title: 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.
authors.affiliation: BioTek Instruments, Highland Park, Winooski, VT 05404, USA. larsonb@biotek.com
authors.forename: Brad
authors.initials: B
authors.lastname: Larson
authors.forename: Timothy
authors.initials: T
authors.lastname: Moeller
authors.forename: Peter
authors.initials: P
authors.lastname: Banks
authors.forename: James J
authors.initials: JJ
authors.lastname: Cali
mesh_terms: D057205:Automation, Laboratory; D001688:Biological Products / Q000032:analysis* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015925:Cryopreservation; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004347:Drug Interactions; D004790:Enzyme Induction; D017930:Genes, Reporter; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000032:analysis; D015203:Reproducibility of Results
chemical_list: D001688:Biological Products; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A