Drug safety testing paradigm, current progress ... - Wiley Online Library

Review Article Received: 18 July 2013,

Revised: 8 August 2013,

Accepted: 22 August 2013

Published online in Wiley Online Library: 26 October 2013

(wileyonlinelibrary.com) DOI 10.1002/jat.2935

Drug safety testing paradigm, current progress and future challenges: an overview Varun Ahuja* and Sharad Sharma ABSTRACT: Early assessment of the toxicity potential of new molecules in pharmaceutical industry is a multi-dimensional task involving predictive systems and screening approaches to aid in the optimization of lead compounds prior to their entry into development phase. Due to the high attrition rate in the pharma industry in last few years, it has become imperative for the nonclinical toxicologist to focus on novel approaches which could be helpful for early screening of drug candidates. The need is that the toxicologists should change their classical approach to a more investigative approach. This review discusses the developments that allow toxicologists to anticipate safety problems and plan ways to address them earlier than ever before. This includes progress in the field of in vitro models, surrogate models, molecular toxicology, ‘omics’ technologies, translational safety biomarkers, stem-cell based assays and preclinical imaging. The traditional boundaries between teams focusing on efficacy/ safety and preclinical/ clinical aspects in the pharma industry are disappearing, and translational research-centric organizations with a focused vision of bringing drugs forward safely and rapidly are emerging. Today’s toxicologist should collaborate with medicinal chemists, pharmacologists, and clinicians and these value-adding contributions will change traditional toxicologists from side-effect identifiers to drug development enablers. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: drug safety; toxicity; in vitro; omics; biomarker

Introduction

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In the pharmaceutical industry, the research and development expenditure in the last few years has increased by 80%, whereas productivity has decreased by 43% (Crawford, 2010). A major factor for many of these failures was determined to be the high attrition rate in drug development (DiMasi et al., 2003). The main causes of failure include an inability to predict these failures before human testing or early in clinical trials. For a pharmaceutical company, a 10% improvement in predicting failures before clinical trials could save 100 million dollars in development costs per drug (Sasseville et al., 2004). The current preclinical testing paradigm, established over 30 years ago, has improved drug safety markedly; evidence suggests that 70% of human toxicity seen during clinical trials is predicted by preclinical studies (Olson et al., 2000). A recent report from Japan studying the range of abilities of non-clinical safety assessment for predicting adverse drug reactions (ADRs) in humans showed that 48% of ADRs were predictable based on a comprehensive non-clinical safety assessment (Tamaki et al., 2013). An opportunity for the toxicology discipline to further improve efficiency lies both in leveraging ’best in class‘ technology and improved integration of informative activities during hit-to-lead and early lead optimization stages. The historical paradigm for toxicology in the pharmaceutical industry was as a screening function that would accept molecules from the ‘discovery team’ and then perform a series of screens to provide a simple ‘yes’ or ‘no’ answer for further development. If a molecule had a favorable profile in these screens, it would then move into clinical development. This paradigm evolved during an era when the understanding of target biology was limited, and regulatory authority expectations about the depth of understanding of targets and molecules was less demanding. Today, the biology of targets is understood at a much

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greater level and knowledge about translational biomarkers that was once of only negligible interest is now highly expected. It has become increasingly clear that descriptive toxicology is not sufficient enough to develop safer and more efficacious medicines. There is a need to explore the molecular and cellular perturbations that underlie the gross toxicological and pathological findings induced by candidate drugs across species as this can add considerable value through: (i) elucidation of mechanisms of toxicity, (ii) discrimination between toxic and adaptive responses, (iii) enhanced assessment of safety margins, (iv) earlier termination of unsafe molecules resulting in enhanced candidate selection, (v) enhanced assessment of human relevance for preclinical toxicities, (vii) prediction of reversibility for a given toxicity, and (xi) monitoring of adverse effects in the clinic. The capabilities and impact of investigative toxicology can be expanded by partnering with disease area and discovery platform scientists to better understand target biology and the potential liabilities of targets. A modern investigative toxicology team needs to be oriented towards problem solving and issue resolution not only during early development, but also during late stage development, anticipating or responding to preclinical safety project questions, translational medicine clinicians and/or health authorities. A benefit of actively engaging the research and development organizations across a broad portfolio is to leverage complementary safety knowledge, mechanistic data

*Correspondence to: Varun Ahuja, Research Scientist – Drug Safety Assessment, Novel Drug Discovery and Development, Lupin Limited (Research Park), 46A/47A, Nande Village, MulshiTaluka, Pune – 412115, India. Email: [email protected] Drug Safety Assessment, Novel Drug Discovery and Development, Lupin Limited (Research Park), 46A/47A, Nande Village, MulshiTaluka, Pune 412 115, India

Copyright © 2013 John Wiley & Sons, Ltd.

Developments in the field of non-clinical safety screening of drugs and candidate biomarkers for drug targets spanning multiple nodes of related pathways and for multiple disease indications. There are currently significant needs, but also significant opportunities, for tools that can more reliably and more efficiently determine the safety of a new chemical entity. In this review, we have focused on some emerging opportunities for modern investigative toxicologist, which have come into light in the last few years and are being explored across various Table 1. Routine exploratory safety studies In silico Genetic toxicology

Safety pharmacology

General toxicology

SAR alerts using in silico tools Mutagenicity assay: Bacterial reverse mutation Clastogenicity assay: in vitro mammalian cell assay In vitro: Receptor binding, Ion channel assays Ex vivo: Tissue/organ studies In vivo: cardiovascular, neurobehavioral and respiratory studies Rodent and Non-rodent: MTD and DRF studies

MTD, maximal tolerated dose; DRF, dose-range finding.

pharmaceutical industries and research organizations dealing with drug safety assessment.

Exploratory Safety Testing The goal of exploratory drug safety testing, also referred to as investigative/discovery toxicology or Lead Optimization (LO) safety assessment is to provide data to support the selection of the best discovery candidate for development with the best overall opportunity of achieving marketing authorization (Car and Robertson, 1999; Kramer et al., 2007; Fielden and Kolaja, 2008). Various authors have discussed the strategy for exploratory safety testing (Loget, 2008; Bass et al., 2009; Cavero, 2009; Higgins et al., 2012). The exploratory battery includes in silico, genetic toxicology, general toxicology and safety pharmacology studies. A summarized exploratory safety testing strategy is given in Table 1. Insights into the toxicological potential of a scaffold or series of structures early in the drug discovery process could help medicinal chemists to prioritize particular scaffolds. Computational prediction used for different types of toxicities and various in silico systems available have been discussed in detail by various authors (Egan et al., 2004; Johnson and Wolfgang, 2001; Muster et al., 2008; Snyder and Smith, 2005). Various in silico systems for computational prediction are enlisted in Table 2. The in silico tools offer good guidance on what additional tests may be necessary or whether further characterization is warranted (Kruhlak et al., 2007); however, they also have limitations. When these tools are

Table 2. Selected in silico systems available for safety predictions (Wolfgang and Johnson, 2002; Muster et al., 2008) Product

Short Description

DEREK

Knowledge (rule)-based expert system

TOPKAT

Employs cross-validated Quantitative structure toxicity relationship models for assessing various measures of toxicity; each module consists of a specific database Machine learning approach to identify molecular fragments with a high probability of being associated with an observed biological activity QSAR modeling system to establish structure–property relationships, create new calculators and generate new compound libraries Knowledge (rule)-based expert system

MCASE

MDL

Hazard Expert

COMPACT Cerius

CADD

For rapid identification of potential carcinogenicity or toxicities mediated by CYP450s Molecular modeling software with a ADME/ tox tool package providing computational models for the prediction of ADME properties Computer-aided drug design (CADD) by multi-dimensional QSARs applied to toxicity-relevant targets

Predicted endpoints Mutagenicity, Carcinogenicity, Skin sensitization, Irritancy, Neurotoxicity, Teratogenicity, Respiratory toxicity, skin sensitization etc. Mutagenicity, Carcinogenicity, Teratogenicity, Skin sensitization, Irritancy, rat chronic LOAEL etc. Carcinogenicity, irritation, teratogenicity, MTD, short-term genotoxicity, mutagenicity, Acute toxicity Mutagenicity, Carcinogenicity, hERG inhibition, Acute toxicity

Mutagenicity, Carcinogenicity, Skin sensitization, Irritancy, Neurotoxicity, Immunotoxicity Carcinogenicity, P450-mediated toxicities ADME, Hepatotoxicity

Receptor and CYP450-mediated toxicities, Endocrine disruption

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MTD, maximal tolerated dose.


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V. Ahuja and S. Sharma used to predict more complex events (organ system toxicity, sensitization, carcinogenicity etc.), the predictive value goes down considerably (MacDonald and Robertson, 2009). Natalie et al. (2010) screened compounds for phospholipidosis using an in vitro assay and an in silico prediction model and found their specificity to be 79% and 29%, respectively. The sensitivity and specificity of available in silico models for predicting various types of toxicities have been described elsewhere (Egan et al., 2004; Snyder and Smith, 2005; Valerio, 2012). Continued expansion of these databases will enhance the value of these screening tools but, at the moment, their utility is somewhat restricted as definitive predictors of adverse effects (MacDonald and Robertson, 2009). Genetic toxicology data are used as a surrogate for long-term carcinogenicity data during early drug development. The aim of genotoxicity testing is to identify potentially hazardous drug candidates. The role of genetic toxicology in drug discovery and optimization has been discussed in detail elsewhere (San 2006; Custer and Sweder, 2008). The various exploratory genetic toxicity assays are listed in Table 3. Preclinical safety pharmacology integrates in vitro and in vivo pharmacological data to assess the potential undesirable pharmacodynamic (PD) effects in humans (Williams, 1990; Pugsley, 2004). Early compound profiling by in vitro safety pharmacology can flag for receptor-, enzyme-, transporter- and channel-related liabilities of compounds and interprets these data in conjunction with absorption, distribution, metabolism and excretion (ADME) and toxicity characteristics, determined either in vitro or in vivo. Useful review of the appropriate use of safety pharmacology assays and their predictive value has been provided elsewhere (Guth et al., 2004; Whitebread et al., 2005; Sandow, 2006; Baldrick, 2008a; Valentin and Hammond, 2008; Cavero, 2009; Valentin et al., 2009; Gintant, 2011; Leishman et al., 2012; Hamdam et al., 2013; Parkinson et al., 2013). Table 4 enlists the various tests available for screening compounds for cardiovascular, central nervous and respiratory system-related liabilities. A major knowledge gap for most therapeutics is the range of primary drug–macromolecule interactions that occur in vivo with endogenous components of pharmacologic and toxicologic pathways. This is exemplified by the thalidomide example in which an approximate 50-year time-lag occurred between the first reports of serious adverse events associated with thalidomide treatment (Kim and Scialli, 2011) and the identification of an off-target associated molecular pathway that is likely to be responsible for aberrant limb development in humans (Ito et al., 2010). The

Table 3. Standard genotoxicity testing battery (Custer and Sweder, 2008) Test Ames bacterial mutation assay Mouse lymphoma assay (MLA), Chinese hamster ovary (CHO) chromosomal aberration assay Micronucleus test (MNT)

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Purpose Evaluation of Gene mutation Evaluation of chromosomal damage

management of off-target findings in preclinical drug development usually includes in silico toxicity screens, an in vitro hERG assay, CYP450 assays and compound screening against a broad panel of human recombinant proteins (e.g. receptors, kinases) or cell-based assays. However, the currently available protein interaction and enzyme activity assays cover a very limited proteome space, do not include endogenous protein targets and lack specificity for toxicology species. To some extent, these limitations can be balanced by deploying more complex integrative molecular and biochemical profiling strategies in tissue samples derived from toxicology studies including genomic, proteomic and metabolomic profiling. The first general toxicology studies are maximal tolerated dose (MTD) and dose-range finding (DRF) studies generally performed in rodents and non-rodents. The value of performing exploratory drug safety studies before candidate nomination is to identify unwanted toxicities evident in a study of up to 14 days duration, as well as any potential toxicities anticipated based on a known cause for concern. In the absence of findings or the presence of findings that are judged manageable, these studies provide a greater comfort in the selection of a molecule for advancement into development with the likelihood of success. Additional benefits of these studies are the identification of target organs to monitor in development and the selection of doses for the GLP toxicology studies. In addition, identification of the toxicity profile of a lead compound can be useful for the backup program where the goal is often an improved safety margin. The design and methodology of non-GLP toleration/doserange finding studies used in an early toxicology screening programme have been described in detail by various authors (Baldrick, 2008b; Bass et al., 2009; Herlich et al., 2009). Table 5 gives a more detailed overview of the general toxicology studies. The strategy could vary slightly among various organizations or depending on the discovery target in terms of the type of study selected, the number/gender of animals used in general toxicology studies and number of dose levels selected or other characteristics. The value of in vivo toxicology studies can be enhanced by including the measurement of functional endpoints providing information related to safety pharmacology (Redfern et al., 2013). In addition to adding an extra dimension to the information content of toxicology studies, the inclusion of functional endpoints also have 3Rs (Reduction, Refinement, Replacement) benefit. Therefore, it would assist with reducing the number of animals used, first by minimizing or obviating the need for standalone repeat-dose investigative studies addressing a specific functional endpoint, and second, by providing clearer information to prevent compounds with problematic safety profiles progressing into further, extensive non-clinical in vivo regulatory toxicology evaluations, only to be stopped eventually because of adverse effects. Some of the functional endpoints which could be included in in vivo toxicology studies are ECG, functional observation battery etc. and have been described in details by various investigators (Matsuzawa et al., 1997; Luft and Bode, 2002; Redfern et al., 2013). A reduction of animal use while designing studies without impacting their predictive value should be taken care of (Chapman et al., 2013).

Developments in Safety Testing Paradigm Evaluation of in vivo chromosomal damage in bone marrow polychromatic erythrocytes

Predicting Toxicity Outcomes From Physiochemical Properties The difficulty in developing predictive molecular models for toxicity comes about because of the diversity of mechanisms


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Developments in the field of non-clinical safety screening of drugs Table 4. Tests and parameters available to assess cardiovascular system (CVS), central nervous system (CNS) and respiratory function in safety pharmacology studies (Hamdam et al., 2013) Core

Follow-up

Established techniques

Emerging techniques

CVS assessment -hERG, -Telemetry

Isolated organ preparation

-hERG, assays for other ion channels -Isolated organ preparation: Whole heart preparation, Isolated Purkinje fibres -Telemetry

-Human embryonic stem cell derived cardiomyocytes -Human induced pluripotent stem cell derived cardiomyocytes -High definition oscillometry

-Higher cognitive function -Seizure liability -Drug abuse and dependence

-Modified Irwin’s test, Functional Observation Battery (FOB) -Photoelectric beam interruption systems -Rotarod -Hot plate test, Tail flick, paw pressure -Morris maize and passive avoidance tests -Electrocerebral silence threshold and PTZ seizure tests -EEG -Self administration and drug discrimination lever chamber models -Drug withdrawal: FOB, body temperature, body weight

-Integrated video and EEG systems -In vitro hippocampal brain slice assay -Telemetry

-Airway resistance -Pulmonary arterial pressure -Compliance

-Plethysmography

-Unrestrained video-assisted plethysmography -Telemetry

CNS assessment -Behavior -Locomotor activity -Motor co-ordination -Sensorimotor reflexes: nociception

Respiratory function assessment -Respiratory rate -Tidal volume -Hemoglobin oxygen saturation

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useful in guiding molecular design decisions by medicinal chemistry project teams towards safer regions of chemical space. The importance of physiochemical properties in causing toxicity was investigated by Hughes et al. (2008) from Pfizer who demonstrated that particular physiochemical properties would closely correlate with an increase in adverse in vivo outcomes in preclinical animal species. They compiled a database of diverse compounds whose in vivo studies were performed in rats or dogs and typically involved dose escalation over three or more dose levels and a duration of 4 days or longer. Individually, topological surface area (TPSA) and clogP (lipophilicity) showed a consistent correlation with the incidence rate of adverse in vivo outcomes. The authors also found that compounds with high clogP (> 3) and low TPSA (≤ 75) were ~2.5 times more likely to have an in vivo finding than compounds with low clogP (≤ 3) and high TPSA (> 75) which were ~2.5 times more likely not to have an in vivo finding, which represents an overall odds ratio of greater than 6. These studies led to the ‘3/75’ guideline from Pfizer which was specific to in vivo toxicology attrition (Leeson and Springthrope, 2007). Greene et al. (2010) from Pfizer



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that give rise to toxic outcomes. These mechanisms can be grouped into four broad classes according to the causative molecular features or activities: (1) the primary pharmacology or mechanism of action of the molecule under study, (2) the secondary pharmacology of the molecule, (3) the presence of a well-defined structural fragment or toxicophore in the molecule, and (4) the overall physicochemical properties of the molecule (Blagg, 2006). From a molecular design perspective, little can be done at the outset of a drug discovery project to address toxicity arising from primary pharmacology. Extensive experimentation is often required to establish a clear link between a mechanism of action and a particular toxicity, and this will usually not have been completed at that point. Useful information is emerging about the second and third classes of toxicity, including associations between secondary pharmacology (Krejsa et al., 2003) or defined structural fragments (toxicophores or structural alerts) and adverse outcomes (Kalgutkar and Soglia, 2005; Kalgutkar et al., 2005). For these classes of toxicity, and explicitly for the fourth class, it is reasonable to expect that some general physicochemical trends might be found that would be

V. Ahuja and S. Sharma Table 5. Exploratory general toxicology studies (Bass et al., 2009) Type of Study Single-dose toxicity (Rodents)

Objectives

Characteristics

Single Rising-dose toler ance/ Dose Escalation (Non-Rodents)

-To determine the potential toxicity of test agent based on ante-mortem measures (clinical observations and body weight) -To identify MTD -To provide rationale for selection of doses to be used in 14-day study or safety pharmacology studies -To identify MTD -To provide rationale for selection of doses to be used in 14-day study

14 day toxicity/ Short Term Repeat-Dose Range Finder (Rodents)

-To determine the toxicity based on ante-mortem measures, clinical and anatomic pathology

7-14 day toxicity/ Short Term Repeat-Dose Range Finder (Non-Rodents)

-To determine the toxicity based on antemortem measures, clinical and anatomic pathology -To provide rational for selection of doses to be used in repeated dose GLP studies

-Limited TK assessment 3 (tox) + 3 (TK) animals/sex/dose group 3to 6 dose levels -Doses: escalated to acute MTD/MFD/Limit dose -Limited TK assessment 1 animal/sex/dose group 3to 6 dose levels -Doses: escalated to acute MTD/MFD/Limit dose -Limited TK assessment 6 (tox) + 6 (TK) animals/sex/dose group 3 dose levels + control group -Histopathology limited to 12 target tissues -Dose selection: based on acute/Single Dose with goal of Lower dose = 5–10× of efficacy dose with goal of no toxicity; Medium dose = may produce tolerable toxicity; High dose = would normally lead to frank toxicity -Limited TK assessment 2 animals/sex/dose group 3 dose levels + control group -Dose selection: based on rising-dose tolerance/Dose escalation with goal of Lower dose = 5–10× of efficacy dose with goal of no toxicity; Medium dose = may produce tolerable toxicity; High Dose = would normally lead to frank toxicity

MTD, maximal tolerated dose; MFD, maximal feasible dose; TK, toxicokinetics.

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expanded on this concept by relating the in vitro cytotoxicity (IC50) values to severity findings at a 10 μM maximum serum drug concentration (Cmax) in in vivo preclinical studies. The authors found that compounds which displayed an IC50 value ≤ 50μM were five times more likely to cause adverse events at lower Cmax thresholds than those compounds with an IC50value ≥ 50 μM. Koslov-Davino et al. (2013) from Pfizer showed that compounds causing endoplasmic reticulum (ER) stress activation, which has been implicated in many disease states as well as compound induced organ toxicities, at concentrations below 40 μM in an in vitro model have a more than four times greater chance of causing in vivo toxicity at 10 μM plasma exposure. They further showed that compounds that cause ER stress below 40 μM have a relatively low polar surface area, high lipophilicity and are significantly enriched if both factors are present, and also showed that basic compounds possess more of these properties. Thus, readily calculated physicochemical parameters can be used to help design compounds that are in a lower risk chemical space in terms of their outcomes in an exploratory in vivo safety study and that, a simple high throughput cytotoxicity measurement can further help to select compounds with less potential


for causing adverse findings to progress to in vivo safety studies. Development of further mechanistically refined in vitro assays is clearly the next step forward in reducing attrition through safety related findings. Surrogate Animal Models Surrogate animal models offer an advantage in ease of genetic manipulation and high throughput over traditional mammalian models. Zebrafish is increasingly used as an in vivo model system for the evaluation of novel drug candidates for efficacy and safety testing, and many other studies have confirmed that mammalian and zebrafish toxicity profiles are strikingly similar (Hill et al., 2005; McGrath and Li, 2008). As a vertebrate animal, zebrafish have a high degree of genetic conservation and their morphological and molecular basis of tissue and organ development is either identical or similar to other vertebrates including humans (Chen and Fishman, 1996; Granato and NüssleinVolhard, 1996). The zebrafish genome is fully sequenced and zebrafish genes share a 60–80% homology with their human counterparts. More importantly, the amino acid sequences of functionally relevant protein domains has been proven to be


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Developments in the field of non-clinical safety screening of drugs even more evolutionary conserved (Reimers et al., 2004; Renier et al., 2007). Additional advantages for using larval zebrafish include small size, easy maintenance and breeding and high productivity (Westerfield, 1994). Because of their small size, efficient low-cost pathological evaluation of all major organs can be carried out on a limited number of slides as well. Other advantages of the zebrafish have proven to be its large numbers of offspring, its small size and translucency during embryonic and larval development. The later allows high resolution live imaging over time and for instance the use of fluorescent reporter molecules. In addition, zebrafish develop quickly, the brain, heart, liver, pancreas, kidney, intestines, bones, muscles and the sensory systems are fully functional at 5 days post fertilization (Kimmel et al., 1995). Up to date, direct visual assays in live animals can be carried out using reporter fish lines which express fluorescent proteins as a read-out for active signaling pathways (Perz-Edwards et al., 2001; Dorsky et al., 2002). The use of zebrafish as an alternative animal model for drug screening can greatly accelerate the drug discovery process, decrease costs and provide more accurate results than cell-based assays. Many specific categories of toxicity can be assessed in the zebrafish, including developmental toxicity (Gustafson et al., 2012), cardiotoxicity (Langheinrich et al., 2003; Park et al., 2013), neurotoxicity (Muth-Köhne et al., 2012), nephrotoxicity (Ding and Chen, 2012), hepatotoxicity (He et al., 2013), ototoxicity (Seng et al., 2010), intestinal and visual safety (Berghmans et al., 2008; Richards et al., 2008). Detailed description of the utility of zebrafish as a drug discovery tool is provided elsewhere (Rubinstein, 2006; McGrath and Li, 2008; Sipes et al., 2011; Sukardi et al., 2011). Although it has been shown that zebrafish toxicological assays can attain a good level of predictivity, false-negatives and false-positives have been found to compromise the sensitivity and specificity of the assays used (Milan et al., 2003; Chiu et al., 2008; Mittelstadt et al., 2008; Redfern et al., 2008; Kokel et al., 2010). These studies, while sporadic in nature, underscored a neglected understanding of the ADME profile of drugs between zebrafish and human or other mammalian models which is affected by factors such as the route of administration and physiology of the fish. To fully realize the potential of zebrafish as a drug toxicological model, the knowledge gap in ADME needs to be addressed (Sukardi et al., 2011). Recently, the intra-peritoneal route of administration was used for optimization of the zebrafish model for cardiovascular safety assessment (Chaudhari et al., 2013). The authors found their method reproducible for the recording of stable zebrafish ECGs to facilitate its routine application in preclinical investigation of QTc-prolonging drugs with reliable estimation of NOAEL. Further validation of the test system is required until it is routinely incorporated into screening strategy. Cell-Based Screening

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Most pharmaceutical companies use some type of cell-based safety screens, but applications and strategies may differ. For cytotoxicity screening, there are two basic approaches, a universal screening approach using one or a few cell lines or a target-organ-based approach, using cells with more specialized functions. This could be for various purposes, first being for rapidly screening the compounds during initial discovery phase and second for more mechanistic or detailed studies during the developmental phase. A list of various in vitro models used by

various groups as available in the literature is given in Table 6. A critical evaluation of in vitro cell culture models for drug screening and toxicity has been done by Astashkina et al. (2012). Single cytotoxic endpoint strategies (Bugelski et al., 2000) may provide valuable information within a class of compounds, but may be misleading when testing diverse compounds for diverse indications. Benbow et al. (2010) found that a probabilistic correlation exists between a compound’s cytotoxic potential in the THLE assay with its overall safety toleration in rodent exploratory toxicity studies. Evans et al. (2001) found that the in vitro IC35 results were comparable to the LD50 values, the resulting correlation being statistically significant. Their work further led to a high throughput screen capable of giving a ‘Yes’, ‘No’ or ‘Borderline’ classification as to whether a compound has a high acute in vivo toxic potential. Drug toxicity (on the cellular and/or organ level) can be caused by a variety of mechanisms such as mitochondrial dysfunction, oxidative stress, ER stress, apoptosis, etc. (Jones et al., 2010; Roberts et al., 2010; Dara et al., 2011; Ahmad et al., 2012). A single experimental approach is therefore unlikely to capture the complexity involved in cellular toxicity. Weyermann et al. (2005) found differences in the viability of the treated cells depending on the test agent and cytotoxicity assay used, and concluded that it is important to choose a suitable cytotoxicity assay depending on the supposed cell death mechanism. Pohjala et al. (2007) also confirmed that assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays. In practice, the mechanism of the possible harmful effects caused by the drug usually is not known beforehand; therefore, the confirmation of the results using more than one cell viability assay is required (Miret et al., 2006). Mingoia et al. (2007) showed that conducting in vitro cytotoxicity screening using a combination of permanent cell lines and cultured hepatocytes would allow mechanistic insight on bioactivation to be obtained, as well as improving the predictability of metabolism-mediated toxicity. Various groups have worked using multiplexed assays, which was found to be cost-effective, more sensitive than a single endpoint assay, providing mechanistic cues of toxicity, is reproducible and amenable for higher throughput screening (Gerets et al., 2009; Wu et al., 2009). In recent years, there has seen the development and commercialization of high content screening (HCS) approaches. Various groups (O’Brien et al., 2006; Xu et al., 2008) screened several compounds with known hepatotoxic liabilities and found the HCS assays utilized to be 50% predictive. Another group (Abraham et al., 2008) utilized high content mechanistic screening to prioritize compounds by relating the high content minimum toxic concentration values for each measured mechanistic endpoint to the compounds in vitro efficacy value. It is crucial to recognize the limitations of cell models and incorporate that knowledge into decisions. No individual cellbased screening strategy is going to cover all situations; thus, a well-defined strategy focused on high value target organs generating sound biological data that is appropriately positioned in the drug development paradigm will offer the best opportunity to improve safety. During the past few years, sufficient progress has been made in some of the fields such as in vitro testing for hepatic toxicity, mitochondrial toxicity, stem-cell based toxicity assays and a lot of publications from industries as well as academics have stressed on these themes, which have been reviewed below:

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Neurons from embryonal carcinoma stem cells and ESCs

Primary and immortalized cell cultures of human and animal origin, organ models also available Primary and immortalized cell lines

Lung

Nervous system

Primary, immortalized and transgenic cell cultures of human and animal origin Cardiomyocytes from ESCs and iPSCs

Primary, immortalized and transgenic cell cultures of human and animal origin, co-culture systems and 3D-cell culture models also available Hepatocyte-like cells from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) Primary cell cultures, immortalized cell line

In vitro system available

Heart

Kidney

Liver

Target organ/ system

Table 6. Availability of in vitro assays for toxicity testing

Cell-lines: SH-SY5Y (Neuroblastoma, Human), U-373 MG (Astrocytoma, Human), -

Calu-3 (Bronchial epithelium, Human)

-

Cell-lines: Hek293 (Human), LLC-PK1 (Tubular epithelium, Porcine) Cell lines: H9c2 (Cardiomyoblast, rat)

-

Cell-lines: HepG2 (Hepatocellular carcinoma, Human), HepaRG (Hepatoma, Human)

Cell-lines commonly used

- Stem-cell-based protocols are the focus of current industrial initiatives

Kuegler et al. (2010); Coyne et al. (2011)

Breier et al. (2010); Moser (2011)

Bérubé et al. (2010) Kostadinova et al., (2013)

Sartipy and Björquist (2011)

Watkins et al. (2011)

- System routinely used in toxicity testing - Stem-cell-based protocols are the focus of current industrial initiatives -Available cell-lines are not much effective, organ models under development showing better results - Systems are used to a limited extent for prescreens

Snykers et al. (2009); Greenhough et al. (2010); Mandenius et al. (2011) Ellis et al. (2011)

Lübberstedt et al. (2011); O’Brien et al., 2004

Selected References

- Stem-cell-based protocols are the focus of current industrial initiatives - No organ models available

- Assays frequently used for toxicity evaluation

Comments

V. Ahuja and S. Sharma

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Developments in the field of non-clinical safety screening of drugs In vitro Hepatic Liability Panel. Hepatotoxicity is the most frequent reason cited for labeling drugs with a black box warning, and for withdrawal of an approved drug (Fung et al., 2001). Human hepatotoxicity has not been predictable because of its low concordance with either standard in vitro cytotoxicity screening assay results (O’Brien et al., 2003; Xu et al., 2004) or regulatory animal study findings (Olson et al., 1998, 2000). Along with hypersensitivity and cutaneous reactions, hepatotoxicity has the poorest correlation (about 50%) with regulatory animal toxicity tests (Olson et al., 1998, 2000). HCS has been applied with 80% sensitivity and 90% specificity for human hepatotoxicity potential (O’Brien et al., 2006). Drug-induced liver injury (DILI) arises via complex multi-step mechanisms, which are initiated by chemical insult to cells. Formation of chemically reactive metabolites, impairment of mitochondrial function and inhibition of the activity of the bile salt export pump (BSEP) and/or other biliary efflux transporters (Lee, 2003; Pauli-Magnus and Meier, 2006) are especially important in initiating processes. The use of cell-based approaches for the evaluation of the propensity of candidate drugs to cause DILI has been proposed by various investigators (Dambach et al., 2005; Greer et al., 2009; Benbow et al., 2010; Gómez-Lechón et al., 2010). The European Medicines Agency has reviewed current approaches to the detection of drug-induced hepatotoxicity alerts in non-clinical, regulatory toxicity studies and proposed their integrated risk assessment (EMEA, 2010). Since DILI can be initiated by various mechanisms, a variety of assays as mentioned below have been proposed. CYPinhibition/ THLE cell toxicity. The assay is used for evaluating CYP independent and CYP dependent cell toxicity. Dambach et al. (2005) have described the use of a panel of human hepatocyte-derived cell lines (THLE) which express high activities of individual human CYP isoforms, or no CYP activity, as a routine screen to support drug discovery. This screening assay discriminates between marketed drugs which cause (idiosyncratic) DILI in man or no DILI in man with very high specificity (>99%) and good sensitivity (69%). The use of a broad panel of CYP expressing and non-CYP expressing THLE cells lines enables investigation of the role of individual CYP-mediated metabolic pathways in cellular injury and detoxification (Greer et al., 2009). Recent data from Pfizer has demonstrated that in vitro cell toxicity data obtained using a non-CYP expressing THLE cell line provided a good prediction of candidate drugs to cause dose-dependent DILI in preclinical species (Benbow et al., 2010). HepG2 cytotoxicity in galactose vs. glucose medium. This assay is useful for assessing mitochondrial impairment. Mitochondrial impairment is considered to be a key mechanism which can in itself cause DILI and potentially also other organ toxicities (Dykens and Will, 2007; Pessayre et al., 2010). A convenient approach for the evaluation of the contribution of mitochondrial impairment to cell toxicity involves comparison of the potency of cell toxicity in galactose vs. glucose media (Mannargudi et al., 2009). This exploits the Crabtree effect, i.e. the differential impact of mitochondrial respiration vs. glycolysis on ATP production in the two media.

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Steatosis assay. One of the major mechanisms involved in steatosis, or accumulation of fatty acids (i.e. ’fatty liver‘), is the inhibition of beta-oxidation of long-chain fatty acids (resulting in lipid accumulation), either by direct inhibition or indirect inhibition such as CoA sequestration or mitochondrial DNA damage, and increased reactive oxygen generation (resulting in lipid peroxidation) (Fromenty and Pessayre, 1995; Jaeschke et al., 2002). The resulting fatty acid accumulation can be detected and quantified by staining primary hepatocytes with neutral lipid stains such as Oil redO (Amacher and Martin, 1997; McMillian et al., 2001). Both of these biochemical endpoints can be measured relatively rapidly using either hepatocytes (Ivanov et al., 1992) or isolated liver mitochondria (Berson et al., 1998; Pessayre et al., 1999). Phospholipidosis assay. Phospholipidosis, defined as the accumulation of excess phospholipids in cells, is often accompanied by various associated or coincidental toxicities, especially in the lung and liver (Halliwell, 1997). Cationic amphiphilic drugs can often induce this phenomenon in vivo. While phospholipidosis per se does not constitute frank toxicity (Reasor and Kacew, 2001), it is reportedly predictive of drug or metabolite accumulation in affected tissues (Hruban, 1984), and as such, possibly associated with toxicities that may require further in vivo investigation (e.g. longer-term animal toxicity studies, reversibility and biomarker studies, etc.). In vitro, the accumulation of phospholipids in cells can be monitored by staining cells with fluorescent phospholipid analogs such as NBDPE (Gum et al., 2001) or NBD-PC (Ulrich et al., 1991). Mitochondrial Toxicity Testing. Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities. Mitochondrial impairment figures prominently in the etiology of hepatoxicity, myopathy, cardiomyopathy, rhabdomyolysis and other serious side effects of various contemporary therapeutics (Amacher, 2005; Chan et al., 2005; Dykens et al., 2007). Of the 50 or so drugs removed from the market owing to safety concerns between 1960 and 1996, at least five have subsequently been associated with mitochondrial toxicity (Dykens and Will, 2007). In addition to post-market drug withdrawals, mitochondrial liabilities have also been associated with many drugs carrying a black box label for hepatic or cardiac toxicity (Dykens et al., 2007), highlighting again the importance of mitochondrial toxicity when considering off-target effects. Mitochondrial injury can occur though several mechanisms, such as (i) uncoupling of electron transport from ATP synthesis, (ii) redox-cycling, (iii) opening of the mitochondrial permeability transition pore, (iv) depletion of mtDNA, (v) inhibition of the Krebs cycle, (vi) inhibition of β-fatty acid oxidation, (vii) inhibition of transporters and (viii) inhibition of the OXPHOS complexes. Not surprisingly, this multitude of mechanisms has precluded the identification of simple structure–activity relationships (SAR) by which to predict mitochondrial liability of compounds (Nadanaciva et al., 2007a). The animals used in regulatory toxicology studies are generally young and healthy and are thus unsuitable for identifying compounds that may cause sub-lethal reductions of mitochondrial capacity. The developments in the field of mitochondrial toxicity testing have been reviewed elsewhere (Dykens and Will, 2007). There are several screens of mitochondrial toxicity that have been used in the early screening of compounds (Hynes et al.,



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Cholestasis assay/inhibition of human BSEP. The assay is used for assessing impairment of bile formation. Bile Salt Export Pump (BSEP) is the transport protein responsible for efflux of bile acids from hepatocytes into bile and genetically acquired defects in BSEP expression resulting in cholestatic liver injury in man, which is believed to arise due to intracellular accumulation of cytotoxic bile salts (Kullak-Ublick et al., 2004). Numerous drugs which cause cholestatic DILI in man inhibit BSEP activity in vitro; therefore, potent BSEP inhibition by

marketed drugs is a potential risk factor for idiosyncratic cholestatic DILI in man (Greer et al., 2009). Kis et al. (2012) have summarized appropriate in vitro methods that could predict BSEP–drug candidate interactions in humans.

V. Ahuja and S. Sharma 2006; Nadanaciva et al., 2007b and Nadanaciva et al., 2007a). Recently, Hynes et al. (2013) found that a combination of a dual parameter assay capable of measuring cellular oxygen consumption and extracellular acidification in cells and the isolated mitochondrial oxygen consumption assay is quite sensitive in identifying compounds that caused mitochondrial impairment. Promising animal models are heterozygous knockout mouse where expression of the mitochondrial isoform of the antioxidant manganese superoxide dismutase (MnSOD+/_) is halved (Dykens and Will, 2007) or heterozygous Sod2+/_ knockout mouse with superoxide dismutase-2 deficiency (Boelsterli and Hsiao, 2008). Stem Cell-Based Assays. The potential of stem cells to grow indefinitely while maintaining pluripotency with stable genotype and phenotype offers the unique opportunity to use these cells as unlimited cells source for various toxicological applications (Vojnits and Bremer, 2010). Human embryonic stem cells (hESCs) are derived from the inner cell mass of pre-implantation embryos and are capable of self-renewal and differentiation to all three germ layers (Thomson et al., 1998; Reubinoff et al., 2000). Induced pluripotent stem cells (iPSCs) are generated by the reprogramming of somatic cells through the introduction of four-key transcription factors: Sox2, Klf4, Oct4 and c-Myc (Takahashi and Yamanaka, 2006). They possess a number of advantages over hESCs, and are free from the ethical complications associated with the use of blastocysts (Dalgetty et al., 2009). Stem cells (ESCs and iPSCs) are being used widely in cardiotoxicity (Cohen et al., 2011), neurotoxicity (Meamer et al., 2010), hepatotoxicity (Greenhough et al., 2010; GuguenGuillouzo et al., 2010), neurotoxicity (Krug et al., 2013), cytotoxicity (May et al., 2012), acute toxicity (Scanu et al., 2011) and embryotoxicity or teratogenicity (Genschow et al., 2002; Hettwer et al., 2010) testing. The use of an embryonic stem cell test (EST), employing murine embryonic stem cells, for developmental toxicity testing has been validated by ECVAM (Spielmann et al., 1997; Genschow et al., 2004). However, EST is the only developmental toxicity test that does not require pregnant animals and is based on a mammalian system (Bremer and Hartung, 2004). At present, realization of the full potential of stem cells is hampered by the difficulty in routinely directing stem cell differentiation in vitro to generate fully functional, specific cell types of choice. The ability of stem cells to differentiate to multiple mature cells can be problematic in terms of obtaining high yield and pure populations of a particular cell type. Being able to do this at a large scale in a reproducible and cost effective manner is even more of a challenge (Hook, 2012). The ethical situation on the use of toxicity tests based on hESCs is still under debate, and recent reports on the establishment of induced pluripotent stem cells (iPSC) are pointing to a way out of this dilemma (Vojnits and Bremer, 2010). Thus, hiPSCs will probably become a key asset in the quest to improve the safety profiles of candidate drugs. High-Content Imaging

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Microscopy in cell biology has long been a descriptive technology limiting its utility in quantitative studies. However, the development of automated, epifluorescence imaging platforms and robust image analysis algorithms, together termed high-


content imaging (HCI), has provided the means to do highthroughput, quantitative analysis of cellular phenotypic assays (Rausch, 2006). These assays gather data at the single cell level and report information on many parameters. Among the many parameters that can be quantified are cell signaling pathways, protein expression levels, cell cycle status, receptor internalization, cytoskeletal integrity, energy metabolism status, nuclear morphology, post-translational protein modifications, cell movement and cell differentiation. Application of this technology to toxicology has paralleled its development for drug discovery uses in recent years. The application of HCI has been discussed in detail elsewhere (Houck and Kavlock, 2008). A commonly employed application of HCI is to determine the effect of chemicals on a set of parameters measuring cell homeostasis. By combining measurement of cell number and nuclear morphology, intra-cellular calcium content, mitochondrial membrane potential and membrane permeability, sensitivity in detecting hepatotoxic chemicals was 93% (O’Brien et al., 2006). Additional HCI endpoints would probably provide insight into specific mechanisms of toxicity. For example, reactive oxygen species generation (Phillips et al., 2005), interference with the cell cycle can be measured by monitoring the expression of cell cycle-specific proteins, e.g. cyclins (Gasparri et al., 2006), mitotic index assays (Gasparri et al., 2004), apoptosis can be detected with multiplexed endpoints including nuclear condensation and caspase activation (Fennell et al., 2006), out-growth of neurites from neuronal precursor cells (Richards et al., 2006) or cell lines can be monitored and used to determine potential neurotoxicity demonstrated by selective activity against neurites relative to other cytotoxic effects. ‘Omics’ Technologies Toxicogenomics involves investigating the response to xenobiotics at the gene expression level. The application of toxicogenomics for profiling toxicant-induced biological perturbations has been describe elsewhere (Blomme et al., 2009; Fabre et al., 2009; Kiyosawa et al., 2010; Afshari et al., 2011; Dalmas et al., 2011). The key examples of toxicogenomics applications include: (i) clustering of compounds in similar mechanistic classes, (ii) generation of hypotheses regarding compound action, (iii) revelation of mechanisms of compound action, (iv) classification of blinded compounds, (v) ranking and categorization of drug candidates by toxicogenomics signature and (vi) discovery of biomarkers of toxicity (Afshari et al., 2011). To date, the liver has been the focus of most of the research. Profiles are being established with known classes of hepatotoxicants in the hope that these profiles can then be referenced when profiling novel compounds (Bulera et al., 2001; Waring et al., 2001). Hrach et al. (2011) used a rat hepatocyte culture in combination with a toxicogenomic classification method to generate a predictive in vitro toxicity classification model and showed the utility of this model for improved mechanistic understanding, refinement of predictivity of toxicological studies and to the reduction of animal usage in toxicology and drug discovery. The concept of gene expression profiling is the same for all target organs and data are now emerging for other organs including kidney (Huang et al., 2001; Goodsaid, 2004). The improvement of the use of toxicogenomics technology in decision making needs to complete the knowledge about how far the outcomes of the gene expression change and what is the impact of the changes


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Developments in the field of non-clinical safety screening of drugs

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Robertson et al. (2000) demonstrated the utility of this technique in discriminating renal and hepatic toxins and pointed out that metabonomics is applicable to rapid-throughput in vivo screening. In a study using 13 model toxins affecting primarily the liver and kidney, Holmes et al. (2001) showed that they could distinguish the affected organ, the site of injury and even strain differences in rats. Clarke and Haselden (2008) demonstrated the application of metabolic profiling in safety assessment as a tool to identify biomarkers for phospholipidosis and peroxisome proliferation. A particular challenge for the application of metabolic profiling is the limited database of normal and affected profiles. More work is required to populate this to the level of those supporting other technologies, but the potential value of a comprehensive endogenous metabolite database will be very high. Some of the recent work where ‘omics’ technology has been used in various aspects for drug safety testing are cited in Table 7. The EU Framework 6 Project: Predictive Toxicology (PredTox), a consortium of pharmaceutical companies, mediumscale enterprises (SMEs) and universities, studied the effects of 16 test compounds using conventional toxicological parameters and; ’omics‘ technologies. The three major observed toxicities, liver hypertrophy, bile duct necrosis and/or cholestasis, and kidney proximal tubular damage were analyzed in detail. The results of the study showed that ’omics‘ technologies can help toxicologists to make better informed decisions during exploratory toxicological studies (Suter et al., 2011). As per FDA perspective on the non-clinical use of the ’omics‘ technologies and the safety of new drugs, the most promising use in the near future would be to clarify pathways for the various types of toxicity and carcinogenicity and get biomarkers for these pathways, to help assess relevance of nonclinical findings to humans (Jacobs, 2009). Translational Safety Biomarkers There is a general notion in the scientific community that research in the field of biomarkers is quite slow, and there is no significant outcome. However, this is a complex field and four distinct stages for the biomarker discovery and development process identified are: discovery, qualification, verification and validation. The three steps in biomarker discovery include identification of markers, prioritization of identified markers and the preliminary qualification of prioritized markers (Gao et al., 2005). Qualification refers to the process whereby a biomarker is linked to a preclinical or clinical end point or to a biological process in a specific context (Goodsaid and Frueh, 2007; Wagner, 2008). The verification of a candidate biomarker requires demonstration of the reproducibility and transferability of these assays between laboratories, an arduous process. Elucidating the molecular basis of tissue injury provides mechanistically anchored safety and efficacy biomarkers that may be useful in a clinical setting. For decades, the assessment of nephrotoxicity has relied primarily on the detection of impaired kidney function by a rise in serum creatinine or blood urea nitrogen (BUN). However, it has long been recognized that as a result of the functional reserve of the kidney, these commonly used clinical markers appear late and are, therefore, unreliable indicators of acute kidney injury. An illustrative and; ’door opening‘ safety biomarker success story is the recent recognition of kidney safety biomarkers for pre-clinical and limited translational contexts by FDA and EMEA. This milestone achieved for kidney biomarkers and the ’know how‘ acquired is being transferred to other organ toxicities, namely the liver,



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in the already described pathways. While data from highly toxic or well-described reference compounds are available (Uehara et al., 2008), results from compounds under development are rather limited and, obviously, this reduces the capacity to use these technologies. Thus, efforts should be made to generate public databases ensuring, at the same time, confidentiality on the products evaluated. Enrichment in toxicogenomics databases, refinements of biomarker gene sets and scoring algorithms and the development of user-friendly integrative software will lead to better evaluation of toxicant-elicited biological perturbations (Kiyosawa et al., 2010). Toxicoproteomics applies global protein measurement technologies to toxicology testing and research. Aims of the field are the discovery of mechanisms governing key proteins in critical biological pathways creating adverse effects, the development of biomarkers and eventual prediction of toxicity based upon pharmacogenomics knowledge (Wetmore and Merrick, 2004; Merrick and Witzmann, 2009). The role of toxicoproteomics in assessing organ-specific toxicity has been described in detail by various authors (Merrick and Witzmann, 2009; Amacher, 2010; Van Summeren et al., 2012). It has been used to examine the differential expression of cortical and medullary proteins in rat kidney (Witzmann et al., 1998), where it was found that 127 proteins differed in abundance between the two regions and 30 proteins were unique to one region or the other. Large-scale application of proteomics is on the horizon with advances in technology and the advent of protein microarrays (Steiner and Witzmann, 2000; Haab, 2001). It is anticipated that the use of toxicoproteomics will enable identification of gene expression profiles and molecular mechanisms early in drug development leading to more accurate assessments of pharmacological activity, and to better-targeted animal studies using fewer animals (Snodin, 2002). Challenges for toxicoproteomics in preclinical risk assessment are: (i) the use as a discovery tool for specific proteins affected by drug and toxicant action; (ii) a better understanding of biochemistry and cell biology; and (iii) biomarker development. The discipline of proteome mapping will be a different and more complex enterprise from the high-throughput, linear-sequencing activities that have been so useful in mapping of the human genome. While the immensity of mapping and measuring the attributes in any one proteome is a large undertaking, biofluid proteomes such as serum/plasma, urine and cerebrospinal fluid hold the most immediate promise for preclinical assessment in terms of better biomarkers (Merrick and Witzmann, 2009). In spite of the presence of many challenges for toxicoproteomics, the opportunities are also close at hand for a greater understanding of toxicant action, the linkage to accompanying dysfunction and pathology, and the development of predictive biomarkers and signatures of toxicity (Merrick and Witzmann, 2009). Metabolomics and metabonomics are metabolic profiling methods that offer the opportunity to identify biomarkers or patterns of biomarker changes related to drug toxicity in biofluid samples, such as urine and blood, using techniques such as NMR and Mass spectrometry. Metabolomics refers to the measurement of the metabolite pool that exists within a cell under a particular set of conditions (Fiehn, 2002) whereas metabonomics describes ’the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification‘ (Nicholson et al., 1999). The application of metabonomics in toxicology has been discussed elsewhere (Beger et al., 2010; Beyoğlu and Idle, 2013).

V. Ahuja and S. Sharma Table 7. Selected references using ‘omics’ technologies in drug discovery for preclinical safety testing Technology Metabolomics

Toxicoproteomics Toxicogenomics

Study objective/ Outcome Study of biomarkers of drug induced hepatotoxicity and nephrotoxicity Identification of developmental toxicity pathways Discovery of new biomarkers Neurotoxicity testing using in vitro method Prediction of proximal tubule kidney toxicity and phospholipidosis in rats following single dose administration Identification of in vitro protein biomarkers of idiosyncratic liver toxicity Understanding mechanism of drug induced hepatotoxicity Validation of biomarkers of nephrotoxicity in rats Multigene biomarker for predicting the future onset of proximal tubular injury in rats Identification of novel set of biomarkers for evaluating phospholipidosisinducing potential of compounds following single dose administration Biomarkers for renal papillary injury in rats

586

heart and vascular system (Marrer and Dieterle, 2010). Some of the approved safety biomarkers are enlisted in Table 8, whereas some exploratory biomarkers are enlisted in Table 9. Various groups are further validating the usefulness of these biomarkers in preclinical studies (Hoffmann et al., 2010; Ozer et al., 2010; Tonomura et al., 2010, 2012). There is also a significant ongoing effort to characterize and qualify a panel of safety biomarkers by various groups, and their clinical bringing validation is also underway (Hoffmann et al., 2010; John-Baptiste et al., 2012; Wadey et al., 2012; Xie et al., 2013). Measurement of a panel of safety biomarkers in parallel would help maximally capture all potential safety signals for a more informative decision to be made in drug research and development as well as for optimal selection of the drug and its dose in clinical practice. In pre-clinical safety studies, drug-induced vascular injury is an issue of concern. As vascular injury involves multiple mediators and cell types, evaluation of a panel rather than a single biomarker may be more useful in monitoring early and severe progressive vascular injury. Evaluation of potential novel markers for clinical monitoring with a mechanistic underpinning would add value in risk assessment and risk management, and various authors have described the progress in this field (Brott et al., 2005; Kerns et al., 2005; Louden et al., 2006). The overall aim of the development of safety biomarkers for use in preclinical studies is to support safety evaluation in clinical trials of new drugs; in parallel some of them (KIM-1, B2M, CysC) are also being widely investigated to determine their utility as diagnostic and prognostic indicators to improve the management of human kidney disease. In the view of the regulators (Dieterle et al., 2010), none of the approved kidney biomarkers can be recommended for monitoring nephrotoxicity in the clinical setting in drug development, at least until further data become available that demonstrate the correlation of these biomarkers with the evolution of drug-induced lesions and their reversibility (Xie et al., 2013). It is therefore expected that pharmaceutical companies submit data for these biomarkers as part of IND enabling studies and clinical studies to regulatory authorities, which would lead to their wider qualification in a translational context.


Reference Beger et al., 2010 Kleinstreuer et al., 2011 van Ravenzwaay et al., 2007 vanVliet et al., 2008 Lienemann et al., 2008 Gao et al., 2004 Blomme et al., 2009 Wang et al., 2008 Minowa et al., 2012 Yudate et al., 2012 Uehara et al., 2013

Preclinical Imaging In vivo imaging is a potentially transformative platform, particularly as it contributes to translational strategies able to both enhance confidence in decision-making and accelerate clinical development (Matthews et al., 2011). It can make a broad range of structural, functional and molecular information relevant to characterizing the pharmacology and toxicology of new drug candidates. Preclinical imaging will play an important and growing role in early phases of drug development. Value generally will be highest when preclinical imaging validates or otherwise enables a translational imaging strategy to enhance confidence or speed decision-making in later clinical development. Modern preclinical imaging technologies such as magnetic resonance imaging (MRI), computerized tomography (CT), positron emission tomography (PET) or single- photon emission computed tomography (SPECT) can add value to preclinical studies by enabling dynamic pharmacological observations on the same animal and because of possibilities for relatively direct clinical translation (Matthews et al., 2012). Examples of the use of preclinical imaging technologies in drug safety assessment studies include the use of SPECT as a dynamic monitor of renal function (Melis et al., 2010), CT has been used for detailed characterization of bone and its pathologies (Badea et al., 2008; Winkelmann and Wise, 2009) and PET for assessing neurotoxicity in NHPs (Ricaurte et al., 2000). The technologies available for preclinical imaging in drug development and their applications have been described elsewhere in detail (Matthews et al., 2012; Comley and Kallend, 2013). Alternative Methods In the development of guidelines for preclinical safety assessment of pharmaceutical products undertaken at the European level by the Safety Working Party-a sub-group of the Committee on Proprietary Medicinal Products (CPMP) and at the global level by ICH, the 3Rs (Replacement, Reduction and Refinement) are extremely important considerations (CPMP, 1997). It has been estimated that the introduction of ICH guidelines led to an


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Heart

Necrosis of heart muscle

Acute tubular alteration

Renal Papillary Antigen (RPA-1)

Troponins: cardiac troponin T (cTnT), and I (cTnI)


Trefoil factor-3 (TFF3)

Acute glomerular alteration

Cystatin-C (CysC)


Acute glomerular alteration

β2-microglobulin (B2M)

Clusterin (CLU)


Kidnye injury molecule-1 (Kim-1)

Kidney

Pathology monitored

Biomarker

Organ

Reference

-Can be included as biomarkers of drug induced acute kidney Biomarker Qualification Program: FDA, tubular alterations in GLP rat studies to support clinical trials EMEA, 2008; Dieterle et al., 2010; Vaidya -Outperforms traditional biomarkers of kidney injuiry et al., 2010 Biomarker Qualification Program: FDA, Can be included as biomarkers of acute drug induced glomerular EMEA, 2008; Dieterle et al., 2010 alteration/damage and/or impairment of kidney tubular reabsorption in GLP rat studies used to support clinical trials Biomarker Qualification Program: FDA, Can be included as biomarkers of acute drug induced glomerular EMEA, 2008; Dieterle et al., 2010 alteration/damage and/or impairment of kidney tubular reabsorption in GLP rat studies used to support clinical trials -Can be included as biomarkers of drug induced acute kidney Biomarker Qualification Program: FDA, tubular alterations in GLP rat studies to support clinical trials EMEA, 2008; Dieterle et al., 2010 -Has better sensitivity and specificity than BUN and creatinine in male rats Can be included as biomarkers of drug induced acute kidney Biomarker Qualification Program: tubular alterations in GLP rat studies to support clinical trials FDA, EMEA, 2008; Dieterle et al., 2010 Biomarker Qualification Program: FDA -Can be included as biomarkers of drug induced acute kidney tubular alterations, particularly in the collecting duct, in male rats -Has better sensitivity and specificity than BUN and creatinine in male rats For safety assessment studies in rats and dogs for following Biomarker Qualification Program: FDA context of use: -When there is previous indication of cardiac structural damage with a particular drug, cardiac troponin testing can help estimate a lowest toxic dose or a highest non-toxic dose to help choose doses for human testing -When there is known cardiac structural damage with a particular pharmacologic class of a drug and histopathologic analyses do not reveal structural damage, circulating cardiac troponins may be used to support or refute the inference of low cardiotoxic potential -When unexpected cardiac structural toxicity is found in a nonclinical study, the retroactive examination of serum or plasma from that study for cardiac troponins can be used to help determine a no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL).

Qualification level

Table 8. Overview of some of the recently qualified safety biomarkers for pre-clinical context




V. Ahuja and S. Sharma Table 9. Overview of some of the exploratory biomarkers Organ Kidney

Liver

Heart Vascular system

Biomarker NGAL

NAG GST-α GGT PNP MDH Thiostatin NT-proBNP vWF, vWFpp, VEGF, ET, ADMA, NO Caveolin-1

Pathology monitored by biomarker Proximal tubular injury (drug-induced) and distal tubular /collecting duct Injury (ischemia) Proximal tubular injury Necrosis in centrilobular region Hepato-biliary injury Hepatocyte necrosis Hepatocyte necrosis Hepatobiliary injuiry Cardiac hypertrophy Endothelial cell injury Endothelial and smooth muscle cell injury

approximate 50% reduction in the number of animals required in the normal core battery of toxicological tests for a ‘standard’ new active substance (Van Cauteren and Lumley, 1997). National and international bodies such as the European Centre for the Validation of Alternative Methods (ECVAM) and the Interagency Co-ordinating Committee on the Validation of Alternative Methods (ICCVAM) in the USA undertake the required validation assessments of alternative methods. Only those methods that are deemed acceptable by ECVAM and/or ICCVAM are normally

Reference Marrer and Dieterle, 2010

Marrer and Dieterle, 2010 Marrer and Dieterle, 2010 Marrer and Dieterle, 2010 Marrer and Dieterle, 2010 Marrer and Dieterle, 2010 Adler et al., 2010 Marrer and Dieterle, 2010 Marrer and Dieterle, 2010; Louden et al., 2006; Kerns et al., 2005; Brott et al., 2005

considered by regulatory agencies as candidates for incorporation into safety testing guidelines. Reproductive toxicity testing is characterized by high animal use. The reproductive cycle is complex and involves hundreds of signaling pathways, and not all of these mechanisms of development and toxicity can readily be mimicked in vitro in a comprehensive way. Inclusion of multiple non-animal complementary assays in a testing strategy might result in enhanced predictive power as compared with individual assays. Table 10 lists various assays that have been optimized for reproductive

Table 10. In vitro tests along with the corresponding targeted segments of the reproductive cycle Test Endocrine Disruption -AR-binding assay (ARBA) -AR Chemically Activated LUciferase eXpression assay (AR CALUX) -PC-3-androgen receptor-Luciferase-MMTV assay (PALM) -ER-binding assay (ERBA) -ER Chemically Activated LUciferase eXpression assay (ER CALUX) -MELN assay Fertility -Follicle bioassay (FBA) -Bovine in vitro maturation assay (bIVM) Bovine in vitro fertilization assay (bIVF) Mouse embryonic peri-implantation assay (MEPA) Ishikawa cell test Embryonic development -Whole embryo culture (WEC) -Embryonic stem cell test (EST) -ReProGlo assay

588

Others PSA secretion assay Placental Perfusion system


Segment of reproductive cycle

Reference

(Anti-) androgenic activity

Schenk et al., 2010

(Anti)-estrogenic activity

Schenk et al., 2010; Witters et al., 2010

Folliculogenesis and Oogenesis

Lazzari et al., 2008; Luciano et al., 2010 Schenk et al., 2010; Lazzari et al., 2008 Schenk et al., 2010

Fertilization Peri-implantation: development from zygote to blastocyst Implantation

Schenk et al., 2010

Embryonic development

Schenk et al., 2010

For assessing prostate functional effects To model placental transfer in humans

Lorenzetti et al., 2010 Myllynen et al., 2010


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Developments in the field of non-clinical safety screening of drugs toxicity screening. Besides a number of other in vitro tests for dermal irritation, sensitization, ocular toxicity, etc. have been approved by ECVAM (http://ihcp.jrc.ec.europa.eu/our_labs/eurlecvam/validation-regulatory-acceptance) and ICCVAM (http:// iccvam.niehs.nih.gov/methods/methods.htm), and the detailed information is available on their websites.

Conclusion Various initiatives have been taken in the last few years by governments and public–private partnerships to foster research in the field of drug safety. Examples are Innovative Medicines Initiative (IMI), which is collaboration between the pharmaceutical industry and the European Commission (http: //imi.europa.eu\index_en.html), and aims at finding solutions to address research bottlenecks in the drug development process. Some of the examples of projects going on in this field with IMI are: mechanism-based integrated systems for the prediction of drug-induced liver injury (MIP-DILI), stem cells for biological assays of novel drugs and predictive toxicology (STEMBANCC), integrating bioinformatics and chemoinformatics approaches for the development of expert systems allowing the in silico prediction of toxicities (eTOX) and SAFE-T (potential biomarker candidates for drug-induced injury of the kidney, liver and vascular system). Further collaborations like this are needed which will be able to provide drug safety scientists with tools to enhance drug screening approaches rapidly and more effectively. During the past 5 years, there has been a paradigm shift in the field of Drug Safety Sciences including an emerging consensus on the need for a flexible, innovative and interdisciplinary science-based approach for Investigative Toxicology. Regulatory acceptance of the plethora of emerging technologies being deployed for investigational safety studies remains a significant challenge but may ultimately be resolved via practical, iterative and fit-for-purpose approaches that have previously been proposed for biomarker method development and validation. The accessibility of broad panels of assays covering an array of protein families permits evaluation of chemicals for their ability to directly modulate many potential targets of toxicity. In addition, advances in cell-based screening have yielded tools capable of reporting the effects of chemicals on numerous critical cell signaling pathways and cell health parameters. Novel, more complex cellular systems are being used to model mammalian tissues and the consequences of compound treatment. Finally, high-throughput technology is being applied to model organism screens to understand mechanisms of toxicity. Integration of successful approaches will contribute towards building a systems approach to toxicology that will provide mechanistic understanding of the effects of chemicals on biological systems and aid in rationale risk assessments. It will also help in implementing the 3Rs approach in preclinical safety testing.

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