title: Validation of a semi-automated human hepatocyte assay for the determination and prediction of intrinsic clearance in discovery.
abstract: An automated high throughput human hepatocyte assay has been established with a 96-well format using a Tecan Genesistrade mark Workstation. Validation of this assay was performed with nine commercially available compounds and an additional 10 Pfizer compounds with varying hepatic extraction ratios (E(H)) ranging from 0.02 to approximately 1. The incubation conditions in the automated assay are readily and precisely controlled and cell viability of over 80% was achieved in the automated assay further confirming its utility for absorption, distribution, metabolism, and excretion (toxicity) (ADME (T)) screening. The results of the nine commercial compounds correlate with both manually executed (R(2)=0.97) and literature reported experimental results (R(2)=0.93). Overall, measured E(H)s were within two-fold of the literature values for approximately 90% of the 19 compounds tested. Additionally, good inter- and intra-day reproducibility was observed for all the 19 compounds. In conclusion, an automated and robust assay suitable for simultaneously testing up to 48 compounds with multiple time points has been validated. Throughput of 192 compounds per run can be achieved using 384-well plates to meet increasing needs in drug discovery. Currently, this automated assay is used to support early discovery profiling towards lead optimization of various discovery targets/programs.
authors.affiliation: Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Reddy
authors.forename: Tycho
authors.initials: T
authors.lastname: Heimbach
authors.forename: Sascha
authors.initials: S
authors.lastname: Freiwald
authors.forename: Danielle
authors.initials: D
authors.lastname: Smith
authors.forename: Roger
authors.initials: R
authors.lastname: Winters
authors.forename: Steven
authors.initials: S
authors.lastname: Michael
authors.forename: Narayanan
authors.initials: N
authors.lastname: Surendran
authors.forename: Hongliang
authors.initials: H
authors.lastname: Cai
mesh_terms: D002470:Cell Survival; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D015507:Drugs, Investigational / Q000493:pharmacokinetics*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008658:Inactivation, Metabolic; D008657:Metabolic Clearance Rate; D012015:Reference Standards
chemical_list: D015507:Drugs, Investigational