Studies on evolution of drug resistance in P. falciparum malaria in vivo and in vitro

The global burden of Malaria remains high with a 2.2 billion people estimated to live in endemic areas (Snow et al, 2005). Prompt and efficacious chemotherapy remains an important cornerstone in the fight against malaria. Development of resistance is the parasite’s mechanisms to respond to massive drug pressure. In this study I analysed drug-parasites interactions in vivo and in vitro using discreet experimental approaches. I started by studying parasite survival in vivo, 7 days after treatment intervention. In the following, I analysed parasites isolates in vitro that had been exposed to various drugs over the last years in the community. In the final part of the project, I attempted to gain insight in the evolution of drug resistance in vitro. For this work, I set up induction of resistance experiments in vitro and analysed in a time course mechanisms of resistance. Of particular interest was here the relationship between gene amplification and the emergence of single nucleotide polymorphisms. The results presented in this work show that 7 days after start of treatment (artemisinin combination therapy or amodiaquine) subpatent parasite populations were detected in 64/156 patients (41%) with high sensitivity. However, persistence of parasites was not associated with recrudescent infection at the individual level but showed instead correlation with initial parasite density in potentially less immune patients. Analysis of field isolates revealed high prevalence of anti-folate resistance due to triple and double mutant Pfdhfr. In theses parasites, no gene copy number variations were detected for genes involved in the folate pathway. However, induction of pyrimethamine resistance in vitro, lead to Pfdhfr amplification in two laboratory strains 3D7 (31 fold) and FCR3 (9 fold) associated with high cross-resistance to cycloguanil and trimethoprim but not to chloroquine. Further, I was able to identify gene amplification as a mechanism for acquisition of S108N in Pfdhfr. My findings of persistent subpatent parasitaemia after treatment underline the need of understanding clearance rate, persistence and elimination of parasites after anti- malarial treatment, particularly in regard to recent reports of emerging artemisinin resistance in western Cambodia (Dondorp et al, 2009), My results also suggest that resistant phenotypes may persist in parasite populations, once introduced. And finally, my data suggest gene amplification as an intermediate step in the acquisition of resistance conferring point mutations in P. falciparum

Plasmodium falciparum K13 Mutations Differentially Impact Ozonide Susceptibility and Parasite Fitness In Vitro

The emergence and spread in Southeast Asia of Plasmodium falciparum resistance to artemisinin (ART) derivatives, the cornerstone of first-line artemisinin-based combination therapies (ACTs), underscore the urgent need to identify suitable replacement drugs. Discovery and development efforts have identified a series of ozonides with attractive chemical and pharmacological properties that are being touted as suitable replacements. Partial resistance to ART, defined as delayed parasite clearance in malaria patients treated with an ART derivative or an ACT, has been associated with mutations in the P. falciparum K13 gene. In light of reports showing that ART derivatives and ozonides share similar modes of action, we have investigated whether parasites expressing mutant K13 are cross-resistant to the ozonides OZ439 (artefenomel) and OZ227 (arterolane). This work used a panel of culture-adapted clinical isolates from Cambodia that were genetically edited to express variant forms of K13. Phenotypic analyses employed ring-stage survival assays (ring-stage survival assay from 0 to 3 h [RSA0–3h]), whose results have earlier been shown to correlate with parasite clearance rates in patients. Our results document cross-resistance between OZ277 and dihydroartemisinin (DHA), a semisynthetic derivative of ART, in parasites carrying the K13 mutations C580Y, R539T, and I543T. For OZ439, we observed cross-resistance only for parasites that carried the rare K13 I543T mutation, with no evidence of cross-resistance afforded by the prevalent C580Y mutation. Mixed-culture competition experiments with isogenic lines carrying modified K13 revealed variable growth deficits depending on the K13 mutation and parasite strain and provide a rationale for the broad dissemination of the fitness-neutral K13 C580Y mutation throughout strains currently circulating in Southeast Asia

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment

A conserved metabolic signature associated with response to fast-acting anti-malarial agents

Characterizing the mode of action of anti-malarial compounds that emerge from high-throughput phenotypic screens is central to understanding how parasite resistance to these drugs can emerge. Here, we have employed untargeted metabolomics to inform on the mechanism of action of anti-malarial leads with different speed of kill profiles being developed by the Novartis Institute of Tropical Diseases (NITD). Time-resolved global changes in malaria parasite metabolite profiles upon drug treatment were quantified using liquid chromatography-based mass spectrometry and compared to untreated controls. Using this approach, we confirmed previously reported metabolomics profiles of the fast-killing (2.5 h) drug dihydroartemisinin (DHA) and the slower killing atovaquone. A slow-acting anti-malarial lead from NITD of imidazolopiperazine (IZP) class, GNF179, elicited little or no discernable metabolic change in malaria parasites in the same 2.5-h window of drug exposure. In contrast, fast-killing drugs, DHA and the spiroindolone (NITD246), elicited similar metabolomic profiles both in terms of kinetics and content. DHA and NITD246 induced peptide losses consistent with disruption of hemoglobin catabolism and also interfered with the pyrimidine biosynthesis pathway. Two members of the recently described class of anti-malarial agents of the 5-aryl-2-amino-imidazothiadiazole class also exhibited a fast-acting profile that featured peptide losses indicative of disrupted hemoglobin catabolism. Our screen demonstrates that structurally unrelated, fast-acting anti-malarial compounds generate similar biochemical signatures in Plasmodium pointing to a common mechanism associated with rapid parasite death. These profiles may be used to identify and possibly predict the mode of action of other fast-acting drug candidates

A G358S mutation in the Plasmodium falciparum Na⁺ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na(+) pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4(G358S) parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na(+) regulation. The G358S mutation reduces the affinity of PfATP4 for Na(+) and is associated with an increase in the parasite’s resting cytosolic [Na(+)]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

Characterization of Novel Antimalarial Compound ACT-451840: Preclinical Assessment of Activity and Dose-Efficacy Modeling.

BACKGROUND: Artemisinin resistance observed in Southeast Asia threatens the continued use of artemisinin-based combination therapy in endemic countries. Additionally, the diversity of chemical mode of action in the global portfolio of marketed antimalarials is extremely limited. Addressing the urgent need for the development of new antimalarials, a chemical class of potent antimalarial compounds with a novel mode of action was recently identified. Herein, the preclinical characterization of one of these compounds, ACT-451840, conducted in partnership with academic and industrial groups is presented. METHOD AND FINDINGS: The properties of ACT-451840 are described, including its spectrum of activities against multiple life cycle stages of the human malaria parasite Plasmodium falciparum (asexual and sexual) and Plasmodium vivax (asexual) as well as oral in vivo efficacies in two murine malaria models that permit infection with the human and the rodent parasites P. falciparum and Plasmodium berghei, respectively. In vitro, ACT-451840 showed a 50% inhibition concentration of 0.4 nM (standard deviation [SD]: ± 0.0 nM) against the drug-sensitive P. falciparum NF54 strain. The 90% effective doses in the in vivo efficacy models were 3.7 mg/kg against P. falciparum (95% confidence interval: 3.3-4.9 mg/kg) and 13 mg/kg against P. berghei (95% confidence interval: 11-16 mg/kg). ACT-451840 potently prevented male gamete formation from the gametocyte stage with a 50% inhibition concentration of 5.89 nM (SD: ± 1.80 nM) and dose-dependently blocked oocyst development in the mosquito with a 50% inhibitory concentration of 30 nM (range: 23-39). The compound's preclinical safety profile is presented and is in line with the published results of the first-in-man study in healthy male participants, in whom ACT-451840 was well tolerated. Pharmacokinetic/pharmacodynamic (PK/PD) modeling was applied using efficacy in the murine models (defined either as antimalarial activity or as survival) in relation to area under the concentration versus time curve (AUC), maximum observed plasma concentration (Cmax), and time above a threshold concentration. The determination of the dose-efficacy relationship of ACT-451840 under curative conditions in rodent malaria models allowed prediction of the human efficacious exposure. CONCLUSION: The dual activity of ACT-451840 against asexual and sexual stages of P. falciparum and the activity on P. vivax have the potential to meet the specific profile of a target compound that could replace the fast-acting artemisinin component and harbor additional gametocytocidal activity and, thereby, transmission-blocking properties. The fast parasite reduction ratio (PRR) and gametocytocidal effect of ACT-451840 were recently also confirmed in a clinical proof-of-concept (POC) study

Efficacy of Cipargamin (KAE609) in a Randomized, Phase II Dose-Escalation Study in Adults in Sub-Saharan Africa With Uncomplicated Plasmodium falciparum Malaria.

BACKGROUND: Cipargamin (KAE609) is a potent antimalarial in a phase II trial. Here we report efficacy, pharmacokinetics, and resistance marker analysis across a range of cipargamin doses. These were secondary endpoints from a study primarily conducted to assess the hepatic safety of cipargamin (hepatic safety data are reported elsewhere). METHODS: This phase II, multicenter, randomized, open-label, dose-escalation trial was conducted in sub-Saharan Africa in adults with uncomplicated Plasmodium falciparum malaria. Cipargamin monotherapy was given as single doses up to 150 mg or up to 50 mg once daily for 3 days, with artemether-lumefantrine as control. Key efficacy endpoints were parasite clearance time (PCT), and polymerase chain reaction (PCR)-corrected and uncorrected adequate clinical and parasitological response (ACPR) at 14 and 28 days. Pharmacokinetics and molecular markers of drug resistance were also assessed. RESULTS: All single or multiple cipargamin doses ≥50 mg were associated with rapid parasite clearance, with median PCT of 8 hours versus 24 hours for artemether-lumefantrine. PCR-corrected ACPR at 14 and 28 days was >75% and 65%, respectively, for each cipargamin dose. A treatment-emerging mutation in the Pfatp4 gene, G358S, was detected in 65% of treatment failures. Pharmacokinetic parameters were consistent with previous data, and approximately dose proportional. CONCLUSIONS: Cipargamin, at single doses of 50 to 150 mg, was associated with very rapid parasite clearance, PCR-corrected ACPR at 28 days of >65% in adults with uncomplicated P. falciparum malaria, and recrudescent parasites frequently harbored a treatment-emerging mutation. Cipargamin will be further developed with a suitable combination partner. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov (NCT03334747)

Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 μM) and achieves artemisinin-like kill kinetics in vitro with a parasite clearance time of \u3c24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials

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