Metabolic assessment in alamethicin-activated liver microsomes: Co-activating CYPs and UGTs

Abstract

The methods and materials described in this chapter are for a medium throughput screening assay for the study of parallel CYP- and UGT-mediated metabolic pathways in microsomes. Alamethicin, a pore-forming peptide, was used to activate UGTs in human liver microsomes. An alamethicin-microsomal activated system in which both CYPs and UGTs were active can be used for studies of metabolic stability and in vitro metabolite profiling. For compounds with minor or no glucuronidation, the metabolic stability remained similar between the co-activating CYPs and UGTs microsomal system and the conventional CYPs microsomal incubation procedure. However, for compounds in which glucuronidation is possible, the microsomal stability of the co-activating CYPs and UGTs microsomal system and the conventional CYPs microsomal incubation procedure are completely different. Literature validation studies addressing if the presence of CYP and UGT in microsomes induce experimental artifacts were summarized and indicated no major issues. Results clearly suggest that the co-activating CYPs and UGTs microsomal system using alamethicin is a valuable in vitro model in drug discovery for the study of parallel CYP- and UGT-mediated metabolic pathways. © 2014 Springer Science+Business Media New York.