MECHANISM-BASED INACTIVATION OF HUMAN RECOMBINANT P450 2C9 BY THE NONSTEROIDAL ANTI-INFLAMMATORY DRUG SUPROFEN

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Abstract
The nonsteroidal anti-inflammatory agent (±)-suprofen [α-methyl-4-(2-thienylcarbonyl)benzeneacetic acid] was evaluated as a P450 2C9 inactivator. (±)-Suprofen inactivated the diclofenac-4-hydroxylase activity of baculovirus-expressed P450 2C9 in a time- and concentration-dependent manner, which was consistent with mechanism-based inactivation. The loss of activity followed pseudo-first-order kinetics and was suprofen- and NADPH-dependent. The kinetic parameters for inactivation kinact and KI were 0.091 min-1 and 3.7 μM, respectively, and the partition ratio was 101. Although P450 2C9 substrate S-warfarin partially protected against inactivation, reactive oxygen scavengers such as superoxide dismutase and catalase did not prevent inactivation. Extensive dialysis did not regenerate enzyme activity, suggesting that inactivation proceeded via covalent modification. Inactivated P450 2C9 lost 24H28N3O9S2. The mass spectrum indicated that conjugation had occurred on the intact thiophene ring, presumably via a thioether linkage. Further evidence for the formation of an electrophilic intermediate in suprofen-P450 2C9 incubations was obtained via the characterization of a novel pyridazine adduct upon addition of semicarbazide to the microsomal mixtures. The pyridazine derivative had a protonated monoisotopic mass of 257.0895 that was consistent with an elemental composition of C14H13O3N2. The formation of the stable pyridazine adduct suggested the generation of an electrophilic γ-thioketo-α, β-unsaturated aldehyde, analogous to that observed during the cytochrome P450-mediated bioactivation of furan. This electrophilic α, β-unsaturated aldehyde represents a possible reactive intermediate that bioalkylates P450 2C9.