Pharmacokinetic Evaluation Of Immunotherapeutic Drugs

Part Ⅰ Studies on the pharmacokinetic of ipilimumabObjective:This study aims to investigate the pharmacokinetic of ipilimumab in C57BL/6 mice,CTLA4 humanized mice,and tumor-bearing CTLA4 humanized mice.Methods:The mice were intravenously(i.v.)or intraperitoneally(i.p.)administrated with Ipilimumab.The concentration of Ipilimumab in the mouse plasma was determined by ABC-enzyme-linked immunosorbent assay(BA-ELISA).The pharmacokinetic parameters were analyzed using Phoenix WinNolin software.Results:When the C57BL/6 mice were i.p.injected with ipilimumab at a dose of 0.3,1.0,10.0 mg/kg,the Cmax and AUC0-last were increased along with increasing dosage.When the mice were i.p.injected with Ipilimumab at 10.0 mg/kg,the AUC0-last was significantly higher in the tumor-bearing CTLA4 humanized mice(12925.7 h*μg/mL)compared with the wild type C57BL/6J mice(9385.7 h*μg/mL)and CTLA4 humanized mice(7691.6 h*μg/mL).The t1/2 in C57BL/6J mice(175.5 h)was significantly longer than those in the CTLA4 humanized mice(26.7 h)and the tumor-bearing CTLA4 humanized mice(27.0 h).Furthermore,the Cmax was significantly higher in the tumor-bearing humanized mice(95.8 μg/mL)compared with the wild-type C57BL/6J mice(31.6 ug/mL)when 3.0 mg/kg Ipilimumab was adminstrated.In addition,ti/2 in the wild-type C57BL/6J mice(462.3 h)was significantly longer than that in the CTLA4 humanized mice(26.8 h).The clearance rate in the CTLA4 humanized mice(0.98 mL/h/kg)was significantly higher than that in the wild-type C57BL/6J mice(0.48 mL/h/kg).These results indicate that the specific binding of ipilimumab to CTLA4 may affect the clearance of ipilimumab in vivo.Conclusions:The pharmacokinetic of Ipilimumab are different in C57BL/6 mice,CTLA4 humanized mice,and tumor-bearing CTLA4 humanized mice.The mechanisms related to targeted drug-mediated drug disposal(TMDD)are still under investigation.Part Ⅱ The anti-tumor activities and pharmacokinetics of miR-34a mutantsObjective:miR-34a has potent anti-tumor activity,but it is terminated in Phase I clinic trial due to severe immune-related adverse reactions.Thus,this study aims to explore the anti-tumor activities of modified miR-34a,a well as the pharmacokinetics of miR-34a.Methods:The 3’-terminal sequence of miR-34a was single-point mutated.Then the anti-tumor activities of miR-34a mutants were tested by using CCK-8 assay and xenograft experiments.The target genes of miR-34a mutant were identified by using transcriptome sequencing method and dual-luciferase reporter assay.In addition,we established a qPCR method for quantitative determination of miR-34a and applied it to pharmacokinetic study of miR-34a in vivo.Results:MRX21,with a G-to-A mutant at 8 locus of the 3’-terminal of miR-34a,showed the most potent anti-tumor activity in cancer cells.The IC50 values of MRX21 were 1.727±1.10 nM,13.53±1.07 nM,and 10.66±1.08 nM,in HCT116,HCT-8,and SGC-7901 cells respectively.The results of nude mice xenograft experiments showed that MRX21 had a significant inhibitory effect on tumor growth.MRX21 down-regulated 59 genes and up-regulated 99 genes in HCT116 cells.Nine down-regulated genes including AURKA,AURKB,CDK1,CKS2,PDGFA,PLK1,JAK2,KIF11 and PTCH1 were also verified by qPCR assays.The pharmacokinetic study of miR-34a showed that the AUC0-last was 68132.61 h*ng/ml,and the MRT0-last was 4.67 h in C57BL/6J mice.Conclusion:MRX21 has potent in vitro and in vivo anti-tumor activity.MRX21 exerts its anti-tumor effect by regulating various oncogenes.