Innovative CiPA Cardiac Assays for Proarrhythmia Safety

New cardiac safety testing guidelines are being finalised as part of the FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, which aims to remove the over-reliance on screening against the hERG channel by expanding the panel to include Na_v1.5, Ca_v1.2, K_v­4.3/KChiP2.2, K_ir2.1 and K_v7.1/KCNE1 human cardiac ion channels.  In addition, the CiPA working groups have recently identified two additional in vitro assays required for in silico models to reliably predict proarrhythmia. The first is a ‘late’ sodium current assay, as inhibition of persistent inward current can affect repolarisation and mitigate proarrhythmia (e.g. Ranolazine). The second assay determines the degree of drug trapping in the hERG channel using the Milnes voltage protocol (Milnes et al, 2010), which can improve the prediction of proarrhythmia risk (Li et al. 2018).

We have validated these additional cardiac assays on the gigaseal quality QPatch48 automated patch clamp platform. Rather than rely on pharmacological activators such as ATX-II or Veratridine to induce late openings of the Na_v1.5 channel, we created a HEK cell line expressing the LQT3 DKPQ hNav1.5 mutant which promotes activation of persistent sodium currents (Chandra, Starmer & Grant, 1998). Late sodium current was measured using step pulse, step-ramp, ramp, and action potential waveform voltage protocols in proprietary solutions to minimise rundown and increase whole-cell seal duration.

Late hNav1.5 DKPQ cells exhibited Ranolazine-sensitive late sustained inward currents that were ~1-3% magnitude of the peak current (>4 nA) in single hole Qplate recordings. A CHO cell line expressing the hERG  channel and published Milnes hERG kinetic protocols were optimised for the QPatch to create a biophysically and pharmacologically validated ‘dynamic’ hERG assay. We compensated for slower activation kinetics at room temperature (compared to manual patch at 37^oC) and optimised liquid addition and voltage protocols to create a stable assay during prolonged vehicle applications. Our QPatch Milnes hERG assay correctly reports the drug trapping profiles of Cisapride (10%) and Dofetilide (95%) as well as several other CiPA toolbox reference compounds.

We developed a late Na_v1.5 DKPQ mutant cell line and a Milnes dynamic hERG kinetic assay on a gigaseal quality screening platform that meet the additional requirements of the CiPA initiative, and can provide high quality HTS automated patch clamp data for more accurate cardiac safety assessment.