Assessment of drug–drug interactions caused by metabolism-dependent cytochrome P450 inhibition

This study was designed to develop methods for detecting metabolism-dependent reversible, quasi-irreversible, and irreversible cytochrome P450 (CYP) inhibition using pooled human liver microsomes and a liquid chromatography/tandem mass spectrometry (LC–MS/MS) system. Metabolism-dependent inhibition (MDI) was identified based on IC50 shifts after pre-incubation of the tested compounds with NADPH. To distinguish reversible MDI from mechanism-based inhibition (MBI), R-fluoxetine and ticlopidine were used as positive inhibitors for reversible MDI and MBI of CYP2C19, respectively. R-fluoxetine and ticlopidine inhibited CYP2C19 activity, as determined using S-mephenytoin as a substrate, and caused 8.7- and 2.3-fold IC50 shifts, respectively, after pre-incubation. Inhibition of CYP2C19 by R-fluoxetine, but not ticlopidine, was markedly reversed by ultracentrifugation, and two or three ultracentrifugations were not more effective than one, indicating that ultracentrifugation only once may be sufficient to reverse the reversible MDI. To distinguish between quasi-irreversible and irreversible inhibition, diltiazem and mifepristone were used as quasi-irreversible and irreversible inhibitors of CYP3A4, respectively, and CYP3A4 activity was measured using midazolam and testosterone as substrates. After pre-incubation, CYP3A4 IC50 shifts caused by diltiazem and mifepristone were greater than 2.5- and 3.7-fold, respectively. Incubation with 2 mM potassium ferricyanide for 10 min reversed the MDI of CYP3A4 by diltiazem, but not mifepristone. Increases in potassium ferricyanide concentration and incubation time reduced the recovery of CYP3A4 activity. The established methods were confirmed using three CYP3A4 inhibitors including diltiazem, mifepristone and amiodarone (a reversible metabolism-dependent inhibitor). We consider these methods to be useful tools for discriminating between reversible MDI and MBI.

► We developed two-step protocol for distinguishing among three types of MDI of CYP.
► The MDI was identified based on the IC50 shifts after pre-incubation with NADPH.
► The ultracentrifugation method discriminates between reversible MDI and MBI.
► The CYP oxidation method distinguishes quasi-irreversible MDI from irreversible MDI.
► Our results provide useful tools for evaluating the mechanism of MDI of CYP.