Compounds for the treatment of age-related hearing loss and use thereof

Abstract

The application discloses a compound shown in a formula I, or a pharmaceutically acceptable salt or ester thereof can activate the PINK1 / Parkin ubiquitination pathway and / or the FUNDC1 receptor pathway, and then shows a significant protection effect on age-related hearing loss. The compound of the application provides a translational medicine candidate drug for treating age-related hearing loss and maintaining auditory health aging, and thus has a positive social significance and economic value.

Description

Technical Field

[0001] This invention relates to the medical field. Specifically, this invention relates to compounds for the prevention or treatment of age-related hearing loss, and methods for using said compounds to prevent or treat age-related hearing loss. Background Technology

[0002] Age-related hearing loss (ARHL) is one of the most common chronic sensorineural defects among the elderly. It refers to irreversible sensorineural hearing loss caused by the gradual degeneration of the inner ear structure with age, characterized by symmetrical, progressive, and high-frequency hearing loss in both ears. ARHL is the third most common health problem affecting the elderly after heart disease and arthritis. A WHO report indicates that more than 1.5 billion people worldwide (nearly 20% of the global population) suffer from hearing loss, and approximately 30% of adults over 60 years of age have hearing loss. ARHL is a leading cause of hearing disability.

[0003] Furthermore, age-related hearing loss often leads to communication difficulties, social isolation, and cognitive decline, increasing the risk of Alzheimer's disease and significantly reducing the quality of life for older adults. ARHL also has a range of adverse effects on the mental, psychological, and physical health of older adults, increasing the social burden. Currently, there are no effective treatments for this disease; treatment options include hearing aids, cochlear implants, and hearing rehabilitation programs to help people with ARHL improve their verbal communication skills and quality of life.

[0004] Age-related hearing loss has complex causes, including genetic predisposition, natural aging, noise exposure, ototoxic drugs, oxidative stress, inflammatory responses, and dietary habits. The main pathological changes include degeneration of hair cells and spiral ganglion neurons, atrophy of the stria vascularis, and lesions in the central auditory pathway.

[0005] The free radical theory of aging can explain the aforementioned degenerative changes: aging stems from the continuous accumulation of oxidative stress. Under physiological conditions, mitochondrial respiration produces reactive oxygen species (ROS), a metabolic byproduct that the body's antioxidant system can neutralize. However, aging disrupts this homeostasis, causing a large accumulation of ROS that exceeds the cell's detoxification capacity. Excessive oxidative damage severely impairs mitochondrial DNA and metabolic function, ultimately inducing irreversible hearing impairment. Clinical studies have also confirmed a direct correlation between plasma ROS levels and the degree of hearing loss in the elderly.

[0006] Mitophagy is crucial for cell survival and cochlear function. Mitophagy refers to the selective degradation of damaged mitochondria through the autophagic pathway to maintain homeostasis, and it is an important mitochondrial quality control mechanism that plays a significant role in the pathological changes of age-related hearing loss. Mitophagy is associated with aging; with increasing age, the expression of mitophagy-related genes and proteins decreases, leading to a decline in mitophagy levels. However, currently, effective treatments for age-related hearing loss remain very limited, and relevant in vivo research models are relatively scarce. Furthermore, the regulatory mechanisms of mitophagy differ significantly among different tissues and cell types, and the signaling pathways and targets involved are highly specific, posing numerous challenges to drug development.

[0007] Therefore, elucidating the regulatory mechanism of mitochondrial autophagy in cochlear tissue and developing novel therapeutic drugs that can target and regulate mitochondrial autophagy and effectively protect auditory function is not only of significant theoretical research value, but also has remarkable clinical application prospects and social significance. Summary of the Invention

[0008] The purpose of this invention is to provide a drug that can effectively treat age-related hearing loss.

[0009] Another object of the present invention is to provide an effective method for treating age-related hearing loss.

[0010] In a first aspect, the present invention provides the use of compounds of Formula I, or pharmaceutically acceptable salts or esters thereof, in the preparation of PINK1 / Parkin ubiquitination pathway and / or FUNDC1 receptor pathway activators. I In the formula, X is selected from S or O; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-6 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-6 Alkyl, optionally substituted C 1-6 Alkyl group.

[0011] In a specific implementation, in formula I, X is S; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-3 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-3 Alkyl, optionally substituted C 1-3 Alkyl group.

[0012] In a preferred embodiment, "optional substitution" refers to substitution by one or more (e.g., 1, 2, or 3) substituents selected from the group consisting of: hydroxyl, halogen, C 1-3 Alkyl, C 1-3 Alkyl groups and amino groups.

[0013] In a specific embodiment, the compound represented by Formula I is selected from the following group of compounds: , ; Preferably, the compound is a compound with the following formula: .

[0014] In a specific implementation, the PINK1 / Parkin ubiquitination pathway and / or FUNDC1 receptor pathway activator is a drug for the prevention or treatment of age-related hearing loss.

[0015] In a second aspect, the present invention provides compounds of Formula I, or pharmaceutically acceptable salts or esters thereof, for use as activators of the PINK1 / Parkin ubiquitination pathway and / or the FUNDC1 receptor pathway. I In the formula, X is selected from S or O; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-6 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-6 Alkyl, optionally substituted C 1-6 Alkyl group.

[0016] In a specific implementation, in formula I, X is S; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-3 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-3 Alkyl, optionally substituted C 1-3 Alkyl group.

[0017] In a specific embodiment, the compound represented by Formula I is selected from the following group of compounds: , ; Preferably, the compound is a compound with the following formula: .

[0018] In a specific implementation, the PINK1 / Parkin ubiquitination pathway and / or FUNDC1 receptor pathway activator is a drug for the prevention or treatment of age-related hearing loss.

[0019] In a third aspect, the present invention provides a method for activating the PINK1 / Parkin ubiquitination pathway and / or the FUNDC1 receptor pathway in a subject, the method comprising administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt or ester thereof, to the subject in need.

[0020] In a specific implementation, the method is used to prevent or treat age-related hearing loss.

[0021] In a preferred embodiment, the method comprises giving an effective amount of the compound represented by Formula I, or a pharmaceutically acceptable salt or ester thereof, to a subject whose hearing has declined.

[0022] In a preferred embodiment, the method is used to reverse low-frequency and mid-frequency hearing loss in a subject.

[0023] In a preferred embodiment, the low-frequency and mid-frequency hearing loss is hearing loss below 24 kHz.

[0024] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0025] Figure 1 This study demonstrates that compound TJ0113 can protect auditory function and hair cell integrity in vivo. (A) shows the molecular structure of TJ0113; (B) shows the experimental protocol corresponding to Figure C; (C) shows the auditory brainstem response threshold of 12-18 month old mice treated with TJ0113; (DF) shows a confocal image of hair cells (Myosin7a, gray) and cell nuclei (DAPI staining, blue), scale bar: 10 μm; (G) shows the quantitative statistical results of Figure DF; (H, I) show the morphology of hair cell styli observed under scanning electron microscopy, scale bar: 20 μm. Data are expressed as mean ± standard error; Figures C and G were analyzed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0026] Figure 2(A) shows the experimental protocols corresponding to Figures C and D; (B) shows the results of auditory brainstem response threshold detection in 6-week-old mice; (C and D) show the auditory brainstem response threshold analysis of 6-15 month old mice treated with TJ0113; (E) shows the NF (green) confocal image, scale bar: 10 μm; (F) shows the quantitative statistical results of Figure E; (G) shows the differential expression of key targets in transcriptome sequencing: comparison between the D-galactose group and the D-galactose + TJ0113 group. Figures C, D, and F were analyzed using one-way ANOVA, and Figure G was analyzed using a t-test; data are expressed as mean ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0027] Figure 3 The results demonstrate that TJ0113 can protect synaptic structures, cochlear tissue morphology, and mitochondrial ultrastructure. Figures (AC) show confocal images of CtBP2 (red), Myosin7a (gray), and DAPI (blue) in different cochlear transduction zones, scale bar: 10 μm; (D) shows the quantitative statistical results of Figure AC; (E) shows hematoxylin-eosin staining of spiral ganglion neurons and stria vascularis in the apical (blue), mid-transduction (green), and basal (red) transduction zones of the cochlea, scale bar: 100 μm; (F) shows the quantitative statistical results of Figure E; (G) shows transmission electron microscopy images of hair cells in different groups, with red arrows indicating mitochondrial morphology and yellow arrows indicating phagocytic vesicle membranes surrounding mitochondria, suggesting early autophagosome formation, scale bar: 0.1 μm; (H) shows transmission electron microscopy images of spiral ganglion neurons in different groups, with red arrows indicating mitochondrial morphology, scale bar: 0.5 μm. Figures D and F were analyzed using one-way ANOVA, and data are expressed as mean ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0028] Figure 4The results show that TJ0113 can improve D-galactose-induced degenerative damage in cochlear explants. (A) shows the experimental protocol of treating cultured cochlea with D-galactose combined with TJ0113; (B) shows that TJ0113 improves the damage of spiral ganglion neurons in the transit area of ​​cochlear explants, scale bar: 20 μm; (C) shows the quantitative analysis results of Figure B; (D) shows the immunofluorescence staining of PHA (green) and Myosin7a (gray), with yellow arrows indicating broken styloid cilia, scale bar: 20 μm; (E) shows the staining of hair cells in the transit area with mitochondrial superoxide probe MitoSOX Red (red) and Myosin7a (gray), scale bar: 20 μm; (F) shows the quantitative statistical results of Figure D; (G) shows the staining and relative expression levels of aging-related β-galactosidase in different groups, scale bar: 20 μm; (H) shows the quantitative statistical results of Figures E and G. Figures C, F, and H were analyzed using one-way ANOVA. Data are expressed as mean ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0029] Figure 5 This study demonstrates that TJ0113 can alleviate D-galactose-induced HEI-OC1 cell death and senescence. Figures (A) show the treatment protocol for the D-galactose-induced simulated senescence model; (BD) show cell viability as determined by the CCK-8 assay; (E) show the staining results of senescence-related β-galactosidases after TJ0113 intervention in the senescence model (overall field scale bar: 10 μm; magnified field scale bar: 5 μm); (F) show the p16 protein expression level after D-galactose treatment and TJ0113 intervention; (G) show the quantitative statistical results of Figure E; (H) show the quantitative statistical results of Figure F; (I) show the principal component analysis plot, indicating clear separation among the sample groups; (J) show the differentially expressed gene volcano plot; and (K) show the mRNA expression levels of senescence-related secretory phenotype genes in the two groups. Figures BD, G, H, and K were analyzed using one-way ANOVA, and data are expressed as mean ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0030] Figure 6This study demonstrates how TJ0113 alleviates age-related ototoxicity through FUNDC1 and PINK1 / Parkin-dependent mitophagy. (A) shows representative images of HEI-OC1 cells stained with MitoSOX Red, CellROX Green, and Rhodamine 123, scale bar: 20 μm; (B) shows flow cytometry detection and quantitative analysis of MitoSOX Red, CellROX Green, and Rhodamine 123 fluorescence signals; (C) shows images of Parkin and the mitochondrial tracer MitoTracker co-localization, overall field of view scale bar: 20 μm, local magnified field of view scale bar: 5 μm; (D) shows the fluorescence intensity distribution curves of Parkin and MitoTracker in stimulated emission depletion microscopy images (normalized to the maximum value); (E) shows the quantitative statistical results of Figure C; (F) shows the detection of FUNDC1 and PINK1 protein expression by Western blotting; and (G) shows the semi-quantitative analysis results of protein band grayscale. Figures E and G were analyzed using one-way ANOVA, and Figure B was analyzed using a t-test; data are expressed as mean ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0031] Figure 7 This study demonstrates the potential mechanism of action of TJ0113 in a D-galactose-induced HEI-OC1 cell senescence model.

[0032] Figure 8 (A) shows a heatmap of differentially expressed genes for aging-related secretory phenotypes between the D-galactose group and the D-galactose + TJ0113 group; (B) shows a heatmap of differentially expressed genes for the Sirt factor family between the D-galactose group and the D-galactose + TJ0113 group. Detailed Implementation

[0033] Through extensive and in-depth research, the inventors unexpectedly discovered that compound TJ0113 has a significant protective effect against age-related hearing loss, thus showing promise as a translational medicine candidate drug for preventing and treating age-related hearing loss and maintaining healthy auditory aging. Based on this, the present invention was completed.

[0034] definition The scientific and technical terms used in this document are the same as or similar to those commonly understood by those skilled in the art. For clarity, some terms used in this document are defined as follows: Optional replacement As used herein, the term "optional substitution" means that the group modified by this term may be unsubstituted or may be substituted by one or more substituents. In specific embodiments, the group modified by "optional substitution" may be substituted by one or more (e.g., 1, 2, or 3) substituents selected from the group consisting of: hydroxyl, halogen, C 1-3 Alkyl, C 1-3 Alkoxy or amino groups are acceptable as long as the substitution meets the valence requirement.

[0035] PINK1 / Parkin ubiquitination pathway The “PINK1 / Parkin ubiquitination pathway” described in this article refers to the classic ubiquitin-dependent mitophagy pathway. PINK1 (PTEN-induced pseudokinase 1) is a mitochondrial kinase, and Parkin is an E3 ubiquitin ligase. Specifically, this pathway involves the upregulation of PINK1 protein expression, which promotes the recruitment of Parkin protein to depolarized, damaged mitochondria. The recruited Parkin then ubiquitinates mitochondrial outer membrane proteins, thereby promoting the recruitment of mitophagosomes and initiating selective mitochondrial isolation and clearance.

[0036] FUNDC1 receptor pathway In addition to the PINK1 / Parkin ubiquitination pathway, there are also PINK1 / Parkin-independent mitophagy pathways, such as FUNDC1, NIX, and BNIP3, which can recruit LC3 to promote the formation of mitophagosomes without relying on PINK1 / Parkin.

[0037] The “FUNDC1 receptor pathway” mentioned in this article refers to the receptor-mediated (non-ubiquitin-dependent) mitophagy pathway, which is also a non-ubiquitin-dependent mitophagy pathway. FUNDC1 (containing the FUN14 domain protein 1) is a highly conserved mitochondrial outer membrane protein that functions as a mitophagy receptor. It can directly recruit LC3 to damaged mitochondria, providing a parallel pathway for mitochondrial isolation independent of ubiquitination.

[0038] The compounds of the present invention In this document, "the compound of the present invention" refers to the compound represented by Formula I: I In the formula, X, Y1, Y2, R1, R2, R3, and R4 are as described above.

[0039] In specific embodiments, the compound of the present invention is either compound UMI-77 or compound TJ0113; preferably compound TJ0113. UMI-77, TJ0113.

[0040] UMI-77 is a classic selective mitophagy inducer that maintains mitochondrial integrity and targets and eliminates damaged mitochondria. TJ0113 is a small molecule derivative derived from the naphthylamine core structure of UMI-77. Similar to UMI-77, TJ0113 selectively induces autophagy in damaged mitochondria without significantly affecting normal mitochondria. Furthermore, TJ0113 exhibits superior metabolic stability, pharmacokinetic properties, and lower toxicity.

[0041] Use of the compounds of the present invention in the treatment of age-related hearing loss In today's society, an aging population is exacerbating the global social and economic burden. Therefore, the development of anti-aging treatments is urgently needed. Due to the complex structure and diverse cell types of the cochlea, there is currently no cure for age-related hearing loss.

[0042] Through systematic in vitro and in vivo experiments, the inventors have demonstrated that the compound shown in Formula I can delay the progression of age-related hearing loss and inhibit cell aging in the long term, thereby exhibiting a significant protective effect against age-related hearing loss.

[0043] Specifically, long-term intervention observations in aging C57BL / 6J mice revealed that compound TJ0113 significantly delayed the increase in hearing threshold with age and mitigated structural degeneration of the auditory pathway. Transmission electron microscopy results confirmed that, in vivo, compound TJ0113 effectively protected the number and morphological integrity of mitochondria in cochlear hair cells and spiral ganglion neurons; consistent results were obtained in basilar membrane explant culture and D-galactose-induced cell model experiments.

[0044] A nine-month animal follow-up study showed that initiating drug intervention at 12 months of age was more effective than at 6 months, with a maximum difference in hearing threshold exceeding 20 dB. This suggests that this mitophagy activator does not require early intervention; its effectiveness is better in middle-aged and elderly individuals who have already experienced hearing loss. This may be because compound TJ0113 activates mitophagy through reactive oxygen species (ROS) pathways. Middle-aged and elderly individuals with pre-existing ROS levels can achieve more significant therapeutic benefits, a characteristic that also helps improve clinical medication adherence.

[0045] Simultaneously, experiments revealed that the TJ0113 compound can protect low- and mid-frequency hearing in middle-aged and elderly individuals, but cannot reverse high-frequency hearing loss at 24kHz and 32kHz. The inventors hypothesize that the cochlear structure degeneration mechanisms differ across frequency bands, and further analysis using single-cell sequencing and basilar membrane segmentation is needed to elucidate the specific mechanisms underlying these frequency band-specific damage.

[0046] Existing research has confirmed that during the aging process of the cochlea in primates, the aging-related molecular characteristics of hair cells and spiral ganglion neurons are the most significant. In this invention, electron microscopy results directly demonstrate the typical damage of mitochondria in the aging cochlea, including swelling, cristae loss, and vacuolation. The inventors have found that compound TJ0113 can comprehensively improve age-related cochlear damage, including lesions in hair cells, band synapses, afferent nerve fibers, spiral ganglion neurons, and stria vascularis; among these, hair cell stomatal cilia, spiral ganglion neurons, and stria vascularis are most sensitive to aging and drug intervention. Compound TJ0113 can repair mitochondrial ultrastructure, and early autophagosome formation was observed. This protective effect covers the auditory epithelium and neural pathways, achieving systemic mitochondrial homeostasis repair, representing an interventional approach that can alter disease progression.

[0047] The inventors' discovery provides new ideas for targeted treatment strategies. For example, subsequent research can focus on the aforementioned sensitive structures to narrow the research scope and more quickly elucidate the core pathological mechanisms.

[0048] From a mechanistic perspective, compound TJ0113 maintains mitochondrial integrity and function by activating mitophagy, while simultaneously reducing inflammation and oxidative stress. This aligns closely with the core pathological features of age-related hearing loss, demonstrating its potential for clinical translation, though further clinical trials are needed for validation. Specifically, transcriptome sequencing revealed that compound TJ0113 can partially reverse age-related transcriptome alterations and inhibit age-related secretory phenotypic pathways. Mechanistic studies indicate that compound TJ0113 primarily maintains mitochondrial homeostasis and mitigates cochlear aging-related damage by synergistically activating mitophagy mediated by the FUNDC1 and PINK1 / Parkin pathways. Therefore, compound TJ0113, which targets and enhances mitophagy, holds promise as a translational medicine candidate for preventing and treating age-related hearing loss and maintaining healthy auditory aging.

[0049] Specifically, TJ0113 can synergistically activate two major mitophagy pathways: the PINK1 / Parkin ubiquitination pathway and the FUNDC1 receptor pathway. In aging cochlea, PINK1 and Parkin expression declines, autophagosome formation is inhibited, and damaged mitochondria accumulate. TJ0113 can upregulate PINK1 expression, promote Parkin recruitment to damaged mitochondria, initiate ubiquitination modification, and subsequently recruit the autophagy membrane to complete mitochondrial clearance. Based on proteomics data, it is speculated that the family of silencing information regulators may be located upstream of PINK1 and participate in regulating this pathway.

[0050] FUNDC1 is a key mitophagy receptor that can directly recruit autophagy-related proteins without ubiquitination, complementing the PINK1 / Parkin pathway. The two pathways work synergistically to significantly enhance the recognition, encapsulation, and clearance efficiency of damaged mitochondria, reduce abnormal mitochondria producing reactive oxygen species, and alleviate intracellular oxidative stress.

[0051] Reduced oxidative stress can further suppress age-related secretory phenotypes and block the cellular senescence process. Cellular senescence is a core link in cochlear degeneration and the progression of age-related hearing loss: senescent cells exhibit permanent cell cycle arrest, upregulate the senescence marker protein p16, and release a large number of pro-inflammatory factors and chemokines through age-related secretory phenotypes, triggering local chronic inflammation and accelerating tissue damage.

[0052] Pharmaceutical Composition Based on the teachings of this invention, those skilled in the art will know that the compounds of this invention can be prepared into pharmaceutical compositions for the treatment of age-related hearing loss.

[0053] In a specific embodiment, the pharmaceutical composition comprises a preventive or therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable excipient.

[0054] Based on the structure of the compounds of this invention, those skilled in the art can formulate the compounds of this invention into various pharmaceutically acceptable salts, thereby improving the relevant properties of these compounds, such as water solubility. Examples of pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, and calcium salts. In a specific embodiment, compound TJ0113 can be formulated into a sodium salt.

[0055] Similarly, the compounds of the present invention can also be formulated into various pharmaceutically acceptable esters to obtain the prodrug form of the compounds of the present invention. After administration to a subject, the prodrug can be hydrolyzed by esterases in the subject's body to obtain the compounds of the present invention. In specific embodiments, the prodrug may be an acetate ester of the compounds of the present invention, etc.

[0056] Those skilled in the art can formulate the compounds or pharmaceutical compositions of the present invention into dosage forms suitable for various routes of administration, including but not limited to those formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal, or topical administration. A dosage is an amount of medicine that effectively improves or eliminates one or more symptoms. For the treatment of a specific disease, an effective amount is an amount sufficient to improve or, in some way, alleviate the symptoms associated with the disease. Such a dosage may be administered as a single dose or may be administered according to an effective treatment regimen. A dosage may cure the disease, but administration is generally intended to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement. The dosage of the medicine will be determined based on the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

[0057] The pharmaceutical formulations of this invention can be administered to any mammal, provided they can obtain the therapeutic effects of the compounds of this invention. Humans, especially the elderly, are of paramount importance among these mammals.

[0058] The pharmaceutical formulations of the present invention can be manufactured using known methods. For example, they can be manufactured by conventional mixing, granulation, tableting, dissolving, or freeze-drying processes. In the manufacture of oral formulations, solid excipients and active compounds can be combined, and the mixture can be selectively ground. If necessary or required, appropriate excipients can be added, and the granular mixture can be processed to obtain capsules, tablets, or tablet cores.

[0059] Suitable excipients, especially fillers, include sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or dicalcium phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, disintegrants, such as the starches mentioned above, as well as carboxymethyl starch, croscarmellose, agar, or alginate or its salts, such as sodium alginate, can be added. Adjuvants, especially flow conditioners and lubricants, include silica, talc, stearates such as calcium magnesium stearate, stearic acid, or polyethylene glycol. If necessary, a suitable coating that resists gastric juices can be provided to the tablet core. For this purpose, a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, varnish solutions, and suitable organic solvents or solvent mixtures. To prepare a coating resistant to gastric juice, a suitable cellulose solution, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, can be used. Dyes or pigments can be added to the coating of the tablet or lozenge core, for example, to identify or characterize combinations of active ingredient dosages.

[0060] Method of using the compound of the present invention Based on the above-mentioned compounds, the present invention further provides a method for activating the PINK1 / Parkin ubiquitination pathway and / or the FUNDC1 receptor pathway in a subject, comprising administering an effective amount of the compound of the present invention, or a pharmaceutically acceptable salt or ester thereof, to the subject in need of doing so.

[0061] Based on the teachings of this invention, those skilled in the art will recognize that the above-described method is used to prevent or treat age-related hearing loss, and is particularly suitable for subjects whose hearing has already declined. In a specific embodiment, the method is used to reverse low-frequency and mid-frequency hearing loss in subjects, such as hearing loss below 24 kHz.

[0062] The administration methods of the compounds of the present invention include, but are not limited to, various administration methods known in the art, and can be determined according to the actual condition of the patient.

[0063] Advantages of the present invention 1. This invention is the first to discover that the compounds shown in Formula I, especially compound TJ0113, exhibit significant protective effects against age-related hearing loss; 2. The compounds shown in Formula I, especially compound TJ0113, have good systemic safety. Acute and long-term oral administration has not caused animal deaths or genotoxicity, and repeated administration has not resulted in drug accumulation. 3. The compounds of this invention provide translational medicine candidates for treating age-related hearing loss and maintaining healthy auditory aging; 4. This invention has completed endpoint indicator detection, and through in vivo experiments at multiple time points and with multiple dosing regimens, it has fully demonstrated the hearing protective effect of the compounds of this invention; and 5. In the context of a rapidly aging global population, this invention has positive social significance and economic value.

[0064] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0065] Example 1. TJ0113 delays the progression of age-related hearing loss. The C57BL / 6J mouse strain is a classic strain for constructing age-related hearing loss models. This strain of mice begins to show high-frequency hearing loss at 6 months of age, progressing to full-range hearing impairment by 12 months. This experiment used an alternate-day gavage administration method, setting two intervention starting points at 6 months and 12 months of age. Figure 1 , Figure 2 ).

[0066] The hearing level of mice in each group was assessed using auditory brainstem response. The results showed that the hearing threshold of mice gradually increased with age, and the baseline hearing of the 6-week-old young control group was significantly better than that of the 6-month-old and 12-month-old aged mice.

[0067] In the experiment starting at 12 months of age, two dosages were set: 5 mg / kg and 10 mg / kg. After 1.5 months of intervention, no significant hearing protection effect was observed in the 10 mg / kg group; therefore, subsequent observations were only conducted between the 5 mg / kg group and the elderly control group. Figure 1 B, C).

[0068] After 1.5 months of intervention, the 5 mg / kg dose group began to show a trend of hearing protection, with the protective effect being particularly significant after 3 months of intervention. At this time, the hearing thresholds in the short sound, 4 kHz, 8 kHz, and 16 kHz frequency bands in this group were significantly lower than those in the elderly control group. Figure 1 C).

[0069] When the intervention reached 15 months of age, the difference in hearing thresholds between the two groups reached its peak: the thresholds in the aforementioned frequency bands decreased by 20.19 dB, 16.78 dB, 13.35 dB, and 18.8 dB, respectively. Figure 1 C); This protective effect can be maintained until 18 months of age, with the threshold differences in each frequency band remaining at 7.49 dB, 11.42 dB, 15.7 dB, and 10.35 dB, respectively. However, in the 24 kHz and 32 kHz high-frequency bands, there was no significant difference in hearing thresholds between the two groups. Figure 1 C).

[0070] In the experiment with 6 months of age as the intervention starting point, two doses were set up: 2.5 mg / kg and 5 mg / kg. Figure 2 A). Long-term monitoring revealed no significant difference between the 2.5 mg / kg dose group and the elderly control group; the 5 mg / kg dose group gradually showed a protective effect after 3 months of intervention, and after 9 months of continuous administration, the short-tone and 16 kHz hearing thresholds were reduced by 19.44 dB and 18.56 dB, respectively, compared with the elderly control group. Figure 2 BD).

[0071] The above results confirm that long-term use of TJ0113 can significantly delay the progression of age-related hearing loss and has a clear protective effect on hearing.

[0072] Example 2. TJ0113 maintains the structural integrity of aging cochlear hair cells and neurofilaments. To clarify the protective effect of TJ0113 on auditory pathway tissue structures, this study performed staining and quantitative analysis on cochlear tissue. Cochlear samples were selected from children who started intervention at 12 months of age and whose cochlear tissue was harvested at 15 months of age (the time point at which the difference in hearing between the two groups was most significant). Myosin7a was used to label hair cells and NF was used to label neurofilaments.

[0073] Compared with the young control group, the elderly control group showed significant hair cell loss in the basal, mid, and apical turns of the cochlea, with damage to outer hair cells being more severe than that to inner hair cells, and the basal turn showing the most severe damage; while the TJ0113 administration group significantly improved the age-related hair cell reduction. Figure 1 DG). However, consistent with the hearing results, the number of basal hair cells did not show a statistically significant improvement after drug intervention (DG). Figure 1 G).

[0074] Observation of cochlear hair cells by co-staining with NF and Myosin7a ( Figure 1 DF) and neurofilaments Figure 2 E) Quantity and Morphology: Compared to young mice, the number of nerve fibers between inner and outer hair cells in aged mice was significantly reduced. The nerve fiber density in the TJ0113-treated group was greater than that in the aged control group, but there was no statistically significant difference in the middle and lower turns of the cochlea. Figure 2 E, F).

[0075] Scanning electron microscopy was used to observe the morphology of the apical cilia of hair cells (the core structure for auditory signal perception and transmission): In the elderly control group, the hair cell bundles were disordered, and the apical structure of the inner hair cells was damaged and disordered; in the TJ0113 drug-treated group, three rows of cilia were observed in the outer hair cells, and the height and thickness of each row of cilia in the inner hair cells were uniform. Figure 1 H, I).

[0076] The above results indicate that TJ0113 can partially protect the innervation of the cochlear nerve and reduce the loss of hair cells and neurofilaments caused by aging in C57BL / 6J mice.

[0077] Example 3. TJ0113 reduces age-related cochlear synapse loss. CtBP2 was used to label the nuclei and band-like synaptic structures of inner hair cells. Figure 3 AC). Quantitative analysis of the number of synapses per inner hair cell in each group revealed that, compared to the young control group, the number of synapses in all cochlear rotations of the elderly control group was significantly reduced; TJ0113 effectively protected the synapses in the middle and basal rotations of the cochlea and delayed their loss, but there was no statistically significant difference in the number of synapses in the apical rotation between the two groups. Figure 3 D).

[0078] The results confirmed that TJ0113 can reduce the loss of cochlear synapses in aged mice, with the protective effect mainly concentrated in the mid-rotation and basal rotation of the cochlea.

[0079] Example 4. TJ0113 inhibits the degeneration of neurons and stria vascularis in the spiral ganglion of the cochlea during aging. Hair cell and spiral ganglion neuron degeneration are the core pathological features of age-related hearing loss, while thinning and atrophy of the stria vascularis are also typical manifestations. Stria vascularis lesions can lead to ion transport disorders, inflammation, and vascular lesions, resulting in cochlear dysfunction.

[0080] Hematoxylin-eosin staining was performed on frozen sections of the cochlea, and the density of neurons in the spiral ganglion and the thickness of the stria vascularis were statistically analyzed. Figure 3 E: In the elderly control group, the density of spiral ganglion neurons and the thickness of the stria vascularis in each rotation of the cochlea were decreased, with the most severe damage in the basal rotation; after TJ0113 intervention, the density of spiral ganglion neurons in each rotation of the cochlea increased significantly, and the thickness of the stria vascularis also improved significantly. Figure 3 F).

[0081] Example 5. TJ0113 Repairs the Ultrastructure and Integrity of Mitochondria in the Aging Cochlea Transmission electron microscopy was used to observe the morphology of hair cell mitochondria: In the aged control group, mitochondria showed typical damage such as swelling, disappearance of cristae, and vacuolation; after treatment with TJ0113, the mitochondrial morphology was significantly restored, and phagocytic vesicle-like membrane structures were observed to encapsulate the damaged mitochondria, indicating the early formation of autophagosomes and the initiation of the process of clearing damaged mitochondria. Figure 3 G).

[0082] Electron microscopy results of mitochondria in spiral ganglion neurons were consistent with those in hair cells: in the elderly control group, mitochondrial cristae were broken, severely swollen, and the matrix was vacuolated; in the TJ0113-treated group, the mitochondrial structure was intact, and the cristae were clearly distinguishable. Figure 3 H).

[0083] In summary, the above ultrastructural results demonstrate that TJ0113 can maintain the mitochondrial integrity of cochlear sensory cells and nerve cells, improve age-related mitochondrial dysfunction, and restart the mitochondrial autophagy quality control function.

[0084] Example 6. TJ0113 alleviates D-galactose-induced cellular senescence and mitochondrial oxidative stress This study used D-galactose to intervene in the cochlear explants of newborn mice to construct an in vitro aging model. Figure 4 A, B). The results showed that D-galactose intervention led to atrophy of spiral ganglion neuronal cell bodies and a significant decrease in the density of spiral ganglion neurons and nerve fibers; while TJ0113 intervention significantly salvaged the density of the above two structures (A, B). Figure 4 B, C).

[0085] D-galactose disrupts hair cell arrangement and reduces cell number; TJ0113 can improve this phenomenon (no statistical difference between groups). PHA staining showed that D-galactose causes breakage of hair cell stereocilia bundles and destruction of the typical V-shaped structure; TJ0113 can effectively maintain the continuity of stereocilia bundles (significant statistical difference between groups), which is consistent with the scanning electron microscopy results. Figure 4 D, F).

[0086] β-galactosidase staining (the gold standard staining for senescence) results showed that the proportion of senescent cells in the D-galactose treatment group was significantly increased, indicating that the degree of senescence of cochlear hair cells significantly increased after D-galactose intervention; TJ0113 could significantly inhibit the proportion of positive staining for senescence in cochlear hair cells ( Figure 4 G, H).

[0087] Detection of mitochondrial reactive oxygen species (ROS) levels using a mitochondrial superoxide fluorescent probe: D-galactose induces a burst of ROS in hair cell mitochondria, while TJ0113 can significantly inhibit the degree of this oxidative stress. Figure 4 E, H). The above results indicate that TJ0113 mainly alleviates D-galactose-induced cochlear damage by reducing mitochondrial reactive oxygen species accumulation and inhibiting cell senescence.

[0088] Example 7. TJ0113 improves the damaged and senescent phenotype of HEI-OC1 cell line A mouse hair cell line, HEI-OC1, was used to construct a cell senescence model using D-galactose. Cell viability assays determined that subsequent experiments would use 20 mg / mL D-galactose for modeling. Figure 5 A, B); Drug intervention experiments confirmed that pretreatment with 2 μM TJ0113 for 24 hours had the best protective effect (A, B); Figure 5 A, C, D).

[0089] The results of aging-related β-galactosidase staining and aging marker protein p16 detection showed that D-galactose significantly upregulated the proportion of senescent cells and p16 protein expression; while both indicators decreased significantly after combined treatment with TJ0113. Figure 5 EH).

[0090] Transcriptome sequencing and principal component analysis showed significant differences in gene expression profiles between the D-galactose model group and the TJ0113 administration group. Figure 5 I) identified 149 differentially expressed genes (71 upregulated and 78 downregulated). Among them, D-galactose group showed significantly increased expression of aging-related secretory phenotype molecules, while TJ0113 downregulated the expression of these molecules. Figure 5 J). Real-time quantitative PCR further validated the expression trends of aging-related secretory phenotype genes such as Nfkb1, Ccl5, and Cxcl1. Figure 5K).

[0091] The above experiments demonstrate that TJ0113 can antagonize D-galactose-induced cytotoxicity and alleviate auditory cell aging.

[0092] Example 8: TJ0113 activates mitophagy, reduces reactive oxygen species levels, and increases mitochondrial membrane potential. In the HEI-OC1 cell senescence model, the results of mitochondrial and intracellular reactive oxygen species (ROS) assays showed that D-galactose significantly increased ROS levels, and TJ0113 pretreatment effectively reversed this change. Figure 6 A, B).

[0093] Rhodamine 123 staining for mitochondrial membrane potential: D-galactose causes a decrease in mitochondrial membrane potential, while TJ0113 can restore membrane potential levels and improve mitochondrial function. Figure 6 A, B).

[0094] However, after blocking mitophagy with Mdivi-1, the protective effect of TJ0113 was significantly counteracted: cellular reactive oxygen species increased again, and mitochondrial membrane potential decreased. This confirms that the protective effect of TJ0113 depends on mitophagy. Figure 6 A, B).

[0095] Example 9. TJ0113 synergistically activates FUNDC1 and PINK1 / Parkin-mediated mitophagy D-galactose inhibits Parkin recruitment to mitochondria, hindering mitochondrial quality control; TJ0113 can significantly restore the colocalization of Parkin and mitochondria, increase the total number of mitochondria, and maintain the integrity of mitochondrial structure. Figure 6 CE).

[0096] Western blot analysis showed that D-galactose downregulated the expression of PINK1 and FUNDC1 proteins, while TJ0113 significantly upregulated their levels. Figure 6 F, G).

[0097] In summary, TJ0113 can simultaneously activate two mitophagy pathways: the FUNDC1 receptor pathway and the PINK1 / Parkin ubiquitination pathway, maintaining mitochondrial quantity and function and inhibiting D-galactose-induced cellular senescence.

[0098] Example 10. Safety, tolerability, and pharmacokinetic studies of compound TJ0113 The inventors prepared compound TJ0113 into capsules and evaluated the safety, tolerability, and pharmacokinetic properties of the capsules after single, multiple, and postprandial oral administration in healthy subjects.

[0099] in conclusion: The TJ0113 compound not only showed a significant trend in delaying age-related hearing loss in animal models, but also demonstrated no significant toxic side effects in humans and exhibited good safety, tolerability, and pharmacokinetic properties in preclinical studies and Phase I clinical trials.

[0100] Table 1 Primer sequences for real-time quantitative PCR All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of the compound of Formula I, or a pharmaceutically acceptable salt or ester thereof, in the preparation of PINK1 / Parkin ubiquitination pathway and / or FUNDC1 receptor pathway activators. I In the formula, X is selected from S or O; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-6 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-6 Alkyl, optionally substituted C 1-6 Alkyl group.

2. The use as described in claim 1, characterized in that, In formula I, X is S; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-3 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-3 Alkyl, optionally substituted C 1-3 Alkyl group.

3. The use as described in claim 2, characterized in that, The compounds shown in Formula I are selected from the following group: 、 ； Preferably, the compound is a compound with the following formula: 。 4. The use as described in any one of claims 1-3, characterized in that, The PINK1 / Parkin ubiquitination pathway and / or FUNDC1 receptor pathway activators are drugs for the prevention or treatment of age-related hearing loss.

5. The compound shown in Formula I, or a pharmaceutically acceptable salt or ester thereof, used as an activator of the PINK1 / Parkin ubiquitination pathway and / or the FUNDC1 receptor pathway. I In the formula, X is selected from S or O; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-6 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-6 Alkyl, optionally substituted C 1-6 Alkyl group.

6. The compound of formula I as claimed in claim 5, or a pharmaceutically acceptable salt or ester thereof, characterized in that, In formula I, X is S; Y1 and Y2 are each independently selected from either non-existent or 0; R1, R2, and R3 are each independently selected from: H, or optionally substituted C. 1-3 alkyl; R4 is selected from: H, halogen, or optionally substituted C. 1-3 Alkyl, optionally substituted C 1-3 Alkyl group.

7. The compound of formula I as claimed in claim 6, or a pharmaceutically acceptable salt or ester thereof, characterized in that, The compounds shown in Formula I are selected from the following group: 、 ； Preferably, the compound is a compound with the following formula: 。 8. The compound of formula I as claimed in any one of claims 5-7, or a pharmaceutically acceptable salt or ester thereof, characterized in that, The PINK1 / Parkin ubiquitination pathway and / or FUNDC1 receptor pathway activators are drugs for the prevention or treatment of age-related hearing loss.

9. A method for activating the PINK1 / Parkin ubiquitination pathway and / or the FUNDC1 receptor pathway in a subject, the method comprising administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt or ester thereof, to a subject in need of such activation.

10. The method as described in claim 9, characterized in that, The method is used to prevent or treat age-related hearing loss.

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