Title:A High Throughput, 384-Well, Semi-Automated, Hepatocyte Intrinsic Clearance Assay for Screening New Molecular Entities in Drug Discovery
VOLUME: 18 ISSUE: 5
Author(s):Lance Heinle, Vincent Peterkin, Sonia M. de Morais, Gary J. Jenkins and Ilaria Badagnani
Affiliation:Department of Drug Metabolism, AbbVie Inc., North Chicago, IL 60064, USA.
Keywords:384-well, clearance, fraction unbound, hepatocyte stability, high throughput, metabolism.
Abstract:A high throughput, semi-automated clearance screening assay in hepatocytes was developed
allowing a scientist to generate data for 96 compounds in one week. The 384-well format assay utilizes
a Thermo Multidrop Combi and an optimized LC-MS/MS method. The previously reported LCMS/
MS method reduced the analytical run time by 3-fold, down to 1.2 min injection-to-injection. The
Multidrop was able to deliver hepatocytes to 384-well plates with minimal viability loss. Comparison
of results from the new 384-well and historical 24-well assays yielded a correlation of 0.95. In
addition, results obtained for 25 marketed drugs with various metabolism pathways had a correlation
of 0.75 when compared with literature values. Precision was maintained in the new format as 8
compounds tested in ≥39 independent experiments had coefficients of variation ≤21%. The ability to predict in vivo
clearances using the new stability assay format was also investigated using 22 marketed drugs and 26 AbbVie
compounds. Correction of intrinsic clearance values with binding to hepatocytes (in vitro data) and plasma (in vivo data)
resulted in a higher in vitro to in vivo correlation when comparing 22 marketed compounds in human (0.80 vs 0.35) and
26 AbbVie Discovery compounds in rat (0.56 vs 0.17), demonstrating the importance of correcting for binding in
clearance studies. This newly developed high throughput, semi-automated clearance assay allows for rapid screening of
Discovery compounds to enable Structure Activity Relationship (SAR) analysis based on high quality hepatocyte stability
data in sufficient quantity and quality to drive the next round of compound synthesis
