Nutrition and Physical Activity in British Army Officer Cadet Training Part 2-Daily Distribution of Energy and Macronutrient Intake.

Dietary intake and physical activity impact performance and adaptation during training. The aims of this study were to compare energy and macronutrient intake during British Army Officer Cadet training with dietary guidelines and describe daily distribution of energy and macronutrient intake and estimated energy expenditure. Thirteen participants (seven women) were monitored during three discrete periods of military training for 9 days on-camp, 5 days of field exercise, and 9 days of a mixture of the two. Dietary intake was measured using researcher-led food weighing and food diaries, and energy expenditure was estimated from wrist-worn accelerometers. Energy intake was below guidelines for men (4,600 kcal/day) and women (3,500 kcal/day) during on-camp training (men = -16% and women = -9%), field exercise (men = -33% and women = -42%), and combined camp and field training (men and women both -34%). Carbohydrate intake of men and women were below guidelines (6 g·kg-1·day-1) during field exercise (men = -18% and women = -37%) and combined camp and field training (men = -33% and women = -39%), respectively. Protein intake was above guidelines (1.2 kcal·kg-1·day-1) for men and women during on-camp training (men = 48% and women = 39%) and was below guidelines during field exercise for women only (-27%). Energy and macronutrient intake during on-camp training centered around mealtimes with a discernible sleep/wake cycle for energy expenditure. During field exercise, energy and macronutrient intake were individually variable, and energy expenditure was high throughout the day and night. These findings could be used to inform evidenced-based interventions to change the amount and timing of energy and macronutrient intake around physical activity to optimize performance and adaptations during military training.

Discovery of a Thiadiazole-Pyridazine-Based Allosteric Glutaminase 1 Inhibitor Series That Demonstrates Oral Bioavailability and Activity in Tumor Xenograft Models.

Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell's ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.

Rapid activation of epithelial-mesenchymal transition drives PARP inhibitor resistance in Brca2-mutant mammary tumours.

Tumours defective in the DNA homologous recombination repair pathway can be effectively treated with poly (ADP-ribose) polymerase (PARP) inhibitors; these have proven effective in clinical trials in patients with BRCA gene function-defective cancers. However, resistance observed in both pre-clinical and clinical studies is likely to impact on this treatment strategy. Over-expression of phosphoglycoprotein (P-gp) has been previously suggested as a mechanism of resistance to the PARP inhibitor olaparib in mouse models of Brca1/2-mutant breast cancer. Here, we report that in a Brca2 model treated with olaparib, P-gp upregulation is observed but is not sufficient to confer resistance. Furthermore, resistant/relapsed tumours do not show substantial changes in PK/PD of olaparib, do not downregulate PARP1 or re-establish double stranded DNA break repair by homologous recombination, all previously suggested as mechanisms of resistance. However, resistance is strongly associated with epithelial-mesenchymal transition (EMT) and treatment-naïve tumours given a single dose of olaparib upregulate EMT markers within one hour. Therefore, in this model, olaparib resistance is likely a product of an as-yet unidentified mechanism associated with rapid transition to the mesenchymal phenotype.

Irreversible Inhibition of EGFR: Modeling the Combined Pharmacokinetic-Pharmacodynamic Relationship of Osimertinib and Its Active Metabolite AZ5104.

Osimertinib (AZD9291) is a potent, selective, irreversible inhibitor of EGFR-sensitizing (exon 19 and L858R) and T790M-resistant mutation. In vivo, in the mouse, it is metabolized to an active des-methyl metabolite, AZ5104. To understand the therapeutic potential in patients, this study aimed to assess the relationship between osimertinib pharmacokinetics, the pharmacokinetics of the active metabolite, the pharmacodynamics of phosphorylated EGFR reduction, and efficacy in mouse xenograft models of EGFR-driven cancers, including two NSCLC lines. Osimertinib was dosed in xenografted models of EGFR-driven cancers. In one set of experiments, changes in phosphorylated EGFR were measured to confirm target engagement. In a second set of efficacy studies, the resulting changes in tumor volume over time after repeat dosing of osimertinib were observed. To account for the contributions of both molecules, a mathematical modeling approach was taken to integrate the resulting datasets. The model was able to describe the pharmacokinetics, pharmacodynamics, and efficacy in A431, PC9, and NCI-H1975 xenografts, with the differences in sensitivity described by the varying potency against wild-type, sensitizing, and T790M-mutant EGFR and the phosphorylated EGFR reduction required to reduce tumor volume. It was inferred that recovery of pEGFR is slower after chronic dosing due to reduced resynthesis. It was predicted and further demonstrated that although inhibition is irreversible, the resynthesis of EGFR is such that infrequent intermittent dosing is not as efficacious as once daily dosing. Mol Cancer Ther; 15(10); 2378-87. ©2016 AACR.

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models.

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.

Inhibition of PI3Kß signaling with AZD8186 inhibits growth of PTEN-deficient breast and prostate tumors alone and in combination with docetaxel.

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kß isoform. Inhibitors of PI3Kß have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kß and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 µmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kß with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs.

Recruitment of participants to a multiple sclerosis trial: the CombiRx experience.

BACKGROUND: and purpose Participant recruitment is central to all clinical trials. Any delay in recruitment affects the completion and ultimate success of the trial. We report our experience with patient screening and randomization in CombiRx, which may inform the design of other trials. CombiRx was a multicenter, phase III, double-blind, randomized clinical trial comparing the combined use of interferon beta-1a and glatiramer acetate to either agent alone in patients with relapsing-remitting multiple sclerosis (RRMS). This trial was launched in January 2005 in 69 centers in the United States and Canada under a co-operative agreement with the National Institute of Neurological Disorders and Stroke (NINDS). The goal was to recruit 1000 patients over 1.5 years after a 6-month start-up period. Instead, the investigators required 4.25 years to enroll 1008 patients. METHODS: During this trial, we assessed the effectiveness of various recruitment strategies, utility of rescreening prior screen failures, and potential factors and strategies used in study conduct, research, and infrastructure, all of which affected recruitment of participants and ultimately time to completion of CombiRx. We particularly were interested in the variability in time to site initiation between academic centers and private practice sites. RESULTS: Physicians who were directly involved in the medical care of patients with RRMS were the primary source of patients recruited to CombiRx. A flexible study design that allowed for rescreening of the initial screen failures after a period of time was useful due to the relapsing/remitting course of the disease. Academic centers took longer to implement the trial than the private practice centers, but once sites were approved for enrollment, there was no important difference in the number of participants enrolled. LIMITATIONS: The CombiRx trial was conducted during a period when multiple new medications were being tested, thus affecting the pace of recruitment and limiting ability to generalize our experiences. However, the lessons we learned about process are relevant. CONCLUSION: Participants can be enrolled successfully in a clinical trial for RRMS, but factors affecting the time to achieve the requirements needed to start screening can be unpredictable and problematic. Prospective planning by the sponsors and investigators, use of central institutional review boards (IRBs), master trial agreements and secure remote desktop access to the trial database may expedite trial implementation and participant recruitment. A good scientific research question with flexible study design and active involvement of the clinicians are important factors driving recruitment. Clinical trials can be implemented successfully both in private practices and at academic centers, a consideration when selecting sites.

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers.

Expression of stromal genes associated with the angiogenic response are not differentiated between human tumour xenografts with divergent vascular morphologies.

Human tumour xenografts have commonly been used to explore the mechanisms of tumour angiogenesis and the interaction of tumour cells with their microenvironment, as well as predict potential utility of anti-angiogenic inhibitors across different tumour types. To investigate how well human tumour xenografts can be used to differentiate the effects of stromal targeting agents we performed a comparative assessment of the murine angiogenic response across a panel of pre-clinical tumour xenografts. By analysing a panel of 22 tumour xenografts with a range of vascular morphologies, micro-vessel densities and levels of fibroblast and inflammatory infiltrate, we have examined the relationship between angiogenic stroma and human tumour models. These models were studied using a combination of immunohistochemistry and species specific mRNA profiling to differentiate the tumour and stromal transcript mRNA profiles. Principal Component Analysis (PCA) and regression analysis was used to investigate the transcriptional relationships between the individual models and the correlation with the stromal architecture. We found the human tumour cell expressed factors to be independent of the murine host responses such as microvessel density, and fibroblast or macrophage cellular infiltrate. Moreover mRNA profiling of the mouse stroma suggested that the host response to the different tumours was relatively uniform despite differences in stromal structures within the tumour. Supporting this, models with different stromal compositions responded similarly to cediranib, a small molecule inhibitor of VEGF signalling. The data indicate that although the angiogenic response to the tumour results in reproducible stromal architectures, these responses are not differentiated at the level of gene expression.

Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types.

Mutations in the chromatin remodeling gene ARID1A have recently been identified in the majority of ovarian clear cell carcinomas (OCCCs). To determine the prevalence of mutations in other tumor types, we evaluated 759 malignant neoplasms including those of the pancreas, breast, colon, stomach, lung, prostate, brain, and blood (leukemias). We identified truncating mutations in 6% of the neoplasms studied; nontruncating somatic mutations were identified in an additional 0.4% of neoplasms. Mutations were most commonly found in gastrointestinal samples with 12 of 119 (10%) colorectal and 10 of 100 (10%) gastric neoplasms, respectively, harboring changes. More than half of the mutated colorectal and gastric cancers displayed microsatellite instability (MSI) and the mutations in these tumors were out-of-frame insertions or deletions at mononucleotide repeats. Mutations were also identified in 2-8% of tumors of the pancreas, breast, brain (medulloblastomas), prostate, and lung, and none of these tumors displayed MSI. These findings suggest that the aberrant chromatin remodeling consequent to ARID1A inactivation contributes to a variety of different types of neoplasms.

Induction of heart valve lesions by small-molecule ALK5 inhibitors.

Aberrant signaling by transforming growth factor-ß (TGF-ß) and its type I (ALK5) receptor has been implicated in a number of human diseases and this pathway is considered a potential target for therapeutic intervention. Transforming growth factor-ß signaling via ALK5 plays a critical role during heart development, but the role of ALK5 in the adult heart is poorly understood. In the current study, the preclinical toxicology of ALK5 inhibitors from two different chemistry scaffolds was explored. Ten-week-old female Han Wistar rats received test compounds by the oral route for three to seven days. Both compounds induced histopathologic heart valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells. The pathology was observed in all animals, at all doses tested, and occurred in all four heart valves. Immunohistochemical analysis of ALK5 in rat hearts revealed expression in the valves, but not in the myocardium. Compared to control animals, protein levels of ALK5 were unchanged in the heart valves of treated animals. We also observed a physeal dysplasia in the femoro-tibial joint of rats treated with ALK5 inhibitors, a finding consistent with a pharmacological effect described previously with ALK5 inhibitors. Overall, these findings suggest that TGF-ß signaling via ALK5 plays a critical role in maintaining heart valve integrity.

Early phase TGFß receptor signalling dynamics stabilised by the deubiquitinase UCH37 promotes cell migratory responses.

TGFß signals through serine/threonine kinase receptors and intracellular Smad transcription factors. An important regulatory step involves ubiquitination of Smads and/or TGFß receptors by specific ubiquitin ligases, in a process that can be reversed by the deubiquitinating enzyme UCH37. Here, to explore the physiological role of UCH37 in TGFß signalling we have generated stable and inducible HaCAT keratinocyte and Colo-357 pancreatic carcinoma cell lines mis-expressing UCH37. We show that UCH37 knockdown significantly inhibits the activity of a TGFß-dependent gene reporter and selectively decreases levels of some TGFß-dependent target genes, notably p21 and PAI-1, but only during the early phase of TGFß receptor activation. Interestingly, UCH37 knockdown in Colo-357 cells had no effect on TGFß-dependent cell proliferation and epithelial-mesenchymal transition, yet significantly impaired cell migration. Collectively, our data indicate that UCH37 sustains early TGFß pathway activation kinetics that determines threshold-specific gene expression patterns, and that opposing actions of ubiquitin ligases and deubiquitinases influences distinct biological TGFß-dependent biological responses. Moreover, we suggest that UCH37 could represent a viable target for novel and selective cancer therapeutics.

The use of intravascular ultrasound imaging to improve use of inferior vena cava filters in a high-risk bariatric population.

OBJECTIVE: Pulmonary embolism is the leading cause of death after gastric bypass procedures for obesity, approximating 0.5% to 4%. All bariatric patients, but especially the super-obese, which have a body mass index (BMI) >50 kg/m(2), are at significant risk for postoperative venous thromboembolism (VTE). Visualization and weight limitations of fluoroscopy tables exclude most bariatric and all super-obese patients from inferior vena cava (IVC) filter placement using fluoroscopy. Intravascular ultrasound (IVUS)-guided IVC filter placement is the only modality that allows these high-risk patients to have an IVC filter placed. METHODS: Hospital and outpatient records of the 494 patients who underwent gastric bypass procedures from January 1, 2004, to May 31, 2006, were reviewed. All patients who had concurrent IVC filter placement with the use of IVUS guidance were selected. Comorbidities, outcomes, and complications were recorded. RESULTS: We identified 27 patients with mean BMI of 70 +/- 3 kg/m(2); of these, 25 were super-obese (BMI >50 kg/m(2)). Procedures included five laparoscopic and 22 open gastric bypass operations. All patients underwent concurrent IVC filter placement using IVUS guidance. In addition to super-obesity, indications for IVC filter placement included history of VTE (n = 4), known hypercoagulable state (n = 2), and profound immobility (n = 21). Mean follow up was 293 +/- 40 days. Technical success rate was 96.3%. There were no catheter site complications. In one surviving patient, a nonfatal pulmonary embolism was detected by computed tomography 2 months postoperatively. Two patients died, and autopsy excluded VTE as the cause of death in both. CONCLUSION: This study suggests efficacy of IVUS-guided IVC filter placement in preventing mortality from pulmonary embolism in high-risk bariatric patients, including the super-obese. IVUS-guided IVC filter placement can be safely performed with an excellent success rate in all bariatric patients, including the super-obese, who otherwise would not be candidates for IVC filter placement due to the limitations imposed by their large body habitus.

Mid-term results with laser atherectomy in the treatment of infrainguinal occlusive disease.

BACKGROUND: Laser atherectomy offers a potential intervention for multivessel infrainguinal disease in patients with poor revascularization options. Despite promising early results reported in the literature, the proper patient population who might benefit from laser atherectomy has yet to be determined. METHODS: From July 2004 to June 2006, patients undergoing laser atherectomy were retrospectively reviewed and assessed for comorbidities, operative and follow-up variables potentially associated with the end points of nondefinitive therapy, and limb salvage. RESULTS: During the study period, 40 patients (21 women, 19 men) underwent laser atherectomy, and the average follow-up was 461 +/- 49 days (range, 17 to 1050 days). Their average age was 68 +/- 2 years (range, 43 to 93 years). The indication for laser atherectomy was critical limb ischemia in 26 (65%) and lower limb claudication in 11 (35%). A total of 47 lesions were treated in the following arterial segments: 34 femoropopliteal and 13 infrapopliteal. Femoropopliteal distribution by the Trans-Atlantic Society Classification (TASC) was A in 3, B in 17, C in 10, D in 4, and infrapopliteal lesions distribution was A in 1, B in 3, C in 4, and D in 5. Adjunctive angioplasty was used in 75% of cases. The overall technical success rate (<50% residual stenosis) was 88%. Laser atherectomy-based treatment was the definitive therapy for 23 patients (58%), and the overall 12-month primary patency was 44%. The limb salvage rate at 12 months in 26 patients with critical limb ischemia was 55%. Renal failure was a risk factor for amputation (P < .001) and failed primary patency (P < .05), type 2 diabetes mellitus was a risk factor for amputation (P < .05), and poor tibial runoff was associated with failed primary patency and amputation (P < .05). Outcome was associated with the number of patent infrapopliteal runoff vessels. CONCLUSION: These data demonstrate that laser atherectomy can be used with high initial technical success rate. Chronic renal failure and diabetes are risk factors for a negative outcome. Poor results in patients with diabetes and renal failure necessitate careful case selection in this subgroup, in which laser atherectomy is less likely to provide a definitive revascularization result or limb salvage.

Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human cancers.

Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high-throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients.