Optical measurements of electrical activity from hiPSC-derived cardiomyocytes is a robust and high-throughput method for measuring NCE-effects on the cardiac action potential

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Target Identification

Target Validation

Compound Screening

Lead Optimization

Preclinical Trials

Clinical Trials

Godfrey Smith1,2, Margaret Anne Craig2, Blake Anson3, Maria Hortigon-Vinagre1, Francis Burton1, Robert Wallis2 and Iffath Ghouri1

1Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom, 2Clyde Biosciences, Glasgow, United Kingdom,3Cellular Dynamics International Inc, Madison, WI, USA

All new chemical entities (NCEs) need to be screened to evaluate for cardiac and potential arrhythmogenic effects as part of their toxicological and safety pharmacological profile. Current pre-clinical in vitro drug testing involves the use of isolated tissue from animal models and non-muscle expression systems. However, interspecies variability exists in the ionic currents governing cardiac repolarisation and additional pre-clinical testing on human cell and tissue preparations would be beneficial in the early detection of pro-arrhythmic effects. The development of human stem cell technology has introduced a potentially valuable preparation which can be used in the safety pharmacology screening process. The cardiac action potential (AP) arises from the co-ordinated interplay of multiple ion channels and transporters; drug interactions with any of these can promote a pro-arrhythmic condition. Traditional AP assessment has been by patch clamp technique, which is laborious, requires highly skilled scientists, and neither the technique nor the established tissue cells have been readily amenable to higher throughput techniques. Here, we present data characterizing a novel optical platform, CellOPTIQ (Clyde Biosciences, Glasgow, UK), used in conjunction with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (iCell® Cardiomyocytes Cellular Dynamics Incorporated, Madison, WI, USA), which offers a solution that overcomes the bottlenecks of low throughput and suitable tissue cells associated with traditional cardiac AP studies. We investigate the effects of a range of pharmacological compounds on the cardiac AP, and demonstrate the suitability of these technologies for assessing NCE-mediated pro-arrhythmogenicity by comparing measured responses to published effects in dog purkinje fiber and effective therapeutic concentrations in humans.

Introduction

Conclusions

Results Figure 1. Consistency of AP shape over time. Figure 5. Effect of overnight As2O3 exposure on AP response to E4031

Methods iCell Cardiomyocytes were seeded in gelatin-coated plastic 48 well plates at a density of 100000 cardiomyocytes/well and maintained in culture for 10 days. Experiments were carried out on days 10-14. The cardiomyocytes were washed in serum-free medium and exposed transiently to 3 µM Di-4-ANEPPS. The plate was placed in a stage incubator on an inverted microscope and the spontaneous electrical activity was recorded as the Di-4-ANEPPS fluorescence signal from areas of iPSCs in individual wells using a 40x (NA 0.6) objective. Fluorescence signals were digitized at 10kHz and the records subsequently analyzed off-line. Acute effects of the pharmacological compounds E4031, Mexiletine and Nifedipine on hERG, Na+ and Ca2+ channels were assessed by exposure to cumulative increases in drug concentrations allowing 10 mins incubation at each concentration. The procedure was repeated up to 9 times and parallel measurements were done on cardiomyocytes with equivalent concentrations of vehicle. Incrementing the drug concentration by 0.5 log units, a threshold was established as the first concentration at which detectable differences between the drug and control group were observed. Effects of Arsenic Trioxide (As2O3), an inhibitor of hERG protein trafficking into the cell surface membrane (thus recapitulating the Long QT Syndrome), were assessed by exposure to 10µM As2O3 in serum free medium for 24 hours. The acute response to increasing concentrations of E4031 in cardiomyocytes treated with As2O3 was then compared to control cardiomyocytes untreated with As2O3. Action potential parameters were measured, including 10%-90% rise time (Trise) and action potential duration at 50, 75 and 90% repolarization (APD50, APD75, and APD90 respectively). Data are expressed as % change from baseline for either the vehicle or the drug groups.

Electrical activity was recorded in the same well over the experimental period of 10-14 days. No change in AP shape was recorded, demonstrating the consistency of the electrical activity throughout the experimental period.

Figure 2. Effect of E4031 on AP characteristics.

Effect of increasing concentrations of E4031 on (i) AP shape and (ii) APD50, APD75 and APD90, demonstrating the effect of the drug on the hERG channel. (iii) % change from baseline of E4031 and vehicle control at increasing concentrations. The decrease in APD at the highest concentration (0.1µM) was due to the cardiomyocytes becoming tachyarrhythmic. (n=9,* p<0.05 vs. control, ** p<0.01 vs. control, *** p<0.001 vs. control).

Figure 3. Effect of Mexiletine on AP characteristics.

Effect of increasing concentrations of Mexiletine on (i) AP shape (with upstroke phase magnified) and (ii) Trise, demonstrating the effect of the drug on the Na+ channel. (iii) % change from baseline of mexiletine and vehicle control at increasing concentrations. (n=9, * p<0.05 vs. control, ** p<0.01 vs. control, *** p<0.001 vs. control).

Figure 4. Effect of Nifedipine on AP characteristics.

Effect of increasing concentrations of Nifedipine on (i) AP shape and (ii) APD50, APD75 and APD90, demonstrating the effect of the drug on the Ca2+ channel. (iii) % change from baseline of Nifedipine and vehicle control at increasing concentrations. (n=4, ** p<0.01 vs. control, *** p<0.001 vs. control).

Results

Effect of increasing concentrations of E4031 on APD75 in cardiomyocytes treated with and without As2O3 overnight. Baseline APD75 pre-E4031 treatment showed a tendency to be longer than in cardiomyocytes untreated with As2O3. Significant lengthening of APD75 compared to controls occurred at lower concentrations of E4031 in As2O3 treated cardiomyocytes compared to untreated cardiomyocytes. (n=2 * p<0.05 vs. vehicle control)

Table 1. Comparison of CellOPTIQ results with published ETPCu values and results from dog purkinje fiber

Compound ETPCu hERG IC50(nM)

Nav1.5 IC50(nM)

Cav1.2 IC50(nM)

iCell Cardiomyocytes Dog Purkinje Human [] to ↑QT

Human [] to ↑QRS

TRise APD50 APD75 APD90 Vmax APD50 APD90 E4031 33 8-121 >1000 ↑3 ↑3 ↑3 ↑3 ↑107 ↑107 3

Mexilitine 1869-41294

500009 257005 1000009 ↑3000 ↓3000 >30000 ↑20000 ↓110006 ↓11006 ↓110006 NE 1121-97184

Nifedipine 3.5-82 3002 335004 228 ↓<100 ↓<100 ↓<100 ↓100005 ↓105 ↓100005 NE2 NE4 >8

ETPCu – Effective therapeutic plasma concentration (nM) unbound taken from Kirsch 2004 1 Kirsch et al. J. Pharmacol. Toxicol. Methods (2004) 50: 93-101; 2 Redfern et al. Cardiovasc. Res. (2003) 58: 32-45; 3 Fujiki et al. J Cardiovasc. Pharmacol. (1994) 23: 374-378; 4 Harmer et al. Br. J. Pharmac. (2011) 164: 260–273; 5 Terrar et al. J. Pharmacol. Toxicol. Methods (2007) 56: 171–185; 6Arita et al Br. J. Pharmac. (1979), 67: 143-152; 7 Peng et al. J. Pharmacol. Toxicol. Methods (2010) 61: 277–286; 8 Balasubramanian et al. J. Pharmacol. Toxicol. Methods (2009) 59: 62–72; 9 Mirams et al. Cardiovasc. Res. (2011), 91: 53-61

This study has demonstrated the detection of a range of drug effects on AP characteristics using the CellOPTIQ optical system in conjunction with iCell Cardiomyocytes. Comparison with published responses in dog purkinje fiber and effective therapeutic concentrations in humans highlights the effectiveness of using these technologies to detect drug effects at therapeutically relevant concentrations. The medium/high-throughput nature of the CellOPTIQ system (~20 assays/hour/instrument) and lower technical skill required for its operation (compared to traditional patch clamp techniques), together with the availability of reproducible human cardiac tissue preparation, make this a valuable addition to the drug development process for assessing NCE-mediated pro-arrhythmogenicity. ACKNOWLEDGEMENTS This work was supported by the Biotechnology and Biological Sciences Research Council (UK), Clyde Biosciences (UK) and Cellular Dynamics International. M.H-V. is recipient of a postdoctoral fellowship from Fundacion Alfonso Martin Escudero (Spain)