Substrate inhibition potential of arachidonic acid on 14,15-epoxidation, a biological drug developmental target, mediated by recombinant human cytochrome P450 2J2 and 2C8 enzymes

Abstract

Arachidonic acid is metabolized to biologically active (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET), and suppression of this step is believed to be a drug developmental target in cancer treatment. Although the key 14,15-epoxidation mediated by human cytochrome P450 (P450 or CYP) 2J2 has been demonstrated, inconsistencies in reported in vitro reaction kinetics have been observed worldwide. The purpose of this study was to clarify the detailed kinetics of arachidonic acid 14,15-expoxidation by recombinant human CYP2J2 and CYP2C8 using precise liquid chromatography–tandem mass spectrometry. In the absence of an NADPH-generating system, substrate-dependent 14,15-EET was detected in multiple batches of commercially available arachidonic acid at minor but constant levels, regardless of incubation period or P450 concentration. Recombinant CYP2J2 and CYP2C8 mediated minor and extensive arachidonic acid 14,15-epoxidation in a substrate inhibition manner after subtracting 14,15-EET impurities from total 14,15-EET formation. CYP2J2 exhibited similar Michaelis (K_m) and substrate inhibition (K_s) constants; however, CYP2C8 had a lower K_s value, indicating more potent substrate inhibition in CYP2C8 than that in CYP2J2. To determine the effects of a given drug on arachidonic acid 14,15-epoxidation during drug development, experiments should be recommended with arachidonic acid of <20 μM for CYP2J2 and <5.0 μM for CYP2C8.