Use of maslinic acid as a cisplatin ototoxicity protective agent

Abstract

The application belongs to the field of biological pharmacy, and particularly relates to application of maslinic acid as a cisplatin ototoxicity protective agent. The application discloses that maslinic acid can prevent cisplatin-induced ototoxicity by restoring the activity of mouse cochlea hair cells SLC7A11, and can repair cisplatin-induced hearing impairment in mice. Experimental results show that maslinic acid can restore the reduced activity of SLC7A11 caused by cisplatin, thereby reducing the intracellular LPO level, increasing the HEI-OC1 cell survival rate, and not affecting the anticancer effect of cisplatin; animal experiments also show that maslinic acid can inhibit cisplatin-induced hearing impairment in mice. Therefore, maslinic acid can reduce the ototoxicity of cisplatin and play an inner ear protection role.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to the application of hawthorn acid as a protective agent against cisplatin ototoxicity. Background Technology

[0002] Platinum-based drugs are common anti-tumor agents used to treat various human cancers, including bladder cancer, head and neck cancer, lung cancer, ovarian cancer, and testicular cancer, due to their broad-spectrum and high efficacy. Among the many chemotherapy drugs widely used in cancer treatment, cisplatin is the most noteworthy and was the first platinum-based compound approved by the FDA for cancer treatment. During tumor treatment, repeated intravenous administration of cisplatin can lead to its accumulation in specific areas of the body, resulting in toxicity, including nephrotoxicity, ototoxicity, hepatotoxicity, peripheral neuropathy, bone marrow suppression, and retinopathy. Of all these toxic side effects, sensorineural hearing loss caused by cisplatin is irreversible and has therefore received the greatest attention.

[0003] Maslinic acid (MA) belongs to the pentacyclic triterpenoid class of compounds and is mainly found in natural plants such as olive and hawthorn. It is also abundant in traditional herbs such as platycodon and patchouli. Maslinic acid is mostly extracted from olives, but can also be semi-synthesized using oleanolic acid as a raw material. Maslinic acid has anti-inflammatory, antioxidant, antitumor, hypoglycemic, and neuroprotective effects. This invention is the first to discover that maslinic acid has a protective effect against cisplatin-induced cochlear hair cell death in mice.

[0004] Literature reports that cisplatin can induce ferroptosis in cochlear hair cells, leading to hearing loss. This invention found that ursolic acid can protect against cisplatin-induced cochlear hair cell death and, by restoring SLC7A11 activity, reduce intracellular lipid peroxidation levels, protecting mouse cochlear hair cells from damage without affecting the anticancer effects of cisplatin. Simultaneously, in vivo experiments showed that ursolic acid can inhibit cisplatin-induced hearing loss. Therefore, ursolic acid has the potential to be developed as a cisplatin-protective agent against hearing loss. Currently, there are no reports on the use of ursolic acid to intervene in cisplatin-induced hearing loss; therefore, this invention is proposed. Summary of the Invention

[0005] This invention overcomes the shortcomings of the prior art and provides the following technical solution:

[0006] The first aspect of the present invention is to provide the use of maslinic acid (MA) and its derivatives in the preparation of ototoxic protective agents.

[0007] Furthermore, the ototoxicity is caused by chemotherapy drugs for tumors.

[0008] Furthermore, the tumor chemotherapy drug is cisplatin.

[0009] Furthermore, the hawthorn acid derivative is a pharmaceutically acceptable salt.

[0010] Furthermore, the structural formula of the hawthorn acid is:

[0011]

[0012] Furthermore, the dosage form of the protective agent includes at least one of the following: suspension, granules, capsules, powders, tablets, pellets, injections, suppositories, and drops.

[0013] Furthermore, the administration route of the protective agent includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

[0014] Furthermore, the dosage of the protective agent is 100-200 mg / kg / Day, calculated based on the effective amount of crataegolic acid per kilogram of body weight, and the administration period is 5-10 days; preferably, the protective agent is administered by gavage, and the dosage is 150 mg / kg / Day.

[0015] A second aspect of the present invention is to provide the use of hawthorn acid in the preparation of formulations for preventing and treating the reduction of reduced glutathione (GSH) in vivo.

[0016] Furthermore, the decrease in GSH was caused by tumor chemotherapy drugs.

[0017] Furthermore, the tumor chemotherapy drug is cisplatin.

[0018] Furthermore, the hawthorn acid derivative is a pharmaceutically acceptable salt.

[0019] Furthermore, the structural formula of the hawthorn acid is:

[0020]

[0021] Furthermore, the dosage form of the preparation includes at least one of suspension, granules, capsules, powders, tablets, pellets, injections, suppositories, and drops.

[0022] Furthermore, the administration route of the formulation includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

[0023] Further, the dosage of the preparation is 100-200 mg / kg / Day based on the effective amount of crataegolic acid per kilogram of body weight, and the administration period is 5-10 days; preferably, the protective agent is administered by gavage, and the dosage is 150 mg / kg / Day.

[0024] A third aspect of the invention is to provide the use of hawthorn acid in the preparation of formulations that reverse the decrease in activity of SLC7A11 cochlear hair cells.

[0025] Furthermore, the reduced activity of the cochlear hair cells SLC7A11 was caused by tumor chemotherapy drugs.

[0026] Furthermore, the tumor chemotherapy drug is cisplatin.

[0027] Furthermore, the hawthorn acid derivative is a pharmaceutically acceptable salt.

[0028] Furthermore, the structural formula of the hawthorn acid is:

[0029]

[0030] Furthermore, the dosage form of the preparation includes at least one of suspension, granules, capsules, powders, tablets, pellets, injections, suppositories, and drops.

[0031] Furthermore, the administration route of the formulation includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

[0032] Further, the dosage of the preparation is 100-200 mg / kg / Day based on the effective amount of crataegolic acid per kilogram of body weight, and the administration period is 5-10 days; preferably, the protective agent is administered by gavage, and the dosage is 150 mg / kg / Day.

[0033] A fourth aspect of the present invention is to provide the use of hawthorn acid in the preparation of a cochlear hair cell protectant, wherein the use is an in vitro application.

[0034] Furthermore, the protection described herein is to increase the level of lipid peroxidation in cochlear hair cells.

[0035] Furthermore, the protection is the inhibition of mouse cochlear hair cell death induced by the SLC7A11 inhibitor sulfasalazine (SAS).

[0036] Furthermore, the protection described herein is to reverse cisplatin-induced cochlear hair cell damage.

[0037] The beneficial effects of this invention include:

[0038] 1. This invention is the first to discover that hawthorn acid can protect against cisplatin-induced hearing damage in mice.

[0039] 2. This invention is the first to discover that hawthorn acid can inhibit cisplatin-induced cochlear hair cell death in HEI-OC1 mice and can reverse the increase in lipid peroxidation level in cisplatin-induced mouse cochlear hair cells.

[0040] 3. This invention is the first to discover a new mechanism by which ursolic acid protects against cisplatin-induced ototoxicity, namely by restoring SLC7A11 activity to exert a protective effect. Attached Figure Description

[0041] Figure 1 MA can restore cisplatin (CP)-induced hearing loss in mice;

[0042] Note: (A) Changes in Click ABR hearing thresholds in mice of the control group (Con), treatment group (high MA), model group (M), and model treatment group (M+high MA). (B, C, D) Changes in hearing thresholds at different frequencies (8kHz, 16kHz, 24kHz) in mice of the control group (Con), treatment group (high MA), model group (M), and model treatment group (M+high MA). Data are expressed as mean ± standard deviation. Compared with the control group, **P<0.01, ***P<0.001; compared with the model group, **P<0.01, ***P<0.001.

[0043] Figure 2 MA can restore the decrease in GSH (reduced glutathione) in mice induced by cisplatin (CP);

[0044] Note: Changes in serum GSH levels in mice of the control group (Con), treatment group (high MA), model group (M), and model treatment group (M+high MA). Compared with the control group, **P<0.01, ***P<0.001; compared with the cisplatin-treated group alone, **P<0.01, ***P<0.001.

[0045] Figure 3 MA can inhibit cisplatin (CP)-induced HEI-OC1 cell death;

[0046] Note: (A) Cell viability of HEI-OC1 cells after 24 h of treatment with CP (20 μM) alone or in combination with different concentrations of MA; (B) Staining of HEI-OC1 cells after 24 h of treatment with CP (20 μM) or MA (30 μM) alone or in combination with CP and MA, followed by crystal violet staining. Data are expressed as mean ± standard deviation, n = 3. Compared with the control group, **P < 0.01, ***P < 0.001; compared with the cisplatin-only group, **P < 0.01, ***P < 0.001.

[0047] Figure 4 MA can restore cisplatin (CP)-induced SLC7A11 activity inhibition;

[0048] Note: (AB) Cystine uptake and intracellular cysteine ​​levels in HHEI-OC1 cells after treatment with CP (20 μM) or MA (30 μM) alone or in combination with CP and MA for 17 h. Data are expressed as mean ± standard deviation, n = 3. Compared with the control group, *P < 0.05, **P < 0.01; compared with the CP-only group, **P < 0.01, ***P < 0.001.

[0049] Figure 5 MA can alleviate cisplatin (CP)-induced elevation of lipid peroxidation (LPO) levels in HEI-OC1 cells;

[0050] Note: LPO levels in HEI-OC1 cells were determined by AB flow cytometry after 10 h of treatment with MA (30 μM) or CP (20 μM) alone or in combination. Data are expressed as mean ± standard deviation, n = 3. Compared with the control group, **P < 0.01, ***P < 0.001; compared with the cisplatin-only group, **P < 0.01, ***P < 0.001.

[0051] Figure 6 MA can inhibit sulfasalazine (SAS)-induced HEI-OC1 cell death;

[0052] Note: (A) Cell viability of HEI-OC1 cells after 24 h of treatment with SAS (300 μM) alone or in combination with different concentrations of MA; (B) Effect of SAS (300 μM) alone or in combination with different concentrations of MA (10 μM, 20 μM, 30 μM) on the morphology of HEI-OC1 cells. Data are expressed as mean ± standard deviation, n = 3. Compared with the control group, **P < 0.01, ***P < 0.001; compared with the cisplatin-only group, **P < 0.01, ***P < 0.001.

[0053] Figure 7 MA can alleviate the SAS-induced increase in lipid peroxidation (LPO) levels in HEI-OC1 cells;

[0054] Note: LPO levels in HEI-OC1 cells were determined by AB flow cytometry after 10 h of treatment with MA (30 μM) or SAS (300 μM) alone or in combination. Data are expressed as mean ± standard deviation, n = 3. Compared with the control group, **P < 0.01, ***P < 0.001; compared with the cisplatin-only group, **P < 0.01, ***P < 0.001.

[0055] Figure 8 MA had no effect on the anticancer activity of cisplatin (CP) on A549 cells;

[0056] Note: Cell viability of A549 cells after 24 h of treatment with CP (20 μM) alone or in combination with MA (30 μM). Data are expressed as mean ± standard deviation, n = 3. Compared with the control group, **P < 0.01; compared with the CP-treated group alone, there was no statistically significant difference (ns). Detailed Implementation

[0057] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0058] The term "SLC7A11" refers to solute carrier family 7 member 11 (SLC7A11). SLC7A11 is located on the cell membrane and transports cysteine ​​into the cell in a 1:1 ratio while exporting glutamate to the extracellular space. After entering the cell, cysteine ​​is reduced to cysteine ​​by NADPH. Under enzymatic action, cysteine ​​combines with glycine and glutamate to synthesize GSH. GSH can reduce lipid peroxides to non-toxic lipid alcohols under the action of GPX4, thereby inhibiting ferroptosis.

[0059] The term "SAS" stands for sulfasalazine, an azo compound of salicylic acid and sulfapyridine. SAS inhibits SLC7A11, a key antioxidant in the ferroptosis regulatory network, leading to GSH depletion and inhibition of GPX4 activity, causing the intracellular ferroptosis defense system to collapse and resulting in ferroptosis.

[0060] Example 1: Effect of MA on cisplatin-induced hearing loss in mice

[0061] Animal grouping: 40 male C57BL / 6J mice, 6-8 weeks old, were randomly divided into 5 groups of 8 mice each: control group (Con), drug administration group (MA), model group (CP), and model drug administration group (CP+MA).

[0062] Hearing impairment model: A mouse hearing impairment model induced by intraperitoneal injection of cisplatin was used. The model group and the treatment group were injected intraperitoneally with 5 mg / kg cisplatin (CP) for 4 consecutive days, while the control group and the treatment group were injected intraperitoneally with the corresponding volume of physiological saline.

[0063] Experimental Methods: After the adaptation culture was completed, the drug administration group (MA) and the model drug administration group (CP + high MA) were administered 150 mg / kg of hawthorn acid by gavage for eight consecutive days, while the control group was administered a solvent (5% DMSO + 95% physiological saline) by gavage for eight consecutive days. Modeling was initiated on the ninth day after the adaptation culture was completed, using the intraperitoneal injection method. The model group (CP) was injected intraperitoneally with 5 mg / kg cisplatin for four consecutive days; the model drug administration group (CP + MA) was first administered hawthorn acid by gavage, followed by intraperitoneal injection of cisplatin two hours later; the control group was injected intraperitoneally with physiological saline for four consecutive days.

[0064] After modeling, auditory brainstem response (ABR) was performed on mice using a brainstem evoked potential instrument (TDT, RZ6) to measure hearing and compare changes in hearing thresholds among different groups. Following the hearing test, whole blood was collected from the mice via ocular sampling. After the blood settled and separated into layers, it was centrifuged, and serum was collected. The GSH content in the mouse serum was then measured using a micro-reduced glutathione (GSH) assay kit.

[0065] like Figure 1 As shown in (A), the hearing threshold of the model group (CP) increased by 70% compared with that of the control group (Con); the hearing threshold of the model drug group (CP+MA) decreased by 37.5% compared with that of the model group (CP).

[0066] like Figure 1 As shown in (B), at a frequency of 8 kHz, the hearing threshold of the model group (CP) increased by 62% compared to the control group (Con); the hearing threshold of the model drug administration group (CP+MA) decreased by 31.5% compared to the model group (CP). Figure 1 As shown in (C), at a frequency of 16 kHz, the hearing threshold of the model group (CP) increased by 69.7% compared to the control group (Con); the hearing threshold of the model drug administration group (CP+MA) decreased by 46% compared to the model group (CP). Figure 1 As shown in (D), at a frequency of 24 kHz, the hearing threshold of the model group (CP) increased by 69.7% compared with the control group (Con); the hearing threshold of the model drug group (CP+MA) decreased by 46% compared with the model group (CP).

[0067] like Figure 2As shown, compared with the control group (Con), the GSH content in the model group (CP) decreased by 78.8%; compared with the model group (CP), the GSH content in the model drug treatment group (CP+MA) increased by 25.8%. Conclusion: MA can restore CP-induced hearing loss in mice.

[0068] Effects of 2MA and CP on HEI-OC1 cell survival

[0069] Experimental Methods: HEI-OC1 cells were cultured in DMEM containing 10% FBS at 37°C with 5% CO2. When the confluence reached 80-90%, the cells were passaged or plated proportionally. 24 hours after plated cell division, when the cells reached approximately 30-40% confluence, HT22 cells were treated with CP (20 μM) alone or in combination with MA (30 μM) for 48 hours. Cell viability was determined using crystal violet staining.

[0070] like Figure 3 As shown in Figures AB, crystal violet staining revealed a significant difference between CP treatment alone and the combined treatment with CP and MA. Treatment of HEI-OC1 cells with 20 μM CP for 48 h reduced cell viability by 55.47% compared to the control group, while treatment with 30 μM MA and CP together increased HEI-OC1 cell viability by 19.00% compared to the CP-only treatment group.

[0071] Conclusion: MA can inhibit CP-induced HEI-OC1 cell death.

[0072] Example 3: Effects of MA and CP on SLC7A11 activity in HEI-OC1 cells

[0073] Experimental methods: The cystine uptake capacity and intracellular cysteine ​​content of HT22 cells after treatment with MA (30 μM) and CP (20 μM) alone or in combination for 17 h were determined according to the kit instructions.

[0074] Depend on Figure 4 As shown in AB, CP treatment significantly reduced the cysteine ​​uptake capacity and intracellular cysteine ​​content of HEI-OC1 cells. MA treatment, however, inhibited these changes, increased the cysteine ​​uptake capacity of HEI-OC1 cells, and increased intracellular cysteine ​​content.

[0075] Conclusion: MA can exert a protective effect by restoring CP-induced inhibition of SLC7A11 activity and reducing LPO levels, thereby preventing cochlear hair cell damage.

[0076] Case Study 4: Effects of MA and CP on Intracellular LPO Levels in HEI-OC1 Cells

[0077] Experimental method: The LPO level of HEI-OC1 cells was determined by Liperfluo fluorescent probe method after 12 h of treatment with CP (20 μM) and MA (30 μM) alone or in combination.

[0078] like Figure 5 As shown in Figures AB, the LPO level increased by 21.89% in the CP-treated group compared to the control group, while the LPO level decreased by approximately 24.14% after MA treatment compared to the CP-treated group. MA significantly inhibited the CP-induced increase in LPO levels in HEI-OC1 cells, alleviated lipid peroxidation, and thus increased the cells' antioxidant capacity, ultimately preventing CP-induced cell death. Conclusion: MA can alleviate the CP-induced increase in lipid peroxidation in HEI-OC1 cells.

[0079] Example 5: Effects of MA and SAS on the survival and morphology of HEI-OC1 cells

[0080] Experimental Methods: HEI-OC1 cells were cultured in DMEM containing 10% FBS at 37°C with 5% CO2. When the confluence reached 80-90%, the cells were passaged or plated proportionally. After plated cell division, when the cells reached approximately 30-40% confluence, HT22 cells were treated with SAS (300 μM) alone or in combination with different concentrations of MA (10, 20, 30 μM) for 24 hours. Cell viability was determined using crystal violet staining.

[0081] like Figure 6 As shown in Figures AB, HEI-OC1 cells in the control group adhered firmly, were spindle-shaped, and had tight intercellular connections. However, after 24 hours of SAS treatment, a large number of cells lost their normal morphology, becoming shrunken, rounded, and even appearing as black dots. Cell density decreased significantly, and dead cells were observed floating. Compared to the SAS group, the co-treatment group of SAS and MA showed significantly improved cell density and morphology, indicating that MA had a significant protective effect against SAS-induced damage to HEI-OC1 cells. Analysis after crystal violet staining revealed that HT22 cell viability decreased by 63.04% after 24 hours of treatment with 300 μM SAS compared to the control group. However, co-treatment with 10, 20, and 30 μM MA and SAS increased HEI-OC1 cell viability by 12.77%, 28.9%, and 59.79%, respectively, compared to the SAS-only treatment group. MA treatment alone had no significant effect on HEI-OC1 cell viability.

[0082] Conclusion: MA can inhibit SAS-induced HEI-OC1 cell death.

[0083] Implementation Case 6: Effects of MA and SAS on LPO Levels in HEI-OC1 Cells

[0084] Experimental method: The LPO level of HEI-OC1 cells was determined by Liperfluo fluorescent probe method after 12 h of treatment with SAS (300 μM) or MA (30 μM) alone or in combination.

[0085] like Figure 7 As shown in AB, the LPO level in the SAS-treated group increased by 38.79% compared with the control group, while the LPO level in the cells decreased by about 42.94% after MA treatment compared with the SAS-treated group. MA significantly inhibited the increase of LPO level in HEI-OC1 cells induced by SAS, reduced lipid peroxidation, and thus increased the antioxidant capacity of cells, ultimately protecting cells from SAS-induced death.

[0086] Conclusion: MA can alleviate the increase in intracellular lipid peroxidation level induced by SAS in HEI-OC1 cells.

[0087] Case Study 7: The Effect of MA on the Anticancer Effect of CP

[0088] Experimental method: A549 cells were treated with CP (20 μM) alone or in combination with MA (30 μM) for 24 h, and cell viability was determined by crystal violet staining.

[0089] The results are as follows Figure 8 As shown in AB, the cell viability of A549 cells treated with 30 μM CP alone for 24 h decreased by 45.11% compared with the control group, while MA treatment had no effect on CP-induced cell death in A549 cells.

[0090] Conclusion: MA has no effect on the anticancer effect of CP.

[0091] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. Application of crataegolic acid and its pharmaceutically acceptable salts in the preparation of protective agents against cisplatin-induced hearing loss.

2. Use according to claim 1, characterized in that, The structural formula of the hawthorn acid is: 。 3. Use according to claim 1, characterized in that, The dosage form of the protective agent includes at least one of suspension, granules, capsules, powders, tablets, pellets, injections, suppositories, and drops; the route of administration of the protective agent includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

4. Use according to claim 3, characterized in that, The dosage of the protective agent is 100-200 mg of effective amount of hawthorn acid per kilogram of body weight per day, and the administration period is 5-10 days.

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