Protecting Human and Animal Health: The Road from Animal Models to New Approach Methods.

Animals and animal models have been invaluable for our current understanding of human and animal biology, including physiology, pharmacology, biochemistry, and disease pathology. However, there are increasing concerns with continued use of animals in basic biomedical, pharmacological, and regulatory research to provide safety assessments for drugs and chemicals. There are concerns that animals do not provide sufficient information on toxicity and/or efficacy to protect the target population, so scientists are utilizing the principles of replacement, reduction, and refinement (the 3Rs) and increasing the development and application of new approach methods (NAMs). NAMs are any technology, methodology, approach, or assay used to understand the effects and mechanisms of drugs or chemicals, with specific focus on applying the 3Rs. Although progress has been made in several areas with NAMs, complete replacement of animal models with NAMs is not yet attainable. The road to NAMs requires additional development, increased use, and, for regulatory decision making, usually formal validation. Moreover, it is likely that replacement of animal models with NAMs will require multiple assays to ensure sufficient biologic coverage. The purpose of this manuscript is to provide a balanced view of the current state of the use of animal models and NAMs as approaches to development, safety, efficacy, and toxicity testing of drugs and chemicals. Animals do not provide all needed information nor do NAMs, but each can elucidate key pieces of the puzzle of human and animal biology and contribute to the goal of protecting human and animal health. SIGNIFICANCE STATEMENT: Data from traditional animal studies have predominantly been used to inform human health safety and efficacy. Although it is unlikely that all animal studies will be able to be replaced, with the continued advancement in new approach methods (NAMs), it is possible that sometime in the future, NAMs will likely be an important component by which the discovery, efficacy, and toxicity testing of drugs and chemicals is conducted and regulatory decisions are made.

Gaps and challenges in nonclinical assessments of pharmaceuticals: An FDA/CDER perspective on considerations for development of new approach methodologies.

Previously, we provided an FDA/CDER perspective on nonclinical testing strategies and briefly discussed the opportunities and challenges of using new approach methodologies (NAMs) in drug development, especially for regulatory purposes. To facilitate the integration of NAMs into nonclinical regulatory testing, we surveyed the CDER Pharmacology/Toxicology community to identify the nonclinical challenges faced by CDER review staff, including gaps and areas of concern underserved by current nonclinical testing approaches, and to understand how development of NAMs with specific contexts of use (COUs) could potentially alleviate them. Survey outcomes were coalesced into CDER-identified needs for which NAMs with specific COUs could potentially be developed to address gaps and challenges in nonclinical safety assessments. We also discussed the current FDA procedure for validation and qualification of NAMs intended to inform regulatory decisions. This manuscript is intended to facilitate productive discussions and collaborations with regulatory, government, and academic stakeholders within the drug development community regarding the development and regulatory use of NAMs and their role in safety and efficacy assessment of pharmaceuticals.

Co-Culture of Human Primary Hepatocytes and Nonparenchymal Liver Cells in the Emulate® Liver-Chip for the Study of Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a significant public health issue, but standard animal tests and clinical trials sometimes fail to predict DILI due to species differences and the relatively low number of human subjects involved in preapproval studies of a new drug, respectively. In vitro models have long been used to aid DILI prediction, with primary human hepatocytes (PHHs) being generally considered the gold standard. However, despite many efforts and decades of work, traditional culture methods have been unsuccessful in either fully preserving essential liver functions after isolation of PHHs or in emulating interactions between PHHs and hepatic nonparenchymal cells (NPCs), both of which are essential for the development of DILI under in vivo conditions. Recently, various liver-on-a-chip (Liver-Chip) systems have been developed to co-culture hepatocytes and NPCs in a three-dimensional environment on microfluidic channels, enabling better maintenance of primary liver cells and thus improved DILI prediction. The Emulate® Liver-Chip is a commercially available system that can recapitulate some in vivo DILI responses associated with certain compounds whose liver safety profile cannot be accurately evaluated using conventional approaches involving PHHs or animal models due to a lack of innate immune responses or species-dependent toxicity, respectively. Here, we describe detailed procedures for the use of Emulate® Liver-Chips for co-culturing PHHs and NPCs for the purpose of DILI evaluation. First, we describe the procedures for preparing the Liver-Chip. We then outline the steps needed for sequential seeding of PHHs and NPCs in the prepared Liver-Chips. Lastly, we provide a protocol for utilizing cells maintained in perfusion culture in the Liver-Chips to evaluate DILI, using acetaminophen as an example. In all, use of this system and the procedures described here allow better preservation of the functions of human primary liver cells, resulting in an improved in vitro model for DILI assessment. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Liver-Chip preparation Basic Protocol 2: Seeding primary human hepatocytes and nonparenchymal cells on Liver-Chips Basic Protocol 3: Perfusion culture for the study of acetaminophen-induced liver injury.

Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate).

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

FutureTox IV Workshop Summary: Predictive Toxicology for Healthy Children.

FutureTox IV, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in November 2018. Building upon FutureTox I, II, and III, this conference focused on the latest science and technology for in vitro profiling and in silico modeling as it relates to predictive developmental and reproductive toxicity (DART). Publicly available high-throughput screening data sets are now available for broad in vitro profiling of bioactivities across large inventories of chemicals. Coupling this vast amount of mechanistic data with a deeper understanding of molecular embryology and post-natal development lays the groundwork for using new approach methodologies (NAMs) to evaluate chemical toxicity, drug efficacy, and safety assessment for embryo-fetal development. NAM is a term recently adopted in reference to any technology, methodology, approach, or combination thereof that can be used to provide information on chemical hazard and risk assessment to avoid the use of intact animals (U.S. Environmental Protection Agency [EPA], Strategic plan to promote the development and implementation of alternative test methods within the tsca program, 2018, https://www.epa.gov/sites/production/files/2018-06/documents/epa_alt_strat_plan_6-20-18_clean_final.pdf). There are challenges to implementing NAMs to evaluate chemicals for developmental toxicity compared with adult toxicity. This forum article reviews the 2018 workshop activities, highlighting challenges and opportunities for applying NAMs for adverse pregnancy outcomes (eg, preterm labor, malformations, low birth weight) as well as disorders manifesting postnatally (eg, neurodevelopmental impairment, breast cancer, cardiovascular disease, fertility). DART is an important concern for different regulatory statutes and test guidelines. Leveraging advancements in such approaches and the accompanying efficiencies to detecting potential hazards to human development are the unifying concepts toward implementing NAMs in DART testing. Although use of NAMs for higher level regulatory decision making is still on the horizon, the conference highlighted novel testing platforms and computational models that cover multiple levels of biological organization, with the unique temporal dynamics of embryonic development, and novel approaches for estimating toxicokinetic parameters essential in supporting in vitro to in vivo extrapolation.

An FDA/CDER perspective on nonclinical testing strategies: Classical toxicology approaches and new approach methodologies (NAMs).

Nonclinical testing of human pharmaceuticals is conducted to assess the safety of compounds to be studied in human clinical trials and for marketing of new drugs. Although there is no exact number and type of nonclinical studies required for safety assessments, as there is inherent flexibility for each new compound, the traditional approach is outlined in various FDA and ICH guidance documents and involves a combination of in vitro assays and whole animal testing methods. Recent advances in science have led to the emergence of numerous new approach methodologies (NAMs) for nonclinical testing that are currently being used in various aspects of drug development. Traditional nonclinical testing methods can predict clinical outcomes, although improvements in these methods that can increase predictivity of clinical outcomes are encouraged and needed. This paper discusses FDA/CDER's view on the opportunities and challenges of using NAMs in drug development especially for regulatory purposes, and also includes examples where NAMs are currently being used in nonclinical safety assessments and where they may supplement and/or enhance current testing methods. FDA/CDER also encourages communication with stakeholders regarding NAMs and is committed to exploring the use of NAMs to improve regulatory efficiency and potentially expedite drug development.

"Natural" is not synonymous with "Safe": Toxicity of natural products alone and in combination with pharmaceutical agents.

During the 25 years since the US Congress passed the Dietary Supplement Health and Education Act (DSHEA), the law that transformed the US Food and Drug Administration's (FDA's) authority to regulate dietary supplements, the dietary supplement market has grown exponentially. Retail sales of herbal products, a subcategory of dietary supplements, have increased 83% from 2008 to 2018 ($4.8 to $8.8 billion USD). Although consumers often equate "natural" with "safe", it is well recognized by scientists that constituents in these natural products (NPs) can result in toxicity. Additionally, when NPs are co-consumed with pharmaceutical agents, the precipitant NP can alter drug disposition and drug delivery, thereby enhancing or reducing the therapeutic effect of the object drug(s). With the widespread use of NPs, these effects can be underappreciated. We present a summary of a symposium presented at the Annual Meeting of the Society of Toxicology 2019 (12 March 2019) that discussed potential toxicities of NPs alone and in combination with drugs.

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

The US Federal Tox21 Program: A strategic and operational plan for continued leadership.

The traditional approaches to toxicity testing have posed multiple challenges for evaluating the safety of commercial chemicals, pesticides, food additives/contaminants, and medical products.The challenges include number of chemicals that need to be tested, time and resource intensive nature of traditional toxicity tests, and unexpected adverse effects that occur in pharmaceutical clinical trials despite the extensive toxicological testing.Over a decade ago, the U.S. Environmental Protection Agency (EPA), National Toxicology Program (NTP), National Center for Advancing Translational Sciences (NCATS), and the Food and Drug Administration (FDA) formed a federal consortium for "Toxicology in the 21st Century" (Tox21) with a focus on developing and evaluating in vitro high-throughput screening (HTS) methods for hazard identification and providing mechanistic insights.The Tox21 consortium generated data on thousands of pharmaceuticals and datapoor chemicals, developed better understanding of the limits and applications of in vitro methods, and enabled incorporation of HTS data into regulatory decisions. To more broadly address the challenges in toxicology, Tox21 has developed a new strategic and operational plan that expands the focus of its research activities. The new focus areas include developing an expanded portfolio of alternative test systems, addressing technical limitations of in vitrotest systems, curating legacy in vivo toxicity testing data, establishing scientific confidence in the in vitrotest systems, and refining alternative methods for characterizing pharmacokinetics and in vitro assay disposition.The new Tox21 strategic and operational plan addresses key challenges to advance toxicology testing and will benefit both the organizations involved and the toxicology community.

A hybrid gene selection approach to create the S1500+ targeted gene sets for use in high-throughput transcriptomics.

Changes in gene expression can help reveal the mechanisms of disease processes and the mode of action for toxicities and adverse effects on cellular responses induced by exposures to chemicals, drugs and environment agents. The U.S. Tox21 Federal collaboration, which currently quantifies the biological effects of nearly 10,000 chemicals via quantitative high-throughput screening(qHTS) in in vitro model systems, is now making an effort to incorporate gene expression profiling into the existing battery of assays. Whole transcriptome analyses performed on large numbers of samples using microarrays or RNA-Seq is currently cost-prohibitive. Accordingly, the Tox21 Program is pursuing a high-throughput transcriptomics (HTT) method that focuses on the targeted detection of gene expression for a carefully selected subset of the transcriptome that potentially can reduce the cost by a factor of 10-fold, allowing for the analysis of larger numbers of samples. To identify the optimal transcriptome subset, genes were sought that are (1) representative of the highly diverse biological space, (2) capable of serving as a proxy for expression changes in unmeasured genes, and (3) sufficient to provide coverage of well described biological pathways. A hybrid method for gene selection is presented herein that combines data-driven and knowledge-driven concepts into one cohesive method. Our approach is modular, applicable to any species, and facilitates a robust, quantitative evaluation of performance. In particular, we were able to perform gene selection such that the resulting set of "sentinel genes" adequately represents all known canonical pathways from Molecular Signature Database (MSigDB v4.0) and can be used to infer expression changes for the remainder of the transcriptome. The resulting computational model allowed us to choose a purely data-driven subset of 1500 sentinel genes, referred to as the S1500 set, which was then augmented using a knowledge-driven selection of additional genes to create the final S1500+ gene set. Our results indicate that the sentinel genes selected can be used to accurately predict pathway perturbations and biological relationships for samples under study.

Metabolic Syndrome and Associated Diseases: From the Bench to the Clinic.

Metabolic Syndrome and Associated Diseases: From the Bench to the Clinic, a Society of Toxicology Contemporary Concepts in Toxicology (CCT) workshop was held on March 11, 2017. The meeting was convened to raise awareness of metabolic syndrome and its associated diseases and serve as a melting pot with scientists of multiple disciplines (eg, toxicologists, clinicians, regulators) so as to spur research and understanding of this condition. The criteria for metabolic syndrome include obesity, dyslipidemia (low high-density lipoprotein and/or elevated triglycerides), elevated blood pressure, and alterations in glucose metabolism. It can lead to a greater potential of type 2 diabetes, lipid disorders, cardiovascular disease, hepatic steatosis, and other circulatory disorders. Although there are no approved drugs specifically for this syndrome, many drugs target diseases associated with this syndrome thus potentially increasing the likelihood of drug-drug interactions. There is currently significant research focusing on understanding the key pathways that control metabolism, which would be likely targets of risk factors (eg, exposure to xenobiotics, genetics) and lifestyle factors (eg, microbiome, nutrition, and exercise) that contribute to metabolic syndrome. Understanding these pathways could also lead to the development of pharmaceutical interventions. As individuals with metabolic syndrome have signs similar to that of toxic responses (eg, oxidative stress and inflammation) and organ dysfunction, these alterations should be taken into account in drug development. With the increasing frequency of metabolic syndrome in the general population, the idea of a "normal" individual may need to be redefined. This paper reports on the substance and outcomes of this workshop.

Proteomic analysis of acetaminophen-induced hepatotoxicity and identification of heme oxygenase 1 as a potential plasma biomarker of liver injury.

PURPOSE: Overdose of acetaminophen (APAP) is a major cause of acute liver failure. This study was aimed to identify pathways related to hepatotoxicity and potential biomarkers of liver injury. EXPERIMENTAL DESIGN: Rats were treated with low (100 mg/kg) and high (1250 mg/kg) doses of APAP, and liver tissues at 6 and 24 h post-treatment were analyzed using a proteomic approach of 16O/18O labeling and 2D-LC-MS/MS. RESULTS: Molecular pathways evolved progressively from scattered and less significant perturbations to more focused and significant alterations in a dose- and time-dependent manner upon APAP treatment. Imbalanced expression of hemeoxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA) was associated with hepatotoxicity. Protein abundance changes of a total of 31 proteins were uniquely correlated to liver damage, among which a dramatic increase of HMOX1 levels in plasma was observed. Liver injury-associated significant elevation of plasma HMOX1 was further validated in mice treated with APAP. CONCLUSIONS AND CLINICAL RELEVANCE: This study unveiled molecular changes associated with APAP-induced liver toxicity at the pathway levels and identified HMOX1 as a potential plasma biomarker of liver injury.

FutureTox III: Bridges for Translation.

Future Tox III, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in November 2015. Building upon Future Tox I and II, Future Tox III was focused on developing the high throughput risk assessment paradigm and taking the science of in vitro data and in silico models forward to explore the question-what progress is being made to address challenges in implementing the emerging big-data toolbox for risk assessment and regulatory decision-making. This article reports on the outcome of the workshop including 2 examples of where advancements in predictive toxicology approaches are being applied within Federal agencies, where opportunities remain within the exposome and AOP domains, and how collectively the toxicology community across multiple sectors can continue to bridge the translation from historical approaches to Tox21 implementation relative to risk assessment and regulatory decision-making.

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides.

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. This algorithm can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.

Molecular Docking for Identification of Potential Targets for Drug Repurposing.

Using existing drugs for new indications (drug repurposing) is an effective method not only to reduce drug development time and costs but also to develop treatments for new disease including those that are rare. In order to discover novel indications, potential target identification is a necessary step. One widely used method to identify potential targets is through molecule docking. It requires no prior information except structure inputs from both the drug and the target, and can identify potential targets for a given drug, or identify potential drugs for a specific target. Though molecular docking is popular for drug development and repurposing, challenges remain for the method. In order to improve the prediction accuracy, optimizing the target conformation, considering the solvents and adding cobinders to the system are possible solutions.

Adverse outcome pathways: From research to regulation scientific workshop report.

An adverse outcome pathway (AOP) helps to organize existing knowledge on chemical mode of action, starting with a molecular initiating event such as receptor binding, continuing through key events, and ending with an adverse outcome such as reproductive impairment. AOPs can help identify knowledge gaps where more research is needed to understand the underlying mechanisms, aid in chemical hazard characterization, and guide the development of new testing approaches that use fewer or no animals. A September 2014 workshop in Bethesda, Maryland considered how the AOP concept could improve regulatory assessments of chemical toxicity. Scientists from 21 countries, representing industry, academia, regulatory agencies, and special interest groups, attended the workshop, titled Adverse Outcome Pathways: From Research to Regulation. Workshop plenary presentations were followed by breakout sessions that considered regulatory acceptance of AOPs and AOP-based tools, criteria for building confidence in an AOP for regulatory use, and requirements to build quantitative AOPs and AOP networks. Discussions during the closing session emphasized a need to increase transparent and inclusive collaboration, especially with disciplines outside of toxicology. Additionally, to increase impact, working groups should be established to systematically prioritize and develop AOPs. Multiple collaborative projects and follow-up activities resulted from the workshop.

Machine Learning Methods for Predicting HLA-Peptide Binding Activity.

As major histocompatibility complexes in humans, the human leukocyte antigens (HLAs) have important functions to present antigen peptides onto T-cell receptors for immunological recognition and responses. Interpreting and predicting HLA-peptide binding are important to study T-cell epitopes, immune reactions, and the mechanisms of adverse drug reactions. We review different types of machine learning methods and tools that have been used for HLA-peptide binding prediction. We also summarize the descriptors based on which the HLA-peptide binding prediction models have been constructed and discuss the limitation and challenges of the current methods. Lastly, we give a future perspective on the HLA-peptide binding prediction method based on network analysis.

Biomarker-based drug safety assessment in the age of systems pharmacology: from foundational to regulatory science.

Improved biomarker-based assessment of drug safety is needed in drug discovery and development as well as regulatory evaluation. However, identifying drug safety-related biomarkers such as genes, proteins, miRNA and single-nucleotide polymorphisms remains a big challenge. The advances of 'omics' and computational technologies such as genomics, transcriptomics, metabolomics, proteomics, systems biology, network biology and systems pharmacology enable us to explore drug actions at the organ and organismal levels. Computational and experimental systems pharmacology approaches could be utilized to facilitate biomarker-based drug safety assessment for drug discovery and development and to inform better regulatory decisions. In this article, we review the current status and advances of systems pharmacology approaches for the development of predictive models to identify biomarkers for drug safety assessment.

Circulating mitochondrial biomarkers for drug-induced liver injury.

Liver mitochondria affected by drugs can be released into circulation and serve as biomarkers for drug-induced liver injury (DILI). The tissue specificity of ALT was improved by differentiating cytosolic ALT1 and mitochondrial ALT2 isoforms released in circulation. Prior to ALT elevation, mitochondrial cytochrome c, OCT, GLDH, CPS1 and DNA were increased in circulation following DILI. The baseline expression of mt-Nd6 was predictive of individual DILI susceptibility in animals. As mitochondrial DILI biomarkers appeared to be drug or species dependent, they might have value in clinical scenarios when culprit drugs are established, but may not be ideal tools to assess DILI potentials of new drugs.

HLADR: a database system for enhancing the discovery of biomarkers for predicting human leukocyte antigen-mediated idiosyncratic adverse drug reactions.

AIM: To establish a database for the associations between idiosyncratic drug reactions (IDRs) and human leukocyte antigens (HLAs) and to systematically assess the characteristics of the drug-HLA associations. MATERIALS & METHODS: Electronic databases were searched to extensively identify drug-HLA association studies from 1966 to present. RESULTS: A drug-HLA-IDR database, HLADR, was created. The drug-HLA relationship network clearly reflected an ethnicity dependency of the associations. The positive predictive values and the negative predictive values demonstrated that other potential factors may also regulate the occurrence of HLA-specific IDRs. CONCLUSIONS: Constructing studies with samples from homogeneous ethnic groups and identifying cofactors that affect negative predictive values and positive predictive values will become necessary to enhance the predictability of HLA biomarkers for future research on IDRs.