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

Abstract

The first step in the oxidative metabolism of cocaine is N-demethylation to norcocaine, which is further N-hydroxylated to more toxic N-hydroxynorcocaine. In this study we examined the kinetics of norcocaine N-hydroxylation mediated by cytochrome P450 (CYP) in mouse and human liver microsomes. N-hydroxynorcocaine was identified by analytical HPLC-MS after incubation of norcocaine with mouse liver microsomes in the presence of NADPH. In mouse liver microsomes, there was no apparent difference in Km values for norcocaine N-hydroxylation between male and female microsomes, while the Vmax rate was approximately two times higher in female than in male microsomes (34+/-10 v. 16+/-4 pmol/min per mg protein). The Km value for norcocaine N-hydroxylation in human liver microsomes was approximately three times higher than that observed in comparable incubations using mouse liver microsomes, whereas the Vmax rate was ten times lower. Both cocaine and norcocaine induced type I difference spectra upon interaction with CYP in mouse liver microsomes. In contrast, in human microsomes both type I and type II spectra were recorded. In the 0.01 to 1 mM concentration range, cocaine and norcocaine inhibited mouse microsomal testosterone 6alpha-, 7alpha- and 16alpha-hydroxylation reactions by 20% to 30%. Testosterone 6beta- and 15alpha-hydroxylations were blocked by 60% and 50%, respectively, by 1 mM norcocaine, while only 40% inhibition was obtained with 1 mM cocaine. Coumarin 7-hydroxylation and pentoxyresorufin O-deethylation were inhibited by 50% by 1 and 0.4 mM norcocaine, respectively. In contrast, 10 and 2 mM cocaine, respectively, were needed to obtain the same degrees of inhibition. In human liver microsomes, 1 mM norcocaine and cocaine blocked testosterone 6beta-hydroxylase by 60% and 40%, respectively. Coumarin 7-hydroxylation was inhibited by only 30% by norcocaine (5.4 mM) and cocaine (10 mM). Norcocaine N-hydroxylation in mouse and human liver microsomes was blocked by 30% and 60%, respectively, by alpha-naphthoflavone (0.1 mM). The reaction was inhibited by 30-40% by metyrapone, cimetidine and gestodene at a concentration of 1 mM in mouse microsomes, while in human microsomes, 70% inhibition was obtained with 1 mM metyrapone and cimetidine. Taken together, these results indicate that (1) norcocaine N-hydroxylation is at least partly a CYP-mediated reaction, (2) the rate of reaction is considerably lower in human liver microsomes than in mouse liver microsomes and (3) several CYP subfamilies including 1A, 2A, 3A and possibly 2B may contribute to the formation of N-hydroxynorcocaine.

Effects of the H3 receptor inverse agonist thioperamide on cocaine-induced locomotion in mice: role of the histaminergic system and potential pharmacokinetic interactions

“…Since intraperitoneal administered cocaine is largely taken up by the portal circulation, the likely mechanism is inhibition of hepatic first pass metabolism by thioperamide through the blockade of cytochrome P450 enzymes. Thioperamide is an imidazole-based compound and, like many drugs that bear an imidazole group, strongly inhibits P450 cytochromes (Yang et al 2002), the primary enzymes involved in the N-demethylation of cocaine in mice, rats, and humans (Pellinen et al 1994(Pellinen et al , 2000Poet et al 1996). It is thus possible that thioperamide increased plasma cocaine concentrations in the present study through the blockade of the cytochrome P450 enzymes that contribute to the elimination of cocaine and that the increased cocaine concentration produced more locomotor stimulation.…”

Fluoxetine, but not sertraline or citalopram, potentiates the locomotor stimulant effect of cocaine: possible pharmacokinetic effects

“…Cocaine undergoes N-demethylation via CYP enzymes, and in particular members of the CYP2B and 3A subfamilies, in both rats and humans (Pellinen et al 1994(Pellinen et al , 2000Poet et al 1996). Fluoxetine, and its major metabolite norfluoxetine, are inhibitors of CYP3A activity (von Moltke et al 1994;Hemeryck and Belpaire 2002).…”

Catalyzation of cocaine N‐demethylation by cytochromes P4502B, P4503A, and P4502D in fish liver

“…Cocaine also causes hepatotoxicity in humans and laboratory animals [11][12][13][14][15]. The cocaine-induced hepatotoxicity has been shown to be associated with the metabolites of cocaine, that is norcocaine and N-hydroxycocaine [12,[16][17][18]. The first step in the bioactivation of cocaine is the N-demethylation of cocaine to norcocaine by cytochrome P450 isozymes [16,17,[19][20][21].…”