A Critical Evaluation of the Experimental Design of Studies of Mechanism Based Enzyme Inhibition, with Implications for In Vitro-In Vivo Extrapolation

Abstract

The published literature on mechanism based inhibition (MBI) of CYPs was evaluated with respect to experimental design, methodology and data analysis. Significant variation was apparent in the dilution factor, ratio of preincubation to incubation times and probe substrate concentrations used, and there were some anomalies in the estimation of associated kinetic parameters (k(inact), K(I), r). The impact of the application of inaccurate values of k(inact) and K(I) when extrapolating to the extent of inhibition in vivo is likely to be greatest for those compounds of intermediate inhibitory potency, but this also depends on the fraction of the net clearance of substrate subject to MBI and the pre-systemic and systemic exposure to the inhibitor. For potent inhibitors, the experimental procedure is unlikely to have a material influence on the maximum inhibition. Nevertheless, the bias in the values of the kinetic parameters may influence the time for recovery of enzyme activity following re-synthesis of the enzyme. Careful attention to the design of in vitro experiments to obtain accurate kinetic parameters is necessary for a reliable prediction of different aspects of the in vivo consequences of MBI. The review calls for experimental studies to quantify the impact of study design in studies of MBI, with a view to better harmonisation of protocols.

Mechanism-Based Inactivation of Human Cytochrome P450 Enzymes and the Prediction of Drug-Drug Interactions

“…In the present work, values for k deg were used that were derived from modeling of the time course of reversal of DDIs caused by induction or inactivation of P450 enzymes in human study subjects. The values obtained were substantially lower than the rat value, were within values reported in Ghanbari et al (2006), and when used generally led to over-prediction of the magnitude of DDIs (i.e., lower k deg leads to greater DDI). Actual measurements of P450 enzyme k deg values in humans in vivo are not obtainable with technology presently available, leaving this parameter as one important source of potential error in DDI predictions for inactivators.…”

“…Also, there are aspects of in vitro experimental design that can yield variability and inaccuracy. These have been discussed in a recent article by Ghanbari et al (2006). The four parameters that are most important to making accurate predictions of DDIs caused by inactivators are 1) the in vivo rate of enzyme degradation, k deg , 2) the relevant in vivo concentration of the inactivator, [I], available to the target enzyme, 3) the in vitro inactivation kinetic parameters (K I and k inact ), and 4) the fraction of the clearance of the affected drug that is mediated by the inactivated enzyme.…”

“…An explanation for the discrepancy between the value for [I] that is most useful for DDI prediction for reversible inhibitors versus irreversible inactivators is not readily apparent and merits further exploration. The experimental design used in inactivation experiments can have an impact on the measured parameters (Yang et al, 2005;Ghanbari et al, 2006). In the present studies, an approach was used wherein the inactivator was incubated with enzyme and cofactor for periods of time before dilution of the mixture into incubations in which the marker activity was measured.…”

Rofecoxib Is a Potent, Metabolism-Dependent Inhibitor of CYP1A2: Implications for in Vitro Prediction of Drug Interactions

“…In a previous study, k e estimates ranging from 0.0005 to 0.00026 min Ϫ1 yielded the most accurate in vivo predictions of CYP1A2 inhibition by the mechanism-based inhibitor zileuton . In our simulations, a CYP1A2 half-life of 38.6 h (i.e., k e ϭ 0.0003 min Ϫ1 ) was assumed, based on a previous estimate of the in vivo CYP1A2 half-life (Faber and Fuhr, 2004;Ghanbari et al, 2006). This value is similar to the in vitro half-life estimate of 36 h determined for CYP1A2 in liver slices (Renwick et al, 2000).…”

In Vitro Evaluation of Reversible and Irreversible Cytochrome P450 Inhibition: Current Status on Methodologies and their Utility for Predicting Drug–Drug Interactions

“…Ghanbari et al have shown that many published studies of TDI which followed this method were limited by insufficient enzyme dilution and over-long incubation times (36). However, with increased LC-MS/MS method sensitivity it is possible to minimise the Fig.…”