Complex In Vitro Model Characterization for Context of Use in Toxicologic Pathology: Use Cases by Collaborative Teams of Biologists, Bioengineers, and Pathologists.

Complex in vitro models (CIVMs) offer the potential to increase the clinical relevance of preclinical efficacy and toxicity assessments and reduce the reliance on animals in drug development. The European Society of Toxicologic Pathology (ESTP) and Society for Toxicologic Pathology (STP) are collaborating to highlight the role of pathologists in the development and use of CIVM. Pathologists are trained in comparative animal medicine which enhances their understanding of mechanisms of human and animal diseases, thus allowing them to bridge between animal models and humans. This skill set is important for CIVM development, validation, and data interpretation. Ideally, diverse teams of scientists, including engineers, biologists, pathologists, and others, should collaboratively develop and characterize novel CIVM, and collectively assess their precise use cases (context of use). Implementing a morphological CIVM evaluation should be essential in this process. This requires robust histological technique workflows, image analysis techniques, and needs correlation with translational biomarkers. In this review, we demonstrate how such tissue technologies and analytics support the development and use of CIVM for drug efficacy and safety evaluations. We encourage the scientific community to explore similar options for their projects and to engage with health authorities on the use of CIVM in benefit-risk assessment.

Considerations for the Identification and Conveyance of Clinical Pathology Findings in Preclinical Toxicity Studies: Results From the 9th ESTP International Expert Workshop.

The European Society of Toxicologic Pathology (ESTP) organized a panel of 24 international experts from many fields of toxicologic clinical pathology (e.g., industry, academia, and regulatory) that came together in 2021 to align the use of terminology to convey the importance of clinical pathology findings in preclinical toxicity studies. An additional goal consisted of how to identify important findings in standard and nonstandard clinical pathology associated endpoints. This manuscript summarizes the information and opinions discussed and shared at the ninth ESTP International Expert Workshop, April 5 to 6, 2022. In addition to terminology usage, the workshop considered topics related to the identification and conveyance of the importance of test item-related findings. These topics included sources of variability, comparators, statistics, reporting, correlations to other study data, nonstandard biomarkers, indirect/secondary findings, and an overall weight-of-evidence approach.

Considerations from an International Regulatory and Pharmaceutical Industry (IQ MPS Affiliate) Workshop on the Standardization of Complex In Vitro Models in Drug Development.

In May 2022, there is an International Regulatory and Pharmaceutical Industry (Innovation and Quality [IQ] Microphysiological Systems [MPS] Affiliate) Workshop on the standardization of complex in vitro models (CIVMs) in drug development. This manuscript summarizes the discussions and conclusions of this joint workshop organized and executed by the IQ MPS Affiliate and the United States Food and Drug Administration (FDA). A key objective of the workshop is to facilitate discussions around opportunities and/or needs for standardization of MPS and chart potential pathways to increase model utilization in the context of regulatory decision making. Participation in the workshop included 200 attendees from the FDA, IQ MPS Affiliate, and 26 global regulatory organizations and affiliated parties representing Europe, Japan, and Canada. It is agreed that understanding global perspectives regarding the readiness of CIVM/MPS models for regulatory decision making and potential pathways to gaining acceptance is useful to align on globally. The obstacles are currently too great to develop standards for every context of use (COU). Instead, it is suggested that a more tractable approach may be to think of broadly applicable standards that can be applied regardless of COU and/or organ system. Considerations and next steps for this effort are described.

Effects of 2,3-DPG knockout on SCD phenotype in Townes SCD model mice.

Sickle cell disease (SCD) is a severe, multisystemic hematological disorder that impacts nearly every major organ in adults. The current approved treatments for SCD directly target mutant hemoglobin or address downstream disease pathology. Several compounds targeting reduction of 2,3-DPG by activation of Pyruvate Kinase-R are currently being evaluated in SCD patients. In this study, we genetically engineered a mouse lacking 2,3-DPG on the Townes SCD mouse model background and evaluated the effects of 2,3-DPG loss on disease pathology. Animals lacking 2,3-DPG showed improvements in hematological markers and reductions in RBC sickling relative to native Townes mice, however, minimal difference in organ damage was observed in 2,3-DPG deficient mice compared to native Townes animals. When animals lacking 2,3-DPG were dosed with a compound designed to increase hemoglobin oxygen affinity, oxygen delivery related toxicity was observed.

Fibroblast activation in response to TGFß1 is modulated by co-culture with endothelial cells in a vascular organ-on-chip platform.

Background: Tissue fibrosis is a major healthcare burden that affects various organs in the body for which no effective treatments exist. An underlying, emerging theme across organs and tissue types at early stages of fibrosis is the activation of pericytes and/or fibroblasts in the perivascular space. In hepatic tissue, it is well known that liver sinusoidal endothelial cells (EC) help maintain the quiescence of stellate cells, but whether this phenomenon holds true for other endothelial and perivascular cell types is not well studied. Methods: The goal of this work was to develop an organ-on-chip microvascular model to study the effect of EC co-culture on the activation of perivascular cells perturbed by the pro-fibrotic factor TGFß1. A high-throughput microfluidic platform, PREDICT96, that was capable of imparting physiologically relevant fluid shear stress on the cultured endothelium was utilized. Results: We first studied the activation response of several perivascular cell types and selected a cell source, human dermal fibroblasts, that exhibited medium-level activation in response to TGFß1. We also demonstrated that the PREDICT96 high flow pump triggered changes in select shear-responsive factors in human EC. We then found that the activation response of fibroblasts was significantly blunted in co-culture with EC compared to fibroblast mono-cultures. Subsequent studies with conditioned media demonstrated that EC-secreted factors play at least a partial role in suppressing the activation response. A Luminex panel and single cell RNA-sequencing study provided additional insight into potential EC-derived factors that could influence fibroblast activation. Conclusion: Overall, our findings showed that EC can reduce myofibroblast activation of perivascular cells in response to TGFß1. Further exploration of EC-derived factors as potential therapeutic targets in fibrosis is warranted.

Animal models for studying COVID-19, prevention, and therapy: Pathology and disease phenotypes.

Translational models have played an important role in the rapid development of safe and effective vaccines and therapeutic agents for the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Animal models recapitulating the clinical and underlying pathological manifestations of COVID-19 have been vital for identification and rational design of safe and effective vaccines and therapies. This manuscript provides an overview of commonly used COVID-19 animal models and the pathologic features of SARS-CoV-2 infection in these models in relation to their clinical presentation in humans. Also discussed are considerations for selecting appropriate animal models for infectious diseases such as COVID-19, the host determinants that can influence species-specific susceptibility to SARS-CoV-2, and the pathogenesis of COVID-19. Finally, the limitations of currently available COVID-19 animal models are highlighted.

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.

A high-throughput microfluidic bilayer co-culture platform to study endothelial-pericyte interactions.

Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.

An orthogonal methods assessment of topical drug concentrations in skin and the impact for risk assessment in the viable epidermis.

Systemic toxicity assessments for oral or parenteral drugs often utilize the concentration of drug in plasma to enable safety margin calculations for human risk assessment. For topical drugs, there is no standard method for measuring drug concentrations in the stratum basale of the viable epidermis. This is particularly important since the superficial part of the epidermis, the stratum corneum (SC), is nonviable and where most of a topically applied drug remains, never penetrating deeper into the skin. We investigated the relative concentrations of a prototype kinase inhibitor using punch biopsy, laser capture microdissection, and imaging mass spectrometry methods in the SC, stratum basale, and dermis of minipig skin following topical application as a cream formulation. The results highlight the value of laser capture microdissection and mass spectrometry imaging in quantifying the large difference in drug concentration across the skin and even within the epidermis, and supports use of these methods for threshold-based toxicity risk assessments in specific anatomic locations of the skin, like of the stratum basale.

Microphysiological systems in early stage drug development: Perspectives on current applications and future impact.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.

Experiments in the EpiDerm 3D Skin In Vitro Model and Minipigs In Vivo Indicate Comparatively Lower In Vivo Skin Sensitivity of Topically Applied Aneugenic Compounds.

Risk management of in vitro aneugens for topically applied compounds is not clearly defined because there is no validated methodology to accurately measure compound concentration in proliferating stratum basale keratinocytes of the skin. Here, we experimentally tested several known aneugens in the EpiDerm reconstructed human skin in vitro micronucleus assay and compared the results to flow cytometric mechanistic biomarkers (phospho-H3; MPM2, DNA content). We then evaluated similar biomarkers (Ki-67, nuclear area) using immunohistochemistry in skin sections of minipigs following topical exposure the potent aneugens, colchicine, and hesperadin. Data from the EpiDerm model showed positive micronucleus responses for all aneugens tested following topical or direct media dosing with similar sensitivity when adjusted for applied dose. Quantitative benchmark dose-response analysis exhibited increases in the mitotic index biomarkers phospho-H3 and MPM2 for tubulin binders and polyploidy for aurora kinase inhibitors are at least as sensitive as the micronucleus endpoint. By comparison, the aneugens tested did not induce histopathological changes, increases in Ki-67 immunolabeling or nuclear area in skin sections from the in vivo minipig study at doses in significant excess of those eliciting a response in vitro. Results indicate the EpiDerm in vitro micronucleus assay is suitable for the hazard identification of aneugens. The lack of response in the minipig studies indicates that the barrier function of the minipig skin, which is comparable to human skin, protects from the effects of aneugens in vivo. These results provide a basis for conducting additional studies in the future to further refine this understanding.

Serum glutamate dehydrogenase activity enables early detection of liver injury in subjects with underlying muscle impairments.

Serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury. Since ALT and AST lack liver specificity, the diagnosis of the onset of hepatocellular injury in patients with underlying muscle impairments is severely limited. Thus, we evaluated the potential of glutamate dehydrogenase (GLDH) as a liver specific alternative biomarker of hepatocellular injury. In our study, serum GLDH in subjects with Duchene muscular dystrophy (DMD) was equivalent to serum GLDH in age matched healthy subjects, while serum ALT was increased 20-fold in DMD subjects. Furthermore, serum GLDH in 131 subjects with variety of muscle impairments was similar to serum GLDH of healthy subjects while serum ALT corelated with serum creatine kinase, a widely accepted biomarker of muscle impairment. In addition, significant elevations of ALT, AST, and CK were observed in a case of a patient with rhabdomyolysis, while serum GLDH stayed within the normal range until the onset of hypoxia-induced liver injury. In a mouse model of DMD (DMDmdx), serum GLDH but not serum ALT clearly correlated with the degree of acetaminophen-induced liver injury. Taken together, our data support the utility of serum GLDH as a liver-specific biomarker of liver injury that has a potential to improve diagnosis of hepatocellular injury in patients with underlying muscle impairments. In drug development, GLDH may have utility as a biomarker of drug induced liver injury in clinical trials of new therapies to treat muscle diseases such as DMD.

Recommendations for Clinical Pathology Data Generation, Interpretation, and Reporting in Target Animal Safety Studies for Veterinary Drug Development.

Clinical pathology testing is routinely performed in target animal safety studies in order to identify potential toxicity associated with administration of an investigational veterinary pharmaceutical product. Regulatory and other testing guidelines that address such studies provide recommendations for clinical pathology testing but occasionally contain outdated analytes and do not take into account interspecies physiologic differences that affect the practical selection of appropriate clinical pathology tests. Additionally, strong emphasis is often placed on statistical analysis and use of reference intervals for interpretation of test article-related clinical pathology changes, with limited attention given to the critical scientific review of clinically, toxicologically, or biologically relevant changes. The purpose of this communication from the Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathology is to provide current recommendations for clinical pathology testing and data interpretation in target animal safety studies and thereby enhance the value of clinical pathology testing in these studies.

Polyomavirus-associated Prostatitis in Wistar Han Rats Following Immunosuppression in a Chronic Toxicity Study.

Chronic prostatitis characterized on light microscopic examination by moderate, multifocal, predominantly lymphocytic inflammation associated with epithelial atypia and intranuclear and cytoplasmic inclusion-like material was identified in the prostate gland of 2 Wistar Han rats administered an immunomodulatory test article in a 6-month chronic toxicity study. Transmission electron microscopy of the prostate glands identified 45-nm, nonenveloped, icosahedral virions arranged in paracrystalline array within the cell nuclei in 1 of the 2 rats. The size, shape, location, and array pattern were most consistent with a polyomavirus. The light and electron microscopic findings after immunosuppression in our case have a resemblance to a polyomavirus recently reported to affect prostate gland epithelium in a colony of immunocompromised X-linked severe combined immune deficiency rats. To the best of our knowledge, this is the first report of light and electronic microscopic lesions in the reproductive tract associated with polyomavirus following chronic immunosuppression in a widely used, wild-type Wistar Han rat.

Principles for Assessing Adversity in Toxicologic Clinical Pathology.

There is limited direction in the literature or regulatory guidance on determination of adversity for clinical pathology (CP) biomarkers in preclinical safety studies. Toxicologic clinical pathologists representing the American Society for Veterinary Clinical Pathology-Regulatory Affairs Committee and Society of Toxicologic Pathology-Clinical Pathology Interest Group identified principles, overall approach, and unique considerations for assessing adversity in CP data interpretation to provide a consensus opinion. Emphasized is the need for pathophysiologic context and a weight-of-evidence approach. Most CP biomarkers do not have the potential to be adverse in isolation, regardless of magnitude of change. Rather, they quantify or describe the impact of effects, provide adjunct or supportive information regarding a process or pathogenesis, and provide translational biomarkers of effect. Most often, CP changes are part of a constellation of findings that collectively are adverse. Thus, most CP changes must be interpreted in conjunction with other study findings and require contextual and integrative interpretation. Exceptions include critical CP changes without correlates that indicate a health risk in the tested species. Overall, CP changes should not be interpreted in isolation and their adversity is best addressed with an integrated approach.

Scientific and Regulatory Policy Committee Points to Consider Review: Inclusion of Reproductive and Pathology End Points for Assessment of Reproductive and Developmental Toxicity in Pharmaceutical Drug Development.

Standard components of nonclinical toxicity testing for novel pharmaceuticals include clinical and anatomic pathology, as well as separate evaluation of effects on reproduction and development to inform clinical development and labeling. General study designs in regulatory guidances do not specifically mandate use of pathology or reproductive end points across all study types; thus, inclusion and use of these end points are variable. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current guidelines and practices on the use of reproductive, anatomic pathology, and clinical pathology end points in general, reproductive, and developmental toxicology studies. The Working Group constructed a survey sent to pathologists and reproductive toxicologists, and responses from participating organizations were collected through the STP for evaluation by the Working Group. The regulatory context, relevant survey results, and collective experience of the Working Group are discussed and provide the basis of each assessment by study type. Overall, the current practice of including specific end points on a case-by-case basis is considered appropriate. Points to consider are summarized for inclusion of reproductive end points in general toxicity studies and for the informed use of pathology end points in reproductive and developmental toxicity studies.

Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop.

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

STP Best Practices for Evaluating Clinical Pathology in Pharmaceutical Recovery Studies.

The Society of Toxicologic Pathology formed a working group in collaboration with the American Society for Veterinary Clinical Pathology to provide recommendations for the appropriate inclusion of clinical pathology evaluation in recovery arms of nonclinical toxicity studies but not on when to perform recovery studies. Evaluation of the recovery of clinical pathology findings is not required routinely but provides useful information on risk assessment in nonclinical toxicity studies and is recommended when the ability of the organ to recover is uncertain. The study design generally requires inclusion of concurrent controls to separate procedure-related changes from test article-related changes, but return of clinical pathology values toward baseline may be sufficient in some cases. Evaluation of either a select or full panel of standard hematology, coagulation, and serum and urine chemistry biomarkers can be scientifically justified. It is also acceptable to redesignate dosing phase animals to the recovery phase or vice versa to optimize data interpretation. Assessment of delayed toxicity during the recovery phase is not required but may be appropriate in development programs with unique concerns. Evaluation of the recovery of clinical pathology data for vaccine development is required and, for efficacy markers, is recommended if it furthers pharmacologic understanding.

Scientific and Regulatory Policy Committee Review: Review of the Organisation for Economic Co-operation and Development (OECD) Guidance on the GLP Requirements for Peer Review of Histopathology.

In 2014, the Organisation for Economic Co-operation and Development (OECD) issued guidance no. 16, Guidance on the GLP Requirements for Peer Review of Histopathology. The stated purpose of the guidance document is "to provide guidance to pathologists, test facility management, study directors and quality assurance personnel on how the peer review of histopathology should be planned, managed, documented, and reported in order to meet Good Laboratory Practice (GLP) expectations and requirements." On behalf of and in collaboration with the global societies of toxicologic pathology, the Society of Toxicologic Pathology initiated a review of OECD guidance no. 16. The objectives of this review are to provide a unified interpretation of the guidance, to recommend compliant processes for organizations to implement, and to avoid inconsistent process adaptations across the industry. This review of the guidance document is the product of a global collaboration with other societies of toxicologic pathology and provides a section-by-section international consensus view and interpretation of the OECD guidance on peer review.

Toxicity of hydroxyurea in rats and dogs.

The toxicity of hydroxyurea, a treatment for specific neoplasms, sickle-cell disease, polycythemia, and thrombocytosis that kills cells in mitosis, was assessed in repeat-dose, oral gavage studies in rats and dogs and a cardiovascular study in telemetered dogs. Hydroxyurea produced hematopoietic, lymphoid, cardiovascular, and gastrointestinal toxicity with steep dose response curves. In rats dosed for 10 days, 50 mg/kg/day was tolerated; 500 mg/kg/day produced decreased body weight gain; decreased circulating leukocytes, erythrocytes, and platelets; decreased cellularity of thymus, lymph nodes, and bone marrow; and epithelial degeneration and/or dysplasia of the stomach and small intestine; 1,500 mg/kg/day resulted in deaths on day 5. In dogs, a single dose at ≥ 250 mg/kg caused prostration leading to unscheduled euthanasia. Dogs administered 50 mg/kg/day for 1 month had decreased circulating leukocytes, erythrocytes, and platelets; increased bone marrow cellularity with decreased maturing granulocytes; increased creatinine kinase activity; and increased iron pigment in bone marrow and hepatic sinusoidal cells. In telemetered dogs, doses ≥ 15 mg/kg decreased systolic blood pressure (BP); 50 mg/kg increased diastolic BP, heart rate, and change in blood pressure over time (+dP/dt), and decreased QT and PR intervals and maximum left ventricular systolic and end diastolic pressures with measures returning to control levels within 24 hr.