Abstract

Metabolism-dependent inhibition (MDI) of cytochrome P450 is usually assessed in vitro by examining whether the inhibitory potency of a drug candidate increases following a 30-min incubation with human liver microsomes (HLM). To augment the IC₅₀ shift, many researchers incorporate a dilution step whereby the samples, after being pre-incubated for 30 min with a high concentration of HLM (with and without NADPH), are diluted prior to measuring P450 activity. In the present study, we show that the greater IC₅₀ shift associated with the dilution method is a consequence of data processing. With the dilution method, IC₅₀ values for direct-acting inhibitors vary with the dilution factor unless they are based on the final (post-dilution) inhibitor concentration whereas the IC₅₀ values for MDIs vary with the dilution factor unless they are based on the initial (pre-dilution) concentration. When the latter data are processed on the final inhibitor concentration, as is commonly done, the IC₅₀ values for MDI (shifted IC₅₀ values) decrease by the magnitude of the dilution factor. The lower shifted IC₅₀ values are a consequence of data processing, not enhanced P450 inactivation. In fact, for many MDIs, increasing the concentration of HLM actually leads to considerably less P450 inactivation because of inhibitor depletion and/or binding of the inhibitor to microsomes. A true increase in P450 inactivation and IC₅₀ shift can be achieved by assessing MDI by a non-dilution method and by decreasing the concentration of HLM. These results have consequences for the conduct of MDI studies and the development of cutoff criteria.