Proposed reductive metabolism of artemisinin by glutathione transferases in vitro

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. I t is a promising new antimalarial and is particularly useful against the dr ug resistant strains of Plasmodium falciparum. It has unique antimalarial p roperties since it acts through the generation of free radicals that alkyla te parasite proteins. Since the antimalarial action of the drug is antagoni sed by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical re ductive reaction in the presence of glutathione transferases. Cytosolic glu tathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysi ng the formation of glutathione (GSH) conjugates and exhibit glutathione pe roxidase activity towards hydroperoxides. Artemisinin was incubated with gl utathione, NADPH and glutathione reductase and GSTs in a coupled assay syst em analogous to the standard assay scheme with cumene hydroperoxide as a su bstrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cyto sols from rat liver, kidney, intestines and in affinity purified preparatio ns of GSTs from rat liver. Using human recombinant GSTs hetelorogously expr essed in Escherichia coli, artemisinin was similarly shown to stimulate NAD PH oxidation with the highest activity observed with GST Ml-l. Using recomb inant GSTs the activity of GSTs with artemisinin was at least two fold high er than the reaction with CDNB. Considering these results, it is possible t hat GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH result ing in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter react ion may then result in the depletion of NADPH via GSSG-reductase. The abili ty of artemisinin to react with GSH in the presence of GST may be responsib le for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.