Alok Kumar Ravi

Evaluation of pharmacological activities and assessment of intraocular penetration of an ayurvedic polyherbal eye drop (Itone™) in experimental models

BackgroundThe polyherbal eye drop (Itone™) is a mixture of aqueous distillates of nineteen traditionally used ingredients that sum up to impart potency to the formulation and make it a useful adjunct in various ocular pathologies. However, as there have been no controlled experimental studies accounting to the above claim, therefore, the present study was designed to evaluate the polyherbal formulation (PHF) for antiangiogenic, anti-inflammatory, anticataract, antioxidant and cytotoxicity in addition to the evaluation of intraocular penetration of PHF in rabbit eyes using LC-MS/MS.Materials and methodsAntiangiogenic activity of the PHF was evaluated using in ovo chick chorio-allantoic membrane (CAM) assay and in vivo cautery induced corneal neovascularization assay in rats. Anticataract potential was evaluated using steroid induced cataract in developing chick embryos, sodium selenite induced cataract in rat pups and galactose induced cataract in rats. The antioxidant activity was evaluated using di-phenyl picryl hydrazyl (DPPH) radical scavenging assay. Anti-inflammatory activity was evaluated in vitro using inhibition of LTB4 formation in human WBCs and in vivo using carrageenan induced paw edema assay in rats. The cytotoxicity was evaluated against HeLa cancer cell lines using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore evaluation of the intraocular penetration of the PHF was carried out in rabbit eyes via aqueous humor paracentesis and further analysis using LC-MS/MS.ResultsPHF significantly inhibited VEGF induced proliferation of new blood vessels in CAM assay and inhibited the cautery induced corneal neovascularization in rats. Additionally, PHF showed noticeable delay in the progression of cataract in the selenite and galactose induced cataract models whereby the PHF treated lenses were graded for stages II and III respectively. However, the PHF did not show any anticataract activity in the hydrocortisone induced cataract model. Moreover, PHF exhibited anti-inflammatory activity whereby it showed 39.34% inhibition of LTB4 formation and significantly inhibited carrageenan induced paw edema in rats. Eight compounds of PHF viz. camphor, casticin, curcumin-II, quercetin, rosmarinic acid, γ-terpinene, β-pinene and dipentene exhibited transcorneal penetration in rabbit eyes.ConclusionThe significant antiangiogenic and anti-inflammatory activities evinced by the PHF merits further investigation for ocular neovascular and inflammatory diseases in humans.

Dynamics of microcystin production and quantification of potentially toxigenic Microcystis sp. using real-time PCR

Cyanobacterial blooms in eutrophied water body are generally composed of various genotypes with or without microcystin-producing genes (mcy gene cluster). Thus there is a need for quantification of potent toxin producing strains. The present study aimed at identifying microcystin variants and its producer strains in Durgakund pond, Varanasi, India, based on quantification of cpcBA-IGS and mcyA (condensation domain) genes using real-time PCR and LC-MS. Increase in microcystin concentrations was correlated with increase in mcyA copy number and the level of pigments (chlorophyll a, phycocyanin and carotenoids). Also, selected environmental factors (water temperature, light irradiance, rainfall, pH, N and P) and the concentration of microcystin variants (MC-LR, -RR and -YR) were also assessed in samples during May 2010 to April 2011 to establish the possible correlation among these parameters. Nutrients favored cyanobacterial bloom but it could not be correlated with the levels of microcystin variants and seemed to be geographically specific. Microcystis sp. dominant in the pond comprised potentially toxigenic cells. The ratio of potentially toxigenic Microcystis sp. to that of total Microcystis sp. ranged from 0% to 14%. Such studies paved the way to identify and quantify the most potent microcystin producer in the tropical aquatic body.

Toxicological investigation of surface engineered fifth generation poly (propyleneimine) dendrimers in vivo

Dendrimers are three dimensional polymers, nanoscopic in size, most widely explored in the field of drug delivery in recent times. In order to establish these polymers as controlled and targeted drug delivery systems, they should be non-toxic, biocompatible and biodegradable. The purpose of the present study is to investigate the toxicological profile of fifth generation poly (propyleneimine) dendrimers (PPI) and some of its surface engineered derivatives. Functionalized PPI dendrimers (TPPI, MPPI and TuPPI) were synthesized to mask the primary amino groups responsible for the positive charge and associated toxicity. Each polymer is administered in three different doses: 2.5 mg/kg, 25 mg/kg and 250 mg/kg (i.e., low, intermediate and high dose) to Wister rats, and blood as well as tissue samples were collected after 24 h and 15 days. Decrease in RBC count and hemoglobin content after 24 h, in case of animals administered with PPI suggests hemolytic activity of PPI. Significant increase in SGOT, SGPT and LDH indicates that PPI causes severe damage to the membranes of the various tissues of the body, especially that of the liver leading to the leakage of these marker enzymes in blood. Sections of liver of animals administered with PPI showed signs of tissue degeneration after 24 h. No signs of toxicity were observed in case of animals administered with functionalized PPI. Neither PPI nor its surface engineered derivatives showed any signs of immunogenicity. It can be concluded that functionalization of dendrimers leads to drastic reduction of toxicity and increases biocompatibility.

Retinal toxicity of intravitreally injected plain and liposome formulation of fluconazole in rabbit eye

PURPOSE Candidal endophthalmitis is a sight-threatening ocular infection that most frequently occurs as a complication of candidemia. Fluconazole has been effective against Candida albicans in various animal models. Our objective was to evaluate retinal toxicity of plain and liposome formulation of fluconazole at various dose levels after intravitreal injection. MATERIALS AND METHODS Twelve New Zealand albino rabbits weighing 2-2.5 kg were used. Two rabbits were used for every dose level. Liposome formulation containing 100 and 200 microg of fluconazole in sterile phosphate buffer solution and plain fluconazole at concentrations of 100, 200, 400 and 800 microg in 0.1 ml of sterile normal saline were injected intravitreally into the right eyes. The left eyes received 0.1 ml normal saline or 0.1 ml of liposome formulation without fluconazole. One week later, the animals were sacrificed, their eyes enucleated and processed for light microscopy and scanning electron microscopy. RESULTS It showed that plain fluconazole at a concentration of 100 microg and above caused retinal changes, with disorganization of the photoreceptor outer segments. However, liposome formulation of fluconazole (200 microg/0.1 ml) did not show any significant microscopic changes of the retina. CONCLUSION The liposome formulation decreased the retinal toxicity of fluconazole up to the studied concentration of 200 microg/0.1 ml.

Development and validation of a LC–MS/MS method for homocysteine thiolactone in plasma and evaluation of its stability in plasma samples

The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC-MS/MS. The gradient elution of HCTL was achieved within 5min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5-32.5nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at -86°C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers.

Stability of latanoprost in generic formulations using controlled degradation and patient usage simulation studies.

Abstract Purpose: To evaluate the stability of latanoprost in generic formulations by using controlled degradation and patient usage simulation studies Methods: Standard latanoprost was subjected to controlled degradation studies. Latanoprost content was assessed by using MRM, and generated Degradation Products (DP) were analysed by using the Information Dependent Acquisition (IDA) protocol of positive ESI-LC-MS/MS. Latanoprost content and formation of DP were assessed in generic formulations and were compared with Xalatan® in a controlled patient usage simulation studies. The last few drops of latanoprost, present in containers used by patients were also evaluated. Results: Extreme pH conditions, oxidation, light and heat were found to be the significant factors for high degree of latanoprost degradation. Systematic analysis of 7 selected generics revealed that the latanoprost content varied from 90–330%. Concentration of the latanoprost in Xalatan was found to be 97% of the label claim. Degradation studies showed the formation of 3 novel and 3 already known impurities. Upon simulated patient usage, 2 of the generic formulations showed significant degradation of latanoprost. Generic formulations having thermally sealed gas tight packing showed good stability during patient usage. Overage of latanoprost was observed in generics with other than thermal sealing. Latanoprost bottles used by patients showed concentrations ranging from 20 to 250% of label claim (144% median). Conclusion: This study revealed the presence of overage of latanoprost in some generic formulations and formation of degradation products. Packaging with gas tight containers may be one of the important factors for latanoprost stability, along with its storage at low temperature during patient usage.

Understanding the Charge Issues in Mono and di-Quaternary Ammonium Compounds for Their Determination by LC/ESI-MS/MS

Chromatographers often develop problems while optimizing a method for the quantification of quaternary ammonium compounds in ESI-MS/MS. Intransigency observed in quaternary ammonium compounds to undergo the classical molecular adduct formation [M+H]+ in ESI-MS/MS reduces confidence among chromatographers while working with unit mass resolution. In this study, we provide the evidence for an exceptional rule followed by mono- and di-quaternary ammonium compounds in ESI-MS/MS in the precursor ion formation. Under ESI conditions mono- and di-quaternary ammonium compounds form molecular ions with the formula of m q / z q rather than ( m + z )/ z . Formation of m q / 2 is observed for di-quaternary ammonium compounds in precursor ion scan and m q / 1 in product ion scan, if loss of one of the quaternary charge occurs during CID. In di-quaternary ammonium compounds, this process can also result in the formation of fragment ions with higher mass as compared to precursor ion. Hydrophilic interaction liquid chromatographic separation has been used to demonstrate the elution of quaternary ammonium compounds in a single run in the ESI-MS/MS. This work concludes that the analyst must realize and consider these charge issues while dealing with positively charged compounds in LC/ESI-MS/MS.

Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes.

Roles of nutrients and other environmental variables in development of cyanobacterial bloom and its toxicity are complex and not well understood. We have monitored the photoautotrophic growth, total microcystin concentration, and microcystins synthetase gene (mcyA) expression in lab-grown strains of Microcystis NIES 843 (reference strain), KW (Wangsong Reservoir, South Korea), and Durgakund (Varanasi, India) under different nutrient regimes (nitrogen, phosphorus, and boron). Higher level of nitrogen and boron resulted in increased growth (avg. 5 and 6.5 Chl a mg/L, resp.), total microcystin concentrations (avg. 1.185 and 7.153 mg/L, resp.), and mcyA transcript but its expression was not directly correlated with total microcystin concentrations in the target strains. Interestingly, Durgakund strain had much lower microcystin content and lacked microcystin-YR variant over NIES 843 and KW. It is inferred that microcystin concentration and its variants are strain specific. We have also examined the heterotrophic bacteria associated with cyanobacterial bloom in Durgakund Pond and Wangsong Reservoir which were found to be enriched in Alpha-, Beta-, and Gammaproteobacteria and that could influence the bloom dynamics.

Complex disposition of an enigmatic pathogen: rare electron microscopic manifestations in nasal rhinosporidiosis

Abstract Rhinosporidiosis is a polypoidal disease of the nose and mucocutaneous tissues, the diagnosis of which is based on the presence of round bodies believed to be causative agents of the disease. Historically, the round body has been considered to be a sporangium of a fungus Rhinosporidium seeberi but without any convincing evidence. Round bodies contain numerous daughter cells, which are likely in the infective stage and are shed through a rupture in the wall of the round body. The released single-celled organisms eventually develop into round bodies on availability of suitable transformative trigger and favourable environment. Surgical excision of the polyp by electrocautery is the only effective treatment; however, recurrence may occur due to spillage of infective endospores in the surrounding mucosa during removal. There are many enigmatic features of the causative agent of this disease, which have been baffling researchers for more than a century. Here we present some rare electron microscopic and previously unreported features of the coat of the round body and single-celled organism in nasal rhinosporidiosis.

Symmetry patterning by the causative organism of rhinosporidiosis in culture

Abstract Rhinosporidiosis is a chronic polypoidal infection of the nose, conjunctiva and other sites, believed to be caused by a fungus, Rhinosporidium Seeberi, with a doubtful taxonomy. Polyps contain histological round bodies and the exact mode of infection is not known. The round bodies are filled up with spherules. In tissue the organism forms spherical round bodies approaching 50-500µ in diameter that contain innumerable single-celled organisms that mature at different rates. Mature organisms are approximately 7-9µ in size and escape through a pore that develops in the wall of the round body. The round body does not exist in nature outside the host. The organism in rhinosporidiosis was believed to be uncultivable, until we cultured it for the first time in our laboratory. We further modified the culture medium and succeeded in culturing the causative agent of the disease in CBEML (Cell Biology and Electron Microcopy Laboratory) medium. Here we present some of the peculiar conspicuous features of the organism in culture leading to symmetry patterning.