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

F.      Ghanbari; K.      Rowland-Yeo; J.   C.   Bloomer; S.   E.   Clarke; M.   S.   Lennard; G.   T.   Tucker; A.      Rostami-Hodjegan

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 (kinact, K I, r). The impact of the application of inaccurate values of kinact and KI 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.

Keywords: Mechanism-based inhibition, CYP inhibition, drug-drug interactions, suicide inhibition, predicting metabolic drug, interactions, enzyme kinetics

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