**1. Introduction**

Various drugs are being introduced into market for generating beneficial therapeutic effects in humans. The pharmaceutical industry invests about \$1.5 billion over the time period of 10-15 years to take a candidate drug from primary screen to market. Unfortunately, many drugs are withdrawn due to side effects associated with off-and on-target toxicity [1]. For example, as many as nine out of ten promising candidates beginning clinical phase I will not achieve marketing approval [2] and only 20% of agents that show efficacy against cardiovascular diseases in preclinical development are licensed after demonstrating sufficient efficacy in phase III testing [3]. The success rate in anticancer drug development process is with 5% of licensed agents even lower. Off-target cardiac toxicity is the most common cause of regulatory delay in approval and market withdrawal of newly developed pharmaceuticals [4, 5]. Druginduced sudden cardiac death and ventricular arrhythmia caused the withdrawal of more drugs in recent years than any other adverse drug reaction. Moreover, over 100 non-cardiac drugs are suspected to be of high-risk and carry cardiovascular-related black box warnings [6]. Similar considerations are raised concerning arrhythmia and toxicity induced by environ‐ mental factors, including industrial chemicals, food additives, cosmetics, and others, as outlined in the European REACH initiative (Registration, Evaluation, Authorization and Restriction of Chemical substances).

Current drug safety evaluation processes that are required for regulatory purposes mostly rely on animal studies and immortalized cell-based assays due to lack of suitable human in vitro cell systems. In Europe, almost 10 million vertebrate animals are used annually for research. Although highly predictive assays involving whole heart or slice preparations and in vivo animal testing remain the standard for preclinical safety pharmacology, this extensive use of

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animals and their tissues does not eliminate high attrition rates of novel drugs. One of the major reasons for this is limited predictability of existing preclinical animal (and cellular) models for assessment of drug safety and efficacy. Animal models do not always predict the toxicity in humans with sufficient accuracy because of inter-species differences [7]. For example, murine and human hearts greatly differ in some aspects of electrophysiological properties [8]. In addition, inbred animals that are frequently used in these analyses do not mimic the genetic diversity of human population required for accurate prediction of drug responses [9]. Therefore, identification of reliable and robust human cell systems for toxicity assessment has become a driving interest for pharmaceutical industries.

and need for the development of multidisciplinary integrated approaches consisting of human in vitro models for risk assessment as an alternative to animal models as they better mimic the

Human Pluripotent Stem Cell Applications in Drug Discovery and Toxicology – An overview

http://dx.doi.org/10.5772/58485

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In this chapter, we will summarize the latest developments in applications of PSCs and their tissue-specific derivatives for toxicity testing. We will outline the recent developments in toxicogenomic technologies which are employed to develop and investigate human biomark‐ ers for toxicity in PSC based models accelerating drug development process. We mainly focus

**2. Human pluripotent stem cells for repeated dose toxicity assessment**

such test systems by using derivatives of human ESCs or iPSCs.

Human pluripotent stem cells (PSC) offer with their ability to recapitulate the most essential steps of embryonic development and give rise to different mature cell types in vitro an optimal human cellular model, which could help in increasing the safety and predictability of RDT testing leading to low late stage attrition of compounds. Combined with this cell model, toxicogenomic technologies would help predict biomarkers in an evidence-based approach. So far, the safety assessment for novel drug candidates includes in vivo RDT tests in rodent and non-rodent models. The drawbacks of RDT studies include false negative results and unexpected humans toxicity of compounds that were judged to be safe in preclinical studies [15, 16]. Such unexpected toxicity is one of the major reasons for the withdrawal of a drug from the market. The heart and liver are often target organs in toxicology. Novel in vitro screening methods are, thus, required to classify toxic compounds earlier in development, which would lead to safer drugs, more efficient drug discovery process, lower costs and reduced laboratory animal use [17]. There is an increasing interest from biopharmaceutical industry to develop

The iPSCs have a clear advantage over ESCs as they do not involve ethical issues. The generation of iPSCs involves reorganization of condensed chromatin to open state chromatin, which is aided by histone acetylation. Epigenetic factors are crucial for iPSC generation and maintenance of their pluripotent state. Although the epigenetic state of iPSCs largely resembles that of ESCs, iPSCs also have a unique DNA methylation patterns they retain epigenetic memory of the respective somatic tissue of origin which might influence their differentiation potential and affect the quality and quantity of cells for RDT [18]. On the other side, it is also well known that different agents, so called epimutagens, can cause DNA methylation and histone modification changes leading to disease [19]. These epigenetic modifications directly affect transcription factors and other chromatin binding proteins that regulate cell type-specific gene expression. The detection of biomarkers related to epigenetic modifications in RDT would be of great importance, but until now there are no systematic studies conducted in this direction. In addition, employing of iPSCs and their derivatives for this purpose poses a great challenge because genetic and epigenetic variations in iPSCs associated with reprogramming and in vitro manipulation may compromise their utility for downstream applications [20]. The lesser the variation in epigenetic changes in iPSCs the greater will be the specificity in in vitro

human in vivo system [14].

on the application of PSCs in RDT testing.

In cardiac area different types of tests are already playing an important role in reducing costs and drug attrition rates. These strategies involve a tiered system which starts with in vitro single cell analyses followed by tests with ex vivo tissues and organs and progresses to in vivo animal models and, finally, clinical trials [10]. The most important in vitro test consists of automated patch-clamp recordings of Chinese hamster ovary (CHO) cells expressing human Ether-à-go-go-Related Gene (hERG) channel. This test is being used to identify compounds that block hERG channel and prolong cardiac action potential (AP) duration (i.e. the QT interval) predisposing to Torsade de pointes tachycardia and sudden cardiac death [11]. The assessment of the torsadogenic potential of each compound in the drug discovery process also includes determination of drug's ability to prolong the AP in isolated, arterially perfused rabbit ventricular wedge preparations or canine Purkinje fibers and monitoring of heart rates and occurrence of arrhythmia in animals. Each of these endpoints has it's own specificity and sensitivity [12]. For example, hERG-expressing CHO cells lack the complexity of native CMs and cannot accurately predict the organ toxicity or lethal and arrhythmogenic side effects of compounds that block other channels or signaling pathways. Therefore, additional in vitro assays that better recapitulate human pathophysiology and diversity are needed to better predict all potential on-and off-target toxicities, reduce drug attrition rates and avoid use of animals for testing drugs that would never reach clinical application.

Pluripotent stem cells (PSCs) have unrestricted proliferation capacity, are able to differentiate into any differentiated cell type thus offering a cost-effective unlimited and invaluable source of organotypic differentiated cells relevant to assess human long-term organ toxicity. The ethical issues associated with human embryonic stem cells (ESCs) were a major concern in their application in toxicity studies. However, the Nobel prize-winning discovery that transient expression of a few transcription factors can stably convert an adult somatic cell into an early embryonic stage, i.e. into so called induced pluripotent stem cells (iPSCs) [13], has opened new possibilities in drug discovery circumventing ethical issues and problematic accessibility.

Repeated dose toxicity (RDT) occurs after repeated exposure to a substance over certain period of time. In the context of cosmetics, which are generally used for months and years, long-term RDT testing is of particular importance and forms the integral part of the quantitative risk assessment. The prediction of endpoints and hazard identification of both newly developed and existing cosmetic ingredients in humans is mainly based on the animal systems as they allow simultaneous evaluation of multiple organ systems. However, there is a great demand and need for the development of multidisciplinary integrated approaches consisting of human in vitro models for risk assessment as an alternative to animal models as they better mimic the human in vivo system [14].

animals and their tissues does not eliminate high attrition rates of novel drugs. One of the major reasons for this is limited predictability of existing preclinical animal (and cellular) models for assessment of drug safety and efficacy. Animal models do not always predict the toxicity in humans with sufficient accuracy because of inter-species differences [7]. For example, murine and human hearts greatly differ in some aspects of electrophysiological properties [8]. In addition, inbred animals that are frequently used in these analyses do not mimic the genetic diversity of human population required for accurate prediction of drug responses [9]. Therefore, identification of reliable and robust human cell systems for toxicity

In cardiac area different types of tests are already playing an important role in reducing costs and drug attrition rates. These strategies involve a tiered system which starts with in vitro single cell analyses followed by tests with ex vivo tissues and organs and progresses to in vivo animal models and, finally, clinical trials [10]. The most important in vitro test consists of automated patch-clamp recordings of Chinese hamster ovary (CHO) cells expressing human Ether-à-go-go-Related Gene (hERG) channel. This test is being used to identify compounds that block hERG channel and prolong cardiac action potential (AP) duration (i.e. the QT interval) predisposing to Torsade de pointes tachycardia and sudden cardiac death [11]. The assessment of the torsadogenic potential of each compound in the drug discovery process also includes determination of drug's ability to prolong the AP in isolated, arterially perfused rabbit ventricular wedge preparations or canine Purkinje fibers and monitoring of heart rates and occurrence of arrhythmia in animals. Each of these endpoints has it's own specificity and sensitivity [12]. For example, hERG-expressing CHO cells lack the complexity of native CMs and cannot accurately predict the organ toxicity or lethal and arrhythmogenic side effects of compounds that block other channels or signaling pathways. Therefore, additional in vitro assays that better recapitulate human pathophysiology and diversity are needed to better predict all potential on-and off-target toxicities, reduce drug attrition rates and avoid use of

Pluripotent stem cells (PSCs) have unrestricted proliferation capacity, are able to differentiate into any differentiated cell type thus offering a cost-effective unlimited and invaluable source of organotypic differentiated cells relevant to assess human long-term organ toxicity. The ethical issues associated with human embryonic stem cells (ESCs) were a major concern in their application in toxicity studies. However, the Nobel prize-winning discovery that transient expression of a few transcription factors can stably convert an adult somatic cell into an early embryonic stage, i.e. into so called induced pluripotent stem cells (iPSCs) [13], has opened new possibilities in drug discovery circumventing ethical issues and problematic

Repeated dose toxicity (RDT) occurs after repeated exposure to a substance over certain period of time. In the context of cosmetics, which are generally used for months and years, long-term RDT testing is of particular importance and forms the integral part of the quantitative risk assessment. The prediction of endpoints and hazard identification of both newly developed and existing cosmetic ingredients in humans is mainly based on the animal systems as they allow simultaneous evaluation of multiple organ systems. However, there is a great demand

assessment has become a driving interest for pharmaceutical industries.

182 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

animals for testing drugs that would never reach clinical application.

accessibility.

In this chapter, we will summarize the latest developments in applications of PSCs and their tissue-specific derivatives for toxicity testing. We will outline the recent developments in toxicogenomic technologies which are employed to develop and investigate human biomark‐ ers for toxicity in PSC based models accelerating drug development process. We mainly focus on the application of PSCs in RDT testing.
