A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.

Examination of the cytotoxic and embryotoxic potential and underlying mechanisms of next-generation synthetic trioxolane and tetraoxane antimalarials.

Semisynthetic artemisinin-based therapies are the first-line treatment for P. falciparum malaria, but next-generation synthetic drug candidates are urgently required to improve availability and respond to the emergence of artemisinin-resistant parasites. Artemisinins are embryotoxic in animal models and induce apoptosis in sensitive mammalian cells. Understanding the cytotoxic propensities of antimalarial drug candidates is crucial to their successful development and utilization. Here, we demonstrate that, similarly to the model artemisinin artesunate (ARS), a synthetic tetraoxane drug candidate (RKA182) and a trioxolane equivalent (FBEG100) induce embryotoxicity and depletion of primitive erythroblasts in a rodent model. We also show that RKA182, FBEG100 and ARS are cytotoxic toward a panel of established and primary human cell lines, with caspase-dependent apoptosis and caspase-independent necrosis underlying the induction of cell death. Although the toxic effects of RKA182 and FBEG100 proceed more rapidly and are relatively less cell-selective than that of ARS, all three compounds are shown to be dependent upon heme, iron and oxidative stress for their ability to induce cell death. However, in contrast to previously studied artemisinins, the toxicity of RKA182 and FBEG100 is shown to be independent of general chemical decomposition. Although tetraoxanes and trioxolanes have shown promise as next-generation antimalarials, the data described here indicate that adverse effects associated with artemisinins, including embryotoxicity, cannot be ruled out with these novel compounds, and a full understanding of their toxicological actions will be central to the continuing design and development of safe and effective drug candidates which could prove important in the fight against malaria.

The pharmacokinetic evaluation of artemisinin drugs for the treatment of malaria in paediatric populations.

INTRODUCTION: The use of artemisinin combination therapies to treat uncomplicated malaria is growing and, therefore, so is the number of children exposed to these agents. As a result, there is a huge drive to develop paediatric formulations. However, relatively limited data exist regarding the pharmacokinetic properties of these drugs in this vulnerable population. AREAS COVERED: The article reviews the pharmacokinetic data for artemisinin drugs used for the treatment of malaria in paediatric populations. The authors discuss how developmental and environmental factors can produce significant variation in the pharmacokinetic properties of artemisinin drugs. The authors also discuss how this variation may lead to suboptimal therapeutic drug concentrations with implications on efficacy, safety and the development of parasite resistance to these drugs. EXPERT OPINION: There is currently a lack of published studies on the pharmacokinetics of artemisinin drugs in children and this subject is complicated by several interdependent variables. Therefore, the construction of a systems-based model of this subject should be a priority area in order to identify gaps in current knowledge to ensure their continued effective and safe use.

The role of heme and the mitochondrion in the chemical and molecular mechanisms of mammalian cell death induced by the artemisinin antimalarials.

The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ(0) cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC(50) values, 48 h: HeLa cells, 6 ± 3 µM; and HeLa ρ(0) cells, 34 ± 5 µM) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC(50) value and biomarker of bioactivation levels, respectively: 10ß-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 µM and 11 ± 5%; and with succinylacetone, >100 µM and 2 ± 5%).

Synthesis, in vitro and in vivo antimalarial assessment of sulfide, sulfone and vinyl amide-substituted 1,2,4-trioxanes prepared via thiol-olefin co-oxygenation (TOCO) of allylic alcohols.

Thiol-Olefin Co-Oxygenation (TOCO) methodology has been applied to the synthesis of a small library of weak base and polar 1,2,4-trioxanes. The 1,2,4-trioxane units synthesised exhibit remarkable stability as they survive base catalysed hydrolysis and mixed anhydride/amine coupling reactions. This unique stability feature has enabled a range of novel substitution patterns to be incorporated within the spiro 1,2,4-trioxane unit. Selected analogues express potent in vitro nM antimalarial activity, low cytotoxicity and oral activity in the Plasmodium berghei mouse model of malaria.

Design, synthesis and antimalarial/anticancer evaluation of spermidine linked artemisinin conjugates designed to exploit polyamine transporters in Plasmodium falciparum and HL-60 cancer cell lines.

A series of artemisinin-spermidine conjugates designed to utilise the upregulated polyamine transporter found in cancer cells have been prepared. These conjugates were evaluated against human promyelocytic leukaemia HL-60 cells and chloroquine-sensitive 3D7 Plasmodium falciparum and several show promising anticancer and antimalarial activity. Although some limitations in this vector-based approach are apparent, a number of high potency Boc-protected analogues were identified with activity against malaria parasites as low as 0.21nM.

Functional and toxicological consequences of metabolic bioactivation of methapyrilene via thiophene S-oxidation: Induction of cell defence, apoptosis and hepatic necrosis.

UNLABELLED: Methapyrilene, [N,N-dimethyl-N'-pyridyl-N'(2-thienylmethyl)-1,2-ethanediamine] (MP) was withdrawn from, clinical use due to reported periportal hepatic necrosis and hepatocarcinogenicity in the rat, via S-oxidation of the thiophene group. In this study MP is used as a model hepatotoxin to further characterise the functional consequences of S-oxidation of the thiophene group in vivo, in rat models and in vitro, in freshly isolated rat hepatocyte suspensions. In vivo histological studies revealed the early depletion of glutathione (GSH), which was confined to the damaged periportal area, in contrast to an increase in GSH levels in the centrilobular region. Additionally, the induction of cell defence was demonstrated by an increase in the protein levels of heme-oxygenase 1 (HO-1) and glutamate cysteine ligase, catalytic subunit (GCLC) in vivo. Histological examination demonstrated that cytotoxicity progresses initially via apoptosis before an increase in necrosis over the 3-day administration. An apoptotic-like mechanism was observed in vitro via the measurement of cytochrome c release and caspase activation. CONCLUSION: This study provides evidence for a complex pathway of MP-induced hepatotoxicity which progresses through early adaptation, apoptosis, necrosis and inflammation, all underpinned by the zonal induction and depletion of GSH within the liver.

Antitumour and antimalarial activity of artemisinin-acridine hybrids.

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.

Synthesis and biological evaluation of extraordinarily potent C-10 carba artemisinin dimers against P. falciparum malaria parasites and HL-60 cancer cells.

A series of artemisinin dimers incorporating a metabolically stable C-10 carba-linkage have been prepared, several of which show remarkable in vitro antimalarial activity (as low as 30 pM) versus Plasmodium falciparum and in vitro anticancer activity in the micromolar to nanomolar range versus HL-60 cell lines.

The role of bioactivation in the pharmacology and toxicology of the artemisinin-based antimalarials.

Artemisinin and artemisinin-based antimalarials are currently recommended as the frontline antimalarial treatment, with over 100 million courses administered annually. Despite possessing excellent therapeutic activity and tolerability, neurotoxicity and embryotoxicity have been reported in cross-species animal models. The endoperoxide group contained within the artemisinins is essential for pharmacologic and toxicological activity and it is hypothesized that its bioactivation to toxic C-centered radical species is the mechanistic basis for activity. The chemical nature of this activation and the ultimate toxic species remain controversial and this review will focus upon studies published on this topic over the past year. Furthermore, the possibility of chemical dissociation of pharmacology and toxicology in novel artemisinins will be discussed.

Evidence for the involvement of carbon-centered radicals in the induction of apoptotic cell death by artemisinin compounds.

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10beta-(p-bromophenoxy)dihydroartemisinin, and 10beta-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10beta-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G(0)/G(1) DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.

Antimalarial and antitumor evaluation of novel C-10 non-acetal dimers of 10beta-(2-hydroxyethyl)deoxoartemisinin.

Four series of C-10 non-acetal dimers were prepared from key trioxane alcohol 10beta-(2-hydroxyethyl)deoxoartemisinin (9b). All of the dimers prepared displayed potent low nanomolar antimalarial activity versus the K1 and HB3 strains of Plasmodium falciparum. The most potent compound assayed was phosphate dimer 14a, which was greater than 50 times more potent than the parent drug artemisinin and about 15 times more potent than the clinically used acetal artemether. In contrast to their potent activity versus malaria parasites, virtually all of the dimers expressed poor anticancer activity apart from the trioxane phosphate ester dimers 14a and 14b, which expressed nanomolar growth inhibitory (GI50) values versus a range of cancer cell lines in the NCI 60 human cell line screen. Further detailed studies on these dimers in vitro in HL60 cells demonstrate that both phosphate ester dimers (14a and 14b) are more potent than the anticancer agent doxorubicin. Interestingly, phosphate ester monomers 9c and 9d, antimalarially active in the low nanomolar region versus P. falciparum, are inactive as anticancer agents even at concentrations in the millimolar region. This observation emphasizes the importance of two trioxane units for high antiproliferative activity, and we propose that the nature of the linker in dimers of this type plays a crucial role in imparting potent anticancer activity.