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The development and spread of drug-resistant phenotypes substantially threaten malaria control efforts. Combination therapies have the potential to minimize the risk of resistance development but require intensive preclinical studies to determine optimal combination and dosing regimens. To support the selection of new combinations, we developed a novel in vitro-in silico combination approach to help identify the pharmacodynamic interactions of the two antimalarial drugs in a combination which can be plugged into a pharmacokinetic/pharmacodynamic model built with human monotherapy parasitological data to predict the parasitological endpoints of the combination. This makes it possible to optimally select drug combinations and doses for the clinical development of antimalarials. With this assay, we successfully predicted the endpoints of two phase 2 clinical trials in patients with the artefenomel-piperaquine and artefenomel-ferroquine drug combinations. In addition, the predictive performance of our novel in vitro model was equivalent to that of the humanized mouse model outcome. Last, our more informative in vitro combination assay provided additional insights into the pharmacodynamic drug interactions compared to the in vivo systems, e.g., a concentration-dependent change in the maximum killing effect ( E max ) and the concentration producing 50% of the killing maximum effect (EC 50 ) of piperaquine or artefenomel or a directional reduction of the EC 50 of ferroquine by artefenomel and a directional reduction of E max of ferroquine by artefenomel. Overall, this novel in vitro-in silico -based technology will significantly improve and streamline the economic development of new drug combinations for malaria and potentially also in other therapeutic areas.

Plasmodium falciparum sporozoite (PfSPZ) direct venous inoculation (DVI) using cryopreserved, infectious PfSPZ (PfSPZ Challenge [Sanaria, Rockville, Maryland]) is an established controlled human malaria infection model. However, to evaluate new chemical entities with potential blood-stage activity, more detailed data are needed on safety, tolerability, and parasite clearance kinetics for DVI of PfSPZ Challenge with established schizonticidal antimalarial drugs. This open-label, phase Ib study enrolled 16 malaria-naïve healthy adults in two cohorts (eight per cohort). Following DVI of 3,200 PfSPZ (NF54 strain), parasitemia was assessed by quantitative polymerase chain reaction (qPCR) from day 7. The approved antimalarial artemether-lumefantrine was administered at a qPCR-defined target parasitemia of ≥ 5,000 parasites/mL of blood. The intervention was generally well tolerated, with two grade 3 adverse events of neutropenia, and no serious adverse events. All 16 participants developed parasitemia after a mean of 9.7 days (95% CI 9.1-10.4) and a mean parasitemia level of 511 parasites/mL (95% CI 369-709). The median time to reach ≥ 5,000 parasites/mL was 11.5 days (95% CI 10.4-12.4; Kaplan-Meier), at that point the geometric mean (GM) parasitemia was 15,530 parasites/mL (95% CI 10,268-23,488). Artemether-lumefantrine was initiated at a GM of 12.1 days (95% CI 11.5-12.7), and a GM parasitemia of 6,101 parasites/mL (1,587-23,450). Mean parasite clearance time was 1.3 days (95% CI 0.9-2.1) and the mean log10 parasite reduction ratio over 48 hours was 3.6 (95% CI 3.4-3.7). This study supports the safety, tolerability, and feasibility of PfSPZ Challenge by DVI for evaluating the blood-stage activity of candidate antimalarial drugs.

The rate at which parasitemia declines in a host after treatment with an antimalarial drug is a major metric for assessment of antimalarial drug activity in preclinical models and in early clinical trials. However, this metric does not distinguish between viable and nonviable parasites. Thus, enumeration of parasites may result in underestimation of drug activity for some compounds, potentially confounding its use as a metric for assessing antimalarial activity in vivo . Here, we report a study of the effect of artesunate on Plasmodium falciparum viability in humans and in mice. We first measured the drug effect in mice by estimating the decrease in parasite viability after treatment using two independent approaches to estimate viability. We demonstrate that, as previously reported in humans, parasite viability declines much faster after artesunate treatment than does the decline in parasitemia (termed parasite clearance). We also observed that artesunate kills parasites faster at higher concentrations, which is not discernible from the traditional parasite clearance curve and that each subsequent dose of artesunate maintains its killing effect. Furthermore, based on measures of parasite viability, we could accurately predict the in vivo recrudescence of infection. Finally, using pharmacometrics modeling, we show that the apparent differences in the antimalarial activity of artesunate in mice and humans are partly explained by differences in host removal of dead parasites in the two hosts. However, these differences, along with different pharmacokinetic profiles, do not fully account for the differences in activity. (This study has been registered with the Australian New Zealand Clinical Trials Registry under identifier ACTRN12617001394336.).

Background: Interventions that effectively target Plasmodium vivax are critical for the future control and elimination of malaria. We conducted a P. vivax volunteer infection study to characterize the antimalarial activity of artefenomel, a new drug candidate., Methods: Eight healthy, malaria-naive participants were intravenously inoculated with blood-stage P. vivax and subsequently received a single oral 200-mg dose of artefenomel. Blood samples were collected to monitor the development and clearance of parasitemia, and plasma artefenomel concentration. Mosquito feeding assays were conducted before artefenomel dosing to investigate parasite transmissibility., Results: Initial parasite clearance occurred in all participants after artefenomel administration (log10 parasite reduction ratio over 48 hours, 1.67; parasite clearance half-life, 8.67 hours). Recrudescence occurred in 7 participants 11-14 days after dosing. A minimum inhibitory concentration of 0.62 ng/mL and minimum parasiticidal concentration that achieves 90% of maximum effect of 0.83 ng/mL were estimated, and a single 300-mg dose was predicted to clear 109 parasites per milliliter with 95% certainty. Gametocytemia developed in all participants and was cleared 4-8 days after dosing. At peak gametocytemia, 75% of participants were infectious to mosquitoes., Conclusions: The in vivo antimalarial activity of artefenomel supports its further clinical development as a treatment for P. vivax malaria., Clinical Trials Registration: NCT02573857., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Ensuring continued success against malaria depends on a pipeline of new antimalarials. Antimalarial drug development utilizes preclinical murine and experimental human malaria infection studies to evaluate drug efficacy. A sequential approach is typically adapted, with results from each stage informing the design of the next stage of development. The validity of this approach depends on confidence that results from murine malarial studies predict the outcome of clinical trials in humans. Parasite clearance rates following treatment are key parameters of drug efficacy. To investigate the validity of forward predictions, we developed a suite of mathematical models to capture parasite growth and drug clearance along the drug development pathway and estimated parasite clearance rates. When comparing the three infection experiments, we identified different relationships of parasite clearance with dose and different maximum parasite clearance rates. In Plasmodium berghei -NMRI mouse infections, we estimated a maximum parasite clearance rate of 0.2 (1/h); in Plasmodium falciparum -SCID mouse infections, 0.05 (1/h); and in human volunteer infection studies with P. falciparum , we found a maximum parasite clearance rate of 0.12 (1/h) and 0.18 (1/h) after treatment with OZ439 and MMV048, respectively. Sensitivity analysis revealed that host-parasite driven processes account for up to 25% of variance in parasite clearance for medium-high doses of antimalarials. Although there are limitations in translating parasite clearance rates across these experiments, they provide insight into characterizing key parameters of drug action and dose response and assist in decision-making regarding dosage for further drug development., (Copyright © 2021 Burgert et al.)

With artemisinin-resistant Plasmodium falciparum parasites emerging in Africa, the need for new antimalarial chemotypes is persistently high. The ideal pharmacodynamic parameters of a candidate drug are a rapid onset of action and a fast rate of parasite killing or clearance. To determine these parameters, it is essential to discriminate viable from nonviable parasites, which is complicated by the fact that viable parasites can be metabolically inactive, whilst dying parasites can still be metabolically active and morphologically unaffected. Standard growth inhibition assays, read out via microscopy or [3H] hypoxanthine incorporation, cannot reliably discriminate between viable and nonviable parasites. Conversely, the in vitro parasite reduction ratio (PRR) assay is able to measure viable parasites with high sensitivity. It provides valuable pharmacodynamic parameters, such as PRR, 99.9% parasite clearance time (PCT99.9%) and lag phase. Here we report the development of the PRR assay version 2 (V2), which comes with a shorter assay duration, optimized quality controls and an objective, automated analysis pipeline that systematically estimates PRR, PCT99.9% and lag time and returns meaningful secondary parameters such as the maximal killing rate of a drug (Emax) at the assayed concentration. These parameters can be fed directly into pharmacokinetic/pharmacodynamic models, hence aiding and standardizing lead selection, optimization, and dose prediction. [ABSTRACT FROM AUTHOR]