Automated Triplexed Hepatocyte-Based Viability and CYP1A and -3A Induction Assays

Abstract

Cytochrome P450 (CYP) enzymes are key players in drug metabolism. Therefore, it is essential to understand how these enzymes can be affected by xenobiotics with regards to induction and toxicity to avoid potential drug-drug interactions. Typically, information has been gathered by combining data from multiple experiments, which is time-consuming and labor intensive, and interassay variability may lead to misinterpretation. Monitoring CYP induction and cytotoxicity by xenobiotics using an automated, multiplexed format can decrease workload and increase data confidence. Here the authors demonstrate the ability to monitor CYP1A and CYP3A4 induction, combined with a cytotoxicity measurement, from a single microplate well using cryopreserved human hepatocytes. The assay procedure was automated in a 384-well format, including cell manipulations, compound titration and transfer, and reagent dispensing, using simple robotic instrumentation. EC(50) and E(max) values were derived for multiple known CYP1A and -3A4 inducers. Induction and toxicological responses in the triplex system were validated based on literature values from conventional single-parameter assays. Validation and pharmacology data confirm that multiplexed cell-based CYP assays can simplify workload, save time and effort, and generate biologically relevant data.

An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D

“…The CYP3A4 fold induction values were plotted against the compound concentration, and a one-half hyperbola equation, Y = E max *X/(EC 50 + X), was fit to the ascending portion of the data set (Y = fold induction, E max = maximum fold induction, X = inducer concentration, and EC 50 = half maximal induction) using SigmaPlot software. 23…”

“…Multiplexed assay systems to evaluate toxicity (i.e., ATP depletion) and metabolism (i.e., cytochrome P450 1A [CYP1A] and CYP3A4 activity) from a single well have been described previously. 23 Multiplexing can result in a reduction in experimental variability, reagent cost, and labor time. Larson et al 23 developed a multiplexed assay in 2D hepatocytes proving that the system could predict CYP induction from known CYP1A and CYP3A4 inducers.…”

“…Table 1). 23,24 Test articles were prepared at nine concentrations in a twofold dilution scheme, spanning a concentration range inclusive of the therapeutic maximum plasma concentration (C max ). Compounds were prepared as stock solutions in 100% DMSO, excluding tetracycline hydrochloride, which was prepared in sterile water due to solubility constraints.…”

“…23 Multiplexing can result in a reduction in experimental variability, reagent cost, and labor time. Larson et al 23 developed a multiplexed assay in 2D hepatocytes proving that the system could predict CYP induction from known CYP1A and CYP3A4 inducers.…”

Proluciferin Acetals as Bioluminogenic Substrates for Cytochrome P450 Activity and Probes for CYP3A Inhibition

“…The CYP3A compound 1 was also applied to hepatocytes in a three-part multiplex format that provided luminescent CYP3A, a fluorescent CYP1A, and a luminescent viability measurement from a single culture well (Larson et al, 2011).…”