-04-02 2008-10-02 National Taiwan University Pharmaceutical compositions for the treatment of hearing loss
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US20080242703A1 (en) * 2007-04-02 2008-10-02 National Taiwan University Pharmaceutical compositions for the treatment of hearing loss
KR20110033295A (en) * 2008-07-21 2011-03-30 오토노미, 인코포레이티드 Controlled Release Antimicrobial Compositions and Methods for the Treatment of Ear Diseases
US20150150793A1 (en) * 2008-07-21 2015-06-04 Otonomy, Inc. Controlled Release Antimicrobial Compositions and Methods for the Treatment of Otic Disorders
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