Active pharmaceutical ingredients (APIs) can be prepared in many different solid forms and phases that affect their physicochemical properties and suitability for oral dosage forms. The development ...and commercialization of dosage forms require analytical techniques that can determine and quantify the API phase in the final drug product.
C solid-state NMR (SSNMR) spectroscopy is widely employed to characterize pure and formulated solid APIs; however,
C SSNMR experiments on dosage forms with low API loading are often challenging due to low sensitivity and interference from excipients. Here, fast magic angle spinning
H SSNMR experiments are shown to be applicable for the rapid characterization of low drug load formulations. Diagnostic
H SSNMR spectra of APIs within tablets are obtained by using combinations of frequency-selective saturation and excitation pulses, two-dimensional experiments, and
H spin diffusion periods. Selective saturation pulses efficiently suppress the broad
H SSNMR signals from the most commonly encountered excipients such as lactose and cellulose, allowing observation of high-frequency API
H NMR signals.
H SSNMR provides a 1-3 orders of magnitude reduction in experiment time compared to standard
C SSNMR experiments, enabling diagnostic SSNMR spectra of dilute APIs within tablets to be obtained within minutes. The
H SSNMR spectra can be used for quantification, provided calibrations are performed on a standard sample with known API loading.
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Despite numerous challenges in their theoretical description and practical implementation, amorphous drugs are of growing importance to the pharmaceutical industry. One such challenge is to gain ...molecular level understanding of the propensity of a molecule to form and remain as a glassy solid. In this study, a series of structurally similar diarylamine compounds was examined to elucidate the role of supramolecular aggregation on crystallization kinetics from supercooled liquid state. The structural similarity of the compounds makes it easier to isolate the molecular features that affect crystallization kinetics and glass forming ability of these compounds. To examine the role of hydrogen-bonded aggregation and motifs on crystallization kinetics, a combination of thermal and spectroscopic techniques was employed. Using variable temperature FTIR, Raman, and solid-state NMR spectroscopies, the presence of hydrogen bonding in the melt and glassy state was examined and correlated with observed phase transition behaviors. Spectroscopic results revealed that the formation of hydrogen-bonded aggregates involving carboxylic acid and pyridine nitrogen (acid–pyridine aggregates) between neighboring molecules in the melt state impedes crystallization, while the presence of carboxylic acid dimers (acid–acid dimers) in the melt favors crystallization. This study suggests that glass formation of small molecules is influenced by the type of intermolecular interactions present in the melt state and the kinetics associated with the molecules to assemble into a crystalline lattice. For the compounds that form acid–pyridine aggregates, the formation of energy degenerate chains, produced due to conformational flexibility of the molecules, presents a kinetic barrier to crystallization. The poor crystallization tendency of these aggregates stems from the highly directional hydrogen-bonding interactions needed to form the acid–pyridine chains. Conversely, for the compounds that form acid–acid dimers, the nondirectional van der Waals forces needed to construct a nucleus promote rapid assembly and crystallization.
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Bruton’s tyrosine kinase (Btk) is thought to play a pathogenic role in chronic immune diseases such as rheumatoid arthritis and lupus. While covalent, irreversible Btk inhibitors are approved for ...treatment of hematologic malignancies, they are not approved for autoimmune indications. In efforts to develop additional series of reversible Btk inhibitors for chronic immune diseases, we sought to differentiate from our clinical stage inhibitor fenebrutinib using cyclopropyl amide isosteres of the 2-aminopyridyl group to occupy the flat, lipophilic H2 pocket. While drug-like properties were retainedand in some cases improveda safety liability in the form of hERG inhibition was observed. When a fluorocyclopropyl amide was incorporated, Btk and off-target activity was found to be stereodependent and a lead compound was identified in the form of the (R,R)- stereoisomer.
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The purpose of this study was to identify and characterize precipitates obtained from a liquid formulation of GNE068.HCl, a Genentech developmental compound, and lipophilic excipients, such as ...propylene glycol monocaprylate, and monolaurate. Precipitates were characterized using powder X-ray diffractometry (PXRD), differential scanning calorimetry, thermogravimetry, microscopy, nuclear magnetic resonance spectroscopy (NMR; solution and solid-state) and water sorption analysis. PXRD and NMR revealed the precipitates to be crystalline solvates of propylene glycol esters. The solvates (capryolate and lauroglycolate) were isomorphic and stable up to 70 °C, beyond which melting of the lattice occurred with subsequent dissolution of the active ingredient in the melt (microscopy and variable temperature PXRD). They were found to be mechanically stable (no change in PXRD pattern upon compression) and were nonhygroscopic up to ∼70% RH (25 °C). Our results highlight the outcome of inadvertent drug-excipient interactions in two separate lipid solution formulations with good solid-state properties and, thus, potential for further development.
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BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of ...the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.
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We demonstrate that natural isotopic abundance 2D heteronuclear correlation (HETCOR) solid-state NMR spectra can be used to significantly reduce or eliminate the broadening of
H and
C solid-state NMR ...spectra of organic solids due to anisotropic bulk magnetic susceptibility (ABMS). ABMS often manifests in solids with aromatic groups, such as active pharmaceutical ingredients (APIs), and inhomogeneously broadens the NMR peaks of all nuclei in the sample. Inhomogeneous peaks with full widths at half maximum (FWHM) of ∼1 ppm typically result from ABMS broadening and the low spectral resolution impedes the analysis of solid-state NMR spectra. ABMS broadening of solid-state NMR spectra has previously been eliminated using 2D multiple-quantum correlation experiments, or by performing NMR experiments on diluted materials or single crystals. However, these experiments are often infeasible due to their poor sensitivity and/or provide limited gains in resolution. 2D
H-
C HETCOR experiments have previously been applied to reduce susceptibility broadening in paramagnetic solids and we show that this strategy can significantly reduce ABMS broadening in diamagnetic organic solids. Comparisons of 1D solid-state NMR spectra and
H and
C solid-state NMR spectra obtained from 2D
H-
C HETCOR NMR spectra show that the HETCOR spectrum directly increases resolution by a factor of 1.5 to 8. The direct gain in resolution is determined by the ratio of the inhomogeneous
C/
H linewidth to the homogeneous
H linewidth, with the former depending on the magnitude of the ABMS broadening and the strength of the applied field and the latter on the efficiency of homonuclear decoupling. The direct gains in resolution obtained using the 2D HETCOR experiments are better than that obtained by dilution. For solids with long proton longitudinal relaxation times, dynamic nuclear polarization (DNP) was applied to enhance sensitivity and enable the acquisition of 2D
H-
C HETCOR NMR spectra. 2D
H-
C HETCOR experiments were applied to resolve and partially assign the NMR signals of the form I and form II polymorphs of aspirin in a sample containing both forms. These findings have important implications for ultra-high field NMR experiments, optimization of decoupling schemes and assessment of the fundamental limits on the resolution of solid-state NMR spectra.
Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid ...cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.
In our continued effort to discover and develop best-in-class Bruton’s tyrosine kinase (Btk) inhibitors for the treatment of B-cell lymphomas, rheumatoid arthritis, and systemic lupus erythematosus, ...we devised a series of novel tricyclic compounds that improved upon the druglike properties of our previous chemical matter. Compounds exemplified by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated, and efficacious in an animal model of arthritis.
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The capacity of the neonate to respond to nonself antigens was evaluated in infant monkeys born after normal and disturbed pregnancies. Mixed lymphocyte cultures were used to test the infants' ...proliferative responses to mitomycin-treated stimulator cells, either from a genetically unrelated animal or from a virally transformed monkey cell line. Periods of daily stress for 6 weeks in mid-late pregnancy (months 3.0-4.5) resulted in a significant decrease in proliferative responses, whereas the same stressor early in pregnancy (months 1.5-3.0) increased responses by the neonate's cells. Similar to the late stress effect, an inhibition of proliferative responses by neonatal cells was induced by dexamethasone administered for 2 days late in pregnancy at 4.5 months after conception, 1 month before term. These findings demonstrate that certain immune responses at birth are extremely sensitive to prior prenatal events. Further, the bidirectional changes indicate that there may be critical periods in gestation when the same extrinsic events have radically different effects on the fetus.
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