Protein binding and α:β anomer ratio of dihydroartemisinin in vivo

To determine the ratio of α : β anomers and the protein binding of dihydroartemisinin (DHA) in vivo. 10-[³H]-DHA was synthesized by reduction of artemisinin with sodium boro-[³H]-hydride and purified with preparative thin layer chromatography. A solution of ³H-DHA (2000 ng in 20 µl) was added to 2 ml whole blood from 15 healthy volunteers and 22 Vietnamese patients with falciparum or vivax malaria. The blood was centrifuged and the plasma stored at −25 °C until analysed by HPLC with radiochromatographic detection. Protein-free ultrafiltrate of the plasma was assayed to determine the free fraction of DHA and the in vivo ratio of α-DHA : β-DHA. The DHA fraction unbound (mean ± SD) was 0.068 ± 0.032 in Vietnamese patients with falciparum malaria ( n = 17), 0.065 ± 0.009 in Vietnamese patients with vivax malaria ( n = 5), 0.117 ± 0.015 in Vietnamese volunteers ( n = 7) and 0.092 ± 0.020 in Caucasian volunteers ( n = 8). The ratios of α-DHA : β-DHA for the four groups were 6.3 ± 0.9, 6.9 ± 0.8, 6.9 ± 0.6 and 5.4 ± 0.8, respectively. DHA is approximately 93% protein-bound in patients with malaria infection and there is a preferential existence in vivo of the α-DHA anomer. Knowledge of this stereochemistry may be valuable in elucidation of the mechanisms of DHA action and/or toxicity, and in the synthesis of new trioxane antimalarials.