Pharmacokinetics and Ex Vivo Antimalarial Activity of Artesunate-Amodiaquine plus Methylene Blue in Healthy Volunteers

Abstract

High rates of artemisinin-based combination therapy (ACT) failures in the treatment of Plasmodium falciparum malaria in Southeast Asia have led to triple-drug strategies to extend the useful life of ACTs. In this study, we determined whether methylene blue [MB; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride hydrate] alters the pharmacokinetics of artesunate-amodiaquine (ASAQ) and enhances the ex vivo antimalarial activity of ASAQ. In an open-label, randomized crossover design, a single oral dose of ASAQ (200 mg AS/540 mg AQ) alone or with MB (325 mg) was administered to 15 healthy Vietnamese volunteers. Serial blood samples were collected up to 28 days after dosing. Pharmacokinetic properties of the drugs were determined by noncompartmental analysis. After drug administration, plasma samples from seven participants were assessed for ex vivo antimalarial activity against the artemisinin-sensitive MRA1239 and the artemisinin-resistant MRA1240 P. falciparum lines, in vitro MB significantly increased the mean area under the curve of the active metabolite of AS, dihydroartemisinin (1,246 ± 473 versus 917 ± 405 ng·h/ml, P = 0.009) but did not alter the pharmacokinetics of AQ, AS, or desethylamodiaquine. Comparing the antimalarial activities of the plasma samples from the participants collected up to 48 h after ASAQ plus MB (ASAQ+MB) and ASAQ dosing against the MRA1239 and MRA1240 lines, MB significantly enhanced the blood schizontocidal activity of ASAQ by 2.0-fold and 1.9-fold, respectively. The ring-stage survival assay also confirmed that MB enhanced the ex vivo antimalarial activity of ASAQ against MRA1240 by 2.9-fold to 3.8-fold, suggesting that the triple-drug combination has the potential to treat artemisinin-resistant malaria and for malaria elimination. (This study has been registered in the Australian New Zealand Clinical Trials Registry [https://anzctr.org.au/] under registration number ACTRN12612001298808.).

Keywords: artesunate-amodiaquine; ex vivo antimalarial activity; malaria; methylene blue; pharmacokinetics.

Copyright © 2020 American Society for Microbiology.

Figures

FIG 1

Mean (SD) plasma concentration-time profiles of artesunate (AS) and dihydroartemisinin (DHA) (A) and of amodiaquine (AQ) and desethylamodiaquine (DAQ) (B) after either artesunate-amodiaquine (ASAQ) or artesunate-amodiaquine plus methylene blue (ASAQ+MB) administration in 15 healthy volunteers or of methylene blue (MB) and azure B (AZB) (C) after ASAQ+MB administration. The single oral dose of ASAQ (200 mg AS/540 mg AQ) either alone or with MB (325 mg MB). (Note the different time and concentration scales for the analytes).

FIG 2

(Left panel) Parasite growth profile of the artemisinin-sensitive MRA1239 and artemisinin-resistant MRA1240 Plasmodium falciparum lines after exposure to DHA (reference drug) for 6 h in media containing 50% plasma, followed by 66 h of incubation in drug-free media under standard growth conditions. (Right panel) Parasite growth of MRA1239 and MRA1240 parasites in media containing 50% plasma collected from seven healthy volunteers at 0 h (before dosing) and 0.5 or 2 h after the administration of either artesunate-amodiaquine (ASAQ) or artesunate-amodiaquine plus methylene blue (ASAQ+MB) for 6 h, followed by 66 h of incubation in drug-free media under standard growth conditions.