Cysteamine, the molecule used to treat cystinosis, potentiates the antimalarial efficacy of artemisinin

Abstract

Malaria continues to be a major threat to global health. Artemisinin combination therapy (ACT) is the recommended treatment for clinical malaria; however, recent reports of parasite resistance to artemisinin in certain areas where malaria is endemic have stressed the need for developing more efficacious ACT. We report that cysteamine (Cys), the aminothiol used to treat nephropathic cystinosis in humans, strongly potentiates the efficacy of artemisinin against the Plasmodium parasite in vivo. Using a mouse model of infection with Plasmodium chabaudi AS, we observe that Cys dosing used to treat cystinosis in humans can strongly potentiate (by 3- to 4-fold) the antimalarial properties of the artemisinin derivatives artesunate and dihydroartemisinin. Addition of Cys to suboptimal doses of artemisinin delays the appearance of blood parasitemia, strongly reduces the extent of parasite replication, and significantly improves survival in a model of lethal P. chabaudi infection. Cys, the natural product of the enzyme pantetheinase, has a history of safe use for the clinical management of cystinosis. Our findings suggest that Cys could be included in novel ACTs to improve efficacy against Plasmodium parasite replication, including artemisinin-resistant isolates. Future work will include clinical evaluation of novel Cys-containing ACTs and elucidation of the mechanism underlying the potentiation effect of Cys.

FIG. 1.

Effect of cysteamine on replication of Plasmodium chabaudi in vivo. (A) The plasma levels of cysteamine-free base (measured by HPLC) following either intraperitoneal (i.p.) or subcutaneous (s.c.) injections (120 mg/kg) were measured in 3 mice and used to calculate C_max and AUC pharmacokinetic parameters (see text). Error bars indicate standard deviation from the mean. (B) A/J female mice were infected with P. chabaudi (10⁵ pRBC i.v.) and treated daily (either s.c. or i.p.) with cysteamine (120 mg/kg) starting at day −1 to day 10. Blood parasitemia was monitored on days 5, 6, and 7 and is plotted. The % inhibition of parasite replication was calculated by comparison to the blood parasitemia measured in PBS-treated controls and is indicated below the graphs. Each dot represents a mouse. Levels of statistical significance are represented by asterisks; ***, P < 0.01; **, P < 0.05 (compared to PBS control group).

FIG. 2.

Effect of cysteamine dosing used for treatment of cystinosis on replication of Plasmodium chabaudi in vivo. (A) The plasma levels of cysteamine-free base (measured by HPLC) following subcutaneous (s.c.) injection (50 mg/kg) were measured in 3 mice and used to calculate C_max and AUC pharmacokinetic parameters (see text). Error bars indicate standard deviation from the mean. (B) A/J female mice were infected with P. chabaudi (10⁵ pRBC i.v.) and treated daily with cysteamine (s.c.) from day −1 to day 10, with the indicated dosing: 1 × 150 mg/kg, 3 × 50 mg/kg, or 4 × 50 mg/kg, given at 1 or 2 h intervals. Blood parasitemia was monitored on days 5, 6, and 7 and is plotted. The % inhibition of parasite replication was calculated by comparison to the blood parasitemia measured in PBS-treated controls and is indicated below the graphs. Each dot represents a mouse. Levels of statistical significance are represented by asterisks; ***, P < 0.01; **, P < 0.05 (compared to PBS control group).

FIG. 3.

Synergistic effect of cysteamine on artemisinin efficacy against replication of Plasmodium chabaudi in vivo. Groups (n = 6) of female A/J (A and B) or C57BL/6 (C) mice were infected with P. chabaudi (10⁷ pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with indicated doses (in mg/kg) of artesunate (A and C) or dihydroartemisinin (DHA) (B) and/or cysteamine (170 mg/kg, i.p.), and blood parasitemia (expressed as percentage of parasitized erythrocytes) was determined at days 4 (left) and 5 (right) postinfection. In all experiments, control groups were treated with PBS. The presence or absence of cysteamine is indicated by a plus or a minus, respectively, and doses of artemisinin derivatives in mg/kg are indicated below. Each dot represents a mouse and bars indicate the mean of the group.

FIG. 4.

Dose-dependent synergistic effect of cysteamine on artemisinin efficacy against replication of Plasmodium chabaudi in vivo. Groups (n = 6) of female A/J mice were infected with P. chabaudi (10⁷ pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with increasing doses (indicated) of artesunate (C) and/or cysteamine (A and B) given i.p. Blood parasitemia was determined at days 4 and 5 postinfection, and the inhibitory effects of the different drug treatments on blood-stage P. chabaudi replication were calculated for each animal compared to the mean of PBS-treated controls (expressed as a percentage). The presence or absence of drug is indicated by a plus or minus, respectively, and all doses are in mg/kg. Errors bars represent standard error of the mean.

FIG. 5.

Effect of cysteamine and artesunate combinations on progression and resolution of P. chabaudi infection in vivo. Groups (n = 6) of female A/J mice were infected with P. chabaudi (10⁶ pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with PBS (A), cysteamine (60 mg/kg, A), or cysteamine (60 mg/kg) combined with increasing doses of artesunate (0.5, 1.0, 5, or 10 mg/kg) (B), all given i.p. Blood parasitemia was measured daily up to day 20 (expressed as percentage of pRBC), and death was recorded (indicated by a cross). Solid and dashed lines represent mice receiving artesunate doses alone or in combination with cysteamine, respectively; artesunate doses are depicted by different color groups, as indicated. Error bars represent standard deviation of the mean, and arrows represent drug treatment days. (C) Kaplan-Meier survival plot for experimental treatment groups for which lethality was observed. Color codes and dashed versus solid lines are as described for panel B. (D) Parasitemia levels at day 6 postinfection for all experimental groups are shown, with each dot representing a mouse. Mean levels are shown as bars.

FIG. 6.

Effect of cysteamine and artesunate combinations on progression of P. chabaudi in pantetheinase-sufficient B6 mice. Groups (n = 6) of female B6 mice were infected with P. chabaudi (10⁶ pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with either PBS or artesunate (1.0 or 30 mg/kg) combined with, or without, cysteamine (60 mg/kg) (A), all given i.p. Blood parasitemia was measured daily up to day 22 (expressed as percentage of pRBC). Solid and dashed lines represent mice receiving artesunate doses alone or in combination with cysteamine, respectively. Error bars represent standard deviation of the mean, and arrows represent drug treatment days. (B) Parasitemia levels at day 6 postinfection for all experimental groups are shown, with each dot representing a mouse. Mean levels are shown as bars.