title: In vitro metabolism of chloroquine: identification of CYP2C8, CYP3A4, and CYP2D6 as the main isoforms catalyzing N-desethylchloroquine formation.
abstract: In humans, the antimalarial drug chloroquine (CQ) is metabolized into one major metabolite, N-desethylchloroquine (DCQ). Using human liver microsomes (HLM) and recombinant human cytochrome P450 (P450), we performed studies to identify the P450 isoform(s) involved in the N-desethylation of CQ. In HLM incubated with CQ, only DCQ could be detected. Apparent Km and Vmax values (mean +/- S.D.) for metabolite formation were 444 +/- 121 microM and 617 +/- 128 pmol/min/mg protein, respectively. In microsomes from a panel of 16 human livers phenotyped for 10 different P450 isoforms, DCQ formation was highly correlated with testosterone 6beta-hydroxylation (r = 0.80; p < 0.001), a CYP3A-mediated reaction, and CYP2C8-mediated paclitaxel alpha-hydroxylation (r = 0.82; p < 0.001). CQ N-desethylation was diminished when coincubated with quercetin (20-40% inhibition), ketoconazole, or troleandomycin (20-30% inhibition) and was strongly inhibited (80% inhibition) by a combination of ketoconazole and quercetin, which further corroborates the contribution of CYP2C8 and CYP3As. Of 10 cDNA-expressed human P450s examined, only CYP1A1, CYP2D6, CYP3A4, and CYP2C8 produced DCQ. CYP2C8 and CYP3A4 constituted low-affinity/high-capacity systems, whereas CYP2D6 was associated with higher affinity but a significantly lower capacity. This property may explain the ability of CQ to inhibit CYP2D6-mediated metabolism in vitro and in vivo. At therapeutically relevant concentrations ( approximately 100 microM CQ in the liver), CYP2C8, CYP3A4, and, to a much lesser extent, CYP2D6 are expected to account for most of the CQ N-desethylation.
authors.affiliation: AstraZeneca R & D Montréal, 7171 Frederick-Banting, Ville Saint-Laurent, Québec, Canada, H4S 1Z9.
authors.forename: Denis
authors.initials: D
authors.lastname: Projean
authors.forename: Bruno
authors.initials: B
authors.lastname: Baune
authors.forename: Robert
authors.initials: R
authors.lastname: Farinotti
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Flinois
authors.forename: Philippe
authors.initials: P
authors.lastname: Beaune
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Taburet
authors.forename: Julie
authors.initials: J
authors.lastname: Ducharme
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D001711:Biotransformation; D002478:Cells, Cultured; D002738:Chloroquine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid; D065727:Cytochrome P-450 CYP2C8; D019389:Cytochrome P-450 CYP2D6 / Q000096:biosynthesis*; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007313:Insecta; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D021241:Spectrometry, Mass, Electrospray Ionization; D014162:Transfection
chemical_list: D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D002738:Chloroquine; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; C104464:CYP3A protein, human; D065727:Cytochrome P-450 CYP2C8; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C007278:desethylchloroquine