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

Eight to twelve weeks old female A/J mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA) and were housed at McGill University Animal Care Center according to the guidelines of the Canadian Council on Animal Care. Mice experimentation protocol was approved by the McGill Facility Animal Care Committee (P. Gros, Principal Investigator; protocol number: 5287), and include procedures to minimize distress and improve welfare. An isolate of P. chabaudi AS was provided by Dr. Mary M. Stevenson (McGill University Health Center Research Institute, Montreal), and was maintained by weekly passage in A/J mice for a maximum of four consecutive passages, as previously described [27]. Plasmodium berghei ANKA parasites were initially obtained from the Malaria Reference and Research Reagent Resource Center (MR4); P. berghei was passaged in C57BL/6J (B6) mice for a maximum of three consecutive passages. In passage mice, blood parasitaemia was monitored daily, and the percentage of parasitized red blood cells (pRBCs) was determined on thin blood smears stained with Dif-Quik (Dade Behring, Newark, DE, USA), as described [28]. Infected blood was diluted in pyrogen-free saline for infection of experimental animals and also for preparation of cryo-preserved stocks for long-term storage.

Cysteamine hydrochloride (Cys) and N-acetyl cysteine (NAC) (Sigma, Burlington ON, Canada) were prepared fresh in PBS. Artesunate and artemether were generously provided by Dafra Pharma International (Turnhout, Belgium). Artesunate was prepared fresh as a 1 mg/mL stock in 5 % sodium bicarbonate solution which was diluted in PBS immediately before use. Artemether (Artesiane™) prepared from fractionated coconut oil stock (20 mg/mL, injectable) was diluted in sesame oil to desired concentration. Beta-Arteether (Artecef™, Artemotil; 50 mg/mL injectable in sesame oil) was diluted in sesame oil to desired concentration. Beta-Arteether was generously provided by Artecef BV (Zeewolde, The Netherlands). Drugs were administered via intraperitoneal (i.p) injection and according to a four-days treatment schedule. Mice were weighed prior to treatment to determine appropriate doses and injection volumes ranged from 100–400 μL per mouse. In the case of animals treated with two drugs, Cys was administered first, followed by artemisinin derivatives 15 min later on alternate sides of the mouse abdomen. Untreated control animals were injected with PBS alone.

For in vivo infections, mice were infected i.v. (0.2 mL) with 10⁷ P. chabaudi pRBCs into the tail vein, and monitored under BSL2 containment conditions. One hour post-infection, mice received either PBS or different drugs or drug combinations (i.p), and this treatment was repeated at 24, 48, and 72 h post-infection. At day 4 and 5 post-infection, blood was sampled, and blood parasitaemia was determined (between 4–10 fields of 100 erythrocytes were counted per mouse, and results are expressed as percentage of parasitized erythrocytes). For experiments involving ex vivo drug exposure followed by in vivo infections, parasitized erythrocytes were exposed to certain compounds for 1 h at 37 °C. Treated pRBCs were then rinsed free of drug, and resuspended in drug-free PBS (at 1 × 10⁶ pRBC/mL), and were then used to infect naïve mice (1 × 10⁵ pRBC). The ability of the treated parasites to induce a productive infection was monitored by measuring blood parasitaemia daily, and by monitoring appearance and progression of lethal and irreversible symptoms of cerebral malaria, as we described [29].

Mice were injected with Cys (140 mg/kg, 0.2 mL, i.p.) in the lower left abdomen, and exactly 15 min later, ART (0.5 mg/kg, 0.2 mL, i.p.) was injected in the lower right abdomen. At 5, 10, and 20 min, mice were euthanized and bled by cardiac puncture and blood was collected in EDTA-containing tubes to isolate plasma. Plasma samples showing hemolysis were not used for ART determination as haemolysis interfered with ART measurement. Control plasma from untreated mice was also included as a placebo for spiked calibration references covering a range between 5 and 2000 ng/mL for ART as well as DHA, and QC samples (10 and 100 ng/mL). The method consisted of a sample preparation step, followed by UPLC with MS (MRM-mode) detection. Briefly, a protecting solution consisting of potassium oxalate and sodium fluoride was added to ice-cold mouse plasma, followed by the addition of the ART-d3 and DHA-d3 internal standard solutions. After mixing, the diluted plasma samples were loaded on preconditioned µ-HLB-SPE wells, which are washed with water followed by 5 % acetonitrile in water. Elution of the compounds was done with a methanol–acetonitrile mixture, and diluted with water before injection in the UPLC system, consisting of an Acquity BEH shield RP18 column with a mobile phase consisting of water-acetonitrile-formic acid (50/50/0.1) in isocratic mode. The MRM transitions used for quantification were m/z 283→265, resp 286→268, for ART and its internal standard, and m/z 323→240, resp 326→243, for DHA and its internal standard. The method was validated and found to comply for linearity, selectivity, recovery, accuracy and precision.

Six days post-infection, mice were exsanguinated and perfused as above; brains were harvested and homogenized in RPMI media-containing 0.5 mg/mL collagenase (Gibco LifeTechnologies), 0.01 mg/mL DNAse I (Roche) and 2 mM EDTA. Infiltrating cells were separated on a 33.3 % Percoll solution. Cells were stained for flow cytometry analyses with the following; Zombie Aqua Dye-V500 (BioLegend), anti-CD45-APC-eFluor780 (clone 30-F11, eBioscience), anti-CD4-PerCP Cyanine5.5 (clone RM4-5, eBioscience), anti-CD8alpha-PE (clone 53-6.7, eBioscience), anti-TCRbeta-FITC (clone H57-597, eBioscience), anti-CD19-BV421 (clone 6D5, BioLegend), F4/80-eFluor450 (clone BM8, eBioscience), anti-CD11b-APC (clone M1/70, eBioscience), anti-CD11c-PerCP Cyanine5.5 (clone N418, eBioscience), anti-Ly6C-PE (clone HK1.4, eBioscience), and anti-Ly6G-FITC (clone 1A8, BioLegend). Leukocytes were gated as CD45^hi cells.

Groups with normally distributed data points were compared using parametric unpaired t tests, while groups with non-Gaussian distributions were compared using non-parametric Mann–Whitney tests. Survival differences were analysed using the Log-Rank test. Standard error of percent inhibition was calculated from individual mice compared to the mean parasitaemia level of the control group.

The capacity of Cys to potentiate the anti-plasmodial activity of different ADs present in clinically-used ACT, including ART, ARTM, and β-ARTE (Fig. 1a) was investigated. ADs were given at sub-optimal concentrations (doses that have little effect on parasitaemia and survival on their own) to distinguish between the lack of an effect and possible additive or synergistic effects of Cys on the ADs, as previously described [26]. Synergy is defined as a total activity of the two compounds given together being greater than the sum of the independent activities of the two compounds given alone. The effect of Cys on ADs anti-plasmodial activity was determined using a standardized four-day test using blood parasitaemia as a quantitative measure of anti-plasmodial activity (Fig. 1b). In these experiments, mice were infected with P. chabaudi (10⁷ pRBC through i.v. route), and 1 h later, they received either PBS, Cys (140 mg/kg) and/or sub-optimal doses of ADs (between 0.05 or 0.5 mg/kg) given i.p. Parasitaemia levels were subsequently measured on day 4 and survival time was monitored.

For ART (Fig. 2a), sub-optimal dosing of 0.2 mg/kg (ART0.2) caused a modest ~10 % reduction of parasitaemia compared to untreated controls, while a higher dose (0.5 mg/kg; ART0.5) produced ~70 % inhibition; Addition of Cys to these two regimens resulted in further strong reduction of parasitaemia, with either a synergistic (ART0.2/Cys) or additive effect (ART0.5/Cys) (Fig. 2a). In addition, mice receiving both ART/Cys showed fewer symptoms of disease, and had a significant increase in survival time: from day six post-infection for control mice (PBS) and mice receiving either Cys alone or ART low dose (ART0.2), to day 7 for those receiving ART high dose (ART0.5) and the ART0.2/Cys combination, to day 10 for mice receiving ART0.5/Cys (Additional file 1A). For ARTM, the strongest effect on parasite replication was seen for the ARTM (0.2 mg/kg)/Cys combination (98 %), compared to Cys (33 %) or ARTM (29 %) alone (Fig. 2b). The ARTM (0.2 mg/kg)/Cys combination produced a synergistic effect and completely blocked parasite replication (<1 % parasitaemia at day 4), and completely protected against lethality with 100 % survival of mice in this group compared to mice receiving either PBS, Cys or ARTM alone (Additional file 1B). Dose–response studies (Fig. 2c) showed that the potentiating effect of Cys was dose-dependent, and that a minimum dosing of 60 mg/kg was required for clear potentiation of ARTM effect on blood parasitaemia. Maximum potentiation was seen at 80 and 100 mg/kg Cys (Fig. 2c), with animals in these two groups surviving past day 7 (Additional file 1C). Cys was also able to potentiate the anti-plasmodial activity of ARTE; while single dosing of Cys (140 mg/kg) or ARTE (0.05; 0.2 mg/kg) caused a maximum reduction of ~20 % in blood parasitaemia, the addition of Cys to high dose ARTE caused a nearly ~80 % reduction in parasitaemia (Fig. 2d), and increased survival to day 8 (Additional file 1D) compared to PBS-treated controls (100 % mortality by day 5). Similar to the effect of Cys on ARTM activity, potentiation of ARTE was dose-dependent, with optimal effects seen at a dose of 100 mg/kg Cys (Additional file 2).

Taken together, these results (representative of three independent experiments) indicate that Cys can potentiate the anti-plasmodial activity of chemically distinct artemisinin molecules in vivo, reducing blood stage parasite replication and increasing survival following acute infection.

A series of experiments were conducted to determine whether the Cys potentiation of ADs anti-plasmodial activity noted against P. chabaudi in vivo is due to (a) increased drug induced anti-plasmodial activity, or (b) indirect static or cidal mechanisms involving the parasite replication niche (RBC) and/or host innate defenses. For this, P. chabaudi pRBCs were exposed to drugs in vitro, and the effect of this exposure on the ability of the treated preparations to induce infection in a naïve host in vivo was tested (Fig. 1c). Plasmodium chabaudi pRBCs were exposed for 1 h at 37 °C to either PBS, Cys (50 μM), ART (5, 10 μM) or both, which was subsequently washed off; The equivalent of 1 × 10⁵ pRBCs (calculated prior to drug exposure) was then injected into mice without any additional exposure to drugs in vivo, and parasite replication was followed over time (Fig. 3a). Results show that although a 1 h treatment with Cys alone or with ART alone (at both doses) had minimal effect on time of appearance and extent of blood stage replication, parasites treated with Cys (50 μM)/ART(10 μM) combination showed (a) considerable delay in onset of parasitaemia, (b) striking >90 % inhibition of parasite replication measured at day 9 and day 10, compared to PBS treated controls, and (c) significant survival (80 %) to infection compared to all other treatment groups which succumbed to infection by day 10 (Fig. 3b). These results indicate that Cys can strongly potentiate the anti-plasmodial activity of ART in a single 1 h exposure of pRBCs, significantly reducing parasite viability. The Cys enhancement of anti-parasitic activity is detected ex vivo, in the absence of the host immune system, strongly pointing at a direct enhancement of ART potency.

Cys is a naturally occurring amino-thiol that contains a thiol group which confers anti-oxidant properties (Fig. 1a). To determine whether the potentiating effect of Cys on artemisinin anti-plasmodial activity is specific, Cys was compared to another closely related aminothiol with similar anti-oxidant properties, N-acetyl-Cysteine (NAC; Fig. 1a). Mice were infected by 10⁷ P. chabaudi pRBCs, and the infected mice were treated with either PBS, ARTM (0.2 mg/kg), Cys (MW 113) or NAC (MW 163) used at the same dose (70 mg/kg) alone or in combinations with ARTM (Fig. 4). The effect of drugs on blood stage replication of P. chabaudi was measured on day four post-infection (Fig. 4a). Results show that at the 70 mg/kg dose used, neither Cys alone nor NAC alone had a significant effect on P. chabaudi replication, and at the sub-optimal dosing of 0.2 mg/kg tested, ARTM had only a moderate effect on blood parasitaemia on day 4. As expected, the Cys/ARTM combination showed potentiation and caused a >90 % inhibition of parasite replication. By contrast, adding NAC to ARTM did not improve the anti-plasmodial effect of ARTM (~40 % inhibition). Cys/ARTM also improved survival to infection (up to 8 days), compared to mice from all other groups and that succumbed from infection between day 5 and 6 (Fig. 4b). Therefore, potentiation by Cys is specific and cannot be achieved with NAC.

Experimental cerebral malaria (ECM) can be induced in mice by infection with P. berghei. In this model, clinical symptoms occur at relatively low levels of blood parasitaemia (<20 %), and are not driven by haemolytic anaemia, but rather, by host pathological inflammatory reactions. Indeed, brain endothelial cells activated by pRBCs produce cytokines and chemotactic factors that recruit neutrophils and activated T lymphocytes. Release of cytotoxic molecules by inflammatory leukocytes leads to loss of integrity of the blood–brain barrier, micro-thrombosis, hypoxia of the brain parenchyma, leading to neurological symptoms including seizures and coma, and ultimately death [21, 22]. To determine whether Cys could be of therapeutic benefit against cerebral malaria, its capacity to potentiate the anti-plasmodial activity of ART against P. berghei was tested using the ex vivo exposure: in vivo infection model (Fig. 1c). Briefly, P. berghei-parasitized RBCs were exposed to different Cys/ADs combinations for one hour at 37 °C, and 10⁵ of the treated pRBC were injected into naïve mice. These were then followed for daily blood parasitaemia, for appearance of clinical symptoms, and survival was also recorded. Treatment of pRBCs in vitro with combinations of Cys (50 μM) plus ART (5, 10 μM) caused a significant delay in the onset of parasitaemia, and on the extent of parasite replication compared to pRBCs treated with either PBS, or with either Cys or ART alone (Fig. 5a). There was also a dramatic increase in the survival of mice infected with pRBCs treated with either Cys/ART combinations (80–100 % survival beyond day 13) (Fig. 5b). The enhanced anti-parasitic activity of the Cys/ART combination over either drug alone used to treat pRBCs in vitro was still evident when dosing as low as 25 μM Cys (Fig. 5c, d) or even 15 μM (Fig. 5e) were used. Together, these results (representative of 3 independent experiments) show that (a) Cys alone has significant activity against P. berghei; (b) Cys can strongly potentiate the anti-plasmodial activity of ART in the experimental cerebral malaria model; and (c) potentiation by Cys occurs at doses that are below those currently approved for life-long clinical treatment of cystinosis in humans.

The integrity of the blood brain barrier (BBB) of mice infected with P. berghei parasites (pRBCs) treated ex vivo with either PBS, Cys, ART or ART/Cys was also investigated, using an Evans blue extravasation assay [30, 31]. For this, mice at day six post-infection are injected with Evans blue, and the dye is allowed to circulate for 1 h prior to sacrifice. Brains of the mice were harvested, and the Evans blue dye was quantified (Fig. 6a). Mice infected with Cys/ART-treated P. berghei displayed preserved BBB integrity with significant dye exclusion, and this was clearly distinct from mice infected with P. berghei pRBCs treated with either PBS, Cys, or ART alone which demonstrated compromised BBB function (Fig. 6a). In parallel experiments, P. berghei-infected mice were sacrificed at day 6 post-infection, exsanguinated, and perfused with PBS; isolated cells from the brain were analysed by fluorescence activated cell sorting (FACS) for the presence and number of infiltrating inflammatory cells (Fig. 6b, c). Mice infected with P. berghei parasites treated with Cys/ART combination showed reduced infiltration of total CD45^hi leukocytes, including macrophages (CD11b⁺F4/80⁺), neutrophils (CD11b⁺Ly6G⁺), CD19⁺ B lymphocytes, and CD4⁺ and CD8⁺ T lymphocytes (TCRβ⁺) (Fig. 6b, c). Taken together, these results indicate that improved clinical symptoms and overall survival in mice infected with P. berghei parasites treated with Cys/ART is associated with improved BBB function, reduced infiltration of pro-inflammatory myeloid and lymphoid cells, and absence of cerebral pathology.