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Drug Metabolism and Disposition Fast Forward
First published on December 17, 2004; DOI: 10.1124/dmd.104.002873

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Annalise Di Marco
Isabella Marcucci
Maria Verdirame
Jose Perez
Manuel Sanchez
Fernando Pelaez
Ashok Chaudhary
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Received for publication November 5, 2004.
Revised December 6, 2004.
Accepted for publication December 10, 2004.

Development and validation of a high throughput radiometric CYP3A4 inhibition assay using tritiated testosterone

Annalise Di Marco 1, Isabella Marcucci 1, Maria Verdirame 1, Jose Perez 2, Manuel Sanchez 2, Fernando Pelaez 2, Ashok Chaudhary 3, Ralph Laufer 4*

1 IRBM - MRL Rome 2 MSD Espana 3 MRL 4 Istituto di Ricerche di Biologia Molecolare (IRBM)

* 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 P-450 (CYP) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6{beta}-hydroxylation assay, the new method does not require HPLC separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6{beta}-hydroxylation of testosterone labelled with tritium in the 6{beta} position. The radiolabelled product is separated from the substrate using 96-well solid phase extraction plates. Using commercially available [1,2,6,7-3H]testosterone as substrate, we demonstrated that the reaction is NADPH-dependent, sensitive to CYP3A4/5 inhibitors and a CYP3A4/5-specific inhibitory monoclonal antibody, but not to inhibitors of or antibodies against other CYP enzymes. The method was further improved by synthesis of testosterone specifically tritiated in the 6{beta} position, which displayed greatly improved conversion rate with an ensuing increase in assay sensitivity. Competition experiments using tritiated and unlabelled testosterone indicated that CYP3A4-mediated 6{beta}-hydroxylation exhibits positive cooperativity and a modest kinetic isotope effect. IC50 values for more than 40 structurally diverse chemical inhibitors were not significantly different from those determined in the testosterone 6{beta}-hydroxylation assay, using HPLC-MS/MS analysis. 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 thus represents a high throughput version of the classical testosterone 6{beta}-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities.


Key words: CYP inhibition, CYP3A, cytochrome P450, high throughput screening, inhibition, microsomes


This article has been cited by other articles:


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A. Di Marco, A. Cellucci, A. Chaudhary, M. Fonsi, and R. Laufer
High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin
Drug Metab. Dispos., October 1, 2007; 35(10): 1737 - 1743.
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A. Di Marco, I. Marcucci, A. Chaudhary, M. Taliani, and R. Laufer
DEVELOPMENT AND VALIDATION OF A HIGH-THROUGHPUT RADIOMETRIC CYP2C9 INHIBITION ASSAY USING TRITIATED DICLOFENAC
Drug Metab. Dispos., March 1, 2005; 33(3): 359 - 364.
[Abstract] [Full Text] [PDF]


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