Prediction of CYP3A-Mediated Drug-Drug Interactions Using Human Hepatocytes Suspended in Human Plasma

Abstract

ABSTRACT:Cryopreserved human hepatocytes suspended in human plasma (HHSHP) represent an integrated metabolic environment for predicting drug-drug interactions (DDIs). In this study, 13 CYP3A reversible and/or time-dependent inhibitors (TDIs) were incubated with HHSHP for 20 min over a range of concentrations after which midazolam 1-hydroxylation was used to measure CYP3A activity. This single incubation time method yielded IC 50 values for the 13 inhibitors. For each CYP3A inhibitor-victim drug pair, the IC 50 value was combined with total average plasma concentration of the inhibitor in humans, fraction of the victim drug cleared by CYP3A, and intestinal availability of the victim drug to predict the ratio of plasma area under the curve of the victim drug in the presence and absence of inhibitor. Of 52 clinical DDI studies using these 13 inhibitors identified in the literature, 85% were predicted by this method within 2-fold of the observed change, and all were predicted within 3-fold. Subsequent studies to determine mechanism (reversible and time-dependent inhibitors) were performed by using a range of incubation periods and inhibitor concentrations. This system differentiated among reversible inhibitors, TDIs, and the combination of both. When the reversible and inactivation parameters were incorporated into predictive models, 65% of 52 clinical DDIs were predicted within 2-fold of the observed changes and 88% were within 3-fold. Thus, HHSHP produced accurate DDI predictions with a simple IC 50 determined at a single incubation time regardless of the inhibition mechanism; further if needed, the mechanism(s) of inhibition can be identified.

Pharmacokinetic interactions between drugs and botanical dietary supplements

“…2008;Xu et al, 2009;Chen et al, 2011;Li and Doshi, 2011). We agree with Mao et al, and others who have also suspected that the use of microsomes tends to overestimate P450 enzyme inhibition, and that incorporating cell membrane permeability and phase II enzyme transformation with intact hepatocytes will provide a more reliable prediction of natural product interactions with P450 enzymes Mao et al, 2011). It might be ideal to combine inhibition and induction studies in a single assay by determining both P450 activities and expression changes simultaneously.…”

Time-Dependent Inhibition and Estimation of CYP3A Clinical Pharmacokinetic Drug-Drug Interactions Using Plated Human Cell Systems

“…The ideal in vitro system for comprehensively evaluating drug-drug interactions should theoretically be human hepatocytes (Zhao, 2008), given the full complement of metabolic clearance mechanisms. Even though the ability to conduct in vitro DDI studies has been demonstrated in human hepatocyte suspensions (Mao et al, 2011), there are limitations with the long-term viability and enzymatic activity of the cells in suspension (Zhao, 2008). It has been demonstrated that human plated cell systems offer unique advantages over human cell suspensions for time-dependent inhibition studies (Li and Doshi, 2011).…”

“…Many investigators have evaluated the use of hepatocyte suspensions (Zhao et al, 2005(Zhao et al, , 2007McGinnity et al, 2006;Van et al, 2007;Lu et al, 2008a,b;Baer et al, 2009;Mao et al, 2011) for time-dependent inhibition studies. The use of hepatocytes for timedependent inhibition holds distinct advantages over the use of microsomes, including being more physiologically relevant and offering the ability to conduct longer incubations.…”

Critical Human Hepatocyte-Based In Vitro Assays for the Evaluation of Adverse Drug Effects

“…After cessation of administration with the TDI inhibitor, the patient would continue to have decreased drug metabolizing capacity before the inactivated enzymes are fully replaced 23,36 . While TDI is generally studied using liver microsomes or recombinant CYP 37,38 , there are substantial efforts in the evaluation of this important mechanism of drug-drug interaction in human hepatocytes 39,40 . Human hepatocytes, because of the intact plasma membrane, complete and uninterrupted drug metabolism enzymes and cofactors, represent a desirable in vitro experimental system for the evaluation of human drug properties.…”

“…Traditionally, TDI studies with hepatocytes utilize suspension cultures 40 . The use of hepatocytes in suspension culture is a common practice with cryopreserved cells as most preparations of cryopreserved hepatocytes would have compromised ability to be cultured as monolayer cultures.…”