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Seizure liability remains a significant cause of attrition in drug discovery and development, leading to loss of competitiveness, delays, and increased costs. Current detection methods rely on observations made in in vivo studies intended to support clinical trials, such as tremors or other abnormal movements. These signs could be missed or misinterpreted; thus, definitive confirmation of drug-induced seizure requires a follow-up electroencephalogram study. There has been progress in in vivo detection of seizure using automated video systems that record and analyze animal movements. Nonetheless, it would be preferable to have earlier prediction of seizurogenic risk that could be used to eliminate liabilities early in discovery while there are options for medicinal chemists making potential new drugs. Attrition due to cardiac adverse events has benefited from routine early screening; could we reduce attrition due to seizure using a similar approach? Specifically, microelectrode arrays could be used to detect potential seizurogenic signals in stem-cell-derived neurons. In addition, there is clear evidence implicating neuronal voltage-gated and ligand-gated ion channels, GPCRs and transporters in seizure. Interactions with surrounding glial cells during states of stress or inflammation can also modulate ion channel function in neurons, adding to the challenge of seizure prediction. It is timely to evaluate the opportunity to develop an in vitro assessment of seizure linked to a panel of ion channel assays that predict seizure, with the aim of influencing structure-activity relationship at the design stage and eliminating compounds predicted to be associated with pro-seizurogenic state.

Introduction: Zucker diabetic fatty (ZDF) rats are used widely as an animal model of metabolic syndrome and insulin resistance. Our study focused on the effects of high versus low dietary fat on the development of Type 2 diabetes in obese male ZDF rats (fa/fa), including biomarkers to detect early signs of hypercoagulability and vascular injury in the absence of overt thrombosis. Methods: In this study, male (5/group) 10-week-old CRL:ZDF370(obese) rats were fed low (LFD, 16.7% fat) or high fat (HFD, 60% fat) diet for 12 or 15 weeks. Cohorts of 5 rats within diet groups were scheduled for sample collection after weeks 12 and 15. Results: HFD–fed ZDF rats had oily coats, lower rates of food consumption, more accelerated weight gain and increased serum cholesterol (+15%) and triglyceride concentrations (+75%) vs. LFD–fed ZDF rats. Urinary ketones were observed only in HFD–fed ZDF rats and greater urine glucose and protein concentrations in HFD–fed ZDF vs. LFD–fed ZDF rats were seen. Hemostasis testing showed ~2-fold greater fibrinogen concentration, increased von Willebrand factor concentration, and high thrombin generation in HFD–fed ZDF vs LFD–fed ZDF rats. Increased mortality in the HFD–fed ZDF rat was attributed to exacerbations of altered carbohydrate metabolism as evidenced by ketonuria and nephropathy leading to renal failure. Discussion: This characterization shows that the ZDF rat at the age, sex and weight used in this study is highly sensitive to dietary fat content that can exacerbate prothrombotic, metabolic and renal disturbances and increase mortality.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and ""cellrhythmias"" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define ""fit for purpose"" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable ""fit for purpose"" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug’s cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

Introduction: Based on the ICH S7B and E14 guidance documents, QT interval (QTc) is used as the primary in vivo biomarker to assess the risk of drug-induced torsades de pointes (TdP). Clinical and nonclinical data suggest that drugs that prolong the corrected QTc with balanced multiple ion channel inhibition (most importantly the l-type calcium, Cav1.2, and persistent or late inward sodium current, Nav1.5, in addition to human Ether-à-go-go-Related Gene [hERG] IKr or Kv11.1) may have limited proarrhythmic liability. The heart rate-corrected J to T-peak (JTpc) measurement in particular may be considered to discriminate selective hERG blockers from multi-ion channel blockers. Methods: Telemetry data from Beagle dogs given dofetilide (0.3 mg/kg), sotalol (32 mg/kg), and verapamil (30 mg/kg) orally and Cynomolgus monkeys given medetomidine (0.4 mg/kg) orally were retrospectively analyzed for effects on QTca, JTpca, and T-peak to T-end covariate adjusted (Tpeca) interval using individual rate correction and super intervals (calculated from 0-6, 6-12, 12-18, and 18-24 hours postdose). Results: Dofetilide and cisapride (IKr or Kv11.1 blockers) were associated with significant increases in QTca and JTpca, while sotalol was associated with significant increases in QTca, JTpca, and Tpeca. Verapamil (a Kv11.1 and Cav1.2 blocker) resulted in a reduction in QTca and JTpca, however, and increased Tpeca. Medetomidine was associated with a reduction in Tpeca and increase in JTpca. Discussion: Results from this limited retrospective electrocardiogram analysis suggest that JTpca and Tpeca may discriminate selective IKr blockers and multichannel blockers and could be considered in the context of an integrated comprehensive proarrhythmic risk assessment.

Recent in vitro cardiac safety studies demonstrate the ability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to detect electrophysiologic effects of drugs. However, variability contributed by unique approaches, procedures, cell lines, and reagents across laboratories makes comparisons of results difficult, leading to uncertainty about the role of hiPSC-CMs in defining proarrhythmic risk in drug discovery and regulatory submissions. A blinded pilot study was conducted to evaluate the electrophysiologic effects of 8 well-characterized drugs on 4 cardiomyocyte lines using a standardized protocol across 3 microelectrode array platforms (18 individual studies). Drugs were selected to define assay sensitivity of prominent repolarizing currents (E-4031 for IKr, JNJ303 for IKs) and depolarizing currents (nifedipine for ICaL, mexiletine for INa) as well as drugs affecting multichannel block (flecainide, moxifloxacin, quinidine, and ranolazine). Inclusion criteria for final analysis was based on demonstrated sensitivity to IKr block (20% prolongation with E-4031) and L-type calcium current block (20% shortening with nifedipine). Despite differences in baseline characteristics across cardiomyocyte lines, multiple sites, and instrument platforms, 10 of 18 studies demonstrated adequate sensitivity to IKr block with E-4031 and ICaL block with nifedipine for inclusion in the final analysis. Concentration-dependent effects on repolarization were observed with this qualified data set consistent with known ionic mechanisms of single and multichannel blocking drugs. hiPSC-CMs can detect repolarization effects elicited by single and multichannel blocking drugs after defining pharmacologic sensitivity to IKr and ICaL block, supporting further validation efforts using hiPSC-CMs for cardiac safety studies.

Background and Purpose Translation of nonclinical markers of delayed ventricular repolarization to clinical QTc prolongation (a biomarker for Torsades de Pointes proarrhythmia) remains an issue in drug discovery and regulatory evaluations. A retrospective analysis of 150 submitted drug applications in a US-FDA database was conducted to determine the utility of established nonclinical in vitro iKr current (hERG), action potential duration (APD) and in vivo (QTc) repolarization assays to detect and predict clinical QTc prolongation. Experimental Approach The diagnostic and prognostic performance of three nonclinical assays were compared with clinical thorough QT study outcomes based on free clinical plasma drug concentrations using sensitivity and specificity, receiver operator curves (ROC), positive and negative predictive values, and likelihood ratios. Key Results Nonclinical assays demonstrated robust specificity (ability to identify drugs without clinical QTc prolongation) but poor sensitivity (ability to identify drugs eliciting clinical QTc prolongation) at low to intermediate (1x-30x) clinical exposure multiples. The QTc assay provided the most robust positive and negative predictive values (ability to predict clinical QTc prolongation). Receiver operator curves (defining overall test accuracy) and likelihood ratios (ability to influence post-test probabilities) demonstrated overall marginal performance for hERG and QTc assays (best results at 30x exposures) while the APD assay demonstrated minimal value. Conclusions and Implications The diagnostic and prognostic value of hERG, APD, and QTc assays varies, with drug concentrations strongly affecting translational performance. While useful in guiding preclinical candidates devoid of clinical QT prolongation, the hERG and QTc repolarization assays provide greater value compared to the APD assay.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

Introduction The importance of drug-induced effects on the inotropic state of the heart is well known. Unlike hemodynamic and cardiac electrophysiological methods, which have been routinely used in drug safety testing for years, the non-clinical assessment of drug effects on myocardial contractility is used less frequently with no established translation to humans. The goal of these studies was to determine whether assessment of alternate measures of cardiac inotropy could detect drug-induced changes in the contractile state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. This study also evaluated drug-induced effects on lusitropy (relaxation) parameters of the heart. Methods A double 4 × 4 Latin square study design using Beagle dogs (n = 8) was conducted. Drugs were administrated orally. Arterial blood pressure (BP), left ventricular pressure (LVP) and the electrocardiogram (ECG) were assessed across different laboratories using the same protocol. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control). Animals were instrumented with an ITS telemetry system or DSI's D70-PCTP or PhysioTel™ Digital system. The data acquisition and analysis systems used were Ponemah, Notocord or EMKA. Results The derived inotropic and lusitropic parameters evaluated included peak systolic and end diastolic LVP, LVdP/dtmax, LVdP/dt40, QA interval, LVdP/dtmin and Tau. This study showed that LVdP/dt40 provided essentially identical results to LVdP/dtmax qualifying it as an index to assess drug effects on cardiac contractility. LVdP/dt40 provided an essentially identical assessment to that of LVdP/dtmax. The QA interval did not react sensitively to the drugs tested in this study; however, it did detect large effects and could be useful in early cardiovascular safety studies. The lusitropic parameter, LVdP/dtmin, was modestly decreased, and Tau was increased, by atenolol and itraconazole. At the doses tested, amrinone and pimobendan produced no changes in LVdP/dtmin while Tau was modestly increased. The drugs did not produce effects on BP, HR or the ECG at the doses tested. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. Discussion These findings indicate that this experimental model can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems. While LVdP/dt40 produced responses similar to LVdP/dtmax, the QA interval and lusitropic parameters LVdP/dtmin and Tau were not markedly changed at the dose of drugs tested. Further studies with drugs that affect early diastolic relaxation through calcium handling are needed to better evaluate drug-induced changes on lusitropic properties of the heart.

The goal of this research consortium including Janssen, MSD, Ncardia, FNCR/LBR, and Health and Environmental Sciences Institute (HESI) was to evaluate the utility of an additional in vitro assay technology to detect potential drug-induced long QT and torsade de pointes (TdP) risk by monitoring cytosolic free Ca2+ transients in human stem-cell-derived cardiomyocytes (hSC-CMs). The potential proarrhythmic risks of the 28 comprehensive in vitro proarrhythmia assay (CiPA) drugs linked to low, intermediate, and high clinical TdP risk were evaluated in a blinded manner using Ca2+-sensitive fluorescent dye assay recorded from a kinetic plate reader system (Hamamatsu FDSS/µCell and FDSS7000) in 2D cultures of 2 commercially available hSC-CM lines (Cor.4U and CDI iCell Cardiomyocytes) at 3 different test sites. The Ca2+ transient assay, performed at the 3 sites using the 2 different hSC-CMs lines, correctly detected potential drug-induced QT prolongation among the 28 CiPA drugs and detected cellular arrhythmias-like/early afterdepolarization in 7 of 8 high TdP-risk drugs (87.5%), 6 of 11 intermediate TdP-risk drugs (54.5%), and 0 of 9 low/no TdP-risk drugs (0%). The results were comparable among the 3 sites and from 2 hSC-CM cell lines. The Ca2+ transient assay can serve as a user-friendly and higher throughput alternative to complement the microelectrode array and voltage-sensing optical action potential recording assays used in the HESI-CiPA study for in vitro assessment of drug-induced long QT and TdP risk.

Contractility of the myocardium engines the pumping function of the heart and is enabled by the collective contractile activity of its muscle cells: cardiomyocytes. The effects of drugs on the contractility of human cardiomyocytes in vitro can provide mechanistic insight that can support the prediction of clinical cardiac drug effects early in drug development. Cardiomyocytes differentiated from human-induced pluripotent stem cells have high potential for overcoming the current limitations of contractility assays because they attach easily to extracellular materials and last long in culture, while having human- and patient-specific properties. Under these conditions, contractility measurements can be non-destructive and minimally invasive, which allow assaying sub-chronic effects of drugs. For this purpose, the function of cardiomyocytes in vitro must reflect physiological settings, which is not observed in cultured cardiomyocytes derived from induced pluripotent stem cells because of the fetal-like properties of their contractile machinery. Primary cardiomyocytes or tissues of human origin fully represent physiological cellular properties, but are not easily available, do not last long in culture, and do not attach easily to force sensors or mechanical actuators. Microengineered cellular systems with a more mature contractile function have been developed in the last 5 years to overcome this limitation of stem cell–derived cardiomyocytes, while simultaneously measuring contractile endpoints with integrated force sensors/actuators and image-based techniques. Known effects of engineered microenvironments on the maturity of cardiomyocyte contractility have also been discovered in the development of these systems. Based on these discoveries, we review here design criteria of microengineered platforms of cardiomyocytes derived from pluripotent stem cells for measuring contractility with higher physiological relevance. These criteria involve the use of electromechanical, chemical and morphological cues, co-culture of different cell types, and three-dimensional cellular microenvironments. We further discuss the use and the current challenges for developing and improving these novel technologies for predicting clinical effects of drugs based on contractility measurements with cardiomyocytes differentiated from induced pluripotent stem cells. Future research should establish contexts of use in drug development for novel contractility assays with stem cell–derived cardiomyocytes.

The implementation of the ICH S7B and E14 guidelines has been successful in preventing the introduction of potentially torsadogenic drugs to the market, but it has also unduly constrained drug development by focusing on hERG block and QT prolongation as essential determinants of proarrhythmia risk. The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative was established to develop a new paradigm for assessing proarrhythmic risk, building on the emergence of new technologies and an expanded understanding of torsadogenic mechanisms beyond hERG block. An international multi-disciplinary team of regulatory, industry and academic scientists are working together to develop and validate a set of predominantly nonclinical assays and methods that eliminate the need for the thorough-QT study and enable a more precise prediction of clinical proarrhythmia risk. The CiPA effort is led by a Steering Team that provides guidance, expertise and oversight to the various working groups and includes partners from US FDA, HESI, CSRC, SPS, EMA, Health Canada, Japan NIHS, and PMDA. The working groups address the three pillars of CiPA that evaluate drug effects on: 1) human ventricular ionic channel currents in heterologous expression systems, 2) in silico integration of cellular electrophysiologic effects based on ionic current effects, the ion channel effects, and 3) fully integrated biological systems (stem-cell-derived cardiac myocytes and the human ECG). This article provides an update on the progress of the initiative towards its target date of December 2017 for completing validation.

Introduction Alterations in cardiac contractility can have significant clinical implications, highlighting the need for early detection of potential liabilities. Pre-clinical methods to assess contractility are typically invasive and their translation to human measures of cardiac function are not well defined. Clinically, cardiac function is most often measured non-invasively using echocardiography. The objective of these studies was to introduce echocardiography into standard large animal cardiovascular safety pharmacology studies and determine the feasibility of this combination. Methods A consortia of laboratories combined their data sets for evaluation. At each site, telemetered beagle dogs, in a 4 × 4 Latin square crossover study design (n = 4), were administered either pimobendan (positive inotrope) or atenolol (negative inotrope) orally at clinically relevant dose levels. Standard telemetry parameters were collected (heart rate, mean arterial blood pressure, etc.) continuously over 24 h, as well as derived contractility endpoints: QA interval and LV +dP/dtmax. At Tmax, echocardiography was performed in conscious dogs with minimal restraint to collect contractility parameters: ejection fraction (EF) and fractional shortening (FS). Results Correlations between telemetry and echo contractility endpoints showed that, in general, a change in LV +dP/dtmax of 1000 mmHg/s translates to a 5.2% change in EF and a 4.2% change in FS. Poor correlations were shown between QA interval derived simultaneously, to both EF and FS. Discussion Comparing data from telemetry-only groups to those that included echocardiography collections showed no effect in the ability to interpret test article-related effects, providing the foundation for the inclusion of echocardiography without compromising standard telemetry data quality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Drug-induced effects on cardiac contractility can be assessed through the measurement of the maximal rate of pressure increase in the left ventricle (LVdP/dtmax) in conscious animals, and such studies are often conducted at the late stage of preclinical drug development. Detection of such effects earlier in drug research using simpler, in vitro test systems would be a valuable addition to our strategies for identifying the best possible drug development candidates. Thus, testing platforms with reasonably high throughput, and affordable costs would be helpful for early screening purposes. There may also be utility for testing platforms that provide mechanistic information about how a given drug affects cardiac contractility. Finally, there could be in vitro testing platforms that could ultimately contribute to the regulatory safety package of a new drug. The characteristics needed for a successful cell or tissue-based testing platform for cardiac contractility will be dictated by its intended use. In this article, general considerations are presented with the intent of guiding the development of new testing platforms that will find utility in drug research and development. In the following article (part 2), specific aspects of using human-induced stem cell-derived cardiomyocytes for this purpose are addressed.

SUMMARY To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ~0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSCCMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm., In Brief Blinova et al. tested human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for improving torsades de pointes arrhythmia risk prediction of drugs in the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative. This validation study confirms their utility based on electrophysiologic responses to 28 blinded drugs, with minimal influence from cell lines, test sites, and electrophysiological platforms., Graphical Abstract

Introduction Drug-induced effects on the cardiovascular system remain a major cause of drug attrition. While hemodynamic (blood pressure (BP) and heart rate (HR)) and electrophysiological methods have been used in testing drug safety for years, animal models for assessing myocardial contractility are used less frequently and their translation to humans has not been established. The goal of these studies was to determine whether assessment of contractility and hemodynamics, when measured across different laboratories using the same protocol, could consistently detect drug-induced changes in the inotropic state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. Methods A 4 × 4 double Latin square design (n = 8) design using Beagle dogs was developed. Drugs were administrated orally. Arterial blood pressure, left ventricular pressure (LVP) and the electrocardiogram were assessed. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope) (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control. Animals were instrumented with an ITS telemetry system, DSI's D70-PCTP system or DSI's Physiotel Digital system. Data acquisition and analysis systems were Ponemah, Notocord or EMKA. Results Derived parameters included: diastolic, systolic and mean arterial BP, peak systolic LVP, HR, end-diastolic LVP, and LVdP/dt max as the primary contractility index. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. Across the laboratories, a consistent change in LVdP/dt max was captured despite some differences in the absolute values of some of the hemodynamic parameters prior to treatment. Discussion These findings indicate that this experimental model, using the chronically instrumented conscious dog, can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems, and that data obtained in this model may also translate to clinical outcomes.

Background: There is a recognized need to improve the application of epidemiologic data in human health risk assessment especially for understanding and characterizing risks from environmental and occupational exposures. Although there is uncertainty associated with the results of most epidemiologic studies, techniques exist to characterize uncertainty that can be applied to improve weight-of-evidence evaluations and risk characterization efforts. Methods: This report derives from a Health and Environmental Sciences Institute (HESI) workshop held in Research Triangle Park, North Carolina, to discuss the utility of using epidemiologic data in risk assessments, including the use of advanced analytic methods to address sources of uncertainty. Epidemiologists, toxicologists, and risk assessors from academia, government, and industry convened to discuss uncertainty, exposure assessment, and application of analytic methods to address these challenges. Synthesis: Several recommendations emerged to help improve the utility of epidemiologic data in risk assessment. For example, improved characterization of uncertainty is needed to allow risk assessors to quantitatively assess potential sources of bias. Data are needed to facilitate this quantitative analysis, and interdisciplinary approaches will help ensure that sufficient information is collected for a thorough uncertainty evaluation. Advanced analytic methods and tools such as directed acyclic graphs (DAGs) and Bayesian statistical techniques can provide important insights and support interpretation of epidemiologic data. Conclusions: The discussions and recommendations from this workshop demonstrate that there are practical steps that the scientific community can adopt to strengthen epidemiologic data for decision making. Citation: Burns CJ, Wright JM, Pierson JB, Bateson TF, Burstyn I, Goldstein DA, Klaunig JE, Luben TJ, Mihlan G, Ritter L, Schnatter AR, Symons JM, Yi KD. 2014. Evaluating uncertainty to strengthen epidemiologic data for use in human health risk assessments. Environ Health Perspect 122:1160–1165; http://dx.doi.org/10.1289/ehp.1308062

Introduction:\ud \ud Voltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays.\ud \ud Methods:\ud \ud Three commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites.\ud \ud Results:\ud \ud Baseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition.\ud \ud Discussion:\ud \ud In conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses.

Systemic inflammation co-activates coagulation, which unchecked culminates in a lethal syndrome of multi-organ microvascular thrombosis known as disseminated intravascular coagulation (DIC). We studied an endotoxin-induced inflammatory state in rats to identify biomarkers of hemostatic imbalance favoring hypercoagulability. Intraperitoneal injection of LPS at 15 mg/kg body weight resulted in peripheral leukopenia and widespread neutrophilic sequestration characteristic of an acute systemic inflammatory response. Early indicators of hemostatic pathway activation developed within 4 hours, including increased circulating concentrations of procoagulant extracellular vesicles (EVs), EVs expressing endothelial cell and platelet membrane markers, and high concentration of soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1), and D-dimers. Inflammation persisted throughout the 48-hour observation period; however, increases were found in a subset of serum microRNA (miRNA) that coincided with gradual resolution of hemostatic protein abnormalities and reduction in EV counts. Dose-adjusted LPS treatment in rats provides a time-course model to develop biomarker profiles reflecting procoagulant imbalance and rebalance under inflammatory conditions.

Introduction : The evaluation of cardiovascular side-effects is a critical element in the development of all new drugs and chemicals. Cardiac safety issues are a major cause of attrition and withdrawal due to adverse drug reactions (ADRs) in pharmaceutical drug development. Methods : The evolution of the HESI Technical Committee on Cardiac Safety from 2000-2013 is presented as an example of an effective international consortium of academic, government, and industry scientists working to improve cardiac safety. Results and Discussion : The HESI Technical Committee Working Groups facilitated the development of a variety of platforms for resource sharing and communication among experts that led to innovative strategies for improved drug safety. The positive impacts arising from these Working Groups are described in this article.

Objective: To document the ability of public health laboratories to respond to radiological emergencies.Methods: The Association of Public Health Laboratories developed, distributed, and analyzed two separate surveys of public health laboratories representing the 50 US states and major nonstate jurisdictions. The 2009 All-Hazards Laboratory Preparedness Survey examined overall laboratory capability and capacity, with a subset of questions on radiation preparedness. A 2011 survey focused exclusively on radiation readiness.Results: The 50 state and District of Columbia public health laboratories responded to the 2009 All-Hazards Laboratory Preparedness Survey, representing a 98% response rate. In addition to the above laboratories, environmental and agricultural laboratories responded to the 2011 Radiation Capabilities Survey, representing a 76% response rate. Twenty-seven percent of the All-Hazards Survey respondents reported the ability to measure radionuclides in clinical specimens; 6% reported that another state agency or department accepted and analyzed these samples via a radioanalytical method. Of the Radiation Capabilities Survey respondents, 60% reported the ability to test environmental samples, such as air, soil, or surface water, for radiation; 48% reported the ability to test nonmilk food samples; 47% reported the ability to test milk; and 56% reported sending data for drinking water to the Environmental Protection Agency.Conclusions: Survey data reveal serious gaps in US radiological preparedness. In 2007, federal experts estimated it would take more than 4 years to screen 100 000 individuals for radiation exposure and 6 years to test environmental samples from a large-scale radiological emergency, relying on existing laboratory assets. Although some progress has been made since 2007, public health laboratory radiological test capabilities and capacities remain insufficient to respond to a major event. Adequate preparation requires significant new investment to build and enhance laboratory emergency response networks, as well as investments in the broader public health system in which public health laboratories function.(Disaster Med Public Health Preparedness. 2011;5:213-217)