Simple, direct, and informative method for the assessment of CYP2C19 enzyme inactivation kinetics

Many clinically relevant drug interactions involving cytochrome P450 inhibition are mediated by mechanism-based inactivation (MBI). Time-dependent inhibition is one of the major features distinguishing between reversible inhibition and MBI. It thus provides a useful screening approach for early drug interaction risk assessment. Accordingly, we developed an easy and informative fluorometric method for the assessment of CYP2C19 enzyme inactivation kinetics. Dibenzylfluorescein (DBF) is widely used as a profluorescent probe substrate for P450 activity and inhibition assays, but its use has been considered to be limited to traditional endpoint assays. We monitored CYP2C19-catalyzed metabolism of DBF using synthesized fluorescein benzyl ester and fluorescein benzyl ether along with commercially available fluorescein as intermediate standards. Furthermore, we demonstrated the use of DBF in a kinetic assay as a progress curve analysis for straightforward determination of whether a compound is a time-dependent inactivator of CYP2C19. The recombinant human CYP2C19 inactivation kinetics of isoniazid, ticlopidine, and tranylcypromine were evaluated, and their key kinetic parameters were measured from the same experiment. The known mechanism-based inactivators, isoniazid and ticlopidine, exhibited clear time-dependent inactivation with K(I) and k(inact) values of 250.5 ± 34 μM and 0.137 ± 0.006 min(-1) and 1.96 ± 0.5 μM and 0.135 ± 0.009 min(-1), respectively. Tranylcypromine did not display any time-dependent inhibition, which is consistent with its reported mechanism of competitive inhibition. In summary, DBF is suitable for use in the progress curve analysis approach and can be used as an initial screen to identify compounds that require more detailed investigations in drug interaction optimization.