Effects of Acetylenic Epoxygenase Inhibitors on Recombinant Cytochrome P450s

Abstract

ABSTRACT:Arachidonate epoxidation, which mediates important biological functions in several tissues, is catalyzed by specific cytochrome P450 (P450) enzymes. Two fatty acid derivatives [2-(2-propynyloxy)-benzenehexanoic acid (PPOH) and N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH)] are used as general, mechanism-based P450 epoxygenase inactivators, but the effects of these drugs on nearly all P450 isoforms are unknown. Here, the activity of these compounds on nine human and three rat recombinant P450s was studied. As expected, PPOH inhibited five known epoxygenases [CYP2B1, 2B6, 2C6, 2C9, and 2C11 (IC 50 ‫؍‬ 23-161 M)] but had little or no activity on P450s typically not considered to be epoxygenases (CYP1A1, 1A2, 1B1, 2A6, 2D6, and 2E1). PPOH was only a very weak inhibitor (IC 50 ‫؍‬ ϳ300 M) of CYP2C19, an important human expoxygenase. An unexpected finding was that MS-PPOH (a metabolically stable congener of PPOH) potently inhibited only two P450 epoxygenases (2C9 and 2C11, IC 50 ‫؍‬ 11-16 M) and showed considerably lower activity (IC 50 ‫؍‬ >90 M) on all other P450s tested, including three epoxygenases (CYP2B1, 2B6, and 2C19). In addition, PPOH and MS-PPOH displayed time-and NADPH-dependent inhibition of CYP2C9 and other epoxygenases. These results support the putative mechanism of action of PPOH and MS-PPOH on recombinant P450s and (with one exception) confirm a general epoxygenase inhibitory profile for PPOH. However, the heterogeneity of inhibitory potencies for MS-PPOH on these enzymes suggests caution in the use of this drug as a general epoxygenase inhibitor. These results will facilitate the judicious use of PPOH and MS-PPOH for epoxygenase research.

Changes in gene expression of cytochrome P-450 in liver, kidney and aorta of cirrhotic rats

“…On the other hand, no effects were observed on aortic CYP2J4 and CYP2C12 and on hepatic CYP2C11 gene expression. This could be explained with a heterogeneous inhibitory effect of MS-PPOH on different CYPs [24].…”

Cytochrome P450 2C24: expression, tissue distribution, high-throughput assay, and pharmacological inhibition

“…Predictably, PPOH was found to be a potent inhibitor of the epoxygenases CYP2B1 and CYP2C9, but only a moderate inhibitor of CYP2B6 and CYP2C11. Surprisingly, however, this drug only weakly inhibited the epoxygenases CYP2C6 and CYP2C19 25 (see also Table 1). As compared with PPOH, MS-PPOH inhibited even fewer epoxygenases, with only very weak activity on CYP2B6 25 .…”

“…Best known CYP epoxygenase targets for one or both of these drugs are CYP4A2, CYP4A3 40 , and CYP2C8 39 . PPOH and MS-PPOH have been used extensively in CYP epoxygenase research, and, although these drugs have been widely assumed to be general inhibitors of all CYP epoxygenases, a recent study found this to not be the case 25 . Predictably, PPOH was found to be a potent inhibitor of the epoxygenases CYP2B1 and CYP2C9, but only a moderate inhibitor of CYP2B6 and CYP2C11.…”

“…CYP2C9 activity (1 pmol) was determined with MFC (75 µM) as substrate and incubated at 37° for 30 min as described 24 . CYP2C19 and CYP2C8 (both 1 pmol, 0.5 µM DBF) were assayed identically to the method described 25 for CYP2C11. Briefly, inhibitors (in 100% acetonitrile) were added to the reaction mixtures containing NADPH-regenerating system (final concentrations of 1.3 mM NADP + , 3.3 mM D-glucose-6 phosphate, 0.4 U/mL glucose-6 phosphate dehydrogenase, and 3.3 mM magnesium chloride) in 0.1 mL buffer, followed by pre-incubation at 37° C for 15 min.…”

Aspirin and Probenecid Inhibit Organic Anion Transporter 3–Mediated Renal Uptake of Cilostazol and Probenecid Induces Metabolism of Cilostazol in the Rat

“…In rats, the metabolism of cilostazol to OPC-13015 or OPC-13213 is mediated by CYP3A2 or CYP2C11 (Kamada et al, 2011). CYP3A2 and CYP2C11 expressed in rats are the most similar to CYP3A4 and CYP2C19 in humans (Bogaards et al, 2000;VanAlstine and Hough, 2011). It was previously suggested that CYP3A1, CYP3A2, and CYP2C11 are expressed in the rat kidney (Pfohl-Leszkowicz et al, 1998) and that CYP3A4, CYP3A5, and CYP2C19 are detected in the human kidney (Lasker et al, 2000;Aleksa et al, 2005).…”