Sulabha Pathak

Sirt1 and mir‐9 expression is regulated during glucose‐stimulated insulin secretion in pancreatic β‐islets

MicroRNA mir‐9 is speculated to be involved in insulin secretion because of its ability to regulate exocytosis. Sirt1 is an NAD‐dependent protein deacetylase and a critical factor in the modulation of cellular responses to altered metabolic flux. It has also been shown recently to control insulin secretion from pancreatic β‐islets. However, little is known about the regulation of Sirt1 and mir‐9 levels in pancreatic β‐cells, particularly during glucose‐dependent insulin secretion. In this article, we report that mir‐9 and Sirt1 protein levels are actively regulated in vivo in β‐islets during glucose‐dependent insulin secretion. Our data also demonstrates that mir‐9 targets and regulates Sirt1 expression in insulin‐secreting cells. This targeting is relevant in pancreatic β‐islets, where we show a reduction in Sirt1 protein levels when mir‐9 expression is high during glucose‐dependent insulin secretion. This functional interplay between insulin secretion, mir‐9 and Sirt1 expression could be relevant in diabetes. It also highlights the crosstalk between an NAD‐dependent protein deacetylase and microRNA in pancreatic β‐cells.

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).

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.

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.

Solid microemulsion preconcentrate (NanOsorb) of artemether for effective treatment of malaria

A microemulsion preconcentrate was formulated on the basis of solubility of artemether (ARM) in the various oily phases and surfactants and phase diagrams. Various solid adsorbents were evaluated for their ability yield solid microemulsion preconcentrates (NanOsorb-ARM). NanOsorb-ARM on dilution yielded microemulsion with average globule size of 183 nm and polydispersity index of 0.498 when determined using photon correlation spectroscopy. The antimalarial activity of NanOsorb-ARM, ARM solution and marketed ARM formulation (Larither) was evaluated in Plasmodium berghei infected mice as per Peters four day protocol. The acute lethal dose and the subacute toxicity of NanOsorb-ARM were determined as per the method suggested in Organization for Economic Cooperation and Development (OECD) guidelines. The NanOsorb-ARM exhibited significantly higher antimalarial activity (P<0.05) as compared to the marketed formulation of artemether (Larither). Surprisingly, placebo NanOsorb also showed significantly higher antimalarial activity as compared to Larither indicating that excipients used for the formulation of NanOsorb may have antimalarial activity. Subacute toxicity studies demonstrated that NanOsorb-ARM is comparatively safer than artemether oily solution with respect to survival, gross pathology, hematology and serum biochemistry in mice of both the genders.

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

BackgroundPlasmodium 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.MethodsNMR 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.ResultsThe 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.ConclusionsThe 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.

Age-dependent sex bias in clinical malarial disease in hypoendemic regions.

Background and Objectives Experimental models show a male bias in murine malaria; however, extant literature on biases in human clinical malaria is inconclusive. Studies in hyperendemic areas document an absence of sexual dimorphism in clinical malaria. Data on sex bias in clinical malaria in hypoendemic areas is ambiguous—some reports note a male bias but do not investigate the role of differential mosquito exposure in that bias. Moreover, these studies do not examine whether the bias is age related. This study investigates whether clinical malaria in hypoendemic regions exhibits a sex bias and whether this bias is age-dependent. We also consider the role of vector exposure in this bias. Methods Retrospective passive clinical malaria datasets (2002–2007) and active surveillance datasets (2000–2009) were captured for the hypoendemic Mumbai region in Western India. To validate findings, passive retrospective data was captured from a primary malaria clinic (2006–2007) in hypoendemic Rourkela (Eastern India). Data was normalized by determining percent slide-positivity rates (SPRs) for males and females, and parasite-positivity distributions were established across age groups. The Mann–Whitney test, Wilcoxon Signed Rank test, and Chi-square analysis were used to determine statistical significances. Results In both the Mumbai and Rourkela regions, clinical malaria exhibited an adult male bias (p<0.01). A sex bias was not observed in children aged ≤10. Post-puberty, male SPRs were significantly greater than females SPRs (p<0.01). This adult male bias was observed for both vivax and falciparum clinical disease. Analysis of active surveillance data did not reveal an age or sex bias in the frequency of parasite positivity. Conclusion This study demonstrates an age-dependent sex bias in clinical malaria in hypoendemic regions and enhanced incidence of clinical malaria in males following puberty. Possible roles of sex hormones, vector exposure, co-infections, and other factors in this enhanced susceptibility are discussed.

Clotrimazole nanoemulsion for malaria chemotherapy. Part II: Stability assessment, in vivo pharmacodynamic evaluations and toxicological studies

The aim of present investigation was to evaluate the potential of clotrimazole as antimalarial drug. Due to poor aqueous solubility and high lipophilicity, it was previously formulated in a nanoemulsion based system. The intrinsic effects of nanoemulsion on improvement of antimalarial activity of clotrimazole were assessed in mice infected with Plasmodium berghei and compared to its suspension formulation. In four-day suppressive test, mice treated with 10mg/kg clotrimazole nanoemulsion showed the highest suppression of parasitemia and; parasitemia was significantly lower than that of 10mg/kg clotrimazole suspension. In onset of activity and recrudescence test, percent reduction of parasitemia was significantly higher in 10 and 15 mg/kg clotrimazole nanoemulsion groups compared to 15 mg/kg suspension group. In both murine models, survival of mice treated with nanoemulsion was significantly prolonged compared to suspension at equivalent doses. The inhibition of parasite growth by clotrimazole in the nanoemulsion was dose dependent as determined by test for linear trend. In repeated dose oral toxicity, levels of serum liver enzymes and biomarkers of hepatotoxicity did not vary significantly from control. Six-month stability testing of the clotrimazole nanoemulsion exhibited no changes in various physiochemical attributes of drug product compared to initial analysis.

Formulation and characterization of atovaquone nanosuspension for improved oral delivery in the treatment of malaria

AIM The objective of the present study was to develop an atovaquone (ATQ) nanosuspension and evaluate its ability to improve the pharmacokinetic and therapeutic efficacy on oral administration. MATERIALS & METHODS The ATQ nanosuspension was prepared by a combination of microprecipitation and high-pressure homogenization. It was freeze dried and characterized for various physiochemical properties. In vivo pharmacokinetics was performed in rats whereas antimalarial efficacy was assessed in mice using a 4-day suppressive test. RESULTS The ATQ nanosuspension stabilized with Solutol(®) HS 15 (BASF India Ltd, Mumbai, India) and Capryol™ 90 (Gattefosse, Mumbai, India) exhibited a z-average diameter of 371.50 nm and a polydispersity index of 0.19. X-ray diffraction and differential scanning calorimetry analysis indicated no substantial changes in the crystalline state of ATQ nanocrystals. The aqueous solubility and in vitro dissolution rate were significantly increased by reducing the particle size. An in vivo pharmacokinetics study of the nanosuspension compared with a drug suspension and Malarone(®) (GlaxoSmithKline, Brentford, UK) exhibited an approximately 4.6-3.2-fold improvement in area under plasma concentration. A significant increase in Cmax and decrease in time to reach peak plasma concentration after administration was also observed. ATQ in nanosized form, even at one-quarter lower doses, exhibited greater reduction in parasitemia and prolonged survival compared with its reference formulations. CONCLUSION Results of this pilot study highlight the potential of nanosuspension as an efficient and commercially viable strategy for improving delivery of ATQ for malaria treatment.

Parasite impairment by targeting Plasmodium-infected RBCs using glyceryl-dilaurate nanostructured lipid carriers.

Antimalarial therapy is a major contributor to declining malaria morbidity and mortality. However, the high toxicity and low bioavailability of current antimalarials and emerging drug resistance necessitates drug-delivery research. We have previously developed glyceryl-dilaurate nanolipid carriers (GDL-NLCs) for antimalarial drug delivery. Here, we show evidence that GDL-NLCs themselves selectively target Plasmodium-infected red blood cells (iRBCs), and cause severe parasite impairment. The glyceryl-dilaurate lipid-moiety was important in the targeting. GDL-NLCs localized to the parasite mitochondrion and uptake led to mitochondrial-membrane polarization and Ca(2+) ion accumulation, ROS release, and stage-specific iRBC lysis. GDL-NLC treatment also resulted in externalization of iRBC-membrane phosphatidylserine and enhanced iRBC clearance by macrophages. GDL-NLC uptake disrupted the parasite-induced tubulovesicular network, which is vital for nutrient import by the parasite. Laser optical trap studies revealed that GDL-NLCs also restored iRBC flexibility. Such restoration of iRBC flexibility may help mitigate the vasculature clogging that can lead to cerebral malaria. We demonstrate the suitability of GDL-NLCs for intravenous delivery of antimalarial combinations artemether-clindamycin and artemether-lumefantrine in the murine model. Complete parasite clearance was achieved at 5-20% of the therapeutic dose of these combinations. Thus, this nanostructured lipid formulation can solubilize lipophilic drugs, selectively target and impair the parasite-infected red cell, and therefore constitutes a potent delivery vehicle for antimalarials.