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Structural dependency of iron- and blood-mediated degradation and heme alkylation for a series of novel ozonides
thesisposted on 14.02.2017, 01:46 by Stingelin, Lukas
Malaria is a devastating disease prevalent in (sub)tropical regions and causes considerable morbidity and mortality. Infection is caused by the hematoprotozoan Plasmodium parasite and transmitted by female anopheline mosquitoes. Drug therapy is critical for the treatment of malaria, however many well-established antimalarial drugs have become ineffective due to parasite resistance. Newer treatments, such as artemisinin combination therapies, are highly effective, but cost, availability and poor biopharmaceutical properties hamper their utility. Alarming reports have also suggested that resistance to the artemisinins may be emerging. A new class of fully synthetic antimalarial compounds, the 1,2,4-trioxolanes or ozonides, overcome many of the limitations seen with the artemisinins, and two compounds of this class, OZ277 and OZ439, have advanced to clinical trials. There is still controversy surrounding the mechanism of action of peroxide antimalarials. The peroxide is required for activity, and many investigators believe that it is activated in the presence of high concentrations of iron(II) species liberated upon hemoglobin digestion within the parasite. Radical species formed are then thought to alkylate parasite targets, leading to parasite death. Peroxides are known to be unstable in environments rich in iron(II), suggesting a link between the processes leading to peroxide degradation and those required for activation and ultimately, antimalarial activity. Based upon this fundamental hypothesis, the goals of this thesis were to characterize the stability and partitioning of representative peroxide antimalarials, to define structural trends in iron- and blood-mediated degradation, to further confirm the link between heme alkylation and biological activity and to elucidate some of the mechanisms involved in these degradation processes and link these to the proposed mechanisms of action. Using a selection of ozonides across a wide range of structures and antimalarial activity, clear structural relationships were defined for their stability in iron(II) sulphate solutions as well as in a biologically-relevant blood-like matrix. Previous correlations between the extent of heme alkylation and in vitro potency were confirmed using the more extensive and structurally diverse set of compounds. Partitioning and degradation studies with OZ277 (as a model ozonide) in a blood-like matrix demonstrated that the degradation rate depended on hematocrit, parasitemia, and compound concentration. Using structurally diverse compounds, there was no evidence of selective accumulation in infected erythrocytes under the conditions used, and degradation increased significantly in infected compared to non-infected blood. Blood-mediated degradation of OZ277 was substantially reduced in presence of an iron chelating agent suggesting a role of chelatable iron which was further supported by the correlation between iron- with blood-mediated degradation. Conversely, the chelating agent only partially reduced the degradation rate in infected blood, indicating additional degradation mechanisms (possibly linked to the mechanism of action). The reduced recovery of one of the metabolites, adamantane lactone, in infected blood is also consistent with this hypothesis. OZ277 rapidly and extensively degraded with digested hemoglobin, but was stable in the presence of non-degraded hemoglobin. This degradation was unaffected by inclusion of the iron chelator, but was completely eliminated in presence of an oxidizing agent. These results are consistent with non-chelatable iron(II) heme released upon hemoglobin digestion being at least partly responsible for OZ277 degradation in infected blood. These studies provide further insight into biological processes related to iron-mediated reactivity of this novel series of compounds.