A RAPID, SENSITIVE AND VALIDATED ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY METHOD FOR DETERMINATION OF PAROMOMYCIN IN MICE PLASMA: APPLICATION TO PHARMACOKINETIC STUDY

Objective: To develop and validate simple, sensitive, accurate and selective UPLC-MS/MS method for quantification of paromomycin (PARO) in mice plasma.Methods: Precipitation method was used for the extraction of plasma samples, an aliquot of 25 µl plasma samples was extracted using 10% perchloric acid in water. Chromatographic separation was performed using waters acquity ultra-performance liquid chromatography (UPLC) columns, BEH HILIC (50 mm× 2.1 mm, 1.7 µm) by a gradient mixture of acetonitrile and water (both containing 0.005% v/v trifluro acetic acid) as a mobile phase at the flow rate of 0.2 ml/min. The analyte was protonated in the positive electrospray ionization (ESI) interface and detected in multiple reactions monitoring (MRM) modes using the transition m/z 308.60-455.30.Results: The method had a short chromatographic run time of 3 min. Calibration curves were linear over wide ranges of 50.51-5019.22 ng/ml. The between and within-batch precision and accuracy of the method was determined by using 4 quality control samples, the highest % CV observed was 11.06. The mean recovery values are 78.17, 101.17 and 92.58 at low, medium and high-quality control levels; respectively.Conclusion: It was concluded that the developed and validated UPLC-MS/MS method was rapid, sensitive, accurate, precise, linear, and specific. Therefore, this method can be used for quantification of PARO in mice plasma with various advantages over the reported methods

Computer aided data acquisition tool for high-throughput phenotyping of plant populations

<p>Abstract</p> <p>Background</p> <p>The data generated during a course of a biological experiment/study can be sometimes be massive and its management becomes quite critical for the success of the investigation undertaken. The accumulation and analysis of such large datasets often becomes tedious for biologists and lab technicians. Most of the current phenotype data acquisition management systems do not cater to the specialized needs of large-scale data analysis. The successful application of genomic tools/strategies to introduce desired traits in plants requires extensive and precise phenotyping of plant populations or gene bank material, thus necessitating an efficient data acquisition system.</p> <p>Results</p> <p>Here we describe newly developed software "<b>PHENOME" </b>for high-throughput phenotyping, which allows researchers to accumulate, categorize, and manage large volume of phenotypic data. In this study, a large number of individual tomato plants were phenotyped with the "PHENOME" application using a Personal Digital Assistant (PDA) with built-in barcode scanner in concert with customized database specific for handling large populations.</p> <p>Conclusion</p> <p>The phenotyping of large population of plants both in the laboratory and in the field is very efficiently managed using PDA. The data is transferred to a specialized database(s) where it can be further analyzed and catalogued. The "PHENOME" aids collection and analysis of data obtained in large-scale mutagenesis, assessing quantitative trait loci (QTLs), raising mapping population, sampling of several individuals in one or more ecological niches etc.</p

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (&gt;18,000 designs), crystallographic data (&gt;490 ligand-bound x-ray structures), assay data (&gt;10,000 measurements), and synthesized molecules (&gt;2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property–free knowledge base for future anticoronavirus drug discovery

THE IMPACT OF PERMEABILITY ENHANCERS ON ASSESSMENT FOR MONOLAYER OF COLON ADENOCARCINOMA CELL LINE (CACO-2) USED IN IN VITRO PERMEABILITY ASSAY

The leading goal in new drug discovery is to have orally bioavailable drug. Poor permeability is one of the reasons for the poor bioavailability of several New Chemical Entities (NCEs). Permeability assessment using in vitro Caco-2 cell monolayer model is considered to be excellent model for screening of NCEs. Permeability enhancers can increase the permeability of compounds by paracellular or transcellular route. There are limitations to use the permeability enhancers due to concentration dependent toxic effect on cell monolayer, several times wrong interpretation can be made due to disrupted cell monolayer integrity. This study was performed on Caco-2 cell monolayer to identify optimal levels of commonly used permeability enhancers which does not cause any damage to the cell monolayer. The assessment involved pre and post TEER measurement and leak test using Lucifer yellow (LY) rejection assay. Total 16 permeability enhancers were tested and optimum levels were as per parenthesis: peanut oil (10%), Cremphore EL,  Miglyol 812, Oleic acid, Propylene glycol (1%), Capmul MCM C8 EP, glycerol, Labrasol, MC8-2, PEG 400, Polysorbate 80, Sporiol TPGS, Transcutol (0.1% ), Capmul , Solutol (0.01% ) and for PPS (0 .0001%). It was important to determine the optimal levels of each permeability enhancer to avoid any false positive results. Keywords: Permeability enhancers, Caco-2 cell line, TEER, % LY rejection, Tight Junction

A RAPID, SENSITIVE AND VALIDATED ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY METHOD FOR DETERMINATION OF PAROMOMYCIN IN MICE PLASMA: APPLICATION TO PHARMACOKINETIC STUDY

Objective: To develop and validate simple, sensitive, accurate and selective UPLC-MS/MS method for quantification of paromomycin (PARO) in mice plasma.Methods: Precipitation method was used for the extraction of plasma samples, an aliquot of 25 µl plasma samples was extracted using 10% perchloric acid in water. Chromatographic separation was performed using waters acquity ultra-performance liquid chromatography (UPLC) columns, BEH HILIC (50 mm× 2.1 mm, 1.7 µm) by a gradient mixture of acetonitrile and water (both containing 0.005% v/v trifluro acetic acid) as a mobile phase at the flow rate of 0.2 ml/min. The analyte was protonated in the positive electrospray ionization (ESI) interface and detected in multiple reactions monitoring (MRM) modes using the transition m/z 308.60-455.30.Results: The method had a short chromatographic run time of 3 min. Calibration curves were linear over wide ranges of 50.51-5019.22 ng/ml. The between and within-batch precision and accuracy of the method was determined by using 4 quality control samples, the highest % CV observed was 11.06. The mean recovery values are 78.17, 101.17 and 92.58 at low, medium and high-quality control levels; respectively.Conclusion: It was concluded that the developed and validated UPLC-MS/MS method was rapid, sensitive, accurate, precise, linear, and specific. Therefore, this method can be used for quantification of PARO in mice plasma with various advantages over the reported methods.</jats:p

In vitro metabolism, disposition, preclinical pharmacokinetics and prediction of human pharmacokinetics of DNDI-VL-2098, a potential oral treatment for Visceral Leishmaniasis

AbstractThe in vitro metabolism and in vivo pharmacokinetic (PK) properties of DNDI-VL-2098, a potential oral agent for Visceral Leishmaniasis (VL) were studied and used to predict its human pharmacokinetics. DNDI-VL-2098 showed a low solubility (10μM) and was highly permeable (>200nm/s) in the Caco-2 model. It was stable in vitro in liver microsomes and hepatocytes and no metabolite was detectable in circulating plasma from dosed animals suggesting very slow, if any, metabolism of the compound. DNDI-VL-2098 was moderate to highly bound to plasma proteins across the species tested (94–98%). DNDI-VL-2098 showed satisfactory PK properties in mouse, hamster, rat and dog with a low blood clearance (<15% of hepatic blood flow except hamster), a volume of distribution of about 3 times total body water, acceptable half-life (1–6h across the species) and good oral bioavailability (37–100%). Allometric scaling of the preclinical PK data to human gave a blood half-life of approximately 20h suggesting that the compound could be a once-a-day drug. Based on the above assumptions, the minimum efficacious dose predicted for a 50kg human was 150mg and 300mg, using efficacy results in the mouse and hamster, respectively