Use of Immortalized Human Hepatocytes to Predict the Magnitude of Clinical Drug-Drug Interactions Caused by CYP3A4 Induction

Abstract

ABSTRACT:Cytochrome P4503A4 (CYP3A4) is the principal drug-metabolizing enzyme in human liver. Drug-drug interactions (DDIs) caused by induction of CYP3A4 can result in decreased exposure to coadministered drugs, with potential loss of efficacy. Immortalized hepatocytes (Fa2N-4 cells) have been proposed as a tool to identify CYP3A4 inducers. The purpose of the current studies was to characterize the effect of known inducers on CYP3A4 in Fa2N-4 cells, and to determine whether these in vitro data could reliably project the magnitude of DDIs caused by induction. Twenty-four compounds were chosen for these studies, based on previously published data using primary human hepatocytes. Eighteen compounds had been shown to be positive for induction, and six compounds had been shown to be negative for induction. In Cytochrome P4503A4 (CYP3A4) is the major drug-metabolizing enzyme in human liver and is responsible for the clearance of many commonly used drugs, including benzodiazepines, statins, calcium channel blockers, and HIV protease inhibitors. Certain drugs can modulate the level of CYP3A4 activity, thereby causing changes in clearance of coadministered drugs that are CYP3A4 substrates. Levels of CYP3A4 activity can be decreased by inhibition of enzyme activity, or increased by induction of new protein synthesis. Changes in CYP3A4 activity, either through inhibition or induction, can result in potentially serious drug-drug interactions (DDIs). Whereas assays for evaluating inhibition of CYP3A4 are routine and the relationship between in vitro data and in vivo effects relatively well understood (Bjornsson et al., 2003), assays for evaluating induction are less well characterized and the relationships between in vitro and in vivo data less clear.Induction of CYP3A4 is thought to occur primarily through transcriptional activation of the gene. The 5Ј-regulatory region of the CYP3A4 gene contains elements that bind various transcription factors that can up-or down-regulate transcription. One such transcription factor is the pregnane X receptor (PXR). PXR is a ligand-activated transcription factor that is activated by a variety of drugs and endogenous compounds to increase transcription of CYP3A4 as well as other drug-metabolizing enzymes and transporters (Kliewer et al., 2002). Most drugs that induce levels of CYP3A4 are thought to do so primarily via PXR activation, e.g., rifampicin, phenytoin, hyperforin, and clotrimazole (Lehmann et al., 1998). There are several in vitro tools for assessment of CYP3A4 induction. To this point, primary human hepatocyte cultures have been considered the gold standard for in vitro induction assessment (Madan et al., 2003). Primary hepatocytes are typically treated with test compound for 2 or 3 days; then, CYP3A4 levels are compared with those in vehicle-treated cells. CYP3A4 expression can be evaluated by measuring enzymatic activity with a selective probe substrate or by measuring CYP3A4 mRNA. Primary hepatocytes are considered a reliable way to assess induction; however, ability to atta...

A Comprehensive in Vitro and in Silico Analysis of Antibiotics That Activate Pregnane X Receptor and Induce CYP3A4 in Liver and Intestine

“…In our transactivation studies in DPX-2 cells, a wide enough array of antibiotic concentrations was used to permit EC 50 and E max estimates that could then be used to compute a RIS (Ripp et al, 2006). By using this strategy, we identified seven of 21 (33%) antibiotics with RIS values that would predict clinically significant enzyme induction (i.e., decrease in target drug area under the curve) using the following RIS criteria (ours): likely, if an RIS score was from 0.1 to 0.5.; possible, for an RIS from 0.05 to 0.1; and not likely for an RIS Ͻ0.05.…”

“…Relative induction scores (RIS) were computed as described by Ripp et al (2006), using prior published values for C max and fraction unbound to calculate C max unb .…”

Comparison of Immortalized Fa2N-4 Cells and Human Hepatocytes as in Vitro Models for Cytochrome P450 Induction

“…In addition, expression of PXR and AhR, which are important transcription factors involved in the regulation of drug-metabolizing enzymes and transporters, have been detected in Fa2N-4 cells (Mills et al, 2004). Ripp et al (2006) further characterized Fa2N-4 cells by evaluating the effect of selected compounds on CYP3A4 induction and comparing in vitro induction data with in vivo induction response. It was suggested that Fa2N-4 cells could be used to identify CYP3A4 inducers as well as to predict the magnitude of clinical drug-drug interactions.…”

“…For instance, CYP3A4 induction by selective CAR activators was not evaluated in Fa2N-4 cells (Ripp et al, 2006). Furthermore, the expression of major drug-metabolizing enzymes, nuclear receptors, and hepatic transporters in these cells are also not known.…”

The Time to Move Cytochrome P450 Induction into Mainstream Pharmacology Is Long Overdue

“…The fact that appropriate pharmacology terminology and concepts apply to P450 induction studies is demonstrated in a recent publication. Ripp et al (2006) related in vitro induction data (E max and EC 50 values) of 24 drugs, obtained in induction studies using Fa2N-4 cells, with their efficacious free plasma concentrations and calculated a relative induction score. This score correlated highly (r 2 values Ͼ0.92) with decreases in area under the plasma concentration versus time curve values for coadministered CYP3A4 object drugs (midazolam or ethinylestradiol) from previously published clinical drug-drug interaction studies.…”