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Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update
Journal of Pharmacological Sciences xxx (xxxx) xxx
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Current Perspective
Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update Yasunari Kanda a, b, *, Daiju Yamazaki a, b, Tomoharu Osada b, c, Takashi Yoshinaga b, d, Kohei Sawada b, e a
Division of Pharmacology, National Institute of Health Sciences (NIHS), Kanagawa, 210-9501, Japan Japan iPS Cardiac Safety Assessment (JiCSA), Japan LSI Medience Corporation, Chiyoda-ku, Tokyo, 101-8517, Japan d Eisai Co., Ltd, Tsukuba, Ibaraki, 300-2635, Japan e The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, 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 1 October 2018 Received in revised form 18 October 2018 Accepted 19 October 2018 Available online xxx
Cardiac safety assessment is challenging because a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation is necessary for patient safety. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a new human cell-based platform to assess cardiac safety in non-clinical testing during drug development. The multi-electrode array (MEA) platform is a promising electrophysiological technology to assess QT interval prolongation and proarrhythmic potential of drug candidates using hiPSC-CMs. The Japan iPS Cardiac Safety Assessment (JiCSA) has established an MEA protocol to evaluate the applicability of hiPSC-CMs for assessing the torsadogenic potential of compounds and completed a large-scale validation study using 60 compounds. During our study, an international multi-site study of hiPSC-CMs was performed by the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative using 28 compounds. We have comparatively analyzed our JiCSA datasets with those of CiPA using the CiPA logistical and ordinal linear regression model. Regardless of the protocol differences, the evaluation results of the 28 compounds were very similar and highly predictable for torsadogenic risks. Thus, an MEA-based approach using hiPSC-CMs would be a standard testing method to evaluate proarrhythmic potentials. This review paper would provide new insights into the hiPSC-CMs/MEA method required for its regulatory use. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Cardiac safety CiPA Human iPS cells JiCSA Standardization
1. Introduction
Abbreviations: CiPA, Comprehensive in Vitro Proarrhythmia Assay; EAD, early after depolarization; fETPC, free effective therapeutic plasma concentration; FPD, field potential duration; FPDcF, FPD with Fridericia's correction; hERG, human -go-go-related gene; hiPSC-CMs, human induced pluripotent stem cellether-a derived cardiomyocytes; JiCSA, Japan iPS Cardiac Safety Assessment; MEA, multielectrode array; TdP, torsade de pointes. * Corresponding author. Division of Pharmacology, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan. Fax: þ81 44 270 1065. E-mail address: [email protected] (Y. Kanda). Peer review under responsibility of Japanese Pharmacological Society.
Cardiotoxicity is one of the major causes of drug attrition at later stages of drug development.1 Therefore, it is important to evaluate the potential for QT interval prolongation in humans before clinical trials. The risk of drug-induced QT interval prolongation has been assessed by evaluating the ability to block the human ether- a-go-go related gene (hERG) channel and prolong ventricular repolarization and the QT interval of the electrocardiogram (ECG) in non-clinical and clinical studies.2,3 Numerous studies have been performed towards implementation of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B guideline, which includes the “QT PRODACT” (Database Construction for the Evaluation of the Risk of QT Interval
https://doi.org/10.1016/j.jphs.2018.10.010 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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Prolongation by Drugs in Japan) organized by the Japan Pharmaceutical Manufacturers Association, and in vivo and in vitro experiments. The importance of the studies is to show validated data of the assessment of drug-induced QT interval prolongation under common protocols with multi-sites as safety pharmacology studies. Currently, the ICH S7B and E14 have been successful. There have not been any withdrawals of marketed drugs for torsadogenic concerns since they were adopted. However, limitations of the protocols implemented under the guidelines have been widely recognized. Assessment of QT interval prolongation under the current protocols is unduly sensitive and not specific for predicting the risk of ventricular proarrhythmia. Therefore, drugs with inhibitory effects on hERG, with QT interval prolongation, and minimal actual TdP risk are deprioritized or excluded from further development or, even if approved, their clinical use might be limited by inappropriate warnings on their product label. Therefore, it is necessary to explore a new paradigm for a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation. Thus, the current cardiac safety paradigm is considered to be inappropriate for assigning TdP liability. The degree of QT interval prolongation, except for pure hERG blockers, is largely drugspecific, and the QT interval can be influenced by many factors (for example, drugedrug interaction, drug metabolism, autonomic perturbation, glucose levels, and sex difference). Since the ICH S7B guideline defines the safety risk associated with hERG or QT signals, the detection of even a small inhibitory effect under the hERG assay may result in adverse internal or regulatory consideration during drug development. Thus, many pharmaceutical companies may have unnecessarily terminated the development of potentially useful drug candidates.4 Human induced pluripotent stem cell (hiPSC) technology paves the way for the establishment of new in vitro testing technologies using human cells in drug development. Because human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can express hERG and other types of cardiac ion channels, the technology can be applied for safety evaluation at early or late-stage non-clinical testing during drug development. The Plan for Promotion of Medical Research and Development was approved by the Headquarters for Healthcare Policy in 2014 in Japan.5 The Japanese government has decided to establish next-generation safety evaluation methods for drugs using iPSC technology. In particular, it proposes to implement a nationwide collaboration among industry, academia, and government focused on the development of standardized cells and testing methods for evaluating cardiotoxicity. To establish new reliable testing methods using hiPSC-CMs, validation studies are required to evaluate their reproducibility, reliability, and robustness. The validated methods can then be implemented in the reviewing processes of the regulatory consideration on drug candidates. Here, we review the current status of large-scale validation studies conducted in Japan and a parallel international study including additional analysis results using free drug concentrations of JiCSA study. Further activities toward the implementation of a validated method to assess cardiotoxicity in the drug development are also discussed. 2. JiCSA validation study 2.1. Compound selection The Japan iPS Cardiac Safety Assessment (JiCSA) was established in 2014 to develop a new paradigm for assessing drug-induced proarrhythmia risk, based on the emergence of cutting-edge iPSC technology.6 Based on a formal validation process (such as OECD guidance document #34), National Institute of Health Sciences
(NIHS) brought experts from regulatory, industry, and academic fields to develop and validate a new testing method that allowed a more precise prediction of clinical proarrhythmia risk as a lead laboratory. To examine sensitivity to key currents in the hiPSC-CMs that affect ventricular repolarization and minimize site-to-site variability prior to the validation study, we performed a pilot study using E-4031 and cisapride as cardiac delayed rectifier Kþ channel (IKr) blockers, the IKs blocker chromanol 293B, and DMSO, vehicle control) as a set of training compounds.7 Then, following discussions, the JiCSA selected 60 compounds with different TdP risks in collaboration with the Japanese Safety Pharmacology Society to perform a large-scale validation. The Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm has been proposed to improve cardiac safety for regulatory approval in the US.8 It aims to replace the non-clinical hERG assay required in the ICH S7B with more translationally relevant assessments of proarrhythmia risk. The international consortium CiPA working groups selected and categorized 28 compounds as having high, intermediate, and low risks of TdP based on published reports, analysis of the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database, other data sources, and clinical opinions (Table 1). The JiCSA 60 compounds overlapped with almost all of the 28 CiPA compounds except for azimilide, which was not available in Japan when the study commenced. The data of azimilide were added to the JiCSA study later and are discussed in this paper with the 28 compounds. 2.2. Large-scale validation study using multi-electrode array (MEA) platform JiCSA developed a standardized protocol using a multi-electrode array (MEA) platform, which can easily record the electrophysiological activity of CM monolayer sheets, and has shown the interfacility validation among laboratories in the industry, government, and academia.9 Based on the pre-validation study, protocols were optimized and improved for the large-scale validation study.10 Furthermore, the clinical properties of the hiPSC-CMs, such as QTRR relationship, were electrophysiologically characterized. The relationship between field potential duration (FPD) and inter-spike interval (ISI) in hiPSC-CMs was very similar with that between QT and RR of human ECG in the Framingham heart study derived from data of approximately 5000 people.7 Moreover, responses of E4031 in hiPSC-CMs were similar to those of the human heart.7 Therefore, hiPSC-CMs are considered to share similar electrophysiological properties to human hearts and be applicable for the assessment of drug effects in humans. Next, a large-scale validation study was performed for 60 compounds with different TdP risks using commercially available hiPSC-CMs (iCell-CMs) and an MEA platform (MED64). We have proposed the TdP scoring system by a two-dimensional (2D) map. Based on the previous paper,1 we calculated margins of each drug between free fraction in medium and free effective therapeutic plasma concentration (fETPC). We also provided relative TdP score to each compound according to the extent of FPDcF change or early after depolarization (EAD) occurrence. EAD is one of the mechanisms by which drugs cause TdP after QT interval prolongation.11 When EAD triggers upstrokes in tissue and repolarization is sufficiently disturbed, drugs will initiate TdP. As shown in Fig. 1A, the 28 compounds were categorized as high-, intermediate-, or low-risks using a 2D map of relative TdP scores and the margins. The data using iCell-CMs results showed good concordance with a public TdP database CredibleMeds (Fig. 1B, Table 2).12 Because druginduced arrhythmia has been extensively examined by the MEA assays in the single cell-line (iCell), we also examined the
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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Table 1 Compound lists of JiCSA study. List of compounds used in the JiCSA study. Colored compounds overlapped with those in the CiPA study (red; high risk, pink; intermediate risk, blue; low risk in the CiPA risk categorization).
Ajmaline
Diltiazem
JNJ303
Quinidine
Amiodarone
Diphenhydramine
Levocetirizine
Quinine sulfate
Amitryptiline
Disopyramide
Loratadine
Ranolazine
Aspirin
Dofetilide
Metoprolol
Risperidone
Astemizole
Dolasetron
Mexiletine
Sematilide
Azimilide
Domperidone
Mibefradil
Sertindole
Bepridil
Dronedarone
Moxifloxacin
Sparfloxacin
Chlorpheniramine
Droperidol
Nifedipine
Tamoxifen
Chlorpromazine
E-4031
Nilotinib
Terfenadine
Chromanol 293B
Erythromycin IV
Nitrendipine
Terodiline
Cilostazole
Famotidine
Ondansetron
Thioridazine
Cisapride
Flecainide
Paliperidone
Tolterodine
Clarithromycin
Gatifloxacin
Pimozide
Vandetanib
Clozapine
Haloperidol
Prenylamine
Verapamil
D,L-sotalol
Ibutilide
Procainamide
Ziprasidone
A
B
CredibleMeds label
hiPS-CMs
Risk of TdP
No report
High True positive False positive +Inter Low
High +Inter Low
False negative True negative Sensitivity
Specificity
Risk of TdP
No report
17
1
4
6
81%
85.7%
Accuracy
availability of another cell-line (Cor.4U) under the same protocols conducted in the JiCSA study.13 We found similar predictability of proarrhythmia risk using Cor.4U-CMs and the iCell.13 Based on these findings, we have proposed a new paradigm for cardiotoxicity risk assessment of drugs using hiPSC-CMs. Although iCell-CMs and Cor.4U-CMs have several different properties, a high predictability was obtained by concordance analysis of our high- and low-risk drugs in the TdP risk categorization against the CredibleMeds using both cell types. Because there are other commercially available hiPSC-CMs and new hiPSCCMs from the academia, it is necessary to validate the between lineto-line differences and define the “fit-for-purpose” cells for testing. From the view point of iPSC technology, there are many critical points that affect iPSC phenotype such as media, matrix, and passage. Recently, good cell culture practices (GCCP) of stem cells and stem cell-derived cells were discussed at the international expert group, which included the National Institutes for Health (NIHS),14 and then a guidance document on Good In Vitro Method Practices (GIVIMP) was prepared for the development and implementation of in vitro methods for regulatory use in human safety assessment by the OECD. Based on the concept of GCCP, further efforts should be made to ascertain the robustness of hiPSC preparations, such as quality check testing standards, validation of iPSC culture media and reagents, iPSC line-to-line differences, optimization of differentiation protocols, and functional assays of differentiated cells. 3. Comparison of JiCSA and CiPA data
82.1%
Fig. 1. Two-dimensional map and concordance analysis. (A) Two-dimensional map of 28 common compounds between JiCSA and CiPA. Compound names, the values of ratio, and the TdP risk score are shown in Table 2. (B) 2 2 Contingency table for TdP risk prediction by hiPSC-CMs (high plus intermediate vs. low risk in JiCSA study based on the 2D map) and CredibleMeds information (risk of TdP vs. no reports). The sensitivity, specificity, and accuracy were calculated for all drugs.
In addition to the Japan validation study, we participated in the CiPA international validation study with 28 blinded compounds (these were included in the JiCSA study) conducted by the FDAHESI and compared the data across HESI Myocyte Subteam sites. The data demonstrates the overall utility of the MEA method across multiple sites and hiPSC-CM cell lines.15 The international validation study investigated the site-to-site variability of hiPSC-CM-
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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Table 2 Result of 28 compounds. Ratio (free concentration in medium/fETPC), TdP risk score, risk in 2D map and CredibleMeds categorization in each compound. Red, pink, and blue characters indicate high and known risks, intermediate risk and possible/conditional risks, and low and no reported risks, respectively.
based assays and identified hiPSC-CM assay endpoints associated with high, intermediate, and low TdP risk using linear regression models. Three predictors (induction of arrhythmia-like events at any concentration, extent of delayed repolarization at any concentration, and extent of drug-induced repolarization prolongation at clinical Cmax for free drug) were sufficient to categorize drugs with reasonable accuracy (area under the curve [AUC] values ~0.8), suggesting the more accurate predictions of clinical TdP risk, rather than just focusing on hERG block and QT interval prolongation, in hiPSC-CMs. The difference in the protocols between JiCSA and CiPA is shown in Table 3. Because JiCSA uses our original two-dimensional map and CiPA performed their original logistics analysis, it is difficult to compare these two large datasets. To resolve this problem, we reanalyzed our data using the CiPA data analysis method. As shown in Fig. 2A, JiCSA data show a good agreement with the FDA-HESI Myocyte Subteam study data,15 regardless of the difference in the data analysis method. As discussed above, we have shown the usefulness of hiPSC-CMs to detect drug-induced proarrhythmia risk. There are some limitations in the myocyte study. According to the CiPA data, bepridil and ranolazine were outliers. When we used free concentration of
the drugs, bepridil was classified into the high-risk category, suggesting the importance of free concentration of drugs in safety risk assessment at a non-clinical level (Fig. 2B). Ranolazine was classified into the low-risk category, indicating that late sodium-blocking Table 3 Protocol differences between JiCSA and CiPA. Several conditions differ between JiCSA and CiPA protocol. Free fraction of compounds in the culture medium was measured in the JiCSA study. CiPA used total fraction of compounds in the culture medium. CiPA classified four arrhythmia types, whereas JiCSA did not classify any. JiCSA classified compounds into three risk groups using 2D map. CiPA used logistic analysis for TdP risk prediction.
Cell type Platform Drug administration Drug concentration Drugs Type of arrhythmia Statistical analysis TdP Risk prediction Clinical reference
JiCSA
CiPA
iCell MED64 (alpha MED) Accumulatively Free Unblinded (60 compounds) None by type None 2D map CredibleMeds
iCell and Cor.4U Maestro, MCS, MED64 etc Single dose per well Total Blinded (28 compounds) Type A, B, C, D Performed Logistic analysis CiPA derived risk categorization
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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A
Risk probability
High and intermediate risk
5
Low risk
Predicted risk probability
1 0.8 0.6 0.4
0
Ibutilide D,l-sotalol Azimilide Dofetilide Quinidine Disopyramide Vandetanib Bepridil Domperidone Droperidol Ondansetron Astemizole Cisapride Pimozide Clarithromycin Risperidone Terfenadine Chlorpromazine Clozapine Ranolazine Metoprolol Mexiletine Loratadine Tamoxifen Nitrendipine Nifedipine Diltiazem Verapamil
0.2
High
Intermediate
Low
1
Sensitivity
0.75
0.5
0.25
AUC=0.928 0 0
0.25
0.5
0.75
1
1-specificity
Risk probability
1
High and intermediate risk
Low risk
0.8 0.6 0.4 0.2 0 Ibutilide D,l-sotalol Adimilide Dofetilide Quinidine Disopyramide Vandetanib Bepridil Domperidone Droperidol Ondansetron Astemizole Cisapride Pimozide Clarithromycin Risperidone Terfenadine Chlorpromazine Clozapine Ranolazine Metoprolol Mexiletine Loratadine Tamoxifen Nitrendipine Nifedipine Diltiazem Verapamil
Predicted risk probability
B
Intermediate
High
Low
1
Sensitivity
0.75
0.5
0.25
AUC=0.978 0 0
0.25
0.5
1-specificity
0.75
1
Fig. 2. Logistic model prediction from JiCSA dataset. Dichotomous model prediction of JiCSA dataset using (A) total and (B) free fraction of compounds in the culture medium. Receiver operating characteristic (ROC) curve from logistic regression model using (A) total and (B) free fraction of compounds in the culture medium (high and intermediate vs. low).
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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drugs might not have been evaluated appropriately. Late sodium currents have been well studied using cell-line expressing mutant human SCN5A or animal disease models. However, late sodium currents have not been well studied in human samples and hiPSCCMs. Future studies would be required to elucidate the late sodium current in hiPSC-CMs toward safety risk assessment, although the data suggest the potential of “fit-for-purpose” use of hiPSC-CMs to assess the proarrhythmia risk of candidate drugs.
those technologies, the gap between in silico and hiPSC-CMs needs to be verified in vitro. Another approach is to construct an in silico model of hiPSC-CMs22 to bridge the gap between “adult” human CMs and immature hiPSC-CMs. In the near future, development of mature hiPSC-CMs will clarify the role of hiPSC-CMs in the CiPA initiative from confirming in silico reconstruction and will act as a standalone assay for drug safety evaluation. 4.3. Personalized medicine
4. Future perspectives 4.1. Maturation Towards the implementation of hiPSC-CMs for regulatory use, the overall immaturity of hiPSC-CMs available on the market has been discussed. Actually, differences between hiPSC-CMs and adult CMs have been reported.16 For example, most hiPSC-CMs display relatively depolarized maximum diastolic membrane potentials.6 In terms of between line differences, hiPSC-CMs from different sources and vendors have been reported to exhibit different electrophysiological phenotypes, which may be caused by different levels of maturation.17,18 Such differences may affect pharmacologic responses and thereby influence assay sensitivity and subsequent TdP risk assessments. In relation to cell maturity, assay timing is also critical to obtain reliable results. Continuous efforts have been made to create more mature hiPSC-CMs by engineering approaches that include more physiological substrates, long-term culture, electrical stimulation, cocultures with endothelial cells or fibroblasts or both, organoids, and 3D structures designed to mimic the morphology. More mature 3D constructs using hiPSC-CMs would recapitulate electrophysiological, contractile, and metabolic properties of adult ventricular myocytes, which can be further refined using “organs-on-a-chip” platforms.19e21 Since new engineered hiPSC-CMs are available, electrophysiological responses to the key ion channel inhibitor would be useful to determine the assay sensitivity of present and future hiPSC-CMs in the next-generation cardiac safety paradigm. In mature CMs, the large conductance of cardiac inward rectifier Kþ channels (IK1) is responsible for stabilizing resting membrane potentials. There is concern about the low expression level of IK1 in hiPSC-CMs, which also affects the function of other ion channels. Transduction of IK1 has been shown to facilitate the electrophysiological maturation of hiPSC-CMs by converting spontaneously action potential-firing cells into quiescent cells. The transduced cells are excitable and electrophysiologically mature CMs.22 Another way to induce hiPSC-CM maturation is to use stimulation.16 Furthermore, hiPSC-CMs are known to lack a regular structure and T-tubule network.23 This immature structure results in poor co-localization of calcium channels and ryanodine receptors, as well as non-uniform distribution of calcium release. The upstroke and decline rates of the Ca2þ signals in hiPSC-CMs are considered to be slower than those in adult human CMs are. Thus, development of electrophysiologically and structurally mature hiPSC-CMs is expected to provide a more accurate clinical prediction. Future studies would be necessary to perform drug evaluation with or without maturation using the same hiPSC-CMs. 4.2. In silico and iPSC-CMs Roles of hiPSC-CMs within the CiPA paradigm remains to be discussed. One possible role of hiPS-CM is to confirm in silico reconstructions of the electrophysiological effects of drugs. In silico modeling, using recombinant cell lines is expected to provide more comprehensive and robust assessments earlier in the drug discovery process.24 Toward the integrated assessment strategy with
Collecting data on hiPSC-CMs from healthy donors might pave the way to personalized medicine, which might make it possible to predict an individual's sensitivity to drugs in the future.25 It can also be applied to the development of drugs for specific populations. For example, hiPSC-CMs from healthy subjects recapitulate susceptibility to moxifloxacin-induced QT interval prolongation.26 In this scenario, drug safety or efficacy may be assessed using cells established from a representative sample of human patients, before moving into clinical trials. This “Clinical trial in a dish” approach could contribute to reducing drug attrition and ensuring patient safety. Moreover, hiPSC-CMs might be useful for evaluating arrhythmogenicity in disease models such as hypokalemia. Low extracellular Kþ concentration has been shown to induce FPD prolongation in hiPSC-CMs generated from a healthy control subject and EAD in hiPSC-CMs generated from symptomatic patient with type 1 of Long QT syndrome.27 4.4. Other differentiated cells from iPSCs As discussed above, the shared scientific experience among regulatory, industry, academic members would allow development of a good model for the use of the hiPSC-based in vitro assays to better predict drug safety.28 Human iPSC technology can be applied to anticancer agents, such as anthracyclines and tyrosine kinase inhibitors. We evaluated the contractility of hiPSC-CMs in vitro using a motion vector system29 involving high-speed imaging. Similar to the strategies used for the implementation of hiPSC-CM in the early drug development and for regulatory enforcement, test methods using various types of differentiated cells from hiPSCs could be developed to bridge the gap between non-clinical tests and clinical trials and facilitate 3R as alternative testing methods. For example, we have used the MEA protocol for neurons in an attempt to understand seizure liability using iPSC-derived neurons in collaboration with HESI NeuTox MEA Subteam.30 We also characterized hiPSC-derived hepatocytes and determined the protocols for liver toxicity. Further validation studies using standardized cells that show stable pharmacological responses are needed. Thus, standardized protocols using hiPSC-CMs would lead to the realization of a new paradigm for safety assessment. 5. Conclusions This paper reviews our JiCSA activities to validate a novel method using hiPSC-CMs and compares the results with those obtained by the CiPA myocyte working stream. The standardized MEA protocol enabled successful data collection with high reproducibility and reliability among laboratories. A collaboration between JiCSA and CiPA would provide scientific evidence for the international acceptance as a novel test method adopted in the ICH guidelines. Finally, the JiCSA/CiPA validation studies provides avenues for the potential use of hiPSC-CMs according to the “fit for purpose” strategy in drug development. In addition, the data clarify the best practice of hiPSC-CMs use. The results would be worth discussing
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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with the ICH S7B Discussion Group. Our efforts address and highlight strategies for standardized implementation of testing methods using hiPSC-CMs. Conflicts of interest There are no conflicts of interest to declare. Acknowledgements We gratefully acknowledge the support of the Japanese Safety Pharmacology Society and JiCSA members. We would like to thank Dr. Ksenia Blinova (FDA) and Dr. Gary Gintant (Abbvie) for reanalyzing the JiCSA data and Dr. David Strauss (FDA) and other CiPA members for their thoughts. We also thank HESI for their continuous support. This study was supported by the Research on Regulatory Harmonization and Evaluation of Pharmaceuticals, Medical Devices, Regenerative and Cellular Therapy Products, Gene Therapy Products, and Cosmetics from Japan Agency for Medical Research and Development (JP18mk0104117). References 1. Redfern WS, Carlsson L, Davis AS, et al. 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. 2003;58:32e45. 2. ICH guidance on S7B. The non-clinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals; 2005. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Safety/S7B/Step4/S7B_Guideline.pdf. 3. ICH guidance on E14. Clinical evaluation of QT/QTc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs; availability; 2005. http:// www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/ E14/E14_Guideline.pdf. 4. Zhang J, Chen H, Tsong Y, Stockbridge N. Lessons learned from hundreds of Thorough QT studies. Ther Innov Regul Sci. 2015;49:392e397. 5. https://www.kantei.go.jp/jp/singi/kenkouiryou/en/pdf/2017_plan.pdf. 6. Kanda Y, Yamazaki D, Kurokawa J, Inutsuka T, Seksino Y. Points to consider for a validation study of iPS cell-derived cardiomyocytes using a multi-electrode array system. J Pharmacol Toxicol Methods. 2016;81:196e200. 7. Yamamoto W, Asakura K, Ando H, et al. Electrophysiological characteristics of human iPSC-derived cardiomyocytes for the assessment of drug-Induced proarrhythmic potential. PLoS One. 2016;11, e0167348. 8. Sager PT, Gintant G, Turner JR, Pettit S, Stockbridge N. Rechanneling the cardiac proarrhythmia safety paradigm: meeting report from the Cardiac Safety Research Consortium. Am Heart J. 2014;167:292e300. 9. Nakamura Y, Matsuo J, Miyamoto N, et al. Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an iPS cell-derived cardiomyocyte sheet: multi-site validation study. J Pharmacol Sci. 2014;124: 494e501.
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10. Asakura K, Hayashi S, Ojima A, et al. Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes. J Pharmacol Toxicol Methods. 2015;75:17e26. 11. Kannankeril P, Roden DM, Darbar D. Pharmacol Rev. 2010;62:760e781. 12. Ando H, Yoshinaga T, Yamamoto W, et al. A new paradigm for drug-induced torsadogenic risk assessment using human iPS cell-derived cardiomyocytes. J Pharmacol Toxicol Methods. 2017;84:111e127. 13. Yamazaki D, Kitaguchi T, Ishimura M, et al. Proarrhythmia risk prediction using human induced stem cell-derived cardiomyocytes. J Pharmacol Sci. 2018;136: 249e256. 14. Pamies D, Bal-Price A, Simeonov A, et al. Good Cell Culture Practice for stem cells and stem-cell-derived models. ALTEX. 2017;34:95e132. 15. Blinova K, Dang Q, Millard D, et al. International multisite study of human induced pluripotent stem cell derived cardiomyocytes for drug proarrhythmic potential assessment. Cell Rep. 2018;24:3582e3592. 16. Yang X, Pabon L, Murry CE. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ Res. 2014;114:511e523. 17. van den Heuvel NH, van Veen TA, Lim B, Jonsson MK. Lessons from the heart: mirroring electrophysiological characteristics during cardiac development to in vitro differentiation of stem cell derived cardiomyocytes. J Mol Cell Cardiol. 2014;67:12e25. 18. Liu J, Laksman Z, Backx PH. The electrophysiological development of cardiomyocytes. Adv Drug Deliv Rev. 2016;96:253e273. re D, et al. Human engineered heart 19. Mannhardt I, Breckwoldt K, Letuffe-Brenie tissue: analysis of contractile force. Stem Cell Rep. 2016;7:29e42. 20. Lemoine MD, Mannhardt I, Breckwoldt K, et al. Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density. Sci Rep. 2017;7:5464. 21. Ulmer BM, Stoehr A, Schulze ML, et al. Contractile work contributes to maturation of energy metabolism in hiPSC-derived cardiomyocytes. Stem Cell Rep. 2018;10:834e847. 22. Li M, Kanda Y, Ashihara T, et al. Overexpression of KCNJ2 in induced pluripotent stem cell-derived cardiomyocytes for the assessment of QT-prolonging drugs. J Pharmacol Sci. 2017;134:75e85. 23. Parikh SS, Blackwell DJ, Gomez-Hurtado N, et al. Thyroid and glucocorticoid hormones promote functional T-tubule development in human-induced pluripotent stem cell-derived cardiomyocytes. Circ Res. 2017;121:1323e1330. 24. Li Z, Ridder BJ, Han X, et al. Assessment of an in silico mechanistic model for proarrhythmia risk prediction under the CiPA initiative. Clin Pharmacol Ther. 2018 (in press). 25. Burridge PW, Li YF, Matsa E, et al. Human induced pluripotent stem cellderived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity. Nat Med. 2016;22:547e556. 26. Shinozawa T, Nakamura K, Shoji M, et al. Recapitulation of clinical individual susceptibility to drug-induced QT prolongation in healthy subjects using iPSCderived cardiomyocytes. Stem Cell Rep. 2017;8:226e234. €l€ 27. Kuusela J, Larsson K, Shah D, Prajapati C, Aalto-Seta a K. Low extracellular potassium prolongs repolarization and evokes early after depolarization in human induced pluripotent stem cell-derived cardiomyocytes. Biol Open. 2017;6:777e784. 28. Wallis R, Benson C, Darpo B, et al. CiPA challenges and opportunities from a nonclinical, clinical and regulatory perspectives. An overview of the safety pharmacology scientific discussion. J Pharmacol Toxicol Methods. 2018;93:15e25. 29. Hayakawa T, Kunihiro T, Ando T, et al. Image-based evaluation of contractionrelaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: correlation and complementarity with extracellular electrophysiology. J Mol Cell Cardiol. 2014;77:178e191. 30. http://hesiglobal.org/committee-on-translational-biomarkers-ofneurotoxicity/.
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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Current Perspective
Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update Yasunari Kanda a, b, *, Daiju Yamazaki a, b, Tomoharu Osada b, c, Takashi Yoshinaga b, d, Kohei Sawada b, e a
Division of Pharmacology, National Institute of Health Sciences (NIHS), Kanagawa, 210-9501, Japan Japan iPS Cardiac Safety Assessment (JiCSA), Japan LSI Medience Corporation, Chiyoda-ku, Tokyo, 101-8517, Japan d Eisai Co., Ltd, Tsukuba, Ibaraki, 300-2635, Japan e The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, 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 1 October 2018 Received in revised form 18 October 2018 Accepted 19 October 2018 Available online xxx
Cardiac safety assessment is challenging because a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation is necessary for patient safety. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a new human cell-based platform to assess cardiac safety in non-clinical testing during drug development. The multi-electrode array (MEA) platform is a promising electrophysiological technology to assess QT interval prolongation and proarrhythmic potential of drug candidates using hiPSC-CMs. The Japan iPS Cardiac Safety Assessment (JiCSA) has established an MEA protocol to evaluate the applicability of hiPSC-CMs for assessing the torsadogenic potential of compounds and completed a large-scale validation study using 60 compounds. During our study, an international multi-site study of hiPSC-CMs was performed by the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative using 28 compounds. We have comparatively analyzed our JiCSA datasets with those of CiPA using the CiPA logistical and ordinal linear regression model. Regardless of the protocol differences, the evaluation results of the 28 compounds were very similar and highly predictable for torsadogenic risks. Thus, an MEA-based approach using hiPSC-CMs would be a standard testing method to evaluate proarrhythmic potentials. This review paper would provide new insights into the hiPSC-CMs/MEA method required for its regulatory use. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Cardiac safety CiPA Human iPS cells JiCSA Standardization
1. Introduction
Abbreviations: CiPA, Comprehensive in Vitro Proarrhythmia Assay; EAD, early after depolarization; fETPC, free effective therapeutic plasma concentration; FPD, field potential duration; FPDcF, FPD with Fridericia's correction; hERG, human -go-go-related gene; hiPSC-CMs, human induced pluripotent stem cellether-a derived cardiomyocytes; JiCSA, Japan iPS Cardiac Safety Assessment; MEA, multielectrode array; TdP, torsade de pointes. * Corresponding author. Division of Pharmacology, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan. Fax: þ81 44 270 1065. E-mail address: [email protected] (Y. Kanda). Peer review under responsibility of Japanese Pharmacological Society.
Cardiotoxicity is one of the major causes of drug attrition at later stages of drug development.1 Therefore, it is important to evaluate the potential for QT interval prolongation in humans before clinical trials. The risk of drug-induced QT interval prolongation has been assessed by evaluating the ability to block the human ether- a-go-go related gene (hERG) channel and prolong ventricular repolarization and the QT interval of the electrocardiogram (ECG) in non-clinical and clinical studies.2,3 Numerous studies have been performed towards implementation of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B guideline, which includes the “QT PRODACT” (Database Construction for the Evaluation of the Risk of QT Interval
https://doi.org/10.1016/j.jphs.2018.10.010 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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Prolongation by Drugs in Japan) organized by the Japan Pharmaceutical Manufacturers Association, and in vivo and in vitro experiments. The importance of the studies is to show validated data of the assessment of drug-induced QT interval prolongation under common protocols with multi-sites as safety pharmacology studies. Currently, the ICH S7B and E14 have been successful. There have not been any withdrawals of marketed drugs for torsadogenic concerns since they were adopted. However, limitations of the protocols implemented under the guidelines have been widely recognized. Assessment of QT interval prolongation under the current protocols is unduly sensitive and not specific for predicting the risk of ventricular proarrhythmia. Therefore, drugs with inhibitory effects on hERG, with QT interval prolongation, and minimal actual TdP risk are deprioritized or excluded from further development or, even if approved, their clinical use might be limited by inappropriate warnings on their product label. Therefore, it is necessary to explore a new paradigm for a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation. Thus, the current cardiac safety paradigm is considered to be inappropriate for assigning TdP liability. The degree of QT interval prolongation, except for pure hERG blockers, is largely drugspecific, and the QT interval can be influenced by many factors (for example, drugedrug interaction, drug metabolism, autonomic perturbation, glucose levels, and sex difference). Since the ICH S7B guideline defines the safety risk associated with hERG or QT signals, the detection of even a small inhibitory effect under the hERG assay may result in adverse internal or regulatory consideration during drug development. Thus, many pharmaceutical companies may have unnecessarily terminated the development of potentially useful drug candidates.4 Human induced pluripotent stem cell (hiPSC) technology paves the way for the establishment of new in vitro testing technologies using human cells in drug development. Because human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can express hERG and other types of cardiac ion channels, the technology can be applied for safety evaluation at early or late-stage non-clinical testing during drug development. The Plan for Promotion of Medical Research and Development was approved by the Headquarters for Healthcare Policy in 2014 in Japan.5 The Japanese government has decided to establish next-generation safety evaluation methods for drugs using iPSC technology. In particular, it proposes to implement a nationwide collaboration among industry, academia, and government focused on the development of standardized cells and testing methods for evaluating cardiotoxicity. To establish new reliable testing methods using hiPSC-CMs, validation studies are required to evaluate their reproducibility, reliability, and robustness. The validated methods can then be implemented in the reviewing processes of the regulatory consideration on drug candidates. Here, we review the current status of large-scale validation studies conducted in Japan and a parallel international study including additional analysis results using free drug concentrations of JiCSA study. Further activities toward the implementation of a validated method to assess cardiotoxicity in the drug development are also discussed. 2. JiCSA validation study 2.1. Compound selection The Japan iPS Cardiac Safety Assessment (JiCSA) was established in 2014 to develop a new paradigm for assessing drug-induced proarrhythmia risk, based on the emergence of cutting-edge iPSC technology.6 Based on a formal validation process (such as OECD guidance document #34), National Institute of Health Sciences
(NIHS) brought experts from regulatory, industry, and academic fields to develop and validate a new testing method that allowed a more precise prediction of clinical proarrhythmia risk as a lead laboratory. To examine sensitivity to key currents in the hiPSC-CMs that affect ventricular repolarization and minimize site-to-site variability prior to the validation study, we performed a pilot study using E-4031 and cisapride as cardiac delayed rectifier Kþ channel (IKr) blockers, the IKs blocker chromanol 293B, and DMSO, vehicle control) as a set of training compounds.7 Then, following discussions, the JiCSA selected 60 compounds with different TdP risks in collaboration with the Japanese Safety Pharmacology Society to perform a large-scale validation. The Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm has been proposed to improve cardiac safety for regulatory approval in the US.8 It aims to replace the non-clinical hERG assay required in the ICH S7B with more translationally relevant assessments of proarrhythmia risk. The international consortium CiPA working groups selected and categorized 28 compounds as having high, intermediate, and low risks of TdP based on published reports, analysis of the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database, other data sources, and clinical opinions (Table 1). The JiCSA 60 compounds overlapped with almost all of the 28 CiPA compounds except for azimilide, which was not available in Japan when the study commenced. The data of azimilide were added to the JiCSA study later and are discussed in this paper with the 28 compounds. 2.2. Large-scale validation study using multi-electrode array (MEA) platform JiCSA developed a standardized protocol using a multi-electrode array (MEA) platform, which can easily record the electrophysiological activity of CM monolayer sheets, and has shown the interfacility validation among laboratories in the industry, government, and academia.9 Based on the pre-validation study, protocols were optimized and improved for the large-scale validation study.10 Furthermore, the clinical properties of the hiPSC-CMs, such as QTRR relationship, were electrophysiologically characterized. The relationship between field potential duration (FPD) and inter-spike interval (ISI) in hiPSC-CMs was very similar with that between QT and RR of human ECG in the Framingham heart study derived from data of approximately 5000 people.7 Moreover, responses of E4031 in hiPSC-CMs were similar to those of the human heart.7 Therefore, hiPSC-CMs are considered to share similar electrophysiological properties to human hearts and be applicable for the assessment of drug effects in humans. Next, a large-scale validation study was performed for 60 compounds with different TdP risks using commercially available hiPSC-CMs (iCell-CMs) and an MEA platform (MED64). We have proposed the TdP scoring system by a two-dimensional (2D) map. Based on the previous paper,1 we calculated margins of each drug between free fraction in medium and free effective therapeutic plasma concentration (fETPC). We also provided relative TdP score to each compound according to the extent of FPDcF change or early after depolarization (EAD) occurrence. EAD is one of the mechanisms by which drugs cause TdP after QT interval prolongation.11 When EAD triggers upstrokes in tissue and repolarization is sufficiently disturbed, drugs will initiate TdP. As shown in Fig. 1A, the 28 compounds were categorized as high-, intermediate-, or low-risks using a 2D map of relative TdP scores and the margins. The data using iCell-CMs results showed good concordance with a public TdP database CredibleMeds (Fig. 1B, Table 2).12 Because druginduced arrhythmia has been extensively examined by the MEA assays in the single cell-line (iCell), we also examined the
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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Table 1 Compound lists of JiCSA study. List of compounds used in the JiCSA study. Colored compounds overlapped with those in the CiPA study (red; high risk, pink; intermediate risk, blue; low risk in the CiPA risk categorization).
Ajmaline
Diltiazem
JNJ303
Quinidine
Amiodarone
Diphenhydramine
Levocetirizine
Quinine sulfate
Amitryptiline
Disopyramide
Loratadine
Ranolazine
Aspirin
Dofetilide
Metoprolol
Risperidone
Astemizole
Dolasetron
Mexiletine
Sematilide
Azimilide
Domperidone
Mibefradil
Sertindole
Bepridil
Dronedarone
Moxifloxacin
Sparfloxacin
Chlorpheniramine
Droperidol
Nifedipine
Tamoxifen
Chlorpromazine
E-4031
Nilotinib
Terfenadine
Chromanol 293B
Erythromycin IV
Nitrendipine
Terodiline
Cilostazole
Famotidine
Ondansetron
Thioridazine
Cisapride
Flecainide
Paliperidone
Tolterodine
Clarithromycin
Gatifloxacin
Pimozide
Vandetanib
Clozapine
Haloperidol
Prenylamine
Verapamil
D,L-sotalol
Ibutilide
Procainamide
Ziprasidone
A
B
CredibleMeds label
hiPS-CMs
Risk of TdP
No report
High True positive False positive +Inter Low
High +Inter Low
False negative True negative Sensitivity
Specificity
Risk of TdP
No report
17
1
4
6
81%
85.7%
Accuracy
availability of another cell-line (Cor.4U) under the same protocols conducted in the JiCSA study.13 We found similar predictability of proarrhythmia risk using Cor.4U-CMs and the iCell.13 Based on these findings, we have proposed a new paradigm for cardiotoxicity risk assessment of drugs using hiPSC-CMs. Although iCell-CMs and Cor.4U-CMs have several different properties, a high predictability was obtained by concordance analysis of our high- and low-risk drugs in the TdP risk categorization against the CredibleMeds using both cell types. Because there are other commercially available hiPSC-CMs and new hiPSCCMs from the academia, it is necessary to validate the between lineto-line differences and define the “fit-for-purpose” cells for testing. From the view point of iPSC technology, there are many critical points that affect iPSC phenotype such as media, matrix, and passage. Recently, good cell culture practices (GCCP) of stem cells and stem cell-derived cells were discussed at the international expert group, which included the National Institutes for Health (NIHS),14 and then a guidance document on Good In Vitro Method Practices (GIVIMP) was prepared for the development and implementation of in vitro methods for regulatory use in human safety assessment by the OECD. Based on the concept of GCCP, further efforts should be made to ascertain the robustness of hiPSC preparations, such as quality check testing standards, validation of iPSC culture media and reagents, iPSC line-to-line differences, optimization of differentiation protocols, and functional assays of differentiated cells. 3. Comparison of JiCSA and CiPA data
82.1%
Fig. 1. Two-dimensional map and concordance analysis. (A) Two-dimensional map of 28 common compounds between JiCSA and CiPA. Compound names, the values of ratio, and the TdP risk score are shown in Table 2. (B) 2 2 Contingency table for TdP risk prediction by hiPSC-CMs (high plus intermediate vs. low risk in JiCSA study based on the 2D map) and CredibleMeds information (risk of TdP vs. no reports). The sensitivity, specificity, and accuracy were calculated for all drugs.
In addition to the Japan validation study, we participated in the CiPA international validation study with 28 blinded compounds (these were included in the JiCSA study) conducted by the FDAHESI and compared the data across HESI Myocyte Subteam sites. The data demonstrates the overall utility of the MEA method across multiple sites and hiPSC-CM cell lines.15 The international validation study investigated the site-to-site variability of hiPSC-CM-
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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Table 2 Result of 28 compounds. Ratio (free concentration in medium/fETPC), TdP risk score, risk in 2D map and CredibleMeds categorization in each compound. Red, pink, and blue characters indicate high and known risks, intermediate risk and possible/conditional risks, and low and no reported risks, respectively.
based assays and identified hiPSC-CM assay endpoints associated with high, intermediate, and low TdP risk using linear regression models. Three predictors (induction of arrhythmia-like events at any concentration, extent of delayed repolarization at any concentration, and extent of drug-induced repolarization prolongation at clinical Cmax for free drug) were sufficient to categorize drugs with reasonable accuracy (area under the curve [AUC] values ~0.8), suggesting the more accurate predictions of clinical TdP risk, rather than just focusing on hERG block and QT interval prolongation, in hiPSC-CMs. The difference in the protocols between JiCSA and CiPA is shown in Table 3. Because JiCSA uses our original two-dimensional map and CiPA performed their original logistics analysis, it is difficult to compare these two large datasets. To resolve this problem, we reanalyzed our data using the CiPA data analysis method. As shown in Fig. 2A, JiCSA data show a good agreement with the FDA-HESI Myocyte Subteam study data,15 regardless of the difference in the data analysis method. As discussed above, we have shown the usefulness of hiPSC-CMs to detect drug-induced proarrhythmia risk. There are some limitations in the myocyte study. According to the CiPA data, bepridil and ranolazine were outliers. When we used free concentration of
the drugs, bepridil was classified into the high-risk category, suggesting the importance of free concentration of drugs in safety risk assessment at a non-clinical level (Fig. 2B). Ranolazine was classified into the low-risk category, indicating that late sodium-blocking Table 3 Protocol differences between JiCSA and CiPA. Several conditions differ between JiCSA and CiPA protocol. Free fraction of compounds in the culture medium was measured in the JiCSA study. CiPA used total fraction of compounds in the culture medium. CiPA classified four arrhythmia types, whereas JiCSA did not classify any. JiCSA classified compounds into three risk groups using 2D map. CiPA used logistic analysis for TdP risk prediction.
Cell type Platform Drug administration Drug concentration Drugs Type of arrhythmia Statistical analysis TdP Risk prediction Clinical reference
JiCSA
CiPA
iCell MED64 (alpha MED) Accumulatively Free Unblinded (60 compounds) None by type None 2D map CredibleMeds
iCell and Cor.4U Maestro, MCS, MED64 etc Single dose per well Total Blinded (28 compounds) Type A, B, C, D Performed Logistic analysis CiPA derived risk categorization
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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A
Risk probability
High and intermediate risk
5
Low risk
Predicted risk probability
1 0.8 0.6 0.4
0
Ibutilide D,l-sotalol Azimilide Dofetilide Quinidine Disopyramide Vandetanib Bepridil Domperidone Droperidol Ondansetron Astemizole Cisapride Pimozide Clarithromycin Risperidone Terfenadine Chlorpromazine Clozapine Ranolazine Metoprolol Mexiletine Loratadine Tamoxifen Nitrendipine Nifedipine Diltiazem Verapamil
0.2
High
Intermediate
Low
1
Sensitivity
0.75
0.5
0.25
AUC=0.928 0 0
0.25
0.5
0.75
1
1-specificity
Risk probability
1
High and intermediate risk
Low risk
0.8 0.6 0.4 0.2 0 Ibutilide D,l-sotalol Adimilide Dofetilide Quinidine Disopyramide Vandetanib Bepridil Domperidone Droperidol Ondansetron Astemizole Cisapride Pimozide Clarithromycin Risperidone Terfenadine Chlorpromazine Clozapine Ranolazine Metoprolol Mexiletine Loratadine Tamoxifen Nitrendipine Nifedipine Diltiazem Verapamil
Predicted risk probability
B
Intermediate
High
Low
1
Sensitivity
0.75
0.5
0.25
AUC=0.978 0 0
0.25
0.5
1-specificity
0.75
1
Fig. 2. Logistic model prediction from JiCSA dataset. Dichotomous model prediction of JiCSA dataset using (A) total and (B) free fraction of compounds in the culture medium. Receiver operating characteristic (ROC) curve from logistic regression model using (A) total and (B) free fraction of compounds in the culture medium (high and intermediate vs. low).
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
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drugs might not have been evaluated appropriately. Late sodium currents have been well studied using cell-line expressing mutant human SCN5A or animal disease models. However, late sodium currents have not been well studied in human samples and hiPSCCMs. Future studies would be required to elucidate the late sodium current in hiPSC-CMs toward safety risk assessment, although the data suggest the potential of “fit-for-purpose” use of hiPSC-CMs to assess the proarrhythmia risk of candidate drugs.
those technologies, the gap between in silico and hiPSC-CMs needs to be verified in vitro. Another approach is to construct an in silico model of hiPSC-CMs22 to bridge the gap between “adult” human CMs and immature hiPSC-CMs. In the near future, development of mature hiPSC-CMs will clarify the role of hiPSC-CMs in the CiPA initiative from confirming in silico reconstruction and will act as a standalone assay for drug safety evaluation. 4.3. Personalized medicine
4. Future perspectives 4.1. Maturation Towards the implementation of hiPSC-CMs for regulatory use, the overall immaturity of hiPSC-CMs available on the market has been discussed. Actually, differences between hiPSC-CMs and adult CMs have been reported.16 For example, most hiPSC-CMs display relatively depolarized maximum diastolic membrane potentials.6 In terms of between line differences, hiPSC-CMs from different sources and vendors have been reported to exhibit different electrophysiological phenotypes, which may be caused by different levels of maturation.17,18 Such differences may affect pharmacologic responses and thereby influence assay sensitivity and subsequent TdP risk assessments. In relation to cell maturity, assay timing is also critical to obtain reliable results. Continuous efforts have been made to create more mature hiPSC-CMs by engineering approaches that include more physiological substrates, long-term culture, electrical stimulation, cocultures with endothelial cells or fibroblasts or both, organoids, and 3D structures designed to mimic the morphology. More mature 3D constructs using hiPSC-CMs would recapitulate electrophysiological, contractile, and metabolic properties of adult ventricular myocytes, which can be further refined using “organs-on-a-chip” platforms.19e21 Since new engineered hiPSC-CMs are available, electrophysiological responses to the key ion channel inhibitor would be useful to determine the assay sensitivity of present and future hiPSC-CMs in the next-generation cardiac safety paradigm. In mature CMs, the large conductance of cardiac inward rectifier Kþ channels (IK1) is responsible for stabilizing resting membrane potentials. There is concern about the low expression level of IK1 in hiPSC-CMs, which also affects the function of other ion channels. Transduction of IK1 has been shown to facilitate the electrophysiological maturation of hiPSC-CMs by converting spontaneously action potential-firing cells into quiescent cells. The transduced cells are excitable and electrophysiologically mature CMs.22 Another way to induce hiPSC-CM maturation is to use stimulation.16 Furthermore, hiPSC-CMs are known to lack a regular structure and T-tubule network.23 This immature structure results in poor co-localization of calcium channels and ryanodine receptors, as well as non-uniform distribution of calcium release. The upstroke and decline rates of the Ca2þ signals in hiPSC-CMs are considered to be slower than those in adult human CMs are. Thus, development of electrophysiologically and structurally mature hiPSC-CMs is expected to provide a more accurate clinical prediction. Future studies would be necessary to perform drug evaluation with or without maturation using the same hiPSC-CMs. 4.2. In silico and iPSC-CMs Roles of hiPSC-CMs within the CiPA paradigm remains to be discussed. One possible role of hiPS-CM is to confirm in silico reconstructions of the electrophysiological effects of drugs. In silico modeling, using recombinant cell lines is expected to provide more comprehensive and robust assessments earlier in the drug discovery process.24 Toward the integrated assessment strategy with
Collecting data on hiPSC-CMs from healthy donors might pave the way to personalized medicine, which might make it possible to predict an individual's sensitivity to drugs in the future.25 It can also be applied to the development of drugs for specific populations. For example, hiPSC-CMs from healthy subjects recapitulate susceptibility to moxifloxacin-induced QT interval prolongation.26 In this scenario, drug safety or efficacy may be assessed using cells established from a representative sample of human patients, before moving into clinical trials. This “Clinical trial in a dish” approach could contribute to reducing drug attrition and ensuring patient safety. Moreover, hiPSC-CMs might be useful for evaluating arrhythmogenicity in disease models such as hypokalemia. Low extracellular Kþ concentration has been shown to induce FPD prolongation in hiPSC-CMs generated from a healthy control subject and EAD in hiPSC-CMs generated from symptomatic patient with type 1 of Long QT syndrome.27 4.4. Other differentiated cells from iPSCs As discussed above, the shared scientific experience among regulatory, industry, academic members would allow development of a good model for the use of the hiPSC-based in vitro assays to better predict drug safety.28 Human iPSC technology can be applied to anticancer agents, such as anthracyclines and tyrosine kinase inhibitors. We evaluated the contractility of hiPSC-CMs in vitro using a motion vector system29 involving high-speed imaging. Similar to the strategies used for the implementation of hiPSC-CM in the early drug development and for regulatory enforcement, test methods using various types of differentiated cells from hiPSCs could be developed to bridge the gap between non-clinical tests and clinical trials and facilitate 3R as alternative testing methods. For example, we have used the MEA protocol for neurons in an attempt to understand seizure liability using iPSC-derived neurons in collaboration with HESI NeuTox MEA Subteam.30 We also characterized hiPSC-derived hepatocytes and determined the protocols for liver toxicity. Further validation studies using standardized cells that show stable pharmacological responses are needed. Thus, standardized protocols using hiPSC-CMs would lead to the realization of a new paradigm for safety assessment. 5. Conclusions This paper reviews our JiCSA activities to validate a novel method using hiPSC-CMs and compares the results with those obtained by the CiPA myocyte working stream. The standardized MEA protocol enabled successful data collection with high reproducibility and reliability among laboratories. A collaboration between JiCSA and CiPA would provide scientific evidence for the international acceptance as a novel test method adopted in the ICH guidelines. Finally, the JiCSA/CiPA validation studies provides avenues for the potential use of hiPSC-CMs according to the “fit for purpose” strategy in drug development. In addition, the data clarify the best practice of hiPSC-CMs use. The results would be worth discussing
Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010
Y. Kanda et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
with the ICH S7B Discussion Group. Our efforts address and highlight strategies for standardized implementation of testing methods using hiPSC-CMs. Conflicts of interest There are no conflicts of interest to declare. Acknowledgements We gratefully acknowledge the support of the Japanese Safety Pharmacology Society and JiCSA members. We would like to thank Dr. Ksenia Blinova (FDA) and Dr. Gary Gintant (Abbvie) for reanalyzing the JiCSA data and Dr. David Strauss (FDA) and other CiPA members for their thoughts. We also thank HESI for their continuous support. This study was supported by the Research on Regulatory Harmonization and Evaluation of Pharmaceuticals, Medical Devices, Regenerative and Cellular Therapy Products, Gene Therapy Products, and Cosmetics from Japan Agency for Medical Research and Development (JP18mk0104117). References 1. Redfern WS, Carlsson L, Davis AS, et al. 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. 2003;58:32e45. 2. ICH guidance on S7B. The non-clinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals; 2005. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Safety/S7B/Step4/S7B_Guideline.pdf. 3. ICH guidance on E14. Clinical evaluation of QT/QTc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs; availability; 2005. http:// www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/ E14/E14_Guideline.pdf. 4. Zhang J, Chen H, Tsong Y, Stockbridge N. Lessons learned from hundreds of Thorough QT studies. Ther Innov Regul Sci. 2015;49:392e397. 5. https://www.kantei.go.jp/jp/singi/kenkouiryou/en/pdf/2017_plan.pdf. 6. Kanda Y, Yamazaki D, Kurokawa J, Inutsuka T, Seksino Y. Points to consider for a validation study of iPS cell-derived cardiomyocytes using a multi-electrode array system. J Pharmacol Toxicol Methods. 2016;81:196e200. 7. Yamamoto W, Asakura K, Ando H, et al. Electrophysiological characteristics of human iPSC-derived cardiomyocytes for the assessment of drug-Induced proarrhythmic potential. PLoS One. 2016;11, e0167348. 8. Sager PT, Gintant G, Turner JR, Pettit S, Stockbridge N. Rechanneling the cardiac proarrhythmia safety paradigm: meeting report from the Cardiac Safety Research Consortium. Am Heart J. 2014;167:292e300. 9. Nakamura Y, Matsuo J, Miyamoto N, et al. Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an iPS cell-derived cardiomyocyte sheet: multi-site validation study. J Pharmacol Sci. 2014;124: 494e501.
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Please cite this article as: Kanda Y et al., Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2018.10.010