title: 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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Kuopio, Finland.
authors.forename: P
authors.initials: P
authors.lastname: Pellinen
authors.forename: L
authors.initials: L
authors.lastname: Kulmala
authors.forename: J
authors.initials: J
authors.lastname: Konttila
authors.forename: S
authors.initials: S
authors.lastname: Auriola
authors.forename: M
authors.initials: M
authors.lastname: Pasanen
authors.forename: R
authors.initials: R
authors.lastname: Juvonen
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002851:Chromatography, High Pressure Liquid; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism*; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008811:Mice, Inbred DBA; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D039181:Steroid 16-alpha-Hydroxylase
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; C008671:norcocaine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D039181:Steroid 16-alpha-Hydroxylase; D003042:Cocaine