One of the most common reasons for failure of compounds in drug discovery programmes is cardiac toxicity. Therefore assessing this in the early stages of any drug discovery programme is key. Currently the earliest indicator for cadio-tox is interaction between compounds and a voltage gated potassium channel hERG (the human Ether-à-go-go-Related Gene) channels. Generally electrophysiological techniques are used in hERG, although fluorescent-based kits are now available. Although hERG is the most common interaction for cardiotoxicity, this does not measure other potential interactions that could result in cardiotox. Sirenko et al. have taken advantage of the recent emergence of inducible pluripotent stem cells (iPSC) to explore the effects of compounds on cardiomyocytes in more detail.
Culture of iPSC is not as straightforward as immortalised cells, however the cells were obtained from a commercial source (Cellular Dynamics) saving the differentiation process and these cells are able to be reproducibly differentiated into a large quantity and cryopreserved until use. The use of iPSC derived cardiomyocytes for toxicity testing has the clear advantage that these cells express full cardiomyocyte functionality and even have the beating characteristic expected of primary cardiomyocytes. Since this beating is analogous to the beating of a heart, and ion channel block can present as drug-induced arrhythmias, it is not hard to see how this can be used to identify cardiotoxic compounds. At this point most groups would have struggled to have the technology available to analyse these beatings, but since the authors were from Molecular Devices, they conveniently had a FLIPR tetra and image xpress high content screening (HCS) platform along with the technical knowledge to enable these beatings to be quantified.
Sirenko et al. were able to quantify these beatings on both systems. On the HCS, they used time-lapse images and quantified the beating using their own differential algorithm that measured the number of beats per minute. On the FLIPR, they used the Calcium 5 Assay Kit which enabled the beating to be measured using Ca2+ influx in beats/min. Since the HCS could not acquire images as quickly as the FLIPR, the HCS approach was less sensitive than using the FLIPR and less work was performed using this method, however, they were still able to obtain IC50’s for known agonists adrenaline and isoproterenol.
The increased sensitivity and throughput possible using the FLIPR, enabled more studies to be performed using it. As such compound-induced dose-dependent atypical beating patterns induced by known cardio-toxic compounds such as hERG, Ca2+ and Na+ channel blockers were measured. They were also able to validate an automated analysis algorithm that was used to calculate the effect of a variety of compounds in high-throughput format, with positive or negative chronographic effects such as digoxin and propranolol. Furthermore, due to the increased sensitivity of the FLIPR it was also possible to quantify components of each individual Ca2+ peak (i.e. amplitude, peak width, decay time etc.) rather than just the number of beats. This meant they were able to measure more compound specific effects that have more physiological deviations such as QT prolongation, i.e. cisapride that increased both peak spacing and peak width.
A large number of drug withdrawals from the market as well as late stage clinical trials are from cardiac toxicity. These withdrawals are clearly very expensive, but more importantly potentially very damaging to health. The use of hERG testing clearly identifies many cardiotoxic compounds, however, the use of iPSC cardiomyocytes and measuring Ca2+ influx and beating enables many more facets of cardiac toxicity to be measured. The study by Sirenko demonstrates the use of this technique to identify compounds already known to effect different facets of cardiac toxicity. The real test, however, would be to put compounds that were withdrawn from the market, or clinical trials due to cardiac toxicity that were missed by other tox screens. If the technique set out by Sirenko et al., were to have picked up this toxicity it would demonstrate a step forward in early determination.