In vitro metabolism of 20(S)-protopanaxadiol and its interaction with cytochrome P450 3A4

20(S)-protopanaxadiol (aPPD) is a ginseng sapogenin and is claimed to be a promising anti-cancer drug candidate. Although bacterial biotransformation of ginsenosides in the gut has been thoroughly studied, few have reported on the metabolism of aPPD. As an orally taken xenobiotic, aPPD must first be absorbed and metabolized in the intestine before it reaches the liver for further metabolism. This thesis compares the metabolite profile and enzyme kinetic profile of aPPD in the human intestinal microsomes (HIM) and liver microsomes (HLM), respectively, and examines the interaction between aPPD and cytochrome P450 (CYP), an essential Phase I xenobiotic metabolizing enzyme. aPPD was incubated with HIM or HLM and NADPH regenerating system. Although we did not perform any detailed NMR structural analysis, three major mono-hydroxylated metabolites and five di-hydroxylated metabolites were identified in HIM and HLM using liquid chromatography mass spectrometry in positive ionization mode. A multiple reaction monitoring method, with m/z 477>459 and 459>441 transitions for mono-hydroxylated metabolites M1-M3, m/z 493>457 and 493>475 transitions for di-hydroxylated metabolites M4-M8, and m/z 443>425 and 425>123 transitions for aPPD, was established for subsequent kinetic study and reaction phenotyping study. aPPD metabolites formation in HIM have a much lower Km value than in HLM. Reaction phenotyping was performed with a panel of specific CYP chemical inhibitors and human recombinant CYP enzymes. CYP3A inhibitors, ketoconazole and troleandomycin, inhibits aPPD monohydroxyl metabolite formation in both HIM and HLM in a concentration dependent manner. Among the human recombinant CYP enzymes assayed, CYP3A4 showed the highest activity in aPPD monohydroxyl metabolite formation followed by CYP3A5. In summary, this study suggests that CYP3A isoforms are the predominant enzymes responsible for aPPD mono-hydroxylation in HLM and HIM.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat