Evaluating the Pharmacodynamic Effect of Antimalarial Drugs in Clinical Trials by Quantitative PCR

Abstract

cThe ongoing development of new antimalarial drugs and the increasing use of controlled human malaria infection (CHMI) studies to investigate their activity in early-stage clinical trials require the development of methods to analyze their pharmacodynamic effect. This is especially so for studies where quantitative PCR (qPCR) is becoming the preferred method for assessing parasite clearance as the study endpoint. We report the development and validation of an analytic approach for qPCR-determined parasite clearance data. First, in a clinical trial with the licensed antimalarial combination sulfadoxine-pyrimethamine (S/P), qPCR data were collected from 12 subjects and used to determine qPCR replicate variability and to identify outliers. Then, an iterative analytic approach based on modeling the log-linear decay of parasitemia following drug treatment was developed to determine the parasite reduction ratio (PRR) and parasite clearance half-life, both measures of parasite clearance. This analytic approach was then validated with data from 8 subjects enrolled in a second study with the licensed antimalarial drug mefloquine. By this method, the PRR and parasite clearance half-lives for S/P and Mefloquine were determined to be 38,878 (95% confidence interval A critical determinant of the success of antimalarial chemotherapy is the speed of clearance of parasites from the blood of infected individuals. Indeed, this pharmacodynamic relationship represents one of the few clinically validated examples in antimicrobial chemotherapy. For example, the superiority of artemisinin antimalarials over comparator drugs, such as quinine, in terms of clinical endpoints can be attributed to the more rapid clearance of parasites from the blood following artemisinin combination chemotherapy (ACT) (1). Indeed, the increasing number of reports from the Greater Mekong Delta of slower clearance of Plasmodium falciparum from the blood following ACT forebodes worsening clinical outcomes (2-5).Quantification of parasitemia has generally relied on counting parasites on blood films. Advantages of this methodology include the ease of collection from finger prick, the low cost of consumables, and well-standardized methods. Additional useful information can be obtained from blood films, including infection with mixed parasite species, the presence of different life cycle stages, and the presence of malaria pigment in white cells. Disadvantages include the need for skilled microscopists who regularly undertake Quality Assurance Programs, the inferior sensitivity of microscopy compared to molecular techniques, and the time-consuming nature of reading multiple slides in the context of an ongoing clinical trial. Although quantitation of biomarkers of parasite biomass, such as histidine-rich protein 2, has some utility in measuring parasite biomass (6), it is not well suited for serial estimation of changes in parasite density in the context of drug treatment due to the kinetics of clearance of circulating parasite antigen differing from that of paras...

Safety, tolerability, pharmacokinetics, and activity of the novel long-acting antimalarial DSM265: a two-part first-in-human phase 1a/1b randomised study

“…In part 2, log 10 PRR 48 and parasite clearance half-life were estimated using the slope of the optimal fit for the log-linear relationship of the parasitaemia decay 21 . Individual log 10 PRR 48 and corresponding 95% CI were calculated using the slope and corresponding SE of the optimal regression model.…”

Pharmacokinetic/pharmacodynamic modelling of the antimalarial effect of Actelion‐451840 in an induced blood stage malaria study in healthy subjects

“…Blood for PK assessment of 451840 was sampled pre-dose, at 0.5, 1, 2, 3, 4, 6,8,10,12,14,16,20,24,48,72,96 and 144 h after drug administration. The bioanalytical methods were the same as previously reported [6].…”

“…Further samples were taken on an individual basis until the last artemether/lumefantrine dosing day and at the final visit (day 28/end of study). Previous studies had indicated that following treatment with some licensed antimalarial drugs, such as sulfadoxine/pyrimethamine [14] and piperaquine [15], gametocytaemia becomes apparent after drug treatment. As an exploratory analysis, to test if 451840 blocks the appearance of gametocytaemia, we undertook PCR analysis for the mature gametocyte-specific transcript pfs25 as previously described [15].…”

“…The subject specific parasite reduction rate over a 48 h period (PRR 48 , i.e. the fold-reduction in parasitaemia over a 48 h period (the asexual life cycle of the parasite) after removing potential lag and tail phases), denoting the drug-specific killing rate [8], its 95% confidence interval (CI) and the parasite clearance half-life (time to 50% reduction in parasite concentration) were derived from the slopes of log-linear regression models of subjects with appropriate overall fit (P value of overall model F test <0.001) as detailed in [14]. The drugspecific slope was derived as weighted average with weights proportional to the inverse variance, from which drug specific PRR 48 and parasite clearance half-life were derived [14].…”

Ensemble modeling highlights importance of understanding parasite-host behavior in preclinical antimalarial drug development

“…We estimated similar value ranges in terms of clearance attributable to host-reactions to infection γ max (0.055-0.44) ( Supplementary Table S7) and drug action E max (0.065-0.33) ( Supplementary Table S9) across all drugs and drug action models. Commonly used measures of drug efficacy such as parasite clearance half-life 39 , summarize all parasite clearance in a single index number 40,41 when in fact parasite clearance is the net effect of multiple parasite clearance mechanisms 23 . Therefore, the inclusion of delayed removal of parasites affected by the drug into mechanistic parasite growth Figure 6.…”