Pharmacotoxicology-related Studies Of Electric Field-responsive Lamotrigine Nanocomposite Formulations

Objective To analyse the cytotoxicity,acute toxicity,long-term toxicity and reproductive toxicity of electric field responsive lamotrigine nanocomposites through cellular and animal in vivo experiments,laying the foundation for further clinical applications.Methods: The value-added inhibitory ability of lamotrigine nanocomposite(LTG-Nano)on b End.3 cells was determined by CCK8 method and its IC50 value was calculated to verify the cytotoxicity of LTGNano.SPF grade Kunming mice were used as the experimental animals.In the acute toxicity test,60 Kunming mice were given LTG-Nano by single gavage at doses of 312mg/kg,412mg/kg,549mg/kg,722mg/kg,950mg/kg and 1250mg/kg respectively.diet and water intake.Body weights were recorded weekly,food intake was recorded daily,and surviving mice were executed at D15 for gross and histopathological examination.Long-term toxicity tests were conducted using 80 Kunming mice given LTG-Nano by repeated gavage for 24 weeks and a recovery period of 4 weeks to evaluate possible toxic effects.Based on the maximum non-lethal dose(312 mg/kg)obtained from the single administration toxicity test,repeated doses of 40mg/kg,80 mg/kg and 160 mg/kg were determined.mice were observed for body weight gain,food intake,organ weights,organ coefficients,blood cell counts,blood biochemical tests and histological examination of major organs.In the reproductive toxicity assay,the corresponding donor was given by gavage daily according to body weight from day 6 of pregnancy until day 18 of pregnancy at doses of 30mg/kg,60mg/kg and 120mg/kg,which were converted to human doses of 210mg/person,420mg/person and840mg/person based on body surface area,starting from day 6 of pregnancy until day 18 of pregnancy,equivalent to the dose given from mid-pregnancy until late gestation.Analysis of mortality and near-death,body weight,number of fetal rats and absorbed fetuses,placenta,fetal rat weight,analysis of fetal rat development,and placental pathology.The safety of LTG-Nano was assessed in conjunction with cytotoxicity assays and in vivo toxicity assays.Results1.Cytotoxicity assay of lamotrigine compound:Both LTG nanocomposite and LTG prodrug showed concentrationdependent inhibition of proliferation of bEnd.3 cells.The IC50 values of both were 639.0ug/m L and 614.9ug/m L respectively,which were not significantly different and showed relatively low cytotoxicity.The correlation results of PI staining and flow cytometry also indicated that both LTG nanocomposite and LTG prodrug had low cytotoxicity and good biosafety.2.In vivo toxicity assay of lamotrigine nanocomposite formulation.Acute toxicity assay: Single gavage administration of LTG-Nano to Kunming mice at doses of 312 mg/kg,412 mg/kg,549 mg/kg,722 mg/kg,950mg/kg and 1250 mg/kg.dose-related animal mortality was seen after dosing.The 950mg/kg and 722mg/kg groups showed a reduction in body weight gain and food intake in males D1-D7,and no abnormalities were seen in organ weights and organ coefficients.The LTG-Nano LD50 was 723.2mg/kg under the conditions of this test.Long-term toxicity test: The doses of 40 mg/kg,80 mg/kg and 160 mg/kg in the experimental groups were given to Kunming mice by repeated gavage of LTG-Nano for 24 weeks and 4 weeks after the drug was discontinued.The mice showed no abnormalities in body weight gain,food intake,organ weight,organ coefficient,blood cell count,blood biochemical tests and histological examination of major organs.Pathological examinations of the major organs at24 weeks and 4 weeks after drug withdrawal showed no abnormalities in cardiac muscle fibres,liver,spleen,lung tissue,kidney tissue,cerebral cortex,ovarian tissue,uterus,adrenal glands,thymus,thyroid and testes.Reproductive toxicity assay: Kunming mice were administered by gavage for 13 days from day 6 to day 18 of gestation at doses of 30 mg/kg,60 mg/kg and 120 mg/kg,respectively.no abnormalities were observed in the status of pregnant mice,placental structure and fetal mouse development.Conclusion: The electric field responsive lamotrigine nanocomposite has no significant toxicity and has a good safety profile for further clinical studies.