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

Abstract

ABSTRACT:The ability to use vitro inactivation kinetic parameters in scaling to in vivo drug-drug interactions (DDIs) for mechanism-based inactivators of human cytochrome P450 (P450) enzymes was examined using eight human P450-selective marker activities in pooled human liver microsomes. These data were combined with other parameters (systemic C max , estimated hepatic inlet C max , fraction unbound, in vivo P450 enzyme degradation rate constants estimated from clinical pharmacokinetic data, and fraction of the affected drug cleared by the inhibited enzyme) to predict increases in exposure to drugs, and the predictions were compared with in vivo DDIs gathered from clinical studies reported in the scientific literature. In general, the use of unbound systemic C max as the inactivator concentration in vivo yielded the most accurate predictions of DDI with a mean -fold error of 1.64. Abbreviated in vitro approaches to identifying mechanism-based inactivators were developed. Testing potential inactivators at a single concentration (IC 25 ) in a 30-min preincubation with human liver microsomes in the absence and presence of NADPH followed by assessment of P450 marker activities readily identified those compounds known to be mechanism-based inactivators and represents an approach that can be used with greater throughput. Measurement of decreases in IC 50 occurring with a 30-min preincubation with liver microsomes and NADPH was also useful in identifying mechanismbased inactivators, and the IC 50 measured after such a preincubation was highly correlated with the k inact /K I ratio measured after a full characterization of inactivation. Overall, these findings support the conclusion that P450 in vitro inactivation data are valuable in predicting clinical DDIs that can occur via this mechanism.The prediction of drug-drug interactions (DDIs) using in vitro enzyme kinetic data has been an area of increasing advances and sophistication. This has proven to be a valuable endeavor because DDIs remain an important issue in clinical practice and the discovery and development of new drugs. The earlier that the potential for DDIs can be identified in new compounds being studied as potential drugs, the greater the likelihood that this deleterious property can be removed through improved design of the molecule. Also, for those compounds already undergoing clinical trials, in vitro DDI data can be leveraged in the design of adequate and appropriate clinical DDI studies. With our increased understanding of drug-metabolizing enzymes and their roles in the metabolism of specific drugs, a mechanistic approach to assessing DDIs can be taken. The results of clinical DDI studies with one drug can be extrapolated to other drugs that are cleared by the same enzyme.The alteration of drug-metabolizing enzyme activities can occur by three main mechanisms: reversible inhibition, mechanism-based inactivation, and induction. Confidence in quantitatively extrapolating in vitro results to in vivo varies with these mechanisms. For reversible inhibitio...

Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

“…The values for [I] in vivo were derived from references [17,31] . A k deg(CYP3A) of 0.000321 min -1 was adopted, in accordance with Obach et al [27] . The values of f m(CYP3A) and f m(CYP2C8) were arbitrarily set to be 0.1-1 to predict the DDI risk for all possible coadministered drugs [17] .…”

“…Single point inactivation experiments were used as previously reported to determine NADPH-dependent and preincubationdependent inhibition by erlotinib [27] . Briefly, erlotinib was incubated with pooled HLMs (1 mg/mL) in the absence and presence of the NADPH-generating system for 30 min at 37 °C.…”

“…Equation (10) was used to predict the interaction potential by reversible inhibition of CYP2C8. All equations were adapted as reported [27,29] .…”

Time‐dependent inhibition (TDI) of CYP3A4 and CYP2C9 by noscapine potentially explains clinical noscapine–warfarin interaction

“…Single point inactivation experiments were used to determine NADPH-dependent and preincubation-dependent inhibition of noscapine, as previously reported [13].In brief, pooled HLMs (1 mg ml -1 ) were incubated with noscapine in the absence and presence of NADPH-generating system for 30 min at 37°C. For CYP3A4 and CYP2C9, the concentration of noscapine utilized was 10-fold that which gave 25% inhibition under reversible inhibition situations.…”

In vitro drug–drug interactions of budesonide: inhibition and induction of transporters and cytochrome P450 enzymes

“…This approach allowed the calculation of an IC 50 shift, which is used to determine whether or not a compound is a timedependent inhibitor (Berry & Zhao, 2008;Fowler & Zhang 2008;Obach et al, 2007;Perloff et al, 2009). The CYP reaction was performed using an incubation volume of 200 mL.…”