Study On In Vitro Transmembrane Transport Of 10-methoxycamptothecin And 10-hydroxycamptothechin

Camptothecin(CPT)is a naturally occurring alkaloid first isolated from the native Chinese tree Camptotheca acuminate Dence.(Nyssaceae)in 1960s.CPT has been paid considerable attention because of its significant anti-tumor activity.Both 10-methoxycamptothecin and 10-hydroxycamptothecin are natural bioactive derivatives of camptothecin and have been confirmed to possess high anti-cancer properties.HCPT was found to be one of the major metabolites of MCPT metabolites in rat pasma after intravenous administration of MCPTIn the present thesis,the uptake and transport of MCPT and HCPT in Caco-2 cells were studied.A sensitive and reliable RP-HPLC method with fluorescence detection was developed and validated for analysis of MCPT and HCPT in HBSS.CPT was used as the internal standard.It was demonstrated that the endogenous substances did not interfere with the determination of the analytes.The retention times were 8.87 min,5.60 min and 7.98 min for MCPT,HCPT and CPT,respectively.The calibration curve was linear(R2>0.99)over MCPT and HCPT concentrations ranging from 50 to 160 nM.The intra-day precision was 13.14%or less,and the accuracy was between 95.97%and 103.50%.The recoveries)were between 89.96%and 97.85%.This assay offers advantages of expediency,recovery of analytes,and suitability for the analysis of MCPT and HCPT in HBSS.This method was also successfully applied in the intestinal absorption kinetics studies of MCPT and HCPT.The Caco-2 cell model was used to study the transport kinetics of MCPT and HCPT.The effects of time,concentration,temperature and pH on the transport of MCPT were investigated.The results showed that both MCPT and HCPT in Caco-2 cell uptake were time-dependent and concentration-dependent.There is nearly no transport of both MCPT and HCPT at 4?.The neutral condition was good for the uptake of MCPT and HCPT at pH 7.4.Overall,the current study suggests that concentration,temprature and pH are capable of mediating the absorption/transportion of both MCPT and HCPT.Our studies also find that MCPT could metabolize to HCPT in the Caco-2 cell model,which proved that the metabolic enzymes in the Caco-2 cells were similar with that in vivo.