Application of a Rat Liver Drug Bioactivation Transcriptional Response Assay Early in Drug Development that informs Chemically Reactive Metabolite formation

Transcriptional responses in rat liver following treatment with compounds with a range of in vitro covalent binding measurements Rats received repeted daily dosing of pharmaceuticals at doses and durations indicated. Approximately 24hrs after dosing, animals were sacrificed, livers removed, a section (~50-100mg) frozen on dry ice and stored at ~-80C until total RNA was isolated. Frozen liver was pulverized in Trizol reagent, extracted with Chloroform, and further purified using the RNEasy column RNA isolation procedure (Qiagen, Chatsworth, CA). 50 ng of each purified RNA sample was amplified and labeled using a custom automated version of the NuGEN Ovation WB protocol. Hybridization to custom rat Affymetrix arrays (containing 43,686 probesets, GEO platform GPL28473), labeling and scanning were completed following the manufacturer's recommendations and profiles were normalized using robust multi-array average followed by ratio’ing to corresponding vehicle control samples within each study.