| Objective:To establish the ras H2 transgenic mouse carcinogenicity model, and to conduct a short-term carcinogenicity study for F3 SM, a new PPARβ/δ agonist, in which the assessment of the carcinogenic susceptibility of F3 SM and its metabolism profile during the trial need to be characterized, and possible relationship between the carcinogenic susceptibility and drug exposure of F3 SM be established.6 Method: 1. Four-week repeated dose toxicity test in C57BL/6 mice: C57 mice received 28 days’ continuous oral administration of F3 SM with 4 dosage(0, 40, 120, 360 mg/kg), daily record of the toxicity and death status of the mice was kept, a batch of mice at the end of the administration session was sacrificed, organ coefficient,blood biochemical indexes, indexes of hematology and histopathological examination were adopted to determine the drug’s no observed adverse effect level(NOAEL) and maximum tolerated dose(MTD) for mice in this condition; The remaining animals were observed for a two-week convalescence, to figure out the reversibility of drug-induced injury; simultaneously satellite groups were set up to provide the drug pharmacokinetics parameters after the first and last administration. 2. ras H2 mice and C57 mice toxicokinetics comparison test: After a single administration with 60 mg/kg F3SM(ig.), blood samples(40 μL) of ras H2 and C57 mice were collected at 5min, 15 min, 30 min, 1h, 3h, 10 h, 24 h. Concentrations of F3 SM in the blood samples were determined with LC-MS-MS. DAS 2.1.1 was used to calculate the toxicokinetic parameters. Preliminar assessment of the consistency of the drug’s metabolism between two genetically different mice was made. 3. 6-month transgenic mice carcinogenicity test: In Tg ras H2 mouse carcinogenicity studies, mice of both sexes were treated with F3 SM for 6 month. Treatment groups include mice treated with several dosages of the drug(30, 60, 120, 240 mg/kg), a blank control(no treatment), a negative(vehicle) control and a positive control was allocated to demonstrate the responsiveness of the model to a known carcinogen. 4 paralleled satellite groups(15 mice /sex/dosage) were set for toxicokinetics study. The positive control mice were administered with 3 intraperitoneal urethane injections(1000 mg/kg), mice of the rest treatment groups was given a daily oral gavage, 6 days a week for consecutive 6 months. Observations on the mice’s toxicity and death situation, particularly the occurrence of the tumor were made cautiously. Mortality, organ coefficient, biochemical and hematological indexes, and histopathological examination information were collected. The incidence of tumors in various organs of each group, and the potential carcinogenic toxicity of F3 SM were evaluated.Result: 1. Four-week repeated dose toxicity test on C57BL/6 mice: Organ coefficient statistics showed that the liver organ coefficients of the 4 F3 SM dosage group animals’ were significantly higher than that of the control group(p<0.01), and in a dose-dependent manner. 2 weeks after the withdrawal of F3 SM, each group was significantly restored, and had no significant difference when compared with the control group. After the first administration, the exposure of F3 SM in vivo was basically proportional to the dose. The residence time was not significantly prolonged; After 4 weeks of continuous administration, drug absorption was reduced in all 3 dose groups, the amount of exposure was declined, accumulation phenomenon did not appear. Blood biochemical parameters and hematological indicators showed that F3 SM primarily elevated mice’ serum ALT, ALP, Tchol, decreased Glu, TG, there was a dose-response relationship. 2 weeks after discontinuation, each indicator was back to normal or tended to recover. Pathology results showed that mice repeatedly fed(ig.) F3 SM for 4 weeks in a dosage of 360, 120, 40 mg/kg could cause significantly liver cell hypertrophy, slight liver cell hyperplasia in dosage of 360 mg/kg. Liver cells could completely recover from hypertrophy and hyperplasia after 2 weeks of withdrawal. 2. Ras H2 mice and C57 mice toxikinetics comparison test:The concentration-time curve of the genetically different two mice’ plasma F3 SM was basically the same after a single gavage of 60 mg/kg of the drug. The difference of AUC(0-t)、MRT(0-t)、t1/2、Tmax and Cmax were not statistically significant as well(p>0.05). 3. 6-month transgenic mice carcinogenicity test:At the end of the trial, there were 3 cases spontaneous splenic tumor in the control group(one of splenic hemangioma, two of splenic angiosarcoma). All the positive control animals died during the experiment, in which lung adenoma incidence was up to 90%. The survival rate, tumor type, location and incidence in the control group and the positive group were in consistence with the literature. The survival rate of the rest experiment groups were over 85%. Weight loss was basically less than 10%. Biochemical and hematological indicators suggested that F3 SM could cause liver damage and stress; organ coefficient results clearly demonstrated liver enlargement with a significant dose-response relationship. Histopathology report detailed the lesions and tumors of various organs, statistical analysis of the tumors occurrence derived that in dosage of 60, 120 mg/kg, F3 SM had led to liver adenomas in male animals. Continuous administration for 26 weeks of the 30, 60, 120 mg/kg dosage of F3 SM in ras H2 mice showed no significant accumulation, while the exposure increased after 240 mg/kg dosage administered for 13 weeks, female animals tended to have accumulated the drug of F3 SM.Conclusion: 1. Four-week repeated dose toxicity test on C57BL/6 mice: under this test condition(0, 40, 120, 360 mg/kg 4 dosages, gavage once a day, seven times a week), NOAEL was 40 mg/kg; MTD was 120 mg/kg. The main potential target organ for toxicity is the liver, the thymus, but the injury had shown some restorability. 2. ras H2 mice andC57 mice toxikinetics comparison test: Plasma concentration-time curves and pharmacokinetics parameters showed no statistically difference in C57 and ras H2 mice after a single administration of F3SM(60 mg/kg), suggesting the result of 4-week repeate dosed toxicity test on C57 mice could provide a favorable basis for the dosage design of ras H2 mice experiment. 3. 6-month transgenic mice carcinogenicity test:Survival rate, tumor type, tumor location and incidence in control and positive control group were in consistence with the literature, meaning that the transgenic mice model had been successfully established. The rest groups’ survival rate and body weight changes were within the allowable range, thus the integrity of the statistical results and good background were ensured. Biochemical and hematological indexes suggested F3 SM could lead to liver damage and animal stress and organ coefficient results clearly revealed liver enlargement, with a significant dose-response relationship. Continuous administration of 30, 60, 120 mg/kg dosage of F3 SM in ras H2 mice showed no significant accumulation, while the exposure increased after 13 weeks’ administered under 240 mg/kg dosage, and drug in female animals tended to have accumulated. Comprehensive judgment about carcinogenicity: in this experimental condition, 60, 12, 240 mg/kg dosage of F3 SM could lead to liver tumors(liver adenoma) but for male mice only, F3 SM at 30 mg/kg and 60 mg/kg dosage is not carcinogenic for female mice. |
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