Abstract
Metabolism-dependent inhibition (MDI) of cytochrome P450 is usually assessed in vitro by examining whether the inhibitory potency of a drug candidate increases after a 30-min incubation with human liver microsomes (HLMs). To augment the IC50 shift, many researchers incorporate a dilution step whereby the samples, after being preincubated for 30 min with a high concentration of HLMs (with and without NADPH), are diluted before measuring P450 activity. In the present study, we show that the greater IC50 shift associated with the dilution method is a consequence of data processing. With the dilution method, IC50 values for direct-acting inhibitors vary with the dilution factor unless they are based on the final (postdilution) inhibitor concentration, whereas the IC50 values for MDIs vary with the dilution factor unless they are based on the initial (predilution) concentration. When the latter data are processed on the final inhibitor concentration, as is commonly done, the IC50 values for MDI (shifted IC50 values) decrease by the magnitude of the dilution factor. The lower shifted IC50 values are a consequence of data processing, not enhanced P450 inactivation. In fact, for many MDIs, increasing the concentration of HLMs actually leads to considerably less P450 inactivation because of inhibitor depletion and/or binding of the inhibitor to microsomes. A true increase in P450 inactivation and IC50 shift can be achieved by assessing MDI by a nondilution method and by decreasing the concentration of HLMs. These results have consequences for the conduct of MDI studies and the development of cut-off criteria.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.038596.
-
ABBREVIATIONS:
- P450
- cytochrome P450
- FDA
- U.S. Food and Drug Administration
- HLMs
- human liver microsomes
- LC/MS/MS
- liquid chromatography/tandem mass spectrometry
- KI
- inhibitor concentration that causes half the maximal rate of enzyme inactivation
- kinact
- maximal rate of enzyme inactivation
- Km
- substrate concentration supporting half the maximal rate of an enzyme-catalyzed reaction
- fumic
- unbound fraction at various concentrations of HLMs
- MDI
- metabolism-dependent inhibition
- PhRMA
- Pharmaceutical Research and Manufacturers of America.
- Received February 7, 2011.
- Accepted April 27, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|