Improved reliability of the rapid microtiter plate assay using recombinantenzyme in predicting CYP2D6 inhibition in human liver microsomes

A higher throughput method of screening for the inhibition of recombinant C YP2D6 using a microtiter plate (MTP) assay was evaluated using 62 new chemi cal entities and compared to data from the dextromethorphan O-demethylase a ssay in human liver microsomes (HLM). The IC50 values for the two assays cl osely matched for 53 compounds (85%), Six of the variant nine compounds had higher IC50 values with the recombinant enzyme, whereas three had lower IC 50 values with the recombinant enzyme. When the inhibition with the recombi nant enzyme was determined at various time points, the IC50 values increase d as the duration of the incubation increased for the six compounds with hi gher IC50 values in the MTP assay. The IC50 values at 10 min matched more c losely the IC50 values in HLM (95% compared with 85%). For three compounds that showed comparable IC50 values in the two assays, and the three compoun ds with lower IC50 values in the MTP assay, the IC50 values did not change over time. These results suggest that the six compounds that showed higher IC50 values in the MTP assay at 45 min are substrates for CYP2D6. Using kno wn CYP2D6 substrates, a similar phenomenon was observed, i.e., inhibition c urves shifted to higher IC50 values as incubation time increased. These res ults indicate that the higher throughput MTP assay is more comparable to HL M if the IC50 values are determined at 10 min rather than the recommended 4 5 min. Furthermore, data acquisition at multiple time points may indicate i f a compound is a potential substrate or metabolism/mechanism-based inhibit or for the enzyme.