Categorizing TdP risk using hERG data obtained from automated patch-clamp using step-ramp hERG protocols and CiPA TMS values

Abstract

The Comprehensive in-vitro Proarrhythmia Assay (CiPA) assesses drug effects on multiple ionic currents and a detailed drug-binding model of HERG to measure pro-arrhythmic risk (TMS value). Adoption of TMS for IND filings is hampered by difficult experimental protocols requiring manual patch-clamp data. Objectives: 1) Assess hERG drug block on currents from a step-ramp protocol using the Sophion QPatch Compact. 2) Determine drug TdP risk categorization from the TMS using CyberQ-qNet analysis software. 3) Determine best practices for data acquisition, current trace data processing, and quality control. hERG currents were recorded at 36 °C using a step-ramp protocol on the Sophion QPatch Compact platform using hERG-expressing CHO cells. Control currents were recorded until hERG current stabilization. Test currents were measured by perfusion with a single test drug concentration until steady-state block. Cells were then perfused with a reference compound (E-4031) resulting in complete block of hERG. Drugs tested were dofetilide, ondansetron, and moxifloxacin. Current traces underwent manual quality control, leak subtraction and reference current trace subtraction. Cytocybernetic's CyberQ-qNET analysis software calculated the qNet and TMS values. Step ramp TMS scores were compared to those obtained by the FDA using the Milnes protocol. CyberQ-qNet analysis software fitted raw HERG data to the CiPA three-state drug block model. The resulting analysis incorporated published values for other currents to obtain TMS. Similar qNET, TMS, and TdP risk categorization were similar with the Step-Ramp protocol and the Milnes protocol: dofetilide (0.0609 vs 0.0524), ondansetron (0.0675 vs 0.0625). Ondansetron was categorized as intermediate risk, but dofetilide was miscategorized as intermediate (although borderline and likely due to small N for low doses). For moxifloxacin (not a CiPA compound), TMS was 0.0552 and was categorized as high-risk TdP. Model fitting was highly sensitive to data quality. One important quality control subtraction of the reference current traces (100% hERG block) to remove non-hERG (i.e. native CHO) currents present in the step pulse. This suggests hERG currents from the Sophion QPatch at physiological temperature using the Step-Ramp could be a viable strategy for assessing arrhythmogenic potential of drugs using our CyberQ-qNet software in compliance with CiPA.