Introduction

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-Artemether (AM)¹ (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).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Artemisinin and derivatives.

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 3-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; Teja-Isavadharm 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 and Hufford, 1990). However, isolated perfused rat livers are reported to metabolize AM to desoxyDHA, 9-hydroxyAM, and 9-hydroxyAM, as well as DHA (Lee and Hufford, 1990). The metabolites of arteether (i.v.) in rat plasma (Chi et al., 1991; Ramu and Baker, 1997) and in rat liver microsomes (Hufford et al., 1990; Leskovac and Theoharides, 1991a) include DHA and several hydroxylated derivatives, one of which, 9-hydroxyarteether, is a highly active antimalarial in vitro (Chi et al., 1991). In contrast, most hydroxyl substituents render the endoperoxide pharmacologically inactive. Because 9- hydroxyAM of unspecified stereochemistry (Lee and Hufford, 1990) and 9-hydroxyarteether O-glucuronide (Ramu and Baker, 1995, 1997) are reported to possess activity in vitro, it is possible that hydroxylated metabolites contribute to the activity of AM in vivo.

We have characterized the biliary metabolites of -[13-¹⁴C]AM ([¹⁴C]AM) in rats with particular reference to isomeric compounds having radical precursors and C-9 hydroxylated derivatives, which might retain antimalarial activity.

	Materials and Methods

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Chemicals. AM was provided by Dr. P. Buchs (SAPEC SA Fine Chemicals, Lugano, Switzerland). [¹⁴C]AM (12.1 mCi/mmol) was provided by Dr. J. A. Kepler (Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, NC). It had a radiochemical purity of 99%: the drug was eluted (R_t 17.4 min) from a Beckman Ultrasphere 5-µm C₁₈ column (25 cm × 0.46 cm i.d.) with acetonitrile/water (3:2, v/v) at a flow rate of 1.2 ml min¹ and the radioactivity was measured using an on-line detector (Radiomatic Flo-Oneeta A250; Packard, Pangbourne, Berkshire, UK). [12-³H]DHA (1.4 Ci/mmol; radiochemical purity >99%) was obtained from Moravek Biochemicals Inc. (Brea, CA). Standards of 9- and 9-hydroxyAM and of 14-hydroxyAM (Abourashed and Hufford, 1996) were generously donated by Dr. C. D. Hufford, The University of Mississippi (Oxford, Mississippi). The sodium salts of -DHA -glucuronide and -DHA -glucuronide were provided by Ultrafine Chemicals (Salford, UK). DesoxyDHA and the furano acetate isomer of DHA were prepared from DHA and artemisinin, respectively, as described previously (Maggs et al., 1997); the direct reaction of DHA with iron (II) chloride tetrahydrate consistently yielded an unidentified isomer. The -glucuronidase preparaton from Helix pomatia (type H-2; 89.4 × 10³ U -glucuronidase/ml) and uridine 5'-diphosphoglucuronic acid (UDPGA) were purchased from Sigma Chemical Co. (Gillingham, Dorset, UK). HPLC grade solvents were products of Fisher Scientific (Loughborough, Leicestershire, UK).

Animal Experiments. Male Wistar rats (210-240 g) were anesthetized with urethane (1.4 g/ml isotonic saline; 1 ml/kg, i.p.). Cannulas were inserted into the jugular vein and common bile duct and the penis was ligated. [¹⁴C]AM (35 µmol/kg) dissolved in dimethyl sulfoxide was administered i.v. (10 µCi/kg; 250 µl). Bile was collected hourly for 5 h at room temperature; it was stored at 30°C until analyzed by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) with parallel radiometric detection. After 5 h, urine was aspirated from the bladder. Radioactivity in aliquots (50-µl) of bile and urine was assayed by liquid scintillation counting. Portions (ca. 50 mg) of the major internal organs and of skin were solubilized in 1 ml of OptiSolv (Wallac UK, Milton Keynes, Buckinghamshire, UK) at 50°C over 16 h. The solutions were decolorized with H₂O₂ (400 µl), neutralized with glacial acetic acid (60 µl), left in darkness overnight, and finally mixed with 20 ml of Ultima Gold (Packard Bioscience BV, Groningen, the Netherlands) for scintillation counting. The complete skin was stripped from each carcass, and the remainder immersed in 3 M KOH (200 ml) in a capped plastic bottle, which was maintained at 50°C for 16 h. An aliquot of the resulting liquid fraction (3 ml) was neutralized with concentrated HCl, and samples (1 ml) were taken for scintillation counting.

Enzymic Hydrolysis. Pooled bile (0- to 2-h collections; 80-200 µl) from two or three rats was diluted with 0.1 M sodium acetate, pH 5.0, (final volume, 1 ml) and incubated with -glucuronidase preparation (ca. 3 × 10³ U -glucuronidase) in a capped glass test tube at 37°C for 16 h. Thereafter, the incubation was extracted with methyl tert-butyl ether (5 volumes × 2). The pooled extracts were evaporated to dry residue at 35°C under a stream of oxygen-free nitrogen, reconstituted in methanol (150 µl), and analyzed immediately by LC-MS. Recovery of biliary radioactivity in the reconstituted extracts was 85 to 95%.

Isomerization of Endoperoxide Glucuronides. Aqueous solutions of synthetic - and -DHA -glucuronides (100 µl, 10 mM) were mixed with iron (II) chloride tetrahydrate in water (50 mM; final concentration, 1 mM) and incubated at 37°C for 3 h. The parent conjugate and its furano acetate and hydroxydesoxy isomers were resolved and identified by LC-MS. Bile (2 µl; 100 × 10³ dpm) from an isolated rat liver perfused with [³H]DHA in a single-pass system (Batty et al., 1998b) contained only [³H]DHA glucuronide (3.8 mM) by radiochromatographic and LC-MS analyses. It was diluted (2:93, v/v) with bile collected from an anesthetized rat before the animal was administered [¹⁴C]DHA. The mixture was incubated with iron (II) chloride in water (40 mM; final concentration, 10 mM) in a capped glass test tube at 37°C for 2 h. Bile (0- to 1-h collections; combined volumes, 120 µl) from two of the rats administered [¹⁴C]AM was treated in the same manner for up to 2.5 h. Each incubation was centrifuged periodically to sediment a precipitate, and the supernatant (30-50 µl) analyzed immediately by LC-MS.

Microsomal Glucuronylation. Hepatic microsomes from adult male Wistar rats were prepared by the standard differential centrifugation technique. They were incubated (final protein concentration, 3.5 mg/ml) with either [³H]DHA (0.1-1.0 mM; 0.1 µCi), desoxyDHA (0.1 mM), or the furano acetate isomer of DHA (0.1 mM) in polypropylene microcentrifuge tubes using a modified form of the method of Batty et al. (1998b). The complete mixtures contained MgCl₂ (5 mM) and UDPGA (3 mM) in Tris-HCl buffer (50 mM, pH 7.4; final volume, 250 µl). Reactions were initiated by adding the UDPGA, performed at 37°C for up to 30 min, and terminated with ice-cold methanol (170 µl). The mixtures were placed on ice. The precipitate was sedimented by centrifugation, and aliquots (100-µl) of the supernatant were analyzed by LC-MS.

LC-MS and LC-MS-MS Analyses. Positive- and negative-ion electrospray mass spectra were obtained by LC-MS using a Quattro II tandem quadrupole instrument fitted with the standard in-line source (Micromass, Manchester, UK). Eluent was delivered by twin Jasco PU-980 pumps (Jasco UK, Great Dunmow, Essex, UK). Samples were eluted from an Ultracarb 5-µm C₈ column (25 cm × 0.46 cm i.d.; Phenomenex, Macclesfield, UK) at 0.9 ml min¹ as follows: bile (20-100 µl), with a gradient of acetonitrile (10 to 20% over 10 min; 20% for 10 min; 20 to 30% over 15 min; 30 to 50% over 10 min) in 0.1 M ammonium acetate, pH 6.9, except that a modified gradient (10 to 30% over 20 min; 30 to 50% over 10 min; 50 to 70% over 5 min) was used to locate nonpolar metabolites; deconjugated biliary metabolites and coinjected standards, with a gradient of methanol (40 to 75% over 30 min) in the acetate buffer; microsomal glucuronylation mixtures, authentic DHA glucuronides, and isomers, with a gradient of acetonitrile (20 to 35% over 15 min; 35 to 70% over 10 min) in the acetate buffer. Eluate was split between a radioactivity flow detector and the LC-MS interface (ca. 40 µl min¹). The source temperature was 70°C; the capillary voltage, 3.9 × 10³ V (3.1 × 10³ V for anion analysis); the high voltage lens and radio frequency lens voltage, 0.2 × 10³ V (0.0 V) and 0.1 V (0.0 V), respectively. Spectra were acquired between m/z 100 to 1050 at one scan/5 s. Analyte fragmentation was achieved by increasing the cone voltage from 30 V (45 V). Collision-induced dissociation (CID) of the ammonium adducts of major metabolites in bile was achieved at a collision energy of 20 eV using argon at 9 × 10³ mBar. The cone voltage was 20 V. Daughter spectra were acquired between m/z 100 to 550 at one scan/5 s. All data were processed via MassLynx 2.1 software (Micromass Ltd., Manchester, UK).

Radiometric HPLC. Eluate was mixed with Ultima Flo AP scintillant (1.0 ml min¹; Packard Bioscience) in a Radiomatic detector, and the radiolabeled components were quantified after background subtraction using A250-1.6 software.

	Results

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Excretion and Distribution of Radioactivity. After the i.v. administration of [¹⁴C]AM (35 µmol/kg) to male rats, 23.4 ± 4.7 (mean ± S.D., n = 6), 11.0 ± 3.2, and 4.2 ± 1.0% of the radioactivity was excreted into bile during the first, second, and third hours, respectively; the biliary recovery over 5 h was 42.3 ± 4.3%. Only 5.4 ± 0.9% of the radioactivity was found in the bladder urine 5 h after dosing.

Microsomal Glucuronylation. [³H]DHA incubated with UDPGA was metabolized to a single glucuronide (R_t 12.5 min; 75% conversion of 0.1 mM [³H]DHA over 30 min), which gave an electrospray spectrum containing m/z 478 ([M + NH₄]⁺, 37), 267 ([478  NH₃  dehydroglucuronic acid (DHG)  H₂O]⁺, 31), 221 ([267  CO  H₂O]⁺, 16) and 163 ([221  (CH₃)₂CO]⁺, 100). DesoxyDHA underwent incomplete turnover to a glucuronide (R_t 12.5 min), which yielded m/z 462 ([M + NH₄]⁺, 46) and m/z 251 ([462  NH₃  DHG  H₂O]⁺, 100). The fragmentation pathways of ammonium adducts of artemisinin derivatives involving loss of ammonia, water, and CO have been characterized by Chi and Baker (1993). DHA furano acetate did not undergo glucuronylation although, in separate incubations, coincubated equimolar DHA was conjugated.

Isomerization of DHA Glucuronides. The synthetic - and -DHA -glucuronides (R_t 12.5 and 12.0 min, respectively) each yielded only two isomeric products when reacted with iron (II) chloride (R_t 5.5 and 10.0 min for the /-, and 4.5 and 10.0 min for the /-glucuronide, respectively). Analogous reactions of artemisinin compounds produce furano acetate and 3-hydroxydesoxy isomers (Jefford et al., 1996). The major furano acetate products were identified by their qualitatively identical electrospray spectra; specifically by the diagnostic loss of the acetate group (Chi and Baker, 1993; Maggs et al., 1997): for the / conjugate, m/z 478 ([M + NH₄]⁺, 100), 401 ([M + NH₄  NH₃  CH₃CO₂H]⁺, 7), 373 ([401  CO]⁺, 8), 267 ([M + NH₄ - NH₃  DHG  H₂O]⁺, 24), 225 ([401  DHG]⁺, 33), 207 ([225  H₂O]⁺, 23). The minor products, taken to be the hydroxydesoxy (epoxide) isomers, gave only one fragment: for the / conjugate, m/z 478 ([M + NH₄]⁺, 27) and m/z 267 (100).

Identification of the Biliary Metabolites of [¹⁴C]AM. The metabolites of [¹⁴C]AM in bile (0- to 3-h pooled collections; 38.6 ± 4.8% of dose) were resolved by HPLC and quantified radiometrically (Table 1). Three major radiolabeled components (III, R_t 14.0 min; V, 22.0 min; VII, 30.5 min) were found (Fig. 2); they and the lesser metabolites I (9.0 min), II (13.0 min), IV (15.5 min), and VI (25.0 min) were all present in the first hourly collection. Small amounts of a nonpolar compound (R_t 41.5 min; 2% chromatographed radioactivity), which was isobaric (m/z 302, [M + NH₄]⁺) with AM (R_t 44 min) were found occasionally.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   HPLC radiochromatogram of the biliary metabolites (pooled 0- to 3-h collections) of [14C]AM (35 µmol/kg, i.v.) in male rats.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   LC-MS selected-ion ([M+NH4]+) chromatograms of (III-VI and VIIa) glucuronides of monohydroxylated [14C]AM (m/z 508) and (VII) the glucuronide of [14C]DHA (m/z 478) in rat bile. The peak for hydroxyAM glucuronide II is not seen because of the facile fragmentation of the metabolite. The minor hydroxyAM glucuronide VIIa coeluted with VII but was characterized by LC-MS-MS.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Electrospray mass spectra of hydroxyAM glucuronides (a) III (assigned to 9-hydroxyAM glucuronide) and (b) V (position of substituent undetermined) in rat bile. Cone voltage was 70 V.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Daughter spectra obtained by CID of [M + NH4]+ for (a) furano acetate isomer of DHA glucuronide (parent ion, m/z 478) and (b) V (hydroxyAM glucuronide; parent ion, m/z 508) in rat bile.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Fragmentation pathways for hydroxyAM glucuronides rationalized from LC-MS-MS spectra of biliary metabolites. m/z 251 was characteristic of V.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Electrospray mass spectra of the major hydroxyAM aglycones recovered from enzymic hydrolysates of rat bile: was identified as 9-hydroxyAM and assigned to III (a); was assigned to V (b). Cone voltage was 50 V.

	Discussion

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The biliary metabolites of AM in male rats given the drug i.v. were predominantly the glucuronylated products of two oxidative pathways, namely O-demethylation and hydroxylation, operating in parallel (Fig. 8). Previous studies have established the role of biliary excretion in the elimination of artemisininoid metabolites from rats (Maggs et al., 1997; Batty et al., 1998b) and mice (China Cooperative Research Group, 1982b). An early report on the distribution of AM in rats noted that the highest levels of the drug were in brain 5 min after i.v. administration (Lee and Hufford, 1990) but the present measurements of total tissue radioactivity after 5 h indicate that this was a transitory concentration.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Metabolic scheme for the proposed biliary metabolites of [14C]AM in male rats. The position of the O-glucuronyl substituent in II, IV to VI, and VIIa, and the positions of the hydroxyl and O-glucuronyl substituents in I, could not be assigned from mass spectral data. Roman numerals refer to peaks in radiochromatograms (Fig. 2).

The glucuronylation of DHA in rats and humans was highly selective for the -epimer: DHA epimerizes rapidly in solution and both epimers are plasma metabolites of artesunate in patients (Batty et al., 1996) but only -DHA -glucuronide was found in human urine and rat bile. Ilett et al. (1998) have partially characterized the glucuronylation of DHA by human liver microsomes. Malaria infection reduces the glucuronylation of DHA by rat liver (Batty et al., 1998a). Notwithstanding the dealkylation of arteether in vivo (Chi et al., 1991; Li et al., 1998) and the conjugation of DHA derived metabolically from AM, DHA glucuronide is not detected in either the plasma or urine of rats given arteether (Ramu and Baker, 1997). In the rat, its excretion appears to be confined to bile. -DHA -glucuronide given i.v. to cannulated rats is eliminated rapidly in bile as a mixture of the parent conjugate and a small amount of furano acetate glucuronide (J.L.M., unpublished observation), and in the absence of hydrolysis by plasma -glucuronidase might undergo direct uptake into the liver.

The lack of sequential dealkylation and hydroxylation of AM, as deduced from the biliary metabolite profile, contrasts with the biotransformations of artemisinin endoperoxides in vitro. Thus DHA is converted extensively to four undefined hydroxylated products by rat liver microsomes (Leskovac and Theoharides, 1991a) and one of the partly characterized metabolites of arteether in a crude hepatic subcellular preparation is an isomer of hydroxyDHA (Baker et al., 1989). Arteether is deethylated by a number of human hepatic P-450s (Grace et al., 1998). A plasma metabolite of i.v. arteether has been assigned tentatively to 9-hydroxyDHA (Chi et al., 1991) but no hydroxylated DHA (glucuronide or aglycone) was located in bile using LC-MS during either this study or earlier studies on the biotransformations of DHA in vivo (Maggs et al., 1997) and in isolated perfused rat livers (Batty et al., 1998b). Subcellular incubations do not provide a balanced representation of the metabolic fate of artemisinin endoperoxides in vivo; in particular, the ratio of dealkylation to hydroxylation can be much higher in vitro (Baker et al., 1989; Chi et al., 1991). Demethylation of AM in mice is reported to be greater after i.v. versus i.m. dosing (Lee and Hufford, 1990).

Unlike microsomal hydroxylation of DHA, the relatively slow (NAD/NADH-dependent) deoxygenation of the endoperoxide function of DHA obtained with rat liver cytosol (Baker et al., 1989; Leskovac and Theoharides, 1991b) is reproduced in the biotransformations of AM in vivo. DHA rather than the parent drug is likely to be the substrate for reduction, as this seems to be the case with arteether (Baker et al., 1989; Chi et al., 1991). DesoxyDHA is also formed from AM by isolated rat livers (Lee and Hufford, 1990), and deoxygenation is a minor pathway for DHA in rats (Maggs et al., 1997) if not in isolated livers (Batty et al., 1998b). Desoxyartemisinin is a urinary metabolite of artemisinin in humans (Lee and Hufford, 1990). These epoxide products are pharmacologically inactive in vitro (Cumming et al., 1997) and in vivo (China Cooperative Research Group, 1982a).

The biliary metabolites of AM were examined for conjugates of two stable DHA isomers, namely 3-hydroxydesoxyDHA and the furano acetate, because their biomimetic iron (II)-mediated synthesis generates free radical intermediates via distinct pathways of one-electron reduction (Jefford et al., 1996; Butler et al., 1998). Examples of these derivatives are minor metabolites of arteether (Chi et al., 1991) and DHA (Maggs et al., 1997) in rats and of arteether (Baker et al., 1989), artelinate (Idowu et al., 1997), and DHA (Baker et al.,1989) in subcellular fractions. Arteflene, an unrelated synthetic antimalarial endoperoxide, undergoes chemically analogous but much more extensive bioactivation in the rat (Bishop et al., 1999). The present failure to locate a biliary glucuronide of either hydroxydesoxyAM or hydroxydesoxyDHA and the excretion of just trace amounts of the furano acetate of DHA as a glucuronide would appear to confirm the essential stability of the artemisininoid peroxide bridge in vivo. This stability is additionally emphasized by the absence from bile of glucuronides of AM and DHA diols, i.e., the products of reduction analogous to those produced by prostaglandin endoperoxide reductase (Burgess and Reddy, 1997); indeed, no diol metabolite of an artemisinin compound has been described to date. The restrained metabolism of the endoperoxides contrasts with the compounds' reactivity toward inorganic iron (II), which exceeds that of simple dialkyl peroxides (Butler et al., 1998). The mechanism(s) of rearrangement of endoperoxides in biological systems is unknown. The prostacyclin and thromboxane synthase hemethiolate enzymes (Ullrich and Brugger, 1994) appear the most likely enzymic candidates (Maggs et al., 1997) but only microsomal P-450 (Idowu et al., 1997) and hemoglobin (Blum et al., 1998) have been implicated experimentally; AM is isomerized when incubated with either blood or hemoglobin (Blum et al., 1998). A predominant role for blood is implied by the observation of Batty et al. (1998b) that isolated rat livers perfused with DHA in a synthetic medium only very rarely excrete isomers of DHA glucuronide into bile. Hepatic cytochrome P-450 seemingly catalyzes extensive hydroxylation and O-dealkylation without obtaining effective access to the peroxide bridge.

The major metabolite of AM in rat bile was identified as 9-hydroxyAM glucuronide. A regioselective hydroxylation at C-9 on the artemisinin skeleton is catalyzed by rat liver microsomes (Leskovac and Theoharides, 1991a) and is also suggested by the plasma metabolites of arteether in male rats (Ramu and Baker, 1997). However, an earlier analysis found a derivative identified tentatively as 3-hydroxyarteether at a higher plasma concentration than 9-hydroxyarteether (Chi et al., 1991). Because the former has a much shorter retention time on a reversed-phase column than the latter, whereas metabolites II to VIIa of AM elute either immediately before or after the isomeric 9-hydroxyAM glucuronide, it is thought improbable that one of the unassigned hydroxyAM conjugates is 3-hydroxyAM glucuronide. Although arteether is metabolized to 2- and 14-hydroxyarteether, as well as 9- and 9-hydroxyarteether, by rat liver microsomes (Hufford et al., 1990; Leskovac and Theoharides, 1991a), neither 2- nor 14-hydroxyAM glucuronide would be accounted prospective metabolite candidates because neither 2- nor 14-hydroxyarteether nor their conjugates has been detected in the plasma of rats given arteether (Chi et al., 1991; Ramu and Baker, 1997). Stereoselective C-9 hydroxylation occurs in rat hepatic microsomes (Leskovac and Theoharides, 1991a), and was first demonstrated in vivo when the - and -epimers of 9-hydroxyarteether were found in plasma at the ratios 3.4 (Ramu and Baker, 1997) and 6.5 (Chi et al., 1991) after i.v. administration of the drug. Similarly, both epimers of 9-hydroxyAM have been recovered from the perfusate of isolated rat livers (Lee and Hufford, 1990), although without quantification. Despite these findings, only the glucuronide of 9-hydroxyarteether could be detected in plasma (Ramu and Baker, 1997) and only the -epimer of 9-hydroxyAM was recovered from biliary conjugates, suggesting that the C-9 hydroxyl group is glucuronylated with a high degree of stereoselectively in rat liver.

The biliary metabolite profile of the rats differed somewhat from that of mice, which includes, in addition to DHA glucuronide (the major metabolite) and III to VI, a hydroxyAM glucuronide substituted on the O-methyl carbon (Bell et al., 1998).

Studies in volunteers suggest that AM and DHA-the only known metabolite of AM in humans-are responsible for the greater part of the plasma antimalarial activity (Teja-Isavadharm et al., 1996). Nevertheless, additional contributions to this activity cannot be discounted because 9-hydroxyarteether, although neither 9-, 2-, nor 14-hydroxyarteether, is strongly parasiticidal in vitro (Chi et al., 1991; Ramu and Baker, 1997). Furthermore, 9-hydroxyarteether glucuronide retains considerable antimalarial activity whereas DHA is essentially inactivated by glucuronylation (Ramu and Baker, 1995, 1997). The C-9 isomer of hydroxyAM is said to be active in vitro but the potency of the individual epimers has not been specified (Lee and Hufford, 1990). It is anticipated that the stereoselectivity obtained with 9-hydroxyarteether will apply to 9-hydroxyAM. In this case, male Wistar rats, which apparently achieve complete -selectivity, i.e., pharmacological deactivation, with AM's C-9 hydroxylated biliary metabolite, are unlikely to form a major pharmacologically active plasma metabolite in addition to DHA.