Application of CY-09 in preparation of medicine for preventing, improving and / or treating night noise induced hearing loss

Abstract

The invention discloses an application of CY-09 in preparation of a medicine for preventing, improving and / or treating night noise induced hearing loss, relates to the field of medicines, and aims to solve the problem that existing medicines for preventing and treating night noise induced hearing loss are rare. The influence of CY-09 on noise-induced hearing loss and synaptic impairment is evaluated through a day / night noise-induced hearing loss animal model, and the result shows that CY-09 has a good protection effect on the number and function of synapses of inner hair cells and the function of cochlea outer hair cells when noise is exposed at night, and the CY-09 has a good protection effect on the number and function of synapses of the inner hair cells and the function of cochlea outer hair cells when noise is exposed at night. The CY-09 can effectively reduce the degree of the night noise-induced hearing loss, the prevention and treatment effect of the CY-09 on the night noise-induced hearing loss is visually verified, and the CY-09 can be used for preparing the medicine for preventing and treating the night noise-induced hearing loss and has good research and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a new use of CY-09, and more particularly to an application of CY-09 in preparing a drug for preventing, improving and / or treating nocturnal noise-induced hearing loss. Background Art

[0002] The latest World Hearing Report shows that about 20% of the world's population suffers from varying degrees of hearing loss, which has become a global public health problem that needs to be urgently addressed. Noise-induced hearing loss is one of the most common sensorineural hearing losses in adults. It is often insidious in onset and difficult to detect. As the disease progresses, it accelerates the occurrence of presbycusis and dementia. Due to the changes in modern people's lifestyles, compared with daytime noise, nighttime occupational noise, traffic noise and entertainment noise are becoming more and more common. Studies have shown that hearing loss caused by nighttime noise exposure is more severe, and the susceptibility of the auditory system to noise at night has increased significantly, which has brought great burdens to individuals, families and society.

[0003] Impaired sound coding at the ribbon synapses between inner hair cells and cochlear afferent nerves is one of the early pathological manifestations of noise-induced hearing loss. The number and function of inner hair cell ribbon synapses reflect the drug effects before and after noise-induced hearing loss. At present, there is no effective drug to prevent and treat noise-induced inner hair cell ribbon synapse damage in clinical practice, not to mention nighttime noise-induced hearing loss. In basic scientific research, only glutamate antagonists, neurotrophic factors, calcium channel blockers, antioxidants, anti-inflammatory drugs and vasodilators have been reported to prevent and treat noise-induced hearing loss, but the effects are poor and none of them can prevent noise-induced inner hair cell ribbon synapse damage.

[0004] CY-09 is a NLRP3 selective inhibitor that directly binds to the ATP binding motif of the NLRP3-NACHT domain and inhibits the NLRP3-ATPase activity, thereby inhibiting the assembly and activation of the NLRP3 inflammasome. However, after reviewing relevant domestic and foreign literature, only research reports on the treatment of type 2 diabetes and atherosclerosis with CY-09 were found, and no research reports on the role of CY-09 in protecting against nighttime noise-induced hearing loss were found. Summary of the invention

[0005] The present invention aims to solve the problem that existing drugs for preventing and treating nocturnal noise-induced hearing loss are scarce, and aims to provide the use of CY-09 in drugs for preventing, improving and / or treating nocturnal noise-induced hearing loss.

[0006] Preferably, the CY-09 is used to protect the number and function of synapses of hair cells in the cochlea under nighttime noise exposure, thereby achieving the purpose of preventing, improving and / or treating nighttime noise-induced hearing loss.

[0007] Preferably, the CY-09 is used alone.

[0008] Preferably, the CY-09 is used in the form of a pharmaceutical composition.

[0009] Preferably, the drug is a pharmaceutical preparation made of CY-09 and pharmaceutical excipients.

[0010] Preferably, the dosage form of the pharmaceutical preparation includes solid dosage forms and liquid dosage forms.

[0011] Preferably, the solid dosage forms include tablets, capsules, granules, pills, suppositories, membranes, glue agents, ointments or powders.

[0012] Preferably, the liquid dosage forms include injections, mixtures, oral liquids, syrups, medicinal wines, sols or emulsions.

[0013] Compared with the prior art, the beneficial effects of the present invention at least include:

[0014] By using a day / night noise-induced hearing loss animal model, the present invention evaluates the effects of CY-09 on noise-induced hearing loss and synaptic damage. The results show that CY-09 has a good protective effect on the number and function of inner hair cell synapses and the function of outer hair cells in the cochlea during nocturnal noise exposure, and can effectively reduce the degree of nocturnal noise-induced hearing loss; while it has no obvious protective effect on the hearing loss caused by daytime noise exposure. The present invention intuitively verifies the preventive and therapeutic effects of the CY-09 on nocturnal noise-induced hearing loss, and can be used to prepare drugs for preventing and treating nocturnal noise-induced hearing loss, having good research and application prospects. Description of the Drawings

[0015] Figure 1 Are the ABR hearing test results of the control group and the CY-09 experimental group of mice during the day and night before and 14 days after noise exposure; wherein:

[0016] A is a schematic diagram of the change in hearing threshold before and after noise exposure in the daytime control group and the CY-09 experimental group;

[0017] B is a schematic diagram of the change in hearing threshold before and after noise exposure in the nocturnal control group and the CY-09 experimental group;

[0018] C is the comparison of the ABR hearing threshold shift between the daytime control group and the experimental group 14 days after noise exposure compared with before noise exposure;

[0019] D is the comparison of the ABR hearing threshold shift between the nocturnal control group and the experimental group 14 days after noise exposure compared with before noise exposure.

[0020] Figure 2Changes in the number of inner hair cell synapses in the control group and the CY-09 experimental group of mice during the day and night on the 14th day after noise exposure.

[0021] Figure 3 Changes in the DPOAE thresholds of the control group and the CY-09 experimental group of mice during the day and night on the 14th day after noise exposure; wherein:

[0022] A shows the results of changes in DPOAE thresholds of the control group and the CY-09 experimental group of mice during the day.

[0023] B shows the results of changes in DPOAE thresholds of the control group and the CY-09 experimental group of mice at night.

[0024] Figure 4 Results of ABR hearing tests on the control group and the Tranilast experimental group of mice during the day and night before noise exposure and on the 14th day after noise exposure; wherein:

[0025] A is a schematic diagram of changes in hearing thresholds of the control group and the Tranilast experimental group of mice during the day before and after noise exposure.

[0026] B is a schematic diagram of changes in hearing thresholds of the control group and the Tranilast experimental group of mice at night before and after noise exposure.

[0027] Figure 5 Results of ABR hearing tests on the control group and the melatonin experimental group of mice at night before noise exposure and on the 14th day after noise exposure. Specific implementation manners

[0028] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] As can be seen from the foregoing, there is a lack of drugs for preventing and treating nocturnal noise-induced hearing loss in the prior art. To solve this problem, through research, we found that the gene expression changes after noise exposure during the day and night are mainly enriched in the "immune response-related signaling pathway". Through qPCR verification, it was found that the expression of the macrophage marker CD68 was significantly upregulated after nocturnal noise exposure. Therefore, it is inferred that nocturnal noise-induced hearing loss is related to the activation of cochlear macrophages.

[0030] Based on the above research, the applicant of the present invention included a large number of gene regulators related to cochlear macrophage activation and drugs involved in the circadian rhythm generation for testing and screening. For example, it was found through literature review that NLRP3 is involved in the recruitment and activation of cochlear macrophages and the subsequent inflammatory response. Therefore, NLRP3 selective inhibitors including but not limited to CY-09 and tranilast were included in the study; drugs involved in the circadian rhythm generation including but not limited to melatonin were included in the study. Finally, it was found that CY-09 has a good protective effect on the number and function of inner hair cell synapses in the cochlea exposed to nocturnal noise, as well as the function of outer hair cells in the cochlea, ultimately achieving the purpose of preventing, improving or treating nocturnal noise-induced hearing loss.

[0031] Therefore, the present invention firstly provides a new use of CY-09, that is, the application of CY-09 in the preparation of drugs for preventing, improving and / or treating nocturnal noise-induced hearing loss.

[0032] When the CY-09 of the present invention is used in the preparation of drugs for preventing, improving and / or treating noise-induced hearing loss, it can be used alone; it can also be used in the form of a pharmaceutical composition; it can also be made into pharmaceutical preparations with pharmaceutical excipients, such as tablets, capsules, granules, pills, suppositories, membranes, glue agents, ointments, powders, injections, mixtures, oral liquids, syrups, medicinal wines, sols or emulsions and other various forms.

[0033] Through experiments on a day / night noise-induced hearing loss mouse model, the present invention evaluated the effect of CY-09 on noise-induced synaptic damage. The results showed that CY-09 has a good protective effect on the number and function of inner hair cell synapses and the function of outer hair cells in the cochlea during nocturnal noise exposure, intuitively verifying the preventive, improving and therapeutic effects of CY-09 on nocturnal noise-induced hearing loss.

[0034] The experimental process and experimental results of the present invention will be described in detail below, so as to elaborate in detail the preventive, improving and therapeutic effects of CY-09 on nocturnal noise-induced hearing loss, and it can be used in the preparation of drugs for preventing and / or treating nocturnal noise-induced hearing loss.

[0035] (I) Experimental materials

[0036] 1. Reagent preparation

[0037] CY-09 solution: CY-09 was purchased from MedChemExpress, catalog number: HY-103666, CAS number: 1073612-91-5. It was dissolved in DMSO to prepare a clear stock solution with a mass concentration of 25 mg / ml and stored at -80 °C for later use. It was freshly prepared on the day of the experiment, and the co-solvent 40% PEG300 → 5% Tween-80 → 50% normal saline was added in sequence.

[0038] Control group solution: Add 5% DMSO → 40% PEG300 → 5% Tween-80 → 50% normal saline of each solvent in sequence.

[0039] Note: The percentage shown before the solvent refers to the volume ratio of the solvent in the final prepared solution.

[0040] Tranilast: Tranilast was purchased from Selleck, catalog number: S1439, and dissolved in 0.5% sodium carboxymethylcellulose.

[0041] Melatonin: Melatonin was purchased from MedChemExpress, catalog number: HY-B0075, dissolved in DMSO, and diluted to the corresponding concentration with normal saline.

[0042] 2. Animals

[0043] A number of male CBA / CaJ mice (Shanghai) aged 6 - 8 weeks were selected, excluding middle ear and inner ear diseases, and ABR auditory threshold detection was performed. Mice with normal ABR auditory threshold were screened as experimental subjects. The experimental animals were maintained at a room temperature of 24°C, given sufficient water and food, with a light / dark cycle of 12 / 12 hours, and the environmental noise was less than 40 dB SPL. All experimental operations were approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine.

[0044] (2) Experimental methods

[0045] 1. Animal grouping and treatment

[0046] (1) For CY-09, the experimental animals were randomly divided into 4 groups, including:

[0047] Experimental group, which was further divided into a daytime group (CY-09-D) and a nighttime group (CY-09-N), with a dosing dose of 5 mg / kg. By intraperitoneal injection, once a day for one week before noise exposure and on the 1st to 3rd days after noise exposure.

[0048] Control group, divided into a daytime group (Ctrl-D) and a nighttime group (Ctrl-N). The control group mice were intraperitoneally injected with the same volume of solvent as the experimental group mice (i.e., the control group solution in the aforementioned reagent preparation).

[0049] (2) For Tranilast, the experimental animals were randomly divided into 4 groups, including:

[0050] Experimental group, which was further divided into a daytime group (Tranilast-D) and a nighttime group (Tranilast-N), with a dosing dose of 50 mg / kg. By oral administration, once a day for one week before noise exposure and on the 1st to 3rd days after noise exposure.

[0051] The control group was divided into a daytime group (Ctrl-D) and a nighttime group (Ctrl-N). The mice in the control group were orally administered the same volume of solvent as the mice in the experimental group.

[0052] It should be noted that as is well known in the art, day generally refers to 6:00 am to 6:00 pm on the same day, and night generally refers to 6:00 pm to 6:00 am the next day. In the embodiments of the present invention, noise exposure was given to the mice in the time period from 8:00 to 10:00 am and named the daytime group; noise exposure was given to the mice in the time period from 8:00 to 10:00 pm and named the nighttime group. In addition, regardless of whether it was the daytime group or the nighttime group, the drug was administered at the same arbitrary time period during the day.

[0053] (3) The melatonin-N experimental animals were randomly divided into 3 groups, including:

[0054] The experimental group was given noise exposure at night and was further divided into a low-dose group (20 mg / kg Melatonin-N) and a high-dose group (40 mg / kg Melatonin-N). By means of intraperitoneal administration, once a day was administered for one week before noise exposure and on the 1st to 3rd days after noise exposure.

[0055] The control group (Ctrl-N) was given noise exposure at night, and the mice were intraperitoneally injected with the same volume of solvent as the mice in the experimental group.

[0056] 2. Noise exposure of experimental animals

[0057] All experimental mice were placed in different intervals of the wire cages, and the mice could move freely in the cage intervals. The speaker was placed at the top directly above the cage. The noise signal was generated by a real-time signal processor (TDT system). Before each noise exposure, the sound of the speaker was calibrated. When calibrating, the probe of the sound level meter was placed inside the cage, and the sound intensity change at each part inside the cage was <1 dB. During noise exposure, noise with a frequency of 2 - 20 kHz and a sound intensity of 107 dB SPL was used for exposure, and the exposure time was 2 hours.

[0058] (3) Detection

[0059] 1. Auditory brainstem response (ABR)

[0060] The baseline values and the changes in hearing thresholds after noise exposure were measured on the 14th day before and after noise exposure, respectively. Specific methods: The experimental mice in each group were selected and weighed, and anesthetized by intraperitoneal injection of 5% chloral hydrate. When the mice had slow and steady breathing, the corneal reflex and auricular pain reflex disappeared and there was no hiccupping, it indicated moderate anesthesia. The anesthetized mice were placed on a thermostatic pad, and the temperature was set at 37 °C and adjusted according to the rectal temperature of the mice to ensure the accuracy of ABR measurement and prevent the death of the mice caused by hypothermia after anesthesia. The sound delivery mode of ABR test was an open sound field, and the speaker was located directly in front of the mice, 10 cm away from the midpoint of the line connecting the two ears. The recording electrode was placed subcutaneously in the middle of the mouse skull, the reference electrode was placed subcutaneously in the mastoid area, and the ground electrode was placed subcutaneously on the shoulder of the upper limb. The stimulating sound of the ABR TDT system was short pure tones with frequencies of 4.0, 8.0, 11.3, 16.0, 22.6, and 32.0 kHz, the stimulating rate was 10 times / s, the stimulating duration was 5 ms, the rise and fall times were both 1 ms, the repetition rate was 21 times / s, the filtering bandwidth was 100 - 3000 Hz, and the number of superimpositions was 400; the sound delivery intensity started from 90 dB SPL and decreased by 5 dB SPL until the disappearance of each waveform was defined as the hearing threshold. When approaching the threshold, generally, it was necessary to repeat the detection more than twice to compare whether the waveforms were consistent. Each index was displayed, recorded, calculated, and stored in real time by the software attached to the test instrument.

[0061] 2. Immunofluorescence staining of the cochlear basilar membrane to count the number of ribbon synapses of inner hair cells

[0062] The experimental mice were sacrificed and the cochleae were quickly removed and fixed thoroughly with 4% paraformaldehyde. The basilar membrane was dissected, the tectorial membrane and vestibular membrane were removed, and immunofluorescence staining of the proteins related to the ribbon synapses of inner hair cells (Ctbp2, GluA2) was performed. A Zeiss LSM880 laser confocal microscope was used to perform layer scanning (Z-stack 0.18 μm) on the specimens, and the number of ribbon synapses in each inner hair cell was counted.

[0063] 3. Distortion product otoacoustic emission (DPOAE)

[0064] Similar to ABR, the baseline values and the changes in DPOAE thresholds after noise exposure were measured 14 days before and after noise exposure, respectively. Specific method: Select the experimental mice in each group and weigh them, and intraperitoneally inject 5% chloral hydrate for anesthesia. When the mouse's breathing is slow and stable, the corneal reflex and auricular pain reflex disappear and there is no hiccupping phenomenon, it indicates moderate anesthesia. Place the anesthetized mouse on a thermostatic pad, set the temperature to 37 °C, and adjust according to the mouse's rectal temperature to ensure the accuracy of DPOAE measurement and prevent the mouse from dying due to a decrease in body temperature after anesthesia. The sound delivery mode for DPOAE testing is a closed sound field. Two consecutive pure tones f1 and f2 are used as the initial signals for testing, f2 / f1 = 1.2, with the same intensity. Take the amplitude corresponding to the frequency point of DPOAE 2f2 - f1, and the number of superimpositions is 512; the sound delivery intensity starts from 80 dB SPL and decreases by 5 dB SPL until the distortion product cannot be elicited. Record the response thresholds at 5 frequency points of 8.0, 11.3, 16.0, 22.6, and 32.0 kHz. Each index is displayed, recorded, calculated, and stored in real time by the software attached to the testing instrument.

[0065] (IV) Statistical methods

[0066] The experimental data results are expressed as mean ± SEM, and statistical analysis and graphing are performed using Prism. Use one-way or two-way ANOVA followed by Bonferroni post-hoc test or use unpaired Student's t-test (the data conforms to normal distribution and homogeneity of variance at the same time, if not, use Mann-Whitney U test). A P < 0.05 is defined as statistically significant difference.

[0067] (V) Experimental results

[0068] 1. Effect of CY-09 on ABR in mice after noise exposure

[0069] As Figure 1 shown, by detecting the changes in ABR before and after noise exposure in each experimental group and the control group, the efficacy of CY-09 on day / night noise-induced synaptic damage was evaluated.

[0070] Comparing the results of Ctrl-D Prenoise, Ctrl-D Ne14d, Ctrl-N Prenoise, and Ctrl-N Ne14d, it can be seen that the ABR thresholds of the control group mice on day and night did not recover 14 days after noise exposure, and the ABR thresholds of the control group mice at night 14 days after noise exposure were significantly higher than those of the control group during the day ( Figure 1A and B), indicating that under the same noise exposure conditions (except for different noise exposure times: noise exposure during the day or at night), compared with daytime noise exposure, night-time noise exposure causes more severe hearing loss, and the noise susceptibility of the auditory system at night increases significantly. The present invention has successfully constructed an animal model of the difference in day / night noise-induced hearing loss.

[0071] In addition, by comparing the Ctrl-D and CY-09-D curves and the ABR hearing threshold shift data of the experimental and control mice in the daytime group after 14 days, it can be seen that on the 14th day after noise exposure, there is no significant difference in the hearing threshold between the CY-09-D group and the control group (Ctrl-D), indicating that CY-09 has no obvious protective or improving effect on the hearing loss caused by daytime noise exposure ( Figure 1 A and C), while on the 14th day after noise exposure, the ABR hearing threshold of the CY-09-N group is significantly restored compared with the control group (Ctrl-N). Especially at the three frequencies of 11.3, 16.0, and 22.6 kHz, the ABR thresholds of the mice in the CY-09-N group are significantly decreased, indicating that CY-09 has an obvious protective effect on the hearing loss caused by night-time noise exposure ( Figure 1 B and D).

[0072] The above results show that CY-09 has a preventive and improving effect on the hearing loss caused by night-time noise exposure, while it has no improving or preventive effect on the hearing loss caused by daytime noise exposure.

[0073] 2. Effect of CY-09 on the number of inner hair cell synapses in mice after noise exposure

[0074] By counting the changes in the number of inner hair cell synapses on the 14th day after noise exposure in each experimental group and the control group, the effect of CY-09 on the number of inner hair cell synapses in mice after noise exposure was evaluated.

[0075] The results are as Figure 2 shown. By comparing the results of the Ctrl-D and Ctrl-N groups, it can be seen that the number of ribbon synapses of inner hair cells in the night-time control group of mice decreased the most after noise exposure, indicating that compared with daytime noise, the loss of the number of ribbon synapses of inner hair cells caused by night-time noise is more serious; 14 days after noise exposure, the number of ribbon synapses of inner hair cells in the CY-09-N group is significantly higher than that of the other three groups, indicating that CY-09 has a protective effect on the ribbon synapses of night-time noise exposure. In addition, by comparing the results of the Ctrl-D and CY-09-D groups, it can be seen that CY-09 has no obvious protective effect on the ribbon synapses of daytime noise exposure.

[0076] The above results show that CY-09 has an obvious protective effect on the synapses of inner hair cells during night-time noise exposure, while it has no protective effect on the synapses of inner hair cells during daytime noise exposure.

[0077] 3. Effects of CY-09 on the function of cochlear outer hair cells in mice after noise exposure

[0078] DPOAE can reflect the function of outer hair cells in mice. By comparing the changes in DPOAE before and after noise exposure in each experimental group and the control group of mice respectively, the effects of CY-09 on the function of the cochlea and outer hair cells in mice after noise exposure were evaluated.

[0079] The results are as Figure 3 shown in Figures A and B. On the 14th day after noise exposure, the DPOAE threshold of mice in the control group at night (Ctrl-N Ne14d) was significantly higher than that in the control group during the day (Ctrl-D Ne14d), which was consistent with the previous ABR results, indicating that night noise could cause more severe hearing loss in mice. Under drug intervention, by comparing the results of Ctrl-N Ne14d and CY-09-N Ne14d, as well as the results of Ctrl-D Ne14d and CY-09-D Ne14d, it can be seen that CY-09 has an obvious protective effect on cochlear outer hair cells during night noise exposure, especially at the frequency of 11.3 kHz; while it has no obvious protective effect on cochlear outer hair cells exposed to noise during the day.

[0080] The above results indicate that CY-09 has an obvious protective effect on the cochlea exposed to night noise, while it has no significant protective effect on the cochlea exposed to day noise.

[0081] 4. Effects of tranilast on ABR in mice after noise exposure

[0082] Tranilast is another NLRP3 inhibitor. In this invention, the effects of this inhibitor on day / night noise-induced hearing loss were evaluated through experiments.

[0083] The results are as Figure 4 shown in Figures A and B. By comparing the ABR thresholds of mice in the experimental groups during the day and at night, it was found that tranilast had no effect on reducing and restoring the increased ABR thresholds caused by noise exposure during the day and at night, indicating that tranilast had no obvious improvement effect on the hearing loss in mice caused by noise exposure during the day or at night.

[0084] 5. Effects of melatonin on ABR in mice after night noise exposure

[0085] Melatonin is involved in the occurrence of the circadian rhythm. In this invention, melatonin was tried to be used to treat night noise-induced hearing loss.

[0086] The results are as Figure 5 shown. By comparing the ABR thresholds of mice in the experimental groups at night (low concentration / high concentration) and the control group, it was found that melatonin had no obvious improvement effect on the hearing loss caused by night noise exposure.

[0087] In summary, CY-09 can effectively protect the number and function of the ribbon synapses of inner hair cells and the function of outer hair cells in the cochlea during nocturnal noise exposure, reduce the degree of nocturnal noise-induced hearing loss, and can be used to prepare drugs for preventing and / or treating nocturnal noise-induced hearing loss.

[0088] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be construed as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and alternatives to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. Use of CY-09 in the preparation of a medicament for preventing, improving, and / or treating nocturnal noise-induced hearing loss.

2. The use according to claim 1, wherein, the CY-09 is used to protect the number and function of inner hair cell synapses in the cochlea during nocturnal noise exposure, so as to achieve the purpose of preventing, improving, and / or treating nocturnal noise-induced hearing loss.

3. The use according to claim 1, wherein, the CY-09 is used alone.

4. The use according to claim 1, wherein, the CY-09 is used in the form of a pharmaceutical composition.

5. The use according to claim 1, wherein, the medicament is a pharmaceutical preparation made of CY-09 and pharmaceutical excipients.

6. The use according to claim 4, wherein, the dosage form of the pharmaceutical preparation includes solid dosage forms and liquid dosage forms.

7. The use according to claim 5, wherein, the solid dosage forms include tablets, capsules, granules, pills, suppositories, films, glue agents, ointments, or powders.

8. The use according to claim 5, wherein, the liquid dosage forms include injections, mixtures, oral liquids, syrups, medicinal wines, sols, or emulsions.

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