RT Journal Article
SR Electronic
T1 DEVELOPMENT AND VALIDATION OF A HIGH-THROUGHPUT RADIOMETRIC CYP3A4/5 INHIBITION ASSAY USING TRITIATED TESTOSTERONE
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 349
OP 358
DO 10.1124/dmd.104.002873
VO 33
IS 3
A1 Annalise Di Marco
A1 Isabella Marcucci
A1 Maria Verdirame
A1 José Pérez
A1 Manuel Sanchez
A1 Fernando Peláez
A1 Ashok Chaudhary
A1 Ralph Laufer
YR 2005
UL http://dmd.aspetjournals.org/content/33/3/349.abstract
AB 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 6β-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 6β-hydroxylation of testosterone labeled with tritium in the 6β 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 6β 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 6β-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 6β-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 6β-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities. The American Society for Pharmacology and Experimental Therapeutics