Comparison of Electrophysiological Data From Human-Induced Pluripotent Stem Cell Derived Cardiomyocytes to Functional Preclinical Safety Assays

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1 toxicological sciences 134(2), doi: /toxsci/kft113 Advance Access publication May 20, 2013 Comparison of Electrophysiological Data From Human-Induced Pluripotent Stem Cell Derived Cardiomyocytes to Functional Preclinical Safety Assays Kate Harris,* Mike Aylott, Yi Cui,* James B. Louttit,* Nicholas C. McMahon,* and Arun Sridhar*,1 *Safety Assessment and Statistical Sciences, GlaxoSmithKline, Ware, Hertfordshire, UK 1 To whom correspondence should be addressed at Safety Assessment, GlaxoSmithKline, Park Road, Ware, SG12 0DP, Hertfordshire, UK. Fax: +44 (0) & arun.x.sridhar@gsk.com. Human-induced pluripotent stem cell cardiomyocytes (hipsc- CMs) are a potential source to develop assays for predictive electrophysiological safety screening. Published studies show that the relevant physiology and pharmacology exist but does not show the translation between stem cell cardiomyocyte assays and other preclinical safety screening assays, which is crucial for drug discovery and safety scientists and the regulators. Our studies are the first to show the pharmacology of ion channel blockade and compare them with existing functional cardiac electrophysiology studies. Ten compounds (a mixture of pure herg [E-4031 and Cisapride], herg and sodium [Flecainide, Mexiletine, Quinidine, and Terfenadine], calcium channel blockers [Nifedipine and Verapamil], and two proprietary compounds [GSK A and B]) were tested, and results from hipsc-cms studied on multielectrode arrays (MEA) were compared with other preclincial models and clinical drug concentrations and effects using integrated risk assessment plots. All ion channel blockers produced (1) functional effects on repolarization and depolarization around the IC 25 and IC 50 values and (2) excessive blockade of herg and/or blockade of sodium current precipitated arrhythmias. Our MEA data show that hipsc-cms demonstrate relevant pharmacology and show excellent correlations to current functional cardiac electrophysiological studies. Based on these results, MEA assays using ipsc- CMs offer a reliable, cost effective, and surrogate to preclinical in vitro testing, in addition to the 3Rs (refine, reduce, and replace animals in research) benefit. Key Words: hipsc-cm; multielectrode array; electrophysiology: ion channel blockers; cardiovascular safety; preclinical. Reducing drug attrition in development has been a major focus of pharmaceutical companies in recent years. Lack of efficacy (30%) and safety (preclinical and clinical 30%) are the two major reasons for drug attrition (Kola and Landis, 2004). Cardiovascular adverse drug reactions have lead to many marketed drug withdrawals and termination of promising preclinical and clinical drug development Received April 9, 2013; accepted May 13, 2013 The Author Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please journals.permissions@oup.com. candidates (Valentin and Hammond, 2008). Drug-induced cardiac electrophysiological alterations, most notably QT prolongation and torsadogenic incidence/risk, have caused widespread regulatory concern and action from both regulators and pharmaceutical companies (Ewart et al., 2012; Redfern et al., 2002; Wakefield et al., 2002). The current strategy that many pharmaceutical companies use to detect and quantify drug-induced cardiac electrophysiological alterations include ion channel screening, in vitro studies, and in vivo studies in either nonrodents and/or rodents (commonly rats). The electrophysiological effect at a given concentration in vitro is compared with an effect obtained at a maximal free (protein unbound) concentration of drug (free C max in vivo), and these two readouts are compared with a projected clinical free concentration to develop a safety margin (Heath et al., 2011; Muller and Milton, 2012). Current safety models employ ion channels heterologously expressed in cell lines and animal models that differ from clinical responses in terms of sensitivity and specificity (Valentin et al., 2009). A myriad of in vivo and in vitro preclinical assays has been applied to identify the risk of drug-induced electrophysiological alterations. There is limited knowledge in the public domain relating the translation of an effect at a given concentration between assays, with the bulk of translative data coming from ion channel assays and two in vitro assays (rabbit ventricular wedge [RVW] or modified langendorff [ScreenIT]) (Harmer et al., 2011; Heath et al., 2011; Lawrence et al., 2006; Liu et al., 2006). Human-induced pluripotent stem cell derived cardiomyocytes (hipsc-cms) have been proposed to be more sensitive than other in vitro assays (Anson et al., 2011; Kamp and Lyons, 2009; Ma et al., 2011) and offer an additional 3Rs benefit (reduce, refine, and replace) for pharmaceutical preclinical electrophysiological safety screening. The hipsc-cms have been employed in single-cell patch clamp, impedence, and multielectrode array (MEA) formats to assess drug-induced electrophysiological alterations. Even though the pharmacological responses of

2 ipsc-cm ELECTROPHYSIOLOGY AND PRECLINICAL TRANSLATION 413 hipsc-cms to drugs have been demonstrated in these studies (Guo et al., 2011a; Kraushaar et al., 2012; Ma et al., 2011), it is difficult to translate and correlate these responses at a given concentration due to lack of comparison to other assays used in preclinical cardiovascular safety studies. The only studies that looked at translation of electrophysiological effects from embryonic stem cell derived cardiomyocytes compared the effects to rabbit and canine Purkinje fibers and isolated rabbit heart and lacked the comparison to in vivo data (Peng et al., 2010; Qu et al., 2013). The translation of an effect at a given drug concentration (e.g., field potential duration (FPD) in MEA to QT interval in RVW study and to in vivo dog telemetry QTc interval) is an important attribute for any assay to be accepted for wider use. This information is critically important for both drug discovery teams and the scientific reviewers of new drug applications at regulatory bodies to precisely understand the direction, magnitude, and sensitivity of an effect between the various preclinical assays. This article outlines this translational assay comparison between the hipsc-cms MEA responses and other preclinical assays used to assess electrophysiological alterations and, therefore, cardiovascular electrophysiological risk. By utilizing concentration-effect comparisons between different assays (Heath et al., 2011; Valentin et al., 2009), one can directly evaluate the utility of the hipsc-cm MEA assay for use in preclinical cardiovascular risk assessment. To address this issue, a total of 10 compounds with a mixture of herg, Na +, and Ca 2+ channel blocking properties were studied, and a comparison of MEA-acquired parameters was made to QT and QRS intervals on ex vivo RVW and in vivo rodent/nonrodent electrocardiograms (ECGs) to determine translation of pharmacological effects at a given concentration. Our results demonstrate a good correlation with existing preclinical assays, thereby justifying the potential use of hipsc-cms for electrophysiological cardiac safety screening. Materials and Methods Cell Culture hipsc-cms were obtained from Cellular dynamics International (CDI), Madison, Wisconsin. Cells were differentiated for 32 days at CDI, cryo frozen, and shipped to our laboratories. Cells were stored in liquid nitrogen until the day they were thawed. hipsc-cms (two different lots) were thawed in plating media (CDI) and plated directly onto fibronectin (50 µg/ml) (Sigma-Aldrich, Poole, Dorset, UK) coated single-well ECO-MEA plates (60EcoMEA-Glasspr-T, Multi-channel systems, Reutlingen, Germany). The number of viable cells plated in each single-well MEA plate was around 350,000 cells per well. Plated cells were maintained in a humidified cell culture incubator at 37 C, 5% CO 2, and 95% air. On day 2 postthaw, plating media (CDI) were removed and replaced with prewarmed (37 C) maintenance media (CDI). Maintenance media were changed every 2 days. These cells are of immature phenotype and lack the microscopic structure of an adult phenotype (Rao et al., 2013). The cells were of a mixed type (atrial, nodal, and ventricular-like action potential phenotype); but the majority of the cells were of the ventricular-like phenotype (Ma et al., 2011). Our selection criteria for the two lots used in our experiments were that beat rates should be between 0.6 and 0.8 Hz and FPD (uncorrected) values between 300 and 500 ms. MEA Assay Cells were used for experiments on days postthaw. Experiments were performed in a dry incubator maintained at 37 C, 5% CO 2, and 95% air. Field potentials (FP) were recorded from spontaneously beating hipsc-cm monolayers on the MEA-1060 system (Multi-channel Systems) using MC_Rack software (Version ). Data were filtered (1 Hz high pass, 3 khz low pass) and sampled at 10 khz. All drugs were dissolved in dimethyl sulfoxide (DMSO) and serially diluted in serum-free maintenance media, with the exception of E-4031, which was dissolved directly in serum-free maintenance media. The primary stock (100 the proposed test concentration) contained 10% DMSO that was serially diluted to yield the test concentrations. Compound solutions were added to the MEA well to give the relevant test concentration. This resulted in a DMSO concentration of 0.1% at the highest test drug concentration used (1:100 from the primary stock). A schematic of the experimental protocol used is shown in Figure 1. At least 1 h before the start of the experiment, maintenance media were exchanged for prewarmed (37 C) serum-free maintenance media. MEA data were continuously recorded during the experiment. After an initial 15-min equilibration period, the test compound was cumulatively added to the MEA Fig. 1. Schematic representation of the experimental protocol.

3 414 HARRIS ET AL. well at increasing concentrations. Four drug concentrations were tested per experiment, and each concentration was recorded for at least 15 min. Timematched vehicle (media or DMSO) control plates were run at the same time as drug plates to correct for any time- and/or vehicle-dependent effects on the field potential. Test compound selection. Ten compounds with a mixture of ion channel blocking properties were selected for investigation. E-4031 and Cisapride were selected for their pure herg channel blockade; Flecainide, Quinidine, Terfenadine, and Verapamil for their mixed ion channel blocking properties; Nifedipine for its pure calcium (Ca 2+ ) channel blocking properties; Mexiletine for sodium (Na + ) channel block; two proprietary GSK compounds (Anonymised; denoted as GSK A and GSK B) were picked due to the abundant preclinical data available from their development. Both GSK A and GSK B had issues with electrophysiological findings during the developmental phase as outlined (in the discussion) and were added to examine utility of the assay to pick up nonreference compounds. The concentration-response curves (CRC) and IC 50 values from the literature are shown in the figure and referenced in the figure legends for the source. Data Analysis Data were analyzed using the MC_Rack software (Multi-channel systems, Version ). The last minute of recording at baseline and following each drug concentration was averaged (to obtain baseline values and drug effects, respectively), and the following parameters were measured: total spike amplitude (in µv), FPD (in ms), and spontaneous beat rate (in Hz). The FPD was measured as the time from the start of the depolarizing spike to the peak of the repolarizing wave. The absolute amplitude of the depolarizing spike (positive and negative) was measured and shown as total spike amplitude. The waveform stability in all electrodes was monitored during the experiment and post hoc analysis. Electrodes were selected for analysis based on the stability of the field potential waveform over the entire duration of the experiments. Electrodes with waveforms that were not stable for the entire experimental duration were excluded from analysis. The FPD was subsequently rate corrected (FPDc) using Fredericia s formula (FPDc = FPD/RR 1/3, where RR = interspike/ interbeat interval). The Fredericia s correction is very linear in a wide range of interspike interval ( ms (beat rates around Hz)) (Batey and Doe, 2002) and in most cases encompassed the range of beat rate changes seen with hipsc-cms post drug addition. At concentrations where arrhythmias (premature triggered activity) and cessation of beating (quiescence) were observed, measurements of FPDc, spike amplitude, and beat rates were not performed. This information is showed alongside the respective bar graphs for each compound. Statistics Eight of the 10 compounds with the exception of GSK A and GSK B were tested on both lots of cells and showed reproducibility between lots. Each endpoint (FPDc, spike amplitude, and beat rate) was analyzed separately for each compound. All data were log transformed, so that the distribution of the unexplained variation in the data was closer to normal and, thus, suitable for a parametric analysis. A series of linear contrasts were run at each dose level, with a mixed-effects ANOVA (accounting for the random variation within plates), to determine whether the change from pre- to posttreatment differed significantly between the treated plates and the corresponding vehicle control plates. Any comparisons that had a linear contrast p value less than 0.05 were considered to be statistically significant. In addition, a separate contingency table analysis (the Mantel-Haenszel chisquare test) was run for each compound separately to compare the number of plates that generated readable data (e.g, not readable in case of arrhythmias or drug-induced quiescence) at each dose level. If this p value was less than 0.05, the readability of the data was considered to relate to the dose. Bar graphs were plotted using Origin (OriginLab, Cary, NC). All results were expressed in terms of percentage (%) change from the respective baseline (vehicle or test compound) and depicted in the bar graphs as mean ± SE% change. Integrated Risk Assessment (IRA) graphs were created for each drug tested to compare and aid translation of hipsc-cm data to other preclinical cardiovascular safety models. Vehicle-corrected % change from baseline values for each drug tested was plotted on all IRA plots using Microsoft Excel Data for other preclinical models. All data shown in the IRA plots were derived from published literature. Ion channel IC 50 values were obtained from published literature (Harmer et al., 2011; Heath et al., 2011; Himmel et al., 2012; Redfern et al., 2003). RVW data for Nifedipine, Cisapride, Terfenadine, Flecainide, Mexiletine and Verapamil were obtained from published papers (Heath et al., 2011; Liu et al., 2006). Quinidine RVW data were obtained from a FDA submission document (FDA Pharmacology review of NDA ; FDA, 2010). In vivo dog data for Cisparide and Terfenadine were obtained from ILSI- HESI and PRODACT studies (Ando et al., 2005; Hanson et al., 2006; Miyazaki et al., 2005; Omata et al., 2005; Tashibu et al., 2005; Toyoshima et al., 2005). Flecainide and Mexiletine in vivo rodent and canine ECG data were obtained from our earlier study (Heath et al., 2011). Quinidine human ECG data were obtained from published literature (Baker et al., 1983; Duff et al., 1985). GSK A and GSK B data were obtained from GSK database and are completely anonymized, but adequate preclinical data are presented for reader interpretation and comparison. Results E-4031 and Nifedipine At first, two ion channel blockers (herg and L-type Ca 2+ channels) known to affect the APD/QT duration were tested. E-4031, a selective herg channel blocker (IC 50 value = 7nM), significantly prolonged the FPDc in a concentration-dependent manner (3 and 10nM) compared with its time-matched vehicle control (p <.05, Figs. 2A and B). At 30nM E-4031, arrhythmic beats were noted that increased in incidence at 100nM. No arrhythmic beats were seen in the vehicle control plates. Nifedipine, an L-type Ca 2+ channel blocker (IC 50 = 0.024µM), significantly shortened the FPDc in a concentration-dependent manner compared with the time-matched vehicle control plates (p <.05; Figs. 2C and D). Nifedipine significantly increased the beating rate of the cells in a concentration-dependent manner (p <.05). The vehicle-corrected % change in beat rates was 36.7, 89, 138, and 198% at 30, 100, 300, and 1000nM, respectively. Cisapride and Terfenadine Cisapride produced a statistically significant prolongation of the FPDc at 100nM compared with the time-matched vehicle controls (p <.05; Figs. 3A and B). No change in FPDc was observed at 3 and 10nM. At 1000nM Cisapride, the presence of arrhythmic beats arising from triggered activity in the monolayers was observed (Fig. 3C). No arrhythmias were seen in vehicle control plates. The IRA for effects of Cisapride on repolarization is shown in Fig. 3D. The in vivo studies were more sensitive to effects of Cisapride. Both RVW and hipsc- CM MEA yielded the same degree of qualitative change with Cisapride, and this was right shifted compared with in vivo preclinical data. Terfenadine significantly prolonged the FPDc at 100nM compared with time-matched vehicle control plates

4 ipsc-cm ELECTROPHYSIOLOGY AND PRECLINICAL TRANSLATION 415 Fig. 2. Panels (A) and (C) show the representative traces of averaged FP recordings overlaid at baseline and with increasing concentrations of E-4031 and Nifedipine, respectively. Panels (B) and (D) depict the bar graphs showing the mean ± SEM percentage change in FPDc at increasing concentrations of E4031 and Nifedipine, respectively, compared with time-matched vehicle control group (*p <.05 vs. control). (p <.05) (Fig. 4B). Further addition of 1000nM Terfenadine shortened the FPDc (Fig. 4B) and decreased the total spike amplitude (data not shown). The IRA for the effects of Terfenadine on repolarization is shown in Figure 4C. As was the case with Cisapride, preclinical in vivo studies were more sensitive to effects of Terfenadine on repolarization compared with the RVW and the hipsc-cm MEA. Flecainide Flecainide (a class Ic antiarrhythmic) exhibits herg and sodium channel blocking properties over a similar concentration range and, therefore, has effects on both repolarization and conduction. At 1µM, Flecainde produced a significant decrease in the total spike amplitude and prolonged the FPDc (Figures 5D and E). At 3µM, Flecainide produced periods of intermittent cessation of beating (quiescence), and when spontaneous beating reappeared after period of quiescence, it degenerated to arrhythmic beats in three out of five plates. Complete cessation of beating with no arrhythmias was seen in the remaining two plates. Addition of 10µM Flecainide produced complete cessation of beating (quiescence) in all plates tested (p <.05 using nonparametric testing). No arrhythmic activity or cessation of beating was observed in the vehicle control plates. The IRA for effects of Flecainide on repolarization and conduction is shown in Figures 5C and F, respectively. The comparison shows that hipsc-cms are able to detect changes in line with RVW and in vivo preclinical studies and the relevant concentrations achieved with clinical studies with Flecainide. Mexiletine Mexiletine is a class Ib antiarrhythmic that exerts its effects through inhibiting Na v 1.5. Mexiletine (10µM) showed a tendency to reduce the total spike amplitude, but this was not significant (p =.06; Fig. 6B). At 30µM, Mexiletine produced cessation of beating in three out of five plates tested. In the remaining two plates, a 50% reduction in spike amplitude was

5 416 HARRIS ET AL. Fig. 3. Panel (A) shows the representative traces of averaged FP recordings overlaid at baseline and with increasing concentrations of Cisapride. Panel (B) depicts the bar graph showing the mean ± SEM percentage change in FPDc at increasing concentrations of Cisapride compared with time-matched vehicle (DMSO) control group. (*p <.05). Panel (C) shows representative raw traces at baseline and of arrhythmias at 1000nM Cisapride. Panel (D) shows the IRA for the repolarization effects of Cisapride in the different functional cardiac safety assays. seen. No siginificant change in FPDc or beat rates was observed at concentrations up to 10µM (data not shown).the IRA for conduction effects of Mexiletine is shown in Figure 6C. The results are in line with free concentrations of Mexiletine in clinical population/human volunteers, while being very comparable to preclinical in vivo and RVW data. Quinidine Quinidine is a class Ia antiarrhythmic, which inhibits Na v 1.5 and Ca v 1.2 at higher concentrations and herg at lower concentrations (values shown in the Fig. 7D). Consistent with its herg blocking potencies, Quinidine significantly prolonged the FPDc from 0.3µM in a concentration-dependent manner (p <.05) (Fig 7A and B). Increase in Quinidine concentrations > 1µM did not produce further increases in FPDc, which is consistent with calcium channel blockade by Quinidine (Mirams et al., 2011). Quinidine significantly reduced the total spike amplitude at 10µM (Figs. 7A and B; p <.05). Quinidine (1µM) produced arrhythmias in two out of the five plates studied; therefore, only data for the remaining three plates (which did not have arrhythmias) are reported at concentrations 1µM. No arrhythmias were identified in the vehicle control plates. The IRA for the effect of Quinidine on both repolarization and conduction effects is summarized in Figure 7D. The results seen with hipsc-cm were in the relevant concentrations achieved in the clinic and were in line with RVW and dog ECG data. Verapamil Verapamil inhibits herg and L-type calcium channels at overlapping concentrations. No significant effect of Verapamil was seen at 0.03µM. Verapamil significantly shortened the FPDc at 0.1 and 0.3µM (p <.05; Fig. 8B) and produced a dosedependent increase in the spontaneous beat rate of hipsc-cm (p <.05). The vehicle-corrected % change in beat rates was 57.1, 111, and 139% at 0.03, 0.1, and 0.3µM respectively. The IRA for effects of Verapamil on repolarization is shown

6 ipsc-cm ELECTROPHYSIOLOGY AND PRECLINICAL TRANSLATION 417 Fig. 4. Panel (A) shows the representative traces of averaged FP recordings overlaid at baseline and increasing concentrations of Terfenadine. Panal (B) depicts bar graph showing the mean ± SEM percentage change in FPDc at increasing concentrations of Terfenadine compared with time-matched vehicle (DMSO) control group (*p <.05). Panel (C) shows the IRA plot for the effect of Terfenadine on repolarization in the different functional cardiac safety assays. in Figure 8D. Compared with RVW data, hipsc-cm showed greater degree of shortening in response to verapamil treatment, which is entirely consistent with the greater degree of shortening seen in other single-cell adult myocytes compared with tissue measurements. GSK A GSK A produced a concentration-dependent prolongation of the FPDc starting at 1µM (p <.05; Fig. 9B). A significant decrease in the vehicle-corrected spontaneous beat rate of 23% was seen at 30µM (p <.05). A significant reduction in total spike amplitude was seen at 100µM (data not shown, p <.05). Arrhythmic beats were observed only at 100µM, but none were observed in vehicle control plates (p <.05). The IRA for translation is shown in Figure 9C. GSK B GSK B, at concentrations between 0.01 and 1µM, had no effect on the FPDc (p = n.s; Fig. 10B). GSK B produced a significant increase in spontaneous beat rates from 0.01µM (p <.05; Fig. 10C). The vehicle-corrected % change in beat rates was 37, 46, 87, ad 67% at 0.01, 0.06, 0.3, and 3µM GSK B, respectively. The IRA for repolarization and heart rate effects of GSK B is shown in Figures 10D and E, respectively. Comparison Between the Current In Vitro Assay (Rabbit Wedge) and the hipsc-cms MEA For acceptance for the use of hipsc-cms as an in vitro assay for drug safety screening, our criterion was that the pharmacological response of the hipsc-cms must be at least as sensitive (effects at a given concentration lie with half a logarithmic unit) as the current in vitro model used, the RVW. Data from hipsc-cms were therefore directly compared with data from the RVW. The translation between RVW and the hipsc-cms MEA is shown in Table 1. QT and QRS from RVW assay were correlated to FPDc and spike amplitude, and the concentration at which a statistically significant effect (p <.05) was seen for the two assays is shown. The last column indicates the fold difference in concentrations at which an effect was seen between the two assays. The hipsc-cms MEA assay was as sensitive (3- to 10-fold difference in most cases) as the wedge (with the exception of Quinidine repolarization effects) and has the same degree of specificity as the RVW. Discussion The effects of drugs on the electrophysiological properties of hipsc-cms were assessed using MEA technology. MEA technology uses electrodes to measure the extracellular FP waveforms generated from spontaneously beating hipsc-cms. The FP waveform is composed of an initial rapid spike corresponding to Na + influx and depolarization, a slow wave/plateau phase corresponding to Ca 2+ influx, followed by a repolarizing wave that corresponds to K + efflux and repolarization. The initial Na + spike has been shown to correlate to the upstroke of the cardiac action potential (Halbach et al., 2003). The FPD is measured from the initial Na + spike to the peak of the repolarizing wave and has been found to correlate to the cardiac action potential

7 418 HARRIS ET AL. Fig. 5. Panel (A) shows the representative traces of averaged FP recordings overlaid at baseline and 0.3 and 1µM Flecainide. Panel (B) depicts the bar graph showing the mean ± SEM percentage change in FPDc at increasing concentrations of Flecainide compared with time-matched vehicle (DMSO) control group (*p <.05). Panel (C) shows the IRA for the effects of Flecainide on repolarization in the different functional cardiac safety assays. Panel (D) shows the averaged Na + spike overlaid at baseline and 0.3 and 1µM Flecainide. Panel (E) bar graph showing the mean ± SEM percentage change in total spike amplitude at increasing concentrations of Flecainide compared with time-matched vehicle (DMSO) control group (*p <.05). Panel (F) shows the IRA for the effect of Flecainide on conduction in the different functional cardiac safety assays. duration and the QT interval of in vitro and in vivo ECG (Guo et al., 2011b; Halbach et al., 2003). It has been shown in both murine and human hipsc-cms and embryonic stem cell derived cardiomyocytes that blockers of herg and Ca 2+ channels prolong and shorten the FPD, respectively, whereas blockers of Na + channels have effects on spike amplitude and/or FPD (depending on drug class classification due to direct Na + current block or dual herg/na + channel block). We sought to validate the use of hipsc-cms for cardiac safety testing using a set of reference compounds with pure and mixed ion channel inhibitory effects and compared results to existing cardiac safety models. Our results show that hipsc-cms demonstrate pharmacologically relevant responses to compounds of known ion channel blocking properties, and these results are comparable to other preclinical data. Although most published literature shows that hipsc-cms have the relevant pharmacological responses (Guo et al., 2011a; Kraushaar et al., 2012; Ma et al., 2011; Peng et al., 2010), our study is the first example of comparison of the data obtained from hipsc-cms to data from other preclinical (in vivo, in vitro) safety assays. This comparison adds to the translational understanding between the various preclinical assays that are used in the pharmaceutical industry to assess cardiac risk in drug development. Our studies show that the hipsc-cms MEA assay has some notable pharmacological properties that make understanding of drug-induced effects and its use in safety pharmacology relevant: Repolarization and depolarization changes occur most commonly between IC 25 and IC 50 for ion channel block; the changes seen at lower concentrations (e.g., FPDc change and/ or spike amplitude reduction) increase in magnitude with incremental concentrations of drug tested, arrhythmias occur only when a single ion channel (like herg) is maximally blocked (e.g., E-4031 and Cisapride) or with mixed ion channel blockade (e.g., Flecainide), and cessation of beating was seen when substantial block of sodium channels occurred (e.g., Mexiletine). Test Compound Effects E-4031 and Cisapride, the two pure herg blockers tested, showed a remarkable degree of similarity on a plate by plate basis. Both compounds prolonged FPD before arrhythmic beats were observed. The incidence of arrhythmic activity increased when the concentration of E-4031 was increased from 30 to

8 ipsc-cm ELECTROPHYSIOLOGY AND PRECLINICAL TRANSLATION 419 Fig. 6. Panel (A) shows the representative traces showing the averaged Na + spike overlaid at baseline and increasing concentrations of Mexiletine. Panel (B) depicts the bar graph showing the mean ± SEM percentage change in total spike amplitude at increasing concentrations of Mexiletine compared with time-matched vehicle (DMSO) control group. Panel (C) shows the IRA for the effects of Mexiletine on conduction in the different functional cardiac safety assays. 100nM. Cisapride prolonged FPDc at 100nM, whereas 1000nM caused the appearance of arrhythmic activity. When comparing the effects of Cisapride with other preclinical assays, both the RVW QT and hipsc-cm FPDc were significant at 100nM. The dog telemetry model is a sensitive model for electrocardiographic effects of Cisapride (QTc increases of 10 20% seen at 0.2 3nM), while human QTc prolongation is seen at µM (human free C max values of nM) (Redfern et al., 2003). Although the RVW assay showed a higher magnitude of QT change (75% QT change compared with 9% FPDc change), arrhythmias occurred at 1000nM in both the RVW (Liu et al., 2006) and hipsc-cm MEA assays. The larger magnitude of change seen in the RVW could be because the rabbit ventricle has a reduced repolarization reserve, relying exclusively on I Kr and very little on I Ks for repolarisation (Liu et al., 2006), whereas hipsc-cms are known to constitutively express I Ks (Ma et al., 2011). Nifedipine, an L-type calcium channel blocker, shortened the FPDc in a concentration-dependent manner. Verapamil, with herg and L-type calcium channel blocking properties at overlapping concentrations, also shortened the FPDc in a concentration-dependent manner. Both compounds increased the spontaneous beat rates of the monolayers, consistent with published literature (Guo et al., 2011a). The mechanisms of beat rate increases seen with calcium channel blockers are not clearly understood but can be hypothesized to be due to the differences in modulation of calcium handling in neonatal cardiac cells compared with adult cardiomyocytes. It has been hypothesized that the increase in the beat rate seen with Ca 2+ channel antagonists could be a consequence of the shortening of the action potential (AP) and increase in diastolic interval, allowing adequate time for cells to fire off spontaneous APs more frequently (Guo et al., 2011a). Compared with the other preclinical models, Verapamil caused a greater degree of FPDc shortening in the hipsc-cms MEA assay. In the conscious dog, Verapamil had no effect on the QTc over the concentration range tested, whereas in the rabbit wedge QT shortening was only seen at 10µM. The greater degree of shortening in the hipsc-cms compared with other models could be due to the fact that our MEA plates had a monolayer of cells, therefore being more akin to a single myocyte electrophysiology studies that show greater degree of shortening with Ca 2+ channel blockers (Terrar et al., 2007). Another factor could be that diffusion of drug to site of action is higher in a monolayer of cells compared with a tissue preparation. In addition, other published data using ex vivo/in vitro models showed a varied response to Verapamil, with some models showing APD/FPD shortening and others showing prolongation over a similar concentration range (Guo et al., 2011b; Ma et al., 2011; Peng et al., 2010). Nonsedating antihistamine Terfenadine was withdrawn from the market due to its torsadogenic potential (Redfern et al., 2003). Recently, Terfenadine has been shown to exhibit herg blocking properties at concentrations below 100nM and inhibits

9 420 HARRIS ET AL. Fig. 7. Panel (A) shows the representative traces showing the averaged FP recordings overlaid at baseline and increasing concentrations of Quinidine. The inset shows the averaged Na + spike overlaid at baseline and increasing concentrations of Quinidine. Panels (B) and (C) depict the bar graphs showing the mean percentage change in spike amplitude and FPDc, respectively, at increasing concentrations of Quinidine compared with time-matched vehicle control group. Panel (D) shows the IRA for the effects of Quinidine on repolarization and conduction in the different preclinical models (*p <.05). Na v 1.5 at higher concentrations (IC 50 = 6.9µM) (Lu et al., 2012). Our results indicate that Terfenadine induced FPDc prolongation at 100nM and caused a reduction in spike amplitude and shortening of the FPDc at 1000nM. However, Terfenadine did not induce any triggered activity/arrhythmias in the monolayers tested. It has been shown that arrhythmogenic effects of Terfenadine are difficult to detect in preclinical models with short exposure times, as it accumulates in the heart of many preclinical species and in man (Cavero et al., 1999; Fish and Antzelevitch, 2003; Hondeghem et al., 2011; Masumiya et al., 2004). Similarly, Terfenadine only induced arrhythmias after longer exposure times ( 12 h) in the hipsc-cm monolayer (Guo et al., 2011a). Our IRA plot indicates that the hipsc-cm MEA assay produced the same degree of change in FPDc as the QT interval in the RVW assay at 100nM and exhibited properties of sodium channel blockade (FPDc shortening and spike amplitude reduction) at a concentration ~10-fold less than in the RVW. This difference in sensitivity to Na + channel block could be attributed to the fact that hipsc-cm MEA is a cellular model, whereas RVW is a tissue model, and compound effects in tissue occur at higher concentrations and/or after longer incubation times. All in vitro models were less sensitive than the conscious dog models at detecting Terfenadine s QTc prolonging effect. Quinidine, Mexiletine, and Flecainide are Class Ia, Ib, and Ic antiarrhythmics, respectively. Quinidine is a more potent inhibitor of herg than of Na v 1.5. Consistent with Quinidine s ion channel inhibiting effects, FPDc prolongation was seen in hipsc-cm s at lower Quinidine concentrations (from 0.3µM) and a reduction in spike amplitude was seen at higher concentrations (from 10µM). In addition, the FPDc data with quinidine correspond to ion channel data. The Na v 1.5 and Ca v 1.2 IC 50 for quinidine is 16.6 and 15.2µM. The prolongation in FPDc at lower concentrations correlates with herg block, and further prolongation at 3 and 10µM is prevented by Ca v 1.2 block. This could be the reason why arrhythmias with Quinidine was

10 ipsc-cm ELECTROPHYSIOLOGY AND PRECLINICAL TRANSLATION 421 Fig. 8. Panel (A) shows the representative traces showing the averaged FP recordings overlaid at baseline and increasing concentrations of Verapamil. Panel (B) depicts the bar graph showing the mean ± SEM percentage change in FPDc at increasing concentrations of Verapamil compared with time-matched vehicle control group (*p <.05). Panel (C) shows the IRA for the effects of Verapamil on repolarization in the different functional cardiac safety assays. Fig. 9. Panel (A) shows the representative traces showing average FP recordings at baseline and increasing concentrations of GSK A. Panel (B) depicts the bar graph showing the mean ± SEM percentage change in FPDc at increasing concentrations of GSK A compared with time-matched vehicle (DMSO) control group (*p <.05). Panel (C) shows the IRA for the effects of GSK A in the different functional cardiac safety assays.

11 422 HARRIS ET AL. Fig. 10. Panel (A) shows the representative traces of averaged FP recordings at baseline and increasing concentrations of GSK B. Panels (B) and (C) depict the bar graphs showing the mean ± SEM percentage change in FPDc and rate, respectively, at increasing concentrations of GSK B compared with time-matched vehicle control group. Panel (D) shows the IRA for the effects of GSK B on repolarization in the different preclinical assays. Panel (E) shows the IRA for the beat rate changes seen in the dog and hipsc-cm MEA assays (*p <.05). Compound Table 1 Concentration at Which a Statistically Significant Change in QT/QRS Was Seen in the Wedge Compared With FPDc/Spike Amplitude change in the hipsc-cm MEA Assay Concentration for p <.05 in the rabbit wedge Concentration for p <.05 in the MEA MEA/wedge fold difference (Log units) Nifedipine repolarization Cisapride repolarization Terfenadine repolarization a Terfenadine conduction a Verapamil repolarization a Flecainide conduction a Flecainide repolarization a Quinidine repolarization b GSK A repolarization a GSK B repolarization 0.6 n/a due to beat rate changes n/a Note. The logarithmic scale difference is shown in the last column. In most cases, MEA fell within half a logarithmic unit of the wedge data and in certain cases was better predictor of an effect. 0, no difference; n/a, not applicable. a MEA > Wedge. b Wedge > MEA. only observed in a subset of plates, as Ca v 1.2 block will reduce calcium influx into the cells and reduce arrhythmic incidence. hipsc-cm data correlate well with the effects on QT and QRS seen in the other preclinical models over a similar concentration range. FPDc change correlates well with herg IC 50 and RVW QT effects and is slightly more sensitive at detecting the effects than the dog model. The change in spike amplitude is in line with Na v 1.5 IC 50 ; however, hipsc-cms were ~10-fold

12 ipsc-cm ELECTROPHYSIOLOGY AND PRECLINICAL TRANSLATION 423 less sensitive at detecting the effects on conduction than the dog model. Mexiletine is a class Ib antiarrhythmic with usedependent sodium channel blocking properties. Our data indicate that there was a trend to reduce spike amplitude at 10µM, and this effect was greater at 30µM where the majority of the plates showed cessation of beating, consistent with blockade of an excitatory current during the action potential upstroke. Our results are in line with other in vivo and in vitro preclinical models that show that effects of Mexiletine are small and occur within the effective free therapeutic concentration range and in some cases difficult to detect in preclinical models (Heath et al., 2011). Flecainide primarily exerts its therapeutic effects through inhibiting Na v 1.5 and slowing conduction in the heart and also inhibits the herg channel (Na v 1.5 and herg IC 50 values lie within threefold of each other). We found that consistent with this effect, a reduction in spike amplitude and FPDc prolongation occurred at 1µM. Flecainide also has proarrhythmic potential, and this was first identified clinically in the Cardiac Arrhythmia Suppression Trial (CAST) where there was an increase in the sudden death of myocardial infarct patients taking Flecainide (The CAST Investigators, 1989). In our experiments, 3µM Flecainide induced periods of cessation of beating (quiescence) and arrhythmic activity (~60% of the plates), consistent with Flecainide s proarrhythmic potential. Although no arrhythmias were seen in the other preclinical models reported in the IRAs, there are several studies where Flecainide produced periods of inexcitability and arrhythmic activity in the RVW and Langendorff perfused heart (Liu et al., 2012). Addition of 10µM caused complete cessation of beating in all plates tested. Our IRA plots show concordance between the various preclinical models. A similar degree of change in both FPDc/QT and spike amplitude/qrs occurred over a similar concentration range and within the free (protein unbound) clinical concentrations of Flecainide. The upstroke velocity of the action potential in hipsc-cms is slow compared with adult ventricular myocardium (28 vs V/s) (Lue and Boyden, 1992; Ma et al., 2011). This slower upstroke velocity is likely a result of the less negative resting membrane potential, which would partially inactivate the Na channel population. It has been previously shown that Na channel blockers show higher magnitude of block at depolarized potentials (studied by microelectrode techniques while voltage clamping the cells to hyperpolarized and depolarized holding potentials; extracellular K + increases) (Campbell et al., 1991a, b; Saint and Tang, 1998). Our spike amplitude reduction data are entirely consistent with higher magnitude of sodium channel block at depolarized potentials seen in previous studies. The possible explanation for this is the fact that at depolarized potentials, smaller number of channels contributes to action potential upstroke, and block of these channels will result in a greater change compared with block at resting potentials around 90 mv. This may also explain the small window between spike amplitude reduction and complete cessation of beating with Flecainide and Mexiletine. In addition, tetrodotoxin (TTX) sensitive peak and late Na + currents (Na v 1.5 and non-na v 1.5 currents) are present in these hipsc- CMs (Ma et al., 2011). In contrast, adult cardiac myocytes, the TTX-sensitive current is predominantly the late Na + current, contributing very little to the peak Na + current (< 10%) (Maltsev et al., 1998, 2009; Maltsev and Undrovinas, 2006; Yang et al., 2012) suggesting that sodium channel distribution in hipsc-cms might be different compared with adult cells contributing to greater effect at beat rates around 0.5 Hz due to expression of TTX-sensitive sodium current. Despite the fact that these cells are relatively immature compared with adult cardiomyocytes, pharmacological data does correlate well with preclinical and clinical data. This is in contrast to a study using embryonic stem cell derived cardiomyocytes that showed less sensitive effects of sodium channel blockade (Qu et al., 2013). The potential difference between ours and by Qu et al. could be due to different cell types used and different times postthaw or the digestion procedures during plating that could impact ion channel function. In our study, we overcame the potential confounding effects of digestive enzymes used in cell culture by directly plating the cells on to MEA plates (Rajamani et al., 2006), thereby reducing processes, time, and experimental errors/variability. GSK A is a proprietary GSK compound that produced significant QT changes in the phase I clinical trial. Prior to the clinical study, herg ion channel data and snapshot ECG recordings in sling trained canines revealed QT prolongation. In the first conscious dog ECG study that was conducted (data obtained via snapshot ECG recordings), QTc prolongation occurred from ~10µM GSK A, and no arrhythmias were seen. When this compound was progressed to phase 1 clinical trial, QTc prolongation was identified at a free plasma concentration of 0.69µM. Follow-up studies were performed in the RVW assay and conscious dog telemetry study. In the in vivo dog telemetry study, QTc prolongation and ventricular premature complexes were seen from a free C max of 13µM. These arrhythmias increased in incidence at 26µM (free concentration). In the RVW, GSK A dose dependently prolonged the QTc from 10µM, no arrhythmias were seen. The QT prolongation seen with GSK A did not follow herg block (IC 50 = 533µM), while the compound did not have any effect on other ion channels. The mechanism of QTc prolongation was not investigated, and the compound was terminated due to arrhythmias in the dog. In the hipsc-cm MEA assay, GSK A produced concentrationdependent prolongation of the FPDc from 1µM, mirroring the QTc prolongation seen in humans. hipsc-cms were more sensitive at detecting the effects on repolarization compared with the other preclinical models. GSK B produced heart rate increases in the dog (~110%, thereby making QTc measurements unreliable). The compound proceeded to clinical testing as the therapeutic free C max at starting dose was > 500-fold lower than the concentrations reached in the dog telemetry study. In the clinical trial,

13 424 HARRIS ET AL. prolongation of QTc occurred at the highest dose of 300 mg/ kg and the free concentration at this dose corresponded to the lowest dose tested in the dog (see IRA in Fig. 10). No change in heart rate was seen in human trials. GSK B MEA data recapitulate the heart rate increases seen in the dog, and this seems to be consistent on the IRA plot (Fig. 10E). No mechanisms for preclinical heart rate increases were found prior to compound termination. Within the pharmaceutical industry, early compound screening is performed to identify preclinical liability, whereas mechanisms for the liabilities are performed later in the drug discovery cycle. Our study demonstrates the utility of the hipsc-cm MEA assay to detect effects seen in preclinical and clinical tests in an assay format that is more amenable to use earlier in the discovery cycle. As stated earlier, arrhythmias occur in the MEA assay when potent herg block is seen (E4031 and Cisapride) or a combinatorial block of multiple ion channels (Flecainide). In each case, a clear precursor (delayed repolarization) to arrhythmias was seen. Cisapride and E-4031 produced FPDc prolongation prior to arrhythmia occurrence. Flecainide produced both FPDc prolongation and spike amplitude reduction prior to arrhythmia induction further validating the scientific view that for mixed ion channel blockers the balance of ion channels controlling repolarization reserve is critical. The concordance with human QTc data shows that the hipsc-cm MEA assay would have raised this hazard earlier in preclinical development, at which stage hazard mitigation steps could have been undertaken. Limitations One potential limitation was that the monolayer was not paced, and it can be argued that pacing might provide rate dependence of FPD. Our compound set was picked with an idea to estimate beat rate changes; hence, the preparations were not paced. Also, some compounds produced beat rate increases, and in such situations, pacing would not have been possible. Our study did not explore testing of negative controls (compound that produce no change in parameters studied). Larger compound numbers are needed to determine sensitivity and specificity values for the assay. It is impossible to assess dispersion of repolarization in a spontaneously beating monolayer. Hence, we had to resort to comparison of QT and FPD effects for repolarization and correlate conduction via measuring spike amplitude in MEA to QRS interval in the RVW. Spike amplitude is obtained from synchronized spiking activity of cells in the vicinity of the electrode. Hence, we used spike amplitude (averaged from many electrodes on the MEA plate) as an index of depolarization and correlated this to depolarization times (QRS interval) in RVW. It is possible that conduction time could be a surrogate for QRS interval, but this could not be performed in our experiments due to limitations in software and will be a focus of a future study. Conclusions Our data show that hipsc-cms act as a reliable surrogate to RVW to study drug-induced electrophysiological changes for early SP cardiac safety screening. Our comparison of the hipsc-cm MEA assay to the RVW shows that MEA assay identifies the same effects as the RVW and that concentrations that produce significant changes lie within half a logarithmic unit of the RVW data. In addition, hipsc-cm MEA assay endpoints correlated well with in vivo QT or QRS changes. The ipsc-cms MEA assay could be a viable alternative to RVW or other in vitro assays used in pharmaceutical development and offer the following advantages: an unlimited supply of human cardiomyocytes, reduction of animal usage, relevant pharmacological responses, and reduced costs. Acknowledgments We thank Ms Amy Nicks for her technical help with cell culture and hardware set up during her internship year at GSK. We thank Dr Blake Anson of Cellular Dynamics International for valuable discussions. References Ando, K., Hombo, T., Kanno, A., Ikeda, H., Imaizumi, M., Shimizu, N., Sakamoto, K., Kitani, S., Yamamoto, Y., Hizume, S., et al. (2005). QT PRODACT: In vivo QT assay with a conscious monkey for assessment of the potential for drug-induced QT interval prolongation. J. Pharmacol. Sci. 99, Anson, B. D., Kolaja, K. L., and Kamp, T. J. (2011). Opportunities for use of human ips cells in predictive toxicology. Clin. Pharmacol. Ther. 89, Baker, B. J., Gammill, J., Massengill, J., Schubert, E., Karin, A., and Doherty, J. E. (1983). Concurrent use of quinidine and disopyramide: Evaluation of serum concentrations and electrocardiographic effects. Am. Heart J. 105, Batey, A. J., and Doe, C. P. (2002). A method for QT correction based on beatto-beat analysis of the QT/RR interval relationship in conscious telemetred beagle dogs. J. Pharmacol. Toxicol. Methods 48, Campbell, T. J., Wyse, K. R., and Hemsworth, P. D. (1991a). Effects of hyperkalemia, acidosis, and hypoxia on the depression of maximum rate of depolarization by class I antiarrhythmic drugs in guinea pig myocardium: Differential actions of class Ib and Ic agents. J. Cardiovasc. Pharmacol. 18, Campbell, T. J., Wyse, K. R., and Pallandi, R. (1991b). Differential effects on action potential duration of class IA, B and C antiarrhythmic drugs: Modulation by stimulation rate and extracellular K+ concentration. Clin. Exp. Pharmacol. Physiol. 18, Cavero, I., Mestre, M., Guillon, J. M., Heuillet, E., and Roach, A. G. (1999). Preclinical in vitro cardiac electrophysiology: A method of predicting arrhythmogenic potential of antihistamines in humans? Drug Saf. 21(Suppl. 1), Duff, H. J., Wyse, D. G., Manyari, D., and Mitchell, L. B. (1985). Intravenous quinidine: Relations among concentration, tachyarrhythmia suppression and electrophysiologic actions with inducible sustained ventricular tachycardia. Am. J. Cardiol. 55,