Validation of a rapid microtiter plate assay to conduct cytochrome P450 2D6 enzyme inhibition studies

doi:10.1016/S1359-6446(98)01248-3

Drug Discovery Today

Volume 3, Issue 10, 1 October 1998, Pages 466-470

https://doi.org/10.1016/S1359-6446(98)01248-3 Get rights and content

Abstract

Cytochrome P450 (CYP) inhibition studies are often performed on new chemical entities during the drug discovery stage. However, advances in combinatorial chemistry mean that an unprecedented number of compounds now require such evaluation. Recently, a microtiter plate assay that utilizes individually expressed CYP enzymes (Supersomes™) has been developed to facilitate this process. The authors discuss some of their experiences with this technology and its potential impact for higher throughput CYP2D6 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 K_i (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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Validation of a rapid microtiter plate assay to conduct cytochrome P450 2D6 enzyme inhibition studies

Abstract

Section snippets

High-throughput approaches for drug discovery in drug metabolism

Conventional approach using HL microsomes and HPLC analysis

Potential of higher-throughput screening to predict CYP2D6 inhibition

Deviation of supersomal IC50 values from HL microsomal IC50 values

Impact of the higher-throughput approach

Conclusions

Drug Discovery Today

Chem.-Biol. Interact.

Anal. Biochem.

Anal. Biochem.

J. Pharmacokinet. Biopharm.

Toxicol. Pathol.

Pharmacol. Rev.

Deviation of supersomal IC₅₀ values from HL microsomal IC₅₀ values