Peroxide Bond-Dependent Antiplasmodial Specificity of Artemisinin and OZ277 (RBx11160)

Abstract

Using nonperoxidic analogs of artemisinin and OZ277 (RBx11160), the strong in vitro antiplasmodial activities of the latter two compounds were shown to be peroxide bond dependent. In contrast, the weak activities of artemisinin and OZ277 against six other protozoan parasites were peroxide bond independent. These data support the iron-dependent artemisinin alkylation hypothesis.The antimalarial artemisinin contains a pharmacophoric peroxide bond within its 1,2,4-trioxane heterocycle. A longstanding hypothesis (17,24,29) to account for the antimalarial specificity of artemisinin is that the peroxide bond undergoes reductive activation by heme released by parasite hemoglobin (Hb) digestion. Although most (95%) of the heme released from parasite digestion is incorporated into the relatively inert hemozoin (12), it is estimated (8) that there is some 100 M of "free" heme/hematin in malaria-infected red blood cells, a more than adequate quantity to react with artemisinin. The irreversible redox reaction between artemisinin and heme produces carbon-centered free radicals or carbocations that alkylate heme (25,32,33) and membrane-associated parasite proteins (2), one of which is the translationally controlled tumor protein (PfTCTP) (4) and another is likely to be PfATP6, a SERCA-type Ca 2ϩ -ATPase (11). Eckstein-Ludwig et al. (11) studied the latter protein in some detail and provided evidence that it may be an important target of the artemisinins.The structural diversity of semisynthetic artemisinins (41) and synthetic peroxides (37) is quite remarkable and, we suggest, has implications for the mechanism of action for this class of antimalarial drugs. In this respect, we thought it might be useful to compare the activities of artemisinin and synthetic peroxide (1,2,4-trioxolane) OZ277 (RBx11160) (40) and their nonperoxidic counterparts deoxyartemisinin (6) and carbaOZ277 (Fig. 1) against a range of protozoan parasites. CarbaOZ277 (1,3-dioxolane) was prepared as a mixture of cis-trans isomers by an acidcatalyzed reaction (9) between the requisite adamantane diol and amino amide cyclohexanone. In vitro antiprotozoal assays for Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, Giardia duodenalis, Babesia divergens, and Neospora caninum were performed as previously described (1,5,16,28,35,38,40) with standard drug controls ( Table 1).The data in Table 1 shows a clear demarcation in the antiplasmodial activities of artemisinin and OZ277 versus their nonperoxidic analogs deoxyartemisinin (6) and carbaOZ277, confirming the peroxide bond-dependent activity of the former. For artemisinin and OZ277, there were losses of activity greater than 3 orders of magnitude against all of the other protozoa compared to activities against P. falciparum. In contrast, for deoxyartemisinin and carbaOZ277, there were only small differences in activity between P. falciparum and the other protozoa. Only for T. b. rhodesiense was there a significant difference between artemisinin and deoxyartemisinin ve...

Artemisinin activity againstPlasmodium falciparumrequires hemoglobin uptake and digestion

“…Artemisinin is highly potent against hemoglobin-digesting stages of malaria parasites (7,30) and against other hemoglobin-degrading pathogens such as Schistosoma species (31), but it is much less potent against other pathogens (32). This suggests that hemoglobin digestion plays a critical role in the mechanism of action of this drug class.…”

“…Similarly, we anticipate that use of endoperoxides with longer exposure in the bloodstream will prevent parasite escape via short-term growth arrest. In this regard synthetic endoperoxides with improved in vivo half-lives that are currently under development hold great promise (32,41,42). A better appreciation of the interaction of artemisinins and partner drugs with the hemoglobin degradation pathway may uncover additional strategies to protect the vital resource of endoperoxide antimalarials.…”

“…To confirm the importance of hemoglobin degradation in the action of artemisinin we examined the effect of pretreatment of parasites with the cysteine protease inhibitors ALLN and E-64. We designed these experiments to focus on events in the mid ring to mid trophozoite stages of infection to distinguish hemoglobinase inhibitor effects from those on schizont rupture and invasion (32). We found that cysteine protease inhibitors potently antagonized artemisinin action.…”

Spiroadamantyl 1,2,4-trioxolane, 1,2,4-trioxane, and 1,2,4-trioxepane pairs: Relationship between peroxide bond iron(II) reactivity, heme alkylation efficiency, and antimalarial activity

“…1 One hypothesis [2][3][4] that accounts for the antimalarial specificity of artemisinin 5 is that the peroxide bond undergoes reductive activation by heme released by parasite hemoglobin digestion to produce carbon-centered free radicals or carbocations that alkylate heme 6 or parasite proteins. Although the 1,2,4-trioxane heterocycle in artemisinin (Figure 1) is the critical pharmacophore, its presence alone 7 is insufficient for high antimalarial activity as most synthetic 1,2,4-trioxanes are less active than artemisinin.…”

Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria

“…1B), also known as RBx11160 or arterolane maleate, was the first synthetic ozonide to be evaluated clinically and is now in Phase III clinical trials as a combination product with piperaquine phosphate (12). Like the artemisinins, OZ277 contains a pharmacophoric peroxide bond, which is essential for activity (13,14), exhibits antimalarial activity against all asexual blood stages of P. falciparum (15,16) and has a rapid onset of action in an established murine model of malaria (11). However, in Phase I clinical trials, the half-life in healthy volunteers was only about two-to threefold longer than that of dihydroartemisinin (DHA) (17), the active metabolite of clinically used semisynthetic ART derivatives.…”