Artemisinin and curcumin show an additive interaction in killing Plasmodium falciparum in culture. In vivo, 3 oral doses of curcumin following a single injection of ␣,␤-arteether to Plasmodium berghei-infected mice are able to prevent recrudescence due to ␣,␤-arteether monotherapy and ensure almost 100% survival of the animals.Artemisinin derivative-based combination therapy (ACT) has been advocated as the therapy of choice to handle widespread drug resistance in Plasmodium falciparum malaria, at the same time preventing recrudescence due to artemisinin monotherapy. However, most of the combinations are less than ideal because of side effects, pharmacokinetic mismatch, and cost (8,15). Studies in this laboratory have shown that curcumin isolated from the roots of Curcuma longa (turmeric) has antimalarial activity in P. falciparum culture and in Plasmodium berghei-infected mice (10). In this study, we propose a novel artemisinin-curcumin therapy to treat malaria.Artemisinin and curcumin (98% curcuminoid content) were purchased from Sigma Chemicals, Bangalore, India. ␣,␤-Arteether (EMAL, a synthetic derivative of artemisinin) developed by Central Drug Research Institute, Lucknow, India, was a kind gift from IPCA Laboratories Ltd., Mumbai, India. [ 3 H]hypoxanthine was purchased from PerkinElmer, Singapore.The P. falciparum FCK strain, a local chloroquine-resistant isolate, was cultured in human O-positive washed erythrocytes using standard protocols (14). Parasites were synchronized using 5% (wt/vol) D-sorbitol, and the cultures were pooled and the parasites released from the erythrocyte with 0.15% (wt/vol) saponin (3) for further processing. The 50% inhibitory concentrations (IC 50 s) for artemisinin and curcumin were determined in P. falciparum cultures by measuring [3 H]hypoxanthine uptake into the parasite as a measure of viability (2). Fractional inhibitory concentrations (FIC) were calculated using the formulawhere A C and B C are the concentrations of A and B in the combination associated with a particular level of effect, e.g., IC 60 , and A E and B E are the concentrations of A and B when used singly to produce the same level of effect, on the basis that a simple additive interaction should lead to a value of 1 (7). FIC were derived from graphs drawn using GraphPad Prism 4.Means Ϯ standard deviations and Student's t test were analyzed using Microsoft Excel. For in vivo studies, Swiss mice (25 to 28 g) were injected intraperitoneally with P. berghei-infected mouse blood (60 to 70% parasitemia) on day 0, such that the animals developed high parasitemia and died in about 5 to 8 days. The artemisinin derivative ␣,␤-arteether was injected intramuscularly at different doses on day 1. This was followed by oral feeding of curcumin in dimethyl sulfoxide on days 1, 2, and 3 (100 mg/kg of body weight). The mice were observed for external symptoms and mortality when the drugs were given alone or in combination. Blood from the tail vein was analyzed on different days for parasitemia using Giemsa to stain the slides.De...

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) and the P. falciparum multidrug resistance 1 (Pfmdr1) mutation (N86Y) were analyzed as markers of chloroquine resistance in the DNAs of 73 blood samples from patients with P. falciparum malaria in India. Seventy of the 73 DNAs had the Pfcrt K76T mutation. Of these, 66 had the SVMNT haplotype and four had CVIET, the African/Southeast Asian haplotype. Only 20 of 69 DNAs had the Pfmdr1 N86Y mutation. It is surprising that the Pfcrt haplotype in India is predominantly SVMNT, rather than that seen in Southeast Asia. The widespread prevalence of the Pfcrt K76T mutation is a cause for concern.

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Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA), a single dose of alpha,beta-arteether (ART) with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE) or ART+curcumin (AC) treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation. But, parasites in liver and spleen were not cleared in AE or AC treatments, perhaps, serving as a reservoir for recrudescence. Parasitemia in blood reached up to 60% in AE-treated mice during the recrudescence phase, leading to death of animals. A transient increase of up to 2–3% parasitemia was observed in AC-treatment, leading to protection and reversal of splenomegaly. A striking increase in spleen mRNA levels for TLR2, IL-10 and IgG-subclass antibodies but a decrease in those for INFγ and IL-12 was observed in AC-treatment. There was a striking increase in IL-10 and IgG subclass antibody levels but a decrease in INFγ levels in sera leading to protection against recrudescence. AC-treatment failed to protect against recrudescence in TLR2−/− and IL-10−/− animals. IL-10 injection to AE-treated wild type mice and AC-treated TLR2−/− mice was able to prolong survival. Blood from the recrudescence phase in AE-treatment, but not from AC-treatment, was able to reinfect and kill naïve animals. Sera from the recrudescence phase of AC-treated animals reacted with several parasite proteins compared to that from AE-treated animals. It is proposed that activation of TLR2-mediated innate immune response leading to enhanced IL-10 production and generation of anti-parasite antibodies contribute to protective immunity in AC-treated mice. These results indicate a potential for curcumin-based combination therapy to be tested for prevention of recrudescence in falciparum and relapse in vivax malaria.

Many reports indicate medicinal value of garlic (Allium sativum), a popular herbal medicine used worldwide, and its therapeutic effect against several diseases. Earlier studies in our laboratory have shown a potential therapeutic role of garlic-artemisinin combination in mice infected with Plasmodium berghei. A single dose of a, barteether with three oral doses of garlic provides almost 95% protection. The present study aims to understand the mode of action of this combination. We have documented the level of nitric oxide (NO), a key molecule of protection and have seen in the reversal of organ morphology caused by malaria infection. The combination effects on the (a) survival rate and degree of parasitemia and (b) NO levels in blood, liver, spleen and thymus of malaria-infected mice were investigated. During the study, liver, spleen and thymus cell suspensions were assessed for immunobiochemical alterations of NO levels. The increase in NO level after infection appears to be unable to protect, whereas striking increase in spleen and thymus leads to protection against infection, and is further confirmed by detection of increased inducible nitric oxide synthase mRNA expression levels in different organs by RT-PCR. In addition, the role of T cell subsets during combination treatment was also studied. All these results indicate a potential mechanism of protection through NO pathway in combination-treated animals after malaria infection and may lead to an immunotherapy trial of malaria disease.

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