An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and
Pranav Shah1, Edward Kerns1, Dac-Trung Nguyen1
1Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
Summary
A new high-throughput assay measures intrinsic clearance (CLint) for thousands of compounds using human cytochrome P450 enzymes. This automated method provides crucial metabolic stability data for advancing in silico drug modeling.
Area of Science:
- Pharmacokinetics and Drug Metabolism
- Computational Chemistry and Cheminformatics
Background:
- Advancing in silico tools requires comprehensive data on metabolic reaction rates and intrinsic clearance (CLint) for diverse compound structures.
- Major human cytochrome P450 (P450) isozymes are critical for drug metabolism, necessitating CLint data for a wide range of compounds.
Purpose of the Study:
- To develop and validate a high-throughput, automated, and robust metabolic stability assay for measuring CLint across a large set of compounds.
- To generate CLint data for individual human P450 isozymes to enable advanced computational modeling.
Main Methods:
- A substrate depletion method (in vitro half-life, t1/2) was employed to determine CLint.
- A 384-well format assay integrated a robotic system for incubation/cleanup, dual ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms for parent compound analysis, and an automated data analysis system.
- Assay validation involved testing compounds with known long, moderate, and short half-lives (t1/2) for CYP3A4.
Main Results:
- The developed assay demonstrated acceptable interday and intraday precision and accuracy (∼12%) within the observed linear range.
- CLint and t1/2 values were successfully measured for over 3000 compounds using the CYP3A4 assay.
- The assay proved robust and sensitive for high-throughput screening of metabolic stability.
Conclusions:
- The automated, high-throughput metabolic stability assay enables rapid screening of large compound libraries.
- The generated CLint data supports the advancement of in silico modeling for individual human P450 isozymes.
- This methodology significantly contributes to drug discovery and development by providing essential pharmacokinetic parameters.
