Mitochondrial permeability transition pore-targeting composition for treating hearing loss

Abstract

The present invention relates to a mitochondrial permeability transition pore (mPTP)-targeting composition for treating hearing loss, and, more specifically, uses a compound, or a pharmaceutically / sitologically acceptable salt thereof, which targets the mitochondrial permeability transition pore (mPTP) to inhibit the activity or opening thereof, and thus can effectively prevent, alleviate, or treat hearing loss caused by mPTP opening.

Description

Mitochondrial permeability transition pore-targeting composition for the treatment of hearing loss

[0001] The present invention relates to a treatment for hearing loss, and more particularly, to a composition for treating hearing loss targeting the mitochondrial permeability transition pore.

[0002] Mitochondria play a crucial role in cellular energy supply and contribute to the intrinsic apoptotic pathway, the extrinsic apoptotic pathway, receptor-mediated apoptosis, and various forms of non-apoptotic cell death. When mitochondrial oxidative stress increases ROS, they are released into calcium carriers. This process triggers the persistent opening of the mitochondrial permeability transition pore (mPTP), exacerbating cellular damage.

[0003] The mPTP, a multiprotein complex, opens under conditions such as excessive calcium accumulation in mitochondria or increased oxidative stress. The mPTP complex consists of various components, including a voltage-dependent anion channel located in the outer mitochondrial membrane; an adenine nucleotide translocator, an inorganic phosphate transporter, and an ATP synthase located in the inner mitochondrial membrane; and cyclophilin D located in the mitochondrial matrix. The mPTP acts as a non-selective channel, allowing the accumulation of ions or increased ROS and Ca 2+ Promotes controlled release of newly formed low molecular weight toxic substances, including:

[0004] Irreversible mPTP opening has been linked to various diseases. For example, a knockout (ppif) mouse model encoding cypD exhibited increased mitochondrial calcium retention and decreased cytosolic calcium release in acute pancreatitis. These changes were manifested by higher mitochondrial membrane potential compared to controls, ultimately leading to suppressed cytochrome c (cytC) release, reduced apoptosis, and enhanced cell viability. Interpretation suggests that ppif gene knockout suppresses mPTP opening, thereby maintaining mitochondrial function and enhancing cell survival.

[0005] Additionally, cyclosporine A (CysA), known to prevent mPTP opening, is used in the treatment of Alzheimer's disease (AD), a disease characterized by amyloid-beta aggregates. Studies have shown that CysA effectively prevents mPTP opening, thereby reducing oxidative stress in AD, suggesting that mPTP may be a promising therapeutic target for AD.

[0006] However, little has been reported to date on the protective or therapeutic effects achieved through inhibition of mPTP opening in hearing loss diseases.

[0007] The purpose of the present invention is to provide a compound having an excellent hearing protection effect by targeting the mitochondrial permeability transition pore (mPTP) and a hearing loss treatment using the compound.

[0008] In order to achieve the above purpose, the present invention provides a pharmaceutical composition for treating or preventing hearing loss, containing a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[0009] <Chemical Formula 1>

[0010]

[0011] In the above chemical formula 1, R 1 silver or , and R' and R'' may be the same or different, and are selected from a 4- to 8-membered azacyclic group or a 7- to 9-membered azabicyclic group, and the azacyclic group or azabicyclic group may be substituted or unsubstituted with (C1-C6)alkyl or benzyl, and R 2 and R 3 may be the same or different, and may be selected from hydrogen, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl or (C1-C6)alkoxy.

[0012] In addition, the present invention provides a health functional food composition for improving or preventing hearing loss, containing a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0013] The compound according to the present invention or a pharmaceutically / foodstuffally acceptable salt thereof can effectively prevent, improve or treat hearing loss caused by mPTP opening by targeting the mitochondrial permeability transition pore (mPTP) and inhibiting its activity or opening.

[0014] In particular, the compound has an excellent protective effect against ototoxic hearing loss caused by cisplatin by inhibiting mPTP opening, and thus can be used as an effective treatment for ototoxic hearing loss.

[0015] Figure 1 shows the mitochondrial permeability transition pore (mPTP) inhibitor library. (A) MTT assay was performed on HEI-OC1 cells, a hearing loss model cell line, after treating them with 1 or 10 μM mPTP inhibitor compounds for 30 hours, and cytotoxicity was measured. (B) MTT assay was performed on cells cultured with 1 or 10 μM mPTP inhibitor compounds for 1 hour, then treated with 20 μM cisplatin for 30 hours, and protective effects were measured. # indicates untreated group with high cytotoxicity, and the percentage of viable cells in each group is expressed as mean ± standard deviation.

[0016] Figures 2 to 6 show the results of selecting five hit compounds by confirming the cytotoxicity and protective effects of mPTP inhibitor compounds.

[0017] Figure 7 shows the cell viability of compound 4 (DBP-iPT) in HEI-OC1 cells. (B) shows cells pretreated with various concentrations (0.1, 0.5, 1, 5, 10, 20, 30, or 40 μM) of DBP-iPT for 1 hour and then treated with 20 μM cisplatin for 30 hours, and (C) shows cells incubated with various concentrations of DBP-iPT for 30 hours and then cytotoxicity was measured (the percentage of viable cells in each group is expressed as the mean ± standard deviation, **P < 0.01, ***P < 0.001).

[0018] Figure 8 shows the evaluation of the inhibitory effect of cisplatin treatment on mPTP opening of DBP-iPT in HEI-OC1 cells. The group treated with calcein and CoCl2 together exhibits a degree of mPTP opening, whereas ionomycin acts as a persistent mPTP activator.

[0019] Figure 9 shows the results of evaluating the calcium concentration in mitochondria and cytoplasm. (A) is a representative image of cells stained with Fluo-4AM (500 nM) (nuclei are shown in blue, cytoplasmic calcium is shown in green, Scale bar = 50 μm), and (B) is a representative image of cells stained with Rhod-2AM (400 nM) (nuclei are shown in blue, mitochondrial calcium is shown in red, Scale bar = 50 μm) (The fluorescence intensity values ​​of each group are expressed as the mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001).

[0020] Figures 10a to 10c show the evaluation of the ROS scavenging effect and mitochondrial membrane potential restoration of DBP-iPT in HEI-OC1 cells. 10a is an evaluation of intracellular ROS levels using DCFDA fluorescent dye, 10b is a detection of drug-treated mitochondrial superoxide using MitoSOX, which is stained red and nuclei are stained blue (Scale bar = 50 μm), and 10c is a measurement of mitochondrial membrane potential using TMRE fluorescent dye (the percentage of viable cells in each group is expressed as the mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001).

[0021] Figure 11 shows the analysis of mitochondrial morphology. (A) HEI-OC1 cells were pretreated with 30 μM DBP-iPT for 1 h and then treated with 20 μM cisplatin for 24 h, and then stained with MitoTracker Red (500 nM). The nucleus is shown in blue and the mitochondria in red (Scale bar = 10 μm). (B) HEI-OC1 cells were pretreated with 30 μM DBP-iPT for 1 h and then treated with 20 μM cisplatin for 24 h, and then analyzed by TEM. Red arrows indicate mitochondria, and green arrows indicate cristae (Scale bar = 1 μm). (The proportion of elongated / fragmented mitochondria in each group is expressed as the mean ± standard deviation, *P < 0.05, **P < 0.01).

[0022] Figure 12 evaluates the expression level of apoptosis (cell death) in HEI-OC1 cells. (A) Flow cytometry and FITC-annexin V-PI analysis confirmed the effect of mPTP opening inhibition on cisplatin-induced apoptosis, and the bar graph showing the quantification of early and late apoptosis, * indicates comparison with the control group, and # indicates comparison with the cisplatin-treated group. (B) Western blot analysis of the expression of cleaved caspase-3 in HEI-OC1 cells, and the relative ratio of caspase-3 expression level was measured using the caspase-3:β-actin ratio (The apoptosis ratio values ​​in each group are expressed as the mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01).

[0023] Figure 13 shows the results of measuring the hearing of mice injected with cisplatin to evaluate the protective effect of DBP-iPT pretreatment. (A) The threshold shift change of auditory brainstem response (ABR) is confirmed, and (B) The result of phalloidin staining of the organ of Corti. White asterisks indicate degenerating hair cells. * indicates comparison with the control group, and # indicates comparison with the cisplatin-treated group. (Scale bar = 50 μm) (The apoptosis rate values ​​of each group are expressed as the mean ± standard deviation, and *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01).

[0024] Figure 14 schematically illustrates that the compound (DBP-iPT) according to the present invention has a protective effect against cisplatin-induced hearing loss by targeting mPTP.

[0025] Hereinafter, the present invention will be described in detail.

[0026]

[0027] The present inventors developed a compound targeting the mitochondrial permeability transition pore (mPTP) associated with various diseases, and confirmed that the compound effectively inhibits the activation or opening of mPTP and thus has a protective effect against cisplatin-induced ototoxicity, thereby completing the present invention.

[0028]

[0029] The present invention provides a pharmaceutical composition for treating or preventing hearing loss, containing a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[0030] <Chemical Formula 1>

[0031]

[0032] In the above chemical formula 1,

[0033] R 1 silver or Selected from,

[0034] R' and R'' may be the same or different, and are selected from a 4- to 8-membered azacyclic group or a 7- to 9-membered azabicyclic group, wherein the azacyclic group or azabicyclic group may be substituted or unsubstituted with (C1-C6)alkyl or benzyl, and R 2 and R 3 may be the same or different, and may be selected from hydrogen, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl or (C1-C6)alkoxy.

[0035] Specifically, in the R' and R'', the azacyclic group is pyrrolidinyl and the azabicyclic group is quinuclidinyl, and the azacyclic group or the azabicyclic group may be substituted or unsubstituted with one or more substituents selected from the group consisting of methyl, ethyl, isopropyl, tert-butyl, and benzyl.

[0036] Preferably, R' and R'' are selected from quinuclidin-3-yl or isopropylpyrrolidin-3-yl, and R 2 and R 3 may be selected from hydrogen, fluoro, chloro, or trifluoromethyl.

[0037] More preferably, the compound or a salt thereof may be selected from the group consisting of 2-(4-(trifluoromethyl)benzyloxy)benzaldehyde O-quinuclidin-3-yl oxime, 2-(benzyloxy)benzaldehyde O-(1-isopropylpyrrolidin-3-yl) oxime, 2-((3-fluorobenzyl)oxy)benzaldehyde O-(1-isopropylpyrrolidin-3-yl) oxime, 4-(2-((3,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole and 4-(2-((2,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole, but is not limited thereto.

[0038]

[0039] The above compound can be used in the form of a pharmaceutically or food-wise acceptable salt within the range having the same efficacy.

[0040] As used herein, “pharmaceutically or food-wise acceptable salt” means a salt that is non-toxic to cells or humans exposed to the composition and has a safety and efficacy profile suitable for administration to humans.

[0041] The above salt may be used in the form of either a basic salt or an acid salt that is pharmaceutically or food-wise acceptable. The basic salt may be used in the form of either an organic basic salt or an inorganic basic salt, and may be selected from the group consisting of sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyridinium salt. The acid salt is useful as an acid addition salt formed by a free acid. Inorganic acids and organic acids can be used as free acids, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, diphosphoric acid, nitric acid, etc. can be used, and organic acids such as citric acid, acetic acid, maleic acid, malic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, stearic acid, etc. can be used, but are not limited thereto, and all salts formed using various inorganic acids and organic acids commonly used in the art can be included.

[0042] In addition, the compound may include not only the above salts, but also all salts, hydrates, solvates, derivatives, etc. that can be prepared by conventional methods. Addition salts can be prepared by conventional methods, and can be prepared by dissolving in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, adding an excess amount of organic base, or adding an aqueous base solution of an inorganic base, and then precipitating or crystallizing. Alternatively, the addition salt can be obtained by evaporating the solvent or the excess amount of base from the mixture and then drying, or by suction filtration of the precipitated salt.

[0043]

[0044] The compound or salt thereof can target the mitochondrial permeability transition pore (mPTP) and inhibit its activity or opening, thereby having a protective effect against cisplatin-induced ototoxicity.

[0045] Specifically, mPTP opens under conditions such as excessive calcium accumulation in mitochondria or increased oxidative stress, and cisplatin-induced hearing loss can also be caused by mPTP opening. According to one experimental example of the present invention, the compound reduces intracellular ROS levels, but does not affect the reduction of mitochondrial ROS levels, and the mitochondrial membrane potential does not decrease and the normal morphology of mitochondria is maintained even when the mitochondrial ROS level does not decrease or increases, thereby showing a mechanism different from the conventional mechanism. With this new mechanism, the compound according to the present invention can effectively prevent cisplatin-induced hearing loss by inhibiting mPTP opening. In addition, the compound can inhibit apoptosis induced by cisplatin and alleviate damage to inner / outer hair cells.

[0046]

[0047] The composition may contain the compound or its salt at a concentration of 0.1 to 40 μM, preferably, 10 to 30 μM, but is not limited thereto. Since it has been confirmed that auditory cells can be protected at a high level without cytotoxicity within the above range, the above range is preferred.

[0048]

[0049] Due to these effects, the compound or its salt can be used as a pharmaceutical composition for treating or preventing hearing loss.

[0050] In this specification, “hearing loss” means a disease that causes hearing impairment or hearing loss.

[0051] The above hearing loss may be conductive hearing loss, which occurs when sound waves are not transmitted normally due to a disorder in the organs such as the outer ear, eardrum, or middle ear, or sensorineural hearing loss, which occurs when there is a problem with the function of the cochlea or an abnormality in the auditory nerve or central nervous system that transmits auditory stimuli to the brain.

[0052] Specifically, the hearing loss may be any one selected from ototoxic hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, diabetic auditory neuropathy, traumatic hearing loss, and viral hearing loss, and preferably ototoxic hearing loss.

[0053] The above ototoxic hearing loss may be caused by the use of drugs such as anticancer drugs and antibiotics, chemicals, or radiation, and more preferably, it may be ototoxic hearing loss caused by ototoxicity induced by the anticancer drug cisplatin, but is not limited thereto.

[0054]

[0055] In this specification, "pharmaceutical composition" means a composition administered for the purpose of preventing or treating a specific disease, and for the purpose of the present invention, means a composition administered for preventing or treating hearing loss or at least one symptom thereof.

[0056] The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition can be combined with an appropriate pharmaceutically acceptable carrier depending on the formulation, and, if necessary, can be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The appropriate carrier, etc., can be selected differently depending on the dosage form and formulation as long as it does not inhibit the activity and properties of the compound according to the present invention or a pharmaceutically acceptable salt thereof.

[0057] The above pharmaceutical composition can be applied in any dosage form, and more specifically, it can be formulated and used as an oral dosage form and a parenteral dosage form of an external preparation, a transdermal patch, a suppository, and an injection according to a conventional method.

[0058] Among the oral dosage forms, the solid dosage forms are in the form of tablets, pills, powders, granules, capsules, etc., and can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc., and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of capsule dosage forms, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included. Among the oral dosage forms, the liquid dosage forms include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

[0059] The above parenteral formulations may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, glycerogelatin, and the like. Without limitation, any suitable formulation known in the art may be used.

[0060]

[0061] The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

[0062] As used herein, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing adverse effects.

[0063] The effective dosage level of the pharmaceutical composition may vary depending on the intended use, the patient's age, sex, weight, and health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the duration of treatment, the drugs used in combination or concurrently, and other factors well known in the medical field. For example, although not fixed, it may generally be administered once or several times daily at a dosage of 0.001 to 1000 mg / kg, preferably 0.01 to 100 mg / kg. The above dosage does not limit the scope of the present invention in any way.

[0064] The pharmaceutical composition may be administered to any animal capable of developing hearing loss, and the animal may include, for example, humans and primates, as well as livestock such as cows, pigs, horses, and dogs.

[0065] The above pharmaceutical composition may be administered via an appropriate route of administration depending on the formulation, and may be administered via various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited, and may be administered by conventional methods such as oral, transdermal, subcutaneous, rectal, intravenous, intramuscular, skin application, respiratory inhalation, intrauterine epidural, or intracerebroventricular injection.

[0066] The above pharmaceutical composition can be used alone for the prevention or treatment of hearing loss, or can be used in combination with surgery or other drug treatments.

[0067]

[0068] In addition, the present invention provides a health functional food composition for improving or preventing hearing loss, containing a compound represented by the following chemical formula 1 or a food-related acceptable salt thereof as an active ingredient:

[0069] <Chemical Formula 1>

[0070]

[0071] In the above chemical formula 1, R 1 silver or , and R' and R'' may be the same or different, and are selected from a 4- to 8-membered azacyclic group or a 7- to 9-membered azabicyclic group, and the azacyclic group or azabicyclic group may be substituted or unsubstituted with (C1-C6)alkyl or benzyl, and R 2 and R 3 may be the same or different, and may be selected from hydrogen, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl or (C1-C6)alkoxy.

[0072] The corresponding features can be replaced with the above-described parts.

[0073]

[0074] In this specification, "health functional food" includes food manufactured and processed using raw materials or ingredients with functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means a food with high medical or healthcare effects that is processed to efficiently exhibit bioregulatory functions such as prevention of hearing loss or its symptoms, biodefense, immunity, and recovery, in addition to providing nutrition, for the purpose of the present invention.

[0075] In the health functional food composition according to the present invention, the health functional food may be manufactured in the form of powder, granules, tablets, capsules, syrup, or beverage, etc., for the purpose of preventing or improving hearing loss. There is no limitation on the form that the health functional food may take, and it may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods.

[0076] The above health functional foods may include all foods in the conventional sense. For example, they may include beverages and various drinks, fruits and their processed foods (canned fruits, jams, etc.), fish, meats and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.), and all functional foods in the conventional sense. They may also include foods used as animal feed.

[0077] The above health functional food composition may be manufactured by further including food additives (food additives) commonly used in the art and other appropriate auxiliary ingredients that are acceptable in terms of food science. Unless otherwise specified, suitability as a food additive may be determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. Items listed in the above 'Food Additives Codex' include, for example, chemical synthetics such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sucrose pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0078] The above other auxiliary ingredients may additionally contain, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. In particular, as the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol can be used, and as the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used.

[0079]

[0080] The effective dosage of the compound or its salt contained in the health functional food composition according to the present invention can be appropriately adjusted depending on the purpose of use, such as prevention or improvement of hearing loss.

[0081] The above health functional food composition has the advantage of not having side effects that may occur with long-term use of general medicines as it is made from food, and is highly portable, so it can be taken as a supplement for preventing or improving hearing loss.

[0082] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0083]

[0084] <Experimental Example 1> Selection of compounds that inhibit the mitochondrial permeability transition pore (mPTP).

[0085] A chemical library of mPTP inhibitors was screened and their cytotoxicity and protective effects were investigated in HEI-OC1 cells, a hearing loss model cell line (cochlea cell line) (Fig. 1).

[0086] Among the mPTP inhibitor compounds of Fig. 1, the cytotoxicity and protective effects of 15 compounds (HYSM000030, 32, 37, 39, 41, 42, 43, 55, 58, 62, 63, 65, 66, 67, and 68) were additionally confirmed by varying the concentration of the compounds to 0.1, 1, and 10 μM using the same MTT assay method as in Fig. 1.

[0087] As a result, as shown in FIGS. 2 to 6 and Table 1 below, five compounds (HYSM000032, 41, 43, 65, and 66) exhibited excellent protective effects without cytotoxicity, and among them, HYSM000065 [DBP-iPT; (4-(2-((3,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole)] was confirmed to exhibit particularly excellent effects. Accordingly, in the following experimental examples, experiments were conducted focusing on DBP-iPT.

[0088] Table 1 below shows the analysis results of 15 compounds.

[0089]

[0090]

[0091] The five compounds selected are:

[0092] Compound 1 (HYSM000032): 2-(4-(trifluoromethyl)benzyloxy)benzaldehyde O-quinuclidin-3-yl oxime

[0093]

[0094] 1 H NMR (CDCl3, 300 MHz) δ8.55 (s, 1H), 7.82 (dd,J= 7.7, 1.6 Hz, 1H), 7.66 (d,J= 8.2 Hz, 2H), 7.55 (d,J= 8.1 Hz, 2H), 7.31 (m, 1H), 6.97 (t,J= 3.8 Hz, 1H), 6.91 (d,J= 8.3 Hz, 1H), 5.16 (s, 2H), 4.40 (m, 1H), 3.21 (m, 1H), 2.78-2.95 (m, 5H), 2.25 (m, 1H), 1.86 (m, 1H), 1.73 (m, 1H), 1.58 (m, 1H), 1.37 (m, 1H)

[0095]

[0096] Compound 2 (HYSM000041): 2-(Benzyloxy)benzaldehyde O-(1-isopropylpyrrolidin-3-yl) oxime

[0097]

[0098] 1 H NMR (CDCl3, 300 MHz) δ8.53 (s, 1H), 7.81 (dd,J= 7.9, 1.7 Hz, 1H), 7.28-7.42 (m, 6H), 6.95 (m, 2H), 5.08 (s, 2H), 4.88 (m, 1H), 2.84-2.95 (m, 3H), 2.62 (m, 1H), 2.49 (quin,J= 6.3 Hz, 1H), 2.23 (m, 1H), 2.05 (m, 1H), 1.15 (dd,J= 3.5, 6.3 Hz, 6H)

[0099] 13 C NMR (CDCl3, 75 MHz) δ156.7, 145.2, 136.5, 131.0, 128.6, 128.0, 127.4, 126.5, 121.3, 121.0, 112.4, 82.2, 70.3, 57.5, 55.2, 50.6, 31.0, 21.2

[0100]

[0101] Compound 3 (HYSM000043): 2-((3-fluorobenzyl)oxy)benzaldehyde O-(1-isopropylpyrrolidin-3-yl) oxime

[0102]

[0103] 1 H NMR (CDCl3, 300 MHz) δ8.52 (s, 1H), 7.81 (dd,J= 7.7, 1.7 Hz, 1H), 7.28-7.38 (m, 2H), 7.15 (m, 2H), 6.89-7.05 (m, 3H), 5.07 (s, 2H), 4.89 (m, 1H), 2.85-2.96 (m, 3H), 2.63 (m, 1H), 2.49 (m, 1H), 2.24 (m, 1H), 2.0 (m, 1H), 1.16 (dd,J= 3.6, 6.3 Hz, 6H)

[0104] 13 C NMR (CDCl3, 75 MHz) δ164.6, 156.3, 145.1, 139.0, 131.0, 130.2, 130.1, 126.7, 122.7, 122.6, 121.2, 115.0, 114.8, 114.3, 114.0, 112.3, 82.2, 69.5, 57.4, 55.2, 50.6, 31.0, 21.2

[0105]

[0106] Compound 4 (HYSM000065): 4-(2-((3,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole [DBP-iPT]

[0107]

[0108] 1 H NMR (CDCl3, 400 MHz) δ8.36 (dd,J= 7.7, 1.6 Hz, 1H), 8.10 (s, 1H), 7.48 (d,J= 6.8 Hz, 2H), 7.44-7.35 (m, 3H), 7.30 (t,J= 8.6 Hz, 1H), 7.10 (t,J= 7.4 Hz, 1H), 7.06 (d,J= 8.2 Hz, 1H), 5.18 (m, 1H), 5.15 (s, 2H), 2.83 (m, 1H), 2.75 (m, 2H), 2.45 (m, 2H), 2.30 (s, 3H), 1.99 (m, 1H);

[0109] 13 C NMR (CDCl3, 100 MHz) δ 154.8, 143.3, 136.7, 128.7, 128.6, 128.2, 128.0, 127.7, 127.3, 121.5, 121.3, 120.0, 111.9, 70.6, 62.4, 59.6, 54.9, 41.6, 33.0

[0110]

[0111] Compound 5 (HYSM000066): 4-(2-((2,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole

[0112]

[0113] 1H NMR (CDCl3, 400 MHz) δ8.31 (d,J= 7.6 Hz 1H), 8.08 (s, 1H), 7.45 (s, 1H), 7.41 (d,J= 8.2 Hz, 1H), 7.27 (q,J= 6.9 Hz, 2H), 7.10 (t,J= 3.7 Hz, 1H), 6.98 (d,J= 8.1 Hz, 1H), 5.22 (d,J= 4.8 Hz, 3H), 3.04 (t,J= 8.0 Hz, 1H), 2.84 (t,J= 8.4 Hz, 2H), 2.65 (d,J= 6.7 Hz, 1H), 2.49 (m, 2H), 2.03 (d,J= 4.9 Hz, 1H), 1.04 (t,J= 7.2 Hz 6H);

[0114] 13 C NMR (CDCl3, 100 MHz) δ154.3, 143.2, 134.7, 133.0, 130.6, 129.5, 128.8, 128.0, 127.4, 121.8, 121.4, 112.1, 67.3, 59.1, 57.9, 54.0, 50.3, 32.4, 21.4, 21.2

[0115]

[0116] <Experimental Example 2> Confirmation of mitochondrial function activation following treatment with DBP-iPT (Compound 4)

[0117] 1. Experimental method

[0118] 1-1. Cell culture and cell viability analysis

[0119] HEI-OC1 (House Ear Institute-Organ of Corti 1) auditory cells were cultured in Dulbecco's modified Eagle medium (Hyclone, Logan, UT, USA) for 16 h. The cell culture medium consisted of 10% fetal bovine serum (Hyclone, Logan, UT, USA) and interferon-γ (50 units / mL) (PeproTech EC Ltd., London, UK). Cells were cultured under permissive conditions (33°C, 10% CO2). Cells were pretreated with various concentrations of DBP-iPT (0.1, 0.5, 1, 5, 10, 20, 30, or 40 μM) before exposure to 20 μM cisplatin after 1-h intervals.

[0120] Cell viability assessment was performed 30 h after cisplatin treatment using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO, USA).

[0121]

[0122] 1-2. Flow cytometry

[0123] HEI-OC1 cells were cultured for 16 h (2 × 10 in 6-well plates) 5 (cells / well) were treated with 30 μM DBP-iPT. Afterwards, 20 μM cisplatin was added, and the cells were cultured for 24 h (ROS scavenging assay) or 30 h (mitochondrial membrane potential recovery assay).

[0124] MitoProbe for flow cytometry TMTransition pore assay kit (Invitrogen, Molecular Probes, Eugene, OR, USA, M34153), 2′,7′-dichlorofluorescein diacetate (DCFDA; Invitrogen, Molecular Probes, Eugene, OR, USA), tetramethyl rhodamine ethyl ester (TMRE; Abcam, ab113852), and annexin V (Invitrogen, Molecular Probes) were used. Quantification of cell ratios followed the recommended protocols for each fluorescent dye and was performed using an LSRFortessa X-20 flow cytometer (BD Biosciences, San Diego, CA, USA) in all experiments. Single cells were counted at a density of 1 × 10 4 It was calculated as cells.

[0125]

[0126] 1-3. Cell imaging analysis

[0127] HEI-OC1 cells were cultured for 16 h (2 × 10 in 6-well plates) 5 (cells / well) were treated with 30 μM DBP-iPT. Afterwards, 20 μM cisplatin was added, and the cells were cultured for 24 hours.

[0128] For imaging, Rhod-2AM, Fluo-4AM, MitoSOX, and MitoTracker Red (sourced from Invitrogen-Molecular Probes, Eugene, OR, USA) were used. All fluorescence images were captured using live-cell imaging technology using ImageXpress® Micro Confocal (Molecular Devices).

[0129]

[0130] 1-4. Transmission electron microscope (TEM) analysis

[0131] HEI-OC1 cells were cultured for 16 h (1 × 10 6 (cells / 100 mm cell culture dish) were treated with 30 μM DBP-iPT. Afterwards, 20 μM cisplatin was added, and the cells were cultured for an additional 24 hours.

[0132] After this incubation period, cells were scraped from the cell culture plates, pelleted by centrifugation, and sequentially fixed in 3% glutaraldehyde and 1% osmium tetroxide for 1 h on ice. Further processing included washing the samples with 0.1 M cacodylate buffer (pH 7.2) containing 0.1% CaCl2, followed by dehydration using a series of ethanol and propylene oxide solutions. The samples were then placed in an Epon 812 mixture and polymerized in an oven at 60°C for 36 h. Ultrathin sections of 70 nm thickness were cut using an ULTRACUT UC7 ultramicrotome (Leica, Austria) and mounted on 75-mesh copper grids. After sectioning, sections were counterstained with uranyl acetate for 10 minutes and lead citrate for 7 minutes. Final examination and imaging were performed using a KBSI Bio-High Voltage EM system (JEM-1400 Plus at 120 kV and JEM-1000BEF at 1000 kV; JEOL Ltd., Tokyo, Japan).

[0133]

[0134] 1-5. Expression analysis of apoptotic cell death proteins using Western blot

[0135] HEI-OC1 cells were cultured for 16 h (1 × 10 6(cells / 100 mm cell culture dish) were treated with 30 μM DBP-iPT. Afterwards, 20 μM cisplatin was added, and the cells were cultured for an additional 30 hours.

[0136] HEI-OC1 cells were thoroughly washed with ice-cold phosphate-buffered saline (PBS) and scraped in 100 μL of RIPA buffer (Elpis Biotech, Daejeon, Korea) containing a protease inhibitor cocktail (GenDEPOT, Katy, TX, USA). The supernatant was carefully transferred to an ice-cold tube and centrifuged at 13,000 rpm for 30 min at 4°C. After centrifugation, the supernatant was aspirated and transferred to a new tube kept on ice. Protein extracts from HEI-OC1 cells were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were electrotransferred to a nitrocellulose membrane. The membranes were blocked for 1 h with a solution containing 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.01% Tween-20 (TBS-T) containing 5% skimmed milk. After the blocking step, the membranes were treated with primary antibody (rabbit cleaved caspase-3, 1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Next, the membranes were washed three times with TBS-T for 5 min each and then incubated with secondary antibody (goat anti-rabbit-lgG-HRP, 1:1000 dilution; Cell Signaling Technology) for 1 h at room temperature. The membranes were finally detected using an enhanced chemiluminescence detection system.

[0137]

[0138] 1-6. Measurement of auditory brainstem response (ABR)

[0139] Eight-week-old male mice were purchased from Hyochang Science (Daegu, Republic of Korea). All animal procedures were conducted in compliance with the institutional animal care guidelines issued by the Animal Research Committee of Kyungpook National University.

[0140] DBP-iPT (43 mg / kg) was administered intraperitoneally to mice twice daily at 12-h intervals for 5 days. Cisplatin (20 mg / kg) was administered 12 h after the last DBP-iPT injection, and ABR was measured 4.5 days after cisplatin administration.

[0141] Auditory function was assessed using an ABR workstation (System 3, Tucker Davis Technology, Inc., Alachua, FL, USA) using an attenuated brainwave response (ABR). Stimuli were repeated 500 times at 5-dB increments, starting at a 90-dB sound pressure level and progressing to the acoustic threshold for each frequency. To mitigate artifacts caused by repetitive stimulation, the phase of the stimulus was reversed during each presentation.

[0142]

[0143] 1-7. Statistical Analysis

[0144] Statistical significance was determined using a two-tailed Student's t-test to compare the treated and untreated groups. A significance level of p < 0.05 was adopted.

[0145]

[0146] 2. Experimental results

[0147] 2-1. Confirmation of the protective effect of compound 4 (DBP-iPT) against auditory cell damage caused by cisplatin-induced ototoxicity.

[0148] Among the five selected compounds, DBP-iPT (4-(2-((3,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole) was investigated for its cytotoxicity and protective effects in HEI-OC1 cells. DBP-iPT consists of three components: a triazole core, N-isopropylpyrrolidin-3, and a benzyloxyphenyl group. In this experimental example, the protective effect of DBP-iPT against cisplatin-induced ototoxicity was investigated and the cytotoxicity of DBP-iPT itself was evaluated.

[0149] HEI-OC1 cells derived from the inner ear of mice were pretreated with DBP-iPT at concentrations of 0.1 to 40 μM for 1 hour before cisplatin treatment to evaluate cell viability. As shown in Fig. 7, the cisplatin-treated group showed a viability of 40% compared to the control group, whereas the group pretreated with DBP-iPT at a concentration of 30 μM showed a viability of approximately 80% without any cytotoxic effect (n = 4 per group). Therefore, the next experiment was conducted using DBP-iPT at a concentration of 30 μM.

[0150]

[0151] To evaluate the inhibitory effect of cisplatin treatment on mPTP opening in HEI-OC1 cells using mPTP inhibitor compounds, an mPTP opening assay was performed. As shown in Fig. 8, the mean fluorescence value was found to decrease by approximately 20% in the cisplatin-treated group compared to the cobalt chloride (CoCl2) control group, whereas the group pretreated with DBP-iPT recovered to the control group. In addition, when ionomycin, a substance that promotes calcium influx, was administered, the fluorescence in the cisplatin-treated group decreased compared to the control group, whereas the fluorescence in the DBP-iPT-treated group increased by approximately 1.5 times compared to the control group. These findings suggest that the compound can effectively prevent cisplatin-induced hearing loss by blocking mPTP opening.

[0152]

[0153] 2-2. Confirmation of DBP-iPT's calcium blocking ability

[0154] mPTP opening is intricately linked to cellular calcium concentration. Under stress conditions induced by factors such as gradual calcium loading and increased ROS production, calcium translocates from the endoplasmic reticulum (ER) to mitochondria. Disruption of mitochondrial calcium homeostasis promotes apoptosis through mPTP opening. Conversely, inhibition of mPTP opening increases mitochondrial calcium storage capacity.

[0155] To investigate the effect of mPTP inhibition on mitochondrial calcium changes, mitochondrial and cytosolic calcium concentrations were evaluated using Rhod-2 AM and Fluo-4 AM fluorescent dyes, respectively. As shown in Fig. 9, in the cisplatin-treated group, the cytosolic and mitochondrial calcium levels increased by approximately 2.5-fold and 1.5-fold, respectively, whereas in the DBP-iPT-treated group, the mitochondrial calcium level increased by approximately 2-fold due to the enhancement of mitochondrial calcium capacity, while the amount of calcium released into the cytosol decreased (n = 4 per group).

[0156]

[0157] 2-3. Confirmation of DBP-iPT's ROS removal ability

[0158] DBP-iPT plays an important role in inhibiting calcium efflux and enhancing mitochondrial calcium capacity (Fig. 9).

[0159] Next, the antioxidant capacity of DBP-iPT was investigated by examining the effects of calcium influx and efflux in both the cytosolic and mitochondrial compartments. To assess the potential reduction in ROS accumulation, a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay was performed. As shown in Figure 10a, the cisplatin-treated group showed increased intracellular ROS levels, indicating intracellular stress. In contrast, the DBP-iPT-treated group showed decreased ROS levels, indicating an enhanced ability of the cells to scavenge ROS (n = 4 per group).

[0160] As shown in Fig. 10b, the MitoSOX assay showed that the mitochondrial ROS level increased 1.5-fold in the cisplatin-treated group due to intracellular stress, and surprisingly, both the DBP-iPT pre-treated group and the DBP-iPT-treated group showed a 3-fold increase in mitochondrial ROS (n = 4 per group).

[0161] Considering that the mitochondrial ROS level increased despite a 40% increase in cell viability in the DBP-iPT pre-treated group compared to the cisplatin-treated group, we expected a correlation between mitochondrial ROS accumulation and mitochondrial membrane potential depolarization. As a result of the experiment, as shown in Fig. 10c, contrary to the expectation that the mitochondrial membrane potential would decrease in the cisplatin-treated group, both the DBP-iPT pre-treated group and the DBP-iPT-treated group showed an approximately 3-fold increase in mitochondrial membrane potential compared to the control group (n = 4 per group).

[0162]

[0163] 2-4. Confirmation of the effectiveness of DBP-iPT in improving the restoration of mitochondrial morphology and dynamics.

[0164] To elucidate the underlying factors contributing to the substantial increase in mitochondrial ROS and mitochondrial membrane potential, comprehensive mitochondrial function studies were performed to assess the maintenance of normal mitochondrial morphology. Under stressful conditions, mitochondria typically undergo fission, with an increased prevalence of fragmented mitochondria. Conversely, healthy mitochondria undergo fusion, resulting in a higher proportion of elongated mitochondria.

[0165] Referring to Figure 11A, which visualizes mitochondrial morphology using MitoTracker Red, the cisplatin-treated group experienced mitochondrial fragmentation, with an increased proportion of fragmented mitochondria compared to elongated mitochondria, whereas the DBP-iPT pre-treated group recovered healthy mitochondrial morphology (n = 4 per group).

[0166] TEM provided further insight into the preservation of mitochondrial health. As shown in Figure 11B, a close examination of the mitochondria indicated by red arrows revealed that mitochondrial morphology was restored in the DBP-iPT-treated group, whereas the cisplatin-treated group showed a significant increase in fragmented mitochondria. Furthermore, cristae in the cisplatin-treated group exhibited a damaged morphology, unlike the other three groups, which showed a wide range of mitochondrial sizes. Even when mitochondrial ROS levels were increased, normal morphology was maintained in the DBP-iPT-treated group, contributing to the preservation of mitochondrial shape and cristae. Consequently, the cisplatin-induced mitochondrial morphological changes suggested that mitochondrial recovery was facilitated by mPTP inhibition.

[0167] Quantification of the number of mitochondria in the same region revealed a significant increase in the cisplatin-treated group, possibly due to mitochondrial fragmentation resulting from cell damage.

[0168]

[0169] Additional experiments were conducted to demonstrate that DBP-iPT protects the apoptosis pathway against cisplatin-induced apoptosis.

[0170] First, the presence of early and late stages of apoptosis was confirmed using annexin V-PI staining. FITC-annexin V staining was used to detect early stage apoptosis, and both FITC-annexin V and PI staining were used to confirm late stage apoptosis. Referring to Figure 12A, cisplatin treatment induced the apoptotic pathway, increasing both early and late apoptotic signals, whereas DBP-iPT treatment decreased both early and late apoptotic signals (n = 4 per group).

[0171] Next, the expression levels of the apoptotic pathway were examined using Western blot analysis. Referring to Figure 12B, the expression of Caspase-3 increased approximately 9-fold in the cisplatin-treated group compared to the control group, whereas the expression level of Caspase-3 decreased in the DBP-iPT-treated group. Thus, it was inferred that DBP-iPT inhibited apoptosis and protected against cisplatin-induced ototoxicity (n = 3 per group).

[0172]

[0173] 2-5. Confirmation of the protective effect of mPTP opening inhibition on cisplatin-induced hearing loss in a mouse model.

[0174] To evaluate the applicability of the results of the previous in vitro experiment, an auditory brainstem response (ABR) experiment was performed using a mouse model. Referring to Fig. 13A, the ABR threshold changes induced by cisplatin increased by approximately 50 dB SPL in response to click, 8K, and 32K, and by approximately 70 dB SPL for 16K, compared to the normal hearing group. The DBP-iPT pre-treatment group also showed a significant recovery in hearing, measuring approximately 10-20 dB SPL higher than the elevated cisplatin-treated group. The DBP-iPT treatment group did not show any toxicity and appeared to maintain normal hearing.

[0175] These results confirm the consistent protective effect of DBP-iPT not only at the in vitro cell level but also in the in vivo mouse model (n = 5 per group).

[0176]

[0177] To evaluate the morphology of hair cells located in the Organ of Corti of the inner ear cochlea, phalloidin staining was performed to investigate damage to inner and outer hair cells.

[0178] The middle turn (50% of the entire turn) of the organ of Corti was stained with phalloidin, and the number of inner and outer hair cells was counted. As shown in Figure 13B, both inner and outer hair cells were damaged in the cisplatin-treated group, whereas the application of DBP-iPT to cisplatin-treated hair cells significantly alleviated this severe damage. The DBP-iPT pretreatment group showed a lower effect than the control group, but showed complete recovery in inner hair cells and significant recovery in outer hair cells. The DBP-iPT treatment group did not show cytotoxicity.

[0179] In summary, the protective effect of DBP-iPT against cisplatin-induced ototoxicity was extended not only at the cellular level but also in a mouse model, highlighting its efficacy in alleviating cisplatin-induced ototoxicity.

[0180]

[0181] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for treating or preventing hearing loss, containing a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: <Chemical Formula 1> In the above chemical formula 1, R 1 silver or Selected from, R' and R'' may be the same or different, and are selected from a 4- to 8-membered azacyclic group or a 7- to 9-membered azabicyclic group, wherein the azacyclic group or azabicyclic group may be substituted or unsubstituted with (C1-C6)alkyl or benzyl, R 2 and R 3 may be the same or different and are selected from hydrogen, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl or (C1-C6)alkoxy.

2. In paragraph 1, In the above R' and R'', the azacyclic group is pyrrolidinyl and the azabicyclic group is quinuclidinyl, A pharmaceutical composition, characterized in that the azacyclic group or azabicyclic group is substituted or unsubstituted with one or more substituents selected from the group consisting of methyl, ethyl, isopropyl, tert-butyl, and benzyl.

3. In paragraph 1, The above R' and R'' are selected from quinuclidin-3-yl or isopropylpyrrolidin-3-yl, The above R 2 and R 3 A pharmaceutical composition characterized in that it is selected from hydrogen, fluoro, chloro, or trifluoromethyl.

4. In paragraph 1, The above compound or a salt thereof, A pharmaceutical composition characterized in that it is selected from the group consisting of 2-(4-(trifluoromethyl)benzyloxy)benzaldehyde O-quinuclidin-3-yl oxime, 2-(benzyloxy)benzaldehyde O-(1-isopropylpyrrolidin-3-yl) oxime, 2-((3-fluorobenzyl)oxy)benzaldehyde O-(1-isopropylpyrrolidin-3-yl) oxime, 4-(2-((3,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole and 4-(2-((2,4-dichlorobenzyl)oxy)phenyl)-1-(1-isopropylpyrrolidin-3-yl)-1H-1,2,3-triazole.

5. In paragraph 1, The above composition, A pharmaceutical composition characterized in that the compound or its salt is contained at a concentration of 0.1 to 40 μM.

6. In paragraph 1, The above compound or salt thereof, A pharmaceutical composition characterized by inhibiting the activation or opening of the mitochondrial permeability transition pore (mPTP).

7. In paragraph 1, The above compound or salt thereof, A pharmaceutical composition characterized by having a protective effect against cisplatin-induced ototoxicity.

8. In paragraph 1, The above hearing loss is, A pharmaceutical composition characterized by being any one selected from ototoxic hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, diabetic auditory neuropathy, traumatic hearing loss, and viral hearing loss.

9. A health functional food composition for improving or preventing hearing loss, containing a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof as an active ingredient: <Chemical Formula 1> In the above chemical formula 1, R 1 silver or Selected from, R' and R'' may be the same or different, and are selected from a 4- to 8-membered azacyclic group or a 7- to 9-membered azabicyclic group, wherein the azacyclic group or azabicyclic group may be substituted or unsubstituted with (C1-C6)alkyl or benzyl, R 2 and R 3 may be the same or different and are selected from hydrogen, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl or (C1-C6)alkoxy.

10. In paragraph 9, The above hearing loss is, A health functional food composition characterized by being any one selected from ototoxic hearing loss, noise-induced hearing loss, presbycusis, sudden hearing loss, diabetic auditory neuropathy, traumatic hearing loss, and viral hearing loss.

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