IN VITRO METABOLISM OF CHLOROQUINE : IDENTIFICATION OF CYP 2 C 8 , CYP 3 A 4 , AND CYP 2 D 6 AS THE MAIN ISOFORMS CATALYZING N-DESETHYLCHLOROQUINE FORMATION

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 M 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 6 -hydroxylation (r 0.80; p < 0.001), a CYP3A-mediated reaction, and CYP2C8-mediated paclitaxel -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 cDNAexpressed 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 CYP2D6mediated metabolism in vitro and in vivo. At therapeutically relevant concentrations ( 100 M CQ in the liver), CYP2C8, CYP3A4, and, to a much lesser extent, CYP2D6 are expected to account for most of the CQ N-desethylation. Chloroquine (CQ) is an antimalarial drug that has been used in the treatment and prophylaxis of malaria since the 1950s. In humans, the liver and kidneys contribute approximately equally to its body disposition (Frisk-Holmberg et al., 1983; Gustafsson et al., 1983; Ette et al., 1989). Following oral or i.v. administration, chloroquine is dealkylated into two main metabolites, N-desethylchloroquine (DCQ) and N-bis-desethylchloroquine (BDCQ) (Fig. 1). DCQ is rapidly detected in blood or plasma, its concentrations amounting to 20 to 50% of those of the parent (Gustafsson et al., 1983; Frisk-Holmberg et al., 1984). For BDCQ, plasma or blood concentrations never reach more than 10 to 15% of CQ levels (Frisk-Holmberg et al., 1983, 1984; Augustjins et al., 1992). Although CQ possesses cytochrome P450 (P450)-inhibiting properties, notably on CYP2D6 (Lancaster et al., 1990; Halliday et al., 1995; Masimirembwa et al., 1995), the enzymes catalyzing its N-dealkylation have never been identified. Distribution studies have shown that CQ is extensively sequestered in tissues, the liver, spleen, kidney, and lungs being the main repositories (Grundmann et al., 1971). Even if CQ plasma concentrations rarely exceed the micromolar range (0.25 0.23 M), liver concentrations may be several hundred times higher (McChesney et al., 1967; MacKenzie, 1983; Augustjins et al., 1992). In rats, liver to plasma ratios ranged from 209 to 541 (Adelusi and Salako, 1982). All CQ indications entail long-term administration. For malaria prophylaxis and treatment, CQ may be given for several weeks (Ducharme and Farinotti, 1996). Against rheumatoid arthritis, maintenance doses are 7to 20-fold greater (Maksymowych and Russel, 1987), and patients may take the drug for years to prevent disease progression. In view of its extremely long half-life (8.9–41 days), its extensive distribution, and its P450-inhibiting properties (see Ducharme and Farinotti, 1996, for review), CQ is likely to be implicated in clinically significant drug-drug interactions. To better characterize the biotransformation of CQ in humans, we used a variety of in vitro approaches to assess its metabolic stability and determine which enzymes are involved in its metabolism. Materials and Methods Chemicals and Solutions. CQ sulfate, DCQ, and BDCQ were a generous gift from Aventis (Vitry, France). CQ stock solutions (50 and 100 mM) were prepared in distilled water and stored at 20°C. DCQ and BDCQ stock solutions (100 mM) were prepared in 50:50 (v/v) methanol/distilled water and stored at 20°C. Diluted aqueous solutions were prepared immediately before the incubations. Chemical reagents, P450 substrates, or inhibitors were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France or St. Louis, MO). Human Liver Microsomes (HLM). HLM were prepared and characterized Denis Projean was supported by a Research Studentship from AstraZeneca R&D Montréal. 1 Abbreviations used are: CQ, chloroquine; DCQ, desethylchloroquine; BDCQ, bisdesethylchloroquine; HLM, human liver microsome(s); HPLC, high-performance liquid chromatography; CLint, in vitro intrinsic clearance; CL int, scaled in vivo intrinsic clearance; CLint,r, in vitro intrinsic clearance in recombinant P450i; CLh, hepatic clearance; P450, cytochrome P450; RAF, relative activity factor; RC, relative contribution; TAO, troleandomycin; P-gp, P-glycoprotein. Address correspondence to: Dr. Julie Ducharme, AstraZeneca R & D Montréal, 7171 Frederick-Banting, Ville Saint-Laurent, Québec, Canada, H4S 1Z9. E-mail: julie.ducharme@astrazeneca.com 0090-9556/03/3106-748–754$7.00 DRUG METABOLISM AND DISPOSITION Vol. 31, No. 6 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 1056/1067917 DMD 31:748–754, 2003 Printed in U.S.A. 748 at A PE T Jornals on D ecem er 4, 2017 dm d.aspurnals. org D ow nladed from