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Drug Metabolism and Disposition Fast Forward
First published on June 28, 2007; DOI: 10.1124/dmd.107.016345

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Received for publication April 23, 2007.
Revised June 25, 2007.
Accepted for publication June 26, 2007.

High throughput radiometric CYP2C19 inhibition assay using tritiated (S)-mephenytoin

Annalise Di Marco 1, Antonella Cellucci 1, Ashok Chaudhary 2, Massimiliano Fonsi 1, Ralph Laufer 1*

1 IRBM - Merck Research Laboratories Rome 2 Merck Research Laboratories West Point PA

* Address correspondence to: E-mail: ralph_laufer{at}merck.com

Abstract

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4'-hydroxylation of (S)-mephenytoin labeled with tritium in the 4' position. Since this reaction is subject to an NIH shift, tritium was also introduced into the 3'- and 5'-positions of the tracer in order to enhance formation of tritiated water product. Tritiated water was separated from the substrate using 96-well solid phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC50 values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4'-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in 96-well format, and can be automated. This assay represents a non-HPLC, high throughput version of the classical (S)-mephenytoin 4'-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.


Key words: CYP inhibition, CYP2C, cytochrome P450, enzyme kinetics, human CYP enzymes, liver microsomes, microsomes


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[Abstract] [Full Text] [PDF]


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