Reliability of Antimalarial Sensitivity Tests Depends on Drug Mechanisms of Action

Abstract

In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.Malaria is a major global health problem, with an estimated 250 to 300 million clinical cases annually and 3.3 billion people at risk, causing nearly a million deaths, mostly among children under 5 years old in sub-Saharan Africa (18, 47). The resistance of Plasmodium falciparum, the most deadly malaria parasite to most antimalarial drugs, is a major obstacle to the eradication of this disease (46). It is also of considerable concern in the light of a recent report on decreased sensitivity to artemesinin drugs in Southeast Asia (14, 28). New chemotherapeutic approaches are thus urgently needed, based on optimization of current drugs and, more importantly, on the discovery of new antimalarial drugs. The latter implies systematic screening of drug libraries, a series of natural compounds, or a structure-based drug design targeting novel targets. In all cases, in vitro evaluation of the thousands of new molecules for their antimalarial activity is an early and necessary step. This early step aims at detecting the antimalarial potential of individual or series of compounds. It is performed in vitro against P. falciparum laboratory strains and, at a later stage, against field isolates, including multidrug-resistant strains. Assays must provide a first indication on the potency of the pharmacological activity, usually expressed as the concentration required to inhibit the parasite viability ...

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

“…Alternatively, this may reflect the activity of AR against most P. falciparum blood stages. 27 Median IC 50 values for AR, among IEV and culture-adapted assays, were 2-to 3-fold higher in comparison with earlier radioisotope assays, and a recent report of SYBR Green I assessing freeze-thawed, culture-adapted P. falciparum samples. 7,17 Higher AR IC50s are unlikely to represent resistance, but perhaps methodological variability.…”

Absolute Bioavailability of cis -Mirincamycin and trans -Mirincamycin in Healthy Rhesus Monkeys and Ex Vivo Antimalarial Activity against Plasmodium falciparum

“…Longer incubations with low-potency drugs against malaria have shown increased potency at 96 h and beyond when 2 life cycles are covered. A recent study by Wein et al (21) suggested that drug mechanism of action may have a significant influence on the outcomes and reliability of the HRP2 in vitro method.…”

Mechanism-Based Model of Parasite Growth and Dihydroartemisinin Pharmacodynamics in Murine Malaria

“…Although there are several proposed mechanisms of action for artemisinin drugs, the DHA turnover model utilized in this model encompasses all probable theories associated with its antimalarial activity (49)(50)(51). However, the IC 50 (1.46 ng/ml) was difficult to estimate in this study, because the concentrations of DHA achieved after dosing greatly exceeded its independently determined IC 50 (1 to 4 nM) in vitro (52,53). Given its low in vitro IC 50 estimate and the high plasma protein binding (90% [54]), further experimentation at lower doses is required to elucidate the IC 50 of DHA in vivo.…”