Efficacy and safety evaluation of a novel trioxaquine in the management of cerebral malaria in a mouse model

Despite many years of research and great progress in the fight against malaria [1], cerebral malaria (CM) has remained the most dreadful severe complication of Plasmodium falciparum, with a higher burden greatly felt in sub-Saharan Africa [2], a continent that already battles the effect of other diseases such as tuberculosis and HIV/AIDS [3]. Children 5 years old and younger are the most susceptible, with a staggering 90% CM related fatalities [4]. With no effective vaccine against malaria yet, chemotherapy has remained the mainstay of malaria management [5]. Extremely limited options for chemotherapy in CM have been a great challenge [5]. For decades, quinine has been the drug of choice for management of CM [6]. Concerns over increasing resistance to the quinoline-based drugs such as chloroquine (CQ) prompted World Health Organization (WHO) policy change to use of artemisinin-based drugs, such as artemether and artesunate. In management of CM, preference has been given to parenteral artesunate due to its superior efficacy and survival outcome in comparison to intramuscular artemether [7, 8]. Despite effective CM chemotherapy with artesunate, mortality rate still remains as high as 15–25% among treated children while those that survive the acute episodes of CM suffer long-term neurological sequelae including epilepsy [9].

Plasmodium falciparum, the most lethal pathogen among the Plasmodium species causing up to 90% of all malaria-related deaths in sub-Saharan Africa has shown multi-drug resistance [10]. Reports of parasite resistance against the WHO-recommended artemisinin (ART) derivatives—the first-line anti-malarials—have already been documented, prompting an urgent need for new drugs [10]. Extremely low success rates for new chemotherapy in the development pipeline to proceed to clinical trials [11] has necessitated a rational deployment of the available effective drugs to optimize their useful therapeutic life and also to protect them from being rendered ineffective due to parasite resistance [12]. Currently, ART derivatives are used in combination with longer durational anti-malarials in the artemisinin-based combination therapy (ACT) strategy, in order to protect them against resistance [5]. Unfortunately, in CM, artesunate has often been used as a monotherapy [7], thus calling for a polypharmacology strategy in such a scenario, especially in sub-Saharan Africa.

In the long and difficult search to develop effective, cheaper and urgently needed anti-malarial drugs, many research groups have employed the emerging strategy in medicinal chemistry called hybridization or covalent bitherapy [12‐14]. This is a rational drug design approach based on covalent linking of drug pharmacophores into a single hybrid molecule, termed as hybrid drug, dual drug or conjugates [12]. The resulting hybrid drugs have been reported to possess superior efficacy to either the individual precursors alone or their co-formulations [15]. This approach reduces the risk of treatment failure and also offers resistance protection to the partner drug or to both drugs [16], and is believed to be cheaper since only the active moieties are covalently linked, thus avoiding the long, laborious and expensive drug development procedures of individual drugs before co-formulation [16]. Trioxaquines, which are hybrid drugs arising from conjugation of 1,2,4-endoperoxide bridge of the artemisinins and a quinoline pharmacophore, are demonstrative of the validity of this approach [12].

In this study, a trioxaquine termed N-(7-chloroquinolin-4-ylamino)-ethyl-artesunate-19-carboxamide (Fig. 1) was evaluated for curative effect of CM in mouse model. The dual drug was previously synthesized and demonstrated to possess remarkable antiplasmodial effect against blood stage rodent malaria parasite (ED₅₀ and ED₉₀ of 5.5 and 13.5 mg/kg, respectively) [17]. The dual drug had also shown a remarkable in vitro antiplasmodial activity against CQ-sensitive (D6) (IC₅₀, 6.89 ng/mL) and CQ-resistant (W2) (IC₅₀, 3.62 ng/mL) P. ​falciparum isolates [17].

Infection with P. falciparum often has high chance of progressing into CM, a severe form of malaria that contributes to high malaria mortality in sub-Saharan Africa [9]. Children 5 years old and younger, expectant women at first trimester, and individuals with naive immunity visiting malaria-endemic areas are at increased risk of infection [5].

Plasmodium falciparum has shown a rising trend of evolution and emergence of resistance to the currently used drugs, even to the WHO gold standard anti-malarial, ACT [5, 10]. Also, very few anti-malarial drugs are in the clinical development pipeline. Optimizing use of existing drugs as well as use of rational drug development strategies, such as covalent bitherapy, could lead to enhanced, useful therapeutic lives of existing therapy as well as add new therapy to the existing anti-malarial drug repertoire [12, 13]. In covalent bitherapy, just as in combination therapy, the goal is to either delay or circumvent development of resistance. In trioxaquines, the fast-acting precursor (ART pharmacophore) provides rapid clearance of the bulk of parasite load while the quinoline moiety clears the remnant parasite that survives the effect of the former, until complete clearance is achieved [13]. This strategy has potential to improve the therapeutic effectiveness as well as delaying or circumventing the emergence of resistance to both individual precursors of the hybrid drug, besides overcoming the challenge of a long drug development pipeline for co-formulated ACT drugs [15]. The trioxaquine exhibited a higher efficacy compared to individual precursors alone (Table 1). Based on the curative test using established infection, the trioxaquine though exhibiting activities comparable to artesunate, proved to be more effective than both the parenteral artesunate, quinine and 4,7-dichloroquine (Table 1). The trioxaquine manifested a rapid parasite clearance of greater than 80% within 24 h post-treatment and no parasite was observable under microscope at the 48 h post-treatment for both dosages of 12.5 and 25 mg/kg (Table 1).

Long-term monitoring of animals treated with trioxaquine at both 12.5 and 25 mg/kg showed no recrudescence, with the animals surviving beyond 60 days post-treatment (Fig. 3). This is a clear indication of complete cure in the treated animals since no recrudescence was observed, an indicator that the dual drug could potentially circumvent or delay development of drug resistance.

Although rapid parasite suppression (84.8%) was observed in the first 24 h post-treatment with artesunate and no parasite was observable under microscope 48 h post-treatment, recrudescence was observed in this treatment group with all the animals succumbing to the infection by day 16 post-treatment. Even though the quinoline moiety, on the other hand, did not show any significant parasite reduction in the first 24 h post-treatment, its contribution in trioxaquine was manifested by lack of recrudescence in trioxaquine-treated animals even beyond 60 days post-treatment. The quinoline pharmachophore (4,7-dichloroquinoline) is believed to promote dual drug accumulation in the food vacuole, allowing the artemisinin-based partner (artesunate) to have a longer half-life [25], a possible explanation for the observed improved treatment outcome in the trioxaquine treatment groups compared to the artesunate-treated group. During treatment with 12.5 mg/kg of the trioxaquine, one mouse was lost on day 4 post-treatment. However, it was deemed that the death was not due to drug toxicity since even mice that received 25 mg/kg of trioxaquine had 100% survival. CM leads to a multi-organ dysfunction involving the liver, kidney and brain with the extent of the organs’ damage varying from individual to individual. Damage to these major organs are cited as the major reason behind CM deaths in infected individuals even after chemotherapeutic intervention [26]. This reason could support the loss of one animal in the 12.5-mg/kg trioxaquine treatment group even after parasitaemia monitoring had shown complete clearance. These results are in agreement with other previously related studies using the same experimental animals, which noted manifestation of neurological symptoms of CM between days 6 and 9 pi [18, 19, 27] and death in untreated animals by day 10 pi [28]. Recrudescence was also reported in artesunate-treated animals even at higher doses of 32 and 64 mg/kg, with all the treated animals succumbing to the infection and quinine being effective only at higher doses >120 mg/kg, which were not well tolerated by the experimental animals [29].

However, albeit in blood-stage infection, it was earlier reported that trioxaquines were capable of inhibiting parasites in mice and also affording curative effect in both the 4-day suppressive test and in established infection studies [13].

Two components have been implicated to be participating in the development of cerebral malaria, namely, the parasite-related factors and the host immune factors. Rupture of the hepatocytes leads to accumulation of the iRBCs within the brain microvasculature as the parasite invades the blood stream [30]. On the blood side of the BBB, P. falciparum-infected RBCs (PfRBCs) cytoadhere to the brain endothelium by binding of P. falciparum erythrocyte membrane protein 1 (PfEMP1), a specific cell-surface ligand expressed by iRBCs [31]. The sequestration of iRBCs triggers activation of the endothelial cells (ECs), which in turn lead to inflammatory responses where there is release of pro-inflammatory cytokine tumour necrosis factor (TNF) [32]. This cytokine is known to upregulate endothelial receptor cells, resulting in upregulation of intracellular adhesion molecules 1 (ICAM-1) and vascular cellular adhesion molecules-1 (VCAM–1) [33]. All these factors would eventually lead to numerous downstream vascular effects, such as increased vasoconstriction, reduced cerebral blood flow to several sites in the brain, vascular obstruction, oxygen starvation, and even disruption of the integrity of the BBB [34]. The observed whitening of the susceptible strain brains (Fig. 2a) could therefore be attributed to the reduced or the complete blockage of cerebral blood flow to several sites in the brain, a condition termed as hypoxia.

The BBB is comprised of specialized endothelial cells (ECs) that function as a selective permeable membrane to control nutrients and ion transport into the brain and to bar unwanted molecules or compounds into the brain [35, 36]. The ECs are sealed together by the presence of tight junctions. However, loss of tight junctions has been observed in areas of iRBC sequestration, leading to creation of openings or gaps within the BBB [36]. Such gaps or openings could function as a gateway for the entry of toxic molecules or compounds into the brain, thus a pointer to the dysfunctioning BBB, which is a major pathological event associated with CM [34, 37]. This was well demonstrated in this work through staining of the human cerebral malaria (HCM) mouse models’ brains with the 2% Evans Blue dye (Fig. 2c), an indicator of the impaired BBB integrity in the infected mice. The data of this study are similar to other previously related studies that confirmed impairment of the integrity of BBB in susceptible animal models and subsequent brain staining with Evans Blue dye [27, 38‐40].

Good safety profile was exhibited as the drug was well tolerated in mice even at high dose of 2000 mg/kg administered in an acute oral toxicity test, with 67% survival. The remarkable mice survival indicates that the exact LD₅₀ could be within 2000 mg/kg. However, it should be noted that this is a dosage 40 times the curative dose of 25 mg/kg. Considering the possible LD₅₀/ED₅₀ ratio, a possible TI value >400 reflects a wider margin of safety for the trioxaquine. The same safety profile was manifested in in vitro cytotoxicity studies where the selectivity index (SI) of the trioxaquine was determined by comparing the IC_50s of Hep2 cell line and that of the CQ-resistant parasite (W2) for the trioxaquine [17]. The high SI value obtained (>2762) indicates that the high antiplasmodial activity for the trioxaquine observed during in vitro antiplasmodial evaluation was due to its activity and not due to its cytotoxicity [17]. A similar class of trioxaquine was reported to have antiplasmodial activity against all the erythrocytic forms of the parasite, including the gametocytes with TI value range of 23–100 considered safe [13]. The high safety profile exhibited both in vivo and in vitro by the trioxaquine would therefore be attributed to the fact that the individual precursors for the trioxaquine are already indicated for clinical use in the management of malaria.

The remarkable efficacy and good safety profile of the trioxaquine observed imply that if the findings could be replicated in clinical studies, then the drug has potential for use in management of CM.

The remarkable in vivo results for the trioxaquine obtained by the established infection test, as well as the post-treatment survival data, clearly indicate that covalent bitherapy is a viable strategy for development of future anti-malarial agents for management of CM. An encouraging safety profile of the drug in mice with a therapeutic index (TI) value of >400 is not surprising since the precursors are part of drugs already indicated for clinical use. Overall therefore, the curative effect together with good safety profiles observed with the trioxaquine clearly demonstrate its potential as a drug candidate for management of CM, especially in a time of shrinking anti-malarial armamentarium for management of CM.