The Metabolism Study Of E2 And G2,Novel Camptothecin Analogues Conjugated With Bile Acid,in Rat Liver Microsomes

Camptothecin?CPT?is a highly effective anti-tumour alkaloid isolated by Wall and Wani in 1966 from the extracts of camptotheca acuminate,a tree native from China.However,the instability of the chemical structural,poor water solubility,low plasma protein binding rate,poor and variable bioavailability result in the unpredictable toxicity in vivo.E2 and G2 were synthesized through introduction of a bile acid group at the 20-position of CPT in our fomer study.It has been investigated that they showed fantastic anti-tumor activity both in vitro and in vivo,with lower toxicity compared to CPT.And they have distinct targeting ability to the hepatocyte.Objective:The aim of this study was to investigate the biology stability and the metabolism characteristic of E2 and G2 through some in vitro models,including rat plasma,simulated gastric fluid,simulated intestinal fluid and rat liver microsomes.Methods:Firstly,optimized HPLC methods were established for the quantitation of E2,G2 and CPT,with great precision,specificity and repeatability.Then the biology stabilities of them were inveatigated in rat plasma,simulated gastric fluid and simulated intestinal fluid.And the metabolism study of E2 and G2 in rat liver microsomes was followed,including metabolic stability,enzyme kinetics study,CYP450 isoforms identification and metabolites proposing.Results:The results showed that:?1?the biology stability study demonstrated the camptothecin derivatives E2 and G2 coupling with cholic acid had an obviously improved stability than camptothecin in rat plasma,simulated gastrol fluid and simulated intestinal fluid,and the stability of the lactone ring was also significantly improved.?2?The results of microsomal incubations indicated that the phase?metabolism with NADPH would be the primary metabolism pathway of E2 and G2,and the depletion of E2 showed a higher conversion rate but a slightly lower conversion percent compared to G2.?3?Chmical inhibiting study showed that CYP2D6,CYP1A2 and CYP2B6 isoforms may be involved in E2phase?metabolism,while CYP1A2,CYP3A4,CYP2C8 and CYP2B6isoforms were probably involved in G2 phase?metabolism.And the enzyme kinetics study revealed that E2 and G2 had similar V_maxax value,but the K_m-value of E2 was about three times higher than G2,the CL_intnt value of G2 was about three times higher than E2,indicating that G2 may possess a greater affinity with CYPs compared to E2,and E2 has a great metabolism stability.?4?10 metabolites of E2 were detected,and 14metabolites of G2 were detected.E2 and G2 have extremely similar metabolism pathways.Hydroxylations and hydrolysis are their main phase?transformation pathways,and the phase?metabolites the metabolites can be further metabolized through glucuronidation.But the difference in metabolism stability may result in some discrepancy in metabolites detected.Conclusion:In summary,E2 and G2 have greater biology stability compared to CPT,and would keep the E-lactone form under the physiological environment.Although with different coupling way?the ester bond and amide bond?during CPT and bile acid,E2 and G2 have similar metabolism routes and phase??hydroxylations and hydrolysis?are their major metabolism pathways,the metabolites can be further metabolized through glucuronidation.But some difference also exists in their metabolism stability and the responsible enzymes,which might be related to the diversity of the hydrolysis ability of the ester bond and amino bond in some extent.And their comparatively low intrinsic clearance indicates that they may preferably achieve liver targeting with the parent chemical.And E2 with more stable metabolism characristics gets more research value.