Metabolomic changes in vertebrate host during malaria disease progression.

Metabolomics refers to top-down systems biological analysis of metabolites in biological specimens. Phenotypic proximity of metabolites makes them interesting candidates for studying biomarkers of environmental stressors such as parasitic infections. Moreover, the host-parasite interaction directly impinges upon metabolic pathways since the parasite uses the host metabolite pool as a biosynthetic resource. Malarial infection, although not recognized as a classic metabolic disorder, often leads to severe metabolic changes such as hypoglycemia and lactic acidosis. Thus, metabolomic analysis of the infection has become an invaluable tool for promoting a better understanding of the host-parasite interaction and for the development of novel therapeutics. In this review, we summarize the current knowledge obtained from metabolomic studies of malarial infection in rodent models and human patients. Metabolomic analysis of experimental rodent malaria has provided significant insights into the mechanisms of disease progression including utilization of host resources by the parasite, sexual dimorphism in metabolic phenotypes, and cellular changes in host metabolism. Moreover, these studies also provide proof of concept for prediction of cerebral malaria. On the other hand, metabolite analysis of patient biofluids generates extensive data that could be of use in identifying biomarkers of infection severity and in monitoring disease progression. Through the use of metabolomic datasets one hopes to assess crucial infection-specific issues such as clinical severity, drug resistance, therapeutic targets, and biomarkers. Also discussed are nascent or newly emerging areas of metabolomics such as pre-erythrocytic stages of the infection and the host immune response. This review is organized in four broad sections-methodologies for metabolomic analysis, rodent infection models, studies of human clinical specimens, and potential of immunometabolomics. Data summarized in this review should serve as a springboard for novel hypothesis testing and lead to a better understanding of malarial infection and parasite biology.

Vivax infection alters peripheral B-cell profile and induces persistent serum IgM.

B cell-mediated humoral responses are essential for controlling malarial infection. Studies have addressed the effects of Plasmodium falciparum infection on peripheral B-cell subsets but not much is known for P. vivax infection. Furthermore, majority of the studies investigate changes during acute infection, but not after parasite clearance. In this prospective study, we analysed peripheral B-cell profiles and antibody responses during acute P. vivax infection and upon recovery (30 days post-treatment) in a low-transmission area in India. Dengue patients were included as febrile-condition controls. Both dengue and malaria patients showed a transient increase in atypical memory B cells during acute infection. However, transient B cell-activating factor (BAFF)-independent increase in the percentage of total and activated immature B cells was observed in malaria patients. Naïve B cells from malaria patients also showed increased TLR4 expression. Total IgM levels remained unchanged during acute infection but increased significantly at recovery. Serum antibody profiling showed a parasite-specific IgM response that persisted at recovery. A persistent IgM autoantibody response was also observed in malaria but not dengue patients. Our data suggest that in hypoendemic regions acute P. vivax infection skews peripheral B-cell subsets and results in a persistent parasite-specific and autoreactive IgM response.

Teratogenicity of Artemether-Clindamycin Nanostructured Lipid Carriers in Rats.

Currently, artemisinin-based combination therapy is considered the best option in the treatment of malaria. However, toxicity of artemisinins limits their use in pregnancy. In the absence of sufficient toxicity data, the World Health Organization recommends that artemisinins are not to be used in the first trimester of pregnancy and can be used only in second and third trimesters, when other treatments are not available. We have recently observed that drugs loaded in nanolipid carriers are selectively taken up in Plasmodium-infected erythrocytes with a concomitant reduction in the dose required to cure animals. Thus, 20% of the therapeutic dose of artemether-clindamycin (ARM-CP) loaded in nanostructured lipid carriers (NLCs; mean particle size 55 ± 10 nm) resulted in complete parasite clearance and 100% survival of infected mice. Here, we investigate the teratogenicity of this formulation in rodents (dosing on alternate days from 6th day to 18th day of gestation; 12-15 animals/group). The teratogenicity of drug-free NLCs and artesunate-clindamycin (ARS-CP) solution was also evaluated. We found that the therapeutic dose of ARS-CP caused fetal resorptions (87.5% resorptions in 8 litters), suggesting its unsuitability for use in pregnancy. Artesunate-clindamycin NLCs at therapeutic doses also resulted in ∼90% fetal resorptions in 10 litters examined. However, postimplantation losses or fetal malformations were not observed at the dose of ARM-CP NLCs that was required for complete parasite clearance in preclinical trials (ie, 20% of the therapeutic dose). Our data suggest that the NLCs loaded with 20% of the therapeutic dose of ARM-CP may have potential in treating malaria during pregnancy.

Multifaceted Role of Heme during Severe Plasmodium falciparum Infections in India.

Several immunomodulatory factors are involved in malaria pathogenesis. Among them, heme has been shown to play a role in the pathophysiology of severe malaria in rodents, but its role in human severe malaria remains unclear. Circulating levels of total heme and its main scavenger, hemopexin, along with cytokine/chemokine levels and biological parameters, including hemoglobin and creatinine levels, as well as transaminase activities, were measured in the plasma of 237 Plasmodium falciparum-infected patients living in the state of Odisha, India, where malaria is endemic. All patients were categorized into well-defined groups of mild malaria, cerebral malaria (CM), or severe noncerebral malaria, which included acute renal failure (ARF) and hepatopathy. Our results show a significant increase in total plasma heme levels with malaria severity, especially for CM and malarial ARF. Spearman rank correlation and canonical correlation analyses have shown a correlation between total heme, hemopexin, interleukin-10, tumor necrosis factor alpha, gamma interferon-induced protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1) levels. In addition, canonical correlations revealed that heme, along with IP-10, was associated with the CM pathophysiology, whereas both IP-10 and MCP-1 together with heme discriminated ARF. Altogether, our data indicate that heme, in association with cytokines and chemokines, is involved in the pathophysiology of both CM and ARF but through different mechanisms.

Evidence of IL-17, IP-10, and IL-10 involvement in multiple-organ dysfunction and IL-17 pathway in acute renal failure associated to Plasmodium falciparum malaria.

BACKGROUND: Plasmodium falciparum malaria in India is characterized by high rates of severe disease, with multiple organ dysfunction (MOD)-mainly associated with acute renal failure (ARF)-and increased mortality. The objective of this study is to identify cytokine signatures differentiating severe malaria patients with MOD, cerebral malaria (CM), and cerebral malaria with MOD (CM-MOD) in India. We have previously shown that two cytokines clusters differentiated CM from mild malaria in Maharashtra. Hence, we also aimed to determine if these cytokines could discriminate malaria subphenotypes in Odisha. METHODS: P. falciparum malaria patients from the SCB Medical College Cuttack in the Odisha state in India were enrolled along with three sets of controls: healthy individuals, patients with sepsis and encephalitis (n = 222). We determined plasma concentrations of pro- and anti-inflammatory cytokines and chemokines for all individuals using a multiplex assay. We then used an ensemble of statistical analytical methods to ascertain whether particular sets of cytokines/chemokines were predictors of severity or signatures of a disease category. RESULTS: Of the 26 cytokines/chemokines tested, 19 increased significantly during malaria and clearly distinguished malaria patients from controls, as well as sepsis and encephalitis patients. High amounts of IL-17, IP-10, and IL-10 predicted MOD, decreased IL-17 and MIP-1α segregated CM-MOD from MOD, and increased IL-12p40 differentiated CM from CM-MOD. Most severe malaria patients with ARF exhibited high levels of IL-17. CONCLUSION: We report distinct differences in cytokine production correlating with malarial disease severity in Odisha and Maharashtra populations in India. We show that CM, CM-MOD and MOD are clearly distinct malaria-associated pathologies. High amounts of IL-17, IP-10, and IL-10 were predictors of MOD; decreased IL-17 and MIP-1α separated CM-MOD from MOD; and increased IL-12p40 differentiated CM from CM-MOD. Data also suggest that the IL-17 pathway may contribute to malaria pathogenesis via different regulatory mechanisms and may represent an interesting target to mitigate the pathological processes in malaria-associated ARF.

Single episode of mild murine malaria induces neuroinflammation, alters microglial profile, impairs adult neurogenesis, and causes deficits in social and anxiety-like behavior.

Cerebral malaria is associated with cerebrovascular damage and neurological sequelae. However, the neurological consequences of uncomplicated malaria, the most prevalent form of the disease, remain uninvestigated. Here, using a mild malaria model, we show that a single Plasmodium chabaudi adami infection in adult mice induces neuroinflammation, neurogenic, and behavioral changes in the absence of a blood-brain barrier breach. Using cytokine arrays we show that the infection induces differential serum and brain cytokine profiles, both at peak parasitemia and 15days post-parasite clearance. At the peak of infection, along with the serum, the brain also exhibited a definitive pro-inflammatory cytokine profile, and gene expression analysis revealed that pro-inflammatory cytokines were also produced locally in the hippocampus, an adult neurogenic niche. Hippocampal microglia numbers were enhanced, and we noted a shift to an activated profile at this time point, accompanied by a striking redistribution of the microglia to the subgranular zone adjacent to hippocampal neuronal progenitors. In the hippocampus, a distinct decline in progenitor turnover and survival was observed at peak parasitemia, accompanied by a shift from neuronal to glial fate specification. Studies in transgenic Nestin-GFP reporter mice demonstrated a decline in the Nestin-GFP(+)/GFAP(+) quiescent neural stem cell pool at peak parasitemia. Although these cellular changes reverted to normal 15days post-parasite clearance, specific brain cytokines continued to exhibit dysregulation. Behavioral analysis revealed selective deficits in social and anxiety-like behaviors, with no change observed in locomotor, cognitive, and depression-like behaviors, with a return to baseline at recovery. Collectively, these findings indicate that even a single episode of mild malaria results in alterations of the brain cytokine profile, causes specific behavioral dysfunction, is accompanied by hippocampal microglial activation and redistribution, and a definitive, but transient, suppression of adult hippocampal neurogenesis.

Intravenous ß-artemether formulation (ARM NLC) as a superior alternative to commercial artesunate formulation.

OBJECTIVES: To compare the in vivo pharmacodynamic efficacy of intravenously administered artemether nanostructured lipid carrier (ARM NLC) with commercial artesunate (C-AST) at different dose levels. METHODS: The study compared the in vivo pharmacodynamic efficacy of ARM NLC with C-AST in a murine model. For this study, the Peters 4 day suppressive test was adopted. Plasmodium berghei was the causative organism for inducing malaria in mice. The efficacies of the formulations were evaluated on the basis of percentage parasitaemia in, and survival of, mice. RESULTS: In comparison with the C-AST formulation, ARM NLC demonstrated superior activity in terms of reduction in parasitaemia and increased survival. CONCLUSIONS: Although both formulations were found to be effective in reducing parasitaemia in the murine model, ARM NLC was found to be superior. The study clearly demonstrates the effectiveness of this novel alternative to existing artesunate dosage forms.

Plasmodium riboprotein PfP0 induces a deviant humoral immune response in Balb/c mice.

Passive immunization with antibodies to recombinant Plasmodium falciparum P0 riboprotein (rPfP0, 61-316 amino acids) provides protection against malaria. Carboxy-terminal 16 amino acids of the protein (PfP0C0) are conserved and show 69% identity to human and mouse P0. Antibodies to this domain are found in 10-15% of systemic lupus erythematosus patients. We probed the nature of humoral response to PfP0C0 by repeatedly immunizing mice with rPfP0. We failed to raise stable anti-PfP0C0 hybridomas from any of the 21 mice. The average serum anti-PfP0C0 titer remained low (5.1 ± 1.3 × 104). Pathological changes were observed in the mice after seven boosts. Adsorption with dinitrophenyl hapten revealed that the anti-PfP0C0 response was largely polyreactive. This polyreactivity was distributed across all isotypes. Similar polyreactive responses to PfP0 and PfP0C0 were observed in sera from malaria patients. Our data suggests that PfP0 induces a deviant humoral response, and this may contribute to immune evasion mechanisms of the parasite.

Global host metabolic response to Plasmodium vivax infection: a 1H NMR based urinary metabonomic study.

BACKGROUND: Plasmodium vivax is responsible for the majority of malarial infection in the Indian subcontinent. This species of the parasite is generally believed to cause a relatively benign form of the disease. However, recent reports from different parts of the world indicate that vivax malaria can also have severe manifestation. Host response to the parasite invasion is thought to be an important factor in determining the severity of manifestation. In this paper, attempt was made to determine the host metabolic response associated with P. vivax infection by means of NMR spectroscopy-based metabonomic techniques in an attempt to better understand the disease pathology. METHODS: NMR spectroscopy of urine samples from P. vivax-infected patients, healthy individuals and non-malarial fever patients were carried out followed by multivariate statistical analysis. Two data analysis techniques were employed, namely, Principal Component Analysis [PCA] and Orthogonal Projection to Latent Structure Discriminant Analysis [OPLS-DA]. Several NMR signals from the urinary metabolites were further selected for univariate comparison among the classes. RESULTS: The urine metabolic profiles of P. vivax-infected patients were distinct from those of healthy individuals as well as of non-malarial fever patients. A highly predictive model was constructed from urine profile of malarial and non-malarial fever patients. Several metabolites were found to be varying significantly across these cohorts. Urinary ornithine seems to have the potential to be used as biomarkers of vivax malaria. An increasing trend in pipecolic acid was also observed. The results suggest impairment in the functioning of liver as well as impairment in urea cycle. CONCLUSIONS: The results open up a possibility of non-invasive analysis and diagnosis of P. vivax using urine metabolic profile. Distinct variations in certain metabolites were recorded, and amongst these, ornithine may have the potential of being used as biomarker of malaria. Pipecolic acid also showed increasing trend in the malaria patient compared to the other groups.

Self-microemulsifiyng suppository formulation of ß-artemether.

Parasitic diseases are of immense global significance as around 30% of world's population experiences parasitic infections. Among these, malaria is the most life-threatening disease. Various routes of administration have been explored for delivering antimalarial actives. The present investigation aims at formulating self-microemulsifying suppositories of ß-artemether with faster onset of action and prolonged effect to be administered by rectal route. These were compared with conventional polyethylene glycol suppositories with respect to melting range, rheology, texture analysis, disintegration time, self microemulsification time, particle size, and drug content. In vitro drug release was studied by using USP apparatus II. Further, the suppositories were evaluated in murine model against virulent rodent malaria parasite Plasmodium berghei wherein the developed self-microemulsifying suppositories could sustain the activity (94%) for 20 days post infection. The survival of animals was also better as compared to the conventional formulation.

A history of juvenile mild malaria exacerbates chronic stress-evoked anxiety-like behavior, neuroinflammation, and decline of adult hippocampal neurogenesis in mice.

Children residing in high malaria transmission regions are particularly susceptible to malaria. This early-life window is also a critical period for development and maturation of the nervous system, and inflammatory insults during this period may evoke a persistent increase in vulnerability for psychopathology. We employed a two-hit model of juvenile mild malaria and a two-week chronic unpredictable mild stress (CUMS) regime, commencing 60 days post-parasite clearance, to assess whether a history of juvenile infection predisposed the mice towards mood-related behavioral alterations and neurocognitive deficits. We showed that adult mice with a history of juvenile malaria (A-H/JMAL) exhibited heightened CUMS-associated anxiety-like behavior, with no observable change in cognitive behavior. In contrast, mice with a history of adult malaria did not exhibit such enhanced stress vulnerability. At baseline, A-H/JMAL mice showed increased activated microglia within the hippocampal dentate gyrus subfield. This was accompanied by a decrease in proliferating neuronal progenitors, with total number of immature hippocampal neurons unaltered. This neuroinflammatory and neurogenic decline was further exacerbated by CUMS. At day-14 post-CUMS, hippocampi of A-H/JMAL mice showed significantly higher microglial activation, and a concomitant decrease in progenitor proliferation and number of immature neurons. Taken together, these results suggest that a history of juvenile mild malaria leaves a neuroinflammatory mark within the hippocampal niche, and this may contribute to a heightened stress response in adulthood. Our findings lend credence to the idea that the burden of malaria in early-life results in sustained CNS changes that could contribute to increased vulnerability to adult-onset neuronal insults.

Design and in vivo pharmacodynamic evaluation of nanostructured lipid carriers for parenteral delivery of artemether: Nanoject.

The objective of the present investigation was to explore the potential of nanostructured lipid carriers (NLC) for the intravenous delivery of artemether (ARM), a poorly water-soluble antimalarial agent. The NLC of ARM (Nanoject) were formulated by employing a microemulsion template technique. The NLC were evaluated for particle size, encapsulation efficiency, in vitro drug release and in vitro hemolysis. The antimalarial activity of the Nanoject and conventional ARM injectable formulation was evaluated in Plasmodium berghei infected mice. The average particle size of Nanoject was 63+/-28 nm and the encapsulation efficiency was found to be 30+/-2%. The Nanoject released ARM in a sustained manner. In vitro haemolytic studies showed that Nanoject had lower haemolytic potential (approximately 13%) as compared to all the components when studied individually. Nanoject showed significantly higher (P<0.005) antimalarial activity as compared to the marketed injectable formulation. The antimalarial activity of Nanoject lasted for a longer duration (more than 20 days) indicating that Nanoject may be long-circulating in vivo. Nanoject showed significantly higher survival rate (60%) even after 31 days as compared to marketed formulation which showed 0% survival (100% mortality). This clearly indicates that Nanoject offers several advantages over the currently marketed oily intramuscular formulation (Larither).

Malaria vaccine: a current perspective.

The observation that inactivated Plasmodium sporozoites could protect against malaria is about a hundred years old. However, systematic demonstration of protection using irradiated sporozoites occurred in the nineteen-sixties, providing the impetus for the development of a malaria vaccine. In 1983, the circumsporozoite protein (CSP), a major sporozoite surface antigen, became the first Plasmodium gene to be cloned, and a CSP-based vaccine appeared imminent. Today, 25 years later, we are still without an effective malaria vaccine, despite considerable information regarding the genomics and proteomics of the malaria parasites. Although clinical immunity to malaria has been well-documented in adults living in malaria endemic areas, our understanding of the host-immune responses operating in such malaria immune persons remains poor, and limits the development of immune control of the disease. Currently, several antigen and adjuvant combinations have entered clinical trials, in which efficacy against experimental sporozoite challenge and/or exposure to natural infection is evaluated. This review collates information on the recent status of the field. Unresolved challenges facing the development of a malaria vaccine are also discussed.

Enhanced antimalalarial activity of a prolonged release in situ gel of arteether-lumefantrine in a murine model.

The World Health Organization (WHO) recommends artemisinin-based combination therapy (ACT) for treatment of falciparum malaria. Arteether (ART), an artemisinin derivative, is effective against Plasmodium falciparum, but it is available only as painful oily intramuscular (i.m.) injections. We formulated lyotropic liquid crystalline preconcentrates of ART and Lumefantrine (LUM) ACT with and without biodegradable polymer for antimalarial therapy. Following i.m. injection, both formed intact gels in situ due to rapid transition into liquid crystalline phase (LCP) which was confirmed by small angle neutron scattering (SANS), X-ray diffraction (XRD), polarization optical microscopy (POM) and rheological changes. Ex vivo release studies revealed prolong release of ART-LUM over 72 h from polymeric lyotropic liquid crystalline phases (P-LLCPr). In vitro hemolysis assay and myotoxicity studies confirmed intramuscular safety. Treatment with ART-LUM P-LLCPr conferred complete protection with no mortality at 1/40th of therapeutic dose in modified Peter's four-day suppressive test as compared to marketed ART formulation resulted in 100% mortality within 20 days. In the clinical simulation model, P-LLCPr treatment resulted in complete cure with no recrudescence or mortality at 1/20th of therapeutic dose, while marketed formulation which resulted in 100% mortality. The high efficacy with significantly reduced dose and a single administration with single shot therapy suggest ART-LUM P-LLCPr as a promising new patient friendly alternative for antimalarial therapy.

Atovaquone oral bioavailability enhancement using electrospraying technology.

Atovaquone in combination with proguanil hydrochloride, marketed as Malarone® tablets by GlaxoSmithKline (GSK), is prescribed for the treatment of malaria. High dose and poor bioavailability are the main hurdles associated with atovaquone oral therapy. The present study reports development of atovaquone nanoparticles, using in house designed and fabricated electrospraying equipment, and the assessment of bioavailability and therapeutic efficacy of the nanoparticles after oral administration. Solid nanoparticles of atovaquone were successfully produced by electrospraying and were characterized for particle size and flow properties. Differential Scanning Calorimetry, X-ray Diffraction, Fourier Transform Infrared Spectroscopy studies were also carried out. Atovaquone nanoparticles along with proguanil hydrochloride and a suitable wetting agent were filled in size 2 hard gelatin capsules. The formulation was compared with Malarone® tablets (GSK) and Mepron® suspension (GSK) in terms of in vitro release profile and in vivo pharmacokinetic studies. It showed 2.9-fold and 1.8-fold improved bioavailability in rats compared to Malarone® tablets and Mepron® suspension respectively. Therapeutic efficacy of the formulation was determined using modified Peter's 4-day suppressive tests and clinical simulation studies using Plasmodium berghei ANKA infected Swiss mice and compared to Malarone®. The developed formulation showed a 128-fold dose reduction in the modified Peter's 4-day suppressive tests and 32-fold dose reduction in clinical simulation studies. Given that only one capsule a day of developed formulation is required to be administered orally compared to 4 Malarone® tablets once a day and that too at a significantly reduced dose, this nanoparticle formulation will definitely reduce the side-effects of the treatment and is also likely to increase patient compliance.

Artemether-lumefantrine nanostructured lipid carriers for oral malaria therapy: Enhanced efficacy at reduced dose and dosing frequency.

Artemether-lumefantrine (ARM-LFN) is a World Health Organization (WHO) approved fixed-dose combination having low solubility and poor oral bioavailability. Nanostructured lipid carriers (NLC) were developed to enhance the oral efficacy of this combination using the microemulsion template technique. They were characterized for drug content, entrapment efficiency, size distribution, in vitro release, antimalarial efficacy, and toxicity. The NLC showed sustained drug release. The recommended adult therapeutic dose is 80mg ARM and 480mg LFN (4 tablets) twice a day, which amounts to 160mg ARM and 960mg LFN daily. ARM-LFN NLC given once a day at 1/5 of therapeutic dose (16mg ARM and 96mg LFN) showed complete parasite clearance and 100% survival in Plasmodium berghei-infected mice. 33% of the mice treated with marketed tablets twice a day at the therapeutic dose showed late-stage recrudescence. Thus, NLC showed enhanced efficacy at 1/10 of the daily dose of ARM-LFN. The 10-fold reduced daily dose was formulated in two soft gelatin capsules thus reducing the number of units to be taken at a time by the patient. The capsules showed good stability at room temperature for a year. The NLC were found to be safe in rats. The biocompatible NLC developed using an industrially feasible technique offer a promising solution for oral malaria therapy.

Nanostructured lipid carriers of artemether-lumefantrine combination for intravenous therapy of cerebral malaria.

Patients with cerebral malaria (CM) are unable to take oral medication due to impaired consciousness and vomiting thus necessitating parenteral therapy. Quinine, artemether, and artesunate which are currently used for parenteral malaria therapy have their own drawbacks. The World Health Organization (WHO) has now banned monotherapy and recommends artemisinin-based combination therapy for malaria treatment. However, presently there is no intravenous formulation available for combination therapy of malaria. Artemether-Lumefantrine (ARM-LFN) is a WHO approved combination for oral malaria therapy. However, the low aqueous solubility of ARM and LFN hinders their intravenous delivery. The objective of this study was to formulate ARM-LFN nanostructured lipid carriers (NLC) for intravenous therapy of CM. ARM-LFN NLC were prepared by microemulsion template technique and characterized for size, drug content, entrapment efficiency, drug release, crystallinity, morphology, amenability to autoclaving, compatibility with infusion fluids, stability, antimalarial efficacy in mice, and toxicity in rats. The ARM-LFN NLC showed sustained drug release, amenability to autoclaving, compatibility with infusion fluids, good stability, complete parasite clearance and reversal of CM symptoms with 100% survival in Plasmodium berghei-infected mice, and safety in rats. The biocompatible ARM-LFN NLC fabricated by an industrially feasible technique offer a promising solution for intravenous therapy of CM.

Bioavailability enhancement of atovaquone using hot melt extrusion technology.

Emerging parasite resistance and poor oral bioavailability of anti-malarials are the two cardinal issues which hinder the clinical success of malaria chemotherapy. Atovaquone-Proguanil is a WHO approved fixed dose combination used to tackle the problem of emerging resistance. However, Atovaquone is a highly lipophilic drug having poor aqueous solubility (less than 0.2 µg/ml) thus reducing its oral bioavailability. The aim of the present investigation was to explore hot melt extrusion (HME) as a solvent-free technique to enhance solubility and oral bioavailability of Atovaquone and to develop an oral dosage form for Atovaquone-Proguanil combination. Solid dispersion of Atovaquone was successfully developed using HME. The solid dispersion was characterized for DSC, FTIR, XRD, SEM, and flow properties. It was filled in size 2 hard gelatin capsules. The formulation showed better release as compared to Malarone® tablets, and 3.2-fold and 4.6-fold higher bioavailability as compared to Malarone® tablets and Atovaquone respectively. The enhanced bioavailability also resulted in 100% anti-malarial activity in murine infection model at 1/8(th) therapeutic dose. Thus the developed methodology shows promising potential to solve the problems associated with Atovaquone therapy, namely its high cost and poor oral bioavailability, resulting in increased therapeutic efficacy of Atovaquone.

Heterozygous mutants of TIRAP (S180L) polymorphism protect adult patients with Plasmodium falciparum infection against severe disease and mortality.

Toll-interleukin-1 receptor domain containing adapter protein (TIRAP) plays a crucial role in TLR2 and TLR4 signaling pathways. Glycosylphospatidylinositol (GPI), considered a toxin molecule of Plasmodium falciparum, interacts with TLR2 and 4 to induce an immune inflammatory response. A single nucleotide polymorphism at coding region of TIRAP (S180L) has been reported to influence TLRs signaling. In the present study, we investigated the association of TIRAP (S180L) polymorphism with susceptibility/resistance to severe P. falciparum malaria in a cohort of adult patients from India. TIRAP S180L polymorphism was typed in 347 cases of severe malaria (SM), 232 uncomplicated malaria and 150 healthy controls. Plasma levels of TNF-α was quantified by ELISA. Heterozygous mutation (S/L) conferred significant protection against MOD (multi organ dysfunction), NCSM (non-cerebral severe malaria) as well as mortality. Interestingly, homozygous mutants (L/L) had 16 fold higher susceptibility to death. TIRAP mutants (S/L and L/L) were associated with significantly higher plasma TNF-α levels compared to wild type (S/S). The results of the present study demonstrate that TIRAP S180L heterozygous mutation may protect patients against severe malaria and mortality.