BILIARY METABOLITES OF b-ARTEMETHER IN RATS: BIOTRANSFORMATIONS OF AN ANTIMALARIAL ENDOPEROXIDE

b-Artemether (AM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is an endoperoxide antimalarial. The biliary metabolites of AM in adult male Wistar rats were characterized with particular reference to potential antimalarial compounds and stable derivatives of free radical intermediates. [13-C]-AM (35 mmol kg, i.v.) was administered to anesthetized rats. Within 0 to 3 h, 38.6 6 4.8% (mean 6 S.D., n 5 6) of the radiolabel was recovered in bile; the 0to 5-h recovery was 42.3 6 4.3%. The major metabolites (0–3 h) were the glucuronides of 9a-hydroxyAM (33.4 6 6.8% biliary radioactivity) and a-DHA (22.5 6 4.4%); four stereochemically unassigned monohydroxyAM glucuronides (II, 3.1 6 0.9; IV, 4.4 6 1.7%; V, 21.4 6 3.0%; VI, 3.0 6 1.1%) and a dihydroxyAM glucuronide (6.0 6 2.1%) were also identified. A sixth monohydroxyAM glucuronide (VIIa) and desoxyDHA glucuronide were detected in trace amounts. The furano acetate isomer of DHA glucuronide, indicative of the formation of a radical intermediate, was also found in trace amounts. O-methyl substitution of DHA favors ring hydroxylation in vivo. However, the principal hydroxylated metabolite, 9ahydroxyAM, is unlikely to possess significant antimalarial activity. b-Artemether (AM) (Paluther, Rhone-Poulenc Rorer, Antony, France; Fig. 1) is a semisynthetic polyoxygenated amorphene containing a peroxide bridge that confers potent antimalarial activity (Meshnick et al., 1996). It is the O-methyl ether of dihydroartemisinin (DHA) and a derivative of artemisinin (qinghaosu), the principal antimalarial constituent of the medicinal plant Artemisia annua. AM is active against the erythrocytic stage of multidrug-resistant strains of Plasmodium falciparum (Sowunmi and Odulola, 1997). Oral, rectal, and i.m. regimens are generally rapidly effective and well tolerated treatments for both severe and uncomplicated falciparum malaria (de Vries and Dien, 1996; Murphy et al., 1997), although short-term monotherapy has been associated with high rates of relapse. Combinations with nonendoperoxide antimalarials have been used (de Vries and Dien, 1996). The parasiticidal activity of endoperoxides has been attributed to chemical activation of the drugs within the food vacuole of the intraerythrocytic stage of the parasite: it is proposed that reductive cleavage of the peroxide bridge by heme liberated during digestion of hemoglobin generates free radicals, which induce oxidative stress and alkylate heme and vital parasite proteins (Cumming et al., 1997; Robert and Meunier, 1998). In chemical systems, iron (II) catalyzes the isomerization of artemisininoid peroxides to ring-contracted (furano acetate) and 3a-hydroxydesoxy compounds (Fig. 1) indicative of the sequential intermediacy of alkoxyl and carbon-centered radicals (Jefford et al., 1996; Butler et al., 1998). Such compounds are minor metabolites of DHA and its O-ethyl ether (arteether) in nonparasitized rats (Chi et al., 1991; Maggs et al., 1997) but their putative free radical precursors are not thought to contribute to the drugs’ antimalarial activity. Lacking a peroxide functionality, the stable metabolites are pharmacologically inactive. Although the mechanism(s) of activation in mammalian cells has not been determined, it has clear if unsubstantiated implications for the clinical safety of endoperoxides (Park et al., 1998), and especially with respect to the causation of the neurotoxicity associated with this class of drugs (Smith et al., 1998). AM is regarded as a prodrug of DHA in humans (Teja-Isavadharm et al., 1996) although the two endoperoxides have similar parasiticidal activities in vitro (de Vries and Dien, 1996). Oral doses are absorbed rapidly and converted extensively to DHA in humans (Na Bangchang et al., 1994) and rats (Li et al., 1998). Systemic drug exposure is generally greater when AM is given i.m. (Teja-Isavadharm et al., 1996; Li et al., 1998) but the bioavailability in severe malaria may be highly variable (Murphy et al., 1997). DHA in plasma is the only known human metabolite of AM (Na Bangchang et al., 1994; TejaIsavadharm et al., 1996) and hitherto plasma DHA has been the only identified metabolite in rats (Li et al., 1998). Demethylation of AM also occurs in mice (China Cooperative Research Group, 1982b; Lee This study was supported by the B. and V. Ax:son Johnson Foundation, the Wellcome Trust (L.P.D.B.), and F. Hoffmann-La Roche Ltd. (P.M.O’N.). The LC-MS system was purchased and maintained by means of grants from the Wellcome Trust. B.K.P. is a Wellcome Principal Research Fellow. A preliminary communication was presented at the Spring 1998 meeting of the British Pharmacological Society and published in abstract form [Bell et al. (1998) Br J Pharmacol 124 (Proceedings Suppl)42P]. 1 Abbreviations used are: AM, b-artemether; [C]AM, b-[13-C]AM; CID, collision-induced dissociation; DHA, dihydroartemisinin; DHG, dehydroglucuronic acid; LC-MS, liquid chromatography-mass spectrometry; LC-MS-MS, liquid chromatography-tandem mass spectrometry; UDPGA, uridine 59-diphosphoglu-