Reliable identification of cardiac conduction abnormalities in drug discovery using automated patch clamp II: Best practices for Nav1.5 peak current in a high throughput screening environment

Abstract

Introduction: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including Na_V1.5 and Ca_V1.2. We identified different parameters that might affect IC₅₀s of compounds on Na_V1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices.

Methods: APC instruments SyncroPatch 384 and Patchliner were used to record Na_V1.5 peak and late current. Up to 24 CiPA compounds were used to investigate effects of voltage protocol, holding potential (-80 mV or - 95 mV) and temperature (23 ± 1 °C or 36 ± 1 °C) on IC₅₀ values on hNa_V1.5 overexpressed in HEK or CHO cells either as frozen cells or running cultures.

Results: The IC₅₀s of 18 compounds on the Na_V1.5 peak current recorded on the SyncroPatch 384 using the CiPA step-ramp protocol correlated well with the literature. The use of frozen or cultured cells did not affect IC₅₀s but voltage protocol and holding potential did cause differences in IC₅₀ values. Temperature can affect V_half of inactivation and also compound potency. A compound incubation time of 5-6 min was sufficient for most compounds, however slow acting compounds such as terfenadine required longer to reach maximum effect.

Discussion: We conclude that holding potential, voltage protocol and temperature can affect IC₅₀ values and recommend the use of the CiPA step-ramp protocol at physiological temperature to record Na_V1.5 peak and late currents for cardiac safety. Further recommendations include: a minimum compound incubation time of 5 min, a replicate number of 4 and the use of positive and negative controls for reliable IC₅₀s.

Keywords: Automated patch clamp; Cardiovascular safety; Comprehensive in vitro Proarrhythmia assay (CiPA); HTS; Ion channels; Na(V)1.5; Proarrhythmic risk assessment; Safety assessment; Safety pharmacology.