Transcript Regulation Of 18 ADME Genes By Prototypical Inducers In Human Hepatocytes

By T. Moeller, C. Brown, M. Muchow, and B. Yee

The importance of measuring mRNA to assess induction potential is paramount. The fundamental change from measuring enzyme activities to transcripts will have profound implications for workflow in studying drug–drug interactions (DDI) involving induction. As with previous guidance, representative genes associated with the major induction pathways may be used to measure the induction potential of test articles. CYP1A2 is the marker for aryl hydrocarbon receptor (AhR) activation, CYP2B6 for constitutive androstane receptor (CAR), and CYP3A4 for pregnane X receptor (PXR) with the potential to use CYP2C9 as a secondary marker. However, discrete enzymatic assays for individual CYP enzymes are replaced by a multiplexed assay to measure several genes from a single induction sample. This increase in data from a simplified system allows for basic testing with target markers as well as broader transcript surveys.

In this study, we used Invitrogen™ QuantiGene™ Plex Assays to simultaneously measure 18 ADME transcripts (CYP1A2, 2B6, 2C9, 2D6, 3A4, 3A5; ABCB1, C2, C3, C4; SLC01B1, 01B3, 22A1; UGT1A1, 1A4, 1A9, 2B7; SULT1E1) and two controls (GAPDH and HRPT). We tested human cryoplateable hepatocytes from 11 samples at single concentrations of omeprazole (AhR ligand), phenobarbital (CAR ligand), and rifampicin (PXR ligand) after 48-hour exposure in a 96-well format to observe individual variations in induction and suppression potentials.

The data show the power of the QuantiGene Plex Assay to generate profiles of transcript levels from a single concentration or a concentration response curve of an inducer in a multiplex format. Primary markers such as CYP1A2, CYP2B6, CYP2C9, and CYP3A4 can be used to fulfill regulatory requirements with the potential to add secondary markers such as phase II enzymes or transporters to probe gene regulation from test articles.