Malaria afflicts 300-500 million people in the world and the mortality ranges from 1-2 million, children in Africa being the most susceptible. With a vaccine not being available against malaria and the front line drugs such as chloroquine and antifolates registering widespread parasite resistance, the challenge of malaria treatment is a formidable task. While, research to discover new drugs has become essential, it has also become necessary to identify therapeutic strategies in the short-term. One approach is to examine whether known drugs used for other applications can be used to treat malaria. A second strategy is to look for natural compounds for antimalarial activity either singly or in combination. Combination therapy has assumed considerable importance in the context of artemisinin derivatives being the sole, tested, efficacious antimalarials left in the basket. A combination therapy with artemisinin derivative may prevent recrudescence due to monotherapy, extend the life of the drug and perhaps bring down the cost of therapy as well. A primary requirement to embark on such studies is to assess the status of drug resistance to the front line drugs in use. In India, chloroquine is still used as the front line drug for malaria therapy. Although, there have been indications and sporadic reports on the development of chloroquine resistance in the country, there has not been a detailed molecular or clinical evaluation for resistance. Keeping all these considerations in mind, the objectives of the present study are as follows: 1. Evaluation of chloroquine resistance inP.falciparum isolates from patients using Pfcrt-mutation as marker. 2. Evaluation of the anti-tubercular drugs, rifampicin and isonicotinic acid hydrazide (INH) for antimalarial activity. 3. Evaluation of curcumin from turmeric singly and in combination with α,β- arteether for antimalarial acitivity. Chapter I deals with the review of literature pertaining to scenario of available antimalarials, efforts to discover new antimalarials based on new drug targets, mechanisms of drug resistance and strategies for combination therapies. Chapter II deals with an evaluation of Pfcrt mutation in clinical samples of P.falciparum malaria in India. After several false starts to find molecular markers to identify chloroquine resistance, mutations in the Pfcrt gene of P.falciparum, K76T mutation in particular, has been shown to correlate very well with chloroquine resistance in culture. A study of 109 P.falciparum – infected blood samples from different parts of India has revealed that close to 95% of the isolates carry the K76T mutation. This was shown on the basis of susceptibility to ApoI restriction digestion of the PCR product covering this region (264 nt) and DNA sequencing of the PCR product. Interestingly, the resistant haplotype in this region of 72-76 amino acids was found to be mostly SVMNT, except for 4 samples with CVIET haplotype. SVMNT has all along been considered to be of South American origin, where as CVIET is of South East Asian/African origin. Subsequent studies by another group in the country has also shown that the Pfcrt - K76T mutation is seen at least in 85% of the cases and in addition to the dominant SVMNT haplotype, newer haplotypes are also seen. The present study has also included an analysis of N86Y mutation in the Pfmdr1 gene based on susceptibility to Afl III restriction enzyme digestion and DNA sequencing of the PCR product (603 nt). Pfmdr1 mutations have been extensively studied in literature for possible correlation to CQR. The net conclusion is that it does not contribute directly to CQR but may have an indirect correlation. It has been shown in Mali that there is very good correlation between Pfcrt - K76T mutation and Pfmdr1 - N86Y mutation in the P.falciparum isolates. However, in the present study with Indian isolates only around 30% of the samples were found to carry the Pfmdr1 - N86Y mutation. While, further studies on the clinical relevance of the extensive Pfcrt mutation seen in the Indian isolates are needed, it is clear that the genetic change towards chloroquine resistance has already taken place in the Indian context. Chapter III is devoted to a study of the antimalarial effects of the anti-tubercular drugs, rifampicin and INH. This is on the basis that rifampicin is an inhibitor of prokaryotic and mitochondrial/chloroplast RNA polymerase. P.falciparum harbors the apicoplast, a remnant of chloroplast with a 35kb DNA. It is known that the β, β’- subunits of the apicoplast RNA polymerase are coded by the apicoplast DNA. There is a report that rifampicin is a slow acting antimalarial in cases of P.vivax -nfection. INH is known to act by inhibiting the enoyl-ACP reductase and β - hydroxy ACP synthase in M.tuberculosis. While, M.tuberculosis is known to manifest Fab I and Fab II pathways of fatty acid biosynthesis, it has recently been shown that P.falciparum manifests the FabII (discrete enzymes) pathway. Thus, it was considered possible that INH may also inhibit the fatty acid biosynthetic pathway of P.falciparum leading to inhibition of phospohlipid and membrane biosynthesis. Studies were, therefore, carried out with rifampicin, INH and the combination on the survival of P.falciparum in culture and P.berghei in mice. With P.falciparum, growth was followed by measuring3[H]-Hypoxanthine incorporation and slide detection of parasites using Giemsa stain. The results indicate that while, rifampicin inhibits P.falciparum growth with an IC50 around 25nM, and INH fails to show any effect even at 200µM concentration. The combination of rifampicin (25nM) and INH (100µM) shows enhanced killing effect. In view of these results, studies were undertaken in mice infected with P.berghei. After 72 hr infection, the mice were orally fed with rifampicin (500 µg/40 g body weight) or INH (1 mg/40 g body weight) or a combination of the two orally for 5 days, starting on day 3. Apart from parasite clearance in blood, protection against mortality is a good index, since all the infected mice die in about 7-8 days. The results indicate that rifampicin leads to around 50% protection and INH treatment gives around 10% protection. However, the combination gives around 83% protection with complete clearance of the parasite in blood. Short- term treatment of infected mice with drugs and an assay of rpoB/C transcription in the parasite using appropriate PCR primers reveal a striking inhibition in combination treatment. Again, when such parasites were put into short-term culture and32P- incorporation into phospholipids was measured, there was striking inhibition with combination treatment. Thus, the results indicate that a combination of rifampicin and INH has potent antimalarial activity in P.berghei-infected mice. The results are dramatic in this case when compared to the results obtained with P.falciparum culture. It is not clear whether the differences are due to differences in action in vitro vs in vivo or due to differences in susceptibility between P.falciparum and P. berghei to the treatment provided. Chapter IV deals with the antimalarial activity of curcumin (diferuloyl methane) from turmeric singly or in combination with artemesinin or its derivative. Curcumin is reported to have a wide variety of biochemical effects and its anti-cancer activity is under serious investigation. There is an earlier report that curcumin shows antimalarial activity against chloroquine-sensitive P.falciparum. In the present study, curcumin was tested against a chloroquine-resistant culture of P.facliparum and it inhibits growth with an IC50 of 5-8 µM. When P.berghei-infected mice were orally fed with curcumin for 5 days, there was delay in the development of parasitemia, with about 30% of the animals protected against mortality by day 28. For reasons mentioned earlier curcumin was tested in combination with artemisinin/derivative in P.falciparum culture and P.berghei in mice. The results indicate that artemisinin and curcumin have an additive inhibitory effect on P.falciparum growth, based on a detailed analysis of the isobolograms. In terms of the mechanism of action, curcumin treatment leads to accumulation of45Ca in the parasite cytoplasm. It also has a striking inhibitory effect on32P-incorporation into parasite proteins and phospholipids, suggesting an interference with phosphorylation mechanisms. None of these effects are seen under artemisinin treatment, which has been reported to specifically inhibit PfATP6 (Ca ATPase) in P.falciparum. In view of the possible different modes of action of artemisinin and curcumin, the combination was tested in P.berghei-infected mice. The infected mice received a single injection of α,β-arteether and 3 oral doses of curcumin (5mg/30g body weight). Curcumin treatment was found to dramatically delay the onset of parasitemia seen in animals treated with α,β-arteether alone due to recrudescence. In particular, a combination with a single injection of α,β-arteether (750µg or 1.5mg/30g body weight) followed by 3 oral doses of curcumin leads to complete prevention of recrudescence and 100% protection against mortality. Several combinations with artemisinin derivative are under investigation and they all suffer from toxic side effects, pharmacokinetic mismatch, known resistance to the combining partner and high cost. It is felt that this artemisinin derivative curcumin combination could prove superior in view of the fact that no resistance is known to curcumin and is safe even at very high doses used in the human. Both the drugs are eliminated fast and curcumin is a cheap chemical and available in plenty from natural source (turmeric). In view of these positive attributes, a clinical trial with this combination is recommended. 121

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.