Medicine for preventing and treating salicylic acid induced tinnitus

Abstract

The invention discloses a medicine for preventing and treating salicylic acid-induced tinnitus, which is prepared by the following preparation steps: step 1, single intraperitoneal injection of salicylic acid (350 mg / kg) can stably induce a rat to have reversible tinnitus-like behaviors (the rat has the tinnitus-like behaviors after treatment for 2-8 hours and disappears after 24 hours), and in addition, the salicylic acid (350 mg / kg) is injected into the intraperitoneal injection of salicylic acid (350 mg / kg); the chronic tinnitus can be stably induced by intraperitoneal injection of salicylic acid (200 mg / kg, twice a day and every 12 hours) for seven consecutive days; 3, selecting 8-week-old C57BL / 6J male mice, quantitatively detecting indexes including auditory interstitial pre-pulse inhibition, pre-pulse inhibition and ABR after salicylic acid treatment, 2 hours after salicylic acid treatment (300 mg / kg) treatment, seven days after recovery (excluding the influence of salicylic acid and anesthetics) and 2 hours after salicylic acid treatment and NBI-921352 administration, so as to evaluate the modulation effect of drugs on tinnitus behaviors and auditory functions, and finally evaluating the tinnitus behavior and auditory function. The NBI-921352 is administered in an intragastric administration way, the dosage is 23 mg / kg, and the medicine takes Nav1.6 as a definite action target and has the characteristics of safety and effectiveness.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a drug for the prevention and treatment of salicylic acid-induced tinnitus. Background Technology

[0002] Tinnitus, or abnormal sound perception in the absence of external sound, is a persistent auditory disorder often accompanied by anxiety, depression, and sleep disturbances. The global prevalence of tinnitus is approximately 10%-15%, and is increasing annually, making it a serious public health problem. The etiology and mechanisms of tinnitus are unclear, reflecting the challenges in clinical treatment; currently, there are no effective treatments or approved drugs. Exploring the pathological mechanisms of tinnitus and identifying effective, reliable, and specific drug targets remains a focus of basic and clinical research.

[0003] Tinnitus is an abnormal spontaneous electrical discharge activity in the auditory system, perceived as sound. Central plasticity has become a focus of current research on the mechanisms of tinnitus. Neuronal hyperexcitability stemming from an imbalance in inhibition-excitation homeostasis has been identified as the main pathogenic mechanism of tinnitus. The cochlear nucleus is the first-level center in the auditory pathway, receiving projections from the cochlear auditory nerve fibers. Animal experiments have shown that increased spontaneous electrical activity in the dorsal cochlear nucleus is one of the necessary conditions for tinnitus, and downregulating its electrical activity has been proven to be a feasible strategy to prevent tinnitus. The primary auditory cortex is the highest-level center for sound perception, and increased neural electrical activity in it is a major pathological feature of tinnitus. Clinical transcranial magnetic stimulation (TMS) can reduce abnormal electrical activity in relevant brain regions of tinnitus patients, alleviating some bothersome tinnitus. Therefore, reducing the electrical activity of the auditory cortex is a second feasible strategy for alleviating tinnitus symptoms.

[0004] Voltage-gated sodium channels (VGSCs) are core protein building blocks for action potential formation, playing a crucial role in the generation and rhythm of cellular electrical excitation, determining neuronal excitability and the signal transduction process from synaptic input to axonal output. Subtle mutations or aberrant expression of VGSCs can lead to corresponding functional alterations, directly or indirectly related to central and peripheral pathological manifestations such as pain and epilepsy, making them important targets for related clinical drug development. Our previous research team published a paper showing that the Nav1.6 subtype of voltage-gated sodium channels in the cochlear nucleus of glutamatergic excitatory neurons in salicylic acid-induced acute and chronic tinnitus animal models exhibited an abnormally high expression trend, with the trend returning to normal levels as the tinnitus-like behavior disappeared in the experimental animals. These results suggest that Nav1.6 is one of the important molecular triggers for tinnitus. This patented study demonstrates that Nav1.6 in glutamatergic excitatory neurons of the primary auditory cortex in salicylic acid-induced acute and chronic tinnitus animal models also shows a significant upregulation trend. The above results strongly suggest that the voltage-gated sodium channel Nav1.6 isoform is closely associated with salicylic acid-induced tinnitus, and that Nav1.6 is an important molecular target for inhibiting tinnitus.

[0005] NBI-921352 is a novel drug candidate with oral activity and blood-brain barrier penetration. Preclinical studies have shown that this compound is a VGSC subtype-selective inhibitor with high specificity for the Nav1.6 channel. Mechanistically, NBI-921352 effectively inhibits extracellular sodium ion influx by specifically binding to the fourth voltage-sensing domain of the Nav1.6 channel protein, thereby blocking the persistent and resuscitatory sodium current mediated by the Nav1.6 channel under pathological conditions. In vivo pharmacodynamic experiments further confirmed that in a Scn8aN1768D / + mutant mouse model, NBI-921352 significantly reduced the incidence of electrically stimulated generalized tonic-clonic seizures, demonstrating clear antiepileptic activity. Based on these experimental results, NBI-921352 is currently primarily a candidate for antiepileptic / anticonvulsant drugs, in the preclinical development stage, and possesses promising drug development prospects.

[0006] To date, neither academic research nor patent literature has disclosed or implied that NBI-921352 has the effect of preventing or treating tinnitus. Therefore, we provide a drug for the prevention and treatment of salicylic acid-induced tinnitus. Summary of the Invention

[0007] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0008] Another objective of this invention is to provide a drug for the prevention and treatment of salicylic acid-induced tinnitus, which targets Nav1.6 and is characterized by safety and efficacy. This invention aims to fill the gap in the existing technology for the lack of specific Nav1.6-targeted anti-tinnitus drugs.

[0009] To achieve the above objectives and some other objectives, the present invention adopts the following technical solution: A drug for the prevention and treatment of salicylic acid-induced tinnitus, comprising the following preparation steps: Step 1: A single intraperitoneal injection of salicylic acid (350 mg / kg) can stably induce reversible tinnitus-like behavior in rats (tinnitus-like behavior exists for 2-8 hours after treatment and disappears after 24 hours). In addition, intraperitoneal injection of salicylic acid (200 mg / kg, twice a day, 12 hours apart) for seven consecutive days can also stably induce chronic tinnitus. Step 2: Using Nav1.6 gene knockout mice (C57BL / 6JCya-Scn8aem1 / Cya), we observed the changes in auditory function after acute salicylic acid treatment. Since Nav1.6 knockout homozygous mice die about three weeks after birth, we used 1-month-old wild-type and heterozygous mice for comparison and quantitatively analyzed auditory brainstem response indicators before and 2 hours after salicylic acid treatment. Step 3: Eight-week-old male C57BL / 6J mice were used. Treatment was performed before salicylic acid treatment, 2 hours after salicylic acid (300 mg / kg) treatment, and 2 hours after salicylic acid treatment plus NBI-921352 administration. Quantitative indicators were measured for auditory interictal pulse inhibition (ABR), to evaluate the modulating effect of the drug on tinnitus behavior and auditory function. NBI-921352 was administered via gavage at a dose of 23 mg / kg. Step 4: Eight-week-old male C57BL / 6J mice were selected and divided into two groups: one group received salicylic acid treatment for 7 consecutive days; the other group received the same salicylic acid treatment, but was given NBI-921352 by gavage 2 hours before the behavioral test on day 8. Tinnitus behavioral tests and ABR assays were performed on all mice before salicylic acid treatment and on day 8 after 7 days of treatment. Step 5: To evaluate the systemic safety of NBI-921352 (23 mg / kg, administered by gavage once daily) for 7 consecutive days, this study observed the liver, kidney and brain tissues of mice by HE staining and detected relevant blood biochemical indicators.

[0010] Preferably, 2 hours after salicylic acid treatment in step three, the P2 / P1 amplitude ratio under 22.6 kHz, 16 kHz, 11.3 kHz, and 8 kHz sound stimulation significantly increased, and the P4 / P1 amplitude ratio under 22.6 kHz and 16 kHz sound stimulation conditions significantly increased, suggesting that salicylic acid induced enhanced central nervous system gain. NBI-921352 treatment could partially reverse the increasing trend of the P2 / P1 and P4 / P1 amplitude ratios.

[0011] Preferably, after treatment with salicylic acid in step one, wild-type mice showed a significant increase in the P2 / P1 amplitude ratio at 22.6 kHz and 16 kHz, and a significant increase in the P4 / P1 amplitude ratio at 16 kHz and 8 kHz, suggesting that salicylic acid induced enhanced central nervous system gain, and heterozygous mice could partially reverse the increasing trend of the P2 / P1 amplitude ratio and the P4 / P1 amplitude ratio.

[0012] Preferably, in step two, there are no statistically significant differences in the ABR thresholds, amplitudes, and latencies of waves I, II, and IV between wild-type and heterozygous mice, indicating that there are no significant changes in the peripheral and central auditory phenotypes of heterozygous mice at 1 month of age.

[0013] Preferably, step four is divided into two groups: one group receives salicylic acid treatment for 7 consecutive days; the other group, in addition to the same treatment, is given the Nav1.6 inhibitor NBI-921352 by gavage 2 hours before the behavioral test on day 8. All mice undergo tinnitus behavioral tests and auditory brainstem response tests before and after salicylic acid treatment (with an interval of more than 12 hours from the last salicylic acid treatment). NBI-921352 can inhibit salicylic acid-induced chronic tinnitus-like behavior.

[0014] The present invention has at least the following beneficial effects: 1. This invention reveals and confirms for the first time that the highly selective Nav1.6 inhibitor NBI-921352 can effectively prevent and / or treat salicylic acid-induced tinnitus, opening up a completely new clinical application field for this compound. It not only clearly proposes for the first time that the Nav1.6 sodium channel is a key drug target for salicylic acid-induced tinnitus, but also confirms the effectiveness of this target from a mechanistic perspective through the successful intervention of the highly selective inhibitor, providing a clear direction for the development of targeted therapies.

[0015] 2. The advantages of this invention lie in its clearly defined target and translational pathway. NBI-921352, as a potential anticonvulsant / epileptic drug, has completed preclinical safety assessments. No significant abnormalities were found in its serum liver and kidney function tests or in HE staining of brain tissue sections, demonstrating good safety. Combined with its ongoing clinical trials, this suggests that this product may have significant advantages in terms of short development cycles and low risk when used for tinnitus treatment.

[0016] 3. Through standardized animal behavioral experiments (GPIAS-PPI test) and auditory brainstem response (ABR) testing, this study confirmed that NBI-921352 can significantly improve salicylic acid-induced tinnitus-like behavior, and its effective dose is within the safe range, further highlighting its therapeutic potential.

[0017] In summary, this invention not only provides important theoretical basis for understanding the pathophysiological mechanism of tinnitus, but also provides novel candidate drugs for clinical application: on the one hand, it is expected to be used to prevent and treat tinnitus side effects caused by salicylates; on the other hand, it also provides a promising targeted treatment solution for other types of tinnitus with similar mechanisms, and has significant clinical application value and market potential. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the dynamic evolution of Nav1.6 expression in the primary auditory cortex after salicylic acid treatment according to the present invention. Figure 2 This is a schematic diagram of the selective expression characteristics of Nav1.6 cells in the primary auditory cortex after salicylic acid treatment according to the present invention. Figure 3This is a schematic diagram illustrating that Nav1.6 knockout does not affect the auditory nerve output inhibition induced by acute salicylic acid in this invention; Figure 4 This is a schematic diagram of the Nav1.6 knockout inhibition of acute salicylic acid-induced central nervous system gain according to the present invention; Figure 5 This is a schematic diagram illustrating how NBI-921352 of the present invention can inhibit tinnitus-like behavior induced by acute salicylic acid treatment; Figure 6 This is a schematic diagram illustrating how NBI-921352 of the present invention does not affect the inhibition of auditory nerve output caused by acute salicylic acid treatment; Figure 7 This is a schematic diagram of the central nervous system gain induced by NBI-921352 suppressing acute salicylic acid treatment according to the present invention; Figure 8 This is a schematic diagram illustrating how NBI-921352 of the present invention can inhibit tinnitus-like behavior induced by chronic salicylic acid treatment; Figure 9 This is a schematic diagram showing that chronic salicylic acid treatment and NBI-921352 administration did not significantly affect the auditory nerve output function according to the present invention; Figure 10 This is a schematic diagram showing that chronic salicylic acid treatment of the present invention did not cause an increase in central nervous system gain, suggesting that its mechanism is different from that of the acute model. Figure 11 This is a schematic diagram of the pathological analysis of mouse liver, kidney and brain tissues after administration of NBI-921352 according to the present invention. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings, so that those skilled in the art can implement it after consulting this specification.

[0020] like Figure 1-11 As shown, a drug for the prevention and treatment of salicylic acid-induced tinnitus includes the following preparation steps: Step 1: A single intraperitoneal injection of salicylic acid (350 mg / kg) can stably induce reversible tinnitus-like behavior in rats (tinnitus-like behavior exists for 2-8 hours after treatment and disappears after 24 hours). In addition, intraperitoneal injection of salicylic acid (200 mg / kg, twice a day, 12 hours apart) for seven consecutive days can also stably induce chronic tinnitus. Step 2: Using Nav1.6 gene knockout mice (C57BL / 6JCya-Scn8aem1 / Cya), we observed the changes in auditory function after acute salicylic acid treatment. Since Nav1.6 knockout homozygous mice die about three weeks after birth, we used 1-month-old wild-type and heterozygous mice for comparison and quantitatively analyzed auditory brainstem response indicators before and 2 hours after salicylic acid treatment. Step 3: Eight-week-old male C57BL / 6J mice were selected and treated with salicylic acid before, 2 hours after, and 2 hours after treatment with salicylic acid plus NBI-921352. Quantitative indicators, including pre-pulse inhibition (PPI), pre-pulse inhibition (PPI), and ABR, were measured to evaluate the modulating effect of the drug on tinnitus behavior and auditory function. NBI-921352 was administered via gavage at a dose of 23 mg / kg. Step 4: Eight-week-old male C57BL / 6J mice were selected and divided into two groups: one group received salicylic acid treatment for 7 consecutive days; the other group received the same salicylic acid treatment, but was given NBI-921352 by gavage 2 hours before the behavioral test on day 8. Tinnitus behavioral tests and ABR assays were performed on all mice before salicylic acid treatment and on day 8 after 7 days of treatment. Step 5: To evaluate the systemic safety of NBI-921352 (23 mg / kg, administered by gavage once daily) for 7 consecutive days, this study observed the liver, kidney and brain tissues of mice by HE staining and detected relevant blood biochemical indicators.

[0021] In the above regimen, compared with the control group, the mRNA and protein expression of Nav1.6 in the primary auditory cortex of rats were significantly upregulated 2-8 hours after a single intraperitoneal injection, and the expression level returned to normal by 24 hours. Similarly, in the seven-day treatment group, the mRNA and protein expression of Nav1.6 in the primary auditory cortex were also significantly increased, and after seven days of recovery following drug withdrawal, the expression level tended to be similar to that of the control group. The trend of Nav1.6 expression changes was highly consistent with the occurrence of salicylic acid-induced tinnitus-like behavior in rats, suggesting that Nav1.6 plays a key role in the development of tinnitus. Immunofluorescence double labeling results showed that the co-labeling rate between Nav1.6 positive neurons and VGLUT (a marker of glutamatergic excitatory neurons) positive neurons was significantly increased, while the co-labeling rate with GAD (a marker of GABAergic inhibitory neurons) positive neurons did not show significant changes. Figure 2 The results indicate that the upregulation of Nav1.6 expression mainly originates from glutamatergic excitatory neurons. These results fully demonstrate that the abnormal expression of Nav1.6 in glutamatergic excitatory neurons is a key molecular mechanism by which salicylic acid-induced imbalance of inhibition-excitation homeostasis in the primary auditory cortex and the induction of tinnitus. Combined with the team's previous research on Nav1.6's involvement in salicylic acid-induced abnormal excitation of the cochlear nucleus, this further supports the following viewpoint: targeting the Nav1.6 channel and inhibiting the electrical activity of glutamatergic excitatory neurons in the central auditory system may be an effective strategy for improving tinnitus.

[0022] Following a single salicylic acid treatment, the ABR threshold in wild-type and heterozygous mice increased by approximately 25 dB (frequency range of 4–45 kHz). In wild-type mice, the latency of wave I was prolonged at 22.6 kHz (55–75 dB), 11.3 kHz (55–60 dB), and 8 kHz (55–70 dB) acoustic stimuli; in heterozygous mice, the latency of wave I was prolonged at 22.6 kHz (55–65 dB) acoustic stimuli. The amplitude of wave I was significantly reduced in both wild-type and heterozygous mice. These results indicate that a single intraperitoneal injection of salicylic acid leads to suppression of cochlear auditory nerve output in both wild-type and heterozygous mice. Figure 3 A single intraperitoneal injection of salicylic acid had a limited direct effect on wave II amplitude in both wild-type and heterozygous mice, with only wild-type mice showing a significant increase in wave II amplitude (75-90 dB) at 22.6 kHz. Following intraperitoneal injection of salicylic acid, wave IV amplitude (90 dB) at 22.6-11.3 kHz was significantly increased in both mouse types. Figure 4 After treatment with salicylic acid, wild-type mice showed a significant increase in the P2 / P1 amplitude ratio at 22.6 kHz and 16 kHz, and a significant increase in the P4 / P1 amplitude ratio at 16 kHz and 8 kHz, suggesting that salicylic acid induced enhanced central nervous system gain. However, heterozygous mice could reverse the increasing trend of P2 / P1 and P4 / P1 amplitude ratios. Figure 4 The above results indicate that Nav1.6 gene knockout can eliminate salicylic acid-induced central nervous system enhancement, further confirming that Nav1.6 plays a key role in salicylic acid-induced tinnitus.

[0023] Tinnitus behavioral experiments showed that, compared with baseline values, mice treated with salicylic acid for 2 hours exhibited significant gap detection defects (significantly reduced GPIAS values) under background noise at both 16 kHz (P<0.01) and 12 kHz (P<0.001) frequencies. Figure 5 B). However, under the administration of the Nav1.6 inhibitor NBI-921352, salicylic acid treatment did not alter the gap detection ability in mice. There were no significant differences in PPI values ​​among the three groups of mice. Figure 5A), indicating that the gap detection deficit in tinnitus mice is not caused by insufficient background sound perception. This data confirms that NBI-921352 can inhibit acute salicylic acid-induced tinnitus-like behavior in mice, and the ABR index shows that NBI-921352 can reverse the salicylic acid-induced central gain enhancement. Compared to baseline, 2 hours after salicylic acid treatment, the ABR threshold increased significantly (4-45 kHz frequency range), and the I-wave amplitude (22.6-8 kHz) decreased significantly, verifying the inhibition of cochlear output signals by salicylic acid. Compared to salicylic acid treatment alone, there were no significant changes in the ABR threshold and I-wave amplitude after co-treatment with salicylic acid and NBI-921352, suggesting that NBI-921352 itself has no significant effect on cochlear signals, presumably because the low drug concentration entering the inner ear due to the blood-labyrinthine barrier does not affect the Nav1.6 channel of the auditory nerve (…). Figure 6 Compared to baseline, 2 hours after salicylic acid treatment, the P2 / P1 amplitude ratios under acoustic stimulation at 22.6 kHz, 16 kHz, 11.3 kHz, and 8 kHz were significantly increased, and the P4 / P1 amplitude ratios under acoustic stimulation at 22.6 kHz and 16 kHz were significantly increased, suggesting that salicylic acid induced enhanced central nervous system gain. NBI-921352 treatment partially reversed the increasing trend of the P2 / P1 and P4 / P1 amplitude ratios. Figure 7 The above results indicate that NBI-921352 can suppress salicylic acid-induced tinnitus-related central electrophysiological indicators and significantly inhibit tinnitus-like behavior in mice.

[0024] Tinnitus behavioral results showed that, compared with baseline, continuous 7-day salicylic acid treatment resulted in significant gap detection defects in mice under 20 kHz and 16 kHz background noise (decreased GPIAS values, P < 0.05). Figure 8 B). Administration of NBI-921352 reversed this defect and improved the gap detection ability in mice. There were no significant differences in the prepulse inhibition (PPI) values ​​among the three groups of mice. Figure 8 A) indicates that their auditory startle response is basically normal, and the gap detection defect is not caused by insufficient background sound perception. The above results support the inhibitory effect of NBI-921352 on salicylic acid-induced chronic tinnitus. In terms of ABR detection, compared with baseline, after 7 consecutive days of salicylic acid treatment, the ABR threshold, I-wave latency, and amplitude of mice did not change significantly, with only a decrease in I-wave amplitude at 8 kHz (65–75 dB); NBI-921352 administration also did not have a substantial effect on cochlear function. Figure 9 Further analysis revealed that the amplitude ratios of P2 / P1 and P4 / P1 did not change significantly after treatment with salicylic acid. Figure 10The results indicated no enhancement of central gain, and combined behavioral and physiological findings suggest that salicylic acid-induced acute and chronic tinnitus may involve different neural mechanisms. This study found that in the chronic model, although mice exhibited persistent tinnitus, the ABR (Auditory Brain Response) did not detect enhanced central gain at the brainstem level. This suggests that under long-term salicylic acid exposure, adaptive changes in the nervous system shift the maintenance mechanism of tinnitus from widespread gain upregulation in the early stages to a mechanism potentially dependent on specific frequency synchronization activities in the auditory cortex, or involving circuit reorganization in non-auditory brain regions such as the limbic system. Furthermore, adaptive regulation of neurotransmitter systems such as GABAergic and cholinergic may also normalize ABR indicators in the chronic phase, while preserving abnormal activity in higher centers, thus leading to the separation between tinnitus behavior and ABR results. Notably, the Nav1.6 inhibitor NBI-921352 effectively inhibited salicylic acid-induced chronic tinnitus, and its mechanism of action may be related to inhibiting the Nav1.6 channel function of glutamatergic neurons in the auditory cortex, thereby regulating the excitability and abnormal synchronization activity of neurons in this region.

[0025] This study observed the liver, kidney, and brain tissues of mice by HE staining and detected relevant blood biochemical indicators.

[0026] The results of tissue morphology show that ( Figure 11 ): Liver: In both the control and treatment groups, the liver capsule structure was intact, and the liver lobules were clearly demarcated and regularly arranged. In the treatment group, only a small number of hepatocytes showed mild edema (green arrows), with loose and lightly stained cytoplasm, and no obvious inflammatory cell infiltration was observed.

[0027] Kidneys: The boundary between the renal cortex and medulla was clear in both groups, the glomeruli were evenly distributed, and the cell count and matrix were normal; the renal tubular epithelial cells were full in morphology, and there was no significant proliferation in the renal interstitium or obvious inflammatory cell infiltration.

[0028] Brain tissue: Both groups showed abundant neurons, with hippocampal pyramidal cells arranged neatly and regularly shaped, with large, round nuclei and clearly defined nucleoli. No obvious tissue abnormalities or inflammatory cell infiltration were observed. Further analysis of the number of damaged neurons in the same region near the hippocampus showed that the drug-treated group did not cause significant brain tissue damage.

[0029] Blood biochemistry analysis showed that the key indicators reflecting liver and kidney function in the serum of the drug-treated group were all within the normal range (Table 1).

[0030] In summary, based on the histomorphological and blood biochemical results, it can be determined that NBI-921352 did not cause significant liver, kidney, or brain tissue damage at the stated therapeutic dose and duration, and has good systemic safety.

[0031] In a preferred embodiment, two hours after salicylic acid treatment in step three, the P2 / P1 amplitude ratios under 22.6 kHz, 16 kHz, 11.3 kHz, and 8 kHz acoustic stimulation significantly increased, and the P4 / P1 amplitude ratios under 22.6 kHz and 16 kHz acoustic stimulation conditions significantly increased, suggesting that salicylic acid induced enhanced central nervous system gain. NBI-921352 treatment could partially reverse the increasing trend of the P2 / P1 and P4 / P1 amplitude ratios.

[0032] In a preferred embodiment, after treatment with salicylic acid in step one, wild-type mice showed a significant increase in the P2 / P1 amplitude ratio at 22.6 kHz and 16 kHz, and a significant increase in the P4 / P1 amplitude ratio at 16 kHz and 8 kHz, suggesting that salicylic acid induced enhanced central nervous system gain.

[0033] In a preferred embodiment, there are no statistically significant differences in the ABR thresholds, amplitudes, and latencies of waves I, II, and IV between wild-type and heterozygous mice in step two, indicating that there are no significant changes in the peripheral and central auditory phenotypes of heterozygous mice at 1 month of age.

[0034] In a preferred embodiment, step four is divided into two groups: one group receives salicylic acid treatment for 7 consecutive days; the other group, in addition to the same treatment, is given the Nav1.6 inhibitor NBI-921352 by gavage 2 hours before the behavioral test on day 8. All mice undergo tinnitus behavioral tests and auditory brainstem response tests before and after salicylic acid treatment (with an interval of more than 12 hours from the last salicylic acid treatment).

[0035] like Figure 1 As shown: (AH) Schematic diagram of Nav1.6 distribution in the primary auditory cortex; (IJ) mRNA and protein expression levels of Nav1.6 after acute salicylic acid treatment; (KL) mRNA and protein expression levels of Nav1.6 after chronic salicylic acid treatment; (MN) Statistical analysis of Nav1.6 protein expression in different layers of the primary auditory cortex after acute / chronic salicylic acid treatment. Compared with the control group, *P<0.05; **P<0.01; ***P<0.001.

[0036] like Figure 2As shown: (AH) Representative images of Nav1.6 protein expression in different types of neurons in the primary auditory cortex; (IL) Co-localization analysis of Nav1.6 with GABAergic neurons (IJ) and glutamatergic neurons (KL) after acute / chronic salicylic acid treatment; (MP) Statistical analysis of Nav1.6 expression with GABAergic neurons (MN) and glutamatergic neurons (OP) in different layers of the primary auditory cortex after acute / chronic salicylic acid treatment. Compared with the control group, *P<0.05; **P<0.01; ***P<0.001.

[0037] like Figure 3 As shown: (A) ABR thresholds in wild-type and heterozygous mice 2 hours after salicylic acid treatment. (B, D, F, H) I-wave latency under different frequency sound stimulation conditions. (C, E, G, I) I-wave amplitude under different frequency sound stimulation conditions. Data are presented as mean ± standard error, analyzed by two-way ANOVA with Bonferroni test. Compared with baseline, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Black asterisks indicate differences before and after wild-type salicylic acid treatment, and red asterisks indicate differences before and after heterozygous salicylic acid treatment.

[0038] like Figure 4 As shown: (A, C, E, G) ABR II amplitude values ​​of heterozygous and wild-type mice before and after salicylic acid treatment. (B, D, F, H) ABR II amplitude values ​​of heterozygous and wild-type mice before and after salicylic acid treatment. (I) P2 / P1 amplitude ratio at 75 dB sound intensity before and after salicylic acid treatment. (J) P4 / P1 amplitude ratio at 75 dB sound intensity before and after salicylic acid treatment. Compared with baseline, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0039] like Figure 5 As shown: (A) PPI percentages in the untreated (baseline), salicylic acid-treated, and salicylic acid-treated + NBI-921352-treated groups. PPI calculation formula: [(AvgTstartle - AvgTpre pulse) / AvgTstartle] × 100% (AvgTpre-pulse and AvgTstartle represent the average startle amplitude in the pre-pulse and pre-pulse tests, respectively). (B) GPIAS percentage. Calculation formula: [(AvgTno-gap-AvgTgap) / AvgTno-gap] ×100% (AvgTno-gap and AvgTgap represent the mean startle amplitude in the gapless and gapped tests, respectively). Data are expressed as mean ± standard error. Compared with the control group, **P<0.01, ***P<0.001.

[0040] like Figure 6 As shown: (A) ABR thresholds of the untreated (baseline), salicylic acid-treated, and salicylic acid-treated + NBI-921352-treated groups. (B, D, F, H) I-wave latency under different frequency acoustic stimulation conditions. (C, E, G, I) I-wave amplitude under different frequency acoustic stimulation conditions. Data are expressed as mean ± standard error, and analyzed using two-way ANOVA with Bonferroni test. Compared with baseline, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0041] like Figure 7 As shown: (A, C, E, G) ABR II amplitude values ​​of the untreated (baseline), salicylic acid-treated, and salicylic acid-treated + NBI-921352-treated groups. (B, D, F, H) ABR IV amplitude values ​​of the untreated (baseline), salicylic acid-treated, and salicylic acid-treated + NBI-921352-treated groups. (I) P2 / P1 amplitude ratio at 75 dB sound intensity. (J) P4 / P1 amplitude ratio at 75 dB sound intensity. Compared with baseline, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0042] like Figure 8 Presented: (A) PPI percentages in the untreated (baseline), chronic salicylate-treated, and chronic salicylate-treated + NBI-921352-treated groups. PPI calculation formula: [(AvgTstartle - AvgTprepulse) / AvgTstartle] × 100% (AvgTpre-pulse and AvgTstartle represent the mean startle amplitude in the pre-pulse and pre-pulse tests, respectively). (B) GPIAS percentage. Calculation formula: [(AvgTno-gap - AvgTgap) / AvgTno-gap] × 100% (AvgTno-gap and AvgTgap represent the mean startle amplitude in the gapless and gapped tests, respectively). Data are expressed as mean ± standard error. *P < 0.05 compared to the control group.

[0043] like Figure 9As shown: (A) ABR thresholds after no treatment (baseline), chronic salicylic acid treatment, and chronic salicylic acid treatment + NBI-921352 administration. (B, D, F, H) I-wave latency under different frequency acoustic stimulation conditions. (C, E, G, I) I-wave amplitude under different frequency acoustic stimulation conditions. Data are expressed as mean ± standard error, and analyzed using two-way ANOVA with Bonferroni test. Compared with baseline, *P<0.05, **P<0.01.

[0044] like Figure 10 As shown: (A, C, E, G) Baseline (untreated), chronic salicylic acid treated, and chronic salicylic acid treated + NBI-921352 administered groups ABR amplitude II. (B, D, F, H) Baseline (untreated), chronic salicylic acid treated, and chronic salicylic acid treated + NBI-921352 administered groups ABR amplitude IV. (I) P2 / P1 amplitude ratio at 75 dB sound intensity. (J) P4 / P1 amplitude ratio at 75 dB sound intensity. Compared with baseline, ns indicates no significant difference.

[0045] like Figure 11 The image shows representative HE-stained sections of (A) liver tissue, (B) kidney tissue, and (C) brain tissue (photographed under a 20x objective lens). The left side represents the control group, and the right side represents the NBI-921352-treated group (n=3). As shown, no significant pathological changes were observed in any tissue of either group of mice.

[0046] Table 1. Serum liver and kidney function indicators in mice. Liver function control group NBI-921352 group Normal value ALT (U / L) 55 43.76 10.06-96.47 AST (U / L) 118 145.83 36.31-235.48 DBIL (umol / l) 7.19 6.23 0.45-33.89 TBIL (umol / l) 9.18 9.09 6.09-53.06 ALB (g / L) 37 36 21.22-39.15 ALP (U / L) 227.56 199.73 22.52-474.35 TBA (umol / l) 2.8 4.3 0-8.51 Kidney function control group NBI-921352 group Normal value UREA (umol / L) 20 20 10.91-85.09 CREA (umol / L) 22 18 3.9-12.4 UA (umol / L) 137 106 44.42-224.77

[0047] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A medicament for preventing and treating salicylic acid-induced tinnitus, characterized by, The following preparation steps are included: Step one: A single intraperitoneal injection of salicylic acid (350 mg / kg) can stably induce reversible tinnitus-like behavior in rats (tinnitus-like behavior exists 2-8 hours after treatment, and disappears after 24 hours), in addition, continuous intraperitoneal injection of salicylic acid (200 mg / kg, twice a day, with an interval of 12 hours) can also stably induce chronic tinnitus; Step two: The changes in auditory function of Nav1.6 gene knockout mice (C57BL / 6JCya-Scn8aem1 / Cya) after acute salicylic acid treatment were observed, since Nav1.6 homozygous knockout mice died at about three weeks after birth, 1-month-old wild-type and heterozygous mice were selected for comparison, and the auditory brainstem response indicators before and 2 hours after salicylic acid treatment were quantitatively analyzed; Step three: 8-week-old C57BL / 6J male mice were selected, and the indicators were quantitatively detected before salicylic acid treatment, 2 hours after salicylic acid (300 mg / kg) treatment, 7 days after recovery (to exclude the effects of salicylic acid and narcotics), and 2 hours after salicylic acid treatment+NBI-921352 administration, to evaluate the modulating effects of the drug on tinnitus behavior and auditory function. NBI-921352 was administered by gavage at a dose of 23 mg / kg; Step four: 8-week-old C57BL / 6J male mice were selected and divided into two groups: one group received continuous salicylic acid treatment for 7 days; the other group received NBI-921352 by gavage 2 hours before behavioral testing on the 8th day based on the same salicylic acid treatment; all mice were subjected to tinnitus behavioral testing and / or ABR detection before salicylic acid treatment and on the 8th day after 7 days of continuous treatment; Step five: The systemic safety of NBI-921352 (23 mg / kg, administered by gavage once a day) was evaluated for 7 consecutive days. In this study, HE staining was performed on the liver, kidney and brain tissues of mice, and relevant blood biochemical indicators were detected.

2. The medicament for preventing and treating salicylic acid-induced tinnitus according to Claim 1, wherein In step three, the P2 / P1 amplitude ratio under 22.6 kHz, 16 kHz, 11.3 kHz and 8 kHz sound stimulation significantly increased 2 hours after salicylic acid treatment, and the P4 / P1 amplitude ratio under 22.6 kHz and 16 kHz sound stimulation significantly increased, indicating that salicylic acid induced central gain enhancement, and NBI-921352 treatment partially reversed the increasing trend of P2 / P1 amplitude ratio and P4 / P1 amplitude ratio.

3. The medicament for preventing and treating salicylic acid-induced tinnitus according to Claim 1, wherein In step one, after salicylic acid treatment, the P2 / P1 amplitude ratio of wild-type mice under 22.6 kHz and 16 kHz significantly increased, and the P4 / P1 amplitude ratio under 16 kHz and 8 kHz also significantly increased, indicating that salicylic acid induced central gain enhancement.

4. The medicament for preventing and treating salicylic acid-induced tinnitus according to Claim 1, wherein In step two, there was no statistical difference in ABR threshold and the amplitude and latency of I wave, II wave and IV wave between wild-type mice and heterozygous mice, indicating that there was no obvious change in the hearing phenotype of peripheral and central auditory system of 1-month-old heterozygous mice.

5. The medicament for preventing and treating salicylic acid-induced tinnitus according to Claim 1, wherein The step four is divided into two groups: one group receives salicylic acid treatment for 7 consecutive days; the other group, on the basis of the same treatment, is given the Nav1.6 inhibitor NBI-921352 by gavage 2 hours before the behavior test on the 8th day, and all mice are subjected to tinnitus behavior test and / or auditory brainstem response detection before and after salicylic acid treatment (more than 12 hours apart from the last salicylic acid treatment).

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