Dynamic Modeling of Cytochrome P450 Inhibition In Vitro: Impact of Inhibitor Depletion on IC₅₀ Shift

Abstract

The impact of inhibitor depletion on the determination of shifted IC 50 (IC 50 determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC 50 -shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC 50 and even increases in IC 50 with preincubation were explained by the depletion of inhibitor during the preincubation. The IC 50 -shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC 50 -shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC 50 -shift methods, shifted IC 50 should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC 50 and shifted IC 50 curves. For compounds showing an IC 50 fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC 50 -shift data using the mechanistic model appeared to allow for reasonable estimation of K i , K I , and k inact directly from the IC 50 shift experiments.

Time-dependent enzyme inactivation: Numerical analyses of in vitro data and prediction of drug-drug interactions

“…Inactivator loss: Inactivator depletion due to metabolism can result in concave upward curvature and atypical distribution (with respect to concentration) in the PRA plots (Funaki, et al, 1991;. Inactivator depletion during the primary incubation has been observed previously (Berry, et al, 2013;Parkinson, et al, 2011). As seen in Figure 8, at lower inactivator concentrations, inactivator depletion results in recovery of activity (Figure 8B).…”

Potential for pharmacokinetic interactions between Schisandra sphenanthera and bosutinib, but not imatinib: in vitro metabolism study combined with a physiologically‐based pharmacokinetic modelling approach

“…Evaluations with a more intensive sampling time were performed for those showing significant depletion compared to control (one‐way analysis of variance with Tukey post‐hoc test, GraphPad Prism version 7.02). Potential depletion of the inhibitors was modelled by first‐order kinetics as reported previously 45 . Corrections for k i values accounting for the concentrations of inhibitor at the end 45,46 and mid‐time of incubation 46 have been proposed, the latter of which was adopted for this study.…”

Inhibition of CYP2B6 by Medicinal Plant Extracts: Implication for Use of Efavirenz and Nevirapine-Based Highly Active Anti-Retroviral Therapy (HAART) in Resource-Limited Settings

“…The difference between the calculated IC 50 values for pre-incubation with or without NADPH for each medicinal plant extract and diagnostic inhibitor was compared [56]. A ratio of IC 50 value for pre-incubation without NADPH (−IC 50 ) and pre-incubation with IC 50 with NADPH (+IC 50 ) was then calculated to establish the fold-shift (−IC 50 /+IC 50 ).…”