Shobhona Sharma

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).

Distinct Th1- and Th2-Type Prenatal Cytokine Responses to Plasmodium falciparum Erythrocyte Invasion Ligands

ABSTRACT Prenatal immunity to Plasmodium falciparum merozoite proteins involved in erythrocyte invasion may contribute to the partial protection against malaria that is acquired during infancy in areas of stable malaria transmission. We examined newborn and maternal cytokine and antibody responses to merozoite surface protein-1 (MSP-1), ribosomal phosphoprotein P0 (PfP0), and region II of erythrocyte binding antigen-175 (EBA-175) in infant-mother pairs in Kenya. Overall, 82 of 167 (50%), 106 of 176 (60%), and 38 of 84 (45%) cord blood lymphocytes (CBL) from newborns produced one or more cytokines in response to MSP-1, PfP0, and EBA-175, respectively. Newborns of primigravid and/or malaria-infected women were more likely to have antigen-responsive CBL than were newborns of multigravid and/or uninfected women at delivery. Newborn cytokine responses did not match those of their mothers and fell into three distinct categories, Th1 (21 of 55 CBL donors produced only gamma interferon and/or interleukin 2 [IL-2]), Th2 (21 of 55 produced only IL-5 and/or IL-13), and mixed Th1/Th2 (13 of 55). Newborns produced more IL-10 than adults. High and low levels of cord blood IL-12 p70 production induced by anti-CD40 activation were associated with malaria-specific Th1 and Th2 responses, respectively. Antigen-responsive CBL in some newborns were detected only after depletion of IL-10-secreting CD8 cells with enrichment for CD4 cells. These data indicate that prenatal sensitization to blood-stage Plasmodium falciparum occurs frequently in areas where malaria is holoendemic. Modulation of this immunity, possibly by maternal parity and malaria, may affect the acquisition of protective immunity against malaria during infancy.

Curcumin-Loaded Hydrogel Nanoparticles: Application in Anti-Malarial Therapy and Toxicological Evaluation

The present investigation involved preparation of hydrogel nanoparticles using a combination of hydroxyl propyl methyl cellulose and polyvinyl pyrrolidone. The objective was to exploit the size and hydrophilic nature of the formulated nanocarriers to enhance absorption and prolong the rapid clearance of curcumin due to possible evasion of the reticulo-endothelial system. Reproducible nanoparticles of size around 100 nm, a fairly narrow distribution and encapsulation efficiency of 72%, were produced by the solvent emulsion-evaporation technique. This optimized system was further subjected to freeze-drying. The freeze-dried product was readily reconstituted with distilled water. The reconstituted product exhibited a size and distribution similar to that before freeze-drying, drug content of greater than 99% and presence of amorphous drug when analyzed by differential scanning calorimetry (DSC) which may result in possible improved absorption of curcumin. In vivo anti-malarial studies revealed significant superior action of nanoparticles over curcumin control suggesting the possibility of the formulation being employed as an adjunct anti-malarial therapy along with the standard therapy. Acute and subacute toxicity studies confirmed the oral safety of the formulation. A battery of genotoxicity studies was conducted to evaluate the nongenotoxic potential of the developed formulation thus indicating the possibility of the formulation being employed for prolonged duration.

Development of SMEDDS using natural lipophile: Application to β-Artemether delivery

The objective of the present investigation was to formulate self-microemulsifying drug delivery systems (SMEDDS) using a novel, indigenous natural lipophile (N-LCT) as an oily phase. SMEDDS based on N-LCT and commercially available modified oil (Capryol 90) were formulated and their application in improving the delivery of a lipophilic anti-malarial drug, beta-Artemether (BAM) was also evaluated. BAM-loaded SMEDDS were characterized with respect to mean globule size and in vitro drug release profile in comparison to the marketed formulation (Larither). Comparative in vivo anti-malarial performance of the developed SMEDDS was evaluated against the (Larither in Swiss male mice infected with lethal ANKA strain of Plasmodium berghei. The parameters studied were percent parasitemia, activity against time and animal survival period. Both the BAM-SMEDDS showed excellent self-microemulsification efficiency and released >98% of the drug in just 15 min whereas (Larither) showed only 46% drug release at the end of 1h. The mean globule size for optimized BAM-SMEDDS was <100 nm. The anti-malarial studies revealed that BAM-SMEDDS resulted in significant improvement in the anti-malarial activity (P<0.05) as compared to that of (Larither) and BAM solubilized in the oily phases and surfactant. The developed SMEDDS highlight safety for use and potential applications of indigenous natural lipophile in the development of novel colloidal drug carriers.

Protective properties and surface localization of Plasmodium falciparum enolase.

ABSTRACT The enolase protein of the human malarial parasite Plasmodium falciparum has recently been characterized. Apart from its glycolytic function, enolase has also been shown to possess antigenic properties and to be present on the cell wall of certain invasive organisms, such as Candida albicans. In order to assess whether enolase of P. falciparum is also antigenic, sera from residents of a region of Eastern India where malaria is endemic were tested against the recombinant P. falciparum enolase (r-Pfen) protein. About 96% of immune adult sera samples reacted with r-Pfen over and above the seronegative controls. Rabbit anti-r-Pfen antibodies inhibited the growth of in vitro cultures of P. falciparum. Mice immunized with r-Pfen showed protection against a challenge with the 17XL lethal strain of the mouse malarial parasite Plasmodium yoelii. The antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues. Immunofluorescence as well as electron microscopic examinations revealed localization of the enolase protein on the merozoite cell surface. These observations establish malaria enolase to be a potential protective antigen.

Clotrimazole nanoemulsion for malaria chemotherapy. Part I: Preformulation studies, formulation design and physicochemical evaluation

Clotrimazole was formulated in nanoemulsion based system with the aim of improving its solubility and dissolution, which can further used for its preclinical evaluation. Clotrimazole nanoemulsion was prepared using spontaneous nanoemulsification method. Preformulation studies were preformed to evaluate drug-excipient compatibility, solution state pH stability and pH solubility profile. Solubility of clotrimazole in oils, surfactants and cosurfactants was determined to identify nanoemulsion components. Surfactants and cosurfactants were screened for their ability to emulsify selected oily phases. Phase diagrams were constructed to identify area of nanoemulsification. Influence of clotrimazole and pH of dilution medium on phase behavior were assessed. Drug-excipient chemical compatibility study facilitated to anticipate acid catalyzed degradation of clotrimazole. The pH of nanoemulsion was adjusted to 7.5, which could stabilize clotrimazole. Nanoemulsion composed of Capryol 90, Solutol HS 15 and Gelucire 44/14 enhanced solubility of clotrimazole up to 25mg/ml. The optimized clotrimazole nanoemulsion could withstand the extensive dilution and did not show any phase separation or drug precipitation. The nanoemulsion exhibited mean globule size <25 nm, which was not affected by pH of dilution medium. Dissolution profile of clotrimazole nanoemulsion in various media showed 100% drug release within 15 min irrespective of pH of medium.

Plasmodium falciparum enolase: stage-specific expression and sub-cellular localization

BackgroundIn an earlier study, it was observed that the vaccination with Plasmodium falciparum enolase can confer partial protection against malaria in mice. Evidence has also build up to indicate that enolases may perform several non-glycolytic functions in pathogens. Investigating the stage-specific expression and sub-cellular localization of a protein may provide insights into its moonlighting functions.MethodsSub-cellular localization of P. falciparum enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting.ResultsEnolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (≥85–90%) present in soluble fraction, while in sexual stages it was mostly associated with particulate fraction. Apart from cytosol, enolase was found to be associated with nucleus, food vacuole, cytoskeleton and plasma membrane.ConclusionDiverse localization of enolase suggests that apart from catalyzing the conversion of 2-phosphoglycericacid into phosphoenolpyruvate in glycolysis, enolase may be involved in a host of other biological functions. For instance, enolase localized on the merozoite surface may be involved in red blood cell invasion; vacuolar enolase may be involved in food vacuole formation and/or development; nuclear enolase may play a role in transcription.

Torque-generating malaria-infected red blood cells in an optical trap

We have used optical tweezers to trap normal and Plasmodiuminfected red blood cells (iRBCs). Two different facets of the behavior of RBCs in infrared light fields emerge from our experiments. Firstly, while the optical field modifies both types of RBCs in the same fashion, by folding the original biconcave disk into a rod-like shape, iRBCs rotate with linearly polarized light whereas normal RBCs do not. Secondly, and in the context of known molecular motors, our measurements indicate that the torque of rotating iRBCs is up to three orders of magnitude larger.

Malaria parasite-infected erythrocytes inhibit glucose utilization in uninfected red cells

The erythrocytic stages of the malaria parasite depend on anaerobic glycolysis for energy. Using [2‐13C]glucose and nuclear magnetic resonance, the glucose utilization rate and 2,3‐diphosphoglycerate (2,3‐DPG) level produced in normal RBCs and Plasmodium falciparum infected red blood cell populations (IRBCs, with <4% parasite infected red cells), were measured. The glucose flux in IRBCs was several‐folds greater, was proportional to parasitemia, and maximal at trophozoite stage. The 2,3‐DPG levels were disproportionately lower in IRBCs, indicating a downregulation of 2,3‐DPG flux in non‐parasitized RBCs. This may be due to lowered pH leading to selective differential inhibition of the regulatory glycolytic enzyme phosphofructokinase. This downregulation of the glucose utilization rate in the majority (>96%) of uninfected RBCs in an IRBC population may have physiological implications in malaria patients.

The P Domain of the P0 Protein of Plasmodium falciparum Protects against Challenge with Malaria Parasites

ABSTRACT Monoclonal antibodies (MAbs) specific for the P domain of the Plasmodium falciparum P0 phosphoriboprotein (PfP0) blocked the invasion of RBCs by P. falciparum. Vaccination with this P-domain peptide protected mice upon malaria parasite challenge. The absolute specificity of the MAbs and the PfP0 P peptide makes them potential protective malaria reagents.