Design and evaluation of primaquine-artemisinin hybrids as a multistage antimalarial strategy

Abstract

It is widely accepted that the struggle against malaria depends on the development of new strategies to fight infection. The "magic bullet" thought to be necessary to reach eradication should not only provide treatment for all Plasmodium spp. that infect human red blood cells but should also eliminate the replicative and dormant liver forms of the parasite. Moreover, these goals should ideally be achieved by using different mechanisms of action so as to avoid the development of resistance. To that end, two hybrid molecules with covalently linked primaquine and artemisinin moieties were synthesized, and their effectiveness against the liver and blood stages of infection was compared in vitro and in vivo with those of the parent compounds. Both hybrids displayed enhanced in vitro activities, relative to those of the parent compounds, against Plasmodium berghei liver stages. Both compounds were about as potent as artemisinin against cultured Plasmodium falciparum (50% inhibitory concentration [IC(50)], ∼10 nM). When used to treat a murine P. berghei infection, one of the molecules displayed better efficacy than an equimolar mixture of the parent pharmacophores, leading to improved cure and survival rates. These results reveal a novel approach to the design and evaluation of antimalarials based on the covalent combination of molecules acting on different stages of the parasite life cycle.

Fig. 1.

Syntheses. Shown are the chemical structures of primaquine (compound 1), artemisinin (compound 2), dihydroartemisinin (compound 3), and artelinic acid (compound 4) and synthetic schemes for hybrid compounds 7 and 10.

Fig. 2.

In vitro inhibition of hepatic Plasmodium berghei infection by compounds 7 and 10. (A to C) Compounds were added to Huh7 hepatoma cells 1 h before infection with luciferase-expressing sporozoites. An amount of DMSO equivalent to that in the highest compound concentration tested was used as a control. Forty-eight hours after the addition of P. berghei sporozoites, cell confluence (red lines on bar plots) was assessed by alamarBlue fluorescence, and the infection rate (bars) was measured by quantifying the luciferase activity by luminescence. The effects of different concentrations of compound 7 (A), compound 10 (B), and a 1:1 mixture of primaquine and artemisinin (C) are shown. Results are expressed as means ± standard deviations. (D) For each compound, the IC₅₀ was calculated by sigmoidal fitting. Red, compound 7; orange, compound 10; green, primaquine plus artemisinin.

Fig. 3.

In vivo inhibition of Plasmodium berghei liver-stage infection and blood patency by oral administration of compounds 7 and 10. C57BL/6 mice (n = 6) were infected by intravenous injection of 10,000 GFP-expressing P. berghei sporozoites. (A) Mice were treated by oral administration of the different compounds in the amounts and at the times noted in the text. Parasite liver loads (P. berghei ANKA 18S rRNA; relative quantity normalized against that of the host HGPRT gene) were assessed by quantitative real-time PCR 44 h after infection. (B) Mice were treated by a single administration of the compounds in the amounts and at the time noted in the text, and the appearance of parasites in the blood was monitored daily. The percentage of mice with parasitemia under the detection limit (parasite-free mice) is shown.

Fig. 4.

In vivo treatment of Plasmodium berghei blood infection by oral administration of compounds 7 and 10. C57BL/6 mice (n = 5) were infected by intravenous injection of 10⁵ GFP-expressing sporozoites. Oral administration of the compounds was initiated when parasitemia reached 2 to 3% and was carried out daily for 10 days. (A) Blood parasite patency, monitored daily by flow cytometry. The percentages of mice with parasitemia under the detection limit are shown. (B) Survival plot showing the percentages of live mice throughout the duration of the experiment.