title: Improved reliability of the rapid microtiter plate assay using recombinant enzyme in predicting CYP2D6 inhibition in human liver microsomes.
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 IC50 values 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.
authors: Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
authors: L V
authors: LV
authors: Favreau
authors: J R
authors: JR
authors: Palamanda
authors: C C
authors: CC
authors: Lin
authors: A A
authors: AA
authors: Nomeir
mesh_terms: D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D011237:Predictive Value of Tests; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D013329:Structure-Activity Relationship
chemical_list: D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019389:Cytochrome P-450 CYP2D6