Abstract
A novel relay method has been developed using cryopreserved human hepatocytes to measure intrinsic clearance of low-clearance compounds. The relay method involved transferring the supernatant from hepatocyte incubations to freshly thawed hepatocytes at the end of the 4-h incubation to prolong the exposure time to active enzymes in hepatocytes. An accumulative incubation time of 20 h or longer in hepatoctyes can be achieved using the method. The relay method was validated using seven commercial drugs (diazepam, disopyramide, theophylline, timolol, tolbutamide, S-warfarin, and zolmitriptan) that were metabolized by various cytochrome P450s with low human in vivo intrinsic clearance at approximately 2 to 15 ml · min−1 · kg−1. The results showed that the relay method produced excellent predictions of human in vivo clearance. The difference between in vitro and in vivo intrinsic clearance was within 2-fold for most compounds, which is similar to the standard prediction accuracy for moderate to high clearance compounds using hepatocytes. The relay method is a straightforward, relatively low cost, and easy-to-use new tool to address the challenges of low clearance in drug discovery and development.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS:
- P450
- cytochrome P450
- DMPK
- drug metabolism and pharmacokinetics
- PK
- pharmacokinetics
- LC-MS/MS
- liquid chromatography/tandem mass spectrometry
- fu,inc
- fraction unbound under incubation condition
- MAO
- monoamine oxidase.
- Received April 24, 2012.
- Accepted May 22, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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