Workshop Report: FDA Workshop on Improving Cardiotoxicity Assessment With Human-Relevant Platforms.

Given that cardiovascular safety concerns remain the leading cause of drug attrition at the preclinical drug development stage, the National Center for Toxicological Research of the US Food and Drug Administration hosted a workshop to discuss current gaps and challenges in translating preclinical cardiovascular safety data to humans. This white paper summarizes the topics presented by speakers from academia, industry, and government intended to address the theme of improving cardiotoxicity assessment in drug development. The main conclusion is that to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development. Potential regulatory applications of human-derived cardiomyocytes and future directions in employing human-relevant platforms to fill the gaps and overcome barriers and challenges in preclinical cardiovascular safety assessment were discussed. This paper is intended to serve as an initial step in a public-private collaborative development program for human-relevant cardiotoxicity tools, particularly for cardiotoxicities characterized by contractile dysfunction or structural injury.

Microphysiological Systems: A Pathologist's Perspective.

High-throughput in vitro models lack human-relevant complexity, which undermines their ability to accurately mimic in vivo biologic and pathologic responses. The emergence of microphysiological systems (MPS) presents an opportunity to revolutionize in vitro modeling for both basic biomedical research and applied drug discovery. The MPS platform has been an area of interdisciplinary collaboration to develop new, predictive, and reliable in vitro methods for regulatory acceptance. The current MPS models have been developed to recapitulate an organ or tissue on a smaller scale. However, the complexity of these models (ie, including all cell types present in the in vivo tissue) with appropriate structural, functional, and biochemical attributes are often not fully characterized. Here, we provide an overview of the capabilities and limitations of the microfluidic MPS model (aka organs-on-chips) within the scope of drug development. We recommend the engagement of pathologists early in the MPS design, characterization, and validation phases, because this will enable development of more robust and comprehensive MPS models that can accurately replicate normal biology and pathophysiology and hence be more predictive of human responses.

National Toxicology Program Position Statement on Informed ("Nonblinded") Analysis in Toxicologic Pathology Evaluation.

The National Toxicology Program (NTP) uses histopathological evaluation of animal tissues as a key element in its toxicity and carcinogenicity studies. The initial histopathological evaluations are subjected to a rigorous peer review process involving several steps. The NTP peer review process is conducted by multiple, highly trained, and experienced toxicological pathologists employing standardized terminology. In addition, ancillary data, such as body and organ weights and clinical pathology findings, are used to corroborate the diagnoses. The NTP does employ masked analysis to confirm subtle lesions or severity scores, as needed, and during its Pathology Working Groups. The use of masked analysis can have a negative effect on histopathological evaluation because it is important for the pathologist to compare treated groups to the concurrent controls, which would not be possible in a blinded evaluation. Therefore, the NTP supports an informed approach to histopathological evaluation in its toxicity and carcinogenicity studies.

Non-proliferative and Proliferative Lesions of the Cardiovascular System of the Rat and Mouse.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Japan (JSTP), Europe (ESTP), Great Britain (BSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The primary purpose of this publication is to provide a standardized nomenclature for characterizing lesions observed in the cardiovascular (CV) system of rats and mice commonly used in drug or chemical safety assessment. The standardized nomenclature presented in this document is also available electronically for society members on the internet (http://goreni.org). Accurate and precise morphologic descriptions of changes in the CV system are important for understanding the mechanisms and pathogenesis of those changes, differentiation of natural and induced injuries and their ultimate functional consequence. Challenges in nomenclature are associated with lesions or pathologic processes that may present as a temporal or pathogenic spectrum or when natural and induced injuries share indistinguishable features. Specific nomenclature recommendations are offered to provide a consistent approach.

Integrated approach to early detection of cardiovascular toxicity induced by a ghrelin receptor agonist.

Cardiovascular (CV) safety concerns are among the leading causes of compound attrition in drug development. This work describes a strategy of applying novel end points to a 7-day rodent study to increase the opportunity to detect and characterize CV injury observed in a longer term (ie, 28 days) study. Using a ghrelin receptor agonist (GSK894281), a compound that produces myocardial degeneration/necrosis in rats after 28 days at doses of 0.3, 1, 10, or 60 mg/kg/d, we dosed rats across a range of similar doses (0, 0.3, 60, or 150 mg/kg/d) for 7 days to determine whether CV toxicity could be detected in a shorter study. End points included light and electron microscopies of the heart; heart weight; serum concentrations of fatty acid-binding protein 3 (FABP3), cardiac troponin I (cTnI), cardiac troponin T (cTnT), and N-terminal proatrial natriuretic peptide (NT-proANP); and a targeted transcriptional assessment of heart tissue. Histologic evaluation revealed a minimal increase in the incidence and/or severity of cardiac necrosis in animals administered 150 mg/kg/d. Ultrastructurally, mitochondrial membrane whorls and mitochondrial degeneration were observed in rats given 60 or 150 mg/kg/d. The FABP3 was elevated in rats given 150 mg/kg/d. Cardiac transcriptomics revealed evidence of mitochondrial dysfunction coincident with histologic lesions in the heart, and along with the ultrastructural results support a mechanism of mitochondrial injury. There were no changes in cTnI, cTnT, NT-proANP, or heart weight. In summary, enhancing a study design with novel end points provides a more integrated evaluation in short-term repeat dose studies, potentially leading to earlier nonclinical detection of structural CV toxicity.

Digitalization of toxicology: improving preclinical to clinical translation.

Though the portfolio of medicines that are extending and improving the lives of patients continues to grow, drug discovery and development remains a challenging business on its best day. Safety liabilities are a significant contributor to development attrition where the costliest liabilities to both drug developers and patients emerge in late development or post-marketing. Animal studies are an important and influential contributor to the current drug discovery and development paradigm intending to provide evidence that a novel drug candidate can be used safely and effectively in human volunteers and patients. However, translational gaps-such as toxicity in patients not predicted by animal studies-have prompted efforts to improve their effectiveness, especially in safety assessment. More holistic monitoring and "digitalization" of animal studies has the potential to enrich study outcomes leading to datasets that are more computationally accessible, translationally relevant, replicable, and technically efficient. Continuous monitoring of animal behavior and physiology enables longitudinal assessment of drug effects, detection of effects during the animal's sleep and wake cycles and the opportunity to detect health or welfare events earlier. Automated measures can also mitigate human biases and reduce subjectivity. Reinventing a conservative, standardized, and traditional paradigm like drug safety assessment requires the collaboration and contributions of a broad and multi-disciplinary stakeholder group. In this perspective, we review the current state of the field and discuss opportunities to improve current approaches by more fully leveraging the power of sensor technologies, artificial intelligence (AI), and animal behavior in a home cage environment.

Enabling novel paradigms: a biological questions-based approach to human chemical hazard and drug safety assessment.

Throughput needs, costs of time and resources, and concerns about the use of animals in hazard and safety assessment studies are fueling a growing interest in adopting new approach methodologies for use in product development and risk assessment. However, current efforts to define "next-generation risk assessment" vary considerably across commercial and regulatory sectors, and an a priori definition of the biological scope of data needed to assess hazards is generally lacking. We propose that the absence of clearly defined questions that can be answered during hazard assessment is the primary barrier to the generation of a paradigm flexible enough to be used across varying product development and approval decision contexts. Herein, we propose a biological questions-based approach (BQBA) for hazard and safety assessment to facilitate fit-for-purpose method selection and more efficient evidence-based decision-making. The key pillars of this novel approach are bioavailability, bioactivity, adversity, and susceptibility. This BQBA is compared with current hazard approaches and is applied in scenarios of varying pathobiological understanding and/or regulatory testing requirements. To further define the paradigm and key questions that allow better prediction and characterization of human health hazard, a multidisciplinary collaboration among stakeholder groups should be initiated.

Proceedings of the 2012 National Toxicology Program Satellite Symposium.

The 2012 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was held in Boston in advance of the Society of Toxicologic Pathology's 31st annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting or discussion. Some lesions and topics covered during the symposium include eosinophilic crystalline pneumonia in a transgenic mouse model; differentiating adrenal cortical cystic degeneration from adenoma; atypical eosinophilic foci of altered hepatocytes; differentiating cardiac schwannoma from cardiomyopathy; diagnosis of cardiac papillary muscle lesions; intrahepatocytic erythrocytes and venous subendothelial hepatocytes; lesions in Rathke's cleft and pars distalis; pernicious anemia and megaloblastic disorders; embryonic neuroepithelial dysplasia, holoprosencephaly and exencephaly; and INHAND nomenclature for select cardiovascular lesions.

Integrated and translational nonclinical in vivo cardiovascular risk assessment: gaps and opportunities.

Cardiovascular (CV) safety concerns are a significant source of drug development attrition in the pharmaceutical industry today. Though current nonclinical testing paradigms have largely prevented catastrophic CV events in Phase I studies, many challenges relating to the inability of current nonclinical safety testing strategies to model patient outcomes persist. Contemporary approaches include a spectrum of evaluations of CV structure and function in a variety of laboratory animal species. These approaches might be improved with a more holistic integration of these evaluations in repeat-dose studies, addition of novel endpoints with greater sensitivity and translational application, and use of more relevant animal models. Particular opportunities present with advances in imaging capabilities applicable to rodent and non-rodent species, technical capabilities for measuring CV function in repeat-dose animal studies, detection and quantitation of microRNAs and wider use of alternative animal models. Strategic application of these novel opportunities considering putative CV risk associated with the molecular drug target as well as inherent risks present in the target patient population could tailor or 'personalize' nonclinical safety assessment as a more translational evaluation. This paper is a call to action for the clinical and nonclinical drug safety communities to assess these opportunities to determine their utility in filling potential gaps in our current cardiovascular safety testing paradigms.

Cancer Hazard Evaluations for Contemporary Needs: Highlights From New National Toxicology Program Evaluations and Methodological Advancements.

The National Toxicology Program strives to raise awareness of cancer hazards in our environment. Identifying cancer hazards is key to primary prevention, informing public health decision making, and decreasing the global cancer burden. In December 2021, the US congressionally mandated 15th Report on Carcinogens was released, adding 8 new substances to the cumulative report. Chronic infection with Helicobacter pylori is listed as "known to be a human carcinogen." Antimony trioxide and 6 haloacetic acids found as water disinfection by-products-dichloroacetic acid, dibromoacetic acid, bromochloroacetic acid, tribromoacetic acid, bromodichloroacetic acid, chlorodibromoacetic acid-are listed as "reasonably anticipated to be a human carcinogen." A new dashboard provides interactive visualization and interrogation of the 256 listed substances, their uses, and associated cancers. Also, the National Toxicology Program recently published a Cancer Hazard Assessment Report on exposure scenarios associated with circadian disruption, concluding that persistent night shift work can cause breast cancer and certain lighting conditions may cause cancer. As highlighted in these reports and evaluations, we are evolving our approaches to meet contemporary challenges. These approaches include focusing on real-world exposures and advancing our methods to address challenges in cancer hazard assessments (eg, developing more structured approaches to evaluate mechanistic data and incorporating read-across approaches to assess chemicals lacking adequate human or animal cancer data). To promote public health, we provide information on environmental health disparities and disease prevention. Building on these efforts, we aim to continue our contributions to the war on cancer, declared 50 years ago.

A Novel Mouse Model to Analyze Non-Genomic ERα Physiological Actions.

Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

Troponin measurements during drug development--considerations for monitoring and management of potential cardiotoxicity: an educational collaboration among the Cardiac Safety Research Consortium, the Duke Clinical Research Institute, and the US Food and Drug Administration.

Drug-induced cardiac toxicity is a recognized challenge in development and implementation of pharmacotherapy. Appropriate biomarkers are needed to detect these abnormalities early in development and to manage the risk of potentially cardiotoxic drugs or biologic agents. Circulating cardiac troponin (cTn) is the most widely used biomarker for detection of myocardial injury. Although most commonly used to detect myonecrosis in the setting of ischemia, cTns are also elevated with other acute and chronic disease processes, including heart failure, renal failure, sepsis, pulmonary embolic disease, and many others. High-sensitivity assays for both cTnI and cTnT are now available that achieve acceptable imprecision (coefficient of variation <10%) at the 99th percentile of a normal reference population. Even more sensitive assays are being developed that detect cTn in ranges that are near the level of normal cellular turnover (apoptosis). These properties of cTn and the continuing evolution of highly sensitive assays position cTn as a potentially uniquely informative marker for early detection of cardiac toxicity. This article summarizes collaborative discussions among key stakeholders in the Cardiac Safety Research Consortium about the use of cTn monitoring in drug development.

Toxicologic pathology in a multicultural world--India.

The global practice of drug development is expanding into many different continents and countries. India, in particular, is rapidly emerging as an economic force in this arena by offering ever-expanding opportunities for pharmaceutical market expansion as well as productive drug development partnerships. The key to the country's current socioeconomic success appears to be education, particularly the development of higher and professional education. Also, recent modifications to India's patent laws offer greater protections and incentives for international investment. Increasing numbers of competent contract research organizations create attractive opportunities for large Western pharmaceutical companies with a desire to gain access to burgeoning markets as well as mitigate the rising cost of drug development with less costly services. Well-trained veterinary pathologists are available, appropriate facilities are being constructed, and laboratory capabilities are expanding. Developing a productive partnership with a credible laboratory service in India, as with any new provider, requires due diligence and knowledgeable scrutiny of key elements of the work stream, such as facilities, education and training of laboratory personnel, Good Laboratory Practices, animal care, timelines, and data management. Ultimately and with appropriate management, mutually beneficial drug development partnerships are available in India.

Resolution of a proteinuric nephropathy associated with Babesia gibsoni infection in a dog.

A 4 yr old male castrated Labrador retriever was evaluated for a short history of inappetance, lethargy, small-bowel diarrhea, polyuria, and polydipsia. Clinicopathologic abnormalities were consistent with protein-losing nephropathy and renal azotemia. Expansive infectious disease testing implicated Babesia gibsoni via whole blood polymerase chain reaction. Renal histopathology results were consistent with membranoproliferative glomerulonephritis and immune complex deposition. The dog was treated with azithromycin, atovaquone, and one dose of corticosteroids/cyclophosphamide. Three months after therapy was completed, the dog was clinically healthy, and all clinicopathologic abnormalities (including Babesia species polymerase chain reaction) had resolved. Atypical presentations of Babesia gibsoni should be considered with proteinuric nephropathy.

A Structured Approach to Optimizing Animal Model Selection for Human Translation: The Animal Model Quality Assessment.

Animal studies in pharmaceutical drug discovery are common in preclinical research for compound evaluation before progression into human clinical trials. However, high rates of drug development attrition have prompted concerns regarding animal models and their predictive translatability to the clinic. To improve the characterization and evaluation of animal models for their translational relevance, the authors developed a tool to transparently reflect key features of a model that may be considered in both the application of the model but also the likelihood of successful translation of the outcomes to human patients. In this publication, we describe the rationale for the development of the Animal Model Quality Assessment tool, the questions used for the animal model assessment, and a high-level scoring system for the purpose of defining predictive translatability. Finally, we provide an example of a completed Animal Model Quality Assessment for the adoptive T-cell transfer model of colitis as a mouse model to mimic inflammatory bowel disease in humans.

Organs-on-chips: into the next decade.

Organs-on-chips (OoCs), also known as microphysiological systems or 'tissue chips' (the terms are synonymous), have attracted substantial interest in recent years owing to their potential to be informative at multiple stages of the drug discovery and development process. These innovative devices could provide insights into normal human organ function and disease pathophysiology, as well as more accurately predict the safety and efficacy of investigational drugs in humans. Therefore, they are likely to become useful additions to traditional preclinical cell culture methods and in vivo animal studies in the near term, and in some cases replacements for them in the longer term. In the past decade, the OoC field has seen dramatic advances in the sophistication of biology and engineering, in the demonstration of physiological relevance and in the range of applications. These advances have also revealed new challenges and opportunities, and expertise from multiple biomedical and engineering fields will be needed to fully realize the promise of OoCs for fundamental and translational applications. This Review provides a snapshot of this fast-evolving technology, discusses current applications and caveats for their implementation, and offers suggestions for directions in the next decade.

Key Characteristics of Cardiovascular Toxicants.

BACKGROUND: The concept of chemical agents having properties that confer potential hazard called key characteristics (KCs) was first developed to identify carcinogenic hazards. Identification of KCs of cardiovascular (CV) toxicants could facilitate the systematic assessment of CV hazards and understanding of assay and data gaps associated with current approaches. OBJECTIVES: We sought to develop a consensus-based synthesis of scientific evidence on the KCs of chemical and nonchemical agents known to cause CV toxicity along with methods to measure them. METHODS: An expert working group was convened to discuss mechanisms associated with CV toxicity. RESULTS: The group identified 12 KCs of CV toxicants, defined as exogenous agents that adversely interfere with function of the CV system. The KCs were organized into those primarily affecting cardiac tissue (numbers 1-4 below), the vascular system (5-7), or both (8-12), as follows: 1) impairs regulation of cardiac excitability, 2) impairs cardiac contractility and relaxation, 3) induces cardiomyocyte injury and death, 4) induces proliferation of valve stroma, 5) impacts endothelial and vascular function, 6) alters hemostasis, 7) causes dyslipidemia, 8) impairs mitochondrial function, 9) modifies autonomic nervous system activity, 10) induces oxidative stress, 11) causes inflammation, and 12) alters hormone signaling. DISCUSSION: These 12 KCs can be used to help identify pharmaceuticals and environmental pollutants as CV toxicants, as well as to better understand the mechanistic underpinnings of their toxicity. For example, evidence exists that fine particulate matter [PM ≤2.5µm in aerodynamic diameter (PM2.5)] air pollution, arsenic, anthracycline drugs, and other exogenous chemicals possess one or more of the described KCs. In conclusion, the KCs could be used to identify potential CV toxicants and to define a set of test methods to evaluate CV toxicity in a more comprehensive and standardized manner than current approaches. https://doi.org/10.1289/EHP9321.

A translational approach to detecting drug-induced cardiac injury with cardiac troponins: consensus and recommendations from the Cardiac Troponins Biomarker Working Group of the Health and Environmental Sciences Institute.

Cardiac troponins (cTns) are established biomarkers of ischemic heart disease in humans. However, their value as biomarkers of cardiac injury from causes other than ischemic heart disease is now being explored, particularly in drug development. In a workshop sponsored by the Cardiac Troponin Biomarker Working Group of the Health and Environmental Sciences Institute, preclinical, clinical, and regulatory scientists discussed the application of cTns in their respective environments, issues in translating the preclinical application of cTn to clinical studies, and gaps in our understanding of cTn biology and pathobiology. Evidence indicates that cTns are sensitive and specific biomarkers of cardiac injury from varying causes in both animals and humans. Accordingly, monitoring cTns can help ensure patient safety during the clinical evaluation of new drugs. In addition, preclinical characterization of cardiac risk and cTns as biomarkers of that risk can guide relevant clinical application and interpretation. We summarize here the outcomes of the workshop which included consensus statements, recommendations for further research, and a proposal for a cross-disciplinary group of clinical, regulatory, and drug development scientists to collaborate in such research.

Current challenges in the evaluation of cardiac safety during drug development: translational medicine meets the Critical Path Initiative.

In October 2008, in a public forum organized by the Cardiac Safety Research Consortium and the Health and Environmental Sciences Institute, leaders from government, the pharmaceutical industry, and academia convened in Bethesda, MD, to discuss current challenges in evaluation of short- and long-term cardiovascular safety during drug development. The current paradigm for premarket evaluation of cardiac safety begins with preclinical animal modeling and progresses to clinical biomarker or biosignature assays. Preclinical evaluations have clear limitations but provide an important opportunity to identify safety hazards before administration of potential new drugs to human subjects. Discussants highlighted the need to identify, develop, and validate serum and electrocardiogram biomarkers indicative of early drug-induced myocardial toxicity and proarrhythmia. Specifically, experts identified a need to build consensus regarding the use and interpretation of troponin assays in preclinical evaluation of myocardial toxicity. With respect to proarrhythmia, the panel emphasized a need for better qualitative and quantitative biomarkers for arrhythmogenicity, including more streamlined human thorough QT study designs and a universal definition of the end of the T wave. Toward many of these ends, large shared data repositories and a more seamless integration of preclinical and clinical testing could facilitate the development of novel approaches to both cardiac safety biosignatures. In addition, more thorough and efficient early clinical studies could enable better estimates of cardiovascular risk and better inform phase II and phase III trial design. Participants also emphasized the importance of establishing formal guidelines for data standards and transparency in postmarketing surveillance. Priority pursuit of these consensus-based directions should facilitate both safer drugs and accelerated access to new drugs, as concomitant public health benefits.

Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors.

Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2'-methoxyethyl-modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic beta cell function.