Validation of a rapid microtiter plate assay to conduct cytochrome P450 2D6 enzyme inhibition studies
Section snippets
High-throughput approaches for drug discovery in drug metabolism
Breakthroughs in combinatorial chemistry are generating a vast number of NCEs. Robotics and common end-point assays (e.g. receptor displacement assays) now make it possible to screen hundreds of thousands of compounds relatively quickly for efficacy in in vitro pharmacology models, but there is an urgent need to accelerate drug discovery studies related to drug metabolism by using innovative approaches and/or new technologies. So far, some high- (or `higher') throughput approaches in drug
Conventional approach using HL microsomes and HPLC analysis
Fig. 1 illustrates the procedure that was used for the measurement of HL microsomal CYP2D6 activity. The analysis was performed by HPLC as previously described[11](see Fig. 1 for details).
When the fluorescent marker substrate dextromethorphan is incubated at low micromolar concentrations with HL microsomes and NADPH, CYP2D6 almost exclusively O-demethylates dextromethorphan to its fluorescent metabolite, dextrorphan[11]. Because dextromethorphan and dextrorphan are both fluorescent at the same
Potential of higher-throughput screening to predict CYP2D6 inhibition
Initially, we determined the IC50 for quinidine, a prototype CYP2D6 inhibitor, and compared it to literature values. The IC50 for quinidine in the microtiter plate assay was 9.5 nM, which is consistent with that reported earlier in the same assay[10](8.9 nM) and in agreement with the reported Ki (27 nM) in HL microsomes for CYP2D6 (Ref. [12]).
Application of the rapid microtiter plate assay for estimating CYP2D6 IC50 values showed that, for 53 of the 62 NCEs evaluated, supersomal IC50 values agreed
Deviation of supersomal IC50 values from HL microsomal IC50 values
For nine of the 62 NCEs, supersomal and HL microsomal IC50 values differed by more than fivefold (Table 2). Compared to HL microsomal IC50s, supersomal IC50 values were lower for three NCEs and higher for six other NCEs (Table 2). These deviations resulted in supersomal IC50 curves that either underestimated (Fig. 2d) or overestimated (Fig. 2e) HL microsomal IC50.
Impact of the higher-throughput approach
The utility of the microtiter plate assay for conducting CYP2D6 enzyme inhibition studies using Supersomes is proving to be invaluable in discovery programs that require early assessment of the potential of NCEs to inhibit CYP2D6. The data presented in this review are from such a discovery program. The finding that six of the 62 NCEs (9.7%) are more potent inhibitors of HL microsomal CYP2D6 than of supersomal CYP2D6 is not of major concern (false positives) because these NCEs would be
Conclusions
The results of the present study demonstrate that Supersomes could be used for conducting higher-throughput CYP2D6 inhibition studies. However, human liver microsomes should subsequently be used to confirm the supersomal results on the few selected NCEs that are likely to be recommended for further development.
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