| Autor: |
Marco, Annalise Di, Marcucci, Isabella, Verdirame, Maria, Pérez, José, Sanchez, Manuel, Peláez, Fernando, Chaudhary, Ashok, Laufer, Ralph |
| Zdroj: |
Drug Metabolism and Disposition; March 2005, Vol. 33 Issue: 3 p349-358, 10p |
| Abstrakt: |
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6{szligbeta}-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6{szligbeta}-hydroxylation of testosterone labeled with tritium in the 6{szligbeta} position. The radiolabeled 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, and sensitive to CYP3A4/5/5 inhibitors and a CYP3A4/5/5-specific inhibitory monoclonal antibody, but not to inhibitors of or antibodies against other P450 enzymes. The method was further improved by synthesis of testosterone specifically tritiated in the 6{szligbeta} position, which displayed greatly improved conversion rate with an ensuing increase in assay sensitivity. Competition experiments using tritiated and unlabeled testosterone indicated that CYP3A4/5-mediated 6{szligbeta}-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{szligbeta}-hydroxylation assay, using HPLC-tandem mass spectrometry 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{szligbeta}-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities. |
| Databáze: |
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