Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells

Abstract

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 IC 50 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 [i Kr ], hCav1.2 [L-Type I ca ], peak hNav1.5, [Peak I na ], late hNav1.5 [Late I na ]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC 50 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. The characterization of drug effects on human currents using automated patch clamp (APC) platforms employing single-or multi-hole planar patch techniques 1-5 has revolutionized the assessment of electrophysiologic (and potential proarrhythmic) effects of new drug candidates. This increasingly popular experimental approach

Time for a Fully Integrated Nonclinical–Clinical Risk Assessment to Streamline QT Prolongation Liability Determinations: A Pharma Industry Perspective

“…Both approaches provide a more comprehensive integrated assessment of the potential of a drug to affect myocyte repolarization mechanistically ( in silico ) or phenotypically (human induced pluripotent stem cell derived cardiomyocyte). Progress has been gradual with these models, and best‐practice approaches are presently being described and defined, 34,35 so the Q&A process will need to define principles for validating these new assays such that all stakeholders have confidence in the quality and robustness of the models. Similarly, there may be a need to reevaluate other in vitro and in vivo proarrhythmia models, e.g., ventricular wedge, atrioventricular blockade in nonrodents, methoxamine‐induced TdP in rabbits, and electromechanical window, given their proposed predictive safety value and use by some pharmaceutical companies 36–40 .…”

“…12,13,57 This could contribute to inconsistency in study execution or the interpretation of findings, important issues that emerging drug sponsors (e.g., new or small companies) may encounter without having significant experience using S7B assays. The lack of consensus in hERG assay conduct is likely the primary reason for variance in potency estimates, i.e., IC 50 , 12,35,45,59,60 which confounds the robustness of hERG-based safety margin calculations. Similarly, the absence of a standard in methodologies and protocols for in vivo QTc assays, lack of validation information, QTc sensitivity estimates, etc., make it difficult to assess the overall "quality" of the study data and may reduce the value of in vivo QTc data for human safety assessment.…”

Chloroquine and hydroxychloroquine provoke arrhythmias at concentrations higher than those clinically used to treat COVID‐19: A simulation study

“…This article is protected by copyright. All rights reserved Copenhagen, Denmark) and using the cell lines, assay buffers, and voltage protocols that were described in previous studies 12,16 . Because it was not possible to monitor the drug concentration in the experimental chamber with the QPatch system, drug concentration was instead verified in the stock solution.…”

“…For the accurate evaluation of arrhythmogenic risk, data should be collected at a physiological temperature. However, due to the difficulty of experiments at physiological temperature with an automated patch system, assays were performed at room temperature (25°C) 16 .…”

Ion Channels and Relevant Drug Screening Approaches

“…Historically a laborious, manual, low-throughput technique where single cells are studied one at a time, the last couple of decades have seen the emergence and evolution of automated, 384-well plate-based electrophysiology platforms ranging from the perforated-patch-based IonWorks Barracuda (Molecular Devices, Sunnyvale, CA) to the latest-generation, giga-seal-based instruments such as Qube (Sophion Bioscience, Copenhagen, Denmark) and SyncroPatch (Nanion Technologies, Munich, Germany). 6 These whole-cell patch-clamp platforms offer much higher throughput than manual methods and are capable of recording currents from populations of individual cells, which increases the likelihood of obtaining useful data from each well of an assay plate. They have been used in screening campaigns for libraries approaching 200,000 compounds.…”