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P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action.

Sanz LM, Crespo B, De-Cózar C, Ding XC, Llergo JL, Burrows JN, García-Bustos JF, Gamo FJ - PLoS ONE (2012)

Bottom Line: Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant.We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring.This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters.

View Article: PubMed Central - PubMed

Affiliation: Tres Cantos Medicine Development Campus, Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.

ABSTRACT
Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

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Parasite viability time-course in response to various concentrations of drugs.A. P. falciparum viability time-course profiles for atovaquone, pyrimethamine, and artemisinin at concentrations corresponding to 1×, 3×, 10×, and 100× their respective IC50. Error bars are SEM of at least 4 independent experiments. B. Values represented in panel A. No PRR or 99.9% PCT could be calculated for the 1× IC50 conditions.
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pone-0030949-g004: Parasite viability time-course in response to various concentrations of drugs.A. P. falciparum viability time-course profiles for atovaquone, pyrimethamine, and artemisinin at concentrations corresponding to 1×, 3×, 10×, and 100× their respective IC50. Error bars are SEM of at least 4 independent experiments. B. Values represented in panel A. No PRR or 99.9% PCT could be calculated for the 1× IC50 conditions.

Mentions: Relatively large killing rate differences occurred with the different compounds tested. Considering that they all have been tested at 10× IC50, these results might reflect suboptimal dosing and, consequently, underestimated killing rate for some of the inhibitors. In order to test this hypothesis, in vitro PRR experiments were conducted with a representative subset of antimalarial drugs using concentrations corresponding to multiples of their respective IC50: 1×, 3×, 10×, and 100× (Figure 4). Atovaquone treatment at 1× IC50 displayed a plateau with a constant, or slightly increasing parasitemia, indicating that the rate of killing was compensated by the rate of growth in the treated culture. Increasing atovaquone concentration to 3× IC50 decreases the parasitemia level at which the plateau is reached. This indicates a net killing effect of the drug, as growth of the remaining parasites can compensate only partially the degree of killing, inducing an equilibrium at a parasitemia 2 logs below the starting inoculum. The treatment at 10× IC50 did permit to reach a virtually complete parasite clearance with a log(PRR) of 2.9 and a 99.9% PCT of 90 hours, while a further 10 fold increase of drug concentration did not significantly modify the killing rate profile observed, confirming that the maximal rate of killing was achieved at the previous concentration (10× IC50) (Figure 4). Pyrimethamine showed a similar pattern and, although at 1× IC50, it also failed to clear all the parasites within 120 hours, killing was higher than in the case of atovaquone 1× IC50 and similar to 3× IC50. This last concentration (3× IC50) appears however to be enough to reach the maximal killing rate of this compound, with log(PRR) and 99.9% PCT reaching values similar to the ones seen in response to 10× and 100× IC50 concentrations. For artemisinin, 1× IC50 is similarly not sufficient to clear the parasites within 120 hours. However a killing rate similar to that seen with 10× IC50 is achieved with as low as 3× IC50 and is not significantly different when the parasites are exposed to a dose corresponding to 100× IC50. This set of experiments shows that the maximal killing rate of these compounds is reached with doses corresponding to at most 10× IC50. This appears to be true for slow, medium, as well as fast killing compounds, as illustrated with atovaquone, pyrimethamine, and artemisinin, respectively. This observation is also true for additional compounds, including chloroquine and lumefantrine, which reached maximum killing rate at concentrations below 10× IC50 (Figure S3 and Table S2). Interestingly, in the case of chloroquine maximal rate of killing was observed at concentrations close to 1× IC50. For both, atovaquone and pyrimethamine, varying the compound concentration did not alter the lag phase duration, suggesting that this initial effect does not depend on the intensity of the parasite exposure to the drugs but rather on their intrinsic modes-of-action. Altogether, this suggests 10× IC50 to be an optimal dose to investigate compound speed-of-action at the highest rate of killing and to reflect true variations in compound ability to clear P. falciparum parasites.


P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action.

Sanz LM, Crespo B, De-Cózar C, Ding XC, Llergo JL, Burrows JN, García-Bustos JF, Gamo FJ - PLoS ONE (2012)

Parasite viability time-course in response to various concentrations of drugs.A. P. falciparum viability time-course profiles for atovaquone, pyrimethamine, and artemisinin at concentrations corresponding to 1×, 3×, 10×, and 100× their respective IC50. Error bars are SEM of at least 4 independent experiments. B. Values represented in panel A. No PRR or 99.9% PCT could be calculated for the 1× IC50 conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285618&req=5

pone-0030949-g004: Parasite viability time-course in response to various concentrations of drugs.A. P. falciparum viability time-course profiles for atovaquone, pyrimethamine, and artemisinin at concentrations corresponding to 1×, 3×, 10×, and 100× their respective IC50. Error bars are SEM of at least 4 independent experiments. B. Values represented in panel A. No PRR or 99.9% PCT could be calculated for the 1× IC50 conditions.
Mentions: Relatively large killing rate differences occurred with the different compounds tested. Considering that they all have been tested at 10× IC50, these results might reflect suboptimal dosing and, consequently, underestimated killing rate for some of the inhibitors. In order to test this hypothesis, in vitro PRR experiments were conducted with a representative subset of antimalarial drugs using concentrations corresponding to multiples of their respective IC50: 1×, 3×, 10×, and 100× (Figure 4). Atovaquone treatment at 1× IC50 displayed a plateau with a constant, or slightly increasing parasitemia, indicating that the rate of killing was compensated by the rate of growth in the treated culture. Increasing atovaquone concentration to 3× IC50 decreases the parasitemia level at which the plateau is reached. This indicates a net killing effect of the drug, as growth of the remaining parasites can compensate only partially the degree of killing, inducing an equilibrium at a parasitemia 2 logs below the starting inoculum. The treatment at 10× IC50 did permit to reach a virtually complete parasite clearance with a log(PRR) of 2.9 and a 99.9% PCT of 90 hours, while a further 10 fold increase of drug concentration did not significantly modify the killing rate profile observed, confirming that the maximal rate of killing was achieved at the previous concentration (10× IC50) (Figure 4). Pyrimethamine showed a similar pattern and, although at 1× IC50, it also failed to clear all the parasites within 120 hours, killing was higher than in the case of atovaquone 1× IC50 and similar to 3× IC50. This last concentration (3× IC50) appears however to be enough to reach the maximal killing rate of this compound, with log(PRR) and 99.9% PCT reaching values similar to the ones seen in response to 10× and 100× IC50 concentrations. For artemisinin, 1× IC50 is similarly not sufficient to clear the parasites within 120 hours. However a killing rate similar to that seen with 10× IC50 is achieved with as low as 3× IC50 and is not significantly different when the parasites are exposed to a dose corresponding to 100× IC50. This set of experiments shows that the maximal killing rate of these compounds is reached with doses corresponding to at most 10× IC50. This appears to be true for slow, medium, as well as fast killing compounds, as illustrated with atovaquone, pyrimethamine, and artemisinin, respectively. This observation is also true for additional compounds, including chloroquine and lumefantrine, which reached maximum killing rate at concentrations below 10× IC50 (Figure S3 and Table S2). Interestingly, in the case of chloroquine maximal rate of killing was observed at concentrations close to 1× IC50. For both, atovaquone and pyrimethamine, varying the compound concentration did not alter the lag phase duration, suggesting that this initial effect does not depend on the intensity of the parasite exposure to the drugs but rather on their intrinsic modes-of-action. Altogether, this suggests 10× IC50 to be an optimal dose to investigate compound speed-of-action at the highest rate of killing and to reflect true variations in compound ability to clear P. falciparum parasites.

Bottom Line: Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant.We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring.This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters.

View Article: PubMed Central - PubMed

Affiliation: Tres Cantos Medicine Development Campus, Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.

ABSTRACT
Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

Show MeSH
Related in: MedlinePlus