Liquid chromatography-mass spectrometric methods for trace quantification of potential genotoxic impurities in ivacaftor and lumacaftor.

OBJECTIVE: The objective of the current study was to develop and validate the sensitive LC-MS methods for trace analysis of genotoxic impurities in Ivacaftor and Lumacaftor. The first method is for the trace analysis of 2,4-di-tert-butyl-5-nitrophenol in ivacaftor and the second method is for the trace analysis of 1-(2,2-difluoro-1,3-benzodioxol-5yl)-cyclopropane carboxylic acid and 3-carboxyphenyl boronic acid in lumacaftor. MATERIALS AND METHODS: High pure analytical grade solvents and reagents were used for this study. The chromatographic separation was performed on Luna C18 (250×4.6mm, 5.0µm) at a column temperature of 25°C using eluent consisting of acetonitrile and 0.1% v/v formic acid in water in a gradient elution mode. The eluent was run at a flow of 1.0mL/min and injection volume of 20µL. RESULTS: The linearity, precision and accuracy of the developed methods was validated over the concentration range of 0.35-15.0ppm for 2,4-di-tert-butyl-5-nitrophenol, 0.30-15.0ppm for 1-(2,2-difluoro-1,3-benzodioxol-5yl)-cyclopropane carboxylic acid and 0.23-15.0ppm for 3-carboxyphenyl boronic acid. In both methods, interference was not observed at the retention time of analyte peaks. All the analytes were found to be stable in solution for a period of 48h. CONCLUSION: The proposed methods are reliable, sensitive, precise, accurate, and robust for the trace level quantification of genotoxic impurities in Ivacaftor and Lumacaftor. These methods can be successfully implemented in the quality control lab for routine analysis.

Improved method for post-processing correction of B1 inhomogeneity in glutamate-weighted CEST images of the human brain.

Glutamate-weighted CEST (gluCEST) imaging is nearly unique in its ability to provide non-invasive, spatially resolved measurements of glutamate in vivo. In this article, we present an improved correction for B1 inhomogeneity of gluCEST images of the human brain. Images were obtained on a Siemens 7.0 T Terra outfitted with a single-volume transmit/32-channel receive phased array head coil. Numerical Bloch-McConnell simulations, fitting and data processing were performed using in-house code written in MATLAB and MEX (MATLAB executable). "Calibration" gluCEST data was acquired and fit with a phenomenological functional form first described here. The resulting surfaces were used to correct experimental data in accordance with a newly developed method. Healthy volunteers of varying ages were used for both fitted "calibration" data and corrected "experimental" data. Simulations allowed us to describe the dependence of CEST at 3.0 ppm (gluCEST) on saturation B1 using a new functional form, whose validity was confirmed by successful fitting to real human data. This functional form was used to parameterize surfaces over the space (B1 , T1 ), which could then be used to correct the signal from each pixel. The resulting images show less signal loss in areas of low B1 and greater contrast than those generated using the previously published method. We demonstrate that, using this method with appropriate nominal saturation B1 , the major limitation of correcting for B1 inhomogeneity becomes the effective flip angle of the acquisition module, rather than inability to correct for inhomogeneous saturation. The lower limit of our correction ability with respect to both saturation and acquisition B1 is about 40% of the nominal value. In summary, we demonstrate a more rigorous and successful approach to correcting gluCEST images for B1 inhomogeneity. Limitations of the method and further improvements to enable correction in regions with severe pathology are discussed.

Longitudinal imaging reveals subhippocampal dynamics in glutamate levels associated with histopathologic events in a mouse model of tauopathy and healthy mice.

Tauopathies are neurodegenerative disorders characterized by abnormal intracellular aggregates of tau protein, and include Alzheimer's disease, corticobasal degeneration, frontotemporal dementia, and traumatic brain injury. Glutamate metabolism is altered in neurodegenerative disorders manifesting in higher or lower concentrations of glutamate, its transporters or receptors. Previously, glutamate chemical exchange saturation transfer (GluCEST) magnetic resonance imaging (MRI) demonstrated that glutamate levels are reduced in regions of synapse loss in the hippocampus of a mouse model of late-stage tauopathy. We performed a longitudinal GluCEST imaging experiment paired with a cross-sectional study of histologic markers of tauopathy to determine whether (1) early GluCEST changes are associated with synapse loss before volume loss occurs in the hippocampus, and whether (2) subhippocampal dynamics in GluCEST are associated with histopathologic events related to glutamate alterations in tauopathy. Live imaging of the hippocampus in three serial slices was performed without exogenous contrast agents, and subregions were segmented based on a k-means cluster model. Subregions of the hippocampus were analyzed (cornu ammonis CA1, CA3, dentate gyrus DG, and ventricle) in order to associate local MRI-observable changes in glutamate with histological measures of glial cell proliferation (GFAP), synapse density (synaptophysin, VGlut1) and glutamate receptor (NMDA-NR1) levels. Early differences in GluCEST between healthy and tauopathy mice were measured in the CA1 and DG subregions (30% reduction, P ≤ 0.001). Synapse density was also significantly reduced in every subregion of the hippocampus in tauopathy mice by 6 months. Volume was not significantly reduced in any subregion until 13 months. Further, a gradient in glutamate levels was observed in vivo along hippocampal axes that became polarized as tauopathy progressed. Dynamics in hippocampal glutamate levels throughout lifetime were most closely correlated with combined changes in synaptophysin and GFAP, indicating that GluCEST imaging may be a surrogate marker of glutamate concentration in glial cells and at the synaptic level. © 2016 Wiley Periodicals, Inc.

Gender differential and implications in the epidemiology of stress fractures among cadets of Indian Armed Forces.

BACKGROUND: Most studies on stress fractures in India have been carried out among recruits as against officer trainees and limited to males. With the continuous induction of women in the Armed Forces, it was decided to carry out a study among officer trainees of the three services and compare the epidemiology among genders. METHODS: A prospective study was carried out in 2011-2012 at Training Institutes of the three services where male and female cadets train together. Baseline data was collected for all trainees who joined the academy during the study period. All cadets were followed up for development of stress fractures for which details were taken. Additional information was taken from the Training Institute. RESULTS: A total of 3220 cadets (2612 male and 608 female cadets) were included in the study. Overall 276 cadets were observed to have stress fractures during training - making an incidence of 6.9% for male cadets and 15.8% for female cadets. Females were found to have a significantly higher incidence of stress fractures. Further the distribution and onset of stress fractures in females was observed to be distinct from males. CONCLUSION: The significant gender differential observed in the study indicates differential role of intrinsic and extrinsic risk factors in the causation of stress fractures among male and female. Special consideration needs to be given to these while planning and implementing measures for prevention. Further studies may be carried out on subject and on the effect of interventions in stress fracture prevention.

Implementation of two-dimensional L-COSY at 7 Tesla: an investigation of reproducibility in human brain.

PURPOSE: To evaluate the utility of two-dimensional (2D) Localized Correlated Spectroscopy (L-COSY) in metabolic profiling of the human brain at 7 Tesla (T). MATERIALS AND METHODS: The 2D L-COSY sequence was implemented at 7 T and its reliability was assessed by test-retest studies of a metabolite phantom and a healthy volunteer. L-COSY data were acquired from the occipital lobe of healthy subjects (n = 6; all male; age, 30-72 years) to assess intersubject variability. Additionally, two subjects underwent scans from the parieto-occipital region, basal ganglia, frontal lobe or dorsolateral prefrontal cortex to test the versatility of L-COSY in studying differing anatomy. Integral/volume measurements of L-COSY spectra were used to estimate normalized metabolite-to-creatine concentrations. RESULTS: Phantom test-retest studies revealed coefficients of variation (CVs) of 3-20% for most metabolites. Human 2D L-COSY spectra permitted detection of several metabolite resonances from multiple locations and inter-subject variation studies demonstrated CVs of 4-26%. Cross-peaks from gamma-aminobutyric acid (GABA), isoleucine (Ile), lysine (Lys) and Ethanolamine (Eth) were quantified, which are not readily resolvable with conventional one-dimensional (1D) MR spectroscopy. CONCLUSION: 2D L-COSY at 7 T demonstrated improved sensitivity in detecting additional metabolites with reliability comparable to established techniques at lower fields, which may aid in the metabolic assessment of diseased states.

Design of small molecules that target metal-A{beta} species and regulate metal-induced A{beta} aggregation and neurotoxicity.

The accumulation of metal ions and amyloid-ß (Aß) aggregates found in the brain of patients with Alzheimer's disease (AD) has been suggested to be involved in AD pathogenesis. To investigate metal-Aß-associated pathways in AD, development of chemical tools to target metal-Aß species is desired. Only a few efforts, however, have been reported. Here, we report bifunctional small molecules, N-(pyridin-2-ylmethyl)aniline (L2-a) and N(1),N(1)-dimethyl-N(4)-(pyridin-2-ylmethyl)benzene-1,4-diamine (L2-b) that can interact with both metal ions and Aß species, as determined by spectroscopic methods including high-resolution NMR spectroscopy. Using the bifunctional compound L2-b, metal-induced Aß aggregation and neurotoxicity were modulated in vitro as well as in human neuroblastoma cells. Furthermore, treatment of human AD brain tissue homogenates containing metal ions and Aß species with L2-b showed disassembly of Aß aggregates. Therefore, our studies presented herein demonstrate the value of bifunctional compounds as chemical tools for investigating metal-Aß-associated events and their mechanisms in the development and pathogenesis of AD and as potential therapeutics.

Effect of grapefruit juice and ritonavir on pharmacokinetics of lopinavir in Wistar rats.

Lopinavir (LPV), a newer HIV protease inhibitor, has poor bioavailability being a substrate of both cytochrome P450 3A enzyme system (CYP3A) and permeability-glycoprotein (P-gp). Ritonavir (RTV) is a known inhibitor of both P-gp and CYP3A and is co-administered with LPV in anti-HIV therapy. Grapefruit juice (GFJ) is known to inhibit CYP3A and has conflicting effects, ranging from activation to inhibition, on P-gp. In this research work, the effects of GFJ and RTV on the pharmacokinetics of LPV were compared in rats. A mechanistic evaluation was undertaken using various in vitro and ex vivo studies to support the in vivo pharmacokinetic data. The plasma levels of LPV were found to increase significantly upon co-administration with GFJ in single dose as well as multidose pretreatment studies. Similar, but marginally higher, results were observed upon co-administration of LPV with RTV. No significant change in t(max) was observed in the various treatment groups. The apparent permeability of LPV in the ileum increased significantly after the pre-incubation with GFJ and RTV compared with no pre-incubation. The GFJ and RTV showed a significant and similar inhibitory effect on rat intestinal microsomes in the metabolism of LPV. The GFJ was equally effective as RTV in increasing the bioavailability of LPV.

Cholesterol reduces pardaxin's dynamics-a barrel-stave mechanism of membrane disruption investigated by solid-state NMR.

While high-resolution 3D structures reveal the locations of all atoms in a molecule, it is the dynamics that correlates the structure with the function of a biological molecule. The complete characterization of dynamics of a membrane protein is in general complex. In this study, we report the influence of dynamics on the channel-forming function of pardaxin using chemical shifts and dipolar couplings measured from 2D broadband-PISEMA experiments on mechanically aligned phospholipids bilayers. Pardaxin is a 33-residue antimicrobial peptide originally isolated from the Red Sea Moses sole, Pardachirus marmoratus, which functions via either a carpet-type or barrel-stave mechanism depending on the membrane composition. Our results reveal that the presence of cholesterol significantly reduces the backbone motion and the tilt angle of the C-terminal amphipathic helix of pardaxin. In addition, a correlation between the dynamics-induced heterogeneity in the tilt of the C-terminal helix and the membrane disrupting activity of pardaxin by the barrel-stave mechanism is established. This correlation is in excellent agreement with the absence of hemolytic activity for the derivatives of pardaxin. These results explain the role of cholesterol in the selectivity of the broad-spectrum of antimicrobial activities of pardaxin.

Solid-state NMR and molecular dynamics simulations reveal the oligomeric ion-channels of TM2-GABA(A) stabilized by intermolecular hydrogen bonding.

The second transmembrane (TM2) domain of GABA(A) receptor forms the inner-lining surface of chloride ion-channel and plays important roles in the function of the receptor protein. In this study, we report the first structure of TM2 in lipid bilayers determined using solid-state NMR and MD simulations. The interatomic (13)C-(15)N distances measured from REDOR magic angle spinning experiments on multilamellar vesicles, containing a TM2 peptide site specifically labeled with (13)C' and (15)N isotopes, were used to determine the secondary structure of the peptide. The (15)N chemical shift and (1)H-(15)N dipolar coupling parameters measured from PISEMA experiments on mechanically aligned phospholipid bilayers, containing a TM2 peptide site specifically labeled with (15)N isotopes, under static conditions were used to determine the membrane orientation of the peptide. Our results reveal that the TM2 peptide forms an alpha helical conformation with a tilted transmembrane orientation, which is unstable as a monomer but stable as pentameric oligomers as indicated by MD simulations. Even though the peptide consists of a number of hydrophilic residues, the transmembrane folding of the peptide is stabilized by intermolecular hydrogen bondings between the side chains of Ser and Thr residues as revealed by MD simulations. The results also suggest that peptide-peptide interactions in the tilted transmembrane orientation overcome the hydrophobic mismatch between the peptide and bilayer thickness.

NMR structure in a membrane environment reveals putative amyloidogenic regions of the SEVI precursor peptide PAP(248-286).

Semen is the main vector for HIV transmission worldwide. Recently, a peptide fragment (PAP(248-286)) has been isolated from seminal fluid that dramatically enhances HIV infectivity by up to 4-5 orders of magnitude. PAP(248-286) appears to enhance HIV infection by forming amyloid fibers known as SEVI, which are believed to enhance the attachment of the virus by bridging interactions between virion and host-cell membranes. We have solved the atomic-level resolution structure of the SEVI precursor PAP(248-286) using NMR spectroscopy in SDS micelles, which serve as a model membrane system. PAP(248-286), which does not disrupt membranes like most amyloid proteins, binds superficially to the surface of the micelle, in contrast to other membrane-disruptive amyloid peptides that generally penetrate into the core of the membrane. The structure of PAP(248-286) is unlike most amyloid peptides in that PAP(248-286) is mostly disordered when bound to the surface of the micelle, as opposed to the alpha-helical structures typically found of most amyloid proteins. The highly disordered nature of the SEVI peptide may explain the unique ability of SEVI amyloid fibers to enhance HIV infection as partially disordered amyloid fibers will have a greater capture radius for the virus than compact amyloid fibers. Two regions of nascent structure (an alpha-helix from V262-H270 and a dynamic alpha/3(10) helix from S279-L283) match the prediction of highly amyloidogenic sequences and may serve as nuclei for aggregation and amyloid fibril formation. The structure presented here can be used for the rational design of mutagenesis studies on SEVI amyloid formation and viral infection enhancement.

Comparative Study of Recovery after Sevoflurane versus Halothane Anaesthesia in Adult Patients.

BACKGROUND: Induction and maintenance characteristics of sevoflurane and halothane have been studied, but little work has been done to compare the postoperative recovery of these two agents. METHODS: Sixty adult, ASA I and II patients were allocated randomly into Group A and Group B of 30 each. Group A received sevoflurane and Group B received halothane for maintenance. At the end of surgery early recovery, intermediate recovery and discharge criteria were assessed. RESULTS: Early recovery assessed with the mean time to extubation was 6.7 ± 2.29 min in Group A and 9.07 ± 1.64 min in Group B; eye opening was 7.28 ± 2.3 min in Group A and 10.6 ± 1.77 min in Group B; response to verbal command was 8.52 ± 2.83 min in Group A and 12.33 ± 2.17 min in Group B, while orientation was 10.43 ± 3.15 min in Group A and 14.77 ± 2.66 min in Group B. These differences were statistically significant (p<0.001). The mean time to reach post anaesthesia care unit discharge criteria was shorter for Group A (21.1 ± 4.69 min) as compared to Group B (27.43 ± 6.51 min) and this difference was statistically significant (p<0.001). CONCLUSION: Early recovery time and time taken to achieve discharge criteria were faster with sevoflurane.

Nasal in-situ gels for delivery of rasagiline mesylate: improvement in bioavailability and brain localization.

Intranasal thermosensitive gel for rasagiline mesylate (RM) was developed for effective treatment of Parkinson's disease. Intranasal gels were prepared by combination of poloxamer 407 and poloxamer 188 (1:1) with mucoadhesive polymers (carbopol 934 P and chitosan). The formulations were evaluated for sol-gel transition temperature, in-vitro drug release and in-vivo mucociliary transit time. Further, optimal intranasal gel formulations were tested for in-vivo pharmacokinetic behavior, nasal toxicity studies and brain uptake studies. It was found that optimal formulations had acceptable gelation temperature (28-33 °C) and adequate in-vitro drug release profile. Pharmacokinetic study in rabbits showed significant (p < 0.05) improvement in bioavailability (four- to six-folds) of the drug from intranasal gels than oral solution. Chronic exposure studies in Wistar rats showed that these intranasal gels were non-irritant and non-toxic to rat nasal mucosa. Estimation of RM in rat brain tissue showed significant (p < 0.01) improvement in uptake of RM form intranasal gel formulations than nasal solution.

LC method for determination of rasagiline mesylate in different plasma matrices and its application to oral pharmacokinetic study in rabbits.

A simple, rapid and sensitive reverse-phase liquid chromatographic method is developed and validated for estimation of rasagiline mesylate in different plasma matrices (rat, rabbit and human plasma). The method employs an isocratic elution technique with a Kromasil C18 column and has an optimized mobile phase composition of 10 mM ammonium acetate buffer-acetonitrile (40:60 v/v). The plasma samples displayed linear detector responses in the concentration range of 0.5-20 µg/mL in all plasma matrices when monitored at 265 nm using an ultraviolet detector. Because the simple protein precipitation method yields over 95% recovery of rasagiline mesylate from all plasma matrices, no internal standard was used in this method. A detailed validation study of the method proves the accuracy, precision and selectivity (in estimating rasagiline mesylate) in all plasma matrices. The drug is also stable under various processing and storage conditions in all plasma matrices, as evident from the study. The present method was applied to determine the drug-protein binding ratio in all the plasma matrices. The use of this method in determining pharmacokinetic parameters of rasagiline mesylate by non-compartmental analysis after oral dosing in rabbits is also discussed.