Pharmaceutical composition for preventing hearing loss or preventing or treating tinnitus, comprising visomitin

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hearing loss or tinnitus, comprising visomitin as an active ingredient. In the present invention, visomitin significantly inhibited ROS production induced by gentamicin or cisplatin treatment. In particular, visomitin exhibited an effect of inhibiting the cytotoxicity of gentamicin in an HEI-OC1 cell line and in cochleas extracted from mice in vitro experiments. In addition, in an in vivo experiment using zebrafish, visomitin significantly inhibited ROS generation in hair cells and apoptosis of hair cells caused by gentamicin treatment. The results demonstrate a superior improvement effect compared to antioxidants used as control drugs, and it is considered that visomitin can be used for preventing or treating hearing loss caused by toxicity of antibiotics or anticancer drugs.

Description

Pharmaceutical composition containing bisomitin for the prevention of hearing loss or the prevention or treatment of tinnitus

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus comprising visomitin as an active ingredient.

[0002] In modern society, problems related to hearing loss and / or tinnitus caused by cellular or nerve damage are becoming a major issue due to factors such as increased noise exposure and the aging of the population. To treat hearing loss, many specialists and pharmaceutical industry professionals still aim to treat these conditions using therapies based on conventional antibiotics or anti-inflammatory drugs; however, these cannot be used as appropriate treatments for hearing loss. In particular, the use of antibiotics (e.g., aminoglycosides) poses a significant problem due to ototoxicity (Antimicrobial Agents and Chemotherapy (1989) 33:797-800).

[0003] Against this backdrop, preclinical studies on various drugs have been reported in an effort to identify substances effective for the prevention and treatment of hearing loss and / or tinnitus; however, it has been confirmed that there are limitations to proceeding to clinical trials. Furthermore, to date, there are no approved drugs for the prevention or treatment of hearing loss and / or tinnitus. Moreover, as there are almost no reported drugs developed for the treatment of hearing loss and / or tinnitus that can be used without toxicity or risk to the human body, further research and drug development are currently required.

[0004] Accordingly, the inventors of the present invention completed the present invention by researching methods to prevent or treat hearing loss and / or tinnitus, and confirming through cell and animal experiments that bisomitin inhibits ototoxicity caused by antibiotics or anticancer agents.

[0005] To achieve the above objective, one aspect of the present invention provides a pharmaceutical composition comprising a compound of the following chemical formula 1 as an active ingredient:

[0006] <Chemical Formula 1>

[0007]

[0008] At this time, the above X is a 1 to 3 valent anion.

[0009] Another aspect of the present invention provides a food composition for the prevention or improvement of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient:

[0010] <Chemical Formula 1>

[0011]

[0012] At this time, the above X is a 1 to 3 valent anion.

[0013] Another aspect of the present invention provides a feed composition for the prevention or improvement of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient:

[0014] <Chemical Formula 1>

[0015]

[0016] At this time, the above X is a 1 to 3 valent anion.

[0017] Another aspect of the present invention provides a use of a compound of the following formula 1 for the prevention or treatment of hearing loss or tinnitus:

[0018] <Chemical Formula 1>

[0019]

[0020] At this time, the above X is a 1 to 3 valent anion.

[0021] Another aspect of the present invention provides a method for preventing or treating hearing loss or tinnitus, comprising the step of administering a compound of the following formula 1 to an individual:

[0022] <Chemical Formula 1>

[0023]

[0024] At this time, the above X is a 1 to 3 valent anion.

[0025] In the present invention, bisomitin significantly inhibited the production of reactive oxygen species (ROS) induced by treatment with gentamicin or cisplatin. In particular, bisomitin demonstrated an inhibitory effect on the cytotoxicity of gentamicin in HEI-OC1 cell lines and cochleas extracted from mice in in vitro experiments. Furthermore, in in vivo experiments using zebrafish, it significantly inhibited the production of ROS within hair cells and the apoptosis of hair cells caused by gentamicin treatment. These results represent a superior improvement compared to the antioxidant used as a control drug, and it is believed that it can be used in the future to prevent or treat hearing loss caused by the toxicity of antibiotics or anticancer drugs.

[0026] Figure 1 is a graph showing the results of confirming ROS production over time in HEI-OC1 cell lines after pretreatment with bisomitin or mitoquinone followed by treatment with cisplatin. Mean ± SD (n=3), #p<0.05 vs DMSO, *p<0.05 vs cisplatin (Dunnett test)

[0027] Figure 2 is a graph showing the results of confirming ROS production in HEI-OC1 cell lines after pretreatment with bisomitin or mitoquinone followed by time-dependent treatment with gentamicin. Mean ± SD (n=3), #p<0.05 vs DMSO, *p<0.05 vs gentamicin (Dunnett test)

[0028] Figure 3 is a graph showing the results of confirming ROS production over time in HEI-OC1 cell lines after simultaneous treatment with bisomitin or mitoquinone and gentamicin. Mean ± SD (n=3), #p<0.05 vs DMSO, *p<0.05 vs gentamicin (Dunnett test)

[0029] Figure 4 is a graph showing the results of confirming cell viability in HEI-OC1 cell lines after pretreatment with bisomitin or mitoquinone and treatment with gentamicin. Mean ± SD (n=3), #p<0.05 vs DMSO, *p<0.05 vs gentamicin (Dunnett test)

[0030] Figure 5a shows the results of fluorescent staining of different regions of cochleas extracted from mice after pretreatment with bisomitin or mitoquinone and treatment with gentamicin. Green: Myo7a, Red: Phalloidin, Blue: DAPI;

[0031] DMSO (a~c), gentamicin (d~f), bisomitin 0.25 μM (g~i), bisomitin 0.5 μM (j~l), mitoquinone 1 μM (m~o)

[0032] Figure 5b shows Myo7a by fluorescently staining different regions of the cochlea after pre-treating mouse-extracted cochleas with bisomitin or mitoquinone and gentamicin. + Phalloidin + DAPI + This is a graph showing the results of cell counting. Mean ± SEM (n≥8), #p<0.05 vs DMSO, *p<0.05 vs gentamicin (Bonferroni test)

[0033] Figure 6a shows the results of confirming ROS production using MitoSOX after pre-treating mouse cochleas with bisomitin or mitoquinone and gentamicin. Red: MitoSOX, Blue: DAPI;

[0034] DMSO (a~c), gentamicin (d~f), bisomitin 0.25 μM (g~i), bisomitin 0.5 μM (j~l), mitoquinone 1 μM (m~o)

[0035] Figure 6b is a graph showing the results of confirming ROS production using MitoSOX after pre-treating mouse cochleas with bisomitin or mitoquinone and then treating them with gentamicin. Mean ± SEM (n≥12), #p<0.05 vs DMSO, *p<0.05 vs gentamicin (Dunnett T3 test)

[0036] Figure 7a shows the results of Yo-Pro-1 staining of hair cells by region after pretreatment with bisomitin, L-selenomethionine, N,N'-dimethylurea, or edaravone and subsequent treatment with gentamicin in zebrafish. Green: Yo-Pro-1, SO: supraorbital, O: otic, OC: occipital

[0037] Figure 7b is a graph showing the results of counting hair cells by site after pre-treating zebrafish with bisomitin, L-selenomethionine, N,N'-dimethylurea, or edaravone and then treating with gentamicin. Mean ± SEM (n≥5), #p<0.05 vs DMSO, *p<0.05 vs gentamicin (Dunnett test)

[0038] Pharmaceutical composition

[0039] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient:

[0040] <Chemical Formula 1>

[0041] .

[0042] At this time, X may be a 1 to 3 valent anion. Specifically, X may be a 1 valent, 2 valent, or 3 valent anion. X may be an anion that provides electrical neutrality to the phosphonium cation (bisomitin cation) in Formula 1. For example, X may be an inorganic acid such as hydrochloric acid, nitric acid, phosphoric acid, hydrobromide, hydroiodide, sulfuric acid, etc.; an organic carboxylic acid such as formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, tartaric acid, succinic acid, oxalic acid, maleic acid, citric acid, hippuric acid, orotic acid, nicotinic acid, glutamic acid, ascorbic acid, edicilic acid, sebacic acid, aspartic acid, etc. It may be a monovalent to trivalent anion formed by sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, camphosulfonic acid, etc. Specifically, Cl - , NO3 - , H2PO4 - , HPO4 2- , PO4 3- , Br - , I - , HSO4 - , SO4 2- , HCO2 - , CH3COO - , CCl3COO - , CF3COO - , HOCH2(CHOH)4COO - , C6H5COO - , CH3CH(OH)COO - , C4H2O4 2- , C4H4O6 2- , C6H5CONHCH2COO - , C4N2H3O2COO - , C5NH4COO - , C3H7N(COO)2 2- , C6H7O6 - , C2H4(SO3)2 - , HC8H 16 (COO)2 - , C8H 16 (COO)2 2-, C2H5N(COO)2 2- , CH3SO3 - , CH3CH2SO3 - , C6H5SO3 - , CH3C6H4SO3 - or C9H 13 OCH2SO3 - It may be, but is not limited to.

[0043] Preferably, the X is Br - It could be.

[0044] In one embodiment of the present invention, the compound of Formula 1 may be bisomitin.

[0045] As used herein, the term "bisomitin" refers to 10-(4,5-dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decyl-triphenylphosphonium bromide, represented by Chemical Formula 2 below. Also known as SkQ1, it is a type of antioxidant that targets mitochondria, possessing high membrane permeability and potent antioxidant capacity. Bisomitin inhibits the production of ROS in mitochondria, and its effects in various inflammations, aging, or wound healing have been reported.

[0046] <Chemical Formula 2>

[0047]

[0048] The compound of Formula 1 above may be included in a pharmaceutical composition at a concentration of about 0.1 uM to about 1000 uM. Specifically, it may be included at about 0.1 uM to about 1000 uM, about 0.2 uM to about 500 uM, or about 0.3 uM to about 300 uM. More specifically, it may be included at about 0.5 uM to about 100 uM.

[0049] As used herein, the term "hearing loss" refers to any condition in which hearing is impaired or lost. Hearing loss may include, but is not limited to, conductive hearing loss and sensorineural hearing loss.

[0050] The aforementioned "conductive hearing loss" refers to hearing loss that occurs when the transmission of sound waves is not carried out normally due to disorders in organs such as the outer ear, eardrum, and middle ear. It is hearing loss caused by ear disease, resulting from problems in organs that transmit sound, such as the eardrum and ossicles. This hearing loss may be caused by various factors, such as infection, injury, inflammation, tumors, and adverse reactions to drugs or other chemicals.

[0051] The above "sensorineural hearing loss" refers to hearing loss caused by a dysfunction of the cochlea or by an abnormality in the auditory nerve or central nervous system that transmits auditory stimuli to the brain. The above sensorineural hearing loss may be, but is not limited to, congenital hearing loss, noise-induced hearing loss, sudden hearing loss, infectious hearing loss, traumatic hearing loss, presbycusis, ototoxic hearing loss, autoimmune hearing loss, Meniere's disease, or hearing loss caused by nerve damage. In the present invention, if the above hearing loss corresponds to a state of hearing deterioration or loss, it may be included within the scope of diseases without being limited to the category of hearing loss of the present invention.

[0052] The term "noise-induced hearing loss" as used in this specification is hearing loss caused by damage to the cochlea due to external noise.

[0053] The term “sudden hearing loss” as used in this specification is hearing loss in which a hearing loss of about 30 dB or more occurs at three or more consecutive frequencies within about 3 days.

[0054] As used herein, the term "infectious hearing loss" refers to hearing loss caused by a viral infection, such as rubella virus or congenital cytomegalovirus infection.

[0055] As used in this specification, the term "traumatic hearing loss" refers to hearing loss caused by the development of a perilymphatic fistula or pneumolabyrinth, etc., due to trauma.

[0056] As used in this specification, the term "age-related hearing loss" refers to hearing loss caused by degenerative changes due to aging.

[0057] As used herein, the term "autoimmune hearing loss" refers to hearing loss caused by an autoimmune disease.

[0058] As used herein, the term "Meniere's disease" refers to a disease characterized by the simultaneous occurrence of symptoms such as rotational vertigo, hearing loss, tinnitus, and a sensation of fullness in the ear.

[0059] As used in this specification, the term "hearing loss due to nerve damage" refers to hearing loss caused by an abnormality in the nerve region extending from the auditory cells of the cochlea to the area of ​​the brain responsible for hearing.

[0060] As used herein, the term "ototoxic hearing loss" refers to hearing loss caused by a deterioration of inner ear function due to drugs, chemicals, radiation, etc. In one embodiment, the ototoxic hearing loss may be induced by the use of anticancer agents, antibiotics, or radiation. Specifically, it may occur when the concentration of ROS increases and hair cells are lost due to the use of anticancer agents, antibiotics, or radiation.

[0061] In one embodiment of the present invention, the hearing loss may be sensorineural hearing loss, and preferably ototoxic hearing loss.

[0062] As used herein, the term "anticancer agent" refers to a drug that inhibits the division of or kills cancer cells. The anticancer agent may cause neurotoxicity. The anticancer agent may be one or more selected from the group consisting of taxane anticancer agents, platinum anticancer agents, and vinca alkaloid anticancer agents. Preferably, the anticancer agent may be a platinum anticancer agent.

[0063] The above taxane-based anticancer agent may be one or more selected from the group consisting of docetaxel, oraxol, paclitaxel, ditaxel, taxol, and taxotere. The above platinum-based anticancer agent may be one or more selected from the group consisting of cisplatin, carboplatin, and oxaliplatin. The above vinca alkaloid-based anticancer agent may be one or more selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine. In one embodiment of the present invention, the anticancer agent may be cisplatin.

[0064] As used in this specification, the term "antibiotic" means a drug that inhibits the growth of microorganisms. The antibiotic may kill or inhibit bacteria through one or more mechanisms selected from the group consisting of inhibition of cell wall synthesis, alteration of cell wall permeability, inhibition of protein synthesis, inhibition of nucleic acid synthesis, and inhibition of folic acid synthesis.

[0065] The above antibiotic may be one or more selected from the group consisting of beta-lactams, sulfonamides, aminoglycosides, tetracyclines, glycopeptides, ansamycins, penicillins, macrolides, streptogramins, cephalosporins, and quinolones. Preferably, the antibiotic may be an aminoglycoside. In one embodiment, the antibiotic may be one or more selected from the group consisting of streptomycin, neomycin, kanamycin, neomycin, gentamicin, tobramycin, amikacin, netilmicin, dibekacin, sisomicin, livodomycin, and paromomycin. In one embodiment of the present invention, the antibiotic may be gentamicin.

[0066] As used herein, the term "tinnitus" refers to the perception of sound in the absence of an external source of acoustic signals, and may include, but is not limited to, objective tinnitus, subjective tinnitus, peripheral tinnitus, and central tinnitus. Specifically, it may include, but is not limited to, subjective tinnitus caused by various factors such as noise, drugs, aging, trauma, and viruses.

[0067] The above "objective tinnitus" refers to tinnitus that is heard from the outside, and the above "subjective tinnitus" refers to tinnitus that is heard only by the patient themselves and not by the outside. Additionally, tinnitus can be classified into peripheral tinnitus and central tinnitus based on differences in how it is perceived by the individual afflicted with the disease. The above "peripheral (or cochlear) tinnitus" is presumed to originate from the peripheral nervous system and the cochlea, and the above "central tinnitus" is presumed to originate from the auditory cortex. However, if it corresponds to tinnitus, it may be included within the scope of the disease, not limited to the above category of tinnitus of the present invention.

[0068] As used herein, the term "treatment" may be used to include both therapeutic and preventive treatments. In this context, prevention may be used to mean alleviating or reducing a pathological condition or disease of an individual.

[0069] In the pharmaceutical composition of the present invention, the active ingredient may be included in any amount (effective amount) depending on the use, formulation, purpose of combination, etc., as long as it can exhibit a preventive or therapeutic effect against hearing loss. A typical effective amount will be determined within the range of about 0.0001% by weight to about 20.0% by weight based on the total weight of the composition. Here, "effective amount" refers to the amount of the active ingredient capable of inducing a preventive or therapeutic effect against a disease. Such effective amount may be determined experimentally within the ordinary capacity of a person skilled in the art.

[0070] As used herein, the term "prevention" may comprehensively mean preventing a disease in advance or reducing the likelihood or frequency of occurrence by administering the pharmaceutical composition in a pharmaceutically effective amount. For example, it may mean reducing the probability of occurrence or reducing the probability of recurrence in patients who are at risk of developing the disease or who have previously developed it.

[0071] As used herein, the term "treatment" may comprehensively refer to improving a disease by administering the pharmaceutical composition in a pharmaceutically effective amount, providing relief or healing of disease symptoms within a shorter time compared to natural healing, and improving a single symptom or most symptoms caused by the disease. The pharmaceutical composition of the present invention may serve as a composition for treating a disease itself, or it may be applied as an adjuvant for treatment of a disease by being administered together with other pharmacological components. Accordingly, the term "treatment" includes the meaning of "adjuvant treatment."

[0072] Here, "therapeutically effective amount" or "pharmaceuticalally effective amount" refers to an amount of a compound or composition effective for preventing or treating a target disease, meaning an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The level of the said effective amount may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0073] The above "active ingredient" refers to a component that exhibits activity alone or exhibits activity together with an adjuvant (carrier) that is not active on its own. In one embodiment, the compound of Formula 1 as the active ingredient can inhibit the generation of ROS caused by antibiotics or anticancer agents and inhibit the apoptosis of hair cells.

[0074] In the present invention, the pharmaceutical composition may be administered to an individual in a therapeutically effective amount or a pharmaceutically effective amount.

[0075] As used herein, the term "administration" means introducing a specific substance into an individual by an appropriate method, and the route of administration of the composition may be any general route as long as it can reach the target tissue. It may be administered orally or parenterally, and may be administered directly to the site of the disease.

[0076] The above-mentioned individual refers to all mammals, including humans, that have already developed or may develop hearing loss, and by administering the composition of the present invention to the individual, the said disease can be effectively prevented and treated. Preferably, the individual may be a human.

[0077] Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, method of administration, age, body weight, sex, pathological condition of the patient, food, time of administration, route of administration, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition according to the present invention may be administered in doses ranging from about 0.00001 mg / kg to about 100 mg / kg in adults, divided into one or several doses.

[0078] In addition, the above pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0079] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier and may be formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions.

[0080] The above-mentioned pharmaceutically acceptable carriers may include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical composition of the present invention may include, but is not limited to, fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

[0081] When the pharmaceutical composition of the present invention is formulated into an oral solid dosage form, it includes tablets, pills, powders, granules, capsules, etc., and such solid dosage forms may include at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may include a lubricant such as magnesium stearate or talc, but is not limited thereto.

[0082] When the pharmaceutical composition of the present invention is formulated as an oral liquid, it includes suspensions, oral liquids, emulsions, syrups, etc., and may include diluents such as water and liquid paraffin, humectants, sweeteners, flavorings, preservatives, etc., but is not limited thereto. When the pharmaceutical composition of the present invention is formulated as a parenteral formulation, it includes sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc., and non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc., but is not limited thereto. Witepsol, macrogol, Tween 61, cacao gelatin, laurin gelatin, glycerogelatin, etc., may be used as the base for suppositories, but is not limited thereto. Suitable pharmaceutically acceptable carriers and formulations are described in detail in "Remington's Pharmaceutical Sciences (19th ed, 1995)".

[0083] Another aspect of the present invention provides the use of the compound of Formula 1 for use in the manufacture of a drug for the prevention or treatment of hearing loss or tinnitus.

[0084] Another aspect of the present invention provides a method for preventing or treating hearing loss or tinnitus, comprising the step of administering a compound of Formula 1 to an individual.

[0085] The compound of Chemical Formula 1 above, hearing loss, tinnitus, individual, administration, individual, prevention and treatment are the same as described above.

[0086] Food composition

[0087] Another aspect of the present invention provides a food composition for the prevention or improvement of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient:

[0088] <Chemical Formula 1>

[0089]

[0090] At this time, the above X may be a 1 to 3 valent anion.

[0091] The compound of Chemical Formula 1, hearing loss, tinnitus, and prevention are the same as described above.

[0092] As used herein, the term "improvement" refers to any act in which hearing loss or tinnitus is improved or beneficially altered by the administration of the composition of the present invention.

[0093] The food composition of the present invention may include conventional food additives, and unless otherwise specified, suitability as a "food composition" may be determined in accordance with the specifications and standards for the relevant items in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety.

[0094] Items listed in the above "Food Additives Codex" may include, for example, chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0095] The above food composition may contain a compound of Formula 1 in an amount of about 0.0001% by weight to about 20.0% by weight relative to the total weight of the composition for the purpose of preventing or improving hearing loss or tinnitus.

[0096] In addition, the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving hearing loss or tinnitus.

[0097] The food composition of the present invention may be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term "functionality" refers to consuming the food for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0098] feed composition

[0099] Another aspect of the present invention provides a feed composition for the prevention or improvement of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient:

[0100] <Chemical Formula 1>

[0101]

[0102] At this time, the above X may be a 1 to 3 valent anion.

[0103] The compound of Chemical Formula 1 above, hearing loss, tinnitus, prevention and improvement are the same as described above.

[0104] The term "feed" above refers to any natural or artificial prescribed food, single-meal food, etc., or the ingredients of said single-meal food, intended for or suitable for animals to eat, consume, and digest. The said feed may be feed for fish, birds, or mammals, and preferably, may be feed for livestock or aquatic organisms defined in Article 2, Paragraph 1 of the Livestock Act and each subparagraph of Article 2 of the Enforcement Rule of the same Act, which have domesticated wild habits, are suitable for rearing, and can contribute to increasing farm income. The said livestock may include cattle, horses, mules, donkeys, goats, goats, sheep, deer, pigs, rabbits, poultry, etc., and poultry may include chickens, turkeys, ducks, ostriches, geese, quail, etc., preferably chickens, but is not limited thereto as long as they are suitable for rearing to obtain livestock products. The above "livestock products" means meat, milk, eggs, honey and processed products thereof, raw hides (including raw fur), raw wool, and other livestock products produced from livestock as defined in Article 2, Subparagraph 3 of the Livestock Act, as prescribed by the Ordinance of the Ministry of Agriculture, Food and Rural Affairs. In addition, it may include companion animals such as dogs and cats, but is not limited thereto.

[0105] The above feed composition may contain a compound of Formula 1 in an amount of about 0.0001% by weight to about 20.0% by weight relative to the total weight of the composition for the purpose of preventing or improving hearing loss or tinnitus.

[0106] In addition, the feed composition containing the compound of Chemical Formula 1 may include concentrated feed, roughage, and / or special feed. Concentrated feed includes seed grains such as wheat, oats, and corn; bran, which is a byproduct obtained by refining grains and includes rice bran, wheat bran, and barley bran; oilseed meal, which is a byproduct obtained by extracting oil from soybeans, rapeseed, sesame, flaxseed, coconut, etc.; residues, such as residual starch, which is the main component of starch residue remaining after removing starch from sweet potatoes, potatoes, etc.; animal feeds such as fish meal, fish residue, fish soluble which is a concentrated fresh liquid obtained from fish, meat meal, blood meal, feather meal, skim milk powder, and dried whey, which is the residue obtained when making cheese from milk or casein from skim milk; yeast, Chlorella, and seaweed.

[0107] Roughage includes raw grass feeds such as wild grass, pasture grass, and green cuts; root vegetables such as feed turnips, feed beets, and a type of turnip called lutea bearger; silage, which is a stored feed made by filling a silo with raw grass, green cut crops, and grains and fermenting them with lactic acid; hay made by cutting and drying wild grass and pasture grass; straw from livestock breeding crops; and leaves of legumes. Special feeds include mineral feeds such as oyster shells and rock salt; urea feeds such as urea or its derivatives such as diuretic isobutane; and feed additives, which are substances added in trace amounts to compound feed to supplement components that are prone to being lacking when only natural feed ingredients are mixed, or to improve the shelf life of the feed.

[0108] The present invention will be explained in more detail below through the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0109] I. Confirmation of hearing loss inhibitory activity at the in vitro level

[0110] Example 1. Confirmation of the reactive oxygen species generation inhibitory ability of bisomitin

[0111] The inhibitory effect of the antioxidant bisomitin on ROS increased by antibiotics or anticancer agents in HEI-OC1 (house ear institute-organ of corti 1) cell lines was evaluated.

[0112] Specifically, 6 x 10 HEI-OC1 cells per well in a 96-well plate 3 After inoculating the dogs, they were cultured for 24 hours in a 10% CO2 incubator at 33°C using DMEM medium containing 10% FBS. After replacing the culture medium, the test substance (bisomitin) or control substance (mitoquinone) was pretreated (24 h) to a final concentration of 0.5 μM or 1 μM; or they were simultaneously treated with an antibiotic (gentamicin, 10 mM) or an anticancer agent (cisplatin, 30 μM) and cultured for an additional 6 to 36 hours. Subsequently, the amount of ROS produced was measured at each time point to confirm the ability to inhibit ROS production by bisomitin treatment.

[0113] ROS quantification was performed by treating cells after the above reaction was completed with DCFDA (2′,7′-dichlorofluorescin diacetate) and reacting for 20 minutes, measuring the fluorescence value of the reaction product (Ex 495 nm / Em 520 nm), and then correcting the fluorescence value by staining the nucleus with Hoechst 33342.

[0114] Example 1.1. Inhibitory effect of cisplatin on ROS production after bisomitin pretreatment

[0115] Figure 1 shows the results of confirming ROS production at each time point after pretreatment with a test substance (bisomitin) or a control substance (mitoquinone) followed by treatment with cisplatin. When HEI-OC1 cell lines were treated with cisplatin alone, the amount of ROS produced gradually increased over time, and a significant increase in ROS was confirmed from 12 hours onwards compared to the control group (DMSO) (#p<0.05).

[0116] After treatment with bisomitin at concentrations of 0.5 uM or 1 uM, ROS production was significantly inhibited in the cisplatin-treated groups (*p<0.05, vs. cisplatin. Dunnett test). Meanwhile, in the mitoquinone-treated group used as a control, ROS production was significantly reduced in the 0.5 uM treatment group (6 hours treatment) and the 1 uM treatment group (12 hours treatment). Additionally, under 24-hour culture conditions, ROS production was significantly reduced in both the 0.5 uM and 1 uM treatment groups, but it was confirmed that ROS production was higher compared to bisomitin.

[0117] Example 1.2. Inhibitory effect of gentamicin on ROS generation after bisomitin pretreatment

[0118] Figure 2 shows the results of confirming ROS production by pre-treating with a test substance (bisomitin) or a control substance (mitoquinone), followed by treatment with gentamicin and reaction at each time interval. When gentamicin was treated alone on HEI-OC1 cell lines, the amount of ROS produced gradually increased over time, and ROS production significantly increased from 6 hours onwards compared to the control group (DMSO) (#p<0.05).

[0119] The results of treating with gentamicin for 6, 12, or 36 hours after treatment with bisomitin or mitoquinone at a concentration of 0.5 uM or 1 uM are as follows.

[0120] In the case of the bisomitin treatment group, ROS production was significantly inhibited at all bisomitin treatment concentrations 6 hours after gentamicin treatment. Additionally, 12 hours after gentamicin treatment, significant inhibition of ROS production was observed only in the 1 uM bisomitin treatment group, and 36 hours after gentamicin treatment, no significant inhibitory effect on ROS production was observed in any of the bisomitin treatment groups, but a decreasing trend was observed.

[0121] In the case of the mitoquinone treatment group, no significant ROS production inhibitory effect was observed at any mitoquinone treatment concentration after 6 hours of gentamicin treatment, but ROS production was significantly reduced in the mitoquinone 1 uM treatment group after 12 hours of gentamicin treatment. However, after 36 hours of gentamicin treatment, no ROS production inhibitory effect by mitoquinone treatment was observed again.

[0122] Through the above results, it was confirmed that bisomitin inhibits the production of ROS induced by treatment with the anticancer drug cisplatin or the antibiotic gentamicin, and that bisomitin is superior in this effect.

[0123] Example 1.3. Inhibitory effect of ROS generation by simultaneous treatment with bisomitin and gentamicin

[0124] Figure 3 shows the results of confirming the generation of ROS by simultaneously treating the test substance (bisomitin) or control substance (mitoquinone) and gentamicin and reacting at each time interval.

[0125] As a result, for all reaction times (12, 24, and 36 hours), the production of ROS decreased in a concentration-dependent manner with bisomitin treatment (*p<0.05). However, at the 36-hour reaction time, bisomitin showed a tendency to reduce ROS production in a concentration-dependent manner, but this was not statistically significant. On the other hand, no significant reduction in ROS production was confirmed with the control substance, mitoquinone, at any of the observation times.

[0126] At this time, in the group treated with gentamicin alone, ROS production increased with treatment time, and it was confirmed that ROS production significantly increased starting 12 hours after treatment compared to the control group (DMSO) (#p<0.05).

[0127] Example 2. Confirmation of protective effect against gentamicin-induced apoptosis

[0128] The protective effect of the antioxidant bisomitin against gentamicin-induced apoptosis in HEI-OC1 cells was evaluated.

[0129] Specifically, 5 x 10 HEI-OC1 cells per well in a 96-well plate 3 After inoculating the cells, they were cultured for 24 hours in DMEM medium containing 10% FBS at 37°C in a 5% CO2 incubator. After replacing the culture medium, the test substance (bisomitin) or control substance (mitoquinone) was added to a final concentration of 0.05 μM to 1 μM, followed by an additional 24 hours of culture. Then, gentamicin (10 mM) was added, and the cells were cultured for an additional 48 hours. The inhibitory effect of bisomitin on apoptosis was confirmed using a CCK-8 assay. For the CCK-8 assay, each well was treated with a CCK-8 solution and reacted for 3 hours, after which the OD value was measured at 450 nm. The results are shown in Figure 4.

[0130] As a result, cell viability decreased in the gentamicin-alone treatment group compared to the control group (DMSO) (#p<0.05). On the other hand, in the bisomitin-treated group, cell viability increased in the group treated with a concentration of 250 nM or higher compared to the gentamicin-alone treatment group (*p<0.05). No significant increase in cell viability was observed in the comparison drug, mitoquinone-treated group.

[0131] Example 3. Confirmation of the effects of bisomitin on reducing hair cell degeneration and inhibiting ROS production in the mouse cochlea

[0132] Example 3.1. Confirmation of the inhibitory effect of gentamicin on apoptosis following bisomitin treatment in hair cells

[0133] We confirmed the inhibitory effect of bisomitin on apoptosis of hair cells induced by gentamicin, an ototoxic substance, using hair cells obtained by isolating the cochlea of ​​2-day-old C57BL / 6 mice.

[0134] Cochleas extracted from mice were plated and cultured for 16 hours, after which bisomitin was added to a concentration of 0.25 µM or 0.5 µM. After incubation for 1 hour, gentamicin (100 µg / mL) was added, and the cells were cultured for an additional 36 hours. A DMSO group that was not treated with the test drug or gentamicin was provided as a normal control. After culture, the cells in the plate were fixed with 4% paraformaldehyde, stained sequentially with Myo7a, Phalloidin, and DAPI, mounted with antifade reagent, and the cells were counted.

[0135] At this time, Myo7a, Phalloidin and Cells that are positive for DAPI and exhibit a normal morphology were defined as living hair cells, and the said hair cells were counted. Mitoquinone (1 uM) was used as a control drug, and the cytotoxicity inhibitory effect was confirmed by the above method.

[0136] As a result, as shown in Figures 5a and 5b, hair cell death caused by gentamicin alone varied depending on the region of the cochlea. This result appears to be due to the different distributions of TRPV1 (transient receptor potential vanilloid 1) and TRPV4 within the cochlea. Generally, TRPV1 and TRPV4 are known to be abundant in the base and mid regions of the cochlea, while their distribution is less in the apex region (Leeet et al. 2013. doi:10.1038 / emm.2013.25). Therefore, this result is judged to be caused by the absorption of gentamicin into the hair cells by TRPV1 and TRPV4, resulting in different degrees of drug toxicity caused by gentamicin.

[0137] In contrast, the bisomitin-treated group showed an inhibitory effect on apoptosis in the base and mid regions of the cochlea (*p<0.05 vs. gentamicin). Meanwhile, the mitoquinone-treated group did not show a significant inhibitory effect on apoptosis induced by gentamicin in any region of the cochlea, and it was confirmed that apoptosis actually increased in the apex region (*p<0.05 vs. gentamicin).

[0138] Example 3.2. Confirmation of the inhibitory effect of gentamicin on ROS production following bisomitin treatment in hair cells

[0139] Aminoglycosides such as gentamicin are known to cause auditory toxicity by being absorbed by hair cells and generating ROS in the mitochondria within the hair cells. Accordingly, the inhibitory effect of bisomitin on ROS generation induced by gentamicin was confirmed using the same method as in Example 3.1 above. At this time, gentamicin was administered for 24 hours. In addition, the cells were treated with MitoSOX Red (5 uM) and DAPI, stained at 37°C for 12 minutes, washed three times with BSS (balanced salt solution), mounted with antifade reagent, and MitoSOX-positive hair cells were counted.

[0140] As a result, as shown in Figures 6a and 6b, it was confirmed that ROS production was significantly increased in all regions of the cochlea in the gentamicin-treated group compared to the control group (DMSO) (p<0.05). On the other hand, in the case of the bisomitin-treated group, ROS production decreased in the base region of the cochlea at all treatment concentrations. In particular, in the 0.5 uM bisomitin-treated group, ROS production was significantly reduced in the mid and apex regions as well (p<0.05 vs. gentamicin). Meanwhile, it was confirmed that ROS production was significantly reduced in the mid and apex regions in the control drug mitoquinone-treated group as well (p<0.05). However, the above inhibitory effect was weaker than the inhibitory effect caused by bisomitin treatment, and no ROS production inhibitory effect was observed in the base region; rather, it was confirmed that more ROS was produced compared to the gentamicin-alone-treated group.

[0141] II. Confirmation of hearing loss inhibitory activity at the in vivo level

[0142] Example 4. Confirmation of the hearing loss-improving effect of bisomitin in a gentamicin-induced zebrafish audiotoxicity model

[0143] Zebrafish are known to possess numerous neuromasts, which are clusters of cells such as hair cells and supporting cells, along their lateral line, exhibiting significant structural similarity to the hair cells of the mammalian inner ear. Therefore, the preventive effect against hearing loss induced by the acoustic toxicity of bisomitin was confirmed by treating the zebrafish with an acoustic toxic substance and bisomitin.

[0144] Zebrafish embryos (5–6 dpf) were obtained from healthy zebrafish (Danio rerio) parents of clear origin. 5–10 embryos per well were pretreated with bisomitin at various concentrations for 1 hour in 6-well plates, followed by treatment with gentamicin (5 uM), known as an ototoxic substance, and incubation for an additional 1 hour. The embryos were then stained with Yo-Pro 1 solution. Afterward, the embryos were anesthetized with tricaine, fixed to slide glasses using 1.5% methylcellulose, and the number of hair cells (SO1, SO2, O1, OC1) was counted using a fluorescence microscope. Subsequently, the total number of hair cells identified for each individual was summed to evaluate the inhibitory activity of bisomitin on hair cell apoptosis induced by gentamicin treatment. The test groups are shown in Table 1 below.

[0145] Test Group Test Substance Concentration (uM) Gentamicin 5 uM Normal Group (DMSO) -- Induced Group (DMSO) -- + Bisomitin 0.1, 0.5 + L-Selenomethionine 100, 500 + N,N'-Dimethylurea 500, 1000 + Edaravone 1000 +

[0146] As a result, as shown in Figures 7a and 7b, the number of hair cells was significantly reduced in the induction group (gentamicin-treated group) compared to the normal group (DMSO) (p<0.05). On the other hand, the number of hair cells in the bisomitin-treated group showed a tendency to increase in a concentration-dependent manner, and in particular, a significant increase in the number of hair cells was observed in the 0.5 uM-treated group (p<0.05 vs. induction group). Meanwhile, L-selenomethionine and N,N'-dimethylurea, used as comparator drugs, did not significantly increase the number of hair cells compared to the gentamicin-treated group, even though they were treated at high concentrations (100 uM to 1000 uM). Conversely, a significant increase in the number of hair cells was confirmed in the group treated with edaravone (1000 uM), another control drug (p<0.05).

[0147] The above results demonstrate that bisomitin exhibits excellent toxicity-protective effects against gentamicin toxicity, suggesting that not all antioxidants equally prevent auditory toxicity caused by anticancer drugs or antibiotics.

[0148] From the above results, it was confirmed that bisomitin inhibits the production of ROS induced by gentamicin or cisplatin and inhibits the death of hair cells, thereby preventing auditory toxicity caused by ototoxic drugs.

Claims

1. A pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient: <Chemical Formula 1> At this time, the above X is a 1 to 3 valent anion.

2. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus, wherein the hearing loss is conductive hearing loss or sensorineural hearing loss.

3. In Paragraph 2, A pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus, wherein the sensorineural hearing loss is noise-induced hearing loss, sudden hearing loss, infectious hearing loss, traumatic hearing loss, presbycusis, ototoxic hearing loss, autoimmune hearing loss, Meniere's disease, or hearing loss caused by nerve damage.

4. In Paragraph 2, A pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus, wherein the above-mentioned ototoxic hearing loss is caused by the use of anticancer agents, antibiotics, or radiation.

5. In Paragraph 4, A pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus, wherein the above anticancer agent is one or more selected from the group consisting of taxane-based anticancer agents, platinum-based anticancer agents, and vinca alkaloid-based anticancer agents.

6. In Paragraph 4, A pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus, wherein the above antibiotic is one or more selected from the group consisting of beta-lactams, sulfonamides, aminoglycosides, tetracyclines, glycopeptides, ansamycins, penicillins, macrolides, streptogramins, cephalosporins, and quinolones.

7. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of hearing loss or tinnitus, wherein the tinnitus is any one selected from the group consisting of objective tinnitus, subjective tinnitus, peripheral tinnitus, and central tinnitus.

8. A food composition for the prevention or improvement of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient: <Chemical Formula 1> At this time, the above X is a 1 to 3 valent anion.

9. A feed composition for the prevention or improvement of hearing loss or tinnitus comprising a compound of the following chemical formula 1 as an active ingredient: <Chemical Formula 1> At this time, the above X is a 1 to 3 valent anion.

10. Use of the compound of Chemical Formula 1 below for the prevention or treatment of hearing loss or tinnitus: <Chemical Formula 1> At this time, the above X is a 1 to 3 valent anion.

11. A method for the prevention or treatment of hearing loss or tinnitus comprising the step of administering the following chemical formula 1 to an individual: <Chemical Formula 1> At this time, the above X is a 1 to 3 valent anion.

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