Robust high-throughput automated electrophysiology assay using a monoclonal CHO-hNav1.9 cellular reagent suitable for fully supporting a Nav1.9 discovery program.
Robert W. Kirby, Said El Haou, Edward Humphries, John Ridley and Marc Rogers
Cardiac toxicity remains the leading cause of new drug safety side-effects. Current preclinical cardiac safety assays rely on in vitro cell-based ion channel assays and ex vivo and in vivo animal models⁽¹⁾. These assays provide an indication of acute risk but they do not always predict the effect of chronic compound exposure, as recently seen with oncology drugs⁽²⁾. Therefore, new assays are required to characterise chronic structural and functional effects in human cells earlier in drug discovery⁽²̛ ³⁾. Impedance-based technology can provide more accurate chronic cardiotoxicity measurements in an efficient manner using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)⁽¹̛ ³⁾.
Here we outline validation of chronic cardiotoxicity assays using an impedance platform in combination with commercial hiPSC-CMs:
hiPSC-CMs from commercial vendors (Ncardia and CDI) were plated onto CardioExcyte96 well plates (Nanion) according to manufacturers’ instructions. Plates were incubated at 37 °C (5 % CO2 ) for 7-10 days and 100 % medium exchanges were performed daily. Impedance signals were recorded using the CardioExcyte96 (CE96) platform and the formation of a spontaneous beating syncytial network was monitored with 30 s impedance recording every hour (1 hr) during the first week in culture.
A single concentration of compound was then applied to each well (N ≥ 4) every 24 hrs as a 1:1000 fold dilution into media (final = 0.1 %) for a maximum of 72 hours before a washout period. The outputs (Figure 1) were recorded at 60 minute intervals to assess acute vs. chronic effects (< 24 hrs vs 24 – 72 hrs).
Impedance assay
hiPSC-CMs from a commercial vendor plated onto a CardioExyte96 well plate delivered a high success rate across the plate
(A) Greater than 90% of wells started beating within 6 hours and stabilised within 72 hours
(B) Consistent base impedance (growth phase) parameters were obtained after 5 days
(C) Recordings were suitable for chronic drug screening after 4-7 days
Cardiac ion channel modulator effects on contractility
A. Acute effects of ion channel ligands on cell contraction
B. Chronic cardiotoxicity drug effects
Functional and structural cardiotoxic effect of drugs with different modes of action, including several therapeutic oncology reagents, were tested. Drug mechanisms included DNA intercalation (doxorubicin), proteasome inhibition (bortezomib), myosin disruption (blebbistatin), SERCA Ca2+ pump inhibition (thapsigargin), kinase inhibition (Sunitinib), and an ion channel modulator with mixed functional and structural effects (amiodarone). Aspirin is considered non-cardiotoxic and was used as a negative control⁽⁴⁾.
Figure 4A. Examination of different modalities leading to chronic cardiotoxicity: DNA intercalator. Concentration and time-dependent decreases in base impedance were observed for doxorubicin with > 24 hrs required to resolve the full effect.
Robust high-throughput automated electrophysiology assay using a monoclonal CHO-hNav1.9 cellular reagent suitable for fully supporting a Nav1.9 discovery program.
To overcome seal enhancer limitations, Sophion and Metrion collaborated to determine whether other insoluble salts can act as seal enhancers and how these solution pairs affect the biophysical properties and pharmacology of the investigated ion channels.