Metabolic assessment in liver microsomes by co-activating cytochrome P450s and UDP-glycosyltransferases.

A "dual-activity" microsomal system in which both CYPs and UGTs were active was evaluated for studies of metabolic stability and in-vitro metabolite profiling. In this "dual-activity" system, alamethicin, a pore-forming peptide, was used to activate UGTs in human liver microsomes without affecting CYP activity. Interference studies indicated that CYP cofactors had little effect on UGT surrogate activity as measured by glucuronidation of acetaminophen and trifluoperazine. Further, UGT cofactor, UDPGA (< 2 mM), did not inhibit the marker activity of five major CYPs including 1A2, 2C9, 2C19, 2D6 and 3A4, suggesting that both oxidation and glucuronidation can be co-activated in microsomes. In a comparison study, compounds with significant glucuronidation showed distinct stability profiles in the "dual-activity" system, compared to the conventional microsomal incubation in which only CYPs were active. For compounds with minor or no glucuronidation, the metabolic stability remained similar between the "dual-activity" system and the conventional microsomal incubation. The feasibility of this "dual-activity" system utilized for metabolite profiling was also investigated using tramadol as a model drug. It was found that oxidative metabolites of tramadol generated in the "dual-activity" system matched those detected in the conventional microsomal incubation. However, tramadol glucuronide was observed in the "dual-activity" system but not in the conventional micromosal incubation. Results clearly suggest that the "dual-activity" system is a valuable in vitro model for metabolism studies in drug discovery.