Pharmaceutical compositions containing Sigma agonists and their use in the treatment of tinnitus

Abstract

This invention belongs to the field of medical technology, specifically relating to the use of a Sigma 1 receptor agonist combined with a GABAAR receptor modulator in the preparation of a pharmaceutical composition for the prevention and / or treatment of tinnitus. This type of pharmaceutical composition exerts an anti-tinnitus effect by synergistically regulating the Sigma 1 and GABAAR receptor pathways, improving imbalances in auditory central nervous system excitability, abnormal neuroplasticity, and related neuroinflammatory responses. In particular, the combination therapy provided by this invention can be used to treat neurodegenerative tinnitus and drug-induced tinnitus.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a Sigma-1 agonist and GABA. A Compositions of R modulators and their use in the prevention and / or treatment of tinnitus. Technical Background

[0002] Tinnitus refers to abnormal sounds perceived by patients in the ear or cranium without external acoustic stimulation. Clinically, tinnitus usually refers to non-pulsatile tinnitus (NPT). The pathogenesis of tinnitus is complex and diverse, and is not yet fully understood, thus treatment outcomes are often unsatisfactory. The mechanisms of tinnitus include cochlear dysfunction caused by excessive exposure to environmental noise or cochlear trauma, as well as plasticity changes in the central auditory system due to an imbalance between excitatory and inhibitory inputs to auditory neurons ([J]. Frontiers in neuroscience, 2021, 15: 621145.). Animal tinnitus models show elevated levels of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the cochlea and dorsal cochlear nucleus, accompanied by astrocyte activation, directly affecting synaptic plasticity in the auditory pathway. The serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) are elevated in tinnitus patients, occurring under inflammatory conditions and involved in the adhesion of leukocytes to endothelial cells; elevated levels of intercellular adhesion molecule-1 (ICAM-1) may trigger oxidative stress due to vascular endothelial cell damage ([J]. American Journal of Otolaryngology, 2024, 45(3): 104215.). Genetic studies have shown that dysregulation of brain-derived neurotrophic factor (BDNF) may play a role in the development of tinnitus. As a key regulator of neuroplasticity, BDNF can antagonize pro-inflammatory cytokines and may prevent cell damage and neuronal loss after permanent sensorineural hearing loss ([J]. Frontiersin Neuroscience, 2025, 19:1620894.). Animals treated with high doses of salicylates developed tinnitus and hearing loss. Salicylates increased the sensitivity of NMDARs and overactivated NMDARs, leading to acute tinnitus ([J]. Journal of Neuroscience, 2008, 28(29): 7313-7323.).

[0003] Gamma-aminobutyric acid (GABA) is a major inhibitory endogenous neurotransmitter in the central auditory pathway, playing a crucial role in processing auditory stimuli and central reorganization following peripheral injury. Noise exposure or age-related cochlear damage leads to decreased expression of glutamate decarboxylase in the cochlear nucleus and inferior colliculus. Vigabatrin is a GABA-producing enzyme. A R agonists have shown effectiveness in a rat noise-induced tinnitus model, but the GABA analog gabapentin has not shown any effective therapeutic effect on tinnitus in human studies ([J]. Journal of Otology, 2007, 2(2): 63-69.). There have even been clinical reports of GABA... A The allosteric agent propofol (R-type) may cause tinnitus (J The Journal of Clinical Pharmacology, 2013, 53(3): 356-358.). In summary, regarding GABA... A Reports on the effectiveness of R-agonists or orthoallosteric agents in treating tinnitus are contradictory, and their efficacy is often insufficient.

[0004] In neurodegenerative disease models, decreased σ-1 receptor levels or activity are associated with neurodegeneration, while activation or overexpression of σ-1 receptors is associated with neuroprotective effects. Sustained NMDAR activation leads to excitotoxicity, resulting in Ca²⁺ influx and subsequently activating downstream programmed cell death. σ-1 agonist treatment reduces Ca²⁺ influx through presynaptic voltage-dependent calcium channels and inhibits the protein kinase C (PKC) signaling cascade, thereby reducing glutamate release at nerve endings. σ-1 receptors are expressed on lymphocytes, and their ligands can inhibit CD3 lymphocyte proliferation in vitro and inhibit LPS-induced cytokine release in vivo ([J]. Sigma Receptors: Their Role in Disease and as Therapeutic Targets, 2017: 133-152.). However, the Sigma-1 agonist Cutamesine did not improve hearing loss in aged mice, so whether this signaling pathway plays a key role needs further investigation (J Neurosci Res. 2015 May;93(5):788-95).

[0005] In summary, the present invention aims to provide a solution containing a Sigma agonist and GABA. A The pharmaceutical composition of the R modulator regulates the imbalance between excitatory and inhibitory inputs to auditory neurons, prevents neurodegenerative changes and inflammatory responses, thereby providing a new pharmacological treatment for the prevention and / or treatment of tinnitus. Summary of the Invention

[0006] The purpose of this invention is to provide a product containing Sigma agonist and GABA. A Pharmaceutical compositions of R modulators, and their applications in the medical field, particularly for the treatment and / or prevention of tinnitus.

[0007] The purpose of this invention is to provide a product containing Sigma agonist and GABA. A Pharmaceutical compositions of R modulators, and their applications in the medical field, particularly for the treatment and / or prevention of hearing loss.

[0008] In one embodiment of the present invention, the tinnitus includes acute tinnitus, subacute tinnitus, and chronic tinnitus.

[0009] In one embodiment of the present invention, the tinnitus includes subjective tinnitus and objective tinnitus, with subjective tinnitus being preferred.

[0010] In one embodiment of the present invention, the tinnitus includes unilateral / bilateral tinnitus, high-frequency / low-frequency tinnitus, pulsatile tinnitus, and continuous / intermittent tinnitus.

[0011] In one embodiment of the present invention, the hearing loss is age-related hearing loss.

[0012] This invention provides a combination of Sigma agonist and GABA. A Use of R modulators in the preparation of medicaments for treating tinnitus, wherein the Sigma agonist is preferably a Sigma-1 agonist.

[0013] In one embodiment of the present invention, the Sigma-1 agonist is selected from any one of Famotizole, Cutamesine, or Igmesine, preferably any one of Famotizole or Igmesine, and more preferably Famotizole.

[0014] In one embodiment of the present invention, the Sigma-1 agonist is selected from one of the following: This invention provides a compound containing a Sigma-1 agonist and GABA. A Use of R-modifier pharmaceutical compositions for the treatment of tinnitus.

[0015] In a preferred embodiment of the present invention, the GABA A The R regulator is GABA. A R-allosteric modifier or GABA A R agonist.

[0016] In a preferred embodiment of the present invention, the GABA A R modulators are neurosteroid compounds.

[0017] In one embodiment of the present invention, the neurosteroid compound is selected from Zuranolone, Itruvone, or Fasedienol, more preferably Itruvone.

[0018] In one embodiment of the present invention, the GABA A The R regulator is selected from one of the following: In one embodiment of the present invention, the Sigma-1 agonist is Fabomatizole.

[0019] In one embodiment of the present invention, the Sigma-1 agonist is Cutamesine.

[0020] In one embodiment of the present invention, the GABA A The R regulator is Zuranolone.

[0021] In one embodiment of the present invention, the GABA A The R regulator is Itruvone.

[0022] In one embodiment of the present invention, the Sigma-1 agonist is combined with GABA. A The molar ratio of the R regulator is 1:1.

[0023] This invention provides a Sigma-1 agonist combined with GABA A Use of R modulators in the preparation of medicaments for the prevention and / or treatment of tinnitus, wherein the tinnitus is any one or more of neurodegenerative tinnitus, inflammatory tinnitus, drug-induced tinnitus, and traumatic tinnitus.

[0024] In one embodiment of the present invention, the Sigma-1 agonist is combined with GABA. A The pharmaceutical composition of the R modulator is a combination of Famotizole and Zuranolone, a combination of Famotizole and Itruvone, a combination of Cutamesine and Zuranolone, or a combination of Cutamesine and Itruvone. Preferably, it is a combination of Famotizole and Itruvone, or a combination of Cutamesine and Itruvone.

[0025] This invention provides a method for combining GABA AMethods for increasing the blood-brain ratio of Sigma-1 agonists by administering R-modifiers.

[0026] In one embodiment of the present invention, the GABA A R modulators are neurosteroid compounds.

[0027] In one embodiment of the present invention, the GABA A The R regulator is either Zuranolone or Itruvone.

[0028] In one embodiment of the present invention, the Sigma-1 agonist is Fabomatizole or Cutamesine, preferably Fabomatizole.

[0029] In one embodiment of the present invention, the Sigma-1 agonist and GABA A The pharmaceutically acceptable salts of R modifiers are selected from hydrochloride, hydrobromide, phosphate, sulfate, acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, benzenesulfonate, benzoate, and bisulfate.

[0030] In one embodiment of the invention, the pharmaceutical composition can be formulated using one or more pharmaceutically acceptable carriers in a conventional manner. Therefore, the active compounds of the invention can be formulated into dosage forms for oral, sublingual, intranasal, parenteral (e.g., intravenous, intramuscular, or subcutaneous), or rectal administration, or suitable for inhalation or insufflation. The compounds of the invention or pharmaceutically acceptable salts thereof can also be formulated into sustained-release dosage forms.

[0031] This invention provides a formulation containing Sigma-1 agonist and GABA. A A formulation composition of an R-modifier and a pharmaceutically acceptable excipient, characterized in that the pharmaceutically acceptable excipient is a cyclodextrin derivative.

[0032] In a preferred embodiment of the present invention, the cyclodextrin derivative is hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, etc.

[0033] In one embodiment of the invention, an effective dose of the compound of the invention or a pharmaceutically acceptable salt thereof may be taken orally with an inert diluent or a carrier. According to some embodiments of the invention, the compound of the invention may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic purposes, the compound of the invention may be used with excipients and in the form of tablets, lozenges, capsules, suspensions, syrups, etc. According to embodiments of the invention, the above-described formulations should contain at least 0.5% (w / w) of the active compound of the invention, but this may vary depending on the specific dosage form, wherein 4% to about 70% by weight is convenient. In such pharmaceutical compositions, the amount of the active compound should reach an appropriate dosage.

[0034] In one embodiment of the invention, relating to oral administration, the active compound of the invention can be formulated into tablets or capsules, for example, by conventional means with pharmaceutically acceptable excipients, such as binders, fillers, lubricants, disintegrants, or wetting agents. Tablets can be coated using methods well known in the art. Liquid formulations for oral administration can be solutions, syrups, or suspensions, or evaporated into a dried product, regenerated with water or other suitable carriers before use. Such liquid formulations can be prepared using pharmaceutically acceptable additives by conventional means, such as suspending agents, emulsifiers, non-aqueous carriers, and preservatives.

[0035] In one embodiment of the present invention, when the active compound of the present invention is used for parenteral administration, the compound provided by the present invention can be combined with sterile water or an organic medium to form an injectable solution or suspension.

[0036] In one embodiment of the invention, the active compound of the invention can be formulated into a rectal composition, such as a suppository or retention enema, containing, for example, a conventional suppository base, such as cocoa butter or other glycerides.

[0037] This invention also provides a Sigma-1 agonist and GABA. A The use of R-modifier pharmaceutical compositions in the treatment and / or relief of migraine medications, wherein the pharmaceutical composition optionally comprises one or more other tinnitus-modifying active agents, including but not limited to microcirculation-improving agents such as Ginkgo biloba extract, betahistine, nimodipine, etc.; neurotrophic and repair agents such as methylcobalamin, adenosylcobalamin, neurotrophic factors, etc.; glucocorticoids such as dexamethasone, methylprednisolone, prednisone, etc.; ion channel modulators and anticonvulsants such as carbamazepine, gabapentin, lidocaine, etc.; anti-anxiety agents such as diazepam, alprazolam, etc.; and antidepressants such as fluoxetine, sertraline, etc.

[0038] Instruction manual illustrations Figure 1 shows the changes in hearing loss in each combination drug group and each single drug group at month 9. The threshold shift on the vertical axis in the figure is the change in hearing threshold of each group of animals (control group, single drug group, combination drug group) at three different frequencies of 4 kHz, 8 kHz, and 16 kHz at month 9, which is used to compare the protective / damaging effects of different drugs and combination regimens on hearing. Detailed Implementation

[0039] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects. It should be understood that the term "comprising" can also encompass a closed meaning, meaning "consisting of".

[0040] The term "tinnitus" can range from a few seconds to a few hours, and can also include continuous tinnitus. It is a sudden noise in the ear accompanied by temporary hearing loss, which often occurs randomly on one side without warning and may be accompanied by a feeling of fullness in the ear during an attack.

[0041] The term "subjective tinnitus" refers to the perception of sound without an objective sound source, and it accounts for the vast majority of tinnitus patients. Subjective tinnitus includes primary and secondary tinnitus. Primary subjective tinnitus refers to tinnitus with or without sensorineural hearing loss and for which no clear cause can be found. Secondary subjective tinnitus, in addition to sensorineural hearing loss, has a relatively clear underlying cause, such as cerumen impaction, foreign bodies in the external auditory canal, cholesteatoma of the external auditory canal, eczema of the external auditory canal, secretory otitis media, chronic mastoiditis of the middle ear, cholesteatoma of the middle ear, adhesive otitis media, cholesterol granuloma of the middle ear, otosclerosis, etc. Subjective tinnitus includes physiological subjective tinnitus and pathological subjective tinnitus. This invention prefers pathological tinnitus, which is generally considered to last for more than 5 minutes and recur within 1 week. Subjective tinnitus is classified according to its course into acute tinnitus, chronic compensatory tinnitus (non-annoying tinnitus), and chronic decompensated tinnitus (annoying tinnitus).

[0042] The term "neurodegenerative tinnitus," also known as age-related tinnitus, occurs during the natural decline of hearing. It is a manifestation of aging or damage to the auditory nervous system, resulting in auditory hallucinations caused by the loss of structural integrity of neurons in the auditory conduction pathway and compensatory over-excitation of the central nervous system.

[0043] The term "drug-induced tinnitus," also known as "drug ototoxicity," refers to the chemical damage that drug components cause to the inner ear (cochlea or vestibule) or auditory nerve after the use of certain drugs, thereby triggering tinnitus, hearing loss, or dizziness.

[0044] The term "inflammatory tinnitus" refers to auditory hallucinations caused by inflammatory lesions in the auditory system (outer ear, middle ear, or inner ear) induced by biological, physical, or chemical factors. These hallucinations include, but are not limited to, mucosal edema, exudate accumulation, and Eustachian tube dysfunction caused by inflammation; inflammatory mediators (such as cytokines and prostaglandins) directly invading the cochlear hair cells and auditory nerve endings, inducing oxidative stress and ion channel dysfunction, leading to abnormal neuronal discharge and sensorineural tinnitus; and local inflammatory infiltration causing abnormal permeability of the inner ear microvessels, resulting in local hypoxia of the auditory receptors and producing tinnitus.

[0045] The term "blood-brain ratio" refers to the B / P ratio, which is the ratio of drug concentration in brain tissue to drug concentration in blood plasma.

[0046] The term "age-related hearing loss," also known as presbycusis, is a bilateral sensorineural hearing loss that gradually occurs with age.

[0047] Additionally, it should be noted that, unless otherwise explicitly stated, the description used in this invention as “each independently” should be interpreted broadly. It can mean either that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0048] Beneficial technical effects of the present invention (1) Significant hearing protection and anti-aging deafness effects: Through the synergistic effect between the components, this invention demonstrates significant technical advantages in preventing and treating hearing loss caused by natural aging. In aged mouse models, none of the single-drug groups could effectively inhibit the increase in hearing threshold with age, while the combined administration group of this invention showed significant differences in hearing threshold shift at multiple frequencies compared with the control group, and the hearing level of aged animals was significantly improved, proving its therapeutic potential in preventing and treating age-related deafness and delaying the degeneration of auditory organ function.

[0049] (2) The drug has a significant relieving effect on tinnitus: In the sodium salicylate-induced tinnitus model, the inhibition rate of each single drug group was not statistically significant and did not achieve the substantial therapeutic purpose; the inhibition rate of the combined drug group was significantly improved compared with the model group, showing a significant difference. This indicates that the present invention can effectively intervene in the generation of tinnitus and significantly improve auditory gating ability.

[0050] (3) sigma-1 R / GABA AThe dual-target synergistic effect of R has anti-inflammatory, antioxidant and neuroprotective effects: when the effect of single drugs is not good, the combination dosing regimen of the present invention shows strong repair ability: both L-1 and L-2 groups have significantly improved sVCAM-1 and SOD indicators; in particular, the L-2 group (Fabomotizole + Itruvone) can significantly increase BDNF level, and has a repair effect on auditory nerve by enhancing neurotrophic support, anti-inflammatory, antioxidant and neuroprotective effects.

[0051] (4) Synergistic effect of combined administration on improving brain-blood ratio: After combined administration, the brain penetration rates of Famotizole, Zuranolone, and Itruvone were all improved to varying degrees. The brain-blood ratio of Famotizole under combined administration was significantly increased by 2 times compared to the single-drug group (S-1), while the ratios of Zuranolone and Itruvone in the combined administration group were increased by 1.3 to 1.4 times. By increasing brain exposure and achieving the same or better therapeutic effects, it is expected to reduce the systemic dosage, thereby achieving the effect of reducing toxicity and increasing efficacy in clinical practice.

[0052] Implementation Plan The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Unless otherwise specified, all proportions, percentages, etc., referred to herein are by weight.

[0053] The Famotizole and Zuranolone used in this invention were purchased from Medchemexpress China. The Cutamesine and Itruvone were synthesized by the applicant. The synthesis method of Cutamesine can be found in references DOI:10.1021 / jm301734g and WO2007021545; the synthesis method of Itruvone can be found in WO9814194.

[0054] Preparation of test samples and operation of instruments (1) Use of ABR test equipment and preparation of anesthetic drugs: For specific methods, please refer to Bio Protoc. 2016 Mar 20;6(6):e1768. doi: 10.21769 / BioProtoc.1768. (2) The control group was given 30% beta-cyclodextrin aqueous solution by gavage; the L, S and G groups were given the drug by gavage after being dissolved in 30% beta-cyclodextrin according to the weight of each group.

[0055] Example 1: Mouse Auditory Brainstem Response (ABR): A Test for Natural Aging (1) Animal grouping and administration methods Grouping and Dosage: Male C3H / HeJJcl mice (6-8 weeks old) were randomly divided into control group (Group C) and treatment group (Group D) with Sigma-1 agonist + GABA. A R-modifier combination therapy group (L group), each Sigma-1 agonist single therapy group (S group), each GABA A R-modifier monotherapy group (G group). Each Sigma-1 agonist + GABA A The R-modifier combination therapy groups were Cutamesine + Zuranolone (L-1), Famotizole + Itruvone (L-2), and Cutamesine + Itruvone (L-3); the Sigma-1 agonist monotherapy groups were Famotizole (S-1), Cutamesine (S-2), and each GABA... A The R-regulatory agents were administered alone to the Zuranolone group (G-1) and the Itruvone group (G-1), with 6 mice in each group.

[0056] Administration method: The drug in group L was first mixed at a molar ratio of 1:1, and then each treatment group was administered the drug according to the animal's body weight. The dosage for groups L, S, and G was 10 mg / kg. All treatment groups were administered the drug throughout the study period (up to month 9). Auditory brainstem response (ABR) was measured at 9 months, with n=6 at each time point.

[0057] (2) Test methods Experimental Methods: Before ABR measurements, animals were anesthetized with xylazine and ketamine, and their external auditory canals and tympanic membranes were examined. Recording electrodes consisted of different active needle electrodes, placed subcutaneously below the test ear, on the top of the head, and below the contralateral ear, respectively. Sound stimuli consisted of 15 ms pitch pulses with a rise / fall time of 1 ms and frequencies of 4, 8, or 16 kHz. Generated by an audio generator and connected to a transducer tube, the stimuli were transmitted to the external auditory canal via a closed acoustic system. The sound source was calibrated by coupling a 0.6 mL rubber tube to a 0.25-inch microphone. Initial sound intensity was 100–105 dB, decreased in steps of 10–20 dB to near the threshold level, and then further decreased in steps of 5 dB to determine the threshold level. To obtain waveforms, each measurement point was recorded, averaged, calculated, and analyzed using a data acquisition system and a microcomputer equipped with custom software. (For detailed methods, please refer to Bio Protoc. 2016 Mar 20;6(6):e1768.DOI: 10.21769 / BioProtoc.1768.).

[0058] (3) Test results and analysis For details, please see [link / details]. Figure 1 As shown, the treatment group compared with the control group: P A value * < 0.05 indicates a statistically significant difference. The control group had a higher threshold shift, representing the degree of natural hearing loss. The groups marked with an asterisk (*) had significantly lower threshold shifts than the control group, indicating that these drug regimens (especially the combination therapy group) have a significant protective effect on hearing and reduce hearing loss.

[0059] During natural aging, the hearing threshold of mice exhibits significant individual variability, but the single-drug groups S-1, S-2, G-1, and G-2 failed to counteract age-related hearing loss. The combination-drug groups L-1, L-2, and L-3 demonstrated a protective effect on hearing. In particular, L-3, the Cutamesine + Itruvone group, showed significantly different hearing threshold shifts at 4, 8, and 16 kHz compared to the control group (P<0.05), successfully controlling the level of hearing loss in aged mice to a relatively mild level.

[0060] Example 2: Effects of sodium salicylate-induced tinnitus on inflammatory response and oxidative stress (1) Laboratory animals and grouping Grouping and Dosage: Healthy male SD rats were randomly divided into a control group (C group), a model group (M group), and a group receiving Sigma-1 agonist plus GABA. A R-modifier combination therapy group (L group), each Sigma-1 agonist single therapy group (S group), each GABA A R-modifier monotherapy group (G group). Each Sigma-1 agonist + GABA A The R-modifier combination therapy groups were the Famotizole + Zuranolone group (L-1) and the Famotizole + Itruvone group (L-2); the Sigma-1 agonist monotherapy groups were the Famotizole group (S-1); and the GABA groups were... A The R-regulatory agents were administered alone to the Zuranolone group (G-1) and the Itruvone group (G-1), with 5 rats in each group.

[0061] (2) Modeling and drug administration methods Modeling method: Group C received intraperitoneal injections of 200 mg / kg of normal saline at 8:00 AM and 4:00 PM for 14 consecutive days; Groups M, L, S, and G received intraperitoneal injections of 200 mg / kg of sodium salicylate at 8:00 AM and 4:00 PM for 14 consecutive days.

[0062] Selection of ratio: When the molar ratio is 1:1 (Sigma-1 agonist: GABA)A When the R-regulator is matched with the Sigma-1 receptor and GABA, it is precisely a good match. A The optimal ratio of the functional complex formed by R ensures sufficient activation of the complex by the Sigma-1 agonist while avoiding saturation or off-target effects caused by excessive amounts of any component. This results in the most significant synergistic regulatory effect, stable signal response, and good specificity. Therefore, this ratio was chosen for subsequent experiments.

[0063] Administration method: The L group drug was first mixed at a molar ratio of 1:1, and then each treatment group was administered the drug according to the animal's body weight. The dosage for groups L, S, and G was 10 mg / kg. Administration to each treatment group began 7 days before salicylic acid modeling and continued throughout the study period (14 days after modeling). On day 15, the pre-startle reflex impulse inhibition rate (PPI) and GPIAS inhibition rate were measured to verify the success of modeling.

[0064] The pre-pulse inhibition (PPI) method for startle reflex: Experimental animals were placed in an anechoic chamber with an ambient noise level of 60 dB SPL on a platform equipped with pressure receptors. In each experiment, rats were pre-acclimated to white background noise with an acoustic stimulus intensity of 65 dB SPL. The detection signal was received by sensors and transmitted to a specific computer to collect startle response data. During the testing period, rats were initially given 10 pulse stimuli with an acoustic intensity of 115 dB SPL. Then, 30 sound stimuli were randomly administered to the rats via a loudspeaker, including: 10 pulse stimuli with an acoustic intensity of 115 dB SPL, 10 startle reflex stimuli without a pre-pulse, and 10 startle reflex stimuli with a pre-pulse. The pre-pulse stimulus lasted 100 ms and included a 20 ms white noise pulse stimulus. This stimulus was provided at three different acoustic intensities: 75 dB SPL, 80 dB SPL, and 85 dB SPL (denoted as PPI 2, PPI 4, and PPI 8, respectively), with 27–32 s intervals between startle stimuli. This study used pre-startle impulse inhibition (PPI) and pre-startle interval inhibition (GPIAS) to detect behavioral changes in tinnitus in rats of different groups. PPI was used to assess the rats' response to startle sounds and determine whether tinnitus was caused by hearing loss; GPIAS inhibition rate was used to assess the severity of tinnitus. GPIAS inhibition rate (GPIAS%) = (AvgTnogap - AvgTgap) / AvgTnogap * 100%. Where AvgTnogap is the amplitude of the startle reflex induced without pre-interval stimulation, and AvgTgap is the amplitude of the startle reflex induced with pre-interval stimulation.

[0065] Serum collection: Blood was collected from all rats via vein on day 14 after rat modeling. Serum was separated by centrifugation and the sVCAM-1 level was measured by enzyme-linked immunosorbent assay (ELISA) using a Thermo MK3 microplate reader. SOD level was measured using a fully automated biochemical analyzer (Thermo Scientific Indiko). BDNF level was measured using a double-antibody sandwich ELISA kit and a microplate reader (at 450 nm wavelength).

[0066] (3) Test results and analysis Compared with the normal group: P The values ​​* < 0.05 and ** < 0.01, respectively, indicating statistically significant differences. Compared with the model group, each drug administration group: P The values ​​are respectively @ <0.05, & <0.01 indicates a statistically significant difference. As shown in Table 1, under all tested acoustic intensities, the GPIAS suppression rate of group M (sodium salicylate model) was significantly lower than that of group C (all met the requirements). P <0.05 indicates successful model establishment. GPIAS inhibition rate results showed that although the inhibition rate in the single-drug group rebounded compared to the model group, there was no significant difference, indicating no therapeutically significant improvement. The inhibition rates in the combination therapy groups L-1 (Fabomotizole + Zuranolone) and L-2 (Fabomotizole + Itruvone) were significantly higher than the model group, showing a statistically significant difference. P <0.05).

[0067] As shown in Table 2, the model group indicated that high-dose salicylic acid induced inflammatory responses (increased sVCAM-1), decreased antioxidant capacity (inhibited SOD), and neurological damage (decreased BDNF) in rats. However, no single-drug groups showed significant improvement in the damage caused by salicylic acid; while the combination therapy groups L-1 (Fabomotizole + Zuranolone) and L-2 (Fabomotizole + Itruvone) significantly improved sVCAM-1 and SOD levels. P <0.01 or 0.05), but only the L-2 group showed a significant increase in BDNF levels compared to the control group ( P <0.05).

[0068] Example 3: Brain tissue distribution of a combination of Sigma-1 agonist and GABAAR modulator 1. Animal grouping and experimental methods Test composition or compound grouping: SD male mice (8-12 weeks old) were randomly divided into groups for each Sigma-1 agonist + GABA. A R-modifier combination therapy group (L group), each Sigma-1 agonist single therapy group (S group), each GABA A R-modifier monotherapy group (G group). Each Sigma-1 agonist + GABA A The R-modifier combination therapy groups were the Famotizole + Zuranolone group (L-1) and the Famotizole + Itruvone group (L-2); the Sigma-1 agonist monotherapy groups were the Famotizole group (S-1); and the GABA groups were... A The R-modifier monotherapy groups were the Zuranolone group (G-1) and the Itruvone group (G-2).

[0069] Administration method: The drugs in group L were first mixed at a molar ratio of 1:1, and then each administration group was administered according to the animal's mass / body weight ratio. The dosage for groups L, S and G was 3 mg / kg.

[0070] Experimental Methods: At each time point, blood samples were collected from three rats under isoflurane anesthesia via cardiac puncture. Animals were sacrificed to collect brain tissue. Plasma was separated, and the brain tissue was homogenized. Plasma and brain homogenate were frozen at −20 °C until analysis.

[0071] The concentrations of test compounds in plasma and brain tissue were determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Using appropriate extraction techniques, the test compounds in plasma and brain homogenates were quantified by qualified LC-MS / MS methods within a calibration range of 1–500 ng / mL. Study samples were analyzed together with calibration samples from the batch and quality control samples dispersed throughout the batch, and the brain-plasma ratio (Cp) was calculated. brain / C plasma ( ), where the blood-brain ratio of each component in group L was calculated, and the specific results are shown in Table 3.

[0072] (3) Test results and analysis As shown in Table 3, compared with each drug group, both L-1 and L-2 groups increased the brain penetration rate of Famotizole, Zuranolone, and Itruvone to varying degrees. Especially for Famotizole, the brain-blood ratio in L-1 and L-2 groups was 2 times higher than that in S-1 group. The brain-blood ratio in L-1 group was 1.3 times higher than that in G-1 group, and in L-2 group it was 1.4 times higher than that in G-2 group. This suggests that combined administration of these two drugs synergistically increases the brain exposure of each individual drug, significantly contributing to improved protective or therapeutic efficacy against the auditory nerve.

[0073] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Sigma-1 agonist combined with GABA A Use of R modulators in the preparation of pharmaceutical compositions for the treatment and / or prevention of tinnitus.

2. Sigma-1 agonist combined with GABA A Use of R modulators in the preparation of pharmaceutical compositions for the treatment and / or prevention of hearing loss.

3. The use according to any one of claims 1 to 2, characterized in that, The Sigma-1 agonist is selected from any one of Famotizole, Cutamesine, and Igmesine, preferably Famotizole or Cutamesine.

4. The use according to any one of claims 1 to 2, characterized in that, GABA A The R modulator is a neurosteroid compound; the neurosteroid compound is selected from Zuranolone, Itruvone, or Fasedienol, preferably Zuranolone or Itruvone, and more preferably Itruvone.

5. The use according to claim 4, characterized in that, In the pharmaceutical composition, the Sigma-1 agonist and GABA A The molar ratio of the R modifier is 1:1, 2:1 and 1:2, preferably 1:

1.

6. The use according to any one of claims 1 to 2, characterized in that, The pharmaceutical composition is a combination of Famotizole and Zuranolone, a combination of Famotizole and Itruvone, a combination of Cutamesine and Zuranolone, a combination of Cutamesine and Itruvone, preferably a combination of Famotizole and Itruvone, or a combination of Cutamesine and Itruvone.

7. The use according to claim 1, characterized in that, The tinnitus mentioned is any one or more of the following: acute tinnitus, subacute tinnitus, chronic tinnitus, neurodegenerative tinnitus, tinnitus caused by inflammation, tinnitus caused by drug toxicity, and tinnitus caused by trauma.

8. The use according to claim 2, characterized in that, Its features are, The hearing loss mentioned is selected from age-related hearing loss.

9. A method for increasing the blood-brain ratio of Sigma-1 agonists, characterized in that, with GABA A R-regulatory agents are administered in combination, including GABA. A R modulators are neurosteroid compounds, preferably Zuranolone and Itruvone.

10. A substance comprising the Sigma-1 agonist and GABA as described in any one of claims 1 to 9. A A pharmaceutical composition comprising an R-modifier and pharmaceutically acceptable excipients, characterized in that... The pharmaceutically acceptable excipient is a cyclodextrin derivative, preferably beta-cyclodextrin.

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