Development and application of high-resolution solid-state NMR methods for probing polymorphism of active pharmaceutical ingredients

The objective of the work presented in this thesis is to apply advanced high-resolution solid-state NMR methods for the structural characterisation of organic crystalline systems, specifically active pharmaceutical ingredients (APIs). The determination of the crystal packing is an important stage in the development of new APIs, and solid-state magic angle spinning (MAS) NMR is well suited to complement existing techniques. Improvements in spectral resolution in recent years have led to the development of homonuclear correlation experiments capable of identifying intermolecular proximities between 1H nuclei. These experiments provide a powerful probe of the local environment of each 1H nucleus in the three-dimensional structure, and the majority of the research presented in this thesis is focussed on the development of detailed analysis methods that may be used to extract more detailed structural information from 2D solid-state NMR correlation spectra. Throughout this thesis, experimental solid-state NMR results are analysed alongside computational data, including density matrix simulations of experiments and first principles calculations of shielding tensors. The results of simulations of a 1H DQ (double-quantum) correlation experiment are compared to experiment, in order to investigate the dependence of the DQ build-up (change in peak intensity as a function of the recoupling pulse duration) on the precise nature of the dipolar coupled proton network. It is found (for a simple dipeptide) that quantitative information on the relative H{H distance may be obtained by comparison of the maximum intensity reached in the corresponding 1H DQ build-up curves. This method is then applied to pharmaceutically relevant systems. It is shown that differences between two polymorphs of an API may be identified in the 1H DQ build-up of particular peaks, and, following the analysis for the dipeptide, this difference may be ascribed to differences in specific intermolecular distances. In the study of a second API, -indomethacin, it is shown that the standard 1H DQ experiment provides insufficient resolution to identify specific DQ peaks. A recently developed 1H(DQ){13C correlation experiment is used to exploit the higher resolution in the 13C dimension, hence allowing the extraction of DQ build-up curves which may be used, in conjunction with simulations, to obtain structural data. Finally, a recently discovered polymorph of the API ibuprofen is studied using 13C CPMAS (cross polarisation) solid-state NMR. Through the use of first-principles calculations, the 13C spectra of both the well known and new polymorphs are assigned, and the conversion of an amorphous solid to the new polymorph is monitored through the use of temperature-controlled solid-state NMR experiments

Imipramine blue sensitively and selectively targets FLT3-ITD positive acute myeloid leukemia cells.

Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200-300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75-150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD+ AML cells compared to FLT3/ITDneg cells and spared normal umbilical cord blood cells. Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML

The Sloan Digital Sky Survey Reverberation Mapping Project: Velocity Shifts of Quasar Emission Lines

Quasar emission lines are often shifted from the systemic velocity due to various dynamical and radiative processes in the line-emitting region. The level of these velocity shifts depends both on the line species and on quasar properties. We study velocity shifts for the line peaks of various narrow and broad quasar emission lines relative to systemic using a sample of 849 quasars from the Sloan Digital Sky Survey Reverberation Mapping (SDSS-RM) project. The coadded (from 32 epochs) spectra of individual quasars have sufficient signal-to-noise ratio (SNR) to measure stellar absorption lines to provide reliable systemic velocity estimates, as well as weak narrow emission lines. The sample also covers a large dynamic range in quasar luminosity (~2 dex), allowing us to explore potential luminosity dependence of the velocity shifts. We derive average line peak velocity shifts as a function of quasar luminosity for different lines, and quantify their intrinsic scatter. We further quantify how well the peak velocity can be measured for various lines as a function of continuum SNR, and demonstrate there is no systematic bias in the line peak measurements when the spectral quality is degraded to as low as SNR~3 per SDSS pixel. Based on the observed line shifts, we provide empirical guidelines on redshift estimation from [OII]3728, [OIII]5008, [NeV]3426, MgII, CIII], HeII1640, broad Hbeta, CIV, and SiIV, which are calibrated to provide unbiased systemic redshifts in the mean, but with increasing intrinsic uncertainties of 46, 56, 119, 205, 233, 242, 400, 415, and 477 km/s, in addition to the measurement uncertainties. These more realistic redshift uncertainties are generally much larger than the formal uncertainties reported by the redshift pipelines for spectroscopic quasar surveys, and demonstrate the infeasibility of measuring quasar redshifts to better than ~200 km/s with only broad lines.Comment: matched to the published version; minor changes and conclusions unchange

A Field-Based Approach for Determining ATOFMS Instrument Sensitivities to Ammonium and Nitrate

Aerosol time-of-flight mass spectrometry (ATOFMS) instruments measure the size and chemical composition of individual particles in real-time. ATOFMS chemical composition measurements are difficult to quantify, largely because the instrument sensitivities to different chemical species in mixed ambient aerosols are unknown. In this paper, we develop a field-based approach for determining ATOFMS instrument sensitivities to ammonium and nitrate in size-segregated atmospheric aerosols, using tandem ATOFMS-impactor sampling. ATOFMS measurements are compared with collocated impactor measurements taken at Riverside, CA, in September 1996, August 1997, and October 1997. This is the first comparison of ion signal intensities from a single-particle instrument with quantitative measurements of atmospheric aerosol chemical composition. The comparison reveals that ATOFMS instrument sensitivities to both NH_4^+ and NO_3^- decline with increasing particle aerodynamic diameter over a 0.32−1.8 μm calibration range. The stability of this particle size dependence is tested over the broad range of fine particle concentrations (PM_(1.8) = 17.6 ± 2.0−127.8 ± 1.8 μg m^(-3)), ambient temperatures (23−35 °C), and relative humidity conditions (21−69%), encountered during the field experiments. This paper describes a potentially generalizable methodology for increasing the temporal and size resolution of atmospheric aerosol chemical composition measurements, using tandem ATOFMS-impactor sampling

The intrinsic and interstellar broad-band linear polarization of nearby FGK dwarfs

We present linear polarization measurements of nearby FGK dwarfs to parts-per-million (ppm) precision. Before making any allowance for interstellar polarization, we found that the active stars within the sample have a mean polarization of 28.5 ± 2.2 ppm, while the inactive stars have a mean of 9.6 ± 1.5 ppm. Amongst inactive stars, we initially found no difference between debris disc host stars (9.1 ± 2.5 ppm) and the other FGK dwarfs (9.9 ± 1.9 ppm). We develop a model for the magnitude and direction of interstellar polarization for nearby stars. When we correct the observations for the estimated interstellar polarization, we obtain 23.0 ± 2.2 ppm for the active stars, 7.8 ± 2.9 ppm for the inactive debris disc host stars and 2.9 ± 1.9 ppm for the other inactive stars. The data indicate that whilst some debris disc host stars are intrinsically polarized most inactive FGK dwarfs have negligible intrinsic polarization, but that active dwarfs have intrinsic polarization at levels ranging up to ∼45 ppm. We briefly consider a number of mechanisms, and suggest that differential saturation of spectral lines in the presence of magnetic fields is best able to explain the polarization seen in active dwarfs. The results have implications for current attempts to detect polarized reflected light from hot Jupiters by looking at the combined light of the star and planet

Deglaciation of Fennoscandia

To provide a new reconstruction of the deglaciation of the Fennoscandian Ice Sheet, in the form of calendar-year time-slices, which are particularly useful for ice sheet modelling, we have compiled and synthesized published geomorphological data for eskers, ice-marginal formations, lineations, marginal meltwater channels, striae, ice-dammed lakes, and geochronological data from radiocarbon, varve, optically-stimulated luminescence, and cosmogenic nuclide dating. This 25 is summarized as a deglaciation map of the Fennoscandian Ice Sheet with isochrons marking every 1000 years between 22 and 13 cal kyr BP and every hundred years between 11.6 and final ice decay after 9.7 cal kyr BP. Deglaciation patterns vary across the Fennoscandian Ice Sheet domain, reflecting differences in climatic and geomorphic settings as well as ice sheet basal thermal conditions and terrestrial versus marine margins. For example, the ice sheet margin in the high-precipitation coastal setting of the western sector responded sensitively to climatic variations leaving a detailed record of prominent moraines and ice-marginal deposits in many fjords and coastal valleys. Retreat rates across the southern sector differed between slow retreat of the terrestrial margin in western and southern Sweden and rapid retreat of the calving ice margin in the Baltic Basin. Our reconstruction is consistent with much of the published research. However, the synthesis of a large amount of existing and new data support refined reconstructions in some areas. For example, we locate the LGM extent of the ice sheet in northwestern Russia further east than previously suggested and conclude that it occurred at a later time than the rest of the ice sheet, at around 17-15 cal kyr BP, and propose a slightly different chronology of moraine formation over southern Sweden based on improved correlations of moraine segments using new LiDAR data and tying the timing of moraine formation to Greenland ice core cold stages. Retreat rates vary by as much as an order of magnitude in different sectors of the ice sheet, with the lowest rates on the high-elevation and maritime Norwegian margin. Retreat rates compared to the climatic information provided by the Greenland ice core record show a general correspondence between retreat rate and climatic forcing, although a close match between retreat rate and climate is unlikely because of other controls, such as topography and marine versus terrestrial margins. Overall, the time slice reconstructions of Fennoscandian Ice Sheet deglaciation from 22 to 9.7 cal kyr BP provide an important dataset for understanding the contexts that underpin spatial and temporal patterns in retreat of the Fennoscandian Ice Sheet, and are an important resource for testing and refining ice sheet models

Ultrasmall silica nanoparticles directly ligate the T cell receptor complex.

The impact of ultrasmall nanoparticles (<10-nm diameter) on the immune system is poorly understood. Recently, ultrasmall silica nanoparticles (USSN), which have gained increasing attention for therapeutic applications, were shown to stimulate T lymphocytes directly and at relatively low-exposure doses. Delineating underlying mechanisms and associated cell signaling will hasten therapeutic translation and is reported herein. Using competitive binding assays and molecular modeling, we established that the T cell receptor (TCR):CD3 complex is required for USSN-induced T cell activation, and that direct receptor complex-particle interactions are permitted both sterically and electrostatically. Activation is not limited to αβ TCR-bearing T cells since those with γδ TCR showed similar responses, implying that USSN mediate their effect by binding to extracellular domains of the flanking CD3 regions of the TCR complex. We confirmed that USSN initiated the signaling pathway immediately downstream of the TCR with rapid phosphorylation of both ζ-chain-associated protein 70 and linker for activation of T cells protein. However, T cell proliferation or IL-2 secretion were only triggered by USSN when costimulatory anti-CD28 or phorbate esters were present, demonstrating that the specific impact of USSN is in initiation of the primary, nuclear factor of activated T cells-pathway signaling from the TCR complex. Hence, we have established that USSN are partial agonists for the TCR complex because of induction of the primary T cell activation signal. Their ability to bind the TCR complex rapidly, and then to dissolve into benign orthosilicic acid, makes them an appealing option for therapies targeted at transient TCR:CD3 receptor binding.These studies were supported by grants from the UK Medical Research Council (Grant number MR/R005699/1) and the Natural Sciences and Engineering Research Council of Canada, as well as through sponsorship from HS Pharmaceuticals LLC

Left Ventricular Ejection Fraction in Patients With Ovarian Cancer Treated With Avelumab, Pegylated Liposomal Doxorubicin, or Both

In the phase III JAVELIN Ovarian 200 trial, 566 patients with platinum-resistant/refractory ovarian cancer were randomized 1:1:1 to receive avelumab alone, avelumab plus pegylated liposomal doxorubicin (PLD), or PLD alone. Cardiac monitoring was included for all patients. We report left ventricular ejection fraction (LVEF) data from the trial. Grade ≥3 cardiac adverse events (AEs) occurred in 4 (2.1%), 1 (0.5%), and 0 patients in the avelumab, combination, and PLD arms, respectively. LVEF decreases of ≥10% to below institutional lower limit of normal at any time during treatment were observed in 1 (0.8%), 3 (1.9%), and 2 (1.5%) patients, respectively; 4 had subsequent assessments, and these showed transient decreases. No patient had a cardiovascular AE related to LVEF decrease. This analysis is, to our knowledge, the first analysis of LVEF in patients receiving immune checkpoint inhibitors. CLINICALTRIALS.GOV IDENTIFIER: NCT02580058

Comparison of Non-Exercise Cardiorespiratory Fitness Prediction Equations in Apparently Healthy Adults

Aims: A recent scientific statement suggests clinicians should routinely assess cardiorespiratory fitness using at least non-exercise prediction equations. However, no study has comprehensively compared the many non-exercise cardiorespiratory fitness prediction equations to directly-measured cardiorespiratory fitness using data from a single cohort. Our purpose was to compare the accuracy of non-exercise prediction equations to directly-measured cardiorespiratory fitness and evaluate their ability to classify an individual\u27s cardiorespiratory fitness. Methods: The sample included 2529 tests from apparently healthy adults (42% female, aged 45.4 ± 13.1 years (mean±standard deviation). Estimated cardiorespiratory fitness from 28 distinct non-exercise prediction equations was compared with directly-measured cardiorespiratory fitness, determined from a cardiopulmonary exercise test. Analysis included the Benjamini-Hochberg procedure to compare estimated cardiorespiratory fitness with directly-measured cardiorespiratory fitness, Pearson product moment correlations, standard error of estimate values, and the percentage of participants correctly placed into three fitness categories. Results: All of the estimated cardiorespiratory fitness values from the equations were correlated to directly measured cardiorespiratory fitness (p \u3c 0.001) although the R2 values ranged from 0.25-0.70 and the estimated cardiorespiratory fitness values from 27 out of 28 equations were statistically different compared with directly-measured cardiorespiratory fitness. The range of standard error of estimate values was 4.1-6.2 ml·kg-1·min-1. On average, only 52% of participants were correctly classified into the three fitness categories when using estimated cardiorespiratory fitness. Conclusion: Differences exist between non-exercise prediction equations, which influences the accuracy of estimated cardiorespiratory fitness. The present analysis can assist researchers and clinicians with choosing a non-exercise prediction equation appropriate for epidemiological or population research. However, the error and misclassification associated with estimated cardiorespiratory fitness suggests future research is needed on the clinical utility of estimated cardiorespiratory fitness. Keywords: Prognosis; cardiopulmonary exercise test; exercise test; fitness algorithm; maximum oxygen consumption