Low-dose CT: A safe and effective imaging modality in post-operative pelvic & acetabular fixation.

INTRODUCTION: Post-operative pelvic & acetabular fixation patients are conventionally imaged using 3-view radiographs (AP, inlet and outlet). The efficacy of such radiographs is inconsistent due to technical difficulties capturing an adequate view, often necessitating repeat radiographs and therefore increasing radiation exposure. Radiographs can be difficult to interpret, limiting the assessment of fracture reduction and fixation, especially with respect to metalwork positioning around articular surfaces. Traditionally, post-operative pelvic & acetabular fixation patients undergo repeat 3-view radiographs post-operatively, at 6 weeks, followed by at 3, 6, 12, 18 and 24 months. We propose a new pathway, in which patients have one low-dose pelvic CT immediately post-operatively, followed by one radiograph (AP pelvis) at the same time points. METHODS: A new pelvic CT protocol was created to provide high quality 3D imaging whilst delivering a 5 times lower radiation dose (compared to normal pelvic CT). Data for all pelvic radiographs and CTs between January 2021 and March 2022 was exported. Using dose area product values, effective radiation dose and attributable lifetime cancer risk were calculated. RESULTS: There were 42 patients included in the analysis (age range 15 to 87).The average effective dose for the 3-view pelvic X-rays was 0.6mSv (range 0.2 to 2.8mSv), and 1.1mSv (range 0.5 to 2.2mSv) for the low-dose pelvic CT. Traditional 7 × 3-view post-operative radiographs: 7 × 0.6mSv = 4.2mSv (corresponding to 1 in 11,000 cancer risk) Low dose post-operative CT and 6 × 1-view radiographs: 1.1mSv + (6 × 0.6mSv / 3) = 2.3mSv (corresponding to 1 in 20,000 cancer risk) CONCLUSION: Low-dose CT scanning (in conjunction with 1-view radiographs) is an effective and safe imaging modality in the post-operative assessment of pelvic & acetabular fracture fixation, conferring a lower radiation burden, easier logistics, and higher quality images when compared to the traditional pathway of 3-view radiographs.

Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral agent that is responsible for the coronavirus disease-2019 (COVID-19) pandemic. One of the live virus vaccine candidates Merck and Co., Inc. was developing to help combat the pandemic was V590. V590 was a live-attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV) in which the envelope VSV glycoprotein (G protein) gene was replaced with the gene for the SARS-CoV-2 spike protein (S protein), the protein responsible for viral binding and fusion to the cell membrane. To assist with product and process development, a quantitative Simple Western (SW) assay was successfully developed and phase-appropriately qualified to quantitate the concentration of S protein expressed in V590 samples. A strong correlation was established between potency and S-protein concentration, which suggested that the S-protein SW assay could be used as a proxy for virus productivity optimization with faster data turnaround time (3 h vs 3 days). In addition, unlike potency, the SW assay was able to provide a qualitative profile assessment of the forms of S protein (S protein, S1 subunit, and S multimer) to ensure appropriate levels of S protein were maintained throughout process and product development. Finally, V590 stressed stability studies suggested that time and temperature contributed to the instability of S protein demonstrated by cleavage into its subunits, S1 and S2, and aggregation into S multimer. Both of which could potentially have a deleterious effect on the vaccine immunogenicity.

Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids Using Capillary Western.

A prototype strain of Coxsackievirus A21 (CVA21) is being evaluated as an oncolytic virus immunotherapy. CVA21 preferentially lyses cells that upregulate the expression of intercellular adhesion molecule 1, which includes some types of tumor cells. CVA21 has an icosahedral capsid structure made up of 60 protein subunits encapsidating a viral RNA genome with a particle diameter size of 30 nm. Rapid and robust analytical methods to quantify CVA21 total, empty, and full virus particles are important to support the process development, meet regulatory requirements, and validate manufacturing processes. In this study, we demonstrate the detection of all four CVA21 capsid proteins, VP1, VP2, VP3, and VP4, as well as VP0, a surrogate for empty particles, using in-house-generated antibodies. An automated and quantitative capillary Western blot assay, Simple Western, was developed using these antibodies to quantify CVA21 total particles through VP1, empty particles through VP0, relative ratio of empty to full particles through VP0 and VP4, and the absolute ratio of empty to total particles through VP0 and VP1. Finally, this Simple Western method was used to support CVA21 cell culture and purification process optimization as a high-throughput analytical tool to make rapid process decisions.

Spinal-Pelvic Dissociation in Pregnancy: Surgical Fixation of Culture-Negative Extrapulmonary Tuberculosis.

A 33-year-old gravid female from Pakistan presented to the Emergency Department with persistent, intractable low back pain and neuropathic left L5 leg pain, associated with a left foot drop. There was a notable history of weight loss for 1 year. Investigations revealed a large collection in the right posterior paraspinal muscles tracking from a large bony defect in the right half of her sacrum extending into the pelvis. The collection was suggestive of an abscess and underwent US-guided aspiration. Culture, PCR examination, and bone biopsy were culture-negative for tuberculosis (TB). Samples taken from the placenta showed two small granulomata in the chorionic villi only. A multidisciplinary approach commenced with initiation of empirical TB treatment and attempted normal vaginal delivery. An urgent caesarean section for the delivery of the baby was required for failure to proceed. Spinal-pelvic stabilization in two stages was performed for the unstable fracture pattern, followed by pharmacotherapy and physiotherapy rehabilitation. At 12-month follow-up, the patient showed resolving TB and eradication of the paraspinal abscess. There was bony union and stability of the spinal-pelvic reconstruction. Back pain and sciatica can be common in pregnancy. However, this case highlights a rare occurrence of culture-negative extrapulmonary TB leading to an unstable spinal-pelvic fracture requiring a multidisciplinary approach for careful obstetric and orthopaedic treatment with empirical treatment by the infectious disease team and microbiology.

Disulfide Stress Targets Modulators of Excitotoxicity in Otherwise Healthy Brains.

Oxidative stress is a long-hypothesized cause of diverse neurological and psychiatric disorders but the pathways by which physiological redox perturbations may detour healthy brain development and aging are unknown. We reported recently (Foley et al., Neurochem Res 39:2030-2039, 2014) that two-electron oxidations, to disulfides, of protein vicinal thiols can vary markedly in association with more modest oxidations of the glutathione redox couple in brains from healthy adolescent rats whereas levels of protein S-glutathionylation were low and unchanged. Here, we demonstrate that the selective oxidations of protein vicinal thiols, occurring only in the more oxidized brains under study, were linked specifically to a peroxide stress as evidenced by increased oxidations, to disulfides, of the presumed catalytic vicinal thiols of peroxiredoxins 1 and 2. Moreover, we identify the catalytic subunit(s) of Na+, K+-ATPase, tubulins, glyceraldehyde-3-phosphate dehydrogenase, and protein phosphatase 1, all of which can modulate glutamate neurotransmission and the vulnerability of neurons to excitotoxicity, as non-peroxidase proteins exhibiting prominent oxidations of vicinal thiols. The two-electron pathway, demonstrated here, linking physiological redox perturbations in otherwise healthy brains to protein determinants of excitotoxicity, suggests an alternative to free radical pathways by which oxidative stress may impact brain development and aging.

Discovery of novel non-carboxylic acid 5-amino-4-cyanopyrazole derivatives as potent and highly selective LPA1R antagonists.

High throughput screening (HTS) of our chemical library identified 3-alkylamino-2-aryl-5H-imidazo[1,2,b]pyrazol-7-carbonitrile 1 as a potent antagonist of the LPA1 receptor (LPA1R). Further evaluation of this class of compounds indicated that LPA1R antagonist activity originated from the degradation of the parent molecule in DMSO during the assay conditions. Here, we describe the isolation and characterization of the degradation products and their LPA1R antagonist activity. We further profiled these novel non-carboxylic acid LPA1R antagonists and demonstrated their inhibition of LPA-induced proliferation and contraction of normal human lung fibroblasts (NHLF).

Protein vicinal thiol oxidations in the healthy brain: not so radical links between physiological oxidative stress and neural cell activities.

Reversible oxidations of protein thiols have emerged as alternatives to free radical-mediated oxidative damage with which to consider the impacts of oxidative stress on cellular activities but the scope and pathways of such oxidations in tissues, including the brain, have yet to be fully defined. We report here a characterization of reversible oxidations of glutathione and protein thiols in extracts from rat brains, from two sources, which had been (1) frozen quickly after euthanasia to preserve in vivo redox states and (2) subjected to alkylation upon tissue disruption to trap reduced thiols. Brains were defined, relatively, as Reduced and Moderately Oxidized based on measured ratios of reduced (GSH) to oxidized (GSSG) glutathione. Levels of protein disulfides formed by the cross-linking of closely-spaced (vicinal) protein thiols, but not protein S-glutathionylation, were higher in extracts from the Moderately Oxidized brains compared to the Reduced brains. Moreover, the oxidized vicinal thiol proteome contains proteins that impact cellular energetics, signaling, neurotransmission, and cytoskeletal dynamics among others. These findings argue that kinetically-competent pathways for reversible, two-electron oxidations, of protein vicinal thiols can be activated in healthy brains in response to physiological oxidative stresses. We propose that such oxidations may link oxidative stress to adaptive, but also potentially deleterious, changes in neural cell activities in otherwise healthy brains.

Discovery of camphor-derived pyrazolones as 11ß-hydroxysteroid dehydrogenase type 1 inhibitors.

Starting from screening hit, (4S,7R)-1,7,8,8-tetramethyl-2-phenyl-1,2,4,5,6,7-hexahydro-4,7-methano-indazol-3-one (7), we optimized the potency and pharmacokinetic properties. This led to the identification of compounds with good in vivo activity in a mouse pharmacodynamic model of inhibition of 11ßHSD1.

Discovery of pyrazolo[3,4-d]pyrimidine derivatives as GPR119 agonists.

We designed and synthesized a novel series of phenylamino- and phenoxy-substituted pyrazolo[3,4-d]pyrimidine derivatives as GPR119 agonists. SAR studies indicated that electron-withdrawing substituents on the phenyl ring are important for potency and full efficacy. Compound 26 combined good potency with a promising pharmacokinetic profile in mice, and lowered the glucose excursion in mice in an oral glucose-tolerance test.

Potent MCH-1 receptor antagonists from cis-1,4-diaminocyclohexane-derived indane analogs.

Benzimidazole and indane are the two key fragments in our potent and selective MCH-1 receptor (MCHR1) antagonists. To identify novel linkers connecting the two fragments, we investigated diamino-cycloalkane-derived analogs and discovered highly potent antagonists with cis-1,4-diaminocyclohexane as a unique spacer in this chemical class. Structural overlay suggested that cis-1-substituted-4-aminocyclohexane functions as a bioisostere of 4-substituted-piperidine and that the active conformation adopts a U-shaped orientation.

Design and synthesis of 2-N-substituted indazolone derivatives as non-carboxylic acid glycogen synthase activators.

Glycogen synthase (GS) catalyzes the transfer of glucose residues from UDP-glucose to a glycogen polymer chain, a critical step for glucose storage. Patients with type 2 diabetes normally exhibit low glycogen levels and decreased muscle glucose uptake is the major defect in whole body glucose disposal. Therefore, activating GS may provide a potential approach for the treatment of type 2 diabetes. In order to identify non-carboxylic acids GS activators, we designed and synthesized a series of 2-N-alkyl- and 2-N-aryl-indazolone derivatives and studied their activity in activating human GS.

Identification of RO4597014, a Glucokinase Activator Studied in the Clinic for the Treatment of Type 2 Diabetes.

To resolve the metabolite redox cycling associated with our earlier clinical compound 2, we carried out lead optimization of lead molecule 1. Compound 4 showed improved lipophilic ligand efficiency and demonstrated robust glucose lowering in diet-induced obese mice without a liability in predictive preclinical drug safety studies. Thus, it was selected as a clinical candidate and further studied in type 2 diabetic patients. Clinical data suggests no evidence of metabolite cycling, which is consistent with the preclinical profiling of metabolism.

Discovery of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors.

Through high throughput screening and subsequent hit identification and optimization, we synthesized a series of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as the first reported potent and reversible GFAT inhibitors. SAR studies of this class of compounds indicated significant impact on GFAT inhibition potency by substitutions on the A-ring and C-ring. The ketone group was found to be necessary for high potency. Compound 28 (RO0509347) demonstrated potent GFAT inhibition (IC(50)=1µM) with a desirable pharmacokinetic profile in rats, and showed significant efficacy in reducing the glucose excursion in an OGTT test in ob/ob mice.

Discovery and optimization of 2-phenyloxazole derivatives as diacylglycerol acyltransferase-1 inhibitors.

In a discovery effort to find safe and effective DGAT-1 inhibitors, we have identified 2-phenyloxazole 4-carboxamide 1 as a conformationally constrained analog of a hydrazide hit, which was previously identified from high-throughput screening. Further optimization of this series has led to chemically more stable 2-phenyloxazole-based DGAT-1 inhibitor 25 with improved solubility, cell-based activity, and pharmacokinetic properties. Compound 25 also demonstrated in vivo efficacy in a diet-induced obesity (DIO) rat model.

Transcriptional repression of SKP2 is impaired in MYCN-amplified neuroblastoma.

The cell cycle regulator, SKP2, is overexpressed in various cancers and plays a key role in p27 degradation, which is involved in tumor cell dedifferentiation. Little is known about the mechanisms leading to impaired SKP2 transcriptional control in tumor cells. We used neuroblastoma as a model to study SKP2 regulation because SKP2 transcript levels gradually increase with aggressiveness of neuroblastoma subtypes. The highest SKP2 levels are found in neuroblastomas with amplified MYCN. Accordingly, we found 5.5-fold (range, 2-9.5) higher SKP2 core promoter activity in MYCN-amplified cells. Higher SKP2 core promoter activity in MYCN-amplified cells is mediated through a defined region at the transcriptional start site. This region includes a specific E2F-binding site that makes SKP2 activation largely independent of mitogenic signals integrated through the SP1/ELK-1 site. We show by chromatin immunoprecipitation that SKP2 activation through the transcriptional start site in MYCN-amplified cells is associated with the low abundance of pRB-E2F1 complexes bound to the SKP2 promoter. Transcriptional control of SKP2 through this regulatory mechanism can be reestablished in MYCN-amplified cells by restoring pRB activity using selective small compound inhibitors of CDK4. In contrast, doxorubicin or nutlin-3 treatment-both leading to p53-p21 activation-or CDK2 inhibition had no effect on SKP2 regulation in MYCN-amplified cells. Together, this implies that deregulated MYCN protein levels in MYCN-amplified neuroblastoma cells activate SKP2 through CDK4 induction, abrogating repressive pRB-E2F1 complexes bound to the SKP2 promoter.

Discovery of novel and potent leukotriene B4 receptor antagonists. Part 1.

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

A selective phosphatase of regenerating liver phosphatase inhibitor suppresses tumor cell anchorage-independent growth by a novel mechanism involving p130Cas cleavage.

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.