A Role for In Vitro Disease Models in The Landscape of Preclinical Cardiotoxicity and Safety Testing

Drug-induced cardiotoxicity is one of the predominant reasons for drug attrition and withdrawals. This is of critical concern when potentially cardiotoxic drugs are administered to individuals with inherited arrhythmogenic cardiac diseases or with metabolic diseases such as obesity and diabetes, which are key risk factors for cardiovascular diseases. Pathophysiological alteration prevalent under such conditions can alter or exacerbate cardiotoxic responses. The growing incidence of obesity, diabetes and metabolic syndrome subject a significant percentage of the population to drug treatments, thereby augmenting their risk for drug-induced cardiovascular toxicity. Hence, screening for drug-induced cardiotoxicity early in the preclinical stages of drug development, by using appropriate human disease models, can be effective in ensuring safety in clinical trials and preventing late stage and post-marketing drug withdrawals owing to cardiotoxicity. The advent of human pluripotent stem cells (hPSC) and induced pluripotent stem cell (iPSC)-derived cardiomyocytes are revolutionizing safety/toxicity screening in human cells by providing relevant human-specific, renewable model systems to explore human drug toxicity. The ability to generate patient-specific iPSCs that can model cardiac diseases, now offers a valuable option that can further improve drug safety assessments and enable a more accurate prediction of toxicity that occurs in the representative population that are prescribed the drugs. Use of appropriate disease models will not only provide cost savings by decreasing potential drug attrition and withdrawals, seen with many drugs, but will also be a promising option to advance precision medicine DOI : 10.14302/issn.2574-4372.jesr-17-1705 Corresponding author: Vijayalakshmi Varma, Division of Systems Biology, Biomarkers and Alternate Models Branch, National Center for Toxicological Research, U.S. Food and Drug Administration, 3900, NCTR Road, Building 54, Rm#126, Jefferson AR-72079. E-Mail: Vijayalakshmi.varma@fda.hhs.gov; Tel.: +1-870-543-7115; Running Title: In vitro disease models in preclinical cardiotoxicity and safety testing


Introduction
The drug development pipeline is an arduous, labor-intensive and expensive process that entails stringent regulations by the FDA, in order to ensure the final marketed drug will be safe and efficacious for use in all patients that are prescribed the drugs [1][2][3].
Although rigorous screening and testing of drugs occur as part of preclinical studies and clinical trials, safety and toxicity continue to be leading causes for the withdrawal of drugs from the markets [1,4,5].
Amongst specific organ toxicities, cardiac toxicity is one of the predominant reasons for late-stage attrition of drugs [6][7][8].A potential reason for late-stage drug attrition and post-marketing withdrawals may be attributed to drug-induce cardiotoxicity augmented by cardiovascular risk factors that are prevalent in many patients with metabolic disorders such as obesity (36% of the population [9] type II diabetes (9.3% of the population [10], and metabolic syndrome (~35% of the population) [11].Such patients are usually not part of clinical trials, unless the clinical trial is specifically for anti-diabetic or anti-obesity drugs.
Thus, drugs prescribed to a significant percentage of the population displaying cardiovascular risk factors put them at a higher risk for drug-induced cardiovascular toxicity.
Cardiovascular adverse events can result when drugs impact either the structural and functional aspects of the different components of the cardiovascular system including cardiac myocytes, fibroblasts, vascular smooth muscle cells and endothelial cells lining the vasculature, or affect the electrical conductivity of the heart or a combination of both, resulting in cardiac dysfunction [12].Conditions that mediate drug-induced cardiotoxicity include 1) Cardiac arrhythmias: which results from the altered electrical conductivity of the heart causing tachycardia, brachycardia, ventricular fibrillation or asystole.[13] Torsade de pointes, is a dangerous form of ventricular arrhythmia, that is commonly triggered by cardiotoxic drugs [14].It is characterized by tachycardia and associated with a prolonged QT interval in an electrocardiogram [12].
2) Cardiac hypertrophy: This results from the increased size of the cardiomyocytes consequent to decreased or compromised cardiac function [15].3) Cardiomyopathy: This is a condition that manifests due to structural and functional alterations in the heart resulting in decreased cardiac output [16].4) Congestive heart failure: Is a condition which occurs due to dysfunctional systoly, diastoly and myocardial contractility resulting in an inability to maintain cardiac output [17] and 5) Vascular toxicity: Is a condition which results from structural and functional alterations of vascular endothelial cells induced by oxidative stress or accumulation of toxins in these cells [18].Obesity is also associated with other risk factors for developing cardiac failure, such as hypertension, hyperlipidemia [26] and inflammation [24].The presence of excess circulating free fatty acids (FFAs) in obesity can increase the delivery of FFAs to the heart resulting in cadiomyocyte lipotoxicity, augment reactive oxygen species (ROS) production and increase oxidative stress.[27].Studies have shown that the accumulation of ROS in the myocardium consequent to hyperglycemia in the diabetic state can trigger myocardial apoptosis leading to diabetic cardiomyopathy [28].Excess circulating FFAs in obesity activate toll receptors stimulating the downstream activation of the NFκB pathway resulting in augmented production of pro-inflammatory cytokines including TNFα and IL-6 [29].TNFα can further increase the production of IL-6 and macrophage chemoattractant protein, (MCP-1), which plays a role in macrophage recruitment [24,30].These pro-inflammatory cytokines are prominent in stimulating the formation of atherosclerotic plaques [31].The consequent state of chronic low-grade inflammation in obesity further plays a key role in the development of insulin resistance [24,32] .Obesity and inflammation are associated with the development of endothelial dysfunction [33].
Diabetes is strongly associated with cardiovascular diseases and symptoms including atrial fibrillation, atrial flutter, coronary artery disease and left ventricular hypertrophy [34] and can contribute to the development of diabetic cardiomyopathy [35].Thus, the pathophysiological changes accompanying obesity and diabetes exert their effects both at the cellular and systemic levels resulting in cardiac dysfunction [23,36], which over time can alter myocardial structure and function causing heart failure [22].Hence pre-existence of these cardiovascular risk factors in patients can augment drug-induced cardiotoxicity.

Current preclinical testing strategies and model systems used in in vitro drug testing
The model systems frequently used in investigating cardiotoxicities, in particular, proarrhythmic risk are transgenic in vitro systems such as Human Embryonic Kidney (HEK) cells and Chinese Hamster Ovary (CHO) cells expressing heterologous ion channel systems [37] with a focus on identifying mainly the arrhythmogenic effects of drugs.Although these systems to a large extent have contributed to understanding ion channel defects and arrhythmogenic mechanisms, they have limitations in accurately predicting toxicities in humans, due to the inability of these cell systems to accurately reproduce the human cardiac physiology and the clinical manifestations of cardiac toxicities.The rodent cell line H9C2 from rat heart [38] is another model that has been used to examine drug-induced toxicities by chemotherapeutic agents [39,40].It is noted that the H9C2 cells exhibit features that are morphologically distinct from human cardiomyocytes and are also less mature than the human adult cardiomyocytes [37].Primary adult human ventricular cardiomyocytes are appropriate model systems for toxicity testing to recapitulate human physiologically functional cardiomyocytes of the human heart [41].However, these cells are difficult to obtain and cannot be maintained and propagated long term in culture [42].Alternatively, human pluripotent stem cells, such as embryonic stem (ES) cells, which are obtained from the blastocyst embryonic stage [43] as well as the iPSCs, which are derived from reprograming somatic cells [44] can be induced to differentiate into any somatic cell type including cardiomyocytes [45].The particular advantage of the iPSCs over the ES cells is twofold i) they overcome the ethical concerns associated with ES cells ii) while both ES and iPSCs can serve as an infinite source of cells, iPSCs can also be generated from somatic cells from individuals with disease enabling the modeling of the disease phenotype in a dish [46].In vivo model systems including rodent and more recently Zebrafish [37,47] have also been used as models to test for cardiotoxicity.However, given the varied structure and morphology of the cardiomyocytes and the electrophysiological characteristics and profiles of the various repolarizing and depolarizing currents channels in these non-human model systems, they may not effectively predict cardiotoxicity in humans.
To enable safety pharmacology efforts, several guidelines for preclinical safety using in vitro and in vivo Hence, the strategy of only examining the blockage of a single ion channel is increasingly being recognized as an imperfect measure ventricular repolarization [48,49].
Also, this much relied on in vitro assay for hERG, although highly sensitive, has only low specificity [49].
Furthermore, nonclinical QT prolongation assays are not fully predictive of the QT prolongation in humans [48].
Hence, although these guidelines have proven useful in decreasing drug-induced cardiotoxicity by enabling the early detection of potentially torsadogenic drugs, incidences of false positive results have led to the incorrect/inappropriate assignment of some drugs as torsadogenic [48].In the case of some drugs, these approaches have also resulted in false negative results, leading to drug attrition [50][51][52].In order to address these issue, partnered efforts by multidisciplinary scientists, representing international regulatory groups, industry and academia are underway for the development of more newer approaches to assess proarrhythmic risk under the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiatives [49,53].

Limitations of current models and strategies
The move from animal and non-cardiac human cell lines to human human iPSC-derived cardiomyocytes are enabling the assessment of cardiotoxicity due to proarrhythmic cardiotoxic risk in more relevant human models.A significant limitation to the use of iPSC-derived cardiomyocytes is that they are recognized to exhibit a more neonatal phenotype as opposed to the preferred adult-like phenotype [54] .Many studies are addressing this issue to obtain a more adult-like phenotype in the iPSC-derived cardiomyocytes [54].
However, a caveat in the use of approaches have been put forth by the US Food and Drug Administration and International Coalition for Harmonization (ICH) including i) ICH S7(A) Safety Pharmacology Studies for Human Pharmaceuticals ii) ICH S7(B) Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals, iii) ICH S9 Nonclinical Evaluation for Anticancer Pharmaceuticals, in Freely Available Online www.openaccesspub.org| JESR CC-license DOI : 10.14302/issn.2574-4372.jesr-17-1705Vol-1 Issue 2 Pg.no. 30 order to minimize the risks that may be associated with cardiovascular toxicity during drug development.These guidelines mainly place emphasis on assessing proarrhythmic risk, which is the most common risk associated with drug-induced cardiotoxicity.The approaches currently used for cardiotoxicity determination are i) an in vitro assay for the Ether-a-go-go-Related gene (hERG), which examines the blockage of the repolarizing potassium channel current (hERG/I kr ) by the drug, and ii) non-clinical in vivo assessment of QT prolongation.However, blockage of hERG constitutes only one out of several cardiac current channels which result in proarrhythmia, when blocked.
cardiovascular toxicity has been documented with several classes of therapeutic drugs, necessitating focus on cardiac safety studies early in drug development.While the field has come a long way in the detection and identification of drug-induced cardiotoxicity particularly arrhythmia using various in vitro model systems, including animal, non-cardiac human cell lines, primary human ventricular cardiomyocytes and more recently human iPSC-derived cadiomyocyte models, drug attrition, withdrawals and non-approvals due to cardiac side effects in patients continue to be of concern.While the development of newer tests and methodologies as part of the CiPA initiative will continue to improve safety pharmacology testing, the use of in vitro human iPSC-derived cells that model cardiovascular disease due to both genetic and non-genetic causes can be beneficial in detecting safety and/or increased susceptibility to toxicity.Human in vitro disease models employed early in preclinical stages will, therefore, be a valuable addition in making the clinical trials safer for patients, enhancing safety testing approaches and in further reducing and preventing late stage drug attrition and post market withdrawals.Disclaimer: This document has been reviewed in accordance with the United States Food and Drug Administration (FDA) policy and approved for publication.The views presented in this article do not necessarily represent the views of the Food and Drug Administration

Cardiovascular risk factors, obesity and diabetes, can increase the potential for drug-induced cardiotoxicity
[23]aintain a constant energy supply to the heart[23].Freely Available Online www.openaccesspub.org| JESR CC-license DOI : 10.14302/issn.2574-4372.jesr-17-1705Vol-1 Issue 2 Pg.no.29 Issue 2 Pg.no. 31 capture of the phenotype in this model.Similarly, Lan et al. [59] modeled hypertrophic cardiomyopathy, which is a common form of congenital cardiac dysfunction resulting from the missense mutation of the MYH7 in vitro iPSC-derived cardiomyocytes used in safety pharmacology studies and early drug development is that these iPSC-derived cardiomyocytes are derived from healthy individuals and are less representative of the pathophysiological state seen in patients to whom drugs are prescribed.The phenotype observed was corrected by treatment with the β blocker, metoprolol demonstrating the precise Freely Available Online www.openaccesspub.org| JESR CC-license DOI : 10.14302/issn.2574-4372.jesr-17-1705Vol-1 cardiovascular risk, can improve the identification of toxicity early in preclinical studies.Ultimately, clinical trials can be made safer by identifying risks in phenotypically-relevant iPSC models in nonclinical