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

Aims To determine the ratio of alpha : beta anomers and the protein binding of dihydroartemisinin (DHA) in vivo. Methods 10-[H-3]-DHA was synthesized by reduction of artemisinin with sodium boro-[H-3]-hydride and purified with preparative thin layer chromatography. A solution of H-3-DHA (2000 ng in 20 mul) 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 degreesC 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 alpha-DHA : beta-DHA. Results 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 alpha-DHA : beta-DHA for the four groups were 6.3 +/- 0.9, 6.9 +/- 0.8, 6.9 +/- 0.6 and 5.4 +/- 0.8, respectively. Conclusions DHA is approximately 93% protein-bound in patients with malaria infection and there is a preferential existence in vivo of the alpha-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.