Supporting an integrated QTc risk assessment using the hERG margin distributions for three positive control agents derived from multiple laboratories and on multiple occasions.

BACKGROUND: Determination of a drug's potency in blocking the hERG channel is an established safety pharmacology study. Best practice guidelines have been published for reliable assessment of hERG potency. In addition, a set of plasma concentration and plasma protein binding fraction data were provided as denominators for margin calculations. The aims of the current analysis were five-fold: provide data allowing creation of consistent denominators for the hERG margin distributions of the key reference agents, explore the variation in hERG margins within and across laboratories, provide a hERG margin to 10 ms QTc prolongation based on several newer studies, provide information to use these analyses for reference purposes, and provide recommended hERG margin 'cut-off' values. METHODS: The analyses used 12 hERG IC50 'best practice' data sets (for the 3 reference agents). A group of 5 data sets came from a single laboratory. The other 7 data sets were collected by 6 different laboratories. RESULTS: The denominator exposure distributions were consistent with the ICH E14/S7B Training Materials. The inter-occasion and inter-laboratory variability in hERG IC50 values were comparable. Inter-drug differences were most important in determining the pooled margin variability. The combined data provided a robust hERG margin reference based on best practice guidelines and consistent exposure denominators. The sensitivity of hERG margin thresholds were consistent with the sensitivity described over the course of the last two decades. CONCLUSION: The current data provide further insight into the sensitivity of the 30-fold hERG margin 'cut-off' used for two decades. Using similar hERG assessments and these analyses, a future researcher can use a hERG margin threshold to support a negative QTc integrated risk assessment.

Test article concentrations in the hERG assay: losses through the perfusion, solubility and stability.

INTRODUCTION: Drug-induced prolongation of the electrocardiographic QT interval (long QT syndrome) has been associated with increased risk of a serious ventricular arrhythmia, torsade de pointes. Inhibition of hERG, a cardiac potassium channel that controls action potential repolarization, is the most common cause of QT prolongation by non-cardiac drugs. The ICH S7B describes preclinical safety testing required for new drugs, including the determination of the hERG IC(50). Actual and target concentrations may differ due to solubility, stability, or loss of compound. Significant differences will invalidate quantitative concentration-response curves which may be critical to interpretation of drug safety. To examine the frequency and significance of these differences, we conducted an analysis of studies where both the electrophysiology and the dose solution analysis were conducted in-house. We have investigated the actual concentrations of test article in vehicle solution as compared to the target concentrations in an attempt to determine the reasons behind differences between the two values. METHODS: Studies that involved both electrophysiology and dose solution analysis performed at ChanTest Corporation were evaluated. The effects of stability, solubility and loss through the perfusion apparatus on actual dosing concentrations were investigated. RESULTS: There was a large range in the loss of the test article attributed to the perfusion apparatus (range from 0 to 74% loss). For 12 of the 22 studies evaluated, the IC(50) was>2-fold more potent when using actual values as determined by HPLC versus the target concentrations. Twenty-two percent of the test articles were not stable 24 h after room temperature storage; 16% after 24 h frozen conditions. DISCUSSION: The best practices when considering dose solution concentration verification of test article solutions are to: determine the solubility of the compound in a physiological buffer, analyze samples collected from the perfusion chamber, and analyze samples the same day as sample collection (e.g., same day as hERG assay).

Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern.

INTRODUCTION: In vitro evaluation of drug effects on hERG K(+) channels is a valuable tool for identifying potential proarrhythmic side effects in drug safety testing. Patch-clamp recording of hERG K(+) current in mammalian cells can accurately evaluate drug effects, but the methodology has not been standardized, and results vary widely. Our objective was to evaluate two potential sources of variability: the temperature at which recordings are performed and the voltage pulse protocol used to activate hERG K(+) channels expressed in HEK293 cells. METHODS: A panel of 15 drugs that spanned a broad range of potency for hERG inhibition and pharmacological class was evaluated at both room and near-physiological temperatures using several patch-clamp voltage protocols. Concentration-response analysis was performed with three stimulus protocols: 0.5- and 2-s step pulses, or a step-ramp pattern. RESULTS: Block by 2 of the 15 drugs tested, d,l-sotalol (antiarrhythmic) and erythromycin (antibiotic), was markedly temperature sensitive. hERG inhibition measured using a 2-s step-pulse protocol underestimated erythromycin potency compared with results obtained with a step-ramp protocol. Using conservative acceptance criteria and the step-ramp protocol, the IC(50) values for hERG block differed by less than twofold for 15 drugs. DISCUSSION: Data obtained at near-physiological temperatures using a step-ramp pattern are highly repeatable and provide a conservative safety evaluation of hERG inhibition.