Category: Assay Development and Screening
Cardiotoxicity is a key reason for drug attrition from the clinic. To identify potential toxicity in drug candidates as early in drug development as possible, predictive high throughput assays are needed. Current in vitro cardiotoxicity testing platforms has two main drawbacks. The physiologically relevant models such as primary human cardiomyocytes or organ on a chip cultures are limited to low/medium throughput. On the other hand, high throughput compatible models like hERG overexpressing cell lines lack the relevant physiological responses, such as sensitivity to changes in cardiac contractility. The availability of hiPSC-derived cardiomyocytes (hiPSC-CMs) has led to the development of several high-throughput compatible assays allowing detection of changes in cardiac contractility with a high sensitivity, therefore, applicable for early phases of drug discovery.
Using proprietary hiPSC-derived ventricular cardiomyocytes (Pluricyte® Cardiomyocytes) that recapitulate a human cardiomyocyte’s contractile and electrophysiological profile, we developed an assay to assess effects of cardiotoxic compounds on the Ca2+-flux (a surrogate for contractility) of Pluricyte® Cardiomyocytes using the fast kinetic fluorescence imaging system FLIPR Tetra® and the FLIPR® calcium-6 kit. The assay is fully automated using 384-well plate format enable multi-parameter analysis including beating frequency, average peak amplitude, and peak width. Data analysis were further performed automated by using Vortex Dotmatic® software.
To validate the assay, we tested known cardioactive compounds such as β adrenoreceptor agonist (Isoproterenol), hERG blocker (E4031), L-type Ca channel blocker (Nifedipine) and Ca channel agonist (BayK 8644) in 8 steps concentration-response curves. We profiled a selective panel of 60 commercially available compounds representing different mechanisms of toxicity 30 minutes post-treatment.
Our data show that optimal cell handling and assay optimization is crucial to establish a reproducible assay with low intra-well and intra-plate coefficient variation (%CV) of peak amplitude and beat rate frequency. The highly reproducible assay we have established can in the future be used as a sensitive assay for toxicity screening.
Greg Luerman– Technical Director, Ncardia, Plymouth Meeting, PA