This study aims to determine the association between heart rate and skeletal muscle oxygen saturation (SmO₂) during 30 minutes of self-paced walking on 4 different days. It was hypothesized that there would be a significant association between heart rate and SmO₂, as increased exercise intensity would result in increased peripheral muscle O₂ consumption and increased heart rate. Participants (age= 18-40) were recruited from Clarkson University as part of an ongoing larger study and informed consent was signed. Heart rate was measured using a pulse oximeter after every five minute interval of the 30 minute walking period and was recorded in an online survey on surveymonkey.com. The muscle oxygen monitor (MOXY) near infrared sensors were placed on the left and right medial gastrocnemius heads as well as on the left biceps brachii muscle to record SmO₂ levels. Data was analyzed using a bivariate Pearson correlation to determine if there was a significant correlation between SmO₂ and heart rate while walking. Heart rate was negatively associated with SmO₂ in the left medial gastrocnemius head (R= -0.139, p= 0.044, n= 211) while there was no significant relationship seen in the right medial gastrocnemius head (p> 0.05). In the left biceps brachii muscle, there was a positive association between heart rate and SmO₂ (R= 0.148, p= 0.035, n= 204). The findings of our study suggest that as walking intensity increases, O₂ consumption in the upper and lower extremities has a weak, but significant association with heart rate. These findings suggest that as blood is shunted from the upper extremity to lower extremity to meet lower extremity demands during walking, O₂ utilization by muscles in the upper extremity is decreased. Further research is needed to elucidate how these relationships change when performing activities that require both upper and lower extremity muscles as well as with varying intensities of exercise.

Despite its common use as a laboratory model, little is known about the in-vivo forces and moments applied to the bovine caudal intervertebral disc. This is particularly critical, as intervertebral disc tissue is known to remodel in response to repeated loading. We hypothesized that the magnitude of loading from muscle contraction during a typical lateral bending motion varies between caudal levels and is accompanied by variations in tissue microstructure. This hypothesis was tested by estimating level-wise forces and bending moments using two independent approaches: a dynamic analytical model of the motion and analysis of muscle cross sections obtained via computed tomography. Microstructure was assessed by measuring collagen fiber crimp period in the annulus fibrosus, and composition was assessed via quantitative histology. Both the analytical model and muscle cross-sections estimated peak bending moments of over 3Nm and peak compressive force of over 125N at the c1c2 level, decreasing distally. There was a significant downward trend from proximal to distal in outer annulus fibrosus collagen crimp period only in the anterior and posterior regions, suggesting remodeling in response to the highest lateral bending moments. There were no observed trends in composition. Our results suggest that while the proximal discs in the bovine tail are subjected to forces and moments from muscle contraction which are comparable (relative to disc size) to those acting on human lumbar discs, the distal discs are not. The resulting pattern of microstructural microstructure suggests that level-wise differences should be considered when using bovine discs as a research model.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TN0155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

Purpose: The purpose of this study was to determine the accuracy of 5 different commercially available activity monitors (AMs) and 1 smartphone application in identifying mean number of steps, mean distance traveled, estimated caloric expenditure, and heart rate (HR). Methods: Subjects (N = 120) performed two 6-minute walks (MWs), one at a comfortable pace (C6MW) and the other at a fast pace (F6MW), around an indoor track wearing all 6 AMs and a StepWatch Activity Monitor in the community. Accuracy and agreement between AM-estimated metrics and actual metrics were examined using the intraclass correlation coefficient (ICC2,1) and Bland–Altman technique (BAM). Results: The ICC2,1 and BAM varied for all 3 conditions with the Fitbit Zip being the most accurate. Conclusions: The AMs exhibited varying degrees of accuracy for identifying steps, calories expended, HR, and distance walked across all different walking conditions. No single AM was accurate across conditions or metrics. Clinicians should be cautious when using data from these AMs to estimate patient activity levels.

SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.

Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator because of its role in the programmed cell death PD-L1/PD-1 pathway. In the preceding manuscript, we described the optimization of a fused, bicyclic screening hit for potency, selectivity, and physicochemical properties in order to further expand the chemical diversity of allosteric SHP2 inhibitors. In this manuscript, we describe the further expansion of our approach, morphing the fused, bicyclic system into a novel monocyclic pyrimidinone scaffold through our under-standing of SAR and use of structure-based design. These studies led to the identification of SHP394 (1), an orally efficacious inhibitor of SHP2, with high lipophilic efficiency, improved potency, and enhanced pharmacokinetic properties. We also report other pyrimidinone analogues with favorable pharmacokinetic and potency profiles. Overall, this work improves upon our previously described allosteric inhibitors and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism.

Purpose The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Methods Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. Results sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. Conclusions MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.

Soluble guanylate cyclase (sGC), the endogenous receptor for nitric oxide (NO), has been implicated in several diseases associated with oxidative stress. In a pathological oxidative environment, the heme group of sGC can be oxidized becoming unresponsive to NO leading to a loss in the ability to catalyze the production of cGMP. Recently a dysfunctional sGC/NO/cGMP pathway has been implicated in contributing to elevated intraocular pressure associated with glaucoma. Herein we describe the discovery of molecules specifically designed for topical ocular administration, which can activate oxidized sGC restoring the ability to catalyze the production of cGMP. These efforts culminated in the identification of compound (+)-23, which robustly lowers intraocular pressure in a cynomolgus model of elevated intraocular pressure over 24 h after a single topical ocular drop and has been selected for clinical evaluation.

Lumbar spinal column laxity contributes to instability, increasing the risk of low back injury and pain. Until the laxity increase due to the cyclic loads of daily living can be quantified, the associated injury risk cannot be predicted clinically. This work cyclically loaded 5-vertebra lumbar motion segments (7 skeletally-mature cervine specimens, 5 osteoporotic human cadaver specimens) for 20000cycles of low-load low-angle (15 degrees) flexion. The normalized neutral zone lengths and slopes of the load-displacement hysteresis loops showed a similar increase in spinal column laxity across species. The intervertebral kinematics also changes with cyclic loading. Differences in the location and magnitude of surface strain on the vertebral bodies (0.34%+/- 0.11% in the cervine specimens, and 3.13%+/- 1.69% in the human cadaver specimens) are consistent with expected fracture modes in these populations. Together, these results provide biomechanical evidence of spinal column damage during high-cycle low-load low-angle loading.

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

AIMS:Graduate education in the healthcare professions can be stressful and mentally taxing. The objective of this study was to identify differences in health and health-related behaviors among graduate allied health students based on sex and curriculum stage (i.e., didactic or clinical education).METHODS:Participants (n=77) were assessed for body fat, BMI, resting metabolic rate (RMR), and peripheral arterial-venous O2 (AVO2), as well as a series of cognitive tasks and self-reported health and health-related behaviors. Independent sample t-tests and tests of proportions were used to assess differences between groups.RESULTS:There was no evidence that the didactic and clinical students were meaningfully different upon entering their programs. Didactic students reported significantly higher tension/anxiety, depression, anger, confusion, fatigue, total mood disturbance, trait mental fatigue, total intensity of mental work performed, and time spent sitting (p<0.05). Didactic students also reported significantly lower RMR (p=0.033), but not after normalizing for fat-free mass. Males reported lower intensity of mental work performed on non-work days compared to females (p=0.009).CONCLUSIONS:Findings indicate students in didactic stages have worse mood and decreased health relative to their counterparts in clinical stages. Opportunities exist to integrate physical and mental health-related education, resources, and self-management programming into graduate allied health curricula to improve wellness among students.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

The poor solubility of molecules has always limited one's ability to formulate pharmaceutical molecules. Formulation technologies commonly used today emerged from specific solubility-related problems. Solid dispersions were developed to stabilize the amorphous phase of a molecule when crystalline material did not show sufficient solubility or dissolution. This chapter presents several cases studies where understanding molecule properties contributed greatly to the successful selection of a preclinical or clinical formulation. These case studies demonstrate how careful selection of excipients, novel approaches toward solubility enhancement, understanding the principles of supersaturation, determining mechanism of permeability, and in silico modeling can all contribute to successful formulation development prior to extensive in vivo evaluation and clinical trials. To understand how solubility and transporter-mediated efflux work together to limit oral exposure, suspension formulations were developed to evaluate the impact of dissolution via particle size reduction.

A fast method was developed to determine the amount of anions and cations present in discovery samples using a reagent free ion chromatography system. The system separates and quantifies counterions present in novel discovery compounds from an accurately weighed sample of 1 mg. The advantage of using these methods in the discovery phase and early development to accurately characterize salt identity and stoichiometry has been demonstrated. Likewise, a further advantage is the ability to accurately quantify undesirable counterions that may be present due to process related manipulation of material and to quantify salt equivalents for “mixed” salt samples. The instrumentation described also has the ability to utilize a reagent-free automated eluent generation approach which in addition to saving time, eliminates the variability that can occur with manually prepared eluents. Several examples have been collated to highlight the usefulness of this approach within discovery.

Salt screening and selection is a well established approach for improving the properties of drug candidates, including dissolution rate and bioavailability. Typically during early development only small amounts of compound are available for solid state profiling, including salt screening. In order to probe large areas of experimental space, high-throughput screening is utilized and is often designed in a way to search for suitable crystallization parameters within hundreds or even thousands of conditions. However, the hit rate in these types of screens can be very low. In order to allow for selection of a salt form early within the drug development process whilst using smaller amounts of compounds, a screening procedure taking into account the compounds properties and the driving forces for salt formation is described. Experiments were carried out on the model compounds clotrimazole, cinnarizine itraconazole and atropine. We found an increase in crystalline hit rate for water-insoluble drugs crystallized from solutions that included at least 10% aqueous content. Conversely it was observed that compounds with greater water solubility did not benefit from aqueous content in salt screening, instead organic solvents lead to more crystalline screening hits. Results from four model compounds show that the inclusion of an aqueous component to the salt reaction can enhance the chance of salt formation and significantly improve the crystalline hit rate for low water soluble drugs.

Amorphous solids and crystalline salts are both of interest as a means of improving the dissolution characteristics and apparent solubility of poorly water soluble active pharmaceutical ingredients which have low bioavailability in humans. The theory and selection of both crystalline drug substance salt forms and amorphous products have been extensively studied. However, less is known about the impact of different counterions on the properties of amorphous drug substance salts. In this study, several salts of either nicardipine or propranolol were prepared and characterized with respect to glass transition temperature, crystallization tendency and moisture sorption behavior. Although the moisture sorption behavior and crystallization tendency varied depending on the counterion used, no trends were readily apparent. The glass transition temperature was found to be dependent on the counterion used to form the salt, and was higher in all instances for the salts than for the neutral compound. Several molecular descriptors were calculated for the various counterions, and multivariate analysis was used to build a model that successfully correlated T-g with a number of these parameters. Important parameters which influenced T-g included counterion pK(a) and electrophilicity index. In conclusion, it is apparent that, as for crystalline salts, the counterion has an effect on the properties of amorphous materials.