Ho‐Ning Wong

Facile oxidation of leucomethylene blue and dihydroflavins by artemisinins: relationship with flavoenzyme function and antimalarial mechanism of action.

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin‐dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH2 initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADH2 for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N‐benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two‐electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AA–MB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two‐electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhanced by artemisinins, especially under aerobic conditions. Recombinant human GR is not affected. Artemisinins thus may act as antimalarial drugs by perturbing the redox balance within the malaria parasite, both by oxidising FADH2 in parasite GR or other parasite flavoenzymes, and by initiating autoxidation of the dihydroflavin by oxygen with generation of ROS. Reduction of the artemisinin is proposed to occur via hydride transfer from LMB or the dihydroflavin to O1 of the peroxide. This hitherto unrecorded reactivity profile conforms with known structure–activity relationships of artemisinins, is consistent with their known ability to generate ROS in vivo, and explains the synergism between artemisinins and redox‐active antimalarial drugs such as MB and doxorubicin. As the artemisinins appear to be relatively inert towards human GR, a putative model that accounts for the selective potency of artemisinins towards the malaria parasite also becomes apparent. Decisively, ferrous iron or carbon‐centered free radicals cannot be involved, and the reactivity described herein reconciles disparate observations that are incompatible with the ferrous iron–carbon radical hypothesis for antimalarial mechanism of action. Finally, the urgent enquiry into the emerging resistance of the malaria parasite to artemisinins may now in one part address the possibilities either of structural changes taking place in parasite flavoenzymes that render the flavin cofactor less accessible to artemisinins or of an enhancement in the ability to use intra‐erythrocytic human disulfide reductases required for maintenance of parasite redox balance.

Synthesis of Artemiside and Its Effects in Combination with Conventional Drugs against Severe Murine Malaria

ABSTRACT This research describes the use of novel antimalarial combinations of the new artemisinin derivative artemiside, a 10-alkylamino artemisinin. It is a stable, highly crystalline compound that is economically prepared from dihydroartemisinin in a one-step process. Artemiside activity was more pronounced than that of any antimalarial drug in use, both in Plasmodium falciparum culture and in vivo in a murine malaria model depicting cerebral malaria (CM). In vitro high-throughput testing of artemiside combinations revealed a large number of conventional antimalarial drugs with which it was additive. Following monotherapy in mice, individual drugs reduced parasitemias to nondetectable levels. However, after a period of latency, parasites again were seen and eventually all mice became terminally ill. Treatment with individual drugs did not prevent CM in mice with recrudescent malaria, except for piperaquine at high concentrations. Even when CM was prevented, the mice developed later of severe anemia. In contrast, most of the mice treated with drug combinations survived. A combination of artemiside and mefloquine or piperaquine may confer an optimal result because of the longer half life of both conventional drugs. The use of artemiside combinations revealed a significant safety margin of the effective artemiside doses. Likewise, a combination of 1.3 mg/kg of body weight artemiside and 10 mg/kg piperaquine administered for 3 days from the seventh day postinfection was completely curative. It appears possible to increase drug concentrations in the combination therapy without reaching toxic levels. Using the drug combinations as little as 1 day before the expected death of control animals, we could prevent further parasite development and death due to CM or anemic malaria. Earlier treatment may prevent cognitive dysfunctions which might occur after recovery from CM.

Preparation of N-Sulfonyl- and N-Carbonyl-11-Azaartemisinins with Greatly Enhanced Thermal Stabilities: in vitro Antimalarial Activities

As the clinically used artemisinins do not withstand the thermal stress testing required to evaluate shelf life for storage in tropical countries where malaria is prevalent, there is a need to develop thermally more robust artemisinin derivatives. Herein we describe the attachment of electron‐withdrawing arene‐ and alkanesulfonyl and ‐carbonyl groups to the nitrogen atom of the readily accessible Ziffer 11‐azaartemisinin to provide the corresponding N‐sulfonyl‐ and ‐carbonylazaartemisinins. Two acylurea analogues were also prepared by treatment of the 11‐azaartemisinin with arylisocyanates. Several of the N‐sulfonylazaartemisinins have melting points above 200 °C and possess substantially greater thermal stabilities than the artemisinins in current clinical use, with the antimalarial activities of several of the arylsulfonyl derivatives being similar to that of artesunate against the drug‐sensitive 3D7 clone of the NF54 isolate and the multidrug‐resistant K1 strain of P. falciparum. The compounds possess relatively low cytotoxicities. The carbonyl derivatives are less crystalline than the N‐sulfonyl derivatives, but are generally more active as antimalarials. The N‐nitroarylcarbonyl and arylurea derivatives possess sub‐ng ml−1 activities. Although several of the azaartemisinins possess log P values below 3.5, the compounds have poor aqueous solubility (<1 mg L−1 at pH 7). The greatly enhanced thermal stability of our artemisinins suggests that strategic incorporation of electron‐withdrawing polar groups into both new artemisinin derivatives and totally synthetic trioxanes or trioxolanes may assist in the generation of practical new antimalarial drugs which will be stable to storage conditions in the field, while retaining favorable physicochemical properties.

Artemisone Uptake in Plasmodium falciparum-Infected Erythrocytes

ABSTRACT Artemisone is one of the most promising artemisinin derivatives in clinical trials. Previous studies with radiolabeled artemisinin and dihydroartemisinin have measured uptake in Plasmodium falciparum-infected erythrocytes. Uptake is much greater in infected than in uninfected erythrocytes, but the relative contributions of transport, binding, and metabolism to this process still await definition. In this study, we characterized mechanisms by which [14C]artemisone is taken up into uninfected and P. falciparum-infected human erythrocytes in vitro. Radiolabeled artemisone rapidly enters uninfected erythrocytes without much exceeding extracellular concentrations. Unlabeled artemisone does not compete in this process. Radiolabeled artemisone is concentrated greatly by a time- and temperature-dependent mechanism in infected erythrocytes. This uptake is abrogated by unlabeled artemisone. In addition, the uptake of artemisone into three subcellular fractions, and its distribution into these fractions, is examined as a function of parasite maturation. These data are relevant to an understanding of the mechanisms of action of this important class of drugs.