Abstract

A higher throughput method of screening for the inhibition of recombinant CYP2D6 using a microtiter plate (MTP) assay was evaluated using 62 new chemical entities and compared to data from the dextromethorphan O-demethylase assay in human liver microsomes (HLM). The IC₅₀ values for the two assays closely matched for 53 compounds (85%). Six of the variant nine compounds had higher IC₅₀ values with the recombinant enzyme, whereas three had lower IC₅₀ values with the recombinant enzyme. When the inhibition with the recombinant enzyme was determined at various time points, the IC₅₀ values increased as the duration of the incubation increased for the six compounds with higher IC₅₀ values in the MTP assay. The IC₅₀ values at 10 min matched more closely the IC₅₀ values in HLM (95% compared with 85%). For three compounds that showed comparable IC₅₀ values in the two assays, and the three compounds 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 known CYP2D6 substrates, a similar phenomenon was observed, i.e., inhibition curves shifted to higher IC₅₀values as incubation time increased. These results indicate that the higher throughput MTP assay is more comparable to HLM if the IC₅₀ values are determined at 10 min rather than the recommended 45 min. Furthermore, data acquisition at multiple time points may indicate if a compound is a potential substrate or metabolism/mechanism-based inhibitor for the enzyme.