METABOLISM OF b-ARTEETHER TO DIHYDROQINGHAOSU BY HUMAN LIVER MICROSOMES AND RECOMBINANT CYTOCHROME P450

b-Arteether (AE) is an endoperoxide sesquiterpene lactone derivative currently being developed for the treatment of severe, complicated malaria caused by multidrug-resistant Plasmodium falciparum. Studies were undertaken to determine which form(s) of human cytochrome P-450 catalyze the conversion of b-arteether to its deethylated metabolite, dihydroqinghaosu (DQHS), itself a potent antimalarial compound. In human liver microsomes, AE was metabolized to DQHS with a Km of 53.7 6 29.5 mM and a Vmax of 1.64 6 1.78 nmol DQHS/min/mg protein. AE biotransformation to DQHS was inhibited by ketoconazole and troleandomycin. Ketoconazole was a competitive inhibitor, with an apparent Ki of 0.33 6 0.11 mM. Because AE is being developed for patients who fail primary treatment, it is possible that AE may be involved in lifethreatening drug-drug interactions, such as the associated cardiotoxicity of mefloquine and quinidine. Coincubation of AE with other antimalarials showed mefloquine and quinidine to be competitive inhibitors with a mean Ki of 41 and 111 mM, respectively. Metabolism of AE using human recombinant P450s provided evidence that cytochrome P450s 2B6, 3A4, and 3A5 were the primary isozymes responsible for its deethylation. CYP3A4 metabolized AE to dihydroqinghaosu at a rate approximately 10 times that of CYP2B6 and ;4.5-fold greater than that of CYP3A5. These results demonstrate that CYP3A4 is the primary isozyme involved in the metabolism of AE to its active metabolite, DQHS, with secondary contributions by CYP2B6 and -3A5. Malaria is endemic in most tropical and subtropical regions of the world. It is estimated that 300–500 million people are at risk of contracting malaria, with 900,000 new cases diagnosed each year (Murray and Lopez, 1994; Olliaro et al., 1996). There are 1–2 million deaths reported annually due to severe, cerebral malaria; with the majority of these deaths being children in Africa (Zuker and Campell, 1993). Qinghaosu (QHS), a unique sesquiterpene lactone endoperoxide, is the active antimalarial moiety isolated from the Chinese medicinal herb, Artemisia annua (Klaymann, 1985). Arteether (see fig. 1), the ethyl ether derivative of the reduced lactol of QHS, dihydroqinghaosu (DQHS), is currently being developed for use in severe and multidrug-resistant malaria, including cerebral malaria. Recent studies have established a dose-dependent neurotoxicity in rats and dogs after repeated im administrations of high doses of AE (Brewer et al., 1994a, 1994b). DQHS, known to be more neurotoxic and efficacious than AE in vitro and in vivo (Brewer et al., 1993; Wesche et al., 1994), has been identified as a major metabolite in rat liver microsomes (Leskovac and Theoharides, 1991a). Large scale human studies with related artemisinin analogs have not shown any neurotoxic side effects (Hien and White, 1993; Looareesuwan, 1994). However, isolated case reports have implicated possible neurological dysfunction in humans after the administration of related artemisinin compounds (Miller and Panosian, 1997; Senanayake and de Silva, 1994; van Hensbroek et al., 1996). Arteether has been shown to be extensively metabolized using various in vitro and in vivo animal models (Chi et al., 1991; Leskovac and Theoharides, 1991a, 1991b). In the isolated perfused rat liver, AE biotransformation pathways include deethylation and hydroxylation followed by glucuronidation (Peggins et al., 1990). In rat liver microsomes, the NADPH-dependent, cytochrome P450-mediated Odeethylation of AE to DQHS has been identified as a major metabolic pathway (Leskovac and Theoharides, 1991a). The positive therapeutic effect of DQHS was determined in humans for a related artemisinin analog, artemether. The plasma level of the active metabolite, DQHS, was measured by HPLC analysis, and the plasma antimalarial activity was assessed in vitro by bioassay for the same sample. The study demonstrated that the plasma concentration and the antimalarial activity profile for DQHS was similar, suggesting that other unidentified metabolites contributed little to the antimalarial activity of DQHS in vivo (Teja-Isavadharm, 1996). A related study in humans administered AE shows an identical trend in the DQHS plasma concentration and antimalarial effect profile (D. Kyle, personal communication). The current study was undertaken to determine which human cytochrome P450 isozyme(s) catalyze the conversion of AE to its active metabolite, DQHS. Because AE is being developed for the treatment of multidrugresistant malaria, it is not unusual for patients to receive multiple antimalarial drugs prior to the administration of AE. In general, antimalarial drugs have long elimination half-lives (t1/2) that can range from days to several weeks; consequently, clinically significant This work was supported by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). 1 Abbreviations used are: QHS, qinghaosu; DQHS, dihydroqinghaosu; AE, b-arteether; BHT, butylated hydroxytoluene; HPLC, high performance liquid chro-