Unit

UNIT 9.18 A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS

  1. Xiaofeng Li,
  2. Tom Suhar,
  3. Lateca Glass,
  4. Ganesh Rajaraman

Published Online: 3 MAR 2014

DOI: 10.1002/0471141755.ph0918s64

Current Protocols in Pharmacology

Current Protocols in Pharmacology

How to Cite

Li, X., Suhar, T., Glass, L. and Rajaraman, G. 2014. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS. Current Protocols in Pharmacology. 9:9.18:9.18.1–9.18.10.

Author Information

  1. AbbVie, Drug Metabolism, North Chicago, Illinois

Publication History

  1. Published Online: 3 MAR 2014

Abstract

Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro–in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Curr. Protoc. Pharmacol. 64:9.18.1-9.18.10. © 2014 by John Wiley & Sons, Inc.

Keywords:

  • enzyme reaction phenotyping;
  • recombinant human CYP;
  • cytochrome P450