Use of amiloride mesylate in the preparation of a drug for resisting cisplatin ototoxicity

Abstract

The application discloses application of amiloride mesylate in preparation of a drug for resisting cisplatin ototoxicity, and belongs to the field of drug use. It is found for the first time that amiloride mesylate has great potential in inhibiting cisplatin ototoxicity, and it is proved that after treatment of amiloride mesylate, application of cisplatin can effectively reduce cisplatin-induced ototoxicity in a HEI-OC1 cell line and a zebra fish model. The combined use of amiloride mesylate and cisplatin has a simple scheme and good effectiveness. Firstly, amiloride mesylate is used to pretreat an object, and then cisplatin is used, so that the cisplatin-induced ototoxicity can be reduced, thereby providing a new idea and scheme for clinically treating cisplatin-induced ototoxicity. Moreover, amiloride mesylate is an approved marketed drug, so that the time for drug research and approval can be greatly shortened, and the clinical transformation process can be accelerated.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical applications, and particularly relates to the application of amiotricazone mesylate in the preparation of anti-cisplatin ototoxic drugs. Background Technology

[0002] Drug therapy is a crucial component of cancer treatment, and the use of effective anti-cancer drugs can help patients achieve longer survival times. Cisplatin, a platinum-based complex, is widely used to treat testicular cancer, ovarian cancer, uterine cancer, bladder cancer, cervical cancer, prostate cancer, and brain cancer due to its broad anti-cancer spectrum, effectiveness against hypoxic cells, strong efficacy, and relatively low price, with significant therapeutic effects. However, cisplatin also has certain side effects. It is a highly cumulative drug, prone to causing nephrotoxicity, and gastrointestinal reactions are common. Some patients experience granulocytopenia, but this usually recovers within 7-14 days after discontinuation. Cisplatin also has ototoxicity, often manifesting as temporary or permanent tinnitus, high-frequency hearing loss, and in severe cases, transitioning to low-frequency hearing loss or even full-frequency hearing loss.

[0003] Chinese patent CN114848836B discloses a conjugate and its application in treating inner ear diseases. This conjugate combines an inner ear-targeting peptide with a main drug for diagnosing, preventing, or treating inner ear diseases. The inner ear-targeting peptide assists the main drug in targeting specific structures and cells within the inner ear, achieving drug enrichment with precision and high efficiency, thus improving the bioavailability of the drug in the inner ear. Chinese patent CN106551930B discloses the application of corydaline in the preparation of anti-cisplatin toxicity drugs. Chinese patent CN201610257853.2 discloses a pharmaceutical composition of amitriazine dimethsulfate and its pharmaceutical use. In this composition, amitriazine dimethsulfate is mainly used for ischemic cochlear vestibular dysfunction, a condition characterized by hypofunction or loss of vestibular function and overactivity, primarily manifested as dizziness, nausea, vomiting, and diarrhea.

[0004] Currently, there are still very few reports on drugs for treating cisplatin ototoxicity. Therefore, there is an urgent need to find a safe drug that can inhibit its toxicity and be used in combination with cisplatin, so as not to affect the anti-cancer effect of cisplatin while eliminating or reducing the side effects of cisplatin. Summary of the Invention

[0005] To overcome the above shortcomings, the present invention aims to provide the application of amitriazine mesylate in the preparation of anti-cisplatin ototoxic drugs, so as to solve the problem of cisplatin-induced ototoxicity.

[0006] To achieve the above objectives, the technical solution provided in this application is as follows:

[0007] The use of amitriazine mesylate in the preparation of an anti-cisplatin ototoxic drug, wherein the anti-cisplatin-induced ototoxic drug comprises amitriazine mesylate.

[0008] Furthermore, the drug can be administered orally, topically, or by injection.

[0009] Furthermore, the drug is made from a pharmaceutically acceptable carrier or excipient.

[0010] Furthermore, the drug dosage forms include soft capsules, pellets, emulsions, tablets, lotions, liniments, ointments, powder for injection, injections, or suppositories.

[0011] The beneficial effects of this application, based on the above technical solutions, are as follows:

[0012] (1) The method of the present invention is simple, convenient and effective. Pre-treatment with amiotriazine mesylate followed by cisplatin can reduce its ototoxicity.

[0013] (2) The method of the present invention has high safety, and amiotriazine mesylate does not have cytotoxicity within the effective concentration; (3) Amiotriazine mesylate of the present invention is an FDA-approved drug, has been approved for marketing, is easy to obtain, and can accelerate the clinical translation process. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the ototoxic effect of amitriazine mesylate (Alm) on cisplatin (CDDP) according to the present invention. (a) Cell viability of HEI-OC1 cells treated with different concentrations of Alm, (b) Cell viability of hair cells treated with Alm and CDDP together;

[0015] Figure 2 This is a schematic diagram of zebrafish lateral line hair cells treated with different drug concentrations according to the present invention. (a) is a normal zebrafish lateral line hair cell cluster, (b) is a schematic diagram of zebrafish lateral line hair cell loss after treatment with 600 μM cisplatin, (ce) is a schematic diagram of inhibiting cisplatin-induced hair cell loss after zebrafish were treated with 1 μM, 10 μM and 20 μM Alm, and (f) is a bar chart showing the number of cells in different groups of lateral line hair cell clusters. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0017] This invention provides the application of amitriazine mesylate in the preparation of anti-cisplatin ototoxic drugs, with specific embodiments as follows:

[0018] (1) Conditions for establishing cisplatin ototoxicity cell models:

[0019] A. Cell seeding: Take a suspension of HEI-OC1 cells and adjust the concentration to 5 x 10⁻⁶ cells / mL. 4 / mL, add 100uL of cell suspension to each well of a 96-well plate using a pipette, and incubate at 33°C in a 10% CO2 incubator.

[0020] B. Cisplatin treatment: When the cells covered 80% of the bottom of the well, the cells in different wells were treated with different concentrations of cisplatin. The cisplatin concentration gradient was set at 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM and 0 μM, with 6 replicates for each concentration. Blank control wells with added cells were also set up. Cell viability was measured at 24 h, 36 h and 48 h.

[0021] C. CCK-8 assay for cell viability: Take cisplatin-treated 96-well plates at the above time points, add 10 μL of CCK-8 solution to each well, put them back into the incubator and incubate for 1 h, and then measure the absorbance of each well at 450 nm using a microplate reader.

[0022] Calculation of cell viability:

[0023] Let As be the absorbance of the experimental group, Ac be the absorbance of the control group (0 μM), and Ab be the absorbance of the blank group (only culture medium added, no cells added).

[0024] Cell viability = [(As-Ab) / (Ac-Ab)] x 100%

[0025] The results showed that treating HEI-OC1 cells with 50 μM cisplatin for 24 h could increase cell viability to 50%, which is the half-inhibitory concentration (IC50) of cisplatin, i.e., the conditions used for modeling.

[0026] (2) Detection of the inhibitory effect of the drug on cisplatin ototoxicity:

[0027] A. Cell seeding: Take a suspension of HEI-OC1 cells and adjust the concentration to 5 x 10⁻⁶ cells / mL. 4 / mL, add 100uL of cell suspension to each well of a 96-well plate using a pipette, and incubate at 33°C in a 10% CO2 incubator.

[0028] B. Drug treatment: When the cells have covered about 50% of the bottom of the well, add 10 μM amiotriazine mesylate into the cell wells of HEI-OC1, with nine replicates for each drug, and incubate in a 33°C, 10% CO2 incubator for 24 h.

[0029] C. Cisplatin treatment: Amitriazine mesylate added to the 96-well plate was removed and 50 μM cisplatin was added for 24 h. At the same time, nine wells with only cisplatin added and no amitriazine mesylate were set up as a control group.

[0030] D. Cell viability detection: Using the CCK-8 assay described above, the absorbance of cells in each well was first measured. The calculation formula is as follows:

[0031] Let As be the absorbance of the experimental group (treated with both drug and cisplatin), Ac be the absorbance of the control group (treated with cisplatin only), and Ab be the absorbance of the blank group (treated with culture medium only, without cells).

[0032] Cell viability = [(As-Ab) / (Ac-Ab)] x 100%

[0033] E. Optimal drug treatment conditions: Different concentrations of amiodarone mesylate (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 0 μM) were set to treat cells for 24 h, 36 h, and 48 h to obtain the concentration and time patterns of amiodarone mesylate inhibiting cisplatin ototoxicity.

[0034] F. Result Determination: (e.g.) Figure 1 By statistically analyzing cell viability values, amitriazine mesylate showed that it inhibited the ototoxicity of cisplatin within the concentration range of 0.1 μM-50 μM.

[0035] (3) Verification of the effectiveness of amitriazine mesylate in zebrafish:

[0036] A. Obtaining zebrafish embryos: The night before egg retrieval, four pairs of female and male zebrafish with autofluorescent hair cells were placed in four separate fish tanks, and the female and male fish were separated by a transparent baffle. On the morning of egg retrieval, the baffle was removed to allow the female and male fish to reunite. After waiting for 30 minutes, the fertilized eggs at the bottom of the fish tank were collected and placed in a 37°C incubator for culture.

[0037] B. Treatment with amitriazine mesylate: After three days of incubation in an incubator, zebrafish hatched. For zebrafish whose eggs had not yet hatched, the eggs could be pricked with a needle to help them hatch. The zebrafish were cultured in seawater containing amitriazine mesylate for 2 hours, and then in seawater containing 600 μM cisplatin for 6 hours. Four groups were set up according to drug concentrations of 0 μM, 1 μM, 10 μM, and 20 μM. The group without amitriazine mesylate and cisplatin was the positive control group, the 0 μM group was the negative control group, and the rest were the experimental groups.

[0038] C. Embedding and fixation: The zebrafish were anesthetized with anesthetic and embedded in agarose gel in a "side-lying" position;

[0039] D. Detection method: The lateral line hair cells of zebrafish were observed and counted using a laser confocal fluorescence microscope to confirm the inhibitory effect of the drug on cisplatin ototoxicity in zebrafish.

[0040] E. Result determination: such as Figure 2 By counting the number of cells in the lateral line hair cell clusters of zebrafish, amitriazine mesylate can inhibit cisplatin-induced hair cell loss.

[0041] It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. The application of amitriazine mesylate in the preparation of an anti-cisplatin ototoxic drug, characterized in that, The anti-cisplatin-induced ototoxicity drug contains amitriazine mesylate.

2. The application of amitriazine mesylate according to claim 1 in the preparation of an anti-cisplatin ototoxicity drug, characterized in that, The drug can be administered orally, topically, or by injection, and its dosage forms include soft capsules, pellets, emulsions, tablets, lotions, liniments, ointments, powder for injection, injections, or suppositories.

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