GLUCURONIDATION OF DIHYDROARTEMISININ IN VIVO AND BY HUMAN LIVER MICROSOMES AND EXPRESSED UDP-GLUCURONOSYLTRANSFERASES

The aim of this study was to elucidate the metabolic pathways for dihydroartemisinin (DHA), the active metabolite of the artemisinin derivative artesunate (ARTS). Urine was collected from 17 Vietnamese adults with falciparum malaria who had received 120 mg of ARTS i.v., and metabolites were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Human liver microsomes were incubated with [12- 3 H]DHA and cofactors for either glucuronidation or cytochrome P450-catalyzed oxidation. Human liver cytosol was incubated with cofactor for sulfation. Metabolites were detected by HPLC-MS and/or HPLC with radiochemical detection. Metabolism of DHA by recombinant human UDP-glucuronosyltransferases (UGTs) was studied. HPLC-MS analysis of urine identified α-DHA-β-glucuronide (α-DHA-G) and a product characterized as the tetrahydrofuran isomer of α-DHA-G. DHA was present only in very small amounts. The ratio of the tetrahydrofuran isomer, α-DHA-G, was highly variable (median 0.75; range 0.09–64). Nevertheless, α-DHA-G was generally the major urinary product of DHA glucuronidation in patients. The tetrahydrofuran isomer appeared to be at least partly a product of nonenzymic reactions occurring in urine and was readily formed from α-DHA-G by iron-mediated isomerization. In human liver microsomal incubations, DHA-G (diastereomer unspecified) was the only metabolite found ( V max 177 ± 47 pmol min −1 mg −1 , K m 90 ± 16 μM). α-DHA-G was formed in incubations of DHA with expressed UGT1A9 ( K m 32 μM, V max 8.9 pmol min −1 mg −1 ) or UGT2B7 ( K m 438 μM, V max 10.9 pmol mg −1 min −1 ) but not with UGT1A1 or UGT1A6. There was no significant metabolism of DHA by cytochrome-P450 oxidation or by cytosolic sulfotransferases. We conclude that α-DHA-G is an important metabolite of DHA in humans and that its formation is catalyzed by UGT1A9 and UGT2B7.