Understanding the Tabletability Differences between Indomethacin Polymorphs Using Powder Brillouin Light Scattering.

PURPOSE: The unconventional tabletability of the indomethacin polymorphs - α and γ - are investigated from a topological and mechanical perspective using powder Brillouin light scattering (p-BLS) to identify the specific structure-performance relationship in these materials. METHOD: Indomethacin (γ-form) was purchased and used to prepare the α polymorph. Powder X-ray diffraction was used to confirm phase identity, while p-BLS was used to obtain the mechanical properties. Energy frameworks were determined with Crystal Explorer to visualize the interaction topologies. Using a Carver press and a stress-strain analyzer, the tableting performance of each polymorph was determined. RESULTS: Polymorph-specific acoustic frequency distributions were observed with distinct, zero-porosity, aggregate elastic moduli determined. The p-BLS spectra for α-indomethacin display a population of low-velocity shear modes, indicating a direction of facilitated shear. This improves slip-mediated plasticity and tabletability. Our p-BLS spectra experimentally indicates that a low-energy slip system is available to α-indomethacin which supports ours and previous energy framework calculations. Despite a 2d-layered crystal motif favorable for shear deformation, the γ-form displays a higher shear modulus that is supported by our hydrogen-bonding analysis of γ-indomethacin. CONCLUSION: Our experimental, mechanical data is consistent with the predicted interaction topologies and these two inputs combined permit a comprehensive, molecular understanding of polymorph-specific tabletability.

Discriminating the Interaction Anisotropy in Polymorphs Using Powder Brillouin Light Scattering.

Drug product performance is polymorph specific, and it is imperative that solid phase stability be monitored throughout the manufacturing process to ensure final product quality and performance. PXRD remains the gold standard for polymorph identification, but due to a growing interest in continuous manufacturing, a need has emerged for alternative process analytical technologies (PATs) that can provide fast, reliable, and non-destructive polymorph discrimination amenable to in situ process monitoring. Herein we demonstrate an original application of powder Brillouin light scattering (p-BLS) for the discrimination of polymorphic molecular solids. We hypothesize that the anisotropic sound velocities directly reflect the strength and orientation of the intermolecular forces in molecular solids. Redistributing these forces upon polymorphic conversion should thus clearly be reflected in the sound frequency distributions obtained by p-BLS. To test this hypothesis, three model compounds - resorcinol, sulfamerazine and furosemide - were selected. Distinct, polymorph-specific, acoustic frequency distributions were observed, and these p-BLS spectra were interpreted using a hydrogen-bond analysis and energy frameworks calculated from CrystalExplorer. In conclusion, this study clearly demonstrates that the sound frequencies measured in p-BLS are sensitive to the interaction forces in molecular solids, and p-BLS is a novel optical technique capable of reliably discriminating polymorphs. Extending this study further, we fully expect that many pharmaceutically relevant processes - e.g., hydrate formation, co-crystallization, or amorphous instability - could potentially be monitored using p-BLS.

Modulation of pituitary adenylate cyclase-activating polypeptide (PACAP) expression in explant-cultured guinea pig cardiac neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) expression was quantified in explant-cultured guinea pig cardiac ganglia neurons. In explant culture, both the percentage of PACAP-immunoreactive neurons and pro-PACAP transcript levels increased significantly. Treatment with neurturin or glial-derived neurotrophic factor significantly suppressed the percentage of PACAP-IR neurons, but not pro-PACAP transcript levels.

Calcium influx through channels other than voltage-dependent calcium channels is critical to the pituitary adenylate cyclase-activating polypeptide-induced increase in excitability in guinea pig cardiac neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) effects on intracellular calcium ([Ca2+]i) and excitability have been studied in adult guinea pig intracardiac neurons. PACAP increased excitability, but did not elicit Ca2+ release from intracellular stores. Exposure to a Ca2+-deficient solution did not deplete [Ca2+]i stores but did eliminate the PACAP-induced increase in excitability. We postulate that Ca2+ influx is required for the PACAP-induced increase in excitability.

PACAP expression in explant cultured mouse major pelvic ganglia.

The major pelvic ganglia (MPG) contain both parasympathetic and sympathetic postganglionic neurons and provide much of the autonomic innervation to urogenital organs and components of the lower bowel. Whereas many parasympathetic neurons were found to express vasoactive intestinal polypeptide (VIP), no MPG neurons exhibited immunoreactivity for pituitary adenylate cyclase-activating polypeptide (PACAP). However, in 3-day cultured MPGs, numerous PACAP-IR cells and nerve fibers were present, and transcript levels for PACAP increased significantly. In 3-day cultured MPGs, PACAP immunoreactivity was seen in cells that were also immunoreactive for VIP or neuronal nitric oxide synthase, but not tyrosine hydroxylase, indicating that PACAP expression occurred preferentially in MPG parasympathetic postganglionic neurons. Transcript levels for the VPAC2, but not VPAC1 or PAC1 receptor, also increased significantly following 3 days in culture. Transcript levels of activating transcription factor 3 (ATF-3), a marker of cellular injury, were increased 64-fold in 3-day explants, and ATF-3-IR nuclei were evident in both TH-IR and nNOS-IR neurons as well as in non-neuronal cells. In sum, these results demonstrate that, although only the parasympathetic neurons in explant cultured MPGs increase expression of PACAP, both sympathetic and parasympathetic postganglionic neurons in the cultured MPG whole-mount increase expression of ATF-3.

Neurturin suppresses injury-induced neuronal activating transcription factor 3 expression in cultured guinea pig cardiac ganglia.

Cultured guinea pig atrial whole mounts containing the intrinsic cardiac ganglia were used as an in vitro model to investigate the induction of the stress/injury marker activating transcription factor 3 (ATF-3). ATF-3 expression was quantified by using immunocytochemical labeling and real-time PCR. In freshly isolated ganglia, no neuronal or Schwann cell nuclei exhibited ATF-3 immunoreactivity. In 2-hour cultures, the induction of ATF-3 expression was evident in many Schwann cell nuclei, whereas no neuronal nuclei were ATF-3 immunoreactive. Beginning at 4 hours, the percentage of neurons with ATF-3-immunoreactive nuclei increased progressively, and, by 48 hours in culture, approximately 95% of the cardiac neurons had ATF-3-immunoreactive nuclei. Neurturin significantly suppressed ATF-3 expression in 48-hour-cultured neurons without effect on ATF-3 expression in Schwann cell nuclei. Neuturin also could reverse neuronal ATF-3 expression after its induction. The suppression of ATF-3 induction by neurturin was mediated by activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Glial-derived neurotrophic factor (GDNF) also suppressed neuronal ATF-3 induction during culture. However, culture in serum-free media, presence of nerve growth factor, or addition of pituitary adenylate cyclase-activating polypeptide had no effect on ATF-3 induction in the 48-hour-cultured cardiac neurons. By 4 hours in culture, there was a significant increase in ATF-3 transcript levels, and neurturin partially suppressed ATF-3 transcript levels in 48-hour cultures. It is proposed that the loss of target-derived neurturin is a potential mechanism stimulating injury-induced expression of ATF-3 in cardiac neurons.

Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue.

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.

Physiologic profile of the fitness status of collegiate cheerleaders.

Cheerleading, traditionally considered a nonathletic activity, has evolved into a competitive sport requiring high levels of fitness. Despite the trend of cheerleaders performing increasingly difficult and athletic skills, very little is known about their fitness levels. The purpose of this study was to provide a physiological profile of the fitness status of a squad of collegiate cheerleaders. Eighteen cheerleaders (11 men and 7 women) participated in this study. Each subject completed a Bruce protocol maximal treadmill test, underwater weighing, 1 repetition maximum bench press, sit-and-reach test, push-ups, curl-ups, and isokinetic strength testing. The mean and SD were calculated to provide the physical fitness profile for each parameter. A comparison to normative data demonstrated that cheerleaders have a high level of fitness and scores similar to other collegiate athletes.