Kinetic characteristics of norcocaine N-hydroxylation in mouse and human liver microsomes: involvement of CYP enzymes

The first step in the oxidative metabolism of cocaine is N-demethylation to norcocaine, which is further N-hydroxylated to more toxic N-hydroxynorcoca ine. In this study we examined the kinetics of norcocaine N-hydroxylation m ediated by cytochrome P450 (CYP) in mouse and human liver microsomes. N-hyd roxynorcocaine was identified by analytical HPLC-MS after incubation of nor cocaine with mouse liver microsomes in the presence of NADPH. In mouse live r microsomes, there was no apparent difference in K-m values for norcocaine N-hydroxylation between male and female microsomes, while the V-max rate w as approximately two times higher in female than in male microsomes (34+/-1 0 v 16+/-4 pmol/min per mg protein). The K-m value for norcocaine N-hydroxy lation in human liver microsomes was approximately three times higher than that observed in comparable incubations using mouse liver microsomes, where as the V-max rate was ten times lower. Both cocaine and norcocaine induced type I difference spectra upon interaction with CYP in mouse liver microsom es. In contrast, in human microsomes both type I and type II spectra were r ecorded. In the 0.01 to 1 mM concentration range, cocaine and norcocaine in hibited mouse microsomal testosterone 6 alpha-, 7 alpha- and 16 alpha -hydr oxylation reactions by 20% to 30%. Testosterone 6 beta- and 15 alpha -hydro xylations were blocked by 60% and 50%, respectively, by 1 mM norcocaine, wh ile only 40% inhibition was obtained with 1 mM cocaine. Coumarin 7-hydroxyl ation and pentoxyresorufin O-deethylation were inhibited by 50% by 1 and 0. 4 mM norcocaine, respectively. In contrast, 10 and 2 mM cocaine, respective ly, were needed to obtain the same degrees of inhibition. In human liver mi crosomes, 1 mM norcocaine and cocaine blocked testosterone 6 beta -hydroxyl ase by 60% and 40%, respectively. Coumarin 7-hydroxylation was inhibited by only 30% by norcocaine (5.4 mM) and cocaine (10 mM). Norcocaine N-hydroxyl ation in mouse and human liver microsomes was blocked by 30% and 60%, respe ctively, by alpha -naphthoflavone (0.1 mM). The reaction was inhibited by 3 0-40% by metyrapone, cimetidine and gestodene at a concentration of 1 mM in mouse microsomes, while in human microsomes, 70% inhibition was obtained w ith 1 mM metyrapone and cimetidine. Taken together, these results indicate that (1) norcocaine N-hydroxylation is at least partly a CYP-mediated react ion, (2) the rate of reaction is considerably lower in human liver microsom es than in mouse liver microsomes and (3) several CYP subfamilies including 1A, 2A, 3A and possibly 2B may contribute to the formation of N-hydroxynor cocaine.