Analysis of Mechanism-Based Inhibition of Human Cytochrome P450 2D6 by Schering 66712

Abstract

Cytochrome P450 2D6 (P450 2D6) makes up less than 5% of P450s found in the liver but is responsible for metabolism of 20-30% of drugs currently on the market. Inhibition of P450 2D6 due to adverse drug reactions or drug-drug interactions can lead to side effects from taking the recommended drug dosage or to side effects of taking more than one drug at once, respectively. Adverse drug reactions and drug-drug interactions are potentially fatal and therefore understanding inhibition is vital. The inhibition of human P450 2D6 by human D4 dopamine receptor antagonist, Schering 66712 ([5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine]) was analyzed to determine the mechanism for inactivation. By measuring loss of bufuralol 1’hydroxylation activity following coincubation of P450 2D6 with SCH 66712 a partition ratio of ~305 was determined. P450 2D6 inactivation was not protected by the addition of an exogenous nucleophile, potassium cyanide. The time dependent inactivation of P450 2D6 by SCH 66712 showed ~100% decrease in 1’OH bufuralol formation after 60 minutes in addition to an unequivocal loss of ~40% ability to bind carbon monoxide after 40 minutes. These findings support that inactivation of P450 2D6 by SCH 66712 occurs by adduction to protein rather than to the heme. Examination of possible SCH 66712 metabolites produced by P450 3A4 yielded no leads. Initial mass spectrometry analysis of P450s 3A4 and 2D6 gave intact protein masses of 56894 and 56936 Da, respectively.