Mechanism-based inactivation of human recombinant P450 2C9 by the nonsteroidal anti-inflammatory drug suprofen.
The nonsteroidal anti-inflammatory agent (+ or -)-suprofen [alpha-methyl-4-(2-thienylcarbonyl)benzeneacetic acid] was evaluated as a P450 2C9 inactivator. (+ or -)-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 microM, 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 <10% of its ability to form a CO-reduced complex, suggesting that inactivation may have resulted from covalent modification of apoprotein. Addition of exogenous nucleophiles such as glutathione and semicarbazide partially protected against inactivation. Apart from the metabolism of suprofen to 5-hydroxysuprofen, the formation of a suprofen-glutathione conjugate was also discernible in microsomal mixtures containing glutathione. Time of flight mass spectrometry revealed a protonated monoisotopic mass of 566.1304 for this conjugate, consistent with an elemental composition of C24H28N3O9S2. 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 gamma-thioketo-alpha, beta-unsaturated aldehyde, analogous to that observed during the cytochrome P450-mediated bioactivation of furan. This electrophilic alpha, beta-unsaturated aldehyde represents a possible reactive intermediate that bioalkylates P450 2C9.
O'Donnell
John P
JP
Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA. odonnelljp@groton.pfizer.com
Dalvie
Deepak K
DK
Kalgutkar
Amit S
AS
Obach
R Scott
RS
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D013496:Suprofen / Q000737:chemistry / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004791:Enzyme Inhibitors; D013496:Suprofen; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases