PDF file, 428K, The combination of sorafenib and diclofenac causes cell death.

The temporal dynamics of accumulating and mitigated risk, as measured in trials undergoing RBM management, have not been well described. Using 78,000+ historical total risk observations, made serially across 30+ studies, 50+ countries, and 4000+ sites, we have developed a novel statistical framework for inference of parameters that closely model observed risk flux. We have used this framework to a) provide insight into many operational aspects of RBM management, including study-, country-, and time-specific effects on risk flux; b) identify underlying metrics that drive (and/or respond) to changes in site monitoring effort; and c) allow opportunities for predictive risk forecasting. Here we describe the statistical framework for risk flux modeling and risk flow simulation, and summarize our results on operational aspects that significantly affect the dynamics of risk flux. We will also demonstrate how such models may be used to forecast the cost/benefit effects of competing risk-adaptive monitoring plans.

Purpose: Clinical trial monitoring is an essential component of drug development aimed at safeguarding subject safety, data quality, and protocol compliance by focusing sponsor oversight on the most important aspects of study conduct. In recent years, regulatory agencies, industry consortia, and nonprofit collaborations between industry and regulators, such as TransCelerate and International Committee for Harmonization, have been advocating a new, risk-based approach to monitoring clinical trials that places increased emphasis on critical data and processes and encourages greater use of centralized monitoring. However, how best to implement risk-based monitoring (RBM) remains unclear and subject to wide variations in tools and methodologies. The nonprescriptive nature of the regulatory guidelines, coupled with limitations in software technology, challenges in operationalization, and lack of robust evidence of superior outcomes, have hindered its widespread adoption. Methods: We describe a holistic solution that combines convenient access to data, advanced analytics, and seamless integration with established technology infrastructure to enable comprehensive assessment and mitigation of risk at the study, site, and subject level. Findings: Using data from completed RBM studies carried out in the last 4 years, we demonstrate that our implementation of RBM improves the efficiency and effectiveness of the clinical oversight process as measured on various quality, timeline, and cost dimensions. (C) 2018 The Author(s). Published by Elsevier Inc.

Nonalcoholic steatohepatitis (NASH) is an emerging health crisis with no approved therapies. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, shows promise in NASH trials. However, the precise mechanisms mediating OCA effects and impact on cholesterol metabolism are not fully understood. We explored the pharmaco-toxicological effects of OCA on patho-physiological pathways in hepatocytes using a previously described perfused organotypic liver system that allows culture in near-physiological insulin/glucose milieus, and exhibits drug responses at clinically-relevant concentrations. Primary hepatocytes experienced 48-hour exposure to OCA at concentrations approximating therapeutic (0.5μM) and supratherapeutic (10μM) levels. Global transcriptomics by RNAseq was complimented by cellular viability (MTT), CYP activity assays, and secreted FGF19 levels in the media. Dose-dependent, transcriptional effects suggested suppression of bile acid synthesis (↓CYP7A1, ↓CYP27A1) and increased bile efflux (↑ABCB4, ↑ABCB11, ↑OSTA, ↑OSTB). Pleiotropic effects included suppression of TGFβ and IL-6 signaling pathways, and signatures suggestive of HDL suppression (↑SCARB1, ↓ApoAI, ↓LCAT) and LDL elevation (↑ApoB, ↓CYP7A1). OCA exhibited direct FXR-mediated effects with increased FGF19 secretion. Transcriptomics revealed regulation of metabolic, anti-inflammatory, and anti-fibrotic pathways beneficial in NASH, and predicted cholesterol profiles consistent with clinical findings. Follow-up studies under lipotoxic/inflammatory conditions would corroborate these effects in a disease-relevant environment.

Relational databases can integrate diverse types of information and manage large sets of similarity search results, greatly simplifying genome-scale analyses. By focusing on taxonomic subsets of sequences, relational databases can reduce the size and redundancy of sequence libraries and improve the statistical significance of homologs. In addition, by loading similarity search results into a relational database, it becomes possible to explore and summarize the relationships between all of the proteins in an organism and those in other biological kingdoms. This unit describes how to use relational databases to improve the efficiency of sequence similarity searching and demonstrates various large-scale genomic analyses of homology-related data. It also describes the installation and use of a simple protein sequence database, seqdb_demo, which is used as a basis for the other protocols. The unit also introduces search_demo, a database that stores sequence similarity search results. The search_demo database is then used to explore the evolutionary relationships between E. coli proteins and proteins in other organisms in a large-scale comparative genomic analysis. © 2017 by John Wiley & Sons, Inc.

Background Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established.Results Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 degrees C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi.Conclusions CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. (C) 2016 John Wiley Sons, Ltd.

Background Rheumatoid arthritis (RA) and atherosclerosis are chronic inflammatory diseases that share pathologic and molecular features. RA doubles the risk of cardiovascular disease (CVD) compared to the non-RA population. Common RA treatments are anti-inflammatory by design, but effects on CVD are unclear. Previously we reported the effect of SIR on IL6 and tumor necrosis factor (TNF) α signaling using a human endothelial cell (EC) and smooth muscle cell (SMC) co-culture system. Here we report additional analyses further characterizing effects of SIR on atherosclerotic (ATH) and oxidative (OX) stress pathways. Objectives To examine if RA drugs decrease ATH signaling and cellular stress in vascular cells under CVD conditions. Methods An in vitro surrogate system that co-cultures human ECs and SMCs was used to assess effects of RA drugs on vascular cells. Fluid flow conditions that drive cardiovascular health and CVD were applied. Atheroprone flow conditions were based on human hemodynamics from the carotid bifurcation, a site prone to developing atherosclerosis. The culture medium contained atherogenic risk factors including in vivo concentrations of oxidized LDL (oxLDL), soluble IL6 receptor (sIL6R), and TNF. Using RNA sequencing and microarray, we performed transcriptomic and biologic pathway analyses of surrogate system response. We compared treatments targeting pathogenic RA pathways, including anti-IL6 (SIR), anti-IL6 receptor (tocilizumab; TCZ), anti-TNF (adalimumab; ADA), and a small molecule JAK inhibitor (tofacitinib; TOF). The magnitude of pathway response was calculated as the L2 norm of the log2 fold change of genes in the pathway. Results The combination of atheroprone flow, sIL6R, TNF, and oxLDL (RA-CVD conditions) induced a robust response of inflammatory and OX stress pathways compared to healthy conditions (atheroprone flow without TNF and with non-oxLDL and sIL6R). The anti-IL6/IL6R treatments (SIR, TCZ) improved RA-CVD conditions by inhibiting key pathogenic pathways, including ATH signaling and NRF2-mediated OX stress. SIR attenuated the magnitude of RA-CVD response in ATH and OX stress pathways vs IgG or vehicle control the most: by 17% (adj P=0.035; Wilcoxon signed-rank test) and 34% (adj P=0.094) in ECs; and 49% (adj P=1.4e-5) and 47% (adj P=1.1e-3) in SMCs, respectively. TCZ was similar to SIR in restoring ECs and SMCs to healthy conditions in both pathways; ADA showed a weaker, similar trend compared to IL6 inhibition. TOF was not effective in suppressing (and tended to exacerbate) ATH and OX stress pathways in ECs. Conclusions IL6 pathway inhibitors SIR and TCZ potently suppressed ATH and cellular stress in vitro. The degree of suppression suggests that these drugs may mitigate the effects of atherogenic factors sIL6R, TNF, and oxLDL. In contrast, TNF inhibitor ADA was a less effective inhibitor of key CVD pathways, while JAK inhibitor TOF tended to exacerbate CVD pathways. Collectively, the data suggest that IL6 inhibition may provide more CVD benefit compared to RA drugs targeting other pathways. Acknowledgements Study sponsored by Janssen Research & Development, LLC, in collaboration with GlaxoSmithKline. Disclosure of Interest R. Feaver Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, S. Collado Employee of: HemoShear, LLC, S. Hoang Employee of: HemoShear, LLC, E. Berzin Employee of: HemoShear, LLC, A. Armstrong Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Gardner Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, H. Liu Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, A. Mackey Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Manka Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Shealy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, B. Blackman Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC

Objectives— The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. Approach and Results— We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. Conclusions— The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 μM) and Ritonavir (3.5 and 62.4 μM) for 48 h. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain with simultaneous mitochondrial uncoupling supporting clinical reports of a lower propensity for steatosis. This in vitro study offers insights into the disparate direct and immune-mediated toxicity mechanisms underlying Nevirapine and Ritonavir toxicity in the clinic.

Over half of BRAFV600E melanomas display intrinsic resistance to BRAF inhibitors, in part due to adaptive signaling responses. In this communication we ask whether BRAFV600E melanomas share common adaptive responses to BRAF inhibition that can provide clinically relevant targets for drug combinations. We screened a panel of 12 treatment-naive BRAFV600E melanoma cell lines with MAP Kinase pathway inhibitors in pairwise combination with 58 signaling inhibitors, assaying for synergistic cytotoxicity. We found enormous diversity in the drug combinations that showed synergy, with no two cell lines having an identical profile. Although the 6 lines most resistant to BRAF inhibition showed synergistic benefit from combination with lapatinib, the signaling mechanisms by which this combination generated synergistic cytotoxicity differed between the cell lines. We conclude that adaptive responses to inhibition of the primary oncogenic driver (BRAFV600E) are determined not only by the primary oncogenic driver but also by diverse secondary genetic and epigenetic changes ("back-seat drivers") and hence optimal drug combinations will be variable. Because upregulation of receptor tyrosine kinases is a major source of drug resistance arising from diverse adaptive responses, we propose that inhibitors of these receptors may have substantial clinical utility in combination with inhibitors of the MAP Kinase pathway.

Genome-wide association studies in human type 2 diabetes (T2D) have renewed interest in the pancreatic islet as a contributor to T2D risk. Chronic low-grade inflammation resulting from obesity is a risk factor for T2D and a possible trigger of β-cell failure. In this study, microarray data were collected from mouse islets after overnight treatment with cytokines at concentrations consistent with the chronic low-grade inflammation in T2D. Genes with a cytokine-induced change of >2-fold were then examined for associations between single nucleotide polymorphisms and the acute insulin response to glucose (AIRg) using data from the Genetics Underlying Diabetes in Hispanics (GUARDIAN) Consortium. Significant evidence of association was found between AIRg and single nucleotide polymorphisms in Arap3 (5q31.3), F13a1 (6p25.3), Klhl6 (3q27.1), Nid1 (1q42.3), Pamr1 (11p13), Ripk2 (8q21.3), and Steap4 (7q21.12). To assess the potential relevance to islet function, mouse islets were exposed to conditions modeling low-grade inflammation, mitochondrial stress, endoplasmic reticulum (ER) stress, glucotoxicity, and lipotoxicity. RT-PCR revealed that one or more forms of stress significantly altered expression levels of all genes except Arap3. Thapsigargin-induced ER stress up-regulated both Pamr1 and Klhl6. Three genes confirmed microarray predictions of significant cytokine sensitivity: F13a1 was down-regulated 3.3-fold by cytokines, Ripk2 was up-regulated 1.5- to 3-fold by all stressors, and Steap4 was profoundly cytokine sensitive (167-fold up-regulation). Three genes were thus closely associated with low-grade inflammation in murine islets and also with a marker for islet function (AIRg) in a diabetes-prone human population. This islet-targeted genome-wide association scan identified several previously unrecognized candidate genes related to islet dysfunction during the development of T2D.