Adjunct Therapy with Curcumin for the Treatment of Malaria : Studies in a Murine Model

Abstract

Malaria accounts for 198 million cases worldwide; with a high mortality rate. 584000 deaths were reported in 2013. Malaria is a re-emerging disease globally due to drug resistance, parasite recrudescence and non-availability of a vaccine. Chloroquine, quinine and antifolates served as frontline antimalarial drugs for decades. Development of resistance to chloroquine and antifolates, and the decreased efficacy of mefloquine, and even quinine, in malaria-endemic regions, has led to artemisinin derivatives evolving as frontline drugs. Artemisinin is a potent antimalarial compound and clears around 104 parasites per cycle. Despite being a potent antimalarial, artemisinin derivatives suffer from poor pharmacokinetic properties and short half lives. This has led to the development of artemisinin-based combination therapies (ACTs) using a partner drug with a longer half-life. However, resistance to ACTs has been reported in the last few years, perhaps due to lack of adherence to prescribed regimens or suboptimal treatment and the use of counterfeit drugs. Therefore there is an urgent need to develop an alternative ACT which overcomes these limitations. This thesis entitled “Adjunct therapy with curcumin for the treatment of malaria: studies in a murine model” describes the antimalarial activity of curcumin and artemisinin and the adjunct role of curcumin in the prevention of parasite recrudescence and cerebral malaria. The thesis is divided into three chapters: The first chapter entitled “Introduction: Malaria and anti-malarial drugs” consists of a brief introduction of malaria, the parasite life cycle and currently known antimalarial drugs. During the course of infection, the Plasmodium undergoes sporogony in the mosquito, and merogony and schizogony in the human host. All these life cycle stages are briefly described with depictions. A major part of this chapter is dedicated to describe antimalarial compounds under the following headings 1. Quinoline derivatives 2. 4-aminoquinolines 3. Antifolates 4. Artemisinin derivatives 5. Antibiotics and 6. Curcumin. The second chapter is aimed at examining the ability of curcumin-arteether (a synthetic derivative of artemisinin) combination therapy in preventing parasite recrudescence in a murine model through immunomodulation employing various immunological, molecular biological, and biochemical techniques. The use of suboptimal doses of antimalarial drugs leads to recrudescence or relapse of malaria (reappearance of the parasite in blood after antimalarial regimen). In the present study we have addressed this issue by the use of curcumin as an adjunct molecule with α,β arteether (a synthetic derivative of artemisinin). We have studied recrudescence in a Swiss mice model. A suboptimal dose was standardized by the use of different doses of α,β arteether (AE) ranging from 250µg to 1500 µg. We found 750 µg to be a suboptimal dose and studied the adjunct nature of curcumin when animals were treated with AE suboptimal dose or AE+curcumin (AC) combination treatment and monitored the survival of animals. Our results clearly demonstrate that ~95% of animals treated with the suboptimal AE dose died of recrudescent malaria but there was almost 100% survival of AC-treated animals; these animals were under observation for at least 3 months. We have studied the effect of curcumin in a recrudescence model at the molecular level. Curcumin by itself has antimalarial activity, but only in combination with α,β arteether prevented recrudescence. Our results indicate that curcumin has immunomodulatory activity. Serum cytokine analysis and spleen mRNA analysis for proinflammatory and anti-inflammatory mediators indicate that AC treatment effectively reduced both mRNA and serum cytokine levels of IFNγ, TNFα, IL-12 and effectively increased both mRNA and serum levels IL-10 and antibodies of the IgG subclass. Using TLR2 and IL-10 knockout animals, we have conclusively demonstrated that TLR2 is involved in the production of IL-10, and IL-10 is required for the AC-mediated protection of animals during the recrudescence period. We conclude that curcumin is able to prevent parasite recrudescence essentially by switching the Th1 response to a Th2 response. The third chapter deals with the study the effect of areether-curcumin (AC) combination therapy in the prevention of Experimental Cerebral Malaria. Although malaria mortality rates have decreased by an impressive 47% between 2000 and 2013, it is still a major affliction of mankind (WHO 2014). Plasmodium falciparum infection causes human cerebral malaria (HCM). The mortality rate in HCM is unacceptably high (15–20%), despite the availability of artemisinin-based therapy. HCM is characterized by a rapid progression from headache, general malaise, and prostration to hemiparesis, ataxia, unrousable coma, and death. Paediatric HCM deaths are mostly due to respiratory arrest. Alternatively, death may be due to parasite-mediated injury to a sensitive location; a small lesion due to parasite in brain stem can cause sudden respiratory arrest. In HCM, cytoadherence of pRBCs in brain microvasculature has been implicated as a major contributing factor for CM pathology. The failure of a large number of adjunct therapies in HCM demands the development of new intervention strategies. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. In this study, we have demonstrated the efficacy of curcumin and PLGA nanocurcumin in the treatment of Experimental Cerebral Malaria (ECM), using the Plasmodium berghei ANKA-infected mouse model (C57BL/6). Curcumin/PLGA nanocurcumin alone can prevent the onset of ECM. We have shown that curcumin/PLGA nanocurcumin can prevent CD8+ T cell, CXCR3+ CD8 T cell and parasite-infected RBC (pRBC) sequestration in the brain. These are also the essential parameters underlying HCM. We have also demonstrated that curcumin effectively inhibits T cell proliferation in spleen. We have explained the anti-inflammatory effects of curcumin by showing the inhibition of NF-B in both brain and spleen, which is a plausible explanation. But, curcumin/PLGA nanocurcumin treated animals died later due to build up of parasitemia in blood and subsequent anemia. Moreover, a combination therapy with arteether and curcumin given even after the onset of neurological symptoms can completely cure and protect the animals against mortality. We have tested AC-combination after the onset of symptoms to mimic patient conditions in HCM, since the murine regimens reported were not successful in the treatment of HCM. Our results clearly demonstrate that AC treatment even after the onset of symptoms ensures 100% survival. Since the bioavailability of curcumin is reported to be poor, we have also tested the efficacy of PLGA nanocurcumin and find that it is superior to native curcumin in terms of therapeutic effects. It is concluded that curcumin would be an ideal adjunct drug to be used with the artemisinin derivatives to treat malaria, including cerebral malaria.