Effect of carbonate anion on cytochrome P450 2D6-mediated metabolism in vitro: the potential role of multiple oxygenating species.
Studies were designed to investigate various anions and their effects on cytochrome P450 2D6-mediated metabolism in vitro. Incubations were initially performed in buffered phosphate, carbonate, sulfate, and acetate solutions (50mM, pH 7.4), with CYP2D6 substrates dextromethorphan, 7-methoxy-4-(aminomethyl)-coumarin (MAMC), (S,S)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride [(-)-OSU6162], and amitriptyline. Dextromethorphan and MAMC O-dealkylation activity in buffered carbonate was approximately 25 and 38%, respectively, relative to phosphate, while activity in sulfate and acetate buffers displayed minor differences. In contrast, N-dealkylation reactions for both (-)-OSU6162 and amitriptyline were unaffected by the presence of carbonate, and the other anions tested. Subsequent kinetic studies revealed that the basis of reduced turnover of dextromethorphan was primarily a V(max) effect, as the V(max) for the rate was 16.9 and 5.6 pmol/min/pmol P450 in phosphate and carbonate, respectively. Interestingly, similar rates of dextromethorphan O-demethylation in phosphate and carbonate were observed when reactions were supported by cumene hydroperoxide (CuOOH). Furthermore, it was observed that while CuOOH could equally support dextromethorphan O-demethylation compared to NADPH, amitriptyline N-demethylation was only minimally supported. Finally, intramolecular kinetic isotope effect (KIE) experiments with amitriptyline-d3 in CuOOH-supported reactions yielded a k(H)/k(D) of 5.2, substantially higher than in phosphate and carbonate supported by NADPH (k(H)/k(D)=1.5). Overall, results suggest that carbonate disrupts the relative ratios of the potential P450 oxygenating species, which differentially catalyze O- and N-dealkylation reactions mediated by CYP2D6.
Hutzler
J Matthew
JM
Department of Global Drug Metabolism, Pharmacia Corporation, 301 Henrietta St., 7265-300-313, Kalamazoo, MI 49007, USA.
Powers
Frank J
FJ
Wynalda
Michael A
MA
Wienkers
Larry C
LC
D000838:Anions / Q000737:chemistry / Q000378:metabolism; D002254:Carbonates / Q000737:chemistry*; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry*; D013379:Substrate Specificity
D000838:Anions; D002254:Carbonates; D017382:Reactive Oxygen Species; D019389:Cytochrome P-450 CYP2D6; D010100:Oxygen