ABSTRACT
Risk stratification in the context of sudden cardiac death has been acknowledged as one of the major challenges facing cardiology for the past four decades. In recent years, the advent of high performance computing has facilitated organ-level simulation of the heart, meaning we can now examine the causes, mechanisms and impact of cardiac dysfunction in silico. As a result, computational cardiology, largely driven by the Physiome project, now stands at the threshold of clinical utility in regards to risk stratification and treatment of patients at risk of sudden cardiac death. In this white paper, we outline a roadmap of what needs to be done to make this translational step, using the relatively well-developed case of acquired or drug-induced long QT syndrome as an exemplar case.
Subject(s)
Arrhythmias, Cardiac/chemically induced , Arrhythmias, Cardiac/complications , Death, Sudden, Cardiac/etiology , Drug-Related Side Effects and Adverse Reactions , Models, Cardiovascular , Animals , Cardiology/methods , Computer Simulation , Heart/physiopathology , Humans , RiskABSTRACT
In February 2014, a group of scientists convened as part of the University of California Davis Cardiovascular Symposium to bring together experimental and mathematical modelling perspectives and discuss points of consensus and controversy on the topic of sodium in the heart. This paper summarizes the topics of presentation and discussion from the symposium, with a focus on the role of aberrant sodium channels and abnormal sodium homeostasis in cardiac arrhythmias and pharmacotherapy from the subcellular scale to the whole heart. Two following papers focus on Na(+) channel structure, function and regulation, and Na(+)/Ca(2+) exchange and Na(+)/K(+) ATPase. The UC Davis Cardiovascular Symposium is a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The focus on Na(+) in the 2014 symposium stemmed from the multitude of recent studies that point to the importance of maintaining Na(+) homeostasis in the heart, as disruption of homeostatic processes are increasingly identified in cardiac disease states. Understanding how disruption in cardiac Na(+)-based processes leads to derangement in multiple cardiac components at the level of the cell and to then connect these perturbations to emergent behaviour in the heart to cause disease is a critical area of research. The ubiquity of disruption of Na(+) channels and Na(+) homeostasis in cardiac disorders of excitability and mechanics emphasizes the importance of a fundamental understanding of the associated mechanisms and disease processes to ultimately reveal new targets for human therapy.
Subject(s)
Brugada Syndrome/metabolism , Heart Arrest/metabolism , Sodium/metabolism , Animals , Brugada Syndrome/physiopathology , Congresses as Topic , Heart Arrest/physiopathology , HumansABSTRACT
There is no refuting that America's population is growing older: for the first time in US history, by 2034 older adults (defined as >65 years of age) are projected to outnumber children under the age of 18, representing approximately 70 million people or almost 25% of the population (Lloyd-Jones et al., 2010). Described as the "silver tsunami", this flood of older adults is driven by the baby boomers (people born after World War II, from 1946 to 1964): they are now reaching old age, living longer due to significant advances in healthcare coupled with a record low birth rate, resulting in a skewed elderly population demographic. Unfortunately, older adults are also becoming increasingly unhealthy. Many often suffer from several chronic disorders requiring the use of multiple medications at a level higher than any other age group, resulting in an increased risk of drug-drug interactions (DDIs) and adverse drug reactions (ADRs). Indeed, because of age-related changes in pharmacokinetics (PK) and pharmacodynamics (PD), older adults are also more vulnerable to drug toxicity. Prescribed drugs certainly improve a range of health outcomes, but also often cause considerable ADRs, leading to devastating consequences for patients, clinicians, and manufacturers. Therefore, safe and effective pharmacotherapy remains one of the greatest growing challenges in geriatric medicine. In this review we examine the effects of aging and its impact on the increased risk of experiencing ADRs, resulting in devastating consequences for patients and manufacturers. We assess the current regulatory considerations related to the development of drugs for this population and highlight issues, concerns, and propose alternatives to the standard battery of tests focused on assessing cardiovascular (CV) safety in an attempt to develop safer and efficient new drugs for the growing elderly demographic.
Subject(s)
Aging , Drug-Related Side Effects and Adverse Reactions , Aged , Child , Drug Interactions , Drug-Related Side Effects and Adverse Reactions/epidemiology , HumansABSTRACT
There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch clamp method, considered the 'gold standard', for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.
Subject(s)
Long QT Syndrome , Ether-A-Go-Go Potassium Channels , Heart , High-Throughput Screening Assays , Humans , Ion Channels , Patch-Clamp TechniquesABSTRACT
There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch clamp method, considered the 'gold standard', for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.
Subject(s)
Long QT Syndrome , Torsades de Pointes , Heart , Humans , Ion Channels , Patch-Clamp TechniquesABSTRACT
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.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Traditional drug discovery is an inefficient process. Human pluripotent stem cell-derived cardiomyocytes can potentially fill the gap between animal and clinical studies, but conventional two-dimensional cultures inadequately recapitulate the human cardiac phenotype. Here, we systematically examined the pharmacological responses of engineered human ventricular-like cardiac tissue strips (hvCTS) and organoid chambers (hvCOC) to 25 cardioactive compounds covering various drug classes. While hvCTS effectively detected negative and null inotropic effects, the sensitivity to positive inotropes was modest. We further quantified the predictive capacity of hvCTS in a blinded screening, with accuracies for negative, positive, and null inotropic effects at 100%, 86%, and 80%, respectively. Interestingly, hvCOC, with a pro-maturation milieu that yields physiologically complex parameters, displayed enhanced positive inotropy. Based on these results, we propose a two-tiered screening system for avoiding false positives and negatives. Such an approach would facilitate drug discovery by leading to better overall success.
Subject(s)
Cardiotonic Agents/pharmacology , Myocardial Contraction/drug effects , Myocytes, Cardiac , Organoids , Cardiovascular Agents/pharmacology , Cells, Cultured , Depression, Chemical , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Humans , Induced Pluripotent Stem Cells , Models, Cardiovascular , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Organoids/drug effects , Organoids/physiology , Stimulation, Chemical , Tissue Engineering/methodsABSTRACT
The cardiac action potential (AP) is vital for understanding healthy and diseased cardiac biology and drug safety testing. However, techniques for high throughput cardiac AP measurements have been limited. Here, we introduce a novel technique for reliably increasing the coupling of cardiomyocyte syncytium to planar multiwell microelectrode arrays, resulting in a stable, label-free local extracellular action potential (LEAP). We characterized the reliability and stability of LEAP, its relationship to the field potential, and its efficacy for quantifying AP morphology of human induced pluripotent stem cell derived and primary rodent cardiomyocytes. Rise time, action potential duration, beat period, and triangulation were used to quantify compound responses and AP morphology changes induced by genetic modification. LEAP is the first high throughput, non-invasive, label-free, stable method to capture AP morphology from an intact cardiomyocyte syncytium. LEAP can accelerate our understanding of stem cell models, while improving the automation and accuracy of drug testing.
Subject(s)
Action Potentials/physiology , Heart/physiology , Microelectrodes , Animals , Animals, Newborn , Electroporation , Humans , Induced Pluripotent Stem Cells/cytology , Ion Channels/antagonists & inhibitors , Ion Channels/metabolism , Myocytes, Cardiac/physiology , Rats , Signal Processing, Computer-Assisted , Time FactorsABSTRACT
The pharmaceutical industry is facing unprecedented challenges as the cost of developing new drugs has reached unsustainable levels, fueled in large parts by a high attrition rate in clinical development. Strategies to bridge studies between preclinical testing and clinical trials are needed to reduce the knowledge gap and allow earlier decisions to be made on the continuation or discontinuation of further development of drugs. The discovery and development of human induced pluripotent stem cells (hiPSCs) have opened up new avenues that support the concept of screening for cell-based safety and toxicity at the level of a population. This approach, termed "Clinical Trials in a Dish" (CTiD), allows testing medical therapies for safety or efficacy on cells collected from a representative sample of human patients, before moving into actual clinical trials. It can be applied to the development of drugs for specific populations, and it allows predicting not only the magnitude of effects but also the incidence of patients in a population who will benefit or be harmed by these drugs. This, in turn, can lead to the selection of safer drugs to move into clinical development, resulting in a reduction in attrition. The current article offers a perspective of this new model for "humanized" preclinical drug development.
Subject(s)
Clinical Trials as Topic , Drug Development , Induced Pluripotent Stem Cells , Stem Cell Transplantation , Animals , Cell Differentiation , Clinical Trials as Topic/standards , Drug Interactions , Drug Repositioning , Health Planning Guidelines , Humans , Research Design , Stem Cell Transplantation/adverse effects , Stem Cell Transplantation/methodsABSTRACT
The ever-increasing cost of drug discovery and development represents a significant challenge for the pharmaceutical industry and new strategies to bridge studies between preclinical testing and clinical trials are needed to reduce the knowledge gap prior to first human exposures, and to allow earlier decisions to be made on the further development of drugs. A number of studies have demonstrated that various cell types differentiated from human induced pluripotent stem cells (iPSCs) do not just respond similarly to human tissues in general, but rather recapitulate the drug response of their specific donor's, when exposed to the same drug in vivo. This recapitulation opens the doors to Clinical Trials in a Dish (CTiD), a platform which involves testing, in vitro, medical therapies for safety on cells collected from a sample of human patients, before moving into clinical trials. However, the science behind CTiD is complex, and every element of the process from tissue acquisition to data generation must be assessed and designed to meet quality metrics and standards. Without such rigorous assessment and design, the basic scientific integrity of CTiD constructs is likely compromised, and the results questionable. Given the lack of standard process and/or quality metrics in place for the use of stem cell-based products for in vitro testing per se, we discuss here the key elements that one needs to consider when designing, implementing and executing CTiD studies, in order to ensure an approach that will reliably mimic clinical trials, and allow obtaining reproducible and reliable experimental data.
Subject(s)
Clinical Trials as Topic/methods , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Induced Pluripotent Stem Cells/drug effects , Cell Differentiation/drug effects , Humans , Induced Pluripotent Stem Cells/cytologyABSTRACT
INTRODUCTION: Cardiac sodium channel antagonists have historically been used to treat cardiac arrhythmias by preventing the reentry of the electrical impulse that could occur following myocardial damage. However, clinical studies have highlighted a significant increase in mortality associated with such treatment. Cardiac sodium channel antagonist activity is now seen as an off-target pharmacology that should be mitigated during the drug development process. The aim of this study was to examine the correlation between in vitro/ex vivo assays that are routinely used to measure Nav1.5 activity and determine the translatability of the individual assays to QRS prolongation in the clinic. METHODS: A set of clinical compounds with known Nav1.5 activity was profiled in several in vitro/ex vivo assays (binding, membrane potential, patch clamp and the Langendorff isolated heart). Clinical data comprising compound exposure levels and changes in QRS interval were obtained from the literature. Sensitivity/specificity analysis was performed with respect to the clinical outcome. RESULTS: The in vitro assays showed utility in predicting QRS prolongation in the clinic. Optimal thresholds were defined for each assay (binding: IC20; membrane potential: IC10; patch clamp: IC20) and sensitivity (69-88%) and specificity (53-84%) values were shown to be similar between assay formats. DISCUSSION: The data provide clear statistical insight into the translatability of Nav1.5 antagonism data generated in vitro to potential clinical outcomes. These results improve our ability to understand the liability posed by such activity in novel development compounds at an early stage.
Subject(s)
Arrhythmias, Cardiac/drug therapy , Myocardial Contraction/drug effects , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Voltage-Gated Sodium Channel Blockers/pharmacology , Animals , Arrhythmias, Cardiac/diagnosis , CHO Cells , Cricetinae , Cricetulus , Dogs , Drug Evaluation, Preclinical/methods , Electrocardiography , Guinea Pigs , Heart/drug effects , Heart/physiology , Humans , Male , Sensitivity and Specificity , Voltage-Gated Sodium Channel Blockers/therapeutic useABSTRACT
During the past decade, a number of non-cardiovascular drugs have had their label revised or have been withdrawn from the market because of unexpected post-marketing reports of sudden cardiac death associated with a prolongation of the QT interval, and an increased propensity to develop a ventricular tachyarrhythmia called Torsades de Pointes. Although a direct link between QT interval prolongation and arrhythmogenesis is still unclear, QT prolongation is now the subject of increased regulatory review and is considered a significant risk factor for predicting human safety of New Chemical Entities. Consequently, pharmaceutical companies are striving to improve the drug discovery and development process to identify, as early as possible, the risk of novel agents, or their metabolites, of causing QT interval prolongation and to make appropriate go/no-go decisions or modify their development programme accordingly.
Subject(s)
Cardiovascular Agents/adverse effects , Long QT Syndrome/chemically induced , Technology, Pharmaceutical/methods , Animals , Cardiovascular Agents/therapeutic use , Humans , Long QT Syndrome/physiopathology , Technology, Pharmaceutical/economics , Technology, Pharmaceutical/legislation & jurisprudenceABSTRACT
Nonclinical studies of drug effects with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide new possibilities for evaluating drug safety and efficacy. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm provides lessons from the cardiac field that also apply to drug studies with other stem cell-based assays.
Subject(s)
Arrhythmias, Cardiac/drug therapy , Arrhythmias, Cardiac/metabolism , Drug Evaluation, Preclinical/methods , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Arrhythmias, Cardiac/pathology , Humans , Induced Pluripotent Stem Cells/pathology , Myocytes, Cardiac/pathologyABSTRACT
Voltage gated ion channels are central in defining the fundamental properties of the ventricular cardiac action potential (AP), and are also involved in the development of drug-induced arrhythmias. Many drugs can inhibit cardiac ion currents, including the Na+ current (INa), L-type Ca2+ current (Ica-L), and K+ currents (Ito, IK1, IKs, and IKr), and thereby affect AP properties in a manner that can trigger or sustain cardiac arrhythmias. Since publication of ICH E14 and S7B over a decade ago, there has been a focus on drug effects on QT prolongation clinically, and on the rapidly activating delayed rectifier current (IKr), nonclinically, for evaluation of proarrhythmic risk. This focus on QT interval prolongation and a single ionic current likely impacted negatively some drugs that lack proarrhythmic liability in humans. To rectify this issue, the Comprehensive in vitro proarrhythmia assay (CiPA) initiative has been proposed to integrate drug effects on multiple cardiac ionic currents with in silico modelling of human ventricular action potentials, and in vitro data obtained from human stem cell-derived ventricular cardiomyocytes to estimate proarrhythmic risk of new drugs with improved accuracy. In this review, we present the physiological functions and the molecular basis of major cardiac ion channels that contribute to the ventricle AP, and discuss the CiPA paradigm in drug development.
Subject(s)
Arrhythmias, Cardiac/chemically induced , Arrhythmias, Cardiac/physiopathology , Cardiotoxins/pharmacology , Ion Channels/physiology , Pharmacology/methods , Animals , Cardiotoxins/adverse effects , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/standards , Heart Conduction System/drug effects , Heart Conduction System/physiology , Humans , Ion Channels/agonists , Ion Channels/antagonists & inhibitors , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Pharmacology/standardsABSTRACT
INTRODUCTION: The Comprehensive in vitro Proarrhythmic Assay (CiPA) aims to update current cardiac safety testing to better evaluate arrhythmic risk. A central theme of CiPA is the use of in silico approaches to risk prediction incorporating models of drug binding to hERG. To parameterize these models, accurate in vitro measurement of potency and kinetics of block is required. The Ion Channel Working Group was tasked with: i) selecting a protocol that could measure kinetics of block and was easily implementable on automated platforms for future rollout in industry and ii) acquiring a reference dataset using the standardized protocol. METHODS: Data were acquired using a 'step depolarisation' protocol using manual patch-clamp at ambient temperature. RESULTS: Potency, kinetics and trapping characteristics of hERG block for the CiPA training panel of twelve drugs were measured. Timecourse of block and trapping characteristics could be reliably measured if the time constant for onset of block was between ~500ms and ~15s. Seven drugs, however had time courses of block faster than this cut-off. DISCUSSION: Here we describe the implementation of the standardized protocol for measurement of kinetics and potency of hERG block for CiPA. The results highlight the challenges in identifying a single protocol to measure hERG block over a range of kinetics. The dataset from this study is being used by the In Silico Working Group to develop models of drug binding for risk prediction and is freely available as a 'gold standard' ambient temperature dataset to evaluate variability across high throughput platforms.
Subject(s)
Anti-Arrhythmia Agents/pharmacokinetics , Arrhythmias, Cardiac/physiopathology , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/physiology , Potassium Channel Blockers/pharmacokinetics , Animals , Anti-Arrhythmia Agents/adverse effects , Arrhythmias, Cardiac/chemically induced , CHO Cells , Cricetinae , Cricetulus , Kinetics , Potassium Channel Blockers/adverse effectsABSTRACT
INTRODUCTION: Although therapeutically beneficial in the treatment of certain diseases, L-type calcium channel antagonism can result in unwanted off-target pharmacology leading to adverse drug reactions and to the termination of the development of otherwise promising compounds. In the present study three marketed calcium channel inhibitors, nifedipine, verapamil and diltiazem were profiled in a series of in vitro and ex-vivo assays in an effort to determine the ability of these assays to discriminate, between dihydropyridine versus non-dihydropyridine-like compounds, and how well they can predict the cardiovascular effects observed in a conscious telemetered rat model. METHODS: Standard calcium channel antagonists were profiled in radioligand binding, patch clamp and calcium flux assays. In addition, cardiovascular endpoints related to calcium channel activity were also examined in ex vivo tissue bath preparations, including relaxation of pre-constricted rat aorta and the guinea pig Langendorff isolated heart model. The data generated were correlated with in vivo blood pressure and heart rate data from conscious telemetered rats. RESULTS: Our results show that the binding, FLIPR and aorta assays allow differentiation of the compounds in two distinct classes of L-type calcium channel antagonists, and are good predictors of in vivo outcomes. DISCUSSION: These results suggest that in vitro and ex vivo profiling remains a valuable tool in predicting potential in vivo cardiovascular safety issues, and can aid in the selection of novel development compounds that show inherent inhibitory activity against L-type calcium channels.
Subject(s)
Calcium Channel Blockers/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/metabolism , Translational Research, Biomedical/methods , Animals , Blood Pressure/drug effects , Blood Pressure/physiology , Cell Line , Diltiazem/metabolism , Diltiazem/pharmacology , Dose-Response Relationship, Drug , Female , Guinea Pigs , Isolated Heart Preparation/methods , Male , Nifedipine/metabolism , Nifedipine/pharmacology , Rabbits , Rats , Rats, Wistar , Verapamil/metabolism , Verapamil/pharmacologyABSTRACT
INTRODUCTION: The Safety Pharmacology Society (SPS) has conducted a survey of its membership to identify industry practices related to testing considered in the Comprehensive In vitro Proarrhythmia Assay (CiPA). METHODS: Survey topics included nonclinical approaches to address proarrhythmia issues, conduct of in silico studies, in vitro ion channel testing methods used, drugs used as positive controls during the conduct of cardiac ion channel studies, types of arrhythmias observed in non-clinical studies and use of the anticipated CiPA ion channel assay. RESULTS: In silico studies were used to evaluate effects on ventricular action potentials by only 15% of responders. In vitro assays were used mostly to assess QT prolongation (95%), cardiac Ca2+ and Na+ channel blockade (82%) and QT shortening or QRS prolongation (53%). For de-risking of candidate drugs for proarrhythmia, those assays most relevant to CiPA including cell lines stably expressing ion channels used to determine potency of drug block were most frequently used (89%) and human stem cell-derived or induced pluripotent stem cell cardiomyocytes (46%). Those in vivo assays related to general proarrhythmia derisking include ECG recording using implanted telemetry technology (88%), jacketed external telemetry (62%) and anesthetized animal models (53%). While the CiPA initiative was supported by 92% of responders, there may be some disconnect between current practice and future expectations, as explained. DISCUSSION: Proarrhythmia liability assessment in drug development presently includes study types consistent with CiPA. It is anticipated that CiPA will develop into a workable solution to the concern that proarrhythmia liability testing remains suboptimal.
Subject(s)
Arrhythmias, Cardiac/chemically induced , Animals , Calcium Channel Blockers/pharmacology , Computer Simulation , Drug Industry , Electrocardiography/drug effects , Humans , Induced Pluripotent Stem Cells , Ion Channels , Long QT Syndrome/chemically induced , Myocytes, Cardiac/drug effects , Sodium Channel Blockers/pharmacology , Surveys and Questionnaires , TelemetryABSTRACT
Inhibition of the delayed-rectifier potassium channel current, human ether-a-go-go (hERG), by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias and sudden death. There remains an unmet need for higher-throughput assays to screen compounds in preclinical development for the potential to block hERG and cause QT prolongation. We evaluated the rubidium efflux assay for its ability to determine block of the hERG potassium channel. hERG-transfected human embryonic kidney-293 cells were cultured on 96-well assay plates and loaded with rubidium ion by incubating in media in which potassium was replaced by 5.4 mM Rb+. Cells were exposed to test compounds and then depolarized with a K+ channel opening buffer containing 50 mM K+. The supernatant was removed, and cells were lysed using 0.1% Triton X-100. Concentration-response curves were generated for test agents by determining the Rb+ efflux using a flame atomic absorption spectrometer. Multiple trials with cisapride yielded 50% inhibitory concentration values between 308.1 +/- 11 nM to 456.3 +/- 24 nM for inhibition of Rb+ efflux and a Z factor of 0.80 +/- 0.07 (n = 5 plates, 12 wells per plate). The values for inhibition of the hERG channel exhibited a rightward shift in potency as compared to those measured using electrophysiological techniques. In addition, we evaluated 19 blinded compounds at 10 microM in the Rb+ efflux assay, and compared results to those using patch clamp electrophysiology and the dofetilide displacement binding assay. The dofetilide displacement binding assay yielded a good correlation with electrophysiological measurements of hERG block. The rubidium efflux assay lacked sensitivity to consistently identify significant channel blockade. In conclusion, the rubidium efflux assay provides a higher-throughput means to identify potent hERG channel blocking agents, but lacks the sensitivity required to accurately determine the potency of blockade.
Subject(s)
Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/physiology , Rubidium/metabolism , Cell Line , Cell Membrane/drug effects , Cell Membrane/physiology , Electrophysiology/methods , Humans , Kidney , Kinetics , Phenethylamines/pharmacology , Sulfonamides/pharmacologyABSTRACT
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.