Agent for preventing age-related hearing loss

Abstract

The present invention provides a means and a system for screening agents for treating or preventing age-related hearing loss. Specifically, the present invention provides an agent for treating or preventing age-related hearing loss, the agent being characterized by containing, as an active ingredient, at least one compound selected from the group consisting of tea polyphenols and salts and hydrates thereof.

Description

Preventive agent for age-related hearing loss

[0001] The present invention relates to an agent for treating or preventing hearing loss, particularly age-related hearing loss. The present invention also relates to an agent for reducing oxidative stress in inner ear cells.

[0002] With the advent of a super-aging society, progressive hearing loss (age-related hearing loss) is steadily increasing. Hearing loss (deafness) is the most common disability in daily life worldwide and is also the biggest risk factor for the development of dementia, with hearing loss accounting for as much as 9% of all dementia cases. Therefore, preventing progressive hearing loss is an urgent issue not only to prevent a decline in quality of life caused by hearing loss, but also to extend healthy life expectancy and reduce social security burdens.

[0003] Age-related hearing loss is a progressive hearing loss caused by multiple factors, including environmental and genetic factors. Most cases of progressive hearing loss result from damage to the inner ear, the organ that receives sound and converts it into neural activity. However, this organ is located deep within the temporal bone, making biopsy virtually impossible and histological and cell biological analysis difficult. Furthermore, hearing loss itself does not cause death, and pathological findings are scarce. For these reasons, analysis of the mechanisms underlying hearing loss progression must rely on animal models. However, creating animal models is time-consuming and labor-intensive, and species differences between animals and humans often result in a disconnect between the animal models and the actual disease.

[0004] In addition, oxidative stress is thought to be one of the causes of age-related hearing loss, and antioxidants are expected to have a preventive effect, but Someya et al. reported that when they tested 17 types of antioxidants using mice, only three types - N-acetylcysteine, α-lipoic acid, and coenzyme Q10 - were found to have a significant effect, while β-carotene, d-α-tocopherol, epigallocatechin gallate (EGCG), etc., did not have a significant effect (Non-Patent Document 1). However, as mentioned above, there is concern about the discrepancy between humans and animal models due to species differences.

[0005] As a research tool to solve these problems, differentiated cells derived from pluripotent stem cells, such as human embryonic stem cells (ES cells) and human induced pluripotent stem cells (iPS cells), have attracted attention. Our laboratory has established several methods for inducing differentiation of inner ear cell-like cells from human iPS cells (Patent Documents 1 and 2, Non-Patent Documents 2 and 3), and is using these methods to search for therapeutic drugs for hereditary cochlear hearing loss. Furthermore, the efficacy of some of the candidate drugs obtained as a result of this search has been confirmed in investigator-initiated clinical trials (Non-Patent Document 4).

[0006] Patent No. 6218152 International Publication No. WO2023 / 033149

[0007] Someya, S. et al. PNAS 106(46):19432-19437, 2009Okura, S. et al., Stem Cell Res. 67:103017, 2023Hosoya, M. et al., Cell Reports 18(1):68-81, 2017Fujioka, M. et al., Medicine 99(19):e19763, 2020

[0008] An object of the present invention is to establish a means and system for screening therapeutic or preventive agents for age-related hearing loss, and to provide effective therapeutic or preventive agents for age-related hearing loss.

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have identified a substance that has the effect of extending survival in long-term culture of inner ear cells derived from iPS cells of hearing-normal individuals and that may serve as a therapeutic or preventive agent for age-related hearing loss. Furthermore, the inventors have discovered that inner ear cells with genetic variants observed in hereditary progressive hearing loss can be produced using pluripotent stem cells, and that therapeutic or preventive agents for age-related hearing loss can be screened in a system in which the produced inner ear cells are subjected to oxidative stress. Furthermore, through screening, the inventors have identified a substance that may serve as a therapeutic or preventive agent for age-related hearing loss.

[0010] Therefore, the present invention includes, for example, the following: [1] An agent for treating or preventing age-related hearing loss, characterized by comprising, as an active ingredient, at least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof. [2] The agent for treating or preventing age-related hearing loss according to [1], comprising, as an active ingredient, at least one compound selected from the group consisting of (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, methyl protocatechuate, (-)-catechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin gallate, and (-)-gallocatechin gallate, and salts and hydrates thereof. [3] The agent for treating or preventing age-related hearing loss according to [1], which is in the form of an oral dosage form. [4] The agent for treating or preventing age-related hearing loss according to [1], which is used in a pharmaceutical or food product. [5] An agent for reducing oxidative stress in inner ear cells, comprising as an active ingredient at least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof.

[0011] [6] A method for treating or preventing age-related hearing loss in a subject, comprising administering to the subject at least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof. [7] A method for reducing oxidative stress in inner ear cells in a subject, comprising administering to the subject at least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof.

[0012] [8] At least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof, for use in treating or preventing age-related hearing loss in a subject. [9] At least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof, for use in reducing oxidative stress in inner ear cells in a subject.

[0013] The present invention provides an agent for treating or preventing hearing loss, particularly age-related hearing loss. The present invention also provides an agent for reducing oxidative stress in human inner ear cells. The agent according to the present invention can reduce oxidative stress in inner ear cells without causing harmful side effects to the human subject. Therefore, the present invention makes it possible to effectively prevent or treat age-related hearing loss.

[0014] This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) and inner ear cells derived from iPS cells of a healthy individual (WD39) after applying oxidative stress by adding H2O2. This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) and inner ear cells derived from iPS cells of a healthy individual (WD39) after applying proteasome inhibition stress by adding epoxomicin. This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (four EYA gene mutation cases) and inner ear cells derived from iPS cells of a healthy individual (WD39) after applying endoplasmic reticulum stress by adding tunicamycin. This graph shows the cell viability of inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) after applying oxidative stress by adding H2O2, as observed visually, and the viability index analyzed using ImageJ after cell photography. 1 is a graph showing an example of the Viability Index after oxidative stress was applied to inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case) in the presence of compounds included in the library. The structures of example compounds that reduce oxidative stress on inner ear cells and may serve as therapeutic or preventive drugs for age-related hearing loss are shown. This is a graph showing the results of a long-term culture experiment using inner ear cells derived from iPS cells of a healthy subject with the addition of 1 μM catechin. This is a graph showing the results of a long-term culture experiment using inner ear cells derived from iPS cells of a healthy subject with the addition of 10 μM catechin. This is a graph showing the cell viability after oxidative stress was applied by tBHP treatment to inner ear cells derived from iPS cells of a patient with hereditary hearing loss (EYA4 gene mutation case).

[0015] The present invention is described in detail below. This specification includes the disclosure of Japanese Patent Application No. 2024-079941, filed on May 16, 2024, from which the present application claims priority.

[0016] The present inventors have discovered that, because the loss of inner ear cells is the cause of age-related hearing loss, substances that prolong the survival of human inner ear cells can be selected as therapeutic or preventative agents for age-related hearing loss. Furthermore, focusing on the fact that highly pathogenic mutations in hearing loss genes (e.g., the EYA4 gene) that cause age-related hearing loss result in hereditary hearing loss (i.e., the causative gene), while the presence of relatively less pathogenic single nucleotide polymorphisms (SNPs) indicates a predisposition to age-related hearing loss, the inventors have discovered that by inducing the differentiation of inner ear cells from pluripotent stem cells carrying genetic variants that cause age-related hearing loss, factors that affect the survival of human inner ear cells after application of oxidative stress can be selected as therapeutic or preventative agents for age-related hearing loss. Furthermore, the inventors have found that the compounds listed in Table 2 below and tea polyphenols, including catechins, particularly reduce oxidative stress on human inner ear cells, and have selected them as therapeutic or preventative agents for age-related hearing loss.

[0017] Therefore, the present invention provides tea polyphenols including the compounds listed in Table 2 below and catechins, for example, (+)-catechin, (-)-epicatechin (compound ID 190), (-)-gallocatechin, (-)-epigallocatechin, methyl protocatechuate (compound ID 137), (-)-catechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin gallate and (-)-gallocatechin gallate, methyl ferulate (compound ID 234), EUK 134 (Compound ID 176), caffeic acid phenethyl ester (Compound ID 274), 7,8-dihydroxyflavone (Compound ID 292), quercitrin (Compound ID 118), lauryl gallate (Compound ID 262), scutellarin (Compound ID 112), mangiferin (Compound ID 111), methoxsalen (Compound ID 26), obacunone (Compound ID 104), forsytoside B (Compound ID 339), 4-methylesculetin (Compound ID 210), NSC 319726 (Compound ID 270), propylthiouracil (Compound ID 27), isoferulic acid (Compound ID 96), baicalin (Compound ID 34), (+)-α-lipoic acid (Compound ID 167), secoisolariciresinol diglucoside (Compound ID 102), trolox (Compound ID 89), sesamol (Compound ID 84), proanthocyanidin derivatives (Compound ID 217), tea polyphenols (Compound ID 216), alizarin (Compound ID 68), tetrahydrocurcumin (Compound ID 147), myricetin (Compound ID 46), rutin (Compound ID 51), diethyl maleate (Compound ID 102), The present invention relates to the use of ginsenoside Re (compound ID 188), ginsenoside Re (compound ID 113), nordihydroguaiaretic acid (NDGA) (compound ID 164), echinacoside (compound ID 103), fisetin (compound ID 40), berberine (compound ID 312), bergenin (compound ID 35), 3,4-dihydroxybenzaldehyde (compound ID 155), tempor (compound ID 73), eupatilin (compound ID 130), and methyl gallate (compound ID 108), which reduces oxidative stress on human inner ear cells and thereby treats or prevents age-related hearing loss.

[0018] As used herein, "tea polyphenols" refers to polyphenols contained in tea, particularly green tea, and are well known in the art. Examples of tea polyphenols include, but are not limited to, (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, methyl protocatechuate, (-)-catechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and gallic acid.

[0019] The present invention relates to the use of the compounds listed in Table 2 and tea polyphenols including catechins, particularly (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, methyl protocatechuate, (-)-catechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin gallate and (-)-gallocatechin gallate, and utilizes these compounds to reduce oxidative stress on inner ear cells and treat or prevent age-related hearing loss.

[0020] In one aspect, the present invention relates to an agent for treating or preventing age-related hearing loss, comprising, as an active ingredient, at least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof.

[0021] In another aspect, the present invention relates to an agent for reducing oxidative stress in inner ear cells, characterized by comprising, as an active ingredient, at least one compound selected from the group consisting of tea polyphenols, and salts and hydrates thereof.

[0022] In one embodiment, the agent comprises tea polyphenols, their salts or hydrates, or mixtures thereof as active ingredients.

[0023] The active ingredient, tea polyphenols, are a subset of catechins or polyphenols found in green tea and other teas, and can be produced using methods known in the art. The compounds may be natural or synthetic, and include, but are not limited to, compounds purified from natural sources or commercially available compounds. The compounds may be, for example, stereoisomers, pharmacologically acceptable salts, or pharmacologically acceptable solvates (e.g., hydrates, alcoholates, etc.). Specific examples of salt forms include salts with alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; organic bases such as ammonium salts; inorganic acids such as hydrochloric acid and phosphoric acid; and organic acids such as acetic acid, citric acid, and succinic acid.

[0024] In the context of the present invention, "age-related hearing loss" refers to a hearing impairment characterized by a progressive age-related decline in auditory function, which has several causes, primarily a decline in inner ear function associated with a decrease in sensory cells in the inner ear. Furthermore, "treatment or prevention of age-related hearing loss" refers to preventing the onset or progression of age-related hearing loss, delaying the onset or progression, or alleviating symptoms. Hearing loss can be assessed by methods known in the art, such as the average hearing level (sound becomes difficult to hear at 25 dB or above) expressed as the average of hearing levels between 500 Hz and 2000 Hz in a pure-tone audiometry test, or the maximum speech intelligibility in a speech audiometry test.

[0025] In the present invention, cells present in the inner ear and the cochlea are referred to as "inner ear cells." For example, inner ear cells include cells of the inner ear sensory epithelium (including supporting cells and hair cells), cochlear ganglion cells (including neurons and glial cells), outer spiral sulcus cells (OSCs), and cells of the stria vascularis of the inner ear (including marginal cells of the stria vascularis of the inner ear).

[0026] The agent for treating or preventing age-related hearing loss or the agent for reducing oxidative stress in inner ear cells according to the present invention (hereinafter also referred to as "the agent") typically contains, as an active ingredient, at least one compound selected from the group consisting of tea polyphenols and their salts and hydrates. The active ingredient can be a salt, solvate, or derivative of the above compound, as long as it has activity for treating or preventing age-related hearing loss or reducing oxidative stress in inner ear cells. In the present invention, "reducing oxidative stress in inner ear cells" refers to reducing or inhibiting cell death of inner ear cells due to oxidative stress.

[0027] Whether a selected compound has an appropriate activity can also be confirmed by methods known in the art. For example, the therapeutic or preventive activity for age-related hearing loss can be confirmed by administering the selected compound to a subject (e.g., a model animal) and determining whether it affects the onset, progression, or symptoms of age-related hearing loss in the subject. Furthermore, for example, the activity of reducing oxidative stress on inner ear cells can be confirmed by applying oxidative stress to inner ear cells (e.g., human inner ear cells) in the presence of the selected compound and determining whether the survival of the inner ear cells is reduced or inhibited.

[0028] The agent may contain one compound or a combination of two or more compounds as an active ingredient. As long as the agent contains at least one compound selected from tea polyphenols as an active ingredient, it will exhibit the above-mentioned effects of treating or preventing age-related hearing loss and reducing oxidative stress on inner ear cells.

[0029] The agent may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, etc. The additives used are appropriately selected from the above or in combination depending on the dosage form.

[0030] When administered orally, this agent may be in the form of tablets, capsules (hard capsules, soft capsules, microcapsules, etc.), granules, powders, pills, lozenges, oral solutions, liquids, suspensions, emulsions, syrups, etc., or may be in the form of a dry preparation that is redissolved before use. When administered parenterally, this agent may be in the form of injections (e.g., solutions, emulsions, suspensions) for intratympanic administration, round window membrane administration, intraaural administration, intravenous injection (including drip infusion), intramuscular injection, intraperitoneal injection, and subcutaneous injection, liquids, suspensions, emulsions, ear drops, ointments, creams, suppositories, poultices, inhalants, liniments, aerosols, and other external preparations, and when administered parenterally, it may be provided in the form of unit-dose ampoules or multi-dose containers.

[0031] The various preparations described above can be produced by a conventional method by appropriately selecting excipients, fillers, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, pH adjusters, preservatives, solubilizers, antiseptics, flavoring agents, absorption enhancers, soothing agents, stabilizers, isotonicity agents, and the like that are commonly used in pharmaceuticals.

[0032] The amount of active ingredient contained in this agent varies depending on the type, use, dosage form, route of administration, etc., but is, for example, 0.01 to 90% by weight, preferably 1 to 50% by weight, based on the total weight.

[0033] The effective amount (dosage or intake amount) of the agent varies depending on the type of active ingredient contained in the agent, the age and weight of the subject, symptoms, route of administration, and frequency and number of administrations, and can vary over a wide range. For example, an effective amount of the active ingredient for an adult per day (e.g., 1 to 1000 mg / day) can be administered once or in divided doses per day for about 1 week to about 1 year, preferably about 1 month to about 12 months.

[0034] The subject for which this agent is used is not particularly limited. For example, it can be administered or ingested by humans and other mammals, such as primates (monkeys, chimpanzees, etc.), livestock animals (cows, horses, pigs, sheep, etc.), pet animals (dogs, cats, etc.), and laboratory animals (mice, rats, rabbits, etc.). Subjects are particularly those for whom treatment or prevention of age-related hearing loss is desired, such as those aged 40 or older, particularly those aged 60 or older, and those with a family history of hearing loss.

[0035] If age-related hearing loss has already developed, the present invention can be combined with an effective method for treating age-related hearing loss known in the art, such as the use of a hearing aid or cochlear implant surgery.

[0036] The use of this agent is not limited to pharmaceutical compositions, and it may also be incorporated into other products, such as food or feed. "Food" and "feed" refer to natural products containing one or more nutrients and their processed products, including all foods and beverages. Foods or feeds containing this agent are useful as health supplements for improving or preventing age-related hearing loss.

[0037] When the agent is incorporated into food, it can be added to various forms of food, such as solid food, jelly food, liquid food, and capsule food.Here, solid food includes bread dough; dough for baked goods such as rice crackers, biscuits, and cookies; noodles such as soba and udon; fish products such as kamaboko and chikuwa; meat products such as ham and sausage; powdered milk, etc.In addition, jelly food includes fruit jelly, coffee jelly, etc.Furthermore, liquid food includes beverages such as soft drinks and fruit drinks (tea, coffee, black tea, fermented milk, lactic acid bacteria drinks, etc.), seasonings, etc. (mayonnaise, dressing, seasoning liquid, etc.).Capsule food includes hard capsules, soft capsules, etc.

[0038] When adding this agent to food, the amount added should be such that the active ingredient content is 0.01-10% by weight of the total food. The amount of intake that can be expected to be effective is determined appropriately for each individual case, taking into consideration factors such as age, weight, sex, and severity of symptoms, and should be taken once a day or in divided doses. It can also be taken continuously over a long period of time.

[0039] As described above, by administering this agent to a subject, it is possible to treat or prevent age-related hearing loss or reduce oxidative stress on inner ear cells in the subject.

[0040] The present invention will be described in more detail below with reference to examples and drawings, but the present invention is not limited to these examples.

[0041] Example 1 In this example, inner ear cells that can serve as a model for age-related hearing loss were established.

[0042] Establishment of EYA4 mutant iPSCs. A 48-year-old woman (45 years old at the time of initial consultation) had a heterozygous mutation in the EYA4 gene (c.960_693del:p.P323Efs*80) and presented with a hearing loss of 72.5 dB in the right ear and 70.0 dB in the left ear (based on a quadrant test) at initial consultation. Mononuclear cells isolated from peripheral blood were electroporated with episomal plasmids encoding OCT3 / 4, SOX2, KLF4, LIN28, L-MYC, and dominant-negative mutant p53 (Hosoya et al., Cell Reports 18(1):68-81, 2017).

[0043] Differentiation of iPS cells into inner ear-like cells Differentiation of iPS cells into outer spiral sulcus cell (OSC)-like cells of the inner ear was performed as previously reported (Non-Patent Document 3). Specifically, iPS cells in the feeder-free culture established as described above were detached using Accutase, passed through a 40 μm cell strainer to obtain single cells, and then plated onto a Matrigel-coated dish at a density of 15,000 to 25,000 cells / cm. 2Cells were seeded at a density of 10 μM (Day 0) and cultured in mTeSR medium containing 10 μM Y27632 for 24 hours, after which the medium was replaced with mTeSR medium without Y27632 (Day 1). The medium was replaced every 24 hours until Day 11. From Day 2 to Day 5, cells were cultured in DMEM / F12 medium containing 1% N2 supplement, 2% B27 supplement, 1× non-essential amino acids, 1× Glutamax, 0.1 mM 2-mercaptoethanol, and 100 μg / mL ampicillin (SF medium). From Day 5 to Day 8, cells were cultured in SF medium supplemented with 25 ng / mL FGF2, 25 ng / mL FGF3, 25 ng / mL FGF10, 25 ng / mL FGF19, and 10 ng / mL BMP4. From Day 8 to Day 11, cells were cultured in SF medium supplemented with 25 ng / mL FGF2, 25 ng / mL FGF3, 25 ng / mL FGF10, and 25 ng / mL FGF19. On Day 11, cells were detached using Accutase and plated on Poly-L-ornithine and fibronectin-coated dishes. They were cultured in DMEM / F12 medium containing 1% N2 supplement, 2% B27 supplement, 25 ng / mL FGF2, 25 ng / mL FGF3, 25 ng / mL FGF10, and 25 ng / mL FGF19. The next day, the medium was replaced with DMEM medium containing 4 ng / mL FGF2, 10 mM HEPES, 6% fetal bovine serum (FBS), and 100 μg / mL ampicillin. The cells were cultured for 2 weeks to 1 month, with medium changes every 2–3 days. If the cells reached confluence, they were detached and reseeded at a 1:3 ratio. The medium was then replaced with DMEM containing 10% FBS, 0.75% NaHCO3, and 50 U / mL penicillin / streptomycin. After culturing for one week with medium changes every other day, the medium was replaced with DMEM containing 10% FBS, 0.375% NaHCO3, and 50 U / mL penicillin / streptomycin. OSC-like cells were obtained after another week of culturing with medium changes every other day.The obtained OSC-like cells were seeded at a density of 10,000 cells / well onto a 96-well plate coated with poly-L-ornithine and fibronectin and used for screening of antioxidants.

[0044] Example 2 In this example, the cellular stress that affects inner ear cells was investigated.

[0045] Various cell stresses were applied to outer spiral sulcus cell (OSC)-like cells induced from iPS cells established from EYA4 patients, as described in Example 1, and the degree of cell death induction was analyzed. Specifically, OSC-like cells induced from iPS cells established from healthy individuals and EYA4 patients were subjected to oxidative stress by adding H2O2, proteasome inhibitory stress by adding epoxomicin, or endoplasmic reticulum stress by adding tunicamycin.

[0046] The results are shown in Figures 1–3. In Figures 1–3, cell viability after each stress is expressed as 1, with the survival rate without stress set at 1. WD39 inner ear cells derived from iPS cells from healthy individuals were not sensitive to oxidative stress induced by the addition of H2O2 (Figure 1), proteasome inhibition stress induced by the addition of epoxomicin (Figure 2), or endoplasmic reticulum stress induced by the addition of tunicamycin (Figure 3). On the other hand, EYA4 mutant inner ear cells (EYA cases) were not sensitive to proteasome inhibition or endoplasmic reticulum stress (Figures 2 and 3), but specifically induced cell death in response to oxidative stress (Figure 1), demonstrating vulnerability to oxidative stress in EYA4 mutant inner ear cells. Therefore, we decided to screen for antioxidants that could reduce oxidative stress induced by the addition of H2O2 to inner ear cells.

[0047] [Example 3] In this example, antioxidants that reduce oxidative stress on inner ear cells caused by the addition of H2O2 were screened. The compounds screened are shown in Table 1.

[0048]

[0049] The day after seeding the inner ear cells prepared in Example 1 onto a 96-well plate, 10 μM of compounds from a library (Table 1) created using Selleck Biotech's compound library creation service (https: / / www.selleck.co.jp / screening-libraries.html) were added and incubated for 2 hours. Then, 200 μM HO was added and incubated for 24 hours. The medium was then removed, and the cells were stained for 10 minutes with PBS containing 1.25 μM Hoechst 33372 and 1 μg / μL 7-amino actinomycin D (7-AAD). After staining, the medium was replaced with dye-free PBS, and blue fluorescent images derived from Hoechst staining and red fluorescent images derived from 7-AAD staining were captured using a Keyence BZ-X810. These images were binarized using the image analysis software ImageJ, and the number of Hoechst-positive cells (total cells) and 7-AAD-positive cells (dead cells) were counted to determine cell viability.

[0050] First, to confirm whether this evaluation system was functioning properly, OSC-like cells were treated with hydrogen peroxide (HO) at concentrations between 0 mM and 1,000 mM, and fluorescent images were taken and analyzed. Visual observation under a fluorescent microscope confirmed that almost all cells were dead at HO concentrations above 250 mM (Figure 4). Images of these cells were then analyzed using ImageJ. Although the values ​​tended to be lower in areas with low cell viability compared to visual viability analysis, the results were generally consistent with visual analysis (Figure 4). Because the analysis using ImageJ differs from the actual viability, we defined the Viability Index (VI).

[0051] Next, we analyzed whether the library compounds inhibited cell death induced by hydrogen peroxide, and each compound inhibited cell death to various degrees (Figure 5). Examples of compounds with a high Viability Index (VI) are shown in Table 2 below: compounds with a VI greater than 0.90, greater than 0.80, greater than 0.70, and greater than 0.60.

[0052]

[0053] Among these compounds, catechins such as (+)-catechin (VI = 0.281), (-)-epicatechin (VI = 0.887), methyl protocatechuate (VI = 0.830), and (-)-epigallocatechin gallate (VI = 0.494) exhibited relatively high VI values. These catechins were selected as potential therapeutic or preventative agents for age-related hearing loss. The structural formulas of these catechins are shown in Figure 6.

[0054] In addition, compounds other than catechins in Table 2, particularly ferulic acid methyl ester (compound ID 234) and 7,8-dihydroxyflavone (compound ID 292), also have the effect of reducing oxidative stress on inner ear cells and can be used as therapeutic or preventive agents for age-related hearing loss.

[0055] Example 4 In this example, a long-term culture system was used to evaluate the effect of candidate antioxidants in reducing oxidative stress on inner ear cells.

[0056] (1) Establishment of iPS cells from hearing individuals and differentiation of iPS cells into inner ear-like cells. iPS cells were established by electroporation of mononuclear cells isolated from the peripheral blood of two hearing individuals (KOMENT-A and KOMENT-B) with episomal plasmids encoding OCT3 / 4, SOX2, KLF4, LIN28, L-MYC, and dominant-negative mutant p53 (Hosoya et al., Cell Reports 18(1):68-81, 2017).

[0057] As in Example 1, iPS cells were differentiated into outer spiral sulcus cell (OSC)-like cells of the inner ear according to a previous report (Non-Patent Document 3). The obtained OSC-like cells were seeded at a density of 10,000 cells / well onto a 96-well plate coated with Poly-L-ornithine and fibronectin and used for the evaluation of antioxidants in a long-term culture system.

[0058] (2) Evaluation of antioxidant candidate catechins The candidate antioxidant catechins evaluated were (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (Cg), (-)-epigallocatechin gallate (EGCg), (-)-epicatechin gallate (ECg), and (-)-gallocatechin gallate (GCg). A mixture of these eight catechins (8Cs) was also evaluated.

[0059] The day after seeding the prepared inner ear cells onto a 96-well plate, they were cultured with 1 μM or 10 μM catechins (8 types of catechins (8Cs) total 8 μM). The medium was changed every 2 to 3 days and the cells were cultured for 6 weeks. The medium was then removed, and the cells were stained for 10 minutes with PBS containing 1.25 μM Hoechst 33372. After staining, the medium was replaced with PBS without the dye, and blue fluorescent images derived from Hoechst staining were captured using a Keyence BZ-X810. These images were binarized using the image analysis software ImageJ, and the number of cells (Hoechst-positive cells) was counted.

[0060] The results are shown in Figures 7 and 8. Figure 7 is a graph showing the results of a long-term culture experiment with the addition of 1 μM catechin. The experiment was performed four times with KOMENT-A and three times with KOMENT-B (n = 6 for each). The significance of the differences was tested using a Dunnett's test. In the graph in Figure 7, the four graphs from the left represent KOMENT-A, and the three graphs from the right represent KOMENT-B. Figure 8 is a graph showing the results of a long-term culture experiment with the addition of 10 μM catechin. The experiment was performed twice with KOMENT-A and once with KOMENT-B (n = 6 for each). The significance of the differences was tested using a Dunnett's test. In the graph in Figure 8, the two graphs from the left represent KOMENT-A, and the graph on the far right represents KOMENT-B. As a result, cells survived after long-term culture at 1 μM for most of the catechins evaluated, and cells survived after long-term culture at 10 μM for all of the catechins.

[0061] Example 5 In this example, induction of cell death in inner ear cells was investigated using an oxidative stress system induced by tBHP.

[0062] As described in Example 1, outer spiral sulcus cell (OSC)-like cells induced from iPS cells established from an EYA4 patient were treated with tert-butyl hydroperoxide (tBHP), and the degree of cell death induction was analyzed. Specifically, the day after seeding the inner ear cells prepared in Example 1 onto a 96-well plate, the medium was removed and replaced with PBS, and green fluorescence images due to autofluorescence were captured using a Keyence BZ-X810. After imaging, the medium was replaced with medium containing 10 μM catechins and cultured for 2 hours. After that, the medium was removed and replaced with medium containing 100 μM tBHP and cultured for 24 hours. The medium was then removed and replaced with PBS, and green fluorescence images were captured in the same manner. These images were binarized using the image analysis software ImageJ, and the cell number was counted. Cell viability was determined, and significant differences were assessed using a Dunnett's test. The catechins evaluated were the same as those in Example 4.

[0063] The results are shown in Figure 9. Figure 9 is a graph showing the cell viability after treatment with catechins and 100 μM tBHP, with the cell viability before treatment taken as 100%. (-) indicates a control without catechins. These results demonstrate that catechins have the effect of reducing oxidative stress on inner ear cells and can be used as a therapeutic or preventative agent for age-related hearing loss.

[0064] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A therapeutic or preventive agent for age-related hearing loss, comprising as an active ingredient at least one compound selected from the group consisting of tea polyphenols, and their salts and hydrates.

2. A therapeutic or preventive agent for age-related hearing loss as described in claim 1, containing as an active ingredient at least one compound selected from the group consisting of (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, methyl protocatechuate, (-)-catechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin gallate and (-)-gallocatechin gallate, and salts and hydrates thereof.

3. The agent for treating or preventing age-related hearing loss according to claim 1, which is in the form of an oral administration formulation.

4. The agent for treating or preventing age-related hearing loss according to claim 1, which is used in medicines or foods.

5. An agent for reducing oxidative stress in inner ear cells, characterized by containing as an active ingredient at least one compound selected from the group consisting of tea polyphenols, and their salts and hydrates.

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