Cysteamine broadly improves the anti-plasmodial activity of artemisinins against murine blood stage and cerebral malaria

Abstract

Background: The potential emergence and spread of resistance to artemisinins in the Plasmodium falciparum malaria parasite constitutes a major global health threat. Hence, improving the efficacy of artemisinins and of artemisinin-based combination therapy (ACT) represents a major short-term goal in the global fight against malaria. Mice defective in the enzyme pantetheinase (Vnn3) show increased susceptibility to blood-stage malaria (increased parasitaemia, reduced survival), and supplementation of Vnn3 mutants with the reaction product of pantetheinase, cysteamine, corrects in part the malaria-susceptibility phenotype of the mutants. Cysteamine (Cys) is a small, naturally occurring amino-thiol that has very low toxicity in vivo and is approved for clinical use in the life-long treatment of the kidney disorder nephropathic cystinosis.

Methods: The ability of Cys to improve the anti-plasmodial activity of different clinically used artemisinins was tested. The effect of different CYS/ART combinations on malarial phenotypes (parasite blood-stage replication, overall and survival from lethal infection) was assessed in a series of in vivo experiments using Plasmodium strains that induce either blood-stage (Plasmodium chabaudi AS) or cerebral disease (Plasmodium berghei ANKA). This was also evaluated in an ex vivo experimental protocol that directly assesses the effect of such drug combinations on the viability of Plasmodium parasites, as measured by the ability of tested parasites to induce a productive infection in vivo in otherwise naïve animals.

Results: Cys is found to potentiate the anti-plasmodial activity of artesunate, artemether, and arteether, towards the blood-stage malaria parasite P. chabaudi AS. Ex vivo experiments, indicate that potentiation of the anti-plasmodial activity of artemisinins by Cys is direct and does not require the presence of host factors. In addition, potentiation occurs at sub-optimal concentrations of artemisinins and Cys that on their own have little or no effect on parasite growth. Cys also dramatically enhances the efficacy and protective effect of artemisinins against cerebral malaria induced by infection with the P. berghei ANKA parasite.

Conclusion: These findings indicate that inclusion of Cys in current formulations of ACT, or its use as adjunct therapy could improve the anti-plasmodial activity of artemisinin, decrease mortality in cerebral malaria patients, and prevent or delay the development and spread of artemisinin resistance.

Keywords: Artemisinins; Blood-stage malaria; Cerebral malaria; Cysteamine; Drug resistance; Plasmodium.

Fig. 1

Experimental framework for testing anti-plasmodial activity of drug combinations. a Molecular structure of drugs used in the study. b Two types of infection protocols were used. For the in vivo/in vivo assay, mice were infected through the i.v. route with 10⁷ Plasmodium parasitized red blood cells (pRBCs). One hour later, drugs were administered through the i.p. route, parasitaemia was monitored on thin blood smears over time, and mortality was recorded. c For the ex vivo/in vivo assay, Plasmodium parasitized red blood cells were exposed for 1 h at 37 °C to different drug combinations. They were then washed free of drugs, counted and naïve mice were infected with 10⁵ treated pRBCs. Parasitaemia was monitored on thin blood smears over time, and mortality is recorded

Fig. 2

Potentiation of anti-plasmodial activity of artemisinin derivatives by Cys in Plasmodium chabaudi AS blood-stage infection (in vivo assays). Groups of A/J mice (minimum of 5 mice per group) were infected with P. chabaudi pRBCs, and were treated with different drug regimens at the indicated doses (in mg/kg). Cys dose was fixed at 140 mg/kg in panels a, b, and d, and at variable doses (20, 40, 60, 80, 100 mg/kg) in panel c. Artemisinin derivatives tested were ART (a), ARTM (b, c), and ARTE (d). The intensity of infection (blood parasitaemia) was measured at day 5 post-infection and is shown as whisker plots (interquartile ranges; mean, with minimum to maximum). The inhibitory activity of different drug treatments is shown as a fraction (% inhibition) of parasitaemia detected in control mice treated with PBS. Statistical significance was calculated by unpaired student t test, *p < 0.05. The data is representative of three independent experiments

Fig. 3

Potentiation of anti-plasmodial activity of ART by Cys in Plasmodium chabaudi blood-stage infection (ex vivo/in vivo assay). P. chabaudi parasitized red blood cells (pRBC) were exposed to different drug combinations in vitro for 1-h at 37 °C, washed free of drugs and then used to infect naïve mice. Groups of A/J mice (minimum of 5 mice per group) were infected with 10⁵ drug-treated P. chabaudi pRBCs, and infected mice were monitored for blood parasitaemia (a) at the indicated times post-infection (days 7–10). Drug concentrations are in micromolar (µM). Mortality was recorded (b). Data is representative of three independent experiments and it is expressed as mean ± SD for each group (*p < 0.05)

Fig. 4

Specificity of Cys versus N-acetyl-Cysteine in potentiation of ARTM in Plasmodium chabaudi blood-stage infection. Mice (minimum of 5 mice per group) were infected by 10⁷ P. chabaudi pRBC through the i.v. route, and blood parasitaemia was monitored (a), and mortality was recorded (b), as described in legend of Fig. 2. The results show that at equivalent concentrations, Cys (70 mg/kg) can strongly potentiate the anti-plasmodial activity of ARTM (0.2 mg/kg), while NAC (70 mg/kg) has no effect. Statistical significance was calculated by unpaired student t test, *p < 0.05. Data are from one of two independent experiments that produced similar results

Fig. 5

Potentiation of anti-plasmodial activity of ART by Cys in Plasmodium berghei cerebral malaria (ex vivo/in vivo assay). P. berghei pRBCs were exposed to different drug combinations (indicated in micromolar, µM) in vitro for 1 h at 37 °C (see Fig. 1). They were then washed free of drugs and used to infect naïve mice. Groups of C57BL/6J (B6) mice (minimum of 5 mice per group) were infected with 10⁵ drug-treated P. berghei ANKA-pRBCs, and monitored for blood parasitaemia (a, c, e) at the indicated times post-infection, and mortality was recorded (b, d). The potentiation of ART by decreasing concentrations of Cys, 50 micromolar (a, b), 25 micromolar (c, d), 15 and 5 micromolar (e) was tested. Mortality on panels a, c, e is indicated by a cross. Data is expressed as mean ± SD for each group (*p < 0.05). The data is representative of three independent experiments

Fig. 6

Reduced cerebral pathogenesis in mice infected with Plasmodium berghei treated with Cys/ART combination (ex vivo/in vivo assay). a Evans blue extravasation was used to assess integrity of the blood–brain barrier (BBB) of mice infected with P. berghei pRBCs treated ex vivo with either PBS, cysteamine (Cys) or artesunate (ART) alone, or in combination (ART/Cys) [see legend to Fig. 4]. Six days post-infection, mice were injected with Evans blue dye, the brains were harvested and photographed, and dye accumulation was quantified. Data is expressed as mean ± SD for each group (*p < 0.05). b, c Six days following P. berghei infection, infiltrating leukocytes were isolated by centrifugation on a 33.3 % Percoll gradient from brain homogenates and were analysed by FACS. b Representative FACS plots of cellular infiltration in the brain indicate reduced infiltration of CD45^hi leukocytes, CD45⁺CD4⁺ T cells, CD45⁺CD8⁺ T cells, TCRb⁻CD19⁺ B cells, F4/80⁺CD11b⁺ macrophages, and Ly6G⁺CD11b⁺ neutrophils in brains of mice that received ex vivo-exposed ART/Cys P. berghei parasites. c Data are expressed as the total number of viable cells in the brain, expressed as mean ± SD for each group (*p < 0.05)