2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes -assessment of inter-facility and cells lot-to-lot-variability-

Regulatory Toxicology and Pharmacology 77 (2016) 75e86

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph

CSAHi study: Validation of multi-electrode array systems (MEA60/ 2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes -assessment of inter-facility and cells lotto-lot-variabilityYumiko Nozaki a, Yayoi Honda a, Hitoshi Watanabe a, Shota Saiki b, g, 1, Kiyotaka Koyabu b, Tetsuji Itoh b, Chiho Nagasawa c, g, 1, Chiaki Nakamori c, Chiaki Nakayama c, Hiroshi Iwasaki c, Shinobu Suzuki d, g, 1, Ikumi Washio d, Etsushi Takahashi e, g, 1, Kaori Miyamoto e, Atsuhiro Yamanishi f, g, 1, Hiroko Endo f, Junko Shinozaki f, Hisashi Nogawa f, Takeshi Kunimatsu a, g, h, *, 1 a

Preclinical Research Laboratories, Sumitomo Dainippon Pharma Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan Research Laboratory for Development, Shionogi & Co., Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan Research Center, Taisho Pharmaceutical Co., Ltd., 1-403, Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan d Nippon Boehringer Ingelheim Co., Ltd., 6-7-5, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan e Research Laboratories, Toyama Chemical Co., Ltd., 4-1, Shimookui 2-chome, Toyama 930-8508, Japan f Toxicology Research Laboratory, Kyorin Pharmaceutical Co., Ltd., 1848, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi, 329-0114, Japan g Consortium for Safety Assessment Using Human iPS Cells (CSAHi), Japan h Non-Clinical Evaluation Expert Committee, Drug Evaluation Committee, Japan Pharmaceutical Manufacturers Association (JPMA), 2-3-11 NihonbashiHoncho, Chuo-ku, Tokyo 103-0023, Japan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 November 2015 Received in revised form 1 February 2016 Accepted 12 February 2016 Available online 13 February 2016

In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of druginduced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia. © 2016 Elsevier Inc. All rights reserved.

Keywords: Consortium for drug safety assessment using human iPS cells (CSAHi) Comprehensive in vitro Proarrthythmia assay (CiPA) Inter-facility variability Multi electrode array (MEA) Human iPS cells-derived cardiomyocytes (hiPS-CMs) Early after depolarization (EAD) or triggered activity (TA)

Abbreviations: CSAHi, Consortium for Safety Assessment using Human iPS Cells; CiPA, Comprehensive in vitro Proarrthythmia Assay; MEA, Multi-electrode array; hiPSCMs, human induced pluripotent stem cells-derived cardiomyocytes; FPD, Field potential duration; TdP, Torsade de Pointes; EAD, Early after depolarization; TA, Triggered activity; MCEAD/TA, Minimum concentration inducing EAD or TA; APD, Action potential duration. * Corresponding author. Preclinical Research Laboratories, Sumitomo Dainippon Pharma Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan. E-mail address: [email protected] (T. Kunimatsu). 1 http://csahi.org/en/. http://dx.doi.org/10.1016/j.yrtph.2016.02.007 0273-2300/© 2016 Elsevier Inc. All rights reserved.

76

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

1. Introduction Risk assessment for cardiovascular adverse events is an important safety pharmacology element in drug development as it allows management of potential arrhythmia including torsade de Pointes (TdP), a fatal polymorphic ventricular tachycardia frequently resulting in ventricular fibrillation and sudden death. Drugs potential for QT prolongation are evaluated in non-clinical studies as a primary end point to predict drug-induced TdP in humans. This assessment, which is conducted according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B guidelines, consists of in vitro -go-go-related gene (hERG) inhibition test, electrohuman ether-a cardiogram in conscious animals, and potentially follow-up tests, including action potential duration (APD) assay. Although in vitro non-clinical assessment of drugs proarrhythmic potential centers initially on hERG channel inhibition, hERG inhibition does not always lead to QT prolongation or arrhythmia, and thus a more accurate predictive model is needed. This is because drug-induced proarrhythmia is known to be evoked by newly-identified mechanisms, including inhibition of channel trafficking and blockage or activation of ion channel current (Takasuna et al., 2009). In addition, interspecies variation can prevent reliable link to clinical relevance. A conference held in July, 2013 the Cardiac Safety Research Consortium (CSRC), Health and Environmental Sciences Institute (HESI), and U.S. Food and Drug Administration (FDA) proposed the replacement of ICH E14 and revision of ICH S7B. Under the new proposed guidelines, risk assessment of drug induced cardiac arrhythmia will be shifted away from QT prolongation and towards arrhythmia. This proposed shift led HESI and FDA to work together to form a consortium to devise the new approach, called comprehensive in vitro proarrhythmia assay (CiPA) (Cavero, 2014; Sager et al., 2014). Assessment of field potential duration using human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) is considered as a valuable tool for detecting potential QT liability and/or arrhythmogenicity in CiPA. In Japan, ahead of other countries, ‘the Consortium for Safety Assessment using Human iPS Cells (CSAHi)', organized by the Non-Clinical Evaluation Expert Committee of Japan Pharmaceutical Manufacturers Association (JPMA), has been engaged in establishing a new test system with standard protocol for comprehensive cardiac toxicity, including druginduced QT prolongation/proarrhythmia. Under this new system, a multi-facility evaluation under a common protocol was carried out to evaluate the effects of test compounds on field potential (FP) and occurrence of arrhythmia-like waveform in hiPS-CMs using a multi-electrode array (MEA) manufactured by Multi Channel Systems. In CSAHi, a similar study with another MEA system (MED64) manufactured by alpha MED scientific was conducted in parallel with our study (Kitaguchi et al., 2016). In this report, we describe inter-facility and cells lot-to-lot variability in a multi-electrode array system for prediction of drug-induced proarrhythmia using human iPS cells-derived cardiomyocytes (hiPS-CMs). 2. Materials and methods 2.1. Test compounds and vehicle E-4031 (Wako, Japan; Enzo Life Science, USA), terfenadine (Sigma-Aldrich, USA), moxifloxacin (Fluka, Japan) and flecainide (Sigma-Aldrich) all known to produce QT prolongation/arrhythmia in clinical practice, as well as chromanol 293B, a selective IKs blocker with potential QT prolongation (Sigma-Aldrich; R&D systems, USA) were used as test compounds. Aspirin (Wako, Japan; Enzo Life Science, USA) and verapamil (Wako; Sigma-Aldrich) were

used as negative controls. Each compound or vehicle was dissolved in dimethyl sulfoxide (DMSO) at 1000-fold the testing concentration to prepare stock solutions. 2.2. Cell culture and plating for FP recording As described in Table 1, commercially available iCell® Cardiomyocytes (Cellular Dynamics International (CDI), USA) were cultured at each testing facility according to a protocol modified from the protocol provided by CDI. In brief, the cells were thawed at 37
C for 4 min and suspended in a Plating Medium (CDI). The cells were then plated onto 6-well tissue culture plates coated with 0.1% Gelatin (Sigma, USA) at a density of 2  106 cells in 2 or 3 mL of Maintenance Medium (CDI) per well. After incubation at 37
C in 5% CO2 for 48 h, the Plating Medium was replaced with a Maintenance Medium (CDI). Dead cells and non-attached cells were carefully rinsed off, and the plated cells were further cultured for an additional 5 days with the Maintenance Medium replaced after two or three days. The cells were next reseeded on MEA dishes, and fibronectin (Corning, USA, or Roche Diagnostics, Japan) was diluted 1:20 with D-PBS() to yield a 50 mg/mL coating solution. A 2 mL bead of coating solution was applied on the electrodes of 1-well or 6-well MEA dishes (200/30iR-Ti or 6wellMEA200/30iR-Ti-tcr, Multi Channel Systems, Germany), and the dishes were incubated at 37
C in 5% CO2 for at least 1 h. The cells were then dissociated from the 6-well plates using TrypLE™ Select (Life technologies, Japan) and reseeded in recording wells at a density of 1.5  104e3.0  104 cells in 2 mL bead of cell suspension per well. After incubation at 37
C in 5% CO2 for 1e3 h, each well was filled with Maintenance Medium that was changed every two or three day throughout the 7e10 day culture period. The gas concentration, frequency and volume of medium replacing and the cell density were modified from the protocol provided by CDI. 2.3. FP recording (MEA assay) An MEA dish was placed in a controlled chamber set at 37
C and perfused with 5% CO2/20% O2/N2. FPs were recorded on spontaneously beating iCell® Cardiomyocytes using an MEA60 or MEA2100 data acquisition system (MC-Rack, Multi Channel Systems) with second low-pass filter at 3000e3500 Hz and analyzed with a high pass filter (HPF) at 1 Hz (Fig. 1). FPs recording were started following an equilibration period of about 20 min. The stock solution of each test compound was adjusted to a proper concentration to achieve an escalating concentration design. Each test solution and vehicle (0.1% DMSO) was directly added to each well, and the medium was gently stirred with a pipette to avoid uneven distribution. The FP stabilized within 10 min upon vehicle and test compound application (data not shown). Thus, following exposure to the vehicle for 10 min to obtain base-values, test solutions were applied every 10 min to each recording well in a graded concentration manner. The final concentration of DMSO in the Maintenance Medium reached up to 0.5%. Each compound was tested in at least 4 wells. 2.4. Cell density variability Cell density variability was assessed using aspirin, E-4031 and terfenadine as test compounds and one cell lot (Lot No. 1033176) in Lab E. The cells were seeded at a density of 1.5  104 or 3.0  104 cells per well.

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

77

Table 1 Protocol for cell culture and FP recording.

Coating Culture conditions Buffer Cell concentration Culture dish Culture period

Pre-plating

Post-plating

0.1% Gelatin 5% CO2, 37
C iCell® Cardiomyocytes Plating Media (48 h), iCell® Cardiomyocytes Maintenance Media (5 days) 2  106 cells/well 6 well plate 7 days

50 mg/mL Fibronectin solution, 2 mL/well 5% CO2, 37
C iCell® Cardiomyocytes Maintenance Media 1.5  104e3.0  104 cells/2 mL 1 well (200/30iR-Ti) or 6 well MEA dish (6wellMEA200/30iR-Ti-tcr) 7e10 days

FP Recording Before recording Experimental conditions Buffer Vehicle Dosing Recording period Data analysis End points of assessment

Incubate for about 20 min to allow equilibration 5% CO2/20% O2/N2, 37
C iCell® Cardiomyocytes Maintenance Media 0.1% DMSO (Final concentration: up to 0.5%) Escalating dose design 10 min Last 30 beats of 10 min recording were analyzed at each drug concentration FPDc, FPDc10, Beat rate, Incidence of arrhythmia-like waveform and beat arrest

Table 2 Base-values of FPDc and beat rate in 0.1% DMSO.

n FPDc Beat rate (bpm)

Lab A

Lab B

Lab C

Lab D

Lab E

32 424 [397e447] 58 [55e65]

45 370 [357e410] 76 [49e81]

39 429 [404e451] 54 [49e61]

30 400 [374e430] 51 [50e55]

32 392 [371e431] 58 [52e62]

2.5. Inter-facility and cells lot-to-lot variability For assessment of inter-facility variability, a total of 5 testing facilities (Lab A, B, C, D and E) carried out the MEA assay on the same lot (Lot No.1091313) of iCell® Cardiomyocytes using all test compounds. Cells lot-to-lot variability was evaluated in two representative facilities (Labs A and E) using 4 lots of iCell® Cardiomyocytes (Lot Nos. 1091313, 1033176, 1093227 and 1094831) and 2 test compounds; E-4031 and terfenadine. Aspirin served as a negative control.

2.6. Data analysis Data acquisition system was standardized so that FP waveform could be analyzed on the same platform in all facilities. The waveform was generated with LabChart® (AD instruments, USA) and filtered at 1 Hz to eliminate electrical noise. FPD and inter-spike interval (ISI) were defined on the waveform as the time interval from the beginning of the sharp spike to the peak of the first positive deflection preceded by the sharp spike and between the peaks of 2 sharp spikes, respectively (Fig. 2a). FPDs were measured with the vehicle and all test compounds at various concentrations from the last 30 beats after 10 min exposure and corrected with ISI according to Fridericia's formula (FPDc ¼ FPD/ISI1/3). Beat rate baseline and FPDc baseline were shown as median, minimum and maximum values. Percentage change in beat rate and FPDc were calculated from the base-value of each trial as follows: percentage change (%) ¼ [measured value e base-value]/[base-value]. Test compound concentration that induced 10% prolongation/shortening of FPD (FPDc10) was calculated by logistic analysis with GraphPad Prism (GraphPad Software, USA). Incidence of arrhythmia-like waveform (EAD: Early after depolarization or TA: Triggered activity) and beat arrest were also obtained (Fig. 2b). Samples that induced arrhythmia-like waveforms and beat arrests

were not included in the data of FPDc and beat rate. 2.7. Statistical analysis All values of FPDc and FPDc10 were presented as mean ± SD. Comparison between all laboratories was conducted using analysis of variance (ANOVA) by Stat Preclinica (Takumi Information Technology, Japan) and values of p < 0.05 or <0.01 were regarded as statistical significance. In a part of data, FPDc10 was not calculated and excluded from statistical analysis in the cases as the following; 1) more than 10% prolongation or shortening of FPDc was not observed at any doses, and accompanied by arrhythmia-like waveforms or arrests, 2) more than 10% prolongation or shortening of FPDc and arrhythmia-like waveforms were not observed up to the highest concentrations, or 3) more than 10% prolongation or shortening of FPDc at all concentrations. 3. Results 3.1. Cell density variability As shown in Fig. 3, base-values for FPDc at the cell density of 1.5  104 were not different from those at the cell density of 3.0  104 (426e433 msec vs. 430e432 msec). Base-values for beat rate were also equal between two densities, 51e53 beats per minutes (bpm) vs. 52 bpm (data not shown). Aspirin had no effect on beat rate or FPDc at both densities. Although E-4031 had no effect on beat rate, it concentration-dependently prolonged FPDc at both cell densities at doses up to 1 nM. Terfenadine at doses up to 100 nM had no effect on beat rate at both cell densities. Although a concentration-dependent increase in FPDc was observed at the cell density of 3.0  104, FPDc-concentration curve was bell-shaped at the cell density of 1.5  104. FPDc change ratio at each concentration was almost the same at both cell densities. There was no

78

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

Fig. 1. MEA60 and MEA2100 systems provided by Multi Channel Systems. a) MEA60 and MEA2100, and b) 1-well and 6-well MEA dishes used in the MEA assay.

Fig. 2. Representative field potential waveforms. a) ISI: Inter-Spike Interval; the interval between the peaks of 2 sharp spikes, FPD: Field Potential Duration; the interval from the beginning of the sharp spike to the peak of the first deflection preceded by the sharp spike. b) Representative arrhythmia-like waveforms (EAD or TA).

statistical difference in FPDc10 of E-4031 and terfenadine between two cell densities.

3.2. Inter-facility variability The effects of test compounds on FP and on the occurrence of EAD or TA were evaluated in five facilities. The test compounds used were aspirin, E-4031, terfenadine, moxifloxacin, flecainide, chromanol 293B and verapamil (Fig. 4 and Table 3).

3.3. Base-value (0.1% DMSO) A slightly higher beat rate and a slightly shortened FPDc were obtained in Lab B as compared to those obtained in the other 4 facilities, where beat rate and FPDc were comparable (Table 2). Excluding Lab B, median base values of beat rate and FPDc were 51e58 bpm and 392e429 msec, respectively.

3.4. Aspirin Aspirin had no effect on beat rate or FPDc, and did not induce arrhythmia-like waveform (EAD or TA) at concentrations up to 100 mM in any facility, except for beat rate in Lab B.

3.5. E-4031 E-4031, a selective IKr blocker, prolonged FPDc in a concentration-dependent manner with FPDc10 of 0.68e5.06 nM, and FPDc10 showed statistical significance in inter-facility variability. In addition, E-4031 induced EAD or TA at 3e30 nM in all facilities, and produced beat arrest at 30 nM in 1 out of the 5 wells tested in Lab A. E-4031 decreased beat rate in Labs A and B. 3.6. Flecainide and moxifloxacin Flecainide and moxifloxacin, two multi-channel blockers, prolonged FPDc in a concentration-dependent manner in all facilities with FPDc10 of 0.03e0.39 mM and 4.71e96.0 mM, respectively. Flecainide showed a statistical significance in inter-facility variability, but moxifloxacin did not. Flecainide resulted in EAD or TA at 0.3e1 mM in all facilities, which was accompanied by beat arrest at 3 mM in 3 out of 3 wells in Lab A. Moxifloxacin produced EAD or TA at 30e300 mM; however, no beat arrest occurred at the same concentration range. Flecainide and moxifloxacin decreased beat rate in Labs B and C, and in Labs A, B, D and E, respectively. 3.7. Chromanol 293B Chromanol 293B, an IKs blocker, prolonged FPDc with FPDc10 of

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

79

Fig. 3. Cell density variability and morphological comparison (40) between cells plated at low and high density. a) The effects of test compounds on FPDc at each cell density. FPDc is presented as the average value ± SD (n ¼ 5e6). Morphological comparison of iCell® Cardiomyocytes at density of b) 0.75  104 cells/well, c) 1.5  104 cells/well and d) 3.0  104 cells/well.

11.2e56.8 mM with statistical significance. Beat arrest was evident at 100 mM in 3 out of 6 wells and 2 out of 4 wells in Labs B and C, respectively. However, chromanol 293B did not induce EAD or TA at concentrations up to 100 mM in any facilities. Chromanol 293B slightly increased beat rate in Labs B and C.

3.8. Verapamil Verapamil, a Ca2þ channel blocker with IKr inhibition, shortened FPDc in a concentration-dependent manner at 30 and 100 nM, but produced no EAD, TA or beat arrest at any concentrations. FPDc10 for FPDc shortening was 4.13e16.3 nM with no statistical significance. Verapamil concentration-dependently increased beat rate in all facilities.

3.9. Terfenadine Terfenadine, a multi-channel blocker with potent IKr inhibition, prolonged FPDc with an FPDc10 of 13.79e169.5 nM with statistical significance in inter-facility variability, however, unlike other IKr blockers, terfenadine did not affect beat rate. Terfenadine produced EAD or TA at 30e300 nM in Labs A, C and E, and beat arrest at 100 and 300 nM in Labs A and D, respectively. Based on the results obtained from all test compounds, variation in FPDc10 among the 5 testing facilities ranged from 3.9- to 20-folds, and it was about 10-folds for minimum concentration that induces EAD or TA (MCEAD/TA). Each value represents the median value in each facility. Values in the brackets represent the minimum and maximum in each facility.

3.10. Cells lot-to-lot variability Provided that no qualitative inter-facility variability was present, assessment of cells lot-to-lot variability was carried out by 2 representative facilities for 2 different lots each (Labs A and E). The results are shown in Table 4. In Lab A where Lot Nos.1093227 and 1094831 were compared, no apparent difference in effects on beat rate or FPDc was noted between the 2 lots for test compounds (aspirin, E-4031 and terfenadine). The variations in FPDc10 following treatment with E-4031 and terfenadine in Lab A were 1.7 and 3.7-folds with no statistical significance, respectively. E-4031 at 10 and 30 nM, but not terfenadine up to 300 nM, induced EAD or TA in both lots. Variability between Lot Nos. 1091313 and 1033176 was evaluated in Lab E. All three test compounds (aspirin, E-4031 and terfenadine) had comparable effects on beat rate and FPDc between two lots. The variations in FPDc10 for E-4031 and terfenadine were 2.3- and 1.1-folds, respectively, and FPDc10 of E-4031 had a statistical significance between two lots. E-4031 induced EAD or TA at 3 nM in both lots. On the other hand, terfenadine induced EAD or TA at 100 nM in Lot No.1091313, but produced no EAD or TA up to 100 nM in Lot No. 1033176. From the results above, cells lot-to-lot variability had no apparent influence on beat rate, FPDc, or EAD/TA. Variation in FPDc10 for E-4031 and terfenadine was within the acceptable range. 4. Discussions 4.1. Standardized protocol for MEA assay with iCell® cardiomyocytes It is imperative that the protocol be standardized when

80

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

Fig. 4. Inter-facility variability. The effects of test compounds on FPDc in each facility: a) aspirin, b) E-4031, c) flecainide, d) moxifloxacin, e) chromanol 293B, f) verapamil and g) terfenadine. FPDc is presented as the average value ± SD in each facility. n ¼ 4e9.

validation studies are carried out in multiple testing facilities. In our MEA assay using iCell® Cardiomyocytes, the protocol provided by CDI was further modified prior to pivotal studies to pay special attention to cells survival rate, seeding density, culture period, dish coating, gas and temperature control for FP measuring and data analysis (not all data are shown).

Cell survival rate can provide information on the quality of cell culture itself. In our assay, cell culture was remarkably improved by removing dead cells and floating/unattached cells 2 days after thawing. iCell® Cardiomyocytes were morphologically found to be larger when plated at a density of 7.5  103 cells/well than at 1.5  104 or

Table 3 Effects of test compounds on beat rate (BR), FPDc, FPDc10 and arrhythmia-like waveform in each facility. Concentration

Aspirin (mM)

E-4031 (nM)

Moxifloxacin (mM)

Chromanol 293B (mM)

FPDc (%)

0.7 0.0 3.4 3.1

0.6 1.1 5.0 2.5

e e e e

Lab C

Lab D

FPDc (%)

EAD or TA

BR (%)

FPDc (%)

EAD or TA

BR (%)

FPDc (%)

10.5 15.8 18.9 19.6

1.5 2.2 2.9 3.6

e e e e

0.9 0.8 0.4 0.4

2.1 2.5 3.1 3.1

e e e e

1.0 1.8 4.7 5.1

0.3 0.3 0.0 0.7

NC

NC

0.1 0.3 1.0 3.0 10.0 30.0

NT NT

NT NT 5.5 7.8 20.9 25.4

NT NT 7.3 7.2 11.2 e

0.5 1.7 12.4 29.4

e e e 2/5 arrest 1/5

3.3 8.5 17.7 17.5

FPDc10

4.16 ± 1.58

0.01 0.03 0.10 0.30 1.00 3.00

NT NT 0.5 3.9 7.5 e

FPDc10

0.389 ± 0.268

1 3 10 30 100 300

NT NT 0.1 2.4 4.4 14.5

FPDc10

96.0 ± 116.6

1 3 10 30 100

NT 0.1 2.4 3.1 3.0

1 3 10 30 100

3 10 30 100 300 FPDc10

e e e e

3.5 3.9 7.2 13.5

3.6 2.4 1.4 14.4

4.9 6.5 0.1 24.9

7.1 10.2 11.9 12.3

56.8 ± 42.2 7.3 14.3 34.2 53.2

e e e e

19.7 38.8 69.3 110.6

7.13 ± 1.42 1.3 2.4 6.7 e

e e 1/4 arrest 3/3

2.1 9.3 10.0 e NT

13.8 ± 10.5

0.6 3.0 0.5 e

e e 1/6 4/5

3.4 5.7 1.6 13.6

NT 1.6 3.0 15.7 56.9 NT

0.4 2.9 9.7 e

22.5 ± 9.3 NT 4.3 8.3 12.4 10.8

e e e arrest 3/6

4.6 3.9 0.4 10.4

NT 8.6 14.9 27.4 47.5

7.9 23.8 57.8 99.4

NT 1.3 3.1 7.6 14.2 170 ± 81

1.0 0.2 4.2 e

2.8 1.3 5.8 8.9

38.3 ± 7.2

2.0 4.1 4.1

e e e

e e e 4/4

3.2 6.7 14.7 e

1.0 1.7 5.4 e

e e e 4/4

2.2 7.6 15.5 e

1.2 4.6 17.5 e

e e 2/4 T unclear 2/2

6.1 15.4 36.6 e

0.5 0.7 5.8 23.7

18.1 ± 3.5 0.9 2.2 3.3 9.3

e e e e

3.5 8.9 15.4 17.9

0.4 0.2 1.6 1.9

15.6 ± 6.0 6.2 18.0 38.3 70.4

2.3 8.7 18.0 30.3

e e e e

8.2 14.9 43.5 76.8

14.61 ± 5.52 NT e e e 1/5

1.6 4.3 4.1 1.2

1.2 5.2 9.3 16.5

<0.01 e e e 4/4

3.1 7.5 17.5 41.0 NT NT

<0.05 e e e 3/4

2.3 5.0 9.9 18.1 NT

NS e e e e

11.2 ± 5.7

NT e e e e

4.7 10.8 21.8 e NT NT

4.71 ± 1.03

NT e e e arrest 2/4

e e e 4/4

0.0280 ± 0.0089

NT NT e 1/6 1/5 4/4

2.4 5.7 13.5 e NT NT

NC

0.681 ± 0.161 0.9 2.3 9.1 e

NT

NT 3.6 11.3 22.7 42.7

NT 4.5 7.2 13.6 11.0

1.7 3.0 2.5

0.161 ± 0.034

16.25 ± 13.15 e e e e

EAD or TA

NT

12.3 e e e e

FPDc (%)

1.73 ± 0.53 e e e 4/6

p

BR (%)

NC

NT

NT NT 6.1 14.6 35.9 e

NT 2.3 6.7 12.0 14.9

6.86 ± 2.92 0.1 0.6 1.4 2.5

e 2/5 e 3/3

NT 3.0 7.4 8.2 12.8 NT

24.0 ± 8.1

e e e e

NT 1.6 1.2 0.2 e

0.181 ± 0.168 e e e 1/6

EAD or TA

NC

5.06 ± 2.33

NT 1.5 6.5 14.5 27.7 NT

26.9 ± 15.1

NT 12.4 30.7 67.7 108.4

e e 2/6 3/4

0.192 ± 0.047

e e e 1/5

2.4 5.6 15.7 33.6

3.6 7.2 0.6 11.0

3.59

e e 2/5 arrest 3/3

2.7 9.8 18.2 e

Lab E

BR (%)

NC

FPDc10 Terfenadine (nM)

EAD or TA

FPDc10

FPDc10 Verapamil (nM)

Lab B

BR (%)

e e e arrest 1/6

95.7 ± 47.6

4.4 6.9 20.0 36.3 NT

<0.05 e e e e

4.13 ± 1.75 0.3 3.3 5.4 6.8

2.4 8.8 17.1 6.8 NT 14.2 ± 2.3

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

Flecainide (mM)

3 10 30 100

Lab A

NS e e e 2/5 <0.01

81

BR and FPDc: average percentage change from base-value, EAD or TA: incidence of arrhythmia-like waveform or arrest [number of incidence/number of wells], FPDc10: average concentration±SD. P values <0.05 and <0.01 are considered statistically significant in inter-facility variability of FPDc10. n ¼ 4e9. NT: Not tested, -: Not detected, N.C.: Not calculated, and N.S.: Not significant. T unclear: T wave was unrecognizable because of deformed waveforms.

82

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

Table 4 Cells lot-to-lot variability -Effects of test compounds on FPDc, FPDc10 and arrhythmia-like waveform in Lab A and E. Concentration

Lab A

Lab E

#1093227

#1094831

FPDc (%) Aspirin (mM)

3 10 30 100 FPDc10

E-4031 (nM)

0.1 0.3 1 3 10 30

EAD or TA

0.16 ± 4.69 0.0.99 ± 3.44 1.45 ± 3.62 1.91 ± 4.46

e e e e

3 10 30 100 300 FPDc10

FPDc (%) 1.77 2.64 2.44 1.52

± ± ± ±

EAD or TA

1.18 1.68 2.23 1.81

NC

NC

NT NT 4.23 ± 1.76 6.32 ± 2.26 10.07 ± 2.18 22.42

NT NT 3.83 ± 3.37 12.27 ± 6.96 15.64 28.55

e e 1/5 2/4

NT 3.40 ± 2.67 5.28 ± 2.61 7.46 ± 3.78 12.04 ± 2.69 160.33 ± 99.20

e e e e

NT 5.25 ± 3.55 8.44 ± 3.49 11.79 ± 2.75 14.86 ± 6.79 43.45 ± 29.33

#1091313

#1033176

FPDc (%)

e e e e

NT 2.03 ± 2.52 4.08 ± 1.92 4.14 ± 2.72 NC

NC

NS

FPDc (%)

e e e

NT 1.22 ± 2.64 3.55 ± 2.07 4.62 ± 2.25

14.17 ± 2.33

p EAD or TA e e e NC

e e e 4/4

0.66 ± 0.16 2.44 ± 1.52 8.82 ± 1.70 17.07 ± 3.37 6.84 ± 10.83 NT

e e e e

EAD or TA

NC 2.36 ± 0.71 5.71 ± 0.89 13.46 ± 2.49 e NT NT

e e 4/6 1/2

3.59 ± 2.59

FPDc10 Terfenadine (nM)

p

4.75 ± 1.58 10.01 ± 1.82 17.30 ± 6.09 e NT NT

NC e e e 8/8

0.30 ± 0.07 e e e 2/5

3.56 ± 2.34 10.26 ± 3.00 18.78 ± 6.77 3.67 ± 8.28 NT 12.63 ± 6.07

<0.01 e e e e NS

FPDc: average percentage change from base-value±SD, EAD or TA: incidence of arrhythmia-like waveform or arrest [number of incidence/number of wells], FPDc10: average concentration±SD. P value < 0.01 is considered statistically significant in lot-to-lot variability of FPDc10. n ¼ 4e9. NT: Not tested, -: Not detected, NC: Not calculated, and NS: Not significant.

3.0  104 cells/well (Fig. 3). hiPS-CMs plated at low density (3.75  103e7.5  103 cells/well) were enlarged showing significant change in gene expression level (Uesugi et al., 2013).This is a characteristic associated with progression of cardiac hypertrophy. Therefore, enlarged iCell® Cardiomyocytes are not representative of normal cardiomyocytes per se, and may respond to test compounds in a different manner as is the case with hypertrophic heart which is highly responsive to treatment. In addition, iCell® Cardiomyocytes show significantly lower beat rate and longer FPDc at a seeding density of 0.75  104 cells/well than at 1.5  104 and 3.0  104 cells/well. Although the seeding density recommended by CDI is 2.0  104 cells/well, a seeding density of 1.5  104e3.0  104 cells/well is generally used in CSAHi studies since no enlarged hiPS-CMs have been reported at densities 1.5  104 cells/well and since there was no apparent difference in the response to E-4031 and chromanol 293B when the cells were seeded at densities of 1.5  104 and 3.0  104 cells/well. In this CSAHi study, we also confirmed that the effects of test compounds (aspirin, E-4031 and terfenadine) on beat rate and FPDc at a cell density of 1.5  104 were not different from those at a density of 3.0  104 cells/well. According to CDI protocol, cells need to be cultured for 6e7 days for the pre-plating phase. In our CSAHi studies, the iCell® Cardiomyocytes were further reseeded on MEA dishes and cultured for another 7e10 days. This allowed the cells to have stable beat rate within 7 days of culture. iCell® Cardiomyocytes survival rate substantially decreased when the cells were plated on non-coated dishes. Thus, coating MEA dishes with fibronectin is an indispensable step. However, caution needs to be taken with the volume of coating solution as the cells may be washed away with too much coating solution, resulting in low cell density. MEA dishes were coated prior to seeding for more than 1 h at 37
C with 2 mL fibronectin solution (50 mg/mL). This volume of fibronectin was found to be appropriate. iCell® Cardiomyocytes need to be maintained under strictly controlled conditions to keep beat rate and FP stable. A time setting of equilibration period is absolutely essential to stabilize

beat rate before the start of FP recording because beat rate yields an appreciable influence on the base FP values. Slightly deviated data in Lab B may be a result from insufficient time setting of beat rate equilibration. Our standardized protocol requests 20 min to stabilize beat rate prior to initiation of FP recording. In our CSAHi studies, beat rate and FP were recorded over a 10 min period at 37
C and 5% CO2, since both parameters were stabilized within 10 min after compound application. Moreover, the strictly controlled conditions allowed longer recording (over one hour) for cumulative dosing. Since sharp spike and deflection on FP waveform are the result of depolarization and repolarization of iCell® Cardiomyocytes, respectively, FPD and ISI are regarded to reflect electrocardiogram QT interval and RR interval, respectively. Thus, FPD may better be corrected with ISI, and FPDc can be contrasted to QTc values. In our CSAHi studies, FPDc were obtained according to Fridericia's formula though those obtained according to Bazett's formula were comparable, probably due to the facts that beat rate was stable and varied within 49e81 bpm, a range similar to that in humans. However, accurate assessment of FPDc may await specific and validated formula in pursuit of predicting human relevance of cardiovascular liability on the basis of in vitro testing with hiPSCMs. 4.2. Validation of reference compounds Aspirin is known not to be arrhythmogenic and not to be associated with QT prolongation in clinical practice (Clements and Thomas, 2014). Thus, we included aspirin in our CSAHi studies as a negative control. Aspirin did not affect beat rate or FPD at any concentration (Fig. 4). These findings support the appropriate quality of the test system in that no false positive is expected for these endpoints and that iCell® Cardiomyocytes are stable throughout the evaluation period. E-4031, a selective IKr blocker and flecainide, a multi-channel blocker and potent IKr blocker, are reported to induce TdP in clinical practice (Clements and Thomas, 2014; Redfern et al., 2003). Our

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

results for these two test compounds are consistent with those reported in a hiPS-CMs study (Guo et al., 2011; Mehta et al., 2013). Both E-4031 and flecainide induced unequivocal FPDc prolongation and arrhythmia (EAD or TA waveform) even at low concentrations. Moxifloxacin, another multi-channel, including IKr blocker, is known to induce QT prolongation in clinical practice (Demolis et al., 2000). Although moxifloxacin also evoked FPDc prolongation and arrhythmia in our CSAHi study, these findings were confined to high concentrations. As moxifloxacin was used as a positive control in a thorough QT study due to its low potential for TdP, the arrhythmia induced by moxifloxacin in our assay is most likely due to a higher exposure than in clinical practice. Or, it may be possible that the arrhythmia-like waveform observed at high concentration is produced by converting with high HPF frequency, because the shape of the second peak, which contained the slow components of the current, was influenced by HPF frequency (Asakura et al., 2015). With regards to cell characteristics, the balance of ion channel expression, including hERG and Ca2þ channel may be distinct in iCell® Cardiomyocytes from that in adults cardiomyocytes, leading to a gap in the concentrations associated with arrhythmia between hiPS-CMs and humans. This hypothesis would warrant further investigation. Chromanol 293B is a selective IKs blocker, and IKs channel is known to be associated with QT syndrome (Volders et al., 2003). FPDc was clearly prolonged in a dose-dependent manner and beat arrest was also evident at 100 mM. It was indicated that our test system is valid for detecting even IKs blockers potential for QT prolongation in clinical setting. In clinical practice, verapamil, a potent Ca2þ channel blocker and IKr blocker, is known to decrease heart rate. In our study, on the other hand, verapamil increased beat rate and shortened FPDc. It is of interest to note that verapamil-induced increase in beat rate was accompanied by shortening of APD in hiPS-CMs (Harris et al., 2013), and that verapamil also increases beat rate in cardiomyocytes derived from human embryonic stem (hES) cells (Clements and Thomas, 2014). Both of these findings are in line with our results. Verapamil decreases beat rate by suppressing the rate of impulse generation in the sinoatrial node (Stark et al., 1988). This nodal effect was not present in the CSAHi study, implying that beat impulse may not be initiated by nodal-like cells in iCell® Cardiomyocytes. Looking at these results from a different perspective, hiPS-CMs may be considered as immature cells with similar gene expression patterns to fatal cardiomyocytes (Guo et al., 2011). It may be possible that hiPS-CMs distinct from that in adult cardiomyocytes. Terfenadine is a multi-channel blocker with potent IKr inhibition. This drug is unique in that its arrhythmogenicity can hardly be detected both in vitro and in vivo non-clinical studies. In the CSAHi study, terfenadine prolonged FPDc and produced EAD or TA in all facilities (except for LabB), although FPDc-concentration curve was bell-shaped in the iCell® Cardiomyocytes used in two facilities (Lab C and E). In studies using hES cell-derived cardiomyocytes, terfenadine prolonged FPDc in a dose-dependent manner at doses up to 300 nM, although this prolonging effect was attenuated at 1000 nM, resulting in bell-shaped response curve similar to that obtained with iCell® Cardiomyocytes (Clements and Thomas, 2014). However, this response might be induced by converting the waveforms with HPF frequency (Asakura et al., 2015). In that report, terfenadine showed concentration-dependent FPDc prolongation with 0.1-Hz HPF, but bell-shaped concentration-response curve with 1-Hz HPF. Distortion of the second peak in the FP at 1-Hz might have influenced the concentration of EAD or TA, as well as FPDc prolongation. The reason for this discrepancy remains unclear, and further studies are needed to elucidate terfenadine interaction with iCell® Cardiomyocytes.

83

Average FPDc10s [a] for E-4031, flecainide, moxifloxacin and terfenadine in all testing facilities were comparable with the unbound plasma concentration leading to QT prolongation or TdP in humans [b] (Table 5). The [a]/[b] ratio for each compound was: [0.2e0.6] for E-4031, [0.9] for flecainide, [3.3] for moxifloxacin, and [27.5], as the highest ratio, for terfenadine. In our previous report, terfenadine significantly prolonged FPDc at 10 and 30 nM and induced arrhythmia at 30 nM following 20-min exposure (Nozaki et al., 2014). These concentrations are lower than FPDc10 of 66.4 nM obtained in this study where only a 10-min exposure was used. When FPDc prolongation was compared between these 2 protocols, the effect was stronger at 20-min exposure than at 10min exposure (data not shown). Thus, it is suggested that terfenadine, due to its pharmacological characteristics, needs long exposure to exert prominent cardiovascular effects. Overall, the results of our CSAHi study reflect well the known proarrhythmic effects of all test compounds in clinical practice, indicating that combination of multi-electrode array (MEA60/2100) and hiPs-CMs is a valid test system for in vitro evaluation of druginduced proarrhythmia. 4.3. Inter-facility variability A new test system needs to provide consistent outcome at various testing facilities. This is particularly critical for new drug development from both scientific as well as regulatory perspectives. MEA assay for detecting drug-induced proarrhythmia in hiPSCMs is one of the promising candidates expected to be integrated in the revised S7B guidelines. A multisite CSAHi study would enable assessment of inter-facility variability. No meaningful qualitative inter-facility variability was observed for all compounds that induced FPD prolongation or shortening, and EAD or TA. Our quantitative inter-facility variability was assessed based on FPDc10 and MCEAD/TA obtained for each test compound at each testing facility (Fig. 5). Deviation in FPDc10 among test compounds that prolonged or shortened FPD ranged from 3.9- to 20-folds, and multi-channel blockers showed a tendency to have relatively larger variability than the others. In the statistical analysis, E-4031, flecainide, chromanol 293B and terfenadine had a statistical significance in FPDc10 in inter-facility variability. It was conceivable that this assay could keep the value for the reasons mentioned below; 1) this assay could detect the potential effects of reference compounds on FPDc with no qualitative inter-facility variability, 2) more than 10-folds differences in IC50 of hERG assay were reported in several compounds, however, it has been used for an essential part of arrhythmogenicity risk assessment in combination with in vivo telemetry study (Kirsch et al., 2004). The variability in hERG assay must be produced by the different protocols in each facility, whereas, that in MEA assay might be influenced by the experiment conditions even under the same protocol. It was considered that this assay would be acceptable for a new platform to assess the risk of arrhythmogenicity and QT liability. The deviation in MCEAD/TA was small when EAD or TA occurred within the same- or 2 next-concentration range. Since an escalated concentration scheme was used with a common ratio of 3, it is concluded that MCEAD/TA inter-facility variability remained within approximately 10-folds for all test compounds that induced EAD or TA. It may be argued whether these ranges of variation are within acceptable limits for the proposed in vitro test system. Taking into account that the test protocol was standardized during the CSAHi study, that open practice was carried out in laboratories to demonstrate details of the testing procedures, and that welltrained individuals were assigned to the study, one can conclude that the inter-facility variability found in this CSAHi study may

84

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

Table 5 Relation between test compound concentrations that induced FPDc prolongation and effective free therapeutic plasma concentrations. in vitro iPS (nM) FPDc10 Min-Max [Ave.] Negative control Aspirin NC IKr blocker E-4031 0.68e5.06 [3.05] IKs blocker Chromanol 11,200e56,800 293B [24,600] 2þ Ca channel blocker Verapamil 4.13e16.3 (Shortened) [9.80] Naþ channel blocker Flecainide 28e389 [190] Multichannel blockers Moxifloxacin 4710e96,970 [33100] Terfenadine 13.79e169.6 [66.38]

in vitro IC50 (nM)

Reference

NE

ND

ND

ND

>10,000 mM

NT

ND

b), d)

3e30/30

5e20, 400

ND

ND

20a

5.95

6.38e19.3

b), d), g), l)

NE/100,000

>100,000 IKs IC50 ¼ 1800

ND

ND

ND

ND

ND

b), d), f)

32500

>100,000 (Shortened)

NE

ND

b), d)

6200

>9800

NE (>1900)

278

b), c), d), i), j)

173000 1112000

40,000b

5916c

7221

a), b), c), d), h), k)

930

>20,000

2.40

2.73e3.63

b), d), e), g), l)

300e1000/3000

4000, 1500

30,000e300,000/NE

86,200

30e300/100-300

50, 20-200

27100

2000

GP APD90

Unbound plasma/serum compound conc. in humans with QT QT prolongation prolongation or TdP in conscious dog (nmol/L)

hERG

250, 100-800 200

Nav1.5

in vivo EC10 unbound (nM)

Range of Arrhythmia/Arrest

NE/NE

Cav1.2

in vitro EC10 (nM)

NC: Not calculated, ND: No data, NT: Not tested, NE: No effect. a) Chaves et al., 2007; b) Kramer et al., 2013; c) Morissette et al., 2013; d) Omata et al., 2005; e) Redfern et al., 2003; f) Sun et al., 2001; g) Toyoshima et al., 2005, h) Hagiwara et al., 2001; i) Heath et al., 2011; j) Sallstrom et al., 2014; k) Siefert et al., 1999; l) Webster et al., 2001. a Increase in APD90 of more than 10% was observed for at least one concentration level. b Ventricular myocardia of guinea pigs were used in this assay. EC10 value was estimated from the results at 10 and 100 mM. c 12% QT prolongation was observed in conscious dogs.

Fig. 5. Comparison of concentrations producing FPDc prolongation, arrhythmia, APD90 prolongation, QT prolongation, or TdP in MEA assay, in vitro or in vivo assay. Blue diamond and aqua bar, FPDc10 and concentration range for FPDc10 in MEA assay; red square, EC10 for APD90 in guinea pig papillary muscles; yellow triangle, EC10 for QTc prolongation in conscious dogs; purple bar, the concentration range for arrhythmia in MEA assay. The reference data in Table 5 were used for data of EC10 in guinea pigs APD assay and QTc EC10 in conscious dogs. y; Ventricular myocardia of guinea pig were used for the assay, and the EC10 value was estimated from the results at 10 and 100 mM. #: The EC10 value was higher than 9800 nM for flecainide and 20,000 nM for terfenadine. x: The EC10 for QTc in conscious dogs was higher than 1900 nM as flecainide had no effect on QTc at free plasma concentration of 1900 nM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

establish a standard for variation range in future studies. Yet, there may still be room to improve the proposed protocol, especially waveform analysis, so that inter-facility variability can further be minimized. 4.4. Cells lot-to-lot variability Along with inter-facility variability, we also validated cells lotto-lot variability to adopt the MEA assay to the new guidelines. A

comparison of the effects of aspirin, E-4031 and terfenadine on different lots of iCell® Cardiomyocytes in each facility resulted in no substantial difference except for E-4031 in Lab E, supporting the notion that iCell® Cardiomyocytes are qualitatively uniform among lots when evaluating QT risk and/or arrhythmogenicity of new drug candidates. In Lab E, E-4031 showed a statistical significance, but the difference of FPDc10 between two lots was 2.3-folds and it was not supposed to be a meaningful difference. As E-4031 induced EAD or TA in 3 out of 5 wells without more than 10% FPDc prolongation

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

in Lab A for Lot No.1093227, FPDc10 was described as average value with no standard deviation. 4.5. Comparison of MEA assay with APD assay, and with in vivo assay To further validate MEA assay, E-4031, flecainide, moxifloxacin and terfenadine, all of which induce FPDc prolongation were subjected to comparison of FPDc10 and MCEAD/TA with effective plasma concentration of 10% prolongation (EC10) of QT in conscious dogs and EC10 of APD90 in guinea pig papillary muscle (Fig. 5). FPDc10 and MCEAD/TA were comparable with EC10s of APD90 prolongation in guinea pigs and with QT prolongation in conscious dogs for E-4031 and moxifloxacin, whereas, FPDc10 was lower than EC10 of APD90 in guinea pigs for flecainide and terfenadine. In contrast, FPDc10 and MCEAD/TA were higher than EC10 of QT prolongation in conscious dogs for terfenadine, and EC10 was not detectable due to flecainide weak effect in conscious dogs. Proarrhythmia of multichannel blockers is generally difficult to detect in the currently available non-clinical test system. EC10 of APD90 in guinea pig papillary muscle has not yet been determined as high as 9.8 and 20 mM for flecainide and terfenadine, respectively, and no proarrhythmic waveform was observed in an in vitro study (Omata et al., 2005). Terfenadine induced evident QT prolongation which was not accompanied by TdP or proarrhythmia in in vivo telemetry studies in dogs or monkeys (Ando et al., 2005; Toyoshima et al., 2005). On the other hand, flecainide is reported to induce neither QT prolongation nor arrhythmia in a telemetry dog study (Toyoshima et al., 2005). The present MEA assay has resolved this dilemma where FPDc prolongation and EAD or TA waveforms were present in both terfenadine and flecainide for which even EC10 of APD90 cannot be established in guinea pig papillary muscle. Our CSAHi study using hiPS-CMs also overcomes a drawback for IKs blockers. Chromanol 293B showed no influence on APD of cardiomyocytes in dogs (Varro et al., 2000) in spite of clinical manifestation of QT syndrome (Volders et al., 2003). Another IKs blocker, HMR-1556, prolonged APD in human cardiomyocytes under sympathetic nervous activation with adrenaline and IKr inhibition with dofetilide (Jost et al., 2005). These differences are most likely attributed to interspecies IKs current variation between dog and human (Dorian and Newman, 2000). Taken all together, the MEA assay is by far superior to the currently available non-clinical test systems in light of concordance of clinical relevance. 5. Conclusion MEA system in combination with hiPS-CMs is becoming a standard tool for assessment of QT risk and/or arrhythmogenicity. Compared to in vivo assessment of cardiovascular events, the advantages of this system are: 1) non-invasive long-term recording, 2) saving on the amount of test compounds, 3) no concern with interspecies variation and 4) better predictability of QT liability for multi-ion channel blockers, which affect cardiac net current complexity. CSAHi study using MEA60/2100 has revealed that the test system is valuable for in vitro arrhythmogenic assessment due to the following four reasons: 1) this assay can detect arrhythmogenicity and/or QT liability for pure hERG blockers and multichannel blockers that induce TdP in clinical practice, 2) FPDc10 in inter-facility variability ranged from 3.9- to 20-folds, but qualitative difference in the effects of compounds was not observed, 3) there is no substantial cells lot-to-lot variability in iCell® Cardiomyocytes, and 4) this assay can predict arrhythmogenic risks that cannot be detected by currently available non-clinical test systems. Thus, we

85

conclude that this test system would be promising for risk assessment of arrhythmogenicity and QT liability in safety pharmacology studies. Conflicts of interest We confirmed that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. The manuscript has been read and approved by all named authors. Acknowledgement We thank iPS PORTAL, Inc. for providing iCell® Cardiomyocytes, and Bio Research Center Co., Ltd. for their technical assistance. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2016.02.007. References Ando, K., Hombo, T., Kanno, A., Ikeda, H., Imaizumi, M., Shimizu, N., , et al.Yamamoto, K., 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 (5), 487e500. Asakura, K., Hayashi, S., Ojima, A., Taniguchi, T., Miyamoto, N., Nakamori, C., , et al.Sawada, K., 2015. Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes. J. Pharmacol. Toxicol. Methods. http://dx.doi.org/10.1016/j.vascn.2015.04.002. Cavero, I., 2014. 13th annual meeting of the safety pharmacology society: focus on novel technologies and safety pharmacology frontiers. Expert Opin. Drug Saf. 13 (9), 1271e1281. http://dx.doi.org/10.1517/14740338.2014.940310. Chaves, A.A., Zingaro, G.J., Yordy, M.A., Bustard, K.A., O'Sullivan, S., GalijatovicIdrizbegovic, A., , et al.Briscoe, R.J., 2007. A highly sensitive canine telemetry model for detection of QT interval prolongation: studies with moxifloxacin, haloperidol and MK-499. J. Pharmacol. Toxicol. Methods 56 (2), 103e114. http:// dx.doi.org/10.1016/j.vascn.2007.04.007. Clements, M., Thomas, N., 2014. High-throughput multi-parameter profiling of electrophysiological drug effects in human embryonic stem cell derived cardiomyocytes using multi-electrode arrays. Toxicol. Sci. 140 (2), 445e461. http:// dx.doi.org/10.1093/toxsci/kfu084. Demolis, J.L., Kubitza, D., Tenneze, L., Funck-Brentano, C., 2000. Effect of a single oral dose of moxifloxacin (400 mg and 800 mg) on ventricular repolarization in healthy subjects. Clin. Pharmacol. Ther. 68 (6), 658e666. http://dx.doi.org/ 10.1067/mcp.2000.111482. Dorian, P., Newman, D., 2000. Rate dependence of the effect of antiarrhythmic drugs delaying cardiac repolarization: an overview. Europace 2 (4), 277e285. http://dx.doi.org/10.1053/eupc.2000.0114. Guo, L., Abrams, R.M., Babiarz, J.E., Cohen, J.D., Kameoka, S., Sanders, M.J., , et al.Kolaja, K.L., 2011. Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes. Toxicol. Sci. 123 (1), 281e289. http://dx.doi.org/10.1093/toxsci/kfr158. Hagiwara, T., Satoh, S., Kasai, Y., Takasuna, K., 2001. A comparative study of the fluoroquinolone antibacterial agents on the action potential duration in guinea pig ventricular myocardia. Jpn. J. Pharmacol. 87 (3), 231e234. Harris, K., Aylott, M., Cui, Y., Louttit, J.B., McMahon, N.C., Sridhar, A., 2013. Comparison of electrophysiological data from human-induced pluripotent stem cell-derived cardiomyocytes to functional preclinical safety assays. Toxicol. Sci. 134 (2), 412e426. http://dx.doi.org/10.1093/toxsci/kft113. Heath, B.M., Cui, Y., Worton, S., Lawton, B., Ward, G., Ballini, E., , et al.McMahon, N.C., 2011. Translation of flecainide- and mexiletine-induced cardiac sodium channel inhibition and ventricular conduction slowing from nonclinical models to clinical. J. Pharmacol. Toxicol. Methods 63 (3), 258e268. http://dx.doi.org/ 10.1016/j.vascn.2010.12.004. Jost, N., Virag, L., Bitay, M., Takacs, J., Lengyel, C., Biliczki, P., , et al.Varro, A., 2005. Restricting excessive cardiac action potential and QT prolongation: a vital role for IKs in human ventricular muscle. Circulation 112 (10), 1392e1399. http:// dx.doi.org/10.1161/circulationaha.105.550111. Kirsch, G.E., Trepakova, E.S., Brimecombe, J.C., Sidach, S.S., Erickson, H.D., Kochan, M.C., , et al.Brown, A.M., 2004. Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern. J. Pharmacol. Toxicol. Methods 50 (2), 93e101. http://dx.doi.org/10.1016/ j.vascn.2004.06.003. Kitaguchi, T., Moriyama, Y., Taniguchi, T., Ojima, A., Ando, H., Uda, T., Otabe, K.,

86

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 77 (2016) 75e86

Oguchi, M., Shimizu, S., Saito, H., Morita, M., Toratani, A., Asayama, M., Yamamoto, W., Matsumoto, E., Saji, D., Ohnaka, H., Washio, I., Miyamoto, N., 2016. CSAHi study: evaluation of multi-electrode array in combination with human iPS cell-derived cardiomyocytes to predict drug-induced QT prolongation and arrhythmia with 7 reference compounds at 10 facilities. J. Pharmacol. Toxicol. Methods 78, 93e102. Kramer, J., Obejero-Paz, C.A., Myatt, G., Kuryshev, Y.A., Bruening-Wright, A., Verducci, J.S., Brown, A.M., 2013. MICE models: superior to the HERG model in predicting Torsade de Pointes. Sci. Rep. 3, 2100. http://dx.doi.org/10.1038/ srep02100. Mehta, A., Chung, Y., Sequiera, G.L., Wong, P., Liew, R., Shim, W., 2013. Pharmacoelectrophysiology of viral-free induced pluripotent stem cell-derived human cardiomyocytes. Toxicol. Sci. 131 (2), 458e469. http://dx.doi.org/10.1093/toxsci/ kfs309. Morissette, P., Nishida, M., Trepakova, E., Imredy, J., Lagrutta, A., Chaves, A., , et al.Salata, J.J., 2013. The anesthetized guinea pig: an effective early cardiovascular derisking and lead optimization model. J. Pharmacol. Toxicol. Methods 68 (1), 137e149. http://dx.doi.org/10.1016/j.vascn.2013.04.010. Nozaki, Y., Honda, Y., Tsujimoto, S., Watanabe, H., Kunimatsu, T., Funabashi, H., 2014. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation. Toxicol. Appl. Pharmacol. 278 (1), 72e77. http://dx.doi.org/10.1016/j.taap.2014.04.007. Omata, T., Kasai, C., Hashimoto, M., Hombo, T., Yamamoto, K., 2005. QT PRODACT: comparison of non-clinical studies for drug-induced delay in ventricular repolarization and their role in safety evaluation in humans. J. Pharmacol. Sci. 99 (5), 531e541. Redfern, W.S., Carlsson, L., Davis, A.S., Lynch, W.G., MacKenzie, I., Palethorpe, S., , et al.Hammond, T.G., 2003. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc. Res. 58 (1), 32e45. Sager, P.T., Gintant, G., Turner, J.R., Pettit, S., Stockbridge, N., 2014. Rechanneling the cardiac proarrhythmia safety paradigm: a meeting report from the cardiac safety research consortium. Am. Heart J. 167 (3), 292e300. http://dx.doi.org/ 10.1016/j.ahj.2013.11.004. Sallstrom, J., Al-Saffar, A., Pehrson, R., 2014. Pharmacokinetic-pharmacodynamic modeling of QRS-prolongation by flecainide: heart rate-dependent effects

during sinus rhythm in conscious telemetered dogs. J. Pharmacol. Toxicol. Methods 69 (1), 24e29. http://dx.doi.org/10.1016/j.vascn.2013.10.001. Siefert, H.M., Domdey-Bette, A., Henninger, K., Hucke, F., Kohlsdorfer, C., Stass, H.H., 1999. Pharmacokinetics of the 8-methoxyquinolone, moxifloxacin: a comparison in humans and other mammalian species. J. Antimicrob. Chemother. 43 (Suppl. B), 69e76. Stark, G., Stark, U., Tritthart, H.A., 1988. Modulation of cardiac impulse generation and conduction by nifedipine and verapamil analyzed by a refined surface ECG technique in Langendorff perfused guinea pig hearts. Basic Res. Cardiol. 83 (2), 202e212. Sun, Z.Q., Thomas, G.P., Antzelevitch, C., 2001. Chromanol 293B inhibits slowly activating delayed rectifier and transient outward currents in canine left ventricular myocytes. J. Cardiovasc. Electrophysiol. 12 (4), 472e478. Takasuna, K., Katsuyoshi, Chiba, Manabe, a.S., 2009. Pre-clinical QT risk assessment in pharmaceutical companies - issues of current QT risk assessment -. Biomol. Ther. 17 (1), 1e11. http://dx.doi.org/10.4062/biomolther.2009.17.1.1. Toyoshima, S., Kanno, A., Kitayama, T., Sekiya, K., Nakai, K., Haruna, M., , et al.Yamamoto, K., 2005. QT PRODACT: in vivo QT assay in the conscious dog for assessing the potential for QT interval prolongation by human pharmaceuticals. J. Pharmacol. Sci. 99 (5), 459e471. Uesugi, M., Ojima, A., Taniguchi, T., Miyamoto, N., Sawada, K., 2013. Low-density plating is sufficient to induce cardiac hypertrophy and electrical remodeling in highly purified human iPS cell-derived cardiomyocytes. J. Pharmacol. Toxicol. Methods. http://dx.doi.org/10.1016/j.vascn.2013.11.002. Varro, A., Balati, B., Iost, N., Takacs, J., Virag, L., Lathrop, D.A., , et al.Papp, J.G., 2000. The role of the delayed rectifier component IKs in dog ventricular muscle and Purkinje fibre repolarization. J. Physiol. 523 (Pt 1), 67e81. Volders, P.G., Stengl, M., van Opstal, J.M., Gerlach, U., Spatjens, R.L., Beekman, J.D., , et al.Vos, M.A., 2003. Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation. Circulation 107 (21), 2753e2760. http://dx.doi.org/10.1161/01.cir.0000068344.54010.b3. Webster, R., Allan, G., Anto-Awuakye, K., Harrison, A., Kidd, T., Leishman, D., , et al.Walker, D., 2001. Pharmacokinetic/pharmacodynamic assessment of the effects of E4031, cisapride, terfenadine and terodiline on monophasic action potential duration in dog. Xenobiotica 31 (8e9), 633e650. http://dx.doi.org/ 10.1080/00498250110054632.