Effects of temperature, compound concentration verification, and internal fluoride on hERG

Are automated electrophysiology platform (EP) data appropriately rigorous to be included in regulatory filings? Automated EP platforms have been in use for more than 20 years yet are still primarily used for initial screening of compounds against hERG and other ion channels. In this study we investigated the effects of 20 known compounds with a wide range of potencies against hERG. We conducted all experiments using SyncroPatch 384i and cryo-preserved assay-ready hERG cells. We compare results for temperature effects (25 °C vs. 35 °C) and the presence of fluoride in the internal solution. Verification of well compound concentrations via LC-MS/MS analysis were determined for all variations of the experiment. We used a standard 2-step voltage protocol (Vh = −80 mV, Vs = +20 mV for 5 s, Vt = −50 mV for 5 s, repeated once every 15 s for >5 min). At 25 °C, in the presence of internal fluoride, we saw a range of targeted IC₅₀s from <1 to >30 μM. When we corrected the data after verification of compound well concentrations, the range of IC₅₀s was now 0.1 μM to 38 μM. At 35 °C, in the presence of internal fluoride, the targeted range of IC₅₀s was <1 μM to >30 μM and with correction for verification of concentrations was 0.2 μM to >38 μM. We conclude that for this set of compounds, temperature (25 °C vs 35 °C) has no meaningful effect on IC₅₀s nor on the verification of concentrations. We repeated this set of experiments, now in the absence of internal fluoride. Targeted IC₅₀s were comparable to those determined in the presence of internal fluoride and verification of concentrations did not change that result. We conclude that for this set of compounds, the presence or absence of internal fluoride has no meaningful effect on IC₅₀s nor on the verification of concentrations, although it has a very large effect on experimental success rate. We hope this study will further the conversation on steps needed to implement automated electrophysiology platforms as an accepted tool used in hERG investigations for regulatory filings.