Characterization of the selectivity and mechanism of cytochrome P450 inhibition by dimethyl-4,4 '-dimethoxy-5,6,5 ',6 '-dimethylenedioxybiphenyl-2,2 '-dicarboxylate

Abstract

In vitro studies with human liver microsomes and cytochrome P450 (P450) prototype substrates were performed to characterize the selectivity and mechanism of inhibition of P450 by dimethyl-4,4'dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB). DDB was found to be a strong inhibitor of testosterone 6 beta -hydroxylation activity (CYP3A4) with a K-i value of 0.27 +/-0.21 muM. At higher concentrations, DDB marginally inhibited caffeine N-3-clemethylation (CYP1A2), diclofenac 4'-hydroxylation (CYP2C9), and dextromethorphan O-demethylation (CYP2D6) activities, but this compound had no effect on CYP2A6-, CYP2C19-, and CYP2E1 mediated reactions. Spectral analysis indicated that the formation of metabolite-P450 complex having absorbance at 456 nm was concentration-dependent; 5 to 33% of the total P450 was complexed in rat and human liver microsomes after a 5-min incubation with DDB. In addition, microsomal incubations with DDB in the presence of NADPH resulted in a loss of spectral P450 content, which was restored after adding K3Fe(CN)(6). This complex formation resulted in a time-dependent loss of CYP3A-catalyzed marker activity (testosterone 6 beta -hydroxylation) in human liver microsomes. The inhibition was only partially restored upon dialysis. These results collectively suggest that formation of a metabolite-CYP3A complex with DDB was responsible for the CYP3A-selective time-dependent loss of catalytic function of CYP3A.