Received for publication March 17, 2008.
Revised July 23, 2008.
Accepted for publication July 23, 2008.

Differential Inhibition of P450s 3A4 and 3A5 By the Newly Synthesized Coumarin Derivaties, 7-Coumarin Propargyl Ether and 7-(4-Trifluoromethyl)coumarin Propargyl Ether

Abstract

The abilities of 7-coumarin propargyl ether (CPE) and 7-(4-trifluoromethyl)coumarin propargyl ether (TFCPE) to act as mechanism-based inactivators of P450 3A4 and 3A5 in the reconstituted system have been investigated using 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and testosterone as probes. CPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner characteristic of a mechanism-based inactivator with a K_I of 112 µM, a K _inact of 0.05 min^-1, and a t_1/2 of 13.9 min. Similarly, TFCPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner with a K_I of 14 µM, a K _inact of 0.04 min^-1 and a t_1/2 of 16.5 min. Parallel losses of P450 3A4 enzymatic activity and heme were observed with both compounds as measured by HPLC and reduced-CO spectra. Interestingly, neither compound inhibited the BFC O-debenzylation activity of P450 3A5. Reactive intermediates of CPE and TFCPE formed by P450 3A4 were trapped with glutathione and the resulting adducts were identified using tandem mass spectral analysis. Metabolism studies using TFCPE resulted in the identification of a single metabolite that is formed by P450 3A4 but not by P450 3A5 and that may play a role in the mechanism-based inactivation.

Key words: CYP3A, cytochrome P450, human CYP enzymes, mass spectrometry